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A Kumar

Publications and source records attributed to A Kumar.

At least 865 records · Page 48Linked to original sources
PubMed1997

The evolution of Ty1-copia group retrotransposons in eukaryote genomes.

The Ty1-copia group of LTR retrotransposons has been studied extensively in yeast and Drosophila, the organisms in which they were first discovered, and more recently in higher plant and vertebrate species. Their properties, such as copy number, sequence homogeneity, transcriptional and transpositional activity vary greatly between these different hosts. We will try to resolve these apparent discrepancies between these properties, explain any fundamental differences in the biology of the Ty1-copia group between hosts, and propose a general model for LTR retrotransposon evolution.

Base Sequence↗
PubMed1997

The chromosomal distributions of Ty1-copia group retrotransposable elements in higher plants and their implications for genome evolution.

Retrotransposons make up a major fraction--sometimes more than 40%--of all plant genomes investigated so far. We have isolated the reverse transcriptase domains of the Ty1-copia group elements from several species, ranging in genome size from some 100 Mbp to 23,000 Mbp, and determined the distribution patterns of these retrotransposons on metaphase chromosomes and within interphase nuclei by DNA:DNA in situ hybridization. With some exceptions, the reverse transcriptase domains were distributed over the length of the chromosomes. Exclusion from rDNA sites and some centromeres (e.g., slash pine, 23,000 Mbp, or barley, 5500 Mbp) is frequent, whereas many species exclude retrotransposons from other sites of heterochromatin (e.g., intercalary and centromeric sites in broad bean). In contrast, in the plant Arabidopsis thaliana, widely used for plant molecular genetic studies because of its small genome (c. 100 Mbp), the Ty1-copia group reverse transcriptase gene domains are concentrated in the centromeric regions, colocalizing with the 180 bp satellite sequence pAL1. Unlike the pAL1 sequence, however, the Ty1-copia signal is also detectable as weaker, diffuse hybridization along the lengths of the chromosomes. Possible mechanisms for evolution of the contrasting distributions are discussed. Understanding the physical distribution of retrotransposons and comparisons of the distribution between species is critical to understanding their evolution and the significance for generation of the new patterns of variability and in speciation.

Chromosome Mapping↗
PubMed1997

The Ty1-copia group of retrotransposons in plants: genomic organisation, evolution, and use as molecular markers.

The genomic organisation and diversity of the Ty1-copia group retrotransposons has been investigated in several crop plants and their relatives from both dicotyledonous and monocotyledonous families, including potato (Solanum tuberosum), faba beans (Vicia faba), Vicia melanops, Vicia sativa, barley (Hordeum vulgare), rye (Secale cereale), and onion (Allium cepa). Extreme heterogeneity in the sequence of the Ty1-copia retrotransposons from all these plants was revealed following sequence analysis of reverse transcriptase fragments. The estimated copy numbers of the Ty1-copia group retrotransposons for the genomes of S. tuberosum, L. esculentum, A. cepa, S. cereale, and V. faba is highly variable, ranging from a few hundred to approximately a million copies per genome. In situ hybridisation data from metaphase and prophase chromosomes of V. faba, S. cereale, and H. vulgare suggest that retrotransposon sequences are dispersed throughout the euchromatic regions of the genome but are almost undetectable in most heterochromatic regions. In contrast, similar data from metaphase chromosomes of A. cepa suggests that although retrotransposon sequences are dispersed throughout the euchromatic regions of the genome, they are predominantly concentrated in the terminal heterochromatin. These results are discussed in the context of the role played by the Ty1-copia group retrotransposons in the evolution of the plant genome. Lastly, the application of retrotransposon sequences as genetic markers for mapping genomes and for studying genetic biodiversity in plants is presented.

Chromosome Mapping↗
PubMed1997

Interleukin-6 receptor-interleukin-6 fusion proteins with enhanced interleukin-6 type pleiotropic activities.

