Evidence supporting the recognition of Cole latent virus as a distinct carlavirus

Some molecular properties are described of Cole latent virus (CoLV), hitherto designated a tentative species of the Carlavirus genus. CoLV genomic RNA (Ribonucleic acid) of 8.3 Kb is polyadenylated. Two unencapsidated polyadenylated subgenomic RNAs (2.6 and 1.3 Kb) and three double-stranded RNAs (dsRNAs) (8.3, 2.6 and 1.3 Kbp), which are twice the size of the genomic and subgenomics ssRNAs, are produced in CoLV-infected plants, two additional dsRNAs (7.2 and 6.3 Kbp) were also detected plant extracts. By using a Carlavirus specific primer and a CoLV cDNA, a-3'-terminus fragment of 116 bp was amplified; it had homology with the carlaviruses Potato virus M (62%)., Hop latent virus (37%) and Blueberry scorch virus (36%) but no significant homology with 11 other carlaviruses. These results support the classification of CoLV as a distinct species of the Carlavirus genus.

Identification and characterization of a potyvirus isolated from siratro plants

The present work describes the identification and characterization of a potyvirus isolated from siratro (Macroptilium atropurpureum Urb.) in the north-west region of the State of Sdo Paulo, Brazil. The virus was transmitted by mechanical inoculation. Its host range was restricted mainly to members of the Fabaceae. A cDNA fragment of about 930 bp was amplified by RT/PCR, cloned and sequenced. The fragment, which included the coat protein gene, had amino acid identity percentages between 88 and 98% with isolates of Bean common mosaic virus (BCMV). Phylogenetic analysis grouped the. siratro potyvirus and BCMV isolates in 99% of the replicates, including Azuki mosaic virus, Dendrobium mosaic virus, Blackeye cowpea mosaic virus and Peanut stripe virus, which have been classified as BCMV strains. This is the first citation on the presence of BCMV in siratro plants in Brazil.

Detecção do Grapevine virus A e Grapevine virus B por hibridização dot-blot com sondas moleculares não radioativas

O vírus A da videira (Grapevine virus A, GVA) e o vírus B da videira (Grapevirus virus B, GVB) estão associados à acanaladura do lenho de Kober (Kober stem grooving) e ao fendilhamento cortical da videira (grapevine corky bark), respectivamente. Este trabalho descreve o uso de sondas moleculares de cDNA na detecção de isolados do GVA (GVA-SP) e do GVB (GVB-C-SP e GVB-I-SP) em videiras (Vitis spp.) e fumo (Nicotiana occidentalis). As sondas marcadas com digoxigenina foram produzidas por RT-PCR utilizando oligonucleotídeos específicos para os genes da proteína capsidial. Os RNA totais foram extraídos de 45 plantas de diversas variedades de videira e de 13 plantas de fumo inoculadas mecanicamente com o GVB. Os RNA extraídos das plantas infetadas, indexadas biologicamente, hibridizaram com as sondas, não se verificando reação com plantas sadias. Para confirmar os resultados de hibridização, foram também feitos testes de RT-PCR. A utilização de hibridização dot-blot com sondas de cDNA mostrou-se eficaz na detecção dos vírus com especificidade e sensibilidade, ressaltando-se que, preferencialmente, folhas maduras e ramos dormentes devem ser utilizados nos testes diagnósticos para o GVB e GVA, respectivamente.

Label-free DNA detection of hepatitis C virus based on modified conducting polypyrrole films at microelectrodes and atomic force microscopy tip-integrated electrodes

