Blood transfusion and iatrogenic risks in Mexico City. Anti-Trypanosoma cruzi seroprevalence in 43,048 blood donors, evaluation of parasitemia, and electrocardiogram findings in seropositive

Iatrogenous transmission of Trypanosoma cruzi by blood transfusion was suggested as a potential risk by Pellegrino (1949). Seropositive blood donors in Mexico were first reported in 1978, however, limited information is available due to small sampling, the use of heterogeneous serologic assays, and geographically limited studies. A wide survey carried out in 18 out of the 32 states of Mexico, showed a national mean of 1.6% seropositive among 64,969 donors, ranging from 0.2 to 2.8%. In the present study, we have screened 43,048 voluntary blood donors in a period of five years at the Instituto Nacional de Cardiología I. Chávez, a concentration hospital located in Mexico city which serves mainly the metropolitan area and accepts from all over the country. Standardized ELISA and IIF were used to identify seropositive individuals in addition to hemoculture, PCR and standard 12 lead ECG tests that were applied to a group of seropositive patients (29/161). The result showed a seropositivity of 0.37% (161/43,048). From the group of seropositive individuals 40% (12/29) were potential carriers of T. cruzi at the donation time and 5/29 had subclinical ECG abnormalities. Parasitological tests performed in 70 erythrocyte and platelet fractions from seropositive units (70/161) showed negative results. Our findings strongly support T. cruzi screening in the transfusion medicine practice and identify subclinical heart disease among seropositive blood donors.     

Effect of grazing on community structure and productivity of a Uruguayan grassland

Grasslands and their grazers provide some of the most compelling examples for studying the relationship between diversity, productivity, and disturbance. In this study, we analyzed the impact of grazing-induced changes in species composition and community structure upon the productivity of a grassland in the Campos region, Uruguay. We compared three treatments: a continuously grazed area, a 9-year old exclosure to domestic herbivores, and grazing-simulated plots inside the exclosure, which were clipped so that their standing biomass resembled that of the grazed area. We studied the community composition of the grazed and ungrazed situations, and determined biomass and above-ground net primary production (ANPP) of the three treatments during 1 year. Grazed plots had higher species richness and diversity than the exclosure. Grazing resulted in the replacement of some cool-season, tussock grasses by warm-season, prostrate grasses. ANPP was 51% higher under grazing than in the exclosure, but the grazing-simulated plots inside the exclosure were the most productive treatment, 29% higher than the grazed plots. Thus, two components of grazing effect may be postulated for this grassland. The structural component resulted in higher ANPP, probably due to the elimination of standing dead biomass. The species composition component resulted in lower ANPP once the structural component was controlled, probably due to the shift to warm-season phenology and prostrate habit. Our findings contrast with a similar experiment carried out in the neighbouring Flooding Pampa region, which suggests that the relationship between grazing and community structure and function is difficult to generalize.     

Epoxy resin as fixative during freeze-substitution

An alternative protocol for freeze-substitution is described. Araldite/Epon embedding medium (20% in acetone) is first used as a stabilizer (as e.g., OsO(4)) and then as embedding medium. The major components of the Araldite/Epon resin formulation react with proteins and lipids and provide for an excellent preservation and reasonable visualisation of the ultrastructure. The ultrastructural appearance can be deliberately influenced with the standard freeze-substitution procedure [Van Harreveld, A., Crowell, J., 1964. Electron microscopy after rapid freezing on a metal surface and substitution fixation. Anat. Rec. 149, 381-386.] using OsO(4) as stabilizing agent by protocols which degrade cytoplasmic and membrane proteins. Epoxy stabilized and embedded samples may become an important tool to get information about the effects of different reagents and protocols used in freeze-substitution. We believe that an in-depth understanding of the procedures is required to correctly interpret images and to complement studies of dynamic processes by light microscopy with reliable, highly detailed ultrastructural information. The block face of epoxy stabilized samples after ultrathin sectioning is highly suited for the analysis of the ultrastructure by AFM.     

Trans-arachidonic acids generated during nitrative stress induce a thrombospondin-1-dependent microvascular degeneration

Nitrative stress has an important role in microvascular degeneration leading to ischemia in conditions such as diabetic retinopathy and retinopathy of prematurity. Thus far, mediators of nitrative stress have been poorly characterized. We recently described that trans-arachidonic acids are major products of NO(2)(*)-mediated isomerization of arachidonic acid within the cell membrane, but their biological relevance is unknown. Here we show that trans-arachidonic acids are generated in a model of retinal microangiopathy in vivo in a NO(*)-dependent manner. They induce a selective time- and concentration-dependent apoptosis of microvascular endothelial cells in vitro, and result in retinal microvascular degeneration ex vivo and in vivo. These effects are mediated by an upregulation of the antiangiogenic factor thrombospondin-1, independently of classical arachidonic acid metabolism. Our findings provide new insight into the molecular mechanisms of nitrative stress in microvascular injury and suggest new therapeutic avenues in the management of disorders involving nitrative stress, such as ischemic retinopathies and encephalopathies.     

