Serum High-density Lipoprotein (hdl) Inhibits In Vitro Enterohemolysin (ehly) Activity Produced By Enteropathogenic Escherichia Coli.

Enterohemolysin (EHly) produced by Escherichia coli shows hemolytic activity towards washed erythrocytes from different animal species on blood agar plates. It has been shown recently that EHly activity is inhibited by normal mammalian serum and by cholesterol in vitro. Plasma lipoproteins can interact with bacterial toxins, such as endotoxin, to reduce their toxicity. In this work, we examine the ability of human purified chylomicrons, very low-density lipoproteins, intermediate-density, low-density and high-density lipoproteins, to inhibit the hemolytic activity of EHly. Our results show that these lipoproteins are hemolysin inactivators, and that high-density lipoprotein is the most potent inhibitor of enterohemolytic activity.3853-

A New Member Of The Aldo-keto Reductase Family From The Plant Pathogen Xylella Fastidiosa.

The Xylella fastidiosa genome program generated a large number of gene sequences that belong to pathogenicity, virulence and adaptation categories from this important plant pathogen. One of these genes (XF1729) encodes a protein similar to a superfamily of aldo-keto reductase together with a number of structurally and functionally related NADPH-dependent oxidoreductases. In this work, the similar sequence XF1729 from X. fastidiosa was cloned onto the pET32Xa/LIC vector in order to overexpress a recombinant His-tag fusion protein in Escherichia coli BL21(DE3). The expressed protein in the soluble fraction was purified by immobilized metal affinity chromatography (agarose-IDA-Ni resin). Secondary structure contents were verified by circular dichroism spectroscopy. Small angle X-ray scattering (SAXS) measurements furnish general structural parameters and provide a strong indication that the protein has a monomeric form in solution. Also, ab initio calculations show that the protein has some similarities with a previously crystallized aldo-keto reductase protein. The recombinant XF1729 purified to homogeneity catalyzed the reduction of dl-glyceraldehyde (K(cat) 2.26s(-1), Km 8.20+/-0.98 mM) and 2-nitrobenzaldehyde (K(cat) 11.74 s(-1), Km 0.14+/-0.04 mM) in the presence of NADPH. The amino acid sequence deduced from XF1729 showed the highest identity (40% or higher) with several functional unknown proteins. Among the identified AKRs, we found approximately 29% of identity with YakC (AKR13), 30 and 28% with AKR11A and AKR11B, respectively. The results establish XF1729 as the new member of AKR family, AKR13B1. Finally, the first characterization by gel filtration chromatography assays indicates that the protein has an elongated shape, which generates an apparent higher molecular weight. The study of this protein is an effort to fight X. fastidiosa, which causes tremendous losses in many economically important plants.453143-5

Isolation and characterization of selenate resistant mutants of Acremonium chrysogenum

Mutants unable to convert exogenous sulfate to sulfite were isolated using the toxic analogue selenate. Three of twenty-eight isolated mutants were chromate sensitive. They showed a possible lesion in the gene that codes the ATP sulfurylase. The others were chromate resistant, and probably had a lesion in one or both of the genes that code the sulfate permease. Methionine increased the resistance levels to selenate. In addition, the frequency of spontaneous mutants obtained in a medium containing methionine was higher (between 2.4 x 10-6 and 18.0 x 10-6) than that obtained using a medium without any intentional source of sulfur (between 0.7 x 10-6 and 5.0 x 10-6). The original strain, as well as the mutants, were able to grow in a sulfur-free liquid medium even after 4 consecutive inoculation procedures. These results indicated the existence of sulfur traces in the medium and/or an efficient intracellular storage system. There was no significant difference between cephalosporin C production in mutants and the original strain.<br>Mutantes incapazes de converter o sulfato extracelular em sulfito foram isolados utilizando o análogo tóxico selenato. De 28 mutantes isolados, apenas 3 foram sensíveis ao cromato, provavelmente apresentando lesão no gene que codifica a ATP sulfurilase. Os demais foram resistentes ao cromato e devem conter lesão no gene sB ou também no gene sC. A metionina elevou os níveis de resistência ao selenato e a freqüência de mutantes espontâneos obtida em meio contendo este aminoácido foi maior (entre 2,42 x 10-6 e 18,04 x 10-6) do que a obtida no meio sem a adição de qualquer fonte intencional de enxofre (entre 0,71 x 10-6 e 5,0 x 10-6). A linhagem original e os mutantes foram capazes de crescer, mesmo depois de quatro etapas de inóculo, fato que pode ser explicado pela existência de traços do referido elemento no meio e/ou a presença de um sistema eficiente de estocagem intracelular. A produção de cefalosporina C foi estudada e a análise dos dados revelou que não houve diferença significativa entre os níveis produzidos pelos mutantes e os produzidos pela linhagem original