The Effects of Mild Hypothermia on Patients with Severe Traumatic Brain Injury

Chinese Journal of Traumatology = Zhonghua Chuang Shang Za Zhi / Chinese Medical Association. Nov, 1998  |  Pubmed ID: 11904054

OBJECTIVE: To investigate the protective effects of mild hypothermia (33-35 degrees C) on the outcome of patients with severe traumatic brain injury (TBI) (GCS<8). METHODS: Patients in the mild hypothermia group were cooled to 33-35 degrees C by cooling blanket with muscular relaxant, and patients in the normothermia group were maintained at 37-38 degrees C. RESULTS: The result showed that the mortality was 26.1% (6/23) in the mild hypothermia group and 58.3% (14/24) in the normothermia group respectively (P<0.05). The mild hypothermia also markedly reduced intracranial pressure (P<0.01 and inhibited hyperglycermia (P<0.05). No significant side-effects were found during hypothermic treatment. CONCLUSIONS: Our clinical data have demonstrated that mild hypothermia is a useful method for management of patients with severe traumatic brain injury.

Clinical Experience of the Management for the Most Severely Head-injured Patients with GCS Score of 3

Chinese Journal of Traumatology = Zhonghua Chuang Shang Za Zhi / Chinese Medical Association. Nov, 1999  |  Pubmed ID: 11900658

OBJECTIVE: To summarize the therapeutic experience of 24 patients of traumatic head injuries with GCS score of 3. METHODS: Twenty-four most severely head-injured patients with GCS score of 3 who were admitted to our department from Jan 1995 to Mar 1998 were retrospectively analyzed. RESULTS: Twelve cases (50.0%) survived, of which 7 cases (29.2%) had good recovery or moderate disability and 5 cases with severe deficits (20.8%), and the other 12 died (50.0%) after therapy. CONCLUSIONS: The prognosis of most severely head-injured patients with GCS score of 3 could be improved by early intracranial hematoma removal with large decompressive craniotomies, early moderate hypothermia therapy, early assistant ventilation and effective prevention and treatment of complications.

Alterations of Bcl-2, Bcl-x and Bax Protein Expressions in Area CA-3 of Rat Hippocampus Following Fluid Percussion Brain Injury

Chinese Journal of Traumatology = Zhonghua Chuang Shang Za Zhi / Chinese Medical Association. Nov, 1999  |  Pubmed ID: 11900665

OBJECTIVE: To investigate the alterations of bcl-2 gene family in the area of CA-3 in rats and the molecular mechanism of neuronal apoptosis following traumatic brain injury. METHODS: To investigate the alterations of bcl-2 gene family in the area of CA-3 in rats and the molecular mechanism of neuronal apoptosis following traumatic brain injury. RESULTS: The immunoreactivity of bcl-2 and bcl-x proteins decreased in the hippocampus ipsilateral impact site at 6 hours after injury, and this was the main cause of down-regulation of the value of (bcl-2+bcl-x)/ bax. During the period of 1-3 days after injury, bax protein expression increased significantly, while bcl-2 and bcl-x protein expressions decreased relatively slowly. The decreased value of (bcl-2+bcl-x)/ bax was mainly due to the bax up-regulation. CONCLUSIONS: The bcl-2 gene family is involved in neuronal apoptosis after traumatic brain injury, and the protein-expression alterations of the bcl-2 gene family members lead to apoptosis of the neuronal cells.

Effect of Hyperventilation on Brain Tissue Oxygen Pressure, Carbon Dioxide Pressure, PH Value and Intracranial Pressure During Intracranial Hypertension in Pigs

Chinese Journal of Traumatology = Zhonghua Chuang Shang Za Zhi / Chinese Medical Association. Nov, 2000  |  Pubmed ID: 11874678

OBJECTIVE: To study the effect of hyperventilat ion on brain tissue oxygen pressure (P(ti)O(2)), brain tissue carbon dio xide pressure (P(ti)O(2)), pH value and intracranial pressure (ICP) dur ing intracranial hypertension in pigs. METHODS: Autologous arterial blood (5.5 mlplus minus0.5 ml) was injected into the left frontal lobe by micropump to establish the model of intr acerebral hematoma in pigs. After blood injection, the animals were hyperventila ted for 15 minutes to decrease the pressure of carbon dioxide in arterial blood (P(a)CO(2)) to 27.35 mm Hgplus minus11.97 mm Hg (1 mm Hg=0.133 kPa). The mean arterial pressure (MAP), intracranial pressure (ICP), cerebral perfusion pressure (CPP), P(ti)O(2), (P(ti)CO(2)), pH value and [HCO(3)(-)] were continuously monitored and the blood gas was analyzed. RESULTS: After hyperventilation, the ICP significantly decr eased (P<0.01), the CPP significantly increased (P<0.05), while the P(ti)O(2) greatly decreased to t he ischemic level (8.20 mm Hgplus minus2.50 mm Hg) (P<0.01), the P(ti)CO(2) decreased (P<0.01) and the pH value increased (P<0.01). At the same time, bl ood gas analysis showed that the P(a)CO(2) greatly decreased and the pH valu e increased. CONCLUSIONS: Hyperventilation can decrease the ICP and the P(ti)O(2) significantly. Therefore, hyperventilation should be avoided earl y after brain injury. The P(ti)O(2) monitoring will be helpful for detec ting cerebral ischemia early.

Expression of Vascular Endothelial Growth Factor in Rat Ovary During Pregnancy and Postpartum

Science in China. Series C, Life Sciences / Chinese Academy of Sciences. Aug, 2002  |  Pubmed ID: 18759025

The expressions of vascular endothelial growth factor (VEGF) in rat ovarian follicles and corpus luteum (CL) during different stages of pregnancy and postpartum were investigated. In addition, the effect of tumor necrosis factor-alpha (TNF-alpha) on VEGF expression was examined. VEGF mRNA was localized in granulosa cells of preantral follicles, as well as in the granulosa and theca cells of small antral follicles, but not in large antral follicles. VEGF transcripts were amplified in rat CL during pregnancy and postpartum using RT-PCR. The mRNA expression reached the maximal level on day 7, maintained it on days 9-18, decreased after day 18, and was minimal on day 1 postpartum. Western blot showed a 23 ku VEGF protein band in rat CL. Expression of VEGF protein varied in a similar way to that of its mRNA. Treatment with TNF-alpha at a dosage of 3000 IU/kg on day 4 of pregnancy significantly increased VEGF expression in rat CL. It is suggested that the down-regulation of VEGF expression in large antral follicles may be one factor that contributes to the termination of ovulation in rat ovary during pregnancy. VEGF may be implicated in the CL formation and maintenance via regulating angiogenesis, and its expression is regulated by TNF-alpha.

Recombinant CD16A-Ig Forms a Homodimer and Cross-blocks the Ligand Binding Functions of Neutrophil and Monocyte Fcgamma Receptors

Molecular Immunology. Jan, 2002  |  Pubmed ID: 11750654

Receptors for the Fc region of IgG (FcgammaR) are expressed as membrane-bound and soluble forms in inflammatory cells. These receptors mediate a variety of immunoregulatory functions. FcgammaR-bearing neutrophils and macrophages play a major role in mediating immune complex induced inflammatory diseases and antibody-mediated tissue injury in autoimmune diseases. To delineate the biological role of the soluble FcgammaR, a recombinant soluble CD16A (CD16A-Ig) was expressed and characterized. CD16A-Ig is secreted as a disulfide-linked homodimer of 70kDa. The purified CD16A-Ig bound to human IgG1 and IgG3 immobilized onto ELISA plates and on IgG1- and IgG3-coated erythrocytes. Saturation binding studies and Scatchard plot analysis showed that immune complex bound to the purified CD16A-Ig with high avidity. Moreover, CD16A-Ig competitively blocked the binding of cell surface-expressed CD16A-CHO cells to IgG-coated plates. The dimeric CD16A-Ig also efficiently cross-blocked the binding of IgG-coated target cells to other FcgammaR expressed on neutrophils and monocytes. These results demonstrate that CD16A-Ig forms a high avidity dimer and is capable of blocking cell-cell interactions mediated by inflammatory cells expressing FcgammaR and IgG-coated target cells. These findings suggest that the dimeric FcgammaR molecules could be used therapeutically for the intervention of immune complex-mediated inflammatory disease.

Scope and Diastereoselectivity of the "interrupted" Feist-Bénary Reaction

Organic Letters. Jan, 2002  |  Pubmed ID: 11796051

[reaction: see text] The base-promoted condensation of beta-dicarbonyl compounds with alpha-haloketones, the Feist-Bénary reaction, conveniently produces highly substituted dihydrofurans. We show here that this reaction is quite general with respect to the nature of the beta-dicarbonyl compound, proceeding with beta-ketoesters, beta-oxopropionates, beta-diketones, and beta-dialdehydes. We also show that the diastereoselectivity of this reaction depends on the acidity of the nucleophile.

Rapid Synthesis of the 7-deoxy Zaragozic Acid Core

Organic Letters. Jan, 2002  |  Pubmed ID: 11796052

[reaction: see text] We have developed an efficient synthesis of the 7-deoxy zaragozic acid core. The synthesis begins with a Feist-Bénary reaction that assembles all three carbons of the polycarboxylic acid portion of the core. This reaction is followed by highly diastereoselective aldol and dihydroxylation reactions that set the remaining stereocenters of the core. The synthesis finishes with lactol oxidation and lactone alcoholysis/ketal formation reactions to construct the bicyclic ring system of the core.

Spatial and Temporal Profile of Apoptosis Following Lateral Fluid Percussion Brain Injury

Chinese Journal of Traumatology = Zhonghua Chuang Shang Za Zhi / Chinese Medical Association. Feb, 2002  |  Pubmed ID: 11835752

To investigate the spatial and temporal profile of neural cell apoptosis following traumatic brain injury (TBI).

Colonoscopic Screening and Follow-up for Colorectal Cancer in the Elderly

World Journal of Gastroenterology : WJG. Apr, 2002  |  Pubmed ID: 11925605

To improve the prevention and treatment of senile patients with colorectal cancer by evaluating the importance of colonoscopy in clinical screening and follow-up.

Direct Gastroscopy for Detecting Gastric Cancer in the Elderly

Chinese Medical Journal. Jan, 2002  |  Pubmed ID: 11930642

To evaluate the safety and effectiveness of direct gastroscopy for detecting gastric cancer.

Expression of Matrix Metalloproteinase -2, -9 and Tissue Inhibitors of Metalloproteinase -1, -2, -3 MRNAs in Rat Uterus During Early Pregnancy

Molecular Reproduction and Development. Jun, 2002  |  Pubmed ID: 11984824

Zymography and in situ hybridizition were used to investigate matrix metalloproteinase-2, -9 (MMP-2, -9) activities, and expression of mRNAs for MMP-2, -9 and tissue inhibitors of matrix metalloproteinases (TIMP-1, -2, -3) in the rat uterus during early pregnancy (day 1-7). The zymography results showed two forms of MMP-2 (64 and 67 kDa) in the rat uteri during early pregnancy. The 64-kDa MMP-2 activity was the highest on day 2 (P < 0.01) and higher on day 5 and 6 (P < 0.05). The 67-kDa MMP-2 activity reached the highest on day 5 and 6 (P < 0.01). The 64-kDa MMP-2 activity at the implantation sites was higher than those at interimplantation sites (P < 0.05). Furthermore, the 67 kDa MMP-2 can be converted to 64 kDa forms by incubation with p-aminophenylmercuric acetate (APMA) and trypsin in vitro. The 92-kDa MMP-9 activity was only detected on day 5 and 6 of pregnancy (P < 0.01). In situ hybridization showed that on day 1-4 of pregnancy, both MMP-2 and TIMP-2 mRNAs were evidently localized in the basal stromal cells. On day 5, MMP-2 mRNA signals were decreased in the basal stromal cells and mRNA for TIMP-2 was expressed in the epithelial cells and subepithelial stromal cells. The mRNAs for MMP-9, TIMP-1, and -3 were mainly expressed in epithelial cells on day 1-5. At the implantation site on day 6, the mRNAs for MMP-2, -9, TIMP-1, -2, and -3 were highly expressed in the primary decidual zone surrounding the implanting embryo, and in the whole decidualized stromal cells (the primary and secondary decidual zones) at the implantation site on day 7. The intensities of mRNAs for the TIMPs in decidualized stromal cells at the implantation site on day 6 and 7 were stronger than those for the MMPs. The weak mRNAs for MMP-2, -9, TIMP-1, and -3 but not TIMP-2 were also observed in the ectoplacental cone/trophoblastic cells of the implanting embryos. However, at the interimplantation sites on day 6 and 7, MMP-2, -9, TIMP-1, -2, and -3 mRNAs were weakly expressed in the epithelial cells, subepithelial stromal cells, and myometrium. The results suggested that the implanting rat embryo strongly induced MMP-2 and -9 proteins and gene expression for decidulization and embryo invasion, which were strictly controlled and balanced by the simultaneous expression of TIMP-1, -2 and -3.

Measuring Receptor/ligand Interaction at the Single-bond Level: Experimental and Interpretative Issues

Annals of Biomedical Engineering. Mar, 2002  |  Pubmed ID: 12051616

There is increased interest in measuring kinetic rates, lifetimes, and rupture forces of single receptor/ligand bonds. Valuable insights have been obtained from previous experiments attempting such measurements. However, it remains difficult to know with sufficient certainty that single bonds were indeed measured. Using exemplifying data, evidence supporting single-bond observation is examined and caveats in the experimental design and data interpretation are identified. Critical issues preventing definitive proof and disproof of single-bond observation include complex binding schemes, multimeric interactions, clustering, and heterogeneous surfaces. It is concluded that no single criterion is sufficient to ensure that single bonds are actually observed. However, a cumulative body of evidence may provide reasonable confidence.

Distinct Molecular and Cellular Contributions to Stabilizing Selectin-mediated Rolling Under Flow

The Journal of Cell Biology. Aug, 2002  |  Pubmed ID: 12177042

Leukocytes roll on selectins at nearly constant velocities over a wide range of wall shear stresses. Ligand-coupled microspheres roll faster on selectins and detach quickly as wall shear stress is increased. To examine whether the superior performance of leukocytes reflects molecular features of native ligands or cellular properties that favor selectin-mediated rolling, we coupled structurally defined selectin ligands to microspheres or K562 cells and compared their rolling on P-selectin. Microspheres bearing soluble P-selectin glycoprotein ligand (sPSGL)-1 or 2-glycosulfopeptide (GSP)-6, a GSP modeled after the NH2-terminal P-selectin-binding region of PSGL-1, rolled equivalently but unstably on P-selectin. K562 cells displaying randomly coupled 2-GSP-6 also rolled unstably. In contrast, K562 cells bearing randomly coupled sPSGL-1 or 2-GSP-6 targeted to a membrane-distal region of the presumed glycocalyx rolled more like leukocytes: rolling steps were more uniform and shear resistant, and rolling velocities tended to plateau as wall shear stress was increased. K562 cells treated with paraformaldehyde or methyl-beta-cyclodextrin before ligand coupling were less deformable and rolled unstably like microspheres. Cells treated with cytochalasin D were more deformable, further resisted detachment, and rolled slowly despite increases in wall shear stress. Thus, stable, shear-resistant rolling requires cellular properties that optimize selectin-ligand interactions.

Early Indicators of Prognosis in 846 Cases of Severe Traumatic Brain Injury

Journal of Neurotrauma. Jul, 2002  |  Pubmed ID: 12184856

A number of factors, including Glasgow coma scale (GCS) score, age, pupillary response and size, hypoxia, hyperthermia, and high intracranial pressure, may play an important role in predicting the outcome of traumatic brain injury. Eight hundred forty-six cases of severe traumatic brain injury (GCS < or = 8) were analyzed retrospectively to clarify the effects of multiple factors on the prognosis of patients. At 1 year after injury, the outcomes in these cases were as follows: good recovery, 31.56%; moderate disability, 14.07%; severe disability 24.35%; vegetative status, 0.59%; and death, 29.43%. The outcomes were strongly correlated (p < 0.05) with GCS score, age, pupillary response and size, hypoxia, hyperthermia, and high intracranial pressure (ICP). These findings indicate that prevention of hypoxia, control of high ICP, and prevention of hyperthermia may be useful means for improving the outcome of patients with severe head injury.

Expression of Gelatinases and Their Tissue Inhibitors in Rat Corpus Luteum During Pregnancy and Postpartum

Molecular Reproduction and Development. Nov, 2002  |  Pubmed ID: 12237942

Extensive tissue remodeling occurs in the corpus luteum (CL) during both formation and luteolysis. Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are believed to play pivotal roles in these processes. In the present study, to evaluate the potential roles of matrix degrading proteases in luteal development and regression, we examined gelatinases and TIMP-1, -2, -3 mRNA expressions, as well as gelatinase activity in rat CL during pregnancy and postpartum using Northern blot, in situ hybridization, and gelatin zymography, respectively. The results showed that MMP-2 mRNA was only expressed at the early stages of pregnancy; TIMP-2 mRNA was highly expressed at the early and late pregnancy and day 1 postpartum, but could not be detected during the mid-phase of pregnancy; TIMP-3 mRNA expression was abundant during early pregnancy and peaked at day 7, but was absent from other time points examined. MMP-9 and TIMP-1 mRNAs in rat CL were below detectable level in the current study. Furthermore, the active MMP-2 was only present during the early stages of pregnancy, and no MMP-9 activity was observed in the zymogram. Taken together, our results suggest that MMP-2 and TIMP-3 may have functional roles in rat luteal formation, while TIMP-2 may be implicated in both formation and regression of the pregnant CL.

Changes of Bcl-x(L) and Bax MRNA Expression Following Traumatic Brain Injury in Rats

Chinese Journal of Traumatology = Zhonghua Chuang Shang Za Zhi / Chinese Medical Association. Oct, 2002  |  Pubmed ID: 12241642

To investigate the changes of bcl-2 gene family and the molecular mechanism of neuronal apoptosis following traumatic brain injury (TBI) in rats.

Identification and Specific Expression of Matrix Metalloproteinase-26 in Rhesus Monkey Endometrium During Early Pregnancy

Molecular Human Reproduction. Oct, 2002  |  Pubmed ID: 12356944

Matrix metalloproteinases (MMPs) and their tissue inhibitors may play important roles in tissue remodelling processes of the uterus. This study identified MMP-26 (endometase/matrilysin-2) in the endometrium of pregnant rhesus monkeys (Macaca mulatta) and monitored the spatial and temporal expression of the transcript and protein in the uteri on days 12, 18 and 26 of pregnancy. The partial monkey MMP-26 gene sequence of 289 nucleotides was 98% identical to that of its human homologue and its protein fragment contained a PHCGVPDGSD sequence in the prodomain identical to that in human MMP-26. RT-PCR analysis demonstrated that the average level of MMP-26 mRNA in the endometrium was high on day 12 of pregnancy, but significantly decreased on days 18 and 26 (P < 0.05). In-situ hybridization confirmed that MMP-26 mRNA is specifically localized in the endometrial compartments, with intense signals in the glandular epithelium on day 12 and in the walls of spiral arterioles adjacent to the implantation site on day 26. The hybridization signal for MMP-26 mRNA in the glandular epithelium decreased dramatically on day 18 and was undetectable on day 26. No MMP-26 mRNA transcripts were detected in the placental villi on days 18 and 26. Immunohistochemistry showed that the expression pattern of MMP-26 protein was similar to that of its mRNA. The restricted expression pattern of MMP-26 in the monkey uterus implies that this new MMP is involved in the highly regulated tissue remodelling processes of the glandular epithelium and spiral arteries during early pregnancy.

Expression and Localization of RAP250 MRNA in Rat Ovary: Possible Implications in Follicular Development and Ovulation

Endocrine. Jul, 2002  |  Pubmed ID: 12374465

The expression levels of nuclear receptor coregulators in specific tissue compartments and cells are thought to influence the expression of hormone-responsive genes involved in metabolism, development, and reproduction. RAP250 is a novel nuclear receptor coactivator highly expressed in brain and reproductive organs. To investigate the possible involvement of RAP250 in tissue-specific regulation of ovarian function, untreated immature, pregnant mare's serum gonadotropin luteinizing hormone (PMSG-LH)-primed, cycling, and pregnant rat models were used to study the localization and expression of RAP250 mRNA in rat ovary by in situ hybridization (ISH) and reverse transcriptase polymerase chain reaction (RT-PCR). The results showed that RAP250 mRNA was primarily localized to granulosa cells of healthy follicles in immature, cycling, and pregnant rats and increased during PMSG-induced follicular development. In the preovulatory and ovulatory follicles from the LH-primed rats of 48-h post-PMSG administration, the signals for RAP250 mRNA increased further and remained high until early luteal formation. Only a subset of corpora lutea during diestrus 1, diestrus 2, and initiation of pregnancy was weakly positive, and atretic follicles were largely negative. The RT-PCR results confirmed the presence of RAP250 mRNA in the rat ovary and strengthen the data from ISH. These findings suggest that RAP250 may play potential roles in follicular development and ovulation.

[Expression of Matrix Metalloproteinase-2, 9 and It's Tissue Inhibitor-1, 2 in Endometrial Carcinoma]

Zhonghua Fu Chan Ke Za Zhi. Oct, 2002  |  Pubmed ID: 12487935

To study the expression of matrix metalloproteinase (MMP)-2, 9 and tissue inhibitor of metalloproteinase (TIMP)-1, 2 protein in human endometrial carcinoma tissue and its relation to the invasion and metastasis of endometrial carcinoma.

Preliminary Exploration of the Clinical Effect of Bleomycin on Craniopharyngiomas

Stereotactic and Functional Neurosurgery. 2002  |  Pubmed ID: 12566834

To investigate the antitumor effect of bleomycin on craniopharyngiomas.

[Embryonic Regulation in Human Embryo Implantation]

Sheng Li Ke Xue Jin Zhan [Progress in Physiology]. Oct, 2002  |  Pubmed ID: 12650078

Matrix Metalloproteinase-28 Transcript and Protein Are Expressed in Rhesus Monkey Placenta During Early Pregnancy

Molecular Human Reproduction. Apr, 2003  |  Pubmed ID: 12651902

The role of matrix metalloproteinases (MMP), especially newly described MMP, in trophoblast invasion during human embryo implantation is poorly understood. In this report, using a model of early pregnancy in the rhesus monkey, we have examined the expression and localization of the most recently identified MMP, MMP-28/epilysin, transcript and protein in macaque uterine samples on days 12, 18 and 26 of pregnancy. MMP-28 mRNA expression was shown by in-situ hybridization after day 12 of pregnancy, and both the syncytial and the cytotrophoblastic cell layers of placental villi, the cytotrophoblast cells of the trophoblastic column, and the extravillous trophoblast cells of trophoblastic shell were primary producers of MMP-28 transcript. Expression of MMP-28 mRNA was undetectable in the endovascular trophoblast cells, decidual cells, luminal and glandular epithelium, arterioles, and myometrium. RT-PCR analysis amplified a fragment of 258 nucleotides from rhesus monkey uterine samples containing implantation sites on days 18 and 26. The cDNA fragment, following sequencing, was confirmed to be part of the haemopexin-like domain of MMP-28. It has 95% identity with the corresponding region of human MMP-28 gene. Immunohistochemical analysis further demonstrated that the localization of MMP-28 protein was similar to that of its mRNA. The restricted distribution pattern of this novel MMP in the villous and extravillous trophoblasts during rhesus monkey early pregnancy suggests a potential role in trophoblast invasion associated with embryo implantation.

