ATP increases head volume in capacitated human sperm via a purinergic channel.

Scanning ion-conductance microscopy allowed us to document an external Ca2+ dependent ATP driven volume increase (ATPVI) in capacitated human sperm heads. We examined the involvement of purinergic receptors (PRs) P2X2R and P2X4R in ATPVI using their co-agonists progesterone and Ivermectin (Iver), and Cu2+, which co-activates P2X2Rs and inhibits P2X4Rs. Iver enhanced ATPVI and Cu2+ and 5BDBD inhibited it, indicating P2X4Rs contributed to this response. Moreover, Cu2+ and 5BDBD inhibited the ATP-induced acrosome reaction (AR) which was enhanced by Iver. ATP increased the concentration of intracellular Ca2+ ([Ca2+]i) in >45% of individual sperm, most of which underwent AR monitored using FM4-64. Our findings suggest that human sperm P2X4R activation by ATP increases [Ca2+]i mainly due to Ca2+ influx which leads to a sperm head volume increase, likely involving acrosomal swelling, and resulting in AR.

Vitrification of Iberian wolf (Canis lupus signatus) sperm: A possible alternative to conventional cryopreservation.

Sperm vitrification is a simple, inexpensive method that allows the cryopreservation of sperm in the field and for endangered species is a useful alternative to conventional freezing. The study, therefore, is focused on the suitability of vitrification for cryopreserving Iberian wolf sperm and utilizing plasma testosterone concentration as a marker for procedure efficacy. Sperm and blood samples were collected from 17 wolves. There were 14 samples suitable for cryopreservation (12 ejaculated and two epididymal). Immediately after collection, these samples were proportioned into two aliquots for conventional freezing using a Tris-citric acid-glucose based extender (TCG) or vitrification utilizing an animal protein free extender (HTF®). Vitrification occurred by directly plunging a sperm suspension into liquid nitrogen. Sperm were assessed for motility, membrane integrity, acrosomal status and DNA integrity before and after cryopreservation. With both techniques, there were similar post-thaw/warming results (P > 0.05) with respect to progressive motility, kinetic variables VCL, VSL, VAP and BCF, DNA fragmentation, sperm membrane functionality and morphological abnormalities. Total motile sperm, progression ratios LIN, STR, and WOB, the ALH, sperm viability and sperm with intact membrane and acrosome were greater (P < 0.05) in the conventional frozen-thawed sperm than vitrified-warmed sperm. Plasma testosterone concentrations varied from 0.0 ng/mL to 7.7 ng/mL. For epididymal sperm, sperm motility and viability following thawing were greater in vitrified-warmed samples than conventionally-frozen samples; however, small sample numbers precluded statistical analysis. When considered together, these results indicate vitrification may be a possible alternative for wolf sperm cryopreservation.

Effect of shortening the prefreezing equilibration time with glycerol on the quality of chamois (Rupicapra pyrenaica), ibex (Capra pyrenaica), mouflon (Ovis musimon) and aoudad (Ammotragus lervia) ejaculates.

The present study reports the effect of shortening the prefreezing equilibration time with glycerol on the quality of frozen-thawed ejaculated sperm from four Mediterranean mountain ungulates: Cantabrian chamois (Rupicapra pyrenaica), Iberian ibex (Capra pyrenaica), mouflon (Ovis musimon) and aoudad (Ammotragus lervia). Ejaculated sperm from these species were divided into two aliquots. One was diluted with either a Tris-citric acid-glucose based medium (TCG-glycerol; for chamois and ibex sperm) or a Tris-TES-glucose-based medium (TTG-glycerol; for mouflon and aoudad sperm), and maintained at 5°C for 3h prior to freezing. The other aliquot was diluted with either TCG (chamois and ibex sperm) or TTG (mouflon and aoudad sperm) and maintained at 5°C for 1h before adding glycerol (final concentration 5%). After a 15min equilibration period in the presence of glycerol, the samples were frozen. For the ibex, there was enhanced (P<0.05) sperm viability and acrosome integrity after the 3h as compared with the 15min equilibration time. For the chamois, subjective sperm motility and cell membrane functional integrity were less (P<0.05) following 15min of equilibration. In the mouflon, progressive sperm motility and acrosome integrity was less (P<0.05) when the equilibration time was reduced to 15min. For the aoudad, the majority of sperm variables measured were more desirable after the 3h equilibration time. The freezing-thawing processes reduced the sperm head size in all the species studied; however, the equilibration time further affected the frozen-thawed sperm head variables in a species-dependent fashion. While the equilibration time for chamois sperm might be shortened, this appears not to be the case for all ungulates.

