The laminin-induced acrosome reaction in human sperm is mediated by Src kinases and the proteasome

The aim of this work was to determine whether laminin (Ln), an extracellular matrix protein, induces the intracellular events that may be involved in producing the acrosome reaction in human sperm. To this end, we evaluated the effect of Ln on tyrosine phosphorylation, intracellular calcium concentration, proteasome activity, and phosphorylation in human sperm. Aliquots of highly motile sperm selected with a Percoll gradient, were incubated with different concentrations of Ln (0-20 μg/ml) for different periods (0-18 h). The percentage of viable acrosome-reacted sperm was evaluated using fluorescein isothiocyanate-labeled Pisum sativum agglutinin and Hoechst 33258 DNA dye. Tyrosine phosphorylation was evaluated by Western blot analysis. The chymotrypsin-like activity of the proteasome was evaluated with a fluorogenic peptide, and intracellular calcium concentration was measured with fura-2. The results indicate that Ln stimulated the acrosome reaction of human sperm in a dose-dependent manner. This increase was drastically inhibited in the presence of herbimycin A, SU6656, and epoxomicin. In addition, Ln increased proteasome activity and phosphorylation; both events were inhibited by herbimycin A and SU6656. Finally, Ln induced an increase in intracellular calcium concentration, which was inhibited by SU6656 and epoxomicin. These results suggest that Ln is able to induce the acrosome reaction. This effect may be mediated by Src kinase and the proteasome, with the consequent induction of a calcium influx.     

Influence of trophic status and conductivity on zooplankton composition in lakes and ponds of Torres del Paine National Park (Chile)

The Torres del Paine National Park is located in the southern Chilean Patagonia. This park has numerous and heterogeneous lakes and ponds with different trophic status and zooplankton composition. The aim of this study was to analyze the influence of trophic status and conductivity on zooplankton assemblages in lakes and ponds within the Torres del Paine National Park. The water bodies described in the present study were previously classified in three groups. The first group consisted of large, deep and oligotrophic lakes with fish populations, low zooplankton species diversity and high predominance of calanoid copepods of small body size. The second group contained small mesotrophic lakes with fish populations and relatively high predominance of small sized daphnids. The third group consisted of fishless ponds of different trophic status, wide conductivity gradient and with zooplankton species of relatively large body size. The results show that Daphnids abundance was directly related to chlorophyll-a concentration and inversely associated with conductivity. Calanoids abundance was also directly associated with conductivity.     

Overexpression of MEOX2 and TWIST1 Is Associated with H3K27me3 Levels and Determines Lung Cancer Chemoresistance and Prognosis

Lung cancer is the leading cause of death from malignant diseases worldwide, with the non-small cell (NSCLC) subtype accounting for the majority of cases. NSCLC is characterized by frequent genomic imbalances and copy number variations (CNVs), but the epigenetic aberrations that are associated with clinical prognosis and therapeutic failure remain not completely identify. In the present study, a total of 55 lung cancer patients were included and we conducted genomic and genetic expression analyses, immunohistochemical protein detection, DNA methylation and chromatin immunoprecipitation assays to obtain genetic and epigenetic profiles associated to prognosis and chemoresponse of NSCLC patients. Finally, siRNA transfection-mediated genetic silencing and cisplatinum cellular cytotoxicity assays in NSCLC cell lines A-427 and INER-37 were assessed to describe chemoresistance mechanisms involved. Our results identified high frequencies of CNVs (66–51% of cases) in the 7p22.3–p21.1 and 7p15.3–p15.2 cytogenetic regions. However, overexpression of genes, such as MEOX2, HDAC9, TWIST1 and AhR, at 7p21.2–p21.1 locus occurred despite the absence of CNVs and little changes in DNA methylation. In contrast, the promoter sequences of MEOX2 and TWIST1 displayed significantly lower/decrease in the repressive histone mark H3K27me3 and increased in the active histone mark H3K4me3 levels. Finally these results correlate with poor survival in NSCLC patients and cellular chemoresistance to oncologic drugs in NSCLC cell lines in a MEOX2 and TWIST1 overexpression dependent-manner. In conclusion, we report for the first time that MEOX2 participates in chemoresistance irrespective of high CNV, but it is significantly dependent upon H3K27me3 enrichment probably associated with aggressiveness and chemotherapy failure in NSCLC patients, however additional clinical studies must be performed to confirm our findings as new probable clinical markers in NSCLC patients.     

