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Biomedical subjects

R K Sharma

Publications and source records attributed to R K Sharma.

At least 235 records · Page 13Linked to original sources
PubMed1996

Expression of human N-myristoyltransferase in Escherichia coli. Comparison with N-myristoyltransferases expressed in different tissues.

Myristoyl CoA:protein N-myristoyltransferase catalyzes the addition of myristate to the amino-terminal glycine residue of a number of eukaryotic proteins. Escherichia coli transformed with human NMT expression construct produced high levels of N-myristoyltransferase. Using the combination of ammonium sulfate precipitation, chromatography on SP-Sepharose fast flow and fast protein liquid chromatography on Mono-S, the enzyme was purified more than 100 fold with 40% yield. The hNMT fusion protein exhibited an apparent molecular weight of 53 kDa on SDS-polyacrylamide gel electrophoresis. Upon cleavage by the Enterokinase [(Asp)4-Lys], the hNMT exhibited an apparent molecular mass of 49 kDa without loss of catalytic activity. The hNMT activity could be greatly activated severalfold with the use of Tris, SDS, ethanol and acetonitrile. The catalytic activity of hNMT was potently inhibited in a concentration dependent manner by NIP71, a bovine brain NMT inhibitory protein with a half maximal inhibition of 31.0 nM. The E. coli expressed hNMT was homogeneous and showed enzyme activity.

Acyltransferases↗
PubMed1996

Overexpression of human N-myristoyltransferase utilizing a T7 polymerase gene expression system.

Myristoyl CoA:protein N-myristoyltransferase catalyzes the addition of myristate to the amino-terminal glycine residue of a number of eukaryotic proteins. The gene encoding human N-myristoyltransferase (hNMT) was cloned into the overexpression vector pT7-7 which utilizes the T7 RNA polymerase gene expression system. The hNMT enzyme was purified to near homogeneity with more than 95% recovery using a single-step purification method involving SP-Sepharose fast flow column chromatography. The specific activity of the purified NMT was 220 nmol/min/mg of protein in the presence of oncoprotein-derived peptide substrate pp60src. The hNMT exhibited an apparent molecular weight of 49 kDa on SDS-polyacrylamide gel electrophoresis. Antibodies to Escherichia coli-expressed hNMT specifically recognize hNMT from crude bacterial lysates. The over-expressed hNMT was homogeneous and showed enzyme activity.

Acyltransferases↗
PubMed1996

Inhibition of bovine brain calmodulin-dependent cyclic nucleotide phosphodiesterase isozymes by deprenyl.

Intracellular concentrations of cyclic nucleotides is regulated by cyclic nucleotide phosphodiesterases and calmodulin-dependent cyclic nucleotide phosphodiesterases (CaMPDE), one of the most intensively studied and best characterized phosphodiesterases. In the present study, the effect of an antiparkinsonian agent, deprenyl (selegeline hydrochloride) which is believed to be a selective inhibitor of monoamine oxidase-B, on bovine brain calmodulin-dependent cyclic nucleotide phosphodiesterase (CaMPDE) isozymes have been investigated. The findings indicated that deprenyl inhibited brain 60 kDa isozyme, however the inhibition for brain 63 kDa CaMPDE was observed to a lesser extent. The inhibition of brain 60 kDa CaMPDE was overcome by increasing the concentration of calmodulin suggesting that deprenyl may be calmodulin antagonist or act specifically and reversibly on the action of calmodulin. The 60 kDa CaMPDE isozyme is predominantly expressed in brain and its inhibition can result in increased intracellular levels of cAMP. The increased intracellular levels of cAMP have a protective role for dopaminergic neurons. Therefore, deprenyl may be a valuable tool to investigate the physiological roles of brain CaMPDE isozymes in progression of Parkinson's disease and gives a new insight into the action of this drug.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1996

Use of dopamine in prevention of contrast induced acute renal failure--a randomised study.

