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C M Hart

Publications and source records attributed to C M Hart.

35 records · Page 2Linked to original sources
PubMed1995

Oxidized low density lipoprotein-mediated activation of phospholipase D in smooth muscle cells: a possible role in cell proliferation and atherogenesis.

Low density lipoproteins (LDL) are risk factors in atherosclerosis and oxidative modification of LDL to oxidized LDL (OX-LDL) increases its atherogenicity. Development of atherosclerosis likely involves OX-LDL-mediated smooth muscle cell (SMC) proliferation. However, the mechanism(s) of SMC proliferation by OX-LDL is unknown. We hypothesized that OX-LDL may mediate SMC proliferation by activation of phospholipase D (PLD) through the generation of the second-messenger, phosphatidic acid (PA). To test this hypothesis, activation of PLD by OX-LDL was investigated in [3H]myristic acid- or [32P]orthophosphate-labeled rabbit femoral artery smooth muscle cells (RFASMC) in the presence of 0.5% ethanol or 0.05% butanol. Phospholipase D activation, as measured by labeled phosphatidylethanol (PEt) or phosphatidylbutanol (PBt) formation, was enhanced (3- to 5-fold) by OX-LDL. This activation of PLD was specific for OX-LDL, as native LDL or acetylated LDL had no effect. Further, OX-LDL-mediated [32P]PEt formation was dose- and time-dependent. To determine the mechanism(s) of OX-LDL-induced PLD activation, the role of protein kinase C (PKC) and Ca2+ was investigated. Pretreatment of [32P]orthophosphate-labeled RFASMC with known inhibitors of PKC such as staurosporine, calphostin-C, or H-7, had no effect on OX-LDL-induced PLD activation. Also, down-regulation of PKC by 12-O-tetradecanoylphorbol 13-acetate (TPA) (100 nM, 18 h) did not alter the OX-LDL-mediated [32P]PEt formation. However, pretreatment of RFASMC with genistein, a putative inhibitor of tyrosine kinases, attenuated the OX-LDL-mediated [32P]PEt formation. In addition, exposure of RFASMC to sodium orthovanadate, an inhibitor of phosphatases, enhanced the OX-LDL-mediated PLD activation. The effects of genistein and vanadate on PLD activation were specific for OX-LDL as these agents did not alter the TPA-induced [32P]PEt formation. Treatment of quiescent RFASMC with OX-LDL increased [3H]thymidine incorporation into DNA. This enhanced incorporation of [3H]thymidine into DNA was also mimicked by exogenously added phosphatidic acid (PA) or lysophosphatidic acid (LPA). These findings suggest that OX-LDL is a potent activator of the PLD pathway in SMC. The activation of PLD by OX-LDL generates second-messengers like PA and/or LPA which modulate mitogenesis. Thus, these results indicate that OX-LDL, in atherosclerotic lesions, may enhance SMC proliferation through the modulation of signal transduction pathways including activation of PLD.

Animals↗
PubMed1994

Deletion analysis of the lambda tR1 termination region. Effect of sequences near the transcript release sites, and the minimum length of rho-dependent transcripts.

In order to determine how much of the natural sequence is required for function of the lambda tR1 rho-dependent terminator, and to determine the minimum length required, we made two deletion series of a tR1 derivative that contains mostly foreign DNA, encoding C-rich RNA, substituted for the natural upstream sequences of tR1. We find the minimum transcript length to be 85 to 90 nucleotides, although an additional 10 to 25 nucleotides provide more efficient termination. Sequences as close as ten nucleotides to the release sites could be replaced without destroying termination, although termination efficiency was reduced by substitution of these proximal regions with DNA encoding the cytidine-rich RNA that is active at upstream sites. These results suggest that sequences proximal and distal to release sites have different functions and optimal structures for rho activation. We also show that two potential stem-loop structures in the tR1 region are not essential for terminator function, or for pausing at the release region. The results are consistent with the model that any pause site downstream of DNA encoding unstructured C-rich RNA is a potential rho-dependent terminator.

