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Biomedical subjects

M S Runge

Publications and source records attributed to M S Runge.

At least 91 records · Page 5Linked to original sources
PubMed1995

Linoleic acid and its metabolites, hydroperoxyoctadecadienoic acids, stimulate c-Fos, c-Jun, and c-Myc mRNA expression, mitogen-activated protein kinase activation, and growth in rat aortic smooth muscle cells.

Previous studies from other laboratories suggest that linoleic acid and its metabolites, hydroperoxyoctadecadienoic acids, play an important role in modulating the growth of some cells. A correlation has been demonstrated between hydroperoxyoctadecadienoic acids and conditions characterized by abnormal cell growth such as atherosclerosis and psoriasis. To determine if linoleic acid and its metabolites modulate cell growth in atherosclerosis, we measured DNA synthesis, protooncogene mRNA expression, and mitogen-activated protein kinase (MAPK) activation in vascular smooth muscle cells (VSMC). Linoleic acid induces DNA synthesis, c-fos, c-jun, and c-myc mRNA expression and MAPK activation in VSMC. Furthermore, nordihydroguaiaretic acid, a potent inhibitor of the lipoxygenase system, significantly reduced the growth-response effects of linoleic acid in VSMC, suggesting that conversion of linoleic acid to hydroperoxyoctadecadienoic acids (HPODEs) is required for these effects. HPODEs also caused significant induction of DNA synthesis, protooncogene mRNA expression, and MAPK activation in growth-arrested VSMC, suggesting that linoleic acid and its metabolic products, HPODEs, are potential mitogens in VSMC, and that conditions such as oxidative stress and lipid peroxidation which provoke the production of these substances may alter VSMC growth.

Animals↗
PubMed1994

Alternative splicing of the mRNA encoding baboon glycoprotein receptor GPIIb.

The cloning and characterization of cDNAs encoding the cell-surface-specific integrin receptors of baboon platelets has been undertaken to provide species-specific probes. These will be used to investigate the expression and distribution of these receptors among primate species. Clones GPIIb-16 and GPIIb-3, encoding portions of the baboon glycoprotein GPIIb, were isolated from a cDNA library derived from baboon platelet mRNA. GPIIb-3 includes an insert of 43 bp, when compared to GPIIb-16 or human GPIIb. This insert is the result of alternative processing of mRNA. The probable origin of the inserted bases is the 3' end of the intron preceeding exon 28 of the gene. A different product of alternative splicing has been reported in this same region of the human GPIIb sequence, suggesting that this location is susceptible to wobble in the intron-exon junctions. The projected shift in the reading frame of the baboon GPIIb-3 cDNA would give a radically altered C terminus of the deduced amino-acid sequence, and the possibility of a novel functional peptide on the platelet surface.

Alternative Splicing↗
PubMed1994

Activation of mitogen-activated protein kinases by arachidonic acid and its metabolites in vascular smooth muscle cells.

Previous studies from this laboratory and others suggest that arachidonic acid and its metabolites play important roles in a variety of biological processes such as signal transduction, contraction, chemotaxis, and cell growth and differentiation. Here we studied the effect of arachidonic acid on mitogen-activated protein (MAP) kinases in vascular smooth muscle cells (VSMC). Arachidonic acid activated MAP kinases in VSMC in a time- and dose-dependent manner. Nordihydroguaiaretic acid (NDGA), a potent inhibitor of the lipoxygenase system, significantly blocked the arachidonic acid-induced activation of MAP kinases, whereas indomethacin, an inhibitor of cyclooxygenase, had no effect. In VSMC, arachidonic acid was converted to 15-hydroxyeicosatetraenoic acid (15-HETE); NDGA inhibited the formation of this HETE. Exogenous addition of 15-HETE to VSMC caused stimulation of MAP kinases. Depletion of protein kinase C attenuated both the arachidonic acid- and 15-HETE-induced activation of MAP kinases in VSMC. Together these results suggest that 1) arachidonic acid activates MAP kinases in VSMC; 2) 15-HETE, a 15-lipoxygenase product of arachidonic acid, at least in part, mediates the arachidonic acid effect on MAP kinases; and 3) protein kinase C appears to be important in arachidonic acid activation of MAP kinases. Therefore, MAP kinases may play an important role in arachidonic acid signaling of VSMC growth and function.

Animals↗
PubMed1994

Elimination of a major inhibitor epitope in factor VIII.

