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Biomedical subjects

D A Troyer

Publications and source records attributed to D A Troyer.

At least 37 records · Page 2Linked to original sources
PubMed1997

Increased tumor proliferation and genomic instability without decreased apoptosis in MMTV-ras mice deficient in p53.

We have used an in vivo tumor model to evaluate the consequences of p53 tumor suppressor protein deficiency in a tissue-specific context. By breeding MMTV-ras transgenic mice, which are highly susceptible to the development of mammary and salivary tumors, with p53(-/-) mice, we generated three classes of animals which contained the MMTV-ras transgene but differed in their p53 functional status (ras/p53(+/+), ras/p53(+/-), or ras/p53(-/-)). ras/p53(-/-) mice developed tumors more rapidly than animals of the other two genotypes; however, the distribution of tumors was unexpectedly altered. Whereas the most frequently observed tumors in ras/p53(+/+) and ras/p53(+/-) mice were of mammary origin, ras/p53(-/-) mice developed primarily salivary tumors. In addition, the mammary and salivary tumors from ras/p53(-/-) mice consistently exhibited a number of unfavorable characteristics, including higher histologic grades, increased growth rates, and extensive genomic instability and heterogeneity, relative to tumors from ras/p53(+/+) mice. Interestingly, the increased growth rates of ras/p53(-/-) tumors appear to be due to impaired cell cycle regulation rather than decreased apoptosis, suggesting that p53-mediated tumor suppression can occur independent of its role in apoptosis.

Aneuploidy↗
PubMed1996

Induction of proinflammatory cytokine expression in experimental acute Chagasic cardiomyopathy.

One of the hallmarks of Chagas' disease (caused by Trypanosoma cruzi) is progressive cardiomyopathy. The disease is associated with increased serum TNF-alpha levels, and TNF-alpha is known to depress cardiac function. It is, however, not known whether the cytokines are produced within the infected myocardium. One-month-old male Lewis rats were injected with cell culture-derived T. cruzi trypomastigotes and killed 15 days post-infection. As compared to normal animals, histologic analysis of infected animals revealed dense infection with amastigotes within myocytes and a minimal inflammatory infiltrate in the myocardium. Northern blot analysis of total RNA revealed no signal for IL-1beta or TNF-alpha, and a weak signal for IL-6 in the control rat hearts, and high levels of expression for the three genes in the infected rats. Western blots revealed results similar to that of mRNA levels, suggesting that, in addition to mechanical damage, infection by T. cruzi induces proinflammatory cytokine production in the myocardium itself, which may further exacerbate the pathology, and affect adversely myocardial function.

Animals↗
PubMed1996

Modulation of antioxidant enzymes and programmed cell death by n-3 fatty acids.

Studies from our laboratory indicate that n-3 (fish oil, FO) lipids at 10% (w/w) in a nutritionally adequate, semipurified diet, and supplemented with equal levels of antioxidants, extended the life span of lupus-prone (NZB/NZW)F1 (B/W) female mice as compared to n-6 (corn oil, CO) lipids. The early rise of autoimmune disease in CO-fed mice was closely linked to the loss of T-cell function. Both IL-2 production and IL-2 receptor expression were reduced due to the loss of naive T-cells and a rise in memory T-cells. Proliferative response to both mitogens and superantigens (staphylococcal enterotoxins A and B) was higher in FO-fed 6.5-mon-old mice. These changes paralleled decreased PGE2 production by splenic cells from FO-fed mice. Analysis of mRNA expression in different organs revealed differential effects of dietary lipids. In FO-fed mice, transforming growth factor beta 1 (TGF beta 1) expression was decreased in kidneys, but splenic tissues had higher expression of TGF beta mRNA. As TGF beta promotes programmed cell death (PCD), we studied the effects of CO and FO on PCD rates in lymphocytes. Both propidium iodide staining and DNA fragmentation were elevated in lymphocytes of FO-fed mice when compared to CO-fed mice of similar age. Also, increased PCD correlated closely with increased Fas gene expression. Thus, in addition to various other antiinflammatory effects, dietary FO appears to increase PCD and prevent accumulation of self-reactive immune cells in lymphoid organs. Further studies are required to dissect the pro- and antiinflammatory mechanisms associated with dietary n-3 and n-6 lipids in modulating autoimmune disorders or malignancy during aging.