We report the use of dopamine in renal doses (5 micrograms/kg/min) to prevent contrast induced nephropathy (CIN). Forty patients with diabetes mellitus who were undergoing coronary angiography were randomly divided into two groups. Gr I (20 patients) was infused with dopamine starting 30 min before cardiac catheterization and continued for 6 h thereafter. Gr II (20 patients) did not receive dopamine. Baseline blood chemistry was performed before catheterization and then repeated 24 h after the procedure. The mean age and sex distribution were similar in both the groups. Urograffin (76%; 120-150 ml) was used in all the cases. The mean serum creatinine and blood urea nitrogen (BUN) levels in Gr I patients before catheterization were 1.5 +/- 0.32 mg % and 16.3 +/- 8.05 mg %, respectively. The corresponding values for Gr II were 1.52 +/- 0.68 mg % and 19.6 +/- 13.4 mg %, respectively. After angiography, Gr I patients did not show significant changes in renal parameters (serum creatinine, 1.37 +/- 0.25 mg % and BUN, 14.7 +/- 5.5 mg %) while Gr II patients showed a significant rise (serum creatinine, 1.96 +/- 1.2 mg % and BUN, 23.25 +/- 12.7 mg %; P = 0.01 and P = 0.05, respectively). Ten patients in Gr II (50%) developed a 25% rise in serum creatinine levels within 24 h of injection of the contrast. None of the patients developed renal failure severe enough to warrant dialysis. Hence alterations of renal function are common after cardiac catheterization. Dopamine in renal doses appears to be an effective means to prevent deterioration in renal function induced by contrast.

Acute Kidney Injury↗
PubMed1996

Role of reactive oxygen species in male infertility.

Human spermatozoa exhibit a capacity to generate ROS and initiate peroxidation of the unsaturated fatty acids in the sperm plasma membrane, which plays a key role in the etiology of male infertility. The short half-life and limited diffusion of these molecules is consistent with their physiologic role in key biological events such as acrosome reaction and hyperactivation. The intrinsic reactivity of these metabolites in peroxidative damage induced by ROS, particularly H2O2 and the superoxide anion, has been proposed as a major cause of defective sperm function in cases of male infertility. The number of antioxidants known to attack different stages of peroxidative damage is growing, and it will be of interest to compare alpha-tocopherol and ascorbic acid with these for their therapeutic potential in vitro and in vivo. Both spermatozoa and leukocytes generate ROS, although leukocytes produce much higher levels. The clinical significance of leukocyte presence in semen is controversial. Seminal plasma confers some protection against ROS damage because it contains enzymes that scavenge ROS, such as catalase and superoxide dismutase. A variety of defense mechanisms comprising a number of anti-oxidants can be employed to reduce or overcome oxidative stress caused by excessive ROS. Determination of male infertility etiology is important, as it will help us develop effective therapies to overcome excessive ROS generation. ROS can have both beneficial and detrimental effects on the spermatozoa and the balancing between the amounts of ROS produced and the amounts scavenged at any moment will determine whether a given sperm function will be promoted or jeopardized. Accurate assessment of ROS levels and, subsequently, OS is vital, as this will help clinicians both elucidate the fertility status and identify the subgroups of patients that respond or do not respond to these therapeutic strategies. The overt commercial claims of antioxidant benefits and supplements for fertility purposes must be cautiously looked into, until proper multicentered clinical trials are studied. From the current data it appears that no single adjuvant will be able to enhance the fertilizing capacity of sperm in infertile men, and a combination of the possible strategies that are not toxic at the dosage used would be a feasible approach.

Antioxidants↗
PubMed1996

Sperm kinematics of cryopreserved normozoospermic specimens after artificial stimulation.

OBJECTIVES: Cryopreservation of semen is associated with reduced motility after thawing, resulting in a decreased pregnancy rate. Artificial stimulation of motility has been used in fresh semen samples. This study measured the effect of motility stimulants on various motion characteristics and other sperm functions using cryopreserved semen. METHODS: Frozen semen samples from healthy donors were thawed, and motility stimulants were added in vitro and incubated for 60 minutes. The percentages of motile spermatozoa in each specimen and other motion characteristics were measured. In addition, spermatozoon's viability, membrane integrity, and ability to penetrate bovine cervical mucus were studied after addition of stimulants. RESULTS: Percentage motility and all other motion characteristics improved after stimulation with pentoxifylline, caffeine, or 2-deoxyadenosine. Linearity did not significantly differ in the control samples after adding any of the stimulants. Viability, membrane integrity, and penetration ability did not improve significantly and were comparable with control values. CONCLUSIONS: Pentoxifylline, caffeine, and 2-deoxyadenosine can stimulate sperm motility and other motion characteristics. This may be beneficial in the cryopreservation of sperm from normal donors and oligozoospermic patients for use in assisted reproduction.

Caffeine↗
PubMed1996

Calmodulin-dependent protein kinase II from bovine cardiac muscle: purification and differential activation by calcium.