Carbonic anhydrases and their functional differences in human and mouse sperm physiology.

Fertilization is a key reproductive event in which sperm and egg fuse to generate a new individual. Proper regulation of certain parameters (such as intracellular pH) is crucial for this process. Carbonic anhydrases (CAs) are among the molecular entities that control intracellular pH dynamics in most cells. Unfortunately, little is known about the function of CAs in mammalian sperm physiology. For this reason, we re-explored the expression of CAI, II, IV and XIII in human and mouse sperm. We also measured the level of CA activity, determined by mass spectrometry, and found that it is similar in non-capacitated and capacitated mouse sperm. Importantly, we found that CAII activity accounts for half of the total CA activity in capacitated mouse sperm. Using the general CA inhibitor ethoxyzolamide, we studied how CAs participate in fundamental sperm physiological processes such as motility and acrosome reaction in both species. We found that capacitated human sperm depend strongly on CA activity to support normal motility, while capacitated mouse sperm do not. Finally, we found that CA inhibition increases the acrosome reaction in capacitated human sperm, but not in capacitated mouse sperm.

Comparative efficacy of two interventions to discontinue long-term benzodiazepine use: cluster randomised controlled trial in primary care.

BACKGROUND: Benzodiazepines are extensively used in primary care, but their long-term use is associated with adverse health outcomes and dependence. AIMS: To analyse the efficacy of two structured interventions in primary care to enable patients to discontinue long-term benzodiazepine use. METHOD: A multicentre three-arm cluster randomised controlled trial was conducted, with randomisation at general practitioner level (trial registration ISRCTN13024375). A total of 532 patients taking benzodiazepines for at least 6 months participated. After all patients were included, general practitioners were randomly allocated (1:1:1) to usual care, a structured intervention with follow-up visits (SIF) or a structured intervention with written instructions (SIW). The primary end-point was the last month self-declared benzodiazepine discontinuation confirmed by prescription claims at 12 months. RESULTS: At 12 months, 76 of 168 (45%) patients in the SIW group and 86 of 191 (45%) in the SIF group had discontinued benzodiazepine use compared with 26 of 173 (15%) in the control group. After adjusting by cluster, the relative risks for benzodiazepine discontinuation were 3.01 (95% CI 2.03-4.46, P<0.0001) in the SIW and 3.00 (95% CI 2.04-4.40, P<0.0001) in the SIF group. The most frequently reported withdrawal symptoms were insomnia, anxiety and irritability. CONCLUSIONS: Both interventions led to significant reductions in long-term benzodiazepine use in patients without severe comorbidity. A structured intervention with a written individualised stepped-dose reduction is less time-consuming and as effective in primary care as a more complex intervention involving follow-up visits.

The anti-inflammatory drug celecoxib inhibits T-type Ca2+ currents in spermatogenic cells yet it elicits the acrosome reaction in mature sperm.

Celecoxib (Cx), an anti-inflammatory drug designed to inhibit COX2, can affect some ion channels. T-type (CaV3) channels have been implicated in sperm physiology. Here we report and characterize the Cx induced inhibition of T-type channels in mouse spermatogenic cells. Unexpectedly, Cx can also induce the acrosome reaction (AR), an intracellular Ca(2+) ([Ca(2+)]i) increase and a sperm depolarization. This [Ca(2+)]i increase possibly results from the ability Cx has to alkalinize intracellular pH (pHi), which is known to activate the sperm specific Ca(2+) channel CatSper. As the Cx induced [Ca(2+)]i increase is sensitive to mibefradil, a CatSper blocker, this channel may mediate the Cx-induced Ca(2+) entry leading to the AR. Our observations demonstrate that Cx can compromise fertilization.

Sorption of tetracyclines onto natural soils: data analysis and prediction.

Residues of tetracyclines reach soils as a result of animal waste application. Sorption is a key process in transport, fate, and effects of contaminants in the environment. In this work, we have attempted to predict the sorption of four widely used tetracyclines (oxytetracycline, tetracycline, chlortetracycline, and doxycycline) from soil physicochemical properties. Batch sorption experiments were performed on 15 natural soils with a broad range of physicochemical properties, and the data were fitted to several isotherm models. Multivariate analysis methods were conducted to identify the main factors affecting the sorption distribution coefficients (K (d)) of the tetracyclines at two aqueous concentration levels (100 and 400 µg L(-1)). All four tetracycline sorption isotherms in alkaline and acidic soils were well described by the Freundlich and Langmuir equation, respectively. At intermediate soil pH (from 5.3 to 7), oxytetracycline and tetracycline exhibited Freundlich behavior, whereas chlortetracycline and doxycycline followed a Langmuir model. Two partial least squares (PLS) models were developed. The first one uses five soil descriptors as input variables; the second uses, pH, cation exchange capacity (CEC), and log K (d,OTC). Both models satisfactorily predicted distribution coefficients within a factor of 1.5. Sorption of tetracyclines in soil is governed by several factors, in the following order of importance: solution speciation, CEC (dominant at acidic-neutral soil pH), transition metal content, and texture. The PLS models indicated that tetracycline sorption can be predicted using a minimal set of soil descriptors including oxytetracycline sorption data.

