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Biomedical subjects

R Kumar

Publications and source records attributed to R Kumar.

At least 397 records · Page 22Linked to original sources
PubMed1999

Catalytic mechanism of nucleoside diphosphate kinase investigated using nucleotide analogues, viscosity effects, and X-ray crystallography.

Nucleoside diphosphate (NDP) kinases display low specificity with respect to the base moiety of the nucleotides and to the 2'-position of the ribose, but the 3'-hydroxyl is found to be important for catalysis. We report in this paper the enzymatic analysis of a series of derivatives of thymidine diphosphate (TDP) where the 3'-OH group was removed or replaced by fluorine, azido, and amino groups. With Dictyostelium NDP kinase, kcat decreases 15-200-fold from 1100 s-1 with TDP, and (kcat/Km)NDP decreases from 12 x 10(6) to 10(3) to 5 x 10(4) M-1 s-1, depending on the substrate. The poorest substrates are 3'-deoxyTDP and 3'-azido-3'-deoxyTDP, while the best modified substrates are 2',3'-dehydro-3'-deoxyTDP and 3'-fluoro-3'-deoxyTDP. In a similar way, 3'-fluoro-2',3'-dideoxyUDP was found to be a better substrate than 2',3'-dideoxyUDP, but a much poorer substrate than 2'-deoxyUDP. (kcat/Km)NDP is sensitive to the viscosity of the solution with TDP as the substrate but not with the modified substrates. To understand the poor catalytic efficiency of the modified nucleotides at a structural level, we determined the crystal structure of Dictyostelium NDP kinase complexed to 3'-fluoro-2',3'-dideoxyUDP at 2.7 A resolution. Significant differences are noted as compared to the TDP complex. Substrate-assisted catalysis by the 3'-OH, which is effective in the NDP kinase reaction, cannot occur with the modified substrate. With TDP, the beta-phosphate, which is the leaving group when a gamma-phosphate is transferred to His122, hydrogen bonds to the 3'-hydroxyl group of the sugar; with 3'-fluoro-2',3'-dideoxyUDP, the beta-phosphate hydrogen bonds to Asn119 and moves away from the attacking Ndelta of the catalytic His122. Since all anti-AIDS nucleoside drugs are modified at the 3'-position, these results are relevant to the role of NDP kinase in their cellular metabolism.

Animals↗
PubMed1999

Trimethylamine N-oxide-induced cooperative folding of an intrinsically unfolded transcription-activating fragment of human glucocorticoid receptor.

A number of biologically important proteins or protein domains identified recently are fully or partially unstructured (unfolded). Methods that allow studies of the propensity of such proteins to fold naturally are valuable. The traditional biophysical approaches using alcohols to drive alpha-helix formation raise serious questions of the relevance of alcohol-induced structure to the biologically important conformations. Recently we illustrated the extraordinary capability of the naturally occurring solute, trimethylamine N-oxide (TMAO), to force two unfolded proteins to fold to native-like species with significant functional activity. In the present work we apply this technique to recombinant human glucocorticoid receptor fragments consisting of residues 1-500 and residues 77-262. CD and fluorescence spectroscopy showed that both were largely disordered in aqueous solution. TMAO induced a condensed structure in the large fragment, indicated by the substantial enhancement in intrinsic fluorescence and blue shift of fluorescent maxima. CD spectroscopy demonstrated that the TMAO-induced structure is different from the alpha-helix-rich conformation driven by trifluoroethanol (TFE). In contrast to TFE, the conformational transition of the 1-500 fragment induced by TMAO is cooperative, a condition characteristic of proteins with unique structures.

Circular Dichroism↗
PubMed1999

The gain-of-function Chinese hamster ovary mutant LEC11B expresses one of two Chinese hamster FUT6 genes due to the loss of a negative regulatory factor.