Bacteriophage lambda↗
PubMed1994

Developmental, hormonal, and pathogenesis-related regulation of the tobacco class I beta-1,3-glucanase B promoter.

The class I beta-1,3-glucanases are antifungal vacuolar proteins implicated in plant defense that show developmental, hormonal, and pathogenesis-related regulation. The tobacco enzymes are encoded by a small gene family with members derived from ancestors related to the present-day species Nicotiana sylvestris and N. tomentosiformis. We studied the expression in transgenic tobacco plants of a chimeric beta-glucuronidase (GUS) reporter gene fused to 1.6 kb of upstream sequence of the tobacco class I beta-1,3-glucanase B (GLB) gene, which is of N. tomentosiformis origin. Expression of the GUS reporter gene and the accumulation of class I beta-1,3-glucanase and its mRNA showed very similar patterns of regulation. In young seedlings the reporter gene was expressed in the roots. In mature tobacco plants it was preferentially expressed in lower leaves and roots and was induced in leaves by ethylene treatment and by infection with tobacco mosaic virus (TMV). Furthermore, it was down-regulated in cultured leaf discs by combinations of the hormones auxin and cytokinin. Histological studies of GUS activity showed that the GLB promoter shows highly localized expression in roots of seedlings. It is also expressed in a ring of cells around necrotic lesions induced by TMV infection, but not in cells immediately adjacent to the lesions or in the lesions themselves. The results of deletion analyses suggest that multiple positive and negative elements in the GLB promoter regulate its activity. The region from -1452 to -1193 containing two copies of the heptanucleotide AGCCGCC, which is highly conserved in plant-stress and defense-related genes, is necessary for high level expression in leaves. Additional regions important for organ-specific and regulated expression were: -568 to -402 for ethylene induction of leaves; -402 to -211 for expression in lower leaves and cultured leaf discs and for TMV induction of leaves; and -211 to -60 for expression in roots.

Cytokinins↗
PubMed1993

Oleic acid supplementation reduces oxidant-mediated dysfunction of cultured porcine pulmonary artery endothelial cells.

We have previously shown that supplementing cultured porcine pulmonary artery endothelial cells (PAEC) with exogenous oleic acid (18:1 omega 9) alters the fatty acid composition of the cells and reduces oxidant-mediated cytotoxicity. Because the mechanisms by which lipid alterations modulate oxidant susceptibility have not been defined, the ability of 18:1 to reduce hydrogen peroxide (H2O2)-mediated PAEC dysfunction was evaluated. PAEC monolayers on polycarbonate filters were incubated for 3 h in maintenance medium supplemented with either 0.1 mM 18.1 in ethanol vehicle (ETOH) or with an equivalent volume of vehicle alone. Twenty-four hours later monolayers were treated for 30 min with 50 or 100 microM H2O2 in Hanks' balanced salt solution (HBSS) or with HBSS alone (nonoxidant control). As a functional index of PAEC monolayer integrity, the permeability of monolayers to albumin was then measured for 3 h. Treatment with 100 microM H2O2 caused cytotoxicity and progressive increases in PAEC monolayer permeability that were attenuated by 18:1 supplementation, whereas 50 microM H2O2 caused only a transient increase in permeability without cytotoxicity. Supplementation with 18:1 also attenuated H2O2-induced reductions in PAEC adenosine triphosphate (ATP) content and disruption of PAEC microfilament architecture. The ATP content of PAEC monolayers was reversibly reduced in the absence of oxidant stress by incubation with glucose-depleted medium containing deoxyglucose and antimycin A. Metabolic inhibitor-induced ATP depletion increased monolayer permeability and altered cytoskeletal architecture, alterations that resolved during recovery of PAEC ATP content. These results demonstrate that ATP depletion plays a critical role in barrier dysfunction and suggests that the ability of 18:1 to reduce oxidant-mediated PAEC dysfunction and injury may relate directly to its ability to preserve PAEC ATP content.

