A Macromolecular Complex of Beta 2 Adrenergic Receptor, CFTR, and Ezrin/radixin/moesin-binding Phosphoprotein 50 is Regulated by PKA

Proceedings of the National Academy of Sciences of the United States of America. Jan, 2003  |  Pubmed ID: 12502786

It has been demonstrated previously that both the cystic fibrosis transmembrane conductance regulator (CFTR) and beta(2) adrenergic receptor (beta(2)AR) can bind ezrinradixinmoesin-binding phosphoprotein 50 (EBP50, also referred to as NHERF) through their PDZ motifs. Here, we show that beta(2) is the major adrenergic receptor isoform expressed in airway epithelia and that it colocalizes with CFTR at the apical membrane. beta(2)AR stimulation increases CFTR activity, in airway epithelial cells, that is glybenclamide sensitive. Deletion of the PDZ motif from CFTR uncouples the channel from the receptor both physically and functionally. This uncoupling is specific to the beta(2)AR receptor and does not affect CFTR coupling to other receptors (e.g., adenosine receptor pathway). Biochemical studies demonstrate the existence of a macromolecular complex involving CFTR-EBP50-beta(2)AR through PDZ-based interactions. Assembly of the complex is regulated by PKA-dependent phosphorylation. Deleting the regulatory domain of CFTR abolishes PKA regulation of complex assembly. This report summarizes a macromolecular signaling complex involving CFTR, the implications of which may be relevant to CFTR-dysfunction diseases.

Molecular Assembly of Cystic Fibrosis Transmembrane Conductance Regulator in Plasma Membrane

The Journal of Biological Chemistry. Jun, 2004  |  Pubmed ID: 15060073

Based on electrophysiological measurements, it has been argued that the active form of cystic fibrosis trans-membrane conductance regulator (CFTR) Cl(-) channel is a multimer. It has also been demonstrated that this multimerization is likely due to PDZ domain-interacting partners. Here we demonstrate that although CFTR in vitro can self-associate into multimers, which depends on PDZ-based interactions, this may not be the case in cell membrane. Using chemical cross-linking, we demonstrated that CFTR exists as a higher order complex in cell membrane. However, this higher order complex is predominantly CFTR dimers, and the PDZ-interacting partners (Na(+)/H(+) exchanger regulatory factor-1 (NHERF1) and NHERF2) constitute approximately 2% of this complex. Interestingly solubilizing membrane expressing CFTR in detergents such as Triton X-100, Nonidet P-40, deoxycholate, and SDS tended to destabilize the CFTR dimers and dissociate them into monomeric form. The dimerization of CFTR was tightly regulated by cAMP-dependent protein kinase-dependent phosphorylation and did not depend on the active form of the channel. In addition, the dimerization was not influenced by either the PDZ motif or its interacting partners (NHERF1 and NHERF2). We also demonstrated that other signaling-related proteins such as Gbeta and syntaxin 1A can be in this higher order complex of CFTR as well. Our studies provide a deeper understanding of how the CFTR assembly takes place in native cell membrane.

Acute Activation and Phosphorylation of Endothelial Nitric Oxide Synthase by HMG-CoA Reductase Inhibitors

American Journal of Physiology. Heart and Circulatory Physiology. Aug, 2004  |  Pubmed ID: 15087285

3-Hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors, statins, provide beneficial effects independent of their lipid-lowering effects. One beneficial effect appears to involve acute activation of endothelial nitric oxide (NO) synthase (eNOS) and increased NO release. However, the mechanism of acute statin-stimulated eNOS activation is unknown. Therefore, we hypothesized that eNOS activation may be coupled to altered eNOS phosphorylation. Bovine aortic endothelial cells (BAECs), passages 2-6, were treated with either lovastatin or pravastatin from 0 to 30 min. eNOS phosphorylation was examined by Western blot by use of phosphospecific antibodies for Ser-1179, Ser-635, Ser-617, Thr-497, and Ser-116. Statin stimulation of BAECs increased eNOS phosphorylation at Ser-1179 and Ser-617, which was blocked by the phosphatidylinositol 3-kinase (PI3-kinase)/Akt inhibitor wortmannin, and at Ser-635, which was blocked by the protein kinase A (PKA) inhibitor KT-5720. Statin treatment of BAECs transiently increased NO release by fourfold, measured by cGMP accumulation, and was attenuated by N-nitro-l-arginine methyl ester, wortmannin, and KT-5720 but not by mevalonate. In conclusion, these data demonstrate that eNOS is acutely activated by statins independent of HMG-CoA reductase inhibition and that in addition to Ser-1179, eNOS phosphorylation at Ser-635 and Ser-617 through PKA and Akt, respectively, may explain, in part, a mechanism by which eNOS is activated in response to acute statin treatment.

Verrucous Hemangioma

International Journal of Dermatology. Oct, 2004  |  Pubmed ID: 15485532

A 13-year-old female presented complaining of swelling of the skin and purplish red papules and nodules on her left leg. These lesions had been present from early childhood and had slowly enlarged, increased in number, and become more verrucous. At the age of 8 years, one of the nodules had been excised by laser, but recurrence was noted within a few months. There was occasional pain and bleeding from the lesion. Physical examination revealed a group of several well-circumscribed, hyperkeratotic, blue-red, vascular plaques arranged linearly along the inside aspect of her left lower extremity, ranging in size from 0.5 to 4.0 cm in diameter (Fig. 1). Smaller, discrete satellite nodules with a similar appearance were noted in the vicinity. Histopathologic examination showed hyperproliferation and hyperkeratosis of the epidermis. The superficial dermis showed multiple, thin-walled, dilated blood-filled spaces (Fig. 2). Similar spaces were present in the lower dermis and subcutaneous tissue. A diagnosis of verrucous hemangioma was made.

Interaction of the Endothelial Nitric Oxide Synthase with the CAT-1 Arginine Transporter Enhances NO Release by a Mechanism Not Involving Arginine Transport

The Biochemical Journal. Mar, 2005  |  Pubmed ID: 15743275

eNOS (endothelial nitric oxide synthase) catalyses the conversion of L-arginine into L-citrulline and NO. Evidence has been presented previously that eNOS is associated with the CAT (cationic amino acid transporter)-1 arginine transporter in endothelial caveolae, and it has been proposed that eNOS-CAT-1 association facilitates the delivery of extracellular L-arginine to eNOS. Definitive proof of a protein-protein interaction between eNOS and CAT-1 is lacking, however, and it is also unknown whether the two proteins interact directly or via an adaptor protein. In the present study, we raised a polyclonal antibody against CAT-1, and show using reciprocal co-immunoprecipitation protocols that eNOS and CAT-1 do indeed form a complex in BAECs (bovine aortic endothelial cells). In vitro binding assays with GST (glutathione S-transferase)-CAT-1 fusion proteins and eNOS show that the two proteins interact directly and that no single CAT-1 intracellular domain is sufficient to mediate the interaction. Overexpression of CAT-1 in BAECs by adenoviral-mediated gene transfer results in significant increases in both L-arginine uptake and NO production by the cells. However, whereas increased L-arginine transport is reversed completely by the CAT-1 inhibitor, L-lysine, increased NO release is unaltered, suggesting that NO production in this in vitro model is independent of CAT-1-mediated transport. Furthermore, eNOS enzymic activity is increased in lysates of CAT-1-overexpressing cells accompanied by increased phosphorylation of eNOS at Ser-1179 and Ser-635, and decreased association of eNOS with caveolin-1. Taken together, these data suggest that direct interaction of eNOS with CAT-1 enhances NO release by a mechanism not involving arginine transport.

Endostatin Induces Acute Endothelial Nitric Oxide and Prostacyclin Release

Biochemical and Biophysical Research Communications. Apr, 2005  |  Pubmed ID: 15752737

Chronic exposure to endostatin (ES) blocks endothelial cell (EC) proliferation, and migration and induces EC apoptosis thereby inhibiting angiogenesis. Nitric oxide (NO) and prostacyclin (PGI(2)), in contrast, play important roles in promoting angiogenesis. In this study, we examined the acute effects of ES on endothelial NO and PGI(2) production. Unexpectedly, a cGMP reporter cell assay showed that ES-induced acute endothelial NO release in cultured bovine aortic endothelial cells (BAECs). Enzyme immunoassay showed that ES also induced an acute increase in PGI(2) production in BAECs. These results were confirmed by ex vivo vascular ring studies that showed vascular relaxation in response to ES. Immunoblot analysis showed that ES stimulated acute phosphorylation of endothelial nitric oxide synthase (eNOS) at Ser116, Ser617, Ser635, and Ser1179, and dephosphorylation at Thr497 in BAECs, events associated with eNOS activation. Short-term exposure of EC to ES, therefore, unlike long-term exposure which is anti-angiogenic, may be pro-angiogenic.

Clinical Analysis of 48 Cases of Inverse Psoriasis: a Hospital-based Study

European Journal of Dermatology : EJD. May-Jun, 2005  |  Pubmed ID: 15908302

Inverse psoriasis, rare in clinical practice, refers to psoriasis only or mainly occurring at flexural sites, such as the axilla, antecubital fossae, popliteal fossae, and inguinal creases. It is also known as flexural psoriasis. With a total collection of psoriatic cases from September 2002 to December 2003 at Xijing hospital, we made a retrospective analysis of the disease history, clinical characteristics, and treatment of the patients affected with inverse psoriasis. The results showed that the major clinical manifestations of inverse psoriasis were sharply demarcated erythematous plaques with varying degrees of infiltration and minimal or no scales. Affected areas often involve the groin, axilla, genitals, and umbilicus. The disease responds well to the narrow band UVB therapy. Compared with common psoriasis, inverse psoriasis has similar and unique characteristics in terms of the affected areas, clinical symptoms, and responses to the treatment.

Macromolecular Complexes of Cystic Fibrosis Transmembrane Conductance Regulator and Its Interacting Partners

Pharmacology & Therapeutics. Nov, 2005  |  Pubmed ID: 15936089

The cystic fibrosis transmembrane conductance regulator (CFTR) is the product of the gene mutated in patients with cystic fibrosis (CF). CFTR is a cAMP-regulated chloride channel localized primarily at the apical or luminal surfaces of epithelial cells lining the airway, gut, exocrine glands, etc., where it is responsible for transepithelial salt and water transport. CFTR chloride channel belongs to the superfamily of the ATP-binding cassette (ABC) transporters, which bind ATP and use the energy to drive the transport of a wide variety of substrates across extra- and intracellular membranes. A growing number of proteins have been reported to interact directly or indirectly with CFTR chloride channel, suggesting that CFTR might regulate the activities of other ion channels, receptors, or transporters, in addition to its role as a chloride conductor. The molecular assembly of CFTR with these interacting proteins is of great interest and importance because several human diseases are attributed to altered regulation of CFTR, among which cystic fibrosis is the most serious one. Most interactions primarily occur between the opposing terminal tails (N- or C-) of CFTR and its binding partners, either directly or mediated through various PDZ domain-containing proteins. These dynamic interactions impact the channel function as well as the localization and processing of CFTR protein within cells. This review focuses on the recent developments in defining the assembly of CFTR-containing complexes in the plasma membrane and its interacting proteins.

Src Kinase Activates Endothelial Nitric-oxide Synthase by Phosphorylating Tyr-83

The Journal of Biological Chemistry. Oct, 2005  |  Pubmed ID: 16123043

The endothelial nitric-oxide synthase (eNOS) is regulated in part by serine/threonine phosphorylation, but eNOS tyrosine phosphorylation is less well understood. In the present study we have examined the tyrosine phosphorylation of eNOS in bovine aortic endothelial cells (BAECs) exposed to oxidant stress. Hydrogen peroxide and pervanadate (PV) treatment stimulates eNOS tyrosine phosphorylation in BAECs. Phosphorylation is blocked by the Src kinase family inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Moreover, eNOS and c-Src can be coimmunoprecipitated from BAEC lysates by antibodies directed against either protein. Domain mapping and site-directed mutagenesis studies in COS-7 cells transfected with either eNOS alone and then treated with PV or cotransfected with eNOS and constitutively active v-Src identified Tyr-83 (bovine sequence) as the major eNOS tyrosine phosphorylation site. Tyr-83 phosphorylation is associated with a 3-fold increase in basal NO release from cotransfected cells. Furthermore, the Y83F eNOS mutation attenuated thapsigargin-stimulated NO production. Taken together, these data indicate that Src-mediated tyrosine phosphorylation of eNOS at Tyr-83 modulates eNOS activity in endothelial cells.

Lysophosphatidic Acid Inhibits Cholera Toxin-induced Secretory Diarrhea Through CFTR-dependent Protein Interactions

The Journal of Experimental Medicine. Oct, 2005  |  Pubmed ID: 16203867

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel localized primarily at the apical or luminal surfaces of epithelial cells that line the airway, gut, and exocrine glands; it is well established that CFTR plays a pivotal role in cholera toxin (CTX)-induced secretory diarrhea. Lysophosphatidic acid (LPA), a naturally occurring phospholipid present in blood and foods, has been reported to play a vital role in a variety of conditions involving gastrointestinal wound repair, apoptosis, inflammatory bowel disease, and diarrhea. Here we show, for the first time, that type 2 LPA receptors (LPA2) are expressed at the apical surface of intestinal epithelial cells, where they form a macromolecular complex with Na+/H+ exchanger regulatory factor-2 and CFTR through a PSD95/Dlg/ZO-1-based interaction. LPA inhibited CFTR-dependent iodide efflux through LPA2-mediated Gi pathway, and LPA inhibited CFTR-mediated short-circuit currents in a compartmentalized fashion. CFTR-dependent intestinal fluid secretion induced by CTX in mice was reduced substantially by LPA administration; disruption of this complex using a cell-permeant LPA2-specific peptide reversed LPA2-mediated inhibition. Thus, LPA-rich foods may represent an alternative method of treating certain forms of diarrhea.

A Parasitism Gene from a Plant-parasitic Nematode with Function Similar to CLAVATA3/ESR (CLE) of Arabidopsis Thaliana

Molecular Plant Pathology. Mar, 2005  |  Pubmed ID: 20565649

SUMMARY The Hg-SYV46 parasitism gene is expressed exclusively in the dorsal oesophageal gland cell of parasitic stages of the soybean cyst nematode, Heterodera glycines, and it encodes a secretory protein that contains a C-terminal motif of the CLAVATA3/ESR-related (CLE) family in Arabidopsis thaliana. In shoot and floral meristems of Arabidopsis, the stem cells secret CLV3, a founding member of the CLE protein family, that activates the CLV1/CLV2 receptor complex and negatively regulates WUSCHEL expression to restrict the size of the stem cell population. Mis-expression of Hg-SYV46 in Arabidopsis (ecotype Columbia-0) under control of the CaMV35S promoter resulted in a wus-like phenotype including premature termination of the shoot apical meristem and the development of flowers lacking the central gynoecium. The wus-like phenotype observed was similar to reports of over-expression of CLV3 and CLE40 in Arabidopsis, as was down-regulation of WUS expression in the shoot apices of 35S::Hg-SYV46/Col-0 plants. Expression of 35S::Hg-SYV46 in a clv3-1 mutant of Arabidopsis was able partially or fully to rescue the mutant phenotype, probably dependent upon localization and level of transgene expression. A short root phenotype, as reported for over-expression of CLV3, CLE40 and CLE19 in roots, was also produced in primary 35S::Hg-SYV46/Col-0 transgenic plants. The results suggest a functional similarity of HG-SYV46 to plant-secreted CLE ligands that may play a role in the differentiation or division of feeding cells induced in plant roots by parasitic nematodes.

