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N M Kumar

Publications and source records attributed to N M Kumar.

54 records · Page 3Linked to original sources
PubMed1992

Mitogenic, chemotactic, and synthetic responses of rat periodontal ligament fibroblastic cells to polypeptide growth factors in vitro.

The mitogenic, chemotactic, and synthetic responses of rat periodontal ligament (PDL) fibroblastic cells to epidermal growth factor (EGF), transforming growth factor-beta (TGF-beta), recombinant human platelet-derived growth factor (rhPDGF)-AB, rhPDGF-BB, natural (n) PDGF-AB, and insulin-like growth factor-I (IGF-I) were examined in vitro using PDL cells obtained from the coagulum of healing tooth sockets. PDGFs and IGF-I have potent and comparable mitogenic effects on PDL fibroblastic cells. The maximum mitogenic effect of PDGFs was observed at the concentration of 10 ng/ml, whereas that of IGF-I was seen at concentrations higher than 100 ng/ml. In contrast, EGF induced moderate, and TGF-beta inhibitory mitogenic responses. The combination of rhPDGF-AB with either EGF or TGF-beta demonstrated comparable mitogenic potency, equivalent to the level of PDGF alone regardless of the mitogenic effect of other growth factors. The combination of rhPDGF-AB and IGF-I, however, showed a synergistic effect revealing the highest mitogenic effect among all individual growth factors as well as any combinations of the growth factors tested. Similarly, PDL fibroblastic cells demonstrated strong chemotactic responses to both IGF-I and PDGFs. The maximum effect was observed by IGF-I at concentrations higher than 10 ng/ml, followed by rhPDGF-BB at 0.1 ng/ml, rhPDGF-AB and nPDGF at concentrations ranging from 0.1 to 1 ng/ml. TGF-beta revealed no, and EGF slightly increased, chemotactic effects. IGF-I slightly enhanced the synthesis of total protein, whereas other factors had no significant effect. However, both rhPDGF-AB and TGF-beta stimulated collagen synthesis. On the other hand, IGF-I showed no effect on collagen synthesis, while EGF suppressed collagen synthesis. These findings suggest that rhPDGF-BB and IGF-I stimulate proliferation and chemotaxis of PDL fibroblastic cells. In addition, the combination of these growth factors further increases the mitogenic effect. rhPDGF-AB also stimulates collagen synthesis by PDL fibroblastic cells. Thus, rhPDGF-BB and IGF-I may have important roles in promotion of PDL healing, and consequently, may be useful for clinical application in periodontal regenerative procedures.

Animals↗
PubMed1992

Expression and cellular distribution of the alpha 1 gap junction gene product in the ocular pigmented ciliary epithelium.

The expression of four different gap junction (GJ) transcripts, corresponding to the alpha 1, alpha 3, beta 1 and beta 2 gene products, has been examined in the ciliary epithelium of human and bovine eyes, and in cultures of ciliary epithelial cells. Northern blot analysis revealed that alpha 1 mRNA, 3.6-kb in size, was the predominant transcript expressed in intact tissue and in cultures of pigmented ciliary epithelial cells (PE). No transcripts from the alpha 3, beta 1 or beta 2 gap junction genes were detected in intact tissue or ciliary epithelial cells as demonstrated by Northern blotting. When the levels of alpha 1 gap junction mRNA were compared between PE and NPE in primary cultures, a striking difference was observed in the level of alpha 1 transcripts: there was about a 6 to 8-fold increase in alpha 1 levels in PE cells, relative to the NPE cells. To verify the differential level of expression of alpha 1 GJ mRNA in the two cell types, indirect immunofluorescence localization studies were performed on semithin cryostat sections of ciliary processes. These studies revealed that alpha 1 gap junctions are present at the apical and lateral borders of PE cells, i.e. at the apical plasma membranes domains of PE-NPE cells, and at the lateral plasma membrane regions of PE-PE cells borders. Further analysis by immunoblotting confirmed that the 43 kD alpha 1 gap junction protein was the major gap junction gene product in the ciliary epithelium.