Herpes simplex virus 1 envelopment follows two diverse pathways

Herpesvirus envelopment is assumed to follow an uneconomical pathway including primary envelopment at the inner nuclear membrane, de-envelopment at the outer nuclear membrane, and reenvelopment at the trans-Golgi network. In contrast to the hypothesis of de-envelopment by fusion of the primary envelope with the outer nuclear membrane, virions were demonstrated to be transported from the perinuclear space to rough endoplasmic reticulum (RER) cisternae. Here we show by high-resolution microscopy that herpes simplex virus 1 envelopment follows two diverse pathways. First, nuclear envelopment includes budding of capsids at the inner nuclear membrane into the perinuclear space whereby tegument and a thick electron dense envelope are acquired. The substance responsible for the dense envelope is speculated to enable intraluminal transportation of virions via RER into Golgi cisternae. Within Golgi cisternae, virions are packaged into transport vacuoles containing one or several virions. Second, for cytoplasmic envelopment, capsids gain direct access from the nucleus to the cytoplasm via impaired nuclear pores. Cytoplasmic capsids could bud at the outer nuclear membrane, at membranes of RER, Golgi cisternae, and large vacuoles, and at banana-shaped membranous entities that were found to continue into Golgi membranes. Envelopes originating by budding at the outer nuclear membrane and RER membrane also acquire a dense substance. Budding at Golgi stacks, designated wrapping, results in single virions within small vacuoles that contain electron-dense substances between envelope and vacuolar membranes.     

Morphological and molecular systematics of Rocky Mountain alpine Laccaria

The alpine zone is comprised of habitats at elevations above treeline, and macromycetes play important ecological roles as decomposers and mycorrhizal symbionts here as elsewhere. Laccaria is an important group of ectomycorrhizal basidiomycetes widely used in experimental and applied research. A systematic study of alpine Laccaria species using morphological, cultural and molecular (ribosomal DNA internal transcribed spacer) data revealed five taxa in the Rocky Mountain alpine zone: L. laccata var. pallidifolia, L. nobilis (the first published report for arctic-alpine habitats), L. pumila, L. montana and L. pseudomontana (a newly described taxon similar to L. montana with more ellipsoidal, finely echinulate basidiospores). All occur in the southern Rocky Mountains of Colorado; however, only L. pumila and L. montana were found on the Beartooth Plateau in the northern Rocky Mountains of Montana and Wyoming. All are associated with dwarf and shrub Salix species, with L. laccata var. pallidifolia also associated with Dryas octopetala and Betula glandulosa. Maximum-parsimony phylogenetic analysis of rDNA-ITS sequences for 27 Laccaria accessions supports the morphological species delineations.     

The SOL Genomics Network: a comparative resource for Solanaceae biology and beyond

The SOL Genomics Network (SGN; http://sgn.cornell.edu) is a rapidly evolving comparative resource for the plants of the Solanaceae family, which includes important crop and model plants such as potato (Solanum tuberosum), eggplant (Solanum melongena), pepper (Capsicum annuum), and tomato (Solanum lycopersicum). The aim of SGN is to relate these species to one another using a comparative genomics approach and to tie them to the other dicots through the fully sequenced genome of Arabidopsis (Arabidopsis thaliana). SGN currently houses map and marker data for Solanaceae species, a large expressed sequence tag collection with computationally derived unigene sets, an extensive database of phenotypic information for a mutagenized tomato population, and associated tools such as real-time quantitative trait loci. Recently, the International Solanaceae Project (SOL) was formed as an umbrella organization for Solanaceae research in over 30 countries to address important questions in plant biology. The first cornerstone of the SOL project is the sequencing of the entire euchromatic portion of the tomato genome. SGN is collaborating with other bioinformatics centers in building the bioinformatics infrastructure for the tomato sequencing project and implementing the bioinformatics strategy of the larger SOL project. The overarching goal of SGN is to make information available in an intuitive comparative format, thereby facilitating a systems approach to investigations into the basis of adaptation and phenotypic diversity in the Solanaceae family, other species in the Asterid clade such as coffee (Coffea arabica), Rubiaciae, and beyond.     

Formation of 19-norsteroids by in situ demethylation of endogenous steroids in stored urine samples

The formation of 19-norsteroids by demethylation of endogenous steroids in stored urine samples was observed. Suspicious urine samples (i.e. containing trace amounts of 19-norandrosterone and 19-noretiocholanolone) were selected and spiked with deuterated analogues of androsterone and etiocholanolone at concentrations corresponding to high endogenous levels (4 microg/mL). After incubation, respective 19-norsteroids (19-norandrosterone-d4 and 19-noretiocholanolone-d5) were identified in these samples by high-resolution mass spectrometry. The transformation of the 5 beta-isomer (etiocholanolone) yields about three-fold higher concentrations, compared to the 5 alpha-isomer. A significant temperature dependence was observed by comparison of reaction kinetics at room temperature (23+/-2 degrees C) and 37 degrees C. Concentrations of 19-norandrosterone-d4 and 19-noretiocholanolone-d5, respectively, were 2.7 and 3.6 times higher at elevated temperature. The conversion of androsterone-d4 to 19-norandrosterone-d4 did not exceed a relative amount of 0.1%. Incubation of the urine samples with androsterone-d4-glucuronide led to the production of 19-norandrosterone-d4-glucuronoide. A partial stabilization was observed after addition of metabolic inhibitors (e.g. EDTA). The application of the incubation experiments described may contribute to the clarification of adverse analytical findings regarding low levels of 19-norsteroid metabolites.