Primary mesenteric fibromatosis.
Primary mesenteric fibromatosis is a rare condition. We report a 46-year-old man with this condition.
Fibromatosis, Abdominal↗
Biomedical subjects
R K Sharma
Publications and source records attributed to R K Sharma.
Suitability of the hypo-osmotic swelling test for assessing the viability of cryopreserved sperm.
OBJECTIVE: To determine whether the hypo-osmotic swelling test can assess viability of frozen spermatozoa. DESIGN: Fresh semen specimens were divided into two aliquots: one was not processed and the other was processed by swim-up. Both aliquots were then cryopreserved by the liquid nitrogen vapor method. SETTING: Andrology laboratory at a tertiary care facility. PATIENT(S): Eleven volunteers with proven fertility. INTERVENTION(S): The hypo-osmotic swelling test and the sperm viability assessment by Hoechst 33258 (Sigma Chemical Co., St. Louis, MO) and eosin-nigrosin strains were performed in each aliquot. MAIN OUTCOME MEASURE(S): The results of hypo-osmotic swelling test as indicated by swelling of sperm tails were compared with the viability results obtained by Hoechst 33258 stain before freezing and by Hoechst-33258 and eosin-nigrosin stains after freezing. RESULT(S): The hypo-osmotic swelling test and Hoechst 33258 viability results were correlated highly in unprocessed and processed fresh specimens (r = 0.95). After cryopreservation, there was no correlation between hypo-osmotic swelling test and Hoechst 33258 or between hypo-osmotic swelling test and eosin-nigrosin. Prefreeze sperm processing did not influence the post-thaw results. Viability assessed by Hoechst 33258 and eosin-nigrosin was well correlated (r = 0.72). CONCLUSION(S): The hypo-osmotic swelling test can accurately evaluate viability in fresh human spermatozoa but not in cryopreserved ones. Thus, this test cannot be used to select viable cells in cryopreserved samples for assisted reproductive procedures, such as intracytoplasmic sperm injection.
Aniline Compounds↗
The prevalence of transfusion transmitted diseases in renal transplant recipients.
Majority of renal transplant patients have history of blood transfusion. Out of a total of 120 renal transplant patients, 20 (16.6%) patients showed raised alanine aminotransferase (ALT) levels (> 45 IU) on 2 or more occasions at 2 weeks interval. This study was undertaken to estimate the risk of transfusion transmitted diseases (TTD) in these 20 renal transplant patients. Another 200 voluntary blood donors were also included as control. Both subject groups were screened by ELISA for HBsAg, anti HBc (IgG & IgM), anti HCV, anti CMV (IgM), anti HIV and VDRL tests for syphilis. A total of 11 (55%) patients and 3 (1.5%) blood donors showed the evidence of HBsAg positivity (p < 0.01). Anti HBc antibody was present in 12 (60%) patients and 39 (19.5%) donors which was significantly (p < 0.01) different. Anti HCV antibody was detected in 5 patients and in one blood donor (p < 0.01). However, anti CMV antibody was present in 4 (20%) patients and 3 (1.5%) donors, respectively. There was no evidence of HIV and syphilis infection in both these groups. High incidence of hepatitis markers were observed in renal transplant patients.
Biomarkers↗
Blunt trauma abdomen: a study of 63 cases.
63 cases of blunt abdominal trauma were studied. It was more common in males and in the age group 21-30 years. Majority of the injuries were due to automobile accidents. Commonly presenting feature was pain abdomen and vomiting. Abdominal paracentesis revealed haemoperitoneum in 40 cases which was subsequently confirmed on laparotomy in all the cases. 40 cases had no visible external injury but subsequent laparotomy revealed internal visceral injury in 29 cases. Exploratory laparotomy was carried out in 43 cases, remaining were treated conservatively. Liver was found to be the commonest organ injured. Post operative complications developed in 5 cases and deaths occurred in 7 cases mainly due to associated extra-abdominal injuries, poor pre-operative general condition, delayed diagnosis and management. We conclude that a multipronged approach towards early diagnosis and vigorous management should be adopted to reduce the morbidity and mortality in patients with blunt abdominal trauma.
Abdominal Injuries↗
Detection of M. leprae by gene amplification; combined ethidium-bromide staining and probe hybridization.