We present a new strategy for the label-free electrochemical detection of DNA hybridization for detecting hepatitis C virus based on electrostatic modulation of the ion-exchange kinetics of a polypyrrole film deposited at microelectrodes. Synthetic single-stranded 18-mer HCV genotype-1-specific probe DNA has been immobilized at a 2,5-bis(2-thienyl)-N-(3-phosphoryl-n-alkyl)pyrrole film established by electropolymerization at the previously formed polypyrrole layer. HCV DNA sequences (244-mer) resulting from the reverse transcriptase-linked polymerase chain reaction amplification of the original viral RNA were monitored by affecting the ion-exchange properties of the polypyrrole film. The performance of this miniaturized DNA sensor system was studied in respect to selectivity, sensitivity, and reproducibility. The limit of detection was determined at 1.82 x 10(-21) mol L-1. Control experiments were performed with cDNA from HCV genotypes 2a/c, 2b, and 3 and did not show any unspecific binding. Additionally, the influence of the spacer length of 2,5-bis(2-thienyl)-N-(3-phosphoryl-n-alkyl)pyrrole on the behavior of the DNA sensor was investigated. This biosensing scheme was finally extended to the electrochemical detection of DNA at submicrometer-sized DNA biosensors integrated into bifunctional atomic force scanning electrochemical microscopy probes. The 18-mer DNA target was again monitored by following the ion-exchange properties of the polypyrrole film. Control experiments were performed with 12-base pair mismatched sequences.

PR gene families of citrus: their organ specific-biotic and abiotic inducible expression profiles based on ESTs approach

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Phytophthora parasitica transcriptome, a new concept in the understanding of the citrus gummosis

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

Biochemical characterization of Neurospora crassa glycogenin (GNN), the self-glucosylating initiator of glycogen synthesis

Glycogenin acts in the initiation step of glycogen biosynthesis by catalyzing a self-glucosylation reaction. In a previous work [de Paula et al., Arch. Biochem. Biophys. 435 (2005) 112-124], we described the isolation of the cDNA gnn, which encodes the protein glycogenin (GNN) in Neurospora crassa. This work presents a set of biochemical and functional studies confirming the GNN role in glycogen biosynthesis. Kinetic experiments showed a very low GNN K-m (4.41 mu M) for the substrate UDP-glucose. Recombinant GNN was produced in Escherichia coli and analysis by mass spectroscopy identified a peptide containing an oligosaccharide chain attached to Tyr196 residue. Site-directed mutagenesis and functional complementation of a Saccharomyces cerevisiae mutant strain confirmed the participation of this residue in the GNN self-glucosylation and indicated the Tyr198 residue as an additional, although less active, glucosylation site. The physical interaction between GNN and glycogen synthase (GSN) was analyzed by the two-hybrid assay. While the entire GSN was required for full interaction, the C-terminus in GNN was more important. Furthermore, mutation in the GNN glucosylation sites did not impair the interaction with GSN. (c) 2005 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

Transcriptome profiling of Paracoccidioides brasiliensis yeast-phase cells recovered from infected mice brings new insights into fungal response upon host interaction

Paracoccidioides brasiliensis is a fungal human pathogen with a wide distribution in Latin America. It causes paracoccidioidomycosis, the most widespread systemic mycosis in Latin America. Although gene expression in P. brasiliensis had been studied, little is known about the genome sequences expressed by this species during the infection process. To better understand the infection process, 4934 expressed sequence tags (ESTs) derived from a non-normalized cDNA library from P. brasiliensis (isolate Pb01) yeast-phase cells recovered from the livers of infected mice were annotated and clustered to a UniGene (clusters containing sequences that represent a unique gene) set with 1602 members. A large-scale comparative analysis was performed between the UniGene sequences of P. brasiliensis yeast-phase cells recovered from infected mice and a database constructed with sequences of the yeast-phase and mycelium transcriptome (isolate Pb01) (https://dna.biomol.unb.br/Pb/), as well as with all public ESTs available at GenBank, including sequences of the P. brasiliensis yeast-phase transcriptome (isolate Pb18) (http:// www.ncbi.nlm.nih.gov/). The focus was on the overexpressed and novel genes. From the total, 3184 ESTs (64.53%) were also present in the previously described transcriptome of yeast-form and mycelium cells obtained from in vitro cultures (https://dna.biomol.unb.br/Pb/) and of those, 1172 ESTs (23.75% of the described sequences) represented transcripts overexpressed during the infection process. Comparative analysis identified 1750 ESTs (35.47% of the total), comprising 649 UniGene sequences representing novel transcripts of P. brasiliensis, not previously described for this isolate or for other isolates in public databases. KEGG pathway mapping showed that the novel and overexpressed transcripts represented standard metabolic pathways, including glycolysis, amino acid biosynthesis, lipid and sterol metabolism. The unique and divergent representation of transcripts in the cDNA library of yeast cells recovered from infected mice suggests differential gene expression in response to the host milieu.

