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Biomedical subjects

L T Williams

Publications and source records attributed to L T Williams.

At least 145 records · Page 8Linked to original sources
PubMed1988

Acidic and basic fibroblast growth factors stimulate tyrosine kinase activity in vivo.

We assessed the ability of acidic and basic fibroblast growth factor (FGF) to stimulate tyrosine kinase activity in intact cells. Immunoblot with polyclonal antiphosphotyrosine antibodies detected a 90-kDa phosphotyrosine-bearing protein in lysates of Swiss 3T3 cells exposed to pituitary-derived FGF, recombinant acidic FGF, or recombinant basic FGF, but not from unstimulated cells or cells exposed to epidermal growth factor or platelet-derived growth factor. Phosphotyrosine and its analogue phenyl phosphate, but not phosphoserine, phosphothreonine, or tyrosine itself, blocked recognition of the 90-kDa protein by antiphosphotyrosine antiserum. A monoclonal antiphosphotyrosine antibody also recognized the 90-kDa protein and was used to partially purify the protein by immunoaffinity chromatography. Phosphoamino acid analysis of the 90 kDa band revealed that it contained 20% phosphotyrosine, 35% phosphothreonine, and 45% phosphoserine. Tyrosine phosphorylation of the 90-kDa protein was detectable within 30 s and reached a plateau within 10 min of FGF addition. The addition of suramin, which blocks the interaction of FGF with its receptor, caused rapid disappearance of the 90 kDa band. Cell fractionation experiments were consistent with the 90-kDa protein being membrane-associated, but cross-linking studies revealed that the FGF receptor had an Mr between 145 and 210 kDa in Swiss 3T3 cells, distinct from the 90-kDa major substrate for tyrosine phosphorylation. These data demonstrate that both acidic and basic FGF activate a tyrosine kinase in vivo leading to phosphorylation of a unique 90-kDa substrate, and they suggest that protein modification by phosphorylation at tyrosine is involved in eliciting the mitogenic effect of FGF.

Animals↗
PubMed1988

Role of tyrosine kinase and membrane-spanning domains in signal transduction by the platelet-derived growth factor receptor.

Three types of mutations were introduced into the platelet-derived growth factor (PDGF) receptor to cause a loss of PDGF-stimulated tyrosine kinase activity: (i) a point mutation of the ATP-binding site, (ii) a deletion of the carboxyl-terminal region, and (iii) replacement of the membrane-spanning sequences by analogous transmembrane sequences of other receptors. Transfectants expressing mutated receptors bind, 125I-labeled PDGF with a high affinity but had no PDGF-sensitive tyrosine kinase activity, phosphatidylinositol turnover, increase in the intracellular calcium concentration, change in cellular pH, or stimulation of DNA synthesis. However, PDGF-induced receptor down regulation was normal in the mutant cells. These results indicate that the transmembrane sequence has a specific signal-transducing function other than merely serving as a membrane anchor and that the receptor kinase activity is necessary for most responses to PDGF but is not required for receptor down regulation.

Animals↗
PubMed1988

Platelet-derived growth factor mRNA detection in human atherosclerotic plaques by in situ hybridization.

Platelet-derived growth factor (PDGF) mRNA, and mRNA for its receptor, have been localized to specific cell types within the human atherosclerotic plaque, using in situ hybridization. The predominant cell types found to express PDGF A and B chain mRNA are mesenchymal-appearing intimal cells and endothelial cells, respectively, with little or no expression detected in macrophages. The distribution of PDGF receptor mRNA containing cells was also examined and found to be localized predominantly in the plaque intima.

Arteriosclerosis↗
PubMed1988

Arachidonic acid and cyclic adenosine monophosphate stimulation of c-fos expression by a pathway independent of phorbol ester-sensitive protein kinase C.

