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Biomedical subjects

R K Sharma

Publications and source records attributed to R K Sharma.

At least 199 records · Page 11Linked to original sources

Immunocytochemical localisation of neuronal nitric oxide synthase in developing and transplanted rabbit retinas.

Nitric oxide (NO) acts as a modulator of neuronal transmission in mature neuronal systems, including the retina. Recently, NO has also been suggested to have a trophic function during development. We examined immunocytochemically the distribution of NO-producing cells in developing and transplanted rabbit retinas. An antibody detecting the neuronal isoform of its biosynthetic enzyme, nitric oxide synthase (NOS), was used on normal developing retinas [starting at embryonic day (E) 15] and on rabbit retinal transplants after various survival times (1-139 days after surgery). Weakly stained cell bodies were first observed in the proximal margin of the neuroblastic layer at E 29. Stained processes projecting towards a developing inner plexiform layer were also visible at this time point. Immunoreactive cells were located at later stages in the innermost part of the inner nuclear layer and in the ganglion cell layer, and are likely to correspond mainly to amacrine cells. NOS-labelled cells were also found in retinal transplants. The first NOS-labelled cells appeared, as in normal developing retinas, in ages corresponding to E 29 and were still detected in transplants corresponding to postnatal day 123. NOS-labelled cells were seen in areas between rosettes, where amacrine cells are located. NOS-labelled processes were at times seen to project for long distances, forming very distinct plexuses. NOS-containing amacrine cells thus appear both in the transplants and in developing retinas in the embryonic stages, long before synaptic function involving these cells can be expected, suggesting a role for NO not only in neuromodulation but also in retinal development.

Age Factors↗

Factors associated with the quality before freezing and after thawing of sperm obtained by microsurgical epididymal aspiration.

OBJECTIVE: To identify whether the cause, site of ductal obstruction, and characteristics of fluid aspirates are associated with the cryosurvival and fertility after thawing of sperm obtained during reconstruction of the excurrent ducts with microsurgical epididymal sperm aspiration, vasal sperm aspiration, or both. DESIGN: Prospective study. SETTING: Andrology center at a tertiary care institution. PATIENT(S): Men undergoing reconstruction of the excurrent duct and sperm aspiration (n = 42) or microsurgical epididymal sperm aspiration (n = 11). INTERVENTION(S): Sperm were tested for an association with the cause and site of obstruction. Fertilization and pregnancy rates after sperm aspiration and intracytoplasmic sperm injection (ICSI) were evaluated for fresh and frozen aspirates. MAIN OUTCOME MEASURE(S): Motile sperm count and percentage motility after thawing. RESULT(S): The motile sperm count before freezing was significantly higher in the caput epididymis than in the corpus. The motile sperm count before freezing was related inversely to the distance from the caput where the sperm were aspirated. Sperm from clear and opaque fluid aspirates had better percent motility than those from cloudy and creamy fluid aspirates. High fertilization and pregnancy rates were achieved using both fresh and frozen epididymal sperm. CONCLUSION(S): None of the factors studied was associated with cryosurvival of aspirated epididymal or vasal spermatozoa. Because motility is low after thawing, these specimens are best used with ICSI.

Adult↗

Importance of reactive oxygen species in the peritoneal fluid of women with endometriosis or idiopathic infertility.

