Factors associated with self-reported use of mouthwashes in southern Brazil in 1996 and 2009.

OBJECTIVE: To evaluate the association between sociodemographic factors and self-reported use of mouthwashes in a city in the south of Brazil over a time span of 13 years. METHODS: The study involves a comparison of two household surveys conducted in 1996 and 2009. Respectively, 850 and 982 households were included in each year. The mother of the family answered to a structured questionnaire to provide sociodemographic data. Self-reported use of mouthwash was evaluated in a binary manner (yes/no). Multivariate Poisson regression models were used to estimate proportion ratios (PR) and 95% confidence intervals (95% CI). RESULTS: The proportion of mouthwash use increased significantly from 10.8% to 24.2% after 13 years. The probability of using mouthwashes increased more than two times from 1996 to 2009 (PR = 2.25 95% CI 1.80-2.82). Higher probabilities for using mouthwashes were observed in households with higher income (PR = 2.63 95% CI 1.97-3.51) and in those households where the mothers had higher educational levels (PR = 1.46 95% CI 1.05-2.03). CONCLUSION: Higher family income and higher maternal educational level were associated with self-reported use of mouthwashes in a higher proportion of households.

Leptospirosis following a flood in the Veneto area, North-east Italy.

In August 2002, an exceptional flood devastated a suburban area in the surroundings of Vicenza, North-east Italy. A fatal case of haemorrhagic pneumonia, which was presumptively diagnosed as leptospirosis, was observed as a consequence of the inundation. A local seroepidemiological survey was activated thereafter, with the principal aim of evaluating the risk of infection with Leptospirae in the population exposed to the flood. A 6.8% seroconversion rate was found in the population studied; however, the case previously observed remained unique, since an overt outbreak of leptospirosis did not occur.

An essential role for the caspase dronc in developmentally programmed cell death in Drosophila.

Dronc is a caspase recruitment domain-containing Drosophila caspase that is expressed in a temporally and spatially restricted fashion during development. Dronc is the only fly caspase known to be regulated by the hormone ecdysone. Here we show that ectopic expression of dronc in the developing fly eye leads to increased cell death and an ablated eye phenotype that can be suppressed by halving the dosage of the genes in the H99 complex (reaper, hid, and grim) and enhanced by mutations in diap1. In contrast to previous reports, we show that the dronc eye ablation phenotype can be suppressed by coexpression of the baculoviral caspase inhibitor p35. Dronc also interacts, both genetically and biochemically, with the CED-4/Apaf-1 fly homolog, Dark. Furthermore, extracts made from Dark homozygous mutant flies have reduced ability to process Dronc, showing that Dark is required for Dronc processing. Finally, using the RNA interference technique, we show that loss of Dronc function in early Drosophila embryos results in a dramatic decrease in cell death, indicating that Dronc is important for programmed cell death during embryogenesis. These results suggest that Dronc is a key caspase mediating programmed cell death in Drosophila.

Debcl, a proapoptotic Bcl-2 homologue, is a component of the Drosophila melanogaster cell death machinery.

Bcl-2 family of proteins are key regulators of apoptosis. Both proapoptotic and antiapoptotic members of this family are found in mammalian cells, but no such proteins have been described in insects. Here, we report the identification and characterization of Debcl, the first Bcl-2 homologue in Drosophila melanogaster. Structurally, Debcl is similar to Bax-like proapoptotic Bcl-2 family members. Ectopic expression of Debcl in cultured cells and in transgenic flies causes apoptosis, which is inhibited by coexpression of the baculovirus caspase inhibitor P35, indicating that Debcl is a proapoptotic protein that functions in a caspase-dependent manner. debcl expression correlates with developmental cell death in specific Drosophila tissues. We also show that debcl genetically interacts with diap1 and dark, and that debcl-mediated apoptosis is not affected by gene dosage of rpr, hid, and grim. Biochemically, Debcl can interact with several mammalian and viral prosurvival Bcl-2 family members, but not with the proapoptotic members, suggesting that it may regulate apoptosis by antagonizing prosurvival Bcl-2 proteins. RNA interference studies indicate that Debcl is required for developmental apoptosis in Drosophila embryos. These results suggest that the main components of the mammalian apoptosis machinery are conserved in insects.