Studies on the extraction of sulfonamides from agricultural soils.

The extraction of six sulfonamides (sulfadiazine, sulfadimidine, sulfathiazole, sulfachloropiridazine, sulfadimethoxine, and sulfaquinoxaline) from soils with different physicochemical characteristics and at several aging times was investigated. Conventional mechanical shaking, microwave-assisted extraction, ultrasound probe-assisted extraction and pressurized liquid extraction techniques were evaluated. The four techniques provided similar results when applied to freshly contaminated soils. However, microwave-assisted extraction was the most suitable to extract sulfonamide aged residues from soils. Microwave-assisted extraction was applied to eight soils aged for 3 months, using acetonitrile:buffer pH 9 (20:80) as the extraction solvent, and recoveries ranged from 15-25% for STZ to 42-64% for SDM.

Determination of pK(a) values of some hydroxylated benzoic acids in methanol-water binary mixtures by LC methodology and potentiometry.

An accurate estimation of pK(a) values in methanol-water binary mixtures is very important for several separation techniques such as liquid chromatography and capillary electrophoresis that use these solvent mixtures. In this study, the pK(a) values of 11 polyphenolic acids have been determined in methanol-water binary mixtures (10%, 20% and 30% (v/v)) by potentiometry, liquid chromatography (LC) and LC-DAD methodology. The results show a similar trend for the pK(a) values of all the studied compounds, as they increase with increasing concentration of organic modifier, which allows a linear relationship between pK(a) values and mole fraction of methanol to be obtained. The pK(a) values obtained in aqueous medium have been compared with those given in the literature, and also with the values predicted by the SPARC on-line pK(a) calculator. The data obtained have been used to test the feasibility of an estimation of dissociation constants in a methanol-water medium from the relationship between pK(a) values and the organic cosolvent fraction in the mixtures.

Kinetic parameters estimation in an anaerobic digestion process using successive quadratic programming.

In this work, an optimization method is implemented in an anaerobic digestion model to estimate its kinetic parameters and yield coefficients. This method combines the use of advanced state estimation schemes and powerful nonlinear programming techniques to yield fast and accurate estimates of the aforementioned parameters. In this method, we first implement an asymptotic observer to provide estimates of the non-measured variables (such as biomass concentration) and good guesses for the initial conditions of the parameter estimation algorithm. These results are then used by the successive quadratic programming (SQP) technique to calculate the kinetic parameters and yield coefficients of the anaerobic digestion process. The model, provided with the estimated parameters, is tested with experimental data from a pilot-scale fixed bed reactor treating raw industrial wine distillery wastewater. It is shown that SQP reaches a fast and accurate estimation of the kinetic parameters despite highly noise corrupted experimental data and time varying inputs variables. A statistical analysis is also performed to validate the combined estimation method. Finally, a comparison between the proposed method and the traditional Marquardt technique shows that both yield similar results; however, the calculation time of the traditional technique is considerable higher than that of the proposed method.

Fast-scanning fluorescence spectroscopy as a detection system in liquid chromatography for confirmatory analysis of flumequine and oxolinic acid.

Oxolinic acid and flumequine were analysed by reversed-phase liquid chromatography after extraction from the sample matrix with dichloromethane and partitioning with NaOH. The detection system consisted of a fast-scanning fluorescence detector, which provides the full spectra of the eluting peaks and can thus be used to confirm the identity of analytes. Determination was performed by partial least squares (PLS) and three-way PLS over the three-dimensional data, i.e. fluorescence intensity versus retention time and excitation wavelength. In both cases, similar results, with prediction errors around 4%, were obtained. The method was successfully applied to the analysis of salmon, pork and chicken muscle spiked up to 300 ng g(-1).

Calmodulin antagonists inhibit T-type Ca(2+) currents in mouse spermatogenic cells and the zona pellucida-induced sperm acrosome reaction.