OBJECTIVE: To determine whether reactive oxygen species in peritoneal fluid might be a factor in infertility. DESIGN: Prospective study. SETTING: Andrology laboratory and gynecology clinic at a tertiary care facility. PATIENT(S): Women with endometriosis (n = 15) or idiopathic infertility (n = 11) who underwent laparoscopy for infertility. Patients undergoing tubal ligation served as controls (n = 13). INTERVENTION(S): Aspiration of peritoneal fluid. MAIN OUTCOME MEASURE(S): Reactive oxygen species levels, presence of polymorphonuclear granulocytes, and leukocyte distribution in peritoneal fluid. RESULT(S): Reactive oxygen species were present in the peritoneal fluid of patients with endometriosis, idiopathic infertility, and tubal ligation. Levels of reactive oxygen species did not show a statistically significant difference between patients with endometriosis and the control group in either unprocessed or processed (cell-free) peritoneal fluid, but did differ significantly between patients with idiopathic infertility and controls in processed peritoneal fluid. Polymorphonuclear granulocytes (> 1 x 10(6)/mL) were not present in the peritoneal fluid of any patient. Macrophage concentrations of peritoneal fluid did not differ significantly between controls and patients with endometriosis or idiopathic infertility. CONCLUSION(S): Reactive oxygen species in the peritoneal fluid may not affect fertility directly in women with endometriosis; however, they may have a role in patients with idiopathic infertility.

Adult↗

Seminal reactive oxygen species and sperm motility and morphology in men with spinal cord injury.

OBJECTIVE: To assess the generation of reactive oxygen species and its relation to semen characteristics in men with spinal cord injury. DESIGN: Cross-sectional study. SETTING: Andrology laboratory at a tertiary care facility and research laboratory at a major medical center. PATIENT(S): Men with spinal cord injury and normal men. INTERVENTION(S): Collecting ejaculates from men with spinal cord injury by electroejaculation and vibratory stimulation and from normal men by masturbation. MAIN OUTCOME MEASURE(S): Measurement of reactive oxygen species before and after stimulation with 50 microM N-formyl-methionyl-leucylphenylalanine (FMLP) and 100 nM 12-myristate 13-acetate phorbol ester (PMA), white blood cell (WBC) concentration, sperm motility and morphology, and ejaculation method. RESULT(S): Compared with controls, levels of reactive oxygen species in men with spinal cord injury were significantly higher in unstimulated, f-MLP-stimulated, and PMA-stimulated specimens. The WBC concentration was significantly elevated in patients with spinal cord injury. Sperm motility in men with spinal cord injury was inversely related to the level of reactive oxygen species. The percentage of morphologically normal spermatozoa was significantly lower in men with spinal cord injury. Levels of seminal reactive oxygen species did not differ when comparing specimen type (antegrade versus retrograde) or method of ejaculation in men with spinal cord injury. CONCLUSION(S): Men with spinal cord injury had elevated levels of reactive oxygen species in their semen. Levels of reactive oxygen species were negatively correlated with sperm motility. Levels of reactive oxygen species were independent of the method of ejaculation or the type of specimen.

Adult↗

Effects of cancer on spermatozoa quality after cryopreservation: a 12-year experience.

OBJECTIVE: To determine whether type of cancer and response to treatment was related to prefreeze or post-thaw semen quality and to predict post-thaw sperm motility from prefreeze motility. DESIGN: Retrospective study. SETTING: Tertiary care institution. PATIENT(S): One hundred six cancer patients cryopreserving their semen specimens. INTERVENTION(S): Computer-assisted semen analysis was performed before and after cryopreservation on each patient specimen. MAIN OUTCOME MEASURE(S): The relationship of sperm motility and motion characteristics to type of cancer and patient's response to treatment. RESULT(S): Prefreeze and post-thaw semen quality did not differ between patients presenting with testicular cancer and Hodgkin's disease. Patients with leukemia or advanced soft tissue cancer had a higher prefreeze and post-thaw motility and higher total and motile sperm count than testicular and Hodgkin's disease patients. A prefreeze sperm motility of > or = 15% could predict a post-thaw motility of > 10%. CONCLUSION(S): Prefreeze or post-thaw semen quality in cancer patients is not affected (except the prefreeze motile sperm count within the testicular cancer patients) by the type of disease. Prefreeze motility can predict post-thaw motility. Cryopreservation of semen should be offered to cancer patients irrespective of the type of disease.

Adult↗

Demonstration and purification of a myristoyl-CoA binding protein from bovine cardiac muscle.