Animals↗
PubMed1991

Distribution of genes for gap junction membrane channel proteins on human and mouse chromosomes.

Gap junctions are widely distributed structures that mediate communication between cells. The channels that allow passage of small molecules between adjacent cells are made up of oligomeric proteins (connexins) that are encoded by a family of related genes. By probing somatic cell hybrid DNA on Southern filters with rat or human cDNAs or human genomic fragments, we have mapped four functioning gap junction genes, (alpha 1, beta 1, beta 2, and alpha 3), to different sites on human chromosomes: GJA1 (connexin43) to 6p21.1-q24.1; GJB1 (connexin32) to Xcen-q22; GJB2 (connexin26) to 13; and GJA3 (connexin46) also to 13, probably near GJB2. The GJA3 probe also hybridized to a restriction fragment that was mapped to chromosome 1. A GJA1-related pseudogene GJA1P was assigned to chromosome 5. The homologous loci in mouse were assigned to regions of known conserved syntenic groups: Gja-1 to chromosome 10; Gjb-1 to XD-F4 and Gjb-2 to 14. Of two sites of hybridization with the GJA3 probe, on mouse 14 and 5, we assume that the site on 14 corresponds to the GJA3 locus on human 13. Based on these data, additional members of this family of related genes can be isolated and characterized, and possible human and mouse mutations can be identified.

Animals↗
PubMed1991

Developmental regulation of gap junction gene expression during mouse embryonic development.

The expression of products from three different gap junction genes (alpha 1, beta 1 and beta 2) was studied in pre- and postimplantation mouse embryos, during organogenesis, during differentiation of F9 teratocarcinoma cells, and in cultured embryonic stem (ES) cells. In this analysis, the following results were obtained. 1) Pre- and postimplantation mouse embryos. The alpha 1 transcript was the earliest gap junction RNA detected (in the 4 cell stage embryo) and its abundance increased significantly throughout subsequent development. 2) Organogenesis. Evidence was obtained for developmental expression of these three different gap junction genes during early embryogenesis and throughout the late stages of organogenesis. The expression patterns for these genes may be related to differences in gap junctional communication requirements for fetal organ development versus neonatal and adult organ function, or the utilization of different genes by different cell types during organogenesis. 3) During the differentiation of F9 cells in culture, expression of these three genes was modulated. Thus, this is the first evidence for modulation of gap junction gene expression during the differentiation of a single cell type in culture. 4) In an ES cell culture line, alpha 1 was the only gap junction gene product detected. This is consistent with the findings of alpha 1 expression in the embryonic inner cell mass region and in undifferentiated teratocarcinoma cells.

Amino Acid Sequence↗
PubMed1991

Isolation and purification of gap junction channels.

This paper reports methods we have developed to solubilize gap junction channels, or connexons, from isolated gap junctions and to purify them in milligram quantities. Two sources of material are used: rat liver gap junctions and gap junctions produced by infecting insect cells with a baculovirus containing the cDNA for human liver beta 1 protein (connexin 32). Complete solubilization is obtained with long chain detergents (lauryl dimethyl amineoxide, dodecyl maltoside) and requires high ionic strength and high pH as well as reducing conditions. The purification involves chromatography on hydroxylapatite and gel filtration on Superose 6. A homogeneous product is indicated by a single band on a silver-stained gel and a homogeneous population of doughnut-shaped particles under the electron microscope. These particles have hexameric symmetry. The purified connexons have a tendency to form aggregates: filaments and sheets. The filaments grow by end-to-end association of connexons and are nonpolar, suggesting that the connexons are paired as in the cell-to-cell channel. The sheets grow by lateral association of the filaments.

Animals↗
PubMed1991

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate and the ras oncogene modulate expression and phosphorylation of gap junction proteins.