Animals↗
PubMed1996

Tyrosine phosphorylation of focal adhesion kinase (p125FAK): regulation by cAMP and thrombin in mesangial cells.

Stress fibers, composed of actin filaments, converge upon and associate with a number of proteins, including focal adhesion kinase (p125FAK), and integrin receptors to form areas of close contact between cells and the extracellular matrix referred to as focal adhesions. Treatment of mesangial cells with cAMP-elevating agents causes a loss of focal adhesions, fragmentation of stress fibers, and decreased tyrosine phosphorylation of p125FAK. Thrombin reverses these effects of cAMP, and this model can be used to address some of the cellular mechanisms involved in regulating the loss and formation of focal adhesions. This study reports the effects of cAMP and thrombin on mesangial cell shape, distribution of actin, formation of stress fibers, and tyrosine phosphorylation of p125FAK. cAMP-treated cells display a condensed cell body with slender processes that traverse the area formerly covered by the cell. Addition of thrombin to these cells restores actin filaments (stress fibers) and increases tyrosine phosphorylation of p125FAK, and the cells resume a flattened morphology, even in the continued presence of cAMP-elevating agents. Peptides that mimic the tethered ligand portion of the thrombin receptor have the same effects on cell morphology and stress fiber formation as thrombin. In selected experiments, agents that disrupt either stress fibers (cytochalasin D) or microtubules (nocodazole; Sigma Chemical, St. Louis, MO) were used to examine the role of these cytoskeletal elements in thrombin-induced restoration of focal adhesions. Cytochalasin D blocked the ability of thrombin to restore focal adhesions and phosphorylate p125FAK. The effects of nocodazole, an agent that destabilizes microtubules (but which has no known receptor), are very similar to those of thrombin. The findings discussed in this study indicate that thrombin can modulate the formation of focal adhesions. The organization of stress fibers and microtubules is apparently intimately related to the phosphorylation of p125FAK and can be modulated by soluble receptor agonists such as thrombin or via altered polymerization of microtubules.

Actins↗
PubMed1995

Dietary lipids and calorie restriction affect mammary tumor incidence and gene expression in mouse mammary tumor virus/v-Ha-ras transgenic mice.

We have studied the effects of food restriction (FR) and substitution of fish oil (FO; omega 3) for corn oil (CO; omega 6) on breast tumor incidence and survival in mouse mammary tumor virus/v-Ha-ras transgenic (Onco) mice. The diets were as follows: group 1, 5% (wt/wt) CO fed ad libitum (AL); group 2, 5% CO, restricted calories (40% fewer calories than AL; FR); group 3, 20% CO fed AL; and group 4, 20% FO fed AL. After 3 years, 40% of FR Onco (group 2) mice were alive, whereas there were no survivors in the other three groups. Similarly, tumor incidence was reduced to 27% (5 out of 18) in FR animals (group 2), whereas it was 83% (11 out of 13) in group 1 mice, 89% (16 out of 18) in group 3 mice, and 71% (10 out of 14) in group 4 mice. These protective effects of FR on survival and tumor incidence were paralleled by higher expression of the tumor suppressor gene p53 (wild type) and free-radical scavenging enzymes (catalase and superoxide dismutase) in breast tumors. Immunoblotting showed less ras gene product, p21, and increased p53 levels in the tumors of FR mice. In addition, FR decreased RNA levels of c-erbB-2, interleukin 6, and the transgene v-Ha-ras in tumors. In contrast, analysis of hepatic mRNA from tumor-bearing FR mice revealed higher expression of catalase, glutathione peroxidase, and superoxide dismutase. Survival and tumor incidence were not influenced significantly by dietary supplementation with FO in place of CO. Taken together, our studies suggest that moderate restriction of energy intake significantly inhibited the development of mammary tumors and altered expression of cytokines, oncogenes, and free-radical scavenging enzymes.

