172 results on '"Arnaldo Zaha"'
Search Results
102. Biological cost in Mycobacterium tuberculosis with mutations in the rpsL, rrs, rpoB, and katG genes
- Author
-
Andrezza Wolowski Ribeiro, Pedro Eduardo Almeida da Silva, Elis Regina Dalla Costa, Arnaldo Zaha, Anandi Martin, Daniela Fernandes Ramos, Fernanda Sá Spies, Juan Carlos Palomino, Marta Osório Ribeiro, Maria Lucia Rosa Rossetti, and Andrea von Groll
- Subjects
Microbiology (medical) ,Ribosomal Proteins ,Immunology ,Antitubercular Agents ,Drug resistance ,Microbial Sensitivity Tests ,medicine.disease_cause ,Microbiology ,Mycobacterium tuberculosis ,Antibiotic resistance ,Bacterial Proteins ,Drug Resistance, Multiple, Bacterial ,RNA, Ribosomal, 16S ,Tuberculosis, Multidrug-Resistant ,medicine ,Humans ,Gene ,Genetics ,Mutation ,biology ,DNA-Directed RNA Polymerases ,biology.organism_classification ,rpoB ,Catalase ,Infectious Diseases ,Genes, Bacterial ,Rifampicin ,Bacteria ,medicine.drug - Abstract
summary When bacteria develop drug-resistant mutations, there is often an associated biological cost; however, some strains can exhibit low- or no-cost mutations. In the present study, a quantitative resazurin reduction assay was used to measure the biological cost of Mycobacterium tuberculosis isolates that contained different mutations in the rpsL, rrs, rpoB, and katG genes, and showed different resistance profiles. Biological costs were determined by comparing the growth curves of drug-resistant isolates with drug-susceptible strains. Some strains, such as those with rpoB mutations other than S531L and strains with mutations in all of the studied genes, grew more slowly than did drug-susceptible strains. However, some strains grew more quickly than drug-susceptible strains, such as those that had only the rpsL K43R mutation. Strains with the mutation katG S315T presented heterogeneous biological costs. When analyzed individually, strains with the mutations rpsL43/katG315, rpoB531, and rpoB531/katG315 grew faster than drug-susceptible strains. The results suggest that some strains with the most common mutations correlated to a high resistance toward streptomycin, isoniazid and rifampicin can grow as well as or better than susceptible strains.
- Published
- 2012
103. A transcriptomic analysis of Echinococcus granulosus larval stages : implications for parasite biology and host adaptation
- Author
-
James D. Wasmuth, Matthew Berriman, Gustavo Salinas, Cecilia Fernández, Henrique Bunselmeyer Ferreira, Cristiano Valim Bizarro, Arnaldo Zaha, Mark Blaxter, Rick M. Maizels, John Parkinson, and Chris A. Sanford
- Subjects
Bioinformatics ,Biochemistry ,Transcriptomes ,Pharmacology, Toxicology and Pharmaceutics(all) ,Molecular cell biology ,0302 clinical medicine ,Genome Databases ,Parasite hosting ,Echinococcus granulosus ,Genetics ,Expressed Sequence Tags ,0303 health sciences ,Expressed sequence tag ,biology ,Schistosoma japonicum ,lcsh:Public aspects of medicine ,Intermediate host ,Genomics ,Oxygen Metabolism ,3. Good health ,Infectious Diseases ,Larva ,Metabolic Pathways ,Host adaptation ,Research Article ,lcsh:Arctic medicine. Tropical medicine ,lcsh:RC955-962 ,Gene prediction ,DNA transcription ,030231 tropical medicine ,Molecular Sequence Data ,Sequence Databases ,03 medical and health sciences ,Genome Analysis Tools ,parasitic diseases ,Animals ,Biology ,030304 developmental biology ,Evolutionary Biology ,Public Health, Environmental and Occupational Health ,lcsh:RA1-1270 ,Sequence Analysis, DNA ,Comparative Genomics ,biology.organism_classification ,Metabolism ,Echinococcus ,RNA processing ,Gene expression ,Transcriptome ,Developmental Biology - Abstract
Background The cestode Echinococcus granulosus - the agent of cystic echinococcosis, a zoonosis affecting humans and domestic animals worldwide - is an excellent model for the study of host-parasite cross-talk that interfaces with two mammalian hosts. To develop the molecular analysis of these interactions, we carried out an EST survey of E. granulosus larval stages. We report the salient features of this study with a focus on genes reflecting physiological adaptations of different parasite stages. Methodology/Principal Findings We generated ∼10,000 ESTs from two sets of full-length enriched libraries (derived from oligo-capped and trans-spliced cDNAs) prepared with three parasite materials: hydatid cyst wall, larval worms (protoscoleces), and pepsin/H+-activated protoscoleces. The ESTs were clustered into 2700 distinct gene products. In the context of the biology of E. granulosus, our analyses reveal: (i) a diverse group of abundant long non-protein coding transcripts showing homology to a middle repetitive element (EgBRep) that could either be active molecular species or represent precursors of small RNAs (like piRNAs); (ii) an up-regulation of fermentative pathways in the tissue of the cyst wall; (iii) highly expressed thiol- and selenol-dependent antioxidant enzyme targets of thioredoxin glutathione reductase, the functional hub of redox metabolism in parasitic flatworms; (iv) candidate apomucins for the external layer of the tissue-dwelling hydatid cyst, a mucin-rich structure that is critical for survival in the intermediate host; (v) a set of tetraspanins, a protein family that appears to have expanded in the cestode lineage; and (vi) a set of platyhelminth-specific gene products that may offer targets for novel pan-platyhelminth drug development. Conclusions/Significance This survey has greatly increased the quality and the quantity of the molecular information on E. granulosus and constitutes a valuable resource for gene prediction on the parasite genome and for further genomic and proteomic analyses focused on cestodes and platyhelminths., Author Summary Cestodes are a neglected group of platyhelminth parasites, despite causing chronic infections to humans and domestic animals worldwide. We used Echinococcus granulosus as a model to study the molecular basis of the host-parasite cross-talk during cestode infections. For this purpose, we carried out a survey of the genes expressed by parasite larval stages interfacing with definitive and intermediate hosts. Sequencing from several high quality cDNA libraries provided numerous insights into the expression of genes involved in important aspects of E. granulosus biology, e.g. its metabolism (energy production and antioxidant defences) and the synthesis of key parasite structures (notably, the one exposed to humans and livestock intermediate hosts). Our results also uncovered the existence of an intriguing set of abundant repeat-associated non-protein coding transcripts that may participate in the regulation of gene expression in all surveyed stages. The dataset now generated constitutes a valuable resource for gene prediction on the parasite genome and for further genomic and proteomic studies focused on cestodes and platyhelminths. In particular, the detailed characterization of a range of newly discovered genes will contribute to a better understanding of the biology of cestode infections and, therefore, to the development of products allowing their efficient control.
- Published
- 2012
104. Expression and analysis of the diagnostic value of an Echinococcus granulosus antigen gene clone
- Author
-
Henrique Bunselmeyer Ferreira and Arnaldo Zaha
- Subjects
Antigenicity ,DNA, Complementary ,Recombinant Fusion Proteins ,Immunoblotting ,Enzyme-Linked Immunosorbent Assay ,Sensitivity and Specificity ,Epitope ,Antigen ,Echinococcosis ,parasitic diseases ,Taenia solium ,medicine ,Animals ,Humans ,Genomic library ,Cloning, Molecular ,Echinococcus granulosus ,Gene Library ,Expression vector ,biology ,biology.organism_classification ,Virology ,Molecular biology ,Echinococcus ,medicine.drug_formulation_ingredient ,Infectious Diseases ,Antigens, Helminth ,Electrophoresis, Polyacrylamide Gel ,Parasitology ,Schistosoma mansoni ,Plasmids - Abstract
A pool of 9 sera from Echinococcus granulosus infected patients (PSP) was used to screen an E. granulosus cDNA library constructed in the expression vector lambda gt11. Ten reactive phage clones were isolated and 8 were confirmed in spot-lysis arrays probed with PSP. The insert of 1 of these clones (lambda AgEg4) previously characterized as an E. granulosus cytosolic malate dehydrogenase encoding gene was subcloned into the plasmid vector pGEX-1 and expressed as a fusion with glutathione S-transferase. The fusion peptide (Ag4-GST) was produced in Escherichia coli and its antigenicity was confirmed in colony immunoassay and in immunoblot using nondenaturing conditions. The lack of antigenicity of Ag4-GST in immunoblot using denaturing conditions suggests that the recognized epitopes are conformational. Ag4-GST was purified by affinity chromatography and tested in ELISA and immunodots to access its sensitivity and specificity in the diagnosis of human cystic hydatid disease. An overall sensitivity of 53.6% was obtained. Cross-reactions were observed with some sera from patients infected with Schistosoma mansoni and Wuchereria bancrofti. Ag4-GST was not recognized by any of the sera from Taenia solium infected patients tested. These preliminary results suggest that Ag4-GST could be useful as an accessory antigen to discriminate some cross-reactions with sera from cysticercosis patients, especially in regions like southern Brazil, where schistosomiasis and filariasis are not prevalent.
- Published
- 1994
105. Detection of Mycobacterium avium in Blood Samples of Patients with AIDS by Using PCR
- Author
-
Vivian de F.S. Rodrigues, Maria Lucia Rosa Rossetti, Marta Osório Ribeiro, Fernanda Carvalho de Queiroz Mello, Leila de Souza Fonseca, Arnaldo Zaha, and Afrânio Lineu Kritski
- Subjects
DNA, Bacterial ,Microbiology (medical) ,Opportunistic infection ,AIDS-Related Opportunistic Infections ,Mycobacterium avium-intracellulare infection ,Polymerase Chain Reaction ,Microbiology ,law.invention ,law ,Immunopathology ,medicine ,Humans ,Sida ,Polymerase chain reaction ,Mycobacterium avium-intracellulare Infection ,biology ,fungi ,food and beverages ,Mycobacteriology and Aerobic Actinomycetes ,Mycobacterium avium Complex ,medicine.disease ,biology.organism_classification ,Virology ,Viral disease ,Mycobacterium - Abstract
Sixty-nine blood samples from 47 patients infected with human immunodeficiency virus were analyzed by using PCR to detect Mycobacterium avium . The sensitivity can be up to 95.7%, depending on the detection method used and the number of blood samples analyzed from each patient. The procedure can be helpful in the diagnosis of mycobacterial disease.
- Published
- 2002
106. Echinococcus ortleppi (G5) and Echinococcus granulosus sensu stricto (G1) loads in cattle from Southern Brazil
- Author
-
Ana Cristina Arend, Karen Luisa Haag, Daniel Ângelo Sgranzerla Graichen, Arnaldo Zaha, Guilherme Brzoskowski dos Santos, Helier Balbinotti, and Jeferson Loureiro Badaraco
- Subjects
Pathology ,medicine.medical_specialty ,Veterinary medicine ,Genotype ,Sequence analysis ,Cattle Diseases ,Helminth genetics ,Kidney ,Parasite load ,DNA, Mitochondrial ,Polymerase Chain Reaction ,Parasite Load ,Electron Transport Complex IV ,Sensu ,Echinococcosis ,Cystic hydatid disease ,parasitic diseases ,medicine ,Parasite hosting ,Animals ,Echinococcus granulosus ,Lung ,Echinococcus granulosus sensu stricto ,General Veterinary ,biology ,Haplotype ,Heart ,General Medicine ,Sequence Analysis, DNA ,DNA, Helminth ,biology.organism_classification ,veterinary(all) ,Echinococcus ,Haplotypes ,Liver ,Regression Analysis ,Parasitology ,Cattle ,Echinococcus ortleppi ,Brazil ,Spleen - Abstract
Echinococcus granulosus sensu stricto (G1) and Echinococcus ortleppi (G5) are haplotypes of the parasite formerly known as Echinococcus granulosus sensu lato, which in its larval stage causes cystic hydatid disease, endemic in Southern Brazil. Epidemiological and molecular knowledge about the haplotypes occurring in a region is essential to control the spread of the disease. The aim of this work was to analyze the haplotype frequency and fertility of hydatid cysts in cattle from the state of Rio Grande do Sul. Cysts were collected and classified according to their fertility status. DNA was extracted from protoscoleces and germinal layers and then used as template for the amplification of the cytochrome c oxidase subunit 1 gene by PCR. Amplicons were purified and sequenced, and the sequences were analyzed for haplotype identification. A total of 638 fertile cysts collected in the last ten years were genotyped. On average, G1 (56.6%) was more frequent than G5 (43.4%). In lungs, the G5 haplotype exhibited a higher parasite load (52.8%), whereas in the liver, G1 was more frequent (90.4%). The analysis revealed an increase in the frequency of G5 haplotype cysts during the period of sampling, and an increase in the abundance of fertile cysts has also been observed in the last several years. Most infertile cysts were genotyped as G1. The possible factors involved in the increase in the proportion of E. ortleppi (G5) and the consequences of this increase are discussed. This study suggests that the proportion of E. ortleppi (G5) loads in cattle may be increasing overtime.