Protein myristoylation refers to the co-translational addition of myristoyl group to an amino-terminal glycine residue of a protein by the enzyme myristoyl-CoA:protein N-myristoyltransferase (NMT). The myristoylation reaction depends on the availability of the cellular pools of coenzyme A and myristate and their subsequent formation of myristoyl-CoA, the substrate of NMT. In the present study a myristoyl-CoA binding protein (MCBP) was purified using various column chromatographies: hydroxylapatite, DEAE Sepharose CL-6B and Sephacryl S-300 gel filtration. The purified protein exhibited an apparent molecular mass of 50 kDa on SDS-polyacrylamide gel electrophoresis. Incubation of protein with [1-(14)C]myristoyl-CoA followed by denaturing gel electrophoresis, fluorography and treatment with hydroxylamine yielded results that are highly suggestive of a covalent ester-linked acyl-protein complex. This complex formation was not observed in the crude cytosolic fractions. The addition of cytosolic fraction to a progressing acyl-protein complex, resulted in deacylation suggesting a role for thioesterase or/proteinases in the regulation of the acylation reaction in bovine cardiac muscle. The acyl-protein complex formation was not inhibited by NIP71, a 71 kDa NMT inhibitory protein from bovine brain.

Acyl Coenzyme A↗

Effect of lipid peroxidation on cryopreserved semen quality in patients with testicular or nontesticular cancer.

OBJECTIVES: Although pre-freeze and post-thaw semen quality in patients with cancer is poor, it is not clear whether lipid peroxidation affects semen quality. This study assessed (1) whether poor semen quality in patients with cancer is caused by lipid peroxidation, and (2) whether patient age or sperm motility is associated with lipid peroxidation. METHODS: Lipid peroxidation was measured by determining malonaldehyde levels using the thiobarbituric acid method. Malonaldehyde levels were measured in cryopreserved semen specimens from patients with testicular (n = 15) or nontesticular cancer (n = 16) and normal men (control subjects, n = 20). A computer-assisted semen analyzer was used to determine the sperm concentration and motility before and after cryopreservation. RESULTS: Malonaldehyde levels did not differ in frozen-thawed semen specimens among patients with testicular (25.90 +/- 1.00 nM/10(8) sperm/hr) or nontesticular cancer (24.48 +/- 1.66 nM/10(8) sperm/hr), and control subjects (24.86 +/- 1.43 nM/10(8) sperm/hr). Malonaldehyde levels did not correlate with post-thaw sperm motility or patient age in patients with testicular or nontesticular cancer. Post-thaw sperm motility from patients with testicular or nontesticular cancer was significantly lower than that in normal subjects. CONCLUSIONS: The poor post-thaw semen quality in patients with testicular or nontesticular cancer is not related to lipid peroxidation but may be caused by other factors such as sperm membrane stress induced during the freeze-thaw process.

Adolescent↗

Effect of cryopreservation and sperm concentration on lipid peroxidation in human semen.

OBJECTIVES: To assess (1) the effect of increased lipid peroxidation on sperm membrane damage that occurs during the freeze-thaw process of cryopreservation; (2) the effect of sperm concentration on lipid peroxidation in normal fertile men; and (3) the relation between sperm motility, morphology, and lipid peroxidation. METHODS: Lipid peroxidation was measured as levels of malonaldehyde by the thiobarbituric acid method. Malonaldehyde levels in fresh and frozen-thawed semen specimens from normal donors (n = 20) were compared at a sperm concentration of 20 x 10(6)/mL. Sperm concentrations of 5, 10, 20, and 40 x 10(6)/mL were used to assess the effect of sperm concentration on malonaldehyde levels in frozen specimens. A computer-assisted semen analyzer was used to analyze sperm concentrations and motility. Comparison of the effect of TEST-yolk buffer, glycerol, and Ham's F-10 buffer on lipid peroxidation was studied in fresh semen from normal fertile men (n = 5). RESULTS: Malonaldehyde levels did not differ between fresh and cryopreserved semen specimens (P = 0.99). There was no difference in malonaldehyde levels from TEST-yolk buffer, glycerol, and Ham's F-10 buffer. As sperm concentrations increased, so did malonaldehyde levels, from 1.21 +/- 0.05 nM/hr at 5 x 10(6)/mL to 3.50 +/- 0.46 nM/hr at 40 x 10(6)/mL (P = 0.000007). Sperm motility, morphology, and malonaldehyde levels in freeze-thaw specimens were not correlated before or after removal of the cryoprotective medium. CONCLUSIONS: Malonaldehyde levels do not differ between fresh and frozen semen in normal men. Cryopreservation-induced membrane damage is not related to lipid peroxidation but may be associated with stress induced during freezing and thawing. Malonaldehyde levels increase as sperm concentrations increase. There is no correlation between sperm motility, morphology, and malonaldehyde levels.

