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Biomedical subjects

H C Marsh

Publications and source records attributed to H C Marsh.

35 records · Page 2Linked to original sources
PubMed1992

Cell surface expression of the C3b/C4b receptor (CR1) protects Chinese hamster ovary cells from lysis by human complement.

The C3b/C4b receptor, also known as complement receptor type 1 (CR1, CD35), is a single chain glycoprotein consisting of 30 repeating homologous protein domains known as short consensus repeats (SCR) followed by transmembrane and cytoplasmic domains. A series of recombinant proteins derived from CR1 has been prepared and assessed for the capacity to inhibit complement lysis of the host Chinese hamster ovary (CHO) cells. The full-length recombinant CR1 inhibited human complement-mediated CHO cell lysis, and the efficiency of inhibition was directly proportional to the number of receptors/cell. The SCR 15-18 of CR1, but not SCR 15-16, inhibited complement lysis of the host CHO cell, bound monomeric C3b (Kd,app = 6.5 x 10(-7) M), and dimeric C3b (Kd = 1.8 x 10(-8) M), and served as a cofactor in the proteolysis of C3b by factor I, confirming and extending the observations of Fearon and colleagues (Kalli, K. R., Hsu, P., Bartow, T. J., Ahearn, J. M., Matsumoto, A. K., Klickstein, L. B., and Fearon, D. T. (1991) J. Exp. Med. 174, 1451-1460). The SCR 1-4 of CR1, but not SCR 1-2, also inhibited complement lysis of the host CHO cell, indicating that more than two SCR are necessary and that four SCR are sufficient for optimal C4b binding to CR1. Thus, the structural requirements for C4b binding are analogous to those for C3b binding, namely, four SCR of CR1 form the binding sites for each of these proteins. CR1 has long been recognized to regulate extrinsic complement activation, that is, to bind to and promote the degradation of fluid phase C3b and of C3b attached to immune complex. These results demonstrate that CR1 is also an intrinsic regulator of complement activation in that, under appropriate conditions, CR1 inhibits complement-mediated lysis of the cell on which it is expressed.

Animals↗
PubMed1991

Recombinant soluble human complement receptor type 1 inhibits inflammation in the reversed passive arthus reaction in rats.

The human CR1 was genetically engineered by site directed mutagenesis into a truncated form which was secreted from transfected Chinese hamster ovary cells. This soluble recombinant CR1 (sCR1) was purified from the supernatants of the Chinese hamster ovary cells cultured in a hollow fiber bioreactor. sCR1 inhibits the C3 and C5 convertases of the classical and the alternative pathways in vitro. The ability of sCR1 to inhibit the immune complex-mediated inflammation in vivo was tested in a rat reversed passive Arthus reaction model. Administration of sCR1 at the dermal sites reduced the Arthus vasculitis in a dose-dependent manner as judged by both gross and microscopic examination, as well as by immunohistologic localization of C3 and C5b-9 neoantigen deposits. These data suggest that sCR1 inhibits the Arthus reaction by interrupting the activation of the C cascade, hence limiting the detrimental immune complex-induced tissue damage in vivo.

Animals↗
PubMed1991

The effect of soluble complement receptor type 1 on hyperacute xenograft rejection.