Calmodulin-dependent protein kinase II was purified to apparent homogeneity with a high yield from the total calmodulin-binding protein fraction of bovine cardiac muscle in a single step by gel filtration column chromatography. This procedure is simple and suitable for adaptation to large scale preparations. The purified calmodulin-dependent protein kinase has a specific enzymic activity of 2.4 mumol/min/mg when mixed histone was used as a substrate. The preparation of enzyme appears to be homogeneous when examined by SDS-PAGE. The molecular weight of the enzyme was determined to be 570 kDa by gel filtration. SDS-PAGE of the enzyme subunit showed a single protein band with an apparent molecular weight of 56 kDa. These results suggest that the calmodulin-dependent protein kinase II from bovine heart is composed of 10 identical subunits. Anti-peptide antibody raised against multifunctional calmodulin-dependent protein kinase II from rat brain showed a single immunoreactive band of 56 kDa on Western blot. These results suggested that bovine cardiac muscle calmodulin-dependent protein kinase could resemble the brain isozyme. Calmodulin-dependent protein kinase II undergoes autophosphorylation with a maximal incorporation of 1 mol of phosphate per mol of the subunit of the enzyme and the autophosphorylated enzyme remains active in the absence of Ca2+ and calmodulin. The concentration of Ca2+ required for the activation of calmodulin-dependent protein kinase II depends on the level of calmodulin in the reaction.

Acrylic Resins↗
PubMed1996

Calmodulin-dependent cyclic nucleotide phosphodiesterase in human cerebral cortex and glioblastoma multiforme.

BACKGROUND: Calmodulin-dependent cyclic nucleotide phosphodiesterase (CaMPDE) has been extensively studied and characterized in normal mammalian tissues; however very little is known about this enzyme in human brain tumors. It has been established that high levels of this enzyme exist in non-central nervous system tumors, PDE inhibitors or cAMP analogues have been used to treat them. This study has examined the levels of CaMPDE in glioblastoma multiforme from six patients and has compared these to the levels of CaMPDE in four patients with normal cerebral tissue. In addition, an enzyme immune assay method (EIA) was developed in this study for the detection of CaMPDE in human cerebral tissue. This method is proposed to be used as an adjunct to the spectrophotometric method presently utilized. This would be beneficial in cases where small tissue samples, for example in stereotactic biopsy, are available. METHODS: The CaMPDE activity and corresponding levels of expression in cerebral tissue from temporal lobectomies and both surgical extraction or stereotactic biopsy in patients with primary tumors were determined by spectrophotometric and EIA, respectively. The EIA was developed from the production of a polyclonal antibody against bovine brain 60 kDa CaMPDE isozyme. Cross reactivity of the antibody with human was confirmed using transblot and immunohistochemistry. RESULTS: Utilising the EIA, there was found to be significant reduction in both catalytic activity (p < 0.001) and in quantitative protein expression (p < 0.001) in glioblastoma multiforme from patients when compared to normal cerebral cortex. Immunoblotting experiments and immunohistochemistry demonstrated that CaMPDE in glioblastoma multiforme failed to react with a polyclonal antibody raised against bovine brain 60 kDa CaMPDE isozyme, whereas the enzyme from normal tissue reacted with antibody. CONCLUSIONS: Contrary to other studies on non-CNS tumors, the catalytic activity and the protein expression of CaMPDE is reduced in glioblastoma multiforme. The EIA method is a more sensitive in detecting CaMPDE than in the spectrophotometric method, especially when a small amount of tissue is available. Immunohistochemistry and the EIA may be useful in the future to use as markers for other types of brain tumors and not for glioblastoma multiforme as demonstrated.

Animals↗
PubMed1996

A new design of a cross-lip vermilion flap.

A new technique is described for the treatment of major vermilion defects using a cross-lip vermilion flap as a multistaged procedure. There is minimal disfigurement of the lip. The reconstruction is achieved using tissue of similar quality, texture, and color. The advantages of this method over the currently available techniques are discussed.

Esthetics↗
PubMed1996

Vascular anatomy of the galeal occipitalis flap: a cadaver study.

The vascular anatomy of the galeal occipitalis flap was studied in 10 fresh cadavers by an intraarterial dye injection technique. The scalp flap was based posteriorly, incorporating both the occipital and posterior auricular arteries. A good transmidline anastomosis was demonstrated between the two occipital arteries in the full-thickness scalp flap. However, in the isolated galeal flap, although the axial distribution of the occipital artery was maintained, fewer transmidline connections were seen between the two occipital arteries. The posterior auricular artery was consistently visualized in all dissections, and it showed good connections with the ipsilateral occipital artery. However, there were very few anastomoses between the two posterior auricular arteries across the midline. The rich anastomotic network between the occipital artery and the posterior auricular artery extended well beyond the vertex. This study showed that a large flap can be raised if both the occipital artery and the posterior auricular artery are included in its base. The occipital artery is a vessel of satisfactory size and is potentially a good vascular source for a thin galeal free flap. It is recommended that the flap be raised at the subperiosteal level for ease of dissection and protection of the vessels, which are initially in the subgaleal plane and then arborize in the galea.