Expression and Functional Analysis of Liver Receptor Homologue 1 As a Potential Steroidogenic Factor in Rat Ovary

Biology of Reproduction. Aug, 2003  |  Pubmed ID: 12672674

Liver receptor homologue 1 (LRH-1) is a member of the nuclear receptor superfamily originally found in liver cells. LRH-1 participates in regulation of cholesterol metabolism and bile acid synthesis. Recent studies have shown that LRH-1 is even more highly expressed in the ovary, and LRH-1 has been implicated as a key transcriptional regulator of cytochrome P450 aromatase (P450arom) in vitro. In the present study, we investigated the spatiotemporal expression patterns of LRH-1 using in situ hybridization and immunohistochemistry in ovaries from rats with a 4-day estrous cycle, from pregnant rats, from immature rats treated with eCG to stimulate follicular development, and from eCG-treated rats that were subsequently given hCG to stimulate ovulation and luteinization. To establish a potential connection between the expression of LRH-1 and that of the steroidogenic genes in vivo, we directly compared the localization patterns of LRH-1 and P450arom transcripts in consecutive ovarian sections from these animals. LRH-1 mRNA and protein were primarily localized to granulosa cells and luteinized follicles or newly formed corpora lutea (CLs) of immature and adult rats, and the levels of expression increased during eCG-hCG-induced follicular development and ovulation. In the functional CLs of pregnant rats, a biphasic change in LRH-1 mRNA content occurred throughout the gestation process, whereas LRH-1 protein was persistently detected during the entire pregnancy. In the consecutive ovarian sections, expression of LRH-1 was approximately colocalized with that of P450arom in both tertiary and Graafian follicles and the functional CLs of pregnant rats. LRH-1 mRNA and protein expression preceded those of P450arom during early follicular development. Stage-specific expression of LRH-1 in rat granulosa and luteal cells suggests a role for LRH-1 in the regulation of ovarian function. The overlapping but distinct expression patterns of LRH-1 and P450arom circumstantially support the recent finding that LRH-1 serves as a critical upstream regulator of P450arom gene expression in ovarian cells, but LRH-1 also may be a multifunctional steroidogenic factor in ovarian physiology.

Expression of Vascular Endothelial Growth Factor and Its Receptors in the Rhesus Monkey (Macaca Mulatta) Endometrium and Placenta During Early Pregnancy

Molecular Reproduction and Development. Jun, 2003  |  Pubmed ID: 12704722

Vascular endothelial growth factor (VEGF) is fundamental for development and maintenance of endometrial and placental vascular function during pregnancy. While there are a number of studies on VEGF in the human placenta, they are mostly restricted to late pregnancy. To further understand the role of VEGF in mediating angiogenesis during human early pregnancy, we employed a rhesus monkey early pregnancy model to study the temporal and spatial expression of VEGF and its receptors, fms-like tyrosine kinase (Flt)-1, and kinase-insert domain-containing receptor (KDR) mRNAs and proteins in the uteri on day 12, 18, and 26 of pregnancy using in situ hybridization, RT-PCR, and immunohistochemistry. VEGF mRNA had been identified in the luminal epithelium on day 12, in the glandular epithelium on day 12 and 18, and the highest expression was detected in the walls of some spiral arterioles adjacent to the implantation site on day 18, in the placental villi and in the fetal-maternal border on day 18 and 26. Besides, immunostaining of VEGF was detected in the placental villi and endometrial compartments including spiral arteries walls and the glandular epithelium. The localization of VEGF in the endothelium correlates with the presence of Flt-1 and KDR receptors on vascular structure. All the results above suggest that VEGF-VEGFR pairs were involved in the process of trophoblast invasion, maternal vascular transformation, and fetoplacental vascular differentiation and development during the rhesus monkey early pregnancy. Expression of VEGF, Flt-1, and KDR in the epithelial cells also hints some additionally functional roles of VEGF during early pregnancy.

Direct Observation of Catch Bonds Involving Cell-adhesion Molecules

Nature. May, 2003  |  Pubmed ID: 12736689

Bonds between adhesion molecules are often mechanically stressed. A striking example is the tensile force applied to selectin-ligand bonds, which mediate the tethering and rolling of flowing leukocytes on vascular surfaces. It has been suggested that force could either shorten bond lifetimes, because work done by the force could lower the energy barrier between the bound and free states ('slip'), or prolong bond lifetimes by deforming the molecules such that they lock more tightly ('catch'). Whereas slip bonds have been widely observed, catch bonds have not been demonstrated experimentally. Here, using atomic force microscopy and flow-chamber experiments, we show that increasing force first prolonged and then shortened the lifetimes of P-selectin complexes with P-selectin glycoprotein ligand-1, revealing both catch and slip bond behaviour. Transitions between catch and slip bonds might explain why leukocyte rolling on selectins first increases and then decreases as wall shear stress increases. This dual response to force provides a mechanism for regulating cell adhesion under conditions of variable mechanical stress.

Standard Large Trauma Craniotomy for Severe Traumatic Brain Injury

Chinese Journal of Traumatology = Zhonghua Chuang Shang Za Zhi / Chinese Medical Association. Oct, 2003  |  Pubmed ID: 14514369

To study the effect of standard large trauma craniotomy (SLTC) on outcomes of patients with severe traumatic brain injury (TBI) (GCS<=8).

Effects of Ganglioside GM1 on Reduction of Brain Edema and Amelioration of Cerebral Metabolism After Traumatic Brain Injury

Chinese Journal of Traumatology = Zhonghua Chuang Shang Za Zhi / Chinese Medical Association. Feb, 2003  |  Pubmed ID: 12542960

To observe the effects of ganglioside GM1 on reduction of brain edema and amelioration of cerebral metabolism after traumatic brain injury (TBI).

[Relationship Between Methylenetetrahydrofolate Reductase Gene Polymorphism and Homocysteine in Type 1 Diabetic Microvascular Complications]

Zhonghua Er Ke Za Zhi. Chinese Journal of Pediatrics. Jul, 2003  |  Pubmed ID: 14746692

[Association of Human Leukocyte Antigen Non-classical Genes with Type 1 Diabetes]

Zhonghua Er Ke Za Zhi. Chinese Journal of Pediatrics. Apr, 2003  |  Pubmed ID: 14754527

HLA-DMA and DMB are non-classical genes whose product (DM molecules) plays an important role in antigen presentation. Our present study was designed to investigate the relationship between human leukocyte antigen-DMA, -DMB and clinical status heterogeneity of type 1 diabetes.

[Observation Amyloplasts in the Gravity-insensitive Mutant of Rice Under Gravity and Microgravity Conditions]

Shi Yan Sheng Wu Xue Bao. Dec, 2003  |  Pubmed ID: 14724941

Using histochemistry and optical microscope, we examined the number and the size of amyloplasts in specialized tissues of gravitropically receptive organs-tissues such as the coleoptile and sheath of rice mutant (insensitive to gravity) and wide type (Oryza sativa L. subsp. japonica) (Zhonghua 11). We found there was no statistical difference between the mutant and wide type, both of which grew on earth or on the clinostat respectively. On earth, it was found that amyloplasts sedimented at the distal end of each cell of the special starch sheath tissues and re-sedimentation of amyloplasts toward the direction of gravity was almost completed in 5 minutes after inverting the seedlings. On the clinostat, amyloplasts dispersed in the starch sheath tissue. Such observations indicated that the mutation was not resulted from the starch-deficiency or starch-absence, the further research is going on.

The Smad Pathway in Transforming Growth Factor-beta Signaling

Science in China. Series C, Life Sciences / Chinese Academy of Sciences. Oct, 2003  |  Pubmed ID: 19448985

The Smad pathway is involved in transforming growth factor-beta (TGF-beta) signal transduction. The Smad complex binds with the promoter of target gene to modulate gene transcription. Various transcriptional coactivators and corepressors associate directly with Smads for appropriate binding of Smads to target promoters and regulation of Smads transcriptional activities. The ultimate degradation of Smads mediated by the ubiquitin-proteasome pathway (UPP) has been established as a mechanism to shut off the Smad pathway. In addition to the Smad pathway, TGF-beta can also activate other signaling pathway such as the MAPK pathway. The cross-talk of the Smad pathway with other signaling pathways constitutes an important mechanism for the regulatory network of TGF-beta signaling.

Secreted Brevican MRNA is Expressed in the Adult Rat Pituitary

Biochemical and Biophysical Research Communications. Feb, 2004  |  Pubmed ID: 14741698

Brevican is the most abundant chondroitin sulfate proteoglycan in the extracellular matrix (ECM) of the adult rat brain. It has been found only in the central nervous system (CNS). In this study, we found that secreted brevican transcript was detectable in the pituitary of both male and female adult rats by reverse transcriptase-polymerase chain reaction (RT-PCR) amplification. In posterior lobe of pituitary, pituicytes were heavily labelled. In anterior and intermediate lobes of pituitary, signals for brevican transcripts were observed in cells of various sizes. These data demonstrated that secreted brevican mRNA is expressed in the adult rat pituitary and brevican might not be a CNS-specific ECM.

Forces Required to Initiate Membrane Tether Extrusion from Cell Surface Depend on Cell Type but Not on the Surface Molecule

Mechanics & Chemistry of Biosystems : MCB. Dec, 2004  |  Pubmed ID: 16783921

When a cell adhered to another cell or substratum via surface proteins is forced to detach, lipid membrane tethers are often extruded from the cell surface before the protein bond dissociates. For example, during the inflammatory reaction leukocytes roll on the surface of activated endothelial cells. The rolling adhesion is mediated by interactions of selectins with their ligands, e.g., P-selectin glycoprotein ligand (PSGL)-1, which extrudes membrane tethers from the surfaces of both leukocytes and endothelial cells. Membrane tether extrusion has been suggested to regulate leukocyte rolling. Here we examine several factors that may affect forces required to initiate membrane tethers, or initial tether force. It was found that initial tether forces were similar regardless of the presence or absence of the cytoplasmic tail of P-selectin and regardless of whether the tethers were extruded via binding to PSGL-1 or Fcy receptors. Initial tether forces were found to depend on the cell types tested and were greatly reduced by treatment of latrunculin A, which inhibits actin polymerization. These data provide additional insights to the control of membrane tether extrusion, which should be taken into account when cellular functions such as rolling where tether extrusion plays a regulatory role are compared using different cell types expressing the same molecule.

Normoxic Induction of the Hypoxic-inducible Factor-1 Alpha by Interleukin-1 Beta Involves the Extracellular Signal-regulated Kinase 1/2 Pathway in Normal Human Cytotrophoblast Cells

Biology of Reproduction. Jun, 2004  |  Pubmed ID: 14960485

During early pregnancy, an environment of relative low oxygen tension is essential for normal embryonic and placental vasculature. In low-oxygen conditions, the hypoxic-inducible factor-1 (HIF-1), composed of alpha and beta subunits, controls the expression of a number of genes such as vascular endothelial growth factor (VEGF), a key angiogenic factor. The recent studies in some tumor cells have found that the labile component, HIF-1 alpha, is not only activated by hypoxia but also by peptides such as interleukin-1 (IL-1) in normoxia. In this article, we demonstrated that exposure of normal human cytotrophoblast cells to IL-1 beta stimulated the expression of HIF-1 alpha protein. Meanwhile, IL-1 beta also induced the secretion of VEGF in normal human cytotrophoblast cells. Our data indicated that IL-1 beta induced extracellular signal-regulated kinase (ERK) 1/2 phosphorylation. Moreover, treatment of cells with PD98059, an inhibitor of ERK1/2 signaling, inhibited the stimulation of HIF-1 alpha protein expression and VEGF secretion by IL-1 beta. These data indicate that, in normal human cytotrophoblast cells, IL-1 beta induces HIF- 1 alpha-mediated VEGF secretion and that IL-1 beta-stimulated ERK1/2 activation may be involved in this process.

Effect of Mild Hypothermia on Brain Dialysate Lactate After Fluid Percussion Brain Injury in Rodents

Neurosurgery. Mar, 2004  |  Pubmed ID: 15028148

To investigate the effects of mild hypothermia on brain microdialysate lactate after fluid percussion traumatic brain injury (TBI) in rats.

Expression of Smad2 and Smad4, Transforming Growth Factor-beta Signal Transducers in Rat Endometrium During the Estrous Cycle, Pre-, and Peri-implantation

Animal Reproduction Science. Feb, 2004  |  Pubmed ID: 15036506

SMADs are intracellular signaling molecules that transmit signals elicited by members of transforming growth factor-beta (TGF-beta) superfamily. To decipher the mechanism of TGF-beta signaling during the estrous cycle and implantation, we performed in situ hybridization to investigate the expression patterns of mRNAs for Smad2 and Smad4 in rat endometrium during the estrous cycle and on Days 0.5, 1.5, 2.5, 3.5, 4.5, 5.5, and 6.5 of pregnancy. Intense epithelial expression of Smad2 mRNA at diestrus and proestrus was reduced at estrus and metaestrus, while Smad4 maintained its constitutive expression during the estrous cycle. During pre-implantation, both Smads were accumulated in the luminal epithelium and the glandular epithelium. Contrary to the dramatic Smad4 expression, Smad2 was highly down-regulated on Day 2.5 and was increased on Day 3.5. During peri-implantation, both Smads were expressed in the luminal epithelium, subepithelial stroma, and the primary decidual zone. Smad4 was down-modulated on Day 5.5. These results suggest that (a) both Smads are involved in the tissue remodeling of cycling and pregnant rat uteri; (b) TGF-beta signaling functions mainly in the epithelium during pre-implantation and Smad2 is involved in the endometrial switch from the neutral phase to the receptive phase; (c) TGF-beta signaling is down-regulated at the time when trophoblast invasion begins and both Smads are involved in the formation of the primary decidual zone.

[Survey of Type 1 Diabetes Incidence in Children from 1997 to 2000 in Beijing Area]

Zhonghua Er Ke Za Zhi. Chinese Journal of Pediatrics. Feb, 2004  |  Pubmed ID: 15059486

The incidence of type 1 diabetes varied in different countries, different nations and different regions. This survey was conducted to clarify the incidence of type 1 diabetes of children in Beijing area between 1997 and 2000, to compare and analyze the difference in incidence of type 1 diabetes between the 2 periods of 1988 - 1996 and 1997 - 2000.

Involvement of ERK1/2 Pathway in TGF-beta1-induced VEGF Secretion in Normal Human Cytotrophoblast Cells

Molecular Reproduction and Development. Jun, 2004  |  Pubmed ID: 15095341

Transforming growth factor-beta1 (TGF-beta1) plays a pivotal role in the angiogenesis during the development of placenta, but the intracellular signaling mechanism by which TGF-beta1 stimulates this process remains poorly understood. In this article, we demonstrated that exposure of normal human cytotrophoblast cells to TGF-beta1 stimulated the secretion of the VEGF gene encoding vascular endothelial growth factor, which is a key factor in angiogenesis. Meanwhile, treatment of normal human cytotrophoblast cells with TGF-beta1-induced expression of HIF-1a, the regulated subunit of hypoxia-inducible factor 1, a known transactivator of the VEGF gene. Our data indicated that TGF-beta1 induced extracellular signal- regulated kinase (ERK) 1/2 phosphorylation in normal human cytotrophoblast cells. Moreover, treating cells with PD98059, an inhibitor of ERK1/2 signaling, inhibited TGF-beta1 stimulation of VEGF secretion and HIF-1a protein expression. These data indicated that in normal human cytotrophoblast cells, TGF-beta1 induced HIF-1a-mediated VEGF secretion, and TGF-beta1-stimulated-ERK1/2 activation may be involved in this process.

Cinchona Alkaloid-lewis Acid Catalyst Systems for Enantioselective Ketene-aldehyde Cycloadditions

Journal of the American Chemical Society. May, 2004  |  Pubmed ID: 15113194

Asymmetric cinchona alkaloid-catalyzed acid chloride-aldehyde cyclocondensation (AAC) reactions afford enantioenriched 4-substituted and 3,4-disubstituted beta-lactones with near perfect absolute and relative stereocontrol. These reactions are characterized by the operational simplicity derived from using commercially available or easily obtained (one-step) reaction catalysts and in situ ketene generation from acid chlorides. The range of aldehyde substrates that serve as effective AAC substrates include sterically hindered aldehydes such as cyclohexanecarboxaldehyde and pivaldehyde.

Proliferative Response of Corneal Endothelial Cells from Young and Older Donors

Investigative Ophthalmology & Visual Science. Jun, 2004  |  Pubmed ID: 15161835

To compare the effect of epidermal growth factor (EGF), nerve growth factor (NGF), platelet-derived growth factor-BB (PDGF-BB), bovine pituitary extract, and fetal bovine serum (FBS), alone or in combination, on proliferation of human corneal endothelial cells (HCEC) cultured from young (<30 years old) and older donors (>50 years old).

Expression of Proteasome Subunits Low Molecular Mass Polypeptide (LMP) 2 and LMP7 in the Endometrium and Placenta of Rhesus Monkey (Macaca Mulatta) During Early Pregnancy

Biology of Reproduction. Oct, 2004  |  Pubmed ID: 15201202

Previous studies have demonstrated that the ubiquitin-proteasome pathway plays an important role in embryo implantation. Low molecular mass polypeptide (LMP) 2 and LMP7 are the two subunits of 20S proteasome, which are critical for proteasome activity. To further elucidate the roles of LMP2 and LMP7 in embryo implantation during early pregnancy, we cloned partial sequences of the LMP2 and LMP7 genes and studied the spatiotemporal expression of LMP2 and LMP7 in rhesus monkey (Macaca mulatta) uteri on Days 12, 18, and 26 of pregnancy. The results showed that the 349-base pair (bp) LMP2 fragment and the 340-bp LMP7 fragment were 97% and 99% identical, respectively, to those of human homologues. From the statistical results of reverse transcription-polymerase chain reaction, in situ hybridization, and immunohistochemistry, we found that the expression levels of LMP2 and LMP7 significantly increased with the elongation of pregnancy. The LMP2 and LMP7 mRNAs were mainly expressed in the luminal and glandular epithelium on Day 12 of pregnancy. On Days 18 and 26 of pregnancy, strong signals of LMP2 and LMP7 mRNAs were detected in the placental villi, trophoblastic column, and arterial endothelial cells close to the implantation site, and moderate expressions were found in the trophoblastic shell and glandular epithelium. The LMP2 and LMP7 mRNAs were extensively distributed in the stroma on Day 26 of pregnancy. The expression patterns of LMP2 and LMP7 proteins were similar to those of their transcripts, but weak immunostaining of LMP2 and LMP7 proteins was detected in stroma at all stages of pregnancy. These results suggest that LMP2 and LMP7 may be involved in some key processes of trophoblastic invasion, angiogenesis, degradation of the extracellular matrix, immune tolerance, and glandular secretion.

[Clinical Significance of Pancreatic Beta-cell Function in Obese Children with Acanthosis Nigricans]

Zhonghua Er Ke Za Zhi. Chinese Journal of Pediatrics. Jun, 2004  |  Pubmed ID: 15265420

The strong relation between type 2 diabetes mellitus and obesity with acanthosis nigricans is widely concerned. This study investigated the pancreatic beta-cell function in obese children with acanthosis nigricans, so as to find out the role of insulin secretion and insulin resistance in obese children with acanthosis nigricans.

Mechanical Switching and Coupling Between Two Dissociation Pathways in a P-selectin Adhesion Bond

Proceedings of the National Academy of Sciences of the United States of America. Aug, 2004  |  Pubmed ID: 15277675

Many biomolecular bonds exhibit a mechanical strength that increases in proportion to the logarithm of the rate of force application. Consistent with exponential decrease in bond lifetime under rising force, this kinetically limited failure reflects dissociation along a single thermodynamic pathway impeded by a sharp free energy barrier. Using a sensitive force probe to test the leukocyte adhesion bond P-selectin glycoprotein ligand 1 (PSGL-1)-P-selectin, we observed a linear increase of bond strength with each 10-fold increase in the rate of force application from 300 to 30,000 pN/sec, implying a single pathway for failure. However, the strength and lifetime of PSGL-1-P-selectin bonds dropped anomalously when loaded below 300 pN/sec, demonstrating unexpectedly faster dissociation and a possible second pathway for failure. Remarkably, if first loaded by a "jump" in force to 20-30 pN, the bonds became strong when subjected to a force ramp as slow as 30 pN/sec and exhibited the same single-pathway kinetics under all force rates. Applied in this way, a new "jump/ramp" mode of force spectroscopy was used to show that the PSGL-1-P-selectin bond behaves as a mechanochemical switch where force history selects between two dissociation pathways with markedly different properties. Furthermore, replacing PSGL-1 by variants of its 19-aa N terminus and by the crucial tetrasaccharide sialyl LewisX produces dramatic changes in the failure kinetics, suggesting a structural basis for the two pathways. The two-pathway switch seems to provide a mechanism for the "catch bond" response observed recently with PSGL-1-P-selectin bonds subjected to small-constant forces.

Quantifying the Effects of Molecular Orientation and Length on Two-dimensional Receptor-ligand Binding Kinetics

The Journal of Biological Chemistry. Oct, 2004  |  Pubmed ID: 15299021

Surface presentation of adhesion receptors influences cell adhesion, although the mechanisms underlying these effects are not well understood. We used a micropipette adhesion frequency assay to quantify how the molecular orientation and length of adhesion receptors on the cell membrane affected two-dimensional kinetic rates of interactions with surface ligands. Interactions of P-selectin, E-selectin, and CD16A with their respective ligands or antibody were used to demonstrate such effects. Randomizing the orientation of the adhesion receptor or lowering its ligand- and antibody-binding domain above the cell membrane lowered two-dimensional affinities of the molecular interactions by reducing the forward rates but not the reverse rates. In contrast, the soluble antibody bound with similar three-dimensional affinities to cell-bound P-selectin constructs regardless of their orientation and length. These results demonstrate that the orientation and length of an adhesion receptor influences its rate of encountering and binding a surface ligand but does not subsequently affect the stability of binding.

[Diagnosis and Treatment of Methylmalonic Acidemia in 14 Cases]

Zhonghua Er Ke Za Zhi. Chinese Journal of Pediatrics. Aug, 2004  |  Pubmed ID: 15347443

Methylmalonic acidemia (MMA) is one of the most common disorders of congenital organic acid metabolism. This study aimed at exploring the clinical characteristics and treatment of the disease to help improve our understanding of it.

Catch Bonds Govern Adhesion Through L-selectin at Threshold Shear

The Journal of Cell Biology. Sep, 2004  |  Pubmed ID: 15364963

Flow-enhanced cell adhesion is an unexplained phenomenon that might result from a transport-dependent increase in on-rates or a force-dependent decrease in off-rates of adhesive bonds. L-selectin requires a threshold shear to support leukocyte rolling on P-selectin glycoprotein ligand-1 (PSGL-1) and other vascular ligands. Low forces decrease L-selectin-PSGL-1 off-rates (catch bonds), whereas higher forces increase off-rates (slip bonds). We determined that a force-dependent decrease in off-rates dictated flow-enhanced rolling of L-selectin-bearing microspheres or neutrophils on PSGL-1. Catch bonds enabled increasing force to convert short-lived tethers into longer-lived tethers, which decreased rolling velocities and increased the regularity of rolling steps as shear rose from the threshold to an optimal value. As shear increased above the optimum, transitions to slip bonds shortened tether lifetimes, which increased rolling velocities and decreased rolling regularity. Thus, force-dependent alterations of bond lifetimes govern L-selectin-dependent cell adhesion below and above the shear optimum. These findings establish the first biological function for catch bonds as a mechanism for flow-enhanced cell adhesion.

