Characterization of messenger RNA from epimastigotes and metacyclic trypomastigotes of Trypanosoma cruzi

The cell-free translation products of polyribosomal and post-polyribosomal mRNAs from the non-infective epimastigotes and the infective metacyclic trypomastigotes of the parasitic protozoan Trypanosoma cruzi were compared by two-dimensional polyacrylamide gel electrophoresis. The result show that although many polypeptides are conserved, quantitative and qualitative differences are observed between both differentiation stages. The results also indicate the existence of post-polyribosomal mRNAs in equilibrium with polyribosomal counterparts. The immunoprecipitation of the in vitro synthesized polypeptides with chagasic human serum and the serum raised against an 85-kDa glycoprotein (P2-WGA), potentially involved in the process of T. cruzi penetration into mammalian cells, shows that while the chagasic serum recognizes the same 72-kDa, 68-kDa and 46-kDa polypeptides in both differentiation stages, the anti-P2-WGA serum immunoprecipitates a single 48-kDa polypeptide from in vitro translation products of metacyclic trypomastigotes.     

Assessment of the cryoprotectant concentration inside a bulky organ for cryopreservation using X-ray computed tomography

Cryoprotection of bulky organs is crucial for their storage and for subsequent transplantation. In this work we demonstrate the capability of the X-ray computed tomography (CT) as a non-invasive method to measure the cryoprotectant (cpa) concentration inside a tissue or an organ, specifically for the case of dymethil sulfoxide (Me₂SO). It is remarkable that the use of Me₂SO has been leader in techniques of cells and tissues cryopreservation. Although CT technologies are mainly based in density differences, and many cpas are alcohols with densities similar to water, the use of very low energies as acceleration voltage (∼70 kV) and the sulfur atom in the molecule of Me₂SO makes possible the visualization of this cpa inside tissues. As result we obtain a CT signal proportional to the Me₂SO concentration with a spatial resolution up to 50 μm in the case of our device.     

Environmental regulation of surface conductance for evaporation from vegetation

We examine conductances for evaporation from both vegetation and soil in response to environmental variables. Data from a vertically-structured pristine forest of Nothofagus are presented as an example of the effects of biodiversity on the scaling of conductances between tiers of plant organisation. Available data sets of maximum leaf stomatal conductances (g _lmax ) and bulk vegetation surface conductances (G _smax ) are compared. Overall, the ratio G _smax /g _lmax is consistently close to 3 for seven major vegetation types of diverse structure. An analytical model accounts for this close relationship, and in particular how G _smax is conservative against changes in leaf area index because of the compensating decrease in plant canopy transpiration and increase in soil evaporation as leaf area index diminishes. The model is also successfully tested by comparison with canopy conductances of emergent trees measured in the Nothofagus forest. The constraint of vegetation surface conductance and evaporation via environmental regulation by irradiance, air saturation deficit and root zone water supply are discussed.     

Monitoring the natural attenuation of a sewage sludge toxicity using the Allium cepa test

Appropriate final disposal of sewage sludge (SS) generated by wastewater treatment plants (WWTP) has been considered a serious environmental problem, but also a viable alternative to be applied in agriculture, once SS is rich in organic matter and nutrients. However, SS can be a source of contamination of several toxic agents. Therefore, its use in agriculture requires special care to avoid possible damage to the environment and exposed organisms. Detoxification of toxic wastes can be performed using the monitored natural attenuation, which involves biological, physical and chemical processes that frequently occur in the environment. This study aimed to assess the feasibility of decontaminating SS after different periods of monitored natural attenuation. To this end, samples of SS and associations of soil/SS with proportions of 10, 25 and 50% SS were buried for 0, 2, 6 and 12 months in holes prepared in a place free of contamination. Allium cepa was used as an indicator to assess the efficiency of the natural attenuation process. According to chemical analysis, the SS samples presented a high concentration of m- and p-cresol, especially for samples analyzed after 0 or 2 months of natural attenuation. The microorganisms present in the SS belonged to 17 different genera of bacteria, which varied in the microbial composition among samples. Both, raw SS and aqueous SS extracts induced DNA damage in A. cepa, even when associated with soil. However, this effect was observed to decline during the attenuation period, although significant effects were detected for the highest tested concentration (100% SS) even at the end of this process. These results thus indicated the necessity of applying a stabilization process associating SS and soil for a period of at least 12 months and showed that the studied raw SS is not a viable material for use as a soil reconditioner, even after natural attenuation. A. cepa test proved to be a useful tool to assess the efficiency of SS detoxification process. Therefore, we suggest that the application of SS in agriculture should be approached with caution and that the SS must be previously submitted to methodologies that evaluate its toxic potential.     