An sIL-6R/IL-6 chimera, directly fusing the natural forms of soluble IL-6 receptor and IL-6, as found in human body fluids, was produced in transfected human cells. The secreted p85 glycoprotein was active at a concentration of 120 pM to produce growth-arrest and spindleoid differentiation of murine melanoma F10.9 cells, which do not respond to IL-6 alone. This fusion protein was as active as the yeast-produced p56 fusion protein containing a shortened sIL-6R, linked through a flexible peptide chain to IL-6 (Hyper IL-6). The concentration of Hyper IL-6 needed to arrest the growth of F10.9 cells was much lower than that needed of a combination of IL-6 and sIL-6R, added separately. Hyper IL-6 was also more active than IL-6 in stimulating growth of murine plasmacytoma T1165 cells, the half maximal stimulation being obtained at 2 pM Hyper IL-6 versus 23 pM for IL-6. In order to evaluate the effect of the fused sIL-6R/IL-6 proteins on human hematopoietic primitive progenitor cells, they were added to suspension cultures of CD34+ cells from human cord blood in addition to both flt3/flk2 ligand (FL) and stem cell factor (SCF). Fused sIL-6R/IL-6 produced a marked stimulation of cell expansion and a marked increase in the number of colony forming units when subsequently plated in semi-solid medium with IL-3, GM-CSF, SCF and erythropoietin. Ex-vivo maintenance and expansion of early progenitor cells in bone marrow transplantation protocols may be a potential application for the sIL-6R/IL-6 chimeric glycoproteins.

Animals↗
PubMed1997

Effect of tumor cells on the activation of murine lymphocytes and macrophages by cisplatin and FK565.

Murine peritoneal macrophages on in vitro treatment with cisplatin (5 micrograms/ml) or FK565 (10 micrograms/ml) showed an enhanced production of tumor necrosis factor (TNF) and reactive nitrogen intermediates (RNI). Similarly, treatment of splenic lymphocytes with these agents also led to an enhanced production of TNF. Co-incubation of macrophages or splenic lymphocytes with P815 (a murine mastocytoma) cells in vitro in the absence of cisplatin or FK565 also resulted in an augmented TNF production, however, it had no effect on the RNI production by macrophages. TNF production of cisplatin- or FK565-treated macrophages got synergistically enhanced in the presence of P815 cells in the presence of P815 whereas the production of RNI was inhibited. Incubation of splenic lymphocytes with P815 cells in the presence of cisplatin or FK565 resulted in an inhibition of TNF production. Indomethacin-treated P815 cells were observed to be less effective in inhibiting nitrite production of macrophages compared to untreated tumor cells. Pretreatment of P815 cells with cisplatin or FK565 before co-incubation did not alter the TNF production of macrophages whereas it inhibited the same in lymphocytes. This study shows that activation of macrophages and lymphocytes is independently influenced by P815 tumor cells in combination with chemoimmunotherapeutic drugs cisplatin and FK565.

Adjuvants, Immunologic↗
PubMed1997

Implant diameter and bone density: effect on initial stability and pull-out resistance.

It is well known that the standard diameter dental implant so often utilized is often inadequate in patients with poor bone quality or quantity, and larger diameter implants have been utilized to overcome this deficiency. The objective of this study was to compare the pull-out resistance of small and large diameter (3.25- and 4.5-mm) dental implants and the relationship of these implants to bone density. Two groups of implants, consisting of 18 implants of each diameter, were placed in the mandibles of five embalmed humans. The bone mineral density of the area surrounding the implant site in the coronal cross section was measured by quantitative computed tomography (QCT). Initial implant stability was tested with a periodontium diagnostic device and pull-out resistance was tested with a mechanical testing system. Results showed the same initial stability for the two implants. The maximum pull-out force required for the large diameter implants was 15% greater than that required for the small diameter implants, although given the small number of samples, this difference was not statistically significant. There were significant positive correlations between the pull-out resistance and the bone density for both the large and small diameter implants (p < 0.05, p < 0.01). We conclude that larger diameter implants appear to have advantages over smaller ones; however, more extensive testing is needed to determine quantitatively the increased load-carrying capacity.

