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M Srivastava

Publications and source records attributed to M Srivastava.

At least 19 recordsLinked to original sources
PubMed2000

A transcriptional insulator at the imprinted H19/Igf2 locus.

Igf2 and H19 exhibit parent-of-origin-specific monoallelic expression. H19 is expressed from the maternal chromosome and Igf2 from the paternal. The two genes share enhancer elements and monoallelic expression of both genes is dependent on cis-acting sequences upstream of the H19 promoter. In this work we examine the mechanisms by which this region silences the maternal Igf2 allele and we demonstrate that deletion of this region can result in high levels of activation of both H19 and Igf2 from a single chromosome. Moreover, by inserting this cis element between a promoter and its enhancer at a heterologous position, we demonstrate that the sequences carry both insulator activity and the ability to be stably imprinted. We also characterize the insulator in vitro and show that it is neither enhancer nor promoter specific.

Animals↗
PubMed2000

H19 and Igf2 monoallelic expression is regulated in two distinct ways by a shared cis acting regulatory region upstream of H19.

H19 and Igf2 are expressed in a monoallelic fashion from the maternal and paternal chromosomes, respectively. A region upstream of H19 has been shown to regulate such imprinted expression of both genes in cis. We have taken advantage of a loxP/cre recombinase-based strategy to delete this region in mice in a conditional manner to determine the temporal requirement of the upstream region in initiating and maintaining the imprinted expression of H19 and Igf2. Analysis of allele-specific expression of H19 and Igf2 and DNA methylation at the H19 promoter demonstrates that this region controls the monoallelic expression of the two genes in different ways, suggesting that it harbors two functionally distinct regulatory elements. Continued presence of the region is required to silence maternal Igf2 in accordance with its proposed role as an insulator. However, it does not have a direct role in keeping the paternal H19 promoter silenced. Instead, on the paternal chromosome, the upstream element mediates epigenetic modifications of the H19 promoter region during development, leading to transcriptional silencing of H19. Thereafter, its presence is redundant for preventing transcription. Presently, this temporal requirement of the silencing element appears to be a unique cis activity in the mammalian system. However, it is likely that other cis-acting elements, positive and negative, have the ability to effect stable changes in the chromatin structure and are not constantly required to give signals to the transcriptional machinery.

Alleles↗
PubMed2000

Over-expression of cyclooxygenase-2 in human prostate adenocarcinoma.

Aberrant or increased expression of cyclooxygenase (COX)-2 has been implicated in the pathogenesis of many diseases including carcinogenesis. COX-2 has been shown to be over-expressed in some human cancers. Employing semi-quantitative reverse transcription-PCR, immunoblotting, and immunohistochemistry we assessed COX-2 expression in samples of pair-matched benign and cancer tissue obtained from the same prostate cancer patient. Mean levels of COX-2 mRNA were 3.4-fold higher in prostate cancer tissue (n = 12) compared with the paired benign tissue. The immunoblot analysis demonstrated that as compared to benign tissue COX-2 protein was over-expressed in 10 of 12 samples examined. Immunohistochemical analysis also verified COX-2 over-expression in cancer than in benign tissue. To our knowledge, this is the first in vivo study showing an over-expression of COX-2 in prostate cancer. These data suggest that COX-2 inhibitors may be useful for prevention or therapy of prostate cancer in humans.

Adenocarcinoma↗
PubMed2000

Low in vivo levels of human anx7 (annexin vii) gene expression are due to endogenous inhibitory promoter sequences.

ANX7 is a Ca(2+)/GTP-dependent membrane fusion protein, which is involved in regulating exocytotic secretion. While the nullizygous ANX7 (-/-) knockout mutation in the mouse is lethal, ANX7 is nonetheless the least expressed member of the annexin gene family in mammalian and lower organisms. To investigate the basis for low ANX7 expression level we have studied the human ANX7 promoter. In our early cloning studies we learned that the human ANX7 promoter lacks TATA and CCAAT boxes, but is GC rich and contains several SP1 binding sites. To study how expression of the ANX7 gene is controlled, we fused nested deletion mutants of the 5'-flanking region to a luciferase reporter gene. We find that the maximal level of luciferase activity is obtained by a construct comprising the domain -889/+34. However, a repressor effect observed in the region between -240 and +34 is dramatically eliminated either by the inclusion of 177 bp upstream sequences, or by the presence of an upstream SV40 enhancer sequences. ANX7 gene expression is also cell type specific and is subject to different transcriptional controls in different cellular environments. The fact that the SV40 enhancer sequences not only eliminates the suppression of luciferase activity in the inhibitory region of the ANX7 promoter, but also reverses the inhibitory effect of phorbol ester, suggests that endogenous cis-acting elements or transacting factors may be responsible for the physiologically low level of ANX7 protein expression.

