Evidence for high affinity binding-protein dependent transport systems in gram-positive bacteria and in Mycoplasma

Gram-negative bacteria are surrounded by two membranes. In these bacteria, a class of high affinity transport systems for concentrating substrates from the medium into the cell, involves a binding protein located between the outer and inner membranes, in the periplasmic region. These 'periplasmic binding-proteins' are thought to bind the substrate in the vicinity of the inner membrane, and to transfer it to a complex of inner membrane proteins for concentration into the cytoplasm. We report evidence leading us to propose that a Gram-positive bacterium, Streptococcus pneumoniae, and a mycoplasma, Mycoplasma hyorhinis, which are surrounded by a single membrane and have therefore no periplasmic region, possess an equivalent to the high affinity periplasmic binding-protein dependent transport systems, i.e. extra-cytoplasmic binding lipoprotein dependent transport systems. The 'binding lipoproteins' would be maintained at proximity of the inner membrane by insertion of their N-terminal glyceride-cysteine into this membrane.     

Analysis of the factors that influence the C=N stretching frequency of polyene Schiff bases. Implications for bacteriorhodopsin and rhodopsin.

In this study quantum mechanical calculations of force constants and normal mode analysis are used to elucidate the factors that influence the C=C and C=N stretching frequencies in polyenes and in protonated Schiff bases. The C=N stretching frequency is found to depend on both the C=N stretching force constant and the C=N-H bending force constant. Due to the contributions of these two modes, the C=N stretching frequency is particularly sensitive to the magnitude of the Schiff base counterion interactions and to the hydrogen bonding environment of the Schiff base nitrogen. Models for chromophore-protein interactions in the retinal binding site and for the photochemical transformations of bacteriorhodopsin and rhodopsin are evaluated in light of these results.     

<Emphasis Type="Italic">Candida dubliniensis</Emphasis>: Epidemiology and Phenotypic Methods for Identification

Candida dubliniensis is an emerging pathogen first described in 1995, which shares many phenotypic features with Candida albicans and therefore may be misidentified in microbial laboratories. Despite various phenotypic techniques described in the literature to differentiate the two species, the correct identification of C. dubliniensis remains problematic due to phenotypic similarities between these species. Thus, as the differences between both are best characterized at genetic levels, several molecular methods have been proposed to provide a specific and rapid identification of this species. Epidemiological studies have shown that C. dubliniensis is prevalent throughout the world and it is primarily associated with oral carriage and oropharyngeal infections in patients infected with human immunodeficiency virus (HIV). However, data acquired from its isolation from other healthy and immunocompromised patients are variable, and there is still no real consensus on the epidemiological relevance of this species. In this article, we review the various phenotypic methods used in the identification of C. dubliniensis and the epidemiological impact of this new species.     

Energetics of charge-charge interactions in proteins

Electrostatic interactions between pairs of atoms in proteins are calculated with a model based on the linearized Poisson-Boltzmann equation. The equation is solved accurately by a method that takes into account the detailed shape of the protein. This paper presents applications to several systems. Experimental data for the interaction of ionized residues with an active site histidine in subtilisin BPN' allow the model to be tested, using various assumptions for the electrical properties of the protein and solvent. The electrostatic stabilization of the active site thiolate of rhodanese is analyzed, with attention to the influence of alpha-helices. Finally, relationships between electrostatic potential and charge-charge distance are reported for large and small globular proteins. The above results are compared with those of simpler electrostatic models, including Coulomb's law with both a distance-dependent dielectric constant (epsilon = R) and a fixed dielectric constant (epsilon = 2), and Tanford-Kirkwood theory. The primary conclusions are as follows: 1) The Poisson-Boltzmann model agrees with the subtilisin data over a range of ionic strengths; 2) two alpha-helices generate a large potential in the active site of rhodanese; 3) epsilon = R overestimates weak electrostatic interactions but yields relatively good results for strong ones; 4) Tanford-Kirkwood theory is a useful approximation to detailed solutions of the linearized Poisson-Boltzmann equation in globular proteins; and 5) the modified Tanford-Kirkwood theory over-screens the measured electrostatic interactions in subtilisin.     

