In JoVE (1)

Other Publications (38)

Articles by Janak Gaire in JoVE

Other articles by Janak Gaire on PubMed

Gonadotrophin Release and Meat Consumption in Vegetarian Women

The American Journal of Clinical Nutrition. Jan, 1986  |  Pubmed ID: 3079942

Many factors including diet modify the hypothalamic-pituitary axis and menstrual periodicity. We have determined the effect of a daily meat or a soybean supplement in rural vegetarian Black women on the length of the menstrual cycle and the episodic and luteinizing releasing hormone stimulated release of luteinizing hormone. The daily meat but not soybean supplement increased the length of the menstrual cycle (p less than or equal to 0.01), increased the release of LH (p less than or equal to 0.01), and decreased the stimulated release of LH in the luteal phase (p less than or equal to 0.01). These changes are opposite to those reported previously in the Caucasian women fed a meatless diet. Thus addition of meat in the diet modifies the episodic release of gonadotrophins and follicular maturation. The importance of a carbohydrate diet preferentially maintaining CNS-rhythmicity is suggested.

Characterization of the Estrogen-induced PS2 Protein Secreted by the Human Breast Cancer Cell Line MCF-7

Endocrinology. Nov, 1987  |  Pubmed ID: 3665845

Our laboratory has reported previously the cloning of a complementary DNA termed pS2, corresponding to a messenger RNA (mRNA) whose synthesis is induced by estrogen in the human breast cancer cells MCF-7. Examination of the possible open reading frames of this complementary DNA has led to the prediction that the pS2 protein could be a secreted polypeptide of either 58 or 63 amino acids in length. Using a rabbit antiserum prepared against a synthetic peptide corresponding to the last 31 amino acids of the putative protein, we show that a protein with the expected migration during sodium dodecyl sulfate gel electrophoresis can indeed be immunoprecipitated from either the culture medium of MCF-7 cells grown in the presence of labeled amino acids or the in vitro translation products of MCF-7 poly(A) RNA enriched in pS2 mRNA. Furthermore, in vivo and in vitro differential amino acid labeling allows us to conclude that the mature pS2 protein is probably secreted as a 58 amino acid long peptide. Finally, we show that pS2 protein synthesis is induced in MCF-7 cells by estradiol and phenol red, but not by the antiestrogen tamoxifen, in keeping with our previous results demonstrating estrogen induction of pS2 mRNA synthesis.

Purification and Functional Characterization of a Cellular Transcription Factor That Binds to an Enhancer Element Within the Adenovirus Early EIIa Promoter

Proceedings of the National Academy of Sciences of the United States of America. Apr, 1988  |  Pubmed ID: 2965812

The adenovirus EIa-inducible early EIIa (EIIaE) promoter is comprised of several sequence elements essential for constitutive and induced expression. We report here the purification of the host-cell factor that interacts with the major upstream element of this promoter, extending between positions -90 and -70 with respect to the main EIIaE cap site and exhibiting enhancer properties. The purified factor, which corresponds to a 40- to 43-kDa polypeptide, specifically binds to its recognition site and stimulates EIIaE promoter activity when added to an in vitro transcription system, reconstituted from purified factors and RNA polymerase. The implication of this factor in the control of the other adenovirus early genes is discussed.

[Hepatitis Caused by Non-steroidal Anti-inflammatory Agents. A Cooperative Evaluation of Regional Centers of Drug Surveillance for 1985]

Thérapie. May, 1988  |  Pubmed ID: 3420570

Isolation and Characterization of Two Novel, Closely Related ATF CDNA Clones from HeLa Cells

Nucleic Acids Research. Jun, 1990  |  Pubmed ID: 1694576

ATF or CRE binding proteins are cellular transcription factors involved in the regulation of adenovirus Ela-responsive and cellular cAMP-inducible promoters. We report the isolation from a HeLa cell cDNA library of two clones that encode proteins with specific ATF/CRE DNA binding activity. The two clones differ by a 63 bp element which is retained in one (ATF-a) and deleted from the other (ATF-a delta) and which may correspond to an alternative exon. The peptide sequences (483 and 462 amino acids, respectively) derived from each of these cDNAs are identical, except for the additional 21 amino acids in ATF-a, but clearly differ from the other ATF/CREB proteins reported. All of them, however, share a conserved leucine zipper domain also found in other transcription factors. ATF-a and ATF-a delta therefore represent two closely related members of a larger multigene family of proteins that interact with conserved promoter elements.

[A 30-month Study of the Calls to the Regional Drug Monitoring Center in Lorraine (Nancy)]

Thérapie. 1990 Jan-Feb, 1990  |  Pubmed ID: 2343435

For over a period of 30 months, all the questions to the French "Centre de Pharmacovigilance" of Nancy Lorraine have been analysed. The University Hospital of Nancy is by for the main caller (65%) and the suspicion of adverse drug reaction represents 61% of all the questions. Gastrointestinal and hepatic disorders, cardiovascular drugs arouse the greatest number of questions. Comparison with results previously published by some French Pharmacovigilance Centres points out some regional particularities.