Cryopreservation↗

Laryngeal fistula following electrical burn.

A rare case of electrical burn resulting in laryngeal fistula is presented. The fistula manifested about 3 weeks postburn. This was successfully managed by using a pectoralis major myocutaneous flap. A 1 year follow-up of the patient is presented.

Accidents, Home↗

The bovine alpha 2D-adrenergic receptor gene: structure, expression in retina, and pharmacological characterization of the encoded receptor.

This report describes cloning of the bovine alpha 2D-adrenergic receptor (alpha 2D-AR) gene and determination of the transcription start site, unequivocal presence of the alpha 2D-AR transcript in the retina, and pharmacological characteristics of the encoded product. Furthermore, expression of the gene in selected bovine tissues has also been scrutinized. A genomic clone was isolated from lambda EMBL3 library and a 3 kb fragment was subcloned and sequenced. This fragment contained the putative TATA box and the coding region. The encoded receptor was transiently expressed in COS cells. The recombinant receptor expressed pharmacological characteristics almost identical to the wild-type bovine retinal receptor, which were typical of the alpha 2D-AR subtype. RNase protection analysis confirmed the expression of the gene in the retina. The bovine receptor was structurally close to its rat analogue which also encodes the alpha 2D-AR, but, the highest homology was observed with the porcine receptor expressing alpha 2A-AR pharmacological characteristics. Certain structural features of the bovine gene were unique to itself and not shared by any other alpha2-AR subtype. Among the tissues tested using reverse transcriptase-polymerase chain reaction (RT-PCR), the alpha 2D-AR message was the most abundant in retina, followed by the brain and olfactory lobe. Thus, the availability of the bovine receptor gene probe will become an important additional tool in the elucidation of molecular mechanisms behind the alpha 2D-AR physiology in neurosensory processes such as those occurring in the eye and the brain.

Amino Acid Sequence↗

A role for amino acid residues in the third cytoplasmic loop in defining the ligand binding characteristics of the alpha2D-adrenergic receptor.

In this report, 5 amino acid residues (aa) in the third cytoplasmic loop of the alpha 2D-adrenergic receptor are identified which (individually or together) alter its ligand-binding characteristics. An important structural discrepancy exists in the third cytoplasmic loop of the alpha 2D-ARs encoded by the rat cDNA and the rat gene--five aa are different. The newly identified bovine receptor as well as the mouse receptor contained the 5 aa identical to that encoded by the rat cDNA. Site-directed mutation of these residues to those of the rat gene encoded receptor resulted in alteration of binding characteristics: significant changes in the ability of the mutant receptor to bind to a number of agonists and antagonists were observed--ranging from a decrease by half in the case of oxymetazoline, to near total loss of binding in the case of prazosin. Thus, the mutant receptor was no longer pure alpha 2D-AR. This indicated a hitherto unrealized role of the third cytoplasmic loop in defining the ligand-binding characteristics of the receptor, and also suggested that the rat gene sequence was most probably in error.