- Published
- 2011
107. Echinococcus granulosus antigen B structure: subunit composition and oligomeric states
- Author
-
Daiani Machado de Vargas, Elliot W. Kitajima, Henrique Bunselmeyer Ferreira, Nádya Pesce da Silveira, Cristian Follmer, Arnaldo Zaha, Mateus Borba Cardoso, and Karina Mariante Monteiro
- Subjects
Electrophoresis ,lcsh:Arctic medicine. Tropical medicine ,lcsh:RC955-962 ,Protein subunit ,Lipoproteins ,Molecular Sequence Data ,Biophysics ,Biochemistry ,Mass Spectrometry ,law.invention ,law ,Echinococcosis ,Genetics ,Parasitic Diseases ,Parasite hosting ,Animals ,Humans ,Amino Acid Sequence ,Echinococcus granulosus ,Relative species abundance ,Peptide sequence ,Biology ,chemistry.chemical_classification ,Microscopy ,biology ,Sequence Homology, Amino Acid ,Zoonotic Diseases ,lcsh:Public aspects of medicine ,Public Health, Environmental and Occupational Health ,Proteins ,lcsh:RA1-1270 ,biology.organism_classification ,Molecular biology ,Amino acid ,RELAÇÕES HOSPEDEIRO-PARASITA ,Metacestode ,Protein Subunits ,Infectious Diseases ,chemistry ,Veterinary Diseases ,Recombinant DNA ,Medicine ,Cattle ,Veterinary Science ,Protein Multimerization ,Zoology ,Research Article - Abstract
Background Antigen B (AgB) is the major protein secreted by the Echinococcus granulosus metacestode and is involved in key host-parasite interactions during infection. The full comprehension of AgB functions depends on the elucidation of several structural aspects that remain unknown, such as its subunit composition and oligomeric states. Methodology/Principal Findings The subunit composition of E. granulosus AgB oligomers from individual bovine and human cysts was assessed by mass spectrometry associated with electrophoretic analysis. AgB8/1, AgB8/2, AgB8/3 and AgB8/4 subunits were identified in all samples analyzed, and an AgB8/2 variant (AgB8/2v8) was found in one bovine sample. The exponentially modified protein abundance index (emPAI) was used to estimate the relative abundance of the AgB subunits, revealing that AgB8/1 subunit was relatively overrepresented in all samples. The abundance of AgB8/3 subunit varied between bovine and human cysts. The oligomeric states formed by E. granulosus AgB and recombinant subunits available, rAgB8/1, rAgB8/2 and rAgB8/3, were characterized by native PAGE, light scattering and microscopy. Recombinant subunits showed markedly distinct oligomerization behaviors, forming oligomers with a maximum size relation of rAgB8/3>rAgB8/2>rAgB8/1. Moreover, the oligomeric states formed by rAgB8/3 subunit were more similar to those observed for AgB purified from hydatid fluid. Pressure-induced dissociation experiments demonstrated that the molecular assemblies formed by the more aggregative subunits, rAgB8/2 and rAgB8/3, also display higher structural stability. Conclusions/Significance For the first time, AgB subunit composition was analyzed in samples from single hydatid cysts, revealing qualitative and quantitative differences between samples. We showed that AgB oligomers are formed by different subunits, which have distinct abundances and oligomerization properties. Overall, our findings have significantly contributed to increase the current knowledge on AgB expression and structure, highlighting issues that may help to understand the parasite adaptive response during chronic infection., Author Summary Antigen B (AgB) is the major secretory protein of the Echinococcus granulosus hydatid cyst, the causative agent of cystic hydatid disease. Structurally, AgB is a multisubunit protein formed by 8-kDa subunits, but it is not known which subunits are secreted by a single parasite (cyst) and how they interact in the formation of distinct AgB oligomeric states. Here, we investigated AgB subunit composition and oligomeric states in individual samples from bovine and human cysts. We identified AgB8/1, AgB8/2, AgB8/3 and AgB8/4 subunits in AgB oligomers of all samples analyzed. Quantitative and qualitative differences in the expression of AgB subunits were observed within and between samples. Using recombinant subunits as models, we showed that AgB subunits form distinct oligomeric states, with a rAgB8/3>rAgB8/2>rAgB8/1 maximum size relation. We also demonstrated by different experimental approaches that rAgB8/3 oligomers are more similar, both in size and morphology, to those observed for E. granulosus AgB. Overall, we provided experimental evidences that AgB is composed of different subunits within a single cyst, and that subunits have different abundances and oligomerization properties. These issues are important for the understanding of AgB expression and structure variations, and their impact for the host-parasite cross-talk.
- Published
- 2011
108. Colorimetric microwell plate reverse-hybridization assay for Mycobacterium tuberculosis detection
- Author
-
Rosa Dea Sperhacke, Andrezza Woloski Ribeiro, Elis Regina Dalla Costa, Franciele Rosso, Candice Tosi Michelon, Maria Lucia Rosa Rossetti, Arnaldo Zaha, Leonides Rezende Jr, Mirela Verza, Patrícia Izquierdo Cafrune, Márcia Susana Nunes Silva, Daniele Kuhleis, Karen Barros Schmid, Afrânio Lineu Kritski, and Martha Maria Oliveira
- Subjects
Microbiology (medical) ,DNA, Bacterial ,Reverse ,Tuberculosis ,lcsh:Arctic medicine. Tropical medicine ,Hybridization assay ,lcsh:RC955-962 ,lcsh:QR1-502 ,Biology ,Polymerase Chain Reaction ,Sensitivity and Specificity ,Colorimetry (chemical method) ,lcsh:Microbiology ,law.invention ,Mycobacterium tuberculosis ,Hibridização ,oligonucleotide probe ,law ,molecular diagnosis ,medicine ,Humans ,Tuberculose ,reverse-hybridization assay ,Tuberculosis, Pulmonary ,Polymerase chain reaction ,IS6110 ,Microwell Plate ,Sputum ,Nucleic Acid Hybridization ,medicine.disease ,biology.organism_classification ,Molecular biology ,DNA extraction ,tuberculosis ,Colorimetry ,Reagent Kits, Diagnostic ,medicine.symptom ,Oligomer restriction ,Oligonucleotide Probes - Abstract
Direct smear examination using Ziehl-Neelsen staining for pulmonary tuberculosis (PTB) diagnosis is inexpensive and easy to use, but has the major limitation of low sensitivity. Rapid molecular methods are becoming more widely available in centralized laboratories, but they depend on timely reporting of results and strict quality assurance obtainable only from costly commercial kits available in high burden nations. This study describes a pre-commercial colorimetric method, Detect-TB, for detecting Mycobacterium tuberculosis DNA in which an oligonucleotide probe is fixed onto wells of microwell plates and hybridized with biotinylated polymerase chain reaction amplification products derived from clinical samples. The probe is capable of hybridising with the IS6110 insertion element and was used to specifically recognise the M. tuberculosis complex. When combined with an improved silica-based DNA extraction method, the sensitivity of the test was 50 colony-forming units of the M. tuberculosis reference strain H37Rv. The results that were in agreement with reference detection methods were observed in 95.2% (453/476) of samples included in the analysis. Sensitivity and specificity for 301 induced sputum samples and 175 spontaneous sputum samples were 85% and 98%, and 94% and 100%, respectively. This colorimetric method showed similar specificity to that described for commercially available kits and may provide an important contribution for PTB diagnosis.
- Published
- 2011
109. Detection of rifampin-resistant genotypes in Mycobacterium tuberculosis by reverse hybridization assay
- Author
-
Márcia Susana Nunes Silva, Arnaldo Zaha, Rosa Dea Sperhacke, Harrison Magdinier Gomes, Marta Osório Ribeiro, Raquel de Abreu Maschmann, Philip Noel Suffys, Enrico Tortoli, Mirela Verza, Fiorella Marcelli, and Maria Lucia Rosa Rossetti
- Subjects
Antibiotics ,lcsh:QR1-502 ,Drug resistance ,Polymerase Chain Reaction ,lcsh:Microbiology ,law.invention ,reverse dot blot hybridization ,Hibridização ,law ,Genotype ,polycyclic compounds ,Polymerase chain reaction ,Nucleic Acid Hybridization ,DNA-Directed RNA Polymerases ,Blot ,Blotting, Southern ,tuberculosis ,Rifampin ,rifampin ,DNA, Bacterial ,Microbiology (medical) ,lcsh:Arctic medicine. Tropical medicine ,Tuberculosis ,lcsh:RC955-962 ,medicine.drug_class ,Microbial Sensitivity Tests ,Biology ,Sensitivity and Specificity ,Rifampicina ,Mycobacterium tuberculosis ,Bacterial Proteins ,Drug Resistance, Bacterial ,medicine ,Humans ,Tuberculose ,Renverse dot blot hybridization ,Antibiotics, Antitubercular ,drug resistance ,biochemical phenomena, metabolism, and nutrition ,bacterial infections and mycoses ,rpoB ,medicine.disease ,biology.organism_classification ,Virology ,Molecular biology ,Mutation - Abstract
We used a colorimetric reverse dot blot hybridization (CRDH) assay to detect the presence of mutations in a specific region of the rpoB gene, associated with rifampin (RIF) resistance, in a panel of 156 DNAs extracted from 103 RIF-sensitive and 53 RIF-resistant cultures of Mycobacterium tuberculosis. When compared with the antimicrobial susceptibility test (AST), the sensitivity and specificity of the CRDH were 92.3% and 98.1%, respectively. When compared with sequencing, the sensitivity and specificity of the CRDH were 90.6% and 100%, respectively. To evaluate the performance of the assay directly in clinical specimens, 30 samples from tuberculosis patients were used. For these samples, the results of the CRDH were 100% consistent with the results of the AST and sequencing. These results indicate that the rate of concordance of the CRDH is high when compared to conventional methods and sequencing data. The CRDH can be successfully applied when a rapid test is required for the identification of RIF resistance in M. tuberculosis.
- Published
- 2011
110. Survey of transcripts expressed by the invasive juvenile stage of the liver fluke Fasciola hepatica
- Author
-
Nicolás Dell’Oca, Carlos Carmona, Pablo Smircich, Fernando Alvarez-Valin, Leda Roche, Natalia Ruétalo, Edileuza Danieli da Silva, Martín Cancela, Gabriel Rinaldi, José F. Tort, and Arnaldo Zaha
- Subjects
lcsh:QH426-470 ,lcsh:Biotechnology ,030231 tropical medicine ,Genomics ,Proteomics ,Genome ,Host-Parasite Interactions ,03 medical and health sciences ,0302 clinical medicine ,Hepatica ,lcsh:TP248.13-248.65 ,parasitic diseases ,Genetics ,Animals ,Fasciola hepatica ,030304 developmental biology ,Expressed Sequence Tags ,2. Zero hunger ,0303 health sciences ,Expressed sequence tag ,biology ,Gene Expression Profiling ,Liver fluke ,biology.organism_classification ,lcsh:Genetics ,Functional genomics ,Research Article ,Biotechnology - Abstract
BackgroundThe common liver flukeFasciola hepaticais the agent of a zoonosis with significant economic consequences in livestock production worldwide, and increasing relevance to human health in developing countries. Although flukicidal drugs are available, re-infection and emerging resistance are demanding new efficient and inexpensive control strategies. Understanding the molecular mechanisms underlying the host-parasite interaction provide relevant clues in this search, while enlightening the physiological adaptations to parasitism. Genomics and transcriptomics are still in their infancy inF. hepatica, with very scarce information available from the invasive newly excysted juveniles (NEJ). Here we provide an initial glimpse to the transcriptomics of the NEJ, the first stage to interact with the mammalian host.ResultsWe catalogued more than 500 clusters generated from the analysis ofF. hepaticajuvenile expressed sequence tags (EST), several of them not detected in the adult stage. A set of putativeF. hepaticaspecific transcripts, and a group of sequences conserved exclusively in flatworms were identified. These novel sequences along with a set of parasite transcripts absent in the host genomes are putative new targets for future anti-parasitic drugs or vaccine development.Comparisons of theF. hepaticasequences with other metazoans genomes or EST databases were consistent with the basal positioning of flatworms in the bilaterian phylogeny. Notably, GC content, codon usage and amino acid frequencies are remarkably different in Schistosomes toF. hepaticaand other trematodes.Functional annotation of predicted proteins showed a general representation of diverse biological functions. Besides proteases and antioxidant enzymes expected to participate in the early interaction with the host, various proteins involved in gene expression, protein synthesis, cell signaling and mitochondrial enzymes were identified. Differential expression of secreted protease gene family members between juvenile and adult stages may respond to different needs during host colonization.ConclusionThe knowledge of the genes expressed by the invasive stage ofFasciola hepaticais a starting point to unravel key aspects of this parasite's biology. The integration of the emerging transcriptomics, and proteomics data and the advent of functional genomics tools in this organism are positioningF. hepaticaas an interesting model for trematode biology.