Polymorphisms in the DNA Repair Genes XPC, XPD, and XPG and Risk of Cutaneous Melanoma: a Case-control Analysis

Cancer Epidemiology, Biomarkers & Prevention : a Publication of the American Association for Cancer Research, Cosponsored by the American Society of Preventive Oncology. Dec, 2006  |  Pubmed ID: 17164380

Sunlight causes DNA damage, including bulky lesions that are removed effectively by the nucleotide-excision repair (NER) pathway. There are at least eight core NER proteins participating in the pathway, and genetic variations in their genes may alter NER functions. We hypothesized that some NER variants are associated with risk of cutaneous melanoma. In a hospital-based case-control study of 602 non-Hispanic White patients with cutaneous melanoma and 603 age- and sex-matched cancer-free controls, we genotyped five common non-synonymous single-nucleotide polymorphisms identified to date and assessed their associations with risk of cutaneous melanoma. We found that a significantly increased risk of cutaneous melanoma was associated with XPD 751Lys/Gln [adjusted odds ratio (OR), 1.55 and 95% confidence interval (95% CI), 1.12-2.16] and XPD 751Gln/Gln (OR, 1.66; 95% CI, 1.03-2.68) genotypes compared with the XPD 751Lys/Lys genotype as well as XPD312Asp/Asn (OR, 1.54; 95% CI, 1.11-2.12) and XPD312Asn/Asn (OR, 1.75; 95% CI, 1.05-2.90) genotypes compared with the XPD 312Asp/Asp genotype. This increased risk was not observed in the other three XPC and XPG single-nucleotide polymorphisms. Moreover, the number of the observed XPD at-risk genotypes (i.e., 312Asn/Asn+Asn/Asp and 751Gln/Gln+Lys/Gln) was associated with cutaneous melanoma risk in a dose-response manner (OR, 1.47; 95% CI, 0.97-2.23 for one at-risk genotype; OR, 1.83; 95% CI, 1.29-2.61 for two at-risk genotypes; P(trend) < 0.001). However, we found no evidence of any interaction between XPD genotypes with XPC and XPG genotypes or the known risk factors. We concluded that genetic variants of the XPD gene might serve as biomarkers for susceptibility to cutaneous melanoma.

Simultaneous Determination of Catechin, Rutin, Quercetin Kaempferol and Isorhamnetin in the Extract of Sea Buckthorn (Hippophae Rhamnoides L.) Leaves by RP-HPLC with DAD

Journal of Pharmaceutical and Biomedical Analysis. Jun, 2006  |  Pubmed ID: 16520013

A rapid and specific reversed-phase high performance liquid chromatography (RP-HPLC) method with diode array detection (DAD) at room temperature was used and validated for the simultaneous determination of five flavonoids (catechin, CA; rutin, RU; quercetin, QU; kaempferol, KA; isorhamnetin, IS) in the extract of sea buckthorn (Hippophae rhamnoides L.) leaves. The sample pretreatment process involved ultrasonic extraction with 85% ethanol under the frequency of 80 kHz, at a temperature of 45 degrees C for 30 min and with the ratio of liquor to material of 15 mL g-1, followed by separation on HIQ SIL C18V column with methanol-acetonitrile-water (40:15:45, v/v/v) containing 1.0% acetic acid as a mobile phase. The extract was detected by DAD at the wavelength of 279 nm for CA, 257 nm for RU, 368 nm for QU, KA and IS. Calibration curves were found to be linear with the ranges of 0.011-0.520 mg ml-1 (CA), 0.007-0.500 mg ml-1 (RU), 0.019-0.280 mg ml-1 (QU), 0.010-0.440 mg ml-1 (KA) and 0.008-0.400 mg ml-1 (IS). The correlation coefficients of linear regression analysis and detection limits were between 0.9963-0.9999 and 0.00079-0.00290 mg ml-1. The contents of CA, RU, QU, KA and IS in sea buckthorn leaves were successfully determined with 3.8, 5.2, 7.3, 10.9 and 11.9 min with satisfactory reproducibility and recovery. Recoveries of the five flavonoids were between 97.27 and 99.98%. The method was applied to the determination of flavonoids in sea buckthorn leaves and was found to be simple, rapid and efficient.

Polymorphisms of the FAS and FAS Ligand Genes Associated with Risk of Cutaneous Malignant Melanoma

Pharmacogenetics and Genomics. Apr, 2006  |  Pubmed ID: 16538172

The FAS/FAS ligand (FASLG) system has a key role in regulating cell growth and thus tumorigenesis. Functional promoter polymorphisms of the FAS and FASLG genes alter the transcriptional activities, but no published study has investigated the role of these polymorphisms in the etiology of cutaneous malignant melanoma (CMM). In a hospital-based, case-control study of 602 non-Hispanic white CMM patients and 603 cancer-free age- and sex-matched control subjects, we genotyped FAS-1377G>A, FAS-670A>G, FASLG-844T>C and FASLG-IVS2nt-124G>A polymorphisms and assessed their respective associations with CMM risk. We found that an increased risk of CMM was associated with the FAS-1377GG [adjusted odds ratio (OR)=1.32; 95% confidence interval (CI)=1.00-1.75 for -1377GG] and -670AA (adjusted OR=1.28; 95% CI=1.00-1.65 for -670AA) genotypes compared to the -1377AA/AG and -670AG/GG genotypes, respectively; an increased risk of CMM was associated with the FASLG-IVS2nt-124AG+GG (OR=1.54; 95% CI=1.18-2.01) genotype compared to the AA genotype, but no evident risk was associated with any of the FAS-844T>C genotypes. In the combined analysis of these four variant alleles, we found that, compared to those having 0-3 variants, those having 4-8 variant alleles had a significantly increased risk for CMM (OR=1.38; 95% CI=1.10-1.73), and this risk was more pronounced in subgroups of old (>50 years) males, and those who were at low risk of sunlight-induced CMM, except for having fair skin colour, moles, dysplastic nevi and a family history of cancer. In conclusion, genetic variants in the FAS and FASLG genes may contribute to the etiology of CMM in the general population, particularly in those with a low risk of sunlight-induced CMM.

Genetic Variants of the ADPRT, XRCC1 and APE1 Genes and Risk of Cutaneous Melanoma

Carcinogenesis. Sep, 2006  |  Pubmed ID: 16621887

Sunlight causes various kinds of DNA damage, including oxidative lesions that are removed effectively by the base excision repair (BER) pathway, in which ADPRT, XRCC1 and APE1 play a key role. However, genetic variation in these genes may alter their functions. We hypothesized that ADPRT, XRCC1 and APE1 polymorphisms are associated with risk of cutaneous melanoma (CM). In a hospital-based case-control study of 602 CM patients and 603 cancer-free control subjects frequency matched on age, sex and ethnicity, we genotyped for three non-synonymous single nucleotide polymorphisms (SNPs) (i.e. the ADPRT Val762Ala, XRCC1 Arg399Gln and APE1Asp148Glu) and assessed their associations with risk of CM. We found no significant difference in the allele frequencies between cases and controls for any of these three SNPs. However, we found that, compared with the APE1 Asp/Asp genotype, a significantly decreased risk of CM was associated with the APE1 Asp/Glu [adjusted odds ratio (OR), 0.60; 95% confidence interval (CI), 0.41-0.86], Glu/Glu (OR, 0.58; 95% CI, 0.38-0.88) and combined APE1 Asp/Glu+Glu/Glu (OR, 0.59; 95% CI, 0.42-0.83) genotypes, but not for other XRCC1 variant genotypes. Moreover, there was evidence for a possible gene-gene interaction between XRCC1 and APE1 variants in the association with risk of CM (P=0.030). We conclude that the APE1 Glu variant may have an effect or interact with XRCC1 in the etiology of CM or in linkage disequilibrium with other untyped protective alleles. Larger studies with more SNPs in the BER genes are needed to verify these findings.

CFTR Regulates Phagosome Acidification in Macrophages and Alters Bactericidal Activity

Nature Cell Biology. Sep, 2006  |  Pubmed ID: 16921366

Acidification of phagosomes has been proposed to have a key role in the microbicidal function of phagocytes. Here, we show that in alveolar macrophages the cystic fibrosis transmembrane conductance regulator Cl- channel (CFTR) participates in phagosomal pH control and has bacterial killing capacity. Alveolar macrophages from Cftr-/- mice retained the ability to phagocytose and generate an oxidative burst, but exhibited defective killing of internalized bacteria. Lysosomes from CFTR-null macrophages failed to acidify, although they retained normal fusogenic capacity with nascent phagosomes. We hypothesize that CFTR contributes to lysosomal acidification and that in its absence phagolysosomes acidify poorly, thus providing an environment conducive to bacterial replication.

A Rapid and Sensitive LC-MS/MS Method for Determination of Coenzyme Q10 in Tobacco (Nicotiana Tabacum L.) Leaves

Journal of Separation Science. Jul, 2006  |  Pubmed ID: 16922277

A rapid and sensitive liquid chromatography-tandem mass spectrometry method with multiple reaction monitoring has been proposed for the analysis of coenzyme Q10 in (CoQ10) tobacco leaves. The method used electrospray ionization with detection in positive ion mode. Sample pretreatment involved ultrasonic extraction of fresh tobacco leaves with anhydrous ethanol for 15 min and followed by extraction of the supernatant with hexane. The separation of CoQ10 was performed on a Symmetry Shield RP18 column with a mixture of acetonitrile and isopropanol (8:7, v/v) containing 0.5% formic acid as mobile phase. Quantification of CoQ10 was performed by the standard addition method. The limit of detection and limit of quantitation of CoQ10 were, respectively, 1.2 ng/mL (S/N = 3) and 4.0 ng/mL (S/N = 10). The relative standard deviations of peak area were 0.91% and 1.21% for intra-day and inter-day, respectively. The recoveries of CoQ10 ranged from 98.2 to 99.3% and the corresponding RSDs were less than 2.4%. Analysis took 5 min, making the method suitable for rapid determination of CoQ10 in tobacco leaves. The proposed method has been successfully applied to the analysis of CoQ10 in the leaves from eight varieties of tobacco.

Genetic Variants of the Vitamin D Receptor Gene Alter Risk of Cutaneous Melanoma

The Journal of Investigative Dermatology. Feb, 2007  |  Pubmed ID: 16990805

Sunlight causes DNA damage but also induces production of vitamin D whose metabolite 1,25-(OH)2D3 has antiproliferative and pro-differentiative effects in both melanocytes and cutaneous melanoma (CM) cells mediated through the vitamin D receptor (VDR). We hypothesized that genetic polymorphisms of VDR are associated with risk of CM. In a hospital-based case-control study of 602 non-Hispanic white CM patients and 603 cancer-free control subjects frequency matched by age and sex, we genotyped two VDR polymorphisms (TaqI and FokI) and assessed their association with CM risk. We found that a significantly decreased risk was associated with VDR-TaqI Tt (adjusted odds ratio (OR), 0.70; 95% confidence interval (CI), 0.54-0.90) and Tt+tt (OR=0.70; 95% CI, 0.55-0.89) genotypes, compared with the VDR-TaqI TT genotype, whereas an increased risk was associated with VDR-FokI Ff genotype (OR=1.32; 95% CI, 1.03-1.68), and a borderline significantly increased risk was associated with Ff+ff (OR=1.26; 95% CI, 1.00-1.59) genotypes, compared with the VDR-FokI FF genotype. In conclusion, genetic variants (i.e., TaqI t protective allele and FokI f risk allele) in VDR may alter risk of CM.

Acetaldehyde Dissociates the PTP1B-E-cadherin-beta-catenin Complex in Caco-2 Cell Monolayers by a Phosphorylation-dependent Mechanism

The Biochemical Journal. Mar, 2007  |  Pubmed ID: 17087658

Interactions between E-cadherin, beta-catenin and PTP1B (protein tyrosine phosphatase 1B) are crucial for the organization of AJs (adherens junctions) and epithelial cell-cell adhesion. In the present study, the effect of acetaldehyde on the AJs and on the interactions between E-cadherin, beta-catenin and PTP1B was determined in Caco-2 cell monolayers. Treatment of cell monolayers with acetaldehyde induced redistribution of E-cadherin and beta-catenin from the intercellular junctions by a tyrosine phosphorylation-dependent mechanism. The PTPase activity associated with E-cadherin and beta-catenin was significantly reduced and the interaction of PTP1B with E-cadherin and beta-catenin was attenuated by acetaldehyde. Acetaldehyde treatment resulted in phosphorylation of beta-catenin on tyrosine residues, and abolished the interaction of beta-catenin with E-cadherin by a tyrosine kinase-dependent mechanism. Protein binding studies showed that the treatment of cells with acetaldehyde reduced the binding of beta-catenin to the C-terminal region of E-cadherin. Pairwise binding studies using purified proteins indicated that the direct interaction between E-cadherin and beta-catenin was reduced by tyrosine phosphorylation of beta-catenin, but was unaffected by tyrosine phosphorylation of E-cadherin-C. Treatment of cells with acetaldehyde also reduced the binding of E-cadherin to GST (glutathione S-transferase)-PTP1B. The pairwise binding study showed that GST-E-cadherin-C binds to recombinant PTP1B, but this binding was significantly reduced by tyrosine phosphorylation of E-cadherin. Acetaldehyde increased the phosphorylation of beta-catenin on Tyr-331, Tyr-333, Tyr-654 and Tyr-670. These results show that acetaldehyde induces disruption of interactions between E-cadherin, beta-catenin and PTP1B by a phosphorylation-dependent mechanism.

Repair Capacity for UV Light Induced DNA Damage Associated with Risk of Nonmelanoma Skin Cancer and Tumor Progression

Clinical Cancer Research : an Official Journal of the American Association for Cancer Research. Nov, 2007  |  Pubmed ID: 17975167

To examine the role of suboptimal DNA repair capacity (DRC) for UV light-induced DNA damage in the development of nonmelanoma skin cancer (NMSC) and tumor progression.