Human Corneal Endothelial Cell Proliferation: Potential for Use in Regenerative Medicine

Cornea. Nov, 2004  |  Pubmed ID: 15448474

To review and update the experience of our laboratory in culturing human corneal endothelial cells (HCEC) from young and older donors.

Effect of Ubiquitin-proteasome Pathway on Mouse Blastocyst Implantation and Expression of Matrix Metalloproteinases-2 and -9

Biology of Reproduction. Feb, 2004  |  Pubmed ID: 14561647

Previous studies have documented that ubiquitin-related proteins are present in human, baboon, rhesus monkey, cow, sheep, and mouse pregnant uteri, indicating that the ubiquitin-proteasome pathway (UPP) may be involved in the extensive uterine remodeling during mammalian early pregnancy, but there is still no direct evidence. A mouse intrauterine injection model was employed to study the direct effect of the UPP on mouse embryo implantation and its possible mechanisms. On Day 3 of pregnancy in each mouse, one of the uterine horns in each mouse was injected with different concentrations of lactacystin, a specific proteasome inhibitor, or anti-ubiquitin antibody, and the other side was used as a control. On days 5, 6, and 7, the number of implanted embryos was counted and the expression and gelatinolytic activities of matrix metalloproteinase-2 (MMP-2) and MMP-9 were studied. Results presented here illustrate that injection of lactacystin and anti-ubiquitin antibody significantly inhibited mouse embryo implantation. Further investigations by reverse transcription-polymerase chain reaction and gelatin zymography showed that MMP-2 and MMP-9 mRNA expression, as well as the gelatinolytic activity of MMP-9 in the lactacystin-treated uterine horn, significantly decreased, whereas the activity of MMP-2 was not significantly affected. The results obtained from this study, together with previous reports, suggest that the UPP is involved in mouse embryo implantation, and UPP's effect on embryo implantation is achieved at least in part by regulating MMP-2 and MMP-9 mRNA expression and the gelatinolytic activity of MMP-9.

Low Force Decelerates L-selectin Dissociation from P-selectin Glycoprotein Ligand-1 and Endoglycan

The Journal of Biological Chemistry. Jan, 2004  |  Pubmed ID: 14573602

Selectin-ligand interactions mediate the tethering and rolling of circulating leukocytes on vascular surfaces during inflammation and immune surveillance. To support rolling, these interactions are thought to have rapid off-rates that increase slowly as wall shear stress increases. However, the increase of off-rate with force, an intuitive characteristic named slip bonds, is at odds with a shear threshold requirement for selectin-mediated cell rolling. As shear drops below the threshold, fewer cells roll and those that do roll less stably and with higher velocity. We recently demonstrated a low force regime where the off-rate of P-selectin interacting with P-selectin glycoprotein ligand-1 (PSGL-1) decreased with increasing force. This counter-intuitive characteristic, named catch bonds, might partially explain the shear threshold phenomenon. Because L-selectin-mediated cell rolling exhibits a much more pronounced shear threshold, we used atomic force microscopy and flow chamber experiments to determine off-rates of L-selectin interacting with their physiological ligands and with an antibody. Catch bonds were observed at low forces for L-selectin-PSGL-1 interactions coinciding with the shear threshold range, whereas slip bonds were observed at higher forces. These catch-slip transitional bonds were also observed for L-selectin interacting with endoglycan, a newly identified PSGL-1-like ligand. By contrast, only slip bonds were observed for L-selectin-antibody interactions. These findings suggest that catch bonds contribute to the shear threshold for rolling and are a common characteristic of selectin-ligand interactions.

Preliminary Study on a Gravity-insensitive Rice Mutant

Journal of Zhejiang University. Science. Feb, 2004  |  Pubmed ID: 14674024

A gravity-insensitive mutant was isolated from rice (Oryza sativa L. cv. Zhonghua 11) transformed by Agrobacterium tumefaciens. The mutant's shoot growth (prostrate growth) was insensitive to gravity; whereas root growth displayed a normal positive gravitropism. Histological observation of root caps and leaf sheaths indicated that there was no significant difference in the number and size of amyloplasts in cells of the mutant and cells of the wild type.

Catalytic Asymmetric Acyl Halide-aldehyde Cyclocondensation Reactions of Substituted Ketenes

Journal of the American Chemical Society. Jan, 2004  |  Pubmed ID: 14709036

Catalytic asymmetric acyl halide-aldehyde cyclocondensation (AAC) reactions of alkyl-substituted ketenes with structurally diverse aldehydes provide cis-disubstituted beta-lactones with high enantioselectivity. The AAC reactions utilize a novel Al(III)-triamine catalyst in which the metal's dynamic coordination geometry leads to a highly selective catalyst complex. These AAC reactions represent a functional solution to highly enantioselective substituted ester enolate aldol additions.

Force History Dependence of Receptor-ligand Dissociation

Biophysical Journal. Feb, 2005  |  Pubmed ID: 15556978

Receptor-ligand bonds that mediate cell adhesion are often subjected to forces that regulate their dissociation via modulating off-rates. Off-rates control how long receptor-ligand bonds last and how much force they withstand. One should therefore be able to determine off-rates from either bond lifetime or unbinding force measurements. However, substantial discrepancies exist between the force dependence of off-rates derived from the two types of measurements even for the same interactions, e.g., selectins dissociating from their ligands, which mediate the tethering and rolling of leukocytes on vascular surfaces during inflammation and immune surveillance. We used atomic force microscopy to measure survival times of P-selectin dissociating from P-selectin glycoprotein ligand 1 or from an antibody in both bond lifetime and unbinding force experiments. By a new method of data analysis, we showed that the discrepancies resulted from the assumption that off-rates were functions of force only. The off-rates derived from forced dissociation data depended not only on force but also on the history of force application. This finding provides a new paradigm for understanding how force regulates receptor-ligand interactions.

Expression of Adamalysin 19/ADAM19 in the Endometrium and Placenta of Rhesus Monkey (Macaca Mulatta) During Early Pregnancy

Molecular Human Reproduction. Jun, 2005  |  Pubmed ID: 15901844

A disintegrin and metalloproteinase (ADAM) 19 may contribute to multiple processes including proteolysis, adhesion and intracellular signalling. These processes are also critical for embryo implantation. The aim of this study was to investigate the spatio-temporal expression of the ADAM19 in rhesus monkey uteri on days 12, 18 and 26 of pregnancy. The results showed that in the cloned monkey 346 bp ADAM19 gene fragment and 114 amino acid residues were 98 and 100% identical to those of human homologues, respectively. In-situ hybridization confirmed that the ADAM19 mRNA was located in the luminal and glandular epithelium on day 12 of pregnancy. On day 18 of pregnancy, strong signals of the ADAM19 mRNA were detected in the placental villi, trophoblastic column and glandular epithelium near the myometrium. Moderate expression of the ADAM19 mRNA was seen in the trophoblastic shell and stromal cells. The placental villi and trophoblastic column expressed abundant ADAM19 mRNA, and ADAM19 transcripts were also detected in the trophoblastic shell and fetal-maternal border on day 26 of pregnancy. The expression pattern of the ADAM19 protein was similar to its transcript, but signals for the ADAM19 protein in the stromal cells and trophoblastic shell increased more than its mRNA on day 18 of pregnancy. Statistical analysis demonstrated that the expression level of ADAM19 significantly increased on day 18 of pregnancy. These data suggest that the ADAM19 may be involved in the key processes of glandular secretion, trophoblast invasion and degradation of extracellular matrix during early pregnancy.

Expressions and Regulation of Endothelial and Inducible Nitric Oxide Synthases in Mouse Uterus During the Estrous Cycle and Early Pregnancy

Frontiers in Bioscience : a Journal and Virtual Library. 2005  |  Pubmed ID: 15970571

Nitric oxide (NO) has been implicated in many cellular processes. We examined the temporal and spatial expressions of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) in mouse uteri during the estrous cycle and early pregnancy, as well as the regulation of eNOS and iNOS by estradiol (E2) and progesterone (P4) in ovariectomized mouse uteri using in situ hybridization and immunohistochemistry. Our results showed that positive eNOS and iNOS signals were localized in the uterine luminal epithelium and glandular epithelium during the estrous cycle. In ovariectomized mice, both E2 and P4 regulated the expression of eNOS and iNOS. During early pregnancy, eNOS and iNOS were detected not only in epithelium, but also in the primary decidual zone surrounding implanting embryos on day 6 of pregnancy, and in the whole decidualized stroma on day 7 of pregnancy. In conclusion, the results demonstrated that two NOS isoforms were localized in mouse uteri in specific temporal and spatial patterns during the estrous cycle and early pregnancy, and ovarian hormones can regulate their expression. Furthermore, the data suggest that the expression of NOS during the peri-implantation period might lead to enhance NO production, which could promote embryo implantation.

Expression of Matrix Metalloproteinase-26 (MMP-26) MRNA in Mouse Uterus During the Estrous Cycle and Early Pregnancy

Life Sciences. Nov, 2005  |  Pubmed ID: 15987643

Matrix metalloproteinases (MMPs) and their tissue inhibitors play important roles in the remodeling of extracellular matrix (ECM). MMP-26, also called endometase or matrilysin-2, is a novel member of the MMP family. The present study was to investigate the temporal and spatial expression of MMP-26 mRNA in mouse uterus during the estrous cycle and early pregnancy by using in situ hybridization and semi-quantitative RT-PCR. In this study, MMP-26 mRNA was found to be localized to the luminal and glandular epithelium at proestrus and estrus, and the expression level was decreased significantly from metestrus to dioestrus. During pre-implantation period, MMP-26 mRNA was predominantly expressed in luminal and glandular epithelium at much higher level; whereas it switched to stroma during peri-implantation period, and also appeared in the blastocysts and the implantation sites. The results suggested that MMP-26 might play a role in the cycling changes of mouse uterus during the estrous cycle and embryo implantation.

Catch Bonds: Physical Models and Biological Functions

Molecular & Cellular Biomechanics : MCB. Sep, 2005  |  Pubmed ID: 16708472

Force can shorten the lifetimes of receptor-ligand bonds by accelerating their dissociation. Perhaps paradoxical at first glance, bond lifetimes can also be prolonged by force. This counterintuitive behavior was named catch bonds, which is in contrast to the ordinary slip bonds that describe the intuitive behavior of lifetimes being shortened by force. Fifteen years after their theoretical proposal, catch bonds have finally been observed. In this article we review recently published data that have demonstrated catch bonds in the selectin system and suggested catch bonds in other systems, the theoretical models for their explanations, and their function as a mechanism for flow-enhanced adhesion.

Two-dimensional Kinetics Regulation of AlphaLbeta2-ICAM-1 Interaction by Conformational Changes of the AlphaL-inserted Domain

The Journal of Biological Chemistry. Dec, 2005  |  Pubmed ID: 16234238

The leukocyte integrin alphaLbeta2 mediates cell adhesion and migration during inflammatory and immune responses. Ligand binding of alphaLbeta2 is regulated by or induces conformational changes in the inserted (I) domain. By using a micropipette, we measured the conformational regulation of two-dimensional (2D) binding affinity and the kinetics of cell-bound intercellular adhesion molecule-1 interacting with alphaLbeta2 or isolated I domain expressed on K562 cells. Locking the I domain into open and intermediate conformations with a disulfide bond increased the affinities by approximately 8000- and approximately 30-fold, respectively, from the locked closed conformation, which has similar affinity as the wild-type I domain. Most surprisingly, the 2D affinity increases were due mostly to the 2D on-rate increases, as the 2D off-rates only decreased by severalfold. The wild-type alphaLbeta2, but not its I domain in isolation, could be up-regulated by Mn2+ or Mg2+ to have high affinities and on-rates. Locking the I domain in any of the three conformations abolished the ability of divalent cations to regulate 2D affinity. These results indicate that a downward displacement of the I domain C-terminal helix, induced by conformational changes of other domains of the alphaLbeta2, is required for affinity and on-rate up-regulation.

Comparison of the Proliferative Capacity of Human Corneal Endothelial Cells from the Central and Peripheral Areas

Investigative Ophthalmology & Visual Science. Nov, 2005  |  Pubmed ID: 16249484

To compare the relative proliferative capacity between human corneal endothelial cells (HCECs) cultured from the central and peripheral areas of the cornea.

Catch Bonds: Physical Models, Structural Bases, Biological Function and Rheological Relevance

Biorheology. 2005  |  Pubmed ID: 16369083

Force can shorten the lifetimes of macromolecular complexes (e.g., receptor-ligand bonds) by accelerating their dissociation. Perhaps paradoxical at first glance, bond lifetimes can also be prolonged by force. This counterintuitive behavior was named catch bonds, which is in contrast to the ordinary slip bonds that describe the intuitive behavior of lifetimes being shortened by force. Fifteen years after their theoretical proposal, catch bonds have finally been observed. In this article we review recently published data that have demonstrated catch bonds in the selectin system and suggested catch bonds in other systems, the theoretical models for their explanations, possible structural bases, their relation to flow-enhanced adhesion, and the potential biorheological relevance.

Thermo-mechanical Responses of a Surface-coupled AFM Cantilever

Journal of Biomechanical Engineering. Dec, 2005  |  Pubmed ID: 16502663

Atomic force microscopy (AFM) has been widely used for measuring mechanical properties of biological specimens such as cells, DNA, and proteins. This is usually done by monitoring deformations in response to controlled applied forces, which have to be at ultralow levels due to the extreme softness of the specimens. Consequently, such experiments may be susceptible to thermal excitations, manifested as force and displacement fluctuations that could reduce the measurement accuracy. To take advantage of, rather than to be limited by, such fluctuations, we have characterized the thermomechanical responses of an arbitrarily shaped AFM cantilever with the tip coupled to an elastic spring. Our analysis shows that the cantilever and the specimen behave as springs in parallel. This provides a method for determining the elasticity of the specimen by measuring the change in the tip fluctuations in the presence and absence of coupling. For rectangular and V-shaped cantilevers, we have derived a relationship between the mean-square deflection and the mean-square inclination and an approximate expression for the specimen spring constant in terms of contributions to the mean-square inclination from the first few vibration modes.

T Cells Like a Firm Molecular Handshake

Proceedings of the National Academy of Sciences of the United States of America. Mar, 2006  |  Pubmed ID: 16537367

Effects of E-cadherin on Mouse Embryo Implantation and Expression of Matrix Metalloproteinase-2 and -9

Biochemical and Biophysical Research Communications. May, 2006  |  Pubmed ID: 16564031

E-cadherin is a cell surface glycoprotein, which is responsible for adhesion between epithelial cells. Whether it is involved in embryo implantation is still unknown. In a mouse intrauterine horn injection model, one uterine horn in each mouse was injected with different doses of E-cadherin antibody on day 3 of pregnancy. The results showed that embryo implantation was significantly inhibited in the mice injected with 3 microg E-cadherin antibody. The mouse uteri in this group were collected on days 5, 6, and 7 of pregnancy and expressions of MMP-2 and -9 were studied. In situ hybridization and RT-PCR results showed that the expression of MMP-2 and -9 mRNAs in uteri of E-cadherin antibody treated group was increased on days 5-7. The results of gelatin zymography of MMPs showed that the activities of pro-MMP-2, MMP-2, and pro-MMP-9 were increased significantly on days 5 and 6, and pro-MMP-9 activity was increased on day 7. The present study suggested that E-cadherin was involved in embryo implantation through decreasing the expressions and activities of MMP-2 and -9.

Probabilistic Modeling of Rosette Formation

Biophysical Journal. Jul, 2006  |  Pubmed ID: 16603493

Rosetting, or forming a cell aggregate between a single target nucleated cell and a number of red blood cells (RBCs), is a simple assay for cell adhesion mediated by specific receptor-ligand interaction. For example, rosette formation between sheep RBC and human lymphocytes has been used to differentiate T cells from B cells. Rosetting assay is commonly used to determine the interaction of Fc gamma-receptors (FcgammaR) expressed on inflammatory cells and IgG coated on RBCs. Despite its wide use in measuring cell adhesion, the biophysical parameters of rosette formation have not been well characterized. Here we developed a probabilistic model to describe the distribution of rosette sizes, which is Poissonian. The average rosette size is predicted to be proportional to the apparent two-dimensional binding affinity of the interacting receptor-ligand pair and their site densities. The model has been supported by experiments of rosettes mediated by four molecular interactions: FcgammaRIII interacting with IgG, T cell receptor and coreceptor CD8 interacting with antigen peptide presented by major histocompatibility molecule, P-selectin interacting with P-selectin glycoprotein ligand 1 (PSGL-1), and L-selectin interacting with PSGL-1. The latter two are structurally similar and are different from the former two. Fitting the model to data enabled us to evaluate the apparent effective two-dimensional binding affinity of the interacting molecular pairs: 7.19x10(-5) microm4 for FcgammaRIII-IgG interaction, 4.66x10(-3) microm4 for P-selectin-PSGL-1 interaction, and 0.94x10(-3) microm4 for L-selectin-PSGL-1 interaction. These results elucidate the biophysical mechanism of rosette formation and enable it to become a semiquantitative assay that relates the rosette size to the effective affinity for receptor-ligand binding.

Expression of Tissue Inhibitor of Metalloproteinase-4 (TIMP-4) in Endometrium and Placenta of Rhesus Monkey (Macaca Mulatta) During Early Pregnancy

Life Sciences. May, 2006  |  Pubmed ID: 16375928

The tissue inhibitors of metalloproteinases (TIMPs) are secreted as important regulators of the matrix metalloproteinases (MMPs). TIMP-4 is the most recently characterized member of the TIMPs family. In the present study, we examined the expression and localization of the TIMP-4 transcript and protein in endometrium and placenta of rhesus monkey (Macaca mulatta) on days 12, 18 and 26 of pregnancy using RT-PCR, in situ hybridization, and immunohistochemistry. The fragment of TIMP-4 gene from rhesus monkey uterine samples shared 95% identity with the corresponding region of human homologue. On day 12 of pregnancy, TIMP-4 mRNA was mainly expressed in the glandular and luminal epithelium. On days 18 and 26 of pregnancy, the expression of TIMP-4 mRNA tended to decline in glandular epithelium and there were strong staining in the placental villi. Furthermore, TIMP-4 mRNA was very faint or undetectable in the stromal cells, endothelial cells of arterioles and myometrium at any stages of pregnancy. The results of immunohistochemical analysis were similar to that of its mRNA. These findings indicate that TIMP-4 might play an important role in glandular secretion, endometrial tissue remodeling and invasion of the trophoblast cells by regulating MMPs in a localized manner in the uteri of rhesus monkey during early pregnancy.

Gene Expression of Transforming Growth Factor-beta Receptors Types I and II in Rat Endometrium During the Estrous Cycle and Early Pregnancy

Life Sciences. May, 2006  |  Pubmed ID: 16426643

Roles of transforming growth factor-beta (TGF-beta) receptor types I (TbetaRI) and II (TbetaRII) during the estrous cycle and implantation of rodents are currently unclear. In the present study, the spatial and temporal expressions of TbetaRI and TbetaRII in rat endometrium during the estrous cycle, pre-, and peri-implantation were examined using in situ hybridization and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). With in situ hybridization, TbetaRI and TbetaRII were expressed at weak levels in rat endometrium during the estrous cycle. During pre-implantation, both receptors were expressed in the luminal epithelium and glandular epithelium on Days 0.5 and 1.5 of pregnancy, but were down-regulated on Days 2.5 and 3.5. During peri-implantation, both TGF-beta receptors were localized in the luminal epithelium and subepithelial stroma to facilitate attachment reaction and trophoblast invasion. They were highly expressed on Day 4.5, whereas were down-regulated on Days 5.5 and 6.5. Semi-quantitative RT-PCR analysis confirmed the data obtained by in situ hybridization. These results suggest that during pre-implantation, both TGF-beta receptors are functional in the proliferation of endometrial epithelial cells. During peri-implantation, both TGF-beta receptors play important roles during the onset of the uterine receptivity and the attachment reaction. TGF-beta signaling is down-regulated when trophoblast invasion begins.

Measuring Molecular Elasticity by Atomic Force Microscope Cantilever Fluctuations

Biophysical Journal. Jan, 2006  |  Pubmed ID: 16258054

In single-molecule mechanics experiments the molecular elasticity is usually measured from the deformation in response to a controlled applied force, e.g., via an atomic force microscope cantilever. We have tested the validity of an alternative method based on a recently developed theory. The concept is to measure the change in thermal fluctuations of the cantilever tip with and without its coupling to a rigid surface via the molecule. The new method was demonstrated by its application to the elasticity measurements of L- and P-selectin complexed with P-selectin glycoprotein ligand-1 or their respective antibodies, which showed values comparable to those measured from the slope of the force-extension curve. L- and P-selectin were found to behave as nearly linear springs capable of sustaining large forces and strains without sudden unfolding. The measured spring constants of approximately 4 and approximately 1 pN/nm for L- and P-selectin, respectively, suggest that a physiological force of approximately 100 pN would result in an approximately 200% strain for the respective selectins.

Role and Regulation of Nodal/activin Receptor-like Kinase 7 Signaling Pathway in the Control of Ovarian Follicular Atresia

Molecular Endocrinology (Baltimore, Md.). Oct, 2006  |  Pubmed ID: 16709598

Although the role of the TGF beta superfamily members in the regulation of ovarian folliculogenesis has been extensively studied, their involvement in follicular atresia is not well understood. In the present study, we have demonstrated for the first time that Nodal, a member of the TGF beta superfamily, is involved in promoting follicular atresia as evidenced by the following: 1) colocalization of Nodal and its type I receptor Activin receptor-like kinase 7 (ALK7) proteins in the granulosa cells was only observed in atretic antral follicles, whereas they were present in theca cells and granulosa cells of healthy follicles, respectively; 2) addition of recombinant Nodal or overexpression of Nodal by adenoviral infection induced apoptosis of otherwise healthy granulosa cells; 3) constitutively active ALK7 (ALK7-ca) overexpression mimicked the function of Nodal in the induction of granulosa cell apoptosis. Furthermore, overexpression of Nodal or ALK7-ca increased phosphorylation and nuclear translocation of Smad2, decreased X-linked inhibitor of apoptotic proteins (Xiap) expression at both mRNA and protein level and phospho-Akt content, as well as triggered mitochondrial release of death proteins Smac/DIABLO, Omi/HtrA2, and cytochrome c in the granulosa cells. Dominant-negative Smad2 significantly attenuated ALK7-ca-induced down-regulation of Xiap and thus rescued granulosa cells from undergoing apoptosis. In addition, whereas up-regulation of Xiap significantly attenuated ALK7-ca-induced apoptosis, down-regulation of Xiap sensitized granulosa cells to ALK7-ca-induced apoptosis. Furthermore, ALK7-ca-induced apoptosis was significantly attenuated by forced expression of activated Akt, and Akt rescued granulosa cells from undergoing apoptosis via proteasome-mediated ALK7 degradation. Taken together, Nodal plays an atretogenic role in the ovary where it induces granulosa cell apoptosis through activation of Smad2, down-regulation of the key survival molecules Xiap and phospho-Akt, as well as the activation of mitochondrial death pathway.

