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C Barros

Publications and source records attributed to C Barros.

At least 37 records · Page 2Linked to original sources
PubMed1998

Characterization of the functional domains of boar acrosin involved in nonenzymatic binding to homologous zona pellucida glycoproteins.

During the first steps of the gamete interaction, the proacrosin/acrosin system seems to play a crucial role in the secondary binding, holding acrosome-reacted spermatozoa during their passage through the zona pellucida. To analyze the functional domains of acrosin, we decided to express recombinant boar acrosin proteins in bacteria and to study their binding capacities to zona pellucida glycoproteins (ZPGPs). The expressed proteins were immunodetected by Western blot with a polyclonal antiacrosin antibody. The recombinant truncated beta-acrosin has a typical hyperbolic curve of a zymogen enzymatic activation. Three of the five recombinant forms (truncated beta-acrosin, Ser/Ala222-truncated beta-acrosin, and truncated beta-acrosin "heavy chain") had the ability to bind ZPGPs. The two shorter forms (the amino and carboxy termini of truncated beta-acrosin) failed to bind. The catalytic site mutant (Ser/Ala222) of truncated beta-acrosin does not differ from the recombinant truncated beta-acrosin in its mechanism of interaction to ZPGPs, indicating that this secondary binding is done by a nonenzymatic process. Our results show that binding between acrosin and ZPGPs depends on the secondary and tertiary structures of acrosin and does not depend on an active catalytic site.

Acrosin↗
PubMed1998

The polysulphate binding domain of human proacrosin/acrosin is involved in both the enzyme activation and spermatozoa-zona pellucida interaction.

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 of 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.

Acrosin↗
PubMed1998

Characterization of the bvgR locus of Bordetella pertussis.

Bordetella pertussis, the causative agent of whooping cough, produces a wide array of factors that are associated with its ability to cause disease. The expression and regulation of these virulence factors is dependent upon the bvg locus (originally designated the vir locus), which encodes two proteins: BvgA, a 23-kDa cytoplasmic protein, and BvgS, a 135-kDa transmembrane protein. It is proposed that BvgS responds to environmental signals and interacts with BvgA, a transcriptional regulator which upon modification by BvgS binds to specific promoters and activates transcription. An additional class of genes is repressed by the bvg locus. Expression of this class, the bvg-repressed genes (vrgs [for vir-repressed genes]), is reduced under conditions in which expression of the aforementioned bvg-activated virulence factors is maximal; this repression is dependent upon the presence of an intact bvgAS locus. We have previously identified a locus required for regulation of all of the known bvg-repressed genes in B. pertussis. This locus, designated bvgR, maps to a location immediately downstream of bvgAS. We have undertaken deletion and complementation studies, as well as sequence analysis, in order to identify the bvgR open reading frame and identify the cis-acting sequences required for regulated expression of bvgR. Studies utilizing transcriptional fusions of bvgR to the gene encoding alkaline phosphatase have demonstrated that bvgR is activated at the level of transcription and that this activation is dependent upon an intact bvgAS locus.

Amino Acid Sequence↗
PubMed1997

Trypsin-like enzymes during fertilization in the shrimp Rhynchocinetes typus.

The spermatozoon of Rhynchocinetes typus is atypical because it is nonmotile and lacks head and tail. The body has a rigid spike. Neither an acrosome-like structure nor changes during gamete interaction which could be interpreted as an acrosome reaction have been observed in this species. Nevertheless, the spermatozoon exerts a lytic effect on the extracellular envelope of the egg, and in this way it penetrates through egg-coats, forming a channel. In this research we found that crude spermatozoa extracts analyzed by gelatin SDS-PAGE developed one band of protease activity that was completely inhibited by SBTI (soybean trypsin inhibitor) and pAB (p-aminobenzamidine). In sperm extracts an enzymatic activity was determined, using BAEE (N-benzoil-L-arginine ethyl ester), but not ATEE, as substrate. This activity was inhibited by SBTI and pAB. We observed that in vitro fertilization was inhibited by spermatozoon incubation with the trypsin inhibitors SBTI, PMSF (phenylmethanesulphonyl fluoride), and pAB. Additionally, we observed that when whole isolated egg-coats were incubated with sperm extract and then analyzed by SDS--PAGE, one band of the egg-coats disappeared. These results have been interpreted as sperm trypsinlike enzyme participation in R. typus sperm passage through the egg-coats.