In the guinea pig-to-rat model of hyperacute xenograft (Xg) rejection, the effect of complement inhibition using systemically administered soluble complement receptor type 1 (sCR1) on discordant cardiac Xg survival was investigated. In PBS-treated control Xg recipients (n = 13), hyperacute rejection was rapid, with a mean Xg survival of 17 +/- 4 min. Therapy with sCR1 prolonged survival of cardiac Xgs in a dose-dependent manner. A 3 mg/kg bolus of sCR1 (n = 4) prolonged Xg survival to 64 +/- 29 min (not significant). Increasing the sCR1 dose to 5.9 mg/kg (n = 4) significantly delayed Xg rejection to 71 +/- 17 min (P-0.026, log-rank test vs. control). In 10 recipients treated with 15 mg/kg sCR1, mean Xg survival was further prolonged to 189 +/- 36 min (P-0.0004) with no adverse effects. While 2 of 8 recipients receiving 60 mg/kg sCR1 died with functioning Xgs at 30 and 300 min due to anastomotic bleeding, Xg survival averaged over 12 hr (747 +/- 100 min, P-0.0004) in the remaining 6 recipients. sCR1 administration significantly inhibited serum complement activity in a parallel dose-dependent fashion, with the 60 mg/kg dose reducing complement activity by 95 +/- 1 and 96 +/- 1% five and 30 min following Xg reperfusion, respectively. Immunofluorescence microscopy revealed rat IgM bound to all cardiac Xgs in control as well as sCR1-treated recipients. In addition, serial histologic examination of cardiac Xgs harvested within 21 min of graft reperfusion revealed occlusive platelet aggregates within the coronary vessels as well as interstitial hemorrhage and myocardial necrosis in Xgs from control recipients, all of which were only minimally present in Xgs from recipients treated with sCR1. These studies show that complement inhibition with sCR1 significantly delays hyperacute cardiac Xg rejection in this discordant model and may be an important component in a therapeutic protocol for xenotransplantation.

Animals↗
PubMed1990

Soluble human complement receptor type 1: in vivo inhibitor of complement suppressing post-ischemic myocardial inflammation and necrosis.

The complement system is an important mediator of the acute inflammatory response, and an effective inhibitor would suppress tissue damage in many autoimmune and inflammatory diseases. Such an inhibitor might be found among the endogenous regulatory proteins of complement that block the enzymes that activate C3 and C5. Of these proteins, complement receptor type 1 (CR1; CD35) has the most inhibitory potential, but its restriction to a few cell types limits its function in vivo. This limitation was overcome by the recombinant, soluble human CR1, sCR1, which lacks the transmembrane and cytoplasmic domains. The sCR1 bivalently bound dimeric forms of its ligands, C3b and methylamine-treated C4 (C4-ma), and promoted their inactivation by factor I. In nanomolar concentrations, sCR1 blocked complement activation in human serum by the two pathways. The sCR1 had complement inhibitory and anti-inflammatory activities in a rat model of reperfusion injury of ischemic myocardium, reducing myocardial infarction size by 44 percent. These findings identify sCR1 as a potential agent for the suppression of complement-dependent tissue injury in autoimmune and inflammatory diseases.

Animals↗
PubMed1990

Recombinant soluble CR1 suppressed complement activation, inflammation, and necrosis associated with reperfusion of ischemic myocardium.

In summary, conversion of wild-type CR1 to a soluble form (sCR1) creates a potent inhibitor of complement activation by both the classical and alternative pathways by inhibiting the C3/C5 convertases. In the rat reperfusion infarct model, sCR1 significantly suppresses complement activation at the endothelial surface of capillaries and venules. This suppression of complement activation is accompanied by reduced accumulation of leukocytes within the infarct zone, perhaps because of reduction of the generation of C5a, which promotes expression of leukocyte adhesion receptors and leukocyte chemotaxis. In addition, formation of the C5b-9 attack complex, which may contribute to direct endothelial injury, was suppressed by sCR1. The inhibition of complement activation and leukocyte infiltration by sCR1 explains the observed significant reduction in myocardial necrosis after ischemia and reperfusion. These studies have identified sCR1 as a potential agent for therapeutic intervention in diseases associated with complement-dependent tissue injury.

Amino Acid Sequence↗
PubMed1989

Two distinct immunogenic epitopes on the alpha chain of human T cell antigen receptor.

Monoclonal antibodies (MAb) to human T Cell Antigen Receptor (TCR) have been used to study the structure and function of TCR. Using purified alpha/beta heterodimeric protein, we have generated two MAb against human TCR alpha protein. The two MAb, alpha F1 and alpha F2, recognized amino acid residues 141-159 and 212-231 of the constant region of the alpha chain TCR. Although neither MAb reacted with viable T cells, both antibodies immunoprecipitated TCR alpha/beta heterodimer from HPB-ALL, Jurkat, PBL and a 32 kDa in vitro translation product of alpha chain cDNA. These antibodies have been shown to be useful in the immunohistochemical staining of human tissues. These two MAb, together with other anti-framwork MAb to TCR, should provide valuable reagents in the study of TCR and clinical classification of T cell lineage neoplasms.

