The antibiotics roseoflavin and 8-demethyl-8-amino-riboflavin from Streptomyces davawensis are metabolized by human flavokinase and human FAD synthetase

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Molecular adaptability of nucleoside diphosphate kinase b from trypanosomatid parasites: stability, oligomerization and structural determinants of nucleotide binding

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Production, purification, crystallization and preliminary X-ray diffraction studies of the nucleoside diphosphate kinase b from Leishmania major

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Structural bioinformatics study of cyclin-dependent kinases complexed with inhibitors

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Caracterização da região 3'-terminal do genoma de um isolado brasileiro do Southern bean mosaic virus

O presente trabalho caracteriza a região 3'-terminal do genoma de um isolado do Southern bean mosaic virus encontrado no Estado de São Paulo (SBMV-SP). O RNA foi extraído de partículas virais purificadas e submetido a RT-PCR usando oligonucleotídeos desenhados para amplificar 972 nt da região 3'-terminal do RNA viral. Foi obtido fragmento de tamanho esperado que inclui o gene da proteína capsidial e a região 3'-terminal não codificadora. O gene da proteína capsidial (ORF4) contém 801 nucleotídeos, incluindo-se o códon de terminação UGA, com seqüência deduzida de 266 aminoácidos e massa molecular estimada de 28.800 Da. Sessenta e um aminoácidos terminais da ORF2 estão sobrepostos na ORF4. O sinal de localização nuclear, encontrado dentro do Domínio R na região 5'-terminal da ORF4 de alguns sobemovírus, não foi identificado no SBMV-SP. Esse dado pode explicar a ausência de partículas virais do SBMV-SP no núcleo celular. A seqüência do SBMV-SP apresentou identidade de nucleotídeos e aminoácidos de, respectivamente, 91% e 93% com o isolado Arkansas (SBMV-ARK) descrito nos EUA. Os resultados obtidos indicam que o SBMV-SP e o SBMV-ARK são isolados muito proximamente relacionados.

A degenerate primer allows amplification of part of the 3 '-terminus of three distinct carlavirus species

Sequences of the coat protein amino acids of definitive and tentative species of carlaviruses deposited in GenBank were aligned and a region of seven amino acids (GLGVPTE) was found to be conserved. The corresponding nucleotides were aligned, allowing the design of a degenerate primer that together with an oligo dT anti-sense primer, was effective for the detection of three distinct carlavirus species, two transmitted by aphids and one by whitefly. These primers have the advantage that about 940 nt from the 3'-terminus, comprising part of the CP gene (about 60%), the 11 K gene, and the terminal untranslated region can be amplified for sequencing. The fact that this amino acid sequence is conserved in almost all of the sequenced carlaviruses, allows the prediction that this primer pair will be useful as a diagnostic tool for carlavirus species. (C) 2007 Elsevier B.V. All rights reserved.

Caracterização do gene da proteína capsidial de dois isolados, patologicamente distintos e sorologicamente semelhantes, do Grapevine virus B em videiras no Estado de São Paulo

No presente trabalho, descreve-se a caracterização do gene codificador da proteína capsidial de dois isolados sintomatologicamente distintos do Grapevine virus B (GVB). Para isto, RNA totais foram extraídos de folhas e pecíolos de videiras (Vitis spp.) infetadas, cultivares Rubi (GVB-C SP) e Itália (GVB-I SP) e utilizados para amplificar, por RT/PCR, um fragmento entre as posições 6425 e 7118 (694 nucleotídeos, nt) do RNA do GVB (GenBank, acesso X75448). O fragmento obtido inclui o gene da proteína capsidial (594 nt) codificando 197 aminoácidos com massa molecular estimada em aproximadamente 21.600 Da. A seqüência do GVB-C SP apresentou maior similaridade de nucleotídeos e aminoácidos deduzidos com o isolado italiano (acesso X75448), enquanto que o GVB-I SP foi mais similar a um outro isolado brasileiro do GVB descrito no Rio Grande do Sul (GVB BR1, acesso AF438410). Os dois isolados paulistas do GVB podem ser diferenciados por digestão com a enzima de restrição EcoRI, uma vez que há um sítio interno no GVB-C SP que está ausente no isolado GVB-I SP.

