PubMed Health⌕ Search

Biomedical subjects

S C Howard

Publications and source records attributed to S C Howard.

51 records · Page 3Linked to original sources
PubMed1990

Binding of tissue plasminogen activator to human aortic endothelial cells.

The experiments described in this paper were designed to examine the specific binding of tissue plasminogen activator (tPA) to cultured human aortic endothelial (HAE) cells. When 125I-labelled tPA was incubated with the cells at 4 degrees C, binding was found to plateau within 90 min after incubations were begun. Binding was saturable and the bound enzyme dissociated from the sites with a half-time of approx. 48 min. Scatchard analyses were performed using tPA molecules isolated from human melanoma and colon cells as well as from C127 and Chinese hamster ovary cells that had been transfected with the human tPA gene. These enzymes showed very similar binding characteristics in spite of the fact that they differ substantially in the types of sugars which comprise their side chains. Neither the chainedness of the molecules (one-chain or two-chain) nor the sites at which they are glycosylated (type I or type II) appear to affect their ability to interact with binding sites. The tPA molecules were found to have an average equilibrium dissociation constant of (1.15 +/- 0.10) x 10(-9) M and HAE cells appeared to have a single, homogeneous population of independent binding sites present at a concentration of (1.57 +/- 0.13) x 10(6) sites per cell. Lowering the pH of the binding buffer from 7.4 to 6.5 resulted in a reversible increase in specific binding of between 2-fold and 7-fold depending upon the particular preparation of cells. Preincubation of tPA with plasminogen activator inhibitor 1 (PAI-1) was found to have little effect on binding, suggesting that tPA interacts at sites distinct from surface-bound PAI-1. No evidence for either internalization or degradation of tPA was observed in assays run at 37 degrees C. This suggests that, like urokinase, tPA remains on cell surfaces for an extended period of time.

Aorta↗
PubMed1990

Glycosylation at Asn-184 inhibits the conversion of single-chain to two-chain tissue-type plasminogen activator by plasmin.

Tissue-type plasminogen activator (tPA) is a glycosylated serine protease which is an effective thrombolytic agent. Native single-chain tPA (sc-tPA) is converted to two-chain tPA (tc-tPA) by plasmin, the product of the reaction of plasminogen with tPA. Native sc-tPA occurs as two glycoforms. Type I sc-tPA is fully glycosylated, while type II lacks glycosylation at Asn-184. The rates at which type I and type II human melanoma sc-tPA were converted to type I and type II tc-tPA by plasmin were determined by two different methods. In each case, the second-order rate constant (kcat/Km) for type II sc-tPA (approximately 8 microM-1 s-1) was about twice that for type I sc-tPA (approximately 4 microM-1 s-1). These results indicate that glycosylation at Asn-184 hinders the conversion of sc-tPA to tc-tPA and suggest that under physiological conditions type I sc-tPA may persist in the single-chain form longer than type II sc-tPA. Previous studies have shown that type I tc-tPA has a lower activity than type II tc-tPA and that sc-tPA has a lower activity and susceptibility to inhibition when compared to tc-tPA. The present work provides further evidence that tPA glycosylation serves to modulate activity. The two major glycoforms may represent more persistent but slow acting (type I) and less persistent but faster acting (type II) variants of tPA.

Asparagine↗
PubMed1989

Cell-type-specific and site-specific N-glycosylation of type I and type II human tissue plasminogen activator.