Antibodies, Monoclonal↗
PubMed1985

Mechanism of action of thrombin on fibrinogen: NMR evidence for a beta-bend at or near fibrinogen A alpha Gly(P5)-Gly(P4).

The following synthetic linear A alpha fibrinogen-like peptides were studied by NMR spectroscopy in aqueous solution: Ac-Asp(P10)-Phe(P9)-Leu-Ala-Glu-Gly(P5)-Gly(P4)-Gly(P3)-Val- Arg(P1)-Gly-(P1)-Pro-Arg(P3')-Val-NHCH3 (F-8), Ac-Phe-Leu-Ala-Glu-Gly-Gly(P4)-Gly(P3)-Val-Arg-Gly-Pro-NHCH3 (F-6), Ac-Leu-Ala-Glu-Gly-Gly(P4)-Gly(P3)-Val-Arg-Gly-Pro-NHCH3 (F-7), and Ac-Gly-Gly(P4)-Gly-(P3)-Val-Arg-Gly-Pro-NHCH3 (F-9). The temperature dependence of the amide proton chemical shift is smaller by approximately 22% for the Gly(P3) amide proton in F-9, F-6, and F-8 and is similarly smaller for the Gly(P4) amide proton in F-6 and F-8, but not F-9, relative to the other amide protons in these peptides. The exchange rates with solvent water for the Gly(P3) amide proton in each of these four peptides were determined by solvent spin saturation transfer experiments. The exchange rate constant for the Gly(P3) amide proton of F-8 was half that of the rate constant determined for this proton in F-9, F-7, and F-6. In conjunction with previously reported data for the rate of hydrolysis of the Arg(P1)-Gly(P1') bond by thrombin, these results suggest that there is a beta-bend at Gly(P5)-Gly(P4), possibly stabilized by salt links between Asp(P10) and Arg(P3') and between phosphorylated Ser(P14) and Arg(P7'), which brings Phe(P9) close to the hydrolyzable Arg-Gly bonds.(ABSTRACT TRUNCATED AT 250 WORDS)

Amides↗
PubMed1983

Mechanism of action of thrombin on fibrinogen. Kinetic evidence for involvement of aspartic acid at position P10.

The following peptide was synthesized by classical methods in solution: Ac-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-NHCH3 (F-8). The Michaelis-Menten parameters for the hydrolysis of the Arg-Gly bond in F-8 by thrombin were determined to be Kcat = 31 X 10(-11) M [(NIH unit/L) s]-1 and KM = 310 X 10(-6) M. Comparison of these values with those determined previously for native fibrinogen and for a series of similar synthetic peptides, together with information about the amino acid sequences of this portion of the A alpha chain of abnormal fibrinogens, suggests an important role for Asp at position P10. Differences in the Michaelis-Menten parameters between F-8 and the 51-residue N-terminal CNBr fragment of the A alpha chain of fibrinogen correspond to only 1-2 kcal/mol in binding affinity.

Amino Acid Sequence↗
PubMed1982

Mechanism of action of thrombin on fibrinogen. Direct evidence for the involvement of phenylalanine at position P9.

The following peptides were synthesized by classical methods in solution: Ac-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-NHCH3 (F-6) and Ac-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-NHCH3 (F-7). The rates of hydrolysis of the Arg-Gly bond in these peptides by thrombin were measured, and the rate for the Phe-containing peptide F-6 was found to be much larger than that for F-7. Previous work [van Nispen, J. W., Hageman, T. C., & Scheraga, H. A. (1977) Arch. Biochem. Biophys. 182, 227] has demonstrated the importance of Phe-Leu at positions P9-P8 of the A alpha chain of fibrinogen for the thrombin-fibrinogen interaction. This work demonstrates that the presence of Leu (P8) alone is insufficient to account for the enhanced hydrolysis rates and that the presence of Phe (P9) is essential for normal action of thrombin on the A alpha chain of fibrinogen.

Amino Acid Sequence↗
PubMed1981

Europium(III) binding to bovine prothrombin residues 1-39 and to bovine prothrombin fragment 1.