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.

Structural and functional characterization of neuwiedase, a nonhemorrhagic fibrin(ogen)olytic metalloprotease from Bothrops neuwiedi snake venom

A fibrino(geno)lytic nonhemorrhagic metalloprotease (neuwiedase) was purified from Bothrops neuwiedi snake venom by a single chromatographic step procedure on a CM-Sepharose column, Neuwiedase represented 4.5% (w/w) of the crude desiccated venom, with an approximate Mr of 20,000 and pI 5.9, As regards the amino acid composition, neuwiedase showed similarities with other metalloproteases, with high proportions of Asx, Glx, Leu, and Ser, Atomic absorption spectroscopy showed that one mole of Zn2+ and one mole of Ca2+ were present per mole olf protein. The cDNA encoding neuwiedase was isolated by RT-PCR from venom gland RNA, using oligonucleotides based on the partially determined amino-acid sequences of this metalloprotease. The fall sequence contained approximately 594 bp, which codified the 198 amino acid residues with an estimated molecular weight of 22,375. Comparison of the nucleotide and amino acid sequences of neuwiedase with those of other snake venom metalloproteases showed a high level of sequential similarity, Neuwiedase has two highly conserved characteristics sequences H(142)E(143)XXH(146)XXG(140)XXH(152) and C164I165M166. The three-dimensional structure of neuwiedase was modeled based on the crystal structure of Crotalus adamanteus Adamalysin II. This model revealed that the zinc binding site region showed a I high structural similarity with other metalloproteases,, the proteolyitc specificity, using the B beta-chain of oxidized insulin as substrate, was shown to be directed to the Ala(14)-Leu(15) and Tyr(16)-Leu(17) peptide bonds which were preferentially hydrolyzed. Neuwiedase is a A alpha,B beta fibrinogenase, Its activity upon the A alpha chain of fibrinogen was detected within 15 min of incubation. The optimal temperature and pH for the degradation of both A alpha and B beta chains were 37 degrees C and 7.4-8.0, respectively. This activity was inhibited by EDTA and 1,10-phenantroline, Neuwiedase also showed proteolytic activity upon fibrin and some components of the extracellular matrix. However, it did not show TAME esterase activity and was not able to inhibit platelet aggregation. (C) 2000 Academic Press.

Molecular characterization of hemoglobin alpha-D chains from Geochelone carbonaria and Geochelone denticulata land turtles

In order to help elucidate the evolution of alpha-globins, the complete cDNA and amino acid sequences of Geochelone carbonaria and Geochelone denticulata land turtles alpha-D chains have been described. In G. carbonaria, the cDNA is 539 bp with ATG start codon located at position 46, TGA stop codon at position 469 and AATAAA polyadenylation signal at position 520. In G. denticulata, the cDNA is 536 bp with ATG start codon located at position 46, TGA stop codon at position 469 and AATAAA polyadenylation signal at position 517. Both cDNAs codify 141 amino acid residues, differing from each other in only four amino acid residues. When comparing with human Hb alpha-chain, alterations in important regions can be noted: alpha110 Ala-Gly, alpha114 Pro-Gly, alpha117 Phe-Tyr and alpha122 His-Gln. There is a high homology between the amino acids of these turtles when compared with chicken alpha-D chains, progressively decreasing when compared with human, crocodile, snake, frog and fish alpha-chains. Phylogenetic analysis of alpha-D chains shows that those of turtles are closer to those of birds than to snakes and lizards. (C) 2002 Elsevier B.V. All rights reserved.

Effect of acute heat stress on heat shock protein 70 messenger RNA and on heat shock protein expression in the liver of broilers

1. The synthesis of heat shock protein 70 (Hsp70) mRNA and the expression of Hsp70 in the liver of broiler chickens submitted to acute heat stress (35 degrees C for 5 h) was investigated.2. Hsp70 expression was detected by SDS-PAGE and Western blot analysis using a polyclonal antiserum against Hsp70 of Blastocladiella emersonii. The specific signal of Hsp70 mRNA was analysed by Northern blot using as probe a Hsp70 cDNA of B. emersonii.3. An increase in the amount of Hsp70 was detected from the first up to the fifth hour of acute heat exposure. This increase in the amount of Hsp70 was accompanied by an increase in Hsp70 mRNA which peaked at 3 h.4. This study shows that the heat induced increase in Hsp70 mRNA and protein in broiler liver, in vivo, are time dependent, similar to that in mammals.

