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.

Protein profile of the seminal plasma of collared peccaries (Pecari tajacu Linnaeus, 1758).

This study was conducted to characterize the major proteins of the peccary seminal plasma, based on the semen samples collected from nine adult and reproductively sound animals. Our approach included the use of two-dimensional electrophoresis followed by Coomassie blue staining and analysis of polypeptide maps with PDQuest Software (Bio-Rad). Proteins were identified by tandem mass spectrometry (LC-MS/MS). We detected 179 protein spots per gel and 98 spots were identified by mass spectrometry, corresponding to 23 different proteins. The combined intensity of those spots accounted for 56.2±6% of the intensities of all spots and 60.9% of the intensities of spots presented in every protein map. Protein spots identified as clusterin represented 19.7±8.3% of the integrated optical densities of all spots detected in the seminal plasma maps. There was a negative association (r=-0.87; P<0.05) between the intensity of a clusterin spot and the percentage of sperm with functional membrane. Spermadhesin porcine seminal plasma protein 1 and bodhesin 2 comprised 5.4±1.9 and 8.8±3.9% of the total intensity of all spots respectively. Many proteins appeared in a polymorphic pattern, such as clusterin (27 spots), epididymal secretory glutathione peroxidase (ten spots), inter-α-trypsin inhibitor (12 spots), and IgG-binding protein (ten spots), among others. In conclusion, we presently describe the major seminal plasma proteome of the peccary, which exhibits a distinct high expression of clusterin isoforms. Knowledge of wild species reproductive biology is crucial for an understanding of their survival strategies and adaptation in a changing environment.

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.

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.

Application of a vibration measuring technique to evaluate the dynamic stiffness of porcine periodontal ligament.

The performance of a tooth replacement by using a dental implant relies on the mechanical and biological capability of the anatomical substitute to restore lost physiological functions. The design of an implant device able to properly replace the physiological tooth requires the study of the load transfer mechanism at the implant-bone interface and the understanding of the relevance of the periodontal ligament (PDL) in this mechanism. The PDL is a connective soft tissue that provides the fixation of the tooth in its bone-socket and the attenuation of occlusal loads. It also provides the ground cells that are involved in the remodelling process, induced by a change in the stress-strain pattern of the alveolar bone and also in the cementum of the tooth root. The purpose of this study was to determine the PDL effects on the dynamic load transfer mechanism, from the tooth to the alveolar bone, evaluating the equivalent dynamic stiffness of the ligament structure. A porcine fresh mandible with a tooth was used within the study, applying an experimental procedure to identify the dynamic transmissibility of the entire system. The transmissibility function provided information about the stiffness and damping of the PDL, information that can assist the design of an improved dental implant system.

Proteomic analysis of seminal plasma and sperm cells and their associations with semen freezability in Guzerat bulls.

The objective of the present study was to describe the relationship of seminal plasma and total sperm cell proteins with the semen freezability parameters of Guzerat bulls. Thirteen bulls were subjected to breeding soundness evaluation. Semen samples were collected, cryopreserved, and then post-thawing sperm kinetics were assessed, where high ( = 7) and low ( = 6) freezability groups were defined. Seminal plasma and total sperm proteins from the 2 groups were separated by 2-dimensional SDS-PAGE, and spots were identified by mass spectrometry. Semen parameters post-cryopreservation were as follows in the high and low freezability groups, respectively: mean total motility, 52.4 ± 20.5 and 13.7 ± 3.9; percentage of normal sperm, 89.0 ± 2.6 and 64.7 ± 14.0; and reactivity of hypo-osmotic swelling test, 38.9 ± 4.7 and 13.6 ± 3.7. Three seminal plasma proteins (osteopontin-K, DNase γ precursor, and DNASE1L3) and 6 proteins from sperm cells (acrosome formation-associated factor isoform 2, annexin A1, disintegrin and metalloproteinase domain-containing protein 2, dihydrolipoyl dehydrogenase, and glyceraldehyde-3-phosphate dehydrogenase) were highly expressed ( < 0.05) in the high freezability group. Another 6 seminal plasma proteins (acrosin inhibitor 1, glutathione peroxidase 3, metalloproteinase inhibitor 2, ephrin-A1, annexin A1, and platelet-activating factor acetylhydrolase) were significantly higher ( < 0.05) in the low freezability group. We described the associations of seminal plasma and sperm cell proteins with post-thawing sperm viability of Guzerat bulls raised in a typical semiarid environment. Such associations indicate that specific seminal plasma proteins more abundant in bulls of low semen freezability may be a response to an early oxidative stress that is not detected by conventional prefreezing semen evaluation. Moreover, specific sperm proteins were more associated with good freezability. The results presented here can serve as guidelines for future research aiming to develop better extenders and/or to improve bull semen selection for cryopreservation.

