Genomic characterization of Brazilian hepatitis C virus genotypes 1a and 1b

Parts of 5' non-coding (5' NC) and of E1 envelope regions of the hepatitis C virus (HCV) genome were amplified from sera of 26 Brazilian anti-HCV antibody-positive patients using the reverse transcription-polymerase chain reaction (RT-PCR). Fourteen samples were PCR positive with primers from the 5' NC region and 8 of them were also positive with primers from the E1 region. A genomic segment of 176 bp from the E1 region of 7 isolates was directly sequenced from PCR products. The sequences were compared with those of HCV strains isolated in other countries and the Brazilian isolates were classified by phylogenetic analysis into genotypes 1a and 1b. This could have a clinical importance since it has been shown that individuals infected with type 1 viruses are less likely to respond to treatment with interferon than individuals infected with types 2 and 3 viruses. Two quasispecies isolated from the same patient with an interval of 13 months differed by two base substitutions (1.1%). The sequence of another isolate presented a three-nucleotide deletion at codon 329

A novel polymorphism in the coding region of the vasopressin type 2 receptor gene

Nephrogenic diabetes insipidus (NDI) is a rare disease characterized by renal inability to respond properly to arginine vasopressin due to mutations in the vasopressin type 2 receptor (V2(R)) gene in affected kindreds. In most kindreds thus far reported, the mode of inheritance follows an X chromosome-linked recessive pattern although autosomal-dominant and autosomal-recessive modes of inheritance have also been described. Studies demonstrating mutations in the V2(R) gene in affected kindreds that modify the receptor structure, resulting in a dys- or nonfunctional receptor have been described, but phenotypically indistinguishable NDI patients with a structurally normal V2(R) gene have also been reported. In the present study, we analyzed exon 3 of the V2(R) gene in 20 unrelated individuals by direct sequencing. A C®T alteration in the third position of codon 331 (AGC®AGT), which did not alter the encoded amino acid, was found in nine individuals, including two unrelated patients with NDI. Taken together, these observations emphasize the molecular heterogeneity of a phenotypically homogeneous syndrome

Partial sequence and toxic effects of granulitoxin, a neurotoxic peptide from the sea anemone Bunodosoma granulifera

A neurotoxic peptide, granulitoxin (GRX), was isolated from the sea anemone Bunodosoma granulifera. The N-terminal amino acid sequence of GRX is AKTGILDSDGPTVAGNSLSGT and its molecular mass is 4958 Da by electrospray mass spectrometry. This sequence presents a partial degree of homology with other toxins from sea anemones such as Bunodosoma caissarum, Anthopleura fuscoviridis and Anemonia sulcata. However, important differences were found: the first six amino acids of the sequence are different, Arg-14 was replaced by Ala and no cysteine residues were present in the partial sequence, while two cysteine residues were present in the first 21 amino acids of other toxins described above. Purified GRX injected ip (800 µg/kg) into mice produced severe neurotoxic effects such as circular movements, aggressive behavior, dyspnea, tonic-clonic convulsion and death. The 2-h LD50 of GRX was 400 ± 83 µg/kg.

Time sequence of changes in the responsiveness of glycogen breakdown to adrenergic agonists in perfused liver of rats with insulin-induced hypoglycemia

The time-course changes of the responsiveness of glycogen breakdown to a- and ß-adrenergic agonists during insulin-induced hypoglycemia (IIH) were investigated. Blood glucose levels were decreased prior to the alteration in the hepatic responsiveness to adrenergic agonists. The activation of hepatic glucose production and glycogenolysis by phenylephrine (2 µM) and isoproterenol (20 µM) was decreased in IIH. The changes in the responsiveness of glycogen catabolism were first observed for isoproterenol and later for phenylephrine. Hepatic ß-adrenergic receptors showed a higher degree of adrenergic desensitization than did a-receptors. Liver glycogen synthase activity, glycogen content and the catabolic effect of dibutyryl cyclic AMP (the ß-receptor second messenger) were not affected by IIH.