Aged↗
PubMed1997

Nipple involvement in peripheral breast carcinoma: a prospective study.

Twenty six females patients of carcinoma of the breast were studied prospectively to find out the incidence of microscopic involvement of the Nipple Areolar Complex (NAC). The tumours were situated at minimum of 2.5 cms away from areola and the nipple was grossly normal. Majority of the patients (69.3%) had T.N.M. stage II disease. Tumours were situated within 4 cms of areola in 88.5% of the cases. Axillary lymph node metastasis was present in 38.4% of the cases. Irrespective of the size & stage of the tumour and tumour areola distance, the incidence of the microscopic involvement of NAC was 0%.

Adult↗
PubMed1996

Identification of a novel cis-acting element participating in maximal induction of the human low density lipoprotein receptor gene transcription in response to low cellular cholesterol levels.

In this paper, we present both in vivo and in vitro evidence for the presence of a novel cis-acting regulatory element that is required for maximal induction of the human low density lipoprotein (LDL) receptor gene following depletion of cellular sterols in HepG2 cells. First, in vivo dimethyl sulfate footprinting of the human LDL receptor promoter before and after transcriptional induction in HepG2 cells revealed protection from -145 to -126, 5'-GAGCTTCACGGGTTAAAAAG-3' (referred to as FP1 site). Second, transient transfections of HepG2 cells with promoter luciferase reporter constructs containing the FP1 site resulted in significant enhancement (approximately 375%) of reporter gene expression in response to low levels of sterols compared with parallel plasmid without the FP1 site. In addition, this response was markedly attenuated on nucleotide substitutions within the FP1 site. Third, by electrophoretic mobility shift assays, the FP1 sequence was found to bind protein(s) from HepG2 nuclear extracts in a sequence-specific manner. In vitro binding of the FP1 mutants paralleled the results obtained for their in vivo transcription. On the basis of competition profiles, the FP1-binding factor is different from the known transcription factors binding to the AT-rich CArG and GArC motifs. Furthermore, the FP1-binding protein is not specific to HepG2 cells because nuclear factor(s) with the same specificity was observed in nuclear extracts of non-hepatic HeLa cells. We conclude that transcriptional induction of the LDL receptor gene in response to sterol depletion is mediated, in part, by an highly conserved novel cis-acting element through the binding of specific nuclear protein(s).

Base Sequence↗
PubMed1996

D-glucose-induced dysmorphogenesis of embryonic kidney.

An organ culture system was used to study the effect of D-glucose on embryonic kidneys, and to delineate the mechanism(s) relevant to their dysmorphogenesis. Metanephroi were cultured in the presence of 30 mM D-glucose. A notable reduction in the size and population of nephrons was observed. Ureteric bud branches were rudimentary and the acuteness of their tips, the site of nascent nephron formation, was lost. Metanephric mesenchyme was atrophic, had reduced cell replication, and contained numerous apoptotic cells. Competitive reverse transcriptase-PCR analyses and immunoprecipitation studies indicated a decrease in expression of heparan sulfate proteoglycan (perlecan). Status of activated protein-2 was evaluated since its binding motifs are present in the promoter region of the perlecan gene. Decreased binding activity of activated protein-2, related to its phosphorylation, was observed. D-glucose-treated explants also had reduced levels of cellular ATP. Exogenous administration of ATP restored the altered metanephric morphology and reduced [35S]sulfate-incorporated radioactivity associated with perlecan. The data suggest that D-glucose adversely affects the metanephrogenesis by perturbing various cellular phosphorylation events involved in the transcriptional and translational regulation of perlecan. Since perlecan modulates epithelial/mesenchymal interactions, its deficiency may have led to the metanephric dysmorphogenesis and consequential atrophy of the mesenchyme exhibiting accelerated apoptosis.

Actins↗
PubMed1996

Phorbol ester stimulation increases sickle erythrocyte adherence to endothelium: a novel pathway involving alpha 4 beta 1 integrin receptors on sickle reticulocytes and fibronectin.

