Isolation and purification of an enzyme hydrolyzing ochratoxin A from <Emphasis Type="Italic">Aspergillus niger</Emphasis>

Ochratoxin A is a mycotoxin produced by several Aspergillus and some Penicillium species which may be present in food and feed products. It can be enzymatically hydrolyzed into ochratoxin α and l-β-phenylalanine, thereby decreasing its toxicity. The ochratoxin A degradation capacity of Aspergillus niger is well known and here we report the isolation and purification of a novel enzyme from A. niger that hydrolyzes this mycotoxin. A wheat germ medium supplemented with ochratoxin A was used to produce the enzyme, which was purified from culture filtrate by acetone precipitation and anion exchange chromatography. An overall purification of 2.5-fold with a recovery of 68% and a final specific activity of 36 U/mg was obtained. The enzyme is a metalloenzyme as it was inhibited at 10 mM EDTA, whereas PMSF had no effect. The ochratoxin A hydrolytic enzyme presented a V _max of 0.44 μM/min and a K _m of 0.5 mM when the reaction was carried out at pH 7.5 and 37°C.     

Comparison of two commercial formulations of Bacillus thuringiensis var. israelensis for the control of Anopheles aquasalis (Diptera: Culicidae) at three salt concentrations

Anopheles aquasalis larvae are salt water tolerant, preferring concentrations between 10 and 20 parts per thousand (ppt). The larvicidal efficacy of two formulations of Bacillus thuringiensis var. israelensis (Vectobac-12AS and Bactivec), was investigated against An. aquasalis at salinities of 0, 10, and 20 ppt. A probit analysis was used to calculate the lethal concentrations (LC50 and LC95) for each product at each salinity. The LC50 and LC95 were higher for Bactivec than Vectobac-12AS, and for Bactivec, the LC50 and LC95 increased with salinity. Vectobac-12AS should thus be preferred to Bactivec for An. aquasalis control, especially in saline breeding habitats.     

pRb-dependent cyclin D3 protein stabilization is required for myogenic differentiation

The expression of retinoblastoma (pRb) and cyclin D3 proteins is highly induced during the process of skeletal myoblast differentiation. We have previously shown that cyclin D3 is nearly totally associated with hypophosphorylated pRb in differentiated myotubes, whereas Rb-/- myocytes fail to accumulate the cyclin D3 protein despite normal induction of cyclin D3 mRNA. Here we report that pRb promotes cyclin D3 protein accumulation in differentiating myoblasts by preventing cyclin D3 degradation. We show that cyclin D3 displays rapid turnover in proliferating myoblasts, which is positively regulated through glycogen synthase kinase 3beta (GSK-3beta)-mediated phosphorylation of cyclin D3 on Thr-283. We describe a novel interaction between pRb and cyclin D3 that maps to the C terminus of pRb and to a region of cyclin D3 proximal to the Thr-283 residue and provide evidence that the pRb-cyclin D3 complex formation in terminally differentiated myotubes hinders the access of GSK-3beta to cyclin D3, thus inhibiting Thr-283 phosphorylation. Interestingly, we observed that the ectopic expression of a stabilized cyclin D3 mutant in C2 myoblasts enhances muscle-specific gene expression; conversely, cyclin D3-null embryonic fibroblasts display impaired MyoD-induced myogenic differentiation. These results indicate that the pRb-dependent accumulation of cyclin D3 is functionally relevant to the process of skeletal muscle cell differentiation.     

SET3p monomethylates histone H3 on lysine 9 and is required for the silencing of tandemly repeated transgenes in Chlamydomonas

SET domain-containing proteins of the SU(VAR)3-9 class are major regulators of heterochromatin in several eukaryotes, including mammals, insects, plants and fungi. The function of these polypeptides is mediated, at least in part, by their ability to methylate histone H3 on lysine 9 (H3K9). Indeed, mutants defective in SU(VAR)3-9 proteins have implicated di- and/or trimethyl H3K9 in the formation and/or maintenance of heterochromatin across the eukaryotic spectrum. Yet, the biological significance of monomethyl H3K9 has remained unclear because of the lack of mutants exclusively defective in this modification. Interestingly, a SU(VAR)3-9 homolog in the unicellular green alga Chlamydomonas reinhardtii, SET3p, functions in vitro as a specific H3K9 monomethyltransferase. RNAi-mediated suppression of SET3 reactivated the expression of repetitive transgenic arrays and reduced global monomethyl H3K9 levels. Moreover, chromatin immunoprecipitation (ChIP) assays demonstrated that transgene reactivation correlated with the partial loss of monomethyl H3K9 from their chromatin. In contrast, the levels of trimethyl H3K9 or the repression of euchromatic sequences were not affected by SET3 downregulation; whereas dimethyl H3K9 was undetectable in Chlamydomonas. Thus, our observations are consistent with a role for monomethyl H3K9 as an epigenetic mark of repressed chromatin and raise questions as to the functional distinctiveness of different H3K9 methylation states.     