Transcriptional Activation by the Adenovirus Larger E1a Product is Mediated by Members of the Cellular Transcription Factor ATF Family Which Can Directly Associate with E1a

Molecular and Cellular Biology. Jan, 1993  |  Pubmed ID: 8417352

We recently isolated three cDNA clones encoding closely related proteins (ATFa1, ATFa2, and ATFa3) that belong to the activating transcription factor-cyclic AMP-responsive element family of cellular transcription factors. Using cotransfection experiments, we showed that these proteins mediate the transcriptional activation induced by the adenovirus E1a 13S mRNA gene product and that the zinc-binding domains present in both E1a conserved region 3 and the most N-terminal portion of the ATFa proteins play crucial roles in this activity. Reciprocal coimmunoprecipitation experiments demonstrated direct interactions between these proteins. Neither the conserved region 3 domain of E1a nor the N-terminal metal-binding element of ATFa is essential for these interactions. The simultaneous alteration of both the N-terminal and the C-terminal domains of ATFa abolished E1a binding, while either mutation alone failed to impair these interactions.

Stromal-epithelial Interaction Mediates Steroidal Regulation of Metalloproteinase Expression in Human Endometrium

Proceedings of the National Academy of Sciences of the United States of America. Oct, 1994  |  Pubmed ID: 7937850

The hallmark of the menstrual cycle is extensive steroid-dependent tissue turnover. Estrogen mediates endometrial cell growth and structural remodeling, whereas progesterone suppresses estrogen-dependent proliferation and promotes cellular differentiation. In nonfertile cycles, tissue degradation and menstruation occur as a consequence of steroidal deprivation as the ovarian corpus luteum fails. Stromal-epithelial interactions are recognized as a necessary component in mediating steroid-induced endometrial turnover. Specific mRNAs for metalloproteinases of the stromelysin family are expressed during endometrial growth and menstrual breakdown but are absent in the progestin-dominated secretory phase. This expression pattern suggests involvement of stromelysins in remodeling the extracellular matrix of the endometrium during tissue growth and breakdown and implicates progesterone in the suppression of these enzymes. We examined the regulation of endometrial stromelysins in explant cultures and found no acute effect of estradiol on their expression, whereas progesterone was a potent inhibitor of stromelysin expression. Progesterone also suppressed stromelysin expression in cultures of isolated stromal cells, but epithelial cells were progesterone insensitive. Coculture of recombined stromal and epithelial cells restored steroidal suppression of the epithelial-specific metalloproteinase. Our data confirm that progesterone inhibits endometrial stromelysins and further demonstrate the necessity for a stromal-derived factor(s) as a mediator of steroid suppression of an epithelial metalloproteinase.

Metalloproteinase Expression and Hormonal Regulation During Tissue Remodeling in the Cycling Human Endometrium

Contributions to Nephrology. 1994  |  Pubmed ID: 8004979

Expression and Regulation of Stromelysin and Matrilysin by Growth Factors and Oncogenes

Biochemical Society Transactions. Feb, 1994  |  Pubmed ID: 8206287

Jun and Fos Heterodimerize with ATFa, a Member of the ATF/CREB Family and Modulate Its Transcriptional Activity

Oncogene. Feb, 1994  |  Pubmed ID: 8290251

Three related clones encoding proteins (ATFa1, 2 and 3) with specific ATF/CRE DNA-binding activities have been isolated from HeLa cell cDNA libraries. All three isoforms have weak effects on the basal activity of the adenovirus E2a promoter. We present evidence suggesting that a C-terminal element of the ATFa molecules negatively interferes with the intrinsic activation function of these proteins. We also show that coexpression of ATFa with c-Jun, Jun-B or Jun-D stimulates ATFa-dependent reporter activity, while coexpression of c-Fos has no effect. Deletion analyses indicate that the metal-binding region of ATFa is dispensible for this effect, but that the domain comprising the leucine-zipper region of ATFa is required. Reciprocal co-immunoprecipitation experiments and electrophoretic band-shift assays with in vitro synthesized proteins reveal direct interactions between ATFa and Jun or Fos. The ATFa/c-Jun heterodimers, but not the ATFa/c-Fos complexes, bind efficiently to ATF, CRE or AP1 sites. The detection of ATFa-Jun complexes in crude extracts from HeLa cells transfected with ATFa and c-Jun expression vectors suggests that such ATFa/c-Jun heterodimers also form in vivo. Altogether these results indicate that the ATFa proteins may contribute to the modulation of the activity of the Jun/Fos complexes by altering their DNA-binding and transcriptional properties.

Structure and Expression of the Human Gene for the Matrix Metalloproteinase Matrilysin

The Journal of Biological Chemistry. Jan, 1994  |  Pubmed ID: 8294454

Matrilysin, a member of the matrix metalloproteinase family, is structurally different from the other matrix metalloproteinases by virtue of the absence of a conserved COOH-terminal protein domain. In addition, matrilysin mRNA is regulated in a specific and distinct manner in normal and malignant tissues. Analysis of the genomic structure of the human matrilysin gene revealed that the organization of the first five exons is highly conserved among the different members of the matrix metalloproteinase family, but that matrilysin contains an atypical sixth exon. The promoter region of the matrilysin gene has several features that are conserved among several other matrix metalloproteinase family members, including the presence of TATA, AP-1, and PEA3 elements. Comparison of the expression of the human matrilysin promoter with rat stromelysin promoter/chloramphenicol acetyltransferase constructs in HeLa cells revealed that constructs containing AP-1 and PEA3 elements respond similarly to epidermal growth factor and tumor promoter (12-O-tetradecanoyl-phorbol-13-acetate) induction, but that the addition of upstream stromelysin sequences results in an increased transcriptional activity not observed with upstream matrilysin sequences. The similarities and differences observed between the promoters of matrilysin and the other metalloproteinases may provide insights into the molecular mechanisms that regulate the expression of this family of enzymes as a whole and the factors that distinguish the expression patterns of individual family members.