Sickle-cell adherence to endothelium has been hypothesized to initiate or contribute to microvascular occlusion and pain episodes. Adherence involves plasma proteins, endothelial-cell adhesion molecules, and receptors on sickle erythrocytes. It has previously been reported that sickle reticulocytes express the alpha 4 beta 1 integrin receptor and bind to cytokine-activated endothelium via an alpha 4 beta 1/vascular-cell adhesion molecule-1 (VCAM-1) interaction. To elucidate other roles for alpha 4 beta 1 in sickle-cell adherence, the ability of activated alpha 4 beta 1 to promote adhesion to endothelium via a ligand different than VCAM-1 was explored. Adherence assays were performed under dynamic conditions at a shear stress of 1 dyne/cm2. Preincubation of sickle erythrocytes with phorbol 12,13-dibutyrate (PDBu) increased adherence of sickle cells eightfold as compared with untreated sickle cells. Normal erythrocytes, whether treated with PDBu or not, did not adhere to the endothelium. Activating anti-beta 1 antibodies 4B4 and 8A2 also increased the adhesion of sickle, but not normal, red blood cell (RBC) adhesion to endothelium. Anti-alpha 4 antibodies HP1/2 and HP2/1, inhibitory antibody 4B5, or an RGD peptide inhibited sickle-cell adherence induced by PDBu. Additional studies were undertaken to examine if fibronectin, a ligand for activated alpha 4 beta 1, was involved in PDBu-induced sickle erythrocyte adherence. Adherence of PDBu-treated sickle cells was completely inhibited by the CS-1 peptide of fibronectin. Fibronectin was detected on the surface of washed endothelium using an antifibronectin antibody in enzyme-linked immunosorbent assays. Antifibronectin antibody pretreatment of endothelial cells inhibited PDBu-induced adherence by 79% +/- 17%. Incubation of sickle RBCs with exogenous fibronectin after PDBu treatment inhibited adherence 86% +/- 8%. Taken together, these data suggest that endothelial-bound fibronectin mediates adherence of PDBu-treated sickle cells. Interleukin-8 (IL-8), a chemokine released in response to bacterial infection, viral infection, or other injurious agents, and known to activate integrins, also increased adherence of sickle erythrocytes to endothelial cells via fibronectin. This novel adherence pathway involving sickle-cell alpha 4 beta 1 activated by PDBu or IL-8 may therefore be relevant in vivo at vascular sites that produce IL-8 or similar agonists in response to vascular injury or immune activation. These observations describe ways in which inflammation and immune responses cause vasoocclusive complications in sickle-cell disease.

Amino Acid Sequence↗
PubMed1996

Expression of human glandular kallikrein, hK2, in mammalian cells.

The human kallikrein family consists of three members, hK1, hK2, and hK3 [prostate-specific antigen (PSA)]. PSA is a widely accepted marker for prostate cancer. The mRNAs for both hK2 and PSA are localized predominantly to prostate epithelium and are regulated by androgens. In addition, hK2 has 78% amino acid homology to PSA. Although similarities to PSA make hK2 a potential prostate cancer marker, they also impede biochemical characterization of hK2 in those human tissues and body fluids where PSA is abundant. To study the expression, biosynthesis, and processing of hK2, recombinant hK2 was expressed in the adenovirus-induced Syrian hamster tumor cell line AV12-664 (AV12-hK2). Expression of hK2 was analyzed by Western blots and ELISA using monoclonal antibodies HK1G 464.3 [specific for prohK2 (phK2)] and HK1D 106.4 [specific for phK2 and mature hK2 (hK2)1. Western blot and ELISA analyses showed that phK2 was secreted into the media by AV12-hK2 cells on day 1 and was gradually converted to the mature form of hK2 by day 7. N-terminal amino acid sequencing verified the Western blot and ELISA data. This demonstrates for the first time that hK2 is secreted as phK2 and converted to hK2 extracellularly. In addition, hK2 detected in day 4-7 AV12-hK2-spent media was enzymatically active. Recombinant hK2 was also expressed in human prostate carcinoma cell lines, PC3 (PC3-hK2) and DU145 (DU145-hK2), that do not express endogenous hK2 or PSA. Similar to AV12-hK2 cells, both cell lines secreted phK2 that was converted to hK2 extracellularly. phK2 was the major form detected in the spent media of PC3-hK2 cells, even after 7 days, indicating a slow conversion of phK2 to hK2. hK2 was the predominant form detected in the spent media of DU145-hK2 starting on day 1, indicating the rapid conversion of phK2 to hK2. In this study, we demonstrate that hK2 exists in different forms and is secreted as phK2. phK2 is then converted to enzymatically active hK2 extracellularly.

