Revisiting the expression and purification of MGD1, the major galactolipid synthase in Arabidopsis to establish a novel standard for biochemical and structural studies

Monogalactosyldiacylglycerol, the major lipid of plants and algal plastids, is synthesized by MGDG synthases (MGD). MGDs belong to the large glycosyltransferase family. They catalyze the transfer of a galactose residue from the donor UDP-Gal to a 1,2-sn-diacylglycerol acceptor. MGDs are monotopic proteins localized in the plastid envelope and, as such, they are difficult to purify. This study re-examined previous purification procedures and aimed to set up a standard protocol for expression and purification of recombinant MGD1, addressing problems frequently encountered with the purification of glycosyltransferases, particularly protein aggregation, and enabling crystallization for structural studies. Briefly, His-tagged versions of MGD1 were expressed in Escherichia coli and purified by a two-step procedure, including immobilized metal affinity chromatography and size-exclusion chromatography. We demonstrated that E. coli is an appropriate host cell to produce a soluble and active form of MGD1. We also investigated the effects of various buffers and additives used during the purification and concentration steps on the biochemical behavior of the enzyme. The protocol we developed typically yields milligram quantities of pure and homogenous protein material and proved suitable for crystallization and biochemical studies. We also revisited the conditions for activity tests and effects of known positive effectors of MGD1 such as phosphatidic acid and phosphatidylglycerol.     

High FLT3 expression and IL10 (G1082A) polymorphism in poor overall survival in calla acute lymphoblastic leukemia

Patients with acute lymphoblastic leukemia presenting the immunophenotypic marker CD10+ (calla), usually has treatment profile good. The FLT3 molecular marker is listed as a prognostic factor, an important leukaemogenic marker in acute leukemias, also the polymorphism (G1082A) of the IL10 interleukin can to present pleiotropic effects in many diseases and could is associated to development of ALL. However, the FLT3 expression is variability among patients with calla-ALL. The aim of this study was to determine the FLT3 expression, to associate with the genotypes and allelic of G1082A (IL10) in 50 patients with calla-ALL and assess the overall survival at 98 months follow-up. The expression was assessed by quantitative real time PCR (RT-PCR), the G1082A polymorphism was identified by allele-specific PCR and for immunophenotypic classification was used specific markers of B lineage-calla. We observed that patients who died showed higher FLT3 expression (p = 0.005), worse survival (p = 0.0137) and the IL10G allele may favor the survival, because the IL10 GG and IL10 GA genotypes showed a low FLT3 expression (p < 0.05).     

Influences of sex,ontogeny and body size on the thermal ecology of Liolaemus lutzae (Squamata,Liolaemidae) in a restinga remnant in southeastern Brazil

Variations in body temperature (T_b) of lizards can be partially explained by intrinsic factors such as sex, ontogeny and body size. Liolaemus lutzae is a lizard species restricted to restingas in the Brazilian coast in the state of Rio de Janeiro. Herein, we studied sexual dimorphism and influences of sex, ontogeny, and body size to the T_b of L. lutzae. Adult males were larger than adult females, probably due to both intersexual selection and intra-sexual selection. There was intersexual difference in lizards' T_b (males hotter than females), but T_b did not differ after factored out for the effects of body size. The mean T_b of juvenile lizards was higher than that of adults after factored out for the effect of body mass. It is possible that adults may have excluded juveniles from microhabitats with better thermal regimes. Also, this might have occurred due to requirements of juveniles to maintain high growth rates. Forage searching for prey by juveniles also exposes them to high environmental temperatures. Juveniles also may have higher T_b than co-specific adults (relative to body mass) to favor prey capture. In absolute values, adult lizards tended to use microhabitats with lower temperatures than that used by juveniles, possibly to avoid risks of overheating and death. Body temperature and snout–vent length were positively related, as well as body temperature and body mass, presumably caused by the thermal inertia of the bodies (trend of a body to resist to changes in its temperature). Intrinsic factors such as sex, ontogeny and body size can affect the thermal ecology of L. lutzae, despite coastal habitat features to which they are exposed also influences the body temperature of active lizards in restinga habitats.     

The effects of the Brazilian antDinoponera quadriceps venom on chemically induced seizure models

Arthropod venoms are potential sources of neuroactive substances, providing new tools for the design of drugs. The aim of this study was to evaluate the effects of Dinoponera quadriceps venom (DqV) on seizure models in mice induced by pentylenetetrazole (PTZ), pilocarpine, and strychnine. In the PTZ model, intraperitoneal treatment with DqV (0.5 mg/kg) increased the time until the first seizure and the percentage of survival (155.4 ± 27.7 s/12.5%, p < 0.05) compared to the control group (79.75 ± 3.97 s/0%), whereas endovenous treatment (0.1 and 0.5 mg/kg) decreased the time until the first seizure (0.1 mg/kg: 77.83 ± 5.3 s versus 101.0 ± 3.3 s in the control group; 0.5 mg/kg: 74.43 ± 3.9 s versus 101.0 ± 3.3 s for the control group, p < 0.05). We did not observe significant changes in the pilocarpine- and strychnine-induced seizure models. In assays that measured oxidative parameters in the PTZ model, intraperitoneal treatment with DqV (0.5 and 2.0 mg/kg) only decreased the levels of MDA and nitrite in the cortex. However, endovenous treatment with DqV (0.1 and 0.5 mg/kg) increased the levels of MDA in the cortex and hippocampus and at a dose of 0.5 mg/kg in the striatum. Moreover, increased in nitrite content was observed in all three of the brain regions analyzed. Taken together, the D. quadriceps venom caused both neuroprotective and neurotoxic effects in a PTZ-induced seizure model, and this effect was dependent on the route of administration used.     

