Biomedical subjects

K Janakidevi

Publications and source records attributed to K Janakidevi.

At least 19 recordsLinked to original sources

PubMed1995

H2O2 and tumor necrosis factor-alpha activate intercellular adhesion molecule 1 (ICAM-1) gene transcription through distinct cis-regulatory elements within the ICAM-1 promoter.

We investigated the mechanisms by which H2O2 increases intercellular adhesion molecule 1 (ICAM-1; CD54) expression in endothelial cells. The H2O2-induced increase in ICAM-1 mRNA was inhibited by actinomycin D, by the antioxidant N-acetylcysteine, and by 3-amino-benzamide (which blocks oxidant-induced AP-1 activity), but not by pyrrolidine dithiocarbamate (which blocks oxidant-induced NF-kappa B activity). Nuclear run-on and transient transfections of ICAM-1 promoter constructs indicated that H2O2 stimulated ICAM-1 gene transcription by activation of a distinct region of the ICAM-1 promoter. The H2O2-responsive element was localized to sequences between -981 and -769 (relative to start codon). Located within this region are two 16-base pair repeats, each containing binding sites for the transcription factors AP-1 and Ets. A similar composite AP-1/Ets element isolated from the macrophage scavenger receptor gene conferred H2O2 responsiveness to a minimal promoter. Mutation of the 16-base pair repeats within the ICAM-1 promoter prevented H2O2-induced DNA binding activity, and their deletion abrogated the H2O2-induced transcriptional activity. In contrast, TNF alpha induced ICAM-1 transcription via activation of promoter sequences between -393 and -176, a region with C/EBP and NF-kappa B binding sites. The results indicate that H2O2 activates ICAM-1 transcription through AP-1/Ets elements within the ICAM-1 promoter, which are distinct from NF-kappa B-mediated ICAM-1 expression induced by TNF alpha.

Animals↗

PubMed1995

Growth characteristics of pulmonary artery smooth muscle cells from fawn-hooded rats.

We compared the proliferative rates of vascular smooth muscle cells (VSMC) from pulmonary arteries of pulmonary hypertensive fawn-hooded rats (FHR) with VSMC from normotensive Sprague-Dawley rats (SDR). VSMC from FHR grew at increased rates and reached higher densities at all serum concentrations studied (5-20%) than the VSMC from SDR. The VSMC from FHR also responded to epidermal growth factor (EGF) at low serum concentrations, as evidenced by significantly greater DNA synthetic rates, than the control VSMC. The increased growth in these cells could be due to increased number and/or affinity of EGF receptors because of the higher specific binding of 125I-EGF to the VSMC from FHR. The VSMC from FHR and SDR were equally sensitive to the antiproliferative effects of heparin, suggesting that the heparin-sensitive pathways are not altered in the VSMC from FHR. These results suggest that the development of pulmonary hypertension in FHR may be related to the higher proliferative capacity of the pulmonary VSMC, which may be coupled to increased activity of the EGF receptors on these cells.

Animals↗

PubMed1993

Hydrogen peroxide-induced increase in endothelial adhesiveness is dependent on ICAM-1 activation.

Reactive oxygen radicals (ROS) generated by phagocytes promote human polymorphonuclear leukocyte (PMN) adhesion to human umbilical vein endothelial cells (EC). We determined the effects of hydrogen peroxide (H2O2), a phagocyte-derived ROS, on EC adhesiveness by determining steady-state intracellular adhesion molecule 1 (ICAM-1) mRNA and ICAM-1 protein expression. The adhesion of PMN to H2O2-treated EC was concentration dependent with maximal adhesion achieved at 0.1 mM H2O2. PMN adhesion occurred rapidly, reaching its maximum value within a 1-h treatment time. The PMN adhesion was dependent on the interaction between CD11/CD18 integrins on PMN and ICAM-1 on EC, since either anti-CD18 or anti-ICAM-1 monoclonal antibodies (MAbs) inhibited (by > 90%) the adhesive interaction between PMN and EC. In parallel with the increases in EC adhesivity, we detected a two- to threefold increase in EC surface expression of ICAM-1 between 0.5 and 1 h after H2O2 challenge. Northern analysis revealed an increase in the steady-state ICAM-1 mRNA signal within 0.5 h after H2O2 exposure, and the response was sustained up to 2 h. Inhibition of intracellular catalase in H2O2-treated EC by 3-amino-1,2,3-triazole augmented the PMN adhesion by 20%, whereas enhancement of EC H2O2-scavenging activity by addition of catalase abrogated the H2O2-induced PMN adhesion, indicating that oxidant-antioxidant balance at the EC interface is a critical factor modulating PMN-EC adhesive interactions. The results suggest that H2O2-induced PMN adhesion is dependent on the rapid induction of the ICAM-1 mRNA signal and the surface expression of ICAM-1 on EC.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗

