The role of glutathione in yeast dehydration tolerance

Among the factors that affect cell resistance against dehydration, oxidation is considered to be of great importance. In this work, we verified that both control and glutathione deficient mutant strains were much more oxidized after dehydration. Moreover, cells lacking glutathione showed a twofold higher increase in oxidation and lipid peroxidation than the control strain. While glucose 6-phosphate dehydrogenase and glutathione reductase activities did not change in response to dehydration in the control strain, the mutant strain gsh1 (glutathione deficient) showed a reduction of 50% in both activities, which could explain the high levels of oxidation shown by gsh1 cells. In conformity with these results, the mutant lacking GSH1 showed a high sensitivity to dehydration. Furthermore, the addition of glutathione to gsh1 cells restored survival rates to the levels of the control strain. We conclude that glutathione plays a significant role in the maintenance of intracellular redox balance during dehydration.     

Effects of plasma apolipoproteins on lipoprotein lipase-mediated lipolysis of small and large lipid emulsions

Large (ca. 120 nm) and small (ca. 35 nm) emulsions consisting of triolein (TO) and phosphatidylcholine (PC) were prepared as the primary protein-free models of chylomicrons and their remnants, respectively. Lipoprotein lipase (LPL)-mediated lipolysis of emulsion TO was retarded in chylomicron-free human plasma compared with the hydrolysis activated by isolated apolipoprotein C-II (apoC-II). In 30% plasma, free fatty acid (FFA) release rate was higher for large emulsions than for small ones, while both emulsions were hydrolyzed at similar rates in the presence of isolated apoC-II. Isolated apolipoprotein C-III (apoC-III) or apolipoprotein E (apoE) worked as LPL-inhibitor of the lipolysis activated by apoC-II. It was also observed that apolipoprotein A-I (apoA-I) showed distinct inhibitory effects on the lipolysis of large and small emulsions: more effective inhibition for small emulsions. Kinetic analyses showed that K(m)(app) and V(max)(app) for the lipolysis of emulsions were lower in the presence of 30% plasma than isolated apoC-II. ApoA-I also markedly decreased K(m)(app) and V(max)(app) for LPL-catalyzed hydrolysis of both emulsions. In chylomicron-free serum, the density of bound apoA-I at small emulsion surfaces was about three fold greater than large emulsion surfaces, but the binding densities of apoC-II, apoC-III and apoE were less for small emulsion surfaces than for large ones, suggesting that apoA-I preferentially binds to small particles and displaces other exchangeable apolipoproteins from particle surfaces. These results indicate that, in addition to the well known inhibitory effects of apoC-III and apoE, apoA-I in plasma regulates the lipolysis of triglyceride (TG)-rich emulsions and lipoproteins in a size-dependent manner.     

Histological study of post-pollination events in Spathodea campanulata beauv. (Bignoniaceae), a species with late-acting self-incompatibility

The reproductive biology of Spathodea campanulata was investigated by means of hand-pollination experiments, observations of pollen tube growth using fluorescence microscopy, and serial sections of ovules in selfed and crossed pistils. Only cross-pollinated flowers developed fruits, and all selfed flowers abscised within 3-4 d. However, self pollen tubes grew successfully to the ovary, penetrating and fertilizing the majority of ovules by 48 h, indicating that S. campanulata is a species with late-acting self-incompatibility. The incidences of ovule penetration, fertilization and endosperm initiation were all significantly slower in selfed vs. crossed pistils, although no other signs of malfunctioning were detected. The possible role of such slow self pollen tube effectiveness as a recognition event is discussed within the context of the slow but not entirely suppressed self pollen tube growth reported for some species with conventional homomorphic self-incompatibility.     

Competition between CTL narrows the immune response induced by prime-boost vaccination protocols

Recombinant vaccines encoding strings of virus- or tumor-derived peptides and/or proteins are currently being designed for use against both cancer and infectious diseases. These vaccines aim to induce cytotoxic immune responses against several Ags simultaneously. We developed a novel tetramer-based technique, based on chimeric HLA A2/H-2K(b) H chains, to directly monitor the CTL response to such vaccines in HLA-A2 transgenic mice. We found that priming and boosting with the same polyepitope construct induced immune responses that were dominated by CTL of a single specificity. When a mixture of viruses encoding single proteins was used to boost the polyepitope primed response, CTL of multiple specificities were simultaneously expanded to highly effective levels in vivo. In addition, we show that a preexisting response to one of the epitopes encoded within a polyepitope construct significantly impaired the ability of the vaccine to expand CTL of other specificities. Our findings define a novel vaccination strategy optimized for the induction of an effective polyvalent cytotoxic response.     

The experimental infection of pigs with different numbers of Taenia solium eggs: immune response and efficiency of establishment

Three of 4 pigs inoculated with 10 eggs of Taenia solium became infected. In those pigs infected with larger numbers of eggs, all became infected. Specific antibodies against the metacestodes were found in serum at day 30 postinoculation (PI) in animals that received 1,000 or more eggs and at day 60 in those that received 10 or 100 eggs. The concentration and diversity of antibodies increased up to the day of death in pigs that received 10,000 or 100,000 eggs. All pigs infected with 1,000 or more eggs developed antibodies, but only 40% and 75% of pigs that received 10 and 100 eggs, respectively, developed antibodies. Metacestodes were found in the muscles of 23 of the 27 infected animals. In 35.7% of the pigs that received 1,000 or more eggs, metacestodes were also found in the brain. Most of the metacestodes found in pigs infected with 10 or 100 eggs were caseous, whereas in pigs infected with 1,000 or more eggs the majority of metacestodes were vesicular. This study shows that the severity of T. solium infection and the possible regulation of the immune system-evasion mechanisms depend on the number of metacestodes that succeed in establishing themselves and remain vesicular.     

