Evolution of the ATP-binding-cassette transmembrane transporters of vertebrates

The ATP-binding-cassette transmembrane transporters (ABC transporters) known from vertebrates belong to four major subfamilies: (1) the P- glycoproteins (Pgp); (2) the cystic fibrosis transmembrane conductance regulators (CFTR); (3) the Tap proteins encoded with the major histocompatibility complex of mammals; and (4) the peroxisomal membrane proteins. Both Pgp and CFTR have a structure suggesting a past internal gene duplication; a phylogenetic analysis indicated that these duplications occurred independently, while an independent tandem gene duplication occurred in the case of the Tap family. Both the Pgp and Tap proteins show evidence of relationship to bacterial ABC transporters lacking internal duplication, and both are significantly more closely related to the HlyB and MsbA families of transporters from purple bacteria than they are to ABC transporters from nonpurple bacteria. The simplest hypothesis to explain this observation is that eukaryotic Pgp and Tap genes are descended from a mitochondrial gene or genes that were subsequently translocated to the nuclear genome. The Pgp genes of eukaryotes are characterized by a remarkable degree of convergent evolution between the ATP-binding cassettes of their N- terminal and C-terminal halves, whereas no such convergence is seen between the two halves of CFTR genes or between the duplicated Tap genes. Exon 13 of the CFTR gene, which encodes a putative regulatory domain not found in other ABC transporters apart from CFTR, showed high levels of both synonymous and nonsynonymous difference in comparisons among different mammalian species, suggesting that this region is a mutational hot spot.      

Evidence for the presence of β-lactamase inStreptomyces glaucescens and its inhibition by sodium clavulanate

Streptomyces glaucescens is shown to possess -lactamase activity which is inhibitable by clavulanate. This is important in regard to its use as a cloning host for enzymes of \-lactam biosynthesis.     

Substitution of active-site His-223 in Pseudomonas aeruginosa elastase and expression of the mutated lasB alleles in Escherichia coli show evidence for autoproteolytic processing of proelastase.

The neutral metalloprotease elastase is one of the major proteins secreted into the culture medium by many Pseudomonas aeruginosa strains. Encoded by the lasB gene, the 33-kDa elastase is initially synthesized as a 53-kDa preproenzyme which is processed to the mature form via a 51-kDa proelastase intermediate. To facilitate studies on proteolytic processing of elastase precursors and on secretion, we developed systems for overexpression of lasB in Escherichia coli under the control of the inducible T7 and tac promoters. Although the 51-kDa proelastase form was detectable in E. coli under inducible conditions, most of the elastase produced under these conditions was found in an enzymatically active 33-kDa form. The amino-terminal sequence of the first 15 amino acid residues of this 33-kDa elastase species was identical to that of the mature P. aeruginosa enzyme, suggesting that processing was autocatalytic. To test this possibility, the codon in lasB encoding His-223, a presumed active-site residue, was changed to encode Asp-223 (lasB1) and Tyr-223 (lasB2). The effects of these mutations on enzyme activity and processing were examined. No proteolytic or elastolytic activities were detected in extracts of E. coli cells containing the lasB mutant alleles. Overexpression of the mutated lasB genes in E. coli resulted in the accumulation of the corresponding 51-kDa proelastase species. These were processed in vitro to the respective 33-kDa forms by incubation with exogenous purified elastase, without an increase in proteolytic activity. Molecular modeling studies suggest that the mutations have little or no effect on the conformation of the mutant elastases. In addition, wild-type elastase and the mutant proelastases were localized to the periplasm of E. coli. The present results confirm that His-223 is essential for elastase activity and provide evidence for autoproteolytic processing of proelastase.     

Three-Dimensional Trabecular Alignment Model

The Shear-slip Mesh Update Method (SSMUM) is being used in flow simulations involving large but regular displacements of one or more boundaries of the computational domain. We follow up the earlier discussion of the method with notes on practical implementation aspects. In order to establish a benchmark problem for this class of flow problems, we define and report results from a two-dimensional viscous flow around a rotating stirrer in a square chamber. The application potential of the method is demonstrated in the context of biomedical design problem, as we perform an analysis of blood flow in a centrifugal left ventricular assist device, or blood pump, which involves a rotating impeller in a non-axisymmetric housing.     