Urocortin in the central nervous system of a primate (Cebus apella): Sequencing, immunohistochemical, and hybridization histochemical characterization

The urocortin (UCN)-like immunoreactivity and UCN mRNA distribution in various regions of the nonprimate mammalian brain have been reported. However, the Edinger-Westphal nucleus (EW) appears to be the only brain site where UCN expression is conserved across species. Although UCN peptides are present throughout vertebrate phylogeny, the functional roles of both UCN and EW remain poorly understood. Therefore, a study focused on UCN system organization in the primate brain is warranted. By using immunohistochemistry (single and double labeling) and in situ hybridization, we have characterized the organization of UCN-expressing cells and fibers in the central nervous system and pituitary of the capuchin monkey (Cebus apella). In addition, the sequence of the prepro-UCN was determined to establish the level of structural conservation relative to the human sequence. To understand the relationship of acetylcholine cells in the EW, a colocalization study comparing choline acetyltransferase (ChAT) and UCN was also performed. The cloned monkey prepro-UCN is 95% identical to the human preprohormone across the matched sequences. By using an antiserum raised against rat UCN and a probe generated from human cDNA, we found that the EW is the dominant site for UCN expression, although UCN mRNA is also expressed in spinal cord lamina IX. Labeled axons and terminals were distributed diffusely throughout many brain regions and along the length of the spinal cord. of particular interest were UCN-immunoreactive inputs to the medial preoptic area, the paraventricular nucleus of the hypothalamus, the oral part of the spinal trigeminal nucleus, the flocculus of the cerebellum, and the spinal cord laminae VII and X. We found no UCN hybridization signal in the pituitary. In addition, we observed no colocalization between ChAT and UCN in EW neurons. Our results support the hypothesis that the UCN system might participate in the control of autonomic, endocrine, and sensorimotor functions in primates.

Trypanosoma cruzi: identification and characterization of a novel ribosomal protein L27 (TcrL27) that cross-reacts with an affinity-purified anti-Sm antibody

Small nuclear ribonucleoproteins (snRNPs)are involved in trans-splicing processing of pre-mRNA in Trypanosoma cruzi. To clone T. cruzi snRNPs we screened an epimastigote cDNA library with a purified antibody raised against the Sm-binding site of a yeast sequence. A clone was obtained containing a 507 bp-insert with an ORF of 399 bp and coding for a protein of 133 amino acids. Sequence analysis revealed high identity with the L27 ribosomal proteins from different species including: Canis familiaris, Homo sapiens, Schizosaccharomyces pombe and Saccharomyces cerevisiae. This protein has not been previously described in the literature and seems to be a new ribosomal protein in T. cruzi and was given the code TcrL27. To express this recombinant T. cruzi L27 ribosomal protein in E. coli, the insert was subcloned into the pET32a vector and a 26 kDa recombinant protein was purified. Immunoblotting studies demonstrated that this purified recombinant protein was recognized by the same anti-Sm serum used in the library screening as well as by chagasic and systemic lupus erythemathosus (SLE) sera. Our results suggest that the T. cruzi L27 ribosomal protein may be involved in autoimmunity of Chagas disease.

Molecular characterisation and evolutionary relationships of a potyvirus infecting Crotalaria in Brazil - Brief report

The 3' terminal genomic region of a potyvirus causing mosaic disease in several Crotalaria species has been cloned and sequenced. Comparisons of the nucleotide and deduced amino acid (aa) sequences of the cloned cDNA with those from other potyviruses show that the Crotalaria-infecting virus (designated Crotalaria mosaic virus; CrMV) is closely related to Cowpea aphid-borne mosaic virus (CABMV). Maximum identity (95.4%) at the coat protein (CP) aa level was observed between CrMV and a Brazilian strain of CABMV. Phylogenetic analyses derived from the sequence alignments of the CP and 3' untranslated region confirmed the identification of CrMV as a strain of CABMV and the name CABMV-Cr is suggested.