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U Kumar

Publications and source records attributed to U Kumar.

At least 55 records · Page 3Linked to original sources
PubMed1998

Diminished expression of renal dopamine D1A receptors in the kidney inner medulla of the spontaneously hypertensive rat.

BACKGROUND: Dysfunctional dopamine neurotransmission and greater than normal retention of salt have been found for renal proximal tubules of the spontaneously hypertensive rat OBJECTIVE: To determine whether there are differences between kidney D1A dopamine receptor distributions of spontaneously hypertensive rats and Wistar-Kyoto rats. METHODS: We examined the expression of D1A dopamine receptors in kidneys of spontaneously hypertensive rats and the normotensive Wistar-Kyoto rat through Western blots and immunocytochemistry, using highly specific antipeptide antibodies directed against the receptor. RESULTS: The specificity of the antisera was demonstrated by Western blot studies, using proximal tubules, from Wistar-Kyoto rats. The antiserum recognized a major polypeptide with Mr of 72 kDa and a minor protein of Mr 66 kDa, which were not detected either by antigen-adsorbed or by preimmune sera. In renal cortex of both Wistar-Kyoto rats and spontaneously hypertensive rats, D1A receptors were expressed at equivalent levels. In the inner medulla of Wistar-Kyoto rat, there was diminished (by 60%) expression of D1A receptors compared with that of the renal cortex. However, the expression of D1A receptors in the inner medulla in the spontaneously hypertensive rat was even more diminished (by 83%) relative to levels found in spontaneously hypertensive rat renal cortex. Immunocytochemical studies localized the D1A receptor protein in renal cortex primarily to epithelia of tubules. Relative to renal cortex, there was an overall decrease in staining intensity in the inner medulla both of Wistar-Kyoto rats and of spontaneously hypertensive rats. Compared with that of Wistar-Kyoto rat, the intensity of staining of D1A receptors in the inner medulla of spontaneously hypertensive rats was greatly diminished, confirming the Western blot analyses. The less than normal expression of D1A receptors in the inner medulla of spontaneously hypertensive rats might be of physiologic importance in the etiology of greater than normal retention of salt and hypertension in spontaneously hypertensive rats.

Animals↗
PubMed1998

Audit of clinical judgements and actions in an audiological investigation of school entrant children screened positive for hearing loss.

Of 117 children attending an aural clinic after screening positive by one or both of two tests for hearing loss at school entry, 43 (36.8%) were either seen again for review or listed for surgery. Clinical findings which were significantly associated with either being reviewed or listed for surgery, as opposed to being discharged, were the presence of conductive or sensory neural hearing loss, pronounced negative pressure, reduced compliance volume or absent stapedial reflexes on impedance testing and the presence of an abnormal tympanic membrane associated with effusion on otoscopy. This population of children were then followed up through their school medical records over 4 y to determine what proportion of those discharged from the clinic had subsequently required some form of surgical intervention due to a hearing problem. Note was also taken of all those actually receiving surgery or long term review for sensory neural hearing loss, these cases being designated as true positives in the aural clinic assessment process. This assessment, albeit of a 'high-risk group', gave rise to a sensitivity of 88.9% and a specificity of 68.0%.

Audiometry, Pure-Tone↗
PubMed1997

Distribution of protein inhibitor of neuronal nitric oxide synthase in rat brain.

