Molecular characterization of clinical Saccharomyces cerevisiae isolates and their association with non-clinical strains

We assessed the molecular characterization of 96 clinical isolates of S. cerevisiae from a Spanish medical institution and we compared them with 6 non-clinical strains isolated from wine, beer and bread and 1 S. boulardii strain collected from a commercial preparation. The strains were subjected to HinfI mtDNA restriction analysis and PCR amplification of delta sequences. Although both techniques are appropriate for routine clinical analysis, that based on PCR turned out to be the most discriminating. This study, apart from providing tools for clinical application, deals with the relationships between clinical and non-clinical strains. The two baker's yeasts analysed shared mtDNA and PCR patterns with a group of 31 clinical isolates. An exogenous entry of a strain was also reflected in the case of 19 clinical isolates and the therapeutic strain S. boulardii. Both baker's yeasts and S. boulardii were identified respectively among 32.3% and 19.8% of the clinical isolates and there seemed to be a connection between their ability to colonize humans and their ability to cause vaginal infection. The rest of food isolates were not grouped with clinical strains.     

Identification of species of the genus Candida by analysis of the 5.8S rRNA gene and the two ribosomal internal transcribed spacers

The PCR amplification and subsequent restriction analysis of the ribosomal region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene is applied to the identification of yeasts belonging to the genus Candida. This methodology has previously been used for the identification of some species of this genus, but in the present work this application has been applied to the identification and characterisation of a greater number of species of the genus Candida, with a special survey of species of clinical and biotechnological interest. Among the species of the genus Candida, the high variability observed, both in the length of the amplified region (ranging between 390 and 900 bp) and in their restriction patterns, allows the unequivocal identification to the species level, with the exception of the group of species that comprises C. membranifaciens, C. conglobata, C. atlantica, C. atmosphaerica, and C. oleophila, that required the sequencing of the D1/D2 domain of the 26S rRNA gene or the 5.8S-ITS region for their proper differentiation. The 5.8S-ITS restriction analysis also failed in the differentiation of species within the pairs C.aaseri/C.butyri,C.fructus/C.musae,C.santamariae var. santamariae / C. beechii and C. zeylanoides / C. krissii. In this case, the high sequence similarities obtained for their 26S D1/D2 domain and the 5.8S-ITS region indicate that each pair of species should be considered as a single species. The main purpose of this work is to generate a database for a high number of yeast species, of both biotechnological and clinical interest, and to facilitate their easy, fast, and reliable identification. The present work improves the database available online at the IATA web page (http://motor.edinfo.es/iata/) with the patterns of 75 species belonging to the genus Candida.     

17beta-estradiol prevents oxidative stress and decreases blood pressure in ovariectomized rats

In this study, we tested whether estrogen deficiency is associated with oxidative stress and decreased nitric oxide (NO) production, which could be responsible for an increased blood pressure in ovariectomized rats. Hemodynamic studies were performed on conscious, chronically instrumented rats. Chronic estrogen replacement on ovariectomized rats lowered blood pressure approximately 13 mmHg, from 119 +/- 3 mmHg in ovariectomized rats to 106 +/- 3 mmHg in ovariectomized-treated rats; it was also accompanied by an increase in cardiac index and vascular conductance, achieving hemodynamic values similar to those shown by sham-operated rats. N(G)-nitro-L-arginine methyl ester administration lowered significantly less the vascular conductance (0.14 +/- 0.01 vs. 0.22 +/- 0.03 and 0.26 +/- 0.01 ml. min(-1). mmHg(-1)/100 g; P < 0.05) in ovariectomized rats than in the sham-operated and estrogen-treated ovariectomized rats, respectively. Estrogen replacement prevented the lower plasma levels of nitrites/nitrates observed in ovariectomized rats. The lower plasma total antioxidant status and reduced thiol groups and the increase in plasma lipoperoxides presented in ovariectomized animals were reestablished with the estrogen treatment. These results show that estrogen administration decreases blood pressure and increases vascular conductance in ovariectomized rats. This effect may be related to an increase in NO synthesis and/or preventing oxidative stress, then improving endothelial function.     

