Browsing by Author "Bustamante, E"

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    Egg coats of the rock shrimp Rhynchocinetes typus
    (2002) Palomino, J; Moreno, RD; Bustamante, E; Messen, L; Dupré, E; Barros, C
    The aim of the present work was to characterize structurally and ultrastructurally the egg coats of the rock shrimp, Rhynchocinetes typus, and to describe their functional roles during fertilization. Oocytes fixed directly from the ovary, have a total diameter of 549 mum and are covered by a 10-mum-thick transparent envelope. Electron microscope sections (dehydrated) of the egg envelope revealed an electron-dense external coat of 0.4 mum covered by filamentous processesi and a granular inner coat of 4-mum thickness. Oocytes placed for 5 min in seawater had a significantly larger diameter (573 mum), because of the increase in the thickness of the egg coats (32 mum) and the formation of a 16-mum perivitelline space. The diameter of the egg proper was reduced by the same extent as the size of the perivitelline space. All these changes were associated to the loss of the egg fertilizability. SDS-PAGE of isolated and solubilized egg coats with 20% beta-mercaptoethanol or 25 mM dithiothreitol (DTT) showed bands between 58 and 105 kDa and between 44 and 103 kDa, respectively. During normal fertilization, the sperm undergoes a drastic change in shape after first contact with the egg. We observed a similar change when solubilized egg coats were placed with vas deferens sperms. When the solubilized egg coat proteins were ultrafiltrated with a membrane of 10,000 MWCO (pore size) and then assayed for their effect on fertilization, an inhibitory effect of 30%, 41%, and 59% was found when oocytes were incubated with spermatozoa pre-treated with 30, 60, and 120 mug/ml of proteins solubilized with beta-mercaptoethanol. A similar inhibitory effect was found when egg coat proteins solubilized with 25 mM DTT were used. Our results suggest that, in the shrimp, the egg coats play an active role during the morphological changes of the sperm during their passage through them. (C) 2002 Wiley-Liss, Inc.
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    Inhibition of mouse in vitro fertilization by an antibody against a unique 18-amino acid domain in the polysulfate-binding domain of proacrosin/acrosin
    (2002) Moreno, RD; Bustamante, E; Schatten, G; Barros, C
    Objective: To determine the contribution of the polysulfate-binding domain (PSBD) of acrosin during sperm penetration.
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    Micro-spatial variation of soil metal pollution and plant recruitment near a copper smelter in Central Chile
    (2004) Ginocchio, R; Carvallo, G; Toro, I; Bustamante, E; Silva, Y; Sepúlveda, N
    Soil chemical changes produced by metal smelters have mainly been studied on a large scale. In terms of plant survival, determination of small scale variability may be more important because less toxic microhabitats may represent safe sites for successful recruitment and thus for plant survival. Three dominant microhabitats (open spaces and areas below the canopy of Sphaeralcea obtusiloba and Baccharis linearis shrubs) were defined in a heavily polluted area near a copper smelter and characterised in terms of microclimate, general soil chemistry, total and extractable metal concentrations in the soil profile (A(0) horizon, 0-5 and 15-20 cm depth), and seedling densities. Results indicated a strong variability in microclimate and soil chemistry not only in the soil profile but also among microhabitats. Air/soil temperatures, radiation and wind speed were much lower under the canopy of shrubs, particularly during the plant growth season. Soil acidification was detected on top layers (0-5 cm depth) of all microhabitats while higher concentrations of N, Cu and Cd were detected on litter and top soil layers below shrubs when compared to open spaces; however, high organic matter content below shrubs decreased bioavailability of metals. Plant recruitment was concentrated under shrub canopies; this may be explained as a result of the nursery effect exerted by shrubs in terms of providing a more favourable microclimate, along with better soil conditions in terms of macronutrients and metal bioavailability. (C) 2003 Elsevier Ltd. All rights reserved.
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    Purification and biochemical characterization of a trypsin-like enzyme present in the sperm of the rock shrimp, Rhynchocinetes typus
    (2001) Bustamante, E; Palomino, J; Amoroso, A; Moreno, R; Barros, C
    The aim of the present work was to isolate, purify and characterize a trypsin-like enzyme from the sperm of the rock shrimp, Rhynchocinetes typus. Sperm proteins were extracted with 1 mM HCl in 10% glycerol at pH 3.0. Purification of the trypsin-like substance was effected by affinity chromatography using SBTI-agarose, yielding a specific activity on BAEE as substrate of 787 U/mg, with a recovery rate of 34%. Enzymatic activity was maximal at 27 degreesC, pH of 8.0, 50 mM Ca2+ and 30 mM Mg2+. One hundred percent inhibition of enzymatic activity was obtained at 0.05 mM Zn2+. Kinetic analysis showed that the K-M on BAEE as substrate at pH 8.0 was 2.5 x 10(-5) M and the V-MAX reached was 198 U. It was also found that the enzyme had a substrate inhibition at concentrations higher than 0.06 mM of BAEE. These findings suggest that thin enzyme has similar characteristics to other trypsin-like enzymes including acrosin.