Male fertility is linked to the selenoprotein phospholipid hydroperoxide glutathione peroxidase

The selenoprotein phospholipid hydroperoxide glutathione peroxidase (PHGPx) accounts for almost the entire selenium content of mammalian testis. PHGPx is abundantly expressed in spermatids as active peroxidase but is transformed to an oxidatively inactivated protein in mature sperm, where it is a major constituent of the mitochondrial capsule in the midpiece. Male infertility in selenium-deficient animals, which is characterized by impaired sperm motility and morphological midpiece alterations, is considered to result from insufficient PHGPx content. We studied the relationship between sperm PHGPx, measured as rescued activity, and human fertility. Sperm specimens from 75 infertile men and 37 controls were analyzed for fertility-related parameters according to World Health Organization criteria. The PHGPx protein content was estimated after reductive solubilization of the spermatozoa by measuring the rescued PHGPx activity. Rescued PHGPx activity of infertile men ranged significantly below that of controls (93.2 +/- 60.1 units/mg sperm protein vs. 187.5 +/- 55.3 units/mg) and was particularly low in oligoasthenozoospermic specimens (61.93 +/- 45.42 units/mg; P < 0.001 compared with controls and asthenozoospermic samples). Rescued PHGPx activity was correlated positively with viability, morphological integrity, and most profoundly forward motility (r = 0.35, 0.44, and 0.45, respectively). In isolated motile samples, motility decreased faster with decreasing PHGPx content. In humans, PHGPx appears to be indispensable for structural integrity of spermatozoa and to codetermine sperm motility and viability. Because the content of PHGPx, irrespective of the cause of alteration, is correlated with fertility-related parameters, PHGPx can be considered a predictive measure for fertilization capacity.     

Glutathione transferase isoenzymes from frog (Xenopus laevis) liver and embryo

The expression of glutathione transferase isoenzymes has been investigated in embryo and adult liver of the frog Xenopus laevis. By analysing the GST isoenzymes recovered from GSH-affinity chromatography in terms of electrophoretic mobility, HPLC elution profile, immunological reactivity, N-terminal amino acid sequence and mass spectrometry molecular mass no significant difference in the GST subunit composition between embryos and liver was found. In both tissues the same three subunits, showing similarity to mu, alpha and sigma class GSTs, are present. These results, together with those previously reported for toad (Bufo bufo), strongly support the notion that the transition from an aquatic environment to a terrestrial atmosphere containing high oxygen concentration has accompanied specific GST gene expression.     

Physiological thiols as promoters of glutathione oxidation and modifying agents in protein S-thiolation.

Glutathione is one of the most relevant antioxidants present in cells. It exerts its scavenging action through the involvement of efficient and ubiquitous enzymes. GSH on the other hand, because of its chemical features, can scavenge reactive oxygen species without the involvement of enzymatic systems. The study deals with the mobilization of GSH pool in a nonenzymatic antioxidant system by other physiological thiols (i.e., cysteine and cysteinyl-glycine), which are far more sensitive than GSH to oxidative conditions. These thiol compounds, in the presence of iron/EDTA, can promote oxygen activation through their oxidation to disulfides. GSH, through trans-thiolation reactions, can regenerate Cys and CysGly, which can then recycle, thus inducing a massive GSH oxidation. In these conditions, making use of bovine lens aldose reductase as a protein model, evidence is given that Cys and CysGly promote specific protein S-thiolation reactions. The possibility that GSH may be recruited in controlling cellular oxygen tension is considered.     

Human alpha-thrombin stimulates proliferation of interferon-gamma differentiated,growth-arrested U937 cells,overcoming differentiation-related changes in expression of p21CIP1/WAF1 and cyclin D1