In neuronal cells, nitric oxide (NO) is synthesized by neuronal nitric oxide synthase (nNOS) and performs various functions including neurotransmission, modulation of nociception and long-term potentiation and memory. Recently, a novel 89-amino acid protein, designated PIN, has been shown to interact specifically with nNOS and inhibit nNOS dimerization. In this report, we investigated the distribution and the correlation of PIN with that of nNOS in various brain regions of rats. Amplified PIN cDNA from brain tissues revealed an open reading frame which is identical to that of human PIN. Northern blotting of brain RNA with PIN cDNA produced two transcripts, a major 0.9 kb and a minor 2.5 kb. Expression of PIN cDNA as a fusion protein in E. coli produced a 10 kDa protein which interacted specifically with pure nNOS in an overlay assay. Immunoblotting of rat brain regions with nNOS antibody demonstrated strong expression in the cerebellum, moderate expression in the cerebral cortex, midbrain, medulla and hippocampus with only weak expression in the spinal cord. By comparison, PIN expression was stronger in the cerebral cortex, midbrain, hippocampus and medulla compared with that of cerebellum and spinal cord. We conclude that PIN interacts strongly with nNOS and is constitutively expressed in various brain regions. The dissimilarity between nNOS and PIN expressions in various brain regions may explain the well known differences in NOS activity between these regions. Our results also suggest that PIN may serve other functions other than nNOS inhibition.

Amino Acid Sequence↗
PubMed1997

The traE gene of plasmid RP4 encodes a homologue of Escherichia coli DNA topoisomerase III.

The polypeptide encoded by the plasmid RP4 traE gene shows extensive protein sequence similarity to Escherichia coli topB, the gene encoding DNA topoisomerase III (Topo III). The traE gene product has been cloned into a bacteriophage T7-based transient expression system, and the polypeptide has been expressed and purified. The TraE protein exhibits topoisomerase activity similar to that of Topo III. Relaxation is stimulated by high temperature and low concentrations of Mg2+. In addition, similar to E. coli Topo III, the TraE protein is a potent decatenase and can substitute for Topo III activity in vivo. The biochemical properties of the TraE protein in vitro suggest that the protein may be involved in the resolution of plasmid DNA replication intermediates either during vegetative replication or in conjugative DNA transfer. Putative homologues of Topo III have been found to be encoded by other broad host range, conjugative plasmids isolated from both Gram-negative and Gram-positive organisms, suggesting that Topo III-like polypeptides may have an essential role in the propagation of many promiscuous plasmids.

Amino Acid Sequence↗
PubMed1997

Expression, purification, and partial characterization of HCV RNA polymerase.

The product of the NS5B gene of Hepatitis C Virus (HCV) has been expressed in Escherichia Coli both as a fusion protein with glutathione-S-transferase (GST) of molecular weight 91 KDa and at high level as a single protein of molecular weight 65 KDa. The protein was sequestered within inclusion bodies and a variety of procedures designed to minimize inclusion body formation proved unsuccessful. The method finally adopted involved the purification of inclusion bodies followed by the solubilization, purification, and refolding of the expressed protein. A good recovery and protein purity of the order of 80-90% were achieved. The purified protein was shown to possess RNA polymerase activity in an assay using polyA/oligoU as template. The enzymatic activity is rifampicin resistant, poly A dependent, and requires Mg++. The availability of purified HCV RNA polymerase will allow the study of viral replication and constitute the basis for testing new anti-viral drugs.

Cloning, Molecular↗
PubMed1997

Expression of NMDA receptor-1 (NR1) and huntingtin in striatal neurons which colocalize somatostatin, neuropeptide Y, and NADPH diaphorase: a double-label histochemical and immunohistochemical study.

The subset of striatal neurons which colocalize SS/NPY/NADPH-d are selectively resistant to neurodegeneration in Huntington's Disease (HD) and to excitotoxic cell death induced experimentally with NMDA receptor (NMDAR) agonists. Here we have analyzed the expression of immunoreactive NMDAR-1 (NR1) subunit (as an index of NMDAR protein) and of huntingtin (the normal product of the HD gene) in primary cultures of rat striatum to see if differential expression of the two antigens in the subset of SS/NPY/NADPH-d and other striatal neurons can explain their selective resistance or vulnerability. Double-label histochemical and immunocytochemical studies were carried out using conventional and confocal laser scanning microscopy to characterize the cellular and subcellular expression of NR1 and SS, or NPY or bNOS, together with NADPH-d histochemistry. The percentages of cultured striatal neurons that were positive for NADPH-d, SS, NPY, bNOS, and NRI were, respectively, 3.8, 8.4, 10.2, 5.1, and 80%. The majority of striatal NADPH-d neurons coexpressed SS and NPY; 17% of SS-producing neurons were strongly positive for NR1; the remaining cells (approximately 80%) exhibited only weak NR1 expression. Comparable data were obtained for NPY-positive neurons, 15% of which colocalized NR1 strongly and 70-80% weakly. By double-label immunofluorescence, huntingtin was nonselectively expressed in virtually all striatal neurons including SS/NPY/NADPH-d neurons. These results show that the majority of striatal SS/NPY/NADPH-d neurons express NR1. The relative abundance of NR1 in SS/NPY/NADPH-d neurons, however, varies between a small subset of neurons that are receptor rich and the remainder that express low levels only and may determine susceptibility to NMDAR-mediated neurotoxicity. Huntingtin is nonselectively expressed in virtually all striatal neurons and does not appear to be a determinant of the selective resistance of normal striatal SS/NPY/NADPH-d neurons to NMDA toxicity.