Catalytic Asymmetric Assembly of Stereodefined Propionate Units: an Enantioselective Total Synthesis of (-)-pironetin

Journal of the American Chemical Society. Jun, 2006  |  Pubmed ID: 16756287

Double diastereoselection in alkaloid-catalyzed acyl halide-aldehyde cyclocondensation (AAC) reactions provides a strategy for realizing syn- or anti-selective propionate aldol additions from a common reaction manifold. Matched AAC homologation of enantioenriched aldehydes afford cis-disubstituted beta-lactones as surrogates for syn aldols; the mismatched AAC reactions provide anti-selective aldols in the form of trans-disubstituted 2-oxetanones. The utility of this reaction technology in synthesis activities is exemplified in a catalytic asymmetric total synthesis of (-)-pironetin.

P27kip1 SiRNA Induces Proliferation in Corneal Endothelial Cells from Young but Not Older Donors

Investigative Ophthalmology & Visual Science. Nov, 2006  |  Pubmed ID: 17065491

To determine whether small interfering (si)RNA downregulation of the cyclin-dependent kinase inhibitor p27kip1 overcomes G(1)-phase arrest and promotes cell-cycle progression in human corneal endothelial cells (HCECs) from young (<30 years old) and older (>60 years old) donors.

Expression of Prostasin and Protease Nexin-1 in Rhesus Monkey (Macaca Mulatta) Endometrium and Placenta During Early Pregnancy

The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society. Oct, 2006  |  Pubmed ID: 16801525

Serine proteases have been documented to play key roles in uterine matrix turnover and trophoblastic invasion during implantation. Roles of prostasin serine protease in these processes, however, are currently unclear. The present study was first conducted to investigate the colocalization of prostasin and its cognate serpin, protease nexin-1 (PN-1), in rhesus monkey endometrium and placenta on days 12, 18, and 26 of pregnancy by using in situ hybridization (ISH) and immunohistochemistry. With ISH, expression of prostasin mRNA was intensely localized in the glandular epithelium on days 12 and 18 and in the placental villi, trophoblastic column, trophoblastic shell, and fetal-maternal border on days 18 and 26. With the progress of pregnancy, expression level in the glandular epithelium was significantly decreased, and the accumulation in the placental compartments was further increased. In addition, the stroma and arterioles exhibited modest levels of prostasin signals. However, expression level of PN-1 in these compartments on adjacent sections in the three stages of early pregnancy was weak or below the level of detection. Prostasin protein expression in the endometrium was found to be consistent with the distribution patterns revealed in the ISH experiments. It may be suggested from these results that prostasin is involved in endometrial epithelial morphology establishment, tissue remodeling, and trophoblastic invasion during early pregnancy. The cognate serpin PN-1 was not coordinately expressed along with prostasin, creating a tissue environment favorable for proteolytic activities of prostasin during early pregnancy events.

Accumulation of Copper in Brown Rice and Effect of Copper on Rice Growth and Grain Yield in Different Rice Cultivars

Chemosphere. Dec, 2006  |  Pubmed ID: 16844189

A pot experiment with 38 commonly cultured rice cultivars showed that the effect of Cu (100 mg kg(-1)) on rice growth, grain yield and accumulation of Cu in brown rice varied greatly with different cultivars. Although the average Cu concentration in brown rice of the 38 cultivars was significantly increased (P<0.01) compared with the control, in none of the cultivars did Cu concentration in brown rice exceed the maximum permissible limit of 10 mg Cu kg(-1). This suggests that rice grown in Cu-contaminated paddy soil (100 mg Cu kg(-1)) will not adversely affect human health through the food chain. Because of the significant negative correlation between grain weight and Cu concentration in brown rice with the soil Cu treatment, screening for cultivars with low Cu accumulation in brown rice and high grain yield for Cu-contaminated areas is feasible. The present research led to the recommendation of three such cultivars: Jiahua, Zhenxian 866, Zhe 733. The average grain yield under Cu treatment (100 mg Cu kg(-1) soil) was significantly (P<0.01) reduced compared with the control. The decreases or increases of grain yields mainly resulted from the combined effects of the panicles per pot, spikelets per panicle and filled spikelets per panicle under the soil Cu treatment. Furthermore, there were significant (r=0.869, P<0.01) positive correlations between the RC (relative changes) of spikelets per panicle and filled spikelets per panicle under the soil Cu treatment.

Flow-enhanced Adhesion Regulated by a Selectin Interdomain Hinge

The Journal of Cell Biology. Sep, 2006  |  Pubmed ID: 17000883

L-selectin requires a threshold shear to enable leukocytes to tether to and roll on vascular surfaces. Transport mechanisms govern flow-enhanced tethering, whereas force governs flow-enhanced rolling by prolonging the lifetimes of L-selectin-ligand complexes (catch bonds). Using selectin crystal structures, molecular dynamics simulations, site-directed mutagenesis, single-molecule force and kinetics experiments, Monte Carlo modeling, and flow chamber adhesion studies, we show that eliminating a hydrogen bond to increase the flexibility of an interdomain hinge in L-selectin reduced the shear threshold for adhesion via two mechanisms. One affects the on-rate by increasing tethering through greater rotational diffusion. The other affects the off-rate by strengthening rolling through augmented catch bonds with longer lifetimes at smaller forces. By forcing open the hinge angle, ligand may slide across its interface with L-selectin to promote rebinding, thereby providing a mechanism for catch bonds. Thus, allosteric changes remote from the ligand-binding interface regulate both bond formation and dissociation.

[Liposome-mediated Glial Growth Factor 2 Gene Therapy in Brain Injury: an Experimental Study with Rats]

Zhonghua Yi Xue Za Zhi. Sep, 2006  |  Pubmed ID: 17156635

To explore the protective effect of glial growth factor-2 (GGF2) on brain injury.

Retraction Notice to "Expression of Matrix Metalloproteinase-26 (MMP-26) MRNA in Mouse Uterus During the Estrous Cycle and Early Pregnancy"

Life Sciences. Nov, 2006  |  Pubmed ID: 17176513

Marked Protection by Selective Cerebral Profound Hypothermia After Complete Cerebral Ischemia in Primates

Journal of Neurotrauma. Dec, 2006  |  Pubmed ID: 17184193

Hypothermia has been demonstrated to protect the brain from ischemia or traumatic brain injury. Achieving profound hypothermia has relied on techniques requiring total body cooling, which may result in serious cardiovascular and pulmonary complications. A technique to selectively cool the brain could conceivably exert a marked protection on cerebral structures and provide a relatively bloodless operative surgical field without systemic complications. Accordingly, this approach was tried in 7 rhesus monkeys after induction of general anesthesia. The right internal carotid artery and both internal jugular veins were each occlusively cannulated and connected to a circulation pump. The left internal carotid artery, both external carotid arteries, and both external jugular veins were temporarily clamped to establish severe cerebral ischemia. Using a closed-circuit system, cooled Ringer's lactate liquid (4 degrees C) was infused through right internal carotid artery with outflow draining though both internal jugular veins. Cooled perfusate decreased cerebral temperature to the target temperature of 15 degrees C. Thereafter, pump flow was discontinued, and brains were rewarmed spontaneously, while the temporarily clamped carotid arteries and jugular veins were opened to resume normal cerebral blood circulation. Neurological functions were recorded daily and cerebral histology was examined at the conclusion of the experiment. Magnetic resonance (MR) scans were routinely taken before and 3 weeks after ischemia. In the normothermia control group of five rhesus monkeys, Ringer's solution at 37 degrees C was infused in the same manner as the cold solution with cerebral temperature maintained at 36.7 +/- 0.32 degrees C. Right cerebral temperature decreased from 36.5 +/- 0.49 to 15.5 +/- 2.29 degrees C, and simultaneously the left cerebral temperature decreased from 36.4 +/- 0.38 to 16.3 +/- 2.4 degrees C for 62.8 +/- 9.76 min during selective cerebral cooled Ringer's liquid perfusion. In contrast, rectal temperature was only reduced to 32.4 +/- 0.96 degrees C from a baseline of 37.2 +/- 0.76 degrees C. Internal jugular vein hematocrit was 38.2 +/- 0.31% before perfusion and 2.82 +/- 0.46% at the end of perfusion in profound hypothermia group; hematocrit was 39.7 +/- 0.62% before perfusion and 3.42 +/- 0.38% at the end of perfusion in the normothermia group. In the hypothermic group, neurological functions were normal during 6 months of follow-up, and microscopic examination of brain tissue did not show evidence of pathological changes in hippocampus or medulla. MR scans did not show any cerebral infarction. In contrast, none of the monkeys in normothermia group survived for more than several hours, and microscopic examination of the brain revealed extensive neuronal necrosis within the medulla. Selective cerebral profound hypothermia provides significant histologic and neurologic protection after severe cerebral ischemia. In addition, there were no major complications, and the operative field remained relatively bloodless in the profound hypothermic group.

Involvement of SMAD4, but Not of SMAD2, in Transforming Growth Factor-beta1-induced Trophoblast Expression of Matrix Metalloproteinase-2

Frontiers in Bioscience : a Journal and Virtual Library. 2006  |  Pubmed ID: 16146757

Matrix metalloproteinases (MMPs) play crucial roles in extravillous trophoblast invasion. In the present study, we examined the possible role of Smad4 and Smad2 in transforming growth factor (TGF)-beta1-induced MMP-2 expression, using the well-established invasive extravillous trophoblast cell line HTR-8/SVneo. Recombinant sense Smad4 or Smad2 retroviral vectors were constructed by inserting full-length Smad4 or Smad2 cDNA into pLXSN retroviral vector. Stable PT67 packaging cell clones were isolated and viral supernatants were used to infect HTR-8/SVneo cells. Effects of retroviral expression of Smad4 and Smad2 on TGF-beta1-regulated MMP-2 expression were assessed by semi-quantitative reverse transcription-polymerase chain reaction and gelatin zymography. The results showed that over-expression of Smad4 augmented MMP-2 mRNA abundance and the secretion of pro-MMP-2, and mimicked the inductive effect of TGF-beta1 on the production of MMP-2. However, retrovirus-mediated sense Smad2 gene transfer had no effect. These findings suggest that Smad4, but not Smad2, mediates TGF-beta1-induced MMP-2 expression in invasive extravillous trophoblasts.

Proteasome Subunit LMP2 is Required for Matrix Metalloproteinase-2 and -9 Expression and Activities in Human Invasive Extravillous Trophoblast Cell Line

Journal of Cellular Physiology. Mar, 2006  |  Pubmed ID: 16222703

The ubiquitin-proteasome pathway (UPP) is involved in the degradation of the extracellular matrix (ECM) and trophoblastic invasion during early pregnancy. Our previous studies demonstrated that inhibition of UPP suppresses expression of matrix metalloproteinase (MMP)-2 and -9. LMP2 is an important proteasome subunit that is critical for proteasome activity. This study investigated the regulatory mechanism of LMP2 on the expression and activities of MMP-2 and MMP-9. Our results showed that transfection of LMP2 siRNA plasmid into the human invasive extravillous trophoblast cell line (HTR8/Svneo) could significantly suppress expression of LMP2 mRNA and protein. The mRNA expression of MMP-2 and MMP-9 and their activities were markedly decreased in the LMP2-inhibited cells. Inhibition of LMP2 could also reduce IkappaBalpha mRNA level, although the expression of phosphorylated IkappaBalpha was increased. In the LMP2-inhibited cells, expression of mRNA encoding NF-kappaB subunits p50 and p65 remained normal, but the p50 protein level was significantly decreased in the cytosolic and nuclear extracts, while p65 protein was markedly reduced only in the nuclear extract. We also demonstrated that blockage of the NF-kappaB pathway by the NF-kappaB translocation inhibitor SN50 markedly reduced the expression of MMP-2 and MMP-9 in HTR8/Svneo cells, a result that is fully consistent with the results from the LMP2-inhibited HTR8/Svneo cells. These data suggest that LMP2 contributes to IkappaBalpha degradation and p50 generation, and that inhibition of LMP2 suppresses expression and activities of MMP-2 and MMP-9 by blocking the transfer of active NF-kappaB heterodimers into the nucleus.

Expression of Smad Ubiquitin Regulatory Factor 2 (Smurf2) in Rhesus Monkey Endometrium and Placenta During Early Pregnancy

The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society. May, 2007  |  Pubmed ID: 17189523

Smad ubiquitin regulatory factor 2 (Smurf2) is an E3 ubiquitin ligase that is involved in the Smad-mediated TGF-beta signaling. TGF-beta has been shown to play an important role during normal embryo implantation, but whether Smurf2 is involved in this process has not been reported. This study was first conducted to investigate the expression of Smurf2 transcript and protein in different compartments of the rhesus monkey uteri and placenta during early pregnancy. The results showed that both the cloned partial sequence of Smurf2 gene and the corresponding amino acid residues shared 99% identity with those of human homologs. On day 12 (D12) of pregnancy, strong signals of Smurf2 mRNA were found in basalis glandular epithelium and luminal epithelium, and moderate expressions were detected in functionalis glandular epithelium. During early villi stage and villi placental stage, Smurf2 mRNAs were mainly localized in the placenta villi, trophoblastic column, trophoblastic shell, and basalis glandular epithelium. There appeared strong staining signals in the arterioles on D26 of pregnancy, but faint staining signals on D18 of pregnancy. No specific staining of Smurf2 mRNA was observed in stromal cells and myometrium. The expression pattern of Smurf2 protein was generally similar to that of its mRNA. These results provide the first evidence that Smurf2 may play specific roles in glandular secretion, trophoblastic cell invasion, and placentation through mediating the expression of the related proteins of TGF-beta signaling pathway during early pregnancy.

Affinity and Kinetic Analysis of Fcgamma Receptor IIIa (CD16a) Binding to IgG Ligands

The Journal of Biological Chemistry. Mar, 2007  |  Pubmed ID: 17202140

Binding of pathogen-bound immunoglobulin G (IgG) to cell surface Fc gamma receptors (FcgammaRs) triggers a wide variety of effector functions. The binding kinetics and affinities of IgG-FcgammaR interactions are hence important parameters for understanding FcgammaR-mediated immune functions. We have measured the kinetic rates and equilibrium dissociation constants of IgG binding to a soluble FcgammaRIIIa fused with Ig Fc (sCD16a) using the surface plasmon resonance technique. sCD16a interacted with monomeric human IgG and its subtypes IgG1 and IgG3 as well as rabbit IgG with on-rates of 6.5 x 10(3), 8.2 x 10(3), 1.1 x 10(4) and 1.8 x 10(4) m(-1) s(-1), off-rates of 4.7 x 10(-3), 5.7 x 10(-3), 5.9 x 10(-3), and 1.9 x 10(-2) s(-1), and equilibrium dissociation constants of 0.72, 0.71, 0.56, and 1.1 mum, respectively. The kinetics and affinities measured by surface plasmon resonance agreed with those obtained from real time flow cytometry and competition inhibition binding experiments using cell surface CD16a. These data add to our understanding of IgG-FcgammaR interactions.

[Construction of Plant Expression Vectors with Fusion Gene of Helicobacter Pylori CagA, UreB and Ctb and Its Genetic Transformation in Tobacco]

Wei Sheng Wu Xue Bao = Acta Microbiologica Sinica. Feb, 2007  |  Pubmed ID: 17436619

Helicobacter pylori (Hp) is the principal cause of most chronic active gastric and peptic ulcer disease, and also is closely related with gastric cancer and MALT lymphoma. Current vaccines are expensive to produce and deliver, however, transgenic plants expressing recombinant vaccine immunogens offer an attractive and potential inexpensive alternative to vaccination and injection. In this study, plant expression vectors which harbor Hp related proteins CagA and UreB were constructed. Fusion gene ctb-linker-cagA and ctb-linker-ureB were cut from vectors p1300-WxCLCN and p1300-WxCLUN, and then constructed into vector pCAMBIA2301 which was under the control of the CaMV 35S promoter by series molecular methods. Those reconstructed vectors were named p2301-35SCLCN and p2301-35SCLUN and were introduced into Agrobacterium tumefaciens strain EHA105 .Tobacco was transformed by co-cultivating leaf discs with Agrobacterium strains harboring fusion genes. The regenerated Kanamycin-resistant transforms were selected, elongated, rooted and transferred fdr flowering in greenhouse. Recombinant plant expression vectors were confirmed by digestion and PCR and transgenic plants were analyzed by PCR, GUS histochemical assays, PCR-Southern blot. The results show that more than 80% transgenic plants are confirmed to be positive ones and these results also indicate that ctb-linker-cagA and ctb-linker-ureB are integrated into the genomic DNA of the tobacco which laid a solid foundation for the research of establishing transgenic plants as bioreactors to carry microbe antigen and Hp transgenic plant vaccines.

Gonadotropin and Intra-ovarian Signals Regulating Follicle Development and Atresia: the Delicate Balance Between Life and Death

Frontiers in Bioscience : a Journal and Virtual Library. 2007  |  Pubmed ID: 17485326

Regulation of mammalian follicular development is tightly regulated by both cell death and survival signals, including endocrine (e.g. gonadotropin) and intra-ovarian regulators (e.g. Nodal and GDF9). The destiny of the individual follicle (growth/ovulation or atresia) is dependent on a delicate balance in the expression and action of factors promoting follicular cell proliferation, growth and differentiation, and of those promoting programmed cell death (apoptosis). Development of the follicle from the primordial to preantral stage is regulated by oocyte-derived factors including GDF9 and BMP15, and is not dependent on gonadotropin support (gonadotropin-independent stage). As the follicle transits into the early antral stage it becomes responsive to gonadotropin (gonadotropin-responsive stages) and further development renders the follicle completely dependent on the presence of gonadotropin while modulated by intra-ovarian regulators (gonadotropin-dependent). Follicle fate is also regulated by pro-apoptotic factors such as the intraovarian regulator Nodal, which is secreted by the theca and promotes apoptosis of differentiated granulosa cells through a mechanism involving Smad2 signaling and suppression of the PI3K/Akt pathway. The intracellular protein prohibitin (PHB) appears to have a dual role during folliculogenesis; acting as a cell survival factor in undifferentiated cells, and as a pro-apoptotic factor following differentiation. Further investigations of the interplay between these endocrine and ovarian regulators will lead to a better understanding into the regulation of follicular development and atresia, allowing development of new techniques for assisted reproduction.

Wild Fulvous Fruit Bats (Rousettus Leschenaulti) Exhibit Human-like Menstrual Cycle

Biology of Reproduction. Aug, 2007  |  Pubmed ID: 17494915

We investigated the menstrual cycle of wild fulvous fruit bats (Rousettus leschenaulti), focusing on changes in the endometrial and ovarian structure and pituitary and steroid hormones. The menstrual cycle lasts for 33 days in bats studied in their natural habitat and in captivity. Vaginal bleeding was restricted to a single day (Day 1). A preovulatory follicle was found in the ovary on Day 18 when the levels of LH and FSH reached their maxima, accompanied by a thickened endometrium. On Day 24, serum levels of progesterone and estradiol-17 were also maximal, and uterine glands increased in size. After that, the levels of progesterone dropped precipitously, leading to menstrual bleeding. Both the morphologic and hormonal changes observed in fulvous fruit bats during the menstrual cycle resemble similar changes in humans. Fulvous fruit bats may be useful nonprimate laboratory models to study menstruation and menstrual dysfunction.

Role of PPARgamma and Its Gonadotrophic Regulation in Rat Ovarian Granulosa Cells in Vitro

Neuro Endocrinology Letters. Jun, 2007  |  Pubmed ID: 17627264

The peroxisome proliferator-activated receptors (PPARs), including PPARalpha, PPARbeta/delta, and PPARgamma, are a family of transcription factors belonging to the steroid receptor superfamily. In rat ovary, PPARgamma is mainly expressed in granulosa cells of developing follicles, implying a possible role of PPARgamma in ovarian functions. In the present study, the role of PPARgamma and its gonadotrophic regulation in granulosa cells collected from diethylstilbestrol-treated immature rats were studied. The results showed that PPARgamma could inhibit proliferation and induce apoptosis in primarily cultured granulosa cells. PPARgamma could also stimulate the biosynthesis of estradiol and progesterone after FSH pretreatment, and FSH could regulate the functions of PPARgamma through PKA, ERK1/2, and p38 MAPK signaling pathways. These data suggested that PPARgamma may be involved in follicular atresia and FSH-stimulated steroidogenesis during follicle development.

Expression of Prostasin Serine Protease and Protease Nexin-1 (PN-1) in Rhesus Monkey Ovary During Menstrual Cycle and Early Pregnancy

The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society. Dec, 2007  |  Pubmed ID: 17827166

Serine proteases and their cognate serpin-class inhibitors are involved in the controlled proteolytic events during follicular development, ovulation, formation, and maintenance of the corpus luteum (CL). In this study, we investigated the expression patterns of prostasin serine protease and protease nexin-1 (PN-1), a serine protease inhibitor also called serpin-E2, in rhesus monkey ovaries during the menstrual cycle and early pregnancy, by using in situ hybridization and immunohistochemistry. Expression of prostasin was localized in oocyte, granulosa cells, and/or theca cells of early antral follicles and antral follicles, with high levels observed in preovulatory follicles. Prostasin was also localized at high levels of abundance in the CL during the menstrual cycle and early pregnancy. During the menstrual cycle, PN-1 was coordinately localized with prostasin in oocytes, granulosa cells, and theca cells of antral follicles and preovulatory follicles and in the CL. In addition, the PN-1 expression level in macaque CL during early pregnancy increased as pregnancy proceeded. We propose that prostasin may be involved in follicular development, ovulation, and CL formation, whereas PN-1 may be present to regulate the proteolysis in these processes.

Determination of the Early Time of Death by Computerized Image Analysis of DNA Degradation: Which is the Best Quantitative Indicator of DNA Degradation?

Journal of Huazhong University of Science and Technology. Medical Sciences = Hua Zhong Ke Ji Da Xue Xue Bao. Yi Xue Ying De Wen Ban = Huazhong Keji Daxue Xuebao. Yixue Yingdewen Ban. Aug, 2007  |  Pubmed ID: 17828487

This study evaluated the correlation between DNA degradation of the splenic lymphocytes and the early time of death, examined the early time of death by computerized image analysis technique (CIAT) and identified the best parameter that quantitatively reflects the DNA degradation. The spleen tissues from 34 SD rats were collected, subjected to cell smearing every 2 h within the first 36 h after death, stained by Feulgen-Van's staining, three indices reflecting DNA content in splenic lymphocytes, including integral optical density (IOD), average optical density (AOD), average gray scale (AG) were measured by the image analysis. Our results showed that IOD and AOD decreased and AG increased over time within the first 36 h. A stepwise linear regression analysis showed that only AG was fitted. A correlation between the postmortem interval (PMI) and AG was identified and the corresponding regression equation was obtained. Our study suggests that CIAT is a useful and promising tool for the estimation of early PMI with good objectivity and reproducibility, and AG is a more effective and better quantitative indicator for the estimation of PMI within the first 36 h after death in rats.

A Catch to Integrin Activation

Nature Immunology. Oct, 2007  |  Pubmed ID: 17878912

Transport Governs Flow-enhanced Cell Tethering Through L-selectin at Threshold Shear

Biophysical Journal. Jan, 2007  |  Pubmed ID: 17028146

Flow-enhanced cell adhesion is a counterintuitive phenomenon that has been observed in several biological systems. Flow augments L-selectin-dependent adhesion by increasing the initial tethering of leukocytes to vascular surfaces and by strengthening their subsequent rolling interactions. Tethering or rolling might be influenced by physical factors that affect the formation or dissociation of selectin-ligand bonds. We recently demonstrated that flow enhanced rolling of L-selectin-bearing microspheres or neutrophils on P-selectin glycoprotein ligand-1 by force decreased bond dissociation. Here, we show that flow augmented tethering of these microspheres or cells to P-selectin glycoprotein ligand-1 by three transport mechanisms that increased bond formation: sliding of the sphere bottom on the surface, Brownian motion, and molecular diffusion. These results elucidate the mechanisms for flow-enhanced tethering through L-selectin.

