DETECTION OF PLASMA MEMBRANE PHOSHATIDYLSERINE TRANSLOCATION IN RAM SPERMATOZOA AFTER IN VITRO TREATING WITH DEXAMETAZONE

In vitro induced apoptosis by dexamethazone treating of ram spermatozoa provoked the phospatidylserine exposure which is mainly localized on sperm tail region of plasma membrane and is correlated negatively with the sperm motility and survival rate.Ин витро индуцирана апоптоза чрез третиране с дексаметазон на сперматозоиди от коч провокира експресия на фосфатидилсерин, която се локализира главно в областта на опашката на плазмената мембрана и корелира негативно с подвижността и преживяемостта на сперматозоидите

Negative Supercoiling Creates Single-Stranded Patches of DNA That Are Substrates for AID–Mediated Mutagenesis

Antibody diversification necessitates targeted mutation of regions within the immunoglobulin locus by activation-induced cytidine deaminase (AID). While AID is known to act on single-stranded DNA (ssDNA), the source, structure, and distribution of these substrates in vivo remain unclear. Using the technique of in situ bisulfite treatment, we characterized these substrates—which we found to be unique to actively transcribed genes—as short ssDNA regions, that are equally distributed on both DNA strands. We found that the frequencies of these ssDNA patches act as accurate predictors of AID activity at reporter genes in hypermutating and class switching B cells as well as in Escherichia coli. Importantly, these ssDNA patches rely on transcription, and we report that transcription-induced negative supercoiling enhances both ssDNA tract formation and AID mutagenesis. In addition, RNaseH1 expression does not impact the formation of these ssDNA tracts indicating that these structures are distinct from R-loops. These data emphasize the notion that these transcription-generated ssDNA tracts are one of many in vivo substrates for AID

Effects of chlorogenic acid, ferulic acid, gallic аcid and quercetin on learning and memory in the two-way active avoidance task in young/healthy rats

The phenolic acids chlorogenic (CA), ferulic (FA) and gallic (GA) and the flavonoid quercetin (Q) are polyphenols abundant in natural food sources. Polyphenols exhibit strong antioxidant properties. There are data that they cross the blood-brain barrier and accumulate in the brain. These substances are tested in different models of impaired memory. The aim of the present study was to investigate their effects on learning and memory processes in young/healthy rats. Male Wistar rats were treated in the course of 7, 14, 21 and 30 days. Control groups were treated with saline. The other 4 groups received CA, FA, GA or Q at an equal dose of 20 mg/kg. At the end of each period, learning and memory processes were evaluated using the two-way active avoidance task (shuttle box). The number of avoidances was recorded in two learning sessions on two consecutive days and in a retention test 24 h after the 2nd training session. Administered for 7 days, the experimental substances had no significant effects on rat behavior. Applied for 14 days, GA and Q significantly increased the number of avoidances in both training sessions and on the retention test. After 21 and 30 days of treatment all tested polyphenols significantly improved the recorded indices of memory. The onset of the effect after 14/21 days of treatment may be explained by the accumulation of polyphenols in the brain following a long-term consumption. Our results suggest that CA, FA, GA and Q, applied subchronically, improve memory and cognition of young/healthy rats

Elevated incidence of polyp formation in APC(Min/⁺)Msh2⁻/⁻ mice is independent of nitric oxide-induced DNA mutations.

Gut microbiota has been linked to a number of human diseases including colon cancer. However, the mechanism through which gut bacteria influence colon cancer development and progression remains unclear. Perturbation of the homeostasis between the host immune system and microbiota leads to inflammation and activation of macrophages which produce large amounts of nitric oxide that acts as a genotoxic effector molecule to suppress bacterial growth. However, nitric oxide also has genotoxic effects to host cells by producing mutations that can predispose to colon cancer development. The major DNA lesions caused by nitric oxide are 8oxoG and deamination of deoxycytosine bases. Cellular glycosylases that belong to the base excision repair pathway have been demonstrated to repair these mutations. Recent evidence suggests that the mismatch repair pathway (MMR) might also repair nitric oxide-induced DNA damage. Since deficiency in MMR predisposes to colon cancer, we hypothesized that MMR-deficient colon epithelial cells are incapable of repairing nitric-oxide induced genetic lesions that can promote colon cancer. Indeed, we found that the MMR pathway repairs nitric oxide-induced DNA mutations in cell lines. To test whether nitric oxide promotes colon cancer, we genetically ablated the inducible nitric oxide synthase (iNOS) or inhibited iNOS activity in the APC(Min/+)Msh2(-/-) mouse model of colon cancer. However, despite the fact that nitric oxide production was strongly reduced in the colon using both approaches, colon cancer incidence was not affected. These data show that nitric oxide and iNOS do not promote colon cancer in APC(Min/+)Msh2(-/-) mice

