Molecular characterization of Giardia duodenalis in dogs from Brazil

The intestinal protozoan parasite Giardia duodenalis (syn. Giardia intestinalis and Giardia lamblia) is a widespread enteric pathogen in human and domestic animals. This organism is one of the most common parasites in domestic dogs in Brazil. In this study, we determined the occurrence and genetic characterization of G. duodenalis isolated from dogs from south-central São Paulo state, Brazil. A total of 300 fecal samples were collected. Fecal specimens were screened for the presence of G. duodenalis using microscopy (zinc sulfate solution flotation technique) and polymerase chain reaction (PCR) targeting the small subunit ribosomal (SSU-rDNA) and glutamate dehydrogenase (GDH) genes. Genetic characterization was performed using restriction fragment length polymorphisms (RFLP) and sequencing analysis of the GDH gene. In addition, selected samples were further characterized by RFLP and sequencing of the beta-giardin gene. The overall occurrence of G. duodenalis was 17.3% (52/300). The occurrence was higher in stray dogs (28%) than in household dogs (6.25%). of the 36 PCR-positive samples that were selected for genotyping, only dog-specific genotype C (20 isolates), D (11 isolates) and mixed C+D (five isolates) isolates were detected in the study. This study provides current information on the infection rates of G. duodenalis genotypes in canine populations and describes for the first time the presence of mixed infections within host-specific C and D genotypes in dogs in Brazil. These genotypes were widespread and commonly found in domestic dogs living in urban and suburban environments of the studied area and confirmed the endemic status of Giardia in this region.

Myostatin (GDF8) single nucleotide polymorphisms in Nellore cattle

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

cDNA sequence and molecular modeling of a nerve growth factor from Bothrops jararacussu venomous gland

The complete nucleotide sequence of a nerve growth factor precursor from Bothrops jararacussu snake (Bj-NGF) was determined by DNA sequencing of a clone from cDNA library prepared from the poly(A) + RNA of the venom gland of B.jararacussu. cDNA encoding Bj-NGF precursor contained 723 bp in length, which encoded a prepro-NGF molecule with 241 amino acid residues. The mature Bj-NGF molecule was composed of I 18 amino acid residues with theoretical pI and molecular weight of 8.31 and 13,537, respectively. Its amino acid sequence showed 97%, 96%, 93%, 86%, 78%, 74%, 76%, 76% and 55% sequential similarities with NGFs from Crotalus durissus terrificus, Agkistrodon halys pallas, Daboia (Vipera) russelli russelli, Bungarus multicinctus, Naja sp., mouse, human, bovine and cat, respectively. Phylogenetic analyses based on the amino acid sequences of 15 NGFs separate the Elapidae family (Naja and Bungarus) from those Crotalidae snakes (Bothrops, Crotalus and Agkistrodon). The three-dimensional structure of mature Bj-NGF was modeled based on the crystal structure of the human NGF. The model reveals that the core of NGF, formed by a pair of P-sheets, is highly conserved and the major mutations are both at the three beta-hairpin loops and at the reverse turn. (C) 2002 Societe francaise de biochimie et biologic moleculaire/Editions scientifiques et medicales Elsevier SAS. All rights reserved.

Cyclin D1 gene polymorphism as a risk factor for squamous cell carcinoma of the upper aerodigestive system in non-alcoholics

Squamous cell carcinoma of the upper aerodigestive tract (UADT) is associated with environmental factors, especially tobacco and alcohol consumption. Genetic factors, including cyclin D1 (CCND1) polymorphism have been suggested to play an important rote in tumorigenesis and progression of UADT cancer. To investigate the relationship between CCND1 polymorphism on susceptibility for UADT cancers, 147 cancer and 135 non-cancer subjects were included in this study. CCND1 genotype at codon 242(G870A) in exon 4 was undertaken using denaturing high performance liquid chromatography (DHPLC) and DNA sequencing. Significant odds ratio (OR) of the AA + GA genotypes [OR = 7.5 (95% Cl: 1.4-39.7)] was observed in non-drinkers but for non-smokers a non-significant [OR = 5.4 (95% Cl: 0.9-31.4)] was found in the adjusted model. These results suggest that allele A may be a risk factor for UADT cancer, especially in non-alcoholics. However, further epidemiological studies are needed to establish the exact role of CCND1 polymorphism and the development of UADT cancers. (C) 2004 Elsevier Ltd. All rights reserved.

