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.

Purification and partial characterization of cathepsin D from porcine (Sus scrofa) liver using affinity chromatography

Cathepsin D, a lysosomal aspartic protease, has been purified from porcine liver using a combination of pepstatin-A agarose and Affi-Gel Blue affinity chromatography, followed by size-exclusion chromatography. The purified protein consists of two polypeptide chains of 15 and 30 kDa, and has an isoelectric point of 6.8. Porcine liver cathepsin D has maximum activity at pH 2.5-3.0 as determined by its activity against hemoglobin, with a K-cat of 14.3 s(-1) and a k(cat)/K-M of 2.70 x 10(6) s(-1) M-1 as determined by the hydrolysis of a fluorogenic peptide substrate.

Automated NMR structure determination and disulfide bond identification of the myotoxin crotamine from Crotalus durissus terrificus

Crotamine is one of four major components of the venom of the South American rattlesnake Crotalus durissus terrificus. Similar to its counterparts in the family of the myotoxins, it induces myonecrosis of skeletal muscle cells. This paper describes a new NMR structure determination of crotamine in aqueous solution at pH 5.8 and 20 degrees C, using standard homonuclear (1)H NMR spectroscopy at 900 MHz and the automated structure calculation software ATNOS/CANDID/DYANA. The automatic NOESY spectral analysis included the identification of a most likely combination of the six cysteines into three disulfide bonds, i.e. Cys4-Cys36, Cys11-Cys30 and Cys18-Cys37; thereby a generally applicable new computational protocol is introduced to determine unknown disulfide bond connectivities in globular proteins. A previous NMR structure determination was thus confirmed and the structure refined. Crotamine contains an alpha-helix with residues 1-7 and a two-stranded anti-parallel beta-sheet with residues 9-13 and 34-38 as the only regular secondary structures. These are connected with each other and the remainder of the polypeptide chain by the three disulfide bonds, which also form part of a central hydrophobic core. A single conformation was observed, with Pro13 and Pro21 in the trans and Pro20 in the cis-form. The global fold and the cysteine-pairing pattern of crotamine are similar to the beta-defensin fold, although the two proteins have low sequence homology, and display different biological activities. (c) 2005 Elsevier Ltd. All rights reserved.

Assignment of the disulfide bridges in bothropstoxin-I, a myonecrotic Lys49 PLA(2) homolog from Bothrops jararacussu snake venom

Bothropstoxin-I (BthTX-1), a Lys49 phospholipase A(2) homolog with no apparent catalytic activity, was first isolated from Bothrops jararacussu snake venom and completely sequenced in this laboratory. It is a 121-amino-acid single polypeptide chain, highly myonecrotic, despite its inability to catalyze hydrolysis of egg yolk phospholipids, and has 14 half-cystine residues identified at positions 27, 29, 44, 45, 50, 51, 61, 84, 91, 96, 98, 105, 123, and 131 (numbering according to the conventional alignment including gaps, so that the last residue is Cys 131). In order to access its seven disulfide bridges, two strategies were followed: (1) Sequencing of isolated peptides from (tryptic + SV8) and chymotryptic digests by Edman-dansyl degradation; (2) crystallization of the protein and determination of the crystal structure so that at least two additional disulfide bridges could be identified in the final electron density map. Identification of the disulfide-containing peptides from the enzymatic digests was achieved following the disappearance of the original peptides from the HPLC profile after reduction and carboxymethylation of the digest. Following this procedure, four bridges were initially identified from the tryptic and SV8 digests: Cys50-Cys131, Cys51-Cys98, Cys61-Cys91, and Cys84-Cys96. From the chymotryptic digest other peptides were isolated either containing some of the above bridges, therefore confirming the results from the tryptic digest, or presenting a new bond between Cys27 and Cys123. The two remaining bridges were identified as Cys29-Cys45 and Cys44-Cys105 by determination of the crystal structure, showing that BthTX-1 disulfide bonds follow the normal pattern of group II PLA(2)s.

