In vitro effects of bisphenol A on the quality parameters,oxidative stress,DNA integrity and adenosine triphosphate content in sterlet (Acipenser ruthenus) spermatozoa

Among endocrine disruptors, the xenoestrogen bisphenol A (BPA) deserves particular attention due to widespread human exposure. Besides hormonal effects, BPA has been suspected to be responsible for adverse effect on reproductive ability of various species. In the present study the effect of BPA on the quality parameters, oxidative stress, the DNA integrity and intracellular ATP content of sterlet (Acipenser ruthenus) spermatozoa were investigated in vitro. Fish spermatozoa were exposed to concentrations of BPA possibly occurring in nature (0.5, 1.75, 2.5, 5 and 10 μg/L) for 2 h. Results revealed that BPA significantly decreased spermatozoa motility and velocity of spermatozoa at concentration of BPA 2.5–10 μg/L. Significant positive correlation (r = 0.713, P < 0.05) was found between percent motile spermatozoa and ATP content. Oxidative stress was observed at concentrations 1.75–10 μg/L, as reflected by significantly higher levels of protein and lipid oxidation and superoxide dismutase activity. Intracellular ATP content of spermatozoa decreased with increasing concentrations of BPA. A dramatic increase in DNA fragmentation expressed as percent tail DNA (2.2% ± 0.46) and Olive tail moment (0.37 ± 0.09 arbitrary units) was recorded at concentrations of 1.75 μg/L and above. The present study confirms that concentrations of BPA that can be encountered in nature are capable to induce oxidative stress, leading to impaired sperm quality, DNA fragmentation and intracellular ATP content.     

Characterization of Fibrinogen Binding by Glycoproteins Srr1 and Srr2 of Streptococcus agalactiae

The serine-rich repeat glycoproteins of Gram-positive bacteria comprise a large family of cell wall proteins. Streptococcus agalactiae (group B streptococcus, GBS) expresses either Srr1 or Srr2 on its surface, depending on the strain. Srr1 has recently been shown to bind fibrinogen, and this interaction contributes to the pathogenesis of GBS meningitis. Although strains expressing Srr2 appear to be hypervirulent, no ligand for this adhesin has been described. We now demonstrate that Srr2 also binds human fibrinogen and that this interaction promotes GBS attachment to endothelial cells. Recombinant Srr1 and Srr2 bound fibrinogen in vitro, with affinities of K_D = 2.1 × 10⁻⁵ and 3.7 × 10⁻⁶ m, respectively, as measured by surface plasmon resonance spectroscopy. The binding site for Srr1 and Srr2 was localized to tandem repeats 6–8 of the fibrinogen Aα chain. The structures of both the Srr1 and Srr2 binding regions were determined and, in combination with mutagenesis studies, suggest that both Srr1 and Srr2 interact with a segment of these repeats via a “dock, lock, and latch” mechanism. Moreover, properties of the latch region may account for the increased affinity between Srr2 and fibrinogen. Together, these studies identify how greater affinity of Srr2 for fibrinogen may contribute to the increased virulence associated with Srr2-expressing strains.     

Mechanisms of ADRF release from rat aortic adventitial adipose tissue

Blood vessels are surrounded by variable amounts of adipose tissue. We showed earlier that adventitial adipose tissue inhibits rat aortic contraction by release of a transferable factor, adventitium-derived relaxing factor (ADRF), which activates smooth muscle K(+) channels. However, little is known about the mechanisms of ADRF release. Using isolated rat aortic rings and isometric contraction measurements, we show that ADRF release depends on extracellular [Ca(2+)] (EC(50) approximately 4.7 mM). ADRF effects do not involve neuronal presynaptic N-type Ca(2+) and Na(+) channels or vanilloid, cannabinoid, and CGRP receptors. ADRF release is strongly inhibited by the protein tyrosine kinase inhibitors genistein and tyrphostin A25. In contrast, daidzein, an inactive genistein analog, and the protein tyrosine kinase inhibitor ST638 had no effect. Protein kinase A inhibition by H89 also inhibited ADRF release, whereas the protein kinase G inhibitor KT-5823 had no effect. We propose that ADRF release is Ca(2+) dependent and is regulated by intracellular signaling pathways involving tyrosine kinase and protein kinase A. Furthermore, ADRF release does not depend on perivascular nerve endings.     