A Structure-based Sliding-rebinding Mechanism for Catch Bonds

Biophysical Journal. Mar, 2007  |  Pubmed ID: 17142266

Catch bonds, whose lifetimes are prolonged by force, have been observed in selectin-ligand interactions and other systems. Several biophysical models have been proposed to explain this counterintuitive phenomenon, but none was based on the structure of the interacting molecules and the noncovalent interactions at the binding interface. Here we used molecular dynamics simulations to study changes in structure and atomic-level interactions during forced unbinding of P-selectin from P-selectin glycoprotein ligand-1. A mechanistic model for catch bonds was developed based on these observations. In the model, "catch" results from forced opening of an interdomain hinge that tilts the binding interface to allow two sides of the contact to slide against each other. Sliding promotes formation of new interactions and even rebinding to the original state, thereby slowing dissociation and prolonging bond lifetimes. Properties of this sliding-rebinding mechanism were explored using a pseudoatom representation and Monte Carlo simulations. The model has been supported by its ability to fit experimental data and can be related to previously proposed two-pathway models.

Effects of Exogenous Salicylic Acid on Growth and H2O2-metabolizing Enzymes in Rice Seedlings Under Lead Stress

Journal of Environmental Sciences (China). 2007  |  Pubmed ID: 17913152

Salicylic acid (SA) was an essential component of the plant resistance to pathogens and also plays an important role in mediating plant response to some abiotic stress. The possible effects of SA on the growth and H2O2-metabolizing enzymes in rice seedlings under lead stress were studied. When rice seedlings grown in nutrient solution containing Pb2+ (0, 0.05, 0.15, 0.25 mmol/L) for 18 d, the plant biomass as well as the chlorophyll content of leaves decreased with increasing Pb concentration. The pre-treatment with SA (treated with 0.1 mmol/L SA for 48 h before Pb stress) partially protected seedlings from Pb toxicity. The chlorophyll contents were significant higher in leaves of Pb-exposed with SA pre-treatment seedlings than in Pb-exposed plants at the same Pb intensity. SA pre-treated alone could significantly increase the length of shoot and root of seedlings but the vigour difference was not marked under long-term exposure to Pb toxicity. SA pre-treated influence the H202 level in leaves of seedlings by up-regulating the activity of superoxide dismutase (SOD), repressing the activity of catalase (CAT) and ascorbate peroxidase (APX) depending on the concentrations of Pb2+ in the growth medium. The results supported the conclusion that SA played a positive role in rice seedlings against Pb toxicity.

Memory in Receptor-ligand-mediated Cell Adhesion

Proceedings of the National Academy of Sciences of the United States of America. Nov, 2007  |  Pubmed ID: 17991779

Single-molecule biomechanical measurements, such as the force to unfold a protein domain or the lifetime of a receptor-ligand bond, are inherently stochastic, thereby requiring a large number of data for statistical analysis. Sequentially repeated tests are generally used to obtain a data ensemble, implicitly assuming that the test sequence consists of independent and identically distributed (i.i.d.) random variables, i.e., a Bernoulli sequence. We tested this assumption by using data from the micropipette adhesion frequency assay that generates sequences of two random outcomes: adhesion and no adhesion. Analysis of distributions of consecutive adhesion events revealed violation of the i.i.d. assumption, depending on the receptor-ligand systems studied. These include Markov sequences with positive (T cell receptor interacting with antigen peptide bound to a major histocompatibility complex) or negative (homotypic interaction between C-cadherins) feedbacks, where adhesion probability in the next test was increased or decreased, respectively, by adhesion in the immediate past test. These molecular interactions mediate cell adhesion and cell signaling. The ability to "remember" the previous adhesion event may represent a mechanism by which the cell regulates adhesion and signaling.

Design of a Zoom Lens Without Motorized Optical Elements

Optics Express. May, 2007  |  Pubmed ID: 19546976

A novel design of a zoom lens system without motorized movements is proposed. The lens system consists of a fixed lens and two double-liquid variable-focus lenses. The liquid lenses, made out of two immiscible liquids, are based on the principle of electrowetting: an effect controlling the wetting properties of a liquid on a solid by modifying the applied voltage at the solid-liquid interface. The structure and principle of the lens system are introduced in this paper. Detailed calculations and simulation examples are presented to show that this zoom lens system appears viable as the next-generation zoom lens.

Relationships Between Blood Pressure and Systolic Time-intervals: a Lumped-model Simulation Study

Conference Proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference. 2007  |  Pubmed ID: 18002304

A lumped model of the arterial circulation is applied to the study of the dependencies between blood pressure and systolic time-intervals (PEP, LVET). The left ventricle is handled as a pressure source directly coupled with the varying vascular conditions. Four factors are individually considered: peripheral resistance, LV contractility, end diastolic volume and heart rate. The computed dependence curves of PEP and LVET on systolic and diastolic pressures are in accordance with physiological knowledge. The relations of PEP and LVET with other hemodynamic variables are being enlightened and insight is gained into the use of pulse delays measured from the ECG for predicting non-invasively the arterial blood pressure.

Kinetics of MHC-CD8 Interaction at the T Cell Membrane

Journal of Immunology (Baltimore, Md. : 1950). Dec, 2007  |  Pubmed ID: 18025211

CD8 plays an important role in facilitating TCR-MHC interaction, promoting Ag recognition, and initiating T cell activation. MHC-CD8 binding kinetics have been measured in three dimensions by surface plasmon resonance technique using purified molecules. However, CD8 is a membrane-anchored, signaling kinase-linked, and TCR-associated molecule whose function depends on the cell membrane environment. Purified molecules lack their linkage to the membrane, which precludes interactions with other structures of the cell as well as signaling. Furthermore, three-dimensional binding in the fluid phase is biologically and physically distinct from two-dimensional binding across apposing cell membranes. As a first step toward characterizing the molecular interactions between T cells and APCs, we used a micropipette adhesion frequency assay to measure the adhesion kinetics of single mouse T cells interacting with single human RBCs coated with MHC. Using anti-TCR mAb we isolated and characterized the specific two-dimensional MHC-CD8 binding from the trimolecular TCR-MHC-CD8 interaction. The TCR-independent MHC-CD8 interaction has a very low affinity that depends on the MHC alleles, but not on the peptide complexed to the MHC and whether CD8 is an alphaalpha homodimer or an alphabeta heterodimer. Surprisingly, MHC-CD8 binding affinity varies with T cells from different TCR transgenic mice and these affinity differences were abolished by treatment with cholesterol oxidase to disrupt membrane rafts. These data highlight the relevance and importance of two-dimensional analysis of T cells and APCs and indicate that membrane rafts play an important role in modulating the affinity of cell-cell interactions.

[The Prevalence of Diabetes in Children and Adolescents of Beijing]

Zhonghua Liu Xing Bing Xue Za Zhi = Zhonghua Liuxingbingxue Zazhi. Jul, 2007  |  Pubmed ID: 18069547

To study the prevalence of Diabetes mellitus (DM) in children and adolescents and to describe the characteristics on age, gender and district distribution of schoolchildren, in Beijing.

A Nonsynonymous Functional Variant in Integrin-alpha(M) (encoded by ITGAM) is Associated with Systemic Lupus Erythematosus

Nature Genetics. Feb, 2008  |  Pubmed ID: 18204448

We identified and replicated an association between ITGAM (CD11b) at 16p11.2 and risk of systemic lupus erythematosus (SLE) in 3,818 individuals of European descent. The strongest association was at a nonsynonymous SNP, rs1143679 (P = 1.7 x 10(-17), odds ratio = 1.78). We further replicated this association in two independent samples of individuals of African descent (P = 0.0002 and 0.003; overall meta-analysis P = 6.9 x 10(-22)). The genetic association between ITGAM and SLE implicates the alpha(M)beta2-integrin adhesion pathway in disease development.

Replacing a Lectin Domain Residue in L-selectin Enhances Binding to P-selectin Glycoprotein Ligand-1 but Not to 6-sulfo-sialyl Lewis X

The Journal of Biological Chemistry. Apr, 2008  |  Pubmed ID: 18250165

Selectin-ligand interactions (bonds) mediate leukocyte rolling on vascular surfaces. The molecular basis for differential ligand recognition by selectins is poorly understood. Here, we show that substituting one residue (A108H) in the lectin domain of L-selectin increased its force-free affinity for a glycosulfopeptide binding site (2-GSP-6) on P-selectin glycoprotein ligand-1 (PSGL-1) but not for a sulfated-glycan binding site (6-sulfo-sialyl Lewis x) on peripheral node addressin. The increased affinity of L-selectinA108H for 2-GSP-6 was due to a faster on-rate and to a slower off-rate that increased bond lifetimes in the absence of force. Rather than first prolonging (catching) and then shortening (slipping) bond lifetimes, increasing force monotonically shortened lifetimes of L-selectinA108H bonds with 2-GSP-6. When compared with microspheres bearing L-selectin, L-selectinA108H microspheres rolled more slowly and regularly on 2-GSP-6 at low flow rates. A reciprocal substitution in P-selectin (H108A) caused faster microsphere rolling on 2-GSP-6. These results distinguish molecular mechanisms for L-selectin to bind to PSGL-1 and peripheral node addressin and explain in part the shorter lifetimes of PSGL-1 bonds with L-selectin than P-selectin.

Protein Tyrosine Phosphatase-1B (PTP1B) Helps Regulate EGF-induced Stimulation of S-phase Entry in Human Corneal Endothelial Cells

Molecular Vision. 2008  |  Pubmed ID: 18253097

Human corneal endothelial cells (HCEC), particularly from older donors, only proliferate weakly in response to EGF. The protein tyrosine phosphatase, PTP1B, is known to negatively regulate EGF-induced signaling in several cell types by dephosphorylating the epidermal growth factor receptor (EGFR). The current studies were conducted to determine whether PTP1B plays a role in regulating cell cycle entry in HCEC in response to EGF stimulation.

Mechanisms for Flow-enhanced Cell Adhesion

Annals of Biomedical Engineering. Apr, 2008  |  Pubmed ID: 18299992

Cell adhesion is mediated by specific receptor-ligand bonds. In several biological systems, increasing flow has been observed to enhance cell adhesion despite the increasing dislodging fluid shear forces. Flow-enhanced cell adhesion includes several aspects: flow augments the initial tethering of flowing cells to a stationary surface, slows the velocity and increases the regularity of rolling cells, and increases the number of rollingly adherent cells. Mechanisms for this intriguing phenomenon may include transport-dependent acceleration of bond formation and force-dependent deceleration of bond dissociation. The former includes three distinct transport modes: sliding of cell bottom on the surface, Brownian motion of the cell, and rotational diffusion of the interacting molecules. The latter involves a recently demonstrated counterintuitive behavior called catch bonds where force prolongs rather than shortens the lifetimes of receptor-ligand bonds. In this article, we summarize our recently published data that used dimensional analysis and mutational analysis to elucidate the above mechanisms for flow-enhanced leukocyte adhesion mediated by L-selectin-ligand interactions.

Decreased Expression of Peroxiredoxins in Fuchs' Endothelial Dystrophy

Investigative Ophthalmology & Visual Science. Jul, 2008  |  Pubmed ID: 18378575

To compare the relative expression of peroxiredoxin (Prx) proteins in normal human corneal endothelium with endothelium in corneas affected by Fuchs' endothelial dystrophy (FED) and between normal human endothelium and epithelial/stromal tissue.

Measuring Diffusion and Binding Kinetics by Contact Area FRAP

Biophysical Journal. Jul, 2008  |  Pubmed ID: 18390627

The immunological synapse is a stable intercellular structure that specializes in substance and signal transfer from one immune cell to another. Its formation is regulated in part by the diffusion of adhesion and signaling molecules into, and their binding of countermolecules in the contact area. The stability of immunological synapses allows receptor-ligand interactions to approximate chemical equilibrium despite other dynamic aspects. We have developed a mathematical model that describes the coupled reaction-diffusion process in an established immunological synapse. In this study, we extend a previously described contact area fluorescence recovery after photobleaching (FRAP) experiment to test the validity of the model. The receptor binding activity and lateral mobility of fluorescently labeled, lipid-anchored ligands in the bilayer resulted in their accumulation, as revealed by a much higher fluorescence intensity inside the contact area than outside. After complete photobleaching of the synapse, fluorescence recovery requires ligands to dissociate and rebind, and to diffuse in and out of the contact area. Such a FRAP time course consequently provides information on reaction and diffusion, which can be extracted by fitting the model solution to the data. Surprisingly, reverse rates in the two-dimensional contact area were at least 100-fold slower than in three-dimensional solution. As previously reported in immunological synapses, a significant nonrecoverable fraction of fluorescence was observed with one of two systems studied, suggesting some ligands either dissociated or diffused much more slowly compared with other ligands in the same synapse. The combined theory and experiment thus provides a new method for in situ measurements of kinetic rates, diffusion coefficients, and nonrecoverable fractions of interacting molecules in immunological synapses and other stable cell-bilayer junctions.

A Coupled Diffusion-kinetics Model for Analysis of Contact-area FRAP Experiment

Biophysical Journal. Jul, 2008  |  Pubmed ID: 18390628

Kinetic rates and binding affinity of receptor-ligand interactions are important determinants of cell adhesion. Measurements of these parameters in fluid phase using soluble molecules (i.e., three-dimensionial parameters) do not necessarily correlate with their counterparts measured when both binding partners are respectively anchored to two apposing surfaces (i.e., two-dimensional (2D) parameters). Moreover, 2D affinities measured by different methods can differ by orders of magnitude. Here we describe a coupled diffusion-reaction model for the fluorescence recovery after photobleaching experiment previously used to demonstrate the dynamics of adhesive bonds in the contact area. Applying the mathematical model to the contact area fluorescence recovery after photobleaching experiment enables in situ measurements of 2D kinetic rates of the adhesion molecules and their retarded diffusion in a stable contact area. The mathematical properties of the model are characterized in this article and its experimental validation will be presented in the companion article.

The Differential Effect of Endothelial Cell Factors on in Vitro Motility of Malignant and Non-malignant Cells

Annals of Biomedical Engineering. Jun, 2008  |  Pubmed ID: 18398681

Motility of cancer cells plays a critical role in tumor metastasis, and as such is a target for intervention. The motility of malignant Calu-1 human lung epithelial carcinoma cells is upregulated when placed on a human umbilical vein endothelial cell monolayer, while that of non-malignant L132 human lung epithelial cells is not. To dissect the factor(s) causing such differential behaviors, the motile responses of both cell lines to endothelial cell factors-secreted to the media, on the endothelial cell surface, and secreted to the extracellular matrix-and to individual extracellular matrix proteins were compared. Cell motility was quantified by tracking the cell movement on a surface with time-lapse video microscopy, which was analyzed with the persistent random walk model of motility. None of the factors tested had a remarkable effect on L132 cell motility, but the Calu-1 cell motility was significantly upregulated by endothelial cell extracellular matrix and by laminin, fibronectin, collagen I and collagen VI individually. Flow cytometry analysis revealed significantly higher expression levels of integrin subunits beta1, alpha2, alpha3, and alpha6, which are known receptors for these extracellular matrix proteins, on the Calu-1 than L132 cells, implicating a role of these integrins in the observed motile behaviors of these cell lines.

Integrin Dependence of Calu-1 Cell Motility on Endothelial Extracellular Matrix Proteins

Annals of Biomedical Engineering. Jun, 2008  |  Pubmed ID: 18398683

We recently showed that the motility of the malignant Calu-1 human epidermoid lung carcinoma cells correlates to their expression levels of alpha2, alpha3, alpha6, and beta1 integrin subunits. To determine a causative relationship underlying this correlation, here we measured Calu-1 cell adhesion to and migration on laminin, collagen IV, human umbilical vein endothelial cell monolayers, and endothelial cell extracellular matrix in the presence of function-blocking antibodies against the suspect integrin subunits. Blocking individual alpha subunits did not affect adhesion to or motility on laminin, but when used in pair-wise combinations, monoclonal antibody treatments significantly decreased tumor cell motility on, without diminishing adhesion to, laminin and the other substrates. Blocking all three alpha subunits at once or the beta1 subunit alone abolished migration on laminin; however, the latter treatment also abolished adhesion, whereas the former treatment did not. By contrast, blocking the beta1 subunit significantly reduced motility on collagen IV, endothelial cell monolayers, and endothelial cell extracellular matrix, but always without affecting adhesion. These results suggest a separation of roles and mechanisms of different integrins in adhesion and motility.

Flow-induced Structural Transition in the Beta-switch Region of Glycoprotein Ib

Biophysical Journal. Aug, 2008  |  Pubmed ID: 18441028

The impact of fluid flow on structure and dynamics of biomolecules has recently gained much attention. In this article, we present a molecular-dynamics algorithm that serves to generate stable water flow under constant temperature, for the study of flow-induced protein behavior. Flow simulations were performed on the 16-residue beta-switch region of platelet glycoprotein Ibalpha, for which crystal structures of its N-terminal domain alone and in complex with the A1 domain of von Willebrand factor have been solved. Comparison of the two structures reveals a conformational change in this region, which, upon complex formation, switches from an unstructured loop to a beta-hairpin. Interaction between glycoprotein Ibalpha and von Willebrand factor initiates platelet adhesion to injured vessel walls, and the adhesion is enhanced by blood flow. It has been hypothesized that the loop to beta-hairpin transition in glycoprotein Ib alpha is induced by flow before binding to von Willebrand factor. The simulations revealed clearly a flow-induced loop-->beta-hairpin transition. The transition is dominated by the entropy of the protein, and is seen to occur in two steps, namely a dihedral rotation step followed by a side-group packing step.

[Expression and Clinical Significance of ADAM19 in Endometrial Carcinoma]

Beijing Da Xue Xue Bao. Yi Xue Ban = Journal of Peking University. Health Sciences. Apr, 2008  |  Pubmed ID: 18458692

To investigate the expression of adamalysin19 (ADAM19) in human endometrial carcinoma and to explore its correlation with clinical behavior and pathological characters.

Discovery of Imidazole Carboxamides As Potent and Selective CCK1R Agonists

Bioorganic & Medicinal Chemistry Letters. Aug, 2008  |  Pubmed ID: 18614364

High-throughput screening revealed diaryl pyrazole 3 as a selective albeit modest cholecystokinin 1 receptor (CCK1R) agonist. SAR studies led to the discovery and optimization of a novel class of 1,2-diaryl imidazole carboxamides. Compound 44, which was profiled extensively, showed good in vivo mouse gallbladder emptying (mGBE) and lean mouse overnight food intake (ONFI) reduction activities.

2-Substituted Piperazine-derived Imidazole Carboxamides As Potent and Selective CCK1R Agonists for the Treatment of Obesity

Bioorganic & Medicinal Chemistry Letters. Sep, 2008  |  Pubmed ID: 18684621

The discovery and structure-activity relationship of 1,2-diarylimidazole piperazine carboxamides bearing polar side chains as potent and selective cholecystokinin 1 receptor (CCK1R) agonists are described. Optimization of this series resulted in the discovery of isopropyl carboxamide 40, a CCK1R agonist with sub-nanomolar functional and binding activity as well as excellent potency in a mouse overnight food intake reduction assay.

Platelet Glycoprotein Ibalpha Forms Catch Bonds with Human WT VWF but Not with Type 2B Von Willebrand Disease VWF

The Journal of Clinical Investigation. Sep, 2008  |  Pubmed ID: 18725999

Arterial blood flow enhances glycoprotein Ibalpha (GPIbalpha) binding to vWF, which initiates platelet adhesion to injured vessels. Mutations in the vWF A1 domain that cause type 2B von Willebrand disease (vWD) reduce the flow requirement for adhesion. Here we show that increasing force on GPIbalpha/vWF bonds first prolonged ("catch") and then shortened ("slip") bond lifetimes. Two type 2B vWD A1 domain mutants, R1306Q and R1450E, converted catch bonds to slip bonds by prolonging bond lifetimes at low forces. Steered molecular dynamics simulations of GPIbalpha dissociating from the A1 domain suggested mechanisms for catch bonds and their conversion by the A1 domain mutations. Catch bonds caused platelets and GPIbalpha-coated microspheres to roll more slowly on WT vWF and WT A1 domains as flow increased from suboptimal levels, explaining flow-enhanced rolling. Longer bond lifetimes at low forces eliminated the flow requirement for rolling on R1306Q and R1450E mutant A1 domains. Flowing platelets agglutinated with microspheres bearing R1306Q or R1450E mutant A1 domains, but not WT A1 domains. Therefore, catch bonds may prevent vWF multimers from agglutinating platelets. A disintegrin and metalloproteinase with a thrombospondin type 1 motif-13 (ADAMTS-13) reduced platelet agglutination with microspheres bearing a tridomain A1A2A3 vWF fragment with the R1450E mutation in a shear-dependent manner. We conclude that in type 2B vWD, prolonged lifetimes of vWF bonds with GPIbalpha on circulating platelets may allow ADAMTS-13 to deplete large vWF multimers, causing bleeding.

Two Stage Cadherin Kinetics Require Multiple Extracellular Domains but Not the Cytoplasmic Region

The Journal of Biological Chemistry. Jan, 2008  |  Pubmed ID: 17999960

Micropipette manipulation measurements quantified the pre-steady state binding kinetics between cell pairs mediated by Xenopus cleavage stage cadherin. The time-dependence of the intercellular binding probability exhibits a fast forming, low probability binding state, which transitions to a slower forming, high probability state. The biphasic kinetics are independent of the cytoplasmic region, but the transition to the high probability state requires the third extracellular domain EC3. Deleting either EC3 or EC3-5, or substituting Trp(2) for Ala reduces the binding curves to a simple, monophasic rise in binding probability to a limiting plateau, as predicted for a single site binding mechanism. The two stage cadherin binding process reported here directly parallels previous biophysical studies, and confirms that the cadherin ectodomain governs the initial intercellular adhesion dynamics.

Flow Induces Loop-to-beta-hairpin Transition on the Beta-switch of Platelet Glycoprotein Ib Alpha

Proceedings of the National Academy of Sciences of the United States of America. Sep, 2008  |  Pubmed ID: 18772372

Interaction of glycoprotein Ib alpha (GPIb alpha) with von Willebrand factor (VWF) initiates platelet adhesion to injured vascular wall to stop bleeding. A major contact between GPIb alpha and VWF involves the beta-switch region, which is a loop in the unliganded GPIb alpha but switches to a beta-hairpin in the complex structure. Paradoxically, flow enhances rather than impedes GPIb alpha-VWF binding. Gain-of-function mutations (e.g., M239V) in the beta-switch reduce the flow requirement for VWF binding, whereas loss-of-function mutations (e.g., A238V) increase the flow requirement. These phenomena cannot be explained by crystal structures or energy calculations. Herein we demonstrate that the beta-hairpin is unstable without contacting VWF, in that it switches to a loop in free molecular dynamics simulations. Simulations with a novel flow molecular dynamics algorithm show that the loop conformation is unstable in the presence of flow, as it switches to beta-hairpin even without contacting VWF. Compared with the wild-type, it is easier for the M239V mutant but harder for the A238V mutant to switch to beta-hairpin in the presence of flow. These results elucidate the structural basis for the two mutants and suggest a regulatory mechanism by which flow activates GPIb alpha via inducing a loop-to-beta-hairpin conformational transition on the beta-switch, thereby promoting VWF binding.