The stimulation of cell proliferation by platelet-derived and other growth factors is associated with a rapid increase in the expression of the c-fos protooncogene. We and others have shown that phosphosphoinositide turnover and protein kinase C play a role in the activation of this gene by growth factors, but that a second, kinase C-independent pathway(s) exist. Because cAMP potentiates the actions of a number of growth factors and is elevated in platelet-derived growth factor-stimulated Swiss 3T3 cells, we examined the ability of cAMP to stimulate c-fos expression in this cell type. Forskolin, a direct activator of adenylate cyclase, elicited marked increases in c-fos mRNA levels. Receptor-mediated activation of adenylate cyclase by prostaglandin E1 and stimulation with the cAMP analog 8-bromo-cAMP also enhanced c-fos expression. In cells made protein-kinase C deficient, c-fos induction by phorbol ester was abolished; by contrast, c-fos was still induced by cAMP-elevating agents in protein kinase C-depleted cells. Platelet-derived growth factor causes cAMP accumulation by stimulating arachidonic acid release and the formation of prostaglandins capable of activating adenylate cyclase. The addition of arachidonic acid and the arachidonate metabolite prostaglandin E2 to Swiss 3T3 cultures stimulated c-fos expression. These data suggest the existence of a pathway from growth factor receptor to gene induction that is mediated by cAMP and does not depend on a phorbol ester-sensitive protein kinase C.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1988

Serotonin-induced deoxyribonucleic acid synthesis in vascular smooth muscle cells involves a novel, pertussis toxin-sensitive pathway.

Serotonin-induced DNA synthesis in bovine aortic smooth muscle cells was totally abolished by pretreatment of cultures with 5 ng/ml pertussis toxin. The half maximally effective concentration of toxin was approximately 10 pg/ml. Pertussis toxin did not affect platelet-derived growth factor (PDGF)-stimulated DNA synthesis and actually enhanced the mitogenic effect of the phorbol ester, phorbol 12-myristate 13-acetate. Pertussis toxin did not inhibit serotonin-stimulated inositol phosphate accumulation or increases in intracellular calcium or cAMP concentrations under conditions sufficient to completely inhibit serotonin-induced (3H)thymidine incorporation. These results demonstrate that a novel, pertussis-sensitive pathway is required for serotonin-, but not platelet-derived growth factor-induced DNA synthesis in vascular smooth muscle cells. The pertussis-sensitive step does not involve cAMP, phosphoinositide hydrolysis, mobilization of intracellular calcium, or phorbol ester-sensitive protein kinase C activity.

Animals↗
PubMed1988

Developmental expression of platelet-derived growth factor and its receptor in the human placenta.

To investigate the role of platelet-derived growth factor (PDGF) during human placental development, expression of the genes encoding PDGF, the PDGF-receptor (PDGF-R) and the c-fos protooncogene was measured. Messenger RNAs for these genes were detected throughout pregnancy and peaked coordinately during the second trimester. An identical pattern of PDGF-R protein expression was confirmed by immunoblotting using a specific PDGF-R antiserum, measurement of PDGF-R kinase activity, and [125I]PDGF binding. These findings show that the components of the PDGF pathway are expressed in a concerted fashion throughout human pregnancy and are present at especially high levels during the midtrimester. Our observations suggest that through autocrine and/or paracrine mechanisms, PDGF is likely to play an important role in placental homeostasis.

Female↗
PubMed1988

Ligand activation causes a phosphorylation-dependent change in platelet-derived growth factor receptor conformation.

The effect of ligand binding on PDGF receptor conformation was examined using peptide antisera directed against specific receptor domains. Antiserum 83, which recognizes the receptor's carboxyl terminus, preferentially immunoprecipitated the ligand-activated form of the PDGF receptor from 35S-labeled BALB/c 3T3 cells. By contrast, two antisera directed against other receptor sequences precipitated unactivated and activated receptors equally well. Denatured receptors were recognized equally by all antisera, even 83. Thus, ligand activation caused a change in PDGF receptor conformation that enhanced accessibility of the receptor was induced by the three different forms of PDGF (AA and BB homodimers and AB heterodimer) and was reversed by suramin, a polyanionic compound that dissociates PDGF from the receptor. The inhibitory effect of suramin on receptor conformation was abolished by the phosphatase inhibitor sodium orthovanadate, suggesting that receptor phosphorylation mediated the conformational change. In a cell-free assay, the change in receptor conformation was induced by PDGF only in the presence of ATP and was inhibited by AMP-PNP, a nonhydrolyzable analog of ATP. The functional significance of receptor conformation was examined in CHO fibroblasts transfected with wild type or mutated forms of the PDGF receptor. When receptor tyrosine kinase activity was abolished by a mutation of the ATP binding site, the receptor no longer underwent PDGF-induced conformational change and did not mediate PDGF-induced mitogenesis, even though [125I]PDGF binding was normal. Ligand-induced receptor autophosphorylation was reduced but present in cells expressing receptors that lacked a major site of autophosphorylation and was present in cells expressing receptors that lacked the kinase insert domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Regulated expression of the platelet-derived growth factor A chain gene in microvascular endothelial cells.