Role of ACTH receptor in adrenocortical tumor formation

Adrenocorticotrophin (ACTH) is the major regulatory hormone of steroid synthesis and secretion by adrenocortical cells. The actions of ACTH are mediated by its specific membrane receptor (ACTH-R). The human ACTH-R gene was recently cloned, allowing systematic determination of its sequence, expression and function in adrenal tumorigenesis. The presence of oncogenic mutations of the ACTH-R gene in adrenocortical tumors has been reported. Direct sequencing of the entire coding region of the ACTH-R gene of sporadic adrenocortical adenomas and carcinomas did not reveal constitutive activating mutations, indicating that this mechanism is not frequent in human adrenocortical tumorigenesis. Recent studies demonstrated allelic loss of the ACTH-R gene in a subset of sporadic adrenocortical tumors using a PstI polymorphism located in the promoter region of the ACTH-R gene. Loss of heterozygosity of the ACTH-R was analyzed in 20 informative patients with a variety of benign and malignant adrenocortical tumors. Three of them showed loss of heterozygosity of the ACTH-R gene. In addition, Northern blot experiments demonstrated reduced expression of ACTH-R mRNA in these three tumors with loss of heterozygosity, suggesting the functional significance of this finding at the transcriptional level. Deletion of the ACTH-R gene seems to be involved in a subset of human adrenocortical tumors, contributing to cellular dedifferentiation.

Partial characterization of nif genes from the bacterium Azospirillum amazonense

Azospirillum amazonense revealed genomic organization patterns of the nitrogen fixation genes similar to those of the distantly related species A. brasilense. Our work suggests that A. brasilense nifHDK, nifENX, fixABC operons and nifA and glnB genes may be structurally homologous to the counterpart genes of A. amazonense. This is the first analysis revealing homology between A. brasilense nif genes and the A. amazonense genome. Sequence analysis of PCR amplification products revealed similarities between the amino acid sequences of the highly conserved nifD and glnB genes of A. amazonense and related genes of A. brasilense and other bacteria. However, the A. amazonense non-coding regions (the upstream activator sequence region and the region between the nifH and nifD genes) differed from related regions of A. brasilense even in nitrogenase structural genes which are highly conserved among diazotrophic bacteria. The feasibility of the 16S ribosomal RNA gene-based PCR system for specific detection of A. amazonense was shown. Our results indicate that the PCR primers for 16S rDNA defined in this article are highly specific to A. amazonense and can distinguish this species from A. brasilense.

A protein with amino acid sequence homology to bovine insulin is present in the legume Vigna unguiculata (cowpea)

Since the discovery of bovine insulin in plants, much effort has been devoted to the characterization of these proteins and elucidation of their functions. We report here the isolation of a protein with similar molecular mass and same amino acid sequence to bovine insulin from developing fruits of cowpea (Vigna unguiculata) genotype Epace 10. Insulin was measured by ELISA using an anti-human insulin antibody and was detected both in empty pods and seed coats but not in the embryo. The highest concentrations (about 0.5 ng/µg of protein) of the protein were detected in seed coats at 16 and 18 days after pollination, and the values were 1.6 to 4.0 times higher than those found for isolated pods tested on any day. N-terminal amino acid sequencing of insulin was performed on the protein purified by C4-HPLC. The significance of the presence of insulin in these plant tissues is not fully understood but we speculate that it may be involved in the transport of carbohydrate to the fruit.

Genome features of Leptospira interrogans serovar Copenhageni

We report novel features of the genome sequence of Leptospira interrogans serovar Copenhageni, a highly invasive spirochete. Leptospira species colonize a significant proportion of rodent populations worldwide and produce life-threatening infections in mammals. Genomic sequence analysis reveals the presence of a competent transport system with 13 families of genes encoding for major transporters including a three-member component efflux system compatible with the long-term survival of this organism. The leptospiral genome contains a broad array of genes encoding regulatory system, signal transduction and methyl-accepting chemotaxis proteins, reflecting the organism's ability to respond to diverse environmental stimuli. The identification of a complete set of genes encoding the enzymes for the cobalamin biosynthetic pathway and the novel coding genes related to lipopolysaccharide biosynthesis should bring new light to the study of Leptospira physiology. Genes related to toxins, lipoproteins and several surface-exposed proteins may facilitate a better understanding of the Leptospira pathogenesis and may serve as potential candidates for vaccine.

Rarity of DNA sequence alterations in the promoter region of the human androgen receptor gene

The human androgen receptor (AR) gene promoter lies in a GC-rich region containing two principal sites of transcription initiation and a putative Sp1 protein-binding site, without typical "TATA" and "CAAT" boxes. It has been suggested that mutations within the 5'untranslated region (5'UTR) may contribute to the development of prostate cancer by changing the rates of gene transcription and/or translation. In order to investigate this question, the aim of the present study was to search for the presence of mutations or polymorphisms at the AR-5'UTR in 92 prostate cancer patients, where histological diagnosis of adenocarcinoma was established in specimens obtained from transurethral resection or after prostatectomy. The AR-5'UTR was amplified by PCR from genomic DNA samples of the patients and of 100 healthy male blood donors, included as controls. Conformation-sensitive gel electrophoresis was used for DNA sequence alteration screening. Only one band shift was detected in one individual from the blood donor group. Sequencing revealed a new single nucleotide deletion (T) in the most conserved portion of the promoter region at position +36 downstream from the transcription initiation site I. Although the effect of this specific mutation remains unknown, its rarity reveals the high degree of sequence conservation of the human androgen promoter region. Moreover, the absence of detectable variation within the critical 5'UTR in prostate cancer patients indicates a low probability of its involvement in prostate cancer etiology.

