Major seminal plasma proteome of rabbits and associations with sperm quality.

The present study was conducted to describe the major seminal plasma proteome of rabbits and potential associations between seminal proteins and semen criteria. Semen samples were collected from 18 New Zealand adult rabbits, and seminal plasma proteins were analyzed by 2-D SDS-PAGE and tandem mass spectrometry. Sperm motility, vigor, concentration, morphology and membrane sperm viability were evaluated. Rabbits ejaculated 364 ±â€¯70 million sperm/ml, with 81 ±â€¯6.1% motile cells, 3.8 ±â€¯0.2 vigor and 66.7 ±â€¯2.5% sperm with normal morphology. Based on the viability and acrosome integrity assay, there were 65.8 ±â€¯2.5% live sperm with intact acrosome and most spermatozoa had both intact acrosome and functional membrane. On average, 2-D gels of rabbit seminal plasma had 232 ±â€¯69.5 spots, as determined by PDQuest software (Bio Rad, USA). Mass spectrometry allowed the identification of 137 different proteins. The most abundant proteins in rabbit seminal plasma were hemoglobin subunit zeta-like, annexins, lipocalin, FAM115 protein and albumin. The intensity of the spots associated with these five proteins represented 71.5% of the intensity of all spots detected in the master gel. Multiple regression models were estimated using sperm traits as dependent variables and seminal plasma proteins as independent ones. Also, sperm motility had positive association with beta-nerve growth factor and cysteine-rich secretory protein 1-like and a negative one with galectin-1. The percentage of rabbit sperm with intact membrane was related to seminal plasma protein FAM115 complex and tropomyosin. Then, the population of morphologically normal sperm in rabbit semen was positively linked to carcinoembryonic antigen-related cell adhesion molecule 6-like and down regulated by seminal plasma isocitrate dehydrogenase. Based on another regression model, the variation in the percentage of live sperm with intact acrosome was partially explained by the amount of leukocyte elastase inhibitor and the peptidyl-prolyl cis-trans isomerase A in the rabbit seminal fluid. The current study reports the identification of 137 proteins of rabbit seminal plasma. Major proteins of seminal secretion relate primarily to prevention of damages caused by lipid peroxide radicals and oxidative stress, membrane functionality, transport of lipids to the sperm membrane and temperature regulation. Moreover, finding seminal plasma proteins as indicators of semen parameters will improve assisted reproductive technologies.

Proteomic analysis of seminal plasma from locally-adapted "Curraleiro Pé-Duro bulls" (Bos taurus): identifying biomarkers involved in sperm physiology in endangered animals for conservation of biodiversity.

The present study was aimed at evaluating the seminal plasma proteins and sperm parameters of Curraleiro Pé-Duro bulls. Semen was collected from 10 bulls by electroejaculation, and sperm parameters were evaluated in fresh and frozen-thawed semen. Seminal plasma proteins were analyzed by 2-D SDS-PAGE and mass spectrophotometry. Tools in computational biology were used to generate bioinformatic knowledge and evaluate gene ontology, protein-protein interactions, phylogenetic trees and multiple sequence alignments. Sperm motility in fresh and frozen-thawed semen was 78.8±1.8% and 21.2±1.6%, respectively. Pearson's correlations were evaluated (p<0.05). Sperm motility and vigor in fresh semen were correlated with clusterin, TIMP2 and cathepsin S (r=0.64-0.71) and sperm defects were related to inhibitor of carbonic anhydrase and BSP 5 (r=0.78-0.80). Clusterin, BSP 5, alpha-enolase, creatine kinase M-type, glyceraldehyde-3-phosphate dehydrogenase, BSP 3, albumin, and 5'-nucleotidase and legumain were correlated with acrosome intact live sperm (r=0.80-0.64). Associations were detected between sperm vigor and spermadhesin 1 (r=-0.89), and between sperm defects in fresh semen and spermadhesin 1 and clusterin (r=-0.81). Sperm motility in frozen-thawed semen was associated with BSP 1, spermadhesin 1, clusterin and spermadhesin Z13 (r=0.64-0.85). The percent of motile sperm after freeze-thawing was negatively correlated (r=-0.64) with the amount of spermadhesin 1 in the seminal plasma. Based on in silico analysis, TIMP2 interacted with BSP1, BSP3, BSP5 and metalloproteinases. Molecular functions of proteins associated with sperm parameters were binding, catalytic activity and enzymatic regulation. Amino acid sequences of spermadhesin 1 and BSP 1 from Bos taurus, and other domestic species were similar. Phylogenetic tree analysis demonstrated that clusterin from Bos taurus was related to Ovis aries and domains of clusterin, spermadhesin 1, BSP 1 and inhibitor of carbonic anhydrase were conserved as well. In summary, specific seminal proteins are associated with sperm parameters of locally-adapted bulls. Use of the endangered mammalian as a model may assist in understanding aspects of evolutionary adaptations and could improve assisted reproductive biotechnologies.