In addition to its central role in blood coagulation and hemostasis, human alpha-thrombin is a growth factor for a variety of cell types. We recently demonstrated that interferon-gamma (IFNgamma)-differentiated U937 cells show increased expression of the proteolytically activated receptor for thrombin (PAR-1) relative to undifferentiated U937. In the present study we show that cell proliferation is inhibited in IFNgamma-differentiated cells relative to undifferentiated U937. Addition of thrombin to the differentiated cells, however, overcomes the inhibition and restores the cells to a highly proliferative state. Ribonuclease protection assays indicate that the IFNgamma-induced growth arrest is associated with an increased expression of the cyclin-dependent kinase inhibitor p21(CIP1/WAF1) and downregulation of cyclin D(1). Treatment of cells with thrombin downregulates p21(CIP1/WAF1) expression in these cells and upregulates cyclin D(1) mRNA expression, thus overcoming the differentiation-related effects in a coordinated manner. Treating differentiated cells with the PAR-1 activation peptide, SFLLRN, stimulates proliferation and has effects similar to those of thrombin on expression of p21(CIP1/WAF1). Thus, it appears that these thrombin stimulated proliferative effects are mediated through activation of PAR-1. These results may help explain how thrombin can overcome growth arrest in normal tissue to initiate tissue repair and why thrombin and thrombin-like enzymes may contribute to unrestricted proliferation observed in certain malignancies.     

Evidence for male XO sex-chromosome system in Pentodon bidens punctatum (Coleoptera Scarabaeoidea: Scarabaeidae) with X-linked 18S-28S rDNA clusters

In scarab beetle species of the genus Pentodon, the lack of analysis of sex chromosomes in females along with the poor characterization of sex chromosomes in the males, prevented all previous investigations from conclusively stating sex determination system. In this study, somatic chromosomes from females and spermatogonial chromosomes from males of Pentodon bidens punctatum (Coleoptera: Scarabaeoidea: Scarabaeidae) from Sicily have been analyzed using non-differential Giemsa staining. Two modal numbers of chromosomes were obtained: 2n = 20 and 19 in females and males, respectively. This finding along with other karyological characteristics such as the occurrence of one unpaired, heterotypic chromosome at metaphase-I and two types of metaphase-II spreads in spermatocytes demonstrate that a XO male/XX female sex determining mechanism - quite unusual among Scarabaeoidea - operates in the species investigated here. Spermatocyte chromosomes have also been examined after a number of banding techniques and fluorescent in situ hybridization with ribosomal sequences as a probe (rDNA FISH). The results obtained showed that silver and CMA(3) staining were inadequate to localize the chromosome sites of nucleolus organizer regions (NORs) due to the over-all stainability of both constitutive heterochromatin and heterochromatin associated to the NORs. This suggests that heterochromatic DNA of P. b. punctatum is peculiar as compared with other types of heterochromatin studied so far in other invertebrate taxa. By rDNA FISH major ribosomal genes were mapped on the X chromosome.     

Versatility of selenium catalysis in PHGPx unraveled by LC/ESI-MS/MS

Phospholipid hydroperoxide glutathione peroxidase (PHGPx; EC 1.11.1.12), a broad-spectrum thiol-dependent peroxidase, deserves renewed interest as a regulatory factor in various signaling cascades and as a structural protein in sperm cells. We present a first attempt to identify catalytic intermediates and derivatives of the selenoprotein by liquid chromatography coupled to electrospray tandem mass spectrometry (LC/ESI-MS/MS) and to explain observed specificities by molecular modeling. The ground state enzyme E proved to correspond to position 3-170 of the deduced porcine sequence with selenium being present as selenocysteine at position 46. The selenenic acid form, which is considered to be the first catalytic intermediate F formed by reaction with hydroperoxide, could not be identified. The second catalytic intermediate G was detected as Se-glutathionylated enzyme. This intermediate is generated in the reverse reaction where the active site selenol interacts with glutathione disulfide (GSSG). According to molecular models, specific binding of reduced glutathione (GSH) and of GSSG is inter alia facilitated by electrostatic attraction of Lys-48 and Lys-125. Polymerization of PHGPx is obtained under oxidizing conditions in the absence of low molecular weight thiols. Analysis of MS spectra revealed that the process is due to a selective reaction of Sec-46 with Cys-148' resulting in linear polymers representing dead-end intermediates (G'). FT Docking of PHGPx molecules allowed reactions of Sec-46 with either Cys-66', Cys-107', Cys-168' or Cys-148', the latter option being most likely as judged by the number of proposed intermediates with reasonable hydrogen bonds, interaction energies and interface areas. We conclude that the same catalytic principles, depending on the conditions, can drive the diverse actions of PHGPx, i.e. hydroperoxide reduction, GSSG reduction, S-derivatization and self-incorporation into biological structures.     