Actin Cytoskeleton↗
PubMed1993

Thrombin receptor activating peptides induce Ca2+ mobilization, barrier dysfunction, prostaglandin synthesis, and platelet-derived growth factor mRNA expression in cultured endothelium.

Endothelial cell activation by thrombin is a key event in wound healing, inflammation, and hemostasis. To better define thrombin-endothelial cell interactions we synthesized several peptides of varying length corresponding to the initial 14 amino acid sequence of the cloned human platelet thrombin receptor after cleavage at an arginine41 site (R/SFLLRNPNDKYEPF). Thrombin receptor activating peptides (TRAPs) as short as 5 amino acids induced significant levels of PGI2 synthesis and expression of PDGF mRNA in human endothelium and produced dose-dependent cellular contraction and permeability of confluent human umbilical vein and bovine pulmonary artery endothelial monolayers. To explore whether TRAPs utilized similar signal transducing pathways as alpha-thrombin to accomplish endothelial cell activation, phospholipase C production of the Ca2+ secretagogue IP3 was measured and detected 10 seconds after either TRAP 7 or alpha-thrombin. Furthermore, TRAPs ranging from 5-14 residues induced significant dose-dependent increases in Fura-2 fluorescence indicative of Ca2+(1) mobilization. These results indicate that thrombin-mediated proteolytic cleavage of the human and bovine thrombin receptor initiates stimulus/coupling responses such phospholipase C activation, Ca2+ mobilization, and protein kinase C activation. The functional consequence of this cellular activation via the cleaved receptor is enhanced cellular contraction, barrier dysfunction, PGI2 synthesis, and expression of PDGF mRNA.

Amino Acid Sequence↗
PubMed1993

A 61 bp enhancer element of the tobacco beta-1,3-glucanase B gene interacts with one or more regulated nuclear proteins.

We show that a 61 bp fragment derived from the promoter region of the tobacco class I beta-1,3-glucanase GLB gene enhances transcription in Nicotiana plumbaginifolia protoplasts independent of orientation relative to the start of transcription. This fragment leads to a cooperative stimulation of transcription when combined with the cauliflower mosaic virus 35S as-1 enhancer element. The GLB enhancer contains two copies of the sequence AGCCGCC, which is conserved in several genes showing expression patterns similar to the GLB gene, as well as a sequence identical at 6 of 7 bp. Point mutations in these three sequences eliminate the enhancer activity of the 61 bp fragment. Nuclear extracts prepared from leaves of tobacco plants contain one or more putative transcription factors that interact specifically with the GLB enhancer. This factor was much less abundant in nuclear extracts prepared from upper leaves of untreated tobacco plants than in nuclear extracts prepared from upper leaves of ethylene-treated plants or from lower leaves. Since beta-1,3-glucanase genes are expressed at very low levels in upper leaves of tobacco plants, at higher levels in lower leaves, and are induced in all leaves after treatment of plants with the stress hormone ethylene, we conclude that the enhancer element interacts with one or more transcription factors whose binding activity is correlated with gene expression in vivo.

Base Composition↗
PubMed1992

Quantitative fatty acid analyses in cultured porcine pulmonary artery endothelial cells: the combined effects of fatty acid supplementation and oxidant exposure.

Supplemental fatty acids can modify the oxidant susceptibility of pulmonary artery endothelial cells (PAEC) in monolayer culture. In addition, in vivo dietary modifications have altered tissue and animal susceptibility to a variety of forms of oxidant stress. These modifications of oxidant injury have been attributed to changes in the numbers of fatty acid double bonds in cell lipids. We tested this hypothesis by incubating porcine PAEC in culture medium supplemented with either 0.1 mM oleic acid (18:1 omega 9) or with an equivalent volume of ethanol vehicle alone (ETOH-0.1%) for 3 h. After supplementation, PAEC were exposed to either oxidant stress, 100 microM hydrogen peroxide (H2O2) in Hanks' balanced salt solution (HBSS), or to control condition, HBSS alone, for 30 min. Supplemental PAEC were exposed to HBSS or H2O2 either immediately or 24, 48, or 72 h after supplementation. Supplementation with 18:1 protected PAEC from H2O2-induced injury at all time points. The fatty acid composition of PAEC phospholipid (PL), triglyceride (TG), and free fatty acid (FFA) subclasses was determined using thin layer and gas chromatography. The PL fraction contained the majority of PAEC fatty acids, and H2O2 reduced the polyunsaturates in this fraction regardless of supplementation. Supplementation with 18:1 increased the 18:1 content of PAEC PL, TG, and FFA at all time points, modified other fatty acids to a lesser extent, but failed to alter the overall number of fatty acid double bonds at all time points. These results indicate that modification of double bond number does not fully explain the mechanisms by which changes in lipid composition can modulate oxidant injury.