Tissue plasminogen activator (t-PA) is an important initiator of fibrinolysis. The t-PA polypeptide has four potential N-glycosylation sites of which three are occupied in type I (Asn-117, -184, and -448) and two in type II (Asn-117 and -448). In an effort to elucidate the factors controlling the expression of N-linked oligosaccharides on this polypeptide, we have used a combination of sequential exoglycosidase digestion, methylation analysis, and controlled acetolysis to determine the oligosaccharide structures at each of the N-glycosylation sites of type I and type II t-PA when isolated from a human colon fibroblast cell strain and from a Bowes melanoma cell line. Our results suggest the following: (i) type I and type II t-PA are N-glycosylated in an identical way at Asn-117 and Asn-448, when isolated from the same cell line; (ii) Asn-117 is predominantly associated with oligomannose-type structures in all cases; (iii) Asn-184 and Asn-448 are predominantly associated with complex-type structures when t-PA is isolated from fibroblast cells, but with both complex- and oligomannose-type structures when isolated from melanoma cells; (iv) fibroblast cell derived t-PA is associated with both neutral and sialylated oligosaccharides, while melanoma cell derived t-PA is also associated with sulfated oligosaccharides, which are located exclusively at Asn-448 of type II t-PA; (v) no complex-type structures occur in common between t-PA from the two cell lines. These results indicate that the t-PA glycoprotein is secreted by each cell line as a set of glycoforms, each glycoform being unique with respect to the nature and disposition of oligosaccharides on a common polypeptide. Further, the two cell lines express no glycoform in common, despite expressing the same t-PA polypeptide. The implications of these results for both the control of oligosaccharide processing in different cell lines and the genetic engineering of mammalian glycoproteins are discussed.

Amino Acids↗
PubMed1989

Effects of N-glycosylation on in vitro activity of Bowes melanoma and human colon fibroblast derived tissue plasminogen activator.

Tissue-type plasminogen activator (t-PA), when isolated from human colon fibroblast (hcf) cells, is N-glycosylated differently than when isolated from the Bowes melanoma (m) cell line (Parekh et al., 1988). Both hcf- and m-t-PA can be separated into type I t-PA (with three occupied N-glycosylation sequons, at Asn-117, -184, and -448) and type II t-PA (with two occupied sequons, at Asn-117 and -448). Oligosaccharide analysis of each of these types of t-PA indicates that hcf-t-PA and m-t-PA have no glycoforms in common, despite having the same primary amino acid sequence. We have therefore compared in vitro the enzymatic activities and fibrin binding of type I and type II hcf- and m-t-PA with those of aglycosyl t-PA isolated from tunicamycin-treated cells. Plasminogen activation kinetics were determined by using an indirect amidolytic assay with Glu-plasminogen and a chromogenic plasmin substrate. In the absence of stimulator, there was little difference in activity between type I and type II t-PA, but the activity of aglycosyl t-PA was 2-4-fold higher than that of the corresponding glycosylated t-PA. In the presence of a fibrinogen fragment stimulator, the Kcat value of type II t-PA was approximately 5-fold that of type I t-PA from the same cell line, while the Km values for activation of Glu-plasminogen were similar (0.13-0.18 microM). The stimulated activity of glycosyl t-PA was similar to that of type II t-PA.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗
PubMed1987

Analysis of the polyhedrin gene promoter of the Autographa californica nuclear polyhedrosis virus.

The polyhedrin gene promoter of the Autographa californica nuclear polyhedrosis virus was analysed with respect to which sequences are required upstream of the mRNA transcription initiation (CAP) site for efficient promoter activity. Insertions (8, 95 and 785 nucleotides) were made in this region at an EcoR V site between the CAAT- and TATA-like boxes. When these mutations were introduced into the virus they did not affect the activity of the polyhedrin promoter as judged by expression of the beta-galactosidase (lacZ) gene inserted in lieu of the polyhedrin coding sequences. Deletions were made in the promoter which progressively removed sequences upstream from the CAP site. Removal of the TATA motif did not affect lacZ gene expression. A sequence 69 nucleotides upstream to the normal position of the polyhedrin ATG translation initiation codon was sufficient for maximum promoter activity but this was reduced by 90% when only 56 nucleotides upstream remained. The normal CAP site was utilized by each deletion mutant. Promoter activity was undetectable when the CAP site was deleted. The results are discussed in relation to other eukaryotic promoters.

Cloning, Molecular↗
PubMed1982

Isolation of a bisegmented double-stranded RNA virus from Thirlmere reservoir.