Disturbances in purinergic [Ca2+]i signaling pathways in a transformed rat thyroid cell line

It was previously shown that in rat thyroid PC-Cl3 cell line, a purinergic P2Y receptor increases the concentration of free cytosolic Ca(2+) ([Ca(2+)](i)) via phospholipase C activation. We here studied whether in a transformed cell line (PC-E1Araf) derived from parental PC-Cl3 cells, ATP is still able to transduce the [Ca(2+)](i)-based intracellular signal.We demonstrate the expression of mRNA for P2Y2 in both PC-Cl3 and PC-E1Araf cells; mRNAs for P2Y1, P2Y4, P2Y6 and P2Y11 were absent. In both cell lines activation of P2Y2 receptor provokes a transient increase in [Ca(2+)](i) followed by a lower sustained phase persisting for over 5min in PC-Cl3 and only 1.5 min in PC-E1Araf cells. In both cell lines the [Ca(2+)](i) reached a plateau level significantly higher than the basal [Ca(2+)](i) level persisting for over 10 min. Removal of extracellular Ca(2+) reduced the initial transient response to ATP in PC-Cl3, but not in PC-E1Araf cells, and completely abolished the plateau phase in both cell lines.In the presence of extracellular Ca(2+) thapsigargin (TG) caused a rise in [Ca(2+)](i) significantly higher in PC-Cl3 than transformed PC-E1Araf cells, while in Ca(2+)-free medium the effect of TG was similar in both cell lines. The capacitative Ca(2+)-entry in PC-Cl3 resulted significantly higher than in PC-E1Araf cells.Further studies were performed in order to investigate whether the different effects of ATP on [Ca(2+)](i) was due to variation in divalent cation plasma membrane permeability. PC-E1Araf cells showed a much lower permeability to Ca(2+), Ba(2+), Sr(2+), Mn(2+), and Co(2+) that may be responsible for the differences in purinergic Ca(2+) signaling pathway with respect to parental PC-Cl3 cells.     

Antioxidant activity of oligomeric acylphloroglucinols from Myrtus communis L

The use of myrtle (Myrtus communis L.) as a culinary spice and as a flavoring agent for alcoholic beverages is widespread in the Mediterranean area, and especially in Sardinia. Myrtle contains unique oligomeric non-prenylated acylphloroglucinols, whose antioxidant activity was investigated in various systems. Both semimyrtucommulone (1) and myrtucommulone A (2) showed powerful antioxidant properties, protecting linoleic acid against free radical attack in simple in vitro systems, inhibiting its autoxidation and its FeCl3- and EDTA-mediated oxidation. While both compounds lacked pro-oxidant activity, semimyrtucommulone was more powerful than myrtucommulone A, and was further evaluated in rat liver homogenates for activity against lipid peroxidation induced by ferric-nitrilotriacetate, and in cell cultures for cytotoxicity and the inhibition of TBH- or FeCl3-induced oxidation. The results of these studies established semimyrtucommulone as a novel dietary antioxidant lead.     

Melusin,a muscle-specific integrin beta1-interacting protein,is required to prevent cardiac failure in response to chronic pressure overload

Cardiac hypertrophy is an adaptive response to a variety of mechanical and hormonal stimuli, and represents an early event in the clinical course leading to heart failure. By gene inactivation, we demonstrate here a crucial role of melusin, a muscle-specific protein that interacts with the integrin beta1 cytoplasmic domain, in the hypertrophic response to mechanical overload. Melusin-null mice showed normal cardiac structure and function in physiological conditions, but when subjected to pressure overload--a condition that induces a hypertrophic response in wild-type controls--they developed an abnormal cardiac remodeling that evolved into dilated cardiomyopathy and contractile dysfunction. In contrast, the hypertrophic response was identical in wild-type and melusin-null mice after chronic administration of angiotensin II or phenylephrine at doses that do not increase blood pressure--that is, in the absence of cardiac biomechanical stress. Analysis of intracellular signaling events induced by pressure overload indicated that phosphorylation of glycogen synthase kinase-3beta (GSK-3beta) was specifically blunted in melusin-null hearts. Thus, melusin prevents cardiac dilation during chronic pressure overload by specifically sensing mechanical stress.