Fetal oxygen uptake during uterine contractures

During contractures there are decreases in fetal oxygen tension. In order to determine if there are concomitant changes in fetal oxygen consumption, we calculated the latter during contractures from measurements of the umbilical blood flow and venous arterial oxygen content differences across the umbilical circulation. There were decreases in both the umbilical venous (from 8.8 +/- 0.2 (SEM) to 8.5 +/- 0.2 ml.dl-1, P less than 0.01) and umbilical arterial (5.9 +/- 0.1 to 5.2 +/- 0.2 mg.dl-1, P less than 0.001) oxygen contents. The umbilical venous-arterial oxygen content difference increased from 2.9 +/- 0.1 to 3.3 +/- 0.2 ml.dl-1 (P less than 0.005). Umbilical blood flow was 194.3 +/- 4.5 ml.min-1 kg-1 during relaxation and was unchanged during contractures. Fetal oxygen uptake increased from 5.7 +/- 0.3 to 6.5 +/- 0.4 ml.min-1 kg-1 (P less than 0.005) during contractures. This observation is consistent with our previous speculation that there is increased muscular activity of tone associated with contractures.     

Evidences for the presence of chymotrypsin-like activity in human spermatozoa with a role in the acrosome reaction

The effect of chymotrypsin inhibitors and substrates on the human sperm acrosome reaction stimulated by the human zonae pellucidae or follicular fluid were evaluated. Motile spermatozoa, selected by a Percoll gradient, were incubated at 1 × 10⁷ cells/ml, 37°C, and 5% CO₂, After 4.5 hr, the chymotrypsin inhibitor TPCK (N-Tosyl-L-Phenylalanine-Chloromethyl Ketone) or the substrate ATEE (N-Acetyl-L-Tyrosine Ethyl Ester) were added for 30 min. Then, four oocytes were added and the percentage of acrosome-reacted spermatozoa on the zona was determined. TPCK and ATEE inhibited the zona pellucida-induced acrosome reaction. The chymotrypsin inhibitors TPCK and chymostatin and the chymotrypsin substrates ATEE, BTEE (N-Benzoyl-L-Tyrosine Ethyl Ester), Succinyl-Ala-Ala-Phe-7-Amido-4-Methyl-Coumarin (Suc-Ala-Ala-Phe-AMC), and Succinyl-Leu-Leu-Val-Tyr-7-Amido-4-Methyl-Coumarin (Suc-Leu-Leu-Val-Tyr-AMC) inhibited the human follicular fluid-induced acrosome reaction. Sperm extracts exhibited hydrolytic activity toward Suc-Ala-Ala-Phe-AMC and Suc-Leu-Leu-Val-Tyr-AMC. This enzyme activity was abolished by TPCK and chymostatin, was independent of Ca²⁺, and was not modified by 1,10 phenanthroline. In addition, the activity was present in the supernatant after the acrosome reaction was induced with calcium ionophore and in epididymal spermatozoa recovered from the cauda region. Electron microscopic observations indicated that the inhibitors prevented the membrane events of the acrosome reaction. These data suggest an association between human spermatozoa and chymotrypsin-like activity with a possible role in the acrosome reaction. © 1994 Wiley-Liss, Inc.     

Conditional Synergism between Cryptochrome 1 and Phytochrome B Is Shown by the Analysis of phyA, phyB, and hy4 Simple, Double, and Triple Mutants in Arabidopsis

