Biomedical subjects

V Azevedo

Publications and source records attributed to V Azevedo.

33 records · Page 2Linked to original sources

PubMed1999

In vitro susceptibility of chromoblastomycosis and phaeohyphomycosis agents to antifungal drugs.

The in vitro susceptibility of chromoblastomycosis and phaeohyphomycosis agents to antifungal drugs was appraised using the reference macrodilution method proposed by the National Committee for Clinical Laboratory Standards (NCCLS) for yeasts modified for filamentous fungi. The antifungal drugs amphotericin B, 5-fluorocytosine, itraconazole and fluconazole were tested against one environmental and 18 clinical isolates. This work amended the macrodilution methods proposed by NCCLS and suggests that a conidial suspension free of hyphae leads to a more reliable assay and provides for better reproducibility. The macrodilution method was performed with 10(4) conidia ml-1. The MIC values ranged from 1.0 to 16.0 micrograms ml-1 for amphotericin B and 3.12 to 25.0 micrograms ml-1 for 5-fluorocytosine. A MIC range of 0.06 to 1.95 micrograms ml-1 was determined for itraconazole while 2.0 to 64.0 micrograms ml-1 was detected for fluconazole.

Antifungal Agents↗

PubMed1999

Dematiaceous fungal pathogens: analysis of ribosomal DNA gene polymorphism by polymerase chain reaction-restriction fragment length polymorphism.

Restriction fragment length polymorphisms (RFLP) of ribosomal gene small subunit (SSU rDNA) and internal transcribed spacer (ITS) regions was examined in 12 isolates of dematiaceous agents of chromoblastomycosis and phaeohyphomycosis. The amplicon length of the fragment ITS1-ITS4, comprising the 5.8 rDNA and ITS1-ITS2 spacers, ranged in size from 620 to 690 bp. This result indicated a polymorphism of size in this region. Additionally the RFLP profiles showed a high degree of inter- and intra-specific variability. In contrast, the SSU rDNA amplification, using NS1-NS2 primers, originated a fragment of approximately 570 bp and its restriction profile proved to be well conserved among the species studied and was clustered into only two genetically heterogeneous groups, the first one formed by Fonsecaea pedrosoi and Fonsecaea compacta and the second one formed by Cladophialophora (Cladosporium) carrionii, Cladophialophora (Xylohypha) bantiana, Phialophora verrucosa and Rhinocladiella species.

Base Sequence↗

PubMed1999

Main features of DNA-based immunization vectors.

DNA-based immunization has initiated a new era of vaccine research. One of the main goals of gene vaccine development is the control of the levels of expression in vivo for efficient immunization. Modifying the vector to modulate expression or immunogenicity is of critical importance for the improvement of DNA vaccines. The most frequently used vectors for genetic immunization are plasmids. In this article, we review some of the main elements relevant to their design such as strong promoter/enhancer region, introns, genes encoding antigens of interest from the pathogen (how to choose and modify them), polyadenylation termination sequence, origin of replication for plasmid production in Escherichia coli, antibiotic resistance gene as selectable marker, convenient cloning sites, and the presence of immunostimulatory sequences (ISS) that can be added to the plasmid to enhance adjuvanticity and to activate the immune system. In this review, the specific modifications that can increase overall expression as well as the potential of DNA-based vaccination are also discussed.

Enhancer Elements, Genetic↗

PubMed1999

Immunological properties of gene vaccines delivered by different routes.

Gene vaccines represent a new and promising approach to control infectious diseases, inducing a protective immune response in the appropriate host. Several routes and methods of genetic immunization have been shown to induce antibody production as well as T helper (Th) cell and cytotoxic T lymphocyte activation. However, few studies have compared the nature of the immune responses generated by different gene vaccination delivery systems. In the present study we reviewed some aspects of immunity induced by gene immunization and compared the immune responses produced by intramuscular (i.m.) DNA injection to gene gun-mediated DNA transfer into the skin of BALB/c mice. Using a reporter gene coding for beta-galactosidase, we have demonstrated that i.m. injection raised a predominantly Th1 response with mostly IgG2a anti-beta gal produced, while gene gun immunization induced a mixed Th1/Th2 profile with a balanced production of IgG2a and IgG1 subclasses. Distinct types of immune responses were generated by different methods of gene delivery. These findings have important implications for genetic vaccine design. Firstly, a combination between these two systems may create optimal conditions for the induction of a broad-based immune response. Alternatively, a particular gene vaccine delivery method might be used according to the immune response required for host protection. Here, we describe the characteristics of the immune response induced by gene vaccination and the properties of DNA involved in this process.