Distribution of I-125-labeled crotamine in mice tissues

Crotamine is a strong basic polypeptide from Crotalus durissus terrificus (Cdt) venom composed of 42 amino acid residues tightly bound by three disulfide bonds. It causes skeletal muscle spasms leading to spastic paralysis of hind limbs in mice. The objective of this paper was to study the distribution of crotamine injected intraperitoneally (ip) in mice. Crotamine was purified from Cdt venom by gel filtration, followed by ion exchange chromatography, using a fast-performance liquid chromatography (FPLC) system. Purified crotamine was irradiated at 2 kGy in order to detoxify. Both native and irradiated proteins were labeled with 125, using chloramine T method, and separated by get filtration. Male Swiss mice were injected ip with 0.1 mL (2 x 10(6) cpm/mouse) of I-125 native or irradiated crotamine. At various time intervals, the animals were killed by ether inhalation and blood, spleen, liver, kidneys, brain, lungs, heart, and skeletal muscle were collected in order to determine the radioactivity content. The highest levels of radioactivity were found in the kidneys and the liver, and the lowest in the brain. (c) 2006 Elsevier Ltd. All rights reserved.

Expression and variability of molecular chaperones in the sugarcane expressome

Molecular chaperones perform folding assistance in newly synthesized polypeptides preventing aggregation processes, recovering proteins from aggregates, among other important cellular functions. Thus their study presents great biotechnological importance. The present work discusses the mining for chaperone-related sequences within the sugarcane EST genome project database, which resulted in approximately 300 different sequences. Since molecular chaperones are highly conserved in most organisms studied so far, the number of sequences related to these proteins in sugarcane was very similar to the number found in the Arabidopsis thaliana genome. The Hsp70 family was the main molecular chaperone system present in the sugarcane expressome. However, many other relevant molecular chaperones systems were also present. A digital RNA blot analysis showed that 5'ESTs from all molecular chaperones were found in every sugarcane library, despite their heterogeneous expression profiles. The results presented here suggest the importance of molecular chaperones to polypeptide metabolism in sugarcane cells, based on their abundance and variability. Finally, these data have being used to guide more in deep analysis, permitting the choice of specific targets to study. (c) 2006 Elsevier GmbH. All rights reserved.

Specific components found in circulating immune complexes (CIC) in paracoccidioidomycosis

Circulating immune complexes (CIC) from 15 paracoccidioidomycosis (PCM) patient sera and from 20 healthy control sera were analysed. After CIC precipitation, solubilization and acid treatment, only a little reactivity to P. brasiliensis antigens was found in the free antibodies from PCM-CIC. This result has suggested that there were antibodies with a high affinity bound to fungus components. Dissociated CIC were fractionated in a column of Sephacryl S300 and the fractions that probably contained antigens were pooled and applied to an affinity column, prepared with mouse anti-gp43 monoclonal antibody. Using ECL-Western blotting assay two polypeptide with apparent mass of 43 and 62 kDa were found.

Assignment of the bisulfide bridges in bothropstoxin-I, a Myonecrotic Lys49 PLA2 homolog from bothrops jararacussu snake venom

Bothropstoxin-I (BthTX-I), a Lys49 phospholipase A2 homolog with no apparent catalytic activity, was first isolated from Bothrops jararacussu snake venom and completely sequenced in this laboratory. It is a 121-amino-acid single polypeptide chain, highly myonecrotic, despite its inability to catalyze hydrolysis of egg yolk phospholipids, and has 14 half-cystine residues identified at positions 27, 29, 44, 45, 50, 51, 61, 84, 91, 96, 98, 105, 123, and 131 (numbering according to the conventional alignment including gaps, so that the last residue is Cys 131). In order to access its seven disulfide bridges, two strategies were followed: (1) Sequencing of isolated peptides from (tryptic + SV8) and chymotryptic digests by Edman-dansyl degradation; (2) crystallization of the protein and determination of the crystal structure so that at least two additional disulfide bridges could be identified in the final electron density map. Identification of the disulfide-containing peptides from the enzymatic digests was achieved following the disappearance of the original peptides from the HPLC profile after reduction and carboxymethylation of the digest. Following this procedure, four bridges were initially identified from the tryptic and SV8 digests: Cys50-Cysl31, Cys51-Cys98, Cys61-Cys91, and Cys84-Cys96. From the chymotryptic digest other peptides were isolated either containing some of the above bridges, therefore confirming the results from the tryptic digest, or presenting a new bond between Cys27 and Cys 123. The two remaining bridges were identified as Cys29-Cys45 and Cys44-Cysl05 by determination of the crystal structure, showing that BthTX-I disulfide bonds follow the normal pattern of group II PLA2s. © 2001 Plenum Publishing Corporation.

