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.     

Effects of okadaic acid on insulin-sensitive cAMP phosphodiesterase in rat adipocytes. Evidence that insulin may stimulate the enzyme by phosphorylation.

Okadaic acid, a potent inhibitor of Type 1 and Type 2A protein phosphatases, was used to investigate the mechanism of insulin action on membrane-bound low Km cAMP phosphodiesterase in rat adipocytes. Upon incubation of cells with 1 microM okadaic acid for 20 min, phosphodiesterase was stimulated 3.7- to 3.9-fold. This stimulation was larger than that elicited by insulin (2.5- to 3.0-fold). Although okadaic acid enhanced the effect of insulin, the maximum effects of the two agents were not additive. When cells were pretreated with 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), the level of phosphodiesterase stimulation by okadaic acid was rendered smaller, similar to that attained by insulin. In cells that had been treated with 2 mM KCN, okadaic acid (like insulin) failed to stimulate phosphodiesterase, suggesting that ATP was essential. Also, as reported previously, the effect of insulin on phosphodiesterase was reversed upon exposure of hormone-treated cells to KCN. This deactivation of previously-stimulated phosphodiesterase was blocked by okadaic acid, but not by insulin. The above KCN experiments were carried out with cells in which A-kinase activity was minimized by pretreatment with H-7. Okadaic acid mildly stimulated basal glucose transport and, at the same time, strongly inhibited the action of insulin thereon. It is suggested that insulin may stimulate phosphodiesterase by promoting its phosphorylation and that the hormonal effect may be reversed by a protein phosphatase which is sensitive to okadaic acid. The hypothetical protein kinase thought to be involved in the insulin-dependent stimulation of phosphodiesterase appears to be more H-7-resistant than A-kinase.     

Enzymatic desulfurization of dibenzothiophene by a cell-free system of Rhodococcus erythropolis D-1

Abstract Pseudomonas syringae cells were exposed to Cu²⁺ alone or in the precence of acetate, proline or cysteine, at concentrations that reduced free Cu²⁺ to 1/10 of the total copper. Ligand concentrations (designated as isoeffective) were determined experimentally using a Cu²⁺-selective electrode and confirmed by computer calculations using published stability constants. Exposure of P. syringae cells to Cu²⁺ alone resulted in rapid and pronounced cell death, and binding of most of the copper in solution. The addition of acetate, proline or cysteine, a few minutes after Cu²⁺ treatment, resulted in a significant reduction in cell death, and in the amount of copper bound to the cells. For short exposures to Cu²⁺, cysteine was more effective than acetate or proline, but after 60 min of treatment, similar results were observed with these ligands. The addition of ligands before Cu²⁺ resulted in even more reduced copper toxicity. The results showed that, at isoeffective concentrations, weak and moderate copper-ligands can effectively antagonize copper toxicity, and that this protective effect does not require previously equilibrated copper-ligand solutions and is not very dependent of the nature of the ligand.     

Functional significance of manganese catalase in Lactobacillus plantarum.

A strain of Lactobacillus plantarum which was unable to produce manganese (Mn)catalase (ATCC 8014) grew somewhat more rapidly and to a slightly higher plateau density than did an Mn catalase-positive strain (ATCC 14421), and this was the case during aerobic or anaerobic growth. However, when maintenance of viability was measured during the stationary phase of the growth cycle, the advantage provided by Mn catalase was obvious. Thus, the viability of ATCC 14431 was undiminished over 21 h of aerobic incubation, during the stationary phase, whereas that of ATCC 8014 decreased by seven orders of magnitude. Addition of catalase to the medium or growth in the presence of hemin, which allows catalase synthesis, protected ATCC 8014 against this loss of viability. Suppression of Mn catalase within ATCC 14431 by treatment with NH2OH caused the cells to lose viability when exposed to 4 mM H2O2.     

Cloning of human muscle phosphofructokinase cDNA

Three overlapping cDNA clones for human muscle phosphofructokinase (HMPFK) covering the complete coding sequence were isolated. The sequence included a poly(A) tail, a 399 bp 3'-untranslated region, a 2337 bp coding region for 779 amino acid residues and a part of the 5'-untranslated region. Homologies between HMPFK and rabbit muscle phosphofructokinase (RMPFK) were 96% of the amino acids and 89% of the nucleotides in the coding region. Like RMPFK, HMPFK also possessed the internal homology between C- and N-halves in its primary structure. Cloning of HMPFK cDNA will help to identify the molecular defect in patients with glycogenosis type VII (HMPFK deficiency).     