The A2 and C2 domains of human blood coagulation factor VIII (fVIII) contain the epitopes targeted by most inhibitory allo- and autoantibodies. Human inhibitors usually display limited or no reaction with porcine fVIII. We constructed an active, recombinant hybrid human/porcine fVIII molecule by replacing the putative human fVIII A2 domain epitope with the homologous porcine sequence. The hybrid retained full activity in the presence of antibodies with specificity restricted to the human A2 epitope. In contrast, the hybrid was neutralized by an anti-C2 antibody. These findings provide a basis for fine epitope mapping and for therapy of the inhibitor patient.

Amino Acid Sequence↗
PubMed1994

Epitope mapping of the alpha-chain of the insulin-like growth factor I receptor using antipeptide antibodies.

Insulin-like growth factor I (IGF I) is an important mitogen for vascular smooth muscle cells (VSMC). The IGF I receptor (IGF IR) is a heterotetramer composed of two cross-linked extracellular alpha-chains and two membrane-spanning beta-chains that contain a tyrosine-kinase domain. It has a high degree of sequence similarity to the insulin receptor (IR), and the putative ligand-specific binding site has been localized to a cysteine-rich region (CRR) of the alpha-chain. To obtain insights into antigenic determinants of the IGF IR, we raised a panel of site-specific polyclonal antibodies against short peptide sequences N-terminal to and within the CRR. Several antibodies raised against linear epitopes within the CRR bound to solubilized and native rat and human IGF IR by ELISA, did not cross-react with IR, but unexpectedly failed to inhibit 125I-IGF I binding. A polyclonal antibody directed against a 48-amino acid synthetic peptide, corresponding to a region of the CRR postulated to be essential for ligand binding, failed to react with either solubilized, reduced or intact IGF IR. Three antibodies specific for the N-terminus of the alpha-chain reacted with solubilized and native IGF IR. One of these, RAB 6, directed against amino acids 38-44 of the IGF IR, inhibited 125I-IGF I binding to rat aortic smooth muscle cells (RASM) and to IGF IR/3T3 cells (overexpressing human IGF IR) by up to 45%. Immunohistochemical analysis revealed strong IGF IR staining in the medial smooth muscle cell layer of rat aorta. These findings are consistent with a model wherein conformational epitopes within the CRR and linear epitopes within the N-terminus of the alpha-chain contribute to the IGF I binding pocket. These antibodies should provide a valuable tool to study structure-function relationships and in vivo regulation of the IGF IR.

Amino Acid Sequence↗
PubMed1994

Thrombolytic therapy in acute myocardial infarction--selected recent developments.

Thrombolytic therapy is the established treatment of choice for most eligible patients with acute myocardial infarction. Early initiation of treatment and early, complete and maintained patency of the infarct-related coronary artery are desirable, because these variables correlate with a reduction in mortality. As a consequence, considerable efforts have been undertaken to develop new pharmacological agents that serve these purposes. Among these, new plasminogen activators such as reteplase (r-PA), saruplase (scuPA), and staphylokinase are in clinical development, and DSPA (bat t-PA) and antibody-targeted plasminogen activators (ScuPA-59D8) have undergone extensive animal testing. Anticoagulants such as recombinant hirudin, hirulog, argatrobane, and Factor Xa inhibitors, as well as antiplatelet agents on the basis of monoclonal antibody 7E3 offer promise as adjunctive therapy to thrombolysis or to invasive intracoronary procedures.

Enzyme Precursors↗
PubMed1994

Endothelin A and B receptors are down-regulated in the hearts of hypertensive rats.

