Release of fatty acid-binding protein from ischemic-reperfused rat heart and its prevention by mepacrine.

In an attempt to resolve the issue of whether there is a loss of fatty acid binding protein (H-FABP) from heart during ischemia and reperfusion, and to further examine the role of this protein in ischemic-reperfusion injury, the amount of H-FABP of heart was monitored during ischemia and reperfusion. Excellent correlation was obtained between the loss of H-FABP from heart and its appearance in the perfusate buffer when examined by Western blot using the specific antibody to H-FABP. Further quantitation was achieved by densitometric scanning of the Western blot and rocket electrophoresis. Maximum release of H-FABP was observed within 20 min of reperfusion, the total release being 10% of the H-FABP content of the heart. Mepacrine, a membrane stabilizer and a phospholipase inhibitor, reduced the release of H-FABP from the heart and prevented the accumulation of nonesterified fatty acids in the tissue during ischemia and reperfusion. In view of the established role of H-FABP in the preservation of membrane phospholipids by either scavenging free radicals during ischemia and reperfusion or by modulating the enzymes of phospholipid synthesis, it seems likely that the loss of H-FABP may have some contribution towards the ischemic-reperfusion injury.

Effect of iron limitation on Bacteroides gingivalis.

This study was undertaken to describe the effects of iron limitation on Bacteroides gingivalis. Four strains of B. gingivalis were grown in brain heart infusion broth, substituting protoporphyrin IX for hemin. Culture with protoporphyrin IX resulted in a loss of a 28 kDa membrane protein, but no decrease in growth. Iron-restricted cultural conditions for the growth of B. gingivalis were achieved using alpha/alpha'-dipyridyl, a ferrous iron chelator, at concentrations from 12.5 microM to 300 microM. Total suppression of bacterial growth for strain A7A1-28 and strain 381 was achieved at 200 microM alpha/alpha'-dipyridyl. At 300 microM alpha/alpha'-dipyridyl, strain W50 and Bowden 18/10 showed 100% and 80% suppression of growth, respectively. The ferric iron chelator Desferal did not show suppression of growth in concentrations up to 500 microM. The dipyridyl inhibition of cell growth for strain A7A1-28 could be reversed by adding excess ferrous ammonium sulphate but not by ferric nitrate. Iron regulation of proteolytic enzymes could not be demonstrated. Two new membrane proteins 42 kDa and 24 kDa are expressed with iron limitation, and the 45 kDa membrane protein was decreased with iron limitation.

Proteolytic activity in black-pigmented bacteroides species.

Black-pigmented Bacteroides species are frequently found in dentoalveolar abscesses. One general mechanism of bacterial virulence is the production of extracellular enzymes which degrade connective tissue or molecules associated with host defense. In this study the proteolytic activity of 18 bacterial strains from 9 black-pigmented Bacteroides species was examined. Bacteroides gingivalis degraded the greatest number of substrates studied and produced the highest levels of enzymatic activity. B. gingivalis was the only species that degraded collagen and produced high levels of enzymes that degraded N-benzoyl-DL-arginine (BANA) and N-CBz-glycyl-glycyl-arginine. Bacteroides intermedius degraded several substrates including PZ peptide. Bacteroides endodontalis produced enzymes that degraded beta-naphthylamide derivatives of glycylproline and glycylphenylalanine. There were considerable differences in enzyme production between strains of the same species. Such heterogeneity between strains in the production of proteolytic enzymes may be relevant to the in vivo infections produced in the host.

Purification of an 80,000-Mr glycylprolyl peptidase from Bacteroides gingivalis.

An enzyme from Bacteroides gingivalis SUNYAB A7A1-28 that hydrolyzes the synthetic peptide glycyl-L-proline 4-methoxy-beta-naphthylamide was purified 1,040-fold by urea extraction, gel filtration, ion-exchange chromatography, and fast protein liquid chromatography. The molecular weight of the enzyme was 80,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 75,000 as determined by gel filtration. The optimum pH for the hydrolysis of glycyl-L-proline 4-methoxy-beta-naphthylamide was 7.5 to 8.5. The enzyme activity was inhibited by the serine protease inhibitors diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride by 82.5 and 78%, respectively. The activity was also inhibited by Hg2+ (55.6%) and Zn2+ (45%).

