Bulky DNA adducts in human sperm: relationship with fertility,semen quality,smoking, and environmental factors

The integrity of DNA of spermatogenic cells can be affected by endogenous and exogenous genotoxic factors. Resulting DNA damage in spermatozoa may significantly contribute to impaired fertility. Here, the 32P-postlabeling method was used to analyze the levels of bulky DNA adducts in sperm cells in a group of 179 males, either healthy donors or patients with an impaired fertility. When all donors were analyzed, the levels of bulky DNA adducts was 1.2-fold higher in smokers than in non-smokers, but the difference was not statistically significant (P=0.054). However, a statistically significant difference existed between current smokers and never smokers among the healthy individuals (1.7-fold increase, P=0.008). No correlation between alcohol or coffee consumption and sperm DNA adducts was found. The levels of DNA adducts in sperm seemed to be unaffected by environmental and occupational factors. On the other hand, groups of healthy persons and patients with male-factor infertility differed significantly with respect to the level of bulky DNA adducts (P=0.012). A significant negative correlation between DNA adducts and sperm concentration or sperm motility existed among patients with an impaired fertility (n=93; P<0.029, r(S)=-0.225). These results suggest that DNA adducts in sperm cells can be applied as potential biomarkers in studies of human infertility.     

Selectivity of 4,5,6,7-tetrabromobenzimidazole as an ATP-competitive potent inhibitor of protein kinase CK2 from various sources

Like the previously reported 4,5,6,7-tetrabromobenzotriazole (TBBt), the structurally related 4,5,6,7-tetrabromobenzimidazole (TBBz) is a selective ATP-competitive inhibitor of protein kinase CK2 from such divergent sources as yeast, rat liver, Neurospora crassa and Candida tropicalis, with K(i) values in the range 0.5-1 microM. It is virtually inactive vs. PKA, PKC, and a very weak inhibitor of protein kinase CK1. The corresponding tetrachlorobenzimidazole (TCBz) is a much weaker inhibitor of CK2, like tetrachlorobenzotriazole (TCBt) relative to TBBt. Bearing in mind the similarity of the van der Waals radii of Br (1.95 A) and CH(3) (2.0 A), the corresponding much less hydrophobic 4,5,6,7-tetramethylbenzotriazole (TMeBt) was prepared and found to be a very weak inhibitor of CK2, as well as of CK1. An unexpected, and significant, difference between TBBt and TBBz are their inhibitory activities vs. the yeast protein kinase PK60S, which phosphorylates, both in vitro and in intact yeast cells, three of the five pp13 kDa ribosomal surface acidic proteins in yeast cells. TBBt was previously noted to be a more effective inhibitor of PK60S than of yeast CK2; by contrast, TBBz is a relatively feeble inhibitor of PK60S, hence more selective than TBBt vs. CK2 in yeast cells. TMeBt was virtually inactive vs PK60S. Like TBBt, TBBz is an additional lead compound for development of more potent inhibitors of CK2.     

A minimal physically realistic protein-like lattice model: designing an energy landscape that ensures all-or-none folding to a unique native state

A simple protein model restricted to the face-centered cubic lattice has been studied. The model interaction scheme includes attractive interactions between hydrophobic (H) residues, repulsive interactions between hydrophobic and polar (P) residues, and orientation-dependent P-P interactions. Additionally, there is a potential that favors extended beta-type conformations. A sequence has been designed that adopts a native structure, consisting of an antiparallel, six-member Greek-key beta-barrel with protein-like structural degeneracy. It has been shown that the proposed model is a minimal one, i.e., all the above listed types of interactions are necessary for cooperative (all-or-none) type folding to the native state. Simulations were performed via the Replica Exchange Monte Carlo method and the numerical data analyzed via a multihistogram method.     