Protein Kinase C Isotypes Required for Phorbol-ester Induction of Stromelysin-1 in Rat Fibroblasts

Molecular Carcinogenesis. Feb, 1996  |  Pubmed ID: 8599579

The phorbol-ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) is a potent inducer of the metalloproteinase stromelysin in fibroblasts in vivo and in several cultured cell lines. Rat-1 and Rat-2 fibroblasts, however, do not respond to TPA stimulation by induction of stromelysin gene activity, although collagenase promoter-mediated activity is induced threefold by TPA treatment in these cells. We determined that rat fibroblasts expressed protein kinase C(PKC)alpha, PKCdelta, PKCepsilon, and PKCzeta but neither the mRNA nor the protein for PKCbeta. When Rat-2 fibroblasts were stably transfected with an expression vector producing PKCbeta, however, TPA treatment of these variants resulted in a 3.1-fold induction of stromelysin promoter-mediated luciferase activity compared with a 1.3-fold induction in parental Rat-2 cells (P<0.002). Transient transfection of PKCepsilon produced a small but significant increase in TPA-stimulation of both stromelysin- and collagenase-mediated gene expression. These results suggest that there are PKC isotype-specific signaling pathways that can differentially regulate matrix metalloproteinase gene expression.

The Nine C-terminal Residues of the Grapevine Fanleaf Nepovirus Movement Protein Are Critical for Systemic Virus Spread

The Journal of General Virology. Jun, 1999  |  Pubmed ID: 10374950

The grapevine fanleaf virus (GFLV) RNA2-encoded polyprotein P2 is proteolytically cleaved by the RNA1-encoded proteinase to yield protein 2A, 2B(MP) movement protein and 2C(CP) coat protein. To further investigate the role of the 2B(MP) and 2C(CP) proteins in virus movement, RNA2 was engineered by alternatively replacing the GFLV 2B(MP) and 2C(CP) genes with their counterparts from the closely related Arabis mosaic virus (ArMV). Transcripts of all chimeric RNA2s were able to replicate in Chenopodium quinoa protoplasts and form tubules in tobacco BY-2 protoplasts in the presence of the infectious transcript of GFLV RNA1. Virus particles were produced when the GFLV 2C(CP) gene was replaced with its ArMV counterpart, but systemic virus spread did not occur in C. quinoa plants. In addition, chimeric RNA2 containing the complete ArMV 2B(MP) gene was neither encapsidated nor infectious on plants, probably because polyprotein P2 was incompletely processed. However, chimeric RNA2 encoding ArMV 2B(MP), in which the nine C-terminal residues were those of GFLV 2B(MP), formed virus particles and were infectious in the presence of GFLV but not ArMV 2C(CP). These results suggest that the nine C-terminal residues of 2B(MP) must be of the same virus origin as the proteinase for efficient proteolytic processing of polyprotein P2 and from the same virus origin as the 2C(CP) for systemic virus spread.

Protein 2A of Grapevine Fanleaf Nepovirus is Implicated in RNA2 Replication and Colocalizes to the Replication Site

Virology. Nov, 1999  |  Pubmed ID: 10544127

RNA2 of grapevine fanleaf virus is replicated in trans by the RNA1-encoded replication machinery. Full processing of the RNA2-encoded polyprotein P2 yields protein 2A of unknown function, the movement protein 2B(MP), and the coat protein 2C(CP). Analysis of a set of deletion mutants in the P2-coding sequence revealed that protein 2A is necessary but not sufficient for RNA2 replication. In addition to the 5' and 3' noncoding sequences and the 2A-coding sequence, an additional sequence coding for 2B(MP) and/or 2C(CP) or the green fluorescent protein (GFP) is necessary for RNA2 replication. When 2A fused to GFP (2AGFP) was transiently expressed in uninfected T-BY2 protoplasts, 2AGFP appeared as punctate structures evenly distributed in the cytoplasm. However, in cells cotransfected with grapevine fanleaf virus RNAs and the 2AGFP construct, 2AGFP was predominantly found in a juxtanuclear location along with 1D(pro) and 1C(VPg), two RNA1-encoded proteins involved in RNA replication. Viral RNA replication as traced by 5-bromouridine 5' triphosphate (BrUTP) incorporation into newly synthesized RNA occurred at the same location. This colocalization is consistent with the hypothesis that 2A enables RNA2 replication through its association with the replication complex assembled from RNA1-encoded proteins.