B-cell infiltration and frequency of cytokine producing cells differ between localized and disseminated human cutaneous leishmaniases

Biopsies from human localized cutaneous lesions (LCL n = 7) or disseminated lesions (DL n = 8) cases were characterized according to cellular infiltration,frequency of cytokine (IFN-gamma, TNF-alpha) or iNOS enzyme producing cells. LCL, the most usual form of the disease with usually one or two lesions, exhibits extensive tissue damage. DL is a rare form with widespread lesions throughout the body; exhibiting poor parasite containment but less tissue damage. We demonstrated that LCL lesions exhibit higher frequency of B lymphocytes and a higher intensity of IFN-gamma expression. In both forms of the disease CD8+ were found in higher frequency than CD4+ T cells. Frequency of TNF-alpha and iNOS producing cells, as well as the frequency of CD68+ macrophages, did not differ between LCL and DL. Our findings reinforce the link between an efficient control of parasite and tissue damage, implicating higher frequency of IFN-gamma producing cells, as well as its possible counteraction by infiltrated B cells and hence possible humoral immune response in situ.     

Effects of angiotensins on day-night fluctuations and stress-induced changes in blood pressure

In this study we evaluated by telemetry the effects of ANG II and ANG-(1-7) infusion on the circadian rhythms of blood pressure (BP) and heart rate (HR) and on the cardiovascular adjustment resulting from restraint stress in rats. ANG II or ANG-(1-7) or vehicle were infused subcutaneously for 7 days. Restraint stress was carried out before, during, and after infusion at 7-day intervals. Parallel with an increase in MAP, ANG II infusion produced an inversion of MAP circadian rhythm with a significant MAP acrophase inversion. It also produced bradycardia during the first 3 days of infusion. Thereafter, HR progressively increased, reaching values similar to or above those of the control period at the end of the infusion period. HR circadian variation was not changed by ANG II infusion. Strikingly, ANG II significantly attenuated the increase in MAP induced by restraint stress without altering the HR response. ANG-(1-7) infusion produced a slight but significant decrease in MAP restricted to the daytime period. No significant changes in the MAP acrophase were observed. In addition, ANG-(1-7) infusion produced a small but significant sustained bradycardia. ANG-(1-7) did not change cardiovascular responses to restraint stress. These data indicate that ANG II can influence the activity of brain areas involved in the determination of stress-induced or circadian-dependent variations of blood pressure without changing HR fluctuations. A significant modulatory influence of ANG-(1-7) on basal MAP and HR is also suggested.     

Role of abnormal neutral endopeptidase-like activities in Hyp mouse bone cells in renal phosphate transport

We investigated whether the absence of Phex (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) in the Hyp mouse affects the expression and activity of neprilysin (NEP) and of endothelin-converting enzyme-like endopeptidase (ECEL1/DINE) in bone marrow stromal cells (BMSC) and osteoblasts (Ob). Total NEP-like activity was higher in Ob than in BMSC regardless of genotype, and Hyp cells showed higher activities than normal. Conditioned media (CM) from Hyp BMSC and Ob inhibited inorganic phosphate (P(i)) uptake by mouse proximal tubule cells, and incubating Hyp Ob with phosphoramidon prevented the production of the inhibitor of renal P(i) uptake. A linear relationship was observed between the NEP-like activity of Hyp and normal cells and the inhibition of P(i) uptake. NEP and ECEL1/DINE mRNA levels were higher in Hyp cells than in normal cells, and in situ hybridization of ECEL1/DINE confirmed higher levels of expression in the Hyp mouse than in normal cells. In conclusion, we observed a correlation between the inhibition of P(i) uptake by CM from Hyp cells and elevated NEP-like activities.     

PMA decreases the proliferation of retinal cells in vitro: the involvement of acetylcholine and BDNF

Protein kinase C (PKC) is involved in several cell events including proliferation, survival and differentiation. The aim of this work was to investigate the role of PKC activation on retinal cells proliferation. We demonstrated that PKC activation by phorbol 12-myristate 13-acetate (PMA), a tumor promoter phorbol ester, is able to decrease retinal cells proliferation. This effect was mediated by M1 receptors and dependent on intracellular Ca(2+) increase, tyrosine kinase activity, phosphatidylinositol 3-kinase activity, polypeptide secretion and activation of TrkB receptors. The effect of PMA was not via activation of mitogen-activated protein (MAP) kinase. Carbamylcholine and brain derived neurotrophic factor were both able to decrease retinal cells proliferation to the same level as PMA did. Our results suggest that PKC activation leads to a decrease in retinal cells proliferation through the release of acetylcholine and brain derived neurotrophic factor in the culture, and activation of M1 and TrkB receptors, respectively.