Biomedical subjects
Publications and source records attributed to R Kumar.
We report on an 8-year-old boy with a proximal interstitial deletion of the long arm of chromosome 6 with breakpoints q13 to q14.2. He has a characteristic facial appearance that is seen in several of the previously described cases. Details of his clinical course are reviewed and compared with the nine previous reported cases of the proximal deletion 6q syndrome.
Low levels of telomerase activity have recently been detected in human primitive hematopoietic cells, however, blood cells exhibit telomere shortening on cell proliferation. This challenging observation led us to study telomerase regulation and telomere length in human hematopoietic progenitor cells from fetal liver (FL), cord blood (CB), peripheral blood (PB), and bone marrow (BM). We found telomerase activity in CD34+/CD38+ cells exceeding levels in CD34+/CD38-, CD34- , and mononuclear cells (P < .05). Baseline telomerase activity was highest in BM (n = 5) CD34+ cells, followed by PB (n = 20), CB (n = 11), and FL (n = 1). Within 48 hours to 72 hours of in vitro culture of CD34+ cells in the presence of cytokines (KL, interleukin-3 [IL-3], IL-6, erythropoietin, granulocyte colony-stimulating factor), telomerase activity was upregulated, peaked after 1 week of culture, and decreased to baseline levels or below detection after 3 to 4 weeks. Stimulation of CD34+ cells with single cytokines resulted in no or minor telomerase upregulation, whereas cytokine combinations resulted in a significant telomerase increase (P < .001). There was a correlation between telomerase activity, cell cycle status by BrdU incorporation, and induction of phosphorylated retinoblastoma protein, CDC2, CDK2, cyclin D1, and cyclin A, but not cyclin E and B1 after 72 hours with multiple (but not single) cytokines. In nonexpanding CD34+ cells, telomerase was low or undetectable. Secondary CD34+ cells showed a reduced ability to upregulate telomerase activity. Antiproliferative cytokines such as transforming growth factor-beta1 and high concentrations of all-trans-retinoic acid in cytokine-supported CD34+ cultures downmodulated telomerase activity. Average telomere lengths were 10.4 kbp, 7.4 kbp, and 7.6 kbp in CB, PB, and BM CD34+ cells, respectively. In ex vivo expansion cultures, an average telomeric DNA loss of 1 to 2 kbp over 4 weeks was observed. However, the rate of base pair loss per population doubling was significantly lower during the first 2 weeks, when telomerase was upregulated, than during weeks 3 and 4 of culture. In summary, telomerase is upregulated in response to cytokine-induced proliferation and cell cycle activation in primitive hematopoietic cells. Telomerase is downregulated between weeks 3 and 4 of ex vivo expansion culture linked with decreased proliferation and greater expansion of more mature cell subsets. Our data suggest that telomerase activity in hematopoietic cells reduces, but does not prevent, telomere shortening on proliferation.
1alpha,25-Dihydroxyvitamin D3 (1,25(OH)2D3) alters the proliferation of neuroblastoma cells in culture in part via a nerve growth factor (NGF)-mediated pathway. This suggests that factors other than NGF also play a role in the growth arrest induced by 1,25(OH)2D3. To more fully characterize the effect of 1,25(OH)2D3 on neuroblastoma cells, we treated the cells with 10(-8) M 1,25(OH)2D3 and examined the cells for changes in the expression of N-myc, c-myc, transforming growth factor-beta2 (TGF-beta2), and protein kinase C (PKC) activity. Our results show that 1,25(OH)2D3 causes a decrease in the expression of N-myc and c-myc, as well as a two-fold increase in total PKC activity and a dose-dependent increase in TGF-beta2 expression. These results show that 1,25(OH)2D3 regulates the expression of growth-regulatory factors other than NGF in neuroblastoma cells and that 1,25(OH)2D3 influences the growth of neural cells via multiple growth regulatory pathways.