Differential Protein Expression in Human Corneal Endothelial Cells Cultured from Young and Older Donors

Molecular Vision. 2008  |  Pubmed ID: 18852868

To establish a baseline protein fingerprint of cultured human corneal endothelial cells (HCEC), to determine whether the protein profiles exhibit age-related differences, and to identify proteins differentially expressed in HCEC cultured from young and older donors.

MHC Variant Peptide-mediated Anergy of Encephalitogenic T Cells Requires SHP-1

Journal of Immunology (Baltimore, Md. : 1950). Nov, 2008  |  Pubmed ID: 18981103

Our lab has demonstrated that encephalitogenic T cells can be effectively anergized by treatment with MHC variant peptides, which are analogues of immunogenic peptides containing an amino acid substitution at an MHC anchor residue. The MHC variant peptide of myelin oligodendrocyte glycoprotein (MOG)(35-55) proves an effective treatment as it does not induce symptoms of experimental autoimmune encephalomyelitis and fails to recruit macrophages or MOG(35-55)-specific T cells to the CNS. In this study, we sought to characterize the signaling pathways required for the induction of anergy by building upon the observations identifying the tyrosine phosphatase SHP-1 as a critical regulator of T cell responsiveness. Motheaten viable heterozygous mice, which contain a mutation in the SHP-1 gene resulting in a reduction in functional SHP-1, were challenged with MOG(35-55) or the MOG(35-55) MHC variant 45D. These mice display symptoms of experimental autoimmune encephalomyelitis upon immunization with MHC variant peptide and have significant CNS infiltration of tetramer-positive CD4(+) cells and macrophages, unlike B6 mice challenged with the variant peptide. The effects of SHP-1 are directly on the T cell as Motheaten viable heterozygous mice autoreactive T cells are not anergized in vitro. Lastly, we demonstrate no distinguishable difference in the initial interaction between the TCR and agonist or MHC variant. Rather, an unstable interaction between peptide and MHC attenuates the T cell response, seen in a decreased half-life relative to MOG(35-55). These results identify SHP-1 as a mediator of T cell anergy induced by destabilized peptide:MHC complexes.

Measuring Receptor-Ligand Binding Kinetics on Cell Surfaces: From Adhesion Frequency to Thermal Fluctuation Methods

Cellular and Molecular Bioengineering. Dec, 2008  |  Pubmed ID: 19890486

Interactions between surface-anchored receptors and ligands mediate cell-cell and cell-environment communications in many biological processes. Molecular interactions across two apposing cell membrane are governed by two-dimensional (2D) kinetics, which are physically distinct from and biologically more relevant than three-dimensional (3D) kinetics with at least one interacting molecular species in the fluid phase. Here we review two assays for measuring 2D binding kinetics: the adhesion frequency assay and the thermal fluctuation assay. The former measures the binding frequency as a function of contact duration and extracts the force-free 2D kinetics parameters by nonlinearly fitting the data with a probabilistic model. The latter detects bond formation/dissociation by monitoring the reduction/resumption of thermal fluctuations of a force sensor. Both assays are mechanically based and operate at the level of mostly single molecular interaction, which requires ultrasensitive force techniques. Characterization of one such technique, the biomembrane force probe, is presented.

Binary Time Series Modeling with Application to Adhesion Frequency Experiments

Journal of the American Statistical Association. Sep, 2008  |  Pubmed ID: 22180690

Repeated adhesion frequency assay is the only published method for measuring the kinetic rates of cell adhesion. Cell adhesion plays an important role in many physiological and pathological processes. Traditional analysis of adhesion frequency experiments assumes that the adhesion test cycles are independent Bernoulli trials. This assumption can often be violated in practice. Motivated by the analysis of repeated adhesion tests, a binary time series model incorporating random effects is developed in this paper. A goodness-of-fit statistic is introduced to assess the adequacy of distribution assumptions on the dependent binary data with random effects. The asymptotic distribution of the goodness-of-fit statistic is derived and its finite-sample performance is examined via a simulation study. Application of the proposed methodology to real data from a T-cell experiment reveals some interesting information, including the dependency between repeated adhesion tests.

Monitoring Receptor-ligand Interactions Between Surfaces by Thermal Fluctuations

Biophysical Journal. Jan, 2008  |  Pubmed ID: 17890399

We describe a new method for determining receptor-ligand association/dissociation events across the interface of two surfaces (two-dimensional binding) by monitoring abrupt decrease/resumption in thermal fluctuations of a biomembrane force probe. Our method has been validated by rigorous control experiments and kinetic experiments. We show that cellular on-rate of association can be measured by analysis of intervals from a dissociation event to the next association event (waiting times). Similarly, off-rate of molecular dissociation can be measured by analysis of intervals from an association event to the next dissociation event (bond lifetimes). Different types of molecular bonds could be distinguished by different levels of reduction in thermal fluctuations. This novel method provides a powerful tool to study cell adhesion and signaling mediated by single or multiple receptor-ligand species.

The Role of MicroRNAs in Copper and Cadmium Homeostasis

Biochemical and Biophysical Research Communications. Aug, 2009  |  Pubmed ID: 19501049

Essential heavy metals (e.g., copper) and non-essential metals (e.g., cadmium) are both toxic to plants at high concentrations. Recently, microRNAs (miRNAs) have emerged as important modulators of plants adaptive response to heavy metal stress. Plant miRNAs negatively regulate target mRNAs by post-transcriptional cleavage. miR398 regulates copper homeostasis via down-regulating the expression of Cu,Zn-superoxide dismutase (CSD), a scavenger of superoxide radicals. miR393 and miR171 play an important role in cadmium stress mediation. This review focuses on the recent advance in the involvement of miRNAs in copper and cadmium stress regulatory networks in plants.

Dynamics of the Interaction of Human IgG Subtype Immune Complexes with Cells Expressing R and H Allelic Forms of a Low-affinity Fc Gamma Receptor CD32A

Journal of Immunology (Baltimore, Md. : 1950). Dec, 2009  |  Pubmed ID: 20007585

CD32A, the major phagocytic FcgammaR in humans, exhibits a polymorphism in the ligand binding domain. Individuals homozygous for the R allelic form of CD32A (CD32A(R) allele) are more susceptible to bacterial infections and autoimmune diseases as compared with H allelic CD32A (CD32A(H)) homozygous and CD32A(R/H) heterozygous individuals. To understand the mechanisms behind this differential susceptibility, we have investigated the dynamics of the interaction of these allelic forms of CD32A when they are simultaneously exposed to immune complexes (IC). Binding studies using Ig fusion proteins of CD32A alleles showed that the R allele has significantly lower binding not only to human IgG2, but also to IgG1 and IgG3 subtypes. Competition assays using purified molecules demonstrated that CD32A(H)-Ig outcompetes CD32A(R)-Ig for IC binding when both alleles simultaneously compete for the same ligand. CD32A(H)-Ig blocked the IC binding mediated by both the allelic forms of cell surface CD32A, whereas CD32A(R)-Ig blocked only CD32A(R) and was unable to cross-block IC binding mediated by CD32A(H). Two-dimensional affinity measurements also demonstrated that CD32A(R) has significantly lower affinity toward all three subtypes as compared with CD32A(H). Our data suggest that the lower binding of CD32A(R) not only to IgG2 but also to IgG1 and IgG3 might be responsible for the lack of clearance of IC leading to increased susceptibility to bacterial infections and autoimmune diseases. Our data further suggests that in humans, inflammatory cells from CD32A(R/H) heterozygous individuals may predominantly use the H allele to mediate Ab-coated target cell binding during phagocytosis and Ab-dependent cellular cytotoxicity, resulting in a phenotype similar to CD32A(H) homozygous individuals.

Relationship Among Oxidative Stress, DNA Damage, and Proliferative Capacity in Human Corneal Endothelium

Investigative Ophthalmology & Visual Science. May, 2009  |  Pubmed ID: 19117931

To determine whether human corneal endothelial cells (HCECs) exhibit signs of oxidative DNA damage and to test whether oxidative stress affects the proliferative capacity of HCECs.

Bending Rigidities of Cell Surface Molecules P-selectin and PSGL-1

Journal of Biomechanics. Feb, 2009  |  Pubmed ID: 19144337

P-selectin is a cell adhesion molecule expressed on activated endothelial cells and platelets. P-selectin glycoprotein ligand 1 (PSGL-1) is a mucin expressed on leukocytes. The interaction of P-selectin and PSGL-1 mediates leukocyte tethering to and rolling on the vascular surface, which are initiating events in inflammatory and thrombotic processes. In the hemodynamic environment of the circulation, P-selectin and PSGL-1 are subject to a wide range of forces, which can cause deformation. For P-selectin/PSGL-1 interaction to be physically possible, these molecules may need to project above much of the glycocalyx layers of the respective cell surfaces, suggesting that they are either longer than the thickness of glycocalyx or better able to support compression than the glycocalyx. As such, the mechanical properties of these molecules and their functional implications merit investigation. Here we report determination of the bending rigidities of P-selectin and PSGL-1 by analyzing their thermally excited curvature fluctuations, whose values are of the order of magnitude of 100pNnm(2).

Smurf2 Participates in Human Trophoblast Cell Invasion by Inhibiting TGF-beta Type I Receptor

The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society. Jun, 2009  |  Pubmed ID: 19255252

Successful embryo implantation depends on the ability of the trophoblast cells to invade the endometrium and the receptivity of the endometrium. Unlike tumor invasion, trophoblast invasion is spatio-temporaly restricted. Transforming growth factor (TGF)-beta is a key inhibitory factor in the invasion of early trophoblast cells. Smad ubiquitination regulatory factor 2 (Smurf2), a HECT type E3 ubiquitin ligase, is an important regulator of the TGF-beta signaling pathway, targeting TGF-beta receptors and various Smads for proteasome-mediated degradation. In this context, we wished to determine whether Smurf2 has a physiological role during embryo implantation, especially in trophoblast invasion. We examined the spatio-temporal expression of Smurf2 in human placental villi and the function of Smurf2 in trophoblast cell migration and invasion in a model system involving a human extravillous trophoblast cell line, HTR-8/SVneo. Results from RT-PCR and immunohistochemical studies showed that expression of Smurf2 in placental villi was the highest during the first trimester and decreased as the pregnancy progressed. Overexpression of Smurf2 in HTR-8/SVneo cells reduced TGF-beta type I receptor levels, and enhanced cell migration and invasion. Conversely, RNA interference-mediated downregulation of Smurf2 resulted in a significant increase in TGF-beta type I receptor protein levels. However, the levels of Smad2, another potential target of Smurf2, remained unchanged. In conclusion, the present study suggests that Smurf2 promotes trophoblast cell migration and invasion, and this function may involve downregulation of TGF-beta type I receptor.

Protein Phosphatase 1A (PPM1A) is Involved in Human Cytotrophoblast Cell Invasion and Migration

Histochemistry and Cell Biology. Aug, 2009  |  Pubmed ID: 19404668

Trophoblast invasion is crucial for embryo implantation and placentation. Excessive trophoblast invasion leads to hydatidiform moles and choriocarcinoma. PPM1A is a phosphatase which dephosphorylates and inactivates a broad range of substrates, including TGF-beta, MAP kinases, p38 and JNK kinase cascades, and is involved in tumor suppression. The objective of this study was to investigate the expression of PPM1A in normal and malignant human placenta and its role in trophoblast invasion, which shares many similarities with invasion of tumor cells. By Western blotting and immunocytochemistry, significantly higher expression of PPM1A in human placental villi at term was found as compared with that during the first trimester. Furthermore, the expression level of PPM1A protein in hydatidiform moles was lower compared with that during normal pregnancy. We further investigated the function of PPM1A in extravillous trophoblast cell line HTR8/SVneo. Transwell migration and Matrigel invasion assays demonstrated that PPM1A siRNA significantly promoted the motility and invasiveness of the cells. Gelatin zymography showed that knockdown of PPM1A with siRNA elevated the expression of pro-matrix metalloproteinase pro-(MMP)-9, but down-regulated tissue inhibitors of metalloproteinases (TIMP)-2. The present data indicate that PPM1A plays a critical role in the regulation of normal placentation by inhibiting trophoblast migration and invasion.

Relationship Between Proline and Hg2+-induced Oxidative Stress in a Tolerant Rice Mutant

Archives of Environmental Contamination and Toxicology. May, 2009  |  Pubmed ID: 18787889

There has been little agreement regarding the mechanism by which proline reduces heavy metal stress. The present work examines the relationship between Hg(2+)-induced oxidative stress and proline accumulation in rice and explores the possible mechanisms through which proline protects against Hg(2+) stress. The effect of proline on alleviation of Hg(2+) toxicity was studied by spectrophotography and enzymatic methods. Hg(2+) induced oxidative stress in rice by increasing lipid peroxidation. Pretreatment of the rice with 2 mM proline for 12 h profoundly alleviated Hg(2+)-induced lipid peroxidation and minimized H(2)O(2) accumulation. Proline pretreatment significantly reduced (p < 0.01) the Hg(2+) content in rice leaves. A comparison of the effects of proline pretreatment on H(2)O(2) accumulation by Hg(2+) and aminotrazole suggested that proline protected cells from Hg(2+)-induced oxidative stress by scavenging reactive oxygen species. The present work demonstrates a protective effect of proline on Hg(2+) toxicity through detoxifying reactive oxygen species, rather than chelating metal ions or maintaining the water balance under Hg(2+) stress.

Cadmium Decreases Crown Root Number by Decreasing Endogenous Nitric Oxide, Which is Indispensable for Crown Root Primordia Initiation in Rice Seedlings

Planta. Sep, 2009  |  Pubmed ID: 19557429

Cadmium (Cd) is toxic to crown roots (CR), which are essential for maintaining normal growth and development in rice seedlings. Nitric oxide (NO) is an important signaling molecule that plays a pivotal role in plant root organogenesis. Here, the effects of Cd on endogenous NO content and root growth conditions were studied in rice seedlings. Results showed that similar to the NO scavenger, cPTIO, Cd significantly decreased endogenous NO content and CR number in rice seedlings, and these decreases were recoverable with the application of sodium nitroprusside (SNP, a NO donor). Microscopic analysis of root collars revealed that treatment with Cd and cPTIO inhibited CR primordia initiation. In contrast, although SNP partially recovered Cd-caused inhibition of CR elongation, treatment with cPTIO had no effect on CR elongation. L: -NMMA, a widely used nitric oxide synthase (NOS) inhibitor, decreased endogenous NO content and CR number significantly, while tungstate, a nitrate reductase (NR) inhibitor, had no effect on endogenous NO content and CR number. Moreover, enzyme activity assays indicated that treatment with SNP inhibited NOS activity significantly, but had no effect on NR activity. All these results support the conclusions that a critical endogenous NO concentration is indispensable for rice CR primordia initiation rather than elongation, NOS is the main source for endogenous NO generation, and Cd decreases CR number by inhibiting NOS activity and thus decreasing endogenous NO content in rice seedlings.

Demonstration of Catch Bonds Between an Integrin and Its Ligand

The Journal of Cell Biology. Jun, 2009  |  Pubmed ID: 19564406

Binding of integrins to ligands provides anchorage and signals for the cell, making them prime candidates for mechanosensing molecules. How force regulates integrin-ligand dissociation is unclear. We used atomic force microscopy to measure the force-dependent lifetimes of single bonds between a fibronectin fragment and an integrin alpha(5)beta(1)-Fc fusion protein or membrane alpha(5)beta(1). Force prolonged bond lifetimes in the 10-30-pN range, a counterintuitive behavior called catch bonds. Changing cations from Ca(2+)/Mg(2+) to Mg(2+)/EGTA and to Mn(2+) caused longer lifetime in the same 10-30-pN catch bond region. A truncated alpha(5)beta(1) construct containing the headpiece but not the legs formed longer-lived catch bonds that were not affected by cation changes at forces <30 pN. Binding of monoclonal antibodies that induce the active conformation of the integrin headpiece shifted catch bonds to a lower force range. Thus, catch bond formation appears to involve force-assisted activation of the headpiece but not integrin extension.

Changes in Thermodynamic Stability of Von Willebrand Factor Differentially Affect the Force-dependent Binding to Platelet GPIbalpha

Biophysical Journal. Jul, 2009  |  Pubmed ID: 19619477

In circulation, plasma glycoprotein von Willebrand Factor plays an important role in hemostasis and in pathological thrombosis under hydrodynamic forces. Mutations in the A1 domain of von Willebrand factor cause the hereditary types 2B and 2M von Willebrand disease that either enhance (2B) or inhibit (2M) the interaction of von Willebrand factor with the platelet receptor glycoprotein Ibalpha. To understand how type 2B and 2M mutations cause clinically opposite phenotypes, we use a combination of protein unfolding thermodynamics and atomic force microscopy to assess the effects of two type 2B mutations (R1306Q and I1309V) and a type 2M mutation (G1324S) on the conformational stability of the A1 domain and the single bond dissociation kinetics of the A1-GPIbalpha interaction. At physiological temperature, the type 2B mutations destabilize the structure of the A1 domain and shift the A1-GPIbalpha catch to slip bonding to lower forces. Conversely, the type 2M mutation stabilizes the structure of the A1 domain and shifts the A1-GPIbalpha catch to slip bonding to higher forces. As a function of increasing A1 domain stability, the bond lifetime at low force decreases and the critical force required for maximal bond lifetime increases. Our results are able to distinguish the clinical phenotypes of these naturally occurring mutations from a thermodynamic and biophysical perspective that provides a quantitative description of the allosteric coupling of A1 conformational stability with the force dependent catch to slip bonding between A1 and GPIbalpha.

Exogenous Nitric Oxide Enhances Cadmium Tolerance of Rice by Increasing Pectin and Hemicellulose Contents in Root Cell Wall

Planta. Sep, 2009  |  Pubmed ID: 19626338

To study the mechanisms of exogenous NO contribution to alleviate the cadmium (Cd) toxicity in rice (Oryza sativa), rice plantlets subjected to 0.2-mM CdCl(2) exposure were treated with different concentrations of sodium nitroprusside (SNP, a NO donor), and Cd toxicity was evaluated by the decreases in plant length, biomass production and chlorophyll content. The results indicated that 0.1 mM SNP alleviated Cd toxicity most obviously. Atomic absorption spectrometry and fluorescence localization showed that treatment with 0.1 mM SNP decreased Cd accumulation in both cell walls and soluble fraction of leaves, although treatment with 0.1 mM SNP increased Cd accumulation in the cell wall of rice roots obviously. Treatment with 0.1 mM SNP in nutrient solution had little effect on the transpiration rate of rice leaves, but this treatment increased pectin and hemicellulose content and decreased cellulose content significantly in the cell walls of rice roots. Based on these results, we conclude that decreased distribution of Cd in the soluble fraction of leaves and roots and increased distribution of Cd in the cell walls of roots are responsible for the NO-induced increase of Cd tolerance in rice. It seems that exogenous NO enhances Cd tolerance of rice by increasing pectin and hemicellulose content in the cell wall of roots, increasing Cd accumulation in root cell wall and decreasing Cd accumulation in soluble fraction of leaves.

Onset of Breast and Pubic Hair Development and Menses in Urban Chinese Girls

Pediatrics. Aug, 2009  |  Pubmed ID: 19651567

To determine the current prevalence and mean ages of onset of pubertal characteristics in healthy urban Chinese girls.

Does Nitric Oxide Play a Pivotal Role Downstream of Auxin in Promoting Crown Root Primordia Initiation in Monocots?

Plant Signaling & Behavior. Oct, 2009  |  Pubmed ID: 19826236

Increasing instances prove that nitric oxide (NO) plays a significant role in mediating root growth and development, and it is reported that NO acts as a messenger and mediates the auxin-induced adventitious roots (AR) developing process in cucumber explants. Compared with the current understanding of AR development in dicots, knowledge of the molecular and physiological mechanisms of crown root (CR) development in monocots is limited, and the roles of NO in CR initiation and development are still far from clear. Our recent studies demonstrate that a critical concentration of endogenous NO is indispensable for CR primordia initiation, the reduction of endogenous NO content blocks CR primordia initiation and decreases CR number in rice seedlings. In this addendum, Base on the results of our studies and previous reports, we supposed that CR formtion in monocots and AR formtion in dicots possible take part in the same NO signaling pathway, althoug in dicots, AR are formed under unusual circumstances and belong to the abnormal developmental program, and in monocot cereals, CR are genetically determined roots and belong to the normal developmental program of cereals. At last, we advanced a proposed schematic model showing the NO signaling pathway of CR emergence in monocots.

Rolling Cell Adhesion

Annual Review of Cell and Developmental Biology. Nov, 2010  |  Pubmed ID: 19575676

Rolling adhesion on vascular surfaces is the first step in recruiting circulating leukocytes, hematopoietic progenitors, or platelets to specific organs or to sites of infection or injury. Rolling requires the rapid yet balanced formation and dissociation of adhesive bonds in the challenging environment of blood flow. This review explores how structurally distinct adhesion receptors interact through mechanically regulated kinetics with their ligands to meet these challenges. Remarkably, increasing force applied to adhesive bonds first prolongs their lifetimes (catch bonds) and then shortens their lifetimes (slip bonds). Catch bonds mediate the counterintuitive phenomenon of flow-enhanced rolling adhesion. Force-regulated disruptions of receptor interdomain or intradomain interactions remote from the ligand-binding surface generate catch bonds. Adhesion receptor dimerization, clustering in membrane domains, and interactions with the cytoskeleton modulate the forces applied to bonds. Both inside-out and outside-in cell signals regulate these processes.

Membrane-based Actuation for High-speed Single Molecule Force Spectroscopy Studies Using AFM

European Biophysics Journal : EBJ. Jul, 2010  |  Pubmed ID: 20054686

Atomic force microscopy (AFM)-based dynamic force spectroscopy of single molecular interactions involves characterizing unbinding/unfolding force distributions over a range of pulling speeds. Owing to their size and stiffness, AFM cantilevers are adversely affected by hydrodynamic forces, especially at pulling speeds >10 microm/s, when the viscous drag becomes comparable to the unbinding/unfolding forces. To circumvent these adverse effects, we have fabricated polymer-based membranes capable of actuating commercial AFM cantilevers at speeds >or=100 microm/s with minimal viscous drag effects. We have used FLUENT, a computational fluid dynamics (CFD) software, to simulate high-speed pulling and fast actuation of AFM cantilevers and membranes in different experimental configurations. The simulation results support the experimental findings on a variety of commercial AFM cantilevers and predict significant reduction in drag forces when membrane actuators are used. Unbinding force experiments involving human antibodies using these membranes demonstrate that it is possible to achieve bond loading rates >or=10(6) pN/s, an order of magnitude greater than that reported with commercial AFM cantilevers and systems.