Growth hormone mRNA expression in the pituitary of Bos indicus and Bos taurus x Bos indicus crossbred young bulls treated with recombinant bovine somatotropin

The effects of breed and of recombinant bovine somatotropin (rbST) treatment on growth hormone gene expression were studied in young bulls. The experiment was completely randomized in a [2 × 2]-factorial arrangement, using two levels of rbst (0 or 250 mg/animal/14 days), and two breed groups (Nelore and Simmental x Nelore crossbred). A CDNA encoding Bos indicus growth hormone was cloned and sequenced for use as a probe in Northern and dot blot analyses. Compared to the Bos taurus structural gene, the Bos indicus CDNA was found to begin 21 bases downstream from the transcription initiation site and had only two discrepancies (C to T at position 144-His and T to C at position 354-Phe), without changes in the polypeptide sequence. However, two amino acid substitutions were found for Bubalus spp., which belong to the same tribe. The rbst treatment did not change any of the characteristics evaluated (body and pituitary gland weights, growth hormone MRNA expression level). Crossbred animals had significantly higher body weight and heavier pituitaries than Nelore cattle. Pituitary weight was proportional to body weight in both breed groups. Growth hormone MRNA expression in the pituitary was similar (P>0.075) for both breed and hormonal treatment groups, but was 31.9% higher in the pure Nelore group, suggesting that growth hormone gene transcription regulation differs among these breeds.

Cloning and characterization of the cDNA for the Brazilian Cratomorphus distinctus larval firefly luciferase: Similarities with European Lampyris noctiluca and Asiatic Pyrocoelia luciferases

Studies on firefly (Lampyridae) luciferases have focused on nearctic species of Photinus and Photuris and Euroasiatic species of Lampyris, Luciola, Hotaria, and Pyrocoelia. Despite accounting for the greatest diversity of fireflies in the world, no molecular studies have been carried out on the highly diverse genera from the neotropical region. Here we report the luciferase cDNA cloning for the larva of the Brazilian firefly Cratomorphus distinctus. The cDNA has 1978 bp and codes for a 547-residue-long polypeptide. Noteworthy, sequence comparison as well as functional properties show the highest degree of similarity with Lampyris noctiluca (93%) and Pyrocoelia spp. (91%) luciferases, suggesting a close phylogenetic relationship despite the geographical distance separating these species. The bioluminescence emission spectrum peaks at 550 nm and, as expected, is sensitive to pH, shifting to 605 nm at pH 6. The kinetic properties of the recombinant luciferase were similar to those of other firefly luciferases. © 2004 Elsevier Inc. All rights reserved.

Vertical migration during tidal transport of megalopae of Necora puber in coastal shallow waters during daytime

For meroplanktonic larvae that must settle in coastal areas, their successful return to the shore is determined largely by physical transport processes; however, many organisms perform vertical movements to aid successful recruitment. In this study, daytime tidal vertical migration of megalopae of the velvet swimming crab Necora puber was investigated across two different exposures in the shallow waters of Plymouth Sound. Crabs were collected using a plankton net at the surface and near the bottom during flood and ebb tides. Distribution of the pelagic postlarvae was patchy and the abundance varied spatially in tens and thousands of metres. In temporal scales, the annual pattern was dominated by low occurrence of megalopae, punctuated by episodic peaks of high abundance. Most megalopae were collected at the surface irrespective of the tidal phase. The effect of wave exposure on the vertical migration of megalopae was not clear, although there was a general higher abundance of megalopae on exposed shores. Daytime abundance in the water column appears to be regulated by the tidal cycle, as megalopae were more abundant during flood than ebb tides. Although the megalopae do not appear to make large vertical migrations, this behaviour should produce a net shoreward transport. © 2005 Elsevier Ltd. All rights reserved.

Description of electrophoretic and chromatographic hemoglobin profile of Rhinoclemmys punctularia

Studies of the hemoglobin pattern in Brazilian reptiles are important for determining ecological and phylogenetic relationships, but they are scarce. Peripheral blood samples were obtained from 7 males and 18 females of Rhinoclemmys punctularia. The hematological profile was based on the total hemoglobin and hematocrit values. The hemoglobin profile was obtained using electrophoretic procedures at different pH, isoelectric focusing, globin chain electrophoresis, and HPLC. The hematocrit (31 ± 2%) and total hemoglobin (7.5 ± 0.2 g/dL) values did not indicate gender variations. Alkaline pH electrophoresis of the total blood samples treated with 1% saponin demonstrated the presence of four well-defined hemoglobin fractions, one major component (fraction I), showing cathodic migration and three others faster than fraction I with anodic migration. When the samples were precipitated with chloroform, only two hemoglobin fractions were observed, similar to fractions I and III from the first procedure. Isoelectric focusing and HPLC showed the same pattern. With acid and neutral pH electrophoresis, two fractions with anodic migration were observed. The globin chain identification at alkaline pH showed two fractions, but four fractions were observed at acidic pH, suggesting that different polypeptide chains are involved in the hemoglobin molecule. The chromatographic separation of the total blood sample demonstrated that the major fraction comprised 81.9% and the minor 18.1%. The results obtained demonstrated a similarity between these hemoglobin components and those of some Chelidae reported in the literature for both land and aquatic animals, reflecting the adaptation to environmental conditions. ©FUNPEC-RP.