Amino Acid Sequence↗

In vitro phosphorylation of bovine cardiac muscle high molecular weight calmodulin binding protein by cyclic AMP-dependent protein kinase and dephosphorylation by calmodulin-dependent phosphatase.

High molecular weight calmodulin binding protein (HMWCaMBP) is one of the major proteins expressed in bovine cardiac muscle. In this study, we report the phosphorylation and dephosphorylation of HMWCaMBP in vitro with a view to understand the function of this protein. The HMWCaMBP was phosphorylated by cAMP-dependent protein kinase with the incorporation of 2.30 mol of phosphate/mol of protein in the presence of EGTA. When phosphorylation was carried out in the presence of Ca2+/calmodulin (CaM), the incorporation of phosphate was reduced to 1.40 mol of phosphate/mol of protein. The decrease in the stoichometry of phosphorylation by Ca2+/CaM appears to be substrate directed i.e. due to the interaction of Ca2+/CaM with HMWCaMBP. The phosphorylated HMWCaMBP was unable to compete for free CaM in a CaM-dependent cyclic nucleotide phosphodiesterase (CaMPDE) assay. These results suggest that the phosphorylation sites may reside in or in proximity to the CaM-binding domain on HMWCaMBP since phosphorylated HMWCaMBP did not inhibit CaMPDE activity. HMWCaMBP was dephosphorylated by CaM-dependent phosphatase, calcineurin.

3',5'-Cyclic-AMP Phosphodiesterases↗

Effects of cryopreserved semen quality and timed intrauterine insemination on pregnancy rate and gender of offspring in a donor insemination program.

PURPOSE: Our purpose was to study the relationship among cryopreserved donor semen quality, pregnancy rates, and preconception sex selection after intrauterine insemination. METHODS: We reviewed the records of the 203 women in our donor insemination program from 1987 to 1994 who became pregnant after more than one insemination cycle and had no female-factor infertility. They were categorized according to the number of cycles required for pregnancy. Semen samples from 54 donors were analyzed before freezing and after thawing. Specimens resulting in pregnancy were compared to specimens from the same donor that did not. Semen characteristics were compared to gender of the child. RESULTS: Two hundred fifty two-women became pregnant of the 422 who were enrolled. The pregnancy rate per cycle was 13%. Semen quality was not related to pregnancy outcome or offspring gender. However, more male children (101 vs 83) were born. CONCLUSIONS: Semen characteristics in good-quality cryopreserved donor semen do not affect pregnancy rate or offspring gender.

Cryopreservation↗

Photoreceptor guanylate cyclases: a review.

Almost three decades of research in the field of photoreceptor guanylate cyclases are discussed in this review. Primarily, it focuses on the members of membrane-bound guanylate cyclases found in the outer segments of vertebrate rods. These cyclases represent a new guanylate cyclase subfamily, termed ROS-GC, which distinguishes itself from the peptide receptor guanylate cyclase family that it is not extracellularly regulated. It is regulated, instead, by the intracellularly-generated Ca2+ signals. A remarkable feature of this regulation is that ROS-GC is a transduction switch for both the low and high Ca2+ signals. The low Ca2+ signal transduction pathway is linked to phototransduction, but the physiological relevance of the high Ca2+ signal transduction pathway is not yet clear; it may be linked to neuronal synaptic activity. The review is divided into eight sections. In Section I, the field of guanylate cyclase is introduced and the scope of the review is briefly explained; Section II covers a brief history of the investigations and ideas surrounding the discovery of rod guanylate cyclase. The first five subsections of Section III review the experimental efforts to quantify the guanylate cyclase activity of rods, including in vitro and in situ biochemistry, and also the work done since 1988 in which guanylate cyclase activity has been determined. In the remaining three subsections an analytical evaluation of the Ca2+ modulation of the rod guanylate cyclase activity related to phototransduction is presented. Section IV deals with the issues of a biochemical nature: isolation and purification, subcellular localization and functional properties of rod guanylate cyclase. Section V summarizes work on the cloning of the guanylate cyclases, analysis of their primary structures, and determination of their location with in situ hybridization. Section VI summarizes studies on the regulation of guanylate cyclases, with a focus on guanylate cyclases activating proteins. In Section VII, the evidence about the localization and functional role of guanylate cyclases in other retinal cells, especially in "on-bipolar" cells, in which guanylate cyclase most likely plays a critical role in electrical signaling, is discussed. The review concludes with Section VIII, with remarks about the future directions of research on retinal guanylate cyclases.