PubMed1992

Treatment of porcine aortic smooth muscle cells with phorbol esters prevents nodulation in vitro.

Vascular smooth muscle cells exhibit a unique pattern of growth in culture. They have the capacity for multilayer growth and form large macroscopic nodules. We find that nodulation is inhibited in the presence of phorbol esters and that there is a concomitant decrease in the production of a 38 kd secreted protein associated with nodulation in porcine smooth muscle. Examination of the organization of actin filaments within the cells using a rhodamine phalloidin stain indicates that there is a rearrangement of actin filaments in response to phorbol esters. This rearrangement increases the number of attachment sites to the culture surface and may contribute to the inhibition of nodulation in smooth muscle cells by phorbol esters.

Actins↗

PubMed1992

Endothelin-1 stimulates DNA synthesis and proliferation of pulmonary artery smooth muscle cells.

Endothelin-1 (ET-1), a 21-amino acid peptide released from the endothelium, elicits a variety of biological effects that include vascular smooth muscle cell (VSMC) contraction, release of secondary mediators, and cell proliferation. The present study was undertaken to examine the proliferative potential of ET-1 toward pulmonary artery VSMC in culture. In the presence of low serum and epidermal growth factor (EGF), ET-1 stimulated marked DNA synthesis and proliferation of VSMC. The contributing factor from serum appeared to be platelet-derived growth factor (PDGF) because the antibody to PDGF eliminated the stimulatory activity. The antibody to EGF also prevented the stimulation, suggesting that both PDGF and EGF are required for the full expression of the VSMC growth-promoting activity of ET-1. A paradoxical aspect of ET-1 effect on VSMC was the ability of ET-1 to inhibit the EGF-stimulated DNA synthesis when the two factors were added together to a high baseline DNA synthetic activity. The inhibition was prevented if ET-1 was added 12-18 h after the addition of EGF or if ET-1 and EGF were added to a protein kinase C-depleted VSMC. The inhibition by ET-1 may be mediated by protein kinase C activation followed by inhibition of EGF binding to its receptor. The results indicate that ET-1 under appropriate conditions can modulate the growth of pulmonary artery VSMC in both positive and negative directions.

Animals↗

PubMed1989

Limited cell attachment time as a method to synchronize cells grown in monolayer culture.

A novel method of synchronizing monolayer tissue culture cells is described. By limiting the period of attachment of trypsinized cells and the subsequent removal of unattached cells a G1 population of cells is isolated. Evaluation of the degree of synchrony has been carried out by measuring the labeling index and incorporation of [3H]thymidine into DNA. Further conformation of synchrony, as well as a comparison with synchrony by isoleucine deprivation, was obtained by flow cytometry. The expected peak in DNA synthesis rate following limited attachment was observed. This peak becomes more prominent and shifts to earlier times with shorter attachment intervals. The synchronization method described is simple, rapid, yields a substantial number of cells, and is applicable to many cell lines.

Animals↗

PubMed1988

Fluorometric analysis of DNA in cell cultures.

A simple, reproducible fluorescent assay has been described which can be used confidently at microgram and submicrogram levels. The method is most suitable for cell cycle and cell growth analysis using [3H]thymidine as the radioactive tracer. A modified method is also described for preparing diaminobenzoic acid hydrochloride.

Aminobenzoates↗

PubMed1988

Clonal nature of atherosclerotic plaques.