Arteries↗
PubMed1996

Determination of mycobacterial phylogeny on the basis of immunological relatedness of superoxide dismutases.

Sixteen strains of cultivable mycobacteria were grown in Sauton's medium, and Mycobacterium leprae was purified from armadillo liver. Cell extracts were prepared from log-phase growths of each of the cultivable mycobacterial strains. Superoxide dismutase (SOD) enzyme was purified from all cultivable mycobacterial strains included in the study, and antibodies against purified SOD enzyme were raised in rabbits. Immunological distances (ImDs) between these anti-SOD antibodies and SOD antigens were determined by a previously described immunoprecipitation method and by a recently developed enzyme-linked immunosorbent assay (ELISA) technique. The reciprocal ImDs among mycobacterial strains were constant, reproducible and consistent by these two methods. An evolutionary tree was constructed on the basis of estimated ImDs. Except for M. duvalii and M. terrae, slowly and rapidly growing mycobacterial species appeared to be separately grouped by this analysis. Rapid growers clustered into a group which is near that of some slow-growing mycobacteria. M. avium falls almost in the middle of the evolutionary tree and the position of M. leprae was found to be between those of M. avium and M. bovis BCG. Measurement of immunological relatedness of SODs provides an alternative system with which to study the taxonomical relatedness among mycobacteria.

Animals↗
PubMed1996

Visceral leishmaniasis in a renal transplant recipient: diagnostic and therapeutic problems.

Visceral leishmaniasis is infrequently reported in renal transplant recipients. A 40-year-old renal transplant recipient developed hepatosplenomegaly and pyrexia of unknown origin 5 months after transplantation. Visceral leishmaniasis was confirmed on bone marrow examination. The usual dose of antiparasitic therapy with stibogluconate sodium failed to eradicate Leishmania donovani. High-dose conventional therapy with stibogluconate sodium for an extended period of time was successful in the treatment of a relapse of leishmaniasis.

Adult↗
PubMed1996

Evaluation of clinical and histological prognostic markers in drug-induced acute interstitial nephritis.

We analyzed the case records of 19 patients diagnosed to have drug-induced acute interstitial nephritis to assess the clinical profile and role of steroids in renal recovery, and to correlate histological features to outcome. Patients with underlying glomerular diseases, malignancy, obstructive nephropathy, or systemic infections were excluded. Nonsteroidal anti-inflammatory drugs alone accounted for 6 cases (group A), whereas antibiotics were the major offender in the remaining patients (group B). In 13/19 (69%) cases, renal failure was severe enough to require dialytic support. Overall 14/19 (74%) of the patients recovered normal renal function within 6 weeks of withdrawal of the offending drugs. Neither the extent of renal recovery nor the time required for it was altered by oral steroids. Tubular atrophy and interstitial fibrosis adversely affected renal recovery. Oliguria, tubular necrosis, interstitial edema, and the density/distribution of interstitial infiltrate did not have any effect on the rate/extent of renal recovery.

Acute Disease↗
PubMed1996

Optimal dose and duration of exposure to artificial stimulants in cryopreserved human spermatozoa.

PURPOSE: Poor sperm motility after cryopreservation is associated with infertility. For any pharmacological stimulation to be of clinical value, its effect in enhancing motility and other motion characteristics should be maintained for at least 1 hour. MATERIALS AND METHODS: Three motility stimulants (pentoxifylline, caffeine and 2-deoxyadenosine) were incubated with post-thaw semen samples from 11 healthy donors for 0, 30, 60, 120 and 180 minutes. The final concentrations used were 2.5 mM., 5 mM. and 10 mM. for pentoxifylline; 1 mM., 2 mM. and 5 mM. for caffeine, and 0.5 mM., 1 mM. and 2.5 mM. for 2-deoxyadenosine. Percent motility and changes in motion characteristics were measured on a computer assisted semen analyzer. RESULTS: Compared to controls (0 minutes, no stimulant), an immediate increase in motility and other motion parameters was noted with all 3 stimulants. All stimulants caused a significant increase in percentage motility at all periods studied (p < 0.01). Similarly, pentoxifylline increased other motion parameters at the 2.5 mM. concentration (p < 0.01), caffeine was effective in increasing curvilinear velocity, average path velocity and amplitude of lateral head displacement (p < 0.01) at 5 mM., and 1 and 2.5 mM. of 2-deoxyadenosine increased the curvilinear velocity, straight line velocity, average path velocity and amplitude of lateral head displacement (p < 0.01). Among all stimulants only 2-deoxyadenosine increased linearity only at the 1 mM. concentration. CONCLUSIONS: Our study suggests that these stimulants, when used at optimum concentrations, can maintain the improved sperm quality for durations longer than the minimum needed for fertilization. This finding may be significant in improving the poor semen quality observed after cryopreservation in oligospermic samples and in semen specimens from cancer patients.

