Charge balance in the alpha-hydroxyacid dehydrogenase vacuole: an acid test.

The proposal that the active site vacuole of NAD(+)-S-lactate dehydrogenase is unable to accommodate any imbalance in electrostatic charge was tested by genetically manipulating the cDNA coding for human muscle lactate dehydrogenase to make a protein with an aspartic acid introduced at position 140 instead of the wild-type asparagine. The Asn 140-Asp mutant enzyme has the same kcat as the wild type (Asn 140) at low pH (4.5), and at higher pH the Km for pyruvate increases 10-fold for each unit increase in pH up to pH 9. We conclude that the anion of Asp 140 is completely inactive and that it binds pyruvate with a Km that is over 1,000 times that of the Km of the neutral, protonated aspartic-140. Experimental results and molecular modeling studies indicate the pKa of the active site histidine-195 in the enzyme-NADH complex is raised to greater than 10 by the presence of the anion at position 140. Energy minimization and molecular dynamics studies over 36 ps suggest that the anion at position 140 promotes the opening of and the entry of mobile solvent beneath the polypeptide loop (98-110), which normally seals off the internal active site vacuole from external bulk solvent.     

The PLB measurement for the connector in Phi29 bacteriophage reveals the function of its channel loop

The connector protein, also known as the portal protein, located at the portal vertex in the Phi29 bacteriophage has been found to play a key role in the genome DNA packaging motor. There is a disordered region, composed of 12 sets of 18-residue loops N229–N246, that has been assumed to serve as a “clamp” to retain the DNA within the pressurized capsid when DNA is fully packaged. However, the process remains undefined about how the clamping of DNA occurs and what signal is used to engage the channel loops to clamp the DNA near the end of DNA packaging. In this study, we use the planar lipid bilayer (PLB) membrane technique to study the connector with its loops cleaved. The channel properties are compared with those of the connector with corresponding wild-type loops at different membrane potentials. On the basis of the hypothesis of the Donnan effects in the flashing Brownian ratchet model, we associate the PLB experimental results with the outcomes from the relevant biochemical experiments on the proheads containing the connectors without the loops, which enables us to provide a clear picture about how the DNA clamping occurs. A mathematical relationship between the Donnan potential and the DNA packaging density is established, demonstrating that they are both in essence the same signal that is received and transmitted by the connector to dictate DNA clamping and the termination of DNA packaging. At the end of the study, the PLB technique is proposed as a viral research tool, and its potential use to study the functions of specific domains in a portal protein of the tailed bacteriophages is highlighted.     

Characteristics of Randomized Trials Published in Latin America and the Caribbean According to Funding Source

Introduction

Few studies have assessed the nature and quality of randomized controlled trials (RCTs) in Latin America and the Caribbean (LAC).

Methods and Findings

The aims of this systematic review are to evaluate the characteristics (including the risk of bias assessment) of RCT conducted in LAC according to funding source. A review of RCTs published in 2010 in which the author''s affiliation was from LAC was performed in PubMed and LILACS. Two reviewers independently extracted data and assessed the risk of bias. The primary outcomes were risk of bias assessment and funding source. A total of 1,695 references were found in PubMed and LILACS databases, of which 526 were RCTs (N = 73.513 participants). English was the dominant publication language (93%) and most of the RCTs were published in non-LAC journals (84.2%). Only five of the 19 identified countries accounted for nearly 95% of all RCTs conducted in the region (Brazil 70.9%, Mexico 10.1%, Argentina 5.9%, Colombia 3.8%, and Chile 3.4%). Few RCTs covered priority areas related with Millennium Development Goals like maternal health (6.7%) or high priority infectious diseases (3.8%). Regarding children, 3.6% and 0.4% RCT evaluated nutrition and diarrhea interventions respectively but none pneumonia. As a comparison, aesthetic and sport related interventions account for 4.6% of all trials. A random sample of RCTs (n = 358) was assessed for funding source: exclusively public (33.8%); private (e.g. pharmaceutical company) (15.3%); other (e.g. mixed, NGO) (15.1%); no funding (35.8%). Overall assessments for risk of bias showed no statistically significant differences between RCTs and type of funding source. Statistically significant differences favoring private and others type of funding was found when assessing trial registration and conflict of interest reporting.

Conclusion

Findings of this study could be used to provide more direction for future research to facilitate innovation, improve health outcomes or address priority health problems.