A novel class of microbial phosphocholine-specific phospholipases C

In this report we describe the 1,500-fold purification and characterization of the haemolytic phospholipase C (PLC) of Pseudomonas aeruginosa, the paradigm member of a novel PLC/phosphatase superfamily. Members include proteins from Mycobacterium tuberculosis, Bordetella spp., Francisella tularensis and Burkholderia pseudomallei. Purification involved overexpression of the plcHR1,2 operon, ion exchange chromatography and native preparative polyacrylamide gel electrophoresis. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry confirmed the presence of two proteins in the purified sample with sizes of 17,117.2 Da (PlcR2) and 78,417 Da (PlcH). Additionally, liquid chromatography electrospray mass spectrometry (LCMS) revealed that PlcH and PlcR2 are at a stoichiometry of 1 : 1. Western blot analysis demonstrated that the enzyme purifies as a heterodimeric complex, PlcHR2. PlcHR2 is only active on choline-containing phospholipids. It is equally active on phosphatidylcholine (PC) and sphingomyelin (SM) and is able to hydrolyse plasmenylcholine phospholipids (plasmalogens). Neither PlcHR2 nor the M. tuberculosis homologues are inhibited by D609 a widely used, competitive inhibitor of the Bacillus cereus PLC. PlcH, PlcR2, and the PlcHR2 complex bind calcium. While calcium has no detectable effect on enzymatic activity, it inhibits the haemolytic activity of PlcHR2. In addition to being required for the secretion of PlcH, the chaperone PlcR2 affects both the enzymatic and haemolytic properties of PlcH. Inclusive in these data is the conclusion that the members of this PC-PLC and phosphatase family possess a novel mechanism for the recognition and hydrolysis of their respective substrates.     

Atrial natriuretic factor as marker of myocardial compromise in Chagas' disease

This study investigated the evolution of plasma atrial natriuretic factor (ANF) in patients in different stages of Chagas' disease and analyzed its usefulness as prognostic factor of the development of myocardial compromise in asymptomatic chagasic patients. Chagas' disease, a determinant of heart failure, is caused by the parasite Trypanosoma cruzi. A total of 21 chagasic patients were studied: 9 in the asymptomatic stage, 6 with conduction defects (CD), and 6 with chronic heart failure (CHF); and 31 controls: 16 healthy, 6 with CD, and 9 with CHF. Plasma ANF radioimmunoassay (RIA) and complementary studies were performed twice for each patient, with an interval period of 12 months. First sample: chagasic patients showed higher ANF levels in the CHF group than in CD and asymptomatic subjects; second sample: the peptide levels were higher in CHF patients than in the asymptomatic group. In non-chagasic CHF patients, ANF levels were higher than in CD patients and controls in both samples. ANF levels were not able to differentiate chagasic asymptomatic and CD patients from healthy subjects and CD controls; meanwhile, chagasic CHF patients showed lower plasma ANF than their controls. Furthermore, ANF is a sensitive marker capable of detecting gradual impairments in cardiac function in all patients studied.     

Courtship and other behaviors affected by a heat-sensitive,molecularly novel mutation in the cacophony calcium-channel gene of Drosophila

The cacophony (cac) locus of Drosophila melanogaster, which encodes a calcium-channel subunit, has been mutated to cause courtship-song defects or abnormal responses to visual stimuli. However, the most recently isolated cac mutant was identified as an enhancer of a comatose mutation's effects on general locomotion. We analyzed the cac(TS2) mutation in terms of its intragenic molecular change and its effects on behaviors more complex than the fly's elementary ability to move. The molecular etiology of this mutation is a nucleotide substitution that causes a proline-to-serine change in a region of the polypeptide near its EF hand. Given that this motif is involved in channel inactivation, it was intriguing that cac(TS2) males generate song pulses containing larger-than-normal numbers of cycles--provided that such males are exposed to an elevated temperature. Similar treatments caused only mild visual-response abnormalities and generic locomotor sluggishness. These results are discussed in the context of calcium-channel functions that subserve certain behaviors and of defects exhibited by the original cacophony mutant. Despite its different kind of amino-acid substitution, compared with that of cac(TS2), cac(S) males sing abnormally in a manner that mimics the new mutant's heat-sensitive song anomaly.     