Apoptosis is a physiological mechanism of cell death that plays an important role in the regulation of tissue homeostasis. The regulation of apoptosis is a complex process and involves a number of gene products including the survival factor Bcl-2, which has been found to be frequently deregulated in human cancers. In addition to deregulation of apoptosis, the process of neoplasia is also believed to be driven by the activation of telomerase, a ribonucleoprotein complex that adds telomeric repeats (hexanucleotide 5'-TTAGGG-3') to the ends of replicating chromosomes. Activation of telomerase has been detected in a vast majority of human cancer cells. Although recent studies have demonstrated the wide occurrence of telomerase activation and Bcl-2 deregulation in human cancer cells, it remains unclear whether there is any linkage between the deregulation of Bcl-2 and telomerase activity in cancer cells. In the studies presented here, we report that the stable overexpression of Bcl-2 in human cancer cells with low Bcl-2 expression was accompanied by increased levels of telomerase activity. In addition, using an IL-2-dependent cytotoxic T-cell line, CTLL-2, we demonstrated that IL-2 deprivation (8 h), which is known to down-regulate Bcl-2 expression, also resulted in concurrent inhibition of telomerase activity in the absence of any detectable apoptosis and accumulation of cells in the G0/G1 phase of the cell cycle. Re-exposure of IL-2-deprived CTLL-2 cells to the recombinant IL-2 led to the up-regulation of both Bcl-2 expression and telomerase activity. Taken together, these findings establish a close linkage between the modulation of telomerase activity by survival factor Bcl-2, and provide a model to study regulation of telomerase activity by an anti-apoptotic pathway that is widely deregulated in cancer cells.
We have already reported that human fibroblast 34-kDa hyaluronic acid-binding protein (HABP) is identical with P32, the protein co-purified with splicing factor SF-2 [Deb and Datta (1996) J. Biol. Chem. 271, 2206-2212]. Data search further revealed that it has 92% sequence homology with a murine protein YL2 which interacts with HIV1 Rev. In this paper we have successfully demonstrated that HIV1 Rev binds with labeled 34-kDa HABP which can be competed with excess unlabeled HABP, suggesting this protein can be a cellular factor promoting HIV1 Rev to function. Interestingly, the multifunctional nature of HABP has been elucidated as it has 100% homology with another protein gC1q, the complement protein. The distinct non-overlapping binding motifs for HA and gC1q have been identified in the same protein, suggesting that either the protein can function independently or its activity is regulated by ligand binding, wherein its binding to one of the ligands may modulate the receptor activity of the other ligand.
Atrial natriuretic peptide (ANP) regulates diverse physiological responses by binding to its specific guanylyl cyclase-A receptor (Npra) which synthesizes the intracellular second messenger cGMP. To understand the molecular mechanisms of cellular signaling of ANP, we have studied its effect on the enzymatic activity of overexpressed protein kinase C (PKC) in murine Leydig tumor (MA-10) cells which were transfected with PKC-alpha cDNA. Treatments with 12-O-tetradecanoylphorbol-13-acetate (TPA), angiotensin II (ANG II) and endothelin-1 (ET-1) stimulated the PKC activity by 4-5-fold in PKC-alpha cDNA transfected MA-10 cells. The pretreatment of PKC-alpha transfected cells with ANP significantly inhibited the TPA-, ANG II- and ET-1-stimulated PKC activity. The agonist-stimulated PKC activity was also inhibited in the presence of 8-bromo-cGMP, however, cAMP had no effect on stimulatory PKC activity. The exposure of cells to Npra- antagonist A71915, which blocks the production of cGMP, significantly reduced the inhibitory effect of ANP on agonist-stimulated PKC activity and accumulation of intracellular cGMP in MA-10 cells. Similarly, inhibition of cGMP-dependent protein kinase by KT5823, restored the stimulatory levels of PKC activity in the presence of ANP. These results provide direct evidence that ANP antagonizes the agonist-stimulated PKC activity in MA-10 cells, involving the specific receptor Npra, its second messenger cGMP and cGMP-dependent protein kinase. Together, these findings implicate that ANP may act as a negative mediator of 'cross-talk' between PKC-alpha and Npra signaling pathway in MA-10 cells.