Spatiotemporal Coupling of CAMP Transporter to CFTR Chloride Channel Function in the Gut Epithelia

Cell. Nov, 2007  |  Pubmed ID: 18045536

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel localized at apical cell membranes and exists in macromolecular complexes with a variety of signaling and transporter molecules. Here, we report that the multidrug resistance protein 4 (MRP4), a cAMP transporter, functionally and physically associates with CFTR. Adenosine-stimulated CFTR-mediated chloride currents are potentiated by MRP4 inhibition, and this potentiation is directly coupled to attenuated cAMP efflux through the apical cAMP transporter. CFTR single-channel recordings and FRET-based intracellular cAMP dynamics suggest that a compartmentalized coupling of cAMP transporter and CFTR occurs via the PDZ scaffolding protein, PDZK1, forming a macromolecular complex at apical surfaces of gut epithelia. Disrupting this complex abrogates the functional coupling of cAMP transporter activity to CFTR function. Mrp4 knockout mice are more prone to CFTR-mediated secretory diarrhea. Our findings have important implications for disorders such as inflammatory bowel disease and secretory diarrhea.

Vitiligo Prevalence Study in Shaanxi Province, China

International Journal of Dermatology. Jan, 2007  |  Pubmed ID: 17214719

Recent publications, especially those based on population surveys, show that the presumed vitiligo prevalence of 1-2% is overestimated.

Rapid and Quantitative Determination of Solanesol in Nicotiana Tabacum by Liquid Chromatography-tandem Mass Spectrometry

Journal of Pharmaceutical and Biomedical Analysis. May, 2007  |  Pubmed ID: 17317070

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with multiple reaction monitoring (MRM) was developed for the determination of solanesol in Nicotiana tabacum. Sample preparation was performed by ultrasonic extraction with methanol for 20 min and then supernatant was extracted with hexane. The method used atmospheric pressure chemical ionization (APCI) detection in positive-ion mode. The separation of solanesol was performed on a Symmetry Shield RP18 column with a mixture of acetonitrile and isopropanol (1:1, v/v) containing 2mM ammonium acetate as mobile phase. Quantification of solanesol was performed by the standard addition method. The limit of quantification (LOQ) and limit of detection (LOD) of solanesol were, respectively, 5.0 ng/ml (S/N=10) and 1.5 ng/ml (S/N=3). The relative standard deviations of peak area were 0.89 and 1.12% for intra-day and inter-day, respectively. The recoveries of solanesol ranged from 97.72 to 99.67% and the corresponding R.S.D.s were less than 2.7%. Analysis took 5 min, making the method suitable for rapid determination of solanesol in N. tabacum. The proposed method has been successfully applied to the analysis of solanesol in various organs of N. tabacum.

Polymorphisms of the Neuronal and Inducible Nitric Oxide Synthase Genes and the Risk of Cutaneous Melanoma: a Case-control Study

Cancer. Apr, 2007  |  Pubmed ID: 17328085

Nitric oxide (NO) is a multifunctional molecule that is produced by both neuronal NO synthase (nNOS) and inducible NO synthase (iNOS), and the expression of nNOS and iNOS is up-regulated in various cancer cells, including cutaneous melanoma (CM). The authors hypothesized that selected functional single-nucleotide polymorphisms (SNPs) in the nNOS and iNOS genes are associated with the risk of CM.

The Effect of Air Pressure on Edema and Healing of Scalded Tissue of Rats

Journal of Burn Care & Research : Official Publication of the American Burn Association. Mar-Apr, 2007  |  Pubmed ID: 17351446

To study the effectiveness of using high-pressure air on edema and healing of second-degree scald burns in rats. A self-designed high pressure airtight box in which the air pressure can be controlled is used to observe the edema and healing time of second degree burned tissues in rat under different air pressures. With the air pressure increased by 30 cm H2O, there was a significant reduction of edema, exudation and healing time of the scalded tissue. Increasing air pressure can reduce edema, exudation and healing time of scalded tissue.

In Vitro Expression Levels of Cell-cycle Checkpoint Proteins Are Associated with Cellular DNA Repair Capacity in Peripheral Blood Lymphocytes: a Multivariate Analysis

Journal of Proteome Research. Apr, 2007  |  Pubmed ID: 17362036

DNA repair should occur after cells sense DNA damage signals and undergo cell-cycle arrest to provide sufficient time for DNA repair, and suboptimal DNA repair capacity (DRC) in peripheral lymphocytes has been suggested as a cancer susceptibility marker. Numerous studies showed a functional link between DNA damage sensing, cell-cycle checkpoint, and DNA repair. We hypothesized that in vitro cell-cycle checkpoint-related protein expression levels in stimulated lymphocytes predict DRC levels. To test this hypothesis, we performed the host-cell reactivation assay for DRC by transfecting stimulated peripheral blood lymphocytes from 120 normal donors with transient expression plasmids damaged by benzo[a]pyrene diol epoxide (BPDE). The same cells were assessed for protein expression induction of eight cell-cycle checkpoint-related genes using the reverse-phase protein lysate microarray assay. In multivariate linear regression analysis adjusting for age, sex, blastogenic rate, and sample storage duration, the association between DRC and expression levels of cell-cycle checkpoint-related proteins induced by BPDE-adducts was statistically significant for p27, CCND1, ATM, and MDM2 (P = 0.00, 0.03, 0.03, and 0.03, respectively), borderline for p73 and p21 (P = 0.07 and 0.09, respectively), but not for p53 and p16 (P = 0.13 and 0.18, respectively). Because the relative expression levels of all these eight proteins were highly correlated, we further performed the principal component analysis and identified ATM as the most important predictor of DRC, followed by MDM2 and p27. Our results provide population-based in vitro evidence demonstrating that cell-cycle checkpoint-related proteins play essential roles in regulating DNA repair, at least in unaffected human peripheral blood lymphocytes. Further studies are warranted to investigate the role of interindividual variation in the expression levels of these proteins in cancer susceptibility.

TNF-alpha Gene Promoter -238G>A and -308G>A Polymorphisms Alter Risk of Psoriasis Vulgaris: a Meta-analysis

The Journal of Investigative Dermatology. Aug, 2007  |  Pubmed ID: 17446901

Tumor necrosis factor-alpha (TNF-alpha) is a major proinflammatory cytokine and involved in the etiology of psoriasis. The -238G>A and -308G>A polymorphisms influence the transcription of the TNF-alpha gene and have been implicated in psoriasis risk. However, the results from the published studies on the association between TNF-alpha polymorphisms and psoriasis risk are conflicting. Our meta-analysis of a total of 997 psoriasis cases and 943 control subjects from eight published case-control studies for the -238G>A polymorphism and of 1,156 psoriasis cases and 1,083 control subjects from 10 published case-control studies for the -308G>A polymorphism showed that a significantly increased risk was associated with the variant GA+AA genotypes of -238G>A, compared with the GG genotype (odds ratio (OR) 2.60, 95% confidence interval (95% CI) 1.48-4.56), whereas a significantly reduced psoriasis risk was associated with the variant GA+AA genotypes of the -308G>A compared with the GG genotype (OR 0.57, 95% CI 0.45-0.71). Our findings suggest that TNF-alpha -238G>A and -308G>A polymorphisms might be used as biomarkers for psoriasis risk prediction. A single larger study with thousands of subjects and biochemical and biological characterization is warranted to evaluate further the role of -238G>A and -308G>A polymorphisms and psoriasis risk in a population of various ethnicities.

Nevus Lipomatosus Cutaneous Superficialis with Angiokeratoma

International Journal of Dermatology. Jun, 2007  |  Pubmed ID: 17550561

Genetic Polymorphisms in DNA Base-excision Repair Genes ADPRT, XRCC1, and APE1 and the Risk of Squamous Cell Carcinoma of the Head and Neck

Cancer. Aug, 2007  |  Pubmed ID: 17614107

Tobacco smoke contains numerous carcinogens that cause DNA damage, including oxidative lesions that are removed effectively by the base-excision repair (BER) pathway, in which adenosine diphosphate ribosyl transferase (ADPRT), x-ray repair cross-complementing 1 (XRCC1), and apurinic/apyimidinic endonuclease (APE1) play key roles. Genetic variations in the genes encoding for these DNA repair enzymes may alter their functions. Although there have been several studies that generated mixed results on the association between XRCC1 variants and the risk of squamous cell carcinoma of the head and neck (SCCHN), no reported studies have investigated the association between ADPRT and APE1 variants and SCCHN risk.

In Vitro Benzo[a]pyrene Diol Epoxide-induced DNA Damage and Chromosomal Aberrations in Primary Lymphocytes, Smoking, and Risk of Squamous Cell Carcinoma of the Head and Neck

International Journal of Cancer. Journal International Du Cancer. Dec, 2007  |  Pubmed ID: 17724733

Cigarette smoking is a major risk factor for squamous cell carcinoma of the head and neck (SCCHN), but only a fraction of those exposed to cigarette smoke develops SCCHN, suggesting variation in individual susceptibility. Tobacco smoke contains a number of carcinogens that cause various kinds of damage to DNA. In this study, we simultaneously measured benzo[a]pyrene diol epoxide-induced DNA damage and chromosomal aberrations by the comet assay and the mutagen sensitivity assay, respectively, in cultured primary lymphocytes from newly recruited 123 patients with SCCHN and 136 age- and sex-matched controls. Using the control median as the cut-off, the elevated risk of SCCHN was 2.35 (95% CI, 1.37-4.03), 2.28 (95% CI, 1.34-3.98) and 3.25 (95% CI, 1.85-5.07) for high levels of tail extension, tail length and oliver tail moment of the comet assay, respectively, and 1.75 (95% CI, 1.04-2.94) for high levels of chromosomal aberrations of the mutagen sensitivity assay. The effects of these 2 types of measurements were additive; subjects with high levels of both DNA damage and chromosomal aberrations had a 4.77-fold increased risk (95% CI, 2.73-8.36) of SCCHN. Cigarette smoking further elevated this risk to more than 20-fold (OR 23.6; 95% CI, 8.92-62.3). These data support our previous finding that suboptimal repair contributed to susceptibility to SCCHN and the new data further suggests a possible gene-environment interaction that may play an important role in the etiology of SCCHN. Further validation studies are warranted.

Role of ENOS Phosphorylation at Ser-116 in Regulation of ENOS Activity in Endothelial Cells

Vascular Pharmacology. Nov-Dec, 2007  |  Pubmed ID: 17822962

Endothelial nitric oxide synthase (eNOS) catalyzes the conversion of L-arginine to L-citrulline and nitric oxide (NO), an important modulator of vascular function. eNOS is regulated post-translationally through phosphorylation/dephosphorylation at a number of specific phosphorylation sites including Ser-116 in the bovine eNOS sequence. Whether phosphorylation of eNOS at Ser-116 in endothelial cells is stimulatory or inhibitory has not previously been definitively determined. In this study we show that mimicking phosphorylation of eNOS at Ser-116 by Asp mutation reduces basal NO release from endothelial cells. Preventing phosphorylation at this site by Ala mutation increases the amount of NO release from endothelial cells in response to agonist stimulation. In addition, mimicking phosphorylation of Ser-116 increases eNOS association with caveolin-1 and reduces the vascular reactivity of intact aortic rings. eNOS phosphorylation at Ser-116, therefore, appears to contribute to negative modulation of eNOS activity and hence to regulation of vascular tone.

Biofilm Formation of the Pathogens of Fatal Bacterial Granuloma After Trauma: Potential Mechanism Underlying the Failure of Traditional Antibiotic Treatments

Scandinavian Journal of Infectious Diseases. 2008  |  Pubmed ID: 17852906

The pathogen of a new type of disease - fatal bacterial granuloma after trauma (FBGT) - was found to be Propionibacterium acnes (P. acnes). Although in vitro studies showed that the pathogenic P. acnes are sensitive to conventional antibiotics, treatments of FBGT patients with these antibiotics were ineffective. The underlying mechanisms were not clear. Since P. acnes are able to form biofilm on orthopaedic biomaterials in vitro, and pathogenic P. acnes of acnes vulgaris was known to form biofilm in vivo, we hypothesize that the pathogens of FBGT are also able to form biofilm during the pathogenesis, which may be 1 of the reasons for antibiotics tolerance of FBGT. Biofilm forming capacity of the pathogens of FBGT were examined with XTT reduction method, as well as with scanning electron microscope. The effect of long-term subminimal inhibitory concentration (MIC) lincomycin on the biofilm forming ability of the pathogens was also tested. Our results show that both the type strain (NCTC737) and the pathogenic P. acnes of FBGT can form biofilm in vitro. These data demonstrated the biofilm formation of the FBGT pathogens in vitro, and its acceleration by lincomycin, which may be 1 of the major mechanisms for the failure of antibiotic treatment.

Polymorphisms of TP53 Arg72Pro, but Not P73 G4C14>A4TA4 and P21 Ser31Arg, Contribute to Risk of Cutaneous Melanoma

The Journal of Investigative Dermatology. Jun, 2008  |  Pubmed ID: 18049450

Agonist-stimulated Endothelial Nitric Oxide Synthase Activation and Vascular Relaxation. Role of ENOS Phosphorylation at Tyr83

Circulation Research. Feb, 2008  |  Pubmed ID: 18096817

Tyr83 in endothelial nitric oxide synthase (eNOS) has been identified previously as a site of Src kinase-mediated phosphorylation of eNOS in bovine aortic endothelial cells (BAECs) that is phosphorylated in response to oxidant stress. In the present study, we have used a phospho-specific antibody to show that Tyr83 in eNOS is also phosphorylated in both BAECs and intact blood vessel segments in response to treatment with a variety of different eNOS-activating agonists, including thapsigargin, vascular endothelial growth factor, bradykinin, ATP, sphingosine-1-phosphate, estrogen, angiopoietin, and acetylcholine. Agonist stimulation of eNOS Tyr83 phosphorylation as well as agonist stimulation of endothelial NO release in BAECs is blocked by Src kinase inhibition by either 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4-d] pyrimidine (PP2) or by dominant negative Src. Mutation of Tyr83 to a nonphosphorylatable Phe blocks agonist stimulation of NO release from eNOS-reconstituted eNOS knockdown endothelial cells. Mutation of Tyr83 also attenuates agonist-induced relaxation of eNOS-reconstituted aortic rings from eNOS knockout mice. Phosphorylation of eNOS at Tyr83 thus appears to be a common covalent modification that is induced, not only by oxidant stress but also by other physiologically relevant extracellular signals known to be important in regulation of eNOS activity in vivo. Moreover, our results demonstrate an important role for Src-mediated phosphorylation of eNOS at Tyr83 in agonist stimulation of eNOS activation and vascular relaxation.

