Microvascular oxygen distribution in awake hamster window chamber model during hyperoxia

The microvascular effects and hemodynamic events following exposure to normobaric hyperoxia (because of inspiration of 100% O2) were studied in the awake hamster window chamber model and compared with normoxia. Hyperoxia increased arterial blood Po2 to 477.9 +/- 19.9 from 60.0 +/- 1.2 mmHg (P < 0.05). Heart rate and blood pressure were unaltered, whereas cardiac index was reduced from 196 +/- 13 to 144 +/- 31 ml.min-1.kg-1 (P < 0.05) in hyperoxia. Direct measurements in the microcirculation showed there was arteriolar vasoconstriction, reduction of microvascular flow (83% of control, P < 0.05), and functional capillary density (FCD, 74 +/- 16% of control), the latter change being significant (P < 0.05). Calculations of oxygen delivery and oxygen consumption based on the measured changes in microvascular blood flow velocity and diameter and estimates of oxygen saturation corrected for the Bohr effect due to the lowered pH and increased Pco2 showed that oxygen transport in the microvascular network did not change between normal and hyperoxic condition. The congruence of systemic and microvascular hemodynamics events found with hyperoxia suggests that the microvascular findings are common to most tissues in the organism, and that hyperoxia, due to vasoconstriction and the decrease of FCD, causes a maldistribution of perfusion in the microcirculation.     

Novel natural peptide substrates for endopeptidase 24.15, neurolysin,and angiotensin-converting enzyme

Endopeptidase 24.15 (EC; ep24.15), neurolysin (EC; ep24.16), and angiotensin-converting enzyme (EC; ACE) are metallopeptidases involved in neuropeptide metabolism in vertebrates. Using catalytically inactive forms of ep24.15 and ep24.16, we have identified new peptide substrates for these enzymes. The enzymatic activity of ep24.15 and ep24.16 was inactivated by site-directed mutagenesis of amino acid residues within their conserved HEXXH motifs, without disturbing their secondary structure or peptide binding ability, as shown by circular dichroism and binding assays. Fifteen of the peptides isolated were sequenced by electrospray ionization tandem mass spectrometry and shared homology with fragments of intracellular proteins such as hemoglobin. Three of these peptides (PVNFKFLSH, VVYPWTQRY, and LVVYPWTQRY) were synthesized and shown to interact with ep24.15, ep24.16, and ACE, with K(i) values ranging from 1.86 to 27.76 microm. The hemoglobin alpha-chain fragment PVNFKFLSH, which we have named hemopressin, produced dose-dependent hypotension in anesthetized rats, starting at 0.001 microg/kg. The hypotensive effect of the peptide was potentiated by enalapril only at the lowest peptide dose. These results suggest a role for hemopressin as a vasoactive substance in vivo. The identification of these putative intracellular substrates for ep24.15 and ep24.16 is an important step toward the elucidation of the role of these enzymes within cells.     

Critical residues for the coenzyme specificity of NAD+-dependent 15-hydroxyprostaglandin dehydrogenase

NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a member of the short chain dehydrogenase/reductase (SDR) family, is responsible for the biological inactivation of prostaglandins. Sequence alignment within SDR coupled with molecular modeling analysis has suggested that Gln-15, Asp-36, and Trp-37 of 15-PGDH may determine the coenzyme specificity of this enzyme. Site-directed mutagenesis was used to examine the important roles of these residues. Several single mutants (Q15K, Q15R, W37K, and W37R), double mutants (Q15K-W37K, Q15K-W37R, Q15R-W37K, and Q15R-W37R), and triple mutants (Q15K-D36A-W37R and Q15K-D36S-W37R) were prepared and expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli and purified by GSH-agarose affinity chromatography. Mutants Q15K, Q15R, W37K, W37R, Q15K-W37K, and Q15R-W37K were found to be inactive or almost inactive with NADP+ but still retained substantial activity with NAD+. Mutant Q15K-W37R and mutant Q15R-W37R showed comparable activity for NAD+ and NADP+ with an increase in activity nearly 3-fold over that of the wild type. However, approximately 30-fold higher in K(m) for NADP+ than that of the wild type enzyme for NAD+ was found for mutants Q15K-W37R and Q15R-W37R. Similarly, the K(m) values for PGE(2) of mutants were also shown to increase over that of the wild type. Further mutation of Asp-36 to either an alanine or a serine of the double mutant Q15K-W37R (i.e., triple mutants Q15K-D36A-W37R and Q15K-D36S-W37R) rendered the mutants exhibiting exclusive activity with NADP+ but not with NAD+. The triple mutants showed a decrease in K(m) for NADP+ but an increase in K(m) for PGE(2). Further mutation at Ala-14 to a serine of a triple mutant (Q15K-D36S-W37R) decreased the K(m) values for both NADP+ and PGE(2) to levels comparable to those of the wild type. These results indicate that the coenzyme specificity of 15-PGDH can be altered from NAD+ to NADP+ by changing a few critical residues near the N-terminal end.     

Prevalence of Trypanosoma lewisi in Rattus norvegicus from Belo Horizonte,State of Minas Gerais,Brazil

From April 1984 to March 1985, a Trypanosoma lewisi prevalence of 21.7% was found in 429 Rattus norvegicus trapped in Belo Horizonte, State of Minas Gerais, Brazil. The infection rates were higher in male and young rats and could be attributed to ecological and behavioral factors. T. lewisi was observed in rats measuring between 60 and 250 mm. Data about monthly T. lewisi infections throughout the year are presented for the first time in Brazil, with the highest prevalences observed in the warm-rainy season (October to March).     

