Nucleotide sequence of 5S rDNA and localization of the ribosomal RNA genes to metaphase chromosomes of the Tilapiine cichlid fish, Oreochromis niloticus

In this study, we report the cloning and nucleotide sequence of PCR-generated 5S rDNA from the Tilapiine cichlid fish, Oreochromis niloticus. Two types of 5S rDNA were detected that differed by insertions and/or deletions and base substitutions within the non-transcribed spacer (NTS). Two 5S rDNA loci were observed by fluorescent in situ hybridization (FISH) in metaphase spreads of tilapia chromosomes. FISH using an 18S rDNA probe and silver nitrate sequential staining of 5S-FISH slides showed three 18S rDNA loci that are not syntenic to the 5S rDNA loci.

Genetic polymorphism, molecular characterization and relatedness of Macrobrachium species (Palaemonidae) based on RAPD-PCR.

The prawn genus Macrobrachium belongs to the family Palaemonidae. Its species are widely distributed in lakes, reservoirs, floodplains, and rivers in tropical and subtropical regions of South America. Globally, the genus Macrobrachium includes nearly 210 known species, many of which have economic and ecological importance. We analyzed three species of this genus (M. jelskii, M. amazonicum and M. brasiliense) using RAPD-PCR to assess their genetic variability, genetic structure and the phylogenetic relationship between them and to look for molecular markers that enable separation of M. jelskii and M. amazonicum, which are closely related syntopic species. Ten different random decamer primers were used for DNA amplification, yielding 182 fragments. Three of these fragments were monomorphic and exclusive to M. amazonicum or M. jelskii and can be used as specific molecular markers to identify and separate these two species. Similarity indices and a phylogenetic tree showed that M. amazonicum and M. jelskii are closest to each other, while M. brasiliense was the most differentiated species among them; this may be attributed to the different habitat conditions to which these species have been submitted. This information will be useful for further studies on these important crustacean species.

Evaluation of HPFH and δβ-thalassemia mutations in a Brazilian group with high Hb F levels.

Fetal hemoglobin (Hb F) is characteristic of the fetal development period. However, in some genetic conditions, such as hereditary persistence of fetal hemoglobin (HPFH) and delta-beta thalassemia (δβ-thalassemia), Hb F continues to be produced in adulthood. We evaluated the frequency of two mutations of HPFH, HPFH-1 and HPFH-2 African, and two mutations in δβ-thalassemia, Sicilian and Spanish, in a Brazilian population. Peripheral blood samples were collected from adults from hospitals and blood centers in southeast and northeast Brazil. These individuals were healthy and without complaints of anemia, but had increased Hb F. Samples were submitted to electrophoretic and chromatographic analyses to quantify Hb F values and, subsequently, to molecular analyses to verify the mutations. In the molecular analysis, 16 of the 60 samples showed a heterozygous profile for the HPFH mutations, two for HPFH-1 and 14 for HPFH-2. In the same sample set, three were heterozygous for Spanish δβ-thalassemia and none were heterozygous for Sicilian δβ- thalassemia. The Hb F values in the HPFH-2 heterozygotes differed from those previously reported for this mutation. In this group, the HPFH mutations were more frequent than the δβ-thalassemia mutations. The finding of these mutations in this Brazilian population reflects the mixing process that occurred during its formation.

Proteinograma do soro lácteo de ovelhas da raça santa inês em diferentes fases de lactação

This study aimed to evaluate the influence of lactation phases on the proteinogram of whey protein in Santa Inês ewes. Ewes were accompanied in a semi-intensive system using the same sanitary and nutritional management evaluated at 15, 30, 60 and 90 days postpartum (end of weaning and lactation). Clinical examination of the mammary gland was carried out through and bacteriological culture. The screening of the material resulted in 44 milk samples of healthy glands concurrent negative by CMT and bacteriological culture exam. For obtaining the whey protein renin solution was used. The whey was fractionated into aliquots and kept in the -80C freezer to later separation of protein fractions. For determination of total protein of whey protein was employed the biuret, observing the linearity of the test. Separation of protein fractions was performed, using polyacrylamide gel containing sodium dodecyl sulfate (SDS-PAGE). Eigth protein were observed including lactoferrin, serum albumin, IgA, IgG (heavy chain IgG (IgG CP), light chain IgG (IgG CL), ß-lactoglobulin, a-lactalbumin and proteins identified as PM 15000 and PM 29000. No significant difference was observed at different stages of lactation in the following protein: IgA (P>0.3895), lactoferrin (P>0.1611), PM 29000 (P>0.4879), α-lactalbumin (P>0.0799) and PM15000 (P>0.4494). In total protein (P<0.0022), albumin protein (P<0.0377) and IgG (P<0.0354) it was observed a significant variation in the first moments of observations, in the ß-lactoglobulin protein (P<0.0005) there was significant variation with reduction of 15 to 30 days postpartum with progressive elevation until the last stage of lactation (90 days postpartum). The SDS-PAGE technique allowed the quantification of eigth whey proteins in health ewes. The protein fractions identified reflect the profile of whey to ovine species, with influence of stages of lactation in albumin, IgG and ß-lactoglobulin.

