The heparin binding domain of S-protein/vitronectin binds to complement components C7, C8, and C9 and perforin from cytolytic T-cells and inhibits their lytic activities

S-Protein/vitronectin is a serum glycoprotein that inhibits the lytic activity of the membrane attack complex of complement, i.e., of the complex including the proteins C5b, C6, C7, C8, and C9n. We show that intact S-protein/vitronectin or its cyanogen bromide generated fragments also inhibit the hemolysis mediated by perforin from cytotoxic T-cells at 45 and 11 microM, respectively. The glycosaminoglycan binding site of S-protein/vitronectin is responsible for the inhibition, since a synthetic peptide corresponding to a part of this highly basic domain (amino acid residues 348-360) inhibits complement- as well as perforin-mediated cytolysis. In the case of C9, the synthetic peptide binds to the acidic residues occurring in its N-terminal cysteine-rich domain (residues 101-111). Antibodies raised against this particular segment react 25-fold better with the polymerized form of C9 as compared with its monomeric form, indicating that this site becomes exposed only upon the hydrophilic-amphiphilic transition of C9. Since the cysteine-rich domain of C9 has been shown to be highly conserved in C6, C7, and C8 as well as in perforin, the inhibition of the lytic activities of these molecules by S-protein/vitronectin or by peptides corresponding to its heparin binding site may be explained by a similar mechanism.     

Effect of substrate and pH on the oxidative half-reaction of phenol hydroxylase

The oxidative half-reaction of phenol hydroxylase has been studied by stopped-flow spectrophotometry. Three flavin-oxygen intermediates can be detected when the substrate is thiophenol, or m-NH2, m-OH, m-CH3, m-Cl, or p-OH phenol. Intermediate I, the flavin C(4a)-hydroperoxide, has an absorbance maximum at 380-390 nm and an extinction coefficient approximately 10,000 M-1 cm-1. Intermediate III, the flavin C(4a)-hydroxide, has an absorbance maximum at 365-375 nm and an extinction coefficient approximately 10,000 M-1 cm-1. Intermediate II has absorbance maxima of 350-390 nm and extinction coefficients of 10,000-16,000 M-1 cm-1 depending on the substrate. A Hammett plot of the logarithm of the rates of the oxygen transfer step, the conversion of intermediate I to intermediate II, gives a straight line with a slope -0.5. Fluoride ion is a product of the enzymatic reaction when 2,3,5,6-tetrafluorophenol is the substrate. These results are consistent with an electrophilic substitution mechanism for oxygen transfer. The conversions of I to II and II to III are acid-catalyzed. A kinetic isotope effect of 8 was measured for the conversion of II to III using deuterated resorcinol as substrate. The conversion of III to oxidized enzyme is base-catalyzed, suggesting that the reaction depends on the removal of the flavin N(5) proton. Product release occurs at the same time as the formation of intermediate III, or rapidly thereafter. The results are interpreted according to the ring-opened model of Entsch et al. (Entsch, B., Ballou, D. P., and Massey, V. (1976) J. Biol. Chem. 251, 2550-2563).     

A Yersinia pseudotuberculosis protein which cross-reacts with HLA-B27

The most-debated question in the investigation of the spondyloarthropathies has been whether there is molecular mimicry between host HLA-B27 antigens and the arthritis-causing pathogens. We have generated a monoclonal anti-HLA-B27 antibody in our laboratory and have used a radioimmunoassay to screen a panel of bacterial species. Two strains of Yersinia pseudotuberculosis were found to be highly reactive. The cross-reactive Yersinia component was identified by Western blot to be a 19,000 component. A preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis chromatography apparatus was constructed to isolate milligram quantities of this component. To verify that the component carried the HLA-B27-specific epitope, rabbits were hyperimmunized with the purified materials. Affinity-purified antibodies from one of the immunized rabbits indeed carried anti-HLA-B27 activity. Last, antibodies generated against synthetic peptides derived from the HLA-B27.1 amino acid sequence were tested against the Yersinia component. Positive reactivity was found with antibodies generated against a peptide spanning residues 69-83 of the HLA-B27.1 protein. Since this resides in the segment responsible for the allotypic specificity of the antigen, these experiments establish the presence of molecular mimicry to a high degree of confidence.     

Escherichia coli RecBC pseudorevertants lacking chi recombinational hotspot activity

Pseudorevertants of an Escherichia coli exonuclease V (RecBC enzyme)-negative mutant have been isolated after ethyl methane sulfonate mutagenesis of a recC73 (presumed missense) mutant. The remedial mutations in each of the four pseudorevertants studied in detail map and complement as recC mutations. By several criteria, such as recombination proficiency, support of phage growth, RecBC nuclease activity, and cell viability, the pseudorevertants appear to have regained partially or completely various aspects of RecBC activity. However, chi recombinational hotspots, which stimulate exclusively the RecBC pathway of recombination, have no detectable activity in lambda vegetative crosses in the pseudorevertants. The properties of these mutants, in which the RecBC pathway of recombination is active yet in which chi is not active, are consistent with the hypothesis that wild-type RecBC enzyme directly interacts with chi sites; alternatively, the mutants may block or bypass the productive interaction of another recombinational enzyme with chi.     

Manganese-54 accumulation by Chlorella spp., Daphnia magna and yellow perch (Perca flavescens)

Concentration factor and biological half-life of ⁵⁴Mn were determined in three species representing an ecologically and economically important food chain. Green algae (Chlorella spp.), Daphnia magna and yellow perch (Perca flavescens) were exposed to ⁵⁴Mn in water and assayed for ⁵⁴Mn uptake. Steady state concentration factors computed from the laboratory data for algae, Daphnia and perch were 4230, 17 000 and 11, respectively. Respective biological half-lives were 1.6, 1.2 and 8.3 days.     

Coordinate regulation of ribosomal protein mRNA level in regenerating rat liver. Study with the corresponding mouse cloned cDNAs.

Cloned mouse ribosomal protein (rp) cDNAs exhibit extensive homology with the corresponding rat sequences. The size of the rp-mRNAs and complexity of the rp-genes are very similar in the two species. Using the mouse rp-recombinant DNAs we find that the relative abundance of rat L7, L13, L18, L30, L32/33 and S16 mRNAs increases after partial hepatectomy. Their maximal level is about twice that of normal rat liver, and is achieved 12-18 h after the operation, while the relative abundance of albumin mRNA decreases to half the normal values 12 h after partial hepatectomy. This concomitant increase in the relative content of these rp-mRNAs indicates coordinate regulation of their level in the rat. The dissimilar behavior of L10 and L19 rp-mRNA suggests additional control mechanisms of rp-mRNA levels in the regenerating rat liver.     

Experimental investigations in prolonged myocardial ischaemia. Note I. Biology of the myocardial fiber after transient myocardial fiber after transient myocardial ischaemia

The first ten days' evolution of post-ischaemic lesions of the premonitory or angina pectoris syndrome type was experimentally studied by the challenge of a short-term (10 and 15 min) ischaemia, of an adaptation to ischaemia and an adaptation followed by prolonged ischaemia (20 and 35 min). Worthy of note was the persistence of reversible lesions after short-term ischaemia and adaptation, and the progressive evolution towards cytolysis and cicatrization of some pancicellular foci after adaptation followed by prolonged ischaemia. The role of mitochondrial lesions, of lysosomal hydrolases, the inefficiency of renewed circulation, as well as problems of diagnosis are discussed.