Bacterial diversity of periodontal and implant-related sites detected by the DNA Checkerboard method.

The aim of this study was to compare the microbial composition of the subgingival biofilm from teeth and implant sulci in relation to contents originating from internal parts of the implant, abutment and implant prosthesis. Twenty subgingival biofilm samples from the mesial and distal aspects of each tooth/implant and 29 samples from the internal parts of titanium implants, abutments and implant prostheses were evaluated for the presence of 18 bacterial species using DNA Checkerboard and the differences between samples from teeth and implants were assessed with Pearson's correlation analysis. The periodontal and peri-implantar sulci presented significantly higher bacterial counts than the implant-related sites (p < 0.05 and p < 0.01, respectively). The highest counts were observed for Capnocytophaga gingivalis, Prevotella intermedia, P. nigrescens and P. micra. The correlation between the counts in the periodontal and peri-implantar sulci was r = 0.66 (p < 0.001). Weaker correlations between samples from the internal parts of the implants and periodontal sulcus (r = 0.49; p < 0.001) or peri-implant sulcus (r = 0.42; p < 0.001) were found. All 18 bacterial species were detected to be colonising the subgingival sulcus of teeth and implants, and implant components in the evaluated patients. Significant correlations between the microbiota were found, the strongest being between the periodontal and peri-implantar sulci.

Bradysia hygida (Diptera, Sciaridae) presents two eukaryotic Elongation Factor 1A gene homologues: partial characterization of the eukaryotic Elongation Factor 1A-F1 gene

Elongation factor 1A is a highly conserved protein that participates in translation. We report the occurrence of two genes homologous to the eukaryotic Elongation Factor 1A in Bradysia hygida and describe the partial cloning and characterization of the B. hygida eukaryotic Elongation Factor 1A-F1 (BheEF1A-F1) gene. The pattern of BheEF1A-F1 expression in the salivary gland at the end of the fourth larval instar was investigated using real-time PCR. The results showed that BheEF1A-F1 expression levels are relatively constant at the time when rapid changes in protein synthesis occur in this tissue. In situ hybridization experiments coupled to Southern blot analyses showed that the BheEF1A-F1 gene is located at position 3d of the A chromosome and a second gene homologous to eEF1A is located at position 6a of the X chromosome. Southern blot analyses showed that both the BheEF1A-F1 gene and the second gene homologous to eEF1A constitute non-amplified genes. The present results contribute to the molecular characterization of a sciarid eEF1A gene.

Bradysia hygida (Diptera, Sciaridae) presents two eukaryotic Elongation Factor 1A gene homologues: partial characterization of the eukaryotic Elongation Factor 1A-F1 gene.

Elongation factor 1A is a highly conserved protein that participates in translation. We report the occurrence of two genes homologous to the eukaryotic Elongation Factor 1A in Bradysia hygida and describe the partial cloning and characterization of the B. hygida eukaryotic Elongation Factor 1A-F1 (BheEF1A-F1) gene. The pattern of BheEF1A-F1 expression in the salivary gland at the end of the fourth larval instar was investigated using real-time PCR. The results showed that BheEF1A-F1 expression levels are relatively constant at the time when rapid changes in protein synthesis occur in this tissue. In situ hybridization experiments coupled to Southern blot analyses showed that the BheEF1A-F1 gene is located at position 3d of the A chromosome and a second gene homologous to eEF1A is located at position 6a of the X chromosome. Southern blot analyses showed that both the BheEF1A-F1 gene and the second gene homologous to eEF1A constitute non-amplified genes. The present results contribute to the molecular characterization of a sciarid eEF1A gene.

Broad-Complex, E74, and E75 early genes control DNA puff BhC4-1 expression in prepupal salivary glands.