Animals↗
PubMed2000

Role of clinical, radiological, and neurophysiological changes in predicting the outcome of tuberculous meningitis: a multivariable analysis.

OBJECTIVES: The role of EEG and evoked potentials has not been evaluated in predicting the prognosis of tuberculous (TB) meningitis. The present study was aimed at evaluating the prognostic significance of clinical, radiological, and neurophysiological variables using multi-variable analysis. METHODS: Patients with TB meningitis diagnosed on the basis of clinical, radiological, and CSF criteria have been prospectively evaluated. All the patients were subjected to a detailed neurological evaluation. The outcome was defined 6 months after starting treatment on the basis of the Barthel index (BI) score into poor (BI <12) and good recovery (BI> or =12). Death was included in the poor recovery group for statistical analysis. Thirteen clinical (age, sex, seizure, focal weakness, stage of meningitis, Glasgow coma scale score, methyl prednisolone therapy), CT (infarction, hydrocephalus, tuberculoma) and neurophysiological (EEG, motor and somatosensory evoked potentials) variables were evaluated employing single variable logistic regression followed by multivariable logistic regression analysis. The best set of predictors were obtained by stepdown logistic regression analysis. RESULTS: Fifty four patients were included in the present study. Their age ranged between 5 and 62 years, 11 were children younger than 12 years and 14 were female. Nine patients were in stage I meningitis, 12 in stage II, and 33 in stage III. On single variable logistic regression analysis the significant predictors of 6 months outcome of TB meningitis included focal weakness, Glasgow coma scale (GCS), motor evoked potential (MEP) and somatosensory evoked potential (SEP). On multivariable analysis the best set of predictors comprised focal weakness, GCS, and SEP. CONCLUSIONS: In patients with TB meningitis focal weakness, GCS, and SEP are the best predictors of 6 month outcome.

Adolescent↗
PubMed2000

Radiomodfication of xanthine oxidoreductase system in the liver of mice by phenylmethylsulfonyl fluoride and dithiothreitol.

The widely distributed xanthine oxidoreductase (XOR) system has been shown to be modulated upon exposure of animals to ionizing radiation through the conversion of xanthine dehydrogenase (XDH) into xanthine oxidase (XO). In the present work, radiomodification of the XOR system by phenylmethylsulfonyl fluoride (PMSF) and dithiothreitol (DTT) was examined using female Swiss albino mice which were irradiated with gamma rays at a dose rate 0.023 Gy s(-1). PMSF, a serine protease inhibitor, and DTT, the sulfhydryl reagent, were administered intraperitoneally prior to irradiation. The specific activities of XDH and XO as well as the XDH/XO ratio and the total activity (XDH+XO) were determined in the liver of the mice. The inhibition of XO activity, restoration of XDH activity, and increase in the XDH/XO ratio upon administration of PMSF were suggestive of irreversible conversion of XDH into XO mediated through serine proteases. The biochemical events required for the conversion were probably initiated during the early phase of irradiation, as the treatment with PMSF immediately after irradiation did not have a modulatory effect. Interestingly, DTT was not effective in modulating radiation-induced changes in the XOR system or oxidative damage in the liver of mice. The DTT treatment resulted in inhibition of the release of lactate dehydrogenase. However, the protection appears to be unrelated to the formation of TBARS. On the other hand, the presence of PMSF during irradiation inhibited radiation-induced oxidative damage and radiation-induced increases in the specific activity of lactate dehydrogenase. These findings suggest that a major effect of ionizing radiation is irreversible conversion of xanthine to xanthine oxidase.

Animals↗
PubMed2000

Rapid disappearance of the medial epithelial seam during palatal fusion occurs by multifocal breakdown that is preceded by expression of alpha smooth muscle actin in the epithelium.

