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Biomedical subjects

M Sharma

Publications and source records attributed to M Sharma.

At least 109 records · Page 6Linked to original sources
PubMed2001

Mycobacterium fortuitum wound infection following laparoscopy.

During a six week period in 1999, seven patients who underwent laparoscopic tubectomies at small town health centres near Chandigarh developed chronic discharging sinuses at the site of incision. Mycobacterium fortuitum was isolated from wound discharge of the five patients by standard methods and two patients were smear positive. Environmental samples e.g., tap water, and a variety of fluids did not yield any mycobacteria and swabs from different parts of the laparoscope were sterile. All patients responded to ciprofloxacin and amikacin therapy. Our observation demonstrates that M. fortuitum is a clinically important nosocomial pathogen in setting of surgical wound infection in our country.

Amikacin↗
PubMed2001

Antiepileptic activity of Panax ginseng against pentylenetetrazole induced kindling in rats.

In the present study, Panax ginseng was evaluated for its antiepileptic activity against pentylenetetrazole (PTZ) induced chemical kindling in rats. PTZ was injected at the dose of 30 mg/kg, i.p. on alternate days and the occurrence of generalized tonic clonic convulsions were considered as the end point. One group received Panax ginseng every day, at a dose of 100 mg/kg, 30 min prior to PTZ injection whereas the other group received an equal volume of distilled water to serve as control. In a separate group the rats were evaluated for motor performance tests after Panax ginseng. The rats treated with Panax ginseng showed significant protection as compared to vehicle treated PTZ injected rats. The study suggests to potential of Panax ginseng against seizures.

Animals↗
PubMed2001

Changing pattern of biotypes, phage types & drug resistance of Salmonella typhi in Ludhiana during 1980-1999.

BACKGROUND & OBJECTIVES: Ludhiana, an industrial city of Punjab, has a large floating population where typhoid has become endemic. A retrospective study was carried out over a period of 20 years (1980-1999) at Ludhiana on the biotyping, phage typing and drug resistance pattern of Salmonella typhi. METHODS: Of a total of 1697 S. typhi isolates obtained, phage typing and biotyping were done of only 1243 isolates. Antimicrobial susceptibility pattern of these isolates was also studied. RESULTS: Of the 1243 S. typhi isolates, 963 (77.5%) and 280 (22.5%) were of biotype I and biotype II respectively. Twenty four different S. typhi phage types were prevalent in Ludhiana in the past two decades. Between 1980 and 1989, more prevalent phage types were phage type A (35%), O (17.6%) and E1 (15.1%). During 1990-1999, there was a considerable increase in the incidence of phage type E1 (48.1%). The cumulative analysis of past two decades revealed that the incidence of phage type E1 (38.8%) was most predominant. In the past one decade (1990-1999), 412 S. typhi isolates of 13 different phage types exhibited multidrug resistance (MDR) pattern ACCoT (resistant to ampicillin, chloramphenicol, co-trimoxazole and tetracycline). High chloramphenicol resistance (74.7%) and MDR pattern ACCoT (68.2%) was shown by phage type E1 of S. typhi. INTERPRETATION & CONCLUSION: An association was observed between drug resistance and phage type pattern of S. typhi as 70 per cent isolates of S. typhi phage types E1 and O exhibited ACCoT multidrug resistant pattern. Reemergence of chloramphenicol susceptibility in the last decade emphasizes the need for regular antimicrobial surveillance to minimize the misuse of these drugs.

Anti-Bacterial Agents↗
PubMed2000

Myostatin, a negative regulator of muscle growth, functions by inhibiting myoblast proliferation.

Myostatin, a member of the transforming growth factor-beta (TGF-beta) superfamily, has been shown to be a negative regulator of myogenesis. Here we show that myostatin functions by controlling the proliferation of muscle precursor cells. When C(2)C(12) myoblasts were incubated with myostatin, proliferation of myoblasts decreased with increasing levels of myostatin. Fluorescence-activated cell sorting analysis revealed that myostatin prevented the progression of myoblasts from the G(1)- to S-phase of the cell cycle. Western analysis indicated that myostatin specifically up-regulated p21(Waf1, Cip1), a cyclin-dependent kinase inhibitor, and decreased the levels and activity of Cdk2 protein in myoblasts. Furthermore, we also observed that in myoblasts treated with myostatin protein, Rb was predominately present in the hypophosphorylated form. These results suggests that, in response to myostatin signaling, there is an increase in p21 expression and a decrease in Cdk2 protein and activity thus resulting in an accumulation of hypophosphorylated Rb protein. This, in turn, leads to the arrest of myoblasts in G(1)-phase of cell cycle. Thus, we propose that the generalized muscular hyperplasia phenotype observed in animals that lack functional myostatin could be as a result of deregulated myoblast proliferation.

