Apical Scaffolding Protein NHERF2 Modulates the Localization of Alternatively Spliced Plasma Membrane Ca2+ Pump 2B Variants in Polarized Epithelial Cells

The membrane localization of the plasma membrane Ca²⁺-ATPase isoform 2 (PMCA2) in polarized cells is determined by alternative splicing; the PMCA2w/b splice variant shows apical localization, whereas the PMCA2z/b and PMCA2x/b variants are mostly basolateral. We previously reported that PMCA2b interacts with the PDZ protein Na⁺/H⁺ exchanger regulatory factor 2 (NHERF2), but the role of this interaction for the specific membrane localization of PMCA2 is not known. Here we show that co-expression of NHERF2 greatly enhanced the apical localization of GFP-tagged PMCA2w/b in polarized Madin-Darby canine kidney cells. GFP-PMCA2z/b was also redirected to the apical membrane by NHERF2, whereas GFP-PMCA2x/b remained exclusively basolateral. In the presence of NHERF2, GFP-PMCA2w/b co-localized with the actin-binding protein ezrin even after disruption of the actin cytoskeleton by cytochalasin D or latrunculin B. Surface biotinylation and fluorescence recovery after photobleaching experiments demonstrated that NHERF2-mediated anchorage to the actin cytoskeleton reduced internalization and lateral mobility of the pump. Our results show that the specific interaction with NHERF2 enhances the apical concentration of PMCA2w/b by anchoring the pump to the apical membrane cytoskeleton. The data also suggest that the x/b splice form of PMCA2 contains a dominant lateral targeting signal, whereas the targeting and localization of the z/b form are more flexible and not fully determined by intrinsic sequence features.     

Vagal nerve and the gastric mucosal defense

An essential role for an intact vagal nerve has been proven in the development of gastric mucosal cyto- and general protection. On the other hand, chemically-induced (ethanol, HCl, indomethacin) gastric mucosal damage is enhanced after acute surgical vagotomy. The aims of this paper were to study the possible mechanisms of the vagal nerve in the development of gastric mucosal defense. The following questions were addressed: 1) effect of surgical vagotomy on the development of ethanol- (ETOH), HCl-, and indomethacin (IND)-induced gastric mucosal damage; 2) changes in the gastric mucosal defense by scavengers, prostacyclin and other compounds (small doses of atropine and cimetidine: 3) changes in the gastric mucosal vascular permeability due to chemicals; 4) effect of indomethacin in the ETOH and HCl models with and without surgical vagotomy; 5) changes in the gastric mucosal content of prostacyclin and PGE₂ in the ETOH and HCl models after surgical vagotomy; and 6) changes in the role of SH-groups in gastric mucosal defense after surgical vagotomy. It was found that: 1) the gastric mucosal damage produced by chemicals (ETOH, HCl, and indomethacin) was enhanced after surgical vagotomy; 2) the cyto- and general gastric protective effects of β-carotene, prostacyclin, and small doses of atropine and cimetidine disappeared after surgical vagotomy; 3) the vascular permeability due to chemicals (ETOH, HCl, indomethacin) significantly increased after surgical vagotomy in association with an increase in both number and severity of gastric mucosal lesions; 4) IND alone (in animals with an intact vagus) did not produce gastric mucosal lesions (in 1-h experiments), but it aggravated ETOH-induced gastric mucosal damage (both its number and severity); 5) the gastric mucosal levels of prostacyclin and PGE₂ decreased after surgical vagotomy; 6) IND application (after surgical vagotomy) decreased further the tissue levels of prostacyclin and PGE₂ in association with an increase of gastric mucosal damage; and 7) the gastric mucosal protective effects of SH-groups were abolished by surgical vagotomy.     

Influence of luteinizing hormone-releasing hormone agonists on human mammary carcinoma cell lines and their xenografts

The specific binding of luteinizing hormone-releasing hormone (LH-RH) agonist in estradiol-dependent MCF-7 and estradiol-independent MDA-MB-231 human breast cancer cells has been studied using [3H]Ovurelin [(D-3H-Phe6),des-Gly10-LH-RH- ethylamide]. The results of Scatchard analyses suggest the presence of a single class of receptor sites, both in cell suspensions and membrane fractions. Evaluation of these peptide receptors appears to reflect additional characteristics of biological behaviour of these human breast cancer cells. The synthetic LH-RH agonist Ovurelin [(D-Phe6),des-Gly10-LH-RH-ethylamide] can directly interfere (25-30%) with the proliferation of MDA-MB-231 human breast cancer cells in culture. The inhibitory effect of Ovurelin in vitro was negligible in the MCF-7 cell line. In the in vivo experiments the treated immunosuppressed mice bearing either MCF-7 or MDA-MB-231 xenografts responded to the high-dose LH-RH analogue Zoladex depot and Decapeptyl depot therapy. Since the MDA-MB-231 tumour was found to be ER-negative it seems possible that the regression of this xenograft results from the direct antitumor action of the LH-RH agonist.     