Ubiquitin Ligase Cullin 7 Induces Epithelial-mesenchymal Transition in Human Choriocarcinoma Cells

The Journal of Biological Chemistry. Apr, 2010  |  Pubmed ID: 20139075

Germ line mutations of the ubiquitin ligase cullin 7 (CUL7) are linked to 3-M syndrome and Yakuts short stature syndrome, both of which are characterized by pre- and post-natal growth retardation. CUL7 knock-out mice show placental and embryonic defects similar to intrauterine growth retardation, suggesting a role of CUL7 in placentation. CUL7 was found in this study to be highly expressed in first trimester invasive human placental villi as well as in HTR8/SVneo and B6Tert cells, two cell lines derived from human first trimester trophoblast cells. However, CUL7 levels in term trophoblast cells or JEG-3 cells, which are derived from human choriocarcinoma but exhibit weak invasion capacity, were low or undetectable. Forced expression of CUL7 in JEG-3 cells induced cell morphological changes characteristic of epithelial-mesenchymal transition, which was accompanied by a complete loss of the epithelial markers E-cadherin and P-cadherin and a significant elevation of mesenchymal markers Vimentin and N-cadherin. JEG-3 cells expressing CUL7 exhibited enhanced cell migration and invasion. Conversely, CUL7-specific RNA interference in HTR8/SVneo cells resulted in increased E-cadherin expression and reduced cell migration and invasion. Furthermore, CUL7 expression down-regulated E-cadherin mRNA expression by up-regulating ZEB1 and Slug, two transcriptional repressors of E-cadherin. Finally, CUL7-induced loss of E-cadherin expression was partially reversed by treatment of CUL7-expressing cells with the proteasome inhibitor MG-132. These results suggest that the CUL7 E3 ligase is a key regulator in trophoblast cell epithelial-mesenchymal transition and placental development.

[The Role of MiR398 in Plant Stress Responses]

Yi Chuan = Hereditas / Zhongguo Yi Chuan Xue Hui Bian Ji. Feb, 2010  |  Pubmed ID: 20176556

MicroRNAs (miRNAs) are a new class of small RNAs, which act as post-transcriptional negative regulators of gene expression. Plant miRNAs are important in the regulation of plant growth, development and in response to various abiotic and biotic stresses. miR398 is the first reported miRNA to be down-regulated by oxidative stresses. miR398 plays an impor-tant role in stresses, such as regulating copper homeostasis, in response to abiotic stresses including heavy metals, sucrose, and ozone and biotic stresses via down-regulating the expression of Cu/Zn-superoxide dismutase (CSD). This review fo-cused on the crucial role of miR398 in regulation of different stresses and the transcriptional regulation of MIR398 gene.

Roles of Nitric Oxide in Alleviating Heavy Metal Toxicity in Plants

Archives of Biochemistry and Biophysics. May, 2010  |  Pubmed ID: 20193657

Nitric oxide (NO) is involved in the regulation of multiple plant responses to a variety of abiotic and biotic stresses. Recently, an increasing number of articles have reported the effects of exogenous NO on alleviating heavy metal toxicity in plants. However, compared with the current understanding of the relationships between NO and other abiotic stresses, knowledge of the molecular and physiological mechanisms of NO in alleviating heavy metal toxicity is quite limited, and some results contradict one another. Therefore, to help clarify the roles of NO in heavy metal tolerance, it is valuable to review and discuss the recent advances on this research topic. In this mini-review, the latest advances in understanding the effects of heavy metals on endogenous NO content and the mechanisms and signaling pathways of exogenous NO in alleviating heavy metal toxicity in plants are summarized and discussed. A basic scheme for the roles of NO in alleviating heavy metal toxicity is also proposed.

The Kinetics of Two-dimensional TCR and PMHC Interactions Determine T-cell Responsiveness

Nature. Apr, 2010  |  Pubmed ID: 20357766

The T-cell receptor (TCR) interacts with peptide-major histocompatibility complexes (pMHC) to discriminate pathogens from self-antigens and trigger adaptive immune responses. Direct physical contact is required between the T cell and the antigen-presenting cell for cross-junctional binding where the TCR and pMHC are anchored on two-dimensional (2D) membranes of the apposing cells. Despite their 2D nature, TCR-pMHC binding kinetics have only been analysed three-dimensionally (3D) with a varying degree of correlation with the T-cell responsiveness. Here we use two mechanical assays to show high 2D affinities between a TCR and its antigenic pMHC driven by rapid on-rates. Compared to their 3D counterparts, 2D affinities and on-rates of the TCR for a panel of pMHC ligands possess far broader dynamic ranges that match that of their corresponding T-cell responses. The best 3D predictor of response is the off-rate, with agonist pMHC dissociating the slowest. In contrast, 2D off-rates are up to 8,300-fold faster, with the agonist pMHC dissociating the fastest. Our 2D data suggest rapid antigen sampling by T cells and serial engagement of a few agonist pMHCs by TCRs in a large self pMHC background. Thus, the cellular environment amplifies the intrinsic TCR-pMHC binding to generate broad affinities and rapid kinetics that determine T-cell responsiveness.

Epithelial-mesenchymal Transition to Be or Not to Be? Is the Answer Yes and No at the Same Time?

International Urology and Nephrology. Sep, 2010  |  Pubmed ID: 20364403

Laboratory Culture of the Freshwater Benthic Gastropod Bellamya Aeruginosa (Reeve) and Its Utility As a Test Species for Sediment Toxicity

Journal of Environmental Sciences (China). 2010  |  Pubmed ID: 20397422

This study aimed to develop original laboratory culture and sediment toxicity testing protocols for the freshwater gastropod Bellamya aeruginosa (Reeve), a new potential species for sediment toxicity testing. B. aeruginosa was successfully cultured with an effective culture system under proposed laboratory conditions. Optimal ad libitum feeding levels for larvae, juveniles, and adults were 2.0, 6.0, and 16.0 mg fish food/(snail x day), respectively. Mean survival rates of juveniles were higher than 90%. The snails could be sexed at 9 weeks of age, and their generation time is approximately 4 months. Reproduction continued all year around; the mean fecundity was 0.55 newborn/(female x day). The utility of this species for bioassays was evaluated in both 10-day and 28-day case studies with artificial sediments. The 10-day LC50 of Cu for larvae was 480 gg/g dry weight (dw), and the lowest observed effects concentration of Cu for survival and growth of larvae was 195 microg/g dw. Survival and growth are reliable indicators of acute toxicity. Larvae accumulated more Cu than adults. B. aeruginosa exhibited a higher sensitivity to Cu exposure than standard test species (Hyalella azteca and Chironomus tentans). The 28-day test of sediment toxicity with adults showed that fecundity was a robust endpoint indicator of reproductive toxicity, and the biochemical endpoints of superoxide dismutase, catalase, and glutathione could be used as sensitive biomarkers for Cu-induced oxidative damage. B. aeruginosa can be therefore recommended as a candidate for the standardization of the freshwater sediment toxicity test protocol.

A Model for Single-substrate Trimolecular Enzymatic Kinetics

Biophysical Journal. May, 2010  |  Pubmed ID: 20441760

We developed a kinetic model for a single-substrate trimolecular enzymatic system, where a receptor binds and stretches a substrate to expose its cleavage site, allowing an enzyme to bind and cleave it into product. We demonstrated that the general kinetics of the trimolecular enzymatic system is more complex than the Michaelis-Menten kinetics. Under a limiting condition when the enzyme-substrate binding is in fast equilibrium, the enzymatic kinetics of the trimolecular system reduces to the Michaelis-Menten kinetics. In another limiting case when the receptor dissociates negligibly slowly from the substrate, the trimolecular system is simplified to a bimolecular system, which follows the Michaelis-Menten equation if and only if there is no enzyme-substrate complex initially. We applied this model to a particular trimolecular system important to hemostasis and thrombosis, consisting of von Willebrand factor (substrate), platelet glycoprotein Ibalpha (receptor), and ADAMTS13 (enzyme). Using parameters from independent experiments, our model successfully predicted published data from two single-molecule experiments and fitted/predicted published data from an ensemble experiment.

[Bioaccumulation of Sediment Heavy Metals in Bellamya Aeruginosa and Its Relations with the Metals Geochemical Fractions]

Ying Yong Sheng Tai Xue Bao = The Journal of Applied Ecology / Zhongguo Sheng Tai Xue Xue Hui, Zhongguo Ke Xue Yuan Shenyang Ying Yong Sheng Tai Yan Jiu Suo Zhu Ban. Mar, 2010  |  Pubmed ID: 20560332

A 28-day sediment bioaccumulation test was conducted to study the bioaccumulation of river sediment heavy metals in Bellamya aeruginosa, and its relations with the geochemical fractions of the metals. A higher bioaccumulation of Cd, Pb, Cu, Cr, Zn, and Mn was found in the hepatopancreas of B. aeruginosa, with the greatest accumulation of Zn (84.32% +/- 4.36%), followed by Cu (7.67% +/- 2.84%), Pb (3.62% +/- 1.84%), Cr (2.22% +/- 1.03%), Mn (1.33% +/- 0.15%), and Cd (0.83% +/- 0.53%). No significant correlations were observed between the heavy metals accumulations in B. aeruginosa hepatopancreas, but the significant positive correlation between the metals pollution index of hepatopancreas and the Nemerow pollution index of sediments suggested that B. aeruginosa could be used as a potential bioindicator for sediment heavy metals pollution. The biota-sediment accumulation factors (BSAFs) for Cd, Cr, Zn, and Mn from different sediments showed a higher variability, while the BSAFs for Cu and Pb were relatively constant. The bioaccumulation of Cd had significant correlations with exchangeable Cd, weak acid soluble Cd, and oxidizable Cd; Pb bioaccumulation had significant correlation with reducible Pb; Cu bioaccumulation had significant correlation with oxidizable Cu; while Cr and Mn bioaccumulation had no correlations with the total concentrations and geochemical fractions of the two metals. Therefore, it would be inappropriate to use the BSAF as the indicator for the bioaccumulation of heavy metals in B. aeruginosa.

Molecular Biomechanics: The Molecular Basis of How Forces Regulate Cellular Function

Molecular & Cellular Biomechanics : MCB. Mar, 2010  |  Pubmed ID: 20700472

Recent advances have led to the emergence of molecular biomechanics as an essential element of modern biology. These efforts focus on theoretical and experimental studies of the mechanics of proteins and nucleic acids, and the understanding of the molecular mechanisms of stress transmission, mechanosensing and mechanotransduction in living cells. In particular, single-molecule biomechanics studies of proteins and DNA, and mechanochemical coupling in biomolecular motors have demonstrated the critical importance of molecular mechanics as a new frontier in bioengineering and life sciences. To stimulate a more systematic study of the basic issues in molecular biomechanics, and attract a broader range of researchers to enter this emerging field, here we discuss its significance and relevance, describe the important issues to be addressed and the most critical questions to be answered, summarize both experimental and theoretical/computational challenges, and identify some short-term and long-term goals for the field. The needs to train young researchers in molecular biomechanics with a broader knowledge base, and to bridge and integrate molecular, subcellular and cellular level studies of biomechanics are articulated.

Synergistic Effects of the GATA-4-mediated MiR-144/451 Cluster in Protection Against Simulated Ischemia/reperfusion-induced Cardiomyocyte Death

Journal of Molecular and Cellular Cardiology. Nov, 2010  |  Pubmed ID: 20708014

Among the identified microRNAs (miRs) thus far, ~50% of mammalian miRs are clustered in the genome and transcribed as polycistronic primary transcripts. However, whether clustered miRs mediate non-redundant and cooperative functions remains poorly understood. In this study, we first identified activation of the promoter of miR-144/451 by GATA-4, a critical transcription factor in the heart. Next, we observed that ectopic expression of miR-144 and -451 individually augmented cardiomyocyte survival, which was further improved by overexpression of miR-144/451, compared to control cells in response to simulated ischemia/reperfusion. In contrast, knockdown of endogenous miR-144 and -451 revealed opposite effects. Using luciferase reporter assay and western blot analysis, we also validated that both miR-144 and miR-451 target CUG triplet repeat-binding protein 2 (CUGBP2), a ubiquitously expressed RNA-binding protein, known to interact with COX-2 3'UTR and inhibit its translation. Accordingly, protein levels of CUGBP2 were greatly reduced and COX-2 activity was markedly increased in miR-144-, miR-451-, and miR-144/451-overexpressing cardiomyocytes, compared to GFP cells. Furthermore, inhibition of COX-2 activity by either NS-398 or DUP-697 partially offset protective effects of the miR-144/451 cluster. Together, these data indicate that both partners of the miR-144/451 cluster confer protection against simulated I/R-induced cardiomyocyte death via targeting CUGBP2-COX-2 pathway, at least in part. Thus, both miR-144 and miR-451 may represent new therapeutic agents for the treatment of ischemic heart disease.

Triphasic Force Dependence of E-selectin/ligand Dissociation Governs Cell Rolling Under Flow

Biophysical Journal. Aug, 2010  |  Pubmed ID: 20713000

During inflammation, flowing leukocytes tether to and roll on vascular surfaces through the association and dissociation of selectin/ligand bonds. The interactions of P- and L- selectins with their respective ligands exhibit catch-slip bonds, such that increasing force initially prolongs and then shortens bond lifetimes. In addition, catch-slip bonds have been shown to govern L-selectin-mediated cell rolling. Using a flow chamber and biomembrane force probe, we show a triphasic force dependence of E-selectin/ligand dissociation that initially behaves as slip bonds, then transitions to catch bonds, and finally transitions again to slip bonds as the force increases. These transitions govern the velocities of neutrophils, HL-60 cells, and Colo-205 cells rolling on E-selectin, as evidenced by the fact that their velocities exhibited a triphasic force dependence that inversely matched the triphasic lifetime-force relationship. At low forces, slip bonds may also precede catch bonds for interactions of P- and L-selectin with their ligands.

The Mechanism of VWF-mediated Platelet GPIbalpha Binding

Biophysical Journal. Aug, 2010  |  Pubmed ID: 20713003

The binding of Von Willebrand Factor to platelets is dependent on the conformation of the A1 domain which binds to platelet GPIbalpha. This interaction initiates the adherence of platelets to the subendothelial vasculature under the high shear that occurs in pathological thrombosis. We have developed a thermodynamic strategy that defines the A1:GPIbalpha interaction in terms of the free energies (DeltaG values) of A1 unfolding from the native to intermediate state and the binding of these conformational states to GPIbalpha. We have isolated the intermediate conformation of A1 under nondenaturing conditions by reduction and carboxyamidation of the disulfide bond. The circular dichroism spectrum of reduction and carboxyamidation A1 indicates that the intermediate has approximately 10% less alpha-helical structure that the native conformation. The loss of alpha-helical secondary structure increases the GPIbalpha binding affinity of the A1 domain approximately 20-fold relative to the native conformation. Knowledge of these DeltaG values illustrates that the A1:GPIbalpha complex exists in equilibrium between these two thermodynamically distinct conformations. Using this thermodynamic foundation, we have developed a quantitative allosteric model of the force-dependent catch-to-slip bonding that occurs between Von Willebrand Factor and platelets under elevated shear stress. Forced dissociation of GPIbalpha from A1 shifts the equilibrium from the low affinity native conformation to the high affinity intermediate conformation. Our results demonstrate that A1 binding to GPIbalpha is thermodynamically coupled to A1 unfolding and catch-to-slip bonding is a manifestation of this coupling. Our analysis unites thermodynamics of protein unfolding and conformation-specific binding with the force dependence of biological catch bonds and it encompasses the effects of two subtypes of mutations that cause Von Willebrand Disease.

Forcing Switch from Short- to Intermediate- and Long-lived States of the AlphaA Domain Generates LFA-1/ICAM-1 Catch Bonds

The Journal of Biological Chemistry. Nov, 2010  |  Pubmed ID: 20819952

Binding of lymphocyte function-associated antigen-1 (LFA-1) to intercellular adhesion molecule-1 (ICAM-1) mediates leukocyte adhesion under force. Using a biomembrane force probe capable of measuring single bond interactions, we showed ICAM-1 binding to LFA-1 at different conformations, including the bent conformation with the lowest affinity. We quantify how force and conformations of LFA-1 regulate its kinetics with ICAM-1. At zero-force, on-rates were substantially changed by conditions that differentially favor a bent or extended LFA-1 with a closed or open headpiece; but off-rates were identical. With increasing force, LFA-1/ICAM-1 bond lifetimes (reciprocal off-rates) first increased (catch bonds) and then decreased (slip bonds). Three states with distinct off-rates were identified from lifetime distributions. Force shifted the associated fractions from the short- to intermediate- and long-lived states, producing catch bonds at low forces, but increased their off-rates exponentially, converting catch to slip bonds at high forces. An internal ligand antagonist that blocks pulling of the α(7)-helix suppressed the intermediate-/long-lived states and eliminated catch bonds, revealing an internal catch bond between the αA and βA domains. These results elucidate an allosteric mechanism for the mechanochemistry of LFA-1/ICAM-1 binding.

Force-induced Cleavage of Single VWFA1A2A3 Tridomains by ADAMTS-13

Blood. Jan, 2010  |  Pubmed ID: 19897584

A disintegrin and metalloprotease with a thrombospondin type 1 motifs 13 (ADAMTS-13) regulates hemostasis by cleaving the folded A2 domain of von Willebrand factor (VWF). The cleavage is regulated by forces as it occurs in flowing blood. We tested the hypothesis that force-induced A2 domain unfolding facilitates cleavage using atomic force microscopy to pull single VWF A1A2A3 tridomain polypeptides by platelet glycoprotein Ibalpha or antibodies to measure time, distance, and force. Structural destabilization of A1A2A3 was induced by 5- to 80-pN forces, manifesting as an abrupt molecular length increase distributed around 20 and 50 nm, probably because of uncoupling A1A2A3 (or partially unfolding A2) and fully unfolding A2, respectively. Time required to destabilize A1A2A3 first increased (catch), reaching a maximum of 0.2 seconds at 20pN, then decreased (slip) with increasing force, independent of ADAMTS-13. The time required to rupture A1A2A3 exhibited a similar catch-slip behavior when pulled by glycoprotein Ibalpha but only slip behavior when pulled by antibody, which was progressively shortened by increasing concentration of ADAMTS-13 after (but not before) structural destabilization, indicating that cleavage of A2 requires the force-induced A2 unfolding. Analysis with a model for single-substrate trimolecular enzymatic kinetics estimated a cleavage rate k(cat) of 2.9 (+/- 59) seconds and a K(d) of 5.6 (+/- 3.4) nM for ADAMTS-13/A1A2A3 binding. These findings quantify the mechanical regulation of VWF cleavage by ADAMTS-13 at the level of single A1A2A3 tridomain.

MiR398 and Plant Stress Responses

Physiologia Plantarum. Sep, 2011  |  Pubmed ID: 21496029

Because of their sessile nature, plants are constantly exposed to a multitude of abiotic and biotic stresses. Great progress has been made in elucidating the complex stress response mechanisms in plants. MicroRNAs (miRNAs), recently recognized as important regulators of gene expression at the posttranscriptional level, have been found to be involved in plant stress responses. Most plant miRNAs usually mediate cleavage of their target mRNAs. The observation that some miRNAs are up- or downregulated in response to stress implies that miRNAs play vital roles in plant resistance to abiotic and biotic stresses. Manipulation of miRNA-guided gene regulation may represent a new way to engineer plants with improved stress tolerance. Among stress-responsive miRNAs, miRNA398 (miR398) is a miRNA proposed to be directly linked to the plant stress regulatory network and regulates plant responses to oxidative stress, water deficit, salt stress, abscisic acid stress, ultraviolet stress, copper and phosphate deficiency, high sucrose and bacterial infection. This review highlights recent progress in understanding the crucial role of miR398 in plant stress responses, and also includes a discussion of miR398-centered gene regulatory network.

Drug Repositioning for Orphan Diseases

Briefings in Bioinformatics. Jul, 2011  |  Pubmed ID: 21504985

The need and opportunity to discover therapeutics for rare or orphan diseases are enormous. Due to limited prevalence and/or commercial potential, of the approximately 6000 orphan diseases (defined by the FDA Orphan Drug Act as <200 000 US prevalence), only a small fraction (5%) is of interest to the biopharmaceutical industry. The fact that drug development is complicated, time-consuming and expensive with extremely low success rates only adds to the low rate of therapeutics available for orphan diseases. An alternative and efficient strategy to boost the discovery of orphan disease therapeutics is to find connections between an existing drug product and orphan disease. Drug Repositioning or Drug Repurposing--finding a new indication for a drug--is one way to maximize the potential of a drug. The advantages of this approach are manifold, but rational drug repositioning for orphan diseases is not trivial and poses several formidable challenges--pharmacologically and computationally. Most of the repositioned drugs currently in the market are the result of serendipity. One reason the connection between drug candidates and their potential new applications are not identified in an earlier or more systematic fashion is that the underlying mechanism 'connecting' them is either very intricate and unknown or indirect or dispersed and buried in an ever-increasing sea of information, much of which is emerging only recently and therefore is not well organized. In this study, we will review some of these issues and the current methodologies adopted or proposed to overcome them and translate chemical and biological discoveries into safe and effective orphan disease therapeutics.

Polymorphisms of the Prion Protein Gene and Their Effects on Litter Size and Risk Evaluation for Scrapie in Chinese Hu Sheep

Virus Genes. Aug, 2011  |  Pubmed ID: 21556743

It is well known that scrapie is a fatal, neurodegenerative disease in sheep and goat, which belongs to the group of transmissible spongiform encephalopathies (TSEs) or prion diseases. It has been confirmed that the polymorphisms of prion protein gene (PRNP) at codons 136, 154, and 171 have strong relationship with scrapie in sheep. In the present study, nine polymorphisms of PRNP at codons 136, 154, and 171 and other six loci (at codons 101, 112, 127, 137, 138, and 152) were detected in 180 Chinese Hu sheep. All the alleles at codons 136, 154, and 171 have been identified and resulted in three new genotypes. The frequencies of predominant alleles were 85% (A136), 99.40% (R154), and 37.78% (Q171), respectively. The predominant haplotype ARQ has a relatively high frequency of 57.77%. The frequencies of dominant genotypes of ARR/ARQ and ARQ/ARQ were 30 and 26.67%, respectively. Three new found genotypes named ARQ/TRK, ARQ/TRR, and TRR/TRQ had the same lower frequencies (0.56%). The relationship of PRNP genotype with scrapie risk and litter size showed that the predominant genotypes are corresponded to the risk score of R(1) (1.67%), R(2) (32.22%), and R(3) (42.22%). Just at the first parity, the individuals with ARH/ARH genotype had significantly larger litter size than the mean value and those with ARQ/ARQ and ARR/ARQ genotypes. In short, this study provided preliminary information about alleles and genotypes of PRNP in Chinese Hu sheep. It could be concluded that Hu sheep has a low susceptibility to natural scrapie, and the predominant PRNP genotype at least has no significant effect on litter size.

The Orphan Disease Networks

American Journal of Human Genetics. Jun, 2011  |  Pubmed ID: 21664998

The low prevalence rate of orphan diseases (OD) requires special combined efforts to improve diagnosis, prevention, and discovery of novel therapeutic strategies. To identify and investigate relationships based on shared genes or shared functional features, we have conducted a bioinformatic-based global analysis of all orphan diseases with known disease-causing mutant genes. Starting with a bipartite network of known OD and OD-causing mutant genes and using the human protein interactome, we first construct and topologically analyze three networks: the orphan disease network, the orphan disease-causing mutant gene network, and the orphan disease-causing mutant gene interactome. Our results demonstrate that in contrast to the common disease-causing mutant genes that are predominantly nonessential, a majority of orphan disease-causing mutant genes are essential. In confirmation of this finding, we found that OD-causing mutant genes are topologically important in the protein interactome and are ubiquitously expressed. Additionally, functional enrichment analysis of those genes in which mutations cause ODs shows that a majority result in premature death or are lethal in the orthologous mouse gene knockout models. To address the limitations of traditional gene-based disease networks, we also construct and analyze OD networks on the basis of shared enriched features (biological processes, cellular components, pathways, phenotypes, and literature citations). Analyzing these functionally-linked OD networks, we identified several additional OD-OD relations that are both phenotypically similar and phenotypically diverse. Surprisingly, we observed that the wiring of the gene-based and other feature-based OD networks are largely different; this suggests that the relationship between ODs cannot be fully captured by the gene-based network alone.