Purification of binder of sperm protein 1 (BSP1) and its effects on bovine in vitro embryo development after fertilization with ejaculated and epididymal sperm.

The present study evaluated functional aspects of binder of sperm 1 (BSP1) in the bovine species. In a first experiment, cumulus-oocyte complexes (n = 1274) were incubated with frozen-thawed ejaculated sperm (18 hours) in Fert-TALP medium containing: heparin, 10, 20, or 40 µg/mL BSP1. Heparin followed by gelatin affinity chromatography was used for purification of BSP1 from bovine seminal vesicle fluid. With ejaculated sperm, cleavage rates were similar when Fert-TALP medium was incubated with heparin (74.1 ± 2.7%), 10 µg/mL BSP1 (77.8 ± 3.1%), or 20 µg/mL BSP1 (74 ± 2.0%). Day-7 blastocyst rates were equivalent after incubations with heparin (40.8 ± 5.0%) and 10 µg/mL BSP1 (34.1 ± 4.4%), but reduced after 20 µg/mL BSP1 (22.4 ± 2.9%) and 40 µg/mL BSP1 (19.3 ± 4.1%; P < 0.05). In the second experiment, cumulus-oocyte complexes (n = 1213) were incubated with frozen-thawed cauda epididymal sperm (18 hours) in Fert-TALP medium containing: no heparin, heparin, 10, 20, or 40 µg/mL. Cleavage and blastocyst rates were similar after treatments with heparin (68.5 ± 1.3% and 24.7 ± 3.2%, respectively) or without heparin (65.5 ± 1.8% and 27.3 ± 1.6%, respectively). Cleavage was higher after treatment with any BSP1 concentrations (74.2 ± 2.7%-79.0 ± 1.1%) than without heparin (P < 0.05). Also, cleavage was better after Fert-TALP medium incubation with 40 µg/mL BSP1 (79.0 ± 1.1%) than with heparin (68.5 ± 1.3%; P < 0.05). Embryo development was higher (P < 0.05) after treatment with 20 µg/mL BSP1 (35.6 ± 2.5%) and 40 µg/mL (41.1 ± 2%) than after incubations with heparin (24.7 ± 3.2%) or without heparin (27.3 ± 1.6%). Interestingly, BSP1 did not cause reductions in blastocyst rates after fertilization with epididymal sperm, as observed with ejaculated sperm. On the basis of immunocytochemistry, there was BSP1 binding to frozen-thawed ejaculated but not to epididymal sperm. Also, anti-BSP1 reaction remained on ejaculated sperm (as expected) and appeared on epididymal sperm after incubation with purified BSP1. Acrosome reaction of ejaculated and epididymal sperm was induced after incubation with purified BSP1 as well, indicating an effect of BSP1 on capacitation. In conclusion, purified BSP1 from bull seminal vesicles was able to bind to and induce capacitation of ejaculated and epididymal sperm. Also, BSP1 added to fertilization media and allowed proper cleavage and embryo development, with the effects being modulated by previous exposure or not of spermatozoa to seminal plasma.