Diarréia em bezerros da raça Nelore criados extensivamente: estudo clínico e etiológico

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Estudo da expressão gênica entre cultivares de cana-de-açúcar contrastantes para a tolerância à seca

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Determinação de Cd, Cu, Ni, Pb e Zn em águas marinhas oriundas da Bacia Potiguar utilizando a técnica de difusão em filmes finos por gradiente de concentração (DGT)

The DGT technique has been used to determine and better understand the dynamics and bioavailability of metals in the sea near to marine outfalls. In this work, the DGT was assessed for the determination of Cd, Pb, Cu, Zn and Ni in marine water samples from Potiguar Basin in its conventional aspect (binding agent, Chelex-100 and diffusive agent: Diffusive polyacrylamide gel (D) and restrictive (R)) and determination of Pb and Cd through its alternative aspect (binding agent: Saccharomyces cerevisiae immobilized in agarose gels and diffusive agent: 3MM Chromatography Paper). The deployment curves for long periods of immersion in the sample, showed a good linear correlation for the conventional aspect to Zn (D: R²=0.9586, R: R²=0.9444), Ni (D: R²=0.9789, R: R²=0.9286) and Cu (D: R²=0.764, R: R²=0.8143), and alternative to Pb (R²=0.9228) and Cd (R²=0.9673). The results of the organic and inorganic composition analysis in conventional aspect, showed that for every element there is no significant fraction of organic compounds in the sample. In addition, considering the alternative aspect, the comparison of obtained and expected masses suggests that some of the Pb is not labile and that the main retention mechanism of Pb for S. Cerevisiae occurs through ion exchange

Avaliação da membrana P81 como agente ligante da técnica de difusão em filmes finos por gradiente de concentração (DGT) para determinação de mercúrio em sistemas aquáticos

Worldwide environmental degradation is an undesirable byproduct resulting from the increasing demand for natural resources. Water sources are suffering intense contamination since they usually receive a huge amount of domestic and industrial effluents - which are mostly wasted without proper treatment - inserting a large number of pollutants in the environment, heavy metals included. Mercury holds great toxicological importance because, under some physicochemical conditions in a water environment, Hg (II) ion turns into methylated compounds stemming from this element, such as methylmercury CH3Hg, which is highly toxic for the aquatic community in which bioaccumulation occurs. Nowadays passive sampling techniques are being developed to enable the analytical procedures which are applied in environmental monitoring. Diffusive gradients in thin-films technique (DGT) has been proven an interesting tool for the determination of labile metal species due to its in situ application. The DGT technique consists of a piston-like device on which the following series of agents is disposed: a binding agent (conventionally Chelex 100 resin), a diffusive agent, usually a polyacrylamide gel, and a membrane filter. Nevertheless, the agents conventinally used for this technique don't usually show satisfactory results in mercury sampling. The main goal of this study was to evaluate the phosphate-treated cellulose membrane (Whatman P 81), an alternative material, as binding agent in the DGT to determine labile mercury fractions in aquatic systems. In this context, we conducted a study of the behavior of this material in relation with system variables, pH and ionic strength. Afterwards we performed immersions of the DGT devices in real and enriched samples and in situ aiming the determination of mercury

Characterization of the hemoglobins and globins of Synbranchus marmoratus Bloch, 1795 (Pisces, Synbranchidae)

1. 1. Total hemolysates of Synbranchus marmoratus Bloch, 1795, captured in Vitoriana, district of Botucatu, State of São Paulo, Brazil, were submitted to agar-starch gel electrophoresis on glass slides using 42 mM-Tris 1.7 mM EDTA-6.1 mM borate buffer, pH 8.8, for the gel and 10 mM borate-1.7 mM NaOH buffer, pH 8.6, for the cuvette. 2. 2. Three distinct hemoglobin bands were detected, with Hb I being of the cathodic type. 3. 3. Cellulose acetate electrophoresis in 800 mM Tris-2.1 mM EDTA buffer, pH 8.9, containing 6 M urea and 2.25 mM β-mercaptoethanol indicated the presence of four globin chains denoted α 1, α 2, β and γ. 4. 4. It is suggested that the probable tetrameric constitution of the hemoglobin of Synbranchus marmoratus Bloch, 1795 is Hb I (α 2 2γ 2), Hb II (α 2 1γ 2) and Hb III (α 2 1β 2). © 1986.