Haplotype and Genotypes of the VDR Gene and Cutaneous Melanoma Risk in Non-Hispanic Whites in Texas: a Case-control Study

International Journal of Cancer. Journal International Du Cancer. May, 2008  |  Pubmed ID: 18183598

In a hospital-based case-control study of 805 non-Hispanic whites with cutaneous melanoma and 841 cancer-free age-, sex- and ethnicity-matched control subjects, 3 VDR polymorphisms (i.e., TaqI, BsmI and FokI) were genotyped using blood samples collected between 1994 and 2006. We tested the hypothesis that the haplotypes and combined genotypes of these polymorphisms were associated with melanoma risk by interacting with known risk factors. Haplotypes t-B-F (adjusted odds ratio [OR], 0.52; 95% confidence interval [CI], 0.34-0.80) and t-B-f (adjusted OR, 0.51; CI, 0.27-0.94) were associated with a reduced risk when compared to T-b-f. The combined genotypes Tt+tt/Bb+BB/Ff+ff (adjusted OR, 0.69; CI, 0.52, 0.90) and Tt+tt/Bb+BB/FF (adjusted OR, 0.58; CI, 0.43, 0.78) were also associated with reduced risk, whereas the combined genotype TT/Bb+BB/Ff+ff genotype (adjusted OR, 2.35; CI, 1.13, 4.98) was associated with increased risk when compared to TT/bb/Ff+ff genotypes. On multivariate analysis, only the TaqI polymorphism was an independent risk factor, while the FokI polymorphism interacted with skin color (p = 0.029), moles (p = 0.017) and first-degree relatives with any cancer (p = 0.013) in modifying risk. Together, these findings suggest that VDR polymorphisms may directly affect or modify the risk associated with known melanoma risk factors. Larger, population-based studies are needed to replicate our findings.

Akt1/protein Kinase B Alpha is Involved in Gastric Cancer Progression and Cell Proliferation

Digestive Diseases and Sciences. Jul, 2008  |  Pubmed ID: 18379876

Akt (also known as protein kinase B, PKB) is involved in a variety of biological processes, for example cell development, proliferation, and angiogenesis. Clinical studies in support of the idea that increased activity of Akt could contribute directly to gastric carcinogenesis are rare, however. In this study we discovered that phospho-Akt1 was overexpressed in human gastric cancers and its levels correlated with tumor differentiation and pTNM. Akt1 activation promoted cell survival, because the phosphatidylinositol 3-kinase(PI3K) inhibitor LY294002 inhibited Akt1 phosphorylation and inhibited cell growth, especially in cells with active Akt1. Dominant negative Akt inhibited proliferation of gastric cancer cells and induced G1 cell-cycle arrest whereas constitutively active Akt increased cell proliferation. We have therefore identified Akt1 as an active kinase that contributes to gastric cancer progression and promotes proliferation of gastric cancer cells.

Functional Polymorphisms of the FAS Gene Associated with Risk of Vitiligo in Chinese Populations: a Case-control Analysis

The Journal of Investigative Dermatology. Dec, 2008  |  Pubmed ID: 18548110

The FAS/FASLG system plays a key role in regulating apoptosis. Previous findings have shown that CD4-dependent destruction of melanocytes is partially inhibited by blocking FAS-FASLG interactions in autoimmune vitiligo. Functional polymorphisms of the FAS and FASLG genes can alter their transcriptional activities. In a hospital-based case-control study of 750 vitiligo patients and 756 controls, we genotyped the FAS-1377 G>A, FAS-670 A>G, and FASLG-844 T>C polymorphisms and assessed their association with the risk of vitiligo. We found that a significantly increased risk of vitiligo was associated with the FAS-1377 AA genotype (adjusted odds ratio (OR), 1.49; 95% confidence interval (CI), 1.07-2.08) and the FAS-1377 AG genotype (adjusted OR, 1.31; 95% CI, 1.05-1.63) when compared with the FAS-1377 GG genotype. However, no evident risk was associated with FAS-670 G>A genotypes. In the combined analysis of the two variant alleles of FAS, genotypes with 3 to 4 risk alleles were associated with an increased risk of vitiligo compared with those having 0-2 variants (adjusted OR, 2.87; 95% CI, 1.90-4.32). In conclusion, genetic variants in the FAS gene may affect the risk of vitiligo in Chinese populations.

Genetic Variants and Haplotypes of the Caspase-8 and Caspase-10 Genes Contribute to Susceptibility to Cutaneous Melanoma

Human Mutation. Dec, 2008  |  Pubmed ID: 18563783

Caspase-8 (CASP8) and caspase-10 (CASP10) play key roles in regulating apoptosis, and their functional polymorphisms may alter apoptosis and cancer risk. However, no reported studies have investigated the association between such polymorphisms and the risk of cutaneous melanoma (CM). In a hospital-based study of 805 non-Hispanic white patients with CM and 835 cancer-free age-, sex-, and ethnicity-matched controls, we genotyped three reported putatively functional polymorphisms of CASP8 and CASP10-CASP8 D302 H (rs1045485:G>C), CASP8 -652 6N del (rs3834129:-/CTTACT), and CASP10 I522L (rs13006529:A>T)-and assessed their associations with risk of CM and interactions with known risk factors for CM. We also calculated the false-positive report probability (FPRP) for significant findings. CASP8 302 H variant genotypes (DH: adjusted odds ratio [OR], 0.70; 95% confidence interval [CI], 0.50-0.98; DH+HH: unadjusted OR, 0.78; 95% CI, 0.62-0.98; FPRP, 0.79) and CASP8 -652 6N del variant genotypes (ins/del: OR, 0.74; 95% CI, 0.57-0.97; ins/del+del/del: OR, 0.76; 95% CI, 0.61-0.95; FPRP, 0.61) were associated with significantly lower CM risk than were the DD and ins/ins genotypes, respectively. However, the CASP10 522L variant genotypes were not associated with significantly altered CM risk. Also, the D-del-I haplotype was associated with a significantly lower CM risk (OR, 0.52; 95% CI, 0.37-0.74; FPRP, 0.04) than was the most common haplotype, D-ins-I. Furthermore, multivariate logistic regression analysis revealed that CASP8 D302 H, CASP8 -652 6N del, and CASP10 I522L were independent risk factors for CM. Therefore, these CASP8 and CASP10 polymorphisms may be biomarkers for susceptibility to CM.

The Environmental Light Influences the Circulatory Levels of Retinoic Acid and Associates with Hepatic Lipid Metabolism

Endocrinology. Dec, 2008  |  Pubmed ID: 18669599

Environmental light is involved in the regulation of photochemical reaction in mouse retina. It remains unclear whether light-mediated increase in all-trans retinoic acid (ATRA) synthesis in retina will result in altering the circulatory levels of ATRA and regulating downstream gene expression and physiological function. Here we showed circulatory levels of ATRA decreased in mice under constant darkness and elevated by light exposure. Fat gene pancreatic lipase-related protein 2 (mPlrp2) and its partner procolipase (mClps), but not hepatic lipase (mHl), activated in livers for responding to lack of light illuminating. Light-triggered alterations in circulatory ATRA levels regulated ecto-5'-nucleotidase gene expression by retinoic acid receptor retinoic acid receptor-alpha and modulated 5'-AMP levels in blood and were associated with mPlrp2 and mClps expression in the livers. Mice deficient in adenosine receptors displayed mPlrp2 and mClps expression in livers under 12-h light, 12-h dark cycles. Caffeine blocked adenosine receptors and induced hepatic mPlrp2 and mClps expression in wild-type mice. Mice activated in hepatic mPlrp2 and mClps expression lowered hepatic and serum lipid levels and markedly elevated circulatory levels of all-trans retinol. Our results suggest environmental light influence hepatic lipid homeostasis by light-modulated retinoic acid signaling associated with mPlrp2 and mClps gene expression in livers.

Akt Inhibitor A-443654 Interferes with Mitotic Progression by Regulating Aurora a Kinase Expression

Neoplasia (New York, N.Y.). Aug, 2008  |  Pubmed ID: 18670641

Both Akt and Aurora A kinase have been shown to be important targets for intervention for cancer therapy. We report here that Compound A (A-443654), a specific Akt inhibitor, interferes with mitotic progression and bipolar spindle formation. Compound A induces G(2)/M accumulation, defects in centrosome separation, and formation of either monopolar arrays or disorganized spindles. On the basis of gene expression array studies, we identified Aurora A as one of the genes regulated transcriptionally by Akt inhibitors including Compound A. Inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, either by PI3K inhibitor LY294002 or by Compound A, dramatically inhibits the promoter activity of Aurora A, whereas the mammalian target of rapamycin inhibitor has little effect, suggesting that Akt might be responsible for up-regulating Aurora A for mitotic progression. Further analysis of the Aurora A promoter region indicates that the Ets element but not the Sp1 element is required for Compound A-sensitive transcriptional control of Aurora A. Overexpression of Aurora A in cells treated with Compound A attenuates the mitotic arrest and the defects in bipolar spindle formation induced by Akt inhibition. Our studies suggest that that Akt may promote mitotic progression through the transcriptional regulation of Aurora A.

Single Nucleotide Polymorphisms in Selected Apoptotic Genes and BPDE-Induced Apoptotic Capacity in Apparently Normal Primary Lymphocytes: A Genotype-Phenotype Correlation Analysis

Journal of Cancer Epidemiology. 2008  |  Pubmed ID: 20445773

Apoptotic capacity (AC) in primary lymphocytes may be a marker for cancer susceptibility, and functional single nucleotide polymorphisms (SNPs) in genes involved in apoptotic pathways may modulate cellular AC in response to DNA damage. To further examine the correlation between apoptotic genotypes and phenotype, we genotyped 14 published SNPs in 11 apoptosis-related genes (i.e., p53, Bcl-2, BAX, CASP9, DR4, Fas, FasL, CASP8, CASP10, CASP3, and CASP7) and assessed the AC in response to benzo[a]pyrene-7,8-9,10-diol epoxide (BPDE) in cultured primary lymphocytes from 172 cancer-free subjects. We found that among these 14 SNPs, R72P, intron 3 16-bp del/ins, and intron 6 G>A in p53, -938C>A in Bcl-2, and I522L in CASP10 were significant predictors of the BPDE-induced lymphocytic AC in single-locus analysis. In the combined analysis of the three p53 variants, we found that the individuals with the diplotypes carrying 0-1 copy of the common p53 R-del-G haplotype had higher AC values compared to other genotypes. Although the study size may not have the statistical power to detect the role of other SNPs in AC, our findings suggest that some SNPs in genes involved in the intrinsic apoptotic pathway may modulate lymphocytic AC in response to BPDE exposure in the general population. Larger studies are needed to validate these findings for further studying individual susceptibility to cancer and other apoptosis-related diseases.

Polymorphism of the Catechol-O-methyltransferase Gene in Han Chinese Patients with Psoriasis Vulgaris

Genetics and Molecular Biology. Jan, 2009  |  Pubmed ID: 21637643

PSORIASIS VULGARIS IS DEFINED BY A SERIES OF LINKED CELLULAR CHANGES IN THE SKIN: hyperplasia of epidermal keratinocytes, vascular hyperplasia and ectasia, and infiltration of T lymphocytes, neutrophils and other types of leukocytes in the affected skin. Catechol-O-methyltransferase (COMT) 158 polymorphism can reduce the activity of the COMT enzyme that may trigger defective differentiation of keratinocytes in psoriasis. Immunocytes can degrade and inactivate catecholamines via monamine oxidase (MAO) and COMT in the cells. We hypothesized that the COMT-158G > A polymorphism was associated with the risk of psoriasis vulgaris in Han Chinese people. In a hospital-based case-control study, 524 patients with psoriasis vulgaris and 549 psoriasis-free controls were studied. COMT-158 G > A polymorphism was genotyped using the PCR sequence-specific primer (PCR-SSP) technique. We found no statistically significant association between the COMT-158 allele A and the risk of psoriasis vulgaris (p = 0.739 adjusted OR = 1.03; 95% CI = 0.81-1.31). This suggests that the COMT-158 G > A polymorphism may not contribute to the etiology of psoriasis vulgaris in the Han Chinese population.

Association of COX2 Functional Polymorphisms and the Risk of Vitiligo in Chinese Populations

Journal of Dermatological Science. Mar, 2009  |  Pubmed ID: 19004621

Cyclooxygenase-2 (COX2) plays an important role in the production of prostaglandin E2 (PGE2), which is made by epidermal keratinocytes in response to ultraviolet radiation (UVR). PGE2 is important for the proliferation and melanogenesis of epidermal melanocytes, the loss of which leads to vitiligo. COX2-1195A>G, -765G>C, and -8473T>C polymorphisms may influence the mRNA levels of COX2 and affect the production of PGE2 subsequently. Therefore, we supposed that these polymorphisms may be associated with vitiligo.

The Pharmacological Properties of a Novel MCH1 Receptor Antagonist Isolated from Combinatorial Libraries

European Journal of Pharmacology. Jan, 2009  |  Pubmed ID: 19041642

Melanin-concentrating hormone (MCH) is a neuropeptide that exhibits potent orexigenic activity. In rodents, it exerts its actions by interacting with one receptor, MCH(1) receptor which is expressed in many parts of the central nervous system (CNS). To study the physiological implications of the MCH system, we need to be able to block it locally and acutely. This necessitates the use of MCH(1) receptor antagonists. While MCH(1) receptor antagonists have been previously reported, they are mainly not accessible to academic research. We apply here a strategy that leads to the isolation of a high affinity and selective MCH(1) receptor antagonist amenable to in vivo analyses without further chemical modifications. This antagonist, TPI 1361-17, was identified through the screening of multiple non-peptide positional scanning synthetic combinatorial libraries (PS-SCL) totaling more than eight hundred thousand compounds in conditions that allow for the identification of only high-affinity compounds. TPI 1361-17 exhibited an IC(50) value of 6.1 nM for inhibition of 1 nM MCH-induced Ca(2+) mobilization and completely displaced the binding of [(125)I] MCH to rat MCH(1) receptor. TPI 1361-17 was found specific, having no affinity for a variety of other G-protein coupled receptors and channels. TPI 1361-17 was found active in vivo since it blocked MCH-induced food intake by 75%. Our results indicate that TPI 1361-17 is a novel and selective MCH(1) receptor antagonist and is an effective tool to study the physiological functions of the MCH system. These results also illustrate the successful application of combinatorial library screening to identify specific surrogate antagonists in an academic setting.

