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Biomedical subjects

V Srinivas

Publications and source records attributed to V Srinivas.

At least 19 recordsLinked to original sources
PubMed2007

Expression of HIF prolyl hydroxylase isozymes in growth plate chondrocytes: relationship between maturation and apoptotic sensitivity.

The overall goal of the current study was to examine the functional activity of the prolyl hydroxylases (PHDs) in maturing chondrocytes. Herein, we show for the first time that the PHDs are expressed in the maturing zone of the growth plate, and by a chondrocytic cell line. We determined if this protein and its substrate, hypoxia inducible factor (HIF)-1alpha, modulated the induction of apoptosis. Using a chondrocyte cell line that matured in culture, we inhibited HIF-1alpha expression using siRNA technology and pharmacologically blocked PHD activity. We noted that PHD suppression sensitized the cells to an apoptotic challenge with H(2)O(2). We next examined the interplay between the PHDs and HIF-1alpha by suppressing HIF-1alpha and blocking PHD activity. We noted reduced killing when the mature HIF-silenced cells were challenged with H(2)O(2). In contrast, there was limited change in the viability of immature cells. Based on these differences in chondrocyte susceptibility, it is concluded that HIF-1alpha sensitizes maturing cells to H(2)O(2)-mediated killing. We next determined if this change in the viability of the PHD-inhibited cells was linked to changes in activation of caspase-3. It was noted that there was a minimal change in enzyme activity of the PHD-inhibited HIF-1alpha suppressed cells. Finally, we found that as the chondrocytes mature, the activities of catalase and SOD were significantly reduced and that there was a decrease in the levels of Bcl-2 and Bcl(XL). This loss of protective activity together with the changes mediated by HIF would be expected to generate conditions that would favor the induction of chondrocyte apoptosis.

Amino Acids, Dicarboxylic↗
PubMed2006

Active caspase-3 is required for osteoclast differentiation.

Based on our earlier observation that caspase-3 is present in osteoclasts that are not undergoing apoptosis, we investigated the role of this protein in the differentiation of primary osteoclasts and RAW264.7 cells (Szymczyk KH, et al., 2005, Caspase-3 activity is necessary for RANKL-induced osteoclast differentiation. The Proceedings of the 8th ICCBMT). We noted that osteoclast numbers are decreased in long bones of procaspase-3 knockout mice and that receptor activator of NF-kappaB ligand (RANKL) does not promote differentiation of isolated preosteoclasts. In addition, after treatment with inhibitors of caspase-3 activity, neither the wild-type primary nor the RAW264.7 cells express TRAP or became multinucleated. We found that immediately following RANKL treatment, procaspase-3 is cleaved and the activated protein is localized to lipid regions of the plasma membrane and the cytosol. We developed RAW264.7 procaspase-3 knockdown clonal cell lines using RNAi technology. Again, treatment with RANKL fails to induce TRAP activity or multinucleation. Finally, we evaluated NF-kappaB in procaspase-3 silenced cells. We found that RANKL treatment prevented activation and nuclear translocation of NF-kappaB. Together these findings provide direct support for the hypothesis that caspase-3 activity is required for osteoclast differentiation.

Animals↗
PubMed2005

Arginine hydrochloride enhances the dynamics of subunit assembly and the chaperone-like activity of alpha-crystallin.