First record of Ceratium furcoides (Dinophyta), an invasive species, in a temporary high-altitude lake in the Iron Quadrangle (MG, Southeast Brazil).

Dinoflagellates of the genus Ceratium are generally marine organisms, but rare occurrences in freshwater have been observed in Brazil. In this paper we are recording for the first time the presence of Ceratium furcoides, an invasive species, in a shallow, natural intermittent pool formed at a high-altitude at the southern end of the Iron Quadrangle, an iron-mining district of Minas Gerais State (Southeast Brazil). Samples were collected in October and November of 2010 (rainy period). The population density of this organism observed in Lagoa Seca ("Dry Pool") was very low, at most 4 ind L-1. Mountain lakes are extremely vulnerable to atmospheric deposition of organisms, making them valuable witnesses both of the many forms of impact arising from human activities and of the extended global connections that facilitate the dispersion and introduction of new species over great distances. Studies on the population dynamics of C. furcoides in natural tropical systems are still rare and very recent to the brazilian scenario and hence the monitoring of its dynamics and the potential impact on aquatic communities of its becoming established are essential to an understanding of the process of bioinvasion by this species.

Meat quality assessment from young goats fed for long periods with castor de-oiled cake.

Diet can influence both the qualitative and quantitative traits of ruminant meat. This study evaluated the effects of castor de-oiled cake on the meat of mixed-breed male goat kids. After 165days of diet treatment, no alterations (p>0.05) were observed in the in vivo performance, anatomic components, dissection and proximate composition of the Longissimus dorsi muscle, as well as in the color and pH of the carcasses. However, diet had an effect (p<0.05) on energy metabolites, fatty acid profile, and expression of certain proteins of the Longissimus dorsi muscle. To conclude, this study showed that the establishment of castor de-oiled cake diet for a long period to goats led to alterations in meat quality, without compromising its consumption qualities.

First record of Ceratium furcoides (Dinophyta), an invasive species, in a temporary high-altitude lake in the Iron Quadrangle (MG, Southeast Brazil) / Primeiro registro de Ceratium furcoides (Dinophyta), uma espécie invasora, em um lago temporário de alta altitude no Quadrilátero Ferrífero (MG, Sudeste do Brasil)

Dinoflagellates of the genus Ceratium are generally marine organisms, but rare occurrences in freshwater have been observed in Brazil. In this paper we are recording for the first time the presence of Ceratium furcoides, an invasive species, in a shallow, natural intermittent pool formed at a high-altitude at the southern end of the Iron Quadrangle, an iron-mining district of Minas Gerais State (Southeast Brazil). Samples were collected in October and November of 2010 (rainy period). The population density of this organism observed in Lagoa Seca (“Dry Pool”) was very low, at most 4 ind L–1. Mountain lakes are extremely vulnerable to atmospheric deposition of organisms, making them valuable witnesses both of the many forms of impact arising from human activities and of the extended global connections that facilitate the dispersion and introduction of new species over great distances. Studies on the population dynamics of C. furcoides in natural tropical systems are still rare and very recent to the brazilian scenario and hence the monitoring of its dynamics and the potential impact on aquatic communities of its becoming established are essential to an understanding of the process of bioinvasion by this species.(AU)

Dinoflagelados do gênero Ceratium são principalmente marinhos, porém existem raras ocorrências em água doce no Brasil. Neste estudo registramos pela primeira vez a ocorrência de Ceratium furcoides, uma espécie invasora em um lago altitudinal natural raso localizado na porção sul do Quadrilátero Ferrífero, uma área de extração mineral no estado de Minas Gerais. As coletas foram realizadas nos meses de outubro e novembro de 2010 (período chuvoso). As densidades observadas para C. furcoides na Lagoa Seca foram muito baixas, com máximo de 4 ind L–1. Lagos de altitude são altamente vulneráveis à deposição atmosférica, o que os torna importantes testemunhos não só dos múltiplos impactos derivados de atividades humanas, mas também das extensas conexões globais, que acabam por facilitar a dispersão e introdução de espécies. Estudos sobre a dinâmica de C. furcoides em sistemas tropicais naturais ainda são escassos e por isso, o monitoramento de sua dinâmica e dos impactos potenciais em comunidades aquáticas a partir do seu estabelecimento, torna-se fundamental para o entendimento dos processos relacionados à bioinvasão desta espécie.(AU)