Animals↗
PubMed1992

Regulated inactivation of homologous gene expression in transgenic Nicotiana sylvestris plants containing a defense-related tobacco chitinase gene.

The class I chitinases are vacuolar proteins implicated in the defense of plants against pathogens. Leaves of transgenic Nicotiana sylvestris plants homozygous for a chimeric tobacco (Nicotiana tabacum) chitinase gene with Cauliflower Mosaic Virus (CaMV) 35S RNA expression signals usually accumulate high levels of chitinase relative to comparable leaves of non-transformed plants. Unexpectedly, some transgenic plants accumulated lower levels of chitinase than nontransformed plants. We call this phenomenon silencing. The incidence of silencing depends on the early rearing conditions of the plants. When grown to maturity in a greenhouse, approximately 25% of plants raised as seedlings in closed culture vessels were of the silent type; none of the plants raised from seed in a greenhouse showed this phenotype. Silencing is also developmentally regulated. Plants showed three patterns of chitinase expression: uniformly high levels of expression in different leaves, uniformly low levels of expression in different leaves, and position-dependent silencing in which expression was uniform within individual leaves but varied in different leaves on the same plant. Heritability of the silent phenotype was examined in plants homozygous for the transgene. Some direct descendants exhibited a high-silent-high sequence of activity phenotypes in successive sexual generations, which cannot be explained by simple Mendelian inheritance. Taken together, the results indicate that silencing results from stable but potentially reversible states of gene expression that are not meiotically transmitted. Gene-specific measurements of chitinase and chitinase mRNA showed that silencing results from co-suppression, i.e. the inactivation of both host and transgene expression in trans. The silent state was not correlated with cytosine methylation of the transgene at the five restriction sites investigated.

Base Sequence↗
PubMed1992

Treatment of congenital and acquired hemophilia patients by extracorporeal removal of antibodies to coagulation factors: a review of US clinical studies 1987-1990. Hemophilia Study Group.

This paper reviews the use of extracorporeal immunoadsorption with immobilized Staphylococcal Protein A in attempts to lower the inhibitor titer in 22 patients with either congenital hemophilia or with acquired inhibitors. Eighty-five immunoadsorption procedures were performed at 13 locations in the United States between June, 1987 and February, 1990. In general, immunoadsorption was shown to efficiently remove IgG and, in eight congenital hemophilia patients, it also produced a clinically significant lowering of inhibitors allowing effective conventional factor replacement therapy. Three of thirteen congenital hemophilia patients treated received factor concentrate prior to immunoadsorption and were anamnestic at the time of treatment. Although they experienced substantial lowering of their inhibitor titers, it was not sufficient to allow effective factor replacement. The effectiveness of immunoadsorption therapy in the 9 patients with acquired inhibitors was more difficult to evaluate due to the wide variety of concomitant medications which were employed, although in several patients serious bleeding episodes were substantially improved (or halted) following immunoadsorption. Side effects associated with immunoadsorption were slight. These findings suggest that immunoadsorption can be a significant benefit to patients with inhibitors, particularly if it is instituted prior to factor administration.

Adolescent↗
PubMed1991

Rho-dependent transcription termination. Characterization of the requirement for cytidine in the nascent transcript.