The LEC11 Chinese hamster ovary (CHO) gain-of-function mutant expresses an alpha(1,3)fucosyltransferase (alpha(1,3)Fuc-T) activity that generates the LeX, sialyl-LeX, and VIM-2 glycan determinants and has been extensively used for studies of E-selectin ligand specificity. In order to identify regulatory mechanisms that control alpha(1,3)Fuc-T expression in mammals, mechanisms of FUT gene expression were investigated in LEC11 cells and two new, independent mutants, LEC11A and LEC11B. Northern and ribonuclease protection analyses, using probes that span the coding region of a cloned CHO FUT gene, detected transcripts in each LEC11 mutant but not in CHO cells or other gain-of-function CHO mutants that express a different alpha(1,3)Fuc-T activity. Coding region sequence analysis and alpha(1,3)Fuc-T acceptor specificity comparisons with recombinant human Fuc-TV and Fuc-TVI showed that the cloned FUT gene is orthologous to the human FUT6 gene. Southern analyses identified two closely related FUT6 genes in the Chinese hamster, whose evolutionary relationships are discussed. The blots showed that rearrangements had occurred in LEC11A and LEC11 genomic DNA, consistent with a cis mechanism of FUT6 gene activation in these mutants. By contrast, somatic cell hybrid analyses revealed that LEC11B cells express FUT6 gene transcripts due to the loss of a trans-acting, negative regulatory factor. Sequencing of reverse transcriptase-polymerase chain reaction products identified unique 5'- and 3'-untranslated region sequences in FUT6 gene transcripts from each LEC11 mutant. Northern and Southern analyses with gene-specific probes showed that LEC11A cells express only the cgFUT6A gene (where cg is Cricetulus griseus), whereas LEC11 and LEC11B cells express only the cgFUT6B gene. In LEC11A x LEC11B hybrid cells, the cgFUT6A gene was predominantly expressed, as predicted if a trans-acting negative regulatory factor functions to suppress cgFUT6B gene expression in CHO cells. This factor is predicted to be a cell type-specific regulator of FUT6 gene expression in mammals.

Amino Acid Sequence↗
PubMed1999

Effects of ischemia on discontinuous action potential conduction in hybrid pairs of ventricular cells.

BACKGROUND: Acute ischemia often occurs in cardiac tissue that has prior injury, resulting in spatially inhomogeneous distributions of membrane properties and intercellular coupling. Changes in action potential conduction with ischemia, which can be associated with release of catecholamines, may be particularly important in tissue that has discontinuous conduction resulting from prior infarction, hypertrophy, or myopathy. METHODS AND RESULTS: Isolated guinea pig ventricular myocytes were electrically coupled by a coupling-clamp circuit to a comprehensive computer model of a guinea pig ventricular myocyte to assess alterations in the critical value of coupling conductance required for action potential conduction from the real cell to the model cell when the real cell was exposed to a solution that included hypoxia, acidosis, and an elevated extracellular potassium concentration to simulate acute ischemia. The "ischemic" solution increased critical coupling conductance from 6.2+/-0.1 to 7.4+/-0.2 nS and decreased the associated maximum conduction delay from 31+/-1 to 23+/-1 ms (mean+/-SEM, n=11). The ischemic solution plus 1 micromol/L norepinephrine decreased critical coupling conductance from 5.9+/-0.2 to 5.0+/-0.1 nS and increased maximum conduction delay from 31+/-2 to 54+/-4 ms (mean+/-SEM, n=8). CONCLUSIONS: The release of catecholamines with ischemia, in a setting of partially uncoupled cells, may play a major role in producing long conduction delays, which may allow reentrant pathways.

Action Potentials↗
PubMed1999

Identification of 1 alpha,25-dihydroxyvitamin D3 response elements in the human transforming growth factor beta 2 gene.

Transforming growth factor-beta (TGF-beta) is one of the most abundant growth factors secreted by bone cells, and regulation of TGF-beta expression is crucial for bone development and growth. Previous studies from our laboratory demonstrated that 1 alpha, 25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) inhibits human osteoblast and keratinocyte growth by increasing TGF-beta2 secretion and synthesis. To examine the mechanism by which 1 alpha,25(OH)2D3 regulates TGF-beta 2 transcription in osteoblasts, we ligated segments of the human TGF-beta 2 promoter 5' of a growth hormone reporter gene in a growth hormone reporter plasmid and examined the effects of 1 alpha,25(OH)2D3 administration on the expression of growth hormone in cells transfected with such chimeric promoter-reporter plasmids. The promoter region extending from -973 to +68 bp of the transcription start site elicited a 7-fold increase in reporter gene activity in transiently transfected osteoblasts after 1 alpha,25(OH)2D3 treatment, whereas the region from -553 to +68 bp of the transcription start site showed no response following 1 alpha,25(OH)2D3 treatment. Transfection of osteoblasts with reporter plasmids containing TGF-beta 2 promoter DNA from -869 to -658 bp revealed a 3.8-fold increase in reporter gene activity. DNA fragments from this region (-743 to -676 bp and -786 to -728 bp) ligated into reporter plasmids also showed increases in reporter activity. Gel retardation assay experiments showed that DNA fragments from -774 to -735 bp and -714 to -675 bp both formed a DNA-protein complex with bacterially expressed human retinoic acid X receptor alpha (RXR alpha) and vitamin D receptor (VDR) and with nuclear extracts from human bone cells. Addition of a vitamin D receptor antibody to reactions containing the aforesaid DNA fragments and bone cell nuclear extract resulted in further retardation of the mobility of the DNA-protein complex (super-shifting). Removal of two putative direct repeat DNA fragments in this region abolished VDR-RXR alpha-vitamin D response element complex formation. The TGF-beta 2 promoter contains two imperfect direct repeat DNA sequences: TGTAGAACAAGTAGA and AATGAAGTTGGTGGA that mediate the effect of 1 alpha,25(OH)2D3.