Calbindin D28K, a member of the troponin-C superfamily of calcium-binding proteins, contains six putative EF-hand domains. Calcium-binding studies of the protein by different groups of investigators have yielded discordant results with respect to the stoichiometry of calcium-binding. It has been suggested that the protein binds anywhere from 3-6 mol of calcium/mol of protein. We used negative ion electrospray ionization mass spectrometry in order to definitively determine the exact calcium-binding stoichiometry of calbindin D28K and two mutant forms of the protein, one lacking EF-hand 2 (delta2) and the other lacking EF-hands 2 and 6 (delta2,6). The full-length protein bound 4 mol of calcium/mol of protein, while both of the deletion mutants bound 3 mol of calcium. Since terbium has been used extensively as a probe for the determination of the calcium-binding stoichiometries of calcium-binding proteins, we also examined the binding of terbium to the three proteins under the same conditions. Full-length calbindin D28K bound 4 mol of terbium/mol of protein, while calbindin delta2 and delta2,6 each bound 3 mol. These results clearly show that calbindin D28K binds 4 mol of calcium/mol of protein and that terbium-binding stoichiometry is similar to that of calcium.
To determine the mechanism responsible for the in vivo production of angiostatin that inhibits growth and metastasis in Lewis lung carcinoma (3LL), we implanted 3LL variant cells into the subcutis of syngeneic C57BL/6 mice. The tumors were infiltrated by macrophages and expressed high levels of steady-state mRNA for metalloelastase (MME). Successive passages (more than three) of cultures established from the tumors resulted in complete depletion of macrophages; steady-state MME mRNA, elastinolytic activity, and production of angiostatin (in the presence of plasminogen) were correspondingly reduced. Coculture of macrophages with either 3LL cells or their conditioned media containing granulocyte-macrophage colony-stimulating factor resulted in secretion of MME and production of angiostatin by the macrophages, suggesting that angiostatin is produced by tumor-infiltrating macrophages whose MME expression is stimulated by tumor cell-derived granulocyte-macrophage colony-stimulating factor.
Epitopes of the 1,25-dihydroxyvitamin D(1,25(OH)2D3) receptor have been shown in developing dorsal root ganglia in fetal mice, as well as in cells maintained in culture [Johnson, J.A., Grande, J.P., Windebank, A.J. and Kumar, R., 1,25-Dihydroxyvitamin D3 receptors in developing dorsal root ganglia of fetal rats, Dev. Brain Res., 92 (1996) 120-124]. To investigate a possible role for 1,25(OH)2D3 in neural cell growth and development, a murine neuroblastoma cell line that expresses 1,25(OH)2D3 receptors, was treated with 1,25(OH)2D3. Treatment with 1,25(OH)2D3 resulted in a decrease in cell proliferation, a change in cell morphology, and the expression of protein markers of mature neuronal cells. The decrease in cell proliferation was accompanied by an increase in the expression of nerve growth factor (NGF). Anti-NGF monoclonal antibody added to the growth medium blocked the decrease in cell proliferation caused by 1,25(OH)2D3 treatment. Our results show that the sterol hormone 1,25(OH)2D3, causes a decrease in the proliferation of mouse neuroblastoma cells through alterations in the expression of NGF.
Rat dopamine-producing nerve cells (1RB3AN27) and rat parotid acinar cells (2RSG) were immortalized by insertion of simian virus 40 (SV40) large T-antigen gene (LTa). Both of these cells divided and produced nuclear LTa in vitro. In order to assess the relationship between cell proliferation and expression of LTa in vivo, immortalized dopamine-producing nerve cells and parotid cells were grafted into the striatum and parotid gland of adult Sprague-Dawley rats, respectively. Grafted cells exhibited nuclear LTa at 1 day but not at 7 and 30 days after transplantation. At 30 days after transplantation, no tumor was found, and there was no evidence of cell division as determined by H and E staining. When the striatal areas containing the grafts were cultured, these cells did not express LTa at 4 days after plating; however, after 3 weeks, when most host cells were eliminated, the cultured grafted cells expressed LTa. After 3 months of culturing, only cells exhibiting LTa were present. These cells had the same morphology and divided with the same doubling time as 1RB3AN27 cells before grafting. Results suggest the presence of a LTa-inhibiting factor in vivo, and support the hypothesis that the expression of LTa is directly linked with proliferation of immortalized cells.