- Published
- 2010
111. Proteomic analysis of the Echinococcus granulosus metacestode during infection of its intermediate host
- Author
-
Henrique Bunselmeyer Ferreira, Karina Mariante Monteiro, Arnaldo Zaha, and Marcos Oliveira de Carvalho
- Subjects
biology ,Echinococcus granulosus ,Proteome ,Effector ,Intermediate host ,Helminth Proteins ,biology.organism_classification ,Biochemistry ,Host-Parasite Interactions ,Metacestode ,Immune system ,Antigen ,Immunity ,Echinococcosis ,parasitic diseases ,Immunology ,biology.protein ,Calgranulin ,Animals ,Cattle ,Molecular Biology - Abstract
Cystic hydatid disease (CHD) is caused by infection with the Echinococcus granulosus metacestode and affects both humans and livestock. In this work, we performed a proteomic analysis of the E. granulosus metacestode during infection of its intermediate bovine host. Parasite proteins were identified in different metacestode components (94 from protoscolex, 25 from germinal layer and 20 from hydatid cyst fluid), along with host proteins (58) that permeate into the hydatid cyst, providing new insights into host-parasite interplay. E. granulosus and platyhelminth EST data allowed successful identification of proteins potentially involved in downregulation of host defenses, highlighting possible evasion mechanisms adopted by the parasite to establish infection. Several intracellular proteins were found in hydatid cyst fluid, revealing a set of newly identified proteins that were previously thought to be inaccessible for inducing or modulating the host immune response. Host proteins identified in association with the hydatid cyst suggest that the parasite may bind/adsorb host molecules with nutritional and/or immune evasion purposes, masking surface antigens or inhibiting important effector molecules of host immunity, such as complement components and calgranulin. Overall, our results provide valuable information on parasite survival strategies in the adverse host environment and on the molecular mechanisms underpinning CHD immunopathology.
- Published
- 2010
112. Biochemical analysis of a recombinant glutathione transferase from the cestode Echinococcus granulosus
- Author
-
Cora Chalar, Verónica Fernández, Gabriela Garcia, Paula Arbildi, Cecilia Fernández, Arnaldo Zaha, Leticia Pascovich, and Laura Harispe
- Subjects
Veterinary (miscellaneous) ,Substrate Specificity ,Lipid peroxidation ,chemistry.chemical_compound ,parasitic diseases ,Taenia solium ,Glycosyltransferase ,medicine ,Benzene Derivatives ,Dinitrochlorobenzene ,Animals ,Enzyme Inhibitors ,Echinococcus granulosus ,Glutathione Transferase ,chemistry.chemical_classification ,Aldehydes ,Glutathione Peroxidase ,biology ,Glutathione peroxidase ,biology.organism_classification ,Molecular biology ,Recombinant Proteins ,medicine.drug_formulation_ingredient ,Infectious Diseases ,Enzyme ,Ethacrynic Acid ,chemistry ,Biochemistry ,Cumene hydroperoxide ,Insect Science ,biology.protein ,Parasitology ,Dimerization ,Peroxidase - Abstract
Glutathione transferases (GSTs) are believed to be a major detoxification system in helminths. We describe the expression and functional analysis of EgGST, a cytosolic GST from Echinococcus granulosus, related to the Mu-class of mammalian enzymes. EgGST was produced as an enzymatically active dimeric protein (rEgGST), with highest specific activity towards the standard substrate 1-chloro-2,4-dinitrobenzene (CDNB; 2.5 μmol min−1 mg−1), followed by ethacrynic acid. Interestingly, rEgGST displayed glutathione peroxidase activity (towards cumene hydroperoxide), and conjugated reactive carbonyls (trans-2-nonenal and trans,trans-2,4-decadienal), indicating that it may intercept damaging products of lipid peroxidation. In addition, classical GST inhibitors (cybacron blue, triphenylthin chloride and ellagic acid) and a number of anthelmintic drugs (mainly, hexachlorophene and rafoxanide) were found to interfere with glutathione-conjugation to CDNB; suggesting that they may bind to EgGST. Considered globally, the functional properties of rEgGST are similar to those of putative orthologs from Echinococcus multilcularis and Taenia solium, the other medically important cestodes. Interestingly, our results also indicate that differences exist between these closely related cestode GSTs, which probably reflect specific biological functions of the molecules in each parasitic organism.
- Published
- 2009
113. A peroxiredoxin from Mycoplasma hyopneumoniae with a possible role in H2O2 detoxification
- Author
-
Claudio Xavier Machado, Arnaldo Zaha, Paulo Marcos Pinto, and Henrique Bunselmeyer Ferreira
- Subjects
DNA, Bacterial ,DNA damage ,Molecular Sequence Data ,Gene Expression ,Microbiology ,law.invention ,Mice ,Porcine enzootic pneumonia ,Mycoplasma hyopneumoniae ,law ,Escherichia coli ,Animals ,Amino Acid Sequence ,Cloning, Molecular ,Peptide sequence ,Gene ,Phylogeny ,Antiserum ,Mice, Inbred BALB C ,biology ,Sequence Homology, Amino Acid ,DNA ,Hydrogen Peroxide ,Peroxiredoxins ,biology.organism_classification ,Molecular biology ,Recombinant DNA ,Peroxiredoxin ,Reactive Oxygen Species ,Dimerization ,DNA Damage - Abstract
Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia, which affects pig farms worldwide, causing heavy economic losses. In the infection process, this bacterium is exposed to reactive oxygen species (ROS) from its own metabolism or generated by the host as one of the strategies used to neutralize the pathogen. Although the presence of classical antioxidant enzymes would be expected in M. hyopneumoniae, important genes directly related to protection against ROS, such as superoxide dismutase, catalases and glutathione peroxidase, have not been identified by sequence homology in the genome sequence annotation. Among the few identified M. hyopneumoniae genes coding for proteins possibly involved with suppression of ROS-mediated damage, one (tpx) coding for a peroxiredoxin (MhPrx) has been recognized. The sequence and phylogenetic analyses perfomed in this study indicate that MhPrx is closely related to the atypical 2-Cys peroxiredoxin subfamily, although it has only one cysteine in its sequence. The MhPrx coding DNA sequence was cloned and expressed in Escherichia coli to produce a recombinant MhPrx (rMhPrx), which was purified and used to immunize mice and produce an anti-MhPrx polyclonal antiserum. Probing of M. hyopneumoniae extracts with this antiserum demonstrated that MhPrx is expressed in all three tested strains (J, 7422 and 7448). Cross-linking assays and size-exclusion chromatography indicate that rMhPrx forms dimers, as has been established for atypical 2-Cys peroxiredoxins. Furthermore, a metal-catalysed oxidation system was used to assay the activity of rMhPrx, showing that it can protect DNA from ROS-mediated damage and may play an essential role during infection.
- Published
- 2009
114. In house colorimetric reverse hybridisation assay for detection of the mutation most frequently associated with resistance to isoniazid in Mycobacterium tuberculosis
- Author
-
Enrico Tortoli, Mirela Verza, Márcia Susana Nunes Silva, Fiorella Marcelli, Raquel de Abreu Maschmann, Marta Osório Ribeiro, Elis Regina Dalla Costa, Philip Noel Suffys, Franciele Rosso, Maria Lucia Rosa Rossetti, and Arnaldo Zaha
- Subjects
DNA, Bacterial ,Microbiology (medical) ,isoniazid ,Isoniazida ,lcsh:Arctic medicine. Tropical medicine ,Tuberculosis ,lcsh:RC955-962 ,Mutant ,Antitubercular Agents ,lcsh:QR1-502 ,Microbial Sensitivity Tests ,Drug resistance ,Polymerase Chain Reaction ,lcsh:Microbiology ,Microbiology ,law.invention ,Mycobacterium tuberculosis ,Bacterial Proteins ,Hibridização ,law ,Drug Resistance, Bacterial ,medicine ,Tuberculose ,Polymerase chain reaction ,drug resistance ,biology ,reverse hybridization assay ,Isoniazid ,Nucleic Acid Hybridization ,Catalase ,bacterial infections and mycoses ,medicine.disease ,biology.organism_classification ,Virology ,Multiple drug resistance ,katG315 ,tuberculosis ,Mutation ,Mutation (genetic algorithm) ,Colorimetry ,medicine.drug - Abstract
Mutations in the katG gene have been identified and correlated with isoniazid (INH) resistance in Mycobacterium tuberculosis isolates. The mutation AGC→ACC (Ser→Thr) at katG315 has been reported to be the most frequent and is associated with transmission and multidrug resistance. Rapid detection of this mutation could therefore improve the choice of an adequate anti-tuberculosis regimen, the epidemiological monitoring of INH resistance and, possibly, the tracking of transmission of resistant strains. An in house reverse hybridisation assay was designed in our laboratory and evaluated with 180 isolates of M. tuberculosis. It could successfully characterise the katG315 mutation in 100% of the samples as compared to DNA sequencing. The test is efficient and is a promising alternative for the rapid identification of INH resistance in regions with a high prevalence of katG315 mutants.
- Published
- 2009
115. High prevalence of Neisseria meningitidis hypervirulent lineages and emergence of W135:P1.5,2:ST-11 clone in Southern Brazil
- Author
-
Leonard W. Mayer, Ludmila F. Baethgen, Cecilia C. Klein, Arnaldo Zaha, Sílvia Rios, Luciana de Souza Nunes, Claudete Iris Kmetzsch, Camile Moraes, Maria Lucia Rosa Rossetti, and Luciana Weidlich
- Subjects
Microbiology (medical) ,DNA, Bacterial ,Population ,Porins ,Population biology ,Neisseria meningitidis, Serogroup C ,Meningitis, Meningococcal ,Neisseria meningitidis ,Neisseria meningitidis, Serogroup B ,Meningococcal disease ,medicine.disease_cause ,Neisseria meningitidis, Serogroup W-135 ,medicine ,Prevalence ,Humans ,Typing ,Serotyping ,education ,education.field_of_study ,Molecular Epidemiology ,biology ,Incidence (epidemiology) ,Infant ,Sequence Analysis, DNA ,medicine.disease ,biology.organism_classification ,Virology ,Bacterial Typing Techniques ,Meningococcal Infections ,Infectious Diseases ,Child, Preschool ,Population Surveillance ,Multilocus sequence typing ,Neisseriaceae ,Brazil - Abstract
Summary Objectives The aim of this study was to characterize Neisseria meningitidis strains causing invasive disease in Rio Grande do Sul (RS), during 2003–2005, monitoring the occurrence of hypervirulent lineages, as well as to determine the diversity of PorA VR types for the corresponding isolates and clinical specimens. Methods Isolates and clinical specimens were characterized by MLST and PorA VR typing. Results This study demonstrated high prevalence of some hypervirulent lineages and emergence of new ones, including the emergence of lineages W135:P1.5,2:ST-11 complex, and C:P1.22,14-6:ST-103 complex. These lineages are probably responsible for the increasing incidence of serogroups C and W135, despite the overall decrease in serogroup B cases during the period. The most prevalent complex was serogroup B ST-32/ET-5 complex. The most prevalent PorA types found for serogroup B were P1.19,15, P1.7,16, and P1.18-1,3, representing a different distribution of PorA types compared to other states of Brazil. Conclusions This study highlights the importance of monitoring each population, even within the same country. The different distribution of PorA VR types in RS has implications in vaccine design and efficacy. Detailed and accurate meningococcal characterization is an important element in studies of meningococcal epidemiology, population biology, and evolution and provides information for the design of control strategies.
- Published
- 2008
116. DNA damage, RAD9 and fertility/infertility of Echinococcus granulosus hydatid cysts
- Author
-
Ulf Hellman, Arnaldo Zaha, María Eugenia Cabrejos, Norbel Galanti, Juana Orellana, Carolina Cabezón, Gonzalo Cabrera, and Alessandra Loureiro Morassutti
- Subjects
Infertility ,Models, Molecular ,DNA Repair ,Physiology ,DNA repair ,DNA damage ,Clinical Biochemistry ,Molecular Sequence Data ,Cell Cycle Proteins ,medicine.disease_cause ,Andrology ,Echinococcosis ,parasitic diseases ,medicine ,Animals ,Humans ,Amino Acid Sequence ,Echinococcus granulosus ,Gene ,Phylogeny ,biology ,Cell Biology ,Anatomy ,Helminth Proteins ,Hydrogen Peroxide ,medicine.disease ,biology.organism_classification ,Oxidants ,Protein Structure, Tertiary ,Oxidative Stress ,Fertility ,Apoptosis ,Reactive Oxygen Species ,Oxidation-Reduction ,Sequence Alignment ,Oxidative stress ,DNA Damage - Abstract
Hydatidosis, caused by the larval stage of the platyhelminth parasite Echinococcus granulosus, affects human and animal health. Hydatid fertile cysts are formed in intermediate hosts (human and herbivores) producing protoscoleces, the infective form to canines, at their germinal layers. Infertile cysts are also formed, but they are unable to produce protoscoleces. The molecular mechanisms involved in hydatid cysts fertility/infertility are unknown. Nevertheless, previous work from our laboratory has suggested that apoptosis is involved in hydatid cyst infertility and death. On the other hand, fertile hydatid cysts can resist oxidative damage due to reactive oxygen and nitrogen species. On these foundations, we have postulated that when oxidative damage of DNA in the germinal layers exceeds the capability of DNA repair mechanisms, apoptosis is triggered and hydatid cysts infertility occurs. We describe a much higher percentage of nuclei with oxidative DNA damage in dead protoscoleces and in the germinal layer of infertile cysts than in fertile cysts, suggesting that DNA repair mechanisms are active in fertile cysts. rad9, a conserved gene, plays a key role in cell cycle checkpoint modulation and DNA repair. We found that RAD9 of E. granulosus (EgRAD9) is expressed at the mRNA and protein levels. As it was found in other eukaryotes, EgRAD9 is hyperphosphorylated in response to DNA damage. Our results suggest that molecules involved in DNA repair in the germinal layer of fertile hydatid cysts and in protoscoleces, such as EgRAD9, may allow preserving the fertility of hydatid cysts in the presence of ROS and RNS.