Animals↗
PubMed1996

Early steps of sperm-egg interactions during mammalian fertilization.

Mammalian eggs are surrounded by two egg coats: the cumulus oophorus and the zona pellucida, which is an extracellular matrix composed of sulfated glycoproteins. The first association of the spermatozoon with the zona pellucida occurs between the zona glycoprotein, ZP3 and sperm receptors, located at the sperm plasma membrane, such as the 95 kDa tyrosine kinase-protein. This association induces the acrosome reaction and exposes the proacrosin/acrosin system. Proacrosin transforms itself, by autoactivation, into the proteolytical active form: acrosin. This is a serine protease that has been shown to be involved in secondary binding of spermatozoa to the zona pellucida and in the penetration of mammalian spermatozoa through it. The zona pellucida is a specific and natural substrate for acrosin and its hydrolysis and fertilization can be inhibited by antiacrosin monoclonal antibodies. Moreover, in in vitro fertilization experiments, trypsin inhibitors significantly inhibits fertilization. The use of the silver-enhanced immunogold technique has allowed immunolocalization of the proacrosin/acrosin system in spermatozoa after the occurrence of the acrosome reaction. This system remains associated to the surface of the inner acrosomal membrane for several hours in human, rabbit and guinea-pig spermatozoa while in the hamster it is rapidly lost. In the hamster, the loss of acrosin parallels the capability of the sperm to cross the zona pellucida. Rabbit perivitelline spermatozoa can fertilize freshly ovulated rabbit eggs and retain acrosin in the equatorial and postacrosomal region. These spermatozoa also show digestion halos on gelatin plates that can be inhibited by trypsin inhibitors. This evidence strongly suggests the involvement of acrosin in sperm penetration through the mammalian zona. Recently it was shown, however, that acrosin would not be essential for fertilization. It is likely, then, that such an important phenomenon in the mammalian reproductive cycle would be ensured though several alternative mechanisms.

Acrosin↗
PubMed1996

Erythema elevatum diutinum in human immunodeficiency virus-infected patients--report of a case and review of the literature.

Erythema elevatum diutinum (EED) is a rare chronic disease of unknown origin, part of the spectrum of cutaneous leucocytoclastic vasculitis. A case of EED in a 32-year-old HIV-infected male patient, with no previous opportunistic infections and a CD4+ cell count of less than 200/mm3, is reported. Therapy with oral dapsone (100 mg/day for 15 days) resulted in clinical cure with no relapse after 6 months of follow-up. To our knowledge, only six cases of EED in HIV-positive patients have been reported to date. A brief review of these seven cases is described.

Adult↗
PubMed1996

Proacrosin and acrosin determination during capacitation and acrosome reaction in rabbit spermatozoa.

The proacrosin/acrosin system has been immunolocalized, by the silver enhanced immunogold technique on the acrosomal region of capacitated and perivitelline rabbit spermatozoa. The purpose of the present work was to investigate the kinetics of the activation of acrosin by determining the proportion of proacrosin and acrosin present in the acrosome of the rabbit spermatozoa during capacitation and the induction of the acrosome reaction by the calcium ionophore A23187. Rabbit spermatozoa selected by the percoll gradient technique were incubated for 0, 0.5 and 6 hours and then the acrosome reaction was induced at 0 and 6 hours with 1.9 microM of the calcium ionophore A23187. It was found that 95% of acrosin activity in rabbit spermatozoa at zero time corresponds to proacrosin and after capacitation and acrosome reaction, a diminution of the activity of proacrosin/acrosin system was found. However, proacrosin represents the large majority of the system activity. Western blot prepared with sperm extract obtained at the start of incubation showed the characteristic doublet band about 53-55 kDa that may correspond to proacrosin and alpha-acrosin. After six hours of incubation and with induction with the calcium ionophore A23187 the same doublet was seen in addition to a third band of 49 kDa that could correspond to a transition form between alpha-acrosin to beta-acrosin. In conclusion, rabbit proacrosin/acrosin system remains in the large proportion as proacrosin during capacitation and acrosome reaction.

