The specific transport system for lysine is fully inhibited by ammonium in Penicillium chrysogenum: An ammonium-insensitive system allows uptake in carbon-starved cells

The regulation exerted by ammonium and other nitrogen sources on amino acid utilization was studied in swollen spores of Penicillium chrysogenum. Ammonium prevented the L-lysine, L-arginine and L-ornithine utilization by P. chrysogenum swollen spores seeded in complete media, but not in carbon-deficient media. Transport of L-[¹⁴C]lysine into spores incubated in presence of carbon and nitrogen sources was fully inhibited by ammonium ions (35 mM). However, in carbon-derepressed conditions (growth in absence of sugars, with amino acids as the sole carbon source) L-[¹⁴C]lysine transport was only partially inhibited. Competition experiments showed that L-lysine (1 mM) inhibits the utilization of L-arginine, and vice versa, L-arginine inhibits the L-lysine uptake. High concentrations of L-ornithine (100 mM) prevented the L-lysine and L-arginine utilization in P. chrysogenum swollen spores. In summary, ammonium seems to prevent the utilization of basic amino acids in P. chrysogenum spores by inhibiting the transport of these amino acids through their specific transport system(s), but not through the general amino acid transport system that is operative under carbon-derepression conditions.     

Ecology of mosquitoes (Diptera: Culicidae) in areas of Serra do Mar State Park, State of São Paulo, Brazil. III. Daily biting rhythms and lunar cycle influence

The ecology of mosquito species (Diptera: Culicidae) was studied in areas of the Serra do Mar State Park, State of S?o Paulo, Brazil. The influence of the lunar cycle and the daily biting rhythms of mosquito populations were analyzed. Systematized biweekly human bait collections were made in a silvatic environment for 24 consecutive months (January 1991 to December 1992). A total of 20,591 specimens of adult mosquitoes belonging to 55 species were collected from 545 catches. Sabethini species were captured exclusively during daylight periods, with the exception of Trichoprosopon digitatum, while members of Anophelinae predominated during nocturnal hours. Members of the subfamily Culicinae that were collected primary during nocturnal periods included Culex nigripalpus, Coquillettidia chrysonotum and Cq. venezuelensis while daytime catches included Psorophora ferox and Ps. albipes. Others members of culicines mosquitoes that were collected during both day and night included: Aedes serratus, Ae. scapularis and Ae. fulvus. Lunar cycles did not appear to influence the daily biting rhythms of most mosquito species in the area, but larger numbers of mosquitoes were collected during the new moon. Ae. scapularis were captured mainly during the full moon.     

Leishmania amazonensis infection does not inhibit systemic nitric oxide levels elicited by lipopolysaccharide in vivo

Leishmaniasis is a parasitic disease that leads to chronic inflammation. Macrophages, depending on their activation state, are either hosts or killers of the parasites. Downregulation of nitric oxide (NO) synthesis by the parasite infecting the macrophages has been proposed to be an important evading mechanism based on in vitro studies. We confirmed inhibition of NO release by macrophages infected with Leishmania amazonensis in vitro. To examine the role of the parasite in regulating NO production in vivo, we monitored systemic NO levels elicited by challenging naive and L. amazonensis-infected BALB/c mice with lipopolysaccharide (LPS). Animals were challenged after 1, 2, 6, and 9 wk of infection. NO production was monitored by electron paramagnetic resonance spectroscopy as the levels of hemoglobin nitrosyl complexes (HbNO) present in the animal's blood. No significant differences in HbNO levels were observed between LPS-treated naive and inoculated mice at any time during infection. To control for increased macrophage numbers in infected mice, naive mice were injected with a macrophage cell line before LPS challenge; this treatment did not increase produced NO levels. The results argue against a major role for the parasite in downregulating NO production in vivo.     

Autoxidation and neurotoxicity of 6-hydroxydopamine in the presence of some antioxidants: potential implication in relation to the pathogenesis of Parkinson's disease

6-Hydroxydopamine (6-OHDA) is a dopaminergic neurotoxin putatively involved in the pathogenesis of Parkinson's disease (PD). Its neurotoxicity has been related to the production of reactive oxygen species. In this study we examine the effects of the antioxidants ascorbic acid (AA), glutathione (GSH), cysteine (CySH), and N-acetyl-CySH (NAC) on the autoxidation and neurotoxicity of 6-OHDA. In vitro, the autoxidation of 6-OHDA proceeds rapidly with the formation of H2O2 and with the participation of the H2O2 produced in the reaction. The presence of AA induced a reduction in the consumption of O2 during the autoxidation of 6-OHDA and a negligible presence of the p-quinone, which demonstrates the efficiency of AA to act as a redox cycling agent. The presence of GSH, CySH, and NAC produced a significant reduction in the autoxidation of 6-OHDA. In vivo, the presence of sulfhydryl antioxidants protected against neuronal degeneration in the striatum, which was particularly remarkable in the case of CySH and was attributed to its capacity to remove the H2O2 produced in the autoxidation of 6-OHDA. These results corroborate the involvement of oxidative stress as the major mechanism in the neurotoxicity of 6-OHDA and the putative role of CySH as a scavenger in relation to PD.     