- Published
- 2008
117. A distinctive repertoire of cathepsins is expressed by juvenile invasive Fasciola hepatica
- Author
-
Martín Cancela, Arnaldo Zaha, Rosario Durán, Edileusa Silva, Carlos Carmona, José F. Tort, Gabriel Rinaldi, Leda Roche, and Daniel Acosta
- Subjects
Proteases ,Cathepsin L ,Molecular Sequence Data ,Biochemistry ,Cathepsin B ,Cathepsin K ,Parasite hosting ,Fasciola hepatica ,Animals ,Humans ,Protein Isoforms ,Amino Acid Sequence ,RNA, Messenger ,Cloning, Molecular ,Phylogeny ,chemistry.chemical_classification ,Cathepsin ,biology ,cDNA library ,Gene Expression Regulation, Developmental ,General Medicine ,biology.organism_classification ,Cathepsins ,Cysteine Endopeptidases ,Enzyme ,chemistry ,Cattle ,Cysteine - Abstract
Secreted cysteine proteases are relevant actors in parasite biology, taking part in critical host colonization roles such as traversing tissue barriers, immune evasion and nutrient digestion. In the trematode Fasciola hepatica, the initial step to successful infection of the mammalian host is the excystment of metacercariae and the invasion through the intestinal wall by the newly excysted juveniles (NEJ). While the cathepsin L-like cysteine proteinases secreted by the adult fluke have been extensively characterized, the cataloguing and description of the cathepsins B and L reported in the invasive stages is only sketchy. To identify the cathepsins expressed during excystment and early invasion we constructed cDNA libraries encoding NEJ cathepsins B and L. We found two cathepsin L-like cysteine proteinases (CL3, CL4) and three cathepsins B (CB1, CB2, CB3) which are predominantly expressed in NEJ. Phylogenetic analysis showed that NEJ-expressed cathepsins L constitute a well-defined clade separate from the adult enzymes. Excystment induction resulted in a significant increment in activity towards cathepsin-specific fluorogenic substrates in metacercariae homogenates, consistent with the detection of precursor and mature forms of cathepsins B and L before and after induction. In NEJ culture supernatants, protein and relative activity profiles show subtle changes during the first 48 h, with prevalence of cathepsin L-like activity, although cathepsins CB3 and CL3 were detected by mass spectrometry. Noticeably, the hydrolysis of a substrate with proline in the P2 position was predominant, a property only shared with adult CL2 and vertebrate cathepsin K among the C1A subfamily of cysteine proteases. Collectively these mRNA, protein and enzymatic data demonstrate the existence of a NEJ-specific repertoire of cathepsins expressed early in invasion, distinct to those used by other trematodes, potentially relevant for specific vaccine and chemotherapy design. The diversity of proteases employed by trematodes in the invasion process is discussed.
- Published
- 2008
118. Restriction-modification systems in Mycoplasma spp
- Author
-
Ana Tereza Ribeiro de Vasconcelos, Arnaldo Zaha, and Marcelo Brocchi
- Subjects
Genetics ,Phase variation ,Transposable element ,lcsh:QH426-470 ,biology ,Mycoplasma ,medicine.disease_cause ,biology.organism_classification ,Genome ,Mycoplasma spp ,lcsh:Genetics ,Mycoplasma hyopneumoniae ,Genomic island ,medicine ,restriction-modification systems ,Selfish DNA ,genomes ,Molecular Biology ,Gene - Abstract
Restriction and Modification (R-M) systems are present in all Mycoplasma species sequenced so far. The presence of these genes poses barriers to gene transfer and could protect the cell against phage infections. The number and types of R-M genes between different Mycoplasma species are variable, which is characteristic of a polymorphism. The majority of the CDSs code for Type III R-M systems and particularly for methyltransferase enzymes, which suggests that functions other than the protection against the invasion of heterologous DNA may exist. A possible function of these enzymes could be the protection against the invasion of other but similar R-M systems. In Mycoplasma hyopneumoniae strain J, three of the putative methyltransferase genes were clustered in a region forming a genomic island. Many R-M CDSs were mapped in the vicinity of transposable elements suggesting an association between these genes and reinforcing the idea of R-M systems as mobile selfish DNA. Also, many R-M genes present repeats within their coding sequences, indicating that their expression is under the control of phase variation mechanisms. Altogether, these data suggest that R-M systems are a remarkable characteristic of Mycoplasma species and are probably involved in the adaptation of these bacteria to different environmental conditions.
- Published
- 2007
119. AB0130 Immunomodulatory and Antiinflamatory Properties of Antigen B, A Lipoprotein Secreted on Hydatic Cyst of Echinococcus Granulosus, in Experimental Arthritis
- Author
-
Vanessa Schuck Clarimundo, Henrique Bunselmeyer Ferreira, Ricardo Machado Xavier, Karina Mariante Monteiro, Arnaldo Zaha, P.G. de Oliveira, and Mirian Farinon
- Subjects
biology ,business.industry ,medicine.medical_treatment ,Immunology ,Zymosan ,Arthritis ,biology.organism_classification ,medicine.disease ,General Biochemistry, Genetics and Molecular Biology ,chemistry.chemical_compound ,Immune system ,Cytokine ,Nociception ,Rheumatology ,Antigen ,chemistry ,medicine ,Immunology and Allergy ,business ,Echinococcus granulosus ,Adjuvant - Abstract
Background Antigen B (AgB) is a lipoprotein secreted in hydatic cyst by Echinococcus granulosus larval stage (1) and seems to be responsible to regulate immune balance via Th2 response to promotes survival of the parasite (2). A Th2 response can suppress the pro-inflammatory Th1 response generated in several immunopathologies. Objectives To evaluate the effect of AgB in three animal models of arthritis. Methods In all models, mice were divided into three groups: vehicle (saline) or AgB (2 and 10μg – once a day, intraperitoneal). BALB/c mice (n=21) were treated and then injected with zymosan into knee joint for development of zymosan Induced-arthritis (ZYA). Nociception in 0, 4 and 6 hours and leukocytes articular migration 6 hours after intra-articular (ia) injection were assessed. Antigen Induced-arthritis (AIA) was induced in BALB/c (n=36) with methylated bovine serum albumin (mBSA) and treatment started one day before ia injection of mBSA. Paw nociception in 0, 3, 5, 7 and 24h and neutrophils migration into knee joints 24h after ia injection of mBSA were evaluated. DBA/1J mice had Collagen Induced-arthritis (CIA) and were divided into preventive (n=25, 18 days of treatment) or therapeutic groups (n=27, 10 days of treatment starting after the onset of arthritis). In the preventive group, articular score, nociception, paw edema and body weight were evaluated. In the therapeutic group, articular score and nociception were evaluated and knee joints were collected to analyses cytokine th1/th2/th17 profile. Statistical analysis: ANOVA or Kruskal-Wallis. Results In ZYA, both treatment reduced nociception in 4 and 6h (p Conclusions Treatment with AgB significantly improved acute experimental arthritis, attenuated nociception and immune cells articular migration. On the other hand, AgB had no effect in chronic experimental arthritis, although therapeutic treatment reduced Il-6 and TNF-a levels, two important pro-inflammatory cytokines and prophylactic treatment improved nociception. We believe that the adjuvant use of AgB with a DMARD can lead to an additive effect on amelioration of immune-mediated arthritis short-term symptoms. References Oriol R. et al. Am J Trop Med Hyg. 1971; 20(4):569. Siracusano A. et al. Exp Parasitol. 2008; 119(4):483-9. Acknowledgements FIPE-HCPA, CAPES, CNPq Disclosure of Interest None declared
- Published
- 2015
120. Characterization of pncA Mutations in Pyrazinamide-Resistant Mycobacterium tuberculosis in Brazil
- Author
-
Arnaldo Zaha, Maria Alice da Silva Telles, Patrícia Izquierdo Cafrune, Vivian de F.S. Rodrigues, Maria Lucia Rosa Rossetti, and Marta Osório Ribeiro
- Subjects
Tuberculosis ,Antitubercular Agents ,Microbial Sensitivity Tests ,medicine.disease_cause ,Amidohydrolases ,Mycobacterium tuberculosis ,Mechanisms of Resistance ,Drug Resistance, Bacterial ,medicine ,Pharmacology (medical) ,Gene ,Antibacterial agent ,Pharmacology ,Mutation ,biology ,Nucleic acid sequence ,Pyrazinamide ,biology.organism_classification ,medicine.disease ,Virology ,Infectious Diseases ,PncA ,Erratum ,medicine.drug - Abstract
In this study the nucleotide sequence of the pncA gene from 59 Mycobacterium tuberculosis clinical isolates was analyzed. Mutations in the pncA gene were identified in 29 of 40 pyrazinamide-resistant isolates, and no pyrazinamidase activity was detected in 39 of them. Twelve mutations found in this work have not been described previously.
- Published
- 2006
121. Evaluation and genotyping of multidrug-resistant cases of tuberculosis in southern Brazil
- Author
-
Lia Gonçalves Possuelo, Marta Osório Ribeiro, Andréia Rosane de Moura Valim, Michele Borges, Maria Lucia Rosa Rossetti, Arnaldo Zaha, and Patrícia Izquierdo Cafrune
- Subjects
Microbiology (medical) ,Adult ,Male ,Tuberculosis ,Genotype ,Immunology ,Reference laboratory ,Microbiology ,Mycobacterium tuberculosis ,Polymorphism (computer science) ,Tuberculosis, Multidrug-Resistant ,medicine ,Humans ,Genotyping ,Tuberculosis, Pulmonary ,Pharmacology ,biology ,Marital Status ,Middle Aged ,biology.organism_classification ,medicine.disease ,Virology ,Bacterial Typing Techniques ,Multiple drug resistance ,DNA Transposable Elements ,Female ,Restriction fragment length polymorphism ,Tb treatment ,Brazil ,Polymorphism, Restriction Fragment Length - Abstract
Sixty isolates of Mycobacterium tuberculosis identified as multidrug-resistant (MDR) at a reference laboratory in Rio Grande do Sul State during the years 1999 and 2000 were analyzed using the IS6110-restriction fragment length polymorphism (RFLP) technique. We also genotyped 202 susceptible strains to compare the genotyping results, as well as the clinical and demographic data. Spacer oligotyping (spoligotyping) analysis was performed for isolates presenting low IS6110 copy number. Patients with identical DNA pattern strains were considered clustered. From 262 isolates, 94 (36%) belonged to 20 distinct RFLP clusters, and after spoligotyping analysis, 89 of the isolates (34%) remained in cluster. MDR isolates did not differ statistically in clustering proportion from susceptible strains. A significant association between the occurrence of MDR and previous tuberculosis (TB) treatment was observed (p0.001), as well as failure on TB treatment (p0.001). Human immunodeficiency virus (HIV)-positive patients were associated with susceptible tuberculosis (p = 0.024). We also identified that unmarried patients were more likely to develop TB due to recent transmission than married patients (p0.005). The introduction of directly observed therapy short-course (DOTS) strategy will be important in decreasing default and failure rates and avoiding the development of new MDR strains.
- Published
- 2006
122. Self-assembly and structural characterization of Echinococcus granulosus antigen B recombinant subunit oligomers
- Author
-
Nádya Pesce da Silveira, Paulo Fernando Bruno Gonçalves, Karina Mariante Monteiro, Nilson Ivo Tonin Zanchin, Sandra M.N. Scapin, Marcos V.A.S. Navarro, Arnaldo Zaha, Hubert Stassen, Henrique Bunselmeyer Ferreira, and Mateus Borba Cardoso
- Subjects
Models, Molecular ,Protein Conformation ,Protein subunit ,Proteolysis ,Size-exclusion chromatography ,Molecular Sequence Data ,Static Electricity ,Biophysics ,Biology ,Biochemistry ,Protein Structure, Secondary ,Analytical Chemistry ,law.invention ,Hydrophobic effect ,Biopolymers ,Dynamic light scattering ,law ,medicine ,Animals ,Amino Acid Sequence ,Echinococcus granulosus ,Molecular Biology ,Thermostability ,medicine.diagnostic_test ,Sequence Homology, Amino Acid ,Circular Dichroism ,biology.organism_classification ,Recombinant Proteins ,Spectrometry, Fluorescence ,Antigens, Helminth ,Recombinant DNA ,Chromatography, Gel - Abstract
Echinococcus granulosus antigen B is an oligomeric protein of 120–160 kDa composed by 8-kDa (AgB8) subunits. Here, we demonstrated that the AgB8 recombinant subunits AgB8/1, AgB8/2 and AgB8/3 are able to self-associate into high order homo-oligomers, showing similar properties to that of parasite-produced AgB, making them valuable tools to study AgB structure. Dynamic light scattering, size exclusion chromatography and cross-linking assays revealed ~ 120- to 160-kDa recombinant oligomers, with a tendency to form populations with different aggregation states. Recombinant oligomers showed helical circular dichroism spectra and thermostability similar to those of purified AgB. Cross-linking and limited proteolysis experiments indicated different degrees of stability and compactness between the recombinant oligomers, with the AgB8/3 one showing a more stable and compact structure. We have also built AgB8 subunit structural models in order to predict the surfaces possibly involved in electrostatic and hydrophobic interactions during oligomerization.