Biopsy and skin-scraping specimens from 130 leprosy cases across the disease spectrum (56 TT/BT/I, 73 BB/BL/LL, and 1 neuritic case) and 50 healthy contacts were studied to assess the application of gene amplification. The nucleic acids from these clinical specimens were extracted by an integrated freeze-thawing--optimized lysozyme-/proteinase-k treatment-purification and fractionation procedure. The nucleic acids from cultured organisms were isolated by the stepwise procedure earlier standardized at this laboratory. Gene amplification for a 360-bp fragment of the 18-kDa protein gene was carried out using primer and the procedure described by its developers, and a 360-bp fragment on Southern blot was taken as the yardstick of positivity. The polymerase chain reaction product was analyzed by electrophoresis, ethidium-bromide (EB) staining, and blot (B) hybridization. Overall sensitivity ranged from 71% in specimens with undetectable acid-fast organisms to 100% in specimens with demonstrable acid-fast bacilli. A positivity of 73% in TT/BT/I specimens and 93% in BB/BL/LL specimens was observed. Four combinations were discerned: EB+, B+ (71%); EB-suspicious, B+ (14%); EB-, B+ (3%) and EB-, B- (12%). By combining the blot hybridization with EB staining, the sensitivity could be significantly improved as compared to EB staining alone. The test was found to be absolutely specific by the absence of any false positivity in control specimens as well as with purified DNAs from mycobacterial as well as non-mycobacterial organisms, grown from these specimens. It is recommended that for optimum sensitivity and specificity both EB staining and blot hybridization should be done.
Biopsy↗
An epidemiological study of obesity in Shimla Town.
A cross sectional study of 888 randomly selected subjects in Shimla town was done to determine prevalence of obesity, its relation to age and physical activity. The prevalence of obesity was 21.5% using cut off levels of BMI 25. The prevalence of obesity rose significantly with age. Obesity was less in those having moderate physical activity as compared to those with low and high activity. Primary prevention is required to tackle this disease risk factor through health education focussing on promotion of moderate regular physical activity.
Adult↗
ATP modulation of the ligand binding and signal transduction activities of the type C natriuretic peptide receptor guanylate cyclase.
The type C natriuretic peptide (CNP)-activated guanylate cyclase (CNP-RGC) is a single-chain transmembrane-spanning protein, containing both CNP binding and catalytic cyclase activities. Upon binding CNP to the extracellular receptor domain, the cytosolic catalytic domain of CNP-RGC is activated, generating the second messenger cyclic GMP. Obligatory in this activation process is an intervening signal transduction step which is regulated by ATP binding to the cyclase. This bridges the events of ligand binding and cyclase activation. A defined sequence motif (Gly499-Xa-Xa-Xa-Gly503), termed ATP regulatory module (ARM), is critical for this step. The present study shows that ATP not only amplifies the signal transduction step, it also concomitantly reduces the ligand binding activity of CNP-RGC. Reduction in the ligand binding activity is a consequence of the transformation of the high affinity receptor-form to the low affinity receptor-form. A single ARM residue Gly499 is critical in the mediation of both ATP effects, signal transduction and ligand binding activity of the receptor. Thus, this residue represents an ATP bimodal switch to turn the CNP signal on and off.
Adenosine Triphosphate↗
A novel calcium-dependent activator of retinal rod outer segment membrane guanylate cyclase.
The membrane guanylate cyclase in retinal rod outer segments (ROS-GC) is known to be negatively regulated by calcium; when the calcium concentration is reduced below the dark-adapted level of about 500 nM, the enzyme is activated by a soluble protein. We now report that the enzyme is also positively regulated by calcium; a novel soluble protein is identified and purified from bovine retina which activates ROS-GC, with half-maximal activation occurring at 2-5 microM calcium. The activation is dose-dependent, and at its maximum, cyclase is stimulated up to 25-fold. The activator has a molecular mass of about 40 kDa and is a multimer of a 6-7 kDa peptide.
Animals↗
Increased N-myristoyltransferase activity observed in rat and human colonic tumors.