Comparison of Two Methods for the Measurement of Rat Liver Methylmalonyl-coenzyme A Mutase Activity: HPLC and Radioisotopic Assays

The Journal of Nutritional Biochemistry. Jan, 1999  |  Pubmed ID: 15539251

Methylmalonyl-coenzyme A mutase (MCM) is a 5'-deoxyadenosylcobalamin-linked mitochondrial enzyme that catalyzes the isomerization of L-methylmalonyl-coenzyme A to succinyl-coenzyme A. In vitro assays of total and holo-MCM activities are important tools for investigating the cobalamin pathway. Several methods have been described for measuring MCM activity. The most commonly-used method is a radioassay based on the permanganate oxidation of DL[CH(3)-(14)C]methylmalonyl-coenzyme A, but radiometric methods are insensitive, laborious, and time-consuming. Therefore, we have compared this method with a nonradiometric assay, potentially most sensitive, based on the separation of methylmalonyl-coenzyme A and succinyl-coenzyme A by high performance liquid chromatography (HPLC). We determined the optimal assay conditions and the reproducibility and sensitivity of each technique. The results obtained by the two techniques were very different: the specific activities obtained by the permanganate oxidation method (0.039 +/- 0.013 nmol/min/mg protein for the holo-MCM activity and 1.90 +/- 0.69 nmol/min/mg protein for the total-MCM activity) were threefold lower than those obtained with the HPLC method (0.124 +/- 0.011 nmol/min/mg protein for the holo-MCM activity and 6.15 +/- 0.76 nmol/min/mg protein for the total-MCM activity). The coefficients of variation for the radiometric method (18.4-40.6%) were three to five times greater than those for the HPLC assay (3.5-12.2%). This demonstrates the lack of sensibility and reproducibility of the permanganate radioassay. Thus, the radiometric method is not suitable for measuring low mutase activities such as the holo activities in tissues. The intrinsic inconvenience of the radiometric assay indicates that the HPLC method is a method of choice for measuring MCM activity.

GFLV Replication in Electroporated Grapevine Protoplasts

Plant Science : an International Journal of Experimental Plant Biology. Jun, 2000  |  Pubmed ID: 10814824

Grapevine fanleaf virus (GFLV), responsible for the economically important court-noué disease, is exclusively transmitted to its natural host in the vineyards through Xiphinema nematodes. We have developed direct inoculation of GFLV into grapevine through protoplast electroporation. Protoplasts were isolated from mesophyll of in vitro-grown plants and from embryogenic cell suspensions. Permeation conditions were determined by monitoring calcein uptake. Low salt poration medium was selected. Electrical conditions leading to strong transient gene expression were also tested for GFLV inoculation (isolate F13). GFLV replication was detected with either virus particles (2 µg) or viral RNA (10 ng) in both protoplast populations, as shown by anti-P38 Western blotting. Direct inoculation and replication were also observed with Arabis mosaic virus (ArMV), a closely related nepovirus, as well as with another GFLV isolate. These results will be valuable in grapevine biotechnology, for GFLV replication studies, transgenic plant screening for GFLV resistance, and biorisk evaluation.

Inhibition of Vitamin B12 Metabolism by OH-cobalamin C-lactam in Rat Oligodendrocytes in Culture: a Model for Studying Neuropathy Due to Vitamin B12 Deficiency

Neuroscience Letters. Jul, 2000  |  Pubmed ID: 10889340

Vitamin B12 is implicated in methylation processes. Myelin basic protein is methylated on one arginine group. A defect in methylation could produce an unstable protein, leading to neurological disorders. In order to study myelin basic protein, we have developed the cultures of newborn rat oligodendrocytes in vitamin B12-depleted medium. As these cells do not grow without serum, vitamin B12 is always present. We overcame this problem by using OH-cobalamin c-lactam, an antagonist of B12. To ensure that the system was vitamin B12 deficient, we measured the concentrations of homocysteine and methylmalonic acid whose accumulations reflect a vitamin B12 deficiency. Methylmalonic acid was measured by mass spectrometry and homocysteine by HPLC. We obtained a powerful model for studying the influence of B12 deficiency on the synthesis of myelin compounds.

Rapid Internalization and Recycling of the Human Neuropeptide Y Y(1) Receptor

The Journal of Biological Chemistry. Feb, 2002  |  Pubmed ID: 11741903

Desensitization of G protein-coupled receptors (GPCRs) involves receptor phosphorylation and reduction in the number of receptors at the cell surface. The neuropeptide Y (NPY) Y(1) receptor undergoes fast desensitization. We examined agonist-induced signaling and internalization using NPY Y(1) receptors fused to green fluorescent protein (EGFP). When expressed in HEK293 cells, EGFP-hNPY Y(1) receptors were localized at the plasma membrane, desensitized rapidly as assessed using calcium responses, and had similar properties compared to hNPY Y(1) receptors. Upon agonist challenge, the EGFP signal decreased rapidly (t(1/2) = 107 +/- 3 s) followed by a slow recovery. This decrease was blocked by BIBP3226, a Y(1) receptor antagonist, or by pertussis toxin, in agreement with Y(1) receptor activation. Internalization of EGFP-hNPY Y(1) receptors to acidic endosomal compartments likely accounts for the decrease in the EGFP signal, being absent after pretreatment with monensin. Concanavalin A and hypertonic sucrose, which inhibit clathrin-mediated endocytosis, blocked the decrease in fluorescence. After agonist, intracellular EGFP signals were punctate and co-localized with transferrin-Texas Red, a marker of clathrin-associated internalization and recycling, but not with LysoTracker Red, a lysosomal pathway marker, supporting receptor trafficking to recycling endosomes rather than the late endosomal/lysosomal pathway. Pulse-chase experiments revealed no receptor degradation after internalization. The slow recovery of fluorescence was unaffected by cycloheximide or actinomycin D, indicating that de novo synthesis of receptors was not limiting. Use of a multicompartment model to fit our fluorescence data allows simultaneous determination of internalization and recycling rate constants. We propose that rapid internalization of receptors via the clathrin-coated pits recycling pathway may largely account for the rapid desensitization of NPY Y(1) receptors.

