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Biomedical subjects

Rakesh Sharma

Publications and source records attributed to Rakesh Sharma.

At least 19 recordsLinked to original sources
PubMed2006

Nitrilase and its application as a 'green' catalyst.

Hydrolase-catalyzed reactions have been widely applied in organic synthesis. Nitrilases are an important class of hydrolase that converts naturally occurring, as well as xenobiotically derived, nitriles to the corresponding carboxylic acids and ammonia. Because of their inherent enantio- and regioselectivities and other benefits, nitrilases are attractive as 'green', mild, and selective catalysts for setting stereogenic centers in fine-chemical synthesis and enantiospecific synthesis of a variety of carboxylic acid derivatives. In this review, the literature has been surveyed to provide a comprehensive coverage of the application of nitrilases in organic synthesis. Literature has also been cited to describe the isolation and/or characterization of nitrilases and related enzymes.

Aminohydrolases↗
PubMed2006

Genome-wide prediction of G4 DNA as regulatory motifs: role in Escherichia coli global regulation.

The role of nonlinear DNA in replication, recombination, and transcription has become evident in recent years. Although several studies have predicted and characterized regulatory elements at the sequence level, very few have investigated DNA structure as regulatory motifs. Here, using G-quadruplex or G4 DNA motifs as a model, we have researched the role of DNA structure in transcription on a genome-wide scale. Analyses of >61,000 open reading frames (ORFs) across 18 prokaryotes show enrichment of G4 motifs in regulatory regions and indicate its predominance within promoters of genes pertaining to transcription, secondary metabolite biosynthesis, and signal transduction. Based on this, we predict that G4 DNA may present regulatory signals. This is supported by conserved G4 motifs in promoters of orthologous genes across phylogenetically distant organisms. We hypothesized a regulatory role of G4 DNA during supercoiling stress, when duplex destabilization may result in G4 formation. This is in line with our observations from target site analysis for 55 DNA-binding proteins in Escherichia coli, which reveals significant (P<0.001) association of G4 motifs with target sites of global regulators FIS and Lrp and the sigma factor RpoD (sigma70). These factors together control >1000 genes in the early growth phase and are believed to be induced by supercoiled DNA. We also predict G4 motif-induced supercoiling sensitivity for >30 operons in E. coli, and our findings implicate G4 DNA in DNA-topology-mediated global gene regulation in E. coli.

Conserved Sequence↗
PubMed2006

Identification and characterization of a major Zn(II) resistance determinant of Mycobacterium smegmatis.

A zinc ion-sensitive mutant of Mycobacterium smegmatis was isolated. The transposon insertion was located in zitA (MSMEG0750), a gene coding for a cation diffusion facilitator family protein. Zinc ions specifically induced expression of zitA. In silico analysis revealed that environmental and opportunistic pathogenic species contain higher numbers of cation diffusion facilitator genes than do obligate pathogens.

Anti-Bacterial Agents↗
PubMed2005

Isolation of novel lipolytic genes from uncultured bacteria of pond water.

Metagenomic libraries give access to gene pool of bacteria present in environmental samples avoiding the culture bias. A metagenomic library of pond water microbial assemblage in plasmid vector containing about 532 Mb of community DNA was prepared. Screening of a part of the unamplified library resulted in isolation of 11 unique lipolytic clones with an ability to hydrolyze tributyrin. DNA sequence of the lipolytic genes varied in G+C composition from 57% to 75%. Twelve lipolytic genes encoding proteins with 25-70% amino acid identity with proteins in the databases were identified. Ten of the encoded proteins belonged to seven known lipolytic protein families. One of the proteins was similar to recently identified esterase BioH. A lipolytic protein with high similarity to yet uncharacterized alpha/beta hydrolase protein family abh_upf0017 was identified from one of the clones. Conserved motif for lipolytic enzymes GXSXG, conserved aspartic and histidine residues were identified in this encoded protein.