Breakdown of the medial epithelial seam (MES) is essential to allow bridging of the mesenchyme during palatal fusion. Evidence exists for three mechanisms for this breakdown that are incompatible at the level of individual cells in the seam. To determine if breakdown of the seam was regionally restricted, 3-dimensional reconstructions were generated using volume rendering software from 1 micron serial sections in the sagittal plane of rat palates fixed during the process of fusion. The earliest break detected in electron micrographs was cell separation and in reconstructions was a discrete defect, with a rounded outline, nearer to the nasal than to the oral margin of the seam. Further breakdown produced a pattern of rounded defects along the nasal margin of the seam resulting in interconnected columns of cells preferentially attached to the oral epithelium. Computer generated slicing of reconstructed seams showed that groups of cells evident in cross-sections as islands at this stage of breakdown of the MES could be artifacts. Unequivocal islands of epithelial cells formed later in fusion had a rounded outline, an incomplete basal lamina and a halo of cells containing phagocytosed apoptotic debris. The pattern of breakdown indicated that the MES breaks down under tension. Laser confocal microscopy of sections and whole-mounts of palates demonstrated alpha-smooth muscle actin preferentially localized in the epithelial cells of the palatal shelves immediately before and during formation of the seam. Expression in epithelial cells of the isoform of actin normally restricted to smooth muscle cells engaged in tonic contraction supported an interpretation that the epithelial cells of the seam may be capable of generating tension during the palatal fusion event.

Actins↗
PubMed1999

Defects in inositol 1,4,5-trisphosphate receptor expression, Ca(2+) signaling, and insulin secretion in the anx7(+/-) knockout mouse.

The mammalian anx7 gene codes for a Ca(2+)-activated GTPase, which supports Ca(2+)/GTP-dependent secretion events and Ca(2+) channel activities in vitro and in vivo. To test whether anx7 might be involved in Ca(2+) signaling in secreting pancreatic beta cells, we knocked out the anx7 gene in the mouse and tested the insulin-secretory properties of the beta cells. The nullizygous anx7 (-/-) phenotype is lethal at embryonic day 10 because of cerebral hemorrhage. However, the heterozygous anx7 (+/-) mouse, although expressing only low levels of ANX7 protein, is viable and fertile. The anx7 (+/-) phenotype is associated with a substantial defect in insulin secretion, although the insulin content of the islets, is 8- to 10-fold higher in the mutants than in the normal littermate control. We infer from electrophysiological studies that both glucose-stimulated secretion and voltage-dependent Ca(2+) channel functions are normal. However, electrooptical recordings indicate that the (+/-) mutation has caused a change in the ability of inositol 1,4,5-trisphosphate (IP(3))-generating agonists to release intracellular calcium. The principle molecular consequence of lower anx7 expression is a profound reduction in IP(3) receptor expression and function in pancreatic islets. The profound increase in islets, beta cell number, and size may be a means of compensating for less efficient insulin secretion by individual defective pancreatic beta cells. This is a direct demonstration of a connection between glucose-activated insulin secretion and Ca(2+) signaling through IP(3)-sensitive Ca(2+) stores.

Animals↗
PubMed1999

HIV-1 Gag shares a signature motif with annexin (Anx7), which is required for virus replication.

Genetic and biochemical analyses of the Gag protein of HIV-1 indicate a crucial role for this protein in several functions related to viral replication, including viral assembly. It has been suggested that Gag may fulfill some of the functions by recruiting host cellular protein(s). In our effort to identify structural and functional homologies between Gag and cellular cytoskeletal and secretory proteins involved in transport, we observed that HIV-1 Gag contains a unique PGQM motif in the capsid region. This motif was initially noted in the regulatory domain of synexin the membrane fusion protein of Xenopus laevis. To evaluate the functional significance of the highly conserved PGQM motif, we introduced alanine (A) in place of individual residues of the PGQM and deleted the motif altogether in a Gag expression plasmid and in an HIV-1 proviral DNA. The proviral DNA containing mutations in the PGQM motif showed altered expression, assembly, and release of viral particles in comparison to parental (NL4-3) DNA. When tested in multiple- and single-round replication assays, the mutant viruses exhibited distinct replication phenotypes; the viruses containing the A for the G and Q residues failed to replicate, whereas A in place of the P and M residues did not inhibit viral replication. Deletion of the tetrapeptide also resulted in the inhibition of replication. These results suggest that the PGQM motif may play an important role in the infection process of HIV-1 by facilitating protein-protein interactions between viral and/or viral and cellular proteins.