PURPOSE: Alpha-crystallin, a major eye lens protein, bears homology with small heat shock proteins (sHsps) and exhibits molecular chaperone-like activity. Structural perturbation by temperature or low concentrations of denaturants leads to enhancement of its chaperone-like activity. We have earlier demonstrated similar enhancement of chaperone-like activity using biologically compatible solutes such as arginine hydrochloride and aminoguanidine. The purpose of the present study is to get an insight into the mechanism of the arginine induced enhancement of chaperone-like activity of alpha-crystallin. METHODS: The effect of arginine hydrochloride on the chaperone-like activity of alpha-crystallin at 25 degrees C was studied using DTT induced aggregation of insulin as a model system. Changes in the accessibility of the thiol group near the end of the alpha-crystallin domain in the absence and the presence of arginine hydrochloride were studied using dithiobisnitrobenzoic acid. Fluorescence resonance energy transfer studies were performed to investigate changes in the dynamics of the subunit assembly. Urea induced denaturation studies of alpha-crystallin were carried out to investigate structural destabilization of alpha-crystallin, if any, in the presence of arginine hydrochloride. RESULTS: Arginine hydrochloride increases the chaperone-like activity of alpha-crystallin several fold towards DTT induced aggregation of insulin at room temperature. Our study shows that both the extent and the rate of accessibility of the thiol group are increased in the presence of arginine. Fluorescence resonance energy transfer experiments show that arginine hydrochloride significantly increases the subunit exchange between the oligomers of alpha-crystallin. Arginine induced structural perturbation and loosening of subunit assembly of alpha-crystallin leads to overall destabilization of the protein as reflected by the urea denaturation study. CONCLUSIONS: Arginine perturbs the tertiary and quaternary structure of alpha-crystallin and enhances the dynamics of the subunit assembly leading to enhanced chaperone-like activity. Thus, in addition to size, surface hydrophobicity, and charge distribution, the dynamics of the subunit assembly appears to be one of the critical factors that can modulate the chaperone activity.

Animals↗
PubMed2005

Cling wrap, an innovative intervention for temperature maintenance and reduction of insensible water loss in very low-birthweight babies nursed under radiant warmers: a randomized, controlled trial.

AIM: The value of polythene film ('cling wrap') to improve thermal control and reduce postnatal weight loss in preterm, very low-birthweight babies was investigated. METHODS: Consecutively born babies with birthweights between 750 and 1500 g were stratified by birthweight (<1250 g, 1251-1500 g) and randomised either to the cling wrap (CW) or no cling wrap (NCW) group. The baby bassinette of the RW was covered with cling wrap up to the level of the neck in the CW group for the 1st 7 days. The primary outcome variables were the incidence of hypothermia (axillary temperature < or = 36 degrees C) after initial stabilisation during the first 7 days and cumulative weight loss (percentage of birthweight) at 48 hours of age. RESULTS: Of 51 babies, 26 were randomised to the CW and 25 to the NCW group. None of the babies in the CW group developed hypothermia in the 1st 7 days but 36% in the NCW group (p = 0.001) did. Babies who were hypothermic on admission took less time to reach normal temperature in the CW group. Cumulative weight loss in the 1st 48 hours was 5.0 + 5.6% in the CW group and 8.6 + 7.0% in the NCW group (p = 0.06). CONCLUSION: Use of CW might be a simple method of maintaining temperature in very low-birthweight babies in developing countries.

Bedding and Linens↗
PubMed2003

Quenched disorder and the critical behavior of a partially frustrated system.

We report the direct observation of the effects of quenched disorder on the critical behavior of partially frustrated amorphous FeMnZr alloys by the systematic analysis of high-precision ac susceptibility data and dc magnetization data. Interestingly, the analysis reveals that the presence of short-range quenched disorder does not alter the actual critical behavior. However, it does affect quantities such as the Curie temperature, the peak value of effective exponent gamma, width of the peak, and crossover temperatures. The observed temperature dependence of the effective critical exponent can be understood in terms of the field-theoretical renormalization group approach. Also, the present results would help in identifying the main source of the spread in the exponent values reported in the literature.

Journal Article↗
PubMed2003

Human recombinant resistin protein displays a tendency to aggregate by forming intermolecular disulfide linkages.

Resistin, a small cysteine rich protein secreted by adipocytes, has been proposed to be a link between obesity and type II diabetes by modulating the insulin signaling pathway and thus inducing insulin resistance. Resistin protein, with 11 cysteine residues, was not significantly homologous at the amino acid level to any other known cysteine rich proteins. Resistin cDNA derived from human subcutaneous adipose tissue was expressed in Escherichia coli as an N-terminal six-His-tag fusion protein. The overexpressed recombinant resistin was purified to homogeneity from inclusion bodies, after solubilization in 8 M urea, using a metal affinity column. While MALDI-TOF mass spectrometric analysis of the purified protein generated a single peak corresponding to the estimated size of 11.3 kDa, the protein exhibited a concentration-dependent oligomerization which is evident from size exclusion chromatography. The oligomeric structure was SDS-insensitive but beta-mercaptoethanol-sensitive, pointing to the importance of disulfide linkages in resistin oligomerization. Estimation of free cysteine residues using the NBD-Cl assay revealed a concentration- and time-dependent increase in the extent of formation of disulfide linkages. The presence of intermolecular disulfide bond(s), crucial in maintaining the global conformation of resistin, was further evident from fluorescence emission spectra. Circular dichroism spectra revealed that recombinant resistin has a tendency to reversibly convert from alpha-helical to beta-sheet structure as a direct function of protein concentration. Our novel observations on the biophysical and biochemical features of human resistin, particularly those shared with prion proteins, may have a bearing on its likely physiological function.