Unfolding of globular proteins: monte carlo dynamics of a realistic reduced model

Reduced lattice models of proteins and Monte Carlo dynamics were used to simulate the initial stages of the unfolding of several proteins of various structural types, and the results were compared to experiment. The models semiquantitatively reproduce the approximate order of events of unfolding as well as subtle mutation effects and effects resulting from differences in sequences of similar folds. The short-time mobility of particular residues, observed in simulations, correlates with the crystallographic temperature factor. The main factor controlling unfolding is the native state topology, with sequence playing a less important role. The correlation with various experiments, especially for sequence-specific effects, strongly suggests that properly designed reduced models of proteins can be used for qualitative studies (or prediction) of protein unfolding pathways.     

Oogenesis in Vimba vimba (L. 1758) from Drawieński National Park (NW Poland)

The objective of the studies was to analyse the process of oogenesis in vimba from a non-migratory population living in the waters of Drawieński National Park in north-west Poland. The character of spawning of this species is an obstacle in determining the right moment to catch spawners or developing artificial spawning biotechniques. Previtellogenesis of vimba begins about six months after hatching and lasts three years. The trophoplasmatic growth of oocytes (October-March/April) begins when carbohydrate vesicles appear near the nuclei oocytes of sexually mature females (aged 4+). Just before spawning, granulated, lipoprotein-like substances are cumulated. The resorption of pre-ovulation corpora lutea (non-ejected oocytes) and post-ovulation corpora lutea (ruptured theca folds and follicles) begins in the ovary of vimba in the middle of June. These were observed in histological cross sections for about two to three months. Describing the process of oogenesis can provide a foundation for developing practical applications in aquaculture aimed at preserving the biodiversity of the park's waters and this critically endangered species of the Polish ichthyofauna.     

Biological properties of a new fluorescent biphalin fragment analogue

Previous studies of structure-activity of biphalin defined fragments which expressed the full biological potency of the parent compound. The most simple fragment was Tyr-D-Ala-Gly-Phe-NH-NH<--X, where X=Phe, but it also could be other hydrophobic amino acids. This paper presents data that replacement of the phenylalanine with a dansyl (X=DNS) groups gives an analogue (AA2016) that fully preserves the high affinity of the initial analogue for both mu and delta opioid receptors. In the tail flick test in rats, intrathecal injection of the compound produces strong antinociception, comparable to the parent biphalin. Because AA2016 contains a strong fluorescent group, it can be a very useful tool for prospective studies in vivo, including biological barrier permeability, tissue distribution, metabolism and receptor-ligand complex formation.     

Different DNA methylation pattern in A and B chromosomes of Crepis capillaris detected by in situ nick-translation. Comparison with molecular methods

Experiments were performed on Crepis capillaris callus lines with 0, 1 and 2 B chromosomes and on hairy root lines without or with 1 and 2 B chromosomes. Comparison of HPLC results for DNA from calli differing in number of B chromosomes did not reveal any significant differences in methylation level (30.4 +/- 1.1%, 30.9 +/- 1.2%, 31.7 +/- 1.7% in lines without or with one or two B chromosomes respectively) which could be attributed to the number of B chromosomes. Restriction patterns obtained after DNA digestion with HhaI, HpaII, MspI or HaeIII (i.e. restriction enzymes sensitive to cytosine methylation) were similar in calli and apical root segments and also did not depend on the presence or number of B chromosomes. Methylation of B chromosomes higher than that of A chromosomes was demonstrated by fluorescent in situ nick translation driven by HpaII, MspI or HaeIII in metaphase chromosomes. After short digestion (I and 3 h), B chromosomes, in contrast to A chromosomes, were weakly labelled or not labelled at all, which indicates longer distances between target sequences containing unmethylated cytosine in the former.     

Effects of cytotoxic T lymphocytes (CTL) directed against a single simian immunodeficiency virus (SIV) Gag CTL epitope on the course of SIVmac239 infection

Vaccine-induced cytotoxic T lymphocytes (CTL) have been implicated in the control of virus replication in simian immunodeficiency virus (SIV)-challenged and simian-human immunodeficiency virus-challenged macaques. Therefore, we wanted to test the impact that vaccine-induced CTL responses against an immunodominant Gag epitope might have in the absence of other immune responses. By themselves, these strong CTL responses failed to control SIVmac239 replication.