Semen variables and sperm membrane protein profile of Saanen bucks (Capra hircus) in dry and rainy seasons of the northeastern Brazil (3°S).

The Saanen is a highly productive breed, and for this reason, it has been raised in Brazil, but mostly under climate conditions completely different from where the breed originated. The objective of this study was to investigate variations in semen parameters and sperm membrane proteins from Saanen bucks (n = 7) raised in Northeastern Brazil, during dry season (September, October, and November) and rainy season (March, April, and May). We showed that during the dry season, sperm motility, concentration, and the percentage of normal sperm decreased as compared to the rainy season. Rectal temperatures of bucks had no significant (p > 0.05) variations during the dry and rainy seasons. However, temperatures of left and right skin testis were higher (p < 0.05) during the dry as compared to the rainy season. Expression of three proteins (lysine-specific demethylase 5D, adenosine triphosphate (ATP) synthase subunit d, and radial spoke head protein 9 homolog) in sperm membrane were more intense in rainy season and only one protein (cytosol aminopeptidase) had greater expression in the dry season of the year. Our results show that mechanisms of testicular thermoregulation of Saanen bucks did not prevent a decrease in seminal parameters during the dry season. This deterioration may be related to reduced expression of proteins associated with important functions in sperm membrane.

Membrane-associated proteins of ejaculated sperm from Morada Nova rams.

The objective was to describe the profile of membrane proteins from sperm of tropically adapted Morada Nova rams (N = 5). Samples from protein-enriched fractions of ejaculated sperm (containing 400 µg of protein) were separated by two-dimensional electrophoresis and respective maps analyzed using PDQuest software (version 7.3.0; Bio-Rad). Proteins were identified using tandem mass spectrometry. Also, membrane proteins were incubated with antibodies against binder of sperm protein (BSP) 1 and bodhesin 2 (Bdh-2), components of vesicular gland secretion. For membrane proteins of ejaculated sperm, an average of 133 ± 4.6 spots were detected per gel, of which, 107 spots were consistently present on all gels. Sixty-eight spots and 37 proteins were identified using mass spectrometry, corresponding to 71.6% of the intensity of all spots detected. Three major spots identified as ram seminal vesicle protein (RSVP) 14 represented approximately 30% of the intensity of all spots. Two of the most intense spots in the gel reacted against anti-BSP1, at 14 kDa. In addition, four low molecular weight spots reacted with anti-Bdh-2 antibodies. Proteins RSVP and Bdh-2 belong to the BSP and spermadhesin families, respectively, and were previously reported as major components of ram seminal proteins. Additional proteins identified in the sperm membrane two-dimensional maps included alpha-2-heparan sulfate-glycoprotein, plasma glutamate carboxypeptidase, arylsulfatase A, cathelicidin, heat shock protein 70 kDa, angiotensin-converting enzyme, leucine aminopeptidase, and clusterin. Some proteins were present as multiple isoforms, such as tubulin (12), alpha-2-heparan sulfate-glycoprotein (5), ATP synthase (5), Bdh-2 (4) and RSVP14 (3). Based on gene ontology analysis, the most common biological processes associated with the membrane proteins were cellular processes (34%), response to stimulus (14%), and metabolic processes (11%). Binding (37%) and catalytic activity (32%) corresponded to the most frequent molecular functions for those proteins. In conclusion, we identified a diverse cohort of components of membrane proteins in ram sperm. Major proteins previously reported in seminal plasma, such as RSVP14 and Bdh-2, were also extracted from sperm membranes. Knowledge of sperm proteins is crucial for elucidating mechanisms underlying their association with sperm function.