The DNA puff BhC4-1 gene of the sciarid Bradysia hygida is induced in salivary glands prior to the pupal molt as a secondary response to the increase in ecdysone titers. Previous studies demonstrated that the BhC4-1 promoter is activated in transgenic Drosophila melanogaster salivary glands as a late response to the ecdysone peak that triggers metamorphosis, revealing that this aspect of BhC4-1 transcriptional regulation is conserved in the Drosophila background. To identify regulators of BhC4-1 expression, we utilized a candidate gene approach and tested the roles of the ecdysone-induced genes BR-C, E74, and E75. Our results reveal that the BR-C Z3 isoform is essential for BhC4-1-lacZ induction in prepupal salivary glands and constitute the first demonstration of the participation of early genes products on DNA puff genes regulation.

Is nitric oxide involved in the tolerance of Calomys callosus as a reservoir host towards Trypanosoma cruzi infection?

Trypanosoma cruzi, the agent of Chagas disease, is known to cause enhanced nitric oxide (NO) production, which might be involved in host resistance. The inducible nitric-oxide-synthase (iNOS) is assumed to be responsible for the NO increase after several infections. We studied the potential role of NO in Calomys callosus, a natural reservoir of this protozoan parasite. The concentration of NO was determined in spleen and liver of animals infected with two different T. cruzi strains, BOL and BOL-SB. Furthermore, the iNOS mRNA expression was quantified in the same cell types. NO production was detectable in both tissues exhibiting only slight differences compared to non-infected controls. All measured NO values were significantly lower than those reported for a number of different mouse strains, which displayed extremely enhanced NO levels after T. cruzi infection. Surprisingly, iNOS mRNA expression was induced in infected C. callosus but without subsequent increase of NO levels, indicating a post-transcriptional regulation mechanism. In summary, our results, indicate that the tolerance of C. callosus to T. cruzi is only accompanied by non-toxic NO intracellular concentrations.

Six Trypanosoma cruzi strains characterized by specific gene expression patterns.

The intracellular parasite Trypanosoma cruzi, the causative agent of Chagas disease, is known to comprise heterogeneous populations. One possibility to explain the obviously distinct phenotypes of different T. cruzi strains is differential expression of particular genes. This could result in environmental adaptations of the parasite within host organs, leading to distinct clinical symptoms. With the aim of identifying differentially expressed genes, we examined different T. cruzi strains by suppression subtractive hybridization analysis. The isolated clones were sequenced and Blasted for sequence-homology with known T. cruzi genes. A stage-specific glycoprotein (82gp), an 85-kDa protein with homology to heat-shock proteins, a beta-tubulin gene, a hexosetransporter, a dehydrogenase/ prostaglandin F2alpha-synthase and a cathepsin B-like protease were identified. The expression of these genes was analyzed by RT-PCR. Diverse expression patterns were detected for different T. cruzi strains, but no specific correlation between the gene expression and the classification of groups could be found. We discuss the presumed importance of these T. cruzi gene expression patterns for future strategies of molecular therapy of Chagas disease. For pathological studies, other parameters such as distinct gene/antigen expression could also be of interest, because they probably likewise correlate with distinct phenotypes.

Immunocharacterization of the DNA puff BhC4-1 protein of Bradysia hygida (Diptera: Sciaridae).

The DNA puff BhC4-1 gene is amplified and highly expressed in the salivary gland of Bradysia hygida late larvae. Using affinity-purified polyclonal antibodies we have identified the product of the BhC4-1 gene as a 43 kDa polypeptide which is present in extracts of salivary glands from late fourth instar larvae and in the corresponding gland secretion, but not in glands from earlier stages. We also demonstrate that this protein is produced mainly in the S1 and S3 regions of the salivary gland, where BhC4-1 amplification levels are more pronounced and larger amounts of mRNA are produced. By immunoelectron microscopy the BhC4-1 protein was detected in secretory granules of the S1 and S3 regions, and localized in fibrous structures present in the saliva.

Analysis of the amplification and transcription of the C3-22 gene of Rhynchosciara americana (Diptera: Sciaridae) in transgenic lines of Drosophila melanogaster.