Base Sequence↗
PubMed1999

Fibrin-dependent platelet procoagulant activity requires GPIb receptors and von Willebrand factor.

Thrombin generation in platelet-rich plasma (PRP) involves complex interactions between platelets and coagulation proteins. We previously reported that the addition of fibrin to PRP enhances tissue-factor initiated thrombin generation by approximately 40%, and the current studies were designed to assess the mechanism(s) underlying thrombin generation in the absence and presence of fibrin. Blocking platelet GPIIb/IIIa + alphavbeta3 receptors with a monoclonal antibody (MoAb) inhibited basal thrombin generation, but did not affect the enhancement produced by fibrin. In contrast, blocking GPIb with any of three different MoAbs had no effect on basal thrombin generation, but essentially eliminated fibrin enhancement of thrombin generation. When thrombin generation was tested in PRP deficient in von Willebrand factor (vWF), both basal and fibrin-enhanced thrombin generation were markedly reduced, and the addition of factor VIII did not normalize thrombin generation. Botrocetin, which induces the binding of vWF to GPIb, enhanced thrombin generation. In all studies, the ability of PRP to support thrombin generation correlated with the production of platelet-derived microparticles and serum platelet-derived procoagulant activity. Thus, two separate mechanisms, both of which depend on vWF, appear to contribute to platelet-derived procoagulant activity: one is independent of fibrin and relies primarily on GPIIb/IIIa, but with a minor contribution from alphavbeta3; and the other is fibrin-dependent and relies on GPIb. These data may have implications for understanding the mechanisms of the abnormalities in serum prothrombin times reported in Bernard-Soulier syndrome, hemorrhage in von Willebrand disease (vWD), and the increased risk of thrombosis associated with elevated vWF levels.

Animals↗
PubMed1999

Regulation of cyclooxygenase-2 pathway by HER2 receptor.

Emerging lines of evidence suggest that in addition to growth factors, the process of colorectal tumorigenesis may also be driven by the upregulation of the inducible form of cyclooxygenase-2 (COX-2), an enzyme responsible for the conversion of arachidonic acid to PGEs. The present study was undertaken to investigate the expression and activation of the HER family members, and to explore the regulation of COX-2 expression by the HER2 pathway in human colorectal cancer cells. Here, we report that human colorectal cancer cell lines express abundant levels of HER2 and HER3 receptors, and are growth-stimulated by recombinant neu-differentiation factor-beta 1 (NDF). NDF-treatment of colorectal cancer cells was accompanied by increased tyrosine phosphorylation and heterodimerization of HER3 with HER2. In addition, we demonstrated that HER2 and HER3 receptors in colorectal cancer cells are constitutively phosphorylated on tyrosine residues and form heterodimeric complexes in the absence of exogenous NDF. Inhibition of HER2/HER3 signaling by an anti-HER3 mAb against the ligand binding site resulted in a decrease in the levels of constitutively activated HER2/ HER3 heterodimers, and the unexpected reduction of COX-2 expression. Activation of the HER2/HER3 pathway by NDF induced the activation of COX-2 promoter, expression of COX-2 mRNA, COX-2 protein and accumulation of prostaglandin E2 in the culture medium. Finally, we demonstrated that NDF promotes the ability of colorectal cancer cells to survive in an extracellular matrix milieu, such as Matrigel, and also to invade through a 8 microm porous membrane. These biological activities of NDF and its stimulation of cell proliferation are blocked by a specific inhibitor of COX-2. Taken together, our findings provide the first biochemical evidence of a possible role of the COX-2 pathway in the mitogenic action of NDF in colorectal cancer cells where it may be constitutively upregulated due to the autocrine/paracrine activation of HER2/ HER3 heterodimers.

3T3 Cells↗
PubMed1999

On-line sample clean-up and chromatography coupled with electrospray ionization mass spectrometry to characterize the primary sequence and disulfide bond content of recombinant calcium binding proteins.