Ecology,conservation status,and phylogenetic placement of endemic Pristimantis frogs (Anura: Craugastoridae) in Trinidad and Tobago and genetic affinities to northern Venezuela

Trinidad and Tobago are home to three endemic species in the anuran genus Pristimantis, of which two (Pristimantis charlottevillensis and Pristimantis turpinorum) occur in Tobago alone and the third (Pristimantis urichi) is present on both islands. Earlier, the IUCN assessed the conservation status of these species as: P. urichi, Endangered (EN); P. charlottevillensis, Least Concern (LC); P. turpinorum, Vulnerable (VU). However, these assessments were based on very little field-based evidence. Here, we present survey results which contributed to reassessments as LC, VU and Data Deficient for these three species, respectively. Despite the close proximity of Trinidad to northern Venezuela, the islands do not share any Pristimantis species with the mainland, which holds a rich endemicity of Pristimantis regionally. In this study, we used genetic sequencing from several island populations and compared them to northern Venezuelan endemics to assess genetic divergence for the first time. The time tree analyses found that only the northern Tobago species P. turpinorum is closely related to mainland Pristimantis, with a genetic split dating to the Late Miocene, suggesting a vicariant event of mainland and island species. Pristimantis urichi, although identical between the two islands, remains highly divergent from the mainland species. Similar results were found for P. charlottevillensis. In addition, there was a high level of divergence between P. urichi and P. charlottevillensis. These findings indicate different island colonization events by different lineages. Sequencing other Venezuelan species remains pivotal to unravel the complexity of the colonization episodes in the region, likely influenced by the changing topography and multiple connection and isolation episodes of the islands by eustatic sea-level changes.     

Multicenter randomized trial of cell therapy in cardiopathies – MiHeart Study

Background

Cardiovascular diseases are the major cause of death in the world. Current treatments have not been able to reverse this scenario, creating the need for the development of new therapies. Cell therapies have emerged as an alternative for cardiac diseases of distinct causes in experimental animal studies and more recently in clinical trials.

Method/Design

We have designed clinical trials to test for the efficacy of autologous bone marrow derived mononuclear cell therapies in four different cardiopathies: acute and chronic ischemic heart disease, and Chagasic and dilated cardiomyopathy. All trials are multicenter, randomized, double-blind and placebo controlled. In each trial 300 patients will be enrolled and receive optimized therapy for their specific condition. Additionally, half of the patients will receive the autologous bone marrow cells while the other half will receive placebo (saline with 5% autologous serum). For each trial there are specific inclusion and exclusion criteria and the method for cell delivery is intramyocardial for the chronic ischemic heart disease and intracoronary for all others. Primary endpoint for all studies will be the difference in ejection fraction (determined by Simpson's rule) six and twelve months after intervention in relation to the basal ejection fraction. The main hypothesis of this study is that the patients who receive the autologous bone-marrow stem cell implant will have after a 6 month follow-up a mean increase of 5% in absolute left ventricular ejection fraction in comparison with the control group.

Discussion

Many phase I clinical trials using cell therapy for cardiac diseases have already been performed. The few randomized studies have yielded conflicting results, rendering necessary larger well controlled trials to test for efficacy of cell therapies in cardiopathies. The trials registration numbers at the NIH registry are the following: Chagasic cardiomyopathy (NCT00349271), dilated cardiomyopathy (NCT00333827), acute myocardial infarction (NCT00350766) and Chronic Ischemic Heart Disease (NCT00362388).     

Linking microarray reporters with protein functions

Background  

The analysis of microarray experiments requires accurate and up-to-date functional annotation of the microarray reporters to optimize the interpretation of the biological processes involved. Pathway visualization tools are used to connect gene expression data with existing biological pathways by using specific database identifiers that link reporters with elements in the pathways.     

RPA facilitates telomerase activity at chromosome ends in budding and fission yeasts

In Saccharomyces cerevisiae, the telomerase complex binds to chromosome ends and is activated in late S-phase through a process coupled to the progression of the replication fork. Here, we show that the single-stranded DNA-binding protein RPA (replication protein A) binds to the two daughter telomeres during telomere replication but only its binding to the leading-strand telomere depends on the Mre11/Rad50/Xrs2 (MRX) complex. We further demonstrate that RPA specifically co-precipitates with yKu, Cdc13 and telomerase. The interaction of RPA with telomerase appears to be mediated by both yKu and the telomerase subunit Est1. Moreover, a mutation in Rfa1 that affects both the interaction with yKu and telomerase reduces the dramatic increase in telomere length of a rif1Δ, rif2Δ double mutant. Finally, we show that the RPA/telomerase association and function are conserved in Schizosaccharomyces pombe. Our results indicate that in both yeasts, RPA directly facilitates telomerase activity at chromosome ends.