Endothelins are vasoactive peptides that have been implicated in the development and maintenance of systemic arterial hypertension. The biologic effects of endothelins result from activation of either or both of the two known endothelin receptor subtypes, A and B [ET-R(A) and ET-R(B)], which are present not only in blood vessels but also throughout the cardiovascular and central nervous systems. To investigate the potential role and regulation of myocardial endothelin receptors in hypertension, we examined the expression of ET-R(A) and ET-R(B) receptors in the hearts of normotensive and hypertensive rats. A cDNA probe for the ET-R(A) receptor was obtained by polymerase chain reaction amplification of rat aortic smooth muscle cell mRNA, using degenerate primers specific for intramembrane domains III and VI of G-coupled receptors. Moderate stringency hybridization screening of a rat aortic smooth muscle cell cDNA library yielded a partial clone for the ET-R(B) receptor. These two clones were used to examine expression of the ET-R(A) and ET-R(B) receptors in heart, brain, and kidney tissues from Wistar-Kyoto (normotensive), spontaneously hypertensive, salt-hypertensive sensitive, and salt-hypertensive resistant rats by Northern analysis. ET-R(A) and ET-R(B) mRNA were present in the hearts of normal rats. Spontaneously hypertensive rat hearts did not express either ET-R(A) or ET-R(B) mRNA, whereas both salt-hypertensive sensitive and resistant rats fed a high-salt diet expressed both ET-R(A) and ET-R(B) receptor mRNAs. Conversely, in the brain of spontaneously hypertensive rats, mRNAs for both ET-R(A) and ET-R(B) mRNA were present.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Fibrin-targeted recombinant hirudin inhibits fibrin deposition on experimental clots more efficiently than recombinant hirudin.

BACKGROUND: Although the indirect thrombin inhibitor heparin and the more potent direct inhibitor hirudin are useful in preventing thrombosis, a substantial opportunity remains for improving the thrombus selectivity of thrombin inhibitors. METHODS AND RESULTS: To explore the effect of targeting an antithrombin to the surface of a clot, we covalently linked recombinant hirudin to the Fab' (or IgG) of a monoclonal antibody (59D8) that selectively binds to an epitope on fibrin that becomes exposed only after thrombin cleaves fibrinopeptide B. Antibody-coupled hirudin bound to an immobilized peptide of the fibrin beta-chain amino-terminal sequence and inhibited the peptidolytic activity of thrombin more efficiently than free hirudin. Thrombin inhibition dependent on binding to immobilized fibrin monomer was enhanced 1100-fold (P < .0001). Hirudin-59D8 Fab' was 10 times more effective than hirudin in inhibiting fibrin deposition on experimental clot surfaces in fibrinogen solution (P < .0001) and human plasma (P < .0001). The more effective inhibition of thrombin by the conjugate was supported by significantly diminished concentrations of fibrinopeptide A in the plasma supernatant of the clot (P = .0001). Inhibition of clotting by an uncoupled mixture of hirudin and 59D8 Fab' was indistinguishable from that by hirudin alone, indicating that the conjugate's greater inhibitory activity was due to the covalent linkage between antibody and hirudin. CONCLUSIONS: Fibrin-targeted hirudin (in comparison with unmodified hirudin) significantly reduces fibrin deposition on the surface of experimental clots.

Antibodies, Monoclonal↗
PubMed1994

Agonist-induced phosphorylation of the vascular type 1 angiotensin II receptor.

Agonist-induced receptor phosphorylation plays a role in transmembrane signal transduction systems. Although the cDNA for the rat vascular type 1 angiotensin II receptor (AT1AR) encodes a G protein-coupled receptor with several potential phosphorylation sites for serine/threonine and tyrosine kinases, little is known about the phosphorylation of this receptor. The aim of this study was to determine the effects of angiotensin II (Ang II) on phosphorylation of the AT1AR in rat aortic vascular smooth muscle cells. Using [32P]orthophosphate-labeled cells, immunoprecipitates with anti-AT1AR antibody revealed a labeled band of molecular weight 52 kD, corresponding to the Ang II receptor. Ang II induced a rapid and significant increase in phosphorylation of the Ang II receptor, with a peak at 20 minutes. Phosphoamino acid analysis showed that the major phosphoamino acid is serine, in both the basal and Ang II-stimulated states. Constitutive and agonist-stimulated tyrosine phosphorylation is also observed to a lesser extent. Immunoblotting of anti-phosphotyrosine immunoprecipitates with anti-AT1AR antibody showed that Ang II caused a delayed tyrosine phosphorylation of the receptor with a peak at 20 minutes in a dose-dependent manner. Forskolin increased total phosphorylation of AT1AR but had no effect on tyrosine phosphorylation. Neither phorbol 12-myristate-13-acetate nor ionomycin altered receptor phosphorylation. These findings suggest that Ang II induces the phosphorylation of its own G protein-coupled receptor through both serine and tyrosine kinases and raise the possibility that phosphorylation of the AT1AR is an important regulator of receptor function.

Angiotensin II↗
PubMed1993

Human and baboon integrin beta 5 subunit-encoding mRNAs have alternative polyadenylation sites.

