Search PubMed⌕ Search

Biomedical subjects

S Roy

Publications and source records attributed to S Roy.

At least 199 records · Page 11Linked to original sources
PubMed2000

Phosphospecific antibodies reveal focal adhesion kinase activation loop phosphorylation in nascent and mature focal adhesions and requirement for the autophosphorylation site.

Focal adhesion kinase (FAK) is a key signaling molecule regulating cellular responses to integrin-mediated adhesion. Integrin engagement promotes FAK phosphorylation at multiple sites to achieve full FAK activation. Phosphorylation of FAK Tyr-397 creates a binding site for Src-family kinases, and phosphorylation of FAK Tyr-576/Tyr-577 in the kinase domain activation loop enhances catalytic activity. Using novel phosphospecific antibody reagents, we show that FAK activation loop phosphorylation is significantly elevated in cells expressing activated Src and is an early event following cell adhesion to fibronectin. In both cases, this regulation is largely dependent on Tyr-397. We also show that the FAK activation loop tyrosines are required for maximal Tyr-397 phosphorylation. Finally, immunostaining analyses revealed that tyrosine-phosphorylated forms of FAK are present in both newly forming and mature focal adhesions. Our findings support a model for reciprocal activation of FAK and Src-family kinases and suggest that FAK/Src signaling may occur during both focal adhesion assembly and turnover.

3T3 Cells↗
PubMed2000

Metal-catalyzed oxidation of brain-derived neurotrophic factor (BDNF): selectivity and conformational consequences of histidine modification.

We have studied the metal-catalyzed oxidation (MCO) of brain-derived neurotrophic factor (BDNF) with regard to target sites and potential conformational changes of the protein. The exposure of BDNF to three different levels of ascorbate/Cu(II)/O2 [20 microM Cu(II), 2 mM ascorbate (level 1); 20 microM Cu(II), 4 mM ascorbate (level 2); 40 microM Cu(II), 4 mM ascorbate (level 3)], chosen based on the extent of chemical modification of Met and His, respectively, resulted in the exclusive oxidation of a buried Met residue, Met92, at level 1 but in the predominant oxidation of His at level 3. His modification had a significant impact on the structure of BDNF, as quantified by CD and ANSA fluorescence measurements, while Met oxidation had not, also assessed through complementary oxidation of BDNF through hydrogen peroxide. Our ultimate objective was the correlation of the surface exposure of an oxidized His residue in a protein with potential effects on the conformational integrity of the oxidized protein. In a series of three proteins, human growth hormone (hGH), human relaxin (hR1x), and BDNF, we have now observed that His oxidation is paralleled by significant conformational changes when the target His residue is more surface exposed (hR1x, BDNF) while conformational consequences of His modification are less significant when the target His residues are more buried in the interior of the protein (hGH).

Amino Acid Sequence↗
PubMed2000

Air pollution in Calcutta elicits adverse pulmonary reaction in children.

BACKGROUND & OBJECTIVE: Pulmonary responses of children chronically exposed to ambient air pollution in Calcutta have been investigated. METHODS: A total number of 153 children from Calcutta and 116 from rural West Bengal in the age group of 6-17 yr were included in this study. Respiratory symptom complex, sputum cytology and micronucleus (MN) count of buccal epithelial cells were evaluated. Blood smears were examined for WBC differential count and RBC morphology. RESULTS: Marked rise in respiratory symptoms (43% in urban vs 14% in rural) and sputum alveolar macrophage (AM) number was observed in urban children compared to their rural counterparts (14.2 +/- 1.4 AM/hpf vs 6.7 +/- 1.4 AM/hpf, mean +/- SE, P < 0.001). The urban group also demonstrated increased numbers of neutrophils, eosinophils and iron-laden AM in their sputum. Besides, buccal epithelial cells of urban children exhibited higher MN frequency than their rural counterparts (0.22 vs 0.17%, P < 0.05). While sputum neutrophilia and eosinophilia suggest inflammatory and allergic lung reactions, elevated MN count is indicative of greater genotoxic effect on the exposed tissues of urban children. Hypochromic red cells in peripheral blood smear was a common finding in both urban and rural groups, but eosinophils and monocytes were present in elevated frequencies in the rural children. INTERPRETATION & CONCLUSION: The study demonstrated that children inhaling grossly polluted air of Calcutta suffer from adverse lung reactions and genetic abnormality in the exposed tissues.

