Insights into subtle conformational differences in the substrate-binding loop of fungal 17β-hydroxysteroid dehydrogenase: a combined structural and kinetic approach

The 17β-HSD (17β-hydroxysteroid dehydrogenase) from the filamentous fungus Cochliobolus lunatus (17β-HSDcl) is a NADP(H)-dependent enzyme that preferentially catalyses the interconversion of inactive 17-oxo-steroids and their active 17β-hydroxy counterparts. 17β-HSDcl belongs to the SDR (short-chain dehydrogenase/reductase) superfamily. It is currently the only fungal 17β-HSD member that has been described and represents one of the model enzymes of the cP1 classical subfamily of NADPH-dependent SDR enzymes. A thorough crystallographic analysis has been performed to better understand the structural aspects of this subfamily and provide insights into the evolution of the HSD enzymes. The crystal structures of the 17β-HSDcl apo, holo and coumestrol-inhibited ternary complex, and the active-site Y167F mutant reveal subtle conformational differences in the substrate-binding loop that probably modulate the catalytic activity of 17β-HSDcl. Coumestrol, a plant-derived non-steroidal compound with oestrogenic activity, inhibits 17β-HSDcl [IC50 2.8?μM; at 100?μM substrate (4-oestrene-3,17-dione)] by occupying the putative steroid-binding site. In addition to an extensive hydrogen-bonding network, coumestrol binding is stabilized further by π-π stacking interactions with Tyr212. A stopped-flow kinetic experiment clearly showed the coenzyme dissociation as the slowest step of the reaction and, in addition to the low steroid solubility, it prevents the accumulation of enzyme-coenzyme-steroid ternary complexes.     

Time-course of hypothalamic-pituitary-adrenal axis activity and inflammation in juvenile rat brain after cranial irradiation

Recent studies reported that exposure of juvenile rats to cranial irradiation affects hypothalamic-pituitary-adrenal (HPA) axis stability, leading to its activation along with radiation-induced inflammation. In the present study, we hypothesized whether inflammatory reaction in the CNS could be a mediator of HPA axis response to cranial irradiation (CI). Therefore, we analyzed time-course changes of serum corticosterone level, as well IL-1β and TNF-α level in the serum and hypothalamus of juvenile rats after CI. Protein and gene expression of the glucocorticoid receptor (GR) and nuclear factor kappaB (NFκB) were examined in the hippocampus within 24?h postirradiation interval. Cranial irradiation led to rapid induction of both GR and NFκB mRNA and protein in the hippocampus at 1?h. The increment in NFκB protein persisted for 2?h, therefore NFκB/GR protein ratio was turned in favor of NFκB. Central inflammation was characterized by increased IL-1β in the hypothalamus, with maximum levels at 2 and 4?h after irradiation, while both IL-1β and TNF-α were undetectable in the serum. Enhanced hypothalamic IL-1β probably induced the relocation of hippocampal NFκB to the nucleus and decreased NFκB mRNA at 6?h, indicating promotion of inflammation in the key tissue for HPA axis regulation. Concomitant increase of corticosterone level and enhanced GR nuclear translocation in the hippocampus at 6?h might represent a compensatory mechanism for observed inflammation. Our results indicate that acute radiation response is characterized by increased central inflammation and concomitant HPA axis activation, most likely having a role in protection of the organism from overwhelming inflammatory reaction.     

Effect of age and environmental factors on semen quality, glutathione peroxidase activity and oxidative parameters in simmental bulls

