- Volume 133, Issue 10, 1987

SUMMARY: A bacterial immunopotentiator, LC 9018 (heat-killed Lactobacillus casei), was studied for its protective and therapeutic efficacies against Mycobacterium fortuitum and M. chelonae infections in mice. This agent reduced the incidence of spinning disease and gross renal lesions and enhanced the elimination of organisms at the site of infection in the host mice, when administered intramuscularly six times a week (0.1 mg dry weight per injection, one injection on each day of treatment) from 1 week before to 2 weeks after infection. The LC 9018 injections in this protocol caused a marked increase in the phagocytic function, OF-producing ability and chemiluminescence of host peritoneal macrophages. Moreover, LC 9018 injections using the same schedule resulted in an enhancement of interleukin-1 -producing function of the macrophages, particularly in the infected mice. These findings indicate that LC 9018 administration with the present protocol can activate macrophage functions, in particular those related to microbicidal activity. This would partly explain the protective and therapeutic efficacy of LC 9018 against infection due to M. fortuitum complex.

SUMMARY: The peptide composition and antigenic cross-reactivity of partially purified and concentrated haemolysins of 16 strains of Vibrio vulnificus were examined by SDS-PAGE and immunoblotting analysis, using a monoclonal antibody (MAb), 6F8D, raised against the haemolysin. All strains produced a common peptide of 36 kDa and the MAb reacted with this peptide. In some strains, larger molecules, including a 56 kDa peptide, were produced, but the MAb did not react with this peptide. The haemolytic activity of the strains was effectively neutralized by the MAb, except in the case of strains producing the 56 kDa peptide. These findings indicate that the 36 kDa haemolysin is common to all 16 strains and that V. vulnificus can produce a second haemolysin which differs in molecular mass and antigenicity.

SUMMARY: The haemolytic action of Vibrio vulnificus haemolysin (VVH) was compared to that of streptolysin O (SLO). Both were cholesterol-binding haemolysins, but differed in the release of haemoglobin (Hb). In the first step of haemolysis, the haemolysins were temperature-independently bound to the cholesterol site on the target erythrocyte membrane. This was followed by the rapid release of K+, which is an intra-erythrocyte marker. Hb was then released, in different ways. In the case of VVH, Hb was released slowly after a relatively long lag, whereas with SLO, Hb was released as rapidly as K+. Haemolysis by VVH was inhibited by the addition of 30 mhl-dextran 4 (mean Mr 4000), which is considered to be an effective colloid-osmotic protectant. The results therefore indicated that haemolysis by VVH (like that by Escherichia coli α-haemolysin and Staphylococcus aureus α-toxin) was caused by a colloid-osmotic mechanism. Both K+ and Hb release caused by VVH proceeded temperature-dependently, and the membrane fluidity of liposomes prepared with lipids extracted from sheep red blood cell membranes increased above 20°C. These results suggest that the temperature-dependence of the haemolysis by VVH is due to the requirement for an increase in the membrane fluidity during the formation of a transmembrane pore.

SUMMARY: Two mutant strains of Escherichia coli K12 Doc-S resistant to the uncoupling agents 4,5,6,7- tetrachloro-2-trifluoromethyl benzimidazole and carbonyl cyanide m-cblorophenylhydrazone were isolated. These strains, designated TUV and CUV, were capable of (a) growth, (h) the transport of succinate and L-proline and (c) electron-transport-linked oxidative synthesis of ATP in the presence of titres of uncoupler which inhibited these processes in strain Doc-S. The inhibition of transport of L-proline by a fixed titre of uncoupler was sharply pH dependent in strain Doc-S: uptake was unaffected at pH 7.6 but completely inhibited at pH 5.6. This pH dependence was not shown by the resistant strains. We believe that uncouplers were equally accessible to their site(s) of action in the energy-conserving membrane of the sensitive and resistant strains. We conclude that uncoupler resistance in these strains of E. coli has arisen as a consequence of mutations which directly affect a specific site of uncoupler action within the cytoplasmic membrane, rather than as a consequence of a decrease in the permeability of cells to uncoupler.

SUMMARY: Homoserine strongly inhibited growth of Mycohacterium smegmatis in medium containing glutamate as the sole source of nitrogen but was without effect when asparagine, alanine or glutamine was the sole nitrogen source. It was readily taken up by glutamate-grown cells, reaching an intracellular concentration of over 20 mM after 4 h incubation. The primary site of action of homoserine was deduced to be the non-competitive inhibition of glutamate transport.