The Protective Role of Per2 Against Carbon Tetrachloride-induced Hepatotoxicity

The American Journal of Pathology. Jan, 2009  |  Pubmed ID: 19056852

Period 2 (Per2) is a key component of the core clock oscillator and is involved in regulating a number of different biological processes and pathways. Here we report that Per2 plays a protective role in carbon tetrachloride (CCl(4))-induced hepatotoxicity via the modulation of uncoupling protein-2 (Ucp2) gene expression in mice. Hepatic injury after acute CCl(4) injection was monitored in both wild-type and Per2-null mice. At the 12-hour time point after CCl(4) treatment, many more vacuolations were observed in the liver tissues of Per2-null mice whereas fatty tissue degeneration primarily occurred in the liver tissues of wide-type mice. Serum alanine and aspartate aminotransferase activities were elevated in Per2-null mice compared with wide-type mice at 24 hours after CCl(4) treatment, which was in agreement with the observation of significantly larger areas of centrilobular necrosis in the livers of Per2-null mice. A deficit of the Per2 gene enhanced Ucp2 gene expression levels in the liver. As a consequence, intracellular levels of ATP markedly decreased in the liver, allowing increased production of toxic CCl(4) derivatives. The absence of Per2 expression caused a dramatic elevation of Clock expression and influenced Ucp2 through a mechanism that involved a Clock-controlled PPAR-alpha signal transduction pathway. Our studies suggest that the Per2 gene functions in hepatocyte protection from chemical toxicants via the regulation of hepatic Ucp2 gene expression levels.

DNA Repair Phenotype and Cancer Susceptibility--a Mini Review

International Journal of Cancer. Journal International Du Cancer. Mar, 2009  |  Pubmed ID: 19065660

DNA repair is a complicated biological process, consisting of several distinct pathways, that plays a fundamental role in the maintenance of genomic integrity. The very important field of DNA repair and cancer risk has developed rapidly in the past decades. In this review of selected published data from our laboratory, we describe mostly our work on the study of phenotypic markers of nucleotide excision repair (NER), as measured by the benzo(a)pyrene diol epoxide (BPDE)/ultraviolet (UV)-induced mutagen sensitivity assays, BPDE-induced adduct assay, host cell reactivation (HCR)-DNA repair capacity (DRC) assay, reverse transcription-polymerase chain reaction (RT-PCR) assay and reverse-phase protein lysate microarray (RPP) assay, by using peripheral blood lymphocytes in a series of molecular epidemiological studies. Results of our studies suggest that individuals with reduced DRC have an elevated cancer risk. This finding needs additional validation by other investigators, and we also discussed issues in conducting this kind of research in the future.

A Functional Single-nucleotide Polymorphism in the Catechol-O-methyltransferase Gene Alter Vitiligo Risk in a Chinese Population

Archives of Dermatological Research. Oct, 2009  |  Pubmed ID: 19112571

Vitiligo is an acquired hypomelanotic skin disorder resulting from the loss of functional melanocytes. The COMT-158 polymorphism can reduce COMT enzyme activity and may thus lead to the overproduction of toxic radicals in the melanocyte microenvironment. To determine whether this polymorphism in the COMT gene is associated with an increased risk of vitiligo in Chinese populations, we used a polymerase chain reaction sequence-specific primer (PCR-SSP) technique to determine the frequency of the polymorphism COMT-158 G > A in 749 vitiligo patients and 763 healthy controls. We found that compared to the COMT-158 GG genotype, the COMT-158 GA genotype (adjusted odds ratio [OR], 1.39; 95% confidence interval [CI], 1.13-1.72) and the combined GA + AA genotype (adjusted OR, 1.41; 95% CI, 1.15-1.74) were associated with an increased risk of generalized vitiligo. The association was more pronounced in patients with early-onset vitiligo (adjusted OR, 1.95; 95% CI, 1.45-2.60), those with a family history of vitiligo (adjusted OR, 3.84; 95% CI, 2.47-5.96), and female patients (adjusted OR, 1.74; 95% CI, 1.29-2.36). When we further clinically stratified the vitiligo patients according to their disease types, we found that the combined GA + AA genotype was associated with vitiligo vulgaris (adjusted OR, 1.31; 95% CI, 1.02-1.68), focal vitiligo (adjusted OR, 1.62; 95% CI, 1.17-2.25), and universal vitiligo (adjusted OR, 1.50; 95% CI, 0.98-2.30), but not with acrofacial vitiligo (adjusted OR, 1.53; 95% CI, 0.86-2.73) or segmental vitiligo (adjusted OR, 1.35; 95% CI, 0.72-2.51). In conclusion, this COMT gene polymorphism may have contributed to the etiology of vitiligo in our Chinese population. Larger population-based studies are required to verify our findings.

Blepharochalasis: a Rare Condition Misdiagnosed As Recurrent Angioedema

Archives of Dermatology. Apr, 2009  |  Pubmed ID: 19380685

[Study on Hepatoxicity and Nephrotoxicity of Cinnabar in Rats]

Zhongguo Zhong Yao Za Zhi = Zhongguo Zhongyao Zazhi = China Journal of Chinese Materia Medica. Feb, 2009  |  Pubmed ID: 19445157

To investigate hepatoxicity and nephrotoxicity of cinnabar to provide the scientific basis for safe uses in clinic.

[Omparative Study on Allergen Assessment Animal Models in Brown Norway Rat and Guinea Pig]

Zhongguo Zhong Yao Za Zhi = Zhongguo Zhongyao Zazhi = China Journal of Chinese Materia Medica. Feb, 2009  |  Pubmed ID: 19459315

To compare the sensitivity of Brown Norway rats (BN) with Guinea pigs (GP) as allergen assessment animal models.

Genetic Polymorphisms of Glutathione S-transferase and Risk of Vitiligo in the Chinese Population

The Journal of Investigative Dermatology. Nov, 2009  |  Pubmed ID: 19571817

Vitiligo is a common acquired depigmenting disorder characterized by white areas on the skin. Oxidative stress is a major pathogenesis hypothesis of vitiligo. Glutathione S-transferases (GSTs) are enzymes involved in protecting cells against chemical toxicity and stress. We hypothesized that the GSTM1- and GSTT1-null genotypes and GSTP1 polymorphisms were associated with increased risk for vitiligo. In a hospital-based case-control study of 749 vitiligo patients and 763 age- and sex-frequency-matched healthy controls, GSTM1 and GSTT1 genotypes and GSTP1 (Ile104Val, Ala113Val, Gly169Asp) polymorphisms were analyzed using the multiplex PCR and PCR-restriction fragment length polymorphism technique, respectively. We found that the GSTT1-null genotype was significantly associated with the susceptibility to vitiligo and the GSTM1-null genotype also showed a trend toward vitiligo association. We further analyzed the combined effect of GSTM1-null and GSTT1-null genotypes and showed an increased risk of developing vitiligo. By contrast, no statistically significant association was found between GSTP1 polymorphisms and vitiligo risk. These results suggest that individuals with homozygous deletion of GSTT1 and/or GSTM1 have a greater predisposition to vitiligo.

Clinical and Molecular Characterization of S1118F-CFTR

Pediatric Pulmonology. Oct, 2009  |  Pubmed ID: 19774621

Cystic fibrosis is a lethal autosomal recessive disorder usually associated with lung disease, pancreatic insufficiency and high sweat chloride levels.

[Anti-thrombosis Effect and Its Mechanism of Qingkailing Injection]

Zhongguo Zhong Yao Za Zhi = Zhongguo Zhongyao Zazhi = China Journal of Chinese Materia Medica. Jun, 2009  |  Pubmed ID: 19777844

To investigate the anti-thrombosis effect and its mechanism of Qingkailing injection (QKL).

[Interaction Between Four Herb Compounds and a Western Drug by CYP3A4 Enzyme Metabolism in Vitro]

Zhongguo Zhong Yao Za Zhi = Zhongguo Zhongyao Zazhi = China Journal of Chinese Materia Medica. Jul, 2009  |  Pubmed ID: 19873787

To explore the interaction between herbal medicines and western drugs based on CYP3A4 enzyme metabolism by using testotesrone as a probe in liver microsome metabolism system in vitro.

[Study on Embryo Toxicity of Cinnabaris]

Zhongguo Zhong Yao Za Zhi = Zhongguo Zhongyao Zazhi = China Journal of Chinese Materia Medica. Nov, 2009  |  Pubmed ID: 20209918

To observe the effect of Cinnabaris on mouse embryos after pregnant mice were treated by Cinnabaris in different periods of pregnancy.

[Study of Mercury Cumulation in Cinnabar-treated Rats]

Zhongguo Zhong Yao Za Zhi = Zhongguo Zhongyao Zazhi = China Journal of Chinese Materia Medica. Dec, 2009  |  Pubmed ID: 20222426

To investigate the mercury cumulation following single dose or long-term use of Cinnabar to rats.

Isolation, Culture and Biological Characteristics of Primordial Germ Cells from Beijing Fatty Chicken

The Journal of Reproduction and Development. Jun, 2010  |  Pubmed ID: 20228615

The aim of this study was to explore the isolation and culture process of Beijing fatty chicken primordial germ cells (PGCs) and investigate their biological characteristics. The PGCs isolated from the genital ridges of Beijing fatty chicken (Gallus domesticus) embryos after 5.5 days of incubation were co-cultured with mice embryonic fibroblasts (MEF). The results showed that the PGCs of the Beijing fatty chicken were positive for periodic acid Schiff (PAS) and alkaline phosphatase (AKP) staining. These cells could proliferate for a prolonged time in vitro and maintain diploid karyotype. Immunocytochemical staining showed that they expressed SSEA-1, SSEA-4, TRA-1-60 and TRA-1-81, and real-time PCR showed that they expressed Cvh, CDH and Dazl. They could form simple embryoid bodies and differentiate into osteoblasts in vitro. In addition, after transfected with pEGFP-N3, pEYFP-N1 and pDsRed-N1 vectors by liposomal transfection, enhanced green, yellow and red fluorescent protein-positive cells could be visualized using a laser confocal microscope. The above results suggested that PGCs from the Beijing fatty chicken not only had strong self-renewal ability, but also had the potential to differentiate towards mesoblast cells. These cells are suitable for genetic manipulation as nuclear donors.

Establishment and Characterization of a Fibroblast Line from Landrace

Artificial Cells, Blood Substitutes, and Immobilization Biotechnology. May, 2010  |  Pubmed ID: 20297920

Up to 32 Landrace ear marginal tissue samples were used for establishing a fibroblast cell bank by the means of primary explantation and cryopreservation. Biological analysis suggested that the Population Doubling Time (PDT) of the cell line was approximately 24h. The diploid accounted for 97.2% of the whole population; isozyme analysis of Lactic Dehydrogenase (LDH) and Malic Dehydrogenase(MDH) disproved cross-contamination from other cell lines. The results of bacterium, fungus, virus and mycoplasma tests were all negative. The transfection rates of three fluorescent proteins were high, indicating that the exogenous genes could be effectively expressed in the cells. It had not only preserved the precious germplasm resource of the Landrace on the cell level but also provided valuable material for the researches of genomics, postgenomics, somatic cloning and so on.

[Study on Embryonic Toxicity of Senecio Scandens, Qianbai Biyanpian and Total Alkaloid from S. Scandens in Rats]

Zhongguo Zhong Yao Za Zhi = Zhongguo Zhongyao Zazhi = China Journal of Chinese Materia Medica. Feb, 2010  |  Pubmed ID: 20423009

To investigate the characteristics of embryonic toxicity of Senecio scandens, its total alkaloid and Qianbai Biyanpian so that to provide guidance for the safety of medication during pregnancy.

A Nematode Effector Protein Similar to Annexins in Host Plants

Journal of Experimental Botany. 2010  |  Pubmed ID: 19887499

Nematode parasitism genes encode secreted effector proteins that play a role in host infection. A homologue of the expressed Hg4F01 gene of the root-parasitic soybean cyst nematode, Heterodera glycines, encoding an annexin-like effector, was isolated in the related Heterodera schachtii to facilitate use of Arabidopsis thaliana as a model host. Hs4F01 and its protein product were exclusively expressed within the dorsal oesophageal gland secretory cell in the parasitic stages of H. schachtii. Hs4F01 had a 41% predicted amino acid sequence identity to the nex-1 annexin of C. elegans and 33% identity to annexin-1 (annAt1) of Arabidopsis, it contained four conserved domains typical of the annexin family of calcium and phospholipid binding proteins, and it had a predicted signal peptide for secretion that was present in nematode annexins of only Heterodera spp. Constitutive expression of Hs4F01 in wild-type Arabidopsis promoted hyper-susceptibility to H. schachtii infection. Complementation of an AnnAt1 mutant by constitutive expression of Hs4F01 reverted mutant sensitivity to 75 mM NaCl, suggesting a similar function of the Hs4F01 annexin-like effector in the stress response by plant cells. Yeast two-hybrid assays confirmed a specific interaction between Hs4F01 and an Arabidopsis oxidoreductase member of the 2OG-Fe(II) oxygenase family, a type of plant enzyme demonstrated to promote susceptibility to oomycete pathogens. RNA interference assays that expressed double-stranded RNA complementary to Hs4F01 in transgenic Arabidopsis specifically decreased parasitic nematode Hs4F01 transcript levels and significantly reduced nematode infection levels. The combined data suggest that nematode secretion of an Hs4F01 annexin-like effector into host root cells may mimic plant annexin function during the parasitic interaction.

AMP-activated Protein Kinase: a Stress-responsive Kinase with Implications for Cardiovascular Disease

Current Opinion in Pharmacology. Apr, 2010  |  Pubmed ID: 20060780

AMP-activated protein kinase (AMPK) was initially viewed as energy sensor and activated by increased intracellular concentrations of AMP following nutrient deprivation. Physiological or pathological stimuli that deplete cellular energy activate AMPK that coordinates a cellular program that limits further ATP depletion and promotes compensatory changes that maintain cellular ATP levels. Recent studies revealed novel roles of AMPK independent of energy status, thereby increasing our understanding of multi-functional roles of AMPK in cells important in pathogenesis of cardiovascular diseases. This enzyme represents an attractive therapeutic target for vascular disease treatment in the future.

The Six-nucleotide Deletion/insertion Variant in the CASP8 Promoter Region is Inversely Associated with Risk of Squamous Cell Carcinoma of the Head and Neck

Cancer Prevention Research (Philadelphia, Pa.). Feb, 2010  |  Pubmed ID: 20086182

Caspase 8 (CASP8) is an apoptosis-related cysteine peptidase involved in the death receptor pathway and likely in the mitochondrial pathway. A CASP8 promoter region six-nucleotide deletion/insertion (-652 6N ins/del) variant and a coding region D302H polymorphism are reportedly important in cancer development, but no reported study has assessed the associations of these genetic variations with risk of head and neck cancer. In a hospital-based study of non-Hispanic whites, we genotyped CASP8 -652 6N del and 302H variants in 1,023 patients with squamous cell carcinoma of the head and neck (SCCHN) and 1,052 cancer-free controls. Crude and adjusted odds ratios (OR) and 95% confidence intervals (CI) were estimated using unconditional logistic regression models. The CASP8 -652 6N del variant genotypes or haplotypes were inversely associated with SCCHN risk (adjusted OR, 0.70; 95% CI, 0.57-0.85 for the ins/del + del/del genotypes compared with the ins/ins genotype; adjusted OR, 0.73; 95% CI, 0.55-0.97 for the del-D haplotype compared with the ins-D haplotype). Furthermore, the number of the CASP8 -652 6N del (but not 302H) variant allele tended to correlate with increased levels of camptothecin-induced p53-mediated apoptosis in T lymphocytes from 170 cancer-free controls. We concluded that the CASP8 -652 6N del variant allele may contribute to the risk of developing SCCHN in non-Hispanic white populations. Further validation by population-based case-control studies and rigorous mechanistic studies is warranted.