Assemblage structure,vertical distributions and stable-isotope compositions of anguilliform leptocephali in the Gulf of Mexico

In August 2007, October 2008 and September–October 2010, 241 Tucker trawl and plankton net tows were conducted at the surface to depths of 1377 m at six locations in the northern and eastern Gulf of Mexico (GOM) to document leptocephalus diversity and determine how assemblage structure, larval size, abundance and isotopic signatures differ across the region and with depth. Overall, 2696 leptocephali representing 59 distinct taxa from 10 families were collected. Five families accounted for 96% of the total catch with Congridae and Ophichthidae being the most abundant. The top four most abundant species composed 59% of the total catch and included: Ariosoma balearicum, Paraconger caudilimbatus, Rhynchoconger flavus and Ophichthus gomesii. Four anguilliform species not previously documented in the GOM as adults or leptocephali were collected in this study, including Monopenchelys acuta, Quassiremus ascensionis, Saurenchelys stylura and one leptocephalus only known from its larval stage, Leptocephalus proboscideus. Leptocephalus catches were significantly greater at night than during the day. Catches at night were concentrated in the upper 200 m of the water column and significantly declined with increasing depth. Leptocephali abundances and assemblages were significantly different between sites on the upper continental slope (c. 500 m depth) and sites on the middle to lower continental slope (c. 1500–2300 m). Sites on the lower continental slope had a mixture of deep-sea demersal, bathypelagic and coastal species, whereas upper-slope sites contained several numerically dominant species (e.g., A. balearicum, P. caudilimbatus) that probably spawn over the continental shelf and upper slope of the GOM. Standard lengths of the four dominant species differed between sites and years, indicating heterochronic reproduction and potential larval source pools within and outside of the GOM. Stable-isotope analyses (δ¹³C and δ¹⁵N) conducted on 185 specimens from six families revealed that leptocephali had a wide range of isotopic values at the family and size-class levels. Species in the families Muraenidae, Congridae and Ophichthidae had similar δ¹⁵N values compared with the broad range of δ¹⁵N values seen in the deep-sea families Nemichthyidae, Nettastomatidae and Synaphobranchidae. Stable-isotope values were variably related to length, with δ¹⁵N values being positively size correlated in ophichthids and δ¹³C values being negatively size correlated in A. balearicum and P. caudilimbatus. Results suggest that leptocephali feed in various water depths and masses, and on different components of POM, which could lead to niche partitioning. Ecological aspects of these important members of the plankton community provide insight into larval connectivity in the GOM as well as the early life history of Anguilliformes.     

CURRICULUM GUIDE FOR RESEARCH ETHICS WORKSHOPS FOR COUNTRIES IN THE MIDDLE EAST

To help ensure the ethical conduct of research, many have recommended educational efforts in research ethics to investigators and members of research ethics committees (RECs). One type of education activity involves multi‐day workshops in research ethics. To be effective, such workshops should contain the appropriate content and teaching techniques geared towards the learning styles of the targeted audiences. To ensure consistency in content and quality, we describe the development of a curriculum guide, core competencies and associated learning objectives and activities to help educators organize research ethics workshops in their respective institutions. The curriculum guide is divided into modular units to enable planners to develop workshops of different lengths and choose content materials that match the needs, abilities, and prior experiences of the target audiences. The content material in the curriculum guide is relevant for audiences in the Middle East, because individuals from the Middle East who participated in a Certificate Program in research ethics selected and developed the training materials (e.g., articles, powerpoint slides, case studies, protocols). Also, many of the activities incorporate active‐learning methods, consisting of group work activities analyzing case studies and reviewing protocols. The development of such a workshop training curriculum guide represents a sustainable educational resource to enhance research ethics capacity in the Middle East.     

Virulence factor regulation and regulatory networks in Streptococcus pyogenes and their impact on pathogen-host interactions

Streptococcus pyogenes (group A streptococcus, GAS) is a very important human pathogen with remarkable adaptation capabilities. Survival within the harsh host surroundings requires sensing potential on the bacterial side, which leads in particular to coordinately regulated virulence factor expression. GAS 'stand-alone' response regulators (RRs) and two-component signal transduction systems (TCSs) link the signals from the host environment with adaptive responses of the bacterial cell. Numerous putative regulatory systems emerged from GAS genome sequences. Only three RRs [Mga, RofA-like protein (RALP) and Rgg/RopB] and three TCSs (CsrRS/CovRS, FasBCAX and Ihk/Irr) have been studied in some detail with respect to their growth-phase-dependent activity and their influence on GAS-host cell interaction. In particular, the Mga-, RALP- and Rgg/RopB-regulated pathways display interconnected activities that appear to influence GAS colonization, persistence and spreading mechanisms, in a growth-phase-related fashion. Here, we have summarized our current knowledge about these RRs and TCSs to highlight the questions that should be addressed in future research on GAS pathogenicity.