Diphenyl Diselenide Prevents Cortico-cerebral Mitochondrial Dysfunction and Oxidative Stress Induced by Hypercholesterolemia in LDL Receptor Knockout Mice

Recent studies have indicated a causal link between high dietary cholesterol intake and brain oxidative stress. In particular, we have previously shown a positive correlation between elevated plasma cholesterol levels, cortico-cerebral oxidative stress and mitochondrial dysfunction in low density lipoprotein receptor knockout (LDLr^?/?) mice, a mouse model of familial hypercholesterolemia. Here we show that the organoselenium compound diphenyl diselenide (PhSe)₂ (1 mg/kg; o.g., once a day for 30 days) significantly blunted the cortico-cerebral oxidative stress and mitochondrial dysfunction induced by a hypercholesterolemic diet in LDLr^?/? mice. (PhSe)₂ effectively prevented the inhibition of complex I and II activities, significantly increased the reduced glutathione (GSH) content and reduced lipoperoxidation in the cerebral cortex of hypercholesterolemic LDLr^?/? mice. Overall, (PhSe)₂ may be a promising molecule to protect against hypercholesterolemia-induced effects on the central nervous system, in addition to its already demonstrated antiatherogenic effects.     

Construction and assessment of reaction models between F1F0-synthase and organotin compounds: molecular docking and quantum calculations

Organotin compounds are the active components of some fungicides, which are potential inhibitors of the F₁F₀-ATP synthase. The studies about the reaction mechanism might indicate a pathway to understand how these compounds work in biological systems, however, has not been clarified so far. In this line, molecular modeling studies and density functional theory calculations were performed in order to understand the molecular behavior of those compounds when they interact with the active site of the enzyme. Our findings indicate that a strong interaction with His132 can favor a chemical reaction with organotin compounds due to π–π stacking interactions with aromatic rings of organotin compounds. Furthermore, dependence on molecule size is related to possibility of reaction with the amino acid residue His132. Thus, it can also be noticed, for organotin compounds, that substituents with four carbons work by blocking the subunit a, in view of the high energy transition found characterized by steric hindrance.     

Ultrastructure of spermatozoa of members of Calappidae,Aethridae and Menippidae and discussion of their phylogenetic placement

The families Aethridae and Calappidae were originally considered as part of the same family; however, their morphology and molecular biology separate them into two families. In this context, we describe the ultrastructure of spermatozoa of species of the Calappidae, Aethridae and Menippidae to elucidate the relationships among taxa. The vasa deferentia were submitted to routine protocols for transmission electron microscopy. Our results indicate that the morphology of the spermatozoa of Hepatus pudibundussupports its exclusion from the Superfamily Calappoidea due to the presence of the apical striated layer. The spermatozoa of Menippe nodifrons is very similar to H. pudibundus and corroborates the recent phylogenetic analysis using sequence data of nuclear genes. Moreover, our results evidence two morphological patterns of spermatozoa within Calappidae. Calappa ocellata and C. cinerea show spermatozoa with a wide acrosome vesicle, a thick operculum shaped as a shallow “W” and a large thickened ring. Calappa gallusand C. hepatica show spermatozoa with a longer acrosome vesicle, a pointed operculum and a slender thickened ring. Our ultrastructure results conform with previous molecular proposal and show that spermatozoa ultrastructure can be an effective tool to adjust phylogenetic relationship when used in association with molecular data.     

Simultaneous hydrolysis of cheese whey and lactulose production catalyzed by β-galactosidase from Kluyveromyces lactis NRRL Y1564

Bioprocess and Biosystems Engineering - β-Galactosidase was produced by the yeast Kluyveromyces lactis NRRL Y1564 in cheese whey supplemented with yeast extract under the optimal temperature...     

Whole-cell bioconversion of naringenin to high added value hydroxylated compounds using Yarrowia lipolytica 2.2ab in surface and liquid cultures

The bioconversion process of bioactive naringenin by whole-cells of Yarrowia lipolytica 2.2ab for the production of increased value-added compounds is successfully achieved in surface and liquid cultures. This approach is an alternative to the commercial production of these bioactive compounds from vegetable sources, which are limited due to their low concentrations and the complexity of the purification processes. The experimentation rendered seven value-added compounds in both surface and liquid bioconversion cultures. Some of the compounds produced have not been previously reported as products from the bioconversion processes, such as the case of ampelopsin. Biosynthetic pathways were suggested for the naringenin bioconversion using whole-cells of Y. lipolytica 2.2ab. Finally, the extracts obtained from the naringenin bioconversion in liquid cultures showed higher percentage of inhibition of DPPH· and ABTS· radicals up to 32.88 and 2.08 times, respectively, compared to commercial naringenin.