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Biomedical subjects

A Kumar

Publications and source records attributed to A Kumar.

At least 757 records · Page 42Linked to original sources
PubMed1997

p21ras farnesyltransferase alpha- and beta-subunits are phosphorylated in PC-12 cells: TGF-beta signaling pathway independent phosphorylation.

Farnesyltransferase (FTase) catalyzes the transfer of a farnesyl isoprenoid to the conserved carboxyl-terminal cysteine residue of proteins terminating with the CAAX sequence. Rat brain FTase is a heterodimer consisting of a 49 kDa alpha-subunit and a 46 kDa beta-subunit. In this report, we show, for the first time, that the beta-subunit of FTase is phosphorylated in vivo and the FTase heterodimer contains phosphorylated alpha/beta-subunits in rat adrenal medulla pheocytochroma PC-12 cells. The presence of the phosphorylated FTase subunits as heterodimer in PC-12 cells which are known to be deficient in TGF-beta signaling pathways argues against the involvement of this pathway in their phosphorylation and heterodimerization.

Alkyl and Aryl Transferases↗
PubMed1997

Human immunodeficiency virus-1-tat protein induces the cell surface expression of endothelial leukocyte adhesion molecule-1, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1 in human endothelial cells.

Human vascular endothelial cells (EC) have been implicated in the dissemination of human immunodeficiency virus type-1 (HIV-1). HIV-1-tat, a viral gene product essential for HIV replication, has been shown to interact with different cell types, altering their growth and inducing gene expression. In the present report, we have examined the effect of HIV-tat on the expression of various adhesion molecules in human umbilical vein EC. Our results show that treatment of EC with HIV-tat induces the cell surface expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and endothelial leukocyte adhesion molecule-1 in a time- and dose-dependent manner. Cycloheximide abolished the HIV-tat-dependent induction of all the adhesion molecules, indicating that protein synthesis was required for induction. The effect of HIV-tat on expression of adhesion molecules was potentiated by tumor necrosis factor (TNF), a well-known inducer of adhesion molecules. Like TNF, HIV-tat also enhanced the adhesion of human promyelomonocytic HL-60 cells to EC, and this effect was abolished by treatment with antibodies either against HIV-tat or adhesion molecules. Our results thus indicate that the HIV-tat protein can activate human vascular EC to induce the expression of various adhesion molecules that may play a role in the extravasation of HIV-infected cells.

Cycloheximide↗
PubMed1997

Expression of pro form of prostate-specific antigen by mammalian cells and its conversion to mature, active form by human kallikrein 2.

To study the expression, biosynthesis, and processing of prostate-specific antigen (PSA) in mammalian cells, recombinant PSA was expressed in Syrian hamster tumor cell line AV12-664 (AV12-PSA). Expression of PSA was monitored by the Tandem-MP PSA assay. PSA was secreted into the medium during the logarithmic phase of cell growth at >9 microg/ml and was stable. The PSA purified from spent medium of AV12-PSA cells did not exhibit any enzymatic activity and did not complex with the protease inhibitor, alpha-1-antichymotrypsin. These findings indicated that an inactive form of PSA was expressed by AV12-PSA cells. NH2-terminal sequencing confirmed the identity of the PSA purified from the spent medium of AV12-PSA cells to be pro-PSA. This demonstrates that PSA is expressed as pro-PSA by mammalian cells and suggests that pro-PSA may be present in biological fluids. Human kallikrein 2 (hK2), another member of the hK family, is also expressed predominantly in prostate epithelium. Although hK2 has been shown to exhibit trypsin-like activity, little is known about its natural substrates. Using purified proteins, we show that hK2 can convert pro-PSA to mature, enzymatically active PSA, thus establishing a physiological connection between hK2 and PSA. These findings imply that hK2 may be regulating PSA activity in vivo.

Amino Acid Sequence↗
PubMed1997

Developmental regulation and the role of insulin and insulin receptor in metanephrogenesis.

