TOPS-MODE based QSARs derived from heterogeneous series of compounds. Applications to the design of new anti-inflammatory compounds

A new application of TOPological Sub-structural MOlecular DEsign (TOPS-MODE) was carried out in anti-inflammatory compounds using computer-aided molecular design. Two series of compounds, one containing anti-inflammatory and the other containing nonanti-inflammatory compounds were processed by a k-means cluster analysis in order to design the training and prediction sets. A linear classification function to discriminate the anti-inflammatory from the inactive compounds was developed. The model correctly and clearly classified 88% of active and 91% of inactive compounds in the training set. More specifically, the model showed a good global classification of 90%, that is, (399 cases out of 441). While in the prediction set, they showed an overall predictability of 88% and 84% for active and inactive compounds, being the global percentage of good classification of 85%. Furthermore this paper describes a fragment analysis in order to determine the contribution of several fragments towards anti-inflammatory property, also the present of halogens in the selected fragments were analyzed. It seems that the present TOPS-MODE based QSAR is the first alternate general 'in silico' technique to experimentation in anti-inflammatory discovery.     

Extracellular localization of proteasomes in human sperm

The proteasome, a multienzymatic protease complex is present in human sperm. Here we present evidence indicating that the proteasome has an extracellular localization, on the plasma membrane of the sperm head. Motile sperm (>90%) in PBS were incubated with the proteasome inhibitors clasto-lactacystin beta-lactone or epoxomicin. Then, the substrate Suc-Leu-Leu-Val-Tyr-AMC (SLLVY-AMC) was added and the enzyme activity evaluated in a spectrofluorometer. Other aliquots were resuspended in Tyrode's medium and incubated at different concentrations for various times with or without inhibitors in the presence of 0.4% azocasein. Hydrolysis of azocasein was evaluated at 440 nm. In addition, sperm membrane proteins were obtained incubating the sperm with Triton X-114 or with 0.5 M KCl plus Triton X-100 and removing insoluble material by centrifugation at 5,000g for 40 min. Proteasomal activity was evaluated with SLLVY-AMC and its presence corroborated by Western blotting. Formaldehyde fixed, unpermeabilized sperm were incubated with anti-proteasome monoclonal antibodies and evaluated using indirect immunofluorescence. The effect of proteasome inhibitors upon the progesterone-induced acrosome reaction was also evaluated. Results indicated that (a) whole, intact sperm were able to hydrolyze the proteasome substrates SLLVY-AMC and azocasein; this activity was inhibited by proteasome inhibitors; (b) proteasomal activity was detected in soluble sperm membrane protein preparations and Western blotting revealed the presence of the proteasome in these fractions; (c) indirect immunofluorescence revealed staining of the head region, particularly of the post acrosomal region; and (d) the proteasome plays an important role during the acrosome reaction.     

Immunolocalization of cubilin, megalin, apolipoprotein J, and apolipoprotein A-I in the uterus and oviduct

Spermatozoa maturation and capacitation occurring in the male and female reproductive tracts, respectively, involves the remodeling of the spermatozoa plasma membrane. Apolipoprotein J (apoJ) and apolipoprotein A-I (apoA-I) have been implicated in the process of lipid exchange from the spermatozoa plasma membrane to epithelial cells lining the male reproductive tract. Evidence suggests that this process is mediated by the cooperative action of the endocytic lipoprotein receptors megalin and cubilin, which are expressed at the apical surface of absorptive epithelia in various tissues, including the efferent ducts and epididymis. Here, we investigated the possibility that these receptors and their lipid-binding ligands, apoJ and apoA-I, might function similarly in the female reproductive tract. We show that megalin and cubilin are expressed in the uterine epithelium at all stages of the estrous cycle, maximally during estrous and metestrous stages. In the oviduct, there is pronounced expression of both megalin and cubilin in the nonciliated cells of the proximal oviduct and epithelial cells of the distal oviduct, particularly during estrous and metestrous stages. In both uterine and oviduct epithelial cells, megalin and cubilin were located on the apical regions of the cells, consistent with a distribution at the cell surface and in endosomes. ApoJ and apoA-I were both detected in apical regions of uterine and oviduct epithelial cells. Secretory cells of the uterine glands were found to express apoJ and apoA-I suggesting that the glands are a site of synthesis for both proteins. In summary, our findings indicate that megalin and cubilin function within the female reproductive tract, possibly mediating uterine and oviduct epithelial cell endocytosis of apoJ/apoA-I-lipid complexes and thus playing a role in lipid efflux from the sperm plasma membrane, a major initiator of capacitation.     