Determinants of blood oxygenation during pregnancy in Andean and European residents of high altitude

High altitude decreases birth weight, but this effect is diminished in long vs. short-resident, high-altitude populations. We asked whether women from long vs. short-resident, high-altitude populations had higher arterial oxygenation levels by comparing 42 Andean and 26 European residents of La Paz, Bolivia (3,600 m), serially during pregnancy (weeks 20, 30, and 36) and again 4 mo postpartum. Pregnancy raised hypoxic ventilatory sensitivity threefold, resting ventilation (.Ve), and arterial O(2) saturation (Sa(O2)) in both groups. Ancestry, as identified using 81 genetic markers, correlated with respiratory pattern, such that greater Andean ancestry was associated with higher respiratory frequency and lower tidal volume. Pregnancy increased total blood and plasma volume approximately 40% in both groups without changing red blood cell mass relative to body weight; hence, hemoglobin fell. The hemoglobin decline was compensated for by the rise in .Ve and Sa(O2) with the result that arterial O2 content (Ca(O2)) was maintained near nonpregnant levels in both groups. Birth weights were similar for all Andean and European babies, but after adjusting for variation in gestational age, maternal height and parity, Andeans weighed 209 g more than Europeans. Babies with heavier birth weights and greater ponderal indices were born to Andean women with higher Ve during pregnancy. We concluded that while maternal .Ve and arterial oxygenation were important, some factor other than higher Ca(O2) was responsible for protecting Andeans from altitude-associated reductions in fetal growth.     

Carbonic anhydrase and matrix metalloproteinase inhibitors. Inhibition of human tumor-associated isozymes IX and cytosolic isozyme I and II with sulfonylated hydroxamates

A series of sulfonylated hydroxamates were synthesized and evaluated as dual inhibitors of both human carbonic anhydrases (hCAs) and matrix metalloproteinases (MMPs), two metalloenzyme families involved in carcinogenesis and tumor invasion processes. The new derivatives were tested on three CA isozymes, the cytosolic isozymes I and II, and the transmembrane, tumor-associated isozyme IX, and also on human gelatinases (MMP-2 and MMP-9). Some of the new derivatives proved to be potent and selective inhibitors of CA II, but only compounds 3b and 6b, devoid of the arylsulfonyl moiety, proved to have a better inhibitory activity on hCA IX than on hCA I and II, in the micromolar range.     

How sugars convey information on protein conformation in the endoplasmic reticulum

The N-glycan-dependent quality control of glycoprotein folding prevents endoplasmic reticulum to Golgi exit of folding intermediates, irreparably misfolded glycoproteins and not completely assembled multimeric complexes. It also enhances folding efficiency by preventing aggregation and facilitating formation of proper disulfide bonds. The control mechanism essentially involves four components, resident lectin-chaperones that recognize monoglucosylated polymannose glycans, a lectin-associated oxidoreductase acting on monoglucosylated glycoproteins, a glucosyltransferase and a glucosidase that creates monoglucosylated epitopes in glycans transferred in protein N-glycosylation or removes the glucose units added by the glucosyltransferase. This last enzyme is the only mechanism component sensing glycoprotein conformations as it creates monoglucosylated glycans exclusively in not properly folded species or in not completely assembled complexes. The purpose of the review is to describe the most significant recent findings on the mechanism of glycoprotein folding and assembly quality control and to discuss the main still unanswered questions.     

The role of vitellogenin during gestation of Girardinichthys viviparus and Ameca splendens; two goodeid fish with matrotrophic viviparity

Goodeid fish have matrotrophic viviparity, and unlike lecitotrophic fish, yolk loss forces the female to provide the nutritional requirements for embryonic development. Vitellogenin (VTG) is the yolk precursor protein synthesized in the maternal liver, but there is only circumstantial evidence regarding VTG supply during the ontogenesis of bony fish with matrotrophic viviparity. Therefore, the goal of the present study was to identify and quantify VTG during gestation of the black fin goodeid Girardinichthys viviparus and the butterfly split-fin goodeid Ameca splendens. Females at different gonadic developmental stages were selected in order to evaluate VTG mRNA expression in the maternal liver using RT-PCR; VTG quantification in maternal muscle and liver, as well as in the embryos, was done using ELISA, and immunohistochemical detection of VTG was done in the black fin goodeid. The results suggest that VTG supplies nutrients during embryonic development of both species, which have different life histories. It is possible that the transition from lecitotrophy to matrotrophic viviparity in bony fish with intraluminal gestation involved adaptive transition strategies that included changes in the relationship between oocytes and follicular cells, as well as a gradual loss of VTG synthesis during embryonic development.     

Gender related differences in the oxidative stress response to PCB exposure in an endangered goodeid fish (Girardinichthys viviparus)

Polychlorinated biphenyls (PCBs) are persistent xenobiotics within aquatic environments, which elicit diverse toxic effects such as induction of oxidative stress. Despite numerous earlier studies, no detailed information exists on the toxic response by different sexes in fish. The aim of this study was to determine sex-linked differences in oxidative stress response and antioxidant defenses in Girardinichthys viviparus, an endangered fish endemic to Mexico, when exposed to sub-lethal concentrations of waterborne PCBs. The biological markers evaluated were lipid peroxidation (LPOX), superoxide dismutase (SOD) and catalase (CAT) activity. Adult eight-month-old specimens born in the laboratory were exposed to ½ of the LC₀ (0.92 mg PCBs/L) in semi-hard synthetic water and sacrificed on days 1, 2, 4, 8 and 16 for biomarker assays. Sex-linked differences were observed in the control fish with respect to all three factors assayed. PCBs elicited significant (p < 0.01) time- and sex-dependent LPOX levels which were higher in the case of males. In PCB-treated G. viviparus, SOD activity was depressed in both sexes and appears to return to pre-exposure levels after 16 days in males only. In contrast, CAT was significantly induced (p < 0.01) in both sexes. This enzyme may be responsible for balancing oxidative stress and antioxidant defenses under experimental conditions. PCBs at sub-lethal concentrations are hazardous to both sexes of G. viviparus since these compounds are able to induce liver LPOX and changes in the antioxidant defense activities. The relationship between these biomarkers and cytochrome P450 and CYP1A induction is also discussed.