RESUMO
AIM: To evaluate bacterial leakage of Apexit Plus, a new root canal sealer, in comparison with AH Plus. METHODOLOGY: A total of 56 single-rooted human teeth were randomly divided into two experimental groups of 16 roots and two control groups. Roots were filled by lateral condensation with Gutta-percha and AH Plus or with Gutta-percha and Apexit Plus. A split chamber microbial leakage model was used in which Streptococcus mutans placed in the upper chamber could reach the lower chamber only through the filled canal. Positive controls were filled only with Gutta-percha and tested with bacteria, whereas negative controls were sealed with wax to test the seal between chambers. Additionally, film thickness, solubility and dimensional change were determined. RESULTS: All positive controls leaked within 24 h, whereas none of the negative controls leaked after 30 days. Apexit Plus had significant less bacterial leakage (log-rank test, P < 0.0001) than AH Plus. AH Plus (0.3% solubility) showed a slightly lower solubility than Apexit Plus (0.5% solubility) but a larger film thickness (28 vs. 11 mum) according to ISO 6876:2001. CONCLUSION: Apexit Plus had a better sealing ability in comparison with AH Plus.
Assuntos
Infiltração Dentária/prevenção & controle , Materiais Restauradores do Canal Radicular , Hidróxido de Cálcio , Resinas Epóxi , Guta-Percha , Humanos , Estimativa de Kaplan-Meier , Teste de Materiais , Obturação do Canal Radicular/métodos , Solubilidade , Streptococcus mutansRESUMO
The expression of procyclins is the earliest known marker of differentiation of bloodstream forms of Trypanosoma brucei to procyclic forms. We have generated transgenic bloodstream and procyclic forms in which the coding region of one procyclin gene was replaced by E. coli beta-glucuronidase (GUS). GUS activity can be monitored in a simple one-step colour reaction in microtitre plates; this assay is potentially suitable for large-scale screening for compounds that influence differentiation. GUS was stage-specifically expressed in procyclic forms and its synthesis occurred in parallel with that of procyclin when bloodstream forms were triggered to differentiate by the addition of cis-aconitate. GUS could also be induced by brief treatment with the proteases trypsin, pronase or thermolysin, but not with pepsin or thrombin. Interestingly, a combination of one of the active proteases with cis-aconitate resulted in increased GUS activity relative to either trigger alone. In contrast to cis-aconitate, protease treatment resulted in considerable cell death. Experiments with the pleomorphic strain AnTat 1.1 showed that long slender bloodstream forms were rapidly killed by proteases, whereas stumpy forms were largely resistant. Stumpy forms treated with trypsin differentiated synchronously and expressed procyclin with faster kinetics than when they were triggered by cis-aconitate. As predicted by the GUS assay, differentiation was even more rapid when both inducers were used simultaneously, with all cells expressing maximal levels of procyclin within 3 h.
Assuntos
Glucuronidase/genética , Glicoproteínas de Membrana/genética , Proteínas de Protozoários , Transgenes , Trypanosoma brucei brucei/crescimento & desenvolvimento , Trypanosoma brucei brucei/genética , Ácido Aconítico/farmacologia , Animais , Animais Geneticamente Modificados , Citratos/farmacologia , Endopeptidases/farmacologia , Escherichia coli/enzimologia , Escherichia coli/genética , Citometria de Fluxo , Regulação da Expressão Gênica no Desenvolvimento , Glicoproteínas de Membrana/metabolismo , Trypanosoma brucei brucei/efeitos dos fármacosRESUMO
In this paper we report the first case of antimycin A resistance in a protozoan parasite that is attributable to a mutation in the mitochondrial apocytochrome b (CYb) gene. We selected for, and isolated, a mutant Leishmania tarentolae strain that is resistant to antimycin A. This resistance was evident at the levels of the in vitro growth and enzymatic activity of the cytochrome bc1 complex. Molecular characterisation of the mutant revealed a Ser35Ile mutation in the expected region of the CYb gene. In kinetoplastids, CYb and other structural genes of the mitochondrial genome are located on the maxicircle component of the mitochondrial DNA, which is present in 20-50 copies. Primer-extension analysis confirmed the presence of the mutation at the mRNA level. The phenotypic manifestation of the mutation implies that the CYb mRNA is edited and translated within the mitochondrion. Thus, this finding provides direct evidence that edited RNAs are translated in kinetoplastid mitochondria. Furthermore, a defined mutation conferring drug resistance to a mitochondrial gene product can be exploited for the development of mitochondrial transfection systems for trypanosomatids.
Assuntos
Antibacterianos/farmacologia , Antimicina A/farmacologia , Apoproteínas/genética , Grupo dos Citocromos b/genética , Leishmania/genética , Mitocôndrias/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Citocromos b , Resistência a Medicamentos , Leishmania/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Dados de Sequência Molecular , Mutação Puntual , RNA Mensageiro/análise , Análise de Sequência de DNA , TransfecçãoRESUMO
We characterized the genes coding for the two dedicated enzymes of ethanolic fermentation, alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC), and show that they are functional in pollen. Two PDC-encoding genes were isolated, which displayed reciprocal regulation: PDC1 was anaerobically induced in leaves, whereas PDC2 mRNA was absent in leaves, but constitutively present in pollen. A flux through the ethanolic fermentation pathway could be measured in pollen under all tested environmental and developmental conditions. Surprisingly, the major factor influencing the rate of ethanol production was not oxygen availability, but the composition of the incubation medium. Under optimal conditions for pollen tube growth, approximately two-thirds of the carbon consumed was fermented, and ethanol accumulated into the surrounding medium to a concentration exceeding 100 mM.
Assuntos
Álcool Desidrogenase/metabolismo , Etanol/metabolismo , Fermentação/fisiologia , Nicotiana/enzimologia , Plantas Tóxicas , Piruvato Descarboxilase/metabolismo , Álcool Desidrogenase/genética , Sequência de Aminoácidos , Anaerobiose/fisiologia , Células Cultivadas , DNA Complementar/genética , Fermentação/genética , Regulação da Expressão Gênica de Plantas , Biblioteca Gênica , Genes de Plantas/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Dados de Sequência Molecular , Folhas de Planta/enzimologia , Pólen/enzimologia , Pólen/crescimento & desenvolvimento , Pólen/metabolismo , Piruvato Descarboxilase/genética , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , Nicotiana/genética , Nicotiana/crescimento & desenvolvimentoRESUMO
The mitochondrial genomes of trypanosomatids lack tRNA genes. Instead, mitochondrial tRNAs are encoded and synthesized in the nucleus and are then imported into mitochondria. This also applies for tRNATyr, which in trypanosomatids contains an 11 nt intron. Previous work has defined an exon mutation which leads to accumulation of unspliced precursor tRNATyr. In this study we have used the splicing-deficient tRNATyr as a vehicle to introduce foreign sequences into the mitochondrion of Leishmania tarentolae. The naturally occurring intron was replaced by synthetic sequences of increasing length and the resulting tRNATyr precursors were expressed in transgenic cell lines. Whereas stable expression of precursor tRNAsTyr was obtained for introns up to a length of 76 nt, only precursors having introns up to 38 nt were imported into mitochondria. These results demonstrate that splicing-deficient tRNATyr can be used to introduce short synthetic sequences into mitochondria in vivo. In addition, our results show that one factor which limits the efficiency of import is the length of the molecule.