Amino Acid Sequence↗
PubMed2005

Rapid in vivo Taxotere quantitative chemosensitivity response by 4.23 Tesla sodium MRI and histo-immunostaining features in N-Methyl-N-Nitrosourea induced breast tumors in rats.

BACKGROUND: Sodium weighted images can indicate sodium signal intensities from different features in the tumor before and 24 hours following administration of Taxotere. AIM: To evaluate the association of in vivo intracellular sodium magnetic resonance image intensities with immuno-biomarkers and histopathological features to monitor the early tumor response to Taxotere chemotherapy in Methyl-Nitroso-Urea induced rat xenograft breast tumors. METHODS AND MATERIALS: Methyl-Nitroso-Urea (MNU) induced rat xenograft breast tumors were imaged for sodium MRI and compared with tumor histology, immunostaining after 24 hours chemotherapy. RESULTS: Sodium MRI signal intensities represented sodium concentrations. Excised tumor histological sections showed different in vitro histological end points i.e. single strand DNA content of cell nuclei during cell cycle (G1/S-G2/M), distinct S or M histograms (Feulgen labeling to nuclear DNA content by CAS 200), mitotic figures and apoptosis at different locations of breast tumors. Necrosis and cystic fluid appeared gray on intracellular (IC) sodium images while apoptosis rich regions appeared brighter on IC sodium images. After 24 hours Taxotere-treated tumors showed lower 'IC/EC ratio' of viable cells (65-76%) with higher mitotic index; apoptotic tumor cells at high risk due to cytotoxicity (>70% with high apoptotic index); reduced proliferation index (270 vs 120 per high power field) associated with enhanced IC sodium in vivo MR image intensities and decreased tumor size (3%; p < 0.001; n = 16) than that of pre-treated tumors. IC-Na MR signal intensities possibly indicated Taxotere chemosensitivity response in vivo associated with apoptosis and different pre-malignant features within 24 hours of exposure of cancer cells to anti-neoplastic Taxotere drug. CONCLUSION: Sodium MRI imaging may be used as in vivo rapid drug monitoring method to evaluate Taxotere chemosensitivity response associated with neoplasia, apoptosis and tumor histology features.

Journal Article↗
PubMed2005

The importance of the gastrointestinal system in the pathogenesis of heart failure.

Chronic heart failure (CHF) is a multi-organ disease with increasing evidence for the involvement of the gastrointestinal (GI) system in this syndrome. In recent research, the gut has received very little attention from cardiologists as its role in the pathogenesis of cardiovascular disease is poorly understood. Intestinal ischaemia may play an important role in bacterial translocation by increasing bowel permeability. Decreased cardiac function can reduce bowel perfusion and so clearly impairs the function of the intestinal barrier. There is an increasing evidence to suggest that a 'leaky' bowel wall may lead to translocation of bacteria and/or endotoxin, which may be an important stimulus for inflammatory cytokine activation in CHF. Impaired functioning of the GI system may also contribute to malnutrition and cachexia in CHF. It is hoped that by improving our understanding of the role of the gut in cardiac disease will lead to the development of novel therapeutic strategies in the future.

Bacterial Translocation↗
PubMed2005

Molecular structure of D-hydantoinase from Bacillus sp. AR9: evidence for mercury inhibition.

Stereospecific conversion of hydantoins into their carbamoyl acid derivatives could be achieved by using the enzyme hydantoinase. Specific hydantoinases convert either the D-form or the L-form of the hydantoin and the amino acids responsible for stereospecificity have not been identified. Structural studies on hydantoinases from a few bacterial species were published recently. The structure of a thermostable D-hydantoinase from Bacillus sp. AR9 (bar9HYD) was solved to 2.3 angstroms resolution. The usual modification of carboxylation of the active-site residue Lys150 did not happen in bar9HYD. Two manganese ions were modelled in the active site. Through biochemical studies, it was shown that mercury inhibits the activity of the enzyme. The mercury derivative provided some information about the binding site of the mercuric inhibitors and a possible reason for inhibition is presented.