Proteomic and peptidomic study of proteolysis in quarter milk after infusion with lipoteichoic acid from Staphylococcus aureus

Mastitic milk is associated with increased bovine protease activity, such as that from plasmin and somatic cell enzymes, which cause proteolysis of the caseins and may reduce cheese yield and quality. The aim of this work was to characterize the peptide profile resulting from proteolysis in a model mastitis system and to identify the proteases responsible. One quarter of each of 2 cows (A and B) was infused with lipoteichoic acid from Staphylococcus aureus. The somatic cell counts of the infused quarters reached a peak 6h after infusion, whereas plasmin activity of those quarters also increased, reaching a peak after 48 and 12h for cow A and B, respectively. Urea-polyacrylamide gel electrophoretograms of milk samples of cow A and B obtained at different time points after infusion and incubated for up to 7 d showed almost full hydrolysis of beta- and alpha(S1)-casein during incubation of milk samples at peak somatic cell counts, with that of beta-casein being faster than that of alpha(S1)-casein. Two-dimensional gel electrophoretograms of milk 6h after infusion with the toxin confirmed hydrolysis of beta- and alpha(S1)-casein and the appearance of lower-molecular-weight products. Peptides were subsequently separated by reversed-phase HPLC and handmade nanoscale C(18) columns, and identified by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. Twenty different peptides were identified and shown to originate from alpha(s1)- and beta-casein. Plasmin, cathepsin B and D, elastase, and amino- and carboxypeptidases were suggested as possible responsible proteases based on the peptide cleavage sites. The presumptive activity of amino- and carboxypeptidases is surprising and may indicate the activity of cathepsin H, which has not been reported in milk previously.

Stejnulxin, a novel snake C-type lectin-like protein from Trimeresurus stejnegeri venom is a potent platelet agonist acting specifically via GPVI

Stejnulxin, a novel snake C-type lectin-like protein with potent platelet activating activity, was purified and characterized from Trimeresurus stejnegeri venom. Under non-reducing conditions, it migrated on a SDS-polyacrylamide gel with an apparent molecular mass of 120 kDa. On reduction, it separated into three polypeptide subunits with apparent molecular masses of 16 kDa (alpha), 20 kDa (beta1) and 22 kDa (beta2), respectively. The complete amino acid sequences of its subunits were deduced from cloned cDNAs. The N-terminal sequencing and cDNA cloning indicated that beta1 and beta2 subunits of stejnulxin have identical amino acid sequences and each contains two N-glycosylation sites. Accordingly, the molecular mass difference between beta1 and beta2 is caused by glycosylation heterogenity. The subunit amino acid sequences of stejnulxin are similar to those of convulxin, with sequence identities of 52.6% and 66.4% for the alpha and beta, respectively. Stejnulxin induced human platelet aggregation in a dose-dependent manner. Antibodies against alphaIIbbeta3 inhibited the aggregation response to stejnulxin, indicating that activation of alphaIIbbeta3 and binding of fibrinogen are involved in stejnulxin-induced platelet aggregation. Antibodies against GPIbalpha or alpha2beta1 as well as echicetin or rhodocetin had no significant effect on stejnulxin-induced platelet aggregation. However, platelet activation induced by stejnulxin was blocked by anti-GPVI antibodies. In addition, stejnulxin induced a tyrosine phosphorylation profile in platelets that resembled that produced by convulxin. Biotinylated stejnulxin bound specifically to platelet membrane GPVI.

A 55-kDa protein isolated from human cells shows DNA glycosylase activity toward 3,N4-ethenocytosine and the G/T mismatch

Etheno adducts in DNA arise from multiple endogenous and exogenous sources. Of these adducts we have reported that, 1,N6-ethenoadenine (ɛA) and 3,N4-ethenocytosine (ɛC) are removed from DNA by two separate DNA glycosylases. We later confirmed these results by using a gene knockout mouse lacking alkylpurine-DNA-N-glycosylase, which excises ɛA. The present work is directed toward identifying and purifying the human glycosylase activity releasing ɛC. HeLa cells were subjected to multiple steps of column chromatography, including two ɛC-DNA affinity columns, which resulted in >1,000-fold purification. Isolation and renaturation of the protein from SDS/polyacrylamide gel showed that the ɛC activity resides in a 55-kDa polypeptide. This apparent molecular mass is approximately the same as reported for the human G/T mismatch thymine-DNA glycosylase. This latter activity copurified to the final column step and was present in the isolated protein band having ɛC-DNA glycosylase activity. In addition, oligonucleotides containing ɛC⋅G or G/T(U), could compete for ɛC protein binding, further indicating that the ɛC-DNA glycosylase is specific for both types of substrates in recognition. The same substrate specificity for ɛC also was observed in a recombinant G/T mismatch DNA glycosylase from the thermophilic bacterium, Methanobacterium thermoautotrophicum THF.