The insulin family of peptides and their receptors influence cellular growth in very early preimplantation embryos. In this study their expression and role in renal organogenesis was investigated. By immunofluorescence microscopy and in situ hybridization, insulin receptor (IR) expression was seen in the ureteric bud branches and early nephron precursors in mouse metanephroi harvested at day 13 of gestation. The expression gradually decreased in successive stages of gestation, and it was confined mainly to renal tubules in 1-week-old mice. Similar developmental regulation of the IR and insulin was observed by reverse transcriptase-polymerase chain reaction (RT-PCR) analyses. Addition of insulin into the culture medium at low concentrations, ranging from 40 to 400 ng/ml, induced trophic changes and increased [3H]thymidine incorporation in the embryonic renal explants, and inclusion of IR beta-subunit-specific antisense oligodeoxynucleotide caused marked dysmorphogenesis and growth retardation of the metanephroi. Specificity of the antisense effect was reflected by immunoprecipitation experiments in which translational blockade of the beta subunit of the IR was observed. RT-PCR analyses revealed that the alpha subunit of the IR was unaffected by the antisense treatment of metanephric explants. Concomitantly, de novo synthesis of morphogenetic regulatory extracellular matrix proteins, especially the proteoglycans, was decreased. Gel-shift analyses indicated a failure in the activation of c-fos promoter region binding protein(s) by insulin in the antisense oligodeoxynucleotide-treated explants. These studies suggest that insulin and its putative receptor are developmentally regulated in the murine embryonic metanephros, and they play a role in renal organogenesis, possibly by affecting other modulators of morphogenesis-i.e., extracellular matrix proteins and protooncogenes.

Animals↗
PubMed1997

Isolation and characterization of complement receptor type 1 (CR1) storage vesicles from human neutrophils using antibodies to the cytoplasmic tail of CR1.

Neutrophil (PMN) activation is associated with increased surface expression of several membrane proteins that are translocated from intracellular pools. Indirect evidence suggests that the intracellular storage pools of complement receptor type 1 (CR1) in resting PMN are distinct from traditional granules and may be the secretory vesicles in which albumin is also stored, but it is not known if this compartment is homogeneous or heterogeneous. To isolate and characterize the CR1-containing vesicles, we used antibodies against unique sequences in the cytoplasmic tail of CR1. Affinity-purified IgG was used to adsorb CR1 storage vesicles from the light membrane fraction (gamma-band) of nitrogen cavitates of resting PMN. The immunoadsorbent could quantitatively remove the CR1-containing vesicles, whereas control adsorbents with nonimmune IgG showed no specific binding of CR1. Immunoblots of specifically isolated vesicles also showed enrichment of albumin, decay-accelerating factor, Fc gammaRIII, and CR3; whereas HLA class I was not detectable in these vesicles. Enzyme assay of specifically isolated vesicles after treatment with Triton X-100 showed that these vesicles contained most of the cells' latent alkaline phosphatase. An additional population of vesicles containing albumin, but not CR1, and that did not bind to anti-CR1 adsorbent was also identified. Immunoelectron microscopy showed that the specifically isolated vesicles had mean diameters of 0.086 to 0.1 microm and stained positive for CR1 and albumin. These results indicate that CR1 storage vesicles can be isolated with antibodies against the cytoplasmic tail of CR1 and show that these vesicles also contain albumin as well as glycosylphosphatidyl inositol-anchored proteins. These results are most compatible with the hypothesis that CR1-containing vesicles have arisen by endocytic retrieval of proteins that had been on the plasma membrane.

Adult↗
PubMed1997

Ala217 is important for the catalytic function and autoactivation of prostate-specific human kallikrein 2.