Taking into account that semen quality depends on animal age and climate conditions and that oxidative stress has been reported to be a common cause of infertility, the objective of this study was to monitor indicators of oxidative stress and antioxidant protection during four seasonal periods in service bulls of various age to get better insight into the significance of these factors upon evaluating service bull semen. The research was conducted over a year on 19 Simmental service bulls. Animals were divided into two groups according to age; Group I consisted of younger bulls aged two to four yrs (n = 9), and Group II was comprised of older bulls aged five to ten yrs (n = 10). Semen samples were obtained once in the middle of every seasonal period and blood samples for biochemical analysis were collected by jugular venipuncture immediately after ejaculate collection. The activity of total glutathione peroxidase (T-GSH-Px), selenium-dependent glutathione peroxidase (Se-GSH-Px) and selenium-independent glutathione peroxidase (non-Se-GSH-Px), together with the intensity of lipid peroxidation (thiobarbituric acid reactive substances; TBARS) and oxidative protein damage (protein carbonyl content (PCC)) were measured in seminal plasma. In samples of spermatozoa and blood serum, the activity of Se-GSH-Px and TBARS and PCC concentrations were determined. Older service bulls had significantly higher ejaculate volume in summer in comparison with younger bulls, whereas the number of spermatozoa and progressive motility percentage did not significantly vary with age. Younger animals had lower progressive motility percentage during summer than in spring, with more intensive oxidative processes observed in seminal plasma (TBARS) and spermatozoa (TBARS and PCC). Based on the results presented here, it can be concluded that younger bulls are more sensitive to elevated ambient temperatures during the summer, when intensified prooxidative processes in semen plasma and spermatozoa eventually led to decreased sperm progressive motility with consequential semen quality deterioration.     

Mitochondrial cytochrome <Emphasis Type="Italic">b</Emphasis> sequences resolve the taxonomy of field mice (<Emphasis Type="Italic">Apodemus</Emphasis>) in the western Balkan refugium

Apodemus sylvaticus stankovici, described from the topographically rough landscape of the western Balkan glacial refugium, was recently proposed as being either a junior synonym of Apodemus flavicollis or a species on its own right. To untangle this taxonomic vagueness, we sequenced complete cytochrome b gene in 28 field mice collected at 12 locations in the mountains of Bosnia and Herzegovina, Montenegro, western Macedonia and northern Greece. Samples yielded 27 new haplotypes which clustered into two distinct groups. One of these clades also included the reference haplotype of A. flavicollis, while another cluster emerged as being identical with the reference sample for A. sylvaticus. As is common in Apodemus, both species retrieved in our analysis were characterized by low levels of intraspecific variation (0.4–0.9%) as opposed to a high level of differentiation between them (8.0–10.0%); therefore, the taxonomic classification of our material was without doubt. We found no evidence regarding the presence of an additional cryptic species in the mountains of the western Balkans. The very similar values of genetic variability in the two species imply their common evolutionary history of a long-term coexistence in the western Balkan refugium.     

Steroid-transforming enzymes in fungi

Fungal species are a very important source of many different enzymes, and the ability of fungi to transform steroids has been used for several decades in the production of compounds with a sterane skeleton. Here, we review the characterised and/or purified enzymes for steroid transformations, dividing them into two groups: (i) enzymes of the ergosterol biosynthetic pathway, including data for, e.g. ERG11 (14α-demethylase), ERG6 (C-24 methyltransferase), ERG5 (C-22 desaturase) and ERG4 (C-24 reductase); and (ii) the other steroid-transforming enzymes, including different hydroxylases (7α-, 11α-, 11β-, 14α-hydroxylase), oxidoreductases (5α-reductase, 3β-hydroxysteroid dehydrogenase/isomerase, 17β-hydroxysteroid dehydrogenase, C-1/C-2 dehydrogenase) and C-17-C-20 lyase. The substrate specificities of these enzymes, their cellular localisation, their association with protein super-families, and their potential applications are discussed. Article from a special issue on steroids and microorganisms.     

N-Benzoyl anthranilic acid derivatives as selective inhibitors of aldo-keto reductase AKR1C3

Human aldo-keto reductases AKR1C1-AKR1C3 are involved in the biosynthesis and inactivation of steroid hormones and prostaglandins and thus represent attractive targets for the development of new drugs. We synthesized a series of N-benzoyl anthranilic acid derivatives and tested their inhibitory activity on AKR1C enzymes. Our data show that these derivatives inhibit AKR1C1-AKR1C3 isoforms with low micromolar potency. In addition, five selective inhibitors of AKR1C3 were identified. The most promising inhibitors were compounds 10 and 13, with IC(50) values of 0.31μM and 0.35μM for AKR1C3, respectively.     