A terbenzimidazole that preferentially binds and conformationally alters structurally distinct DNA duplex domains: A potential mechanism for topoisomerase I poisoning

The terbenzimidazoles are a class of synthetic ligands that poison the human topoisomerase I (TOP1) enzyme and promote cancer cell death. It has been proposed that drugs of this class act as TOP1 poisons by binding to the minor groove of the DNA substrate of TOP1 and altering its structure in a manner that results in enzyme-mediated DNA cleavage. To test this hypothesis, we characterize and compare the binding properties of a 5-phenylterbenzimidazole derivative (5PTB) to the d(GA4T4C)2 and d(GT4A4C)2 duplexes. The d(GA4T4C)2 duplex contains an uninterrupted 8-bp A⋅T domain, which, on the basis of x-ray crystallographic data, should induce a highly hydrated “A-tract” conformation. This duplex also exhibits anomalously slow migration in a polyacrylamide gel, a feature characteristic of a noncanonical global conformational state frequently described as “bent.” By contrast, the d(GT4A4C)2 duplex contains two 4-bp A⋅T tracts separated by a TpA dinucleotide step, which should induce a less hydrated “B-like” conformation. This duplex also migrates normally in a polyacrylamide gel, a feature further characteristic of a global, canonical B-form duplex. Our data reveal that, at 20°C, 5PTB exhibits an ≈2.3 kcal/mol greater affinity for the d(GA4T4C)2 duplex than for the d(GT4A4C)2 duplex. Significantly, we find this sequence/conformational binding specificity of 5PTB to be entropic in origin, an observation consistent with a greater degree of drug binding-induced dehydration of the more solvated d(GA4T4C)2 duplex. By contrast with the differential duplex affinity exhibited by 5PTB, netropsin and 4′,6-diamidino-2-phenylindole (DAPI), two AT-specific minor groove binding ligands that are inactive as human TOP1 poisons, bind to both duplexes with similar affinities. The electrophoretic behaviors of the ligand-free and ligand-bound duplexes are consistent with 5PTB-induced bending and/or unwinding of both duplexes, which, for the d(GA4T4C)2 duplex, is synergistic with the endogenous sequence-directed electrophoretic properties of the ligand-free duplex state. By contrast, the binding to either duplex of netropsin or DAPI induces little or no change in the electrophoretic mobilities of the duplexes. Our results demonstrate that the TOP1 poison 5PTB binds differentially to and alters the structures of the two duplexes, in contrast to netropsin and DAPI, which bind with similar affinities to the two duplexes and do not significantly alter their structures. These results are consistent with a mechanism for TOP1 poisoning in which drugs such as 5PTB differentially target conformationally distinct DNA sites and induce structural changes that promote enzyme-mediated DNA cleavage.

Cotranslational folding and calnexin binding during glycoprotein synthesis.

To analyze cotranslational folding of influenza hemagglutinin in the endoplasmic reticulum of live cells, we used short pulses of radiolabeling followed by immunoprecipitation and analysis with a two-dimensional SDS/polyacrylamide gel system which was nonreducing in the first dimension and reducing in the second. It separated nascent glycopolypeptides of different length and oxidation state. Evidence was obtained for cotranslational disulfide formation, generation of conformational epitopes, N-linked glycosylation, and oligosaccharide-dependent binding of calnexin, a membrane-bound chaperone that binds to incompletely folded glycoproteins via partially glucose-trimmed oligosaccharides. When glycosylation or oligosaccharide trimming was inhibited, the folding pathway was perturbed, suggesting a role for N-linked oligosaccharides and calnexin during translation of hemagglutinin.

Particle flux characterisation and sedimentation patterns of protistan plankton during the iron fertilisation experiment LOHAFEX in the Southern Ocean

The taxonomic composition and types of particles comprising the downward particle flux were examined during the mesoscale artificial iron fertilisation experiment LOHAFEX. The experiment was conducted in low-silicate waters of the Atlantic Sector of the Southern Ocean during austral summer (January-March 2009), and induced a bloom dominated by small flagellates. Downward particle flux was low throughout the experiment, and not enhanced by addition of iron; neutrally buoyant sediment traps contained mostly faecal pellets and faecal material apparently reprocessed by mesozooplankton. TEP fluxes were low, <5 mg GX eq/m**2/day, and a few phytodetrital aggregates were found in the sediment traps. Only a few per cent of the POC flux was found in the traps consisting of intact protist plankton, although remains of taxa with hard body parts (diatoms, tintinnids, thecate dinoflagellates and foraminifera) were numerous, far more so than intact specimens of these taxa. Nevertheless, many small flagellates and coccoid cells, belonging to the pico- and nanoplankton, were found in the traps, and these small, soft-bodied cells probably contributed the majority of downward POC flux via mesozooplankton grazing and faecal pellet export. TEP likely played an important role by aggregating these small cells, and making them more readily available to mesozooplankton grazers.