Adolescent↗
PubMed1999

Control of muscle cell-type specification in the zebrafish embryo by Hedgehog signalling.

The specification of different muscle cell types in the zebrafish embryo requires signals that emanate from the axial mesoderm. In previous studies we and others have shown that overexpression of different members of the Hedgehog protein family can induce the differentiation of two types of slow-twitch muscles, the superficially located slow-twitch fibres and the medially located muscle pioneer cells. Here we have investigated the requirement for Hedgehog signalling in the specification of these distinct muscle cell types in two ways: first, by characterising the effects on target gene expression and muscle cell differentiation of the u-type mutants, members of a phenotypic group previously implicated in Hedgehog signalling, and second, by analysing the effects of overexpression of the Patched1 protein, a negative regulator of Hedgehog signalling. Our results support the idea that most u-type genes are required for Hedgehog signalling and indicate that while such signalling is essential for slow myocyte differentiation, the loss of activity of one signal, Sonic hedgehog, can be partially compensated for by other Hedgehog family proteins.

Animals↗
PubMed1999

Adenoviral vector-mediated expression of physiologic levels of human factor VIII in nonhuman primates.

An E1-, E2a-, E3-deleted adenoviral vector (Av3H82) encoding an epitope-tagged B domain-deleted human factor VIII cDNA (flagged FVIII) was evaluated in nonhuman primates. Twelve cynomolgus monkeys received intravenous administration of Av3H82; 6 monkeys received 6 x 10(11) particles/kg and another 6 received 3 x 10(12) particles/kg. Adenoviral vector transduction of the liver was efficient, reproducible, and linearly dose dependent. Physiologic levels of flagged FVIII were readily detected in plasma samples obtained from monkeys that received the higher dose of vector and human FVIII mRNA was detected in their livers. Expression of transgene mRNA was restricted to the liver by the albumin promoter. Although vector DNA was readily detected in the liver of monkeys that received the lower dose, neither human FVIII mRNA nor flagged FVIII protein could be detected. Vector distribution was widespread, with the highest levels observed in liver and spleen. Histopathology, hematology, and serum chemistry analysis identified the liver and blood as major sites of toxicity. Transient mild serum elevations of liver enzymes were observed, along with a dose-dependent inflammatory response in the liver. In addition, mild lymphoid hyperplasia was observed in the spleen. Mild anemia and a transient decrease in platelet count were observed, as was marrow hyperplasia and extramedullary hematopoiesis.

Adenoviridae↗
PubMed1999

Multifunctional enzymes and evolution of biosynthetic pathways: retro-evolution by jumps.

A likely scenario of evolution of biosynthetic pathways is believed to have occurred by retro-evolution through recruitment of existing enzymes rather than generation of de novo classes. It had been proposed that such retro-evolution occurred in steps as a response to depletion of an essential metabolite and availability of another related substance in the environment. In this article, I argue that because of instability of many such extant intermediates, it is unlikely that retro-evolution had occurred in steps. I further propose that such evolution in many cases has taken place by jumps, i.e., by recruitment of a multifunctional enzyme capable of catalyzing several steps at a time, albeit inefficiently. I further speculate that in some cases one primordial multienzyme may have catalyzed the whole sequence of reaction of a biosynthetic pathway, i.e., the pathway may have evolved by a single leap. Gene duplications and further evolution to more efficient enzymes led to extant pathways. Such a mechanism predicts that some or all enzymes of a pathway must have descended from a common ancestor. Sequence and structural homologies among extant enzymes of a biosynthetic pathway have been examined.

Amino Acids↗
PubMed1999

Interactions of c-Raf-1 with phosphatidylserine and 14-3-3.

Activation of Raf-1 occurs at the plasma membrane. We recently showed that 14-3-3 must be complexed with Raf-1 for efficient recruitment to the plasma membrane and activation by Ras, but that 14-3-3 is completely displaced from Raf-1 following plasma membrane binding. We show here that the Raf-1 zinc finger is not absolutely required for 14-3-3 binding but is required to stabilize the interaction between Raf-1 and 14-3-3. Incubation of Raf-1 with phosphatidylserine, an inner plasma membrane phospholipid, results in removal of 14-3-3 and an increase in Raf-1 kinase activity, whereas removal of 14-3-3 from Raf-1 using specific phosphopeptides substantially reduces Raf-1 basal kinase activity. Displacement of 14-3-3 from activated Raf-1 by phosphopeptides has no effect on kinase activity if Raf-1 is first removed from solution, but completely eradicates kinase activity of soluble activated Raf-1. These results suggest a mechanism for the removal of 14-3-3 from Raf-1 at the plasma membrane and show that removal of 14-3-3 from Raf-1 has markedly different effects depending on experimental conditions.