BACKGROUND: Colorectal cancer is one of the leading causes of cancer death in North America. Since treatment of colonic cancer remains difficult because of the lack of effective chemotherapeutic agents, it is important to continue to search for cellular functions that can be disrupted by chemotherapeutic drugs and inhibit the development or progression of this disease. Modification of proteins by myristoylation has been recognized as important in the function of various viral, oncogenic, and signal-transduction proteins and thus has been proposed as a target for chemotherapeutic drug design. However, the activity of the enzyme that catalyzes this modification, N-myristoyltransferase, has not been investigated in cancer relative to normal tissue. PURPOSE: The purpose of this study was twofold: 1) to investigate the activity of N-myristoyltransferase in azoxymethane-induced rat colonic cancer tissue compared with normal and normal-appearing rat colonic tissue and 2) to determine if similar differences would be observed in a small sample of human colonic tumors. METHODS: N-Myristoyltransferase activity was determined in 45 colonic tissue specimens from Sprague-Dawley rats--10 given injections of the colon carcinogen, azoxymethane, and three untreated. Tissue specimens included 35 colonic tumors of varying pathologic stages, seven specimens of normal-appearing adjacent mucosa, and three specimens of normal colonic mucosa. Colectomy specimens from five patients were assayed for N-myristoyltransferase activity. Subcellular distribution of N-myristoyltransferase activity was determined. Synthetic peptides of known myristoylated proteins--pp60src and cyclic adenosine monophosphate-dependent protein kinase--were used in kinetic analyses of N-myristoyltransferase in colonic cancer and normal-appearing colonic tissue. All P values are two-tailed. RESULTS: N-Myristoyltransferase activity was increased in rat colonic tumors compared with normal-appearing adjacent mucosa and normal mucosa (P = .0002). Elevation of N-myristoyltransferase activity was present in all tumors, including colonic polyps. Increased N-myristoyltransferase activity was also observed in human colonic tumors and was predominantly cytosolic. N-Myristoyltransferase of colonic cancer tissues had a similar Michaelis constant but an approximate twofold higher maximum velocity for both the pp60src- and cyclic adenosine monophosphate-dependent protein kinase-derived peptides compared with N-myristoyltransferase of normal-appearing tissue. CONCLUSIONS: This study demonstrates for the first time that N-myristoyltransferase activity is higher in colonic epithelial neoplasms than in normal-appearing colonic tissue and that an increase in N-myristoyltransferase activity appears at an early stage in colonic carcinogenesis.
Acyltransferases↗
Single amino acid residue-linked signaling shifts in the transduction activities of atrial and type C natriuretic factor receptor guanylate cyclases.
The type A (ANF) and the type C (CNP) natriuretic factor-activated guanylate cyclases, respectively termed as ANF-RGC and CNP-RGC, are single-chain transmembrane-spanning proteins, containing ligand binding and catalytic cyclase domains at two opposite ends of the protein. The binding activity resides at the N-terminal extracellular region and the catalytic cyclase activity at the carboxyl end. The ANF-RGC residue Leu-364, residing in the extracellular region, is critical for the ANF-binding activity; the CNP-RGC residue Glu-332 is critical for the CNP-binding activity. The counter part of CNP-RGC-Glu-332 residue is the ANF-RGC residue Gln-338 and of ANF-RGC-Leu-364 residue in CNP-RGC is the Valine-358. The present study shows a remarkable signal switching phenomenon associated with these residues. By changing the ANF-RGC residue Gln-338 to Glu, ANF-RGC switches from no to significant CNP signal transduction activity; similarly, a change from Valine-358 to Leu generates ANF signal transduction activity in CNP-RGC. These acquired signal transduction activities in the cyclases are in addition to their natural signal transduction activities. Thus, these new cyclases show both ANF and CNP signaling activities.
Amino Acid Sequence↗
Regulation of bovine rod outer segment membrane guanylate cyclase by ATP, phosphodiesterase and metal ions.
In vertebrate retina, rod outer segment is the site of visual transduction. The inward cationic current in the dark-adapted outer segment is regulated by cyclic GMP. A light flash on the outer segment activates a cyclic GMP phosphodiesterase resulting in rapid hydrolysis of the cyclic nucleotide which in turn causes a decrease in the dark current. Restoration of the dark current requires inactivation of the phosphodiesterase and synthesis of cyclic GMP. The latter is accomplished by the enzyme guanylate cyclase which catalyzes the formation of cyclic GMP from GTP. Therefore, factors regulating the cyclase activity play a critical role in visual transduction. But regulation of the cyclase by some of these factors--phosphodiesterase, ATP, the soluble proteins and metal cofactors (Mg and Mn)--is controversial. The availability of different types of cyclase preparations, dark-adapted rod outer segments with fully inhibited phosphodiesterase activity, partially purified cyclase without PDE contamination, cloned rod outer segment cyclase free of other rod outer segment proteins, permitted us to address these controversial issues. The results show that ATP inhibits the basal cyclase activity but enhances the stimulation of the enzyme by soluble activator, that cyclase can be activated in the dark at low calcium concentrations under conditions where phosphodiesterase activity is fully suppressed, and that greater activity is observed with manganese as cofactor than magnesium. These results provide a better understanding of the controls on cyclase activity in rod outer segments and suggest how regulation of this cyclase by ATP differs from that of other known membrane guanylate cyclases.
3',5'-Cyclic-GMP Phosphodiesterases↗
Differential activation of bovine brain N-myristoyltransferase(s) by a cytosolic activator.