By substituting template segments encoding AU-rich, GU-rich, and CA-rich transcripts for natural sequences upstream of the phage lambda rho-dependent tR1 termination site, we demonstrate that cytidines are required in the upstream RNA for rho-dependent termination to occur. These results are extended through in vitro mutagenesis of a template encoding an inactive AU-rich upstream sequence: certain mutant templates encoding new cytidines are able to activate rho-dependent termination. Cytidines must be dispersed over a region of the transcript in order for rho to be activated, although no specific pattern of cytidines appears to be required. The results show that no local clustering or regular spacing of cytidines is necessary and that cytidines are not used as a "ruler" to determine the location of termination sites. Rho is somewhat sensitive to the relative positions of cytidines since slightly different nascent transcripts activate rho termination activity to various degrees. A model is presented in which hexameric rho binds 78 nucleotides of contiguous RNA in a primary site, such that each monomer interacts with at least one cytidine somewhere in the 13 nucleotides allotted to the monomeric primary site.

Bacteriophage lambda↗
PubMed1991

Supplemental fatty acids alter lipid peroxidation and oxidant injury in endothelial cells.

The purpose of this study was to determine the effects of supplemental fatty acids on oxidant injury in cultured endothelial cells. Porcine pulmonary artery endothelial cells (PAEC) in monolayer culture were incubated in culture medium supplemented with 0.1 mM fatty acid or with fatty acid vehicle alone for 3 h. Monolayers were then exposed to oxidant stress (100 microM H2O2 in buffer) or to control conditions (buffer alone) for 30 min. Supplementation with stearic acid (18:0) or oleic acid [18:1(n-9)] reduced H2O2-induced PAEC injury measured as release of intracellular lactate dehydrogenase (LDH). In contrast, supplementation with linolenic acid [18:3(n-6)] or eicosatrienoic acid [20:3(n-3)] enhanced H2O2-induced injury to PAEC. Both supplemental cis-vaccenic acid [18:1(n-7)] and 18:1(n-9) reduced the production of lipid peroxidation products in oxidant-stressed PAEC, whereas supplementation with 18:3(n-6) enhanced lipid peroxidation. Supplementation with 18:1(n-9) protected PAEC from H2O2 as long as 72 h after supplementation despite the intracellular redistribution of [18:1(n-9)] from triglycerides to phospholipids. Saturated and monounsaturated supplemental fatty acids protected PAEC from oxidant injury, but polyunsaturated fatty acids enhanced oxidant injury. These results support the hypothesis that supplemental fatty acids replace resident fatty acids, alter the oxidant reactivity of the cellular lipids, and thereby modify the oxidant susceptibility of PAEC.

Animals↗
PubMed1990

Fatty acid supplementation protects pulmonary artery endothelial cells from oxidant injury.

Although supplemental fatty acids have been shown to alter the susceptibility of experimental animals to oxidant gases, the relationship between the degree of tissue fatty acyl unsaturation and resistance to oxidant exposure remains undefined. Because vascular endothelial cells have been demonstrated to be sensitive cellular targets in oxidant-induced lung injury, we evaluated the effects of a supplemental fatty acid on the lipid composition and oxidant susceptibility of pulmonary artery endothelial cells (PAEC) in monolayer culture. PAEC were incubated in culture medium supplemented with an ethanolic solution of 0.1 mM cis-vaccenic acid (CVA), an 18-carbon monounsaturated fatty acid, or with the ethanol vehicle alone for 3 h. Cells were then exposed to either control or oxidant (hyperoxia: 95% O2; or hydrogen peroxide: 100 microM) conditions. Oxidant-induced cell injury was assessed by phase-contrast microscopy and by measuring the release of intracellular lactate dehydrogenase. Incubation with CVA increased the CVA content of PAEC lipids and protected cells from oxidant-induced injury for up to 72 h after supplementation. CVA had no effect on nonoxidant-induced cell injury. Although the mechanism by which CVA protects cells against oxidant injury remains undefined, evidence is presented that indicates the mechanism does not involve induction of antioxidant enzyme activity, alterations in the physical state of PAEC membranes, or enhancement of PAEC nucleic acid repair mechanisms. These results define a useful model for exploring the relationship between lipid composition and oxidant susceptibility and suggest that fatty acid modifications may constitute an important strategy for protecting cells against oxidant injury.

