Microheterogeneity in 16S ribosomal DNA-defined bacterial populations from a stratified planktonic environment is related to temporal changes and to ecological adaptations

Temporal changes of the bacterioplankton from a meromictic lake (Lake Vilar, Banyoles, Spain) were analyzed with four culture-independent techniques: epifluorescence microscopy, PCR-denaturing gradient gel electrophoresis (DGGE) fingerprinting, fluorescence in situ whole-cell hybridization and flow cytometry sorting. Microscopically, blooms of one cyanobacterium (Synechococcus sp.-like), one green sulfur bacterium (Chlorobium phaeobacteroides-like), and one purple sulfur bacterium (Thiocystis minor-like) were observed at different depths and times. DGGE retrieved these populations and, additionally, populations related to the Cytophaga-Flavobacterium-Bacteroides phylum as predominant community members. The analyses of partial 16S ribosomal DNA sequences from the DGGE fingerprints (550 bp analyzed) revealed higher genetic diversity than expected from microscopic observation for most of these groups. Thus, the sequences of two Synechococcus spp. (both had a similarity of 97% to Synechococcus sp. strain PCC6307 in 16S rRNA), two Thiocystis spp. (similarities to Thiocystis minor of 93 and 94%, respectively), and three Cytophaga spp. (similarities to Cytophaga fermentans of 88 and 89% and to Cytophaga sp. of 93%, respectively) were obtained. The two populations of Synechococcus exhibited different pigment compositions and temporal distributions and their 16S rRNA sequences were 97.3% similar. The two Thiocystis populations differed neither in pigment composition nor in morphology, but their 16S rRNA sequences were only 92.3% similar and they also showed different distributions over time. Finally, two of the Cytophaga spp. showed 96.2% similarity between the 16S rRNA sequences, but one of them was found to be mostly attached to particles and only in winter. Thus, the identity of the main populations changed over time, but the function of the microbial guilds was maintained. Our data showed that temporal shifts in the identity of the predominant population is a new explanation for the environmental 16S rRNA microdiversity retrieved from microbial assemblages and support the hypothesis that clusters of closely related 16S rRNA environmental sequences may actually represent numerous closely related, yet ecologically distinct, populations.     

Frequency of Strongyloides stercoralis infection in alcoholics

Several studies have shown that chronic alcoholics have increased susceptibility to infections due to higher exposure to infectious agents as well as breakdown in their immune defenses. As Strongyloides stercoralis infection is usually more relevant in immunocompromised patients, the aim of this study was to evaluate the frequency of S. stercoralis infection in alcoholics. Thus, coproparasitological examination was carried out in 145 subjects, from which 45 were chronic alcoholics (mean age of 45.7 +/- 11.0 years), 10 were nonalcoholic cirrhotic patients (mean age of 50.2 +/- 13.1 years), and 90 were asymptomatic nonalcoholic subjects (mean age of 46.7 +/- 10.1 years), which served as controls. From the alcoholics, 9 had hepatic cirrhosis, 9 had chronic pancreatitis and 27 had neither cirrhosis nor pancreatitis. For the diagnosis of strongyloidiasis, the Baermann-Moraes and Lutz methods were used in three fecal samples from each subject. Samples were collected at alternated days, and three slides of each sample were analyzed for each method, thus totalizing 2,610 slides examined. The frequency of strongloidiasis in the total alcoholic group (33.3%) and in the subgroups of alcoholics, i.e., patients with hepatic cirrhosis (44.4%), with chronic pancreatitis (33.3%), and those with no cirrhosis or pancreatitis (29.6%) was statistically higher than that found in the control group (5.5%). None of the individuals with nonalcoholic hepatic cirrhosis had S. stercoralis infection. Our results showed that the chronic alcoholism itself is an important factor that predisposes to strongyloidiasis.     

In vitro and in vivo anti-Trypanosoma cruzi activity of a novel nitro-derivative

Nitroarylidenemalononitriles and their cyanoacetamide derivatives with remarkable anti-epimastigote properties, were synthesized attempting to obtain new 3,5-diamino-4-(5'-nitroarylidene)-4H-thiadiazine 1,1-dioxide derivatives, which in previous reports had shown anti-Trypanosoma cruzi activity. Tests to evaluate the cytotoxicity of compounds were performed on J774 macrophages. 5-nitro-2-thienyl-malononitrile (5NO2TM), was the only product which maintained a high anti-epimastigote activity at concentrations in which it was no longer cytotoxic, thus it was assayed against intracellular amastigotes. Its anti-amastigote activity was similar to that of nifurtimox. Afterwards in vivo toxicity and anti-chagasic activity were determined. A reduction in parasitemia was observed.     

Hantavirus pulmonary syndrome in Uberaba,Minas Gerais,Brazil

This report describes the epidemiological and clinical-evolutive characteristics of eight patients with hantavirus pulmonary syndrome (HPS) in Uberaba, Minas Gerais, Brazil. A positive history of contact with rodents was present in 100% of the cases. The time between the onset of symptoms and hospital care was, on average, 3.6 days. All patients showed clinical and laboratory findings suggestive of HPS. Elevated urea and creatinine levels were observed in 6 (75%) cases, PO2 was < 60 mmHg in 100% of the cases, and a chest X-ray demonstrated a bilateral interstitial-alveolar infiltrate. The diagnosis was confirmed by the detection of IgM antibodies against Sin Nombre virus by ELISA. Three patients died as a direct consequence of HPS.     