14-3-3 Proteins↗
PubMed1999

GalR mutants defective in repressosome formation.

Transcription repression of the galactose operon of Escherichia coli requires (1) the binding of the GalR repressor to tandem operators flanking the promoters, (2) the binding of histone-like protein, HU, to a site between the GalR-binding sites, and (3) negatively supercoiled DNA. Under these conditions, protein-protein interactions mediate the formation of a nucleoprotein complex in the form of a DNA loop, which we have termed a repressosome. To analyze the structure of the repressosome, we have screened and isolated galR mutants in which single amino acid substitutions in GalR lead to defects in loop formation while the protein's operator-binding activity is retained. The mutant proteins were purified and their properties confirmed in vitro. We verified that in the case of the two stronger mutations, the proteins had secondary structures that were identical to that of wild-type GalR as reflected by circular dichroism spectroscopy. Homology-based modeling of GalR by use of the crystal structures of PurR and LacI has enabled us to place the three sites of mutation in a structural context. They occur in the carboxy-terminal subdomain of the GalR core, are surface exposed, and, therefore, may be involved in protein-protein interactions. On the basis of our model of GalR and its structural alignment with LacI and PurR, we have identified additional residues, the substitution of which leads to a specific defect in repression by looping. The effects of the mutations are the same in the presence of HMG-17, a eukaryotic protein unrelated to HU, which can also mediate GalR-dependent repression of the gal promoter. This observation suggests that the mutations define sites of GalR-GalR interaction rather than HU-GalR interaction in the repressosome.

Bacteriophages↗
PubMed1999

Involvement of caspases in proteolytic cleavage of Alzheimer's amyloid-beta precursor protein and amyloidogenic A beta peptide formation.

The amyloid-beta precursor protein (APP) is directly and efficiently cleaved by caspases during apoptosis, resulting in elevated amyloid-beta (A beta) peptide formation. The predominant site of caspase-mediated proteolysis is within the cytoplasmic tail of APP, and cleavage at this site occurs in hippocampal neurons in vivo following acute excitotoxic or ischemic brain injury. Caspase-3 is the predominant caspase involved in APP cleavage, consistent with its marked elevation in dying neurons of Alzheimer's disease brains and colocalization of its APP cleavage product with A beta in senile plaques. Caspases thus appear to play a dual role in proteolytic processing of APP and the resulting propensity for A beta peptide formation, as well as in the ultimate apoptotic death of neurons in Alzheimer's disease.

Acute Disease↗
PubMed1999

Hsp60 accelerates the maturation of pro-caspase-3 by upstream activator proteases during apoptosis.

The activation of caspases represents a critical step in the pathways leading to the biochemical and morphological changes that underlie apoptosis. Multiple pathways leading to caspase activation appear to exist and vary depending on the death-inducing stimulus. We demonstrate that the activation of caspase-3, in Jurkat cells stimulated to undergo apoptosis by a Fas-independent pathway, is catalyzed by caspase-6. Caspase-6 was found to co-purify with caspase-3 as part of a multiprotein activation complex from extracts of camptothecin-treated Jurkat cells. A biochemical analysis of the protein constituents of the activation complex showed that Hsp60 was also present. Furthermore, an interaction between Hsp60 and caspase-3 could be demonstrated by co-immunoprecipitation experiments using HeLa as well as Jurkat cell extracts. Using a reconstituted in vitro system, Hsp60 was able to substantially accelerate the maturation of procaspase-3 by different upstream activator caspases and this effect was dependent on ATP hydrolysis. We propose that the ATP-dependent 'foldase' activity of Hsp60 improves the vulnerability of pro-caspase-3 to proteolytic maturation by upstream caspases and that this represents an important regulatory event in apoptotic cell death.

Amino Acid Sequence↗
PubMed1999

Effect of hormone replacement therapy on left ventricular hypertrophy.

The mechanisms behind the reduction in cardiovascular morbidity and mortality in postmenopausal women receiving hormone replacement therapy are not fully understood. In this case-control study, we report a statistically significant lower left ventricular mass in a group of elderly women who were receiving hormone replacement therapy for >10 years than in age-matched controls.

Aged↗