Animals↗
PubMed1995

Effects of calorie restriction on transforming growth factor beta 1 and proinflammatory cytokines in murine Sjogren's syndrome.

The present study was carried out to determine whether restricting dietary calories prevents salivary gland abnormalities and modulates expression of transforming growth factor beta and proinflammatory cytokines, IL-6, and TNF alpha in major salivary glands (SG) of autoimmune lupus-prone (NZB x NZW)F1 (B/W) female mice. These mice develop focal lymphocytic interstitial and periductal round cell infiltrates in salivary glands similar to those of humans with Sjogren's syndrome. Weanling B/W mice were fed a nutritionally adequate semipurified diet either ad libitum (AL) or a calorie-restricted (CR; 40% less calories than AL) diet. The mice were sacrificed at 3.5 months (young) and 8.5 months (old) of age. Histopathologic and histomorphometric analyses as well as growth factor and cytokine protein and mRNA expression were carried out in the SG. Histomorphometric analysis of SG from young mice showed no differences between AL and CR mice, but old AL (vs old CR) had a 7.3-fold higher focus score and a 34-fold increase in percentage area inflammation. mRNA analysis revealed significantly higher levels of TGF beta 1 in SG of old CR (6.8-fold) mice. In contrast, CR reduced mRNA expression of proinflammatory cytokines (IL-6, 2.9-fold for young and 4.8-fold for old; TNF alpha, old 3.9-fold). By immunoblotting, significantly higher levels of TGF beta 1 protein was detected in old CR mice (vs old AL; 13.2-fold). IL-6 and TNF alpha proteins were undetectable in both young and old CR groups, whereas an increase in IL-6 (4.7-fold) and TNF alpha (9.3-fold) was observed in old AL mice. These results indicate that amelioration of the histological severity of disease in SG of B/W mice is paralleled and possibly mediated by increased expression of immunosuppressive TGF beta 1 and decreased expression of proinflammatory cytokines.

Animals↗
PubMed1995

Dietary omega-3 lipids delay the onset and progression of autoimmune lupus nephritis by inhibiting transforming growth factor beta mRNA and protein expression.

The present study was carried out to test whether transforming growth factor beta (TGF beta) plays a pathological role in the induction or progression of glomerulonephritis in a murine model of systemic lupus erythematosus (SLE), and whether dietary supplementation with fish oil (FO) can modulate the expression of TGF beta. Weanling female (NZB x NZW) F1 (B/W) mice were divided into three groups. One group was fed an unmanipulated diet (lab. chow; LC) and the other two groups were fed a nutritionally adequate semipurified diet supplemented with 10% CO or FO. Both water and food were provided ad libitum. Proteinuria and serum anti-dsDNA antibody levels were measured to assess disease progression. Mice were killed at 3.5 and 6.5 months of age and renal mRNA levels for TGF beta isoforms, fibronectin-1 (FN-1) and intercellular adhesion molecule-1 (ICAM-1) were studied by Northern blot analysis. TGF beta 1 protein levels were also examined in kidneys by Western blot analysis. Our results indicate that at 3.5 months of age, when urinary protein levels were undetectable and very low levels of anti-dsDNA were detected, no mRNA signal could be detected for TGF beta isoforms, ICAM-1 and FN-1 in either dietary group. However, at 6.5 months, the FO-fed mice, compared to LC and CO, had [1] greatly reduced proteinuria (LC: 2-3+, CO: 2-3+; FO: trace -1+) and serum anti-dsDNA antibodies; [2] improved survival (CO: 100% death (15/15) occurred by 8 months; FO: 50% were alive at 12 months (8/15) and [3] reduced renal TGF beta 1 mRNA and protein levels. TGF beta 2 and beta 3 were not significantly affected by FO diet. Similarly, lower levels of renal FN-1 and ICAM-1 mRNA were observed in FO fed mice. These data indicate that in B/W mice on a FO diet, prolonged survival and amelioration of renal disease may be attributed at least in part to lower levels of TGF beta 1 mRNA and protein in the kidneys.