SUMMARY: Streptomyces griseus strain 13189 produced a large number of submerged spores in response to nitrogen-mediated nutritional shift-down. The pool size of ppGpp increased immediately after shift-down and was accompanied by a dramatic decrease in the GTP pool. In contrast, nutritional shift-down of a relaxed (rel) mutant derived from strain 13189 resulted in a slight increase in ppGpp pool size, a less extensive decrease in GTP pool size and spore titres 10-fold less than produced by the parental strain. Addition of decoyinine, a specific inhibitor of GMP synthetase, to the re1 mutant restored the sporulation frequency to the parental level. IMP dehydrogenase was competitively inhibited by ppGpp with a Ki of 0-05 mM, whereas other nucleotides were less effective inhibitors. The observed intracellular concentration of ppGpp during shift-down was sufficient to inhibit IMP dehydrogenase activity and to result in the decrease in the GTP pool. These results demonstrate that sporulation of S. griseus, like that of Bacillus subtilis, is nutrient-dependent and that the decrease in GTP content caused by the stringent response (ppGpp) is correlated with initiation of submerged spore formation due to amino acid starvation.

SUMMARY: Undercooling and ice nucleation in yeast cells exposed to increasing hypertonic polyvinyl-pyrrolidone (PVP) concentrations were measured. Ice nucleation rates were analysed in terms of classical nucleation theory. Contrary to earlier reports, nucleation was found to be of the catalysed type, by active catalytic sites within the cell. With increasing PVP concentration, the nucleation temperature tends to a limiting value (approximately 236 K), whereas the homogeneous nucleation temperature of the extracellular PVP solution continues to decrease with increasing PVP concentration. The calculated parameters in the nucleation equation indicate that the nucleation mechanism within the cell is unaffected by hypertonic stress. The experimentally determined freezing kinetics of undercooled water (in oil emulsions) were found to parallel closely previously reported death rates of yeast cells as a function of temperature. The observed kinetics are compatible with a slow crystallization or precipitation of emulsifying agent at the oil/water interface, to yield catalytic sites capable of promoting ice nucleation. Experiments with water- (or yeast)-in-oil dispersions containing no emulsifying agents led to long-term freezing resistance and high recoveries of viable cells.

SUMMARY: During apical extension an ion current of about 0.2 μA cm−2 flows into the hyphal tips of Neurospora crassa and out from the distal regions of the hyphae. This current is carried principally by protons and requires phosphate and glucose in the growth medium. Exogenous calcium ions were required for tip extension but not for the current. A permeable pH sensitive dye, bromocresol green, provided evidence that the cytoplasm at sites of proton current entry was made acidic in comparison with the subapical cytoplasm. In a few cases non-growing hyphae were found with a normal current and growing hyphae were found with outward current at the apex. These current patterns, although rare, indicate there is no tight correlation between the vectorial flow of electrical current and hyphal extension in this organism.

SUMMARY: Trypsin-like enzyme activity in spent culture media from 3-d-old batch cultures of Bacteroides gingivalis W50 was measured by using the hydrolysis of Nα-benzoyl-L-arginine-p-nitroanilide. The cell-free culture medium was fractionated by differential centrifugation at 10000 g and 75000 g, yielding two particulate fractions and a soluble supernatant fraction. About 80% of the total recoverable activity was associated with the particulate fractions, the remainder being in the supernatant. Electron microscopy of ruthenium-red/osmium stained ultrathin sections of the pellet fractions showed them to be composed of vesicular particles (extracellular vesicles), between 50 and 250 nm in diameter. Enzyme activity in all three fractions was enhanced by dithiothreitol. Gel-permeation chromatography of the soluble fraction yielded one peak of activity which contained 64 kDa and 58 kDa polypeptides. Enzyme activity from the vesicular fractions could be solubilized by sonication, giving a similar chromatographic profile to the supernatant fraction. The main peak of activity was composed of 64 kDa and 58 kDa polypeptides. In addition, there was a higher molecular mass enzyme activity peak composed of the 64 kDa and 58 kDa components along with 111 kDa, 93 kDa and 70 kDa polypeptides. We conclude that the trypsin-like enzyme of B. gingivalis is released as a soluble protein and is also associated with extracellular vesicles, in which it may exist as a soluble component and also as a protein complex.