We have used on-line sample clean-up, concentration, and chromatography with electrospray ionization mass spectrometry (ESI-MS), to characterize and determine the presence of disulfide bonds in recombinant full-length rat brain calbindin D28K and two deletion mutants of the protein, one lacking EF-hand 2 (calbindin delta 2) and the other lacking EF-hands 2 and 6 (calbindin delta 2,6). The molecular weights of the expressed proteins dissolved in biological buffers were determined with high accuracy using a low-flow, pressurized chamber infusion system, that allows on-line protein clean-up by removing buffers/salts incompatible with ESI-MS. The molecular weight determinations showed that the amino-terminal methionine residues had been cleaved during the expression and isolation of the recombinant proteins. Approximately 85-90% of the protein sequences were confirmed by on-line HPLC-ESI-MS analysis of peptides generated by a lysyl endoproteinase C digestion. Comparisons of ESI-MS spectra of native and reduced calbindin D28K and delta 2 show that the full length- and delta 2 mutant-protein contain one disulfide bond. Molecular mass determinations of calbindin delta 2,6 showed that this protein contains a highly active cysteine residue that covalently binds a mercaptoethanol group, or forms a homodimer via a disulfide bond. The results show surprising differences amongst the deletion mutants of calbindin D28K with respect to the formation of disulfide bonds. These differences are not readily detected by other techniques and show that ESI-MS is a powerful, rapid method by which to detect disulfide linkages for intact proteins.

Amino Acid Sequence↗
PubMed1999

Syringomyelic dystonia and athetosis.

Two patients with movement disorders associated with syringomyelia are described, one of whom developed unusual torticollis, and the other had choreoathetoid-dystonic movements of the hand and arm. In each case, the movements resolved with decompression of the syrinx. The literature is reviewed and possible mechanisms explored.

Adult↗
PubMed1999

GM-CSF-transduced B16 melanoma cells are highly susceptible to lysis by normal murine macrophages and poorly tumorigenic in immune-compromised mice.

Granulocyte-macrophage colony-stimulating factor (GM-CSF)-transduced B16-F10 murine melanoma cells had lower tumorigenicity in both syngeneic and nude mice than parental or LacZ-transduced (control) cells. The subcutaneous (s.c.) tumors producing GM-CSF were densely infiltrated with macrophages, whereas the control tumors were not. In vitro studies showed that GM-CSF-transduced B16 cells were susceptible to lysis mediated by nonactivated murine macrophages, whereas control B16 cells were not. Macrophage-mediated cytotoxicity against GM-CSF-transduced B16 cells was independent of the presence of NO, H2O2, O2-, tumor necrosis factor alpha, and matrix metalloproteinase. Coculture experiments using GM-CSF-producing and -nonproducing B16 cells demonstrated that GM-CSF produced by the transduced B16 cells activated macrophages to kill the bystander non-GM-CSF-producing tumor cells. The results suggest that GM-CSF released by tumor cells can induce macrophage-mediated cytotoxicity, which in turn can inhibit the in vivo growth of GM-CSF-transduced tumor cells.

Animals↗
PubMed1999

Comparative behavioural effects of benzodiazepine and non-benzodiazepine anxiolytics in rhesus monkeys.

Quantitative behavioural assessment of benzodiazepines (chlordiazepoxide, diazepam and lorazepam) and non-benzodiazepines (buspirone) anxiolytics were investigated in unrestrained rhesus monkeys (Macaca mulatta) living in social colonies. The different behaviour, categorised as social, solitary and abnormal were video recorded and analysed. Chlordiazepoxide (2.5-5 mg kg-1, p.o.), diazepam (2.5-5 mg kg-1, p.o.) and lorazepam (0.5-1 mg kg-1, p.o.) induced dose-dependent significant changes in certain social and solitary behavioural responses. Thus increases in social grooming, approach, contact, self grooming, feeding and resting with eyes open and decreased aggressiveness and vigilance. On the other hand buspirone (5-10 mg kg-1, p.o.) produced no significant alteration in social and solitary behavioural patterns. On the basis of the above findings the social and solitary behaviour protocol in non-human primates can be a useful tool for studying the effect of a new anxiolytic compound before clinical trial.

Animals↗
PubMed1999

Necrotizing fascitis after injection sclerotherapy for hemorrhoids: report of a case.

A case report of a patient who underwent submucosal injection sclerotherapy for hemorrhoids is presented. Subsequently developed necrotizing fascitis of the anorectum, perianal region, and scrotum necessitated emergency debridement and defunctioning colostomy. Postoperatively, the patient developed septicemia and renal failure requiring an extended hospital stay. Restoration of bowel continuity was done after three months. A brief review of known complications of this technique was made. It would appear that necrotizing fascitis can be added to this list.

Adult↗
PubMed1999

Omentum autotransplantation in thromboangiitis obliterans: report of three cases.