Animals↗
PubMed1999

Inhibition of microvascular endothelial apoptosis in tissue explants by serum albumin.

Plasma factors appear to inhibit endothelial cell (EC) apoptosis in vivo so that flow influences microvascular form. The identity of these factors has not, however, been established. Earlier, we reported that apoptosis in isolated, serum-deprived human EC is inhibited by albumin (Alb). Here, we demonstrate likely biological relevance of this to vascular remodelling in experiments with tissue explants. Rat skin explants were incubated in medium M199 with or without serum, bovine Alb, or human Alb. EC in paraffin sections of explants were labelled by lectin histochemistry and the relative proportion of apoptotic was EC determined. Apoptosis was confirmed by transmission electron microscopy and terminal deoxynucleotidyl transferase labelling. Serum-free culture induced EC apoptosis (P < 0.02) and this was strongly inhibited by Alb at physiological concentrations (P < 0.01). This was not a nonspecific protein effect, as mercaptoethanol denaturation destroyed the activity and ovalbumin was not protective. Also, protection was not due to serum contaminants, as recombinant human Alb had activity identical to that of native material. The dose response was identical for all Alb preparations tested, with maximal activity at physiological concentrations. Protection was not limited to rat tissue as similar results were obtained with human gingival explants. These data support a role for Alb as a plasma antiapoptotic factor for EC in tissues.

Animals↗
PubMed1999

Effect of radiation on the xanthine oxidoreductase system in the liver of mice.

The xanthine oxidoreductase system is one of the major sources of free radicals in many pathophysiological conditions. Since ionizing radiations cause cell damage and death, the xanthine oxidoreductase system may contribute to the detrimental effects in irradiated systems. Therefore, modulation of the xanthine oxidoreductase system by radiation has been examined in the present study. Female Swiss albino mice (7-8 weeks old) were irradiated with gamma rays (1-9 Gy) at a dose rate of 0.023 Gy s(-1) and the specific activities of xanthine oxidase (XO) and xanthine dehydrogenase (XDH) were determined in the liver of the animals. The mode and magnitude of change in the specific activities of XO and XDH were found to depend on radiation dose. At doses above 3 Gy, the specific activity of XO increased rapidly and continued to increase with increasing dose. However, the specific activity of XDH was decreased. These findings are suggestive of an inverse relationship between the activity of XO and XDH. The ratio of the activity of XDH to that of XO decreased with radiation dose. However, the total activity (XDH + XO) remained constant at all doses. These results indicate that XDH may be converted into XO. An intermediate form, D/O, appears to be transient in the process of conversion. The enhanced specific activity of XO may cause oxidative stress that contributes to the radiation damage and its persistence in the postirradiation period. Radiation-induced peroxidative damage determined in terms of the formation of TBARS and the change in the specific activity of lactate dehydrogenase support this possibility.

Allopurinol↗
PubMed1999

Fertility outcome after repeat vasoepididymostomy.