Animals↗
PubMed1996

Equilibrium and stopped-flow kinetic studies of interaction between T7 RNA polymerase and its promoters measured by protein and 2-aminopurine fluorescence changes.

The mechanism of bacteriophage T7 RNA polymerase binding to its promoter DNA was investigated using stopped-flow and equilibrium methods. To measure the kinetics of protein-DNA interactions in real time, changes in tryptophan fluorescence in the polymerase and 2-aminopurine (2-AP) fluorescence in the promoter DNA upon binary complex formation were used as probes. The protein fluorescence changes measured conformational changes in the polymerase whereas the fluorescence changes of 2-AP base, substituted in place of dA in the initiation region (-4 to +4), measured structural changes in the promoter DNA, such as DNA melting. The kinetic studies, carried out in the absence of the initiating nucleotide, are consistent with a two-step DNA binding mechanism, [formula: see text] where the RNA polymerase forms an initial weak EDa complex rapidly with an equilibrium association constant K1. The EDa complex then undergoes a conformational change to EDb, wherein RNA polymerase is specifically and tightly bound to the promoter DNA. Both the polymerase and the promoter DNA may undergo structural changes during this isomerization step. The isomerization of EDa to EDb is a fast step relative to the rate of transcription initiation and its rate does not limit transcription initiation. To understand how T7 RNA polymerase modulates its transcriptional efficiency at various promoters at the level of DNA binding, comparative studies with two natural T7 promoters, Phi10 and Phi3.8, were conducted. The results indicate that kinetics, the bimolecular rate constant of DNA binding, kon (K1k2), and the dissociation rate constant, koff (k-2), and thermodynamics, the equilibrium constants of the two steps (K1 and k2/k-2) both play a role in modulating the transcriptional efficiency at the level of DNA binding. Thus, the 2-fold lower kon, the 4-fold higher koff, and the 2-5-fold weaker equilibrium interactions together make Phi3.8 a weaker promoter relative to Phi10.

2-Aminopurine↗
PubMed1996

Cloning and sequencing of the genes encoding 2-nitrotoluene dioxygenase from Pseudomonas sp. JS42.

The first step in the metabolism of 2-nitrotoluene (2NT) by Pseudomonas sp. JS42 (JS42) is the addition of dioxygen to the aromatic nucleus of 2NT to form 3-methylcatechol with concomitant release of nitrite. This reaction is catalyzed by the three-component dioxygenase system 2-nitrotoluene 2,3-dioxygenase (2NTDO). We report here the cloning and nucleotide (nt) sequence of a 4912-basepair (bp) SacI DNA fragment from JS42 encoding all of the genes required for 2NTDO activity. Sequence analysis of the 4912-bp SacI DNA fragment revealed five open reading frames (ORFs). The amino acid (aa) sequences of the predicted polypeptides from these ORFs exhibit high homology to the aa sequences of polypeptides from other three-component dioxygenase systems. Based on aa sequence analyses, four of the peptides were designated Reductase2NT, Ferredoxin2NT, ISP alpha 2NT and ISP beta 2NT (ISP for iron-sulfur protein) with gene designations ntdAaAbAcAd. The predicted aa sequence from the remaining ORF (ORF2) had identity to ISP alpha subunits from other three-component dioxygenase systems but had a calculated molecular weight (M(r)) of 21,259, which is uncharacteristically small for ISP alpha subunits.

Amino Acid Sequence↗