- Published
- 2006
123. Proteomic survey of the pathogenic Mycoplasma hyopneumoniae strain 7448 and identification of novel post-translationally modified and antigenic proteins
- Author
-
Luiza Amaral de Castro, Henrique Bunselmeyer Ferreira, Gustavo Chemale, Marilene Henning Vainstein, Paulo Marcos Pinto, Jalusa Deon Kich, Ana Paula Metz Costa, and Arnaldo Zaha
- Subjects
Proteomics ,Swine ,Immunoblotting ,Biology ,Microbiology ,Porcine enzootic pneumonia ,Mycoplasma hyopneumoniae ,Bacterial Proteins ,Tandem Mass Spectrometry ,Heat shock protein ,Animals ,Electrophoresis, Gel, Two-Dimensional ,Mycoplasma Infections ,Gene ,Gel electrophoresis ,General Veterinary ,General Medicine ,Pneumonia of Swine, Mycoplasmal ,biology.organism_classification ,Specific Pathogen-Free Organisms ,Membrane protein ,Proteome ,Protein Processing, Post-Translational - Abstract
Mycoplasma hyopneumoniae is an important pathogen for pigs, being the causative agent of enzootic pneumonia. Recently, the genome sequences of three strains, J, 7448 and 232 have been reported. Here, we describe the results of a proteomic analysis, based on two-dimensional gel electrophoresis of soluble protein extracts, immunoblot and mass spectrometry, which was carried out aiming the identification of gene products and antigenic proteins from the M. hyopneumoniae pathogenic strain 7448. A preliminary M. hyopneumoniae proteome map in two pH ranges (3-10 and 4-7) was produced. A total of 31 different coding DNA sequences (CDSs), including three hypothetical ones, were experimentally verified with the identification of the corresponding protein products by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. According to the Clusters of Orthologous Groups (COG) functional classification, the identified proteins were assigned to the groups of metabolism (13), cellular processes (5) and information and storage processing (4). Nine of the identified proteins were not classifiable by COG, including some related to cytoadherence and possibly involved in pathogenicity. Moreover, at least five highly antigenic proteins of M. hyopneumoniae were identified by immunoblots, including four novel ones (a heat shock protein 70, an elongation factor Tu, a pyruvate dehydrogenase E1-beta subunit and the P76 membrane protein). The now available proteome map is expected to serve as a reference for comparative analyses between M. hyopneumoniae pathogenic and non-pathogenic strains, and for methabolic studies based on cells cultured under modified conditions.
- Published
- 2006
124. Effects of protoscoleces and AgB from Echinococcus granulosus on human neutrophils: possible implications on the parasite's immune evasion mechanisms
- Author
-
A. Ramos, Ana M. Ferreira, Lorena Taroco, Arnaldo Zaha, Ana Hernández, Veridiana Gomes Virginio, and Henrique Bunselmeyer Ferreira
- Subjects
Adult ,Neutrophils ,Neutrophile ,Lipoproteins ,Down-Regulation ,Enzyme-Linked Immunosorbent Assay ,Flow cytometry ,Microbiology ,chemistry.chemical_compound ,Immune system ,Downregulation and upregulation ,parasitic diseases ,medicine ,Animals ,Humans ,Interleukin 8 ,Echinococcus granulosus ,Cells, Cultured ,CD11b Antigen ,General Veterinary ,biology ,medicine.diagnostic_test ,Interleukin-8 ,General Medicine ,Helminth Proteins ,Hydrogen Peroxide ,biology.organism_classification ,Flow Cytometry ,Infectious Diseases ,chemistry ,Integrin alpha M ,Insect Science ,Immunology ,Phorbol ,biology.protein ,Parasitology - Abstract
The factors affecting the innate susceptibility to Echinococcus granulosus infections are largely unknown. We assessed the interaction of healthy human neutrophils with protoscoleces (PSC) and antigen B (AgB) of E. granulosus by analysis of CD11b upregulation and H(2)O(2) production by flow cytometry. PSC induced neutrophil activation, but their viability was not affected. In contrast, no effects were observed with AgB in both assays. Neutrophil-enriched fractions were also incubated with PSC or AgB, and interleukin 8 (IL-8) production was measured by ELISA. Significant increment in IL-8 production was detected only in supernatants from neutrophil-enriched fractions cultured with PSC. The possible effect of a prior incubation with AgB on the phorbol myristate acetate-induced activation was also evaluated. No changes were observed in CD11b expression, but the H(2)O(2) production was significantly reduced in platelet-activating factor (PAF)-primed neutrophils. These results suggest a possible AgB-mediated mechanism of evasion of the host immune response, which would operate upon events of spillage of the fertile hydatid cyst content.
- Published
- 2006
125. Detection of genetic polymorphism among and within Echinococcus granulosus strains by heteroduplex analysis of a microsatellite from the U1 snRNA genes
- Author
-
Roratto, P. A., Bartholomei-Santos, M. L., Gutierrez, A. M., Kamenetzky, L., Rosenzvit, M. C., and Arnaldo Zaha
- Subjects
Echinococcus granulosus ,Repeticiones de Microsatélite ,Ácidos Nucleicos Heterodúplex ,Reacción en Cadena de la Polimerasa ,Polimorfismo Genético - Abstract
Polymerase chain reaction of a pentanucleotide microsatellite in the U1 snRNA gene complex generated a multiple band pattern due to the priming of paralogous sequences. Denaturation and slow renaturation of polymerase chain reaction products allow the formation of heteroduplex DNA that can be detected by its differential mobility in polyacrylamide gel electrophoresis. Heteroduplex analysis was used to determine if the U1 snRNA microsatellite could be a useful genetic marker in Echinococcus granulosus. A U1 snRNA microsatellite fragment from E. granulosus was isolated and characterized by Southern blot and sequencing. Four E. granulosus strains were analyzed: sheep, Tasmanian sheep, cattle, and camel strains. The former two showed polymorphism and shared three of the six patterns found for sheep strain. The cattle strain displayed two patterns, and the camel strain was monomorphic. The electrophoretic profiles were used for statistical analysis in order to determine genetic distance and the relationship among strains. Heteroduplex analysis can be helpful in genotyping E. granulosus strains and is useful in detecting polymorphism within strains. Fil: Roratto, P. A. Universidade Federal de Santa Maria. Departamento de Biologia; Brazil. Fil: Bartholomei-Santos, M. L. Universidade Federal de Santa Maria. Departamento de Biologia; Brazil. Fil: Gutierrez, Ariana M. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Parasitología; Argentina. Fil: Kamenetzky, Laura. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Parasitología; Argentina. Fil: Rosenzvit, Mara C. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Parasitología; Argentina. Fil: Zaha, A. Universidade Federal do Rio Grande do Sul. 3Centro de Biotecnologia; Brazil.
- Published
- 2006
126. Searching for antigen B genes and their adaptive sites in distinct strains and species of the helminth Echinococcus
- Author
-
Karen Luisa Haag, Robin B. Gasser, Arnaldo Zaha, Paolo Marinho de Andrade Zanotto, L. Alves-Junior, and Francisco J. Ayala
- Subjects
Microbiology (medical) ,Lipoproteins ,Molecular Sequence Data ,Echinococcus multilocularis ,Microbiology ,Evolution, Molecular ,Species Specificity ,Phylogenetics ,Molecular evolution ,parasitic diseases ,Gene duplication ,Genetics ,Animals ,Amino Acid Sequence ,Selection, Genetic ,Echinococcus granulosus ,Molecular Biology ,Gene ,Ecology, Evolution, Behavior and Systematics ,Genes, Helminth ,biology ,Phylogenetic tree ,Helminth Proteins ,biology.organism_classification ,Adaptation, Physiological ,Echinococcus ,Infectious Diseases ,Antigens, Helminth ,Multigene Family - Abstract
Twenty-seven PCR-derived antigen B (AgB) nucleotide sequences from four Echinococcus species (Echinococcus granulosus, Echinococcus multilocularis, Echinococcus oligarthrus and Echinococcus vogeli) were aligned with 78 already published sequences, to generate a maximum likelihood phylogeny of the AgB multigene family. The phylogenetic analysis confirms that the family is constituted by four groups of genes present in each one of the four species (AgB1, AgB2, AgB3 and AgB4), and suggests that it originated by ancient duplication events preceding speciation within the genus. AgB5 sequences, which had been formerly suggested to correspond to a putatively new AgB subunit, cluster with AgB3. Likelihood tests suggest that AgB gene evolution may have been driven by heterogeneous selection pressures acting on particular AgB1, AgB3 and AgB4 codons. No selection is detected in AgB2. We discuss implications of our findings in terms of AgB biology and its use as a diagnostic tool.
- Published
- 2005
127. Echinococcus granulosus antigen B hydrophobic ligand binding properties
- Author
-
Henrique Bunselmeyer Ferreira, John Barrett, Arnaldo Zaha, Peter M. Brophy, and Gustavo Chemale
- Subjects
Protein subunit ,Lipoproteins ,Recombinant Fusion Proteins ,Biophysics ,Antibodies, Helminth ,Ligands ,Biochemistry ,Chromatography, Affinity ,Analytical Chemistry ,Palmitic acid ,chemistry.chemical_compound ,Affinity chromatography ,Bacterial Proteins ,Fatty acid binding ,Escherichia coli ,Animals ,Echinococcus granulosus ,Molecular Biology ,chemistry.chemical_classification ,Binding Sites ,biology ,Cyst Fluid ,Fatty Acids ,Fatty acid ,Antibodies, Monoclonal ,Helminth Proteins ,biology.organism_classification ,Ligand (biochemistry) ,Molecular biology ,Dissociation constant ,Kinetics ,Spectrometry, Fluorescence ,chemistry ,Cestoda ,Hydrophobic and Hydrophilic Interactions - Abstract
Antigen B (AgB), an immunodominant component of the cestode parasite Echinococcus granulosus, presents homology to and shares apparent structural similarities with helix-rich hydrophobic ligand binding proteins (HLBPs) from other cestodes. In order to investigate the fatty acid binding properties of AgB, two of its subunit components (rAgB8/1 and rAgB8/2) were expressed in Escherichia coli and purified, and the native antigen was purified from the hydatid cyst fluid by affinity chromatography using a monoclonal antibody raised against rAgB8/1. The interaction of the purified native and recombinant proteins with the fluorescent ligands DAUDA, ANS, DACA and 16-AP was investigated. The palmitic acid derived fluorescent ligand, 16-AP, showed the greatest enhancement in fluorescence when bound to native AgB or to its recombinant subunits, and the dissociation constants for 16-AP binding were determined. Surprisingly, in contrast to HLBPs from other cestodes, interactions with other fatty acids, including palmitic acid, caused an increase in fluorescence instead of competing with 16-AP. Our results suggest that AgB might have evolved different functions in the binding of hydrophobic compounds, dependent on cestode environment.
- Published
- 2004
128. Purification, cloning, and expression of the mitochondrial malate dehydrogenase (mMDH) from protoscolices of Echinococcus granulosus
- Author
-
Fernán Agüero, Arnaldo Zaha, Yolanda Repetto, Ulf Hellman, Juan José Cazzulo, and Griselda Noé
- Subjects
DNA, Complementary ,Molecular Sequence Data ,Gene Expression ,Helminth genetics ,medicine.disease_cause ,Malate dehydrogenase ,law.invention ,law ,Malate Dehydrogenase ,parasitic diseases ,medicine ,Animals ,Amino Acid Sequence ,Cloning, Molecular ,Echinococcus granulosus ,Molecular Biology ,Escherichia coli ,Peptide sequence ,Genes, Helminth ,Cloning ,biology ,Edman degradation ,Base Sequence ,Sequence Homology, Amino Acid ,DNA, Helminth ,biology.organism_classification ,Molecular biology ,Recombinant Proteins ,Mitochondria ,Kinetics ,Biochemistry ,Recombinant DNA ,Parasitology - Abstract
Protoscolices of the parasitic helminth Echinococcus granulosus contain two malate dehydrogenases (EC 1.1.1.37), one cytosolic and one mitochondrial. The latter has been separated from the other isoform and purified to protein homogeneity. Sequencing of tryptic peptides by Edman degradation allowed the design of oligonucleotide primers for PCR, leading to the cloning and sequencing of a full length cDNA. The encoding gene is present as a single copy per haploid genome and codes for a protein with high sequence identity (56-58%) with the similar enzymes from mammals, Caenorhabditis elegans and yeast. Active recombinant mitochondrial malate dehydrogenase was expressed in Escherichia coli, as protein fusions with glutathione S-transferase or a poly-His tail. The purified recombinant enzymes had a kinetic behaviour similar to that of the native enzyme, being inhibited by excess of the substrate oxaloacetate and unaffected by excess L-malate. The results indicate that E. granulosus contains two typical eukaryotic malate dehydrogenases, with relative levels quite different from those present in mammalian tissues like heart, in good agreement with the predominantly fermentative metabolism of the protoscolices.