Animals↗

Mechanisms of inhibition of calmodulin-stimulated cyclic nucleotide phosphodiesterase by dihydropyridine calcium antagonists.

Calmodulin-dependent cyclic nucleotide phosphodiesterase (CaMPDE) is one of the key enzymes involved in the complex interaction between the cyclic nucleotide and Ca2+ second-messenger systems. CaMPDE exists as tissue-specific isozymes, and initially these isozymes were designated according to their respective subunit molecular mass. A variety of pharmacological agents have been used to inhibit CaMPDE, and this inhibition occurs mostly via Ca2+-dependent association with the proteins. We have examined the effect of dihydropyridine Ca2+-channel blockers felodipine and nicardipine on CaMPDE. The results suggest that the 63-kDa (PDE 1B1) and 60-kDa (PDE 1A2) CaMPDE isozymes are inhibited by felodipine and nicardipine by partial competitive inhibition and that these two Ca2+ antagonists appear to counteract each other. This study further demonstrates the existence of a specific site, distinct from the active site on CaMPDE, that exhibits high-affinity binding of these drugs. Felodipine and nicardipine have similar affinities for 60-kDa CaMPDE isozymes but bring about different levels of enzyme inhibition, suggesting the possibility of designing specific drugs that can protect the enzyme from inhibition by dihydropyridine Ca2+-channel blockers.

3',5'-Cyclic-AMP Phosphodiesterases↗

Typhoid enteric perforation.

BACKGROUND: Perforation of the bowel is the most serious complication of typhoid fever. The role of early limited surgery in managing these patients needs to be assessed. METHODS: The records of 110 cases of typhoid enteric perforation treated at JLN Hospital, Ajmer between 1990 and 1995 were reviewed. RESULTS: A total of 42.7% of the patients were in the 21-30-year age group, and 83.6% were male. All patients presented with the classic features of typhoid enteric perforation. A total of 83.6% were operated on within 36 h of perforation. Surgical management consisted of primary closure of the perforation (74.5%), closure with omental graft (14.5%), resection and anastomosis (3.6%), and only drainage (7.3%). A total of 79.1% of patients developed wound infection and 10% of patients developed faecal fistula. The overall mortality rate was 16.4%. Increasing the time interval between perforation and operation significantly increased the mortality (P < 0.05). The mortality was least with early primary closure of the perforation. Patients with postoperative faecal fistula had higher mortality rates (P < 0.001). CONCLUSIONS: Early limited surgery with thorough peritoneal lavage provides optimal results, faecal fistula is a grave complication, and the use of the McBurney incision may provide better results in terms of subsequent wound healing.

Adolescent↗

Influence of technique and transplantation site on rosette formation in rabbit retinal transplants.

In order to determine mechanical and host-graft related interactions in the histogenesis of retinal transplants, a new technique for transplanting flat and comparatively large pieces of embryonic rabbit retina into adult rabbit eyes was elaborated. With the procedure, free-floating grafts in the epiretinal space survive, develop and differentiate largely without rosette formation, suggesting that the dissection and transplantation procedure is adequate for obtaining a normal development. On the other hand, subretinal transplants mature at an apparently faster pace than epiretinal transplants, but do not become regularly laminated. Outer segments do not develop well in the epiretinal transplants, whereas they do so in the subretinal ones, suggesting host-graft interactions by means of yet unknown diffusible factors.

Animals↗