Caffeine↗
PubMed1996

Complete congenital sternal cleft in an adult: repair by autogenous tissues.

We are reporting here a rare case of congenital complete sternal cleft in an adult of 25 years of age, with absent anterior pericardium and "diastasis recti." Successful surgical repair was achieved along with the preservation of the already existing anatomic and mechanical sanctity of thoracic cage, sternal reconstruction, and protection of the underlying heart and great vessels with a good cosmetic result using autogenous tissue i.e., iliac bone and "V-Y" myoplasty of pectoralis major muscles with their functional preservation and correction of associated anomalies.

Adult↗
PubMed1996

Sperm quality improvement in cryopreserved human semen.

PURPOSE: We determined if separation of spermatozoa (washed) on a discontinuous colloidal suspension of silica (Percoll) density gradient before cryopreservation improves post-thaw motility compared to an unprocessed (raw) cryopreserved sample. MATERIALS AND METHODS: Ten normal healthy volunteers recruited into the andrology laboratory donor program were studied. Raw and washed cryopreserved spermatozoa were compared for loss of motility with time, motion characteristics, viability and membrane integrity after incubation for 1, 6 and 24 hours. Within group comparisons were made to baseline measurements (0 hours before incubation). RESULTS: Raw and washed cryopreserved spermatozoa showed statistically significant decreases in motility and other motion characteristics after thawing. There were significant decreases in motility and other motion characteristics after incubation periods of 1, 6 and 24 hours, and significant decreases in viability and membrane integrity at 6 and 24 hours in the unprocessed spermatozoa. Although, motility and motion characteristics of washed samples decreased significantly with longer incubation periods, loss of motility with time (longevity) was greater in raw samples. Washed samples retained greater sperm motility for up to 24 hours (p < 0.03). CONCLUSIONS: Specimens prepared by Percoll separation techniques before freezing offer the possibility of selecting spermatozoa that retain motility for up to 24 hours. This finding can be of benefit for couples undergoing intrauterine insemination to achieve pregnancy.

Cell Survival↗
PubMed1996

In vivo NMR spectroscopic studies on the bioenergetic changes induced by metabolic modulators in Ehrlich ascites tumour cells.

Changes in the energy metabolism of Ehrlich ascites tumour (EAT) cells induced by treatment with 2-deoxy-D-glucose (2-DG) alone and in combination with other metabolic modulators, were studied using in vivo nuclear magnetic resonance (NMR) spectroscopy. Accumulation of 2-DG-6-phosphate (2-DG-6-P) and cellular energy status as reflected in the ratio of beta ATP to inorganic phosphate (beta-ATP/Pi) in cells could be monitored as a function of time using P-31 NMR spectroscopy. Presence of 2-DG induced a significant decrease in the levels of nucleotide triphosphates, which continued to reduce for some time even after removing 2-DG from the vicinity of cells. When administered along with 3-O-methyl-D-glucose (3-O-MG), 2-DG phosphorylated at a slower rate and the resultant 2-DG-6-P accumulated to a lesser extent than that obtained with 2-DG alone. Treatment with Photosan II, a haematoporphyrin derivative (Hpd), has been reported to increase the glucose utilisation and rate of glycolysis. Upon concomitant administration of 2-DG along with Hpd (after Hpd pretreatment for 1-2 hr), the extent of phosphorylation of 2-DG increased. The combination of 2-DG with 3-O-MG or Hpd reduced the energy status (beta-ATP/Pi) to a greater extent and the recovery was considerably less upon removal of the treatment, compared to the effects of either drug administered separately. Since metabolic depletion of ATP is known to inhibit the post-irradiation DNA repair processes, it is hypothesized that the use of these drug combinations, as adjuvants to tumour radiotherapy, should enhance the therapeutic efficacy and selectivity.

Animals↗