Bile acid secretion during rat liver carcinogenesis

Retro-differentiation of liver parenchyma during neoplastic processes is characterized by the expression of tumor antigens, such as alpha-fetoprotein and the placental isoenzyme of glutathione-S-transferase (GST-P). To investigate whether this may also affect a typical liver function such as bile acid secretion was the aim of this work. Rat hepatocarcinogenesis was induced by diethylnitrosamine (i.p., 200 mg/Kg body weight at day 0) and promoted by two-thirds partial hepatectomy (at day 21) plus 2-acetamidofluorene administration (50 mg/Kg body weight, subcutaneously, twice a week from day 14 to day 35). In order to carry out planimetric measurements of neoplastic tissue after immunohistochemical staining, a novel monoclonal antibody (MAb 14.1.3) against GST-P with no cross-reactivity against the major liver isoform of GST (GST-H) was raised. Analysis of total biliary bile acid output using the 3alpha-hydroxysteroid dehydrogenase method indicated that a significant reduction (-26%) occurred during the formation of GST-P-positive foci (12 wk). This was restored to normal values during adenoma formation (16-20 wk), but decreased again during carcinoma transformation (32 wk). These changes were not parallel to that observed in bile flow, which was progressively but slightly decreased throughout the whole period under study. HPLC analysis of bile samples collected for 1 h at different time points during hepatocarcinogenesis revealed that in contrast to what happens during cholestatic disease, a continuous and progressive increase in the cholic acid-to-chenodeoxycholic acid ratio (from 4.4+/-0.5 in control animals to 15.1+/-1.9 in rats with hepatocellular carcinoma) occurs. A significant and transient increase at 16 wk (+120%) in the proportion of bile acids amidated with glycine as compared to those conjugated with taurine was also observed. These results indicate that the mechanisms accounting for the secretion of major bile acids are modified differently at various steps of rat liver tumor development.     

The continuous administration of aspirin attenuates atherosclerosis in apolipoprotein E-deficient mice

Aspirin reduces the incidence of thrombotic occlusive events. Classically this has been thought to be due to the platelet inhibitory action of aspirin but it has recently been shown that inflammation plays a predominant role in the initiation and progression of lesions in atherosclerosis. In humans, treatment with aspirin reduces cardiovascular risk and slows carotid plaque growth in a dose-dependent fashion. We have explored this issue in Apo E-deficient mice on a high-fat, high cholesterol diet which provided these animals with a continuous administration of 500 microg/day of acetylsalicylic acid in the drinking water. After 10 weeks of treatment, the size of the atherosclerotic lesion at the aortic sinus had reduced by 35%. At the end of the trial there were no significant changes in either plasma lipids or in the quantitative distribution among lipoproteins. Likewise, the total antioxidant status and the resistance of plasma to oxidation in vitro was similar and there was no change in the distribution of iron deposits and in the relative composition of plasma pro-oxidants and antioxidants, or in the concentration of plasma in ferritin. Therefore, it is our hypothesis that the antiinflammatory effect is responsible for the reduction in lesion size. We propose that antiinflammatory molecules which do not cause gastrointestinal complications should be tested in humans to determine long-term efficacy in the attenuation of atherosclerosis.     

The Arabidopsis thaliana PPX/PP4 phosphatases: molecular cloning and structural organization of the genes and immunolocalization of the proteins to plastids

The PPX/PP4 Ser/Thr protein phosphatases belong to the type 2A phosphatase subfamily and are present in most eukaryotic organisms. We have previously isolated two closely related DNAs encoding PPX isoforms (PPX-1 and PPX-2) of Arabidopsis thaliana. Here we report the molecular cloning of the genes encoding these proteins. The genes PPX-1 and PPX-2 are composed of eight exons and seven introns located at equivalent positions related to the coding sequences. Whereas the intron-exon organization of the PPX genes is completely different from that of the PP2A-3/PP2A-4 A. thaliana family, specific intron-exon boundaries are conserved among PPX genes from distantly related organisms. Based on GUS expression, both PPX genes show the same spatial and temporal pattern of expression: they are expressed in all the organs and tissues analyzed, and from the earliest stage of development. When PPX proteins were localized to the root in semi-thin methacrylate sections by immunofluorescence, staining was predominantly confined to small organelles, shown to be plastids by co-localization of PPX and ferredoxin. Interestingly, only some ferredoxin-positive plastids were also PPX-positive, and PPX staining was consistently brighter in the epidermis. The localization was confirmed with immunogold and electron microscopy. Our results suggest that, despite its strong sequence conservation, PPX in plants functions differently than in animals.     

