Adherence of Brucella to human epithelial cells and macrophages is mediated by sialic acid residues

The basis for the interaction of Brucella species with the surface of epithelial cells before migration in the host within polymorphonuclear leucocytes is largely unknown. Here, we studied the ability of Brucella abortus and Brucella melitensis to adhere to cultured epithelial (HeLa and HEp-2) cells and THP-1-derived macrophages, and to bind extracellular matrix proteins (ECM). The brucellae adhered to epithelial cells forming localized bacterial microcolonies on the cell surface, and this process was inhibited significantly by pretreatment of epithelial cells with neuraminidase and sodium periodate and by preincubation of the bacteria with heparan sulphate and N-acetylneuraminic acid. Trypsinization of epithelial cells yielded increased adherence, suggesting unmasking of target sites on host cells. Notably, the brucellae also adhered to cultured THP-1 cells, and this event was greatly reduced upon removal of sialic acid residues from these cells with neuraminidase. B. abortus bound in a dose-dependent manner to immobilized fibronectin and vitronectin and, to a lesser extent, to chondroitin sulphate, collagen and laminin. In sum, our data strongly suggest that the adherence mechanism of brucellae to epithelial cells and macrophages is mediated by cellular receptors containing sialic acid and sulphated residues. The recognition of ECM (fibronectin and vitronectin) by the brucellae may represent a mechanism for spread within the host tissues. These are novel findings that offer new insights into understanding the interplay between Brucella and host cells.     

Complete release of adenosine deaminase from mouse lymphocytes stabilized by low-pH acetate

Complete release of adenosine deaminase from mouse lymphocytes takes place when intact cells are stabilized by low-pH acetate buffer. Both the low pH and the acetate affect the enzyme extraction markedly. At pH 5.0 all the adenosine deaminase activity detectable in the whole cell homogenates is released into the acetate buffer in very few minutes, with a total amount of 2% protein being extracted. The complete extraction of the enzyme activity is never observed when, at pH 5.0, the acetate is replaced by glutamate, citrate, succinate or maleate and only 45% and 15% of the adenosine deaminase activity is extracted by the acetate at pH 6.0 and 7.0, respectively. The breakdown of adenosine by the enzyme activity extracted from the stabilized cells is due to deamination alone, since inosine is the only product of the catalyzed reaction and its formation is completely inhibited by coformycin, a selective inhibitor of adenosine deaminase. The enzyme extracted shows a specific activity 50-times higher than that found in the crude homogenates, and a substantial purification of the enzyme extracted is achieved by a single Sephadex G-100 gel filtration.     

Studies on the light-harvesting complexes from the thermotolerant purple bacterium Rhodopseudomonas cryptolactis

The antenna complexes from Rps. cryptolactis have been isolated and purified. Rps. cryptolactis contains two types of variable antenna complex, B800-850 and B800-820 as well as the core B875 antenna complex. The variable antenna complexes contain more than two types of antenna apoprotein, and have a Bchla:carotenoid ratio of 2:1. They can both be crystallised, but the B800-820 complex is the easiest with which to get relatively large single 3-D crystals (up to 0.5 mm in each dimension).     

Interaction of Staphylococcal α-Toxin with Artificial and Natural Membranes

Comparison of hemolytic activity and chromate-releasing activity of partially purified preparations of staphylococcal alpha-toxin indicated the presence of a lytic factor other than alpha-toxin. This lytic release factor (RF) was isolated from the preparations and was shown to be active against both lipid spherules and erythrocytes. Heat-purified alpha-toxin (HP alpha-toxin) disrupted spherules, with the formation of fragments which always showed the presence of ring structures similar in dimensions (ca. 90 A) to pure alpha 12S-toxin. The interaction of HP alpha-toxin with spherules was accompanied by loss of hemolytic activity and adsorption of toxic protein. The alpha 12S-toxin, although only weakly hemolytic, was shown to be lytic for spherules. An alpha 12S-free toxin rapidly disrupted spherules, with formation of fragments with attached rings similar in dimensions to the alpha 12S molecule. Lipid monolayer experiments showed that HP alpha-toxin could penetrate lipid monolayers by virtue of a hydrophobic interaction. Effects of HP alpha-toxin on rabbit and human erythrocyte ghosts were similar to its effects on spherules, in that rings appeared on membrane fragments. Toxin-lysed rabbit erythrocytes showed similar rings on the resulting membrane fragments. However, rings were not seen on toxin-treated rabbit erythrocytes in the prelytic lag phase; this result and the fact that human erythrocytes are largely insensitive to alpha-toxin were interpreted as evidence against a lytic mechanism involving ring formation as the primary event. Rings were interpreted as toxin polymers similar to alpha 12S molecules, formed from specifically orientated active toxin molecules at the surface of lipid structures. Possible mechanisms for toxin lysis of spherules and erythrocytes are discussed.     