Adjuvants, Immunologic↗
PubMed1995

Effects of energy intake on type 1 plasminogen activator inhibitor levels in glomeruli of lupus-prone B/W mice.

Calorie restriction (CR) and/or reduced energy intake ameliorates the progression of autoimmune renal disease in (NZB x NZW)F1 (B/W) female mice and increases life span. Like other forms of glomerulonephritis, the lupus-like kidney disease observed in these animals is frequently accompanied by glomerular deposition of fibrin and increased accumulation of mesangial matrix. Because alterations in plasminogen activator inhibitor type 1 (PAI-1) expression or function may be involved in both fibrin deposition and accumulation of extracellular matrix, we have studied the effects of CR on the expression of PAI-1 in kidneys from female B/W mice fed either ad libitum or on a 40% CR diet. By immunohistochemistry and immunoblotting, we found that the glomerular levels of PAI-1 antigen were highest in older ad lib fed animals with more advanced glomerular disease. Increased levels of PAI-1 protein were paralleled by increased levels of PAI-1 mRNA in total RNA extracted from renal cortex and in diseased glomeruli as detected by in situ hybridization. CR diminished the accumulation of PAI-1 protein and reduced the expression of PAI-1 mRNA. Thus, glomeruli from animals fed ad lib showed much greater deposition of PAI-1 protein, increased expression of PAI-1 mRNA, and more severe histological abnormalities than animals on a CR diet. The differences between CR and ad lib animals were more pronounced in animals studied at 9 to 10 months versus those at 3 to 4 months of age. These observations indicate that the ameliorating effects of CR include diminished PAI-1 gene expression and decreased localization of PAI-1 in glomeruli.

Animals↗
PubMed1993

Two-chain urokinase, receptor, and type 1 inhibitor in cultured human mesangial cells.

Urokinase-type plasminogen activator (u-PA), its receptor (u-PAR), and type 1 inhibitor (PAI-1) in cultured human mesangial cells were investigated. Treatment with phospholipase C (PLC) released plasminogen activators [with relative mol wt (M(r)) of 55,000 and 100,000] and u-PAR into the culture medium. By Western blot, both u-PA and PAI-1 were present in the M(r) 100,000 band. Since PAI-1 binds only active, two-chain u-PA (tcu-PA), formation of the M(r) 100,000 band reflects conversion of the single-chain, proenzyme form of u-PA (scu-PA) to tcu-PA. Immunofluorescence staining of whole cells demonstrated the presence of PAI-1, u-PA, and u-PAR. Immunofluorescence staining and Western blot analysis showed enrichment of PAI-1, u-PA, and u-PAR in a preparation of substratum-attached extracellular matrix and membrane proteins termed adhesion plaques. Using a chromogenic assay, we found that PAI-1 expression in adhesion plaques exceeded that of u-PA. We conclude that cultured human mesangial cells produce receptor-bound u-PA/PAI-1 complexes localized to adhesion plaques.

Blotting, Western↗
PubMed1992

Vasopressin and phorbol ester-stimulated phosphatidylcholine metabolism in mesangial cells.

We have studied the effects of the vasoactive agents phorbol 12-myristate 13-acetate (PMA) and vasopressin (VP) on phosphatidylcholine metabolism in cultured rat glomerular mesangial cells. PMA and VP stimulate the incorporation of [3H]choline into phosphatidylcholine and the release of [3H]choline into the culture medium. VP, but not PMA, also increases the release of phosphorylcholine into the medium. This suggests that PMA specifically stimulates phospholipase D, whereas VP stimulates phospholipases C and D. Experiments were also conducted to look for production of phosphatidic acid and diacylglycerol, products of phospholipase D- and C-mediated breakdown of phosphatidylcholine. Treatment of cells prelabeled with [3H]myristic acid for 2.5 min with PMA or VP increases the content of [3H]myristic acid in diacylglycerol and phosphatidic acid. A dual labeling study ([3H]myristic acid and [14C]arachidonic acid) suggests that phosphatidylcholine is an important source of diacylglycerol in cells treated with VP and PMA. When PMA or VP are added to [3H]myristic acid-labeled cells in the presence of ethanol, increased labeling of phosphatidylethanol is seen as early as 2.5 min. Desensitization of protein kinase C by overnight treatment of cells with PMA blocked subsequent VP-stimulated formation of phosphatidylethanol and release of [3H]choline. When cells were simultaneously treated with VP and PMA, additive effects on phosphatidylethanol formation and [3H]choline release were observed.