A protein phosphatase 1 gamma (PP1γ) of the human protozoan parasite Trichomonas vaginalis is involved in proliferation and cell attachment to the host cell

In this work, evidence for a critical role of Trichomonas vaginalis protein phosphatase 1 gamma (TvPP1γ) in proliferation and attachment of the parasite to the mammalian cell is provided. Firstly, proliferation and attachment of T. vaginalis parasites to HeLa cells was blocked by calyculin A (CA), a potent PP1 inhibitor. Secondly, it was demonstrated that the enzyme activity of native and recombinant TvPP1γ proteins was inhibited by CA. Thirdly, reverse genetic studies confirmed that antisense oligonucleotides targeted to PP1γ but not PP1α or β inhibited proliferation and attachment of trichomonads CA-treated parasites underwent cytoskeletal modifications, including a lack of axostyle typical labelling, suggesting that cytoskeletal phosphorylation could be regulated by a CA-sensitive phosphatase where the role of PP1γ could not be ruled out. Analysis of subcellular distribution of TvPP1γ by cell fractionation and electron microscopy demonstrated the association between TvPP1γ and the cytoskeleton. The expression of adhesins, AP120 and AP65, at the cell surface was also inhibited by CA. The concomitant inhibition of expression of adhesins and changes in the cytoskeleton in CA-treated parasites suggest a specific role for PP1γ -dependent dephosphorylation in the early stages of the host-parasite interaction. Molecular modelling of TvPP1γ showed the conservation of residues critical for maintaining proper folding into the gross structure common to PP1 proteins. Taken together, these results suggest that TvPP1γ could be considered a potential novel drug target for treatment of trichomoniasis.     

Impact of supramolecular interactions of dextran‐β‐cyclodextrin polymers on invertase activity in freeze‐dried systems

β‐Cyclodextrin (β‐CD)‐grafted dextrans with spacer arms of different length were employed to evaluate the impact of supramolecular interactions on invertase activity. The modified dextrans were used as single additives or combined with trehalose in freeze‐dried formulations containing invertase. Enzyme activity conservation was analyzed after freeze‐drying and thermal treatment. The change of glass transition temperature (T_g) was also evaluated and related to effective interactions. Outstanding differences on enzyme stability were mainly related to the effect of the spacer arm length on polymer–enzyme interactions, since both the degree of substitution and the molecular weight were similar for the two polymers. This change of effective interactions was also manifested in the pronounced reduction of T_g values, and were related to the chemical modification of the backbone during oxidation, and to the attachment of the β‐CD units with spacer arms of different length on dextran. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:791–798, 2015     

Loss of sexual dimorphism is associated with loss of lekking behavior in the green manakin Xenopipo holochora

Manakins (Pipridae) are well know for elaborate male sexual displays and ornate plumage coloration, both of which are thought to have evolved as a consequence of lekking breeding, the prevalent mating system in the family. Less attention has been paid to a handful of ‘drab’ manakin species, in which sexual dimorphism appears to be reduced or absent. Using character reconstruction, we show that these ‘exceptions to the rule’ represent phylogenetically independent cases of losses in sexual dimorphism, and as such could provide a focal group to investigate the link between changes in morphology and in life history (e.g. mating system). We take a first step in this direction by focusing on two subspecies of the putatively monomorphic green manakin Xenopipo holochlora to formally confirm that the species is sexually monomorphic in size and plumage color and test the prediction that sexual monomorphism is associated with the loss of lekking behavior in this species. Our results show that size dimorphism is present but limited in the green manakin, with substantial overlap in male and female morphometric measures, and that sexes are largely monochromatic (including from an avian perspective), despite marked coloration differences between subspecies. Behavioral observations indicate that males do not form leks and do not engage in elaborate sexual displays, that there is no stable pair bond formation, and that females provide parental care alone. These findings are consistent with the idea that changes in mating behavior may have driven changes in morphology in Pipridae, and we encourage similar studies on other drab manakins to better understand this relationship.     

