Browsing by Author "Moreno, RD"

Now showing 1 - 8 of 8
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    A basic 18-amino acid peptide contains the polysulfate-binding domain responsible for activation of the boar proacrosin/acrosin system
    (2000) Moreno, RD; Barros, C
    Proacrosin is the zymogen of acrosin, a serine protease localized in the acrosomal matrix of mammalian sperm. Proacrosin/acrosin binds to solubilized zona pellucida glycoproteins (ZPGs) and various polysulfates in a non-enzymatic mechanism. In addition, both polysulfates and ZPGs induce proacrosin activation once they bind to the polysulfate-binding domain (PSBD) of the enzyme. We show here that the peptide (43)IFMYHNNRRYHTCCGILL(60) inhibited the proacrosin activation induced by either fucoidan or ZPGs. In addition, the peptide was recognized by the monoclonal antibody C5F10, which is directed against the PSBD region. Our data suggest that the PSBD is composed of many "subsites" that may or may not interact with each other.
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    Acetylcholinesterase accelerates assembly of amyloid-beta-peptides into Alzheimer's fibrils: Possible role of the peripheral site of the enzyme
    (1996) Inestrosa, N.C.; Alvarez, A; Perez, CA; Moreno, RD; Vicente, M; Linker, C; Casanueva, OI; Soto, C; Garrido, J
    Acetylcholinesterase (AChE), an important component of cholinergic synapses, colocalizes with amyloid-beta peptide (A beta) deposits of Alzheimer's brain. We report here that bovine brain AChE, as well as the human and mouse recombinant enzyme, accelerates amyloid formation from wild-type A beta and a mutant A beta peptide, which alone produces few amyloid-like fibrils. The action of AChE was independent of the subunit array of the enzyme, was not affected by edrophonium, an active site inhibitor, but it was affected by propidium, a peripheral anionic binding site ligand. Butyrylcholinesterase, an enzyme that lacks the peripheral site, did not affect amyloid formation. Furthermore, AChE is a potent amyloid-promoting factor when compared with other A beta-associated proteins. Thus, in addition to its role in cholinergic synapses, AChE may function by accelerating A beta formation and could play a role during amyloid deposition in Alzheimer's brain.
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    Differential effects of polysulphates between mouse and hamster during in vitro fertilization
    (2001) Moreno, RD; Orihuela, PA; Barros, C
    The interaction between zona pellucida polysulphates and sperm receptors appears to be a widespread mechanism used by mammals during gamete interaction. In this work, the effect of hepar in on binding, penetration and fertilization of mouse and hamster oocytes was assessed. We found that heparin inhibited oocyte penetration and fertilization in both species. Heparin as well as fucoidan (a fucose-sulphate polymer) inhibited the proteolytic: activity of acrosomal enzymes in both species. Our results suggest that zona pellucida penetration in both species may be modulated by polysulphates acting on either the proteolytic rate of degradation of the zona and/or its interaction with acrosome-reacted sperm (secondary binding).
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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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    Functional interactions between sulphated polysaccharides and proacrosin: implications in sperm binding and digestion of zona pellucida
    (1999) Moreno, RD; Hoshi, M; Barros, C
    Acrosin is a serine protease located within mammalian acrosome as inactive proacrosin. Sulphated polymers bind to proacrosin and acrosin, to a domain different from the active site. Upon binding, these polymers induce proacrosin activation and some of them, such as fucoidan, inhibit sperm binding to the zona pellucida. In this work we have studied the interaction of solubilised zona pellucida glycoproteins (ZPGs), heparin and ARTS (Acrosome Reaction Inducing Substance of Starfish) with boar and human acrosin. We have found that ARIS, solubilised ZPGs and fucoidan, but not heparin, inhibit the binding of the monoclonal antibody against human acrosin C5F10 to boar or human proacrosin. These results suggest that fucoidan, solubilised ZPGs and ARIS bind to a related domain on the proacrosin surface. Moreover, ARIS was able to induce human proacrosin activation. On the other hand, neither ARIS nor heparin from porcine intestinal mucosa or bovine lung induced hamster sperm acrosome reaction or sperm motility. Recent data showed that acrosin is involved in dispersal of the acrosomal matrix after acrosome reaction. Thus, the control of the ZPG glycan chains over proacrosin activation may regulate both sperm penetration rate and limited proteolysis of zona pellucida proteins.
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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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    SNAREs in mammalian sperm: Possible implications for fertilization
    (2000) Ramalho-Santos, J; Moreno, RD; Sutovsky, P; Chan, AWS; Hewitson, L; Wessel, GM; Simerly, CR; Schatten, G
    Soluble N-ethylmalameide-sensitive factor attachment protein receptor (SNARE) proteins are present in mammalian sperm and could be involved in critical membrane fusion events during fertilization, namely the acrosome reaction. Vesicle-associated membrane protein/synaptobrevin, a SNARE on the membrane of a vesicular carrier, and syntaxin 1, a SNARE on the target membrane, as well as the calcium sensor synaptotagmin I, are present in the acrosome of mammalian sperm (human, rhesus monkey, bull, hamster, mouse). Sperm SNAREs are sloughed off during the acrosome reaction, paralleling the release of sperm membrane vesicles and acrosomal contents, and SNARE antibodies inhibit both the acrosome reaction and fertilization, without inhibiting sperm-egg binding. In addition, sperm SNAREs may be responsible, together with other sperm components, for the asynchronous male DNA decondensation that occurs following intracytoplasmic sperm injection, an assisted reproduction technique that bypasses normal sperm-egg surface interactions. The results suggest the participation of sperm SNAREs during membrane fusion events at fertilization in mammals. (C) 2000 Academic Press.
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    The polysulphate binding domain of human proacrosin/acrosin is involved in both the enzyme activation and spermatozoa zona pellucida interaction
    (1998) Moreno, RD; Sepulveda, MS; de Ioannes, A; Barros, C
    Mammalian acrosin is a protease present as a zymogen in the acrosome of a non-reacted mammalian sperm, and in vitro is able to carry out limited hydrolysis of homologous and heterologous zonae pellucidae. On the other hand, sulphated polymers and zona pellucida glycoproteins bind to acrosin on a domain different from the active site, named the polysulphate binding domain (PSBD). Thus it is believed that acrosome-reacted spermatozoa bind to glycan chains bf the zona pellucida through PSBD participating as secondary binding receptor. The aim of the present work was to study the role of PSBD during both human gamete interaction and acrosin activation. In this work we present evidence that the anti-human acrosin monoclonal antibody C5F10 is directed to an epitope located on or near the PSBD on human proacrosin/acrosin. Moreover, we show that this antibody is able to inhibit both proacrosin activation induced by fucoidan and the sperm binding to the zona pellucida. Our results suggest that the same PSBD is involved in both sperm secondary binding, during zona pellucida penetration, and proacrosin activation.