Animals↗
PubMed1991

High glucose increases diacylglycerol mass and activates protein kinase C in mesangial cell cultures.

We showed previously that glomerular mesangial cells displayed increased fibronectin, laminin, and type IV collagen synthesis and mRNA levels when grown in medium containing 30 mM glucose compared with those cells grown in 10 mM glucose [S. H. Ayo, R. A. Radnik, W. F. Glass II, J. A. Garoni, E. R. Rampt, D. R. Appling, and J. I. Kreisberg. Am. J. Physiol. 260 (Renal Fluid Electrolyte Physiol. 29): F185-F191, 1990]. However, total protein synthesis and actin mRNA were unchanged. In this report, we show that an increase in medium glucose concentration resulted in an increase in diacylglycerol (DAG) mass and transiently increased protein kinase C (PKC) activity as assessed by the translocation of PKC from the soluble to the particulate fraction. Effects of increased glucose on DAG were evident at 30 min and were maintained through 1 wk of growth in medium containing 30 mM glucose. Although total PKC activity (i.e., soluble plus particulate fractions) did not change with high-glucose treatment, the percent activity associated with the particulate fraction (i.e., activated PKC) increased significantly after 60 min in RPMI 1640 medium with 30 mM glucose. The distribution of PKC returned to control values by 24 h. High glucose did not stimulate phosphoinositide hydrolysis, as evidenced by the absence of an increase in the water-soluble inositol phosphates, indicating that DAG was not generated through the action of a phosphoinositide-specific phospholipase C. Cells treated with the cell-permeable DAG analogue 1-oleoyl-2-acetyl glycerol to activate PKC displayed approximately two-fold increases of fibronectin, laminin, and type IV collagen mRNA levels after normalization against actin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Epidermal growth factor stimulates phospholipase A2 in vasopressin-treated rat glomerular mesangial cells.

Epidermal growth factor (EGF) enhances vasopressin- and ionophore-A23187-induced prostaglandin production and arachidonate release by rat glomerular mesangial cells in culture. The purpose of the present study was to delineate the phospholipid pathways involved in this effect. In cells labelled with [14C]arachidonate, EGF significantly enhanced the free arachidonate released in response to A23187 or vasopressin without enhancing the production of [14C]arachidonate-labelled diacylglycerol. EGF increased the [14C]arachidonate-labelled phosphatidic acid formed in response to vasopressin, but to a much smaller extent than it increased free arachidonate release. These results indicate that activation of phospholipase C is not sufficient to explain the increase in free arachidonate release observed on addition of EGF. To examine if EGF enhanced phospholipase A2 activity, mesangial cells were labelled with [2-2H]glycerol and [14C]-arachidonate, and the formation of arachidonate-poor lysophospholipids was studied. When combined with vasopressin, EGF significantly enhanced the formation of arachidonate-poor lysophospholipids as compared with vasopressin alone. The fate of exogenously added lysophosphatidylcholine was not altered after stimulation with vasopressin plus EGF, indicating that decreased deacylation or reacylation of the lysophospholipids was not responsible for their accumulation. Taken together, these results indicate that EGF enhances free arachidonate release by activation of phospholipase A2. The signalling mechanism responsible for the change in phospholipase A2 activity is not known, but could conceivably involve phosphorylation of modulating proteins such as lipocortin or G-proteins.

Animals↗
PubMed1988

Phorbol ester inhibits arginine vasopressin activation of phospholipase C and promotes contraction of, and prostaglandin production by, cultured mesangial cells.