Calcium Oscillations Trigger Focal Adhesion Disassembly in Human U87 Astrocytoma Cells

The Journal of Biological Chemistry. Jul, 2002  |  Pubmed ID: 12011063

Integrin-associated intracellular Ca(2+) oscillations modulate cell migration, probably by controlling integrin-mediated release of the cell rear during migration. Focal adhesion kinase (FAK), via its tyrosine phosphorylation activity, plays a key role in integrin signaling. In human U87 astrocytoma cells, expression of the dominant negative FAK-related non-kinase domain (FRNK) inhibits the Ca(2+)-sensitive component of serum-dependent migration. We investigated how integrin-associated Ca(2+) signaling might be coupled to focal adhesion (FA) dynamics by visualizing the effects of Ca(2+) spikes on FAs using green fluorescent protein (GFP)-tagged FAK and FRNK. We report that Ca(2+) spikes are temporally correlated with movement and disassembly of FAs, but not their formation. FRNK transfection did not affect generation of Ca(2+) spikes, although cell morphology was altered, with fewer FAs of larger size and having a more peripheral localization being observed. Larger sized FAs in FRNK-transfected cells were not disassembled by Ca(2+) spikes, providing a possible explanation for impaired Ca(2+)-dependent migration in these cells. Stress fiber end movements initiated by Ca(2+) spikes were visualized using GFP-tagged myosin light chain kinase (MLCK). Ca(2+)-associated movements of stress fiber ends and FAs had similar kinetics, suggesting that stress fibers and FAs move in a coordinated fashion. This indicates that increases in Ca(2+) likely trigger disassembly of adhesive structures that involves disruption of integrin-extracellular matrix interactions, supporting a key role for Ca(2+)-sensitive inside-out signaling in cell migration. A rapid increase in tyrosine phosphorylation of FAK was found in response to an elevation in Ca(2+) induced by thapsigargin, and we propose that this represents the initial triggering event linking Ca(2+) signaling and FA dynamics to cell motility.

Grapevine Fanleaf Virus Replication Occurs on Endoplasmic Reticulum-derived Membranes

Journal of Virology. Sep, 2002  |  Pubmed ID: 12163601

Infection by Grapevine fanleaf nepovirus (GFLV), a bipartite RNA virus of positive polarity belonging to the Comoviridae family, causes extensive cytopathic modifications of the host endomembrane system that eventually culminate in the formation of a perinuclear "viral compartment." We identified by immunoconfocal microscopy this compartment as the site of virus replication since it contained the RNA1-encoded proteins necessary for replication, newly synthesized viral RNA, and double-stranded replicative forms. In addition, by using transgenic T-BY2 protoplasts expressing green fluorescent protein in the endoplasmic reticulum (ER) or in the Golgi apparatus (GA), we could directly show that GFLV replication induced a depletion of the cortical ER, together with a condensation and redistribution of ER-derived membranes, to generate the viral compartment. Brefeldin A, a drug known to inhibit vesicle trafficking between the GA and the ER, was found to inhibit GFLV replication. Cerulenin, a drug inhibiting de novo synthesis of phospholipids, also inhibited GFLV replication. These observations imply that GFLV replication depends both on ER-derived membrane recruitment and on de novo lipid synthesis. In contrast to proteins involved in viral replication, the 2B movement protein and, to a lesser extent, the 2C coat protein were not confined to the viral compartment but were transported toward the cell periphery, a finding consistent with their role in cell-to-cell movement of virus particles.

Calcium Rises Locally Trigger Focal Adhesion Disassembly and Enhance Residency of Focal Adhesion Kinase at Focal Adhesions

The Journal of Biological Chemistry. Jul, 2004  |  Pubmed ID: 15102844

Focal adhesion kinase (FAK) activity and Ca(2+) signaling led to a turnover of focal adhesions (FAs) required for cell spreading and migration. We used yellow Cameleon-2 (Ycam), a fluorescent protein-based Ca(2+) sensor fused to FAK or to a FAK-related non-kinase domain, to measure simultaneously local Ca(2+) variations at FA sites and FA dynamics. Discrete subcellular Ca(2+) oscillators initiate both propagating and abortive Ca(2+) waves in migrating U87 astrocytoma cells. Ca(2+)-dependent FA disassembly occurs when the Ca(2+) wave reaches individual FAs, indicating that local but not global Ca(2+) increases trigger FA disassembly. An unexpectedly rapid flux of FAK between cytosolic and FA compartments was revealed by fluorescence recovery after photobleaching studies. The FAK-Ycam recovery half-time (17 s) at FAs was slowed (to 29 s) by Ca(2+) elevation. FAK-related non-kinase domain-Ycam had a faster, Ca(2+)-insensitive recovery half-time (11 s), which is consistent with the effect of Ca(2+) on FAK-Ycam dynamics not being due to a general modification of the dynamics of FA components. Because FAK association at FAs was prolonged by Ca(2+) and FAK autophosphorylation was correlated to intracellular Ca(2+) levels, we propose that local Ca(2+) elevations increase the residency of FAK at FAs, possibly by means of tyrosine phosphorylation of FAK, thereby leading to increased activation of its effectors involved in FA disassembly.