Animals↗
PubMed2000

Conformational and temperature-sensitive stability defects of the delta F508 cystic fibrosis transmembrane conductance regulator in post-endoplasmic reticulum compartments.

Deletion of phenylalanine at position 508 (DeltaF508) is the most common cystic fibrosis (CF)-associated mutation in the CF transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel. The consensus notion is that DeltaF508 imposes a temperature-sensitive folding defect and targets newly synthesized CFTR for degradation at endoplasmic reticulum (ER). A limited amount of CFTR activity, however, appears at the cell surface in the epithelia of homozygous DeltaF508 CFTR mice and patients, suggesting that the ER retention is not absolute in native tissues. To further elucidate the reasons behind the inability of DeltaF508 CFTR to accumulate at the plasma membrane, its stability was determined subsequent to escape from the ER, induced by reduced temperature and glycerol. Biochemical and functional measurements show that rescued DeltaF508 CFTR has a temperature-sensitive stability defect in post-ER compartments, including the cell surface. The more than 4-20-fold accelerated degradation rate between 37 and 40 degrees C is, most likely, due to decreased conformational stability of the rescued DeltaF508 CFTR, demonstrated by in situ protease susceptibility and SDS-resistant thermoaggregation assays. We propose that the decreased stability of the spontaneously or pharmacologically rescued mutant may contribute to its inability to accumulate at the cell surface. Thus, therapeutic efforts to correct the folding defect should be combined with stabilization of the native DeltaF508 CFTR.

Animals↗
PubMed2000

Androgen receptor interacts with a novel MYST protein, HBO1.

The androgen receptor (AR), a member of the nuclear receptor superfamily, plays a central role in male sexual differentiation and prostate cell proliferation. Results of treating prostate cancer by androgen ablation indicate that signals mediated through AR are critical for the growth of these tumors. Like other nuclear receptors, AR exerts its transcriptional function by binding to cis-elements upstream of promoters and interacting with other transcriptional factors (e.g. activators, repressors and modulators). To determine the mechanism of AR-regulated transcription, we used the yeast two-hybrid system to identify AR-associated proteins. One of the proteins we identified is identical to the human origin recognition complex-interacting protein termed HBO1. A ligand-enhanced interaction between AR and HBO1 was further confirmed in vivo and in vitro. Immunofluorescence experiments showed that HBO1 is a nuclear protein, and Northern blot analysis revealed that it is ubiquitously expressed, with the highest levels present in human testis. HBO1 belongs to the MYST family, which is characterized by a highly conserved C2HC zinc finger and a putative histone acetyltransferase domain. Surprisingly, two yeast members of the MYST family, SAS2 and SAS3, have been shown to function as transcription silencers, despite the presence of the histone acetyltransferase domain. Using a GAL4 DNA-binding domain assay, we mapped a transcriptional repression domain within the N-terminal region of HBO1. Transient transfection experiments revealed that HBO1 specifically repressed AR-mediated transcription in both CV-1 and PC-3 cells. These results indicate that HBO1 is a new AR-interacting protein capable of modulating AR activity. It could play a significant role in regulating AR-dependent genes in normal and prostate cancer cells.

Acetyltransferases↗
PubMed2000

Determinants of the nuclear localization of the heterodimeric DNA fragmentation factor (ICAD/CAD).