New insights into the complex mixture of latex cysteine peptidases in Calotropis procera.

The latex of Calotropis procera is a rich source of proteolytic activity. This latex is known to contain two distinct cysteine peptidases: procerain and procerain B. In this study, new cysteine peptidases were purified from C. procera latex. The enzymes were purified by two sequential ion-exchange chromatography steps (CM-Sepharose plus Resource S(®)) at pH 5.0 and 6.0. The purified enzymes had molecular mass spectra corresponding to CpCP-1=26,213, CpCP-2=26,133 and CpCP-3=25,086 Da. These enzymes exhibited discrete differences in terms of enzymatic activity at a broad range of pH and temperature conditions and contained identical N-terminal amino acid sequences. In these respects, these three new proteins are distinct from those previously studied (procerain and procerain B). Circular dichroism analysis revealed that the new peptidases contain extensive secondary structures, α(15-20%) and ß(26-30%), that were stabilized by disulfide bonds. The purified enzymes exhibited plasma-clotting activity mediated by a thrombin-like mechanism. The set of results suggest the three isolated polypeptides correspond to different post-translationally processed forms of the same protein.

On the dielectric behaviour of collagen-algal sulfated polysaccharide blends: effect of glutaraldehyde crosslinking.

In this paper, impedance measurements in the frequency range from 10(-2) to 10(6) Hz are presented for collagen and algal sulfated polysaccharide crosslinked films. We are considering the development of new biomaterials which have potential applications in coating of cardiovascular prostheses, support for cellular growth and in systems for controlled drug delivery. The effect of crosslink sulfated polysaccharide on the physical chemical properties of collagen was studied using FT-infrared spectroscopy, differential scanning calorimetry (DSC), dielectric spectroscopy. The resulting films crosslinked with glutaraldehyde (GA) in concentrations of 0.001% and 0.05% when analysed by DSC, showed that the GA treatment not only left the thermal stability of the collagen unaffected, but it also decreased the thermal transition energy. Dielectric spectroscopy shows that the effect of the crosslink on the blend film was associated to the decrease and stabilization of the dielectric permittivity at low frequencies and decreased its conductivity.

Characterization of principal nutritional components of Brazilian oil palm (Eliaes guineensis) fruits.

In spite of the fact that most of the members of Palmaceae contain high concentrations of oil, its potential as a source of oil and protein for human consumption has not been exploited. The pulp and kernels of the Eliaes guineensis palm fruits grown in the Northeast region of Brazil were analyzed only for their proximate composition. The lipid content of the dried pulp and kernels was 73.2% and 32.6%, respectively. Hexane extracted oils from the pulp and kernels yielded similar refractive indices, specific gravity but different peroxide, acid, iodine and saponification values. Gas chromatographic analysis revealed the presence of 24 and 18 fatty acids in pulp and kernel oils, respectively. The principal saturated acid of the pulp oil was palmitic acid (36.9% of the total), and lauric acid (53.3%) for kernel oil. Oleic acid was the predominant monounsaturated fatty acid in both the oils though its concentration in the pulp and kernel oils was 45.29% and 5.5%, respectively. In relation to the essential amino acids, pulp proteins presented a better profile than the kernel proteins. In comparison to the FAO reference protein, the pulp proteins were deficient in methionine, lysine and threonine (16.8%, 51.6% and 93.5% of FAO reference protein) but contained leucine, valine, isoleucine and phenylalanine in optimal concentrations. With exception to phenylalanine and valine (102.2% and 111.4% of reference protein, respectively), the kernel proteins were deficient in all other essential amino acids. The oils from this palm can be used as culinary oil and in margarine manufacture, while pulp could be a supplement for essential amino acids leucine, isoleucine, phenylalanine and valine with other protein sources that are deficient in these amino acids.