Ribonuclease production by Aspergillus species

Ribonuclease production by Aspergillus flavipes. A sulphureus and A. fischeri in semisynthetic medium, after 24-144 hours at 30 degrees C under shaking, was studied. After cultivation, the medium was separated from micelia by filtration and the resultant solution was used as enzymatic extract. The highest amount of biomass and RNase was obtained after 96 hours of cultivation. The enzymes produced by three species presented similar characteristics, with optimum temperature at 55 degrees C and two peaks of activity at pH 4.5 and 7.0. A. flavipes RNases were more sensitive to temperature: 50% of the initial activity was lost after 1 hour at 70 degrees C. After this heat treatment, RNase of A. sulphureus lost 30% of this activity and that of A. fischeri only 16%. The nucleotides released by enzimatic hydrolysis of RNA were separated by ion exchange chromatography in a AG-1X8-formiate column and identified by paper chromatography. This procedure indicated that the raw enzymatic extract of Aspergillus flavipes is able to hydrolyze RNA, releasing 3'-nucleotides monophosphate at pH 4.5 and 3' and 5'-nucleotides monophosphate at pH 7.0 and 8.5. This result suggests that this strain produces two different types of RNase, one acidic and other alcaline, with different specificities.

Improvement in RNA extraction from S. cerevisie by optimization in the autolysis and NH3 hydrolysis

The optimization of autolysis of Saccharomyces cerevisiae from brewery was studied aiming at the maximum ribonucleic acid extraction and yeast extract production. The best conditions for yeast autolysis was 55.2ºC, pH= 5.1 and 9.8% NaCl for 24h of processing, without the NH3 use. In these conditions, the RNA yield was 89.7%, resulting in 51.3% of dehydrated yeast extract with 57.9% protein. The use of 12.2% NH3 at 60ºC after autolysis (8h) and plasmolysis (8h) was not viable due to the reduction in the RNA yield from 89.7to78.4%. on the other hand, the thermal shock at 60ºC for 15 minutes prior to autolysis provided an increase in the yield from 89.7 to 91.4%. The autolysis, including NaCl plasmolysis in the optimized conditions was efficient, economic and with short time, thus usable for industrial purpose to obtain more valuable products such as yeast extract enriched in RNA and/or protein, for different applications.

Nitrogen assimilation in Citrus based on CitEST data mining

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Adsorptive potentiometric stripping analysis of nucleic acids at mercury electrodes

The application of adsorptive stripping potentiometry to the reductive detection of nucleic acids at mercury electrodes is reported. Compared to analogous voltammetric stripping modes, constant current potentiometric stripping analysis (PSA) effectively addresses the hydrogen discharge background problem, and hence greatly improves the characteristics of the superimposed cytosine/adenine (CA) reduction peak. Compared to earlier schemes for trace measurements of nucleic acids at mercury or carbon electrodes that rely on anodic signals arising from the guanine residue, convenient quantitation can now be carried out in connection with the cytosine and adenine residues. Variables influencing the adsorptive PSA response are explored and optimized. With five minute accumulation, the detection limits for tRNA, ssDNA and dsDNA are 30 mu g l(-1), 60 mu g l(-1) and 2 mg l(-1), respectively. Such different values reflect the strong dependence of the PSA CA signal upon the nucleic-acid structure. This allows the quantitation of ssDNA or tRNA in the presence of dsDNA, and offers new possibilities for electrochemical studies of DNA structure and interactions.

Effects of nandrolone decanoate on the neuromuscular junction of rats submitted to swimming