- Published
- 2004
129. 14-3-3 gene characterization and description of a second 14-3-3 isoform in both Echinococcus granulosus and E. multilocularis
- Author
-
Norbert Müller, Claudia Paiva Nunes, Marı́a del Mar Siles-Lucas, Bruno Gottstein, and Arnaldo Zaha
- Subjects
Gene isoform ,DNA, Complementary ,Protein family ,Tyrosine 3-Monooxygenase ,Molecular Sequence Data ,610 Medicine & health ,Complementary DNA ,parasitic diseases ,Animals ,Protein Isoforms ,Amino Acid Sequence ,Echinococcus granulosus ,Gene ,Peptide sequence ,Conserved Sequence ,Genes, Helminth ,Phylogeny ,General Veterinary ,biology ,Sequence Homology, Amino Acid ,630 Agriculture ,General Medicine ,Helminth Proteins ,Sequence Analysis, DNA ,DNA, Helminth ,biology.organism_classification ,Molecular biology ,Introns ,Echinococcus ,Metacestode ,Blotting, Southern ,Infectious Diseases ,14-3-3 Proteins ,Insect Science ,Parasitology ,Sequence Alignment - Abstract
Members of the 14-3-3 protein family have been identified as regulatory molecules in intracellular signaling pathways and cell cycle control. Previously, the first Echinococcus 14-3-3 isoform (E14-3-3.1) was isolated from E. granulosus and E. multilocularis metacestode stages. Hyperexpression of this isoform was claimed to be associated with non-restricted tumor-like growth of the E. multilocularis metacestode. In this report, we describe the characterization of a 14-3-3 cDNA from E. granulosus and E. multilocularis corresponding to a second isoform of this family, E14-3-3.2. The characterized 14-3-3 gene was interrupted by two introns whose sequence and positions were conserved in both Echinococcus species. The deduced amino acid sequence of E14-3-3.2 showed 88% identity to the E14-3-3.1 isoform and 52% identity to a third Echinococcus isoform (E14-3-3.3) described by other authors. These findings, coupled to Southern blot analysis, suggest the presence of more than one 14-3-3 gene in Echinococcus. Phylogenenetically, the Echinococcus 14-3-3.1 and 14-3-3.2 isoforms appeared to cluster with zeta-type (“pro-tumorigenic”) 14-3-3 isoforms from closely related organisms, whereas the E14-3-3.3 isoform grouped with 14-3-3 epsilon isoforms. The presence of more than one 14-3-3 isoform might indicate isoform-specific roles in the different parasite stages of Echinococcus.
- Published
- 2004
- Full Text
- View/download PDF
130. Proteomic analysis of the larval stage of the parasite Echinococcus granulosus: causative agent of cystic hydatid disease
- Author
-
Gustavo Chemale, Arjan J. van Rossum, Peter M. Brophy, Arnaldo Zaha, Henrique Bunselmeyer Ferreira, James R. Jefferies, and John Barrett
- Subjects
Expressed Sequence Tags ,Two-dimensional gel electrophoresis ,biology ,Globulin ,Proteome ,Albumin ,biology.organism_classification ,Biochemistry ,Blood proteins ,Echinococcus ,Metacestode ,Peptide mass fingerprinting ,Echinococcosis ,Larva ,parasitic diseases ,biology.protein ,Animals ,Electrophoresis, Gel, Two-Dimensional ,Bovine serum albumin ,Echinococcus granulosus ,Molecular Biology - Abstract
We describe the preparation of Echinococcus granulosus metacestode protein extracts for two-dimensional electrophoresis (2-DE). Protoscoleces and hydatid fluid were prepared by precipitation using trichloroacetic acid (TCA) to remove nonprotein contaminants. Compared to the untreated control, TCA precipitation improved the 2-DE gel profile of the protoscoleces proteins. Comparison of 2-DE gels from insoluble and soluble fractions of the protoscoleces protein extract showed that most proteins are insoluble after lysis by sonication. Host serum proteins, especially albumin and globulins, caused horizontal streaking problems on the hydatid fluid 2-DE gels due to their high content in this sample. Even after the preparation of a hydatid fluid parasite enriched fraction, the high amount of bovine serum albumin and globulins made parasite-specific proteins difficult to detect by 2-DE. Despite the absence of an E. granulosus genome sequencing or expressed sequence tag (EST) projects, it was possible to identify 15 prominent protein spots from a whole protein protoscoleces 2-DE gel by peptide mass fingerprinting. These include actins, tropomyosin, paramyosin, thioredoxin reductase, antigen P-29, cyclophilin, and the heat shock proteins hsp70 and hsp20. This work demonstrates that 2-DE and PMF are important tools to identify proteins from the hydatid fluid and protoscoleces and for the comparative analysis of cysts from different hosts or between active and resting cysts.
- Published
- 2003
131. A set of recombinant antigens from Echinococcus granulosus with potential for use in the immunodiagnosis of human cystic hydatid disease
- Author
-
Henrique Bunselmeyer Ferreira, A. F. Zandonai, Marilise Brittes Rott, Karina Mariante Monteiro, Arnaldo Zaha, Alberto Nieto, Veridiana Gomes Virginio, and Ana Hernández
- Subjects
Immunology ,medicine.disease_cause ,Subclass ,law.invention ,Epitopes ,Antigen ,law ,Echinococcosis ,parasitic diseases ,Clinical Studies ,medicine ,Escherichia coli ,Immunology and Allergy ,Parasite hosting ,Animals ,Humans ,Serologic Tests ,Echinococcus granulosus ,Antiserum ,biology ,cDNA library ,biology.organism_classification ,Virology ,Recombinant Proteins ,Echinococcus ,ROC Curve ,Antigens, Helminth ,Area Under Curve ,Case-Control Studies ,Immunoglobulin G ,Recombinant DNA - Abstract
SUMMARY Several recombinant clones expressing antigens from Echinococcus granulosus were isolated previously from a parasite cDNA library using cystic hydatid disease (CHD) patients’ sera or rabbit hyperimmune antiserum against a lipoproteic fraction from bovine cyst fluid. Six of these antigens were expressed in Escherichia coli and the purified recombinant proteins were tested in enzyme-linked immunosorbent assay (ELISA) for specific IgG with a panel of sera from patients with surgically confirmed (n = 58) or immunologically diagnosed (n = 71) CHD. Sera from clinically normal individuals (n = 203) and sera from individuals with other helminthic infections (n = 65) were assayed for the assessment of specificity. A cut-off value was determined by receiver-operating-characteristic plots for each antigen. A recombinant antigen B subunit (AgB8/2) presented the highest sensitivity (93·1%), considering the group of sera from patients with CHD surgically confirmed, and specificity (99·5%) and is proposed as the basis for an immunodiagnostic test. The other recombinant antigens tested presented sensitivities between 58·6% and 89·7%, and three of them were considered of complementary value. In subclass-specific ELISA, different IgG isotypes showed dominance in the response for each of the recombinant antigens. There was a clear predominance of IgG4 response for all antigens tested, indicating that this would be the subclass of choice to be assessed for these recombinant proteins.
- Published
- 2003
132. In vitro segmentation induction of Mesocestoides corti (Cestoda) tetrathyridia
- Author
-
Henrique Bunselmeyer Ferreira, Norbel Galanti, Cristiano Valim Bizarro, Arnaldo Zaha, Ingrid Espinoza, Melissa Medeiros Markoski, and Sandra Estrazulas Farias
- Subjects
Mesocestoides corti ,media_common.quotation_subject ,Cestoda ,Biology ,Andrology ,Mice ,Mesocestoides ,Reproduction, Asexual ,medicine ,Parasite hosting ,Animals ,Trypsin ,Rats, Wistar ,Trypsin activity ,Ecology, Evolution, Behavior and Systematics ,media_common ,Mice, Inbred BALB C ,Temperature ,biology.organism_classification ,In vitro ,Culture Media ,Rats ,Larva ,Immunology ,Parasitology ,Cattle ,Female ,Reproduction ,Trypsin Inhibitors ,Fetal bovine serum ,medicine.drug - Abstract
Mesocestoides corti is a suitable model for studying cestode development because of its ability to reproduce asexually and segment in vitro. The cultured parasite is also capable of sexual differentiation and, probably, reproduction. To establish conditions that increase the efficiency of in vitro M. corti larvae (tetrathyridia) segmentation, we tested the effects of an inducing agent and some physical parameters in cultures. We found that a 5% CO2-95% N2 gas phase, an incubation temperature of 39 C (instead of 37 C), and a 24-hr pretreatment with trypsin (10(5) BAEE/ml, BAEE = Na-benzoil-L-arginine ethyl ester unit of trypsin activity) in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 20% fetal bovine serum (FBS) are able to increase individually or synergistically the segmentation rate of tetrathyridia. A segmentation rate of up to 100% was achieved on day 4 of culture, when all these conditions were used simultaneously, in comparison with an average rate of 40% obtained not before day 11 in cultures without any inducing treatment. Fetal bovine serum is essential for segmentation, and a concentration of 20% was established as the standard for induction.
- Published
- 2003
133. Molecular cloning and characterization of an Echinococcus granulosus cDNA encoding malate dehydrogenase
- Author
-
Jaqueline J.S. Rodrigues, Henrique Bunselmeyer Ferreira, and Arnaldo Zaha
- Subjects
chemistry.chemical_classification ,Base Sequence ,Sequence Homology, Amino Acid ,biology ,Molecular Sequence Data ,DNA ,Molecular cloning ,biology.organism_classification ,Malate dehydrogenase ,Echinococcus ,Enzyme ,chemistry ,Biochemistry ,Echinococcosis ,Malate Dehydrogenase ,Complementary DNA ,Animals ,Humans ,Parasite hosting ,Parasitology ,Amino Acid Sequence ,NAD+ kinase ,Cloning, Molecular ,Echinococcus granulosus ,Molecular Biology - Published
- 1993
134. Echinococcus granulosus: Cloning and Functional in Vitro Characterization of an Actin Filament Fragmenting Protein
- Author
-
Jaqueline J.S. Rodrigues, Henrique Bunselmeyer Ferreira, R.R. Yamamoto, Sandra Estrazulas Farias, Arnaldo Zaha, and Elizabeth Cortez-Herrera
- Subjects
DNA, Complementary ,Immunology ,Molecular Sequence Data ,Physarum polycephalum ,macromolecular substances ,Molecular cloning ,Dictyostelium discoideum ,Coding region ,Animals ,Amino Acid Sequence ,Gene ,Peptide sequence ,Actin ,Conserved Sequence ,Sheep ,biology ,Sequence Homology, Amino Acid ,General Medicine ,Helminth Proteins ,biology.organism_classification ,Actins ,Recombinant Proteins ,Echinococcus ,Infectious Diseases ,Biochemistry ,Parasitology ,Cattle ,Gelsolin ,Sequence Alignment - Abstract
We report the isolation and characterization of an Echinococcus granulosus gene that codes for a protein with actin filament fragmenting and nucleating activities (EgAFFP). The genomic region corresponding to the EgAFFP gene presents a coding sequence of 1110 bp that is interrupted by eight introns. The EgAFFP deduced amino acid sequence is about 40% homologous to those of several members of the gelsolin family, such as Physarum polycephalum fragmin, Dictyostelium discoideum severin, and Lumbricus terrestris actin modulator. As do other proteins of the same family, EgAFFP presents three repeated domains, each one characterized by internal conserved amino acid motifs. Assays with fluorescence-labeled actin showed that the full-length recombinant EgAFFP effectively binds actin monomers in both a calcium-dependent and calcium-independent manner and also presents actin nucleating and severing activities.
- Published
- 2001
135. Comparative analysis of the 14-3-3 gene and its expression in Echinococcus granulosus and Echinococcus multilocularis metacestodes
- Author
-
Arnaldo Zaha, Mar Siles-Lucas, and Claudia Paiva Nunes
- Subjects
14-3-3 gene ,Tyrosine 3-Monooxygenase ,Cestoda ,Molecular Sequence Data ,Echinococcus multilocularis ,Complementary DNA ,parasitic diseases ,Parasite hosting ,Animals ,Northern blot ,Amino Acid Sequence ,Echinococcus granulosus ,biology ,Metacestode ,Reverse Transcriptase Polymerase Chain Reaction ,14-3-3 protein ,DNA, Helminth ,biology.organism_classification ,Blotting, Northern ,Molecular biology ,Genética ,Echinococcus ,Infectious Diseases ,14-3-3 Proteins ,Gene Expression Regulation ,Expression level ,Immunology ,Animal Science and Zoology ,Parasitology ,Sequence Alignment - Abstract
It was suggested that the unlimited proliferative capacity of the Echinococcus multilocularis metacestode may be related to overproduction of the 14-3-3 protein. As is known, the proliferative capacities of E. granulosus and E. multilocularis metacestodes are very different. By comparing the expression levels of the 14-3-3 gene between in vitro -obtained E. granulosus and E. multilocularis metacestodes, we were able to provide experimental evidence of the potential relation between 14-3-3 over-expression and tumour-like growth in E. multilocularis metacestodes. RT–PCR and Northern blot experiments indicated that 14-3-3 expression level is about 4-fold higher in the E. multilocularis metacestode. This differential expression was confirmed both by immunoblotting and immunocytochemistry experiments, which allowed detection of the protein in the cyst wall from E. multilocularis but not in the cyst wall from E. granulosus . The alignment of the Echinococcus 14-3-3 cDNA sequence with known 14-3-3 isoforms from other organisms, grouped the parasite sequence into the tumour growth-related isoforms. Nucleotide sequence data reported in this paper are available in the EMBL, Genbank TM and DDJB databases under the acession numbers AF207904 and AF207905. The known relation between over-expression of some 14-3-3 isoforms and tumour-related processes, together with the present results, suggest that the Echinococcus 14-3-3 protein could be one of the molecules responsible for the differences between E. granulosus and E. multilocularis metacestode growth behaviour.