Acrosin↗
PubMed1995

[Non-Hodgkin's lymphomas associated with the acquired immunodeficiency syndrome. A multicenter clinical study of 77 cases].

BACKGROUND: The aim of the present was to study the prevalence of non Hodgkin's lymphoma (NHL) in AIDS patients as well as the clinicopathologic characteristics, response to treatment and survival. METHODS: From January 1984 to January 1991, 77 patients with NHL associated with AIDS diagnosed in 9 hospitals in Madrid were retrospectively studied. RESULTS: Ninety-two per cent of the patients were men (mean age 30 years: range: 9-66 years), 62% were intravenous drug abusers and 20 (26%) homosexuals. Pathologic study determined that 62 (80%) patients had high grade NHL (44% small noncleaved), 17% immunoblastic and 20% unclassifiable, and 15 (20%) had intermediate grade (16% diffuse large cell) being all the cases of the B immunophenotype. Sixty-five per cent were in advanced stages and 69% had B symptoms. Extranodal localizations were present in 88%, bone marrow in 29% and CNS in 29%. Six cases had primary CNS lymphomas. 50% of the patients had less than 200 x 10(6)/l CD4 lymphocytes. Forty-seven patients were evaluable for response to chemotherapy: 12 (26%) showed a complete response 27 (57%) a partial response and 8 (17%) did not respond. Opportunistic infections developed in 18%. The estimated survival at 3 years was 14% (median 6 months). On univariate analysis the parameters related to the worst survival were: primary CNS lymphoma, liver involvement, lack of treatment response, LDH > or = 300 UI/l, alkaline phosphatase > or = 500 UI/l and ESR > or = 70 mm. CONCLUSIONS: Non Hodgkin's lymphomas associated with AIDS usually behave in an "aggressive" way with a high frequency of advanced stages, B symptoms, high grade histologic subtypes and extranodal involvement. Response to treatment is poor, bone marrow toxicity frequent and survival short.

Adolescent↗
PubMed1995

Endocarditis caused by Arcanobacterium haemolyticum.

A 33-year-old, active intravenous drug-abusing male infected with the human immunodeficiency virus developed endocarditis due to Arcanobacterium haemolyticum. Empirical treatment with ampicillin plus gentamicin failed to achieve a marked clinical improvement. When the results of antibiotic susceptibility were available (ampicillin MIC < or = 0.06 mg/l; ampicillin MBC 2 mg/l; MBC:MIC ratio > 32) therapy was changed to vancomycin plus gentamicin. During the following days progressive clinical and radiological improvement was observed. The patient received antibiotics for 30 days and no relapse occurred during a 14-month follow-up. The literature of endocarditis due to this uncommon bacterium is reviewed here.

Actinomycetaceae↗
PubMed1994

Immunolocalization of proacrosin/acrosin in rabbit sperm during acrosome reaction and in spermatozoa recovered from the perivitelline space.

The participation of acrosin in mammalian sperm penetration through the zona pellucida has been amply debated. In this paper we report the immunolocalization--by silver enhanced immunogold technique using ACRO-8C10 monoclonal antibody to human acrosin--of proacrosin/acrosin on ejaculated rabbit spermatozoa incubated in vitro in a capacitating medium and on spermatozoa recovered from the perivitelline space. After incubation in a capacitating medium, four different patterns were observed: (1) no labeling on acrosome intact spermatozoa; (2) labeling on the rim of the head; (3) labeling on the whole acrosome area; and (4) no labeling on acrosome reacted spermatozoa. At the start of incubation, spermatozoa with pattern 1 were the most abundant, whereas at the end of the 32 h incubation period, patterns 2 and 3 were the most frequent. On the other hand, 625 perivitelline spermatozoa were recovered from 17 fertilized rabbit eggs, of which 26% were labeled with the antiacrosin monoclonal antibody ACRO-8C10 in two different areas: (1) only on the equatorial region; and (2) only on the postacrosomal area. These results are consistent with the idea that proacrosin/acrosin remains associated to the acrosome reacted spermatozoa for long periods of time, and that proacrosin/acrosin associated to perivitelline spermatozoa could be responsible for the second penetration of fresh rabbit eggs by perivitelline spermatozoa.