N-Myristoyltransferase (NMT) is an essential eukaryotic enzyme that catalyses the transfer of myristate from myristoyl-CoA to the NH2-terminal glycine residue of a number of important proteins of diverse function. Little is known about the control and regulation of NMT in higher eukaryotes. We have identified a N-myristoyltransferase activator (NAF45) which copurified with one form of bovine brain NMT activity which failed to bind to phosphocellulose column chromatography (NMT.PU) and represented 32 +/- 4% of the total NMT activity applied to this column. The NAF45 and NMT activities were resolved by mono Q column chromatography from the NMT.PU fraction. Resolution resulted in a loss of NMT.PU activity. NAF45 was a nondialysable molecule with an apparent molecular mass of 45 kDa. Reconstitution of the NAF45 and NMT resulted in the recovery of NMT activity, indicating that this form of NMT is dependent on the presence of NAF45. NAF45 was able to stimulate other forms of NMT activity (maximum of 3-4 fold) in a highly cooperative fashion (Hills coefficient of approximately 5.5). Stimulation was in terms of both the rate of myristoylation and the overall production of myristoylpeptide, indicating that the end point of the reaction was shifted in favour of myristoylpeptide production. NAF45 was able to compete with NIP71 for NMT [King, M.J. and Sharma, R.K. (1993) Biochem, J. 291, 635-639], although its affinity appeared lower than the NIP71.
Acyltransferases↗
The magnetic resonance renogram in renal transplant evaluation using dynamic contrast-enhanced MR imaging.
To assess the role of the dynamic gadolinium DTPA (Gd-DTPA) magnetic resonance (MR) renogram in differentiating various causes of renal allograft dysfunction, contrast-enhanced MR imaging studies were performed in 5 normal renal allografts, 5 patients with acute rejection (AR), and 7 patients with cyclosporine nephrotoxicity. Time-versus-signal intensity (SI) curves were plotted. Normal renal allografts showed a rapid increase and slow decay, with a definite peak in cortical (CX) SI curves (peak mean signal intensity (Amax 338.6 +/- 46.5). The outer medullary (OM) (Amax 306.5 +/- 59) and inner medullary (IM) (Amax 263.4 +/- 47.4) curves did not show a definite peak. The OM curve slowly reached a steady state and caught up with the CX curve. AR episodes were characterized by a blunted uprise and delayed peak of CX (Amax 180 +/- 70.9) and a low-amplitude vascular phase with a slow constant uprise of the inner medullary curve (Amax 120.35 +/- 42.4). The OM signal intensity curve (Amax 150.73 +/- 78) failed to catch up with the CX curve. The maximum amplitude of SI curves in CsA-induced allograft dysfunction were low, with no definite peak, and CX, OM, and IM curves ran parallel to each other with a constant gap. Dynamic Gd-DTPA, MR imaging is a noninvasive technique that shows distinct characteristics in acute rejection, cyclosporine nephrotoxicity, and normally functioning renal allografts.
Adult↗
ATP bimodal switch that regulates the ligand binding and signal transduction activities of the atrial natriuretic factor receptor guanylate cyclase.
Atrial natriuretic factor (ANF)-dependent guanylate cyclase (ANF-RGC) is a single-chain transmembrane-spanning protein, containing both ANF binding and catalytic cyclase activity. ANF binding to the extracellular receptor domain activates the cytosolic catalytic domain, generating the second messenger cyclic GMP. Obligatory in this activation process is an intervening step regulated by the ATP binding to the cyclase. This is a signal transduction step that bridges the events of ligand binding and cyclase activation. A defined structural motif (Gly503-Xa-Gly505-Xa-Xa-Xa-Gly509), termed ATP regulatory module (ARM), is critical for this step. The present study shows that the ARM-Gly505 residue acts as an ATP bimodal switch in regulating both the ligand binding and signal transduction activities of ANF-RGC, thus representing a critical site to turn the hormone signal on and off.
Adenosine Triphosphate↗
Structural, genetic and pharmacological identity of the rat alpha 2-adrenergic receptor subtype cA2-47 and its molecular characterization in rat adrenal, adrenocortical carcinoma and bovine retina.