Extensive diversity of intestinal trichomonads of non-human primates

Despite the fact that the non-human primates are our closest relatives and represent a species-rich mammalian group, little is known about their intestinal protistan parasites/commensals. Particularly, the intestinal trichomonads represent a neglected part of the fauna of the primate digestive system. We have established 30 trichomonad strains isolated from feces of 11 primate species kept in 3 Czech zoos and performed an analysis of their SSU rDNA and ITS1-5·8S rDNA-ITS2. Our results showed that intestinal trichomonads are rather common among non-human primates. Molecular phylogenetic analysis showed that the strains are unexpectedly diversified, belonging to 8 or 9 distinct species. Interestingly, the vast majority of the strains from non-human primates belonged to the genus Tetratrichomonas while no member of this genus has been found in the human intestine so far. In addition, hominoid and non-hominoid primates differed in their intestinal trichomonads. Our results suggest that captive primates possibly may be infected by intestinal trichomonads of other vertebrates such as pigs, cattle, birds, tortoises and lizards.     

Biotechnological methods of <Emphasis Type="Italic">in vitro</Emphasis> propagation in willows (<Emphasis Type="Italic">Salix</Emphasis> spp.)

Many populations of high-mountainous relic dioecious willows in Central Europe only consist of female individuals and are thus limited in their reproductive potential. We completed micropropagation experiments with shoot apexes and nodal segments of common and endangered willow (Salix) species, which can help to reintroduce autochthonous genotypes to their natural sites. Until recently, cultivation of green young shoot apexes of S. alba and S. lapponum showed the highest percentage of regeneration. We successfully applied the two-times-sterilisation due to high contamination of natural explants. The OK medium was the most efficient culture medium. In vitro propagation of willows with unisexual catkins, anther and ovule cultures were tested and optimised. Isolated anthers were cultivated on selected media and then microcallus and calluses of S. caprea and calluses of S. viminalis were formed on the A medium. Among various tested and optimised media for the ovule culture, the CP medium was the most efficient one. In this case, only the microcalluses of S. viminalis were observed. We developed biotechnological procedures that can be useful in conserving fragmented populations of high-mountainous willows.     

Age and growth of invasive round goby <Emphasis Type="Italic">Neogobius melanostomus</Emphasis> from middle Danube

Age and growth of the invasive population of round goby Neogobius melanostomus from the Slovak stretch of River Danube was examined. The samples (n=1130) were collected soon after the invasion was recorded (2004–2005), and later, when the population was already established (2008–2010). Invasive round goby in newly-occupied areas were found to reach smaller body size (15–153 mm standard length) compared to native populations. Age from 0+ to 4+, determined from scales, was recorded in both sexes. Annulus formed in April–May, which varied with age. Growth of freshly established gobies was negative allometric, suggesting increased allocation of their sources to reproduction, which corresponds to less specialized life-history. However, positive allometric growth found in longer established individuals suggests a shift in allocation towards somatic growth, which corresponds to more specialized life-history typical for native populations. None of the three parameters predicted by the theory of alternative ontogenies and invasive potential met the expectations, though two parameters, i.e. growth rate and age at maturation remain equivocal. This can be explained by too short of a time span that has elapsed from the beginning of invasion, or by ecological disturbances that have broken up otherwise stable environment in the habitat studied.     

Different reactions to adverse neighborhoods in games of cooperation

In social dilemmas, cooperation among randomly interacting individuals is often difficult to achieve. The situation changes if interactions take place in a network where the network structure jointly evolves with the behavioral strategies of the interacting individuals. In particular, cooperation can be stabilized if individuals tend to cut interaction links when facing adverse neighborhoods. Here we consider two different types of reaction to adverse neighborhoods, and all possible mixtures between these reactions. When faced with a gloomy outlook, players can either choose to cut and rewire some of their links to other individuals, or they can migrate to another location and establish new links in the new local neighborhood. We find that in general local rewiring is more favorable for the evolution of cooperation than emigration from adverse neighborhoods. Rewiring helps to maintain the diversity in the degree distribution of players and favors the spontaneous emergence of cooperative clusters. Both properties are known to favor the evolution of cooperation on networks. Interestingly, a mixture of migration and rewiring is even more favorable for the evolution of cooperation than rewiring on its own. While most models only consider a single type of reaction to adverse neighborhoods, the coexistence of several such reactions may actually be an optimal setting for the evolution of cooperation.