PURPOSE: Historically, epididymal obstruction has been treated with surgical reconstruction. We determine whether it is worthwhile for patients to undergo repeat surgical reconstruction after failed vasoepididymostomy or whether they should be advised only to undergo sperm acquisition for assisted reproductive technique. MATERIALS AND METHODS: A total of 18 patients underwent repeat vasoepididymostomy performed by a single urologist (A. J. T.). Cases were divided based on the etiology of obstruction into groups 1--prior vasectomy (4), 2--congenital (7) and 3--inflammatory (7). Data were available regarding time of obstruction between initial and repeat vasoepididymostomy, quality of epididymal fluid, levels of anastomoses, semen analyses at least 12 months after surgery for all 18 men and pregnancy rates based on more than 18 months of followup in 12. RESULTS: Mean patient age at repeat vasoepididymostomy was 40.6 years (50.5, 36 and 39.4 years for groups 1, 2 and 3, respectively). Mean interval between vasectomy and initial vasoepididymostomy was 12.3 years (range 10 to 18). Mean interval between initial and repeat vasoepididymostomy was 19 months (range 12 to 41). Of the patients 10 underwent unilateral and 8 bilateral anastomoses, for a total of 26 repeat anastomoses. Overall patency rate was 66.7% (12 of 18) with sperm in the ejaculate in 75, 85 and 43% of patients in groups 1, 2 and 3, respectively. The patency rates according to the levels of the anastomosis were 66.7, 62.5 and 100% in the caput, corpus and cauda, respectively. Natural conception occurred in 3 of 12 couples (25%, 2 caput and 1 caudal anastomosis) during a mean followup of 23 months (range 13 to 34). All 3 cases had congenital obstruction. Pregnancy was achieved in 2 group 1 cases with cryopreserved sperm extracted at repeat vasoepididymostomy, and in 1 case each in groups 1 and 2 with microsurgical epididymal sperm aspiration and intracytoplasmic sperm injection. CONCLUSIONS: After repeat vasoepididymostomy two-thirds of men have sperm in the semen. Natural conception occurred in 25% of patients (3 of 12) followed for more than 18 months. Inability to establish pregnancy in the remaining 7 of 9 patients with sperm in the semen with a followup longer than 18 months may be due to epididymal dysfunction or partial obstruction and subsequent poor sperm quality. Aspiration of motile sperm and cryopreservation were possible in 11 of 18 cases at repeat vasoepididymostomy and should be recommended in case azoospermia remains or occurs after surgery. It appears worthwhile to offer patients repeat vasoepididymostomy after a failed initial procedure.

Adult↗
PubMed1999

Molecular dissection of nucleolin's role in growth and cell proliferation: new insights.

Cells require optimum protein synthetic activity in order to support cell proliferation, maintain homeostatic and metabolic integrity, and repair damage. Since growth depends on protein synthesis through ribosome biogenesis, the control of biosynthesis of ribosomes is necessarily a key element for control of growth. Nucleolin is a major nucleolar protein of exponentially growing eukaryotic cells, which is directly involved in the regulation of ribosome biogenesis and maturation. The highly conserved nucleolin contains three major domains through which it controls the organization of nucleolar chromatin, packaging of pre-RNA, rDNA transcription, and ribosome assembly. Numerous reports have implicated the involvement of nucleolin either directly or indirectly in the regulation of cell proliferation and growth, cytokinesis, replication, embryogenesis, and nucleogenesis. Nucleolin, an RNA binding protein, is also an autoantigen, a transcriptional repressor, and a switch region targeting factor. In addition, nucleolin exhibits autodegradation, DNA and RNA helicase activities, and DNA-dependent ATPase activity. An interesting aspect of nucleolin action is that it is a target for regulation by proteolysis, methylation, ADP-ribosylation, and phosphorylation by CKII, cdc2, PKC-xi, cyclic AMP-dependent protein kinase, and ecto-protein kinase. For these and other reasons, nucleolin is fundamental to the survival and proliferation of cells. Considerable progress has been made in recent years with the identification of new nucleolin binding proteins that may mediate these many nucleolin-dependent functions. Nucleolin also functions as a cell surface receptor, where it acts as a shuttling protein between cytoplasm and nucleus, and thus can even provide a mechanism for extracellular regulation of nuclear events. Exploration of the regulation of this multifaceted protein in a remarkable number of diverse functions is challenging.

Animals↗
PubMed1999

Pharmacogenomics of the cystic fibrosis transmembrane conductance regulator (CFTR) and the cystic fibrosis drug CPX using genome microarray analysis.