Cyclic AMP stimulates ouabain-insensitive ion movement in shark rectal gland

Summary When the active sodium-potassium pump (Na–K-ATPase) of shark rectal glands is blocked by ouabain, the concentration of intracellular ions changes in the direction of equilibrium with extracellular fluid. These changes were examined when isolated perfused glands were in the basal state and also when they were stimulated to secrete with cAMP and theophylline, to see whether stimulation affected the passive movement of sodium, potassium and chloride across cell membranes. In basal glands 10^–4M ouabain induced an increase of 30 meq/l in intracellular [Na⁺] and a decrease in intracellular [K⁺] of about 50 meq/l after 30 min, while intracellular [Cl^–] was unchanged. In stimulated glands, these movements were exaggerated. The increase in intracellular [Na⁺] averaged 112 meq/l, and the decrease in intracellular [K⁺], 96 meq/l (P<0.01), while mean intracellular [Cl^–] rose by 80 meq/l. Furosemide, 10^–4M, partially reversed the accelerated changes in intracellular electrolytes seen after ouabain was added to stimulated glands. These results are consistent with an action of cAMP upon a ouabaininsensitive cotransport of sodium, potassium and chloride in the rectal gland, analogous to that described in avian erythrocytes.Some of these results have been previously reported in abstract form in Bull Mt Desert Isl Biol Lab (Silva et al. 1979a).     

Aromatic glycosyl disulfide derivatives: Evaluation of their inhibitory activities against Trypanosoma cruzi

Aromatic oligovalent glycosyl disulfides and some diglycosyl disulfides were tested against three different Trypanosoma cruzi strains. Di-(β-d-galactopyranosyl-dithiomethylene) benzenes 2b and 4b proved to be the most active derivatives against all three strains of cell culture-derived trypomastigotes with IC₅₀ values ranging from 4 to 11 μM at 37 °C. The inhibitory activities were maintained, although somewhat lowered, at a temperature of 4 °C as well. Three further derivatives displayed similar activities against at least one of the three strains. Low cytotoxicities of the active compounds, tested on confluent HeLa, Vero and peritoneal macrophage cell cultures, resulted in significantly higher selectivity indices (SI) than that of the reference drug benznidazole. Remarkably, several molecules of the tested panel strongly inhibited the parasite release from T. cruzi infected HeLa cell cultures suggesting an effect against the intracellular development of T. cruzi amastigotes as well.     

Experimentally Approaching the Solvent-Accessible Surface Area of a Protein: Insights into the Acid Molten Globule of Bovine α-Lactalbumin

Each conformational state of a protein is inextricably related to a defined extent of solvent exposure that plays a key role in protein folding and protein interactions. However, accurate measurement of the solvent-accessible surface area (ASA) is difficult for any state other than the native (N) state. We address this fundamental physicochemical parameter through a new experimental approach based on the reaction of the photochemical reagent diazirine (DZN) with the polypeptide chain. By virtue of its size, DZN is a reasonable molecular mimic of aqueous solvent. Here, we structurally characterize nonnative states of the paradigmatic protein α-lactalbumin. Covalent tagging resulting from unspecific methylene (:CH₂) reaction allows one to obtain a global estimate of ASA and to map out solvent accessibility along the amino acid sequence. By its mild apolar nature, DZN also reveals a hydrophobic phase in the acid-stabilized state of α-lactalbumin, in which there is clustering of core residues accessible to the solvent. In a fashion reminiscent of the N state, this acid-stabilized state also exhibits local regions where increased :CH₂ labeling indicates its nonhomogenous nature, likely pointing to the existence of packing defects. By contrast, the virtual absence of a defined long-range organization brings about a featureless labeling pattern for the unfolded state. Overall, :CH₂ labeling emerges as a fruitful technique that is able to quantify the ASA of the polypeptide chain, thus probing conformational features such as the outer exposed surface and inner cavities, as well as revealing the existence of noncompact apolar phases in nonnative states.