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This study compared the effects of dopamine receptor stimulation in the entorhinal cortex on dopamine release in the nucleus accumbens, measured by in vivo microdialysis in conscious Sprague-Dawley rats, with and without oestradiol and progesterone priming. Nonselective dopamine receptor stimulation with apomorphine reduced dopamine release in the nucleus accumbens, an effect which was prevented by injection of cis-flupenthixol into the entorhinal cortex. Selective D1 receptor stimulation with SKF38393 increased dopamine release, whereas selective D2 receptor stimulation with quinpirole did not affect dopamine release. Combined administration of oestradiol and progesterone potentiated the response to apomorphine and prevented the response to SKF38393. The effects of single hormone administration on the response to apomorphine suggested that the modulation was primarily due to oestradiol enhancing effects of progesterone. Experiments with high [K+] suggested these hormonal effects were exerted predominantly in the entorhinal cortex. The present experiments have demonstrated that dopaminergic modulation of transmission in a cortico-striatal loop linking temporal and prefrontal cortex is regulated by oestradiol and progesterone. Dysfunction in this system in humans may give rise to affective and cognitive symptoms which may, if initiated by a postpartum fall in oestrogen and progesterone concentrations, constitute the core pathophysiology of puerperal psychosis.
A group of 5-ethyl-5-halo-6-alkoxy (or azido)-5,6-dihydro-2'-deoxyuridines, which differ in configuration at the C-5 and C-6 positions, were synthesized by the regiospecific addition of XR (X = I, Br, Cl; R = alkoxyl, azido) to the 5,6-olefinic bond of 5-ethyl-2'-deoxyuridine (EDU). In vitro antiviral (HSV-1, HSV-2, HCMV, VZV) activities were determined. Structure-activity studies showed that the C-5 halogeno (I, Br, Cl) and C-6 alkoxy (OMe, OEt) or azido, substituents were determinants of antiviral activity where the (5R,6R)-5 and (5S,6S)-6 diastereomers of 5-ethyl-5-iodo-6-methoxy-5,6-dihydro-2'-deoxyuridine exhibited greater potency against HSV-1, HSV-2, and HCMV than the related 5-chloro-6-ethoxy and 5-bromo (or chloro)-6-azido diastereomers. The most potent antiviral agents, (+)-trans-(5R,6R)-5 and (-)-trans-(5S,6S)-6 diastereomers of 5-ethyl-5-iodo-6-methoxy-5,6-dihydro-2'-deoxyuridine were approximately 2-to-8 fold more potent than the reference drug EDU against HSV-1 and HSV-2.
The authors present a young boy with severe generalized dystonia treated with bilateral simultaneous pallidotomy. Microelectrode recordings with the patient under propofol anesthesia showed that the mean discharge rate of globus pallidus internus (GPi) neurons was between 21 and 31 Hz. This contrasts sharply with the mean GPi neuronal firing rates of approximately 80 Hz that are characteristic of Parkinson's disease. The patient had no immediate benefit from surgery, but a progressive improvement in both axial and limb dystonia began within 3 days. The Burke-Fahn-Marsden scores were 75 (maximum possible = 120) at baseline, 52 at 5 days, and 16 at 3 months after surgery. The mechanism of action of pallidotomy for dystonia and the reasons for the delayed and progressive improvement are unknown. Nevertheless, the magnitude of the improvement and the safety of the procedure in this one patient warrant a careful evaluation of pallidotomy for dystonia.
Frameless 3D volume registration of Magnetic Resonance (MR) and computed (CT) data sets has been described by Kummar et al. [11]. Its use in 3D volume registration for stereotactic planning in patients undergoing pallidotomy is presented. Pre-operative examinations with the stereotactic frame and postoperative examinations without the stereotactic frame can be co-registered and reviewed for accuracy of planned and lesional coordinates.