Thromboangiitis obliterans (TAO), also known as Buerger's disease, is an important cause in India of chronic arterial occlusion of the limbs. The current modalities of therapy do not provide adequate relief to a large number of these patients. An omentum autotransplantation to the lower limbs was performed in three patients as a limb salvage procedure. The initial results with up to a year of follow-up are so far encouraging.

Adult↗
PubMed1999

Dorsal lumbotomy incision for pediatric pyeloplasty--a good alternative.

This study reviews the value and benefits of the dorsal lumbotomy incision (DL) for exposure of the pelviureteric junction (PUJ) in infantile and paediatric pyeloplasty. Ninety-one children underwent pyeloplasty for confirmed PUJ obstruction between January 1993 and December 1997. The conventional loin incision (CL) (n = 60) was used as the standard, to which DL (n = 31) was compared. Information on the duration of surgery, length of hospital stay, length of time to return to full oral intake, and complications was obtained retrospectively from the hospital chart. The results were analysed using a non-parametrical statistical analysis. Follow-up was between 6 months and 5 years (median 2.4 years). The median age at surgery was 1.5 years (1 month-14 years). The median operating time was similar in both groups (95 min). The median length of stay was 3 days for the DL group compared to 7 days for the CL group (P < 0.001). The length of hospital stay had no correlation to the patient's age at surgery or the type of postoperative analgesia used. The median time to return to full oral intake and unrestricted activity in the DL group was 43 h compared to 83 h for the CL group (P < 0.001). The surgeons found that there was better exposure of the PUJ in infants in the DL group compared to the DL incision. There were no operative complications related to the DL itself. At the time of last follow-up 3 children required re-operation for a failed pyeloplasty, 2 in the DL group. The results suggest that DL is a safe and reasonable alternative to CL in paediatric pyeloplasty and probably the incision of choice in infantile pyeloplasty.

Adolescent↗
PubMed1999

The structure of the nuclear hormone receptors.

The functions of the group of proteins known as nuclear receptors will be understood fully only when their working three-dimensional structures are known. These ligand-activated transcription factors belong to the steroid-thyroid-retinoid receptor superfamily, which include the receptors for steroids, thyroid hormone, vitamins A- and D-derived hormones, and certain fatty acids. The majority of family members are homologous proteins for which no ligand has been identified (the orphan receptors). Molecular cloning and structure/function analyses have revealed that the members of the superfamily have a common functional domain structure. This includes a variable N-terminal domain, often important for transactivation of transcription; a well conserved DNA-binding domain, crucial for recognition of specific DNA sequences and protein:protein interactions; and at the C-terminal end, a ligand-binding domain, important for hormone binding, protein: protein interactions, and additional transactivation activity. Although the structure of some independently expressed single domains of a few of these receptors have been solved, no holoreceptor structure or structure of any two domains together is yet available. Thus, the three-dimensional structure of the DNA-binding domains of the glucocorticoid, estrogen, retinoic acid-beta, and retinoid X receptors, and of the ligand-binding domains of the thyroid, retinoic acid-gamma, retinoid X, estrogen, progesterone, and peroxisome proliferator activated-gamma receptors have been solved. The secondary structure of the glucocorticoid receptor N-terminal domain, in particular the taul transcription activation region, has also been studied. The structural studies available not only provide a beginning stereochemical knowledge of these receptors, but also a basis for understanding some of the topological details of the interaction of the receptor complexes with coactivators, corepressors, and other components of the transcriptional machinery. In this review, we summarize and discuss the current information on structures of the steroid-thyroid-retinoid receptors.

Amino Acid Sequence↗
PubMed1999

Distribution of 1,25-dihydroxyvitamin D3 receptor immunoreactivity in the rat brain and spinal cord.

A complete mapping study on the 1,25-dihydroxyvitamin D3 receptor immunoreactivity within the rat central nervous system was performed with a monoclonal and a polyclonal antibody. Specific immunostaining was observed within both nuclear and cytoplasmic compartments of a variety of cells in the cerebellum, mesopontine area, diencephalon, cortex, spinal cord, and limbic system. Both monoclonal and polyclonal antibodies provided similar staining patterns. The monoclonal antibody stained distinct domains within the nuclei of all and the cytoplasm of specific neuronal cell types, like motor neurons, Purkinje cells, and pyramidal cells of the cortex more clearly than the polyclonal antibody. The expression of vitamin D3 receptor in the rat central nervous system was confirmed by in situ hybridisation. The widespread distribution of vitamin D3 receptor in distinct portions of the sensory, motor, and limbic brain systems suggests multiple functional properties of 1,25-dihydroxyvitamin D3 in the central nervous system.

Animals↗