Tobacco Carcinogen NNK Transporter MRP2 Regulates CFTR Function in Lung Epithelia: Implications for Lung Cancer

Cancer Letters. Jun, 2010  |  Pubmed ID: 20089353

Lung cancer is the leading cause of cancer death in the United States. About 85% of all lung cancers are linked to tobacco smoke, in which more than 50 lung carcinogens have been identified and one of the most abundant is 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). The human lung epithelium constitutes the first line of defense against tobacco-specific carcinogens, in which apically-localized receptors, transporters, and ion channels in the airway may play a critical role in this native defense against tobacco smoke. Here we showed that multidrug resistance protein-2 (MRP2) and cystic fibrosis transmembrane conductance regulator (CFTR), two ATP-binding cassette (ABC) transporters, are localized to the apical surfaces of plasma membrane in polarized lung epithelial cells. We observed that there is a functional coupling between CFTR and MRP2 that may be mediated by PDZ proteins. We also observed the existence of a macromolecular complex containing CFTR, MRP2, and PDZ proteins, which might form the basis for the regulatory cooperation between these two ABC transporters. Our results have important implications for cigarette smoke-associated lung diseases (such as smoke-related emphysema, chronic obstructive pulmonary disease, and lung cancer).

Compartmentalized Cyclic Adenosine 3',5'-monophosphate at the Plasma Membrane Clusters PDE3A and Cystic Fibrosis Transmembrane Conductance Regulator into Microdomains

Molecular Biology of the Cell. Mar, 2010  |  Pubmed ID: 20089840

Formation of multiple-protein macromolecular complexes at specialized subcellular microdomains increases the specificity and efficiency of signaling in cells. In this study, we demonstrate that phosphodiesterase type 3A (PDE3A) physically and functionally interacts with cystic fibrosis transmembrane conductance regulator (CFTR) channel. PDE3A inhibition generates compartmentalized cyclic adenosine 3',5'-monophosphate (cAMP), which further clusters PDE3A and CFTR into microdomains at the plasma membrane and potentiates CFTR channel function. Actin skeleton disruption reduces PDE3A-CFTR interaction and segregates PDE3A from its interacting partners, thus compromising the integrity of the CFTR-PDE3A-containing macromolecular complex. Consequently, compartmentalized cAMP signaling is lost. PDE3A inhibition no longer activates CFTR channel function in a compartmentalized manner. The physiological relevance of PDE3A-CFTR interaction was investigated using pig trachea submucosal gland secretion model. Our data show that PDE3A inhibition augments CFTR-dependent submucosal gland secretion and actin skeleton disruption decreases secretion.

Long-term Follow-up of in Situ Extramammary Paget's Disease in Asian Skin Types IV/V Treated with Photodynamic Therapy

Acta Dermato-venereologica. Mar, 2010  |  Pubmed ID: 20169299

Photodynamic therapy is a potentially advantageous treatment for non-melanoma skin cancers. We evaluated the clinical response, recurrence and adverse events of photodynamic therapy for in situ extramammary Paget's disease in 14 male and 3 female Chinese patients with 21 lesions. Topical 20% 5-aminolevulinic acid was applied for 6 h. Each lesion was irradiated with 633 nm red light three times, 1 week apart, at a total dose of 339 J/cm2, followed by three assessments at 6, 12 and 24 months. Overall complete response (CR) rates were 52.4%, 42.9%, and 33.3% at 6, 12 and 24 months, respectively. The CR rate was significantly higher in scrotal lesions (66.6%) than in non-scrotal lesions (8.3%). The overall recurrence rate was 50%. The highest CR rate was for the lesions < 4 cm in diameter (62.5%), followed by those 4-8 cm (33.3%) and > 8 cm (0%). Most adverse events were well tolerated. In conclusion, photodynamic therapy for extramammary Paget's disease is not recommended as the first option except for scrotal cases or lesions < 4 cm in diameter.

Promoter Variant in the Catalase Gene is Associated with Vitiligo in Chinese People

The Journal of Investigative Dermatology. Nov, 2010  |  Pubmed ID: 20613769

Vitiligo is an acquired depigmentation disorder, and reactive oxygen species have an important role in the physiology of cell damage. Reduced catalase enzyme activity and accumulation of excessive hydrogen peroxide have been observed in vitiligo. In a hospital-based case-control study of vitiligo patients (n=749) and age- and sex-matched healthy controls (n=763), we investigated three catalase (CAT) gene polymorphisms (-89A>T, 389C>T, and 419C>T) to examine whether CAT gene polymorphisms are associated with vitiligo susceptibility in the Chinese population. The case-control analysis revealed a 1.54-fold (95% confidence interval (CI) 1.25-1.91) increased risk of developing vitiligo for -89A>T genotype carriers. No evidence for any association between 389C>T and 419C>T polymorphisms in the catalase gene and vitiligo susceptibility was found. An analysis of haplotypes showed increased risk for T(-89)C(389) (odds ratio (OR) 1.90, 95% CI 1.26-2.86) and T(-89)T(389) (OR 2.80, 95% CI 1.24-6.30). Logistic regression analysis of catalase activity also showed a dose-response relationship between increased risk and decreased activity in CAT -89A>T variant genotype carriers, especially in vitiligo patients (P(trend) <0.001). Our molecular epidemiologic findings suggest that the CAT -89A>T variant genotypes were associated with a significant decrease in catalase enzyme activity and a genetic predisposition for vitiligo in Chinese people.

Premature Sebaceous Hyperplasia of Nipple and Areola

International Journal of Dermatology. Jul, 2010  |  Pubmed ID: 20618501

[Material and Mechanisms Induced Pseudo Allergic Reactions of Yuxingcao Injection]

Zhongguo Zhong Yao Za Zhi = Zhongguo Zhongyao Zazhi = China Journal of Chinese Materia Medica. Jun, 2010  |  Pubmed ID: 20815217

To investigate the characteristics, sensitizin and the mechanism of pseudo allergic reaction induced by Yuxingcao injection.

[Content Detection of Bacterial Endotoxin in Two Kinds of Injection by Gelatin Technique]

Zhongguo Zhong Yao Za Zhi = Zhongguo Zhongyao Zazhi = China Journal of Chinese Materia Medica. Jun, 2010  |  Pubmed ID: 20822008

To detect content of bacterial endotoxin in Yuxingcao and Qingkailing injections by specific and nonspecific tachypleus amebocyte lysate technique for in order to investigate the feasibility of specific tachypleus amebocyte lysate technique for detecting bacterial endotoxin in traditional Chinese drug injections.

[Detection of Bacterial Endotoxin Content in Eight Kinds of Injection by Cytokine Revulsion]

Zhongguo Zhong Yao Za Zhi = Zhongguo Zhongyao Zazhi = China Journal of Chinese Materia Medica. Aug, 2010  |  Pubmed ID: 20931847

By using RAW 264.7 macrophage cell line, we studied the dose-effect relationship of endotoxin induced RAW 264.7 cells to release TNF-alpha, and then detected the content of endotoxin in 8 kinds of injections, so that we can investigate the feasibility and the interference factors of the novel test.

[Pseudoanaphylactoid Reaction Analysis of Chinese Herbal Injections in Beagle Dogs]

Zhongguo Zhong Yao Za Zhi = Zhongguo Zhongyao Zazhi = China Journal of Chinese Materia Medica. Sep, 2010  |  Pubmed ID: 21137349

To investigate the preclinical evaluation method of pseudoanaphylactoid reactions for Chinese herbal injections.

[Pharmacology Experimental Study of New Hematischesis Compounds After Flos Sophorae Carbonized]

Zhongguo Zhong Yao Za Zhi = Zhongguo Zhongyao Zazhi = China Journal of Chinese Materia Medica. Sep, 2010  |  Pubmed ID: 21137353

To search new effective compounds, the different hemostatic effects of Flos Sophorae, Flos Sophorae Carbonisatus and principal constituent were observed.

Kinetics of Liquid Phase Catalytic Hydrogenation of Dicyclopentadiene over Pd/C Catalyst

The Journal of Physical Chemistry. A. Mar, 2010  |  Pubmed ID: 19785473

To investigate the kinetics behaviors of dicyclopentadiene hydrogenation, a series of experiments were performed at different temperatures (323-353 K) under varying hydrogen pressure (0.5-1.5 MPa) with a range of Pd/C catalyst loading (0.25-1.00 wt %) using ethanol as solvent in a batch reactor. The time dependent concentration variations for each component were traced under the conditions of removing both the internal and external diffusion effects. The Langmuir-Hinshelwood mechanism was proposed with the consideration of the noncompetitive adsorption between the organic species with hydrogen, and the surface reaction was the rate-determining step. The kinetic equations for the sequence reaction were derived on the basis of the analysis of mechanisms, and the model parameters were determined by fitting the experimental data in differential temperature using the method of Runge-Kutta. The reaction activation energies for the first and second steps are 3.19 and 31.69 kJ x mol(-1), respectively, and the reliability of the model was verified by these experimental results to change hydrogen pressure, reactant concentration and catalyst loading. The simulation results agreed well with the experimental data.

[Simultaneous Determination of Five Effective Components in Sijunzi Bolus Using High Performance Liquid Chromatography-evaporation Light Scattering Detection]

Se Pu = Chinese Journal of Chromatography / Zhongguo Hua Xue Hui. Jan, 2010  |  Pubmed ID: 20458923

A high performance liquid chromatographic (HPLC) method was developed for the simultaneous determination of lobetyolin, pachymic acid, glycyrrhizic acid, atractylenoide III and atractylenolide I in Sijunzi bolus. The separation was performed on an HIQ SIL C18 V column (250 mm x 4.6 mm, 5 microm) with 0.5% acetic acid-methanol as the mobile phase of gradient elution at a flow rate of 1.0 mL/min. The detection was performed with an evaporation light scattering detector (ELSD) and the sample volume was 10 microL. The temperature of drift tube and heating grade of nebulizer was respectively set at 55 degrees C and 60% at 0.2 MPa of pressure. Nitrogen gas was used as carrier gas. Under the optimized conditions, there were good linear relationships between the logarithm values of mass concentration and the peak areas of lobetyolin, pachymic acid, glycyrrhizic acid, atractylenoide III and atractylenolide I in the ranges of 0.076 - 1.21, 0.048 -0.76, 0.153 - 2.45, 0.045 - 0.72 and 0.098 - 1.56 g/L, respectively. The recoveries of the five components were between 97.13% and 100.25%, the relative standard deviations (RSDs) were between 1.23% and 2.44%. This method is simple, rapid, accurate and suitable for the quality control of Sijunzi bolus.

CFTR Chloride Channel in the Apical Compartments: Spatiotemporal Coupling to Its Interacting Partners

Integrative Biology : Quantitative Biosciences from Nano to Macro. Apr, 2010  |  Pubmed ID: 20473396

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel located primarily at the apical or luminal surfaces of epithelial cells in the airway, intestine, pancreas, kidney, sweat gland, as well as male reproductive tract, where it plays a crucial role in transepithelial fluid homeostasis. CFTR dysfunction can be detrimental and may result in life-threatening disorders. CFTR hypofunctioning because of genetic defects leads to cystic fibrosis, the most common lethal genetic disease in Caucasians, whereas CFTR hyperfunctioning resulting from various infections evokes secretory diarrhea, the leading cause of mortality in early childhood. Therefore, maintaining a dynamic balance between CFTR up-regulating processes and CFTR down-regulating processes is essential for maintaining fluid and body homeostasis. Accumulating evidence suggests that protein-protein interactions play a critical role in the fine-tuned regulation of CFTR function. A growing number of proteins have been reported to interact directly or indirectly with CFTR chloride channel, suggesting that CFTR might be coupled spatially and temporally to a wide variety of interacting partners including ion channels, receptors, transporters, scaffolding proteins, enzyme molecules, signaling molecules, and effectors. Most interactions occur primarily between the opposing terminal tails (amino or carboxyl) of CFTR protein and its binding partners, either directly or mediated through various PDZ scaffolding proteins. These dynamic interactions impact the channel function, as well as localization and processing of CFTR protein within cells. This article reviews the most recent progress and findings about the interactions between CFTR and its binding partners through PDZ scaffolding proteins, as well as the spatiotemporal regulation of CFTR-containing macromolecular signaling complexes in the apical compartments of polarized cells lining the secretory epithelia.

[Evaluation of Vitro Hepatotoxicity of Monocrotaline by Precision-cut Liver Slice Technique]

Zhongguo Zhong Yao Za Zhi = Zhongguo Zhongyao Zazhi = China Journal of Chinese Materia Medica. Mar, 2011  |  Pubmed ID: 21657086

To modify the empirical method of precision-cut liver slice technique, and study the hepatotoxicity of monocrotaline by this technique.

NADPH Oxidase 4 Promotes Endothelial Angiogenesis Through Endothelial Nitric Oxide Synthase Activation

Circulation. Aug, 2011  |  Pubmed ID: 21788590

BACKGROUND- Reactive oxygen species serve signaling functions in the vasculature, and hypoxia has been associated with increased reactive oxygen species production. NADPH oxidase 4 (Nox4) is a reactive oxygen species-producing enzyme that is highly expressed in the endothelium, yet its specific role is unknown. We sought to determine the role of Nox4 in the endothelial response to hypoxia. METHODS AND RESULTS: Hypoxia induced Nox4 expression both in vitro and in vivo and overexpression of Nox4 was sufficient to promote endothelial proliferation, migration, and tube formation. To determine the in vivo relevance of our observations, we generated transgenic mice with endothelial-specific Nox4 overexpression using the vascular endothelial cadherin promoter (VECad-Nox4 mice). In vivo, the VECad-Nox4 mice had accelerated recovery from hindlimb ischemia and enhanced aortic capillary sprouting. Because endothelial nitric oxide synthase (eNOS) is involved in endothelial angiogenic responses and eNOS is activated by reactive oxygen species, we probed the effect of Nox4 on eNOS. In cultured endothelial cells overexpressing Nox4, we observed a significant increase in eNOS protein expression and activity. To causally address the link between eNOS and Nox4, we crossed our transgenic Nox4 mice with eNOS(-/-) mice. Aortas from these mice did not demonstrate enhanced aortic sprouting, and VECad-Nox4 mice on the eNOS(-/-) background did not demonstrate enhanced recovery from hindlimb ischemia. CONCLUSIONS: Collectively, we demonstrate that augmented endothelial Nox4 expression promotes angiogenesis and recovery from hypoxia in an eNOS-dependent manner.

