Immunohistochemical localization of proliferating cell nuclear antigen (PCNA) in the pig ovary

The aim of the study was to determine the expression of proliferating cell nuclear antigen protein (PCNA) in the pig ovary. The localization of PCNA was demonstrated in paraffin sections of pig ovarian tissue using primary mouse monoclonal anti-PCNA antibody. In primordial follicles, no remarkable staining for PCNA either in granulosa cells or in the oocytes was observed. In primary to secondary follicles, positive staining in oocytes and in some granulosa cells was detected. The advanced preantral and particularly actively growing small to large antral follicles showed extensive PCNA labeling in the layers of granulosa and theca cells and in the cumulus cells encircling the oocyte. PCNA labeling was expressed in nuclei of oocytes in preantral and small antral follicles. In atretic follicles, the level of PCNA protein expression was dependent on the stage of atresia. Follicles demonstrating advanced atresia showed only limited or no PCNA labeled granulosa and theca cells. The results of the study demonstrate that follicular growth and development in pig ovary may be effectively monitored by determining the granulosa cell expression of PCNA.     

Structural and functional characterization of a plant S-nitrosoglutathione reductase from Solanum lycopersicum

S-nitrosoglutathione reductase (GSNOR), also known as S-(hydroxymethyl)glutathione (HMGSH) dehydrogenase, belongs to the large alcohol dehydrogenase superfamily, namely to the class III ADHs. GSNOR catalyses the oxidation of HMGSH to S-formylglutathione using a catalytic zinc and NAD⁺ as a coenzyme. The enzyme also catalyses the NADH-dependent reduction of S-nitrosoglutathione (GSNO). In plants, GSNO has been suggested to serve as a nitric oxide (NO) reservoir locally or possibly as NO donor in distant cells and tissues. NO and NO-related molecules such as S-nitrosothiols (S-NOs) play a central role in the regulation of normal plant physiological processes and host defence. The enzyme thus participates in the cellular homeostasis of S-NOs and in the metabolism of reactive nitrogen species. Although GSNOR has recently been characterized from several organisms, this study represents the first detailed biochemical and structural characterization of a plant GSNOR, that from tomato (Solanum lycopersicum). SlGSNOR gene expression is higher in roots and stems compared to leaves of young plants. It is highly expressed in the pistil and stamens and in fruits during ripening. The enzyme is a dimer and preferentially catalyses reduction of GSNO while glutathione and S-methylglutathione behave as non-competitive inhibitors. Using NAD⁺, the enzyme oxidizes HMGSH and other alcohols such as cinnamylalcohol, geraniol and ω-hydroxyfatty acids. The crystal structures of the apoenzyme, of the enzyme in complex with NAD⁺ and in complex with NADH, solved up to 1.9 Å resolution, represent the first structures of a plant GSNOR. They confirm that the binding of the coenzyme is associated with the active site zinc movement and changes in its coordination. In comparison to the well characterized human GSNOR, plant GSNORs exhibit a difference in the composition of the anion-binding pocket, which negatively influences the affinity for the carboxyl group of ω-hydroxyfatty acids.     

Susceptibility of Prussian carp infected by metacercariae of Posthodiplostomum cuticola (v. Nordmann, 1832) to fish predation

The susceptibility of prey fish infected by metacercariae of Posthodiplostomum cuticola (Digenea: Diplostomatidae) to the predation of non-host predators under experimental conditions was investigated. Parasitized young-of-the-year Prussian carp Carassius auratus were consumed significantly more often by perch Perca fluviatilis compared to non-parasitized individuals, independent of Prussian carp density. The proportion of parasitized and non-parasitized fish consumed by the predator remained stable at four different prey densities. The probability of predation did not increase with the intensity of parasite infection. The effect of P. cuticola on the host seems to result mostly from pathological changes (poor condition, black spots). However, our results provide evidence of higher probability of parasitized Prussian carp being consumed by fish predators under experimental conditions.     