Hydrops fetalis caused by intrauterine human parvovirus infection

During a large outbreak of erythema infectiosum in 1987 in Toyama prefecture, Japan, a 32-year-old woman acquired a mild rash on her arms and legs at 18 weeks of gestation. At 26 weeks and 4 days of gestation, the fetus died by hydrops fetalis and pregnancy was terminated. Histological studies of the fetus revealed degeneration of erythroblastic cells in the liver and bone marrow. Extensive extramedullary hematopoiesis and hemosiderin deposits were observed in the liver. Antibody response to human parvovirus B19 virus was demonstrated in maternal sera by ELISA. Furthermore, dot hybridization with the molecularly cloned DNA probe revealed the presence of human parvovirus DNA sequence in the fetal liver, spleen, lung, kidney and placenta. This report describes the first case in Japan of hydrops fetalis caused by human parvovirus B19 infection.     

Insulin-like growth factor I rapidly stimulates tyrosine phosphorylation of a Mr 185,000 protein in intact cells

The type I insulin-like growth factor (IGF) receptor, like the insulin receptor, contains a ligand-stimulated protein-tyrosine kinase activity in its beta-subunit. However, in vivo, no substrates have been identified. We used anti-phosphotyrosine antibodies to identify phosphotyrosine-containing proteins which occur during IGF-I stimulation of normal rat kidney and Madin-Darby canine kidney cells labeled with ortho[32P]phosphate. Both cells provide a good system to study the function of the type I IGF receptors because they contain high concentrations of these receptors but no insulin receptors. In addition, physiological levels of IGF-I, but not insulin, stimulated DNA synthesis in growth-arrested cells. IGF-I stimulated within 1 min of tyrosine phosphorylation of two proteins. One of them, with a molecular mass between 97 and 102 kDa, was supposed to be the beta-subunit of the type I IGF receptor previously identified. The other protein had an approximate molecular mass of 185 kDa, which resembled, by several criteria, pp 185, originally identified during the initial response of Fao cells to insulin binding (White, M. F., Maron, R., and Kahn, C. R. (1985) Nature 318, 183-186). These data suggest that tyrosine phosphorylation of pp 185 may occur during activation of both the type I IGF receptor and the insulin receptor, and it could be a common substrate that transmits important metabolic signals during ligand binding.     

Reassessment of insulin effects on the Vmax and Km values of hexose transport in isolated rat epididymal adipocytes

Effects of insulin on the kinetic parameters of hexose transport in rat epididymal adipocytes were re-examined. The transport activity was assessed by measuring the rate of uptake of 3-O-[3H]methyl-D-glucose (MeGlc) under equilibrium exchange and zero-trans conditions. The incubation was carried out at 37 degrees C in an infant incubator. During the incubation, the cell suspension (25%, v/v, in a total volume of 48 microliter) was mechanically swirled at a rate of 600 rpm (r = 2 mm). The swirling facilitated the rapid uptake of MeGlc without stimulating the basal transport activity by "mechanical agitation". The basal and insulin-treated cells were incubated under identical conditions, except for the length of the incubation period. The incubation was terminated by the addition of 350 microliters of 1 mM phloretin, which inhibited transport in approximately 0.06 s. The time course of MeGlc uptake was consistent with the view that the process was a multiple-phase reaction. The initial phase of the reaction was completed when the intracellular distribution space of MeGlc was approximately 1% of the total cell volume. Insulin (10 nM) increased the Vmax value of MeGlc uptake 16-fold in equilibrium exchange experiments and 18-fold in zero-trans experiments. At the same time, the hormone decreased the Km value of MeGlc uptake from 11.7 to 5.4 mM in equilibrium exchange experiments and from 9.7 to 4.8 mM in zero-trans experiments. It is concluded that the major effect of insulin on MeGlc uptake is to increase the Vmax value, but the hormone has the additional effect of lowering the apparent Km value.