Oxidative stress and antioxidant defenses after prolonged starvation in Dentex dentex liver

The aim of this work was to evaluate the effects of prolonged starvation and refeeding on antioxidant status and some metabolic-related parameters in common dentex (Dentex dentex) liver. Fish deprived of food for 5 weeks showed a significant increase in lipid peroxidation, measured as malondialdehyde (MDA) levels. The activity of the antioxidative enzymes superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPX) in starved fish significantly increased (by 42%, 22%, and 52%, respectively), whereas glutathione reductase (GR) activity was significantly depressed by 53% compared to controls. No qualitative changes in the SOD isoenzymatic pattern were detected by nondenaturing PAGE analysis, but the isoforms corresponding to CuZn-SOD I and II were enhanced in starved fish. The activity of the enzymes indicative of oxidative metabolism, beta-hydroxyacyl CoA dehydrogenase (HOAD) and citrate synthase (CS), significantly increased (by 123% and 28%, respectively), and that of glucose-6-phosphate dehydrogenase (G6PDH) was inhibited by 56%. Oxidative damage under these circumstances is reversible since all biomarkers assayed returned to control values after refeeding. Our results show that prolonged starvation leads to a pro-oxidant situation and oxidative stress despite activation of antioxidant defense mechanisms, and that inhibition of G6PDH activity might be responsible for this failure in cellular antioxidant defenses.     

The Mre11 complex and the metabolism of chromosome breaks: the importance of communicating and holding things together

The conserved Mre11 complex (Mre11, Rad50, and Nbs1) plays a role in each aspect of chromosome break metabolism. The complex acts as a break sensor and functions in the activation and propagation of signaling pathways that govern cell cycle checkpoint functions in response to DNA damage. In addition, the Mre11 complex influences recombinational DNA repair through promoting recombination between sister chromatids. The Mre11 complex is required for mammalian cell viability but hypomorphic mutants of Mre11 and Nbs1 have been identified in human genetic instability disorders. These hypomorphic mutations, as well as those identified in yeast, have provided a benchmark for establishing mouse models of Mre11 complex deficiency. In addition to consideration of Mre11 complex functions in human cells and yeast, this review will discuss the characterization of mouse models and insight gleaned from those models regarding the metabolism of chromosome breaks. The current picture of break metabolism supports a central role for the Mre11 complex at the interface of chromosome stability and the regulation of cell growth. Further genetic analysis of the Mre11 complex will be an invaluable tool for dissecting its function on an organismal level and determining its role in the prevention of malignancy.     

An oestrogen membrane receptor participates in estradiol actions for the prevention of amyloid-beta peptide1-40-induced toxicity in septal-derived cholinergic SN56 cells

Although oestrogen [17 beta-estradiol (E2)]-related neuroprotection has been demonstrated in different models, the involvement of non-classical oestrogen receptors (ERs) remains unexplored. Using the SN56 cholinergic cell line, we present evidence indicating that an ER associated with the plasma membrane participates in oestrogen-dependent inhibition of cell death induced by amyloid-beta peptide (A beta) toxicity. Similarly to E2 alone, a 15-min exposure to estradiol-horseradish peroxidase (E-HRP) significantly reduced A beta-induced cell death. This effect was decreased by the ER antagonist ICI 182,780 as well as by MC-20 antibody directed to a region neighbouring the ligand-binding domain of ER alpha. Using confocal microscopy on unpermeabilized SN56 cells exposed to MC-20 antibody, we identified a protein at the plasma membrane level. Western blot analysis of purified SN56 cell membrane fractions using MC-20 antibody revealed the presence of one band with the same electrophoretic mobility as intracellular ER alpha. Using conjugated forms of the steroid, E-HRP and E2 conjugated to bovine serum albumin-FITC, we demonstrated by confocal microscopy that SN56 cells contain surface binding sites for E2. Binding of both conjugates was blocked by pre-incubation with E2 and decreased by either ICI 182,780 or MC-20 antibody in a concentration-dependent manner. Thus, a membrane-related ER that shares some structural homologies with ER alpha may participate in oestrogen-mediated neuroprotection.     