Circular Dichroism↗
PubMed2002

Incidental appendectomy during radical cystectomy--is it necessary?

OBJECTIVES: To evaluate the rationale behind incidental appendectomy during radical cystectomy. Incidental appendectomy during radical cystectomy is done to avoid the morbidity due to the future development of appendicitis and an additional operation. METHODS: We performed a retrospective review of 105 patients who underwent radical cystectomy during an 8-year period (1986 to 1993). Appendectomy was incorporated in the initial 40 radical cystectomies and was avoided in the next 65 radical cystectomies. RESULTS: We had a median follow-up of greater than 10 years in patients who underwent appendectomy along with radical cystectomy and of more than 7 years in patients in whom appendectomy was avoided. None of the patients from either group had any complication related to the appendicular pathologic findings. CONCLUSIONS: The data from this study suggest that no advantage exists in persisting with incidental appendectomy during radical cystectomy.

Adult↗
PubMed2002

Evaluation of hydrophobicity versus chaperonelike activity of bovine alphaA- and alphaB-crystallin.

Calf lens alphaA-crystallin isolated by reversed-phase HPLC demonstrates a slightly more hydrophobic profile than alphaB-crystallin. Fluorescent probes in addition to bis-ANS, like cis-parinaric acid (PA) and pyrene, show higher quantum yields or Ham ratios when bound to alphaA-crystallin than to alphaB-crystallin at room temperature. Bis-ANS binding to both alphaA- and alphaB-crystallin decreases with increase in temperature. At room temperature, the chaperone-like activity of alphaA-crystallin is lower than that of alphaB-crystallin whereas at higher temperatures, alphaA-crystallin shows significantly higher protection against aggregation of substrate proteins compared to alphaB-crystallin. Therefore, calf lens alphaA-crystallin is more hydrophobic than alphaB-crystallin and chaperone-like activity of alpha-crystallin subunits is not quantitatively related to their hydrophobicity.

Anilino Naphthalenesulfonates↗
PubMed2002

Effect of trifluoroethanol on the structural and functional properties of alpha-crystallin.

Alpha crystallin is an eye lens protein with a molecular weight of approximately 800 kDa. It belongs to the class of small heat shock proteins. Besides its structural role, it is known to prevent the aggregation of beta- and gamma-crystallins and several other proteins under denaturing conditions and is thus believed to play an important role in maintaining lens transparency. In this communication, we have investigated the effect of 2,2,2-trifluoroethanol (TFE) on the structural and functional features of the native alpha-crystallin and its two constituent subunits. A conformational change occurs from the characteristic beta-sheet to the alpha-helix structure in both native alpha-crystallin and its subunits with the increase in TFE levels. Among the two subunits, alphaA-crystallin is relatively stable and upon preincubation prevents the characteristic aggregation of alphaB-crystallin at 20% and 30% (v/v) TFE. The hydrophobicity and chaperone-like activity of the crystallin subunits decrease on TFE treatment. The ability of alphaA-crystallin to bind and prevent the aggregation of alphaB-crystallin, despite a conformational change, could be important in protecting the lens from external stress. The loss in chaperone activity of alphaA-crystallin exposed to TFE and the inability of peptide chaperone--the functional site of alphaA-crystallin--to stabilize alphaB-crystallin at 20-30% TFE suggest that the site(s) involved in subunit interaction and chaperone-like function are quite distinct.

Animals↗
PubMed2002

Retrospective review and long-term follow-up of radical cystectomy in a developing country.