This study addressed the effects of nandrolone decanoate (ND) on contractile properties and muscle fiber characteristics of rats submitted to swimming. Male Wistar rats were grouped in sedentary (S), swimming (Sw), sedentary+ND (SND), and swimming+ND (SwND), six animals per group. ND (3 mg/kg) was injected (subcutaneously) 5 days/week, for 4 weeks. Swimming consisted of 60-min sessions (load 2%), 5 days/week, for 4 weeks. After this period, the sciatic nerve extensor digitorum longus (EDL) muscle was isolated for myographic recordings. Fatigue resistance was assessed by the percent (%) decline of 180 direct tetanic contractions (30 Hz). Safety margin of synaptic transmission was determined from the resistance to the blockade of indirectly evoked twitches (0.5 Hz) induced by pancuronium (5 to 9 x 10(-7) M). EDL muscles were also submitted to histological and histochemical analysis (haematoxylin-eosin (HE); nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR)). Significant differences were detected by two-way ANOVA (p<0.05). ND did not change body mass, fatigue resistance or kinetic properties of indirect twitches in either sedentary or swimming rats. In contrast, ND reduced the safety margin of synaptic transmission in sedentary animals (SND=53.3+/-4.7% vs. S=75.7+/-2.0%), but did not affect the safety margin in the swimming rats (SwND=75.81+/-3.1% vs. Sw=71.0+/-4.0%). No significant difference in fiber type proportions or diameters was observed in EDL muscle of any experimental group. These results indicate that ND does not act as an ergogenic reinforcement in rats submitted to 4 weeks of swimming. on the other hand, this study revealed an important toxic effect of ND, that it reduces the safety margin of synaptic transmission in sedentary animals. Such an effect is masked when associated with physical exercise. (C) 2004 Elsevier B.V. All rights reserved.

Kinetic aspects of rat (Rattus-norvegicus albinus) submandibular glands lactic-dehydrogenase

Submandibular glands of male rats were homogenized with 33 mM sodium potassium phosphate buffer, pH 6.5, containing 1 mM MgCl2 and 0.1 mM DTT and purified with ammonium sulphate, phosphocellulose chromatography, eluted with KC1 0.5 M, followed by Blue Sepharose CL-6B chromatography, eluted with NADH 0.5 mM. The enzyme kepts stable for 60 days when stored at -15-degrees-C in 33 mM phosphate buffer. In other experiment the enzyme was purified by oxamate-agarose chromatography from a crude extract of submandibular gland and the results obtained were better than by phosphocellulose and Sepharose CL-6B chromatography. The Km values for pyruvate. NADH, lactate and NAD+ were established. Sodium oxamate at 0.1 and 0.9 mM concentrations inhibited the LDH activity by 40 and 85%, respectively (competitive); with sodium oxalate the inhibition was of 30% (uncompetitive) and with 3-acetyl pyridine adenine dinucleotide was 80%.

Identification and characterization of novel mutations of the aspartoacylase gene in non-Jewish patients with Canavan disease

Canavan disease, an inherited leukodystrophy, is caused by mutations in the aspartoacylase (ASPA) gene. It is most common among children of Ashkenazi Jewish descent but has been diagnosed in many diverse ethnic groups. Two mutations comprise the majority of mutant alleles in Jewish patients, while mutations in the ASPA gene among non-Jewish patients are different and more diverse. In the present study, the ASPA gene was analysed in 22 unrelated non-Jewish patients with Canavan disease, and 24 different mutations were found. of these,14 are novel, including five missense mutations (E24G, D68A, D249V, C152W, H244R), two nonsense mutations (Q184X, E214X), three deletions (923delT, 33del13, 244delA), one insertion mutation (698insC), two sequence variations in one allele ([10T>G; 11insG]), an elimination of the stop codon (941A>G, TAG-->TGG, X314W), and one splice acceptor site mutation (IVS1 - 2A>T). The E24G mutation resulted in substitution of an invariable amino acid residue (Glu) in the first esterase catalytic domain consensus sequence. The IVS1 - 2A>T mutation caused the retention of 40 nucleotides of intron 1 upstream of exon 2. The results of transient expression of the mutant ASPA cDNA containing these mutations in COS-7 cells and assays for ASPA activity of patient fibroblasts indicated that these mutations were responsible for the enzyme deficiency. In addition, patients with the novel D249V mutation manifested clinically at birth and died early. Also, patients with certain other novel mutations, including C152W, E214X, X314W, and frameshift mutations in both alleles, developed clinical manifestations at an earlier age than in classical Canavan disease.

Morphometry and histochemistry of the semitendinosus muscle of tufted capuchin monkeys (Cebus apella Linnaeus, 1758)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)