Drosophila melanogaster was transformed with an 18 kb fragment of the C3 DNA puff of Rhynchosciara americana, including the C3-22 gene and the origins of replication that direct amplification. Different tissues and developmental stages of five independent transgenic lines were analyzed by quantitative Southern blot hybridization. No indication was found that the transformed fragment was amplified, strongly suggesting that factors involved in DNA puff amplification have not been conserved in Drosophila. Transcription of the C3-22 gene in the transgenic lines was found to be at a low and constitutive level throughout development. These results indicate that, unlike other DNA puff genes, the factors that regulate the C3-22 gene are not conserved in Drosophila.

Identification of regulatory regions in the DNA puff BhC4-1 promoter.

The mechanisms that control DNA puff BhC4-1 expression in the salivary gland of sciarid late larvae have been shown to be conserved in Drosophila. By analysing Drosophila transformed with constructs carrying progressive deletions of the BhC4-1 promoter fragment (-3314/+40) fused to the lacZ reporter gene we show that the elements required for the correct BhC4-1-lacZ developmental regulation in prepupal salivary glands are contained in a 226 bp fragment (-186/+40). Also, interestingly, this study identified a 67 bp fragment (-253/-187) that activates BhC4-1-lacZ expression specifically in the ring gland.

The induction of DNA puff BhC4-1 gene is a late response to the increase in 20-hydroxyecdysone titers in last instar dipteran larvae.

The characterization of DNA puff BhC4-1 expression was extended and its response to 20-hydroxyecdysone investigated in Bradysia hygida and in transgenic Drosophila carrying the BhC4-1 gene. In both organisms the activation of BhC4-1 in salivary glands occurs at the end of the larval stage coinciding with the peak in ecdysone titers which induces metamorphosis. Injections of 20-hydroxyecdysone into mid-fourth instar larvae of B. hygida show that the induction of BhC4-1 expression, as well as amplification and puff C4 expansion, are late events induced by the hormone. This late response of BhC4-1 expression was also observed in transgenic salivary glands cultivated in the presence of 20-hydroxyecdysone. In vitro studies using transgenic Drosophila indicate that both repressor and activator factors regulate the timing of BhC4-1 expression in salivary glands.

The DNA puff BhB10-1 gene is differentially expressed in various tissues of Bradysia hygida late larvae and constitutively transcribed in transgenic Drosophila.

We extended the characterization of the DNA puff BhB10-1 gene of Bradysia hygida by showing that, although its mRNA is detected only at the end of the fourth larval instar, BhB10-1 expression is not restricted to the salivary gland, the tissue in which this gene is amplified. Different amounts of BhB10-1 mRNA were detected in other larval tissues such as gut, Malpighian tubules, fat body, brain and cuticle, suggesting that this gene is expressed differentially in the various tissues analyzed. Analysis of transgenic Drosophila carrying the BhB10-1 transcription unit and flanking sequences revealed that the tested fragment promotes transcription in a constitutive manner. We suggest that either cis-regulatory elements are missing in the transgene or factors that temporally regulate the BhB10-1 gene in B. hygida are not conserved in Drosophila.

The DNA puff BhB10-1 gene is differentially expressed in various tissues of Bradysia hygida late larvae and constitutively transcribed in transgenic Drosophila

We extended the characterization of the DNA puff BhB10-1 gene of Bradysia hygida by showing that, although its mRNA is detected only at the end of the fourth larval instar, BhB10-1 expression is not restricted to the salivary gland, the tissue in which this gene is amplified. Different amounts of BhB10-1 mRNA were detected in other larval tissues such as gut, Malpighian tubules, fat body, brain and cuticle, suggesting that this gene is expressed differentially in the various tissues analyzed. Analysis of transgenic Drosophila carrying the BhB10-1 transcription unit and flanking sequences revealed that the tested fragment promotes transcription in a constitutive manner. We suggest that either cis-regulatory elements are missing in the transgene or factors that temporally regulate the BhB10-1 gene in B. hygida are not conserved in Drosophila

The DNA puff BhB10-1 gene encodes a glycine-rich protein secreted by the late stage larval salivary glands of Bradysia hygida.