L1 sequence of a new human papillomavirus type-58 variant associated with cervical intraepithelial neoplasia

The present study on molecular characterization of a human papillomavirus (HPV) isolated in Central Brazil describes the L1 gene sequence from a new variant of HPV-58, the isolate Bsb-02. The sample was from a smear obtained from a woman with cervical intraepithelial neoplasia grade II. The whole L1 gene from isolate Bsb-02 was sequenced automatically, showing 99.1% nucleotide identity with the gene from the HPV-58 reference. The clustering between Bsb-02 and HPV-58 reference sequence was also supported by phylogenetic analysis. Fourteen nucleotide substitutions were observed: eight were synonymous and six were associated with amino acid substitutions. A10V and V144I have not been previously described. At GenBank, the only complete L1 sequence from HPV-58 in addition to the HPV-58 reference one is that of Bsb-02. These data provide information that may be relevant to HPV diagnosis and to rational vaccine strategies. HPV variants may also be associated with host immune responses and with the risk of cervical neoplasia.

A search for RNA insertions and NS3 gene duplication in the genome of cytopathic isolates of bovine viral diarrhea virus

Calves born persistently infected with non-cytopathic bovine viral diarrhea virus (ncpBVDV) frequently develop a fatal gastroenteric illness called mucosal disease. Both the original virus (ncpBVDV) and an antigenically identical but cytopathic virus (cpBVDV) can be isolated from animals affected by mucosal disease. Cytopathic BVDVs originate from their ncp counterparts by diverse genetic mechanisms, all leading to the expression of the non-structural polypeptide NS3 as a discrete protein. In contrast, ncpBVDVs express only the large precursor polypeptide, NS2-3, which contains the NS3 sequence within its carboxy-terminal half. We report here the investigation of the mechanism leading to NS3 expression in 41 cpBVDV isolates. An RT-PCR strategy was employed to detect RNA insertions within the NS2-3 gene and/or duplication of the NS3 gene, two common mechanisms of NS3 expression. RT-PCR amplification revealed insertions in the NS2-3 gene of three cp isolates, with the inserts being similar in size to that present in the cpBVDV NADL strain. Sequencing of one such insert revealed a 296-nucleotide sequence with a central core of 270 nucleotides coding for an amino acid sequence highly homologous (98%) to the NADL insert, a sequence corresponding to part of the cellular J-Domain gene. One cpBVDV isolate contained a duplication of the NS3 gene downstream from the original locus. In contrast, no detectable NS2-3 insertions or NS3 gene duplications were observed in the genome of 37 cp isolates. These results demonstrate that processing of NS2-3 without bulk mRNA insertions or NS3 gene duplications seems to be a frequent mechanism leading to NS3 expression and BVDV cytopathology.

Hepatitis B virus genotype E detected in Brazil in an African patient who is a frequent traveler

Genotype E of hepatitis B virus (HBV) has not been described in Brazil and is found mainly in Africa. Genotype A is the most prevalent in Brazil, and genotypes B, C, D, and F have already been reported. We report here an HBV genotype E-infected patient and some characterization of surface (S) protein, DNA polymerase (P) and precore/core (preC/C) coding regions based on the viral genome. The patient is a 31-year-old black man with chronic hepatitis B who was born and raised in Angola. He has been followed by a hepatologist in São Paulo, Brazil, since November 2003, and he is a frequent traveler to Latin America, Africa, and Europe. In 2003, he was diagnosed with HBV infection and started treatment with lamivudine with the later addition of adefovir dipivoxil. No known risk factor was identified. Serologically, he is HBsAg and anti-HBe positive, but HBeAg and anti-HBs negative. DNA sequence analysis of the S/P region confirmed that this patient is infected with genotype E, subtype ayw4. The preC/C region showed G1896A and G1899A mutations but no mutations in the basal core promoter. Nucleotide substitutions common in genotype E were also observed (C1772, T1858 and A1757). Although this is not an autochthonous case and there is no evidence of further spread, the description of this case in Brazil highlights the current risk of viral genotypes spreading with unprecedented speed due to constant travel around the world.