Glycoproteome profiling of transforming growth factor-beta (TGFbeta) signaling: nonglycosylated cell death-inducing DFF-like effector A inhibits TGFbeta1-dependent apoptosis

Transforming growth factor-beta (TGFbeta) is a potent regulator of cell growth, differentiation, and apoptosis. TGFbeta binds to specific serine/threonine kinase receptors, which leads to activation of Smad-dependent and Smad-independent signaling pathways. O-Glycosylation is a dynamic PTM which has been observed in many regulatory proteins, but has not been studied in the context of TGFbeta signaling. To explore the effect of TGFbeta1 on protein O-glycosylation in human breast epithelial cells, we performed analyses of proteins which were affinity purified with Helix pomatia agglutinin (HPA). HPA lectin allowed enrichment of proteins containing GalNAc and GlcNAc linked to serine and threonine residues. Using 2-DE and MALDI-TOF-MS, we identified 21 HPA-precipitated proteins, which were affected by treatment of cells with TGFbeta1. Among these proteins, regulators of cell survival, apoptosis, trafficking, and RNA processing were identified. We found that TGFbeta1 inhibited the appearance of cell death-inducing DFF-like effector A (CIDE-A) in 2-D gels with HPA-precipitated proteins. CIDE-A is a cell death activator which promotes DNA fragmentation. We observed that TGFbeta1 did not affect expression of CIDE-A, but inhibited its glycosylation. We found that deglycosylation of CIDE-A correlated with enhanced nuclear export of the protein, and that high level of nonglycosylated CIDE-A inhibited TGFbeta1-dependent cell death. Thus, inhibition of the glycosylation of CIDE-A may be a mechanism to protect cells from apoptosis.     

Differential Effects of Cystathionine-γ-lyase-Dependent Vasodilatory H(2)S in Periadventitial Vasoregulation of Rat and Mouse Aortas

Background

Hydrogen sulfide (H₂S) is a potent vasodilator. However, the complex mechanisms of vasoregulation by H₂S are not fully understood. We tested the hypotheses that (1) H₂S exerts vasodilatory effects by opening KCNQ-type voltage-dependent (K_v) K⁺ channels and (2) that H₂S-producing cystathionine-γ-lyase (CSE) in perivascular adipose tissue plays a major role in this pathway.

Methodology/Principal Findings

Wire myography of rat and mouse aortas was used. NaHS and 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADTOH) were used as H₂S donors. KCNQ-type K_v channels were blocked by XE991. 4-Propargylglycine (PPG) and ß-cyano-l-alanine (BCA), or 2-(aminooxy)-acetic acid (AOAA) were used as inhibitors of CSE or cystathionine-ß-synthase (CBS), respectively. NaHS and ADTOH produced strong vasorelaxation in rat and mouse aortas, which were abolished by KCNQ channel inhibition with XE991. Perivascular adipose tissue (PVAT) exerted an anticontractile effect in these arteries. CSE inhibition by PPG and BCA reduced this effect in aortas from rats but not from mice. CBS inhibition with AOAA did not inhibit the anticontractile effects of PVAT. XE991, however, almost completely suppressed the anticontractile effects of PVAT in both species. Exogenous l-cysteine, substrate for the endogenous production of H₂S, induced vasorelaxation only at concentrations >5 mmol/l, an effect unchanged by CSE inhibition.

Conclusions/Signficance

Our results demonstrate potent vasorelaxant effects of H₂S donors in large arteries of both rats and mice, in which XE991-sensitive KCNQ-type channel opening play a pivotal role. CSE-H₂S seems to modulate the effect of adipocyte-derived relaxing factor in rat but not in mouse aorta. The present study provides novel insight into the interaction of CSE-H₂S and perivascular adipose tissue. Furthermore, with additional technical advances, a future clinical approach targeting vascular H₂S/KCNQ pathways to influence states of vascular dysfunction may be possible.     

Deprotection of centromeric cohesin at meiosis II requires APC/C activity but not kinetochore tension

Genome haploidization involves sequential loss of cohesin from chromosome arms and centromeres during two meiotic divisions. At centromeres, cohesin''s Rec8 subunit is protected from separase cleavage at meiosis I and then deprotected to allow its cleavage at meiosis II. Protection of centromeric cohesin by shugoshin‐PP2A seems evolutionarily conserved. However, deprotection has been proposed to rely on spindle forces separating the Rec8 protector from cohesin at metaphase II in mammalian oocytes and on APC/C‐dependent destruction of the protector at anaphase II in yeast. Here, we have activated APC/C in the absence of sister kinetochore biorientation at meiosis II in yeast and mouse oocytes, and find that bipolar spindle forces are dispensable for sister centromere separation in both systems. Furthermore, we show that at least in yeast, protection of Rec8 by shugoshin and inhibition of separase by securin are both required for the stability of centromeric cohesin at metaphase II. Our data imply that related mechanisms preserve the integrity of dyad chromosomes during the short metaphase II of yeast and the prolonged metaphase II arrest of mammalian oocytes.