Interesse dos estudantes de Farmácia e Biologia por plantas medicinais e fitoterapia

Este trabalho descreve os resultados obtidos em um levantamento realizado com 262 estudantes dos cursos de Farmácia e Biologia, de várias partes do Brasil, sobre o tema Plantas Medicinais e Fitoterapia. Foram aplicados questionários com questões relativas aos dados pessoais de cada estudante e questões relacionadas a temas, como por exemplo: a) por que o estudante se interessava por Plantas Medicinais e Fitoterapia? e b) estudante deveria citar cinco plantas que mais conhecia e/ou utilizava. Os resultados da pesquisa demonstraram que os estudantes de Farmácia e Biologia reconhecem a importância das Plantas Medicinais e da Fitoterapia para o seu futuro desempenho como profissionais e têm interesse em se aprimorar nessa área de conhecimento. A alta freqüência de estudantes que consideraram a fitoterapia mais eficaz do que os medicamentos convencionais e a baixa freqüência daqueles que associaram plantas medicinais à riqueza da biodiversidade brasileira foram resultados preocupantes. Um ponto positivo foi o grande número de citações de plantas medicinais que contam com algum estudo de validação, como sendo as mais conhecidas e utilizadas. Consideramos interessante que as disciplinas dos Cursos de Farmácia e Biologia, envolvidas com o tema, concentrem‑se no estudo de plantas medicinais já conhecidas e utilizadas pelos estudantes, capacitando‑os nos aspectos técnico‑científicos de cada espécie e seus produtos.

Isolation and partial characterization of a lectin from Artocarpus incisa L. seeds.

A lectin was isolated from the saline extract of Artocarpus incisa seed by affinity chromatography on cross-linked Adenanthera pavonina galactomannan in 0.15 M NaCl. The lectin was also retained in a D-gal-agarose resin and had no requirements for divalent metal cations (Ca2+ and Mn2+) for activity. The lectin contains 2.1% of carbohydrate and is characterized by high contents of acidic and hydroxylated amino acids. The lectin presented two protein bands in SDS-PAGE, with M(r) 15.5 and 12 kDa, respectively, and contains no alpha-helix, 64% antiparallel beta-sheet and 21% parallel beta-sheet/beta-turn. When submitted to gel filtration in Superose 12 R (FPLC) and Superdex 75 HR 5/5 (HPLC) columns, the lectin showed an M(r) of 48-49 kDa, suggesting a tetrameric structure.

Isolation and partial characterisation of highly toxic lectins from Abrus pulchellus seeds.

The seeds of Abrus pulchellus, sub-specie tenuiflorus, belonging to the Leguminosae, subfamily Papilionoideae contain highly toxic lectins exhibiting specificity for galactose and galactose-containing structures. The toxins which agglutinate rabbit erythrocytes, present a highly toxic activity in vivo when injected in the peritoneal cavity of mice (LD50=31 microg x kg(-1)) or when tested with the microcrustacean Arthemia salina (LD50=3.5 microg x ml(-1)). The active fraction was purified in a single step, by affinity chromatography on a Sepharose-4B column. The purified toxins migrated as two single bands of Mr 63000 and 61500 Da (SDS-PAGE) and Mr 31500 and 29000 Da (SDS-PAGE with 2-mercaptoethanol), respectively, suggesting the presence of disulphide-bridge interchains as occurs in other plant toxins. The antibodies anti-A. pulchellus toxins did not recognize ricin preparation and only partial identity was observed to A. precatorius toxic lectins prepared in a similar way to ricin and A. pulchellus toxins.

Anti-inflammatory effect of glucose-mannose binding lectins isolated from Brazilian beans.