- Published
- 2001
136. Echinococcus granulosus: molecular cloning and phylogenetic analysis of an inducible glutathione S-transferase
- Author
-
Verónica Fernández, Cecilia Fernández, Claudio Martínez, Héctor Musto, Cora Chalar, and Arnaldo Zaha
- Subjects
medicine.medical_specialty ,Immunology ,Molecular Sequence Data ,Molecular cloning ,Polymerase Chain Reaction ,Phylogenetics ,Molecular genetics ,medicine ,Animals ,Amino Acid Sequence ,RNA, Messenger ,Cloning, Molecular ,Echinococcus granulosus ,Conserved Sequence ,Phylogeny ,Glutathione Transferase ,Genetics ,biology ,Phylogenetic tree ,General Medicine ,biology.organism_classification ,Molecular biology ,Echinococcus ,Infectious Diseases ,Glutathione S-transferase ,Parasitology ,Enzyme Induction ,Phenobarbital ,biology.protein ,Sequence Alignment - Abstract
Fernandez, V., Chalar, C., Martinez, C., Musto, H., Zaha, A., and Fernandez, C. 2000. Echinococcus granulosus: Molecular cloning and phylogenetic analysis of an inducible glutathione S -transferase. Experimental Parasitology 96 , 190–194.
- Published
- 2001
137. Cloning and characterization of Echinococcus granulosus (Cestode) EgactI and EgactII actin gene promoters and their functional analysis in the NIH3T3 mouse cell line
- Author
-
Arnaldo Zaha, Gustavo Chemale, Etel Rodrigues Pereira Gimba, and Sandra Estrazulas Farias
- Subjects
Chloramphenicol O-Acetyltransferase ,Physiology ,Immunology ,Biophysics ,Heterologous ,promoters ,Gene Expression ,Biology ,Biochemistry ,Mice ,Structure-Activity Relationship ,Transcription (biology) ,Genes, Reporter ,Gene expression ,Animals ,Deletions ,General Pharmacology, Toxicology and Pharmaceutics ,cestode ,Cloning, Molecular ,Echinococcus granulosus ,Promoter Regions, Genetic ,Gene ,lcsh:QH301-705.5 ,Actin ,lcsh:R5-920 ,Base Sequence ,General Neuroscience ,deletions ,Promoter ,Cell Biology ,General Medicine ,Transfection ,3T3 Cells ,Helminth Proteins ,biology.organism_classification ,Molecular biology ,Genética ,Actins ,Echinococcus ,lcsh:Biology (General) ,Regulatory sequence ,Promoters ,lcsh:Medicine (General) ,Cestode ,actin - Abstract
We report here for the first time the structure and function of a promoter from a cestode. The ability of DNA fragments respectively encompassing the 935-bp and 524-bp regions upstream from the ATG codon from the EgactI and EgactII actin genes of Echinococcus granulosus to promote transcription was studied in the NIH3T3 mouse cell line. The results of transfection assays showed that both regions have strong promoter activity in these cells. The fragments were tested in both orientations and the 524-bp fragment of EgactII presented a bidirectional promoter activity. Deletion analysis of EgactI and EgactII promoters indicated the presence of regulatory regions containing putative silencer elements. These results indicate that both EgactI and EgactII promoters are functional and that the preliminary functional evaluation of E. granulosus and possibly of other cestode promoters can be performed in heterologous cell lines.
- Published
- 2000
138. Monospecific polyclonal antibodies for immunodetection of Mycoplasma hyopneumoniae in swine pneumonic lungs
- Author
-
David Driemeier, Nelson Morés, Luiza Amaral de Castro, Henrique Bunselmeyer Ferreira, Desirée Cigaran Schuck, and Arnaldo Zaha
- Subjects
General Veterinary ,Mycoplasma hyopneumoniae ,Polyclonal antibodies ,Immunology ,biology.protein ,Biology ,biology.organism_classification ,Virology ,Swine Mycoplasmal Pneumonia - Published
- 2009
139. Breeding systems in Echinococcus granulosus (Cestoda; Taeniidae): selfing or outcrossing?
- Author
-
Karen Luisa Haag, Aldo Mellender de Araújo, Arnaldo Zaha, R.C.A. Thompson, Mar Siles-Lucas, and Bruno Gottstein
- Subjects
Genotype ,Swine ,Population ,Cestoda ,Zoology ,610 Medicine & health ,Outcrossing ,Polymerase Chain Reaction ,Strain ,Animals ,Inbreeding ,Genetic variability ,Horses ,education ,Echinococcus granulosus ,education.field_of_study ,Sheep ,630 Agriculture ,biology ,Ecology ,Selfing ,Genetic Variation ,biology.organism_classification ,Genética ,SSCP ,Echinococcus ,Metacestode ,Infectious Diseases ,Genetics, Population ,Taeniidae ,Animal Science and Zoology ,Parasitology ,Cattle ,Sequence Alignment - Abstract
We used the PCR–SSCP method followed by sequencing in order to assess the genetic variability of coding and non- coding parts of the genome of Echinococcus granulosus (Cestoda; Taeniidae) and to test whether or not the parasite populations are mainly self-fertilizing. For this, we analysed a sample of 110 E. granulosus metacestode isolates collected from different geographical regions (Southern Brazil, Europe and Australia) and from different intermediate hosts (ovine, bovine, human, macropod, swine and equine). Using appropriate controls, we were able to identify 4 strains in that sample (sheep, cattle, pig and horse strains). The high degree of genetic differentiation between strains, but not within, and the monomorphism found in most loci (EgAg4, EgActII, EgHbx2 and EgAg6 – non-coding – EgAgB/1 and EgND1 – coding) indicated that they are largely selfed. On the other hand, outcrossing was also shown to occur, since 5 potential hybrid genotypes between cattle and sheep strains were found in populations of Southern Brazil, but absent in other geographical areas. We suggest that both processes are adaptive. The article also reports, for the first time, the occurrence of the E. granulosus cattle strain in South America.
- Published
- 1999
140. Selection, recombination and history in a parasitic flatworm (Echinococcus) inferred from nucleotide sequences
- Author
-
Bruno Gottstein, Aldo Mellender de Araújo, Arnaldo Zaha, and Karen Luisa Haag
- Subjects
Microbiology (medical) ,lcsh:Arctic medicine. Tropical medicine ,lcsh:RC955-962 ,Swine ,Molecular Sequence Data ,lcsh:QR1-502 ,Helminth genetics ,parasites ,phylogeny ,lcsh:Microbiology ,Negative selection ,Phylogenetics ,parasitic diseases ,Animals ,Horses ,Selection, Genetic ,Gene ,Phylogeny ,Genetics ,Recombination, Genetic ,Sheep ,Phylogenetic tree ,biology ,Base Sequence ,Strain (biology) ,Single-strand conformation polymorphism ,sequencing ,DNA, Helminth ,biology.organism_classification ,recombination ,SSCP ,Echinococcus ,Sequenciamento [Nucleotideo] - Abstract
Three species of flatworms from the genus Echinococcus (E. granulosus, E. multilocularis and E. vogeli) and four strains of E. granulosus (cattle, horse, pig and sheep strains) were analysed by the PCR-SSCP method followed by sequencing, using as targets two non-coding and two coding (one nuclear and one mitochondrial) genomic regions. The sequencing data was used to evaluate hypothesis about the parasite breeding system and the causes of genetic diversification. The calculated recombination parameters suggested that cross-fertilisation was rare in the history of the group. However, the relative rates of substitution in the coding sequences showed that positive selection (instead of purifying selection) drove the evolution of an elastase and neutrophil chemotaxis inhibitor gene (AgB/1). The phylogenetic analyses revealed several ambiguities, indicating that the taxonomic status of the E. granulosus horse strain should be revised.
- Published
- 1998
141. Transposable elements in South American populations of Drosophila simulans
- Author
-
Arnaldo Zaha, Vera L. S. Valente, Élgion Lúcio da Silva Loreto, and Revues Inra, Import
- Subjects
0106 biological sciences ,0303 health sciences ,lcsh:QH426-470 ,Research ,General Medicine ,[SDV.GEN.GA] Life Sciences [q-bio]/Genetics/Animal genetics ,Biology ,biology.organism_classification ,010603 evolutionary biology ,01 natural sciences ,03 medical and health sciences ,lcsh:Genetics ,[SDV.GEN.GA]Life Sciences [q-bio]/Genetics/Animal genetics ,South american ,Genetics ,Genetics(clinical) ,Animal Science and Zoology ,lcsh:Animal culture ,Drosophila (subgenus) ,Humanities ,Ecology, Evolution, Behavior and Systematics ,ComputingMilieux_MISCELLANEOUS ,030304 developmental biology ,lcsh:SF1-1100 - Abstract
Elements transposables dans des populations sud-americaines de Drosophila simulans. Cette etude recherche la presence de quatre elements transposables (mariner. gypsy, hobo et 412) au sein de populations sud-americaines de Drosophila simulans. Le profil d'hybridation genomique de 12 populations differentes a ete determine par Southern Blot. Bien que seul un petit nombre de copies de mariner ait ete observe, chaque population presentait un profil d'hybridation caracteristique, ce qui suggere que l'element etait actif. Le nombre de copies de l'element gypsy s'est avere tres petit; avec un profil d'hybridation similaire dans toutes les lignees, ce qui indique que l'element etait inactif dans les populations etudiees. L'une des lignees ne comportait pratiquement pas d'element gypsy, ce qui n'etait encore jamais arrive chez D. simulans ni D. melanogaster. L'element hobo de 1,1 kb n'a ete detecte dans aucune lignee d'Amerique du Sud alors qu'il existe dans toutes les lignees, originaires d'autres sites geographiques, analysees jusqu'a present. Ces informations en accord avec les donnees de la litterature, suggerent que l'element hobo n'a peut-etre envahi que recemment le genome des populations de D. simulans en Amerique du Sud. Pour l'element 412, les populations etudiees ont montre des bandes specifiques. ce qui suggere que cet element peut avoir des activites de transposition dans le genome de ces mouches.
- Published
- 1998
142. Reduced genetic variability within coding and non-coding regions of the Echinococcus multilocularis genome
- Author
-
Karen Luisa Haag, Aldo Mellender de Araújo, Bruno Gottstein, and Arnaldo Zaha
- Subjects
Swine ,Molecular Sequence Data ,610 Medicine & health ,Variabilidade genética ,Biology ,Echinococcus multilocularis ,Genome ,DNA, Mitochondrial ,Nucleotide diversity ,Strain ,Species Specificity ,Genotype ,Animals ,Genetic variability ,Gene ,Genes, Helminth ,Polymorphism, Single-Stranded Conformational ,Polimorfismo ,Genetics ,Cell Nucleus ,Polymorphism, Genetic ,Sheep ,Base Sequence ,630 Agriculture ,Single-strand conformation polymorphism ,NADH Dehydrogenase ,biology.organism_classification ,Introns ,SSCP ,Echinococcus ,Infectious Diseases ,Antigens, Helminth ,Animal Science and Zoology ,Parasitology ,Cattle - Abstract
Echinococcus multilocularis, a vulpine intestinal tapeworm, is the causative agent of alveolar echinococosis in humans, one of the most severe and lethal parasitic infections in man. To date, there is very little knowledge about the genetical polymorphism of this parasite. To assess sequence polymorphism, we analysed a sample of 33 E. multilocularis isolates from Europe, North America and Asia by PCR-SSCP followed by nucleotide sequencing. This assessment was performed comparatively to sheep, cattle and pig E. granulosus strains. Coding (nuclear antigen B and mitochondrial NADH dehydrogenase genes) and non-coding (introns of actin and homeobox-containing genes) regions of the parasite genome were chosen as targets. Since the estimated nucleotide diversity among genotypes of E. multilocularis were, in general, 10 times lower than among the recognized different strains of E. granulosus, we suggest that the conventional classification of the former species in 2 separated strains (European and North American) should be reviewed.