Animals↗
PubMed1997

Is there a relationship between proximity to industry and the occurrence of otitis media with effusion in school entrant children?

In a study of the relationship between the prevalence of persistent otitis media with effusion (OME) in school entrant children in Redcar and Cleveland and the distance of the homes of these children from known industrial emission points, 1156 school entrant children were screened for the presence of persistent OME. The social disadvantage scores and map references were determined from the postcode area of each of the study entrants and map references were also obtained of known industrial emission points in the locality. Analyses were conducted on the association between the presence of OME and distance from emission sources and between the presence of OME and disadvantage score. A significantly greater proportion of study entrants with OME lived within 1000 meters of an industrial emission point than further away. However, there was no trend established between the proportion of study entrants with OME and increasing distance from an emissions source, nor was there any significant relationship established between the social disadvantage score of the areas of residence of the study entrants and the presence of OME. Further research is required to establish the effect of confounding variables on this relationship.

Air Pollution↗
PubMed1997

A randomized cross-over study comparing the use of a catheter valve and a leg-bag in urethrally catheterized male patients.

OBJECTIVES: To evaluate the use of a catheter valve (CV) and a leg-bag (LB) in men with urethral catheters and to determine the preferred modality. PATIENTS AND METHODS: Eighteen men (mean age 71.4 years, SD 3.4, range 63-76) presenting in acute retention and catheterized urethrally were enrolled in a 6-week randomized cross-over study comparing the CV with the LB (3 weeks with each modality). The outcome was assessed using a questionnaire which quantified the incidence of bladder spasms, nocturnal frequency, the extent of any by-passing episodes and the degree of discomfort. At the end of the study, the patients were asked to express a preference (using a subjective scoring scale) for either the CV or the LB and to give the reasons why. RESULTS: There was no difference in the reported incidence of bladder spasms or discomfort and no difference in positive urine cultures with each modality. However, with the CV, there was a slightly higher incidence in nocturnal frequency and by-passing episodes. Thirteen patients preferred the CV whilst five preferred the LB. With the subjective scoring scale (range 0-10), the CV scored significantly higher than the LB (7.2, SD 3.1 and 5.2, SD 2.8, P < 0.05: paired t-test). CONCLUSIONS: Most patients preferred to use the CV, reporting that it felt more comfortable and discreet, and many patients did not like the sensation of urine 'sloshing about' when using the leg-bag. Some patients using the CV were troubled by nocturnal frequency. In these patients we suggest that a combination of a valve during the day and an overnight bag may be the ideal solution.

Acute Disease↗
PubMed1997

Purification and partial characterization of chromogranin-A from human brain.

Chromogranin A was purified from post-mortem human brain tissue, obtained at autopsy by a three step chromatography procedure to a single band purity on SDS gel electrophoresis at 68 kDa. Purified protein was further characterized by immunoblotting using a mouse anti-chromogranin A monoclonal antibody LK2H10, which recognizes the 68 kDa chromogranin A in human brain. The results demonstrate a simple protocol of preparing CgA from the human brain. Chromogranin plays a very important role in various neurological disorder and has been examined immunohistochemically in Alzheimer's disease, Parkinson's disease, Pick's disease. The procedure of purification may be useful in further defining its biological function, biochemistry and physiological significance in the central nervous system and may be useful in delineating the molecular pathology of certain neurological disorders.