Platelet-derived growth factor (PDGF) is composed of homologous polypeptide chains, termed A and B, that are expressed as mitogenically active A-A, B-B, or A-B dimers. Previous work in our laboratory has demonstrated that PDGF B chain mRNA expression is stimulated in microvascular endothelial cells by phorbol esters (PMA), thrombin, and transforming growth factor-beta (TGF-beta) and blocked by agents that elevate cyclic AMP (cAMP). Here we report the first evidence that the expression of A chain mRNA is also regulated in these cells. PDGF A chain mRNA levels were increased 5-25-fold by phorbol esters, thrombin, and TGF-beta. Transcripts of four different sizes were induced. The increase in A chain mRNA stimulated by TGF-beta was more prolonged (peak 4 h, duration 48 h) than the increase stimulated by PMA and thrombin (peak 4 h, duration 8 h). Among the agents known to increase B chain mRNA levels, PMA was most efficacious, followed in decreasing order by thrombin and TGF-beta. However, for A chain mRNA induction by these same agents, the order was reversed; TGF-beta was most efficacious, followed in decreasing order by thrombin and PMA. Agents that elevate cyclic AMP, known to block induction of B chain mRNA, blocked A chain induction by thrombin but had less effect on A chain mRNA induced by TGF-beta. Thus PDGF A chain mRNA levels are regulated by the same agents that regulate B chain mRNA levels in microvascular endothelial cells. While the changes in A chain mRNA are qualitatively similar to the changes in B chain mRNA in microvascular endothelial cells, there are differences in the relative efficacies of these agents in the regulation of PDGF A and B chain genes. These differences suggest that the forms of PDGF produced by endothelial cells depend on the nature of the inducing stimulus.

Cells, Cultured↗
PubMed1987

Agents that increase cAMP accumulation block endothelial c-sis induction by thrombin and transforming growth factor-beta.

Endothelial cells express the product of the c-sis gene, which encodes the B-chain of platelet-derived growth factor (PDGF). Through local production of growth factors such as PDGF in vascular sites, endothelial cells may stimulate proliferation of adjacent cells through a paracrine mechanism. Previously, we have shown that the expression of c-sis mRNA and release of growth factor activity by human renal endothelial cells is induced by thrombin. We now show that another agent of possible importance in mediating proliferation of cells adjacent to the endothelial cell layer, transforming growth factor-beta (TGF-beta), also induced c-sis expression in these cells. In addition, we have studied the effect of agents that increase intracellular cAMP levels upon the induction of endothelial cell c-sis mRNA. The adrenergic agonists isoproterenol and norepinephrine blocked the elevation of cellular c-sis mRNA accompanying exposure to either thrombin or TGF-beta. This effect was mediated through beta-adrenergic receptors, since propranolol but not phentolamine reversed the inhibition. Forskolin, a direct activator of adenylate cyclase, also blocked induction of c-sis mRNA by thrombin and TGF-beta and inhibited the release of PDGF activity into the media of these cells. Basal, as well as stimulated c-sis mRNA levels were attenuated by these agents that increase cellular cAMP levels. These data suggest that increased cAMP production inhibits the expression of c-sis encoded mitogens by endothelial cells, and that c-sis expression is subject to bidirectional regulation in these cells.

Animals↗
PubMed1987

Biosynthetic and glycosylation studies of cell surface platelet-derived growth factor receptors.

