cDNA cloning and 1.75 angstrom crystal structure determination of PPL2, an endochitinase and N-acetylglucosamine-binding hemagglutinin from Parkia platycephala seeds

Parkia platycephala lectin 2 was purified from Parkia platycephala (Leguminosae, Mimosoideae) seeds by affinity chromatography and RP-HPLC. Equilibrium sedimentation and MS showed that Parkia platycephala lectin 2 is a nonglycosylated monomeric protein of molecular mass 29 407 +/- 15 Da, which contains six cysteine residues engaged in the formation of three intramolecular disulfide bonds. Parkia platycephala lectin 2 agglutinated rabbit erythrocytes, and this activity was specifically inhibited by N-acetylglucosamine. In addition, Parkia platycephala lectin 2 hydrolyzed beta(1-4) glycosidic bonds linking 2-acetoamido-2-deoxy-beta-D-glucopyranose units in chitin. The full-lengthamino acid sequence of Parkia platycephala lectin 2, determined by N-terminal sequencing and cDNA cloning, and its three-dimensional structure, established by X-ray crystallography at 1.75 angstrom resolution, showed that Parkia platycephala lectin 2 is homologous to endochitinases of the glycosyl hydrolase family 18, which share the (beta alpha)(8) barrel topology harboring the catalytic residues Asp125, Glu127, and Tyr182.

Physiological responses of Nile tilapia, Oreochromis niloticus, fed vitamin C- and lipid-supplemented diets and submitted to low-temperature stress

Water temperature alterations can determine harmful physiological modifications in fish, which should be prepared to cope with this, and nutrition strategies seem to be essential. This study evaluated the effects of different levels of vitamin C and lipids on physiological responses of Nile tilapia, Oreochromis niloticus, submitted to temperature stress. There were two phases: Phase I - preparing fish to store vitamin C and lipid at appropriate temperature, and Phase II - evaluating the contributions these reserves make to fish physiology under low-temperature stress. The experiment used a 3 x 2 factorial design with three vitamin C levels (300, 600, and 1200 mg/kg diet) and two lipid levels (8.0 and 12.0%), plus absence of nutrient test and a diet of 6.0% lipids and 125.0 mg/kg vitamin C. In Phase I, 192 fish were kept at 26.0 +/- 1.0 C for 112 d, and in Phase II, 48 fish were kept at 18.0 +/- 0.5 C for 32 d and at 15.0 +/- 0.5 C for 11 d. Fish fed C0L0 diet showed lower erythrocytes values in both phases; higher vitamin C supplement determined higher red blood cell (RBC) number and higher hematocrit (Htc) (Phase II); Htc was significantly lower in Phase II; after temperature stress, fish fed C0L0 diet had higher mean corpuscular volume, lower hemoglobin corpuscular concentration, and significantly lower vitamin C concentration in the liver; and higher supplementation determined a higher concentration in the liver (Phases I and II). Higher plasmatic cortisol concentration was seen in fish fed C0L0 diet. In conclusion, our results show that the absence of vitamin C in diets impairs RBC formation and does not enable fish to cope with stress; excess vitamin C is efficient in mitigating stress and 600 mg/kg diet is economic and physiologically sufficient to prepare fish for coping with low-temperature stress. Lipid supplementation does not determine alterations in stress biochemical parameters.

Immunization of bovines using a DNA vaccine (pcDNA3.1/MSP1b) prepared from the jaboticabal strain of Anaplasma marginale

Anaplasma is a tick-borne ehrlichial pathogen of cattle that causes the disease, anaplasmosis. In the present study, a total of 11 Anaplasma marginale seronegative calves were assigned into two groups: one immunized (G1, n = 6) and one nonimmunized-control (G2, n = 5). Six calves were immunized by using a DNA vaccine containing the gene of a major surface protein, MSP1b, encoded by the plasmid identified as pcDNA3.1/MSPIb. Calves received three intramuscular inoculations of 100 mug of pcDNA3.1/MSP1b at a 20-day interval. The control group received buffer phosphate at the same schedule as the experimental group. The immune response elicited by immunization with pcDNA3.1/MSP1b was evaluated in mice and calves. Twenty days following initial immunization, specific serum antibody from four BALB/c mice bound MSP1b in inummoblots. Sixty days after the last immunization, all calves were challenged with cryopreserved A. marginale at a dose of 10(4) parasites/mL/animal by intravenous injection. Results of packed cell volume (PCV) and detection of infected erythrocytes in all experimental groups revealed that the decrease of PCV and detection of infected erythrocytes occurred at 28 to 42 days after challenge. Mean temperature values did not increase over 39.85degreesC. Antibodies developed by immunized bovines from G2 were detected 14 days after challenge. MSP1b was characterized during the immunization period and MSP2 was the most predominant polypeptide at the challenge period. DNA of A. marginale was detected in all groups just after challenge by nested PCR assay. It can be concluded that all immunized bovines were partially protected against homologous challenge.