MICA-A5.1 allele is associated with atypical forms of celiac disease in HLA-DQ2-negative patients

We selected 38 consecutive celiac disease (CD) patients (from a group of 316 consecutive CD patients) and 91 healthy blood donors, all of whom were HLA-DQ2 (DQA1*0501/DQB1*0201) negative, and investigated the presence of the classically associated alleles HLA-DQ8 and HLA-DRB4. We also studied the distribution of MICA transmembrane alleles in the two clinical forms of the disease. For this reason, these 38 DQ2-negative patients were subdivided into two groups: 18 typical CD patients and 20 atypical CD patients. No differences were found in the distribution of the DRB4 allele between DQ2-negative patients and controls. The HLA-DQ8 heterodimer (DQA1*03xx/DQB1*0302) was increased in CD patients (29%) compared with controls (10%), but no statistical differences were found. No differences were observed in the frequency of these alleles between either group of CD DQ2-negative patients. MICA-A5.1 was increased in atypical CD patients when compared with the typical forms of disease ( P(c)=0.03) and with healthy controls (P(c)=0.002). No other MICA allele was found to be significantly increased in the groups under study. The presence of MICA-A5.1 in atypical CD DQ2-negative patients may indicate a possible role of this allele in the development of CD.     

Identification,cloning, and sequencing of different cytokine genes in four species of owl monkey

Non-human primates could prove to be suitable models for the study of infectious diseases such as malaria, tuberculosis, and hepatitis; the molecules of their immune systems are in the process of being fully characterized. Due to the relevance of cytokines in the modulation of the immune response, a molecular analysis of these proteins in non-human primates from the Aotus genus was carried out. Peripheral blood mononuclear cells from four species of Aotusmonkey were obtained and their mRNAs for interleukin-2 (IL-2), IL-4, IL-6, IL-10, interferon-gamma (IFN), and tumor necrosis factor (TNF)-alpha were characterized. This study shows a high degree of conservation between nucleotide and amino acid sequences of cytokines from different Aotus species and those from humans. The TNF-alpha molecules were identical in amino acid sequences for both.     

Lipase-catalyzed glycerolysis of an oil rich in eicosapentaenoic acid residues

The activities of four immobilized lipases for glycerolysis of a commercially available fish oil (TG500) rich in eicosapentaenoic residues (>58%, w/w) have been characterized in solvent-free systems. The effects of the mole ratio of TG500 to glycerol and temperature have been investigated. The highest conversion was obtained at 60°C with a Candida antarctica fraction B lipase (Chirazyme L-2) and a mole ratio of TG500 (based on fatty acid equivalents) to glycerol of 1.5 to 1.     

Hepatic lipase: structure/function relationship,synthesis, and regulation

Hepatic lipase (HL) is a lipolytic enzyme, synthesized by hepatocytes and found localized at the surface of liver sinusoid capillaries. In humans, the enzyme is mostly bound onto heparan-sulfate proteoglycans at the surface of hepatocytes and also of sinusoid endothelial cells. HL shares a number of functional domains with lipoprotein lipase and with other members of the lipase gene family. It is a secreted glycoprotein, and remodelling of the N-linked oligosaccharides appears to be crucial for the secretion process, rather than for the acquisition of the catalytic activity. HL is also present in adrenals and ovaries, where it might promote delivery of lipoprotein cholesterol for steroidogenesis. However, evidence of a local synthesis is still controversial. HL activity is fairly regulated according to the cell cholesterol content and to the hormonal status. Coordinate regulations have been reported for both HL and the scavenger-receptor B-I, suggesting complementary roles in cholesterol metabolism. However, genetic variants largely contribute to HL variability and their possible impact in the development of a dyslipidemic phenotype, or in a context of insulin-resistance, is discussed.     

PPARalpha suppresses insulin secretion and induces UCP2 in insulinoma cells

Fatty acids may promote type 2 diabetes by altering insulin secretion from pancreatic beta cells, a process known as lipotoxicity. The underlying mechanisms are poorly understood. To test the hypothesis that peroxisome proliferator-activated receptor alpha (PPARalpha) has a direct effect on islet function, we treated INS-1 cells, an insulinoma cell line, with a PPARalpha adenovirus (AdPPARalpha) as well as the PPARalpha agonist clofibric acid. AdPPARalpha-infected INS-1 cells showed PPARalpha agonist- and fatty acid-dependent transactivation of a PPARalpha reporter gene. Treatment with either AdPPARalpha or clofibric acid increased both catalase activity (a marker of peroxisomal proliferation) and palmitate oxidation. AdPPARalpha induced carnitine-palmitoyl transferase-I (CPT-I) mRNA, but had no effect on insulin gene expression. AdPPARalpha treatment increased cellular triglyceride content but clofibric acid did not. Both AdPPARalpha and clofibric acid decreased basal and glucose-stimulated insulin secretion. Despite increasing fatty acid oxidation, AdPPARalpha did not increase cellular ATP content suggesting the stimulation of uncoupled respiration. Consistent with these observations, UCP2 expression doubled in PPARalpha-treated cells. Clofibric acid-induced suppression of glucose-simulated insulin secretion was prevented by the CPT-I inhibitor etomoxir. These data suggest that PPARalpha-stimulated fatty acid oxidation can impair beta cell function.     

Study of the cytotoxic effect of a peptidic inhibitor of the p53-hdm2 interaction in tumor cells

Inhibitors of the p53-hdm2 interaction are attractive molecules for stimulating the p53 pathway in tumors. In this report, an inhibitor of the p53-hdm2 interaction, the AP peptide, is used to activate p53 in tumor cells expressing various levels of hdm2 protein. It induces apoptosis only in cells expressing high endogenous levels of hdm2 protein. The absence of apoptosis in tumor cells with low hdm2 levels is due not to alterations in the p53-dependent apoptotic pathway but to a different regulation of this pathway. The peptide is also less toxic for non-tumor cells than for tumor cells overexpressing the hdm2 protein.