Mechanical Testing of Isolated Amorphous Silicon Slanted Nanorods

Journal of Nanoscience and Nanotechnology. Nov, 2005  |  Pubmed ID: 16433427

Mechanical testing was performed on a new class of nanostructures-amorphous Si slanted nanorods of rectangular cross section, fixed at one end to the substrate. These nanorods were grown spatially well separated on nano-pillars under the oblique angle physical vapor deposition technique. Various samples with different dimensions and inclination angles were tested in bending using an atomic force microscope. The material response was elastic up to large stresses/deflections. The Young's modulus was calculated from the slope of the experimentally observed stiffness versus the geometrical factor common to all the samples and was found to be (94.14 +/- 10.21) GPa. No size effect of this parameter was observed within the accuracy of the present measurement.

Differential Growth Inhibitory Effects of W. Somnifera Root and E. Officinalis Fruits on CHO Cells

Phytotherapy Research : PTR. May, 2007  |  Pubmed ID: 17357174

The Chinese Hamster ovary (CHO) cell line is widely used for measuring drug cytotoxicity and resistance. Therefore, the effects of two major Ayurvedic drugs (W. somnifera root and E. officinalis fruits) on the short and long-term growth of these cells were investigated. A standard 96-well plate assay was used to measure short-term growth. For assessment of long-term growth, the colony formation assay (CFA) was used, which measures clonogenic potential. This assay is the best measure of the cytotoxicity of anticancer drugs and the radio-sensitivity of tumor cells. As reported by others, the aqueous extracts of both herbal drugs were found to have short-term growth inhibitory effects on CHO cells when added to cells at the time of cell plating. However, this is the first report showing that these two herbal drugs have significantly different effects on the long-term growth of CHO cells. Thus, extracts of W. somnifera root, but not E. officinalis fruit, caused a reproducible, dose dependent, inhibition of colony formation of CHO cells.

Determining the Absolute Efficiency of a Delay Line Microchannel-plate Detector Using Molecular Dissociation

The Review of Scientific Instruments. Feb, 2007  |  Pubmed ID: 17578132

We present a method to measure the absolute detection efficiency of a delay-line microchannel-plate detector using the breakup of diatomic molecular ions. This method provides the absolute total detection efficiency, as well as the individual efficiency for each signal of the detector. The method is based on the fact that molecular breakup always yields two hits on the detector, but due to finite detection efficiency some of these events are recorded as single particles while others are detected in pairs. We demonstrate the method by evaluating the detection efficiency for both timing and position signals of a delay-line detector using laser-induced dissociation of molecular ions. In addition, the detection efficiency as a function of position has been determined by dividing the detector into sectors.

Hyaluronidase and Collagenase Inhibitory Activities of the Herbal Formulation Triphala Guggulu

Journal of Biosciences. Jun, 2007  |  Pubmed ID: 17762148

Myrrh (guggulu) oleoresin from the Commiphora mukul tree is an important component of antiarthritic drugs in Ayurvedic medicine. Clinical data suggest that elevated levels of hyaluronidase and collagenase type 2 enzymes contribute significantly to cartilage degradation. Triphala guggulu (TG) is a guggulu-based formulation used for the treatment of arthritis. We assessed the chondroprotective potential of TG by examining its effects on the activities of pure hyaluronidase and collagenase type 2 enzymes. Triphala shodith guggulu (TSG), an intermediate in the production of TG, was also examined. A spectrophotometric method was used to assay Hyaluronidase activity, and to detect potential Hyaluronidase inhibitors. Aqueous and hydro-alcoholic extracts of TSG showed weak but dose-dependent inhibition of hyaluronidase activity. In contrast, the TG formulation was 50 times more potent than the TSG extract with respect to hyaluronidase inhibitory activity. A validated X-ray film-based assay was used to measure the gelatinase activity of pure collagenase type 2. Hydro-alcoholic extracts of the TG formulation were 4 times more potent than TSG with respect to collagenase inhibitory activity. Components of Triphala were also evaluated for their inhibitory activities on hyaluronidase and collagenase. This is the first report to show that the T2 component of Triphala (T.chebula) is a highly potent hyaluronidase and collagenase inhibitor. Thus, the TG formulation inhibits two major enzymes that can degrade cartilage matrix. Our study provides the first in vitro preclinical evidence of the chondroprotective properties of TG.

Distinct Motifs of Neuropeptide Y Receptors Differentially Regulate Trafficking and Desensitization