Oxidative physiology is weakly associated with pigmentation in birds

The mechanistic link between avian oxidative physiology and plumage coloration has attracted considerable attention in past decades. Hence, multiple proximal hypotheses were proposed to explain how oxidative state might covary with the production of melanin and carotenoid pigments. Some hypotheses underscore that these pigments (or their precursors, e.g., glutathione) have antioxidant capacities or function as molecules storing the toxic excess of intracellular compounds, while others highlight that these pigments can act as pro‐oxidants under specific conditions. Most studies addressing these associations are at the intraspecific level, while phylogenetic comparative studies are still scarce, though needed to assess the generality of these associations. Here, we tested whether plumage and bare part coloration were related to oxidative physiology at an interspecific level by measuring five oxidative physiology markers (three nonenzymatic antioxidants and two markers of lipid peroxidative damage) in 1387 individuals of 104 European bird species sampled during the breeding season, and by scoring plumage eumelanin, pheomelanin, and carotenoid content for each sex and species. Only the plasma level of reactive oxygen metabolites was related to melanin coloration, being positively associated with eumelanin score and negatively with pheomelanin score. Thus, our results do not support the role of antioxidant glutathione in driving variation in melanin synthesis across species. Furthermore, the carotenoid scores of feathers and bare parts were unrelated to the measured oxidative physiology parameters, further suggesting that the marked differences in pigmentation across birds does not influence their oxidative state.     

Methods for Cryopreservation of Guinea Fowl Sperm

Conservation of indigenous poultry species is an important part of the new Hungarian agricultural strategy. Semen cryopreservation is the most practical method for the long term storage of poultry genetic material. The objective was to compare four protocols for cryopreservation of guinea fowl sperm (slow and fast programmable, freezing in nitrogen vapor, and pellet) and three cryoprotectants (10% ethylene glycol, 6% dimethyl-formamide and 6% dimethyl-acetamide). The efficiency of the methods was examined by in vitro tests (subjective motility scoring, sperm concentration, morphological and live/dead sperm analysis with eosin-aniline staining). Thereafter, the two most promising methods were tested by artificial insemination of frozen-thawed semen (3 times a week for 3 weeks using 300 million spermatozoa/hen), followed by candling of incubated eggs, assessment of fertilization, embryonic death, and hatching rate. The survival rate of live, intact spermatozoa was greatest (p≤0.05) in pellet method and the slow programmable protocol (with 10% ethylene glycol) (28.6 and 23.5%). The two best protocols (based on in vitro assessment of post-thaw semen quality) were subsequently tested in vivo with artificial insemination. The pellet method yielded a 64% fertility rate compared to slow protocol with only 30% fertility. Regardless, both freezing protocols significantly increased embryonic deaths compared to the control group (16,7; 9,1 and 8,3%, respectively). During the 3-week in vivo trial, fertility increased and early embryonic death decreased over time. According to the results the guinea fowl sperm could tolerate the fast freezing in pellet better than the slower freezing rates and resulted acceptable fertility rate.     

Trait‐based diatom functional diversity as an appropriate tool for understanding the effects of environmental changes in soda pans

Saline lakes, among the most seriously endangered ecosystems, are threatened due to climate change and human activities. One valuable feature of these environments is that they constitute areas of high biodiversity. Ecologists are, therefore, under great pressure to improve their understanding of the effects of natural and anthropogenic disturbances on the biodiversity of saline lakes. In this study, a total of 257 samples from 32 soda pans in Central Europe between 2006 and 2015 were examined. The effects of environmental variables and of geographical and limnoecological factors on functional diversity were analyzed. Furthermore, the explanatory power of the trait‐based approach was assessed, and the applicability of the indices for biomonitoring purposes was determined. It was found that low habitat heterogeneity and harsh environments lead to the selection of a small number of suitable traits, and consequently, to a naturally low level of functional diversity. Anthropogenic activities enhance diversity at functional level due to the shift toward freshwater characteristics. On the regional scale, the effects of the region and status (natural, degraded, reconstructed) on diatom functional diversity were significant and more pronounced than that of the environmental and other limnoecological factors. The degree of variance found in functional diversity ascribed to environmental variables is five times greater in the case of the application of a trait‐based approach, than when a taxonomic one is employed in the literature. Each of the tested functional diversity indices was sensitive to the most important environmental variables. Furthermore, these were type‐specific and proved to be more complex indicators than taxonomic metrics. It is possible to suggest four functional diversity indices (FGR, FRic, FDis, and FDiv) which emphasize their independence from substrate and seasonal variations for ecological status assessment and conservation planning.