Protein phosphatase 2A holoenzyme and its subunits from Medicago sativa

We detected an about 200 kDa holoenzyme of protein phosphatase 2A (PP2A) in the crude extract of Medicago sativa microcallus cells by gel permeation chromatography. By polymerase chain reaction (PCR) we isolated two M. sativa cDNA fragments corresponding to the catalytic (C) subunit, and one each coding for the A and the B regulatory subunits of PP2A. The C subunit sequences were different from that published previously, indicating the existence of at least three different isoforms in M. sativa. Using the PCR fragments as probes, we obtained two distinct full-length clones for both the A and B subunits from an alfalfa cDNA library. Our results demonstrate that the components of the PP2A holoenzyme, namely the catalytic and regulatory subunits, are present in alfalfa in several isoforms and that their sequences are highly similar to their plant, yeast and animal counterparts. The distinct regulatory subunit genes are constitutively expressed during the cell cycle. Interestingly, two A-B subunit pairs had parallel mRNA steady-state levels in different plant tissues suggesting that not all of the possible isoform combinations are present in all tissues. The expression of the MsPP2A B subunit form was induced by abscisic acid indicating a specific function for this protein in the stress response.     

Intracellular Signal Triggered by Cholera Toxin in Saccharomyces boulardii and Saccharomyces cerevisiae

As is the case for Saccharomyces boulardii, Saccharomyces cerevisiae W303 protects Fisher rats against cholera toxin (CT). The addition of glucose or dinitrophenol to cells of S. boulardii grown on a nonfermentable carbon source activated trehalase in a manner similar to that observed for S. cerevisiae. The addition of CT to the same cells also resulted in trehalase activation. Experiments performed separately on the A and B subunits of CT showed that both are necessary for activation. Similarly, the addition of CT but not of its separate subunits led to a cyclic AMP (cAMP) signal in both S. boulardii and S. cerevisiae. These data suggest that trehalase stimulation by CT probably occurred through the cAMP-mediated protein phosphorylation cascade. The requirement of CT subunit B for both the cAMP signal and trehalase activation indicates the presence of a specific receptor on the yeasts able to bind to the toxin, a situation similar to that observed for mammalian cells. This hypothesis was reinforced by experiments with ¹²⁵I-labeled CT showing specific binding of the toxin to yeast cells. The adhesion of CT to a receptor on the yeast surface through the B subunit and internalization of the A subunit (necessary for the cAMP signal and trehalase activation) could be one more mechanism explaining protection against the toxin observed for rats treated with yeasts.     

Effects of Ethanol and Other Alkanols on Transport of Acetic Acid in Saccharomyces cerevisiae

In glucose-grown cells of Saccharomyces cerevisiae IGC 4072, acetic acid enters only by simple diffusion of the undissociated acid. In these cells, ethanol and other alkanols enhanced the passive influx of labelled acetic acid. The influx of the acid followed first-order kinetics with a rate constant that increased exponentially with the alcohol concentration, and an exponential enhancement constant for each alkanol was estimated. The intracellular concentration of labelled acetic acid was also enhanced by alkanols, and the effect increased exponentially with alcohol concentration. Acetic acid is transported across the plasma membrane of acetic acid-, lactic acid-, and ethanol-grown cells by acetate-proton symports. We found that in these cells ethanol and butanol inhibited the transport of labelled acetic acid in a noncompetitive way; the maximum transport velocity decreased with alcohol concentration, while the affinity of the system for acetate was not significantly affected by the alcohol. Semilog plots of V_max versus alcohol concentration yielded straight lines with negative slopes from which estimates of the inhibition constant for each alkanol could be obtained. The intracellular concentration of labelled acid was significantly reduced in the presence of ethanol or butanol, and the effect increased with the alcohol concentration. We postulate that the absence of an operational carrier for acetate in glucose-grown cells of S. cerevisiae, combined with the relatively high permeability of the plasma membrane for the undissociated acid and the inability of the organism to metabolize acetic acid, could be one of the reasons why this species exhibits low tolerance to acidic environments containing ethanol.