Amidohydrolases↗
PubMed2005

Relationship between cytokines and the embryotoxicity of hydrosalpingeal fluid.

OBJECTIVE: The exact chemical composition of hydrosalpingeal fluid is unknown. The objective of this study was to characterize cytokines in hydrosalpingeal fluid (HSF) and examine their possible role in the embryo development. STUDY DESIGN: HSF was aspirated at laparoscopic salpingectomy in eight infertile women. Levels of IL-1beta, IL-13, IL-8, IL-6 and TNF-alpha in the HSF were determined by quantitative immunoassay kits. Two-cell mouse embryos were incubated with 0, 25, 50 and 75% concentrations of HSF. The blastocyst development rate (BDR) of mouse embryos was measured at each HSF concentration. RESULT(S): The embryotoxicity of HSF was concentration dependent. An increase in the HSF concentration resulted in significant decrease in % BDR (p < 0.01). IL-1beta was present in six of the eight HSF samples with a mean (+/-SD) concentration of 0.9 +/- 0.8 pg/mL. IL-13 was not detected in any of the HSF samples. IL-8, IL-6 and TNF-alpha were detected in all samples with a mean (+/-SD) concentration of 4741.2 +/- 6554.4 pg/mL, 204.8+/-132.8 pg/ml and 12 +/- 12.8 pg/mL respectively. IL-6 was positively correlated with BDR (r = 0.53; p < 0.04). CONCLUSION(S): We demonstrated for the first time the presence of IL-1beta, IL-8, IL-6 and TNF-alpha and the absence of IL-13 in human hydrosalpingeal fluid. IL-6 was positively related to the BDR.

Adult↗
PubMed2005

Whole blood endotoxin responsiveness in patients with chronic heart failure: the importance of serum lipoproteins.

BACKGROUND: Endotoxin [lipopolysaccharide (LPS)] may be an important stimulus for cytokine release in patients with chronic heart failure (CHF). We sought to investigate the relationship between whole blood endotoxin responsiveness and serum lipoprotein concentrations. It is not known if low-dose LPS is sufficient to stimulate immune activation. METHODS AND RESULTS: Whole blood from 32 CHF patients (mean age 66+/-2 years, NYHA class 2.7+/-0.2, five female) and 11 healthy control subjects (mean age 47+/-4 years, six female) was stimulated with LPS at nine different concentrations (0.001 to 10 ng/mL), and tumor necrosis factor (TNF-alpha) release was quantified. Reference standard endotoxin at concentrations of 0, 0.6, 1, and 3 EU/ml was added to whole blood from nine CHF patients (age 64+/-9.1 years, all NYHA class II, eight male) and incubated for 6 h, the TNF-alpha production being measured. Serum lipoproteins were quantified using standard techniques. In CHF patients, there was an inverse relationship between whole blood TNF-alpha release and serum cholesterol which was strongest at 0.6 ng/mL of LPS (r=-0.53, p=0.002). A similar although weaker relationship was found for serum HDL. No such correlation was found in healthy subjects or with serum LDL (all r(2)<0.1). Low concentrations of LPS induced a stepwise increase in TNF-alpha release from whole blood to concentrations well above those seen in CHF. CONCLUSIONS: Serum lipoproteins may play an important role in regulating LPS bioactivity in CHF. Very low LPS activity, at levels seen in vivo in CHF, can induce significant TNF-alpha production ex vivo.

Aged↗
PubMed2005

Cellular endotoxin desensitization in patients with severe chronic heart failure.