Gap junctional intercellular communication is inhibited in response to tumor promoters and oncogene transformation, suggesting that loss of this function is an important step in tumor formation. To elucidate the molecular mechanisms responsible for this inhibition, we examined the expression of gap junction proteins and mRNA in mouse primary keratinocytes after treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and/or ras transformation. During normal cell growth, keratinocytes expression the alpha 1 (connexin 43) and beta 2 (connexin 26) proteins. Within 5 min of TPA treatment, the alpha 1 protein became rapidly phosphorylated on serine residues and its expression was dramatically reduced by 24 h. The beta 2 protein, after an initial increase in expression, was also significantly reduced 24 h after treatment with TPA. ras transformation caused changes similar to those induced by TPA. The alpha 1 protein underwent an increase in serine phosphorylation, although its expression declined only slightly, while beta 2 expression was greatly reduced. The effects of TPA and ras on alpha 1 expression were additive; treatment of ras-transformed cells with TPA resulted in increased alpha 1 phosphorylation, with greatly decreased protein levels, much lower than those generated by either agent alone. These data provide a likely explanation for the similar and synergistic inhibition of gap junctional intercellular communication by phorbol esters and ras.

Animals↗
PubMed1990

Differential regulation of the levels of three gap junction mRNAs in Xenopus embryos.

Xenopus mRNAs that potentially encode gap junction proteins in the oocyte and early embryo have been identified by low-stringency screening of cDNA libraries with cloned mammalian gap junction cDNAs. The levels of these mRNAs show strikingly different temporal regulation and tissue distribution. Using a nomenclature designed to stress important structural similarities of distinct gap junction gene products, the deduced polypeptides have been designated the Xenopus alpha 1 and alpha 2 gap junction proteins. The alpha 2 gap junction mRNA is a maternal transcript that disappears by the late gastrula stage. It is not detected in any organ of the adult except the ovary, and resides primarily, if not exclusively, in the oocytes and early embryos. The alpha 1 gap junction mRNA appears during organogenesis, and is detected in RNA from a wide variety of organs. It is also found in full-grown oocytes, but is rapidly degraded upon oocyte maturation, both in vivo and in vitro. The alpha 1 and alpha 2 mRNAs encode proteins with different degrees of amino acid sequence similarity to the predominant gap junction subunit of the mammalian heart (connexin 43). Together with our earlier report of a mid-embryonic (beta 1) gap junction mRNA, the results suggest that intercellular communication during oocyte growth and postfertilization development is a complex phenomenon involving the coordinated regulation of several genes.

Amino Acid Sequence↗
PubMed1988

Topology of the 32-kd liver gap junction protein determined by site-directed antibody localizations.

Synthetic peptides corresponding to sequences in the human liver gap junction protein were chemically synthesized and used for generation of peptide antisera to defined sequences in the protein. The antibodies were affinity purified and characterized by demonstrating that they specifically recognized both their corresponding synthetic peptide (as indicated by dot blot analysis) and the native 32-kd gap junction protein (by immunoblotting). The specificity of a subset of the different site-specific antibodies was subsequently confirmed by demonstration of their binding to specific gap junction fragments produced by treatment with a lysine-specific endoproteinase. Immunoelectron microscopy was used to localize the specific peptide antibody epitopes to either the cytoplasmic or extracellular surfaces of the gap junction. Results indicate a transmembrane orientation for the protein with the amino and carboxyl termini located on the cytoplasmic side of the membrane. Based on these data, a model is proposed for the transmembrane folding of the gap junction protein.

Amino Acid Sequence↗
PubMed1988

Sequence and developmental expression of mRNA coding for a gap junction protein in Xenopus.

Cloned complementary DNAs representing the complete coding sequence for an embryonic gap junction protein in the frog Xenopus laevis have been isolated and sequenced. The cDNAs hybridize with an RNA of 1.5 kb that is first detected in gastrulating embryos and accumulates throughout gastrulation and neurulation. By the tailbud stage, the highest abundance of the transcript is found in the region containing ventroposterior endoderm and the rudiment of the liver. In the adult, transcripts are present in the lungs, alimentary tract organs, and kidneys, but are not detected in the brain, heart, body wall and skeletal muscles, spleen, or ovary. The gene encoding this embryonic gap junction protein is present in only one or a few copies in the frog genome. In vitro translation of RNA synthesized from the cDNA template produces a 30-kD protein, as predicted by the coding sequence. This product has extensive sequence similarity to mammalian gap junction proteins in its putative transmembrane and extracellular domains, but has diverged substantially in two of its intracellular domains.