Screening for ethanol-producing filamentous fungi

Of nineteen Aspergilli and ten Rhizopus strains examined for their ability to ferment simple sugars (glucose, xylose, and arabinose) as well as complex substrates (cellulose, oat-spelt xylan, corn fiber, and corn germ pressing), three Rhizopus strains were identified that could produce more than 31 g ethanol/l under anaerobic stress. By 72 h, glucose , xylose, cellobiose, and corn fiber were fermented with perspective yields of 100, 47, 80, and 40 percent, of theoretical.     

Nα-formyl-norleucyl-leucyl-phenylalanyl chloromethylketone

Summary N-formyl-norleucyl-leucyl-phenylalanine-chloromethyl ketone is chemotactic for, and induces lysosomal enzyme release from rabbit peritoneal neutrophils over essentially the same range of concentrations as does the free acid form of the same peptide (Na-formyl-norleucyl-leucyl-phenylalanine-OH). The chloromethyl ketone derivative does however differ from the free acid in respect to its ability to interact with the neutrophil and cause deactivation or desensitization to cytochalasin B. Neutrophils preincubated in the cold with the chloromethyl ketone followed by washing have cytochalasin B sensitivity conferred upon them, as measured by the release of lysosomal enzymes. The degree of release induced by this pre-treatment appears to be related to the initial responsiveness of the cells. This is in contrast to the free acid where no cytochalasin B sensitivity in conferred under any circumstances. Thus, the chloromethyl ketone, unlike the free acid, appears to irreversibly activate the cell. Desensitization to the late addition of cytochalasin B is also significantly retarded when the chloromethyl ketone derivative is compared to the free acid form of the peptide. These studies suggest that the chloromethyl ketone derivative of the peptide may covalently interact with the neutrophil receptor.     

Biodegradation of Pinus radiata softwood by white- and brown-rot fungi

The weight and component losses of Pinus radiata wood after decay by six species of white-rot and two species of brown-rot fungi for periods varying from 30 to 360 days were evaluated. Three groups of decayed wood samples were identified based on the principal component analysis (PCA) of the data on their weight and component losses. Selective lignin degradation was produced by Ceriporiopsis subvermispora and Punctularia atropurpurascens within different periods, the longest one lasting 90 days, and also by Merulius tremellosus after 90 days of biodegradation. Comparing the data on biodegradation of P. radiata by Trametes versicolor with the ones reported for biodegradation of Eucalyptus globulus and E. grandis indicated that P. radiata is as susceptible to wood decay by this white-rot fungus as the two types of hardwood.     

Evaluation of a combined brown rot decay–chemical delignification process as a pretreatment for bioethanol production from Pinus radiata wood chips

Wood chips of Pinus radiata softwood were biotreated with the brown rot fungus (BRF) Gloeophyllum trabeum for periods from 4 and 12 weeks. Biodegradation by BRF leads to an increase in cellulose depolymerization with increasing incubation time. As a result, the intrinsic viscosity of holocellulose decreased from 1,487 cm³/g in control samples to 783 and 600 cm³/g in 4- and 12-week decayed wood chips, respectively. Wood weight and glucan losses varied from 6 to 14% and 9 to 21%, respectively. Undecayed and 4-week decayed wood chips were delignified by alkaline (NaOH solution) or organosolv (ethanol/water) processes to produced cellulosic pulps. For both process, pulp yield was 5–10% lower for decayed samples than for control pulps. However, organosolv bio-pulps presented low residual lignin amount and high glucan retention. Chemical pulps and milled wood from undecayed and 4-week decayed wood chips were pre-saccharified with cellulases for 24 h at 50°C followed by simultaneous saccharification and fermentation (SSF) with the yeast Saccharomyces cerevisiae IR2-9a at 40°C for 96 h for bioethanol production. Considering glucan losses during wood decay and conversion yields from chemical pulping and SSF processes, no gains in ethanol production were obtained from the combination of BRF with alkaline delignification; however, the combination of BRF and organosolv processes resulted in a calculated production of 210 mL ethanol/kg wood or 72% of the maximum theoretically possible from that pretreatment, which was the best result obtained in the present study.