Antitumor activity of extracts from Peperomia elongata

The cytotoxic potential of ethanol extracts from Peperomia elongata H. B. & K. (Piperaceae) were evaluated against human cancer cell lines by the MTT method. The samples considered cytotoxic were tested for antimitotic activity with the sea urchin egg development test and for hemolytic activity using mice erythrocytes. The extracts from leaves (hexane), stems (ethanol, hexane, hexane: AcOEt, AcOEt, and MeOH: H2O insoluble), and roots (R4) presented potential cytotoxic action. The stems extracts showed the highest toxicity in all tumor cell lines tested, with an IC50 <= 9.0 mu g/mL for ethanol extract, IC50 <= 11.6 mu g/mL for MeOH:H2O insoluble, IC50 <= 7.3 mu g/mL for hexane extract, IC50 <= 11.4 mu g/mL for hexane: AcOEt, and IC50 <= 16.2 mu g/mL for AcOEt extract. All extracts considered cytotoxic for tumoral cell lines presented antimitotic activity. The samples from roots (R4) and stems (ethanol, MeOH: H2O insoluble, and hexane extract from leaves) were found to possess lytic activity in mice erythrocytes but in higher doses (> 125 mu g/mL). Further studies for the isolation and identification of the active principles of these extracts should be undertaken.

CYTOGENETIC AND DNA CONTENT IN 6 GENERA OF THE FAMILY CALLICHTHYIDAE (PISCES, SILURIFORMES)

Cytogenetic studies involving conventional Giemsa staining, C-banding analysis and silver staining of NORs were performed on nine species belonging to six genera of the family Callichthyidae. The diploid number ranged from 2n = 44 to 2n = 100, the number of chromosomal pairs with NORs ranged from 1 to 4 and constitutive heterochromatin was mainly distributed in the centromeric and/or pericentromeric position of the chromosomes. The DNA content of erythrocytes from six species studied ranged from 1.18 +/- 0.07 to 2.77 +/- 0.22 pg/nucleus. The extensive variability in karyotypes and in nuclear DNA content detected are in accordance with the initial hypothesis that chromosome rearrangements and polyploidy have played a significant role in the evolutionary history of Callichthyidae.

Eumenitin, a novel antimicrobial peptide from the venom of the solitary eumenine wasp Eumenes rubronotatus

A novel antimicrobial peptide, eumenitin, was isolated from the venom of the solitary eumenine wasp Eumenes rubronotatus. The sequence of eumenitin, Leu-Asn-Leu-Lys-Gly-Ile-Phe-Lys-Lys-Val-Ala-Ser-Leu-Leu-Thr, was mostly analyzed by mass spectrometry together with Edman degradation, and corroborated by solid-phase synthesis. This peptide has characteristic features of cationic linear a-helical antimicrobial peptides, and therefore, can be predicted to adopt an amphipathic a-helix secondary structure. In fact, the CD spectra of eumenitin in the presence of TFE or SDS showed a high content of alpha-helical conformation. Eumenitin exhibited inhibitory activity against both Gram-positive and Gram-negative bacteria, and moderately stimulated degranulation from the rat peritoneal mast cells and the RBL-2H3 cells, but showed no hemolytic activity against human erythrocytes. This antimicrobial peptide in the eumenine wasp venom may play a role in preventing potential infection by microorganisms during prey consumption by their larvae. (c) 2006 Elsevier B.V. All rights reserved.

Purification and properties of buffalo (Bubalus bubalis) erythrocyte hexokinase

Buffalo erythrocytes contain one isozyme of hexokinase that apparently lacks microheterogeneity as shown by chromatographic properties. A single protein band was detected by means of Western blotting using an antibody raised in rabbits against homogeneous rat brain hexokinase I. The native protein has a molecular weight of 200,000 +/- 2880 by gel filtration. Partial purification of erythrocyte hexokinase by a combination of several procedures, including affinity chromatography, which was previously applied successfully to the purifica tion of other mammalian type I hexokinases, produced a partially purified enzyme that showed several contami nants after SDS-polyacrylamide gel electrophoresis. The affinity of buffalo erythrocyte hexokinase for glucose (K-m = 0.012 +/- 0.001 mM) is lower than most other mammal hexokinases type I. It phosphorylates other sugars, with considerably higher K-m values. This isozyme is able to use MgATP but does not use MgGTP, MgCTP or MgUTP. We used inhibition patterns, obtained with products to elucidate enzyme sequential mechanisms. Our results are clearly in agreement with a random sequential mechanism and in disagreement with an ordered sequential mechanism with either glucose or ATP as the obligatory first substrates. The ADP inhibition was of mixed type with both ATP and glucose as substrates. (C) 1997 Elsevier B.V.