BACKGROUND: Increased levels of bacterial lipopolysaccharide (LPS) have been demonstrated in chronic heart failure (CHF). LPS can induce cellular desensitization, with specific down-regulation of LPS-mediated cellular tumor necrosis factor (TNF-alpha) production which does not affect other cytokine parameters. It is not known if LPS desensitization occurs in CHF. METHODS AND RESULTS: Mononuclear cells from 24 CHF patients (mean age 70+/-2 years, age range 58 to 78 years, NYHA class 3.0+/-0.2) and 11 healthy controls (mean age 53+/-3 years, age range 39 to 75 years) were separated from venous blood and cultured for 24 h with LPS (E. coli, 0-10 ng/mL). Culture supernatants were tested for TNF-alpha and interleukin-1 receptor antagonist (IL-1RA). Patients were subgrouped into mild (n=10), moderate (n=5), and severe (n=9) CHF. Independently of age, mononuclear cells from patients with severe heart failure produced less TNF-alpha than controls (p<0.05) and patients with mild (p<0.001) or moderate CHF (p<0.05). IL-1RA release was higher for CHF patients as a group, compared with controls (p<0.05). There was no significant difference in IL-1RA release between CHF patient subgroups. CONCLUSIONS: Mononuclear cells from patients with severe heart failure produce significantly less TNF-alpha than healthy controls or patients with mild to moderate disease. Production of IL-1RA is not affected. This resembles a picture indicative of LPS desensitization occurring in patients with severe CHF.

Aged↗
PubMed2005

Oxidative stress and its implications in female infertility - a clinician's perspective.

Reactive oxygen species (ROS) have a role in the modulation of gamete quality and gamete interaction. Generation of ROS is inherent in spermatozoa and contaminating leukocytes. ROS influence spermatozoa, oocytes, embryos and their environment. Oxidative stress (OS) mediates peroxidative damage to the sperm membrane and induces nuclear DNA damage. ROS can modulate the fertilizing capabilities of the spermatozoa. There is extensive literature on OS and its role in male infertility and sperm DNA damage and its effects on assisted reproductive techniques. Evidence is accumulating on the role of ROS in female reproduction. Many animal and human studies have elucidated a role for ROS in oocyte development, maturation, follicular atresia, corpus luteum function and luteolysis. OS-mediated precipitation of pathologies in the female reproductive tract is similar to those involved in male infertility. OS influences the oocyte and embryo quality and thus the fertilization rates. ROS appears to play a significant role in the modulation of gamete interaction and also for successful fertilization to take place. ROS in culture media may impact post-fertilization development, i.e. cleavage rate, blastocyst yield and quality (indicators of assisted reproduction outcomes). OS is reported to affect both natural and assisted fertility. Antioxidant strategies should be able to intercept both extracellular and intracellular ROS. This review discusses the sources of ROS in media used in IVF-embryo transfer and strategies to overcome OS in oocyte in-vitro maturation, in-vitro culture and sperm preparation techniques.

Antioxidants↗
PubMed2004

MRI of coronary artery atherosclerosis in rabbits: Histopathology-MRI correlation and atheroma characterization.

BACKGROUND AND OBJECTIVES: We report in vivo magnetic resonance imaging (MRI) characteristics and histopathology correlation of the thrombus formation in atherosclerosis the rabbit animal model. DESIGN AND METHODS: Atherosclerosis was induced in white male rabbits with vegetable ghee followed oxidized diet. Baseline MRI of atherosclerosis-recruited rabbits was done and later animals were used for atheroma histopathology characterization. Contiguous cross-sectional T2-weighted fast spin echo MRI images were compared by coronary histopathology. In all animals, coronary aortic wall thickening and atheroma size was measured using MRI. RESULTS: MRI images and digitized histological sections confirmed intraluminal thrombus in 6 (67%) of the 9 animals. MRI data showed correlation with the histopathology for aortic wall thickness (R2 = 0.82, P < 0.0001), lumen area (R2 = 0.88, P < 0.0001) and plaque size (R2 = 0.77, P < 0.0001). Optimized TE and TR parameters and multicontrast enhancement generated better MRI visibility of vulnerable plaque components. The MRI data evaluated % stenosis, plaque burden. Frequency of plaques, plaque height in aorta and coronary artery atheroma was also assessed by histology. In vivo, MRI determined the presence and size of the thrombus in this animal model of atherosclerosis and histopathology defined the plaque disruption. CONCLUSION: The combination of in vivo MRI and comparison with histopathology images of rabbit coronary thrombus may be a research tool for understanding of the pathogenesis of acute coronary plaques.