Programmed cell death or apoptosis leads to the activation of the caspase-activated DNase (CAD), which degrades chromosomal DNA into nucleosomal fragments. Biochemical studies revealed that CAD forms an inactive heterodimer with the inhibitor of caspase-activated DNase (ICAD), or its alternatively spliced variant, ICAD-S, in the cytoplasm. It was initially proposed that proteolytic cleavage of ICAD by activated caspases causes the dissociation of the ICAD/CAD heterodimer and the translocation of active CAD into the nucleus in apoptotic cells. Here, we show that endogenous and heterologously expressed ICAD and CAD reside predominantly in the nucleus in nonapoptotic cells. Deletional mutagenesis and GFP fusion proteins identified a bipartite nuclear localization signal (NLS) in ICAD and verified the function of the NLS in CAD. The two NLSs have an additive effect on the nuclear targeting of the CAD-ICAD complex, whereas ICAD-S, lacking its NLS, appears to have a modulatory role in the nuclear localization of CAD. Staurosporine-induced apoptosis evoked the proteolysis and disappearance of endogenous and exogenous ICAD from the nuclei of HeLa cells, as monitored by immunoblotting and immunofluorescence microscopy. Similar phenomenon was observed in the caspase-3-deficient MCF7 cells upon expressing procaspase-3 transiently. We conclude that a complex mechanism, involving the recognition of the NLSs of both ICAD and CAD, accounts for the constitutive accumulation of CAD/ICAD in the nucleus, where caspase-3-dependent regulation of CAD activity takes place.

Apoptosis↗
PubMed2000

Analysis of tamoxifen-DNA adducts by high-performance liquid chromatography using postcolumn online photochemical activation.

Tamoxifen, a widely used nonsteroidal antiestrogen in the treatment of breast cancer, forms several metabolites. 4-Hydroxytamoxifen (4-OHTam), a metabolite found in the bloodstream, has much higher affinity for the estrogen receptor than tamoxifen itself. Oxidative activation of 4-OHTam induces DNA damage. DNA isolated from HL-60 cells exposed to 10 microM 4-OHTam in the presence of 1 microM hydrogen peroxide was digested enzymatically to release both normal and modified nucleosides. The modified nucleosides were enriched by butanol extraction. Using UV detection, HPLC analysis of the butanol extract from 200 microg DNA digest detected approximately 4 4-OHTam-dG adducts per 10(7) nucleotides (n = 3). Online postcolumn UV irradiation in HPLC and fluorescence detection improved the detection sensitivity by 3 x 10(2) times. Using 4-OHTam as an example, this report demonstrated for the first time the power of the technique to assay tamoxifen-DNA adducts directly in the DNA digest without relying on postlabeling.

Animals↗
PubMed2000

An alternatively spliced transcript of the rat nociceptin receptor ORL1 gene encodes a truncated receptor.

Opioid receptor-like protein ORL1, the receptor for the neuropeptide nociceptin (also named orphanin FQ), has two alternatively spliced isoforms in the rat. This alternative splicing event is generated by retaining of intron 3, 81 bases in length, in the mRNA region encoding the second extracellular loop of ORL1. A full-length rat ORL1 receptor has 367 amino acid residues. However, as revealed by sequencing of rat ORL1 genomic DNA and cDNA, the insertion of the unspliced intron 3 brings in an in-frame stop codon and, therefore, creates a truncated open-reading frame encoding only the N-terminal half of ORL1 (from the N-terminus to an alternate extracellular tail C-terminal to the fourth transmembrane domain). The two alternatively spliced transcripts are differentially expressed in tissues. In transfected mammalian cells, the full-length ORL1 displays high-affinity and selective binding for nociceptin, and inhibits the production of cyclic AMP. In contrast, the truncated ORL1 binds nociceptin and other opioid peptides very poorly and non-selectively (affinity in micromolar range), and it does not mediate any inhibitory effects on cyclic AMP production. Apparently, this truncated ORL1 does not function as a receptor for nociceptin or other ligands tested. Such alternative splicing to create a truncated ORL1 receptor might be an endogenous mechanism to negatively regulate nociceptin/ORL1 functions.

Alternative Splicing↗
PubMed2000

Caspase-8-mediated intracellular acidification precedes mitochondrial dysfunction in somatostatin-induced apoptosis.

Activation of initiator and effector caspases, mitochondrial changes involving a reduction in its membrane potential and release of cytochrome c (cyt c) into the cytosol, are characteristic features of apoptosis. These changes are associated with cell acidification in some models of apoptosis. The hierarchical relationship between these events has, however, not been deciphered. We have shown that somatostatin (SST), acting via the Src homology 2 bearing tyrosine phosphatase SHP-1, exerts cytotoxic action in MCF-7 cells, and triggers cell acidification and apoptosis. We investigated the temporal sequence of apoptotic events linking caspase activation, acidification, and mitochondrial dysfunction in this system and report here that (i) SHP-1-mediated caspase-8 activation is required for SST-induced decrease in pH(i). (ii) Effector caspases are induced only when there is concomitant acidification. (iii) Decrease in pH(i) is necessary to induce reduction in mitochondrial membrane potential, cyt c release and caspase-9 activation and (iv) depletion of ATP ablates SST-induced cyt c release and caspase-9 activation, but not its ability to induce effector caspases and apoptosis. These data reveal that SHP-1-/caspase-8-mediated acidification occurs at a site other than the mitochondrion and that SST-induced apoptosis is not dependent on disruption of mitochondrial function and caspase-9 activation.

