Dose-dependent effect of heparin on fertilizing ability of goat spermatozoa

Intact bovine oocytes were used to study the effect of heparin on goat IVF. Oocytes were matured in Medium 199 plus estrous sheep serum. Fresh semen was incubated for 4 h at room temperature, and spermatozoa were then resuspended in medium Talp plus serum and incubated further for 1 h at 39 degrees C in 5% CO(2) in air. Later, spermatozoa were resuspended in Talp plus serum and heparin and were then incubated in microdrops until the oocytes were matured. In Experiment 1, the effect of heparin on spermatozoa from individual males was studied by a dose-response curve. In Experiment 2, the timing of sperm penetration in matured oocytes was studied to assess the stage at which the action of heparin could be expressed in the fertilization process. In Experiment 3, heparin from the same source but at different grades of bioactivity was adjusted for bioactivity and its effect on spermatozoa was compared in terms of penetration rates in order to identify heparin-dependent variations on goat IVF. In Experiment 4, the influence of calcium on the effect of heparin at different levels of bioactivity on the fertilizing ability spermatozoa was assessed as in Experiment 3. In Experiment 5, different batches of heparin from the same source and grade of bioactivity were compared as above. The results suggest that 1) heparin stimulates fertilization rates following a comparable pattern between males; 2) the most probable site of action is at the stage of sperm capacitation; and 3) provided that the source and grade of bioactivity is preserved, heparin maintains the efficiency of sperm penetration into matured oocytes.     

Elemental distribution in striated muscle and the effects of hypertonicity: Electron probe analysis of cryo sections

A method of rapid freezing in supercooled Freon 22 (monochlorodifluoromethane) followed by cryoultramicrotomy is described and shown to yield ultrathin sections in which both the cellular ultrastructure and the distribution of diffusible ions across the cell membrane are preserved and intracellular compartmentalization of diffusabler ions can be quantitated. Quantitative electron probe analysis (Shuman, H., A.V. Somlyo, and A.P. Somlyo. 1976. Ultramicros. 1:317-339.) of freeze-dried ultrathin cryto sections was found to provide a valid measure of the composition of cells and cellular organelles and was used to determine the ionic composition of the in situ terminal cisternae of the sarcoplasmic reticulum (SR), the distribution of CI in skeletal muscle, and the effects of hypertonic solutions on the subcellular composition if striated muscle. There was no evidence of sequestered CI in the terminal cisternae of resting muscles, although calcium (66mmol/kg dry wt +/- 4.6 SE) was detected. The values of [C1](i) determined with small (50-100 nm) diameter probes over cytoplasm excluding organelles over nuclei or terminal cisternae were not significantly different. Mitochondria partially excluded C1, with a cytoplasmic/ mitochondrial Ci ratio of 2.4 +/- 0.88 SD. The elemental concentrations (mmol/kg dry wt +/- SD) of muscle fibers measured with 0.5-9-μm diameter electron probes in normal frog striated muscle were: P, 302 +/- 4.3; S, 189 +/- 2.9;C1, 24 +/- 1.1;K, 404 +/- 4.3, and Mg, 39 +/- 2.1. It is concluded that: (a) in normal muscle the "excess CI" measured with previous bulk chemical analyses and flux studies is not compartmentalized in the SR or in other cellular organelles, and (b) the cytoplasmic C1 in low [K](0) solutions exceeds that predicted by a passive electrochemical distribution. Hypertonic 2.2 X NaCl, 2.5 X sucrose, or 2.2 X Na isethionate produced: (a) swollen vacuoles, frequently paired, adjacent to the Z lines and containing significantly higher than cytoplasmic concentrations of Na and Cl or S (isethionate), but no detectable Ca, and (b) granules of Ca, Mg, and P = approximately (6 Ca + 1 Mg)/6P in the longitudinal SR. It is concluded that hypertonicity produces compartmentalized domains of extracellular solutes within the muscle fibers and translocates Ca into the longitudinal tubules.     

Study of yeast populations and their enological properties in Guijoso Appellation of Origin (Spain)

The aim of the present study was to select strains of yeast with good enological qualities which were adapted to the ecological surroundings of Guijoso Appellation of Origin (A.O). For this, 11 white and red vats from different grape varieties and stages of fermentation were studied, making a total of 28 samples, with a selection of 370 isolated yeasts. Yeast cells of the Saccharomyces genus were analysed by DNA mitochondrial restriction for discrimination at the strain level, obtaining a total of 23 different molecular patterns. The pattern most frequently found was of G04, with 56 % of the isolated yeasts, followed by G02 with 15 % and G07 with 9 %. Other patterns found showed percentages close to 3 %, such as G01, G03, G05 and G06, while the remaining patterns were limited one or two isolated yeasts. Microfermentations at 25 and 15 °C were performed using a synthetic must, and the rate of fermentation, SH₂ and foam production and the capacity to consume sugars from the medium were studied. Furthermore, the killer phenotype, flocculation capacity and phenolic off-flavour (POF) characteristics were also analysed. Natural musts from Chardonnay and Cabernet Sauvignon varieties were fermented using preselected strains and the wines obtained were analysed and tasted. Two strains were selected (G01 and G04) to be used as starters in Guijoso A.O.     