Selectins are essential for leukocyte recruitment in inflammation. Because of a lectin domain present in the selectin structure, we investigated the anti-inflammtory activity of six mannose-glucose binding lectins from brazilian beans: Dioclea guianensis-DguiL; D. grandiflora-DgL; Cratylia floribunda-CfL; D. violacea-D.vL; D. virgata-DvirL and Canavalia brasiliensis-ConBr. The lectins were injected intravenously (i.v.) into rats (0.1 and 1.0 mg/kg; 30 min before irritants) and its activities compared to E. coli endotoxin (LPS,30 mug/kg i.v.). Three lectins (DvL, CfL and DguiL), although less intense than LPS, inhibited the neutrophil migration induced by carrageenan (Cg, 300 mug) in a dose-dependent manner (0.1 and 1.0 mg/kg). DvL activity was reversed by 0.1 M alpha-D-methyl-mannoside (alpha-CH3), but not by 0.1 M alpha-D-galactose. The fMLP (44 ng)-induced neutrophil migration was also reduced by these lectins. Endotoxin contamination of lectin samples could be excluded since alpha-CH3 treatment reversed the DvL effect, but did not modify LPS inhibitory activity. Carrageenan (300 mug)-induced paw oedema was also reduced by LPS or lectin treatments. Conversely, none of the tested lectins inhibited dextran (Dex, 300 mug)-induced paw oedema, a classical leukocyte independent model, or zymosan (Zy, 1.0 mg)-induced peritonitis and paw oedema. LPS showed no effect upon Dex-induced paw oedema and barely reduced (25%) the oedematogenic effects of zymosan. As proposed for LPS, the lectin inhibitory activity was better observed on neutrophil-mediated inflammatory reactions. We speculate that the plant lectin antiinflammatory activity is probably due to a competitive blockage of a common leukocyte and/or endothelial selectin carbohydrate ligand.

Characteristics of the histamine release from hamster cheek pouch mast cells stimulated by lectins from Brazilian beans and concanavalin A.

We have characterized the histamine releasing effects of lectins extracted from Brazilian beans, in comparison to concanavalin A, in hamster cheek pouch cell suspensions containing mast cells. The lectins from Dioclea virgata, Canavalia brasiliensis, and Dioclea rostrata induce histamine release in a similar manner to concanavalin A, but appear to differ in potency and efficacy. The effects depended on the temperature, pH, and metabolic energy, demonstrating the non-cytotoxic nature of the histamine release. It is suggested that the lectins studied act by the same mechanism as concanavalin A (interacting with sugars in the antibodies bound to the mast cells), since high concentrations of glucose inhibit the histamine release. The lectins at high concentrations quench the histamine release. This suppression is reversed by increasing calcium concentration, suggesting that the lectins bind to the calcium that is essential for the secretion, thereby confirming and extending our previous data using the lectin from Dioclea virgata in rat peritoneal mast cells.

Purification, physicochemical characterization and biological properties of a lectin from Erythrina velutina forma aurantiaca seeds.

A lectin was purified from seeds of Erythrina velutina forma aurantiaca by affinity chromatography on cross-linked guar gum. The lectin is a potent agglutinin for human (minimal concentration of protein able to cause visible agglutination of a 2% erythrocyte suspension varying from 1 to 4 micrograms/ml), rabbit (4 micrograms/ml) and chicken erythrocytes (8 micrograms/ml) but presented low activity against cow (250 micrograms/ml) or sheep (333 micrograms/ml) blood cells. Hemagglutination of human O+ erythrocytes was inhibited by D-lactose (0.2 mM) > D-galactose (0.8 mM) > D-raffinose (2.1 mM). At pH 7.5, chromatography on a Superose 12 HR 10/30 column showed that the lectin was primarily a dimer (56.0 kDa) composed of two identical subunits (31.6 kDa each). A small amount of a tetrameric form was also apparently present. The lectin is a glycoprotein (7.3% carbohydrate), has a pI of 4.5, contains high levels of acidic (Asp and Glu, 64.2 and 51.6 residues/mol, respectively) and hydroxy amino acids (Ser and Thr, 42.9 and 38.5 residues/mol, respectively) but relatively low amounts of sulfur amino acids (Cys and Met, 1.0 and 5.0 residues/mol, respectively) and has an N-terminal sequence of Val-Glu-Thr-Ile/Leu-Pro-Phe-Ser. Its hemagglutinating activity was abolished by heating at 70 degrees C for 10 min. The activation energy (delta G') required for denaturation measured by loss of hemagglutination activity was 24.87 kcal/mol. In rats, the purified lectin (100 micrograms) induced neutrophil migration into the peritoneal cavity (3.7 +/- 0.6 x 10(6) neutrophils/ml) or into the air pouch (2.75 +/- 0.25 x 10(6) neutrophils/ml), 8 and 10 times greater than the negative control, respectively.