Acrosin↗
PubMed1993

High rates of survival and fertilization of mouse and hamster oocytes after vitrification in dimethylsulphoxide.

A high proportion (> 70%) of mouse and hamster oocytes exposed for 3-5 min to 1.5 M dimethylsulfoxide (DMSO) and washed briefly in 3.9 M DMSO before vitrification in 6.0 M DMSO appeared morphologically normal on recovery. Significantly fewer (< 46%) mouse oocytes appeared normal when the time of exposure to 1.5 M DMSO was reduced to 1 min or less. The rate of fertilization in vitro of vitrified oocytes was reduced compared to the rate for untreated controls (mouse: 79% vs. 94%; hamster: 73% vs. 87%). After removal of the zona pellucida, fertilization was similar in vitrified and control hamster oocytes inseminated with hamster (> 90%) or human (21% vs. 23%) sperm. Sperm nuclear decondensation and pronuclear formation appeared to be delayed in the cytoplasm of vitrified hamster oocytes. Seventy-nine percent of 2-cell-stage mouse embryos derived from vitrified oocytes implanted after transfer to pseudopregnant recipients, but only 40% developed to normal fetuses compared to 61% of controls. The reason for this high rate of postimplantation loss is unknown.

Animals↗
PubMed1992

Immunodetection of acrosin during the acrosome reaction of hamster, guinea-pig and human spermatozoa.

Mammalian sperm acrosomes contain a trypsin-like protease called acrosin which causes limited and specific hydrolysis of the extracellular matrix of the mammalian egg, the zona pellucida. Acrosin was localized on hamster, guinea-pig and human sperm using monoclonal and polyclonal antibodies to human acrosin labelled with colloidal gold. This was visualized directly with transmission electron microscopy, and with light and scanning microscopy after silver enhancement of the colloidal gold probe. Four distinct labelling patterns were found during capacitation and the acrosome reaction in hamster and guinea-pig spermatozoa, and three patterns were found in human spermatozoa. In the hamster, acrosin was not detected on the inner acrosomal surface after the completion of the acrosome reaction, thus correlating with the observation that hamster spermatozoa lose the ability to penetrate the zona after the acrosome reaction. With guinea-pig and human spermatozoa, acrosin was still detected after the completion of the acrosome reaction, thus correlating with the observation that acrosome reacted guinea-pig spermatozoa bind to and penetrate the zona pellucida.

Acrosin↗
PubMed1992

[The results of a program of in vitro fertilization and tubal embryo transfer (ZIFT)].

Twenty-one sterile couples, between September 1989, and August 1991, were treated for ovulation stimulation of their cycles, in order to practice in them, a in vitro fertilization and tube embryo transfer (ZIFT). Two protocols of ovulation induction were used, both with leuprolide acetate (Lupron), one in the luteal phase and the other in follicular phase and since the second or the fourth day of the cycle, respectively, gonadotropins were added (Metrodine and Pergonal). Out of all the twenty-nine initiated cycles, twenty-seven were aspirated (93.1%) and twenty-four reached an embryo transfer (82.8%). Seven clinic pregnancies were obtained (29.17% per transfer) and four deliveries (16.67% per transfer). The sterility period average was 69.64 +/- 36.6 months and the patients age average was 34.1 +/- 4.38 years. The global rate of fertilization was 63.53%. With luteal phase Lupron best results were got (pregnancy rate of 38.46% per transfer) and there were not considerable difference in the number of gonadotropins ampulla employed. When embryos were transfer to the tubes and the uteri the pregnant rate was 50% per transfer, in comparison to 18.75% when transfer was made only in the tubes.

Chile↗
PubMed1992

Golden hamster perivitelline spermatozoa do not show proacrosin/acrosin at the inner acrosomal membrane.

It has been suggested that acrosin may function in penetration of the zona pellucida and of the highly structured extracellular matrix of the perivitelline space. In this study we investigated whether golden hamster perivitelline spermatozoa contain proacrosin/acrosin, as evidenced by the silver enhanced immunogold technique using the monoclonal antibody antiacrosin C2E5. None of the 197 spermatozoa recovered from the perivitelline space showed proacrosin/acrosin associated with the acrosomal region, suggesting that acrosin would not play a role in the penetration of the perivitelline extracellular matrix.

Acrosin↗