Amino Acid Sequence↗
PubMed1986

Cloning and characterization of human and rat liver cDNAs coding for a gap junction protein.

An extended synthetic oligonucleotide (58-mer) has been used to identify and characterize a human liver gap junction cDNA. The cDNA is 1,574 bases long and contains the entire coding region for a gap junction protein. In vitro translation of the RNA products of this cDNA is consistent with it coding for a 32,022-D protein. Southern blot analysis indicates that the gap junction gene is present as a single copy, and that it can be detected in a variety of organisms using the human liver cDNA as a probe. The human cDNA has been used to screen a rat liver cDNA library, and a rat liver junction cDNA clone has been isolated. The rat liver clone is 1,127 bases in length, and it has strong sequence homology to the human cDNA in the protein-coding region, but less extensive homology in the 3'-untranslated region.

Amino Acid Sequence↗
PubMed1982

Transcriptional activity of the uteroglobin gene in rabbit endometrial nuclei during early pregnancy.

In studies on the regulation of rabbit uteroglobin production by progesterone, we have shown previously that the increase in uteroglobin secretion by the uterus in early pregnancy is preceded by an increase in uteroglobin synthesis and a parallel increase in uteroglobin messenger (m) RNA activity, which is accounted for by a rise in the steady state level of this specific mRNA in uterine epithelial cells. To investigate whether similar changes in the transcriptional activity of the uteroglobin gene occur during this period, we have transcribed endometrial nuclei in a "run-off" endogenous transcription system with [32P]uridine triphosphate as the precursor, given as a short pulse over 7 min at 25 C in vitro. Specific uteroglobin RNA transcripts were quantitated by hybridization of [32P]RNA to nitrocellulose filters containing cloned, full length uteroglobin complementary DNA. The percentage of uteroglobin RNA transcripts rose from day 0 (nonpregnant rabbits) to a peak on day 4 and declined on day 6 of pregnancy. No differences in RNA degradation and no preferential degradation of newly transcribed uteroglobin RNA were found during incubation of nuclei from the different days of pregnancy. The pattern of change in rate of transcription of the uteroglobin gene corresponds to the pattern of change in level of uteroglobin mRNA, suggesting that transcriptional controls are important in regulating the production of uteroglobin in pregnant rabbit uterus.

Animals↗
PubMed1982

Hybridization analysis of steady-state levels of uteroglobin mRNA in rabbit uterus and lung during early pregnancy.

Uteroglobin is a predominant protein in the rabbit uterus, where it is induced by progesterone, and occurs also in the lung, where its level is constitutive. A recombinant plasmid containing uteroglobin complementary DNA (cDNA) has been constructed previously from partially purified uteroglobin mRNA. In this study, the cloned uteroglobin cDNA has been used as a probe to determine the cellular content of uteroglobin mRNA at different times in early pregnancy in both rabbit uterus and lung. By RNA-excess hybridization to poly A-enriched RNA and to total nucleic acid extracts an increase in steady-state uteroglobin mRNA level was detected, from approximately 250 molecules/uterine epithelial cell in non-pregnant rabbits to approximately 6800 molecules/cell on day 4 of pregnancy, after which the levels declined progressively up to day 8. The pulmonary level of uteroglobin mRNA was about 400 molecules/cell and did not change significantly with day of pregnancy. The major factor in regulating the production of uteroglobin in the uterus of pregnant rabbits is the accumulation and subsequent depletion of its mRNA.

Animals↗
PubMed1981

The characterisation of a 5-S 'monosome' fraction from chromatin of Physarum polycephalum.