Animals↗

PubMed1999

Use of RAPD (random amplified polymorphic DNA) to analyse genetic diversity of dematiaceous fungal pathogens.

Thirteen strains of chromoblastomycosis and phaeohyphomycosis etiologic agent fungi were obtained from different geographical origins. These strains were genotypically compared by means of the RAPD (Random Amplified Polymorphic DNA) technique. The data generated showed a high degree of polymorphism between distinct species and a low polymorphism between strains of the same species. The results generated by these tests were subjected to a numerical taxonomy analysis, using the unweighted pair-group method. A phenogram was constructed for the set of strains studied. Based on its structure, we concluded that genotypical data provide enough information to us to use the unweighted pair-group method to cluster the strains in accordance to their respective species. The phenogram grouped in a single branch the strains of Fonsecaea pedrosoi and F. compacta species, indicating a great similarity between these fungi, and suggesting that the classification as distinct species may not be appropriate for these species of the genus Fonsecaea.

Chromoblastomycosis↗

PubMed1997

The complete genome sequence of the gram-positive bacterium Bacillus subtilis.

Bacillus subtilis is the best-characterized member of the Gram-positive bacteria. Its genome of 4,214,810 base pairs comprises 4,100 protein-coding genes. Of these protein-coding genes, 53% are represented once, while a quarter of the genome corresponds to several gene families that have been greatly expanded by gene duplication, the largest family containing 77 putative ATP-binding transport proteins. In addition, a large proportion of the genetic capacity is devoted to the utilization of a variety of carbon sources, including many plant-derived molecules. The identification of five signal peptidase genes, as well as several genes for components of the secretion apparatus, is important given the capacity of Bacillus strains to secrete large amounts of industrially important enzymes. Many of the genes are involved in the synthesis of secondary metabolites, including antibiotics, that are more typically associated with Streptomyces species. The genome contains at least ten prophages or remnants of prophages, indicating that bacteriophage infection has played an important evolutionary role in horizontal gene transfer, in particular in the propagation of bacterial pathogenesis.

Bacillus subtilis↗

PubMed1997

Evaluation of cDNA libraries from different developmental stages of Schistosoma mansoni for production of expressed sequence tags (ESTs).

A comparative study of the gene expression profile in different developmental stages of Schistosoma mansoni has been initiated based on the expressed sequence tag (EST) approach. A total of 1401 ESTs were generated from seven different cDNA libraries constructed from four distinct stages of the parasite life cycle. The libraries were first evaluated for their quality for a large-scale cDNA sequencing program. Most of them were shown to have less than 20% useless clones and more than 50% new genes. The redundancy of each library was also analyzed, showing that one adult worm cDNA library was composed of a small number of highly frequent genes. When comparing ESTs from distinct libraries, we could detect that most genes were present only in a single library, but others were expressed in more than one developmental stage and may represent housekeeping genes in the parasite. When considering only once the genes present in more than one library, a total of 466 unique genes were obtained, corresponding to 427 new S. mansoni genes. From the total of unique genes, 20.2% were identified based on homology with genes from other organisms, 8.3% matched S. mansoni characterized genes and 71.5% represent unknown genes.

Animals↗

PubMed1996

Bacillus subtilis can modulate its capacity and specificity for protein secretion through temporally controlled expression of the sipS gene for signal peptidase I.

Bacillus subtilis contains three chromosomally encoded type I signal peptidases (SipS, SipT and SipU), which remove signal peptides from secretory precursor proteins. In the present study the biological function of SipS and the regulation of its synthesis were analysed. Unlike the type I signal peptidase of Escherichia coli, SipS was essential neither for protein secretion nor viability of the cell. However, in the absence of SipS the rate of processing of several preproteins was reduced, and four of the seven major secreted proteins of B. subtilis were hardly detectable in the growth medium. Surprisingly, the processing of Bacillus amyloliquefaciens alpha-amylase and the secretion of at least two endogenous B. subtilis proteins was improved in the absence of SipS. These findings indicate that the substrate preference of SipS differs from that of SipT and SipU, and that SipS is an important factor determining the efficiency of protein secretion in B. subtilis. SipS is transcribed in a growth phase- and medium-dependent manner. In minimal medium, the growth phase-dependent transcription of sipS is controlled by the DegS-DegU two-component regulatory system, indicating that the expression of sipS is regulated by the same factors that control the expression of most genes for secreted degradative enzymes. Our observations suggest that B. subtilis can modulate its capacity and specificity for protein secretion through the controlled expression of sipS.