Acids↗
PubMed2000

Results of modern therapy for patients with mediastinal nonseminomatous germ cell tumors.

BACKGROUND: The aim of this study was to determine the effects of independent prognostic variables, such as prechemotherapy tumor markers, the extent of disease at diagnosis, the tumor markers postchemotherapy (PC), and the pathology of the PC residual mass on the overall survival of patients with primary mediastinal nonseminomatous germ cell tumors (PMNSGCT). METHODS: The authors undertook a retrospective review of 39 patients with PMNSGCT between 1983 and 1997 who received their initial chemotherapy at Indiana University and 36 additional patients referred for PC resection. All patients received chemotherapy based on the combination of cisplatin and etoposide. The median follow-up was 22 months (range, 12-144 months). RESULTS: The prechemotherapy tumor markers did not affect overall survival. Extent of disease (mediastinal only vs. visceral metastases) was an important prognostic factor for survival in univariate analysis (P = 0.042). Sixty-two of 75 patients underwent PC resection of residual disease. Fifteen of the 62 patients achieved a CR with chemotherapy alone, as the PC resection revealed only necrosis. Fourteen of these 15 patients continuously had no evidence of disease (NED). Forty-seven of the 62 patients had NED with chemotherapy and PC resection, including 31 with teratoma and 16 with carcinoma. However, 11 of 31 with teratoma and 11 of 16 with carcinoma subsequently relapsed. Although 18 patients had elevated tumor markers at the time of PC resection, 6 of 18 had only necrosis and 4 had teratoma. The PC tumor markers did not affect survival. The pathology of the resected specimen was the most significant predictor of survival in multivariate analysis (P < 0.001). CONCLUSIONS: Twenty-eight of 39 patients (71.8%) with PMNSGCT treated at Indiana University achieved NED status, but only 16 (41%) continuously had NED. Twenty of 36 (55.5%) referred for resection continuously had NED. Disease confined to the mediastinum and necrosis in the PC specimen were important prognostic factors for survival.

Adolescent↗
PubMed2000

Myostatin regulation during skeletal muscle regeneration.

Myostatin, a member of the TGF-beta superfamily, is a key negative regulator of skeletal muscle growth. The role of myostatin during skeletal muscle regeneration has not previously been reported. In the present studies, normal Sprague-Dawley and growth hormone (GH)-deficient (dw/dw) rats were administered the myotoxin, notexin, in the right M. biceps femoris on day 0. The dw/dw rats then received either saline or human-N-methionyl GH (200microg/100g body weight/day) during the ensuing regeneration. Normal and dw/dw M. biceps femoris were dissected on days 1, 2, 3, 5, 9 and 13, formalin-fixed, then immunostained for myostatin protein. Immunostaining for myostatin revealed high levels of protein within necrotic fibres and connective tissue of normal and dw/dw damaged muscles. Regenerating myotubes contained no myostatin at the time of fusion (peak fusion on day 5), and only low levels of myostatin were observed during subsequent myotube enlargement. Fibres which survived assault by notexin (survivor fibres) contained moderate to high myostatin immunostaining initially. The levels in both normal and dw/dw rat survivor fibres decreased on days 2-3, then increased on days 9-13. In dw/dw rats, there was no observed effect of GH administration on the levels of myostatin protein in damaged muscle. The low level of myostatin observed in regenerating myotubes in these studies suggests a negative regulatory role for myostatin in muscle regeneration.

Animals↗
PubMed2000

Genomic structure and novel variants of myo-inositol monophosphatase 2 (IMPA2).

