Improved Human Sperm Recovery Using Superoxide Dismutase and Catalase Supplementation in Semen Cryopreservation Procedure

The aim of this work was to evaluate the effects of ROS scavenger supplementation in human semen samples undergoing cryopreservation procedures.After screening out andrological pathologies, we selected 25 male partners of infertile couples with the following semen profile: volume >/= 2.0 ml, normal viscosity, sperm count >/=20 x 10(6)/ml, straight progressive motility (classes 1 and 2) >/= 40% (Mazzilli, Rossi, Delfino and Nofroni (1999) Andrologia 31: 187-194), atypical forms 相似文献   

Long-term release of clodronate from biodegradable microspheres

This paper describes the formulation of a biodegradable microparticulate drug delivery system containing clodronate, a bisphosphonate intended for the treatment of bone diseases. Microspheres were prepared with several poly(D,L-lactide-co-glycolide) (PLGA) copolymers of various molecular weights and molar compositions and 1 poly(D,L-lactide) (PDLLA) homopolymer by a water-in-oil-in-water (w/o/w) double emulsion solvent evaporation procedure. Critical process parameters and formulation variables (ie, addition of stabilizing agents) were evaluated for their effect on drug encapsulation efficiency and clodronate release rate from microparticles Well-formed clodronate-loaded microspheres were obtained for all polymers by selecting suitable process parameters (inner water/oil volume ratio 1∶16, temperature-raising rate in the solvent evaporation step 1°C/min, 2% wt/vol NaCl in the external aqueous phase). Good yields were obtained in all batches of clodronate microspheres (above 60%); drug encapsulation efficiencies ranged between 49% and 75% depending on the polymer used. Clodronate release from all copolymer microspheres was completed in about 48 hours, while those from PDLLA microspheres required about 20 days. The change of microsphere composition by adding a surfactant such as Span 20 or a viscosing agent such as carboxymethylcellulose extended the long-term release up to 3 months. Clodronate was successfully entrapped in PLGA and PDLLA microspheres, and drug release could be modulated from 48 hours up to 3 months by suitable selection of polymer, composition, additives, and manufacturing conditions. Published: July 11, 2001.     

Specific methylation of the CpG-rich domains in the promoter of the human tissue transglutaminase gene

The presence of a [Fe]-hydrogenase in the hydrogenosomes of the anaerobic chytridiomycete fungus Neocallimastix sp. L2 has been demonstrated by immunocytochemistry, subcellular fractionation, Western-blotting and measurements of hydrogenase activity in the presence of various concentrations of carbon monoxide (CO). Since the hydrogenosomal hydrogenase activity can be inhibited nearly completely by low concentrations of CO, it is likely that the [Fe]-hydrogenase is responsible for at least 90% of the hydrogen production in isolated hydrogenosomes. Most likely, this hydrogenase is encoded by the gene hydL2 that exhibits all the motifs that are characteristic of [Fe]-hydrogenases. The open reading frame starts with an N-terminal extension of 38 amino acids that has the potential to function as a hydrogenosomal targeting signal. The downstream sequences encode an enzyme of a calculated molecular mass of 66.4 kDa that perfectly matches the molecular mass of the mature hydrogenase in the hydrogenosome. Phylogenetic analysis revealed that the hydrogenase of Neocallimastix sp. L2. clusters together with similar ('long-type') [Fe]-hydrogenases from Trichomonas vaginalis, Nyctotherus ovalis, Desulfovibrio vulgaris and Thermotoga maritima. Phylogenetic analysis based on the H-cluster - the only module of [Fe]-hydrogenases that is shared by all types of [Fe]-hydrogenases and hydrogenase-like proteins - revealed a monophyly of all hydrogenase-like proteins of the aerobic eukaryotes. Our analysis suggests that the evolution of the various [Fe]-hydrogenases and hydrogenase-like proteins occurred by a differential loss of Fe-S clusters in the N-terminal part of the [Fe]-hydrogenase.     