Wild-type or phyA, phyB, or hy4 mutant Arabidopsis seedlings lacking phytochrome A (phyA), phytochrome B (phyB), or cryptochrome 1 (cry1), respectively, and the double and triple mutants were used in combination with blue-light treatments given simultaneously with red or far-red light. We investigated the interaction between phytochromes and cry1 in the control of hypocotyl growth and cotyledon unfolding. Under conditions deficient for cry1 (short exposures to blue light) or phyB (far-red background), these photoreceptors acted synergistically: Under short exposures to blue light (3 h/d) added to a red-light background, cry1 activity required phyB (e.g. the hy4 mutant was taller than the wild type but the phyBhy4 mutant was not taller than the phyB mutant). Under prolonged exposures to blue light (24 h/d) added to a far-red light background, phyB activity required cry1 (e.g. the phyAphyB mutant was taller than the phyA mutant but the phyAphyBhy4 mutant was not taller than the phyAhy4 mutant). Under more favorable light inputs, i.e. prolonged exposures to blue light added to a red-light background, the effects of cry1 and phyB were independent. Thus, the synergism between phyB and cry1 is conditional. The effect of cry1 was not reduced by the phyA mutation under any tested light condition. Under continuous blue light the triple mutant phyAphyBhy4 showed reduced hypocotyl growth inhibition and cotyledon unfolding compared with the phyAphyB mutant. The action of cry1 in the phyAphyB double mutant was higher under the red-light than the far-red-light background, indicating a synergistic interaction between cry1 and phytochromes C, D, or E; however, a residual action of cry1 independent of any phytochrome is likely to occur.     

Involvement of nitric oxide synthase and protein kinase C activation on chagasic antibodies action upon cardiac contractility

We have already demonstrated the presence of antibodies in the sera of chagasic patients with the ability to interact with neurotransmitter receptors triggering several intracellular pathways of transduction signals. Here we show that, chagasic IgG induced protein kinase C (PKC) translocation to rat cardiac membranes and this effect was inhibited by muscarinic cholinergic blockers atropine and AF-DX 116 pointing to the participation of M₂ receptors in this effect. It was also able to stimulate nitric oxide synthase (NOS) activity and this action was blunted by phospholipase C (PLC) and PKC inhibitors indicating that the production of nitric oxide (NO) would be the consequence of the cascade of enzymatic pathways triggered by mAChR activation. PKC and NOS activities were involved in chagasic IgG negative inotropic actions on rat isolated myocardium as its effects were blunted by staurosporine and L-N-monomethyl arginine. Furthermore, low concentrations of chagasic IgG inhibited the cardiac mechanical action of carbachol in a non-competitive manner. These data suggested that PKC activation in myocardium by chagasic IgG would be involved in its physiological actions by modulating NOS activity. The participation of PKC-mediated phosphorylation of mAChR leading to receptor desensitization as one of the causes of dysautonomia is also discussed.     

Role of ABCA1 on membrane cholesterol content,insulin-dependent Akt phosphorylation and glucose uptake in adult skeletal muscle fibers from mice

The ATP-binding cassette transporter A1 (ABCA1) promotes cellular cholesterol efflux, leading to cholesterol binding to the extracellular lipid-free apolipoprotein A-I. ABCA1 regulates lipid content, glucose tolerance and insulin sensitivity in adipose tissue. In skeletal muscle, most GLUT4-mediated glucose transport occurs in the transverse tubule, a system composed by specialized cholesterol-enriched invaginations of the plasma membrane. We have reported that insulin resistant mice have higher cholesterol levels in transverse tubule from adult skeletal muscle. These high levels correlate with decreased GLUT4 trafficking and glucose uptake; however, the role of ABCA1 on skeletal muscle insulin-dependent glucose metabolism remains largely unexplored. Here, we evaluated the functional role of the ABCA1 on insulin-dependent signaling pathways, glucose uptake and cellular cholesterol content in adult skeletal muscle. Male mice were fed for 8?weeks with normal chow diet (NCD) or high fat diet (HFD). Compared to NCD-fed mice, ABCA1 mRNA levels and protein content were lower in muscle homogenates from HFD-fed mice. In Flexor digitorum brevis muscle from NCD-fed mice, shABCA1-RFP in vivo electroporation resulted in 65% reduction of ABCA1 protein content, 1.6-fold increased fiber cholesterol levels, 74% reduction in insulin-dependent Akt (Ser⁴⁷³) phosphorylation, total suppression of insulin-dependent GLUT4 translocation and decreased 2-NBDG uptake compared to fibers electroporated with the scrambled plasmid. Pre-incubation with methyl-β cyclodextrin reestablished both GLUT4 translocation and 2-NBDG transport. Based on the present results, we suggest that decreased ABCA1 contributes to the anomalous cholesterol accumulation and decreased glucose transport displayed by skeletal muscle membranes in the insulin resistant condition.