Purification, physicochemical characterization and biological properties of a lectin from Erythrina velutina forma aurantiaca seeds

A lectin was purified from seeds of Erythrina velutina forma aurantiaca by affinity chromatography on cross-linked guargum. The lectin is a potent agglutinin for human (minimal concentration of protein able to cause visible agglutination of a 2 per cent erythrocyte suspension varying from 1 to 4 mug/ml), rabbit(4 mug/ml) and chicken erythrocytes (8 mug/ml) but presented low activity against cow (250 mug/ml) or sheep (333 mug/ml) blood cells. Hemagglutination of human O+ erythrocytes was inhibited by D-lactose (0.2 mM) > D-galactose(0.8 mM) > D-raffinose (2.1 mM). At pH 7.5, chromatography on a Superose 12 HR 10/30 column showed that the lectin was primarily a dimer (56.0 kDa) composed of two identical subunits (31.6 kDa each). A small amount of a tetrameric form was also apparently present. The lectin is a glycoprotein (7.3 per cent carbohydrate), has a pI of 4.5, contains high levels of acidic (Asp and Glu, 64.2 and 51.6 residues/mol, respectively) and hydroxy amino acids (Ser and Thr, 42.9 and 38.5 residues/mol, respectively) but relatively low amounts of sulfur amino acids (Cys and Met, 1.0 and 5.0 residues/mol, respectively) and has an N-terminal sequence of Val-Glu-Thr-Ile/Leu-Pro-Phe-Ser. Its hemagglutinating activity was abolished by heating at 70 degrees Celsius for 10 min. The activation energy (delta G') required for denaturation measured by loss of hemagglutination activity was 24.87 kcal/mol. In rats, the purified lectin (100 mug) induced neutrophil migration into the peritoneal cavity (3.7 ñ 0.6 x 10(6) neutrophils/ml) or into the air pouch (2.75 ñ 0.25 x 10(6) neutrophils/ml), 8 and 10 times greater than the negative control, respectively.

In vivo protective effect of the lectin from Canavalia brasiliensis on BALB/c mice infected by Leishmania amazonensis.

In vivo administration of Canavalia brasiliensis lectin (at the time of infection, or maintained throughout the infection) reduced the lesions of highly susceptible BALB/c mice infected by Leishmania amazonensis. At the doses used C. brasiliensis lectin (ConBr) does not interfere with penetration or fate of Leishmania in the macrophages in vitro. Since Interferon-gamma (IFN-gamma) is the major macrophage activating factor, and considered a critical element in the successful immune response against leishmaniasis, we explored its participation in this phenomenon. ConBr either in vivo or in vitro induced IFN-gamma production in normal or in leishmania-infected BALB/c mice. However we were unable to change the course of disease by in vivo IFN-gamma administration (although IFN-gamma preparations were effective in inducing a leishmanicidal effect in vitro on L. amazonensis-infected peritoneal macrophages). Additionally, IFN-gamma neutralization with anti-IFN-gamma monoclonal antibody did not alter the protection conferred by ConBr administration. These data show that lectin administration in vivo is protective in the otherwise unchecked L. amazonensis infection of BALB/c mice, and suggest that such effect is not mediated by IFN-gamma.