Journal Article↗
PubMed2004

Physiological basis and image processing in functional magnetic resonance imaging: neuronal and motor activity in brain.

Functional magnetic resonance imaging (fMRI) is recently developing as imaging modality used for mapping hemodynamics of neuronal and motor event related tissue blood oxygen level dependence (BOLD) in terms of brain activation. Image processing is performed by segmentation and registration methods. Segmentation algorithms provide brain surface-based analysis, automated anatomical labeling of cortical fields in magnetic resonance data sets based on oxygen metabolic state. Registration algorithms provide geometric features using two or more imaging modalities to assure clinically useful neuronal and motor information of brain activation. This review article summarizes the physiological basis of fMRI signal, its origin, contrast enhancement, physical factors, anatomical labeling by segmentation, registration approaches with examples of visual and motor activity in brain. Latest developments are reviewed for clinical applications of fMRI along with other different neurophysiological and imaging modalities.

Algorithms↗
PubMed2004

Chemosensitivity assay in mice prostate tumor: Preliminary report of flow cytometry, DNA fragmentation, ion ratiometric methods of anti-neoplastic drug monitoring.

Flow cytometry, DNA fragmentation, ion ratiomateric analysis and NMR peaks characterized drug chemosensitivity of antineoplastic drugs. Hypotheses were: 1. The chemosensitive effect of different cancer cell lines is characteristic; 2. DNA fragmentation, ion ratiometric analysis suggest apoptosis status of tumor cells. METHODS: PC-3 cell lines were compared with DU-145, LNCaP cell lines in culture for the [Na]i and [Ca]i ion sensing dyes, cell death, NMR peaks and apoptosis staining for chemotherapeutic action of different drugs. RESULTS: DNA fragmentation, ratiometric ions and fluorescence endlabelling plots were characteristic for cell lines and drug response. 31P-23Na NMR spectra showed characteristic high phospho-choline and sodium peaks. CONCLUSION: Flow cytometry, DNA fragmentation, ion ratiometric methods and NMR peaks indicated apoptosis and offered in vivo drug monitoring method.

Journal Article↗
PubMed2004

Haemoglobin predicts survival in patients with chronic heart failure: a substudy of the ELITE II trial.

AIMS: The correction of anaemia in chronic heart failure (CHF) has been suggested to be associated with an improvement in symptoms and cardiac function. We aimed to investigate the relationship between the concentration of haemoglobin and survival in CHF. METHODS AND RESULTS: We analysed haemoglobin concentrations in 3044 patients recruited in the Evaluation of Losartan In The Elderly (ELITE II) trial. Patients of mean age 71.5 +/- 6.8 years (+/-SD) and New York Heart Association (NYHA) class 2.5 +/- 0.6 were enrolled from June 1997 to May 1998 and followed-up for survival (range 1-780 days, median 551). In univariate analysis, age, NYHA class, serum creatinine, left ventricular ejection fraction (all P<0.0001) and sex (P=0.046) all predicted survival. Haemoglobin as a continuous variable for all patients was not a significant prognostic marker (P=0.26). However, sub-dividing patients according to 1.0 g/dL increments of haemoglobin revealed that the survival relationship was non-linear. The results from the polynomial regression suggest that the optimal interval is a symmetric one centred around 14.5 g/dL. This was independent of age, sex, NYHA class, left ventricular ejection fraction, creatinine, co-existing chronic obstructive pulmonary disease and treatment allocation (P<0.001). There was a minor fall in plasma haemoglobin at the 12-month follow-up (mean change for all patients 0.3 +/- 2.2 g/dL, P<0.0001), with no difference between captopril and losartan groups (P>0.3). CONCLUSION: Haemoglobin is an independent predictor of mortality in CHF patients, with anaemic and polycythaemic patients having the worst survival.