The sperm acrosome reaction (AR) is a regulated exocytotic process required for gamete fusion. It depends on an increase in [Ca(2+)](i) mediated by Ca(2+) channels. Although calmodulin (CaM) has been reported to regulate several events during the AR, it is not known whether it modulates sperm Ca(2+) channels. In the present study we analyzed the effects of CaM antagonists W7 and trifluoroperazine on voltage-dependent T-type Ca(2+) currents in mouse spermatogenic cells and on the zona pellucida-induced AR in sperm. We found that these CaM antagonists decreased T-currents in a concentration-dependent manner with IC(50) values of approximately 10 and approximately 12 microM, respectively. W7 altered the channels' voltage dependence of activation and slowed both activation and inactivation kinetics. It also induced inactivation at voltages at which T-channels are not activated, suggesting a promotion of inactivation from the closed state. Consistent with this, W7 inhibited the ZP-induced [Ca(2+)](i) transients in capacitated sperm. Likewise, W7 and TFP inhibited the AR with an IC(50) of approximately 10 microM. In contrast, inhibitors of CaM-dependent kinase II and protein kinase A, as well as a CaM-activated phosphatase, had no effect either on T-currents in spermatogenic cells or on the sperm AR. Together these results suggest a functional interaction between CaM and the sperm T-type Ca(2+) channel. They are also consistent with the involvement of T-channels in the AR.

Determination of the dissociation constants of sulfonated azo dyes by capillary zone electrophoresis and spectrophotometry methods.

The dissociation constants of 10 sulfonated azo dyes, six of the most common food colours used as additives (Food Yellow 4, Food Yellow 3, Food Red 9, Food Red 7, Food Red 17 and Food Blue 5), and four commonly used as textile dyes (Acid Orange 7, Acid Orange 12, Acid Red 26 and Acid Red 88), have been determined by two different systems, one by using capillary electrophoresis (CE) with diode array detection and the other by using UV-visible absorption spectrophotometry, which has been used as reference method to obtain the pKa values. The pKa values obtained by CE were determined in two ways, first on the basis of the electrophoretic mobilities (calculated from the migration times), and after we propose a new methodology, in which the dissociation constants are determined from the spectra corresponding to the maxima of electrophoretic peaks. The pKa values obtained by using these CE methods have been compared with those obtained by using the spectrophotometric method. The results show that the pKa values obtained by the CE proposed method are in general closer to the reference values than those obtained from the electrophoretic mobilities. Moreover, the proposed method retains the advantages of CE, as the possibility of working with small amounts of sample, despite its purity.

Inwardly rectifying K(+) channels in spermatogenic cells: functional expression and implication in sperm capacitation.

To fertilize, mammalian sperm must complete a maturational process called capacitation. It is thought that the membrane potential of sperm hyperpolarizes during capacitation, possibly due to the opening of K(+) channels, but electrophysiological evidence is lacking. In this report, using patch-clamp recordings obtained from isolated mouse spermatogenic cells we document the presence of a novel K(+)-selective inwardly rectifying current. Macroscopic current activated at membrane potentials below the equilibrium potential for K(+) and its magnitude was dependent on the external K(+) concentration. The channels selected K(+) over other monovalent cations. Current was virtually absent when external K(+) was replaced with Na(+) or N-methyl-D-glucamine. Addition of Cs(+) or Ba(2+) (IC(50) of approximately 15 microM) to the external solution effectively blocked K(+) current. Dialyzing the cells with a Mg(2+)-free solution did not affect channel activity. Cytosolic acidification reversibly inhibited the current. We verified that the resting membrane potential of mouse sperm changed from -52 +/- 6 to -66 +/- 9 mV during capacitation in vitro. Notably, application of 0.3-1 mM Ba(2+) during capacitation prevented this hyperpolarization and decreased the subsequent exocytotic response to zona pellucida. A mechanism is proposed whereby opening of inwardly rectifying K(+) channels may produce hyperpolarization under physiological conditions and contribute to the cellular changes that give rise to the capacitated state in mature sperm.

Determination of naphthalenesulfonates in water by on-line ion-pair solid-phase extraction and ion-pair liquid chromatography with fast-scanning fluorescence detection.

A fast analytical method for quantifying a mixture of 12 naphthalenesulfonates and naphthalenedisulfonates has been developed. This method consists of on-line ion-pair solid-phase extraction with PLRP-s sorbent and ion-pair liquid-chromatography using fast-scanning fluorescence spectrometer as a detection system and multivariate calibration. As complete separation is unnecessary, the compounds were analysed in isocratic conditions and the chromatographic analysis took only 25 min. Three-way partial least-squares (PLS) was used to carry out multivariate calibration for spiked tap water. In these conditions, quantification limits were between 0.01 and 3 microg x l(-1). Repeatability was also evaluated and relative standard deviations (n=3) were between 0.5 and 4, depending on the compound. Finally, spiked tap and Ebro river waters were analysed to evaluate prediction capability of the method.

