Gerhard Bodo

Inhibition of neuraminidase activity by derivatives of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid.

Eighteen derivatives of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid were assayed for inhibitory activity against neuraminidases from viral and bacterial sources. Twelve of these compounds were active against neuraminidases of Vibrio cholerae, influenza AMel, BLee, and Newcastle disease virus, causing 50% enzyme inhibition in concentrations ranging from 10−3M to 10−6M. The most active of them and the most potent neuraminidase inhibitor described so far is 2-deoxy-2,3-dehydro-N-trifluoroacetylneuraminic acid. This compound has an inhibitor constant (Ki) of 7,9 × 10−7, M for influenza AMel virus neuraminidase whereas the Km of the virus enzyme for the substrate is 1000 times weaker (Km = 6, 9 × 10−4M). The mechanism of inhibition is competitive, and enzyme inhibition is independent of enzyme concentration. 2-Deoxy-2,3-dehydro-N-trifluoroacetylneuraminic acid inhibits hemagglutination by NDV and SV5 but does not inhibit agglutination of red cells by Sendai virus or influenza A and B viruses.

Vascular anticoagulant beta: a novel human Ca2+/phospholipid binding protein that inhibits coagulation and phospholipase A2 activity. Its molecular cloning, expression and comparison with VAC-alpha.

A cDNA was cloned coding for a new member of the human Ca2+-modulated phospholipid-binding protein family termed annexins. Due to its 56% identity to the human vascular anticoagulant (VAC) the new protein is named VAC-beta, renaming the previous VAC as VAC-alpha. Northern analysis detects one hybridizing mRNA species of 2.2 kb in human placenta. Genomic Southern blot analysis shows a VAC-beta gene of comparable complexity to the VAC-alpha gene. The cDNA was expressed in Escherichia coli and the recombinant protein purified to homogeneity. Antiserum raised against VAC-beta weakly cross-reacts with VAC-alpha. The properties of VAC-beta as an anticoagulant and as an inhibitor of phospholipase A2 activity were analyzed and compared to those of VAC-alpha.

Crystallization and preliminary X-ray studies of human vascular anticoagulant protein

The human vascular anticoagulant protein, a 36 kDa member of the annexin/lipocortin family, has been crystallized using polyethylene glycol 20,000, by the vapour diffusion method. The crystals are monoclinic, space group P2(1), cell dimensions a = 83.9 A, b = 80.9 A, c = 71.4 A, beta = 108.7 degrees and diffract to at least 2.2 A resolution.

Methylation of vaccinia virus-specific mRNA in the interferon-treated chick cell☆

Abstract Results obtained with cell-free protein-synthesizing systems imply a mediating role of the methylated 5′-terminal “cap” structure in the regulation of poxvirus-specific protein synthesis. In order to study a possible involvement of methylation in the interferon-induced inhibition of virus-specific protein synthesis in Vaccinia-infected cells, the extent of methylation of Vaccinia mRNA synthesized in interferon-pretreated and control chick embryo fibroblasts was compared. Induction of the antiviral state by pretreatment of chick cells with homologous interferon does not markedly reduce the degree of overall methylation of virus-specific mRNAs. Besides the two predominant “cap I” structures m 7 GpppAmpN and m 7 GpppGmpN, “incomplete” structures m 7 GpppA and m 7 GpppG can be detected in Vaccinia mRNA isolated from interferon-treated as well as from control cells. The ratio of complete to incomplete “cap I” structures is decreased in the viral RNA from interferon-treated cells. This qualitative change in “cap” methylation seems not to be limited to virus-specific mRNA. Possible implications of these findings on translational control in the interferon-treated cell are discussed.

Biological activity of poly rI:poly rC: effect on poxvirus-specific functions.

Abstract Poxvirus-specific functions have been studied during the “early” period of the replication cycle in chick embryo fibroblasts pretreated with poly rI:Poly rC. “Early” viral protein synthesis and formation of DNA was found to be strongly reduced in a similar fashion to that of cells exposed to homologous interferon preparations. “Early” viral RNA of cowpox or vaccinia (WR), on the other hand, was synthesized at the rate expected for cells in which protein synthesis is inhibited. Even at high concentrations, (100 μg/ml), poly rI:poly rC had no inhibitory effect on “early” viral RNA transcribed in vitro by the RNA-polymerase contained in the virion. The results are discussed with respect to the mechanism of action of interferon and poly rI: poly rC.

The human lymphoma cell line NC-37: An alternative source of human lymphoblastoid interferon

Abstract NC-37 lymphoma cells treated with n-butyrate or 5-bromodeoxyuridine (BrdUrd) and induced with Sendai virus produced crude interferon with titers of up to 2 × 105 units/ml (100 units/103 cells) and with specific activities of up to 106 units/mg protein. More than 90% of the interferon activity was neutralized by antiserum against human interferon type α (HuIFN-α). Treatment of the cells with BrdUrd, but not with butyrate, resulted in the production of moderate amounts of interferon without virus induction.

Interferon-induced translation defects in a cell-free protein-synthesizing system from mouse erythroleukemia cells

Abstract The mechanism of the interferon-induced translation inhibition was studied in vitro using a fractionated cell-free protein-synthesizing system prepared from Friend virus-transformed mouse erythroleukemia cells and L cells. The soluble fraction of the system was prepared from nonpreincubated S30 lysates. To lower endogenous protein synthesis, the ribosomes were prepared from extracts treated with micrococcal nuclease at 20°. If the components were prepared from interferon-treated cells, viral and cellular mRNA translation was inhibited. In the cell-free system prepared from interferon-treated mouse erythroleukemia cells, the translation defect could be localized in the high-speed supernatant, while ribosomes from interferon-treated and control cells were of comparable activity. In contrast, ribosomes from interferon-treated L cells prepared by the same method were unable to support translation of exogenous mRNAs. The interferon-induced translational defect in either cell-free system could not be overcome by adding tRNA. Aminoacylation and stability of endogenous leucine-specific tRNAs was not impaired in the high-speed supernatant of both of the cell-free systems from interferon-treated cells.