Bacillus subtilis↗

PubMed1996

Human T lymphotropic virus type II (HTLV-II): epidemiology, molecular properties, and clinical features of infection.

Human T lymphotropic virus, type II (HTLV-II), infection has been shown to be endemic in a number of American Indian populations, and high rates of infection have also been documented in intravenous drug abusers in urban areas throughout the world. Although the role of HTLV-II in human disease has yet to be clearly defined, there is accumulating evidence that like HTLV-I, infection may also be associated with rare lymphoproliferative and neurological disorders. In this article we review and summarize the epidemiology, molecular properties and clinical features of HTLV-II infection.

Amino Acid Sequence↗

PubMed1996

Sequence analysis of the Bacillus subtilis chromosome region between the serA and kdg loci cloned in a yeast artificial chromosome.

The standard strategies of genome sequencing based on lambda-vector or cosmid libraries are only partially applicable to AT-rich Gram-positive bacteria because of the problem of instability of their chromosomal DNA in heterologous hosts like Escherichia coli. One complete collection of ordered clones known for such bacteria is that of Bacillus subtilis, established by using yeast artificial chromosomes (YACs). This paper reports the results of the direct use of one of the YAC clones from the above collection for the sequencing of the region cloned in it. The strategy applied consisted of the following: (i) construction of M13 banks of the partially purified YAC DNA and sequencing of 800 M13 clones chosen at random; (ii) directed selection of M13 clones to sequence by using marginal contig fragments as hybridization probes; (iii) direct sequencing of joining PCR fragments obtained by combinations of primers corresponding to the ends of representative contigs. The complete 104,109 bp insert sequence of this YAC clone was thus established. The strategy used allowed us to avoid resequencing the two largest, previously sequenced, contigs (13,695 and 20,303 bp) of the YAC insert. We propose that the strategy used can be applied to the sequencing of the whole bacterial genome without intermediate cloning, as well as for larger inserts of eukaryotic origin cloned in YACs. Sequencing of the insert of the YAC clone 15-6B allowed us to establish the contiguous sequence of 127 kb from spollA to kdg. The organization of the newly determined region is presented. Of the 138 ORFs identified in the spollA-kdg region, 57 have no clear putative function from their homology to proteins in the databases.

Amino Acid Sequence↗

PubMed1995

The Bacillus subtilis chromosome region encoding homologues of the Escherichia coli mssA and rpsA gene products.

A gene was found in Bacillus subtilis which encodes a protein highly homologous to the Escherichia coli rpsA gene product, the S1 ribosomal protein. The B. subtilis protein contains the domain responsible for binding to ribosomes and two S1 motifs, instead of four as found in the E. coli protein. The B. subtilis protein is similar in this way to the equivalent protein of plant chloroplast ribosomes, supposed to be the counterpart of E. coli S1. The gene is expressed during vegetative growth in B. subtilis at the transcriptional and translational levels, as judged by Northern hybridization and expression in a translational fusion with a reporter gene. In contrast to the E. coli situation, it can be inactivated without dramatic effects on cell viability. Southern hybridization of the B. subtilis DNA fragment encoding this gene revealed specific homologous fragments in all other Gram-positive bacteria tested. The hybridization pattern with B. stearothermophilus suggests the presence of at least two homologous genes in this bacterium. We show that in B. subtilis the ORF preceding the rpsA homologue encodes a protein which is highly similar to the product of the E. coli mssA gene which is located upstream of rpsA. Again, in contrast to the E. coli situation, where these genes are co-transcribed, in B. subtilis they are separated by a transcription terminator and the mssA homologue is transcribed during sporulation. We suggest that during the evolution very similar structures and genetic organization of these two genes were conserved but acquired different functions in Gram-negative and Gram-positive bacteria.

Amino Acid Sequence↗

PubMed1993

An ordered collection of Bacillus subtilis DNA segments cloned in yeast artificial chromosomes.