Prevalence of hepatitis C Virus infection among hemophiliacs in Central Brazil

In order to investigate the hepatitis C virus (HCV) infection prevalence and risk factors in hemophiliacs in Central Brazil, 90 patients were interviewed and serum samples tested for HCV RNA and anti-HCV antibodies. An overall prevalence of 63.3% (CI 95%: 53.0-72.7) was found. Multivariate analysis of risk factors showed that number of blood transfusions was significantly associated with this infection. Most hemophiliacs received locally produced cryoprecipitate. All infected patients were transfused before the screening of blood units for anti-HCV. However, hemophiliacs who received exclusively screened cryoprecipitate were HCV negative. It confirms the expected decline in transfusion-acquired hepatitis C.     

Some factors affecting the adherence of probiotic Propionibacterium acidipropionici CRL 1198 to intestinal epithelial cells

Adhesion to the intestinal mucosa is generally considered an important property of probiotic microorganisms and has been related to many of their health benefits. This study investigated some factors that could affect or be involved in the adherence of Propionibacterium acidipropionici CRL 1198, a dairy strain with suggested probiotic effects and high adherence in vitro and in vivo to intestinal epithelial cells. In vitro adhesion of propionibacteria was decreased by gastric digestion but not affected by bile and pancreatic enzymes. Adherence was also decreased by pretreatment of bacterial cells with protease, sodium metaperiodate, and trichloroacetic acid, revealing that different features of the cell surface, like protein factors, carbohydrates, and teichoic acids, are involved in the process. Adherence to intestinal epithelial cells was enhanced by calcium and was dependent on other divalent cations. Adhesion to intestinal mucus was also demonstrated. The results should explain the metabolic effects in the host previously obtained with this strain and support the potential of Propionibacterium for development of new probiotics.     

Engineering of coordinated up- and down-regulation of two glycosyltransferases of the O-glycosylation pathway in Chinese hamster ovary (CHO) cells

Production of O-linked oligosaccharides that interact with selectins to mediate cell-cell adhesion occurs in one segment of a branched glycan biosynthesis network. Prior efforts to direct the branched pathway towards selectin-binding oligosaccharides by amplifying enzymes in this branch of the network have had limited success, suggesting that metabolic engineering to simultaneously inhibit the competing pathway may also be required.We report here the partial cloning of the CMP-sialic, acid:Galbeta1,3GalNAcalpha2,3-sialyltransferase (ST3Gal I) gene from Chinese hamster ovary (CHO) cells and the simultaneous inhibition of expression of CHO cell ST3Gal I gene and overexpression of the human UDP-GlcNAc:Galbeta1,3GalNAc-R beta1,6-N-acetylglucosaminyltransferase (C2GnT) gene. A tetracycline-regulated system adjoined to tricistronic expression technology allowed "one-step" transient manipulation of multiple enzyme activities in the O-glycosylation pathway of a previously established CHO cell line already engineered to express alpha1,3-fucosyltransferase VI (alpha1,3-Fuc-TVI). Tetracycline-regulated co-expression of a ST3Gal I fragment, cloned in the antisense orientation, and of C2GnT cDNA resulted in inhibition of the ST3Gal I enzymatic activity and increase in C2GnT activity which varied depending on the extent of tetracycline reduction in the cell culture medium. This simultaneous regulated inhibition and activation of the two key enzyme activities in the O-glycosylation pathway of mammalian cells is an important addition to the metabolic engineering field.     

Human infections by black yeasts of genus Exophiala

Clinical and laboratory aspects of the infections caused by Exophiala species are reviewed with regard to its epidemiology, diagnosis and treatment. Exophiala is a genus of dematiaceous hyphomycetes whose taxonomy and nomenclature undergo constant revision. Exophiala species are widely distributed in nature, and they are uncommon human pathogens. In recent years it appears to have increased its frequency as a cause of human infections, mainly in immunocompromised patients. They have been associated with phaeohyphomycosis, chromoblastomycosis, eumycotic mycetoma and disseminated infection. The procedures recommended for diagnosis consist of detection of fungal elements in tissue and growth of the organism in culture. Identification is mostly based upon microscopic observation of morphological characteristics and conidiogenesis, combined with the evaluation of physiological tests and nitrate and carbohydrate assimilations. Antifungal agents such as amphotericin B, itraconazole and voriconazole showed in vitro activity to most of the Exophiala species of clinical interest. The therapeutic recommendations are mainly deduced from the observation of single cases.