Evolutionary and biogeographic history of the subfamily Neoplecostominae (Siluriformes: Loricariidae)

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

A single nucleotide deletion at the C1 inhibitor gene as the cause of hereditary angioedema: insights from a Brazilian family

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Molecular identification of Pichia guilliermondii, Debaryomyces hansenii and Candida palmioleophila

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

Identification of sex in Carica papaya L. using RAPD markers

Brazil is currently the worlds largest producer of papaya (Carica papaya L.), producing fruits for both the domestic market and export. Only fruits from hermaphrodite plants are marketed because they have the necessary commercial characteristics, i.e. they are pear-shaped and have thicker flesh and a smaller internal cavity. Increased papaya yield has been limited mainly by the ratio of female to hermaphrodite (1:2) plants normally occurring in orchards. This ratio causes great losses to papaya producers and the identification of the sex of seedlings during the nursery stage would be an important advance. In our study random amplified polymorphic DNA (RAPD) analysis was used to differentiate between the sexual forms of three commercial C. papaya cultivars belonging to the Solo group. RAPD assays using the BC210 primer were able to detect hermaphrodites in all of the cultivars tested. The BC210(438)molecular marker was much better at papaya sex differentiation than other markers described in the literature.

Morphological and molecular characterization of Colletotrichum spp. from citrus orchards affected by postbloom fruit drop in Brazil

Brazilian isolates of Colletotrichum spp. from citrus orchards affected by postbloom fruit drop were examined for colony colour, mycelial growth, benomyl-resistance, pathogenicity, and genetic variability by random amplified polymorphic DNA (RAPD) analysis. All isolates were obtained from flowers and persistent calyxes from different citrus hosts from São Paulo, Brazil. DNA polymorphisms detected after amplification with random 10-mer primers were used to classify the isolates into two groups. Group I isolates grew rapidly on potato-dextrose agar (PDA) and were sensitive to benomyl, and group II isolates grew slowly on PDA and were benomyl-resistant. Colletotrichum acutatum was analyzed by RAPD and had high genetic similarity with group II isolates of Colletotrichum from citrus. Probably, the group I is C, gloeosporioides and group II is C. acutatum.

pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis clinical isolates from the southeast region of Brazil

Objectives: To investigate the presence of mutations in the pncA gene in 31 pyrazinamide-resistant Mycobacterium tuberculosis and 5 susceptible strains. MICs and pyrazinamidase (PZase) activity were also determined.Methods: All 36 M. tuberculosis clinical isolates were genotyped by mycobacterial interspersed repetitive units (MIRUs) and most were also typed by spoligotyping. The MIC value necessary to inhibit 99% of the resistant mycobacterial isolates was determined by microplate Alamar Blue assay (MABA) and by Lowenstein-Jensen assay (LJA). The PZase activity was measured by pyrazinamide deamination to pyrazinoic acid and ammonia, and the entire pncA sequence including the 410 by upstream from the start codon was determined by DNA sequencing of purified PCR products.Results: of the 31 isolates resistant to pyrazinamide, 26 (83.9%) showed at least one mutation in the pncA gene or in its putative regulatory region: Among the 22 different mutations detected in the pncA gene and in its regulatory region, 9 (40.9%) mutations (consisting of six substitutions, two insertions and one deletion) have not been described in previous studies. Three pyrazinamide-resistant isolates, confirmed by MIC varying from 800 to 1600 mg/L, carried the wild-type pncA sequence and retained PZase activity.Conclusions: These results contribute to the knowledge of the molecular mechanism of pyrazinamide resistance in Brazil and also expand the profile of pncA mutations worldwide. The MABA was successfully used to determine the MICs of pyrazinamide.

Use of primers derived from a new sequence of the bovine Y chromosome for sexing Bos taurus and Bos indicus embryos

A bovine male-specific marker was identified in our laboratory through random amplified polymorphic DNA (RAPD) analysis. This fragment of 3216 bp was cloned, sequenced and mapped by fluorescent in situ hybridization (FISH) on the taurine Yq. Primers derived from this sequence were initially screened by polymerase chain reaction (PCR) for their ability to detect Y-specific segments in zebu and taurine genomic DNA. Two of these primers amplified a 655 bp Y-specific sequence present in taurine and zebu male genomic DNA. These primers were then used for detecting the 655 bp male sequence in DNA from 173 zebu and 30 taurine embryos, which had been previously sexed using primers for the sequence BC 1.2. The results revealed an accuracy of 100%. (C) 2002 Elsevier B.V. All rights reserved.

Aracatuba virus: A vaccinialike m virus associated with infection in humans and cattle

We describe a vaccinialike virus, Aragatuba virus, associated with a cowpoxlike outbreak in a dairy herd and a related case of human infection. Diagnosis was based on virus growth characteristics, electron microscopy, and molecular biology techniques. Molecular characterization of the virus was done by using polymerase chain reaction amplification, cloning, an DNA sequencing of conserved orthopoxvirus genes such as the vaccinia growth factor (VGF), thymidine kinase (TK), and hemagglutinin. We used VGF-homologous and TK gene nucleoticle sequences to construct a phylogenetic tree for comparison with other poxviruses. Gene sequences showed 99% homology with vaccinia virus genes and were clustered together with the isolated virus in the phylogenetic tree. Aragatuba virus is very similar to Cantagalo virus, showing the same signature deletion in the gene. Aragatuba virus could be a novel vaccinialike virus or could represent the spread of Cantagalo virus.