Prostate-specific human kallikrein, hK2, is a serine protease found in prostate tissues that has 78% amino acid sequence identity with prostate-specific antigen (PSA). We have previously reported the affinity purification of hK2 heterologously expressed in a hamster cell line and demonstrated an arginine-restricted substrate specificity. Here, we describe the cloning, expression, purification, and enzymatic activity of a mutant form of hK2 containing an alanine to valine substitution at residue 217 ([Val217]hK2). This mutant form was secreted into the serum-free spent media of recombinant cells as the stable proenzyme form ([Val217]phK2). Mild trypsin treatment was used to convert [Val217]phK2 to the active form, which had reduced catalytic function compared to the wild-type hK2. Kinetic studies using the chromogenic substrate D-H-Pro-Phe-Arg-4-nitroanilide showed that [Val217]hK2 has significantly decreased substrate binding, with a K(m) of 4200 microM compared to 11 microM for wild-type hK2. The k(cat) for [Val217]hK2 was more than 100-fold lower than for hK2. hK2, but not [Val217]hK2, was able to activate [Val217]phK2. [Val217]hK2 also showed altered specificity on a synthetic peptide substrate compared to wild-type hK2, which exhibited partial hydrolysis at a PSA chymotrypsin-like cleavage site as well as the trypsin-like site cleaved by hK2. These results indicate that Ala217 is a key residue affecting the catalytic properties of hK2.

Alanine↗
PubMed1997

Developmental regulation, expression, and apoptotic potential of galectin-9, a beta-galactoside binding lectin.

Galectin-9, a beta-galactoside binding lectin, has recently been isolated from murine embryonic kidney. In this study, its biological functions and expression in embryonic, newborn, and adult mice tissues were investigated. By Northern blot analyses, it was found widely distributed and its expression was developmentally regulated. In situ hybridization studies revealed an accentuated expression of galectin-9 in liver and thymus of embryonic mice. In postnatal mice, antigalectin-9 immunoreactivity was observed in various tissues, including thymic epithelial cells. The high expression of galectin-9 in the thymus led us to investigate its role in the clonal deletion of thymocytes. Fusion proteins were generated, which retained lactose-binding activity like the endogenous galectin-9. Galectin-9, at 2.5 microM concentration, induced apoptosis in approximately 30% of the thymocytes, as assessed by terminal deoxytransferase-mediated dUTP nick end labeling method. The apoptotic effect was dose dependent and lactose inhibitable. At higher concentrations, it induced homotypic aggregation of the thymocytes. Electron microscopy revealed approximately 60% of the thymocytes undergoing apoptosis in the presence of galectin-9. By immunofluorescence microscopy, some of the thymocytes undergoing apoptosis had plasmalemmal bound galectin-9. Galectin-9 failed to induce apoptosis in hepatocytes. Taken together, these findings indicate that galectin-9, a developmentally regulated lectin, plays a role in thymocyte-epithelial interactions relevant to the biology of the thymus.

Aging↗
PubMed1997

Immediate early and early lytic cycle proteins are frequent targets of the Epstein-Barr virus-induced cytotoxic T cell response.

Epstein-Barr virus (EBV), a human gamma-herpesvirus, can establish both nonproductive (latent) and productive (lytic) infections. Although the CD8+ cytotoxic T lymphocyte (CTL) response to latently infected cells is well characterized, very little is known about T cell controls over lytic infection; this imbalance in our understanding belies the importance of virus-replicative lesions in several aspects of EBV disease pathogenesis. The present work shows that the primary CD8+ CTL response to EBV in infectious mononucleosis patients contains multiple lytic antigen-specific reactivities at levels at least as high as those seen against latent antigens; similar reactivities are also detectable in CTL memory. Clonal analysis revealed individual responses to the two immediate early proteins BZLF1 and BRLF1, and to three (BMLF1, BMRF1, and BALF2) of the six early proteins tested. In several cases, the peptide epitope and HLA-restricting determinant recognized by these CTLs has been defined, one unusual feature being the number of responses restricted through HLA-C alleles. The work strongly suggests that EBV-replicative lesions are subject to direct CTL control in vivo and that immediate early and early proteins are frequently the immunodominant targets. This contrasts with findings in alpha- and beta-herpesvirus systems (herpes simplex, cytomegalovirus) where viral interference with the antigen-processing pathway during lytic infection renders immediate early and early proteins much less immunogenic. The unique capacity of gamma-herpesvirus to amplify the viral load in vivo through a latent growth-transforming infection may have rendered these agents less dependent upon viral replication as a means of successfully colonizing their hosts.