Subsequent to the first alpha 2-adrenergic receptor (alpha 2-AR) gene cloning of alpha 2-C10 from human platelet, cloning of the first rodent alpha 2-AR cDNA, cA2-47, was reported. Based on the structural and limited pharmacological comparison, it was concluded that the rodent receptor is a molecular and pharmacological analog of the human receptor, which is pharmacologically classified as the alpha 2A-AR. A later study slightly revised the structure of the human receptor. Thus, the precise structural comparison of the rat receptor to the human platelet receptor is no longer valid. Another rat alpha 2-AR gene, RG20, was then cloned and was also found to be a structural analog of the human alpha 2-C10. It, however, varied slightly from the alpha 2A subtype pharmacology, but matched the newly defined alpha 2D subtype pharmacology. It was, therefore, concluded that RG20 encodes the alpha 2D subtype. The structural and pharmacological relationship of RG20 with cA2-47 is not known, although it has been tacitly assumed that both are the identical alpha 2D receptor subtypes. The present study addresses this and other issues relating to the precise structural, genetic and pharmacological relationship of cA2-47 with the human platelet alpha 2-C10 receptor, and also the localization of cA2-47 transcript in certain rat tissues. The results show that the cA2-47 receptor shows a high degree of sequence identity to the alpha 2-C10 receptor, yet important differences exist between them. The sequence identity of cA2-47 receptor to the RG20 receptor is almost, but not quite complete. The cA2-47 gene is not present in the human and the human gene is not present in the rat; that cA2-47 receptor subtype is pharmacologically similar to the RG20 receptor subtype, both being of the alpha 2D subtype. The cA2-47 receptor transcript in addition to being found in the rat brain is present in the rat adrenal gland, testes, adrenocortical carcinoma and the bovine retina.
Adrenal Cortex Neoplasms↗
Myristoyl CoA:protein N-myristoyltransferase: subcellular localization, activation and kinetic behavior in the presence of organic solvents.
Myristoyl CoA:protein N-myristoyltransferase (NMT) catalyses the addition of myristate to the amino terminal glycine residue of a number of cellular, eukaryotic and viral proteins. The majority of the catalytic activity of spleen NMT was recovered in the soluble cytosolic fraction (98.2%) compared to the particulate fraction (19.3%). Recovery of NMT activity, from both cytosol and particulate fractions, was found to be higher than the total activity in crude homogenates, suggesting that the particulate fraction may contain an inhibitory activity towards NMT. The effects of organic solvents (ethanol and acetonitrile) on bovine spleen NMT were investigated. NMT activity was activated several-fold in a time- and concentration-dependent manner in the presence of cAMP-dependent protein-kinase derived peptide substrate as fatty acyl CoA acceptor, suggesting that the activation by organic solvents is not due to a solvent effect. Similar activation by ethanol and acetonitrile was also observed with pp60src as substrate, suggesting that the effect of solvent is on NMT and not on the substrate. Gel filtration chromatography indicated that the high catalytic activity of NMT was observed only in the presence of organic solvents: removal of organic solvents from the medium drastically reduced the catalytic activity of NMT, suggesting that NMT did not undergo covalent modification. Kinetic data indicated that ethanol enhanced the Vmax without affecting the Km.
Acetonitriles↗
Diverse mechanisms of calcium mobilization by peroxisome proliferators in rat hepatocytes.
The ability of six peroxisome proliferators to modulate Ca2+ homeostasis was studied in freshly isolated rat hepatocytes. Clofibrate and bifonazole (0.5 mM) caused a transient increase in cytosolic-free Ca2+ concentration ([Ca2+]i) by releasing the intracellular inositol 1,4,5-trisphosphate-sensitive Ca2+ pool. However, the mobilization of this pool by clofibrate was only transient; a subsequent exposure of the cells to the endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin resulted in a second release of the same Ca2+ store, indicating that this pool could refill from the cytosol, independently of extracellular Ca2+. By contrast, bifonazole-exposed hepatocytes no longer responded to a stimulation by thapsigargin. Bifonazole also strongly inhibited Ca2+ influx. Ciprofibrate and nafenopin (0.5 mM) produced increases in [Ca2+]i that were sustained, even in the absence of extracellular Ca2+. The [Ca2+]i response was not due to release of the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool and was not inhibited by prior treatment with the protonophore carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone, but was slightly antagonized by prior exposure to the Ca2+ ionophore ionomycin. Pretreating the cells with nafenopin completely abolished the response elicited by ciprofibrate, and vice versa. By contrast to the other peroxisome proliferators, WY-14,643 and bezafibrate (1 mM) increased cytosolic free Ca2+ only by approximately 30 nM. In conclusion, the structurally diverse peroxisome proliferators tested in this study all produced changes in [Ca2+]i in hepatocytes but through the redistribution of different internal Ca2+ pools. Further studies are needed to determine whether any of the observed Ca2+ changes have a role in the pleiotropic effects elicited by peroxisome proliferators.
Animals↗