Coenzyme Q10 deficiency in mitochondrial DNA depletion syndromes

We evaluated coenzyme Q₁₀ (CoQ) levels in patients studied under suspicion of mitochondrial DNA depletion syndromes (MDS) (n = 39). CoQ levels were quantified by HPLC, and the percentage of mtDNA depletion by quantitative real-time PCR. A high percentage of MDS patients presented with CoQ deficiency as compared to other mitochondrial patients (Mann–Whitney-U test: p = 0.001). Our findings suggest that MDS are frequently associated with CoQ deficiency, as a possible secondary consequence of disease pathophysiology. Assessment of muscle CoQ status seems advisable in MDS patients since the possibility of CoQ supplementation may then be considered as a candidate therapy.     

Plant virus infection development as affected by heavy metal stress

The work was focused on the investigation of possible dependencies between the development of viral infection in plants and the presence of high heavy metal concentrations in soil. Field experiments have been conducted in order to study the development of systemic tobacco mosaic virus (TMV) infection in Lycopersicon esculentum L. cv. Miliana plants under effect of separate salts of heavy metals Cu, Zn and Pb deposited in soil. As it is shown, simultaneous effect of viral infection and heavy metals in tenfold maximum permissible concentration leads to decrease of total chlorophyll content in experiment plants mainly due to the degradation of chlorophyll a. The reduction of chlorophyll concentration under the combined influence of both stress factors was more serious comparing to the separate effect of every single factor. Plants' treatment with toxic concentrations of lead and zinc leaded to slight delay in the development of systemic TMV infection together with more than twofold increase of virus content in plants that may be an evidence of synergism between these heavy metal's and virus' effects. Contrary, copper although decreased total chlorophyll content but showed protective properties and significantly reduced amount of virus in plants.     

Fouling communities and degradation of archeological metals in the coastal sea of the Southwestern Gulf of Mexico

Corrosion and biofouling phenomena of cast iron and brass were evaluated under natural conditions to determine the degradation process of archeological artifacts. Field exposure studies of experimental materials were conducted over 15 months at an offshore position in the sea of Campeche in the Gulf of Mexico. Corrosion was determined by gravimetric measurements. The community structure of the benthic assemblage inhabiting the surfaces of both materials was evaluated. A total of 53 species was identified. The community in both cases was composed of a small number of species. Encrusting, attached and erect life forms were dominant on iron. Attached life forms were dominant on brass. Biofouling produced a decrease in the weight loss measurements of cast iron samples. Biofouling provided a beneficial factor for in situ preservation of iron archeological artifacts in wreck sites.     

Heat Treatment and β-Carotene Incorporation Effect in the Interaction of β-Lactoglobulin and Carboxymethylcellulose System

Encapsulation technologies using proteins or polysaccharides can be employed with the purpose of solubilizing and protecting carotenoids. However, information on the role of protein and polysaccharide interactions is still slightly limited. The aim of this work was to investigate the effect of β-carotene linked to protein β-lactoglobulin (BLG) in the interaction carboxymethylcellulose (CMC) using isothermal titration calorimetry (ITC). Firstly, BLG and CMC interaction was assessed by means of turbidity analysis. Based on the results of turbidity, the thermodynamic profile of BLG-CMC complexes at pH 4.0 was obtained using ITC analysis at 25 °C. Afterward, it was evaluated the effect of a thermal treatment applied to the BLG (68 °C for 50 min) in the interaction with CMC also using ITC and circular dichroism (CD). ITC and CD analysis showed that the heat treatment applied on BLG did not cause changes in molecular interactions. The binding isotherm of BLG-CMC complexes incorporated with β-carotene showed an increase in the molar ratio and a slight decrease in enthalpy of the system. Incorporation of β-carotene in the system did not significantly affect the BLG and CMC interaction, suggesting this system can be applied in food application as encapsulation.     

GROmaρs: A GROMACS-Based Toolset to Analyze Density Maps Derived from Molecular Dynamics Simulations

We introduce a computational toolset, named GROmaρs, to obtain and compare time-averaged density maps from molecular dynamics simulations. GROmaρs efficiently computes density maps by fast multi-Gaussian spreading of atomic densities onto a three-dimensional grid. It complements existing map-based tools by enabling spatial inspection of atomic average localization during the simulations. Most importantly, it allows the comparison between computed and reference maps (e.g., experimental) through calculation of difference maps and local and time-resolved global correlation. These comparison operations proved useful to quantitatively contrast perturbed and control simulation data sets and to examine how much biomolecular systems resemble both synthetic and experimental density maps. This was especially advantageous for multimolecule systems in which standard comparisons like RMSDs are difficult to compute. In addition, GROmaρs incorporates absolute and relative spatial free-energy estimates to provide an energetic picture of atomistic localization. This is an open-source GROMACS-based toolset, thus allowing for static or dynamic selection of atoms or even coarse-grained beads for the density calculation. Furthermore, masking of regions was implemented to speed up calculations and to facilitate the comparison with experimental maps. Beyond map comparison, GROmaρs provides a straightforward method to detect solvent cavities and average charge distribution in biomolecular systems. We employed all these functionalities to inspect the localization of lipid and water molecules in aquaporin systems, the binding of cholesterol to the G protein coupled chemokine receptor type 4, and the identification of permeation pathways through the dermicidin antimicrobial channel. Based on these examples, we anticipate a high applicability of GROmaρs for the analysis of molecular dynamics simulations and their comparison with experimentally determined densities.