Susceptibility/resistance of Anticarsia gemmatalis larvae to its nucleopolyhedrovirus (AgMNPV): Structural study of the peritrophic membrane

This investigation compares the peritrophic membrane (PM) morphology along the midgut of susceptible (SL) and resistant (RL) Anticarsia gemmatalis larvae to the AgMNPV. The PM increased the thickness from the anterior to the posterior midgut region in both insects strain; however, the intensity of FITC-WGA reaction of the PM in the RL were greater than in SL. The PM in RL was ultrastructurally constituted by several layers of fibrous/vesicular materials in comparison with the few ones in SL. Our results showed that the structure of PM in the RL could be one of the resistance barriers to AgMNPV. © 2007.

Spatio-temporal distribution of fatty acid-binding protein 6 (fabp6) gene transcripts in the developing and adult zebrafish (Danio rerio)

We have determined the structure of the fatty acid-binding protein 6 (fabp6) gene and the tissue-specific distribution of its transcripts in embryos, larvae and adult zebrafish (Danio rerio). Like most members of the vertebrate FABP multigene family, the zebrafish fabp6 gene contains four exons separated by three introns. The coding region of the gene and expressed sequence tags code for a polypeptide of 131 amino acids (14 kDa, pI 6.59). The putative zebrafish Fabp6 protein shared greatest sequence identity with human FABP6 (55.3%) compared to other orthologous mammalian FABPs and paralogous zebrafish Fabps. Phylogenetic analysis showed that the zebrafish Fabp6 formed a distinct clade with the mammalian FABP6s. The zebrafish fabp6 gene was assigned to linkage group (chromosome) 21 by radiation hybrid mapping. Conserved gene synteny was evident between the zebrafish fabp6 gene on chromosome 21 and the FABP6/Fabp6 genes on human chromosome 5, rat chromosome 10 and mouse chromosome 11. Zebrafish fabp6 transcripts were first detected in the distal region of the intestine of embryos at 72 h postfertilization. This spatial distribution remained constant to 7-day-old larvae, the last stage assayed during larval development. In adult zebrafish, fabp6 transcripts were detected by RT-PCR in RNA extracted from liver, heart, intestine, ovary and kidney (most likely adrenal tissue), but not in RNA from skin, brain, gill, eye or muscle. In situ hybridization of a fabp6 riboprobe to adult zebrafish sections revealed intense hybridization signals in the adrenal homolog of the kidney and the distal region of the intestine, and to a lesser extent in ovary and liver, a transcript distribution that is similar, but not identical, to that seen for the mammalian FABP6/Fabp6 gene. © 2008 The Authors.

Morphological and molecular analysis of the collagen fibers in inflammatory process

Collagen makes up one third of the total protein in humans, being formed by the connection of three polypeptide chains arranged in a triple helix. This protein has fundamental importance in the formation of extracellular matrix of connective tissue. This study aimed to analyze the structural changes of collagen, which are resulting from inflammatory processes in oral mucosa, and to make the comparative analysis between the histopathology and the Raman spectra. The samples of tissues with inflammatory fibrous hyperplasia (IFH) and normal mucosa (NM) were evaluated by Raman Spectroscopy, hematoxylin-eosin and Massons trichrome stain. The histological analysis in both stains showed differences in collagen fibers, which was presented as thin fibers and arranged in parallel direction in NM and as collagen fibers are thick, mature and not organized, showing that these types of stain show morphological changes of collagen in IFH. The Raman Spectroscopy discriminate the groups of NM and IFH based on vibrational modes of proline, hydroxiproline and CH3, CH2. The histological stains only shows information from morphological data, and can be complemented by Raman spectra. This technique could demonstrate that inflammatory process caused some changes in collagen structure which is related to aminoacids such as proline and hidroxyproline. © 2011 SPIE-OSA.