BACKGROUND: Cystic fibrosis (CF) is the most common lethal recessive disease affecting children in the U.S. and Europe. For this reason, a number of ongoing attempts are being made to treat the disease either by gene therapy or pharmacotherapy. Several phase 1 gene therapy trials have been completed, and a phase 2 clinical trial with the xanthine drug CPX is in progress. The protein coded by the principal CFTR mutation, DeltaF508-CFTR, fails to traffic efficiently from the endoplasmic reticulum to the plasma membrane, and is the pathogenic basis for the missing cAMP-activated plasma membrane chloride channel. CPX acts by binding to the mutant DeltaF508-CFTR and correcting the trafficking deficit. CPX also activates mutant CFTR channels. The comparative genomics of wild-type and mutant CFTR has not previously been studied. However, we have hypothesized that the gene expression patterns of human cells expressing mutant or wild-type CFTR might differ, and that a drug such as CPX might convert the mutant gene expression pattern into one more characteristic of wild-type CFTR. To the extent that this is true, a pharmacogenomic profile for such corrective drugs might be deduced that could simplify the process of drug discovery for CF. MATERIALS AND METHODS: To test this hypothesis we used cDNA microarrays to study global gene expression in human cells permanently transfected with either wild-type or mutant CFTR. We also tested the effects of CPX on global gene expression when incubated with cells expressing either mutant or wild-type CFTR. RESULTS: Wild-type and mutant DeltaF508-CFTR induce distinct and differential changes in cDNA microarrays, significantly affecting up to 5% of the total genes in the array. CPX also induces substantial mutation-dependent and -independent changes in gene expression. Some of these changes involve movement of gene expression in mutant cells in a direction resembling expression in wild-type cells. CONCLUSIONS: These data clearly demonstrate that cDNA array analysis of cystic fibrosis cells can yield useful pharmacogenomic information with significant relevance to both gene and pharmacological therapy. We suggest that this approach may provide a paradigm for genome-based surrogate endpoint testing of CF therapeutics prior to human administration.

Cell Line↗
PubMed1998

Diffusion of transferrin receptor clusters.

Two dimensional motion of membrane receptors provides a mechanism for interaction among receptors in the plane of the membrane. In some cases the lateral diffusion leads to formation of clusters which may also be mobile. We have used image cross-correlation (ICCS) spectroscopy technique to measure the translational motion of transferrin receptors in the membrane of 3T3 fibroblasts and HEp2 carcinoma cells. The technique is based on the measurement and analysis of fluctuations in the intensity observed in fluorescence confocal microscope images measured as a function of time. The fluorescence fluctuations arise from stochastic concentration fluctuations about the equilibrium concentration caused by movement of receptors. The amplitude of the fluctuations depend on the number of fluorescent molecules in the observation volume and the dynamics provide the rate of movement. The diffusion observed by this analysis is orders of magnitude slower than that measured by conventional photobleaching techniques. The slower motion corresponds to the diffusion of receptor clusters which provide the more dominant fluctuations.

3T3 Cells↗
PubMed1998

Prognosis of Japanese encephalitis: a multivariate analysis.

To evaluate the role of various clinical neurophysiological laboratory and radiological parameters in the prognosis of Japanese encephalitis (JE), 28 patients were prospectively evaluated. Their age ranged between two and 54 years, with eight of them being female and 11 children. The outcome was defined, at the end of a six-month period, as being poor (bed-ridden), partial (partial dependence for activity for daily life) and complete recovery (independent for activities of daily life). A number of clinical (age, degree of coma, decerebration or decortication and reflex changes), laboratory (CSF cell and protein content, and haemagglutination inhibition titre in the serum), neurophysiological (EEG, EMG, sensory and motor evoked potentials) and the extent of radiological changes were subjected to stepwise multivariate logistic regression analysis. By the end of six months, four patients had died, seven had poor, ten partial and seven had complete recovery. The best set of predictors of outcome included age, Glasgow coma scale and reflex changes.

Adolescent↗
PubMed1998

Failure of macrophage activation in destructive periodontal disease.

Macrophage populations in 22 biopsies of untreated advanced periodontitis were compared with those in 26 biopsies of clinically healthy (minimally inflamed) gingival tissue. The immunohistochemical investigation used high specificity monoclonal antibodies, including a pan-macrophage marker and probes for acute inflammatory, resident histiocytic, and reparative phenotypes. Macrophage expression of the functional activation markers MHC class II, basic fibroblast growth factor (bFGF), acid phosphatase (AP), and tartrate-resistant acid phosphatase (TRAP) was also examined. The study showed that advanced periodontitis and minimally inflamed tissues displayed similar distribution patterns and numbers for the macrophage phenotypic markers: there were, however, regionally-specific differences in the populations. In the advanced periodontitis lesion, there was little evidence of macrophage activation for the expression of HLA-DR, bFGF, and TRAP, although strong expression of HLA-DR and bFGF was observed in association with blood vessels. Macrophages expressing AP showed a distinct regional distribution; this, however, was not associated with foci of degenerate plasma cells. The apparent failure of recruitment and activation of macrophages may in part be both a cause and a consequence of the pathological features of this disease.

Adult↗