Adrenergic beta-Antagonists↗
PubMed2004

Increased concentrations of lipoprotein(a), circadian rhythms and metabolic reactions evoked by acute myocardial infarction, associated with acute reactions in relation to large breakfasts.

Of 54 patients with acute coronary artery disease (CAD) that were included in this study, 41 patients had acute myocardial infarction (AMI), five patients possible MI, four patients unstable angina and the remaining four angina pectoris. The control subjects (n = 85) were randomly selected from the general population of the city of Moradabad of similar age range after exclusion for CAD (n = 9), diabetes (n = 6) and excess intake of trans fatty acids (n = 20). The incidence of lipoprotein(a) excess (> 30 mg/dl; 42.6 vs 24.7%; P < 0.05) and mean concentration of lipoprotein(a) (Lp[a], 6.4 mg/dl, 95% confidence interval: 2.8-10.5; P < 0.05) was significantly greater in the acute CAD group compared with control subjects. Mean total cholesterol and triglycerides were significantly higher and mean nitrite level lower in the study group as compared with the control group.There was a significant greater incidence of cardiac events in the second quarter of the day compared with the fourth quarter. Lp(a), triglycerides, blood glucose, plasma insulin, malondialdehyde, diene conjugates, TBARS and TNF-alpha and IL-6 levels, which were significantly greater during the acute phase, showed a significant decline and serum nitrite and coenzyme Q demonstrated an increase at 4 weeks of follow-up when the acute reactions evoked by MI had been controlled. Large breakfasts were a predisposing factor for cardiac events in the second quarter of the day and it was significantly associated with metabolic reactions. The findings indicate that acute reactions as a result or as circadian rhythms appear to be important in the pathogenesis of AMI-associated complications and that a large breakfast in association with nitrite deficiency may further trigger the circadian rhythms. However, more studies in a larger number of subjects would be necessary in order to confirm our findings.

Acute Disease↗
PubMed2003

The relationship between cholesterol and survival in patients with chronic heart failure.

OBJECTIVES: We sought to describe the relationship between cholesterol and survival in patients with chronic heart failure (CHF). BACKGROUND: Increasing lipoprotein levels are a cardiovascular risk factor. In patients with CHF, the prognostic value of endogenous lipoproteins is not fully clarified. METHODS: A group of 114 patients with CHF recruited to a metabolic study was followed for a minimum of 12 months (derivation study). The results were applied to a second group of 303 unselected patients with CHF (validation study). The relationship between endogenous lipoproteins and survival was explored. RESULTS: In the derivation study, survival at 12 months was 78% (95% confidence interval [CI] 70% to 86%) and 56% (95% CI 51% to 62%) at 36 months. Increasing total serum cholesterol was a predictor of survival (hazard ratio 0.64, 95% CI 0.48 to 0.86), independent of the etiology of CHF, age, left ventricular ejection fraction, and exercise capacity. Receiver-operating characteristic curves demonstrated a best cut-off value of </=5.2 mmol/l (200.8 mg/dl) as being the best predictor of mortality at 12 months (sensitivity 80.0%, specificity 62.9%). In the validation population, one-year survival was 88% (95% CI 84 to 91%) and three-year survival was 68% (95% CI 63 to 73%). The chance of survival increased 25% for each mmol/l increment in total cholesterol. Survival rates above and below the cut-off value for cholesterol in patients with ischemic heart disease (n = 181) were 92% (95% CI 89 to 94) versus 75% (95% CI 64 to 85%) at one year and 72% (95% CI 67 to 76%) versus 50% (95% CI 43 to 56%) at three years. CONCLUSIONS: In patients with CHF, lower serum total cholesterol is independently associated with a worse prognosis.

Age Factors↗