Amino Acid Sequence↗
PubMed1997

Identification of N(G)-methylarginine residues in human heterogeneous RNP protein A1: Phe/Gly-Gly-Gly-Arg-Gly-Gly-Gly/Phe is a preferred recognition motif.

Three sites of N(G),N(G)-arginine methylation have been located at residues 205, 217, and 224 in the glycine-rich, COOH-terminal one-third of the HeLa A1 heterogeneous ribonucleoprotein. Together with the previously determined dimethylated arginine at position 193 [Williams et al., (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5666-5670], it is evident that all four sites fall within a span of sequence between residues 190 and 233 that contains multiple Arg-Gly-(Gly) sequences interspersed with phenylalanine residues. These RGG boxes have been postulated to represent an RNA binding motif [Kiledjian and Dreyfuss (1992) EMBO J. 11, 2655-2664]. Dimethylation of HeLa A1 appears to be quantitative at each of the four positions. Arginines 205 and 224 have been methylated in vitro by a nuclear protein arginine methyltransferase using recombinant (unmethylated) A1 as substrate. This suggests A1 may be an in vivo substrate for this enzyme. Examination of sequences surrounding the sites of methylation in A1 along with a compilation from the literature of sites that have been identified in other nuclear RNA binding proteins suggests a methylase-preferred recognition sequence of Phe/Gly-Gly-Gly-Arg-Gly-Gly-Gly/Phe, with the COOH-terminal flanking glycine being obligatory. Taken together with data in the literature, identification of the sites of A1 arginine methylation strongly suggests a role for this modification in modulating the interaction of A1 with nucleic acids.

Amino Acid Sequence↗
PubMed1997

Rescue of cardiac alpha-actin-deficient mice by enteric smooth muscle gamma-actin.

The muscle actins in higher vertebrates display highly conserved amino acid sequences, yet they show distinct expression patterns. Thus, cardiac alpha-actin, skeletal alpha-actin, vascular smooth muscle alpha-actin, and enteric smooth muscle gamma-actin comprise the major actins in their respective tissues. To assess the functional and developmental significance of cardiac alpha-actin, the murine (129/SvJ) cardiac alpha-actin gene was disrupted by homologous recombination. The majority ( approximately 56%) of the mice lacking cardiac alpha-actin do not survive to term, and the remainder generally die within 2 weeks of birth. Increased expression of vascular smooth muscle and skeletal alpha-actins is observed in the hearts of newborn homozygous mutants and also heterozygotes but apparently is insufficient to maintain myofibrillar integrity in the homozygous mutants. Mice lacking cardiac alpha-actin can be rescued to adulthood by the ectopic expression of enteric smooth muscle gamma-actin using the cardiac alpha-myosin heavy chain promoter. However, the hearts of such rescued cardiac alpha-actin-deficient mice are extremely hypodynamic, considerably enlarged, and hypertrophied. Furthermore, the transgenically expressed enteric smooth muscle gamma-actin reduces cardiac contractility in wild-type and heterozygous mice. These results demonstrate that alterations in actin composition in the fetal and adult heart are associated with severe structural and functional perturbations.

Actins↗
PubMed1997

Destructive spondyloarthropathy of the cervical spine in patients with chronic renal failure.