A cDNA probe, encoding part of the human integrin polypeptide, GPIIIa (beta 3), was used to screen a cDNA library derived from baboon smooth muscle polyadenylated mRNA. One cross-hybridizing clone, lacking the baboon equivalent of 478 bp of the human sequence, was found to be 98% similar to a human cDNA encoding the beta 5-chain of the receptor for vitronectin, a cell adhesion molecule. The baboon mRNA terminates at 3' position 212 nucleotides upstream from the polyadenylation site of the aligned human mRNA.

Amino Acid Sequence↗
PubMed1993

Characterization of a cDNA encoding the beta-chain of baboon receptor glycoprotein GPIb.

A cDNA probe, encoding part of the baboon integrin polypeptide, GPIIb, was used to screen a cDNA library derived from baboon platelet polyadenylated mRNA. One cross-hybridizing clone was found to be 94% similar to a human cDNA encoding the beta-chain of the receptor for von Willebrand factor, a major component of the platelet-subendothelium attachment mechanism.

Amino Acid Sequence↗
PubMed1993

The tissue plasminogen activator finger domain confers fibrin-dependent enhancement of catalytic activity to single-chain urokinase-type plasminogen activator.

To determine whether the fibrin-binding domains of tissue plasminogen activator (tPA) can confer enhanced catalytic activity to single-chain urokinase-type plasminogen activator (scuPA), we constructed, expressed, and characterized the kinetics of five recombinant tPA/scuPA hybrid molecules. The hybrid molecules are: 1) tPA3-50 (tPA finger)/scuPA138-411, 2) tPA177-256 (tPA kringle2)/scuPA140-411 (scuPA catalytic), 3) tPA3-50/tPA177-256/scuPA140-411, 4) scuPA1-47 (scuPA growth factor)/tPA177-256/scuPA140-411, and 5) scuPA1-138 (scuPA growth factor and kringle)tPA127-256/scuPA139-411. The amidolytic activity of all hybrids was comparable, as were the kinetics for conversion from single-chain to two-chain plasminogen activator. We found that 1) the lag time prior to achieving maximal velocity among these hybrids varied, 2) hybrids 2, 3, 4, and 5 were 2-134-fold more potent (by kcat/Km) than hybrid 1, and 3) those hybrid proteins containing the tPA finger domain (hybrids 1 and 3) gave a 2-fold increase in catalytic efficiency in the presence of DESAFIB (reptilase-digested fibrinogen). These kinetic differences are likely mediated by changes in the tertiary structure of the scuPA catalytic domain resulting from interactions between catalytic and noncatalytic domains in the presence of fibrin.

Animals↗
PubMed1993

High-level expression of antibody-plasminogen activator fusion proteins in hybridoma cells.

We show that the mouse gamma 2b heavy chain or human beta-globin 3' untranslated region can greatly enhance protein expression in myeloma cells transfected by genes coding for antibody-plasminogen activator fusion proteins. Expression plasmids were constructed containing a cloned genomic heavy chain variable region from fibrin-specific monoclonal antibody 59D8, a cloned genomic constant region of the mouse gamma 2b heavy chain, and DNA sequence coding for either tissue-type plasminogen activator (tPA) or a segment of urokinase (UK) and their respective 3' untranslated sequences. Cell lines transfected with these constructs, pSVtPA (tPA) and pSVUKG(UK), produced extremely low levels of mRNA and protein (0.008-0.06 micrograms/ml) in comparison with the parental 59D8 myeloma cell line (7.6-10 micrograms/ml). In vitro nuclear run-off analysis indicated that the low steady-state levels of mRNA encoded by pSVUKG(UK) did not result from a lower rate of transcription of the transfected gene (relative to the rate of transcription of the endogenous heavy chain gene in the 59D8 parent cells). In an attempt to increase protein secretion, we assembled the expression plasmids pSVtPA(Ig), pSVUKG(Ig), and pSVUKG(beta), in which the 3' untranslated region of the mouse gamma 2b heavy chain or human beta-globin gene was substituted for the 3' untranslated region of the plasminogen activator gene. Analysis of supernatant media from cell lines transfected with these constructs showed an increase in recombinant protein secretion of 68 to 100 fold in comparison with that from cell lines transfected with pSVtPA(tPA) or pSVUKG(UK).