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.

Growth, testis size, spermatogenesis, semen parameters and seminal plasma and sperm membrane protein profile during the reproductive development of male goats supplemented with de-oiled castor cake.

The present study was conducted to evaluate the effect of de-oiled castor cake on reproductive traits of crossbreed goats. Fourteen males were grouped into two lots (n = 7/group), as described: group without de-oiled castor cake (WCC) and group fed with de-oiled castor cake (CC). Goats received two diets containing a mixture of Bermudagrass hay and concentrates with the same energy (73% total digestive nutrients) and protein content (15% crude protein) during 150 days, corresponding to ages from 40 (puberty) to 60 weeks. Blood plasma concentrations of urea, albumin, lactate dehydrogenase, creatinine, alanine aminotransferase and testosterone were determined. We also evaluated scrotal circumference, sperm parameters, quantitative aspects of spermatogenesis and daily sperm production (DSP), as well as the proteome of seminal plasma and sperm membrane. Seminal fluid and sperm proteins were analyzed by 2D SDS-PAGE and mass spectrometry. After 150 days of castor cake feeding, animals had no changes in the biochemical composition of blood plasma, suggesting the absence of intoxication by ingestion of ricin. There were no alterations in dry mater intake, weight gain, testis size, peripheral concentrations of testosterone, sperm concentration, motility and morphology. Sertoli and germ cell populations in the testis and DSP were not affected either. However, there were significant variations in the expression of five seminal plasma proteins and four sperm membrane proteins. In conclusion, the replacement of soybean meal by castor cake (with ricin concentrations of 50mg/kg) did not interfere with the growth and core reproductive development of male goats. However, the diet with ricin altered the expression of certain seminal plasma and sperm membrane proteins, which play roles in sperm function and fertilization. Lower expression of these proteins may impair the ricin-fed animals to perform as high-fertility sires.

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.

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.

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.

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.

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.

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.

Isolation and characterization of a lectin from the seeds of Dioclea grandiflora (Mart.).

By a combination of solubility fractionation, affinity and molecular-sieve chromatography, a lectin preparation containing several closely related lectin components of different isoelectric point was isolated from the seeds of Dioclea grandiflora Mart. The lectins showed a carbohydrate specificty for D-mannose (D-glucose)-binding and had a requirement for the presence of Ca(2+) and Mn(2+). The results of preliminary characterization studies showed that the D. grandiflora lectins had similar properties to those of concanavalin A, the lectin from the seeds of Canavalia ensiformis, a plant also belonging to the tribe Diocleae. Thus the D. grandiflora lectins contained no covalently bound carbohydrate and had an amino-acid composition characterized by a low content of methionine and the virtual absence of cysteine. Above pH 4.8 they had molecular weight of about 100,000, while below pH 3.1 they were dissociated to half-molecules. Between these two pH values there was a fast association-dissociation equilibrium for the two species. In dissociating solvents, three subunits were obtained of the approximate size of 25-26,000, 13-14,000 and 8-9,000. The lectins from C. grandiflora similar to concanavalin A were more distantly related to the lectins obtained from the members of the tribe Vicieae although these were also specific for D-mannose (D-glucose)-binding.

Rat paw edema and leukocyte immigration induced by plant lectins.

Lectins from Dioclea grandiflora (DG) and Canavalia brasiliensis (CB) were compared with Concanavalin A (ConA) for their ability to induce paw edema and peritoneal cell immigration in rats. ConA caused a slight edema with a peak at 1 h after injection, while DG or CB induced a pronounced and long-lasting edema that reached a maximum at about 6 h. Different antiinflammatory drugs partially inhibited the edema. alpha-D-glucose (GLU) partially blocked the edema caused by ConA and markedly inhibited that due to CB, but had no effect on the edema induced by DG. alpha-Methyl mannoside (alpha-MM) blocked the edema caused by DG and ConA, but did not affect that caused by CB. At doses much lower than those used to induce paw edema, the lectins promoted an intense accumulation of neutrophil and mononuclear cells in the rat peritoneal cavity. CB and DG were more potent than ConA, which also presented a different profile of cell immigration. GLU significantly inhibited leukocyte accumulation caused by all lectins. alpha-MM impaired ConA- and DG-induced cell immigration, but only partially inhibited CB. Thus, despite their physicochemical similarities with ConA, DG and CB have more powerful pro-inflammatory effects. This difference seems to be related to their sugar-binding properties. However, while ConA- and DG-induced effects were inhibited more by alpha-MM than by GLU, CB-induced effects were inhibited more by glucose.