Rapid Determination of Ractopamine in Swine Urine Using Surface-enhanced Raman Spectroscopy

Journal of Agricultural and Food Chemistry. Sep, 2011  |  Pubmed ID: 21846097

Ractopamine is approved for use in swine to improve carcass leanness in the United States, but banned in the European Union and China because ractopamine residue may pose health risks. This study investigated the possibility of applying surface-enhanced Raman spectroscopy (SERS) for analysis of ractopamine in swine urine. Ractopamine (0.1-10 μg mL(-1)) was added to urine samples collected from 20 swine to prepare a total of 240 samples. A simple centrifugation, a liquid-liquid extraction (LLE) method, and a more complicated method involving liquid-liquid extraction and solid-phase extraction (LLE-SPE) were used to extract ractopamine from urine samples. Principal component analysis (PCA) and partial least-squares (PLS) regression were used for spectral data analyses. Although no satisfactory result was obtained with the centrifugation method, ractopamine could be detected at levels of 0.8 and 0.4 μg mL(-1) with the LLE and LLE-SPE extraction methods, respectively. The R2 of the PLS model of actual ractopamine values versus predicted values was 0.74 for the LLE method and 0.73 for the LLE-SPE method. The SERS method with simple sample preparation has great potential for rapid analysis of ractopamine in swine urine.

[Material and Mechanisms for Evaluation of Shuanghuanglian Injection Induced Pseudoanaphylactoid Reactions]

Zhongguo Zhong Yao Za Zhi = Zhongguo Zhongyao Zazhi = China Journal of Chinese Materia Medica. Jul, 2011  |  Pubmed ID: 22016949

To investigate the substance basis and the mechanism of pseudoanaphylactoid reactions (PR) induced by Shuanghuanglian injection (SHLI).

[Toxicity Study of Realgar]

Zhongguo Zhong Yao Za Zhi = Zhongguo Zhongyao Zazhi = China Journal of Chinese Materia Medica. Jul, 2011  |  Pubmed ID: 22016954

To investigate the toxicity of realgar and provide the scientific basis for safety use of realgar in clinic.

[Asenic Accumulation Following Realgar Administration in Rats]

Zhongguo Zhong Yao Za Zhi = Zhongguo Zhongyao Zazhi = China Journal of Chinese Materia Medica. Jul, 2011  |  Pubmed ID: 22016955

To explore arsenic accumulation and toxicity mechanism following long-term use of realgar and provide scientific basis for safety use of realgar in clinic.

Analysis of Inducible Nitric Oxide Synthase Gene Polymorphisms in Vitiligo in Han Chinese People

PloS One. 2011  |  Pubmed ID: 22205923

Vitiligo is a chronic depigmented skin disorder with regional melanocytes depletion. The pathogenesis was not completely clarified. Recently, more and more evidence suggested that polymorphisms of some genes are associated with vitiligo risk. Here, we want to examine the association between the inducible nitric oxide synthase (iNOS) gene polymorphisms and the risk of vitiligo in Chinese populations.

Development and Validation of Chinese Version of Female Sexual Function Index in a Chinese Population-a Pilot Study

The Journal of Sexual Medicine. Apr, 2011  |  Pubmed ID: 21235720

Female sexual dysfunction (FSD) is a prevalent problem that has been continuously overlooked in mainland China. An assessment instrument for FSD is urgently needed.

Inhibition of Keratin 17 Expression with Antisense and RNAi Strategies: Exploring Novel Therapy for Psoriasis

Experimental Dermatology. Jul, 2011  |  Pubmed ID: 21355890

Psoriasis is now considered to be a chronic, immune-mediated and inflammatory skin disease. As the precise cause of psoriasis remains unknown, its treatment is challenging for dermatologists. Keratin 17 (K17), an intermediate filament protein, is highly expressed in psoriatic lesions, while not normally expressed in healthy epidermis. Studies have suggested that K17 plays a role in the pathogenesis of psoriasis. However, no study has been performed to determine the potential application of K17 down-regulation as a treatment option for psoriatic lesions. We hypothesized that anti-K17 interference may suppress the development and progression of psoriasis and potentially serve as a novel strategy for the treatment of psoriasis. Therefore, we down-regulated and silenced K17 gene expression in keratinocytes (KCs) using antisense and RNA interference (RNAi) techniques. We found that K17-specific antisense oligonucleotides (ASODN) or siRNAs inhibited proliferation and induced apoptosis in KCs as well as down-regulated K17 expression at both mRNA and protein levels. For our in vivo study, we constructed the SCID-hu xenogeneic transplantation psoriasis mouse model by grafting psoriatic lesions onto SCID mice and topically applied K17-specific ASODN and liposome-encapsulated siRNA to the grafts. We observed morphological and histological improvement in the treated psoriatic grafts. As a result, K17 mRNA and protein expression was significantly decreased in the grafts of the mouse model. Taken together, we conclude that anti-K17 therapy is an effective treatment option for psoriasis, and the K17 molecule, as a new target, may hold tremendous potential for the treatment of psoriasis in the future.

Chronic Activation of AMP-activated Protein Kinase Prevents 20-hydroxyeicosatetraenoic Acid-induced Endothelial Dysfunction

Clinical and Experimental Pharmacology & Physiology. May, 2011  |  Pubmed ID: 21388435

1. 20-Hydroxyeicosatetraenoic acid (20-HETE) is a potent vasoconstrictor involved in vascular dysfunction and blood pressure regulation. Studies have revealed strong associations between 20-HETE and endothelial dysfunction; however, the signalling mechanisms are largely unknown. Therefore, the aim of the present study was to investigate the effect of 20-HETE on the association between endothelial nitric oxide synthase (eNOS) and heat shock protein 90 (Hsp90). 2. In mouse aortic rings, 20-HETE significantly enhanced the constriction to phenylephrine and inhibited the relaxation to acetylcholine (P=0.05 vs control rings). In mice with chronic AMP-activated protein kinase (AMPK) activation, this protected against the negative effects of 20-HETE (P<0.05). Immunoprecipitation of eNOS in human umbilical vein endothelial cells treated with 20-HETE revealed a decrease in basal and vascular endothelial growth factor-stimulated Hsp90 association with eNOS (P<0.05). Pretreatment of cells with 5'-aminoimidazole-4-carboxyamide-ribonucleoside (AICAR; a chronic activator of AMPK) prevented the loss of Hsp90 association with eNOS following 20-HETE treatment. Treatment with 20-HETE for 24 h induced an increase in eNOS phosphorylation that was not seen following acute treatment (30 min). The increased eNOS phosphorylation was accompanied by transient changes in Akt phosphorylation. 3. In conclusion, 20-HETE impairs eNOS-Hsp90 association, which can be reversed by chronic activation of AMPK. This provides a mechanism for reduced nitric oxide bioactivity and endothelial dysfunction in diseases with elevated 20-HETE levels, such as hypertension.

Heme Oxygenase-1 Protects Human Melanocytes from H2O2-induced Oxidative Stress Via the Nrf2-ARE Pathway

The Journal of Investigative Dermatology. Jul, 2011  |  Pubmed ID: 21412259

Oxidative stress caused by hydrogen peroxide (H(2)O(2)) leads to cell death and has been implicated in the pathogenesis of vitiligo. The nuclear factor E2-related factor 2 (Nrf2)-antioxidant response element (ARE), a major antioxidant pathway, regulates oxidative stress-related cytoprotective genes. We hypothesized that the Nrf2-ARE pathway protects human melanocytes from H(2)O(2)-induced oxidative damage through the induction of downstream antioxidative genes. Thus, we used Nrf2 short interfering RNA (siRNA) and pCMV6-XL5-Nrf2 to downregulate or upregulate Nrf2 expression in immortalized human melanocyte cell line PIG1. The melanocytes were then analyzed under different oxidative stress conditions for cell viability and apoptosis. Our study demonstrated that heme oxygenase-1 (HO-1) was the most induced antioxidant gene in PIG1 cells after treatment with H(2)O(2). Knockdown of Nrf2 or zinc protoporphyrin IX (ZnPP) treatment increased cell death caused by H(2)O(2) in melanocytes, but upregulation of Nrf2 or hemin treatment reduced cell death caused by H(2)O(2) in melanocytes. In addition, the H(2)O(2)-induced Nrf2-ARE/HO-1 pathway was confirmed in primary cultured human melanocytes by examining the expression and translocation of Nrf2 and HO-1. These data suggested that regulation of the Nrf2/HO-1 pathway can reduce H(2)O(2)-induced oxidative damage in human melanocytes. Our data demonstrate that HO-1 protects human melanocytes from oxidative damage via the Nrf2-ARE pathway.

Expression of Arabidopsis Pathogenesis-related Genes During Nematode Infection

Molecular Plant Pathology. May, 2011  |  Pubmed ID: 21453430

The expression pattern of pathogenesis-related genes PR-1 to PR-5 was examined in the roots and leaves of Arabidopsis thaliana plants on infection with beet-cyst (Heterodera schachtii) and root-knot (Meloidogyne incognita) nematodes. During H. schachtii parasitism of Arabidopsis, the expression of PR-1, PR-2 and PR-5, which are considered to be markers for salicylic acid (SA)-dependent systemic acquired resistance (SAR), was induced in both roots and leaves of infected plants. In addition, the expression of PR-3 and PR-4, which are used as markers for jasmonic acid (JA)-dependent SAR, was not altered in roots, but in the leaves of H. schachtii-infected plants, the expression PR-3 was induced, whereas the expression of PR-4 was down-regulated. During M. incognita infection of Arabidopsis, the expression of PR-1, PR-2 and PR-5 was highly induced in roots, as was PR-3 to a lesser extent, but the expression of PR-4 was not altered, indicating that infection with M. incognita activated both SA- and JA-dependent SAR in roots. However, all PRgenes examined (PR-1 to PR-5) were down-regulated in the leaves of M. incognita-infected plants, suggesting the suppression of both SA- and JA-dependent SAR. Furthermore, constitutive expression of a single PR in Arabidopsis altered the transcription patterns of other PR genes, and the over-expression of PR-1 reduced successful infection by both H. schachtii and M. incognita, whereas the over-expression of PR-3 reduced host susceptibility to M. incognita but had no effect on H. schachtii parasitism. The results suggest that fundamental differences in the mechanisms of infection by beet-cyst and root-knot nematodes differentially regulate PR protein production and mobilization within susceptible host plants.

Tumor Thickness Predicts Long-term Complete Response of Facial Basal Cell Carcinomas in Asian Skin Types Iv/v Treated with Methyl Aminolaevulinate Photodynamic Therapy

Photomedicine and Laser Surgery. Jul, 2011  |  Pubmed ID: 21456944

Basal cell carcinoma (BCC) responds well to topical photodynamic therapy (PDT), with high clearance rates of 72-100%, although the therapy showed limited effectiveness for lesions > 2 mm thick. Tumor thickness is thought to be associated with therapeutic response. Objective: The purpose of this study was to investigate the efficacy, safety, and response depth of methyl aminolaevulinate (MAL) PDT for BCC.

Foxp3 Expression in Melanoma Cells As a Possible Mechanism of Resistance to Immune Destruction

Cancer Immunology, Immunotherapy : CII. Aug, 2011  |  Pubmed ID: 21547596

The forkhead transcription factor Foxp3 is the only definitive marker of CD4(+)CD25(+) regulatory T cells (Tregs) and has been identified as a key regulator in the development and function of Tregs. Foxp3 expression has been reported in a variety of solid tumors, including melanoma. In this study, we validated Foxp3 expression in both tumor-infiltrating Tregs and melanoma cells by performing immunohistochemical analysis of human melanoma tissue sections. Further, we assessed Foxp3 expression in melanoma cell lines by performing flow cytometry, confocal microscopic analysis, reverse transcription-polymerase chain reaction (RT-PCR), and Western blotting. Inhibition of Foxp3 expression in melanoma cells using small interfering RNA (siRNA) resulted in downregulation of B7-H1 and transforming growth factor (TGF)-β expression; in contrast, Foxp3 overexpression resulted in the upregulation of the expression of these proteins. Coculture of Foxp3-expressing melanoma cells with naive CD4(+)CD25(-) T cells resulted in strong inhibition of T-cell proliferation. This antiproliferative effect was partially abrogated by specific inhibition of Foxp3 expression and was effectively enhanced by overexpression of Foxp3. We observed an attenuated antiproliferative effect even when melanoma cells and T cells in the coculture were separated using Transwell inserts. These findings indicated that melanoma cells could have Foxp3-dependent Treg-like suppressive effects on T cells and suggested that the mimicking of Treg function by melanoma cells may represent a possible mechanism of tumor resistance to immune destruction in the melanoma tumor microenvironment.

Analysis of CFTR Interactome in the Macromolecular Complexes

Methods in Molecular Biology (Clifton, N.J.). 2011  |  Pubmed ID: 21594790

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel localized primarily at the apical surface of epithelial cells lining the airway, gut, exocrine glands, etc., where it is responsible for transepithelial salt and water transport. A growing number of proteins have been reported to interact directly or indirectly with CFTR chloride channel, suggesting that CFTR might regulate the activities of other ion channels, receptors, and transporters, in addition to its role as a chloride conductor. Most interactions occur primarily between the opposing terminal tails (N or C) of CFTR and its binding partners, either directly or mediated through various PDZ domain-containing proteins. This chapter describes methods we developed to cross-link CFTR into a macromolecular complex to identify and analyze the assembly and regulation of CFTR-containing complexes in the plasma membrane.

[Development of Animal Model for Anaphylactoid Test of Rodent]

Zhongguo Zhong Yao Za Zhi = Zhongguo Zhongyao Zazhi = China Journal of Chinese Materia Medica. Feb, 2011  |  Pubmed ID: 21598549

To establish a simple and feasible method of anaphylactoid test on awaked small animals for screening and assessing anaphylactoid reaction of traditional Chinese medicine (TCM) injection with different concentration of tween 80.

Mutual Enhancement Between Heparanase and Vascular Endothelial Growth Factor: a Novel Mechanism for Melanoma Progression

Cancer Letters. Sep, 2011  |  Pubmed ID: 21624769

Heparanase is closely related to growth factors in the role of promoting tumor progression. Among them, vascular endothelial growth factor (VEGF) is necessary for tumor vascularity and metastasis. Release of VEGF by heparanase can initiate relative signaling pathways, resulting in an up-regulation of transcriptional factors related with heparanase. Therefore, VEGF likely has a potential function as a regulator of heparanase expression in melanoma. We hypothesized that a novel mechanism exists where heparanase and VEGF are mutually enhanced in melanoma. Our study was conducted to validate the hypothetical mutual enhancement and elucidate its effect on melanoma progression. We found that the addition of exogenous VEGF and its cDNA transfection induce heparanase over-expression by means of western-blot and real-time RT-PCR, while anti-VEGF siRNA reduces heparanase expression in A2058 and WM793 melanoma cell lines. Likewise, VEGF expression is also regulated by heparanase in these two cell lines. Additionally, the cells with mutual enhancement phenotypes exhibit higher proliferation and transmigration capacity. PD98059, a specific inhibitor of the MEK/ERK signaling pathway, is involved in this mutual enhancement. These data are the first to show that heparanase and VEGF have a mutual enhancement in melanoma cells, which may be a novel mechanism for promoting melanoma progression.