OBJECTIVE: To retrospectively review the clinical data from patients undergoing radical cystectomy for bladder cancer, and to analyse the complications and survival rates associated with this operation in a developing country. PATIENTS AND METHODS: The study comprised 105 patients who underwent radical cystectomy from 1986 to 1993. Data were collected from retrospective reviews of hospital and physician's office records, and by contact with the patients. Metastatic status was evaluated before surgery and tumours staged using the Tumour-Nodes-Metastasis classification. The indication for surgery was histologically confirmed muscle invasion after transurethral resection biopsy, or endoscopically uncontrollable superficial disease. The data were analysed to assess the perioperative complications and long-term survival, with 5-year survival rates determined using Kaplan-Meier survival curves. RESULTS: The complication rate was 27.6%; most of the complications were managed conservatively with good results and re-operation was required in only two patients. There were two deaths (1.9%) at 15-45 days after surgery, but none during surgery. Patients were divided into node-negative and node-positive groups for analysis and 5-year survival rates determined; for node-negative organ-confined disease (< or =pT3A) the survival was 68% and for nonorgan-confined disease (> or =pT3B) 25%. The 5-year survival rate in the presence of nodal metastases was 13% for N1 and none for N2 disease. Six patients developed urethral recurrence, detected on follow-up urethral-wash cytology. Five of these patients underwent urethrectomy, and four of the six survived for 5 years. Pelvic recurrence occurred in five patients (4.7%), none of whom survived for 5 years. CONCLUSION: Radical cystectomy and pelvic lymph node dissection remains the mainstay of treatment in muscle-invasive bladder cancer. This is a relatively safe procedure with minimal morbidity and mortality; 68% of the present patients with organ-confined disease survived 5 years and 12 patients were alive at 10 years, indicating the effectiveness of this operation in selected cases. However, < 29% of patients with nonorgan-confined and nodal metastatic disease survived 5 years, thereby implying the need for more effective adjuvant therapy in these patients. Radical cystectomy is a viable option in developing countries, with 5-year survival rates comparable with most large published series.

Adult↗
PubMed2001

Studies on the alpha-crystallin target protein binding sites: sequential binding with two target proteins.

PURPOSE: alpha-Crystallin belongs to a class of small heat shock proteins and is shown to prevent aggregation of several proteins. We have shown that the temperature-induced structural perturbation leads to several fold enhanced activity. The purpose of this study was to investigate the availability and specificity of the hydrophobic sites that might become available at elevated temperatures. Specifically, we address the following question: Is there an increased exposure of fixed number of hydrophobic sites as a function of temperature or does a new set of sites become available at elevated temperatures? METHODS: alpha-Crystallin target protein complexes were made at two different temperatures and this complex was investigated for its chaperone-like activity towards the same target protein and also other target proteins. DTT-induced aggregation of insulin, alpha-lactalbumin, thermal aggregation of betaL- and gamma-crystallin, and photo-aggregation of gamma-crystallin were used as model systems. Increased light scattering was used to monitor the progress of aggregation. RESULTS: alpha-Crystallin target protein complex prepared at 37 degrees C temperature was effective against thermal aggregation of betaL-crystallin as well as non-thermal aggregation at elevated temperatures. However, the complex prepared at high temperature was ineffective at lower temperatures as well as with other target proteins at both temperatures. CONCLUSIONS: More target protein binding sites become available at elevated temperatures. The sites available at low temperature are a subset of the total sites available at elevated temperatures.

Animals↗
PubMed2001

Oxygen sensing and HIF-1 activation does not require an active mitochondrial respiratory chain electron-transfer pathway.

Hypoxia induces the stabilization and transcriptional activation of the hypoxia-inducible factor 1alpha (HIF-1alpha) protein, the regulatory member of the HIF-1 complex. The molecular mechanisms that are responsible for oxygen sensing and the downstream pathways utilized by the hypoxic signal are still poorly understood. One hypothesis for oxygen sensing has postulated that reactive oxygen species generated at mitochondrial complex III are the initiators of the hypoxic signal. Here we find that mitochondrial DNA-less (rho(o)) cells have a normal response to hypoxia, measured at the level of HIF-1alpha protein stabilization, nuclear translocation, and its transcriptional activation activity. Furthermore, overexpression of catalase, either in the mitochondria or in the cytosol, fails to modify the hypoxia response indicating that hydrogen peroxide is not a signaling molecule in the hypoxic signaling cascade that culminates with HIF-1 activation.