Progesterone receptor gene and protein expression in the anterior preoptic area and hypothalamus of defeminized rats

Progesterone receptor (PR) plays an important role during sexual differentiation of the rat brain. The objective of the present study was to determine PR protein and gene expression pattern in preoptic-anterior hypothalamic area (POA-AHA) and hypothalamus (HYP), after estradiol or testosterone treatment during the postnatal critical period of sexual differentiation of the rat brain (defeminized animals). Three-day-old female rats were subcutaneously (s.c.) injected with a single dose of 17beta-estradiol (200 microg), or testosterone enanthate (200 microg), or vehicle (corn oil). POA-AHA and HYP were dissected 3 h, 24 h, and 14 days, as well as on the day of vaginal opening (VO) after treatments. Other animals, previously treated as above, were acutely injected with 17beta-estradiol (5 microg) on the day of VO; POA-AHA and HYP were obtained 3 h later. Total RNA was extracted and processed for semiquantitative RT-PCR and tissue slices were prepared for protein detection by immunohistochemistry. We observed that PR mRNA expression was increased in POA-AHA and HYP of the animals treated with estradiol or testosterone 3 hours after treatments, compared with the vehicle-treated control group. We also found a significant increase in PR mRNA and protein expression in POA-AHA and HYP on the day of VO in both estradiol and testosterone defeminized rats. Interestingly, the acute administration of estradiol on the day of VO (VO + E(2)) did not increase PR mRNA or protein expression in POA-AHA and HYP of either estradiol or testosterone defeminized animals, as opposed to the marked induction observed in the intact animals of the control group. The overall results suggest that estradiol and testosterone treatment during the postnatal critical period of sexual differentiation of the brain modifies the regulation of the PR mRNA and protein expression during early onset of maturity.     

P-glycoprotein-like protein contributes to cadmium resistance in<Emphasis Type="Italic"> Euglena gracilis</Emphasis>

Selective pressures from polluted environments have led to the development of resistance systems in aquatic organisms. Using different techniques, this study examined a cadmium defense mechanism of the freshwater unicellular protozoa Euglena gracilis, and found it to be an efflux pump similar to the multidrug resistance P-glycoprotein. Cd²⁺-treated E. gracilis were able to extrude Rhodamine 123 at 21 °C, but not at 4 °C. Furthermore, verapamil, a P-glycoprotein modulator, partially blocked the efflux process (at 21 °C), and enhanced the Cd²⁺ toxic effects on these cells. Western immunoblots of cell lysates, using the anti-P-glycoprotein antibody JSB-1, revealed a 120-KDa protein, which was expressed, in high amounts on Cd²⁺-exposed cells (74% above the control values). Moreover, cells treated with JSB-1 became more sensitive to the harmful effects of cadmium, showing a decreased survival rate. Taken together, these results suggest that a MDR phenotype has evolved in Euglena as one of the mechanisms for cadmium detoxification.Abbreviations DTT dithiothreitol - mAb JSB-1 anti-human P-gp monoclonal antibody JSB-1 - MDR multidrug resistance - MRP MDR-associated protein - PBS phosphate buffered saline - P-gp P-glycoproteinCommunicated by G. HeldmaierA.J.F.O. and F.L.S.S. are undergraduate students under a CNPq special program for research training     

Caseinolysis in cheese by Enterobacteriaceae strains of dairy origin

AIMS: To study the effect of Enterobacteriaceae strains of dairy origin on caseins under cheese manufacture and ripening conditions. METHODS AND RESULTS: Strains belonging to the genera Enterobacter, Escherichia, Hafnia and Serratia were isolated from fresh raw milk cheeses. Residual caseins in cheeses made from milk individually inoculated with 10 strains of Enterobacteriaceae were determined by capillary electrophoresis. Hierarchical cluster analysis of strains based on data of residual caseins grouped together strains from the same genus, excepting Hafnia strains, which were separated into two groups. Serratia was the most proteolytic genus in our study. Preferences for degradation of casein fractions differed among the four genera studied. CONCLUSIONS: Enterobacteriaceae strains posses proteolytic systems active on all casein fractions under cheese manufacture and ripening conditions. The effects on caseins were similar for strains belonging to the same genus. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of Enterobacteriaceae in cheeses may affect proteolysis during ripening. Assays of Enterobacteriaceae proteolytic activity on milk agar plates may underestimate their caseinolytic activity in cheese.