A NOVEL APPROACH TO SOLID PHASE CHEMICAL SYNTHESIS OF OLIGONUCLEOTIDE mRNA CAP ANALOGS

A novel approach for the synthesis of 5′-capped 2′-O-methyloligoribonucleotides on a disulfide-tethered solid support is described. The key step of the synthesis is ZnCl₂ promoted coupling of m⁷GDP imidazolide to a fully deprotected oligonucleotide 5′-phosphate on-support. By this methodology m⁷G⁵′pppm²′Apm²′Upm^2′Ap has been prepared.     

Conventional freezing vs. cryoprotectant-free vitrification of epididymal (MESA) and testicular (TESE) spermatozoa: Three live births

Data of cryoprotectant-free vitrification of human testicular and epididymal spermatozoa are limited. The aim of this investigation was to compare two aseptic technologies of TESE (testicular) and MESA (epididymal) spermatozoa cryopreservation: standard conventional freezing with the use of cryoprotectants and cryoprotectant-free vitrification. Sperm motility, capacitation-like changes, acrosome reaction and the mitochondrial membrane potential of frozen (5% glycerol, −10 °C/min) and vitrified (Human Tubal Fluid + 1% Human Serum Albumin+0.25 M sucrose, plunging into liquid nitrogen of capillaries with spermatozoa isolated from liquid nitrogen (aseptic method) were compared. The quality of the cryoprotectant-free vitrified MESA- and TESE-spermatozoa was higher than that of spermatozoa conventionally frozen with permeable cryoprotectants. Intracellular sperm injection (ICSI) was performed with vitrified spermatozoa. We report the birth of three healthy babies from two women following ICSI with motile MESA- and TESE-spermatozoa vitrified without cryoprotectants. This is the first report of full-term pregnancies and babies born after ICSI with epididymal and testicular spermatozoa vitrified without cryoprotectants. In conclusion, cryoprotectant-free vitrification can be successfully applied for the cryopreservation of motile TESE- and MESA-spermatozoa.     

Point Mutations in Dimerization Motifs of the Transmembrane Domain Stabilize Active or Inactive State of the EphA2 Receptor Tyrosine Kinase

The EphA2 receptor tyrosine kinase plays a central role in the regulation of cell adhesion and guidance in many human tissues. The activation of EphA2 occurs after proper dimerization/oligomerization in the plasma membrane, which occurs with the participation of extracellular and cytoplasmic domains. Our study revealed that the isolated transmembrane domain (TMD) of EphA2 embedded into the lipid bicelle dimerized via the heptad repeat motif L⁵³⁵X₃G⁵³⁹X₂A⁵⁴²X₃V⁵⁴⁶X₂L⁵⁴⁹ rather than through the alternative glycine zipper motif A⁵³⁶X₃G⁵⁴⁰X₃G⁵⁴⁴ (typical for TMD dimerization in many proteins). To evaluate the significance of TMD interactions for full-length EphA2, we substituted key residues in the heptad repeat motif (HR variant: G539I, A542I, G553I) or in the glycine zipper motif (GZ variant: G540I, G544I) and expressed YFP-tagged EphA2 (WT, HR, and GZ variants) in HEK293T cells. Confocal microscopy revealed a similar distribution of all EphA2-YFP variants in cells. The expression of EphA2-YFP variants and their kinase activity (phosphorylation of Tyr⁵⁸⁸ and/or Tyr⁵⁹⁴) and ephrin-A3 binding were analyzed with flow cytometry on a single cell basis. Activation of any EphA2 variant is found to occur even without ephrin stimulation when the EphA2 content in cells is sufficiently high. Ephrin-A3 binding is not affected in mutant variants. Mutations in the TMD have a significant effect on EphA2 activity. Both ligand-dependent and ligand-independent activities are enhanced for the HR variant and reduced for the GZ variant compared with the WT. These findings allow us to suggest TMD dimerization switching between the heptad repeat and glycine zipper motifs, corresponding to inactive and active receptor states, respectively, as a mechanism underlying EphA2 signal transduction.     

Surface charges of the membrane and cell adhesion substances determine the structural integrity of hair bundles from the inner ear of fish

Summary The hairs (stereocilia = stereovilli) of sensory cells from the inner ear of vertebrates are interconnected by several types of connectors, whose role is unknown. They appear to stabilize the hair bundle mechanically, and may be directly involved in mechano-electric transduction. Our transmission electron-microscopical investigation of sensory epithelia from two species of fish (Rutilus rutilus, Scardinius erythrophthalmus, both Leuciscidae) has shown that not only the connectors but also the surface charges of the membrane are important factors for determining the shape of the hair bundle and the spatial interrelation of the stereovilli. A reduction of the ionic strength in the medium leads to an increase in distance between the stereovilli. This may be the result of an extension of the spread of the surface potential of the membrane at low ionic strength. The connectors are not broken by the increase in distance between the stereovilli. They are EDTA (ethylene-diamine-tetra-acetic-acid) resistant as are some cell adhesion molecules such as N-CAM (nerve-cell adhesion molecule) and protein A from Dictyostelium discoideum. The connectors do not prevent polycation-induced fusion of adjacent stereovillar membranes.