A novel bisegmented double-stranded RNA virus has been isolated from water processed from Thirlmere reservoir. The virus is icosahedral, 58 nm in diam., has a buoyant density of 1.32 g/ml in CsCl, has an S value of 400 and a RNA/protein ratio of 0.087. The two linear segments of RNA have approx. mol. wt. of 2.26 X 10(6) and 2.09 X 10(6). The virus contains six polypeptides. The virus was isolated in Drosophila melanogaster cells and fails to replicate in other insect, amphibian, avian, piscine, mammalian and plant cells tested. The virus is biochemically different from infectious pancreatic necrosis virus (IPNV) and Drosophila X virus (DXV). The virus is also serologically unrelated to IPNV (strain Sp) and another invertebrate pathogenic virus, Tellina virus 1. The virus shares common antigens with DXV but is not completely identical.

Animals↗
PubMed1982

Relationship between xenotransplantability and cell surface properties of ascites sublines of a rat mammary adenocarcinoma.

Cell surface properties of several ascites sublines of the 13762 rat mammary adenocarcinoma were compared in an effort to understand factors important to the xenotransplantability of these tumors into C57BL/6J mice. MAT-C, MAT-C1, and MAT-cMR6-S ascites sublines were xenotransplantable; MAT-B1, MAT-C2, and MAT-MR2-S were not. All of these sublines contained a large mucin-type sialoglycoprotein (ASGP-1) as a major cell surface component. Mat-B1 and MAT-cMR6-S ASGP-1 molecules were sulfated, but MAT-C1 and MAT-MR2-S incorporated little sulfate. The major oligosaccharide of MAT-C1 ASGP-1, a disialo-oligosaccharide, was present in very low amounts in the other sublines. ASGP-1 was detected readily in the plasma of animals with MAT-C1 or MAT-MR2-S tumors, but it was not detected readily in animals with MAT-B1 or MAT-cMR6-S tumors. Thus none of these significant features of ASGP-1 that differed among the sublines correlated with xenotransplantation. However, both MAT-C1 and MAT-cMR6-S sublines had branched cell surface microvilli. Moreover, MAT-C grown in mice after xenotransplantation had extensive, branched microvilli, even though only about 10% of the population of the cells grown in the rat had them. These results suggest that the branched microvilli may provide a protective mechanism, possibly by acting as a barrier to the approach of cytotoxic cells.

Adenocarcinoma↗
PubMed1980

Changes in expression of a major sialoglycoprotein associated with ascites forms of a mammary adenocarcinoma.

Glycoproteins of a cultured form (MR) of the 13762 rat mammary adenocarcinoma and its variants have been studied by analyses for peanut agglutinin receptors, [3H]glucosamine labeling, lactoperoxidase labeling and CsCl density gradient centrifugation. The 13762 MR cells, derived from 13762 MAT-B ascites cells, do not contain detectable ASGP-1, the predominant cell surface sialoglycoprotein of the ascites forms of the 13762 tumor. Transplantation and continued passage as ascites cells of MR cells or clonal lines derived from MR results in abrupt expression of ASGP-1 at about passage 16; it is absent in early passages of the ascites tumor. When these ascites cells are transferred to culture, ASGP-1 is again lost. No ASGP-1 is found in solid tumors derived from subcutaneous transplantation of the 13762 MR cells. The results suggest modulation of ASGP-1 content of the 13762 tumor cells.

Adenocarcinoma↗
PubMedDate not supplied

Evidence for a mass community effect of insecticide-treated bednets on the incidence of malaria on the Kenyan coast.

The use of insecticide-treated bednets (ITBNs) has been shown to be effective in reducing mortality and morbidity from malaria. However, there is mixed evidence as to whether or not community-wide use of ITBNs engenders a 'mass effect', such that those not sleeping under bednets are offered protection from widespread ITBN use in the area in which they live. We have analysed data collected in Kilifi, Kenya, from a cohort of children followed from birth to investigate how the degree of net usage in the locality of a child affects the risk of developing malaria. This effect was explored using a Cox proportional hazards model. For those not using ITBNs, we found that an increasing level of ITBN usage within the area surrounding each child was associated with a decreasing risk of developing malaria, thus providing evidence in support of a mass community effect. The size and significance of this effect were found to decrease as non-overlapping areas of increasing distance away from a child's home were considered. The effect was significant for areas at distances of up to 1.5 km away from each child.

Bedding and Linens↗