Genotyping hepatitis C virus from hemodialysis patients in Central Brazil by line probe assay and sequence analysis

The present study examined the distribution of hepatitis C virus (HCV) genotypes and subtypes in a hemodialysis population in Goiás State, Central Brazil, and evaluated the efficiency of two genotyping methods: line probe assay (LiPA) based on the 5' noncoding region and nucleotide sequencing of the nonstructural 5B (NS5B) region of the genome. A total of 1095 sera were tested for HCV RNA by RT-nested PCR of the 5' noncoding region. The LiPA assay was able to genotype all 131 HCV RNA-positive samples. Genotypes 1 (92.4%) and 3 (7.6%) were found. Subtype 1a (65.7%) was the most prevalent, followed by subtypes 1b (26.7%) and 3a (7.6%). Direct nucleotide sequencing of 340 bp from the NS5B region was performed in 106 samples. The phylogenetic tree showed that 98 sequences (92.4%) were classified as genotype 1, subtypes 1a (72.6%) and 1b (19.8%), and 8 sequences (7.6%) as subtype 3a. The two genotyping methods gave concordant results within HCV genotypes and subtypes in 100 and 96.2% of cases, respectively. Only four samples presented discrepant results, with LiPA not distinguishing subtypes 1a and 1b. Therefore, HCV genotype 1 (subtype 1a) is predominant in hemodialysis patients in Central Brazil. By using sequence analysis of the NS5B region as a reference standard method for HCV genotyping, we found that LiPA was efficient at the genotype level, although some discrepant results were observed at the subtype level (sensitivity of 96.1% for subtype 1a and 95.2% for subtype 1b). Thus, analysis of the NS5B region permitted better discrimination between HCV subtypes, as required in epidemiological investigations.

Sequence change in the HS2-LCR and Gg-globin gene promoter region of sickle cell anemia patients

The fetal hemoglobin (HbF) levels and ßS-globin gene haplotypes of 125 sickle cell anemia patients from Brazil were investigated. We sequenced the Gg- and Ag-globin gene promoters and the DNase I-2 hypersensitive sites in the locus control regions (HS2-LCR) of patients with HbF level disparities as compared to their ßS haplotypes. Sixty-four (51.2%) patients had CAR/Ben genotype; 36 (28.8%) Ben/Ben; 18 (14.4%) CAR/CAR; 2 (1.6%) CAR/Atypical; 2 (1.6%) Ben/Cam; 1 (0.8%) CAR/Cam; 1 (0.8%) CAR/Arab-Indian, and 1 (0.8%) Sen/Atypical. The HS2-LCR sequence analyses demonstrated a c.-10.677G>A change in patients with the Ben haplotype and high HbF levels. The Gg gene promoter sequence analyses showed a c.-157T>C substitution shared by all patients, and a c.-222_-225del related to the Cam haplotype. These results identify new polymorphisms in the HS2-LCR and Gg-globin gene promoter. Further studies are required to determine the correlation between HbF synthesis and the clinical profile of sickle cell anemia patients.

cDNA and deduced primary structure of basic phospholipase A2 with neurotoxic activity from the venom secretion of the Crotalus durissus collilineatus rattlesnake

To illustrate the construction of precursor complementary DNAs, we isolated mRNAs from whole venom samples. After reverse transcription polymerase chain reaction (RT-PCR), we amplified the cDNA coding for a neurotoxic protein, phospholipase A2 D49 (PLA2 D49), from the venom of Crotalus durissus collilineatus (Cdc PLA2). The cDNA encoding Cdc PLA2 from whole venom was sequenced. The deduced amino acid sequence of this cDNA has high overall sequence identity with the group II PLA2 protein family. Cdc PLA2 has 14 cysteine residues capable of forming seven disulfide bonds that characterize this group of PLA2 enzymes. Cdc PLA2 was isolated using conventional Sephadex G75 column chromatography and reverse-phase high performance liquid chromatography (RP-HPLC). The molecular mass was estimated using matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. We tested the neuromuscular blocking activities on chick biventer cervicis neuromuscular tissue. Phylogenetic analysis of Cdc PLA2 showed the existence of two lines of N6-PLA2, denominated F24 and S24. Apparently, the sequences of the New World’s N6-F24-PLA2 are similar to those of the agkistrodotoxin from the Asian genus Gloydius. The sequences of N6-S24-PLA2 are similar to the sequence of trimucrotoxin from the genus Protobothrops, found in the Old World.