Changes in the Eu3+ luminescence decay constant observed for Eu3+ bound to prothrombin fragment 1 and the peptide containing residues 1-39 of prothrombin result solely from changes in the number of water molecules in the primary hydration sphere of the ion. Highly coordinated sites which bind Eu3+ in a different manner from simple Gla-containing peptides exist on both the fragment 1 and 1-39 molecules. Metal ions bound at some of these sites do not appear to undergo rapid exchange with metal ions in solution. The binding of other lanthanide ions and calcium ions in the presence of europium ions cause a change in the conformation of the macromolecules, resulting in even tighter coordination of fragment 1 or residues 1-39 to Eu3+. Hydrophilic groups other than the actual Eu3+ binding sites on fragment 1 help to maintain its water solubility when complexes to Eu3+ or Ca2+. Because the 1-39 peptide does not contain as many of these hydrophilic groups, the peptide precipitates upon binding to di- and trivalent cations. Self-association of the 1-39 molecules appears to occur at high peptide concentrations which result in a peptide concentration effect on Eu3+ binding not seen in fragment 1. pH titration yields results which suggest a role for Gla carboxyl groups in Eu3+ complexation to both fragments 1 and 1-39. Although the two molecules are not identical with regard to Eu3+ binding behavior, enough similarities exist that residues 1-39 appear to be a reasonable model of the fragment 1 molecule for the purposes of metal ion complexation studies.

Amino Acid Sequence↗
PubMed1981

Tryptophan exposure in various conformational isomers of bovine prothrombin fragment 1. An acrylamide quenching study.

In order to assess the importance of a variety of environmental factors on the structure of bovine prothrombin fragment 1, we have examined acrylamide quenching of fragment 1 intrinsic fluorescence. Tryptophan exposure, determined from Stern-Volmer plots, is heterogeneous with one or more of the three fragment 1 tryptophans being exposed to solvent. In the presence of Ca2+ or Mg2+ even the most accessible tryptophan(s) are relatively buried. Only small differences in tryptophan exposure may exist between fragment 1-Ca2+ and fragment 1-Mg2+ complexes. Lowering pH, on the other hand, results in increased tryptophan exposure. Finally, structural isomers of fragment 1 which exist in the absence of metal ions have identical tryptophan exposure as determined by acrylamide quenching and fluorescence intensity.

Acrylamides↗
PubMed1979

The nature of the slow metal ion-dependent conformational transition in bovine prothrombin.

Kinetic parameters characterizing the slow structural isomerization observed via metal ion-dependent intrinsic fluorescence quenching of bovine prothrombin Fragment 1 have been determined. From forward and reverse rate constants, an equilibrium constant of approx. 0.25 is calculated. This result is consistent with the hypothesis that there exists, in the absence of metal ions, an equilibrium between two forms of bovine Fragment 1, one of which can interact rapidly with Ca2+ and subsequently with phospholipid. The other form of Fragment 1 cannot interact with Ca2+ in a manner that yields a phospholipid-binding form of the protein. Interconversion of these two forms of Fragment 1 occurs and may involve the isomerization of a proline residue.

Animals↗
PubMed1975

Ultrathin coating by plasma polymerization applied to corneal contact lens.

Plasma coating (deposition of polymer under the influence of plasma) is utilized to modify the surface properties of corneal contact lens. An ultrathin layer (thickness of roughly 200 A) of plasma polymer of acetylene/H20/N2 is applied to poly(methyl methacrylate) (PMMA) corneal contact lenses. The surface becomes highly wettable with water compared to uncoated lens. When coated and uncoated contact lenses are placed on rabbit eyes, a remarkable difference in accumulation of mucous matter is observed. With the control lenses, the accumulation of mucous matter in a week is sufficient to affect the optical clarity of the lenses, whereas the coated lenses show no change after three months continuous wearing. The comparative degree of adhesion of the corneal epithelium cells onto glass, modified glass, PMMA, and coated PMMA surfaces is studied using tissue cultures and phase contrast microscopy. The coated PMMA surface exhibits a degree of tissue adhesion lower than that of control PMMA and higher than that of glass surface, and no sign of toxicity of the coated surface is observed by the tissue cultures.

Acetylene↗