Absence of endogenous interleukin-10 enhances the evolution of acute lung injury

Interleukin-10 (IL-10) exerts a wide spectrum of regulatory activities in the immune and inflammatory response. The aim of this study was to investigate the role of endogenous IL-10 on the modulation of the inflammatory response in mice subjected to carrageenan-induced lung injury. When compared to carrageenan-treated IL-10 wild-type (WT) mice, carrageenan-treated IL-10 knock-out mice (IL-10KO) mice experienced a higher rate of pleural exudation, and polymorphonuclear cell migration. Exudate levels of the pro-inflammatory cytokines tumour necrosis factor, interleukin-1beta and interleukin-6 were also greatly enhanced in IL-10KO mice in comparison to wild-type mice. Lung myeloperoxidase (MPO) activity was significantly reduced in IL-10WT mice when compared to IL-10KO mice-treated with carrageenan. The degree of oxidative and nitrosative damage was significantly higher in IL-10KO mice than in wild-type littermates, as indicated by elevated malondialdehyde levels and formation of nitrotyrosine and poly (ADP-ribose) synthetase (PARS). Staining of lung tissue sections obtained from carrageenan-treated IL-10WT with an anti-COX-2 antibody showed a positive staining of the inflamed tissue. Furthermore, expression of inducible nitric oxide synthase (iNOS) was found mainly in the macrophages of the inflamed lungs from carrageenan-treated IL-10WT mice. The intensity and degree of the staining for COX-2 and iNOS were markedly enhanced in tissue sections obtained from carrageenan-treated IL-10KO mice. Most notably, the degree of lung injury caused by carrageenan was also enhanced in IL-10KO mice. Taken together, our results clearly demonstrate that endogenous IL-10 exerts an anti-inflammatory role during acute inflammation and tissue damage associated with carrageenan-induced pleurisy, possibly by regulating neutrophil recruitment, and the subsequent cytokine and oxidant generation.     

Reduction of 1,4-quinone and ubiquinones by hydrogen atom transfer under UVA irradiation

1,4-Benzoquinone, coenzyme Q 0 and Q 10 were reacted with a series of hydrogen donors in the ESR cavity in the presence or absence of UVA irradiation. The signals of the radicals generated from the hydrogen donors or of those of the semiquinones were detected. The reaction mechanism was interpreted by a hydrogen atom transfer instead of the usual electron transfer mechanism on the basis of the redox potentials of the reactants and the Marcus theory. The hydrogen atom transfer is explained by the excited triplet state of quinones, which, on the basis of quantum mechanic calculations, may be reached even under visible light. In some cases, hydrogen atom transfer was also observed without irradiation, although to a lesser extent.     

The antihypertensive drug carvedilol inhibits the activity of mitochondrial NADH-ubiquinone oxidoreductase

A study is presented on the interaction of carvedilol with mitochondria isolated from several rat organs. It is shown that carvedilol causes a moderate uncoupling effect under non phosphorylating succinate supported respiration of intact mitochondria, as well as a marked inhibition of coupled respiration with NAD-dependent substrates. The inhibitory effect was also found in the bovine heart purified Complex I as well as in experiments with mitochondrial particles, where the individual redox segments of the respiratory chain were analysed. It is also shown that carvedilol, though exhibiting an intrinsic scavenger activity, caused reactive oxygen species to be produced as a consequence of its inhibitory effect on the steady-state respiration. Under these conditions the pro-oxidant activity of carvedilol appears to prevail over its scavenging activity, and a net generation of ROS is promoted.     

Structure and nucleotide sequence of Euphorbia characias copper/TPQ-containing amine oxidase gene

A cDNA encoding for a copper containing amine oxidase has been isolated and sequenced from young leaves of Euphorbia characias, a perennial mediterranean shrub. A single long open reading frame of 2068 pb encodes a protein composed of 653 amino acids with a molecular mass of about 74 kDa. A putative 24-aminoacid signal peptide precedes the sequence of the mature protein, with characteristics of a secretion signal peptide. Alignments of Euphorbia amine oxidase cDNA nucleotide sequence with that of amine oxidase from the seedlings of the pulses lentil, pea, and chickpea reveal several conserved regions, especially in the C-terminus, with a homology 90%–97%. The near 5 region shows several insertions, deletions, and different nucleotide sequence with ca. 60% homology. The enzyme contains 1%–2% carbohydrate deduced by deglycosylation experiments. Five cysteine residues are present in the deduced aminoacid sequence with a single disulfide bridge as judged by titration with cysteine reagents.     

Identification of the human salivary cystatin complex by the coupling of high-performance liquid chromatography and ion-trap mass spectrometry

Human salivary cystatins, five major (S, S1, S2, SA, SN) and two minor (C and D), are multifunctional proteins playing a different role in the oral environment. Salivary cystatin SN is able to effectively inhibit lysosomal cathepsins B, C, H and L and cystatin SA inhibits cathepsins C and L in vitro. These activities suggest, particularly for cystatin SN, an important role in the control of proteolytic events in vivo. Differently, cystatins S are involved, together with statherin, in the mineral balance of the tooth. Due to their distinct role, a reliable method for identification and quantification of the different cystatins, as well as of possible truncated and derived forms, could be helpful for the assessment of the status of the oral cavity. To this purpose high-performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI MS) was applied to the analysis of human saliva obtained from healthy subjects. All known salivary cystatins, with the exception of cystatin C, were detected. Strong evidence was also obtained for the presence in saliva of post-translational modified isoforms of cystatins, which may be related to donor habits. Cystatin SN and cystatins S, S1 and S2 were well separated by HPLC-ESI MS coupling from other components and thus this approach can be successfully applied to their quantification.