Recently, we cloned the human myo-inositol monophosphatase 2 (IMPA2) cDNA and established its map location to chromosome 18p11.2, a region previously implicated in bipolar disorder. Because the myo-inositol monophosphatase enzyme has been shown to be inhibited by lithium, an effective therapeutic agent for bipolar disorder, IMPA2 is a plausible positional and functional candidate gene. To permit comprehensive screening for variants we characterized the genomic structure and isolated the potential promoter of IMPA2. The gene was found to encode eight exons spanning;27 kb. The proximal 1-kb 5' flanking region did not contain an obvious TATA box but multiple potential binding sites for Sp1 and consensus motifs for AP2 and other transcription factors were evident. Sequencing of the coding region and splice junctions in unrelated bipolar disorder patients detected novel variants. A missense mutation in exon 2, His76Tyr, was found in one patient. His76 is evolutionarily conserved and replacement with Tyr introduces a potential site for phosphorylation. The other polymorphisms included an RsaI polymorphism, IVS1-15G>A, and a T --> C silent mutation in the third nucleotide of codon 53 in exon 2. By Fisher's exact test the silent mutation showed a trend for association (P = 0.051) with bipolar disorder suggesting that further scrutiny of this gene is warranted.

Alternative Splicing↗
PubMed2000

Anthrax toxin-mediated delivery of cholera toxin-A subunit into the cytosol of mammalian cells.

The protective antigen (PA) component of anthrax toxin mediates delivery of either lethal factor (LF) or oedema factor into the cytosol of mammalian cells. The N-terminal domain of LF(1-254) (amino acids 1-254 of LF) binds to PA and, when fused to heterologous proteins, delivers such proteins into the cytosol. In the present study, we fused the catalytic subunit of cholera toxin (CT-A) with LF(1-254) and showed that the fusion protein LF(1-254)-CT-A retains ADP-ribosylation activity in solution and increased intracellular cAMP levels in J774A.1 macrophage cells when added together with PA. A mutant fusion protein, in which arginine-7 of CT-A was replaced with lysine, did not show ADP-ribosylation activity in solution and failed to increase cAMP levels in macrophage cells. The data show that LF(1-254)-CT-A retains its catalytic activity in solution as well as when translocated into the cytosol of eukaryotic cells via an alternative pathway to the GM(1) receptor used by CT.

Adenosine Diphosphate↗
PubMed2000

Transforming growth factor-beta1 increases albumin permeability of isolated rat glomeruli via hydroxyl radicals.

BACKGROUND: Transforming growth factor-beta1 (TGF-beta1) is a multifunctional cytokine. Glomerular cells and tubular epithelial cells secrete and respond to TGF-beta1. A close association between elevated levels of TGF-beta1 and the development of glomerulonephritis, glomerulosclerosis, and tubular hypertrophy has been documented. The role of TGF-beta1 in proteinuria is not well understood. METHODS: Isolated rat glomeruli were incubated in medium alone or with TGF-beta1 (1 to 10 ng/mL) and TGF-beta1 + 200 U/mL of superoxide dismutase (SOD) or 1 mmol/L of dimethylthiourea (DMTU) scavengers of superoxide and hydroxyl radicals, respectively, for up to 60 minutes at 37 degrees C. Glomerular albumin permeability (Palb) was calculated from the volumetric response of glomeruli to an oncotic gradient using videomicroscopy. RESULTS: One or 2.5 ng/mL of TGF-beta1 had no effect on Palb (0.18 +/- 0.08, N = 17; 0.18 +/- 0. 079, N = 20 vs. control 0.00 +/- 0.06, N = 25), whereas 5 or 10 ng/mL of TGF-beta1 caused a significant increase in Palb (0.31 +/- 0. 09, N = 20; 0.33 +/- 0.06, N = 23) within 15 minutes. The effect of 10 ng/mL of TGF-beta1 on Palb increased further after 30, 45, or 60 minutes of incubation (0.43 +/- 0.06, N = 24; 0.53 +/- 0.06, N = 25; 0.74 +/- 0.075, N = 21). The TGF-beta1-induced increase in Palb (0. 75 +/- 0.065, N = 15) was blocked by SOD (0.07 +/- 0.14 N = 15) or by DMTU (0.04 +/- 0.13, N = 15). Incubation of glomeruli with the carrier medium (4N HCl) in which TGF-beta1 is dissolved and SOD or DMTU alone did not affect Palb. CONCLUSION: Elevated levels of TGF-beta1 derived from glomerular or extraglomerular sources are capable of increasing glomerular Palb via superoxide and hydroxyl radicals and may lead to proteinuria in vivo.

Animals↗