Animals↗
PubMed1989

Lung serotonin metabolism.

The pulmonary vascular endothelium, a metabolically active tissue, serves as an important site of injury in many types of clinical and experimental lung disease. Removal of 5-HT from the circulation constitutes one of the endothelial metabolic functions that is depressed early in the course of lung injury. Experimental evidence confirms that measuring 5-HT uptake detects alterations in endothelial cell function that precede the abnormalities detected by more conventional diagnostic tests such as radiographs, pulmonary function tests, and arterial blood gases. In addition, depression of 5-HT uptake can lead to increased concentrations of 5-HT in the pulmonary vasculature, which may contribute to the pathogenesis of lung injury. The development of an ideal method for measuring 5-HT uptake accurately in the lungs of critically ill patients has just begun. As yet, numerous variables reviewed in this article confound clinical measurements of 5-HT uptake. However, if investigators can continue to refine and develop the techniques for measuring 5-HT uptake in human patients, clinicians can look forward to the addition of a sensitive tool to their diagnostic armamentarium. Hopefully, the ability to detect diffuse lung injury earlier in its course will enable future clinicians to institute therapy that will prevent the pathologic progression to morbidity and death seen all too frequently in current medical practice.

Chemical Phenomena↗
PubMed1987

Transcription antitermination by phage lambda gene Q protein requires a DNA segment spanning the RNA start site.

The gene Q protein of phage lambda is a transcription antiterminator that modifies RNA polymerase near the phage late gene promoter and thereby causes antitermination at distant sites. To define the site of action of Q protein, we have reconstructed the regulatory system on plasmids that allow the intracellular concentration of Q protein to be regulated, and that allow the effect of Q protein on transcription from variant promoter segments to be measured in vivo and in vitro. We show that DNA sequences essential for Q protein-mediated antitermination span the RNA start site, but do not extend beyond nucleotide 18 of the late RNA coding region. We also show that the modification that permits antitermination persists while RNA polymerase passes at least two terminators in vivo and in vitro.

Bacteriophage lambda↗
PubMedDate not supplied

Oleic acid reduces oxidant stress in cultured pulmonary artery endothelial cells.

Altering the fatty acid composition of cultured porcine pulmonary artery endothelial cells (PAEC) modulates their susceptibility to oxidant stress. This study demonstrates that supplementing PAEC with oleic acid (18:1 omega 9), but not gamma-linolenic acid (18:3 omega 6), provided dose-dependent protection from hydrogen peroxide (H2O2)-induced cytotoxicity. It was hypothesized that 18:1 reduced PAEC susceptibility to oxidant stress by altering H2O2 metabolism. To test this hypothesis, confluent PAEC monolayers were treated with 100-200 microM H2O2 or control conditions 24 h after supplementation with 0.1 mM 18:1, 18:3, or vehicle for 3 h. Intracellular [H2O2] in control cells (14.4-29.0 pM), estimated from the rate of aminotriazole-mediated inactivation of endogenous catalase activity, increased following treatment with 200 microM H2O2 (19.0-37.3 pM). Supplementation with 18:1 attenuated increases in intracellular [H2O2] only in oxidant-exposed cells, whereas supplementation with 18:3 attenuated intracellular [H2O2] only in control cells. Supplementation with 18:1 or 18:3 tended to reduce or enhance PAEC lipid hydroperoxide content following H2O2 exposure, respectively, but did not alter PAEC reduced glutathione content, the activities of glutathione peroxidase or catalase, or H2O2 uptake and release. Alteration of H2O2 metabolism in cultured PAEC may contribute to the ability of fatty acids to modulate cellular oxidant susceptibility.

Animals↗