Traffic (Copenhagen, Denmark). Mar, 2008  |  Pubmed ID: 18088318

Activated human neuropeptide Y Y(1) receptors rapidly desensitize and internalize through clathrin-coated pits and recycle from early and recycling endosomes, unlike Y(2) receptors that neither internalize nor desensitize. To identify motifs implicated in Y(1) receptor desensitization and trafficking, mutants with varying C-terminal truncations or a substituted Y(2) C-terminus were constructed. Point mutations of key putative residues were made in a C-terminal conserved motif [phi-H-(S/T)-(E/D)-V-(S/T)-X-T] that we have identified and in the second intracellular i2 loop. Receptors were analyzed by functional assays, spectrofluorimetric measurements on living cells, flow cytometry, confocal imaging and bioluminescence resonance energy transfer assays for beta-arrestin activation and adaptor protein (AP-2) complex recruitment. Inhibitory GTP-binding protein-dependent signaling of Y(1) receptors to adenylyl cyclase and desensitization was unaffected by C-terminal truncations or mutations, while C-terminal deletion mutants of 42 and 61 amino acids no longer internalized. Substitutions of Thr357, Asp358, Ser360 and Thr362 by Ala in the C-terminus abolished both internalization and beta-arrestin activation but not desensitization. A Pro145 substitution by His in an i2 consensus motif reported to mediate phosphorylation-independent recruitment of beta-arrestins affected neither desensitization, internalization or recycling kinetics of activated Y(1) receptors nor beta-arrestin activation. Interestingly, combining Pro145 substitution by His and C-terminal substitutions significantly attenuates Y(1) desensitization. In the Y(2) receptor, replacement of His155 with Pro at this position in the i2 loop motif promotes agonist-mediated desensitization, beta-arrestin activation, internalization and recycling. Overall, our results indicate that beta-arrestin-mediated desensitization and internalization of Y(1) and Y(2) receptors are differentially regulated by the C-terminal motif and the i2 loop consensus motif.

Enhancing High-order Above-threshold Dissociation of H2+ Beams with Few-cycle Laser Pulses

Physical Review Letters. Apr, 2008  |  Pubmed ID: 18517942

High-order (three-photon or more) above-threshold dissociation (ATD) of H(2)(+) has generally not been observed using 800 nm light. We demonstrate a strong enhancement of its probability using intense 7 fs laser pulses interacting with beams of H(2)(+), HD(+), and D(2)(+) ions. The mechanism invokes a dynamic control of the dissociation pathway. These measurements are supported by theory that additionally reveals, for the first time, an unexpectedly large contribution to ATD from highly excited electronic states.

Development of Automated Brightfield Double in Situ Hybridization (BDISH) Application for HER2 Gene and Chromosome 17 Centromere (CEN 17) for Breast Carcinomas and an Assay Performance Comparison to Manual Dual Color HER2 Fluorescence in Situ Hybridization (FISH)

Diagnostic Pathology. 2008  |  Pubmed ID: 18945356

Human epidermal growth factor receptor 2 (HER2) fluorescence in situ hybridization (FISH) is a quantitative assay for selecting breast cancer patients for trastuzumab therapy. However, current HER2 FISH procedures are labor intensive, manual methods that require skilled technologists and specialized fluorescence microscopy. Furthermore, FISH slides cannot be archived for long term storage and review. Our objective was to develop an automated brightfield double in situ hybridization (BDISH) application for HER2 gene and chromosome 17 centromere (CEN 17) and test the assay performance with dual color HER2 FISH evaluated breast carcinomas.

Benchmark Measurements of H(3)(+) Nonlinear Dynamics in Intense Ultrashort Laser Pulses

Physical Review Letters. Sep, 2009  |  Pubmed ID: 19792303

The H(3)(+) ion is the simplest polyatomic molecule and is destined to play a central role in understanding such molecules in intense ultrashort laser pulses. We present the first measurements of the intense field dissociation and ionization of D(3)(+) using coincidence three-dimensional momentum imaging. Our results show features that are a consequence of this molecule's unique equilateral triangular geometry, providing a fundamentally new system for theoretical development.

Suppressed Dissociation of H(2)(+) Vibrational States by Reduced Dipole Coupling

Physical Review Letters. Sep, 2009  |  Pubmed ID: 19792305

The suppression of H(2)(+) strong-field dissociation has intrigued experimentalists and theorists since the early days of laser-molecular science. We unravel a vibrational suppression effect due to weak dipole-matrix element coupling strengths of certain vibrational states, dependent on the laser frequency-a form of Cooper minima. This effect is demonstrated by our full-dimensional calculations on H(2)(+) dissociation and persists for a broad range of laser conditions including both weak and strong-field dissociation. Using a crossed-beams coincidence, three-dimensional momentum-imaging technique, the vibrational suppression effect is clearly observed for H(2)(+) and HD(+) at 790 and 395 nm, in good agreement with our theory.

MIRAGAA--a Methodology for Finding Coordinated Effects of MicroRNA Expression Changes and Genome Aberrations in Cancer

Bioinformatics (Oxford, England). Jan, 2010  |  Pubmed ID: 19933823

Cancer evolves through microevolution where random lesions that provide the biggest advantage to cancer stand out in their frequent occurrence in multiple samples. At the same time, a gene function can be changed by aberration of the corresponding gene or modification of microRNA (miRNA) expression, which attenuates the gene. In a large number of cancer samples, these two mechanisms might be distributed in a coordinated and almost mutually exclusive manner. Understanding this coordination may assist in identifying changes which significantly produce the same functional impact on cancer phenotype, and further identify genes that are universally required for cancer. Present methodologies for finding aberrations usually analyze single datasets, which cannot identify such pairs of coordinating genes and miRNAs.