The cell surface pool of metabolically labeled platelet-derived growth factor (PDGF) receptors in BALB/c3T3 fibroblasts was studied using an antiphosphotyrosine antibody. Exposure of intact cells to PDGF stimulates autophosphorylation of surface PDGF receptors and allowed immunoaffinity purification of only PDGF-activated receptors. Pulse-chase experiments demonstrated appearance of newly synthesized receptors in a surface activatable pool within 30-45 min of synthesis. In the absence of exogenous PDGF, the apparent half-life of this pool was 2 h. The presence of both N- and O-linked oligosaccharide chains on cell surface PDGF receptors was demonstrated. Enzymatic removal of the N-linked oligosaccharide chains reduced the receptor's apparent Mr by approximately 40 kDa and removal of O-linked oligosaccharide caused approximately a 7-kDa reduction. Activation of receptor tyrosine autophosphorylation by PDGF did not require either processing of high-mannose N-linked oligosaccharides to complex forms or the presence of sialic acid on receptor oligosaccharide chains. Tryptic cleavage of PDGF-activated surface receptors in intact cells yielded two discrete phosphotyrosine-containing fragments of 107 and 85 kDa. Cleveland digest patterns from each fragment indicate that both are derived from the intact PDGF receptor. These data indicate that PDGF receptors are synthesized and turn over rapidly in the absence of ligand. Partial characterization of the extracellular domain oligosaccharide contribution to receptor function and trypsin susceptibility is provided.

Animals↗
PubMed1987

Processing of the platelet-derived growth factor receptor. Biosynthetic and degradation studies using anti-receptor antibodies.

Studies of platelet-derived growth factor (PDGF) receptor biosynthesis and degradation have been limited by the lack of anti-receptor antibodies. In this study, peptides based on the cDNA-predicted amino acid sequence of the PDGF receptor were used to produce antisera that specifically immunoprecipitated the receptor. PDGF receptor biosynthesis was examined by pulse-chase labeling of cultured fibroblasts with [35S]methionine followed by immunoprecipitation. In BALB/c 3T3 fibroblasts the receptor was synthesized as a 160-kDa precursor that was converted to a mature 180-kDa form within 30-45 min. Removal of high mannose oligosaccharides by endo-beta-N-acetylglucosaminidase H treatment reduced the apparent molecular weight of the 160-kDa precursor but did not affect the migration of the 180-kDa mature receptor. When mannosidase II was inhibited by swainsonine, the 160-kDa precursor failed to mature; instead a 168-kDa form of the receptor was observed. Nevertheless, swainsonine-treated cells responded mitogenically to PDGF. The mature 180-kDa form of the receptor had a half-life of approximately 3 h in the absence of ligand. Addition of PDGF reduced the receptor half-life to 45 min. These studies define and characterize a PDGF receptor precursor, show that receptor degradation is enhanced by PDGF, and demonstrate the functional integrity of incompletely processed PDGF receptors.

Animals↗
PubMed1987

The stimulation of paracrine and autocrine mitogenic pathways by the platelet-derived growth factor receptor.

Platelet-derived growth factor (PDGF) and PDGF-like polypeptides stimulate cell proliferation through paracrine and autocrine pathways. Each of these pathways is mediated by the PDGF receptor. Recently, cDNA clones for the receptor have been isolated and sequenced. The receptor gene on chromosome 5 is transcribed into a single 5.2 kb mRNA. The translated product, which is processed through at least one identifiable precursor, is expressed at the cell surface and is rapidly degraded. When activated by PDGF, the receptor mediates a group of diverse intracellular reactions. The receptor domains that mediate tyrosine kinase activity can be identified in the amino acid sequence of the receptor. However, the domains that mediate other PDGF-stimulated responses, such as turnover of phosphatidylinositol and enhanced expression of the c-myc and c-fos genes, have not been determined. Recently, a full-length receptor cDNA clone has been expressed in cells that normally lack PDGF receptors. This expression system should provide an approach to studies of the function of specific receptor domains and should help determine the relationship among the intracellular reactions stimulated by PDGF.

Animals↗
PubMed1987

Microbiology of acute periodontal infection in myelosuppressed cancer patients.