DRONC, an ecdysone-inducible Drosophila caspase.

Caspases play an essential role in the execution of programmed cell death in metazoans. Although 14 caspases are known in mammals, only a few have been described in other organisms. Here we describe the identification and characterization of a Drosophila caspase, DRONC, that contains an amino terminal caspase recruitment domain. Ectopic expression of DRONC in cultured cells resulted in apoptosis, which was inhibited by the caspase inhibitors p35 and MIHA. DRONC exhibited a substrate specificity similar to mammalian caspase-2. DRONC is ubiquitously expressed in Drosophila embryos during early stages of development. In late third instar larvae, dronc mRNA is dramatically up-regulated in salivary glands and midgut before histolysis of these tissues. Exposure of salivary glands and midgut isolated from second instar larvae to ecdysone resulted in a massive increase in dronc mRNA levels. These results suggest that DRONC is an effector of steroid-mediated apoptosis during insect metamorphosis.

Targeted disruption of caspase genes in mice: what they tell us about the functions of individual caspases in apoptosis.

Cysteine proteases of the caspase family are crucial mediators of apoptosis. All mammalian cells contain a large number of caspases. Although many caspases are activated in a cell committed to apoptosis, recent data from caspase gene knockout mice suggest that individual caspases may be involved in the cell and stimulus-specific pathways of cell death. The gene disruption studies also establish the functional hierarchy between two structurally distinct classes of caspases. The present review discusses these recent findings and elaborates on how these mutant mouse models have helped the understanding of the mechanisms that govern programmed cell death in the immune and other systems.

Conversion of procaspase-3 to an autoactivating caspase by fusion to the caspase-2 prodomain.

Caspases are cysteine proteases that play an essential role in apoptosis. Initial activation of caspases defines the key step in apoptotic execution. Based on primary structure, caspases can be divided into two groups, those with long amino-terminal prodomains (class I), and those with relatively short prodomains (class II). On overexpression in mammalian cells, class I caspases can induce cell death that is dependent on their autocatalytic activity. Recent studies suggest that the long prodomains in some class I caspases are able to mediate dimerization of procaspase molecules, thereby promoting autoprocessing. In this communication, we demonstrate that fusion of the prodomain of a class I caspase (Nedd2/caspase-2) with procaspase-3 greatly augments autocatalysis and apoptosis induction by the chimeric caspase-3 molecule. The chimeric caspase-3 molecules were able to form homodimers in Saccharomyces cerevisiae and were efficiently processed in transfected mammalian cells. These results provide direct evidence for a role of a class I caspase prodomain in caspase autoactivation and processing and establish a basis for functional hierarchy among the two classes of caspases.

Prodomain-dependent nuclear localization of the caspase-2 (Nedd2) precursor. A novel function for a caspase prodomain.

Caspases are cysteine proteases that play an essential role in apoptosis by cleaving several key cellular proteins. Despite their function in apoptosis, little is known about where in the cell they are localized and whether they are translocated to specific cellular compartments upon activation. In the present paper, using Aequorea victoria green fluorescent protein fusion constructs, we have determined the localization of Nedd2 (mouse caspase-2) and show that both precursor and processed caspase-2 localize to the cytoplasmic and the nuclear compartments. We demonstrate that the nuclear localization of caspase-2 is strictly dependent on the presence of the prodomain. A caspase-2 prodomain-green fluorescent protein localized to dot- and fiber-like structures mostly in the nucleus, whereas a protein lacking the prodomain was largely concentrated in the cytoplasm. We also show that an amino-terminal fusion of the prodomain of caspase-2 to caspase-3 mediates nuclear transport of caspase-3, which is normally localized in the cytoplasm. These results suggest that, in addition to roles in dimerization and recruitment through adaptors, the caspase-2 prodomain has a novel function in nuclear transport.

Human and Saccharomyces cerevisiae dolichol phosphate mannose synthases represent two classes of the enzyme, but both function in Schizosaccharomyces pombe.

