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A Brandelli

Publications and source records attributed to A Brandelli.

34 records · Page 2Linked to original sources
PubMed2000

Isolation and characterization of a novel feather-degrading bacterial strain.

Feather waste, generated in large quantities as a byproduct of commercial poultry processing, is almost pure keratin, which is not easily degradable by common proteolytic enzymes. Feather-degrading bacteria were isolated from a Brazilian poultry industrial waste. Among these isolates, a strain identified as kr2 was the best feather-degrading organism when grown on basal medium containing 10 g/L of native feather as a source of energy, carbon, and nitrogen. The isolate was characterized according to morphological characteristics and biochemical tests belonging to the Vibrionaceae family. Keratinolytic activity of this isolate was monitored throughout the cultivation of the bacterium on raw feather at different temperatures. The optimum temperature for growth was about 30 degrees C, at which maximum enzyme and soluble protein production were achieved. The enzyme had a pH and temperature optima of 8.0 and 55 degrees C, respectively.

Animals↗
PubMed1999

Purification and characterization of beta-N-acetylhexosaminidase from bovine tick Boophilus microplus (Ixodide) larvae.

beta-N-Acetylhexosaminidase (HEX, E.C. 3.2.1.52) from larvae of the ixodid tick Boophilus microplus was purified to capillary zone electrophoresis homogeneity, and characterized. Enzyme purification was carried out by sequential liquid chromatography on Sephadex G-200, p-aminobenzyl-N-acetyl-beta-D-thioglucosamine affinity, and Mono-Q FPLC columns. Purification was about 1600-fold, with a yield of 10%, as determined with p-nitrophenyl-N-acetylglucosaminide as substrate. The enzyme presented optimum pH 4.7, and optimum temperature 65 degrees C. The molecular weight of non-denatured enzyme was estimated as 127,000 by gel filtration chromatography, and 60,000 in SDS-PAGE. The tick hexosaminidase presented glycosyl residues, as evidenced by binding to Concanavalin-A. Among several p-nitrophenyl glycosides tested as substrate, HEX was active only on p-nitrophenyl-N-acetylglucosaminide and p-nitrophenyl-N-acetylgalactosaminide. The purified enzyme presented immunogenicity in rabbit, and the correspondent antibodies inhibited about 90% of its original, in vitro activity.

Animals↗
PubMed1999

Peritoneal fluid modulates the sperm acrosomal exocytosis induced by N-acetylglucosaminyl neoglycoprotein.

The effect of peritoneal fluid (PF) on the human sperm acrosome reaction (AR) was tested. Sperm was pre-incubated with PF and the AR was induced by calcium ionophore A23187 and a neoglycoprotein bearing N-acetylglycosamine residues (NGP). The AR induced by calcium ionophore was inhibited 40% by PF from controls (PFc) and 50% by PF from the endometriosis (PFe) group, but not by PF from infertile patients without endometriosis (PFi). No significant differences were found in the spontaneous AR. When the AR was induced by NGP, pre-incubation with PFc reduced (60%) the percentage of AR, while PFe and PFi caused no significant differences. The average rates of acrosome reactions obtained in control. NGP- and ionophore-treated sperm showed that NGP-induced exocytosis differed significantly between the PFc (11%) and PFe/PFi groups (17%), and the ionophore-induced AR was higher for PFi (33%) than PFc/PFe (25%). The incidence of the NGP-induced AR was reduced in the first hour of pre-incubation with PFc and remained nearly constant throughout 4 h of incubation. The present data indicate that PF possesses a protective factor which prevents premature AR.

Acetylglucosamine↗
PubMed1998

Effect of antibodies against beta-N-acetylhexosaminidase on reproductive efficiency of the bovine tick Boophilus microplus.

A polyclonal antibody (anti-HEX) was developed against a soluble N-acetylhexosaminidase (HEX) isolated from larval extracts of Boophilus microplus. Purified hexosaminidase was strongly inhibited by the IgG fraction of this antibody. The antibody inhibited the hexosaminidase activity of other sources, such as haemolymph and larval membranes. The antibody reacted with different antigens in the tick haemolymph, but did not recognize any antigen in saliva, as seen by immunoblot analysis. The anti-HEX was inoculated into fully engorged B. microplus females, resulting in a decrease in oviposition of approximately 26%, relative to the effect of pre-immune IgG. These data show the potential of the use of this tick enzyme as an antigen in vaccine development.

Animals↗
PubMed1998

Glycosidases in the peritoneal fluid from infertile women with and without endometriosis.

