The risk of child and adolescent mortality among vulnerable populations in Rio de Janeiro, Brazil

This study investigated the importance of socioeconomic factors such as education, income, religion, family structure and residence in explaining the increased risk of mortality among vulnerable populations aged less than 20 years in Rio de Janeiro, Brazil. Data used were from the 1991 Brazilian Demographic Census and comprised 121,060 women aged 15-49 residing in Rio de Janeiro. Two alternative statistical methods were used to calculate the risk of death: the widely used Brass method (an indirect estimate which assesses population risks) and a case-control study (which assesses individual risks). The study also focused on the importance of indicators of human and social capital, the lack of which may explain the higher risk of death among children and adolescents. Lack of education was found to be a major determinant of mortality at young ages. Residence in a favela (shantytown), families in which mothers were the head of the family, and a lower median level of income were found to be significant determinants of mortality among vulnerable populations in Brazil. However, religion was not found to be as important a predictor of high mortality.     

Inhibition of tumor necrosis factor mRNA translation by a rationally designed immunomodulatory peptide

Based on sequences of immunomodulatory peptides derived from the heavy chain of HLA Class I, novel immunomodulatory peptides with increased potency were developed by computer-aided rational design. Allotrap 1258 was characterized in detail and shown to inhibit cell-mediated immune responses in vitro and in vivo. Immunomodulatory activity was associated with the capability of the peptides to modulate heme oxygenase (HO) activity. In this study we analyzed the effect of Allotrap 1258 on cytokine expression. Allotrap 1258 inhibited concanavalin A- and lipopolysaccharide-induced human and mouse tumor necrosis factor (TNF) production in vitro and in vivo but had no effect on interleukin (IL)-1, IL-2, IL-4, IL-6, or IL-10 expression. Experiments with HO-1/KO and iNOS/KO mice showed that Allotrap 1258-mediated inhibition of TNF was independent of HO-1 and iNOS. Quantitation of TNF protein expression and mRNA steady state levels demonstrated that Allotrap 1258-mediated inhibition occurred at the translational level. Deletion of the AU-rich element in the 3'-untranslated region (UTR) of TNF mRNA, a region known to be involved in TNF mRNA translation, had minimal effect on Allotrap 1258-mediated inhibition. However, replacement of the TNF 3'-UTR with the human globin 3'-UTR rendered the peptide inactive. This demonstrates that besides AU-rich elements, other sequences in the 3'-UTR of TNF mRNA are involved in translational control of TNF expression. Such sequences are necessary for Allotrap 1258-mediated inhibition of TNF production.     

Two forms of UvrC protein with different double-stranded DNA binding affinities

Using phosphocellulose followed by single-stranded DNA-cellulose chromatography for purification of UvrC proteins from overproducing cells, we found that UvrC elutes at two peaks: 0.4 m KCl (UvrCI) and 0.6 m KCl (UvrCII). Both forms of UvrC have a major peptide band (>95%) of the same molecular weight and identical N-terminal amino acid sequences, which are consistent with the initiation codon being at the unusual GTG site. Both forms of UvrC are active in incising UV-irradiated, supercoiled phiX-174 replicative form I DNA in the presence of UvrA and UvrB proteins; however, the specific activity of UvrCII is one-fourth that of UvrCI. The molecular weight of UvrCII is four times that of UvrCI on the basis of results of size exclusion chromatography and glutaraldehyde cross-linking reactions, indicating that UvrCII is a tetramer of UvrCI. Functionally, these two forms of UvrC proteins can be distinguished under reaction conditions in which the protein/nucleotide molar ratio is >0.06 by using UV-irradiated, (32)P-labeled DNA fragments as substrates; under these conditions UvrCII is inactive in incision, but UvrCI remains active. The activity of UvrCII in incising UV-irradiated, (32)P- labeled DNA fragments can be restored by adding unirradiated competitive DNA, and the increased level of incision corresponds to a decreased level of UvrCII binding to the substrate DNA. The sites of incision at the 5' and 3' sides of a UV-induced pyrimidine dimer are the same for UvrCI and UvrCII. Nitrocellulose filter binding and gel retardation assays show that UvrCII binds to both UV-irradiated and unirradiated double-stranded DNA with the same affinity (K(a), 9 x 10(8)/m) and in a concentration-dependent manner, whereas UvrCI does not. These two forms of UvrC were also produced by the endogenous uvrC operon. We propose that UvrCII-DNA binding may interfere with Uvr(A)(2)B-DNA damage complex formation. However, because of its low copy number and low binding affinity to DNA, UvrCII may not interfere with Uvr(A)(2)B-DNA damage complex formation in vivo, but instead through double-stranded DNA binding UvrCII may become concentrated at genomic areas and therefore may facilitate nucleotide excision repair.     