Hydropericardium syndrome (HPS) in broiler birds of 3 to 6 weeks of age was recorded for the first time in the Haldwani area of Nainital district (UP) in India in November, 1994. The overall mortality in 6 poultry farms was 61.62 per cent. The disease was experimentally transmitted by bacteria free infected liver homogenate extract passed through membrane filters of 0.22 and 0.1 mu APD. The aetiological agent was inactivated by heat treatment at 56 degrees C for one hour and 80 degrees C for 10 min. A precipitin band was demonstrated in agar gel immunodiffusion and counter immunoelectrophoresis using infected liver homogenate extract as antigen and homologous antisera raised in the laboratory. The disease was effectively controlled by formalinised and heat inactivated autogenous vaccine prepared from the infected livers of birds which died of natural infection.
Two experiments examined dose-related effects of 200, 400 and 600 micrograms scopolamine (n = 24, s.c.) and 5 and 10 mg diazepam (n = 6, PO) on parallel tests of visual memory and learning taken from the CANTAB battery. Scopolamine significantly impaired accuracy of performance on a delayed matching to sample test of visual recognition memory in a dose- and delay-dependent manner, but had only marginal decremental effects on a test of visuospatial paired associates learning. Scopolamine significantly lengthened decision times in a visual search matching to sample task at the 400 and 600 micrograms doses, without significantly affecting accuracy. The drug also impaired performance on tests of spatial (on accuracy and response time measures) and pattern (on response time only) memory. Most of the deleterious effects on scopolamine were removed by covariance analyses with indices of subjective sedation, but the effects of delayed matching accuracy and latency remained. By contrast, diazepam significantly impaired paired associates learning but affected delayed matching to sample in a delay-independent manner. These results suggest that scopolamine can produce selective deficits in tests of short-term visual recognition memory which do not depend on overall impairments in arousal and which contrast with deficits in visual associative learning produced by diazepam. They have implications for the pharmacological modelling of dementia and memory disorders in man and for the neurochemical substrates of the short-term recognition memory and associative learning for visual stimuli.
OBJECTIVE: Cyclic GMP has been shown to be in some respects an inhibitory modulator of heart function. Various studies on the modulation of cardiac L-type calcium current (ICa) by cGMP in different species show inconsistency and the role of cGMP remains unclear and controversial. The present study was focused on the differences in the modulation of basal ICa by cGMP in adult and newborn rabbit ventricular cells. METHODS: Enzymatically isolated adult and newborn (1-4-day-old) rabbit ventricular myocytes were used to measure ICa under whole-cell voltage clamp conditions with internal perfusion of isolated cells. RESULTS: We have shown that in adult ventricular cells, the intracellular perfusion of 8BrcGMP did not produce any effect on basal ICa, while intracellular perfusion of 8BrcGMP or 8CPT-cGMP in newborn ventricular cells significantly and reversibly increased basal ICa without changing the voltage dependence for activation of ICa. Both methylene blue and LY-83583 (which inhibit guanylyl cyclase and thus lower cGMP levels), in adult ventricular cells, failed to produce any significant effect on basal ICa, while in newborn ventricular cells the application of methylene blue or LY-83583 produced irreversible inhibition of basal ICa. Similarly, KT-5823, an inhibitor of cGMP-dependent protein kinase, also inhibited basal ICa in newborn ventricular cells but not in adult ventricular cells. However, extracellular application of methylene blue during the intracellular perfusion of 8BrcGMP was unable to inhibit ICa. Extracellular application of nitrosoglutathione which releases nitric oxide produced a significant increase in ICa in newborn but not in adult ventricular cells. Intracellular application of a cAMP-dependent protein kinase inhibitor peptide blocked the stimulatory effect of cAMP but not of 8CPT-cGMP, while the stimulatory effect of nitrosoglutathione on ICa was not blocked by the presence of a phosphodiesterase inhibitor (isobutylmethyl-xanthine). CONCLUSIONS: We propose that, for newborn rabbit ventricular cells, cGMP plays a crucial role in maintaining basal ICa by a mechanism mediated via protein-kinase-G-dependent phosphorylation of calcium channels or some associated protein.