We have previously shown that arginine vasopressin (AVP) causes a rapid (5-10 min) contractile response in cultured mesangial cells plated onto slippery substrata such as poly(hydroxyethyl methacrylate)-coated dishes. This contraction is associated with an increase in the levels of inositol trisphosphate (InsP3), diacylglycerol and prostaglandin E2 (PGE2). We now report that agents which are known to activate protein kinase C, i.e. phorbol 12-myristate 13-acetate (PMA) and oleolylacetylglycerol (OAG), also contract mesangial cells; however, the contractile response is slow to develop (15-30 min). The inactive phorbol ester, 4 alpha -phorbol 12,13-didecanoate, did not elicit contraction. PMA and OAG did not increase InsP3 release in mesangial cells. However, pretreatment of mesangial cells with PMA inhibited the formation of InsP3. This inhibition could not be explained by a reduction in AVP binding since PMA treatment did not influence the number or affinity of [3H]AVP binding sites in intact cells. PMA alone stimulated PGE2 production in mesangial cells to a degree similar to AVP. Contrary to what was seen with InsP3, pretreatment of cells with PMA before AVP had an additive effect on arachidonic acid release and PGE2 production. Thus, there is an apparent dissociation of phospholipase C activity from that of phospholipase A2.

Arginine Vasopressin↗
PubMed1987

Elevation of cAMP in cultured mesangial cells diminishes vasopressin-stimulated increases of phosphate uptake and 32P-specific activity in ATP but has no effect on phosphoinositide metabolism.

Agents known to elevate intracellular cyclic AMP (cAMP) in cultured mesangial cells (e.g., isoproterenol with and without isobutylmethylxanthine (MIX] inhibit vasopressin-induced contraction. Since contraction of these cells in response to vasopressin is accompanied by release of inositol trisphosphate and increased intracellular ionized calcium, we wanted to determine whether cAMP is exerting its relaxing effect by altering phosphoinositide metabolism. Isoproterenol and MIX did not diminish the release of inositol trisphosphate in response to vasopressin. However, the stimulated 32P incorporation into phospholipids seen with vasopressin treatment was diminished by prior treatment with isoproterenol-MIX. Since incorporation of 32P into phospholipids is not only dependent on phospholipid synthesis but also on the amount of label in the gamma-phosphate of ATP, we determined the specific activity of 32P in ATP. We found that suppression of 32P incorporation into phospholipids in cells treated with isoproterenol-MIX was paralleled by a decline of specific activity of 32P in ATP. Furthermore, the changes in ATP specific activity were paralleled by similar changes in phosphate uptake into the cells. Thus, diminished phosphate uptake (transport) could account for the decline of 32P content in phospholipids and ATP following treatment of mesangial cells with isoproterenol-MIX.

1-Methyl-3-isobutylxanthine↗
PubMed1986

Lipid alterations in LLC-PK1 cells exposed to mercuric chloride.

We have studied the effects of HgCl2 on the lipids of LLC-PK1 (pig kidney) epithelial cells. Our results show that treatment of cells with HgCl2 caused a rapid accumulation of unesterified fatty acids (particularly arachidonic acid) and lysophospholipids. A 27-fold increase in unesterified arachidonic acid and a 17-fold increase in lysophosphatidylethanolamine (LPE) was accompanied by a 26% decline in the mass of phosphatidylethanolamine as determined by gas chromatography and lipid phosphorus assay. Similar changes were seen following HgCl2 treatment of cells whose lipids were labelled with 14C stearic acid, 3H arachidonic acid, or 14C acetate, but the radiolabelling techniques also identified an increased content of label in lysophosphatidylcholine (LPC) and a corresponding decrease in phosphatidylcholine. These alterations were accompanied by the formation of blebs on the plasma membrane and irreversible injury as indicated by electron microscopy. The possible role of unesterified fatty acids in the pathogenesis of injury was studied by adding fatty acids to the cells. The addition of unsaturated fatty acids (oleic, linoleic, or arachidonic acids) to the cells caused plasma membrane blebbing and loss of viability. Similarly, the addition of LPC or LPE to the cells resulted in cell death; however, plasma membrane blebbing did not result.

Animals↗