Cell Hypoxia↗
PubMed2001

Prediction of mortality by application of PRISM score in intensive care unit.

OBJECTIVE: Prediction of mortality by application of Pediatric Risk of Mortality (PRISM) score in Pediatric Intensive Care Unit (PICU) patients under Indian circumstances. DESIGN: Prospective study. SETTING: PICU of a tertiary care multi-specialty hospital. METHODS: 100 sick pediatric patients admitted consecutively in PICU were taken for this study. PRISM score was calculated. Hospital outcome was recorded as (died/survived). The predicted death was calculated by the formula: RESULTS: Of 100 patients, 18 died and 82 survived. By PRISM score 49 children had the score of 1-9. The expected death in this group was 10.3% (n = 5.03) and the observed death was 8.2% (n = 4). Among 45 children with the score of 10-19, the expected mortality was 21.2% (n = 9.6) and observed was 24.4% (n = 11). There were 3 patients with the score of 20-29, the expected mortality in this group was 39.3% (n = 1.18) and observed mortality 33.3% (n = 1). There were 3 patients with score > or = 30, observed death 66.3% (n = 2) and expected mortality was 74.7% (n = 2.24). There was no significant difference between expected and observed mortality in any group. (p > 0.5). ROC analysis showed area under the curve of 72%. CONCLUSION: PRISM score has good predictive value in assessing the probability of mortality in relation to children admitted to a PICU under Indian circumstances.

Cause of Death↗
PubMed2000

Circulating levels of TNF alpha and TNF receptor superfamily members in lymphoid neoplasia.

We have correlated the serum levels of TNF alpha and soluble TNF receptor superfamily members with clinico-pathologic parameters in patients of Hodgkin's disease (HD, N = 26) and non-Hodgkin's lymphoma (NHLs, N = 35). HD patients had significantly higher levels of TNF alpha, sTNFRI, and sTNFRII in serum while NHL patients had significantly higher levels of sTNFRI, sTNFRII, sCD27, and sFas as compared to controls. In NHL patients the levels of sCD27 correlated directly and significantly with the high-stage disease, bone marrow involvement, lymph nodal presentation, and serum LDH levels. Similarly in NHL patients, levels of sFas also correlated directly and significantly with the presence of high stage disease. HD patients with B symptoms had significantly higher levels of sTNFRII.

Adolescent↗
PubMed2000

Epstein-Barr virus association in classical Hodgkin's disease provides survival advantage to patients and correlates with higher expression of proliferation markers in Reed-Sternberg cells.

BACKGROUND: Most Epstein-Barr virus (EBV) associated lymphoproliferative disorders have high proliferation indices. However, classical Hodgkin's disease (cHD) is heterogeneous, with respect to proliferation index of the Reed-Sternberg cell (RS cell), and EBV association. Hence, we investigated whether cHD with and without EBV-association differ with respect to the proliferation index of the RS cells. Further we investigated whether this would have a bearing on patients survival. PATIENTS AND METHODS: We investigated 110 cases of cHD for: a) EBV association by immunohistochemical demonstration of EBV-latent membrane protein-1 and EBV encoded nuclear RNA 1 by mRNA in situ hybridisation; b) Proliferating cell nuclear antigen (PCNA) expression in the RS cells. RESULTS: EBV association was noted in 86 of 110 cases (78%). Higher PCNA expression (P = 0.004) and younger age (P = 0.001) correlated independently with EBV association. The 10 year relapse free survival (RFS) of EBV+ and EBV- patients were 60% and 44%, respectively (P = 0.03). The 10 year overall survival (OS) of EBV+ and EBV- patients were 85% and 64%, respectively (P = 0.03). EBV association maintained its significant impact on RFS and OS within Cox proportional hazard model. CONCLUSIONS: Our study suggests that EBV is likely to confer a higher PCNA expression and also contribute towards maintaining the RS cells of cHD in cell cycle. Hence, RS cells in EBV associated cHD would be more responsive to chemotherapy and radiotherapy associated DNA damage. Thus, EBV-association provides survival advantage to cHD patients treated with standard chemotherapy and radiotherapy protocols.

Adolescent↗