Heparina e K3EDTA como anticoagulantes para tambaqui (Colossoma macropomum Cuvier, 1816)

The efficacy of sodium heparin and tripotassium EDTA as anticoagulant and their effect on the hematological parameters of tambaqui (Colossoma macropomum) were evaluated in this study. Ten fish weighing 384.9 +/- 85.71 g and measuring 27.90 +/- 2.10 cm were used for heparin 5.000 IU, heparin 100 IU and K3EDTA 10% evaluation. Clotting inhibition after 10 h, erythrogram and osmotic fragility of erythrocytes were observed. The results were submitted to variance analysis and means compared by Tukey test (P < 0.05). Heparin 5.000 IU, heparin 100 IU and K3EDTA 10% were effective in preventing coagulation for more than 10 h. However, tripotassium EDTA caused hemolysis since first moments. In erythrogram there was no difference (P > 0.05) in erythrocyte count, hematocrit, hemoglobin and MCHC. on the other hand, an increase in MCV (P < 0.05) in samples kept with K3EDTA10% was observed. This anticoagulant provoked a significant increase (P < 0.01) in the osmotic fragility of erythrocytes when compared to pure heparin, diluted heparin and the control group. Heparin as an anticoagulant is more appropriate for tambaqui since it was effective in preventing coagulation for more than 10 h, without causing hemolysis, changes on hematological parameters or osmotic fragility of erythrocytes.

Hematological parameters and anaerobic threshold in Brazilian soccer players throughout a training program

We assessed the responses of hematological parameters and their relationship to the anaerobic threshold of Brazilian soccer players during a training program. Twelve athletes were evaluated at the beginning (week 0, T1), in the middle (week 6, T2), and at the end (week 12, T3) of the soccer training program. on the first day at 7:30 AM, before collecting the blood sample at rest for the determination of the hematological parameters, the athletes were conducted to the anthropometric evaluation. on the second day at 8:30 AM, the athletes had their anaerobic threshold measured. Analysis of variance with Newman-Keuls'post hoc was used for statistical comparisons between the parameters measured during the soccer training program. Correlations between the parameters analyzed were determined using the Pearson's correlation coefficient. Erythrocytes concentration, hemoglobin, and hematocrit were significantly increased from T1 to T2. The specific soccer training program led to a rise in erythrocytes, hemoglobin, and hematocrit from T1 to T2. We assumed that these results occurred due to the plasma volume reduction and may be explained by the soccer training program characteristics. Furthermore, we did not observe any correlation between the anaerobic threshold and the hematological parameters.

Transplacental Transfer of Iron in the Water Buffalo (Bubalus bubalis): Uteroferrin and Erythrophagocytosis

The objectives of this investigation were to understand transplacental transport of iron by secreted uteroferrin (UF) and haemophagous areas of water buffalo placenta and clarify the role(s) of blood extravasation at the placental-maternal interface. Placentomes and interplacentomal region of 51 placentae at various stages of gestation were fixed, processed for light and transmission electron microscopy, histochemistry and immunohistochemistry. Haemophagous areas were present in placentomes collected between 4 and 10 months of pregnancy. Perl's reaction for ferric iron was negative in placentomes, but positive in endometrial glands. Positive staining for UF indicated areas in which it was being taken up by phagocytosis and/or fluid phase pinocytosis in areolae of the interplacentomal mesenchyme, with little staining in endometrial stroma. Imunohistochemistry detected UF in trophectoderm of haemophagous regions of placentomes and in other parts of the foetal villous tree, but the strongest immunostaining was in the epithelial cells and lumen of uterine glands. Ultrastructural analyses indicated that erythrophagocytosis was occurring and that erythrocytes were present inside cells of the chorion that also contained endocytic vesicles and caveolae. Results of this study indicate that both the haemophagous areas of placentomes and the areolae at the interface between chorion and endometrial glands are important sites for iron transfer from mother to foetal-placental tissues in buffalo throughout pregnancy.