OBJECTIVES: To investigate the activity and biochemical properties of glycosidases in the peritoneal fluid of infertile patients with or without endometriosis in comparison with fertile women. DESIGN AND METHODS: Peritoneal fluid was collected from 29 women undergoing a laparoscopy. The sample was separated into the following groups: fertile women (n = 11), infertile with endometriosis (n = 11), and infertile without endometriosis (n = 7). The activity of glycosidases was determined with specific p-nitrophenylglycosides as colorimetric substrates. RESULTS: The activity of alpha-fucosidase, alpha-glucosidase, alpha-mannosidase, beta-galactosidase, beta-glucuronidase, and beta-N-acetylhexosaminidase was investigated. Enzymatic activities of alpha-fucosidase and beta-N-acetylglucosaminidase were detected in higher amounts than other glycosidases. The activities of alpha-fucosidase and N-acetylglucosaminidase were increased in the case of infertile patients without endometriosis, while beta-galactosidase was increased in endometriosis patients. Enzyme properties as pH optimum, pH stability, thermal stability and inhibition by specific carbohydrates were similar for both control and infertile samples. Analysis of kinetic parameters indicate that Vmax values of glycosidases were higher for infertile samples than their respective controls. CONCLUSIONS: The results indicate that higher amounts of glycosidases are present in the peritoneal fluid from infertile patients. The elevated activity of these hydrolytic enzymes suggest possible deleterious effect on gametes, and could explain some cases of infertility.

Ascitic Fluid↗
PubMed1998

Isolation, partial characterization and biological activity of mannosyl glycopeptides from seminal plasma.

Affinity chromatography on Concanavalin-A Sepharose, followed by gel filtration and hydrophobic interaction chromatography, permits the isolation of low molecular weight N-glycosidically linked oligomannosidic glycopeptides (MGp) from the autoproteolysis products of human seminal plasma. The monosaccharide composition of MGp showed only mannose, N-acetylglucosamine and a small amount of galactose. Structural studies were carried out by methylation analysis and chromium trioxide oxidation, and results were consistent with the structures accepted for high-mannose N-glycans. MGp was capable of inhibiting the sperm acrosomal exocytosis mediated by sperm-surface receptors. These data suggest that MGp act as a "decapacitation" factor preventing premature sperm exocytosis.

Acrosome Reaction↗
PubMed1998

Purification and characterization of beta-N-acetylhexosaminidase from the phytopathogenic fungus Bipolaris sorokiniana.

N-acetylhexosaminidase (HEX) from the phytopathogenic fungus Bipolaris sorokiniana was isolated and characterized. The production of HEX by B. sorokiniana was not altered by growing on different carbon sources. Enzyme purification was carried out by sequential liquid chromatography on Sephacryl S-200 HR, and p-aminobenzyl-2-acetamido-2-deoxy-beta-D-thioglucopyranoside agarose. The purification was about 70-fold, with a yield of 41%, determined with p-nitrophenyl-N-acetylglucosaminide as substrate. The enzyme had pH and temperature optima of 4.5 and 55 degrees C, respectively. The molecular weight of non-denatured enzyme was estimated as 120,000 Da by gel filtration chromatography, and about 55,000 Da by SDS-PAGE. The fungal HEX had glycosylated residues as evidenced by binding to Concanavalin-A. Bipolaris sorokiniana enzyme was also active with p-nitrophenyl-chitobioside and p-nitrophenyl-N-acetylgalactosaminide as substrates.

Carbohydrates↗
PubMed1997

Modulation of sperm acrosomal exocytosis by guanyl nucleotides and G-protein-modifier agents.

Mammalian sperm must undergo an exocytotic event during fertilization, the acrosome reaction (AR). This process is specifically induced by egg-surface glycoproteins and it involves guanine nucleotide binding proteins (G-proteins). Neoglycoproteins (NGP) with mannose or N-acetylglucosamine residues has been demonstrated to induce the AR in human sperm. Activators of G-proteins, like GTP gamma S, GppNHp, mastoparan and AlF4- were capable of inducing the AR, while other nucleotides or analogues did not. When sperm were preincubated with these agents and then with NGPs, only the G-protein inhibitor GDP beta S decreased the AR rate. The preincubation of sperm with Pertussis toxin resulted in the inhibition of NGP-induced AR, while no effect was observed with cholera toxin. Results indicate that direct activation of G-proteins is sufficient to elicit the AR, and the induction of the AR in human sperm is mediated by N-acetylglucosaminyl and mannosyl binding sites involving PTx-sensitive G-proteins similar to the induction by zona pellucida glycoproteins.

Acrosome↗
PubMed1997

Glycosidic residues involved in human sperm-zona pellucida binding in vitro.