Animals↗
PubMed1997

Ligand binding pocket of the human somatostatin receptor 5: mutational analysis of the extracellular domains.

The ligand binding domain of G protein-coupled receptors for peptide ligands consists of a pocket formed by extracellular and transmembrane domain (TM) residues. In the case of somatostatin (SRIF), however, previous studies have suggested that the binding cavity of the octapeptide analog SMS201-995 (SMS) is lined by residues in TMs III-VII. The additional involvement of the extracellular domains for binding SMS or the natural SRIF ligands (SRIF-14, SRIF-28) has not been clarified. Using a cassette construct cDNA for the human somatostatin 5 receptor (sst5R), we systematically examined the role of exofacial structures in ligand binding by creating a series of mutants in which the extracellular portions have been altered by conservative segment exchange (CSE) mutagenesis for the extracellular loops (ECLs) and by deletion (for the NH2-terminal segment) or truncation analysis (ECL3). CHO-K1 cells were stably transfected with wild type or mutant human sst5R constructs, and agonist binding was assessed using membrane binding assays with 125I-LTT SRIF-28 ligand. Deletion of the NH2 terminus or CSE mutagenesis of ECL1 and ECL3 produced minor 2-8-fold decreases in affinity for SRIF-14, SRIF-28, and SMS ligands. Truncation of ECL3 to mimic the size of this loop in sst1R and sst4R (the two subtypes that do not bind SMS) did not interfere with the binding of SMS, SRIF-14, or SRIF-28. In contrast, both ECL2 mutants failed to bind 125I-LTT SRIF-28. Immunocytochemical analysis of nonpermeabilized cells with a human sst5R antibody revealed that the mutant receptors were targeted to the plasma membrane. Labeled SMS (125I-Tyr3 SMS) also failed to bind to the mutant ECL2 receptors. These results suggest a potential contribution of ECL2 (in addition to the previously identified residues in TMs III-VII) to the SRIF ligand binding pocket.

Amino Acid Sequence↗
PubMed1997

Pure tone audiometry and impedance screening of school entrant children by nurses: evaluation in a practical setting.

BACKGROUND: Screening for hearing loss in English children at entry to school (age 5-6 years) is usually by pure tone audiometry sweep undertaken by school nurses. This study aimed to compare the validity and screening rates of pure tone audiometry with impedance screening in these children. METHODS: Two stage pure tone audiometry and impedance methods of screening were compared in 610 school entry children from 19 infant schools in north east England. Both procedures were completed by school nurses. The results of screening were validated against subsequent clinical assessment, including otological examination and actions taken by an independent assessor. RESULTS: Both methods produced broadly similar validation indices after two stages of screening: sensitivity was 74.4% for both methods; specificity was 92.1% and 90.0%; and predicted values of a positive test 43.2% and 37.6% respectively for pure tone audiometry and impedance methods. Single stage screening in both methods produced higher sensitivity but lower specificity and predictive values of a positive test than two stage screening. Screening rates were appreciably higher with impedance methods than with pure tone audiometry. CONCLUSIONS: In choosing the method to be used, it must be borne in mind that the impedance method is technically more efficient but takes longer than pure tone audiometry screening. However, the latter method allows opportunity for other health inquiries in these children.

Audiometry, Pure-Tone↗
PubMed1997

Expression of the five somatostatin receptor (SSTR1-5) subtypes in rat pituitary somatotrophes: quantitative analysis by double-layer immunofluorescence confocal microscopy.