STUDY DESIGN: Eleven patients with chronic renal failure and destructive spondyloarthropathy of the cervical spine were evaluated with plain radiographs, flexion-extension views, computed tomography myelogram, or magnetic resonance imaging to determine the results of surgical and nonsurgical treatment. OBJECTIVES: To determine if cervical spine fusion is an effective method of treatment for patients with chronic renal failure and destructive spondyloarthropathy. SUMMARY OF BACKGROUND DATA: Several reports have described the pathogenesis and appearance of this condition, but little has been reported about the orthopedic management of destructive spondyloarthropathy of the cervical spine. METHODS: Three patients had no spinal surgery, three patients had laminectomies alone, three patients had laminectomies with anterior fusions, and two patients had laminectomies with posterior fusions. Radiographs, computed tomography myelograms, and magnetic resonance images were evaluated to determine the results of treatment. Histologic examinations were performed in two patients. RESULTS: Patients with laminectomy alone had no improvement in pain or neurologic function (one died in the immediate postoperative period), one of three patients with anterior fusions had some improvement (one died in the immediate postoperative period), and both of those patients with posterior fusions improved, although both died within a year of surgery from unrelated causes. CONCLUSIONS: Even though the osteopenia present in patients with chronic renal failure tends to allow wire pull-out and makes internal fixation of the spine difficult, successful cervical spinal fusion can relieve pain and improve neurologic deficits in selected patients with chronic renal failure and destructive spondyloarthropathy, allowing them to remain more active for longer periods of time.

Aged↗
PubMed1997

Genetic distribution of Bare-1-like retrotransposable elements in the barley genome revealed by sequence-specific amplification polymorphisms (S-SAP).

Retrotransposons are present in high copy number in many plant genomes. They show a considerable degree of sequence heterogeneity and insertional polymorphism, both within and between species. We describe here a polymerase chain reaction (PCR)-based method which exploits this polymorphism for the generation of molecular markers in barley. The method produces amplified fragments containing a Bare-1-like retrotransposon long terminal repeat (LTR) sequence at one end and a flanking host restriction site at the other. The level of polymorphism is higher than that revealed by amplified fragment length polymorphism (AFLP) in barley. Segregation data for 55 fragments, which were polymorphic in a doubled haploid barley population, were analysed alongside an existing framework of some 400 other markers. The markers showed a widespread distribution over the seven linkage groups, which is consistent with the distribution of the Bare-1 class of retrotransposons in the barley genome based on in situ hybridisation data. The potential applicability of this method to the mapping of other multicopy sequences in plants is discussed.

Genome, Plant↗
PubMed1997

Cytotoxic T lymphocyte (CTL) adherence assay (CAA): a non-radioactive assay for murine CTL recognition of peptide-MHC class I complexes.

Cytotoxic T lymphocytes (CTL) form an important immune surveillance system against intracellular pathogens. Here we describe a simple, visual assay for identifying peptides specifically recognized by CTL, based on the discovery that CTL develop increased adhesive properties upon TCR triggering. Several CTL lines were shown to pellet to the bottom of a round bottom 96-well plate in the absence of peptide. In contrast, these same CTL lines incubated with their cognate peptide, allowing them to present peptide to each other, adhered to the sides of the well and were readily distinguished by macroscopic visual examination of the plate after 4-5 h or overnight incubation. This CTL adherence assay (CAA) demonstrated peptide specificity and MHC restriction, and was titratable with peptide concentration. With this technique, a minimal-sized, malaria CTL epitope was correctly identified from a panel of overlapping nonamers, although the adherence pattern of two mono-substituted, variant peptides was less predictive of lytic activity. Also, substitutions in an HIV-1 envelope CTL epitope that reduced lytic activity were correctly predicted. Inhibitors of RNA and protein synthesis, upon preincubation, abrogated the adherence, indicating, at minimum, a need for live cells. Wortmannin, a PI-3 kinase inhibitor, inhibited the peptide specific adherence, consistent with a role for TCR or integrin signal transduction in CAA. Other cytoskeletal and metabolic inhibitors had no effect. Adherence of the T cells may involve low affinity, nonspecific interactions since wells coated with FCS, BSA or milk powder all produced an effective CAA in the presence of peptide under serum free conditions. Consequently, CAA may represent a rapid, simple method for screening large numbers of peptides to find cytolytic epitopes for a given CTL line and may identify additional epitopes causing T cell activation and adherence but not cytolytic activity.