Determination of sulfonated azo dyes in river water samples by capillary zone electrophoresis.

A method based on capillary zone electrophoresis coupled with photodiode-array detection has been developed to determine several sulfonated dyes, including a sulfonated dye (acid yellow 1), and the sulfonated azo dyes acid orange 7, acid orange 12, acid orange 52, acid red 26, acid red 27 and acid red 88. A CElect-FS75 CE column is used. The electrophoresis buffer contains a 1:5 dilution of 10 mM phosphoric acid and tetrabutylammonium hydroxide buffer (pH 11.5), and 25 mM of triethylamine, the final pH being 11.55. The detection limits for the seven dyes ranged from 0.1 to 4.53 microg/ml. Spiked river water samples (100 ml), containing different concentration levels (0.025-0.150 microg/ml) of the dyes were analyzed after acidification (pH 3) and pre-concentration in disposable SPE Oasis HLB, 1 ml cartridges.

Dual regulation of the T-type Ca(2+) current by serum albumin and beta-estradiol in mammalian spermatogenic cells.

This study provides evidence for a novel mechanism of voltage-gated Ca(2+) channel regulation in mammalian spermatogenic cells by two agents that affect sperm capacitation and the acrosome reaction (AR). Patch-clamp experiments demonstrated that serum albumin induced an increase in Ca(2+) T current density in a concentration-dependent manner, and significant shifts in the voltage dependence of both steady-state activation and inactivation of the channels. These actions were not related to the ability of albumin to remove cholesterol from the membrane. In contrast, beta-estradiol significantly inhibited Ca(2+) channel activity in a concentration-dependent and essentially voltage-independent fashion. In mature sperm this dual regulation may influence capacitation and/or the AR.

Determination of sulphonate dyes in water by ion-interaction high-performance liquid chromatography.

An ion-interaction high-performance liquid chromatography method for quick separation and determination of the sulphonated dyeAcid Yellow 1, and the sulphonated azo dyes Acid Orange 7, Acid Orange 12, Acid Orange 52, Acid Red 2, Acid Red 26, Acid Red 27 and Acid Red 88 has been developed. An RP-ODS stationary phase is used, and the mobile phase contains an acetonitrile-phosphate buffer (27:73, v/v) mixture at pH 6.7, containing 2.4 mM butylamine as ion-interaction reagent. Good separations were obtained using isocratic elution and spectrophotometric detection at 460 nm. The detection limits for the eight dyes ranged from 7 to 28 microg/l for an injection volume of 100 microl. Spiked tap water samples (100 ml), containing different concentration levels (0.3-1.2 microg/l) of the dyes were analyzed after acidification (pH 3) and preconcentration in disposable solid-phase extraction C18 cartridges.

Determination of dyes in foodstuffs by capillary zone electrophoresis.

A rapid method based on capillary zone electrophoresis coupled with photodiode-array detection has been developed to determine the dyes Tartrazine E-102, Sunset Yellow FCF E110, Amaranth E-123, New Coccine E-124, Patent Blue V calcium salt E-131 and Allura Red AC E-129 in foodstuffs. Separation was done by using a Bare CElect-FS75 CE column, using a 10 mM phosphate buffer at pH 11.0. Hydrodynamic injections at 0.5 p.s.i. for 4 s (21 nl of sample) and 20 kV separation voltage were used. The quantitation limits for the six dyes ranged from 3 to 6 microg/ml. A linear relationship between 3 to 95 microg/ml, with correlation coefficient better than 0.995 was obtained. This method has been applied to the determination of the studied dyes in beverages, jellies and syrups.

Study on partition equilibria of metal complexes in non-ionic micellar solutions from spectrophotometric data.

The complexation equilibria for Zn(II)-8-quinolinol and Zn(II)-5,7-dichloro-2-methyl-8-quinolinol systems were studied spectrophotometrically in aqueous micellar solutions of the non-ionic surfactant Brij-35 in NaCl 0.1 M medium at 25 degrees C. The partition model, in which the different species involved in the equilibria can distribute themselves between aqueous and micellar pseudophases, was applied. Calculations were performed by means of the spdis program, developed specifically to handle multiwavelength spectrophotometric data in micellar systems. A factor analysis was applied to the spectrophotometric data in order to determine the number of species in equilibrium. A quantitative relationship was found between fluorescence intensity and the micellar solubilization of metal chelates.