We present the molecular characterization of a gene of Bradysia hygida DNA puff B10 whose temporal expression in the salivary gland correlates with the puff expansion. The transcription unit of this gene, named BhB10-1, was mapped in a 2-kb EcoRI genomic fragment that is amplified in the salivary gland of late fourth instar larvae. Its 1.3-kb transcript undergoes poly-A tail shortening during development, indicating that post-transcriptional controls as well as transcription activation are involved in the temporal regulation of the BhB10-1 gene. Analysis of the deduced amino acid sequence from the cDNA indicates that the BhB10-1 protein is a glycine-rich secretory protein. A BhB10-1-fusion protein expressed in bacteria was used to raise polyclonal antibodies. Using an immunopurified antibody, we identified the product of the DNA puff BhB10-1 gene as a 23-kDa polypeptide that is produced mainly by the salivary gland regions S1 and S3 and is present in the saliva of late larvae. This is the first direct identification of a protein encoded by a DNA puff amplified gene.

The DNA puff gene BhC4-1 of Bradysia hygida is specifically transcribed in early prepupal salivary glands of Drosophila melanogaster.

The BhC4-1 gene of the sciarid Bradysia hygida is located at DNA puff C4 and is amplified and actively transcribed in the salivary gland at the end of the last larval instar. We show here that a 3.6 kb fragment from the upstream region of the BhC4-1 gene is able to drive transcription in transgenic Drosophila specifically in prepupal salivary gland in a temporally regulated manner. The mRNA is present in maximal amounts in prepupae +3 h; in prepupae +9 h the levels of BhC4-1 mRNA decline, and it is no longer detected in pupae +24 h. Taken together these results suggest that most, if not all, of the key promoter regulatory elements were included in the DNA fragments employed to transform Drosophila. Moreover, strong expression of the transgenes implies conservation of the regulatory elements involved, since Drosophila transcription factors appear to recognize B. hygida regulatory DNA sequences. Quantitative Southern blot hybridization indicates that the sequences from DNA puff C4 are not amplified at detectable levels in salivary glands of transgenic prepupae when the BhC4-1 gene is transcribed. Transcription of a DNA puff in the absence of amplification indicates that the induction of these processes involves distinct mechanisms.

Molecular characterization of an 18 kb segment of DNA puff C4 of Bradysia hygida (Diptera, sciaridae).

The data presented here are an extension of the molecular characterization of DNA puff C4 of Bradysia hygida. A cDNA related to a gene amplified in this puff and expressed when puff C4 expands was cloned and sequenced. Analysis of the amino acid sequence deduced from the open reading frame present in the cDNA indicate that the encoded protein is secreted and comprises mostly alpha-helical coiled-coil. An 18 kb genomic segment containing the transcription unit of this gene was also cloned and the structure and expression of the 1.4 kb mRNA was determined. Quantitative slot blot hybridization of DNA complementary to the transcription unit shows that this gene is amplified about 21 times in the salivary gland, confirming data previously obtained. Fragments upstream of the 5' end, and beyond the 3' end, of the gene transcription unit were also analysed and shown to be amplified at least eight and five times, respectively. Based on these data we discuss how amplification could occur at DNA puffs.

Sequence of a cDNA encoding bothropstoxin I, a myotoxin from the venom of Bothrops jararacussu.

With the aim of further understanding the structure/function relationships in the membrane-damaging activity of the Lys49 phospholipase A2 (Lys49-PLA2) sub-family, we used PCR (polymerase chain reaction) on total venom gland cDNAs from Bothrops jararacussu with degenerate oligodeoxyribonucleotides encoding the N- and C-termini of myotoxin II, a Lys49-PLA2 from Bothrops asper. A 350-bp cDNA coding for bothropstoxin I (BtxtxI) was amplified. Sequencing of the amplified fragment shows that BtxtxI has a Lys49, and comparison with the known structure of myotoxin II showed that the amino acids involved in the formation of a novel dimeric structure in this protein were also conserved.