Dolichol phosphate mannose (Dol-P-Man), formed upon transfer of Man from GDPMan to Dol-P, is a mannosyl donor in pathways leading to N-glycosylation, glycosyl phosphatidylinositol membrane anchoring, and O-mannosylation of protein. Dol-P-Man synthase is an essential protein in Saccharomyces cerevisiae. We have cloned cDNAs encoding human and Schizosaccharomyces pombe proteins that resemble S. cerevisiae Dol-P-Man synthase. Disruption of the gene for the S. pombe Dol-P-Man synthase homolog, dpm1(+), is lethal. The known Dol-P-Man synthase sequences can be divided into two classes. One contains the S. cerevisiae, Ustilago maydis, and Trypanosoma brucei enzymes, which have a COOH-terminal hydrophobic domain, and the other contains the human, S. pombe, and Caenorhabditis synthases, which lack a hydrophobic COOH-terminal domain. The two classes of synthase are functionally equivalent, because S. cerevisiae DPM1 and its human counterpart both complement the lethal null mutation in S. pombe dpm1(+). The findings that Dol-P-Man synthase is essential in yeast and that the Ustilago and Trypanosoma synthases are in a different class from the human enzyme raise the possibility that Dol-P-Man synthase could be exploited as a target for inhibitors of pathogenic eukaryotic microbes.

The essential Schizosaccharomyces pombe gpil+ gene complements a bakers' yeast GPI anchoring mutant and is required for efficient cell separation.

The Schizosaccharomyces pombe gpil+ gene was cloned by complementation of the Saccharomyces cerevisiae gpil mutant, which has temperature-sensitive defects in growth and glycosyl phosphatidylinositol (GPI) membrane anchoring or protein, and which is defective in vitro in the first step in GPI anchor assembly, the formation of n-acetylglucosaminyl phosphatidylinositol (GlcNAc-PI). S. pombe gpil+ encodes a protein with 29% identity to amino acids 87-609 of the S. cerevisiae protein, and is the functional homolog of the S. cerevisiae Gpil protein, for it restores [3H]inositol-labelling of protein and in vitro GlcNAc-PI synthetic activity to both S. cerevisiae gpil and gpil::URA3 cells. Disruption of gpil+ is lethal. Haploid delta gpil+::his7+ spores germinate, but proceed through no more than three rounds of cell division, many cells ceasing growth as binucleate, septate cells with thickened septa. These results indicate that GPI synthesis is an essential function in fission yeast, and suggest that GPI anchoring is also required for completion of cytokinesis.

Isolation of temperature-sensitive yeast GPI-anchoring mutants.

We are using a genetic approach to explore the synthesis and function of glycosylphosphatidylinositol (GPI). We have developed a novel strategy to isolate Saccharomyces cerevisiae mutants blocked in GPI anchoring by screening colonies of mutagenized yeast cells for those that fail to incorporate [3H]inositol into protein. Among our isolates are strains blocked in mannosylation of the GPI-anchorprecursor, and strains defective in the synthesis of N-acetylglucosaminyl phosphatidylinositol (GlcNAc-PI). We have characterized one mutant, gpi1, further. This strain is defective in GlcNAc-PI synthesis and is temperature-sensitive for growth. Completion of the first step in GPI assembly is therefore required for the growth of the unicellular eukaryote S. cerevisiae. We have isolated plasmids that complement the gpi1 mutation from S. cerevisiae genomic DNA-and fission yeast cDNA libraries.

Isolation of temperature-sensitive yeast GPI-anchoring mutants

We are using a genetic approach to explore the synthesis and function of glycosylphosphatidylinositol (GPI). We have developed a novel strategy to isolate Saccharomyces cerevisiae mutants blocked in GPI anchoring by screening colonies of mutagenized yeast cells for those that fail to incorporate [3H]inositol into protein. Among our isolates are strains blocked in mannosylation of the GPI-anchor precursor, and strains defective in the synthesis of N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI). We have characterized one mutant, gpil, further. This strain is defective in GlcNAC-PI synthesis and is temperature-sensitive for growth. Completion of the first step in GPI assembly is therefore required for the growth of the unicellular eukaryote S. cerevisiae. We have isolated plasmids that complement the gpil mutation from S. cerevisiae genomic DNA- and fission yeast cDNA libraries