Control of Chagas disease in blood banks in Colombia

The screening programs for the Chagas disease agent, Trypanosoma cruzi, were examined in Colombian blood banks and, as a consequence, several procedural improvements in the blood bank network were recommended. Screening strategies and techniques were examined, as well as the action taken when seropositive donors were discovered. From a total of 180 blood banks in 33 departments, 103 banks in 20 departments answered the survey. The 103 banks collected 291, 105 units of blood, corresponding to 66.6% of all units collected in the country in 1997. Of these blood units, 99.6% were screened for Chagas trypanosomes; 3,321 (1.2%) of 287,048 were found positive for anti-T. cruzi. The data were grouped by department; geographical differences for seroprevalence rates varied markedly between 0% and 12.6%. The most commonly used serological technique was ELISA, but only 33.2% of the positive samples for anti-T. cruzi underwent further confirmatory testing, mainly through indirect immunofluorescent test. Most (95.1%) of the blood banks used basic, internal quality control procedures, and 73.8% sent positive samples to other laboratories for external quality control.     

Chrysanthemum chlorotic mottle viroid RNA: dissection of the pathogenicity determinant and comparative fitness of symptomatic and non-symptomatic variants

Chrysanthemum chlorotic mottle viroid (CChMVd) is a small RNA (398-401nt) with hammerhead ribozymes in both polarity strands that mediate self-cleavage of the oligomeric RNA intermediates generated in a rolling-circle mechanism of replication. Within the in vivo branched RNA conformation of CChMVd, a tetraloop has been identified as a major determinant of pathogenicity. Here we present a detailed study of this tetraloop by site-directed mutagenesis, bioassay of the CChMV-cDNA clones and analysis of the resulting progenies. None of the changes introduced in the tetraloop, including its substitution by a triloop or a pentaloop, abolished infectivity. In contrast to observations for other RNAs, the thermodynamically stable GAAA tetraloop characteristic of non-symptomatic CChMVd-NS strains was not functionally interchangeable for other stable tetraloops of the UNCG family, suggesting that the sequence, rather than the structure, is the major factor governing conservation of this motif. In most cases, the changes introduced initially led to symptomless infections, which eventually evolved to be symptomatic concurrently with the prevalence in the progeny of the UUUC tetraloop characteristic of symptomatic CChMVd-S strains. Only in one case did the GAAA tetraloop emerge and eventually dominate the progeny in infected plants that were non-symptomatic. These results revealed two major fitness peaks in the tetraloop (UUUC and GAAA), whose adjacent stem was also under strong selection pressure. Co-inoculations with CChMVd-S and -NS variants showed that only when the latter was in a 100- or 1000-fold excess did the infected plants remain symptomless, confirming the higher biological fitness of the S variant and explaining the lack of symptom expression previously observed in cross-protection experiments.     

Phylogeny of Stemphylium spp. based on ITS and glyceraldehyde-3-phosphate dehydrogenase gene sequences

The phylogenetic relationships among 44 isolates representing 16 species of Stemphylium were inferred from ITS and glyceraldehyde-3-phosphate dehydrogenase (gpd) sequence data. The results generally agree with morphological species concepts. There was strong support for monophyly of the genus Stemphylium. Analysis of the gpd fragment in particular was useful for establishing well-supported relationships among the species and isolates of Stemphylium. Species of Stemphylium that appear to have lost the ability to produce a sexual state are scattered among the species with the ability to reproduce sexually (Pleospora spp.). Species that are pathogenic to alfalfa are resolved into two groups. Stemphylium botryosum and two isolates with morphological characters similar to S. globuliferum had identical sequences at both loci. These two loci in S. vesicarium, S. alfalfae and S. herbarum are nearly identical but differ from S. botryosum. The separation of S. vesicarium, S. herbarum and S. alfalfae into separate species by morphometric evidence was not supported by the molecular data. Morphological and developmental characters such as size and shape of conidia, conidiophores, and ascospores, and size and time of maturation of pseudothecia are useful for diagnosing species. However, other morphological characters such as septum development and small variations in conidial wall ornamentation are not as useful.     

Ethnicity and type 2 diabetes in Rio de Janeiro,Brazil, with a review of the prevalence of the disease in Amerindians

To what extent can ethnic factors contribute to the prevalence of type 2 diabetes and impaired glucose tolerance (IGT) in an urban Brazilian population? Conversely, how can environmental factors such as diet change these prevalences in a given ethnic group, in this case Brazilian Indians? To answer these questions estimates of ethnic admixture in Afro- and Euro-Brazilians from Rio de Janeiro, Brazil, were established using eight genetic systems and compared with the prevalences of these conditions obtained previously. This information was integrated with results obtained inside and outside of Brazil. The similarity of prevalences for type 2 diabetes and IGT in Afro- and Euro-Brazilians may be related to the extensive gene flow that occurred between them and to similar socioeconomic levels in the samples investigated. On the other hand, changes in the traditional diet are probably conditioning the appearance of diabetes among Brazilian and other South American Indians.