Glycosidic residues of the mammalian zona pellucida (ZP) are known to be involved in sperm binding, suggesting the presence of complementary carbohydrate binding sites on spermatozoa. However, in previous studies, in which sperm suspensions were incubated with monosaccharides, no inhibitory effect was observed. Results of studies in which sperm were treated shortly after swim-up suggest that the use of non-capacitated cells may explain the apparently conflicting results. In the present report, we studied the effect of preincubation of capacitated spermatozoa with different monosaccharides on their ability to bind to ZP. After 5 h under capacitating conditions, spermatozoa were incubated in medium with or without a monosaccharide, resuspended in fresh medium and used for hemizona (HZ) binding assay. When ZH were incubated with spermatozoa treated with N-acetyl-D-glucosamine, D-mannose, D-fucose, L-fucose or D-galactose, a significant decrease in the number of spermatozoa bound was observed (level of inhibition: 62, 58, 82, 68 and 48% respectively) while treatment of spermatozoa with D-glucose produced no inhibition. Sugar treatment neither altered sperm motility nor the rate of acrosome reaction. These results suggest that N-acetylglucosamine, mannose, fucose and galactose residues are involved in human sperm-zona pellucida binding in vitro.

Binding Sites↗
PubMed1997

Proteolytic release and partial characterization of human sperm-surface glycopeptides.

Sperm-surface glycopeptides were obtained from intact sperm membranes after proteolytic release by different enzymatic treatments such as autoproteolysis, trypsin, papain and pronase. Glycopeptides were isolated, their properties and composition were examined, and their monosaccharide and amino acid constituents were characterized. The monosaccharides identified were fucose, mannose, galactose, N-acetylglucosamine, and N-acetylgalactosamine, which form part of more than one type of oligosaccharide units. Autoproteolytic treatment mainly provided O-glycosidic type oligosaccharides, while a mixture of O- and N-glycosidic oligosaccharides was obtained in variable proportions when treated with trypsin, papain or pronase. The highest degree of peptide cleavage was obtained with pronase. Despite the higher yields reached with trypsin, these glycopeptides contain the lowest percentage of oligosaccharide chains. Proteolytic treatment provides a simple, rapid procedure for the isolation of glycopeptides from the sperm surface.

Glycopeptides↗
PubMed1996

Human sperm beta-glucuronidase is poorly extractable by Triton X-100.

A part of sperm glycosidase activities was detected as detergent-insoluble after sequential extractions with Triton X-100. Sixty per cent of total beta-glucuronidase activity was found in the detergent-insoluble fraction. This portion of beta-glucuronidase was resistant to extractions in the presence of 1 M KCl, chaotropic agents, colchicine or cytochalasine B, being only partially solubilized by 3 M KCl or DNAse I treatment. Results demonstrate that beta-glucuronidase is tightly associated to the Triton X-100 resistant fraction.

Actins↗
PubMed1995

A new predictive test for in-vitro fertilization based on the induction of sperm acrosome reaction by N-acetylglucosamine-neoglycoprotein.

Neoglycoproteins with N-acetylglucosamine residues (BSA-GlcNAc) induced specifically the acrosome reaction (AR) in human spermatozoa. Our objective was to investigate the relationship between this phenomenon and the invitro fertilization (IVF) rate. Sperm suspensions from IVF protocols were incubated with BSA-GlcNAc (t), using calcium ionophore (i) or medium alone (c) as positive or negative controls. When the normalized AR percentage ratio (STIM) (% ARt-%ARc):(%ARi-%ARc) was compared with fertilization rate in 31 couples from our IVF programme, a positive correlation was found (r = 0.46, P < 0.01). The fertilization rate in patients with STIM > or = 0.2 was higher than in non-responders (STIM < 0.2); 72 +/- 7% compared with 5 +/- 3%. The overall predictive value of this test for adequate fertilization rate (> 30%) was 87%, sensitivity 91% and specificity 78%. False positives were 9% and false negatives 22%. For successful fertilization rates (> 60%), the results were: overall predictive value, 84%; sensitivity 100%; specificity 64%. False positives were 23% and no false negatives were found. The results indicated that the induction of AR in human spermatozoa by GlcNAc-neoglycoproteins could be used to predict their fertilizing ability in vitro.