This study characterized the subgingival microbial flora associated with 27 acute exacerbations of preexistent periodontal disease in 24 patients with chemotherapy-induced myelosuppression. All but two acute periodontal infections developed at low granulocyte levels (less than 1,000/microL). Suspected pathogens were detected in high concentrations in subgingival plaque specimens in 17 episodes of acute periodontal infection; a single pathogen was recovered in ten acute infections, and more than one pathogen was recovered in seven acute infections. Staphylococcus epidermidis, Candida albicans, S aureus, and Pseudomonas aeruginosa predominated, with combinations of these detected in some patients. Concomitant bacteremias developed in two of these patients. The subgingival microflora associated with ten acute periodontal infections was characterized by predominantly indigenous microorganisms, which in nine episodes were in abnormal proportions compared with microbial profiles in noncancer patients with similar degrees of periodontal disease. These data demonstrate that pathogens normally associated with infections in myelosuppressed cancer patients, as well as indigenous oral flora, are associated with acute periodontal infections during granulocytopenia. This finding is important, since this body site has not commonly been recognized as a source for acute infection in these patients.

Acute Disease↗
PubMed1987

Dietary intake of individuals interested in eating a healthy diet: a validated study of intake before and after dietary advice.

The food intake of 37 adult volunteers (12 M, 25 F) interested in eating a healthy diet was measured on two occasions by the 7-day weighed record method. In week 1 subjects consumed their habitual diet and in week 2 attempted to follow dietary recommendations (DR). Intakes were compared with recommended dietary allowances (RDA) and with dietary guidelines (DG). Validation of recorded energy intakes against changes in body weight, and sodium, potassium and nitrogen intakes against urine excretions suggested reliable dietary records in both study weeks. Mean dietary intakes in week 1 met the RDA for all nutrients studied except for energy (which was close) and zinc (in females only). Daily energy intake was 2160 kcal (9.04 MJ) in week 1 and 1810 kcal (7.56 MJ) in week 2, and subjects lost weight during week 2. Absolute intakes of vitamins and minerals were similar in both study weeks, thus increasing nutrient density in week 2 and suggesting improved dietary quality. Mean dietary intakes in week 1 conformed more closely to DG than commonly reported and changed in the direction recommended in week 2. Mean daily intakes in week 1 and week 2 respectively were: fibre, 31 and 34 g; sodium 114 and 90 mmol; Na:K ratio, 1.37 and 1.10; alcohol, 3.3 and 2.3 per cent of total energy; fat, 38.7 and 34.2 per cent of food energy or 37.4 and 33.4 per cent of total energy. Intake of fibre increased with respect to energy but sodium intake per unit energy did not decrease. Energy intake was positively related to sodium, fibre and zinc intakes and influenced ease of conformity to DR for these nutrients. It is concluded that our motivated subjects were able to make short-term dietary changes consistent with DR. Whether or not these changes continue in the long term is the subject of a follow-up study.

Adult↗
PubMed1986

Pertussis toxin-sensitive pathway in the stimulation of c-myc expression and DNA synthesis by bombesin.

The bombesin-like peptides are potent mitogens for Swiss 3T3 fibroblasts, human bronchial epithelial cells, and cells isolated from small cell carcinoma of the lung. The mechanism of signal transduction in the proliferative response to bombesin was investigated by studying the effect of Bordetella pertussis toxin on bombesin-stimulated mitogenesis. At nanomolar concentrations, bombesin increased levels of c-myc messenger RNA and stimulated DNA synthesis in Swiss 3T3 cells. Treatment of the cells with pertussis toxin (5 nanograms per milliliter) completely blocked bombesin-enhanced c-myc expression and eliminated bombesin-stimulated DNA synthesis. This treatment had essentially no effect on the mitogenic responses to either platelet-derived growth factor or phorbol 12,13-dibutyrate. These results suggest that the mitogenic actions of bombesin-like growth factors are mediated through a pertussis toxin-sensitive guanine nucleotide-binding protein. Furthermore they indicate that bombesin-like growth factors act through pathways that are different from those activated by platelet-derived growth factor.

Animals↗