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.     

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.     

Roadsides provide refuge for orchids: characteristic of the surrounding landscape

Seminatural habitats are declining throughout the world; thus, the role of small anthropogenic habitats in the preservation of plants is becoming increasingly appreciated. Here, we surveyed the orchid flora of roadside verges in five Central European countries (Austria, Hungary, Romania, Slovakia, and Slovenia) and tested how the surrounding landscape matrix affects the overall number of species and individuals, and also different functional groups of orchids. We found more than 2,000 individuals of 27 orchid species during our surveys. According to our results, the increasing coverage of agricultural and urban areas negatively affects both the number of orchid species and individuals on roadsides. Our study further suggests that differences in the surrounding habitats affect which species are found on roadsides, since the increasing coverage of grasslands or forested areas around orchid occurrences had a significant positive effect on the number of grassland or forest‐dwelling species and individuals, respectively. Most variance in orchid numerosity and diversity was explained by the cover of the suitable habitat types of the respective taxa in the surrounding landscape of the sampling points. This highlights the importance of roadsides acting as refugia for numerous species and valuable plant communities as well as in supporting biodiversity in general.     

Poxvirus Antigen Staining of Immune Cells as a Biomarker to Predict Disease Outcome in Monkeypox and Cowpox Virus Infection in Non-Human Primates

Infection of non-human primates (NHPs) such as rhesus and cynomolgus macaques with monkeypox virus (MPXV) or cowpox virus (CPXV) serve as models to study poxvirus pathogenesis and to evaluate vaccines and anti-orthopox therapeutics. Intravenous inoculation of macaques with high dose of MPXV (>1–2×10⁷ PFU) or CPXV (>10² PFU) results in 80% to 100% mortality and 66 to 100% mortality respectively. Here we report that NHPs with positive detection of poxvirus antigens in immune cells by flow cytometric staining, especially in monocytes and granulocytes succumbed to virus infection and that early positive pox staining is a strong predictor for lethality. Samples from four independent studies were analyzed. Eighteen NHPs from three different experiments were inoculated with two different MPXV strains at lethal doses. Ten NHPs displayed positive pox-staining and all 10 NHPs reached moribund endpoint. In contrast, none of the three NHPs that survived anticipated lethal virus dose showed apparent virus staining in the monocytes and granulocytes. In addition, three NHPs that were challenged with a lethal dose of MPXV and received cidofovir treatment were pox-antigen negative and all three NHPs survived. Furthermore, data from a CPXV study also demonstrated that 6/9 NHPs were pox-antigen staining positive and all 6 NHPs reached euthanasia endpoint, while the three survivors were pox-antigen staining negative. Thus, we conclude that monitoring pox-antigen staining in immune cells can be used as a biomarker to predict the prognosis of virus infection. Future studies should focus on the mechanisms and implications of the pox-infection of immune cells and the correlation between pox-antigen detection in immune cells and disease progression in human poxviral infection.     

Nonhuman Transferrin Receptor 1 Is an Efficient Cell Entry Receptor for Ocozocoautla de Espinosa Virus

Ocozocoautla de Espinosa virus (OCEV) is a novel, uncultured arenavirus. We found that the OCEV glycoprotein mediates entry into grivet and bat cells through transferrin receptor 1 (TfR1) binding but that OCEV glycoprotein precursor (GPC)-pseudotyped retroviruses poorly entered 53 human cancer cell lines. Interestingly, OCEV and Tacaribe virus could use bat, but not human, TfR1. Replacing three human TfR1 amino acids with their bat ortholog counterparts transformed human TfR1 into an efficient OCEV and Tacaribe virus receptor.     

Insights into β-Lactamases from Burkholderia Species,Two Phylogenetically Related yet Distinct Resistance Determinants

Burkholderia cepacia complex and Burkholderia pseudomallei are opportunistic human pathogens. Resistance to β-lactams among Burkholderia spp. is attributable to expression of β-lactamases (e.g. PenA in B. cepacia complex and PenI in B. pseudomallei). Phylogenetic comparisons reveal that PenA and PenI are highly related. However, the analyses presented here reveal that PenA is an inhibitor-resistant carbapenemase, most similar to KPC-2 (the most clinically significant serine carbapenemase), whereas PenI is an extended spectrum β-lactamase. PenA hydrolyzes β-lactams with k_cat values ranging from 0.38 ± 0.04 to 460 ± 46 s⁻¹ and possesses high k_cat/k_inact values of 2000, 1500, and 75 for β-lactamase inhibitors. PenI demonstrates the highest k_cat value for cefotaxime of 9.0 ± 0.9 s⁻¹. Crystal structure determination of PenA and PenI reveals important differences that aid in understanding their contrasting phenotypes. Changes in the positioning of conserved catalytic residues (e.g. Lys-73, Ser-130, and Tyr-105) as well as altered anchoring and decreased occupancy of the deacylation water explain the lower k_cat values of PenI. The crystal structure of PenA with imipenem docked into the active site suggests why this carbapenem is hydrolyzed and the important role of Arg-220, which was functionally confirmed by mutagenesis and biochemical characterization. Conversely, the conformation of Tyr-105 hindered docking of imipenem into the active site of PenI. The structural and biochemical analyses of PenA and PenI provide key insights into the hydrolytic mechanisms of β-lactamases, which can lead to the rational design of novel agents against these pathogens.