Screening and characterization of mutations in isoniazid-resistant Mycobacterium tuberculosis isolates obtained in Brazil

We investigated mutations in the genes katG, inhA (regulatory and structural regions), and kasA and the oxyR-ahpC intergenic region of 97 isoniazid (INH)-resistant and 60 INH-susceptible Mycobacterium tuberculosis isolates obtained in two states in Brazil: São Paulo and Parana. PCR-single-strand conformational polymorphism (PCR-SSCP) was evaluated for screening mutations in regions of prevalence, including codons 315 and 463 of katG, the regulatory region and codons 16 and 94 of inhA, kasA, and the oxyR-ahpC intergenic region. DNA sequencing of PCR amplicons was performed for all isolates with altered PCR-SSCP profiles. Mutations in katG were found in 83 (85.6%) of the 97 INH-resistant isolates, including mutations in codon 315 that occurred in 60 (61.9%) of the INH-resistant isolates and 23 previously unreported katG mutations. Mutations in the inhA promoter region occurred in 25 (25.8%) of the INH-resistant isolates; 6.2% of the isolates had inhA structural gene mutations, and 10.3% had mutations in the oxyR-ahpC intergenic region (one, nucleotide -48, previously unreported). Polymorphisms in the kasA gene occurred in both INH-resistant and INH-susceptible isolates. The most frequent polymorphism encoded a G(269)A substitution. Although KatG(315) substitutions are predominant, novel mutations also appear to be responsible for INH resistance in the two states in Brazil. Since ca. 90.7% of the INH-resistant isolates had mutations identified by SSCP electrophoresis, this method may be a useful genotypic screen for INH resistance.

Organization of 5S rDNA in species of the fish Leporinus: two different genomic locations are characterized by distinct nontranscribed spacers

To address understanding the organization of the 5S rRNA multigene family in the fish genome, the nucleotide sequence and organization array of 5S rDNA were investigated in the genus Leporinus, a representative freshwater fish group of South American fauna. PCR, subgenomic library screening, genomic blotting, fluorescence in situ hybridization, and DNA sequencing were employed in this study. Two arrays of 5S rDNA were identified for all species investigated, one consisting of monomeric repeat units of around 200 bp and another one with monomers of 900 bp. These 5S rDNA arrays were characterized by distinct NTS sequences (designated NTS-I and NTS-II for the 200- and 900-bp monomers, respectively); however, their coding sequences were nearly identical. The 5S rRNA genes were clustered in two chromosome loci, a major one corresponding to the NTS-I sites and a minor one corresponding to the NTS-II sites. The NTS-I sequence was variable among Leporinus spp., whereas the NTS-II was conserved among them and even in the related genus Schizodon. The distinct 5S rDNA arrays might characterize two 5S rRNA gene subfamilies that have been evolving independently in the genome.

Identification of polymorphisms in the 5 '-untranslated region of the TAFI gene: relationship with plasma TAFI levels and risk of venous thrombosis

Background and Objectives. Thrombin activatable fibrinolysis inhibitor (TAFI) plays an important role in hemostasis, functioning as a potent fibrinolysis inhibitor. TAFI gene variations may contribute to plasma TAFI levels and thrombotic risk.Design and Methods. We sequenced a 2083-bp region of the 5 ' -regulatory region of the TAFI gene in 127 healthy subjects searching for variations, and correlated identified polymorphisms with plasma TAFI levels. TAFI polymorphisms were examined as risk factors for venous thrombosis by determining their prevalence in 388 patients with deep venous thrombosis (DVT) and in 388 controls.Results. Seven novel polymorphisms were identified: -152 A/G, -438 A/G, -530 C/T, -1053 T/C, -1102 T/G, -1690 G/A, and -1925 T/C. -152 A/G, -530 C/T and -1925 T/C were found to be in strong linkage disequilibrium, as were the -438 A/G, -1053 T/C, -1102 T/G and -1690 G/A, Plasma TAFI levels were higher in -43866/-1053CC/-1102GG/-1690AA homozygotes than In -438AG/-1053TC/-1102TG/-1690GA heterozygotes, and -438AA/-1053TT/-1102TT/-1690GG homozygotes had the lowest TAFI levels (p=0.0003). TAFI concentrations in -152AA/-530CC/-1925TT homozygotes were somewhat higher but not significantly different from levels observed for -152AG/-530CT/-1925TC heterozygotes, Taken in combination, -438AG/-1053TC/-1102TG/-1690GA and -438AA/-1053TT/-1102TT/-1690GG yielded an OR for DVT of 0.8 (95%CI: 0.6-1). in subjects aged < 35 years the OR was 0.7 (95%CI: 0.5-1.1), the OR for -152AG/-530CT/-1925TC was 1 (95%CI: 0.5-2.2) in the whole group of patients and controls, whereas in subjects aged <35 years the OR was 0.1 (95%CI: 0.02-0.9).Interpretation and Conclusions. Polymorphisms in the TAFI promoter determine plasma antigen levels and may influence the risk of venous thrombophilia. <(c)>2001, Ferrata Storti Foundation.