Amino Acid Sequence↗
PubMed1993

[Future developments in thrombolytic therapy].

A primary failure rate to achieve reperfusion by thrombolytic therapy in acute myocardial infarction of 20-40%, an early reocclusion rate of 5-20%, as well as an increased risk of bleeding that excludes many patients from this form of therapy are today considered main problems. Better application of conventional thrombolytic agents, new conjunctive therapies and development of improved plasminogen activators are investigated with the aim of minimizing these problems.

Anticoagulants↗
PubMed1993

Inhibition of platelets and thrombin: implications for treatment of coronary artery thrombosis.

Molecular biology approaches have brought considerable progress to the development of novel plasminogen activators and antithrombin agents. While the modification of t-PA itself and the construction of chimeras between t-PA and pro-urokinase by molecular techniques have not resulted in enhanced efficacy, this can be achieved by constructing hybrids consisting of plasminogen activator domains and domains of monoclonal "targeting" antibodies. This and alternative approaches offer the promise of improved therapy in the future. Of equal importance is effective anticoagulant therapy through new antithrombin agents, platelet fibrinogen receptor inhibitors, or alternative approaches, among them gene therapy. Prevention of short-term thrombotic complications of invasive procedures such as PTCA or stent implantation may be better attained with the new antithrombins, while prevention of longer term complications (restenosis) may require inhibition of the thrombin receptor.

Angioplasty, Balloon, Coronary↗
PubMed1992

Molecular cloning of the rat vascular smooth muscle thrombin receptor. Evidence for in vitro regulation by basic fibroblast growth factor.

To study thrombin's receptor-mediated effects on vascular cells, we cloned and characterized a cDNA encoding a rat smooth muscle cell thrombin receptor. A rat aortic smooth muscle (RASM) cell cDNA library was screened with a 500-base pair (bp) sequence from the human thrombin receptor, obtained by polymerase chain reaction (PCR) amplification of cDNA synthesized from human erythropoietic leukemia (HEL) cell mRNA with PCR primers based on the published human thrombin receptor sequence. Clone pRTHR17 contains a 3418-bp insert that includes 50 bp of the 5'-untranslated region and the entire coding and 3'-untranslated regions of the RASM cell thrombin receptor. The sequence of pRTHR17 is 85% similar, at the nucleotide level, and 78% similar, at the deduced amino acid level, to the human thrombin receptor. Although the putative thrombin cleavage and binding sites are present, there are significant differences between the rat and human receptors in their amino-terminal sequences. Detectable signals (consisting of a single band of 3.45 kb) are present by Northern analysis of mRNA from RASM cells, and rat lung, kidney, and testes, but not in aorta or other tissues probed. The results of Southern analysis of rat genomic DNA are consistent with the existence of a single copy of the gene encoding this receptor. The steady state thrombin receptor mRNA level is low in cultured growth-arrested RASM cells and not detectable in rat aorta. To determine whether regulation of the RASM cell thrombin receptor occurs under growth-stimulating conditions, growth-arrested RASM cells were treated with basic fibroblast growth factor (bFGF, recently proposed to be a major mitogen controlling vascular smooth muscle cell growth following injury (Lindner, V., and Reidy, M. A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3739-3743)). There was a significant increase in thrombin receptor mRNA following the addition of bFGF. These data demonstrate that: 1) mRNA for a thrombin receptor similar to that reported from human megakaryocyte and hamster fibroblast cell lines is present in proliferating primary culture rat smooth muscle cells, 2) the most significant sequence differences are present in the amino-terminal tail of the thrombin receptor, and 3) the mRNA level for this receptor is regulated under growth-stimulating conditions in vitro.

Amino Acid Sequence↗
PubMed1992

A rat beta-interferon-induced mRNA: sequence characterization.

Polymerase chain reaction amplification of a cDNA derived from rat aortic smooth muscle cells, using sequences from conserved regions of the intramembrane domains of adrenergic receptors as primers, yielded the clone, rat8. This clone possesses a high degree of sequence similarity to a series of human interferon (IFN)-inducible genes. The rat8 sequence is 70% similar to that derived from the human alpha-IFN-induced gene, 9-27; there is 66% similarity between the deduced amino acid sequences encoded by the rat and the human genes. The rat homologue hybridizes with many bands in Southern analysis of rat DNA, suggesting that it is a member of a large multigene family.

Amino Acid Sequence↗