Animals↗
PubMed1997

Down-regulation of surface receptors for TNF and IL-1 on circulating monocytes and granulocytes during human endotoxemia: effect of neutralization of endotoxin-induced TNF activity by infusion of a recombinant dimeric TNF receptor.

Leukocytes rapidly lose their surface receptors for TNF and IL-1 upon exposure to various stimuli in vitro. We sought to determine by FACS analysis changes in the expression of TNF receptors (TNFR) and type II IL-1R on circulating monocytes and granulocytes during endotoxemia in vivo, and the role of endogenous TNF in these changes. Twelve healthy subjects received an i.v. injection with LPS (2 ng/kg), directly preceded by a 30-min infusion of either a recombinant human dimeric TNFR type II-IgG fusion protein (TNFR:Fc; 6 mg/m2; n = 6) or vehicle (n = 6). LPS administration was associated with decreases in the expression of types I and II TNFR and type II IL-1R on both monocytes and granulocytes. Treatment with TNFR:Fc completely neutralized LPS-induced TNF activity (p < 0.0001 vs LPS only), modestly blunted the decrease in monocyte TNFR (p < 0.05), but did not influence reduced expression of granulocyte TNFR or monocyte/granulocyte type II IL-1R. In separate experiments, rTNF added to whole blood reduced cellular type I and type II TNFR expression by an effect on the type I TNFR; TNF did not (monocytes) decrease or only marginally (granulocytes) decreased type II IL-1R expression. LPS induces down-modulation of monocyte and granulocyte receptors for TNF and IL-1 in humans in vivo. TNF is involved in reduced monocyte TNFR expression during endotoxemia.

Adult↗
PubMed1997

Deficient cytokine signaling in mouse embryo fibroblasts with a targeted deletion in the PKR gene: role of IRF-1 and NF-kappaB.

The interferon (IFN)-induced double-stranded RNA (dsRNA)-activated Ser/Thr protein kinase (PKR) plays a role in the antiviral and antiproliferative effects of IFN. PKR phosphorylates initiation factor eIF2alpha, thereby inhibiting protein synthesis, and also activates the transcription factor, nuclear factor-kappaB (NF-kappaB), by phosphorylating the inhibitor of NF-kappaB, IkappaB. Mice devoid of functional PKR (Pkr(o/o)) derived by targeted gene disruption exhibit a diminished response to IFN-gamma and poly(rI:rC) (pIC). In embryo fibroblasts derived from Pkr(o/o) mice, interferon regulatory factor 1 (IRF-1) or guanylate binding protein (Gbp) promoter-reporter constructs were unresponsive to IFN-gamma or pIC but response could be restored by co-transfection with PKR. The lack of responsiveness could be attributed to a diminished activation of IRF-1 and/or NF-kappaB in response to IFN-gamma or pIC. Thus, PKR acts as a signal transducer for IFN-stimulated genes dependent on the transcription factors IRF-1 and NF-kappaB.

Animals↗
PubMed1997

Coronary artery fistula in children and adults: a review of 25 cases with long-term observations.

We studied 25 patients with coronary artery fistula between 1976 and 1994. Age ranged from 1 to 58 years. Twelve patients were symptomatic; seven had dyspnoea, four had angina, one had palpitation and one had syncope. Coronary arteries in four. Coronary artery fistula drained into right ventricle in 11, right atrium in nine, pulmonary artery in four and left ventricle in two. The Qp/Qs ranged from 1.0 to 2.6 with a mean of 1.39 +/- 0.38. Five patients had associated cardiac anomalies. Two had atrial septal defects, one had patent ductus arteriosus, one had atresia of proximal right coronary artery and in one patient, the right coronary was arising from left coronary artery. Five patients underwent surgery without any operative mortality. Thirteen patients were followed-up medically for a mean period of 6.1 +/- 5.1 years. There were no complications related to coronary artery fistula during follow-up. In one patient coronary artery fistula closed spontaneously.

Adolescent↗