- Published
- 1997
- Full Text
- View/download PDF
143. Genetic structure of natural populations of Dryas iulia (Lepidoptera: Nymphalidae) revealed by enzyme polymorphism and mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLP)
- Author
-
Karen Luisa Haag, Aldo Mellender de Araújo, and Arnaldo Zaha
- Subjects
Male ,Esterases ,General Medicine ,Biochemistry ,DNA, Mitochondrial ,Lepidoptera ,Leucyl Aminopeptidase ,Genetics, Population ,Phosphoglucomutase ,Genetics ,Animals ,Female ,Molecular Biology ,Ecology, Evolution, Behavior and Systematics ,Polymorphism, Restriction Fragment Length - Abstract
Dryas iulia appears to have undergone a mode of evolution different from that of other members of its subfamily (Heliconiinae). While other species constitute highly subdivided and inbred populations, those of D. iulia are thought to be large and uniform. Analyzing six samples from Southern Brazil (state of Rio Grande do Sul) in relation to three enzyme systems (EST, LAP, and PGM) and their mtDNA RFLP patterns, we found that they are very similar at the molecular level. The F statistics for enzyme polymorphism data revealed that inbreeding makes a great contribution to the population homozygosity, since FIS equals 0.1322 and FST equals 0.0023. Since the chi-square test showed that FST is not significant, we conclude that all localities belong to the same population. The mtDNA differentiation was about 12 times greater than for nuclear genes; FST was equivalent to 0.0265. We suggest that this difference is due to a higher dispersal of males, in relation to females.
- Published
- 1993
144. Comparative proteomic analysis of pathogenic and non-pathogenic strains from the swine pathogen Mycoplasma hyopneumoniae
- Author
-
Henrique Bunselmeyer Ferreira, Paulo Marcos Pinto, Catia Silene Klein, and Arnaldo Zaha
- Subjects
Proteômica ,biology ,Strain (chemistry) ,lcsh:Cytology ,Research ,Mycoplasma hyopneumoniae ,biology.organism_classification ,Proteomics ,Virology ,Genome ,Biochemistry ,Microbiology ,Bacterial adhesin ,Porcine enzootic pneumonia ,lcsh:QH573-671 ,Pathogen ,Molecular Biology ,Bacteria - Abstract
Background Mycoplasma hyopneumoniae is a highly infectious swine pathogen and is the causative agent of enzootic pneumonia (EP). Following the previous report of a proteomic survey of the pathogenic 7448 strain of swine pathogen, Mycoplasma hyopneumoniae, we performed comparative protein profiling of three M. hyopneumoniae strains, namely the non-pathogenic J strain and the two pathogenic strains 7448 and 7422. Results In 2DE comparisons, we were able to identify differences in expression levels for 67 proteins, including the overexpression of some cytoadherence-related proteins only in the pathogenic strains. 2DE immunoblot analyses allowed the identification of differential proteolytic cleavage patterns of the P97 adhesin in the three strains. For more comprehensive protein profiling, an LC-MS/MS strategy was used. Overall, 35% of the M. hyopneumoniae genome coding capacity was covered. Partially overlapping profiles of identified proteins were observed in the strains with 81 proteins identified only in one strain and 54 proteins identified in two strains. Abundance analysis of proteins detected in more than one strain demonstrates the relative overexpression of 64 proteins, including the P97 adhesin in the pathogenic strains. Conclusions Our results indicate the physiological differences between the non-pathogenic strain, with its non-infective proliferate lifestyle, and the pathogenic strains, with its constitutive expression of adhesins, which would render the bacterium competent for adhesion and infection prior to host contact.
- Published
- 2009
145. Effects of protoscoleces and AgB from Echinococcus granulosus on human neutrophils: possible implications on the parasite’s immune evasion mechanisms.
- Author
-
Veridiana Virginio, Lorena Taroco, Ana Ramos, Ana Ferreira, Arnaldo Zaha, Henrique Ferreira, and Ana Hernández
- Subjects
ENZYME-linked immunosorbent assay ,IMMUNE response ,GRANULOCYTES ,NEUTROPHILS - Abstract
Abstract??The factors affecting the innate susceptibility toEchinococcus granulosusinfections are largely unknown. We assessed the interaction of healthy human neutrophils with protoscoleces (PSC) and antigen B (AgB) ofE. granulosusby analysis of CD11b upregulation and H
2 O2 production by flow cytometry. PSC induced neutrophil activation, but their viability was not affected. In contrast, no effects were observed with AgB in both assays. Neutrophil-enriched fractions were also incubated with PSC or AgB, and interleukin 8 (IL-8) production was measured by ELISA. Significant increment in IL-8 production was detected only in supernatants from neutrophil-enriched fractions cultured with PSC. The possible effect of a prior incubation with AgB on the phorbol myristate acetate-induced activation was also evaluated. No changes were observed in CD11b expression, but the H2 O2 production was significantly reduced in platelet-activating factor (PAF)-primed neutrophils. These results suggest a possible AgB-mediated mechanism of evasion of the host immune response, which would operate upon events of spillage of the fertile hydatid cyst content. [ABSTRACT FROM AUTHOR]- Published
- 2007
- Full Text
- View/download PDF
146. 14-3-3 gene characterization and description of a second 14-3-3 isoform in both Echinococcus granulosus and E. multilocularis.
- Author
-
Cláudia Paiva Nunes, Arnaldo Zaha, Bruno Gottstein, Norbert Müller, and María del Mar Siles-Lucas
- Abstract
Members of the 14-3-3 protein family have been identified as regulatory molecules in intracellular signaling pathways and cell cycle control. Previously, the first Echinococcus 14-3-3 isoform (E14-3-3.1) was isolated from E. granulosus and E. multilocularis metacestode stages. Hyperexpression of this isoform was claimed to be associated with non-restricted tumor-like growth of the E. multilocularis metacestode. In this report, we describe the characterization of a 14-3-3 cDNA from E. granulosus and E. multilocularis corresponding to a second isoform of this family, E14-3-3.2. The characterized 14-3-3 gene was interrupted by two introns whose sequence and positions were conserved in both Echinococcus species. The deduced amino acid sequence of E14-3-3.2 showed 88% identity to the E14-3-3.1 isoform and 52% identity to a third Echinococcus isoform (E14-3-3.3) described by other authors. These findings, coupled to Southern blot analysis, suggest the presence of more than one 14-3-3 gene in Echinococcus. Phylogenenetically, the Echinococcus 14-3-3.1 and 14-3-3.2 isoforms appeared to cluster with zeta-type (“pro-tumorigenic”) 14-3-3 isoforms from closely related organisms, whereas the E14-3-3.3 isoform grouped with 14-3-3 epsilon isoforms. The presence of more than one 14-3-3 isoform might indicate isoform-specific roles in the different parasite stages of Echinococcus. [ABSTRACT FROM AUTHOR]
- Published
- 2004
147. Segments of chromosomal DNA from Rhynchosciara americana that undergo additional rounds of DNA replication in the salivary gland DNA puffs have only weak ARS activity in yeast
- Author
-
David C. Hayward, Sarah E. Millar, M T Pueyo, Francisco Lara, Michael J. Browne, David M. Glover, Felipe Garcia Vallejo, Christopher A. Read, Arnaldo Zaha, and Roberto Vicente Santelli
- Subjects
DNA Replication ,Saccharomyces cerevisiae ,Biology ,Chromosomes ,Salivary Glands ,law.invention ,Restriction fragment ,chemistry.chemical_compound ,law ,Genetics ,Animals ,Genomic library ,Cloning, Molecular ,Repetitive Sequences, Nucleic Acid ,Polytene chromosome ,Diptera ,DNA replication ,Gene Amplification ,General Medicine ,biology.organism_classification ,Molecular biology ,genomic DNA ,chemistry ,Larva ,Recombinant DNA ,biology.protein ,Rhynchosciara americana ,Replicon ,DNA - Abstract
We have constructed a library of recombinant phage containing DNA from salivary gland chromosomes of Rhynchosciara americana . We have isolated phage from this library that carry sequences homologous to cDNA clones that hybridise in situ to the DNA puffs at the polytene chromosome regions C3 and C8. This has enabled us to demonstrate a 16-fold amplification of the genomic DNA sequences at these regions during DNA-puffing. At the C8 site there is a sequence element that has characteristics of ‘scrambled’ moderately repetitive DNA. This is located within 3 kb from the gene encoding a 1.95-kb mRNA. We have assayed restriction fragments from the two DNA puffs for Ars activity in yeast. The only strong Ars activity is associated with a part of the moderately repetitive DNA element from the C8 puff which is not present at this site in all animals.
- Published
- 1985
148. Characterization of DNA Segments Adjacent to the nifHDK Genes of Azospirillum Brasilense Sp7 by Tn5 Site-Directed Mutagenesis
- Author
-
Irene Silveira Schrank, Elza F. de Araujo, Arnaldo Zaha, and Diógenes Santiago Santos
- Subjects
biology ,EcoRI ,Azospirillum brasilense ,biology.organism_classification ,Molecular biology ,law.invention ,chemistry.chemical_compound ,Plasmid ,chemistry ,law ,biology.protein ,Recombinant DNA ,Genomic library ,Site-directed mutagenesis ,Gene ,DNA - Abstract
Three recombinants containing DNA inserts of 13 kb (λAb5), 15 kb (λAb2c) and 16 kb (λAb4) were isolated from a gene library of A. brasilense Sp7. These DNA inserts are overlapping DNA segments covering approximately 40 kb of the A. brasilense genome in the region of the nifHDK genes. The λAb2c recombinant containing the nifHDK genes localized in a 6.5 kb EcoRI DNA fragment was characterized. The EcoRI DNA fragments of this recombinant (6.5, 4.15, 2.4, 1.2 and 0.8 kb) were subcloned into the plasmid pACYC184 and after Tn5 mutagenesis, recloned into the plasmid pRK290 and transferred to A. brasilense. Five Nif− mutants were obtained, one with Tn5 inserted in the nifHDK genes and four mapping approximately 4 to 5 kb downstream from the nifHDK genes.
- Published
- 1988
149. DNA strand scission in E. coli by electronically excited state molecules generated by enzymatic systems
- Author
-
Sonia M. De Toledo, Arnaldo Zaha, and Nelson Durán
- Subjects
inorganic chemicals ,Biophysics ,Guanosine ,Photochemistry ,environment and public health ,Biochemistry ,chemistry.chemical_compound ,Oxygen Consumption ,Escherichia coli ,A-DNA ,Triplet state ,Molecular Biology ,Bond cleavage ,Horseradish Peroxidase ,Electrophoresis, Agar Gel ,biology ,Indoleacetic Acids ,Chemistry ,Singlet oxygen ,Mutagenesis ,Cell Biology ,DNA ,Excited state ,biology.protein ,Peroxidase ,Plasmids - Abstract
E.coli containing pAT 153 plasmid undergoes strand scission when exposed to the indole-3-acetic acid/peroxidase/D2 system. Neither the initial components of this reaction nor the final stable products are responsible for this effect. Indole-3-aldehyde in its triplet state and singlet oxygen have been recently identified in this system. That singlet oxygen is one of the species acting on the plasmid in E.coli cells was suggested by protective effect of histidine and guanosine which are singlet oxygen quenchers. Similar effect on plasmid with malonaldehyde/peroxidase/O2 system was observed, which is an excellent singlet oxygen generator. This is the first report of a biological system where it is possible to detect a DNA scission in the intact cell by a bioenergized process. This presumably is related to spontaneous mutagenesis.
- Published
- 1982
150. Cloning and characterization of the ribosomal RNA genes of Rhynchosciara americana
- Author
-
Orilio Leoncini, Cornelis P. Hollenberg, Francisco Lara, and Arnaldo Zaha
- Subjects
EcoRI ,Biology ,HindIII ,Salivary Glands ,chemistry.chemical_compound ,Genetics ,Animals ,Cloning, Molecular ,Genetics (clinical) ,Southern blot ,Base Sequence ,Diptera ,Chromosome Mapping ,Nucleic Acid Hybridization ,Ribosomal RNA ,biology.organism_classification ,Molecular biology ,Restriction enzyme ,chemistry ,Genes ,RNA, Ribosomal ,biology.protein ,Rhynchosciara americana ,BamHI ,DNA - Abstract
The ribosomal RNA genes (rDNA) of Rhynchosciara americana were analysed using Southern transfers of DNA cleaved with EcoRI, HindIII, BamHI and PstI. The results show that the rDNA is heterogeneous in structure. Following digestion with EcoRI and hybridization to rRNA three bands corresponding to fragments of 9.5, 7.5 and 5.5 kilobases (kb) were detected. Recombinants containing EcoRI fragments of R. americana DNA were prepared using the vector lambda gt lambda B. Three different recombinants (lambda gt Ra1, lambda gt Ra23 and lambda gt Ra5) were isolated containing the rDNA fragments of 9.5, 7.5 and 5.5 kb, respectively. These fragments were transferred to pBR325 and analysed with restriction enzymes and Southern hybridization with 28 S and 18 S rRNA. The lambda gt recombinants were further analysed by R-loop mapping. The data show that the rDNA occurs in two different repeating gene units. A shorter repeat of 9.5 kb and a longer repeat of 13 kb, in which the 28 S rRNA coding sequence contains an insertion of 3.5 kb.
- Published
- 1982
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.