The clonal nature of atherosclerotic plaques has been examined in diet-induced atherosclerosis of interspecies hybrid hare females (Lepus timidus (female) X Lepus europaeus (male], which exhibit genetic mosaicism with respect to glucose-6-phosphate dehydrogenase (G6P-D). Four diet groups have been used: control pellet diet, hyperlipemic (HL) diet, and HL diet supplemented with two different concentrations of 25-hydroxycholesterol. Lesion and nonlesion tissue samples and primary cultures developed from similar samples were used to determine the G6P-D variant patterns. A close correlation was observed, using regression analysis, between the in vivo and the in vitro phenotype. As has been reported earlier by us, most lesions in this animal model were heterozygous and the few homozygous areas recorded were all in the diet groups supplemented with 25-hydroxycholesterol. However, in tissue culture, homozygosity appeared with greater frequency, still of the T phenotype. In order to rule out maternal dominance as a factor, preliminary experiments involving G6P-D analysis have been carried out using tissue samples and cell cultures derived from a reverse cross. These also show a strong bias toward the T phenotype. The studies presented here highlight the difficulties in interpretation using G6P-D analysis as the sole criterion to determine the clonal nature of the atherosclerotic plaques. It also seems likely that the kind of diet given to induce atherosclerosis might influence the nature of the clonal growth. Studies using transfection are essential to determine whether transforming sequences are present in atherosclerotic lesions.

Animals↗

PubMed1985

Mosaicism in female hybrid hares heterozygous for glucose-6-phosphate dehydrogenase. VII. Evidence for selective advantage of one phenotype over the other in ditypic samples from aortas of hares fed cholesterol oxidation products.

In a previous study the authors demonstrated monotypic foci, all of timidus type, in the atherosclerotic lesions and normal aortic tissue of glucose-6-phosphate dehydrogenase (G-6-PD) mosaic hares fed 25-hydroxycholesterol or triol in addition to a high-cholesterol diet. Other hares fed the high-cholesterol diet without the cholesterol oxidation product additives did not develop monotypic foci. In the current study the question asked was whether ditypic samples from lesions or aortic mediae of the same hares fed the additives would show a higher percentage of timidus type G-6-PD than the hares fed the cholesterol alone. The results showed a significantly higher percentage of timidus type in both lesions and mediae in the groups fed either 25-hydroxycholesterol or triol than in the group fed cholesterol alone. The observations suggest that the change from ditypism to monotypism reported earlier with these cholesterol oxidation products was the result of progressive elimination of the europaeus type to leave only the timidus G-6-PD. This is consistent with the "selective advantage of one phenotype over the other" hypothesis for explaining the observed monotypism as opposed to the "genetic transformation" hypothesis.

Animals↗

PubMed1984

Mosaicism in female hybrid hares heterozygous for glucose-6-phosphate dehydrogenase. V. The recovery of DNA synthesis of hare fibroblasts after ultraviolet irradiation.

The effect of uv irradiation on the recovery of DNA synthesis is examined in a population of hare fibroblasts exhibiting heterozygosity with reference to the X-linked enzyme, glucose-6-phosphate-dehydrogenase (G-6-PD). These cells have been grown from skin explants of a hybrid female cross between Lepus timidus (female) and L. europaeus (male), the former carrying the G-6-PD gene for the slow-moving "T" variant and the latter with the fast-moving "E" variant gene. The hybrid, therefore, exhibits genetic mosaicism due to random inactivation in each cell, of one of the two X chromosomes in the embryonic stage. Exponentially growing cells from 13 fibroblast strains, comprising a wide range of E to T ratios, were exposed to moderately low dose of uv irradiation (6 J/m2). The recovery in DNA synthesis during the 2- to 8-h postirradiation period was calculated as the mean percentage rates of [3H]thymidine incorporated during the time as compared to the unirradiated zero-time controls. The results show a statistically significant positive correlation as determined by linear regression analysis between the levels of E and the rate of recovery in DNA synthesis. This is valid also at the higher dose of uv (21 J/m2). These results strengthen our earlier observations with 25-hydroxycholesterol that in the in vitro system the cell expressing the E variant is perhaps more resistant to cytotoxic agents. This also indicates that various factors contribute to the development of monotypism which include cell growth, cell death, mutation, and selection, to name a few.