Purification of a PHA-Like chitin-binding protein from Acacia farnesiana seeds: A time-dependent oligomerization protein

A lectin-like protein from the seeds of Acacia farnesiana was isolated from the albumin fraction, characterized, and sequenced by tandem mass spectrometry. The albumin fraction was extracted with 0.5 M NaCl, and the lectin-like protein of A. farnesiana (AFAL) was purified by ion-exchange chromatography (Mono-Q) followed by chromatofocusing. AFAL agglutinated rabbit erythrocytes and did not agglutinate human ABO erythrocytes either native or treated with proteolytic enzymes. In sodium dodecyl sulfate gel electrophoresis under reducing and nonreducing conditions, AFAL separated into two bands with a subunit molecular mass of 35 and 50 kDa. The homogeneity of purified protein was confirmed by chromatofocusing with a pI=4.0+/-0.5. Molecular exclusion chromatography confirmed time-dependent oligomerization in AFAL, in accordance with mass spectrometry analysis, which confers an alteration in AFAL affinity for chitin. The protein sequence was obtained by a liquid chromatography quadrupole time-of-flight experiment and showed that AFAL has 68% and 63% sequence similarity with lectins of Phaseolus vulgaris and Dolichos biflorus, respectively.

The 844ins68 cystathionine beta-synthase and C677T MTHFR gene polymorphism and the vaso-occlusive event risk in sickle cell disease

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Purification, Characterization, and Preliminary X-Ray Diffraction Analysis of a Lactose-Specific Lectin from Cymbosema roseum Seeds

The unique carbohydrate-binding property of lectins makes them invaluable tools in biomedical research. Here, we report the purification, partial primary structure, carbohydrate affinity characterization, crystallization, and preliminary X-ray diffraction analysis of a lactose-specific lectin from Cymbosema roseum seeds (CRLII). Isolation and purification of CRLII was performed by a single step using a Sepharose-4B-lactose affinity chromatography column. The carbohydrate affinity characterization was carried using assays for hemagglutination activity and inhibition. CRLII showed hemagglutinating activity toward rabbit erythrocytes. O-glycoproteins from mucine mucopolysaccharides showed the most potent inhibition capacity at a minimum concentration of 1.2 A mu g mL(-1). Protein sequencing by mass spectrometry was obtained by the digestion of CRLII with trypsin, Glu-C, and AspN. CRLII partial protein sequence exhibits 46% similarity with the ConA-like alpha chain precursor. Suitable protein crystals were obtained with the hanging-drop vapor-diffusion method with 8% ethylene glycol, 0.1 M Tris-HCl pH 8.5, and 11% PEG 8,000. The monoclinic crystals belong to space group P2(1) with unit cell parameters a = 49.4, b = 89.6, and c = 100.8 A....

Evidence of zinc deficiency in competitive swimmers

Objective: The aim of this study was to assess the nutritional zinc (Zn) status of elite swimmers during different training periods.Methods: A longitudinal paired study was performed at the University of São Paulo in eight male swimmers 18 to 25 y old who had been swimming competitively at the state and national levels for at least 5 y. The swimmers were evaluated over a total period of 14 wk: before the basic and specific preparatory period (BSPP-baseline), at the end of the basic and specific preparatory period (post-BSPP), and at the end of the polishing period (PP). Levels of Zn were determined in the plasma, erythrocyte, urine, and saliva by atomic absorption spectrophotometry. Anthropometric measurements and a 3-d food record were also evaluated.Results: The median plasma Zn concentration was below the reference value in all training periods (BSPP-baseline 59 mu g/dL, post-BSPP 55.9 mu g/dL, after PP 58.8 mu g/dL, P > 0.05), as were threshold values for erythrocytes (BSPP-baseline 36.5 mu g of Zn/g of hemoglobin, post-BSPP 42 mu g of Zn/g of hemoglobin, after PP 40.7 mu g of Zn/g of hemoglobin, P > 0.05), urinary Zn (BSPP-baseline 280 mu g/24 h, post-BSPP 337 mu g/24 h, after PP 284 mu g/24 h, P > 0.05), and salivary Zn (BSPP-baseline 66.1 mu g/L, post-BSPP 54.1 mu g/L, after PP 79.7 mu g/L, > 0.05). Salivary Zn did not correlate with plasma and erythrocyte Zn levels.Conclusion: The results suggest that the elite swimmers studied presented a possible Zn deficiency and that salivary Zn was not adequate to evaluate the Zn nutritional status. (C) 2012 Elsevier B.V. All rights reserved.