Heart-reactive antibodies in rabbit anti-Streptococcus mutans sera fail to cross-react with Streptococcus mutans.

Immunization of rabbits with Streptococcus mutans antigens results in the production of serum antibodies that bind in vitro to human, rabbit, and monkey cardiac muscle. Antibodies to heart, however, have also been reported to occur at lower titers in the sera of unimmunized rabbits. In this study, the specificities of heart-reactive antibodies (HRA) in sera of unimmunized and S. mutans-immunized rabbits were compared using indirect immunofluorescence, Western blot, and Bio-Dot immunoassays. Both groups of sera gave striational indirect immunofluorescence-staining patterns on thin sections of native human and monkey cardiac muscle. Western blot analyses revealed that antibodies in normal sera bound 9 to 20 components of human, rabbit, and monkey heart. The major bands had Mr of 205,000, 160,000, 135,000, and 70,000. Several of the normal sera did not have antibody activity to S. mutans antigens, indicating that these HRA do not cross-react with these bacteria. Although immunization of rabbits with S. mutans caused increased titers of HRA (two to three doubling dilutions), Western blot assays using anti-S. mutans sera showed banding patterns qualitatively similar to those of normal sera on heart extracts. Antibodies to skeletal muscle myosin were detected in both serum groups. Of eighteen normal rabbit sera sixteen had antimyosin titers of 10 to 40, whereas all eighteen anti-S. mutans sera had titers of 10 to 160. Affinity-purified antimyosin antibodies isolated from anti-S. mutans serum did not bind to S. mutans components. Conversely, affinity-purified antibodies to S. mutans antigens did not bind to myosin or to other cardiac muscle components. Among these were antibodies to the 185-kDa cell wall protein (also known as B, I/II, IF, Spa A, and P1) previously believed to possess antigenic mimicry. HRA were removed from anti-S. mutans sera by absorption with S. mutans but this effect was not specific, because a non-cross-reactive internal standard antibody was also absorbed to the same extent. Because previous evidence for antigenic mimicry between S. mutans and cardiac muscle was based on serum cross-absorption experiments, this immunologic relationship is not substantiated. These results indicated that naturally occurring antibodies to cardiac muscle components are present in the sera of unimmunized rabbits and that immunization with S. mutans does not stimulate production of new heart-reactive antibody, but rather serves to boost antibody production by preexisting clones of self-reactive B-lymphocytes.

Binding of Streptococcus mutans antigens to heart and kidney basement membranes.

Using indirect immunofluorescence, alkali-extracted components of Streptococcus mutans were found to bind in vitro to capillary walls and sarcolemmal sheaths of monkey cardiac muscle and to glomerular and tubular basement membranes of monkey kidney. Adsorption of S. mutans components to tissue fragments was also detected by indirect radioimmunoassay and immunoblotting on nitrocellulose paper. Antibodies did not bind to untreated, control tissues in these experiments, proving that antigens shared by S. mutans and tissue components were not involved. Rabbit and monkey heart and kidney components bound S. mutans antigens of 24,000, 35,000, and 65,000 Mr. Monkey heart also bound molecules of 90,000 and 120,000 Mr. Rabbits immunized by intravenous injection of disrupted S. mutans cells developed severe nephritis that was characterized by the deposition of immunoglobulins, complement component C3, and S. mutans antigens in the glomeruli. Immunoglobulin G eluted from nephritic kidneys reacted in immunoblots with the 24,000, 35,000, and 65,000 Mr components of S. mutans extract, indicating that the antigens that bound to tissue in vitro also bound in vivo and reacted with antibodies in situ. Antibodies to other S. mutans antigens were not detected in the kidney eluate, although they were present in the serum of the same rabbit.

Comparative utilization of paraffins by a Trichosporon species.

Utilization of normal and isoparaffins, separately and in mixtures, by a Trichosporon sp. was investigated. From a mixture of normal paraffins and isoparaffins, the organism consumed straight-chain paraffins, leaving the branched paraffins relatively unchanged. When offered separately, the highest utilization of n-alkanes by the organism was obtained in the range of undecane to octadecane; n-pentadecane was poorly utilized. From a mixture of n-alkanes, the rate of consumption of shorter-chain alkanes, n-decane to n-dodecane, was found to be relatively faster and more uniform than that of longer-chain alkanes.