Animals↗

PubMed1984

Mosaicism in female hybrid hares heterozygous for glucose-6-phosphate dehydrogenase. VI. Production of monotypism in the aortas of 4 of 10 mosaic hares fed cholesterol oxidation products.

The normal aortic tissue of black women heterozygous for glucose-6-phosphate dehydrogenase (G-6-PD) usually consists of two cell phenotypes (mosaicism). By electrophoresis two G-6-PD types are demonstrated (ditypism). Advanced atherosclerotic lesions from such women not infrequently yield samples displaying only one G-6-PD type (monotypism). Possible causes for the monotypism include (1) monoclonal origin of the lesion and (2) selective growth and/or survival advantage of one phenotype over the other. We have been attempting to produce monotypism in the aortas of a hybrid hare model that displays G-6-PD mosaicism in the normal state. In the current study 14 G-6-PD mosaic hares were fed a high-cholesterol diet for 6 to 17 months. All developed extensive moderately severe (as compared to humans) atherosclerotic lesions. No monotypic samples were found. Ten hares were fed (in addition to the standard high-cholesterol diet) small doses of one of two cholesterol oxidation products (25-hydroxycholesterol or triol) for 6 to 21 months. Four of the ten developed monotypic foci in either atherosclerotic lesions or normal aortic tissue or both. The reason for giving the oxidation products was because they are cytotoxic for cells in vitro and one (25-hydroxycholesterol) had been shown to be more toxic for one phenotype than the other with cultured fibroblasts from the mosaic hares (not infrequently resulting in the culture becoming monotypic). We suggest that the monotypism observed in vivo was probably produced by a mechanism similar to that operating in vitro.

Animals↗

PubMed1983

Increased thermal stability of solubilized chromatin after poly(ADP-ribose) synthesis.

A hyperthermic shift in the hyperchromicity curve of thermally denatured swine aortic-smooth-muscle-cell chromatin solubilized by digestion of nuclei with micrococcal nuclease was observed after the chromatin was incubated under conditions to allow poly-(ADP-ribose) synthesis by the endogenous poly(ADP-ribose) polymerase. When the order of solubilization and poly(ADP-ribosyl)ation was reversed, a smaller proportion of the solubilized chromatin exhibited greater thermal stability. Nuclease digestion of nuclei preincubated for poly(ADP-ribose) synthesis revealed no difference in kinetics of digestion or fragment size distribution compared to that of control nuclei. Poly(ADP-ribose) synthesis in these nuclei was proportionately greater in the chromatin fraction most resistant to solubilization by micrococcal nuclease treatment.

Animals↗

PubMed1982

Coronary arterial heart disease in hybrid hares fed alternating cholesterol-enriched and normal diets.

At one-week intervals hybrid hares (Lepus europaeus X Lepus timidus) were fed supplemented or nonsupplemented commercial pellets. Supplements were 1% cholesterol (USP-grade) and 6% peanut oil. In addition, the normal nonsupplemented diet was fed for a month when they appeared weak or when serum cholesterol values exceeded 1,000 mg/dl at monthly examinations. At the end of 1 year on such an alternating regimen, the hares appeared well nourished. Atherosclerotic lesions were severe and involved the epicardial and intramyocardial segments of the coronary arteries as well as the thoracic and abdominal aorta. Coronary arterial lesions were characterized by fibromuscular thickening with stainable lipids, necrosis, and calcification. Intramyocardial arterioles were diseased with fibromuscular plaques rich in foam cells. Transmural infarcts of the left ventricle occurred distal to the stenotic arterial lesions. Exact mechanisms are unknown for the development of such functionally significant coronary atherosclerosis. Among the possible pathogenetic factors, however, the alternating schedule seems most important. Such a regimen resulted in fluctuating, mild to moderate hypercholesterolemia and relatively good nutritional state of hybrid hares.

Animals↗

PubMed1982