Inhibitory Effects of Vitamin E on UVB-induced Apoptosis of Chicken Embryonic Fibroblasts

Cell Biology International. Apr, 2011  |  Pubmed ID: 21054279

Apoptosis research has been focused on several model species in the past decades, whereas studies concerned with non-mammalian vertebrate, particularly birds, have rarely been involved. In accord with requirements to expand the biodiversity of apoptotic research, a chicken embryonic fibroblasts model involving UVB (ultraviolet B) as the death stimulus was established through primary explantation and serial passage. Myriads of antioxidants can inhibit UVB-induced apoptosis by virtue of scavenging reactive oxygen species. To improve our understanding of the possible anti-apoptotic effects and mechanisms of Vitamin E against UVB-induced apoptosis in chicken embryonic fibroblasts, cells treated with Vitamin E after UVB irradiation were stained with AO/EB and Fluo-3/AM to visualize chromatin distribution and calcium homoeostasis, respectively. They were also analysed by flow cytometry to detect mitochondrial transmembrane potential, and cell cycle progression and apoptotic rates were recorded. RT-PCR was used to analyse the expression of some apoptosis-related genes. Typical apoptotic events, including cell shrinkage, blebbing and nuclear condensation, occurred after radiation. In the presence of Vitamin E following irradiation, apoptotic cells were reduced. Ca2+ release was temporarily prevented, and cell cycle arrest at S/G2 checkpoint had almost completely reverted to normal. fas decreased, while procaspase-3 remained nearly unchanged with and without Vitamin E, and bcl2/bax ratio was up-regulated, indicating possible anti-apoptotic mechanisms through the mitochondrial pathway. This new investigation of an apoptosis model involving chicken embryonic fibroblasts expands the database of knowledge across a wider spectrum of vertebrate species.

Vitiligo Autoantigen VIT75 is Identified As Lamin A in Vitiligo by Serological Proteome Analysis Based on Mass Spectrometry

The Journal of Investigative Dermatology. Mar, 2011  |  Pubmed ID: 21085190

VIT75 is a 75-kDa melanocyte membrane antigen (Ag) that had been observed, but not identified until now. Its immunopathogenic role in vitiligo remains unknown. In this study, serological proteome analysis based on mass spectrometry was employed to identify VIT75. Three disparate 75-, 60-, and 45-kDa proteins on two-dimensional (2D) gel were, respectively, identified as lamin A, tyrosinase-related protein 1, and melanin-concentrating hormone receptor 1. The latter two proteins are well-known Ags. Immunoreactivity analysis showed that the 75-kDa protein displayed on the 2D gel was recognized by human anti-lamin A IgG. Antibody (Ab) reactivity to lamin A was positive in 28.6% of patients' sera. Only 3.1% healthy sera reacted with the lamin A. A total of 91.7% of the positive sera was from the active non-segmental vitiligo (NSV). The positive rate and mean titer of anti-lamin A Ab are higher for NSV with autoimmune disease than for NSV without autoimmune disease. These data demonstrate that VIT75 is lamin A. To our knowledge, this is a previously unreported vitiligo Ag. Anti-lamin A Ab may be a potential marker of NSV with autoimmune disease. The study indicates that the targets of autoantibodies in vitiligo patients can be revealed by serological proteome analysis.

Spermine, a Molecular Switch Regulating EGFR, Integrin β3, Src, and FAK Scaffolding

Cellular Signalling. Apr, 2012  |  Pubmed ID: 22227249

Intracellular polyamine levels are highly regulated by the activity of ornithine decarboxylase (ODC), which catalyzes the first rate-limiting reaction in polyamine biosynthesis, producing putrescine, which is subsequently converted to spermidine and spermine. We have shown that polyamines regulate proliferation, migration, and apoptosis in intestinal epithelial cells. Polyamines regulate key signaling events at the level of the EGFR and Src. However, the precise mechanism of action of polyamines is unknown. In the present study, we demonstrate that ODC localizes in lamellipodia and in adhesion plaques during cell spreading. Spermine regulates EGF-induced migration by modulating the interaction of the EGFR with Src. The EGFR interacted with integrin β3, Src, and focal adhesion kinase (FAK). Active Src (pY418-Src) localized with FAK during spreading and migration. Spermine prevented EGF-induced binding of the EGFR with integrin β3, Src, and FAK. Activation of Src and FAK was necessary for EGF-induced migration in HEK293 cells. EGFR-mediated Src activation in live HEK293 cells using a FRET based Src reporter showed that polyamine depletion significantly increased Src kinase activity. In vitro binding studies showed that spermine directly binds Src, and preferentially interacts with the SH2 domain of Src. The physical interaction between Src and the EGFR was severely attenuated by spermine. Therefore, spermine acts as a molecular switch in regulating EGFR-Src coupling both physically and functionally. Upon activation of the EGFR, integrin β3, FAK and Src are recruited to EGFR leading to the trans-activation of both the EGFR and Src and to the Src-mediated phosphorylation of FAK. The activation of FAK induced Rho-GTPases and subsequently migration. This is the first study to define mechanistically how polyamines modulate Src function at the molecular level.

The Interaction of the Novel 30C02 Cyst Nematode Effector Protein with a Plant β-1,3-endoglucanase May Suppress Host Defence to Promote Parasitism

Journal of Experimental Botany. Jun, 2012  |  Pubmed ID: 22442414

Phytoparasitic nematodes secrete an array of effector proteins to modify selected recipient plant cells into elaborate and essential feeding sites. The biological function of the novel 30C02 effector protein of the soybean cyst nematode, Heterodera glycines, was studied using Arabidopsis thaliana as host and the beet cyst nematode, Heterodera schachtii, which contains a homologue of the 30C02 gene. Expression of Hg30C02 in Arabidopsis did not affect plant growth and development but increased plant susceptibility to infection by H. schachtii. The 30C02 protein interacted with a specific (AT4G16260) host plant β-1,3-endoglucanase in both yeast and plant cells, possibly to interfere with its role as a plant pathogenesis-related protein. Interestingly, the peak expression of 30C02 in the nematode and peak expression of At4g16260 in plant roots coincided at around 3-5 d after root infection by the nematode, after which the relative expression of At4g16260 declined significantly. An Arabidopsis At4g16260 T-DNA mutant showed increased susceptibility to cyst nematode infection, and plants that overexpressed At4g16260 were reduced in nematode susceptibility, suggesting a potential role of host β-1,3-endoglucanase in the defence response against H. schachtii infection. Arabidopsis plants that expressed dsRNA and its processed small interfering RNA complementary to the Hg30C02 sequence were not phenotypically different from non-transformed plants, but they exhibited a strong RNA interference-mediated resistance to infection by H. schachtii. The collective results suggest that, as with other pathogens, active suppression of host defence is a critical component for successful parasitism by nematodes and a vulnerable target to disrupt the parasitic cycle.

Theoretical Study on the Mechanism and Kinetics for the Self-reaction of C2H5O2 Radicals

The Journal of Physical Chemistry. A. May, 2012  |  Pubmed ID: 22494036

Oxygen-to-oxygen coupling, direct H-abstraction and oxygen-to-(α)carbon nucleophilic substitution processes have been investigated for both the singlet and triplet self-reaction of C(2)H(5)O(2) radicals at the CCSD(T)/cc-pVDZ//B3LYP/6-311G(2d,2p) level to evaluate the reaction mechanisms, possible products and rate constants. The calculated results show that the title reaction mainly occurs through the singlet oxygen-to-oxygen coupling mechanism with the formation of entrance tetroxide intermediates, and the most dominant product is C(2)H(5)O + HO(2) + CH(3)CHO (P5) generated in channel R5. Beginning from the radical products of P5 (C(2)H(5)O, HO(2)) and reactant (C(2)H(5)O(2)), five secondary reactions HO(2) + HO(2) (a), HO(2) + C(2)H(5)O (b), C(2)H(5)O + C(2)H(5)O (c), HO(2) + C(2)H(5)O(2) (d), and C(2)H(5)O + C(2)H(5)O(2) (e) mainly proceed on the triplet potential energy surface. Among these reactions, (a), (b), and (d) are kinetically favorable because of lower barrier heights. The calculated rate constants of channel R5 between 200 and 295 K are almost independent of the temperature, which is in agreement with the experimental report. With regard to the final products distribution, CH(3)CHO, C(2)H(5)OH, C(2)H(5)OOH, H(2)O(2), and (3)O(2) are predicted to be major, whereas C(2)H(5)OOC(2)H(5) should be in minor amount.

Baicalein Protects Human Melanocytes from H(2)O(2)-induced Apoptosis Via Inhibiting Mitochondria-dependent Caspase Activation and the P38 MAPK Pathway

Free Radical Biology & Medicine. Jul, 2012  |  Pubmed ID: 22569306

The removal of H(2)O(2) by antioxidants has been proven to be beneficial to patients with vitiligo. Baicalein (5,6,7-trihydroxyflavone; BE) has antioxidant activity and has been used in vitiligo therapy in Chinese traditional medicine. In this study, we investigated the potential protective effect and mechanisms of BE against H(2)O(2)-induced apoptosis in human melanocytes. Melanocytes from the PIG1 cell line were pretreated with different concentrations of BE for 1h, followed by exposure to 1.0 mM H(2)O(2) for 24h. Cell apoptosis, reactive oxygen species levels, and mitochondrial membrane potentials were evaluated by flow cytometry, and cell viability was determined by an MTT assay. The expressions of Bax, Bcl-2, caspase-3, total and phosphorylated ERKs, and p38 MAPK were assayed by Western blot to investigate the possible molecular mechanisms. Our results showed that BE significantly inhibited H(2)O(2)-induced apoptosis, intracellular reactive oxygen species generation, and changes in the mitochondrial membrane potential. It also reduced the Bax/Bcl-2 ratio, the release of cytochrome c, the activation of caspase-3, and the phosphorylation of p38 MAPK in a concentration-dependent manner. The results demonstrate for the first time that BE exerts a cytoprotective role in H(2)O(2)-induced apoptosis by inhibiting the mitochondria-dependent caspase activation and p38 MAPK pathway.

Analysis of Large Phenotypic Variability of EEC and SHFM4 Syndromes Caused by K193E Mutation of the TP63 Gene

PloS One. 2012  |  Pubmed ID: 22574117

EEC (ectrodactyly, ectodermal dysplasia, clefting; OMIM 604292) is an autosomal dominant developmental disorder resulting mainly from pathogenic mutations of the DNA-binding domain (DBD) of the TP63 gene. In this study, we showed that K193E mutation in nine affected individuals of a four-generation kindred with a large degree of phenotypic variability causes four different syndromes or TP63-related disorders: EEC, Ectrodactyly-ectodermal dysplasia (EE), isolated ectodermal dysplasia, and isolated Split Hand/Foot Malformation type 4 (SHFM4). Genotype-phenotype and DBD structural modeling analysis showed that the K193-located loop L2-A is associated with R280 through hydrogen bonding interactions, while R280 mutations also often cause large phenotypic variability of EEC and SHFM4. Thus, we speculate that K193 and several other DBD mutation-associated syndromes may share similar pathogenic mechanisms, particularly in the case of the same mutation with different phenotypes. Our study and others also suggest that the phenotypic variability of EEC is attributed, at least partially, to genetic and/or epigenetic modifiers.

[Development of Gastric Precancerous Lesion Animal Model]

Zhongguo Zhong Yao Za Zhi = Zhongguo Zhongyao Zazhi = China Journal of Chinese Materia Medica. Jan, 2012  |  Pubmed ID: 22741469

To establish a model of gastric precancerous lesion by using Aristolochic manshuriensis which contains aristolochic acids.

[Effect of Shaoyao Gancao Tang on Function and Expression of P-glycoprotein in Caco-2 Cells]

Zhongguo Zhong Yao Za Zhi = Zhongguo Zhongyao Zazhi = China Journal of Chinese Materia Medica. Apr, 2012  |  Pubmed ID: 22792804

To investigate the effect of Shao Yao-Gan Cao-Tang on function and expression of P-glycoprotein (P-gp) in Caco-2 cells.

Dual-Function CXCR4 Antagonist Polyplexes To Deliver Gene Therapy and Inhibit Cancer Cell Invasion

Angewandte Chemie (International Ed. in English). Jul, 2012  |  Pubmed ID: 22855422

Giving a one-two punch: A bicyclam-based biodegradable polycation with CXCR4 antagonistic activity shows potential for combining cancer chemotherapy with gene therapy or siRNA therapy. This dual-function polycation prevents cancer cell invasion by inhibiting CXCL12-stimulated CXCR4 activation, while at the same time efficiently and safely delivering therapeutic DNA into cancer cells.

A Chemokine Receptor CXCR2 Macromolecular Complex Regulates Neutrophil Functions in Inflammatory Diseases

The Journal of Biological Chemistry. Feb, 2012  |  Pubmed ID: 22203670

Inflammation plays an important role in a wide range of human diseases such as ischemia-reperfusion injury, arteriosclerosis, cystic fibrosis, inflammatory bowel disease, etc. Neutrophilic accumulation in the inflamed tissues is an essential component of normal host defense against infection, but uncontrolled neutrophilic infiltration can cause progressive damage to the tissue epithelium. The CXC chemokine receptor CXCR2 and its specific ligands have been reported to play critical roles in the pathophysiology of various inflammatory diseases. However, it is unclear how CXCR2 is coupled specifically to its downstream signaling molecules and modulates cellular functions of neutrophils. Here we show that the PDZ scaffold protein NHERF1 couples CXCR2 to its downstream effector phospholipase C (PLC)-β2, forming a macromolecular complex, through a PDZ-based interaction. We assembled a macromolecular complex of CXCR2·NHERF1·PLC-β2 in vitro, and we also detected such a complex in neutrophils by co-immunoprecipitation. We further observed that the CXCR2-containing macromolecular complex is critical for the CXCR2-mediated intracellular calcium mobilization and the resultant migration and infiltration of neutrophils, as disrupting the complex with a cell permeant CXCR2-specific peptide (containing the PDZ motif) inhibited intracellular calcium mobilization, chemotaxis, and transepithelial migration of neutrophils. Taken together, our data demonstrate a critical role of the PDZ-dependent CXCR2 macromolecular signaling complex in regulating neutrophil functions and suggest that targeting the CXCR2 multiprotein complex may represent a novel therapeutic strategy for certain inflammatory diseases.