Acetylglucosamine↗
PubMed1995

Characterization of beta-N-acetylglucosaminidase from human epididymis.

beta-N-acetylglucosaminidase (NAG) activity in human epididymal fluid was separated into two forms (I and II) after HPLC-hydrophobic interaction chromatography. Both forms exhibited maximal activity at a pH of around 4.5 and had a molecular weight of 125 kD when determined by Superose-HPLC. After incubation at 50 degrees C, form I retained only 30% of its activity while form II retained 90% activity. When analysed by non-denaturing electrophoresis, form I displayed higher electrophoretic mobility than did form II. These features indicate that the I and II isoforms found in the human epididymis are the A and B forms present in other tissues. NAG activity was measured in the fluid obtained form the different epididymal regions of 13 different samples. An average four-fold increase in activity between the proximal caput and distal corpus was found. The contribution of each isoform to the total activity was studied. The proximal caput found to be rich in the A isoform (59%), whereas the B form was predominant in the distal corpus (65%). Human spermatozoa contain membrane-associated NAG activity with an isoform distribution similar to that found in cauda epididymal fluid (CEP, 80% B). Finally, enzyme activity in CEP was two-fold greater than in seminal plasma. Taken together these results suggest that NAG may become associated with human spermatozoa during epididymal transit.

Acetylglucosaminidase↗
PubMed1994

Participation of glycosylated residues in the human sperm acrosome reaction: possible role of N-acetylglucosaminidase.

Sperm binding to the egg zona pellucida is mediated by complementary protein-carbohydrate interaction. This binding results in the exocytosis of the sperm acrosome, or acrosome reaction (AR). We report the effect of different neoglycoproteins (sugar residues covalently bound to bovine serum albumin) on the human sperm AR. p-Aminophenyl-N-acetyl-beta-D-glucosaminide-BSA (BSA-GlcNAc) and p-aminophenyl-alpha-D-mannopyranoside-BSA (BSA-Man) at 1 micrograms/ml were capable of inducing the greatest percentages of AR (3-fold stimulation with respect to controls), while other NeoGPs had only a weak effect on this process. The BSA-GlcNAc-induced acrosome reaction was inhibited by N-acetylglucosamine (GlcNAc), p-nitrophenyl-GlcNAc, and purified soluble beta-N-acetylglucosaminidase (beta NAG). The induction of the AR with BSA-Man could be inhibited by mannose, while soluble alpha-mannosidase was only partially effective. These data suggest that binding sites for GlcNAc and mannose may be involved in the induction of the AR in human sperm. The characteristics of the BSA-GlcNAc induction suggest that the beta NAG molecule may be the mediator of this effect.

Acetylglucosaminidase↗
PubMedDate not supplied

Voltage-dependent calcium channels and Gi regulatory protein mediate the human sperm acrosomal exocytosis induced by N-acetylglucosaminyl/mannosyl neoglycoproteins.

Mammalian spermatozoa must undergo an exocytotic event during fertilization, the acrosome reaction (AR). In most species studied this process is induced by specific glycoproteins of the oocyte extracellular matrix, the zona pellucida (ZP), and it involves guanine nucleotide-binding regulatory proteins (G-proteins), resulting in an uptake of extracellular calcium by the sperm. In the bull, this event has been reported to be mediated by voltage-dependent calcium channels (VDCC). Previous observations showed that neoglycoproteins (NGPs) with N-acetylglucosamine or mannose (GlcNAc-BSA or Man-BSA) residues induce the AR in capacitated human spermatozoa. We report here that the pretreatment of spermatozoa with 125 ng/ml pertussis toxin (PTx) inhibited GlcNAc-BSA- or Man-BSA-induced AR, whereas 1 microgram/ml cholera toxin had no effect. These data indicate that the transduction mechanism for GlcNAc-BSA- and Man-BSA-induced AR involves G-proteins of the inhibitory type (GI). An increase in the AR rate was observed when capacitated spermatozoa were incubated with increasing concentrations of potassium ions (K+) in Biggers-Whitten-Whittingham (BWW) modified medium (2.6 +/- 0.3-fold at 80 mM K+). This induction was observed only when the pH was raised to 8.5, and it was inhibited by verapamil, nitrendipine, omega-conotoxin, nickel ions (Ni2+), lanthanum ions (La3+), or cadmium ions (Cd2+) in a concentration-dependent manner, indicating the participation of VDCC activated by membrane depolarization. The GlcNAc-BSA- or Man-BSA-induced AR was completely inhibited by preincubation of spermatozoa with VDCC blockers and calcium antagonists, indicating a link between the binding of sugar residues of the NGPs and channel activation. The AR induced by membrane depolarization with high K+ medium was not inhibited by PTx, suggesting that Ca2+ entry is downstream to GI-protein activation. These data show that the induction of the AR in human spermatozoa by GlcNAc- or Man-NGPs involves VDCC and GI-like regulatory proteins similar to the induction described for ZP in other mammalian species.

Acetylglucosamine↗