A so-called '5-S mononucleosome' fraction was isolated from nuclei of Physarum polycephalum by digestion with micrococcal nuclease and subsequent fractionation by gel filtration. This fraction had electrophoretic and sedimentation properties which were similar to DNA of approximately 140 base pairs in length. It is shown that lysis of the nuclei activates a protease which is resistant to phenyl-methyl sulphonyl fluoride, o-phenanthroline and parachloromercuri benzene sulphonate and which subsequently degrades the nucleosomes.

Chromatin↗
PubMed1980

The binding of histones H1 and H5 to chromatin in chicken erythrocyte nuclei.

The binding curves of histones H1 and H5 to chromatin in nuclei have been determined by a novel method which utilises the differential properties of free and bound histones on cross-linking with formaldehyde. The dissociation is thermodynamically reversible as a function of [NaCl]. The binding curves are independent of temperature over the range 4 degrees - 37 degrees C and independent of pH over the range 5.0 to 9.0. The curves are sigmoid, indicating co-operative dissociation with NaCl. The standard free energy of dissociation in 1 M NaCl for H1 is 0.5 Kcals/mole and for H5 is 3.5 Kcals/mole.

Animals↗
PubMedDate not supplied

Selective effect of alcohol on cellular immune responses of lymphocytes from AIDS patients.

In this study we examined the in vitro effects of alcohol on the proliferative responses of lymphocytes from healthy donors and AIDS patients to a recombinant fusion peptide, env-gag, corresponding to portions of the gp41 envelope (env) and internal core (gag) proteins of HIV. The effects of alcohol (ETOH) on the natural killer (NK) cell activities of lymphocytes from healthy donors and patients with AIDS were also investigated. Peripheral blood mononuclear cells from both normal donors and AIDS patients produced significant levels of lymphocyte proliferative responses to the HIV env-gag peptide; however, these responses were significantly higher in patients with AIDS, showing the specificity of the response. The env-gag-induced proliferative responses of lymphocytes from normal subjects were significantly suppressed when cultures contained only higher levels of ETOH (0.2% and 0.3%), whereas ETOH even at a lower level (0.1%) produced significant suppression of the env-gag-induced proliferation of lymphocytes only from AIDS patients. Direct addition of ETOH at concentrations of 0.1%, 0.2%, and 0.3% to cultures of lymphocytes from normal donors and NK target cells did not produce significant suppression of NK cell activities. However, ETOH at concentrations of 0.2% and 0.3% significantly suppressed the NK activities of lymphocytes from AIDS patients, and the suppressive effect was observed at all E:T cell ratios examined. Control peptide from the Escherichia coli expression vector did not produce any significant effect on lymphocyte proliferative responses or NK activity of both normal donors and AIDS patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Acquired Immunodeficiency Syndrome↗
PubMedDate not supplied

Characterization of gap junction expression in the adrenal gland.

To characterize the presence of gap junctions in adrenal tissue we used immunohistochemical analysis of connexin expression in whole adrenal glands and in primary rat adrenal cortical cell cultures. alpha 1 (connexin 43) gap junction protein was demonstrated in rat adrenal glands and in primary adrenal cell cultures. The alpha 1 gap junction proteins were present in different amounts such that little or no alpha 1 antigen was detected in the zona glomerulosa (ZG), while cells of the zona fasciculata (ZF) and zona reticularis (ZR) stained intensely. There was an inverse relationship between cell division rate and gap junction number in the intact adrenal gland. We placed the cells from the most intensely staining areas (ZF/ZR) of the rat adrenal cortex into culture. These primary adrenal cells expressed alpha 1 gap junction antigen on their cell surfaces in regions of cell-cell contact. ACTH (40mU/ml) treatment resulted in an increase in gap junction expression relative to controls in primary rat adrenal cultures. These results indicate that the abundance of gap junction expression is differential in the cortical zones and can be modulated by hormonal stimulation. These observations are also thought to reflect morphological and functional differences in adrenal cortical zones.

Adrenal Glands↗