Regulation of Catch Bonds by Rate of Force Application

The Journal of Biological Chemistry. Sep, 2011  |  Pubmed ID: 21775439

The current paradigm for receptor-ligand dissociation kinetics assumes off-rates as functions of instantaneous force without impact from its prior history. This a priori assumption is the foundation for predicting dissociation from a given initial state using kinetic equations. Here we have invalidated this assumption by demonstrating the impact of force history with single-bond kinetic experiments involving selectins and their ligands that mediate leukocyte tethering and rolling on vascular surfaces during inflammation. Dissociation of bonds between L-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) loaded at a constant ramp rate to a constant hold force behaved as catch-slip bonds at low ramp rates that transformed to slip-only bonds at high ramp rates. Strikingly, bonds between L-selectin and 6-sulfo-sialyl Lewis X were impervious to ramp rate changes. This ligand-specific force history effect resembled the effect of a point mutation at the L-selectin surface (L-selectinA108H) predicted to contact the former but not the latter ligand, suggesting that the high ramp rate induced similar structural changes as the mutation. Although the A108H substitution in L-selectin eliminated the ramp rate responsiveness of its dissociation from PSGL-1, the inverse mutation H108A in P-selectin acquired the ramp rate responsiveness. Our data are well explained by the sliding-rebinding model for catch-slip bonds extended to incorporate the additional force history dependence, with Ala-108 playing a pivotal role in this structural mechanism. These results call for a paradigm shift in modeling the mechanical regulation of receptor-ligand bond dissociation, which includes conformational coupling between binding pocket and remote regions of the interacting molecules.

Circulating MicroRNAs Are Elevated in Plasma from Severe Pre-eclamptic Pregnancies

Reproduction (Cambridge, England). Dec, 2011  |  Pubmed ID: 22187671

Until recently, the molecular pathogenesis of pre-eclampsia remained largely unknown. Reports have shown that circulating microRNAs are promising novel biomarkers for cancer, pregnancy, tissue injury and other conditions. The objective of present study was to identify differentially expressed microRNAs in plasma from severe pre-eclamptic pregnancies compared with plasma from normal pregnancies. By mature microRNA microarray analysis, 15 microRNAs, including 13 up-regulated and 2 down-regulated microRNAs, were screened to be differentially expressed in plasma from women with severe pre-eclampsia. Seven microRNAs, namely miR-24, miR-26a, miR-103, miR-130b, miR-181a, miR-342-3p, and miR-574-5p, were validated to be elevated in plasma from severe pre-eclamptic pregnancies by using real-time quantitative stem-loop reverse-transcription polymerase chain reaction analysis. Gene ontology and pathway enrichment analysis revealed that these microRNAs were involved in specific biological process categories (including regulation of metabolic processes, regulation of transcription, and cell cycle) and signaling pathways (including the mitogen-activated protein kinase signaling pathway, the transforming growth factor-beta signaling pathway, and pathways in cancer metastasis). This study presents, for the first time, the differential expression profile of circulating microRNAs in severe pre-eclampsia patients. The seven elevated circulating microRNAs may play critical roles in the pathogenesis of severe pre-eclampsia, and one or more of them may become potential markers for diagnosing severe pre-eclampsia.

Engineering a Zinc Binding Site into the De Novo Designed Protein DS119 with a βαβ Structure

Protein & Cell. Dec, 2011  |  Pubmed ID: 22231358

Functional proteins designed de novo have potential application in chemical engineering, agriculture and healthcare. Metal binding sites are commonly used to incorporate functions. Based on a de novo designed protein DS119 with a βαβ structure, we have computationally engineered zinc binding sites into it using a home-made searching program. Seven out of the eight designed sequences tested were shown to bind Zn(2+) with micromolar affinity, and one of them bound Zn(2+) with 1:1 stoichiometry. This is the first time that metalloproteins with an α, β mixed structure have been designed from scratch.

Cold Shock Y-box Protein-1 Participates in Signaling Circuits with Auto-regulatory Activities

European Journal of Cell Biology. Sep, 2011  |  Pubmed ID: 21962637

The cold shock protein Y-box (YB) binding-1 is an example of a highly regulated protein with pleiotropic functions. Besides activities as a transcription factor in the nucleus or regulator of translation in the cytoplasm, recent findings indicate extracellular effects and secretion via a non-classical secretion pathway. This review summarizes regulatory pathways in which YB-1 participates, all iterating auto-regulatory loops. Schematics are developed that elucidate the cold shock protein activities in (i) fine-tuning its own expression level following platelet-derived growth factor-B-, thrombin- or interferon-γ-dependent signaling, (ii) as a component of the messenger ribonucleoprotein (mRNP) complex for interleukin-2 synthesis in T-cell commitment/activation, (iii) pro-fibrogenic cell phenotypic changes mediated by transforming growth factor-β, and (iv) receptor Notch-3 cleavage and signal transduction. Emphasis is put forward on subcellular protein translocation mechanisms and underlying signaling pathways. These have mostly been analysed in cell culture systems and rarely in experimental models. In sum, YB-1 seems to fulfill a pacemaker role in diverse diseases, both inflammatory/pro-fibrogenic as well as tumorigenic. A clue towards potential intervention strategies may reside in the understanding of the outlined auto-regulatory loops and means to interfere with cycling pathways.

Tianeptine Reverses Stress-induced Asymmetrical Hippocampal Volume and N-acetylaspartate Loss in Rats: an in Vivo Study

Psychiatry Research. Dec, 2011  |  Pubmed ID: 22047727

Stress-induced hippocampal volume loss and decrease in N-acetylaspartate (NAA) level have been reported to be associated with impaired neural plasticity and neuronal damage in adults. Accordingly, reversing structural and metabolite damage in the hippocampus may be a desirable goal for antidepressant therapy. The present study investigated the effects of tianeptine on chronic stress-induced hippocampal volume loss and metabolite alterations in vivo in 24 Sprague-Dawley rats. Rats were subjected to a consecutive 28-day forced swimming test stress. Tianeptine (50mg/kg) or saline was administered intragastrically 4h after swimming each day. Spontaneous behaviors, serum corticosterone concentration, hippocampal volume and NAA level were evaluated after stress. Chronic tianeptine treatment counteracted the chronic stress-induced suppression of spontaneous behaviors, elevated serum corticosterone concentration, reduced hippocampal volume and decreased NAA level. Moreover, we found asymmetrical right-left hippocampal volume loss in stressed rats, with the left hippocampus more sensitive to chronic stress than the right hippocampus. In addition, stressed rats showed a decreased level of hippocampal metabolites, without significant loss of hippocampal volume. These findings provide experimental evidence for impaired structural plasticity of the brain being an important feature of depressive illness and suggest that prophylactic tianeptine treatments could reverse structural changes in brain. The structural and neurochemical alterations in the hippocampus may be valuable indexes for evaluating the prophylactic and curative effect of antidepressant treatments in depressive and stress-related disorders.

T Cell Receptor Signaling is Limited by Docking Geometry to Peptide-major Histocompatibility Complex

Immunity. Nov, 2011  |  Pubmed ID: 22101157

T cell receptor (TCR) engagement of peptide-major histocompatibility complex (pMHC) is essential to adaptive immunity, but it is unknown whether TCR signaling responses are influenced by the binding topology of the TCR-peptide-MHC complex. We developed yeast-displayed pMHC libraries that enabled us to identify new peptide sequences reactive with a single TCR. Structural analysis showed that four peptides bound to the TCR with distinct 3D and 2D affinities using entirely different binding chemistries. Three of the peptides that shared a common docking mode, where key TCR-MHC germline interactions are preserved, induced TCR signaling. The fourth peptide failed to induce signaling and was recognized in a substantially different TCR-MHC binding mode that apparently exceeded geometric tolerances compatible with signaling. We suggest that the stereotypical TCR-MHC docking paradigm evolved from productive signaling geometries and that TCR signaling can be modulated by peptides that are recognized in alternative TCR-pMHC binding orientations.

[Heterologous Expression of a Rice Syntaxin-related Protein KNOLLE Gene (OsKNOLLE) in Yeast and Its Functional Analysis in the Role of Abiotic Stress]

Yi Chuan = Hereditas / Zhongguo Yi Chuan Xue Hui Bian Ji. Nov, 2011  |  Pubmed ID: 22120082

Syntaxin-related protein KNOLLE, a multifunctional protein family belonging to the SNARE superfamily, plays an important role in many physiological processes in plants. In order to understand the function of the syntaxin-related protein KNOLLE (OsKNOLLE) in rice (Oryza sativa), the CDS sequence of OsKNOLLE gene isolated from a japonica rice cultivar "Zhonghua 11" was fused into an expression vector pYX212 and transformed into the S. cerevisiae strain BY4741 by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. The transformants with OsKNOLLE showed better survival abilities than the transformants with the empty vectors based on their phenotypes in responses to different abiotic stresses such as salt, Cu2+, H2O2, Cd2+, and Hg2+. These data suggests that OsKNOLLE plays a crucial role in re-sponses to abiotic stresses. This experimental system sets up a method for studying functions of the OsKNOLLE gene in the future and clarifies the relationship between OsKNOLLE and abiotic stresses.

Impact of Ozone Exposure on Prostaglandin Release in Nasal Polyps

European Archives of Oto-rhino-laryngology : Official Journal of the European Federation of Oto-Rhino-Laryngological Societies (EUFOS) : Affiliated with the German Society for Oto-Rhino-Laryngology - Head and Neck Surgery. Dec, 2011  |  Pubmed ID: 22130916

A dysregulation of the cyclooxygenases and a leukotriene/prostaglandin imbalance are assumed to be part of the pathogenesis of the aspirin (ASA) intolerance syndrome. Ozone is an air pollutant with known proinflammatory effects on exposed epithelia, however, its impact on the expression of the cyclooxygenases 1 and 2 (cox1/2) and prostaglandin E(2) (PGE(2)) in the nasal mucosa is not well known. Therefore, we analyzed cox expression and PGE(2) levels after ozone exposure in nasal mucosa and in nasal polyps considering ASA intolerance. Isolated epithelial nasal cells from control subjects without chronic rhinosinusitis (CRS), and those from patients with nasal polyps with and without ASA intolerance were cultured and exposed in vitro to ozone. Cox1/2 expression levels were analyzed by immunohistochemistry and PGE(2) release by ELISA. After ozone exposure cox1/2 expression remained unchanged in all the three groups. PGE(2) release was lowered in cell cultures from controls and from polyps of ASA tolerant but not in those of ASA intolerant patients after ozone exposure. In the latter, PGE(2) expression remained unchanged. Our in vitro data suggest that aspirin tolerant patients with polyps might be more affected by ozone compared to aspirin intolerant ones. The potential clinical impact of impaired PGE(2) expression caused by ozone on the functions of respiratory epithelia remains to be clarified.

Structural Basis and Kinetics of Force-induced Conformational Changes of an αA Domain-containing Integrin

PloS One. 2011  |  Pubmed ID: 22140490

Integrin α(L)β₂ (lymphocyte function-associated antigen, LFA-1) bears force upon binding to its ligand intercellular adhesion molecule 1 (ICAM-1) when a leukocyte adheres to vascular endothelium or an antigen presenting cell (APC) during immune responses. The ligand binding propensity of LFA-1 is related to its conformations, which can be regulated by force. Three conformations of the LFA-1 αA domain, determined by the position of its α₇-helix, have been suggested to correspond to three different affinity states for ligand binding.

Fbxw8 is Involved in the Proliferation of Human Choriocarcinoma JEG-3 Cells

Molecular Biology Reports. Mar, 2011  |  Pubmed ID: 20878477

Fbxw8 is the F-box component of a SCF-like E3 ubiquitin ligase complex. Mice lacking Fbxw8 exhibit pathological defects in placenta and embryo similar to fetal growth retardation, suggesting a role of Fbxw8 in placentation. Proliferative capacity of trophoblast cells is very important in placental development. In this context, we revealed that Fbxw8 was expressed in four different human trophoblast cell lines. Silencing of Fbxw8 expression by siRNA inhibited the growth of choriocarcinoma JEG-3 cells. By Western blotting, cell cycle analysis, we showed that down-regulation of Fbxw8 by RNAi induced cell-growth arrest at G2/M phase through decreasing the levels of CDK1, CDK2, cyclin A and cyclin B1 and up-regulation of p27 at protein level. Conversely, over-expression of Fbxw8 led to the opposite effect. These results suggest that Fbxw8 plays an essential role in the proliferation of human trophoblast cells, especially JEG-3 cells, via G2/M phase transition in association with regulation of CDK1, CDK2, cyclin A, cyclin B1 and p27 expression.

Age-related Gene Response of Human Corneal Endothelium to Oxidative Stress and DNA Damage

Investigative Ophthalmology & Visual Science. Mar, 2011  |  Pubmed ID: 21087955

Nuclear oxidative DNA damage increases with age in human corneal endothelial cells (HCECs) and contributes to their decreased proliferative capacity. These studies investigated whether HCECs respond to this damage by upregulating their expression of oxidative stress and DNA damage-signaling genes in an age-dependent manner.

Molecular Stiffness of Selectins

The Journal of Biological Chemistry. Mar, 2011  |  Pubmed ID: 21216951

During inflammation, selectin-ligand interactions provide forces for circulating leukocytes to adhere to vascular surfaces, which stretch the interacting molecules, suggesting that mechanical properties may be pertinent to their biological function. From mechanical measurements with atomic force microscopy, we analyzed the molecular characteristics of selectins complexed with ligands and antibodies. Respective stiffness of L-, E-, and P-selectins (4.2, 1.4, and 0.85 piconewton/nm) correlated inversely with the number (2, 6, and 9) of consensus repeats in the selectin structures that acted as springs in series to dominate their compliance. After reconstitution into a lipid bilayer, purified membrane P-selectin remained a dimer, capable of forming dimeric bonds with P-selectin glycoprotein ligand (PSGL)-1, endoglycan-Ig, and a dimeric form of a glycosulfopeptide modeled after the N terminus of PSGL-1. By comparison, purified membrane L- and E-selectin formed only monomeric bonds under identical conditions. Ligands and antibodies were much less stretchable than selectins. The length of endoglycan-Ig was found to be 51 ± 12 nm. These results provide a comprehensive characterization of the molecular stiffness of selectins and illustrate how mechanical measurements can be utilized for molecular analysis, e.g. evaluating the multimericity of selectins and determining the molecular length of endoglycan.

High Prevalence of Low Affinity Peptide-MHC II Tetramer-negative Effectors During Polyclonal CD4+ T Cell Responses

The Journal of Experimental Medicine. Jan, 2011  |  Pubmed ID: 21220453

T cell affinity for antigen initiates adaptive immunity. However, the contribution of low affinity cells to a response is unknown as it has not been possible to assess the entire affinity range of a polyclonal T cell repertoire. In this study, we used a highly sensitive two-dimensional binding assay to identify low affinity cells in polyclonal autoreactive and pathogen-reactive CD4(+) T cell populations specific for myelin oligodendrocyte glycoprotein (MOG) and lymphocytic choriomeningitis virus (LCMV) antigens, respectively. Low affinity CD4(+) T cells, below detection with peptide-major histocompatibility complex class II tetramers, were at least as frequent as high affinity responders and contributed significant effector cytokines in both primary antigen-specific responses. We further demonstrated that MOG- and LCMV-specific CD4(+) T cells possessed similarly broad ranges in their affinities (>100-fold wide), only differing in the frequencies of low and high affinity cells. Thus, low as well as high affinity CD4(+) T cells are critical effectors in autoimmune and pathogen-specific responses.

Two-stage Cooperative T Cell Receptor-peptide Major Histocompatibility Complex-CD8 Trimolecular Interactions Amplify Antigen Discrimination

Immunity. Jan, 2011  |  Pubmed ID: 21256056

The T cell receptor (TCR) and CD8 bind peptide-major histocompatibility complex (pMHC) glycoproteins to initiate adaptive immune responses, yet the trimolecular binding kinetics at the T cell membrane is unknown. By using a micropipette adhesion frequency assay, we show that this kinetics has two stages. The first consists of TCR-dominant binding to agonist pMHC. This triggers a second stage consisting of a step increase in adhesion after a one second delay. The second-stage binding requires Src family kinase activity to initiate CD8 binding to the same pMHC engaged by the TCR. This induced trimeric-cooperative interaction enhances adhesion synergistically to favor potent ligands, which further amplifies discrimination. Our data reveal a TCR-CD8 positive-feedback loop involved in initial signaling steps that is sensitive to a single pMHC is rapid, reversible, synergistic, and peptide discriminative.

Microarray-based Analysis of Cadmium-responsive MicroRNAs in Rice (Oryza Sativa)

Journal of Experimental Botany. Jun, 2011  |  Pubmed ID: 21362738

MicroRNAs (miRNAs) are a class of small non-coding RNAs that negatively regulate specific target mRNAs at the post-transcriptional level. Plant miRNAs have been implicated in developmental processes and adaptations to environmental stresses. Cadmium (Cd) is a non-essential heavy metal that is highly toxic to plants. To investigate the responsive functions of miRNAs under Cd stress, miRNA expression in Cd-stressed rice (Oryza sativa) was profiled using a microarray assay. A total of 19 Cd-responsive miRNAs were identified, of which six were further validated experimentally. Target genes were also predicted for these Cd-responsive miRNAs, which encoded transcription factors, and proteins associated with metabolic processes or stress responses. In addition, the mRNA levels of several targets were negatively correlated with the corresponding miRNAs under Cd stress. Promoter analysis showed that metal stress-responsive cis-elements tended to occur more frequently in the promoter regions of Cd-responsive miRNAs. These findings suggested that miRNAs played an important role in Cd tolerance in rice, and highlighted a novel molecular mechanism of heavy metal tolerance in plants.

Molecular Dynamics Simulations of Forced Unbending of Integrin α(v)β₃

PLoS Computational Biology. Feb, 2011  |  Pubmed ID: 21379327

Integrins may undergo large conformational changes during activation, but the dynamic processes and pathways remain poorly understood. We used molecular dynamics to simulate forced unbending of a complete integrin α(v)β₃ ectodomain in both unliganded and liganded forms. Pulling the head of the integrin readily induced changes in the integrin from a bent to an extended conformation. Pulling at a cyclic RGD ligand bound to the integrin head also extended the integrin, suggesting that force can activate integrins. Interactions at the interfaces between the hybrid and β tail domains and between the hybrid and epidermal growth factor 4 domains formed the major energy barrier along the unbending pathway, which could be overcome spontaneously in ~1 µs to yield a partially-extended conformation that tended to rebend. By comparison, a fully-extended conformation was stable. A newly-formed coordination between the α(v) Asp457 and the α-genu metal ion might contribute to the stability of the fully-extended conformation. These results reveal the dynamic processes and pathways of integrin conformational changes with atomic details and provide new insights into the structural mechanisms of integrin activation.

Drought-induced Proline Accumulation is Uninvolved with Increased Nitric Oxide, Which Alleviates Drought Stress by Decreasing Transpiration in Rice

Journal of Plant Research. Jan, 2012  |  Pubmed ID: 21400017

Accumulation of proline is trusted to be an adaptive response of plants against drought stress, and exogenous application of nitric oxide (NO) enhances proline accumulation in Cu-treated algae. In order to investigate whether NO works as a necessary signaling molecule in drought-induced proline accumulation in rice leaves, effects of drought stress on endogenous NO content and proline accumulation were studied in rice leaves, using sodium nitroprusside (SNP, a NO donor) and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO, a NO scavenger). The results showed that drought treatment increased both endogenous NO and proline contents in rice leaves, while foliar spray of various concentrations of SNP failed to induce proline accumulation in the leaves of well-watered rice and foliar spray of cPTIO failed to inhibit proline accumulation in the leaves of drought-stressed rice. These results indicate that increase of endogenous NO is dispensable for proline accumulation in the leaves of rice under drought stress. Further studies indicate that exogenous application of NO alleviates drought-induced water loss and ion leakage by decreasing transpiration rate of rice leaves.

Microarray Analysis of Global Gene Regulation in the Cry1Ab-resistant and Cry1Ab-susceptible Strains of Diatraea Saccharalis

Pest Management Science. Jan, 2012  |  Pubmed ID: 22228544

BACKGROUND: Extensive adoption of transgenic Bt corn in recent years for stalk borer control has increased risk of resistance evolution in the target pest populations. A Bt-resistant strain of the sugarcane borer, Diatraea saccharalis, was approximately 100-fold more tolerant to Cry1Ab toxin than the susceptible counterpart. To gain a better understanding of the molecular mechanisms of Bt resistance, the Cry1Ab-susceptible (Cry1Ab-SS) and Cry1Ab-resistant (Cry1Ab-RR) strains of D. saccharalis were subjected to a microarray analysis. RESULTS: Results showed that the expression levels of many genes were significantly different between the Cry1Ab-RR and Cry1Ab-SS strains. Microarray analysis of 7145 cDNAs revealed 384 differentially expressed genes. A total of 273 genes were significantly upregulated 2-51.6-fold, and 111 genes were significantly downregulated 2-22.6-fold in the Cry1Ab-RR strain. The upregulation of three potential resistance-related genes, coding for a glutathione S-transferase (GST), a chymotrypsin-like protease (CHY) and a lipase (LP), was confirmed using real-time PCR, indicating a reproducibility of the microarray data. Ontology analysis revealed that more than twice the number of metabolic-related genes were upregulated compared with downregulated genes with the same biological function. Up to 35.2% of the upregulated genes in the resistant strain were associated with catalytic activity, while only 9.5% of the downregulated genes were related to the same catalytic molecular function. CONCLUSION: The large portion of metabolic- or catalytic-related genes with significant upregulations indicated a potential large increase in metabolic or catalytic activities in the Cry1Ab-RR strain. This cDNA microarray gene expression data could be used to characterize and identify new genes that may be associated with Bt resistance in D. saccharalis. Copyright © 2012 Society of Chemical Industry.

Tungstate: is It Really a Specific Nitrate Reductase Inhibitor in Plant Nitric Oxide Research?

Journal of Experimental Botany. Jan, 2012  |  Pubmed ID: 21914661

Nitrate reductase (NR) is an enzymatic source of nitric oxide (NO) in plants, and it needs Mo for the Mo-cofactor to be activated. Because NR-deficient mutants are not always available in some species, a cheap and simple pharmacological application of tungstate, which substitutes for Mo in the Mo-cofactor as a competitive antagonist, is widely used as a NR inhibitor in plant NO research. However, evidence indicates that tungstate not only inactivates NR but also inhibits other molybdate-dependent enzymes in plants. In addition, a number of investigations have shown that tungstate also inhibits root growth, affects cortical microtubule formation, and induces programmed cell death (PCD) in plants, just like other heavy metals do. Therefore, tungstate has been shown to exert many other effects that are not connected with the inhibition of NR activity. The origin and mechanism of using tungstate as a NR inhibitor in plants is reviewed here and the progress regarding tungstate toxicity to plants and the possible problems involved in using tungstate as a NR inhibitor in plant NO research are analysed. In summary, the use of tungstate as a NR inhibitor in plant NO research must be treated with caution, keeping in mind that it is not completely specific. It is necessary to search for more NR-deficient mutants and new, specific NR inhibitors. A combination of pharmacological and biochemical analysis with a genetic approach will be necessary in order to investigate the roles of NO in plants.