Adaptations of the helix-grip fold for ligand binding and catalysis in the START domain superfamily

With a protein structure comparison, an iterative database search with sequence profiles, and a multiple-alignment analysis, we show that two domains with the helix-grip fold, the star-related lipid-transfer (START) domain of the MLN64 protein and the birch allergen, are homologous. They define a large, previously underappreciated superfamily that we call the START superfamily. In addition to the classical START domains that are primarily involved in eukaryotic signaling mediated by lipid binding and the birch antigen family that consists of plant proteins implicated in stress/pathogen response, the START superfamily includes bacterial polyketide cyclases/aromatases (e.g., TcmN and WhiE VI) and two families of previously uncharacterized proteins. The identification of this domain provides a structural prediction of an important class of enzymes involved in polyketide antibiotic synthesis and allows the prediction of their active site. It is predicted that all START domains contain a similar ligand-binding pocket. Modifications of this pocket determine the ligand-binding specificity and may also be the basis for at least two distinct enzymatic activities, those of a cyclase/aromatase and an RNase. Thus, the START domain superfamily is a rare case of the adaptation of a protein fold with a conserved ligand-binding mode for both a broad variety of catalytic activities and noncatalytic regulatory functions. Proteins 2001;43:134-144.     

Characterization of protein kinases in mitotic and meiotic cell extracts

A number of protein kinases have been separated and identified in extracts from mitotic and interphase culture cells and from mature and immature amphibian oocytes using nondenaturing polyacrylamide gel electrophoresis followed by in situ phosphorylation assays. Certain of these protein kinase activities appear to correlate with the biological activity of extracts, assayed by their ability to induce meiotic maturation following injection into Xenopus oocytes. These results are consistent with the notion that protein phosphorylation/dephosphorylation may be integral to the mechanisms of both nuclear membrane breakdown and chromosome condensation, events common and distinctive to mitosis and meiosis.     

C-terminal truncated cannabinoid receptor 1 coexpressed with G protein trimer in Sf9 cells exists in a precoupled state and shows constitutive activity

We have investigated the existence of a precoupled form of the distal C-terminal truncated cannabinoid receptor 1 (CB1-417) and heterotrimeric G proteins in a heterologous insect cell expression system. CB1-417 showed higher production levels than the full-length receptor. The production levels obtained in our expression system were double the values reported in the literature. We also observed that at least the distal C-terminus of the receptor was not involved in receptor dimerization, as was predicted in the literature. Using fluorescence resonance energy transfer, we found that CB1-417 and Galpha(i1)beta(1)gamma(2) proteins were colocalized in the cells. GTPgammaS binding assays with the Sf9 cell membranes containing CB1-417 and the G protein trimer showed that the receptor could constitutively activate the Galpha(i1) protein in the absence of agonists. A CB1-specific antagonist (SR 141716A) inhibited this constitutive activity of the truncated receptor. We found that the CB1-417/Galpha(i1)beta(1)gamma(2) complex could be solubilized from Sf9 cell membranes and coimmunoprecipitated. In this study, we have proven that the receptor and G proteins can be coexpressed in higher yields using Sf9 cells, and that the protein complex is stable in detergent solution. Thus, our system can be used to produce sufficient quantities of the protein complex to start structural studies.     

Epithelial Viscoelasticity Is Regulated by Mechanosensitive E-cadherin Turnover

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