Phosphorylation-Induced Activation of the Response Regulator VraR from Staphylococcus aureus: Insights from Hydrogen Exchange Mass Spectrometry

A two-component system consisting of the histidine kinase vancomycin-resistance-associated sensor and the response regulator vancomycin-resistance-associated regulator (VraR) allows Staphylococcus aureus to sense antibiotic-related cell wall stress and to mount a suitable response. An experimental structure of full-length VraR is not available yet, but previous work points to similarities between VraR and the well-characterized NarL. This work employs hydrogen exchange mass spectrometry to gain insights into the phosphorylation-induced activation of VraR, a process that primes the protein for dimerization and DNA binding. Whereas VraR is highly dynamic, phosphorylated VraR shows less extensive deuteration. This rigidification is most dramatic within the receiver domain, which carries the phosphorylation site D55. Alterations in the DNA-binding domain are much less pronounced. Changes in deuteration within the receiver domain are consistent with a Y-T coupling mechanism. In analogy to NarL, the activation of VraR is thought to involve separation and subsequent reorientation of the two domains, thereby allowing the alpha8-turn-alpha9 element to engage in DNA binding. The current work suggests that this structural transition is triggered by a reduction in the effective length of the linker through enhanced hydrogen bonding. In addition, separation of the two domains may be favored by the establishment of noncovalent protein-protein interactions and intradomain contacts at the expense of previously existing interdomain bonds. alpha9 appears to be packed against the receiver domain in nonactivated VraR. Support is presented for alpha1 as a dimerization interface in phosphorylated VraR, whereas protein-protein interactions for nonphosphorylated VraR are impeded by extensive disorder in this region

Staphylococcus aureus methicillin-resistance factor fmtA is regulated by the global regulator SarA.

fmtA encodes a low-affinity penicillin binding protein in Staphylococcus aureus. It is part of the core cell wall stimulon and is involved in methicillin resistance in S. aureus. Here, we report that the transcription factor, SarA, a pleiotropic regulator of virulence genes in S. aureus, regulates the expression of fmtA. In vitro binding studies with purified SarA revealed that it binds to specific sites within the 541-bp promoter region of fmtA. Mutation of a key residue of the regulatory activity of SarA (Arg90) abolished binding of SarA to the fmtA promoter, suggesting that SarA binds specifically to the fmtA promoter region. In vivo analysis of the fmtA promoter using a lux operon reporter fusion show high level expression following oxacillin induction, which was abrogated in a sarA mutant strain. These data suggest that SarA is essential for the induction of fmtA expression by cell wall-specific antibiotics. Further, in vitro transcription studies show that SarA enhances fmtA transcription and suggest that regulation of fmtA could be via a SigA-dependent mechanism. Overall, our results show that SarA plays a direct role in the regulation of fmtA expression via binding to the fmtA promoter

Nitric oxide induces DNA mutations that are repaired by the MMR pathway.

<p>(A) Measurements of the amounts of nitric oxide in culture medium generated by various concentrations of SNAP or by macrophages that were stimulated by LPS and IFN-γ. (B) The mutant frequency at the <i>lac</i>I gene in Msh2^+/− (WT, n = 3) and Msh2^−/− (n = 3) SNAP-treated or non-treated macrophages was measured by the Big Blue mutagenesis screen. Bone marrow-derived macrophages were cultured in presence of 150 µM SNAP for 20 hrs. Genomic DNA was isolated and the mutant frequency at <i>lac</i>I gene was calculated. (C) Mutation spectrum in SNAP-treated macrophages as determined by sequencing analysis. The C:G → T:A transitions are shown. The tranversion mutations include C:G → A:T, C:G → G:C, T:A → G:C and T:A → A:T substitutions. The insertions and deletions are presents as other mutations. (D) The mutant frequency at the <i>hprt</i> gene in Msh2^+/− (WT, n = 8) and Msh2^−/− (n = 8) SNAP-treated or non-treated Pre-B cells. (E) The mutant frequency at the <i>hprt</i> gene in SW680 (n = 8) and DLD-1 (n = 8) SNAP-treated or non-treated human colorectal cancer cell lines.</p