Blending problem-based learning with Web technology positively impacts student learning outcomes in acid-base physiology

World Wide Web (Web)-based learning (WBL), problem-based learning (PBL), and collaborative learning are at present the most powerful educational options in higher education. A blended (hybrid) course combines traditional face-to-face and WBL approaches in an educational environment that is nonspecific as to time and place. To provide educational services for an undergraduate second-year elective course in acid-base physiology, a rich, student-centered educational Web-environment designed to support PBL was created by using Web Course Tools courseware. The course is designed to require students to work in small collaborative groups using problem solving activities to develop topic understanding. The aim of the study was to identify the impact of the blended WBL-PBL-collaborative learning environment on student learning outcomes. Student test scores and satisfaction survey results from a blended WBL-PBL-based test group (n = 37) were compared with a control group whose instructional opportunities were from a traditional in-class PBL model (n = 84). WBL students scored significantly (t = 3.3952; P = 0.0009) better on the final acid-base physiology examination and expressed a positive attitude to the new learning environment in the satisfaction survey. Expressed in terms of a difference effect, the mean of the treated group (WBL) is at the 76th percentile of the untreated (face-to-face) group, which stands for a "medium" effect size. Thus student progress in the blended WBL-PBL collaborative environment was positively affected by the use of technology.     

Small fish use the hypoxic pelagic zone as a refuge from predators

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Structural Basis for the Site-Specific Incorporation of Lysine Derivatives into Proteins

Posttranslational modifications (PTMs) of proteins determine their structure-function relationships, interaction partners, as well as their fate in the cell and are crucial for many cellular key processes. For instance chromatin structure and hence gene expression is epigenetically regulated by acetylation or methylation of lysine residues in histones, a phenomenon known as the ‘histone code’. Recently it was shown that these lysine residues can furthermore be malonylated, succinylated, butyrylated, propionylated and crotonylated, resulting in significant alteration of gene expression patterns. However the functional implications of these PTMs, which only differ marginally in their chemical structure, is not yet understood. Therefore generation of proteins containing these modified amino acids site specifically is an important tool. In the last decade methods for the translational incorporation of non-natural amino acids using orthogonal aminoacyl-tRNA synthetase (aaRS):tRNAaaCUA pairs were developed. A number of studies show that aaRS can be evolved to use non-natural amino acids and expand the genetic code. Nevertheless the wild type pyrrolysyl-tRNA synthetase (PylRS) from Methanosarcina mazei readily accepts a number of lysine derivatives as substrates. This enzyme can further be engineered by mutagenesis to utilize a range of non-natural amino acids. Here we present structural data on the wild type enzyme in complex with adenylated ε-N-alkynyl-, ε-N-butyryl-, ε-N-crotonyl- and ε-N-propionyl-lysine providing insights into the plasticity of the PylRS active site. This shows that given certain key features in the non-natural amino acid to be incorporated, directed evolution of this enzyme is not necessary for substrate tolerance.     

Influence of 1,2-alkanediols on the structure of their intercalates with strontium phenylphosphonate solved by molecular simulation and experimental methods

Strontium phenylphosphonate intercalates with 1,2-diols (from 1,2-ethanediol to 1,2-hexanediol) were synthesized and characterized by X-ray diffraction, thermogravimetry, chemical analysis, and molecular simulation methods. Prepared samples exhibit a very good stability at ambient conditions. Structural arrangement calculated by simulation methods suggested formation of cavities surrounded by six benzene rings. Each cavity contained one molecule of diol and one molecule of water for the 1,2-ethanediol to 1,2-butanediol intercalates. In the case of 1,2-pentanediol two types of cavities alternated: one with diol molecules and another one with two water molecules. In the 1,2-hexanediol intercalate the benzene rings created two types of cavities containing one or two diol molecules, respectively, and this conformational variability led to a more disordered arrangement with respect to the models with shorter alkyl chains. Coordination of the oxygen atoms of the diols to the strontium atoms of the host follows the same pattern for all 1,2-diol intercalates except the 1,2-hexanediol intercalate, where these oxygen atoms can be mutually exchanged at their positions. The calculated basal spacings and structural models are in good agreement with experimental basal spacings obtained from X-ray powder diffraction and with other experimental results.