A collection of 772 Bacillus subtilis DNA segments was obtained by cloning in yeast artificial chromosomes. The B. subtilis inserts of 288 clones were mapped by hybridization using as probes 65 cloned genes and 188 isolated insert ends. In this way, 59 inserts were ordered in four contigs that cover > 98% of the B. subtilis chromosome. This ordered collection is now available for further genetic and physical analysis of the B. subtilis genome.

Bacillus subtilis↗

PubMed1993

The organization of the Bacillus subtilis 168 chromosome region between the spoVA and serA genetic loci, based on sequence data.

Three different lambda phage clones with overlapping inserts of Bacillus subtilis DNA, which cover the region from spoIIAA to serA, have been isolated. The nucleotide sequence of their inserts, starting after spoVAF and ending at serA, has been determined. A contiguous sequence of 35,354 bp was established, including previously analysed overlapping adjacent regions. Within the newly determined sequence 31 open reading frames (ORFs) with putative ribosome-binding sites have been found. Nine of them correspond to previously sequenced and characterized genes: spo-VAF, lysA, sipS, ribG, ribB, ribA, ribH, ribTD and dacB. Comparison of the amino acid sequences of the products encoded by the other ORFs to known proteins allowed putative functions to be assigned to seven of these ORFs. Among these are the following: (i) the ppiB gene, encoding a cytoplasmic peptidylprolyl isomerase; (ii) two pairs of signal-transducers, one homologous to phoR-phoP of B. subtilis, encoding regulators of phosphatase biosynthesis, and the second to the fecI-fecR of Escherichia coli, which is responsible for the regulation of the citrate-dependent iron (III) transport system; (iii) aroC and serA genes, involved in the biosynthesis of aromatic amino acids and serine, respectively, the function of which has been confirmed by constructing corresponding mutants with disrupted ORFs. The organization of putative operons has been postulated on the basis of the sequences of their transcription terminators, promoters and regulatory elements.

Amino Acid Sequence↗

PubMed1993

The transcriptional organization of the Bacillus subtilis 168 chromosome region between the spoVAF and serA genetic loci.

The genetic organization of the spoVAF-serA area of the Bacillus subtilis chromosome and its putative transcription map have been derived from analysis of the nucleotide sequence. In order to confirm this transcription map as regards size of transcripts and to determine growth conditions for their appearance, we undertook Northern hybridization analysis of total RNA from vegetatively growing and sporulating cells. Twenty-three distinct transcripts were thus identified, 14 of which were predicted from sequence analysis and nine of which were not predicted. Eight of the latter are homologous to open reading frames identified by sequence analysis but were not expected, since no obvious promoter or terminator was found in the sequence. The last unexpected transcript does not correspond to an ORF and might identify a novel gene. Three predicted transcripts were not detected. The transcripts were classified in four groups as (i) constitutive, (ii) regulated by nutritional depletion, (iii) specific for sporulation, and (iv) possibly regulated temporally. These studies demonstrate that systematic Northern analysis of a bacterial chromosome region is a useful complement to sequence analysis.

Bacillus subtilis↗

PubMed1993

Physical mapping of stable RNA genes in Bacillus subtilis using polymerase chain reaction amplification from a yeast artificial chromosome library.

A new approach for mapping genes which utilizes yeast artificial chromosome clones carrying parts of the Bacillus subtilis genome and the polymerase chain reaction technique is described. This approach was used to physically map stable RNA genes of B. subtilis. Results from over 400 polymerase chain reactions carried out with the yeast artificial chromosome clone library, using primers specific for the genes of interest and designed from published sequences, were collected. The locations of 10 known rRNA gene regions (rrnO, rrnA, rrnE, rrnD, rrnB, rrnJ-rrnW, and rrnI-rrnH-rrnG) have been determined by this method, and these results correlate with those observed by standard genetic mapping. All rRNA operons, except rrnB, are found between 0 and 90 degrees, while rrnB has been placed in the area of 270 degrees on the chromosome map. Also localized were the tRNA gene clusters associated with the following ribosomal operons: rrnB (21 tRNAs), rrnJ (9 tRNAs), rrnD (16 tRNAs), and rrnO and rrnA (2 internal tRNAs). A previously unmapped four-tRNA gene cluster, trnY, a tRNA gene region that is not associated with a ribosomal operon, was found near the origin of replication. The P-RNA gene, important for processing of tRNAs, was found between map locations 197 and 204 degrees.

Bacillus subtilis↗