Using quantitative double-label fluorescence immunocytochemistry and confocal microscopy, we have analysed the pattern of expression of SSTR1-5 in normal rat pituitary somatotrophes. Antipeptide rabbit polyclonal antibodies were produced against the extracellular domains of SSTR1-5. SSTR antigens were colocalized in GH positive cells using rhodamine conjugated secondary antibody for SSTRs and FITC-conjugated secondary antibody for GH. SSTR5 was the predominant subtype which was expressed in 86 +/- 9.7% of GH cells followed by SSTR2 in 42 +/- 6.4% of GH positive cells. SSTR4 and SSTR3 were modestly expressed in 23 +/- 4.7% and 18 +/- 3.2% of somatotrophes respectively whereas SSTR1 was the least expressed subtype occurring in only 5 +/- 1.2% of somatotrophes. These results demonstrate variable expression of the 5 SSTRs in somatotrophes. The preponderance of the SST-28 preferring SSTR5 subtype correlates with the reported higher potency of SST-28 than SST-14 for inhibiting GH secretion.

Animals↗
PubMed1996

Free radicals in the neurotoxic actions of beta-amyloid.

The fragment of the beta-amyloid protein that contains the peptide sequence 25 to 35 of the parent compound, increases cytosolic calcium and is directly cytotoxic to neurons in tissue culture. The cytotoxic action of beta-amyloid has been considered to be the primary determinant of the neurodegeneration observed in Alzheimer's disease. The cytotoxic effects of beta-amyloid 25-35 or beta-amyloid 1-40 added to primary cultures of rat hippocampal or cortical neurons can be reduced or prevented by anti-oxidant lazaroid drugs such as U-74500A, U-78517F, or U-83836E. beta-amyloid 25-35 evokes an immediate increase in cytosolic calcium when added to suspensions of PC 12 cells, and is cytotoxic to PC 12 cells in tissue culture. Both the calcium-elevating and the cytotoxic actions of beta-amyloid 25-35 on PC 12 cells are reduced or prevented by lazaroid anti-oxidant drugs. Since the lazaroids have been shown to scavenge free radical species, the present results indicate that the actions of beta-amyloid 25-35 on calcium regulation and on cell survival are mediated by free radicals.

Amyloid beta-Peptides↗
PubMed1996

Purification and characterization of chromogranin-A from the adrenal gland of human and bovine.

In the present study, we report a simple, highly reproducible procedure for the purification of chromogranin A. A three step column chromatography of supernatant from human and bovine provided a major single band at M(r) 67 and two minor bands at low molecular mass. Identity of the major and minor bands of CgA was confirmed by western blotting using LK2H10, a monoclonal antibody against chromogranin A. Recognition of smaller fragments in the final preparation in both species by antibody to chromogranin A may suggest that these are the proteolytic breakdown product of the main protein. Bovine adrenal gland was found to be many fold richer in chromogranin than human adrenal gland. The simplification of the purification procedure for CgA may now help in elucidating further physiological function of this protein.

Adrenal Glands↗
PubMed1996

Agonist-dependent regulation of cloned human somatostatin receptor types 1-5 (hSSTR1-5): subtype selective internalization or upregulation.

Agonist regulation of somatostatin receptors (SSTRs) was investigated in stable CHO-K1 cells individually expressing the 5 human (h) SSTR subtypes. hSSTR 2,3,4, and 5 displayed rapid agonist-dependent internalization of [125I] LTT SST-28 ligand in a time- and temperature-dependent manner over 60 min. Maximum internalization of radioligand occurred with hSSTR3 (78%) followed by hSSTR5 (66%), hSSTR4 (29%) and hSSTR2 (20%). In contrast, hSSTR1 displayed virtually no internalization. Prolonged agonist treatment led to differential upregulation of some of the SSTRs. After 22 h, hSSTR1 was upregulated at the membrane by 110%, hSSTR2 and hSSTR4 by 26% and 22% respectively, whereas hSSTR3 and hSSTR5 showed little change. Agonist-induced recruitment of hSSTR1 to the membrane was confirmed by immunocytochemistry with hSSTR1 antibodies. These results show that SST regulates all 5 hSSTRs by differential subtype selective internalization or upregulation. Subtype selectivity for internalization and upregulation is inversely related.

Animals↗