Silver in Situ Hybridization (SISH) for Determination of HER2 Gene Status in Breast Carcinoma: Comparison with FISH and Assessment of Interobserver Reproducibility

The American Journal of Surgical Pathology. Jun, 2010  |  Pubmed ID: 20421783

The importance of HER2 status in breast cancer management has focused attention on the ability of clinical assays to correctly assign HER2 amplification status. There is no consensus as to the best method for assessing HER2 status. Disadvantages of fluorescence in situ hybridization (FISH) testing include longer time required for staining and scoring slides, requirements for specialized training and fluorescence microscopy, and loss of the signal due to quenching of the fluorescent dye. Silver-enhanced in situ hybridization (SISH) is a rapid fully automated assay providing permanently stained slides that are interpreted by conventional bright field microscopy which enables pathologists to evaluate slides within the context of tissue morphology. This study evaluates the concordance between SISH and FISH assays in determining the status of HER2 gene amplification in a cohort of 298 primary invasive breast carcinomas. Furthermore, we assessed in detail the variables contributing to interobserver interpretive reproducibility of HER2 SISH among 10 pathologists. HER2 was quantified using the ratio of HER2 to CHR17 signals using the conventional historical interpretation scale and also by the American Society of Clinical Oncology/College of American Pathologists reporting scheme. For SISH status determined by consensus among 10 pathologists, overall concordance between SISH and FISH was identified in 288 of 298 cases (96.6%) using the conventional Food and Drug Administration approved criteria. Overall agreement was observed in 282 of 285 cases (98.9%) using the American Society of Clinical Oncology/College of American Pathologists result reporting scheme (with equivocal cases removed). In conclusion, SISH represents a novel approach for the determination of HER2 status in breast cancer. The overall concordance between SISH and FISH is excellent, and the interpretation of SISH results by pathologists is most reproducible using the HER2/CHR17 ratio.

Texture Evolution of Vertically Aligned Biaxial Tungsten Nanorods Using RHEED Surface Pole Figure Technique

Nanotechnology. Aug, 2010  |  Pubmed ID: 20639581

Vertically aligned biaxial tungsten nanorods with cubic A15 crystal structure were deposited by DC magnetron sputtering on native oxide covered Si(100) substrates with glancing angle flux incidence (theta approximately 85 degrees) and a two-step substrate rotation mode at room temperature. These vertical nanorods were grown to different thicknesses (10, 25, 50 and 100 nm) and analyzed for biaxial texture evolution using a highly surface sensitive reflection high-energy electron diffraction (RHEED) pole figure technique. The initial polycrystalline film begins to show the inception of biaxial texture with a fiber background between 10 and 25 nm. Biaxial texture development is eventually completed between 50 and 100 nm thicknesses of the film. The out-of-plane crystallographic direction is [002] and the in-plane texture is selected so as to obtain maximum capture area. In a comparison with 100 nm thick inclined tungsten nanorods deposited at 85 degrees without substrate rotation, it is found that the selection of in-plane texture does not maintain maximum in-plane capture area. This anomalous behavior is observed when the [002] texture axis is tilted approximately 17 degrees from the substrate normal in the direction towards the glancing incident flux.

Morphology and Texture Evolution of Nanostructured CaF2 Films on Amorphous Substrates Under Oblique Incidence Flux

Nanotechnology. Nov, 2010  |  Pubmed ID: 20921590

The morphology and biaxial texture of vacuum evaporated CaF(2) films on amorphous substrates as a function of vapour incident angle, substrate temperature and film thickness were investigated by scanning electron microscopy, x-ray pole figure and reflection high energy electron diffraction surface pole figure analyses. Results show that an anomalous [220] out-of-plane texture was preferred in CaF(2) films deposited on Si substrates at < 200 °C with normal vapour incidence. With an increase of the vapour incident angle, the out-of-plane orientation changed from [220] to [111] at a substrate temperature of 100 °C. In films deposited with normal vapour incidence, the out-of-plane orientation changed from [220] at 100 °C to [111] at 400 °C. In films deposited with an oblique vapour incidence at 100 °C, the texture changed from random at small thickness (5 nm) to biaxial at larger thickness (20 nm or more). Using first principles density functional theory calculation, it was shown that [220] texture formation is a consequence of energetically favourable adsorption of CaF(2) molecules onto the CaF(2)(110) facet.

Tunable Bandgap in Graphene by the Controlled Adsorption of Water Molecules

Small (Weinheim an Der Bergstrasse, Germany). Nov, 2010  |  Pubmed ID: 20963796

Increased Insulin-like Growth Factor 1 Receptor Protein Expression and Gene Copy Number in Small Cell Lung Cancer

Journal of Thoracic Oncology : Official Publication of the International Association for the Study of Lung Cancer. Dec, 2010  |  Pubmed ID: 21124078

Identification of new therapies in small cell lung cancer (SCLC) is urgently needed. Insulin-like growth factor 1 receptor (IGF1R) is a tyrosine kinase receptor implicated in the pathogenesis of several malignancies and is potentially an attractive target for anticancer treatment. Knowledge about IGF1R protein expression, gene copy number, and the prognostic relevance of these features in SCLC is limited.

EGFR Protein Expression in Non-small Cell Lung Cancer Predicts Response to an EGFR Tyrosine Kinase Inhibitor--a Novel Antibody for Immunohistochemistry or AQUA Technology

Clinical Cancer Research : an Official Journal of the American Association for Cancer Research. Dec, 2011  |  Pubmed ID: 21994417

Epidermal growth factor receptor (EGFR) protein expression in non-small cell lung cancer (NSCLC) is not recommended for predicting response to EGFR tyrosine kinase inhibitors (TKI) due to conflicting results, all using antibodies detecting EGFR external domain (ED). We tested the predictive value of EGFR protein expression for response to an EGFR TKI with an antibody that detects the intracellular domain (ID) and compared fluorescence-based Automated QUantitative Analysis (AQUA) technology to immunohistochemistry (IHC).

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