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.     

The correlation between specific protein synthesis and tumoricidal function in murine peritoneal macrophages

Macrophages from the lipopolysaccharide (LPS)-responsive C3H/HeN mouse strain and the closely related LPS-nonresponsive C3H/HeJ strain were compared for tumoricidal activation and protein synthetic changes following in vivo and in vitro stimulation, utilizing two-dimensional polyacrylamide gel electrophoresis of [35S]methionine-labeled proteins. Peritoneal macrophages elicited from C3H/HeN mice with heat-killed Propionibacterium acnes exhibited tumoricidal activity in a 16-hr cytolytic assay and expressed cytoplasmic levels of a 23.5-kDa protein during 48 hr of culture. The inability to detect persistent expression of p23.5 in P. acnes-stimulated C3H/HeJ macrophages correlated with the cytolytic impotence of those cells in the 16-hr chromium release assay. C3H/HeN macrophage populations lacking tumoricidal capacity could be rendered lytic, as could P. acnes-elicited C3H/HeJ macrophages, following in vitro stimulation with bacterial lipopolysaccharide. Concomitant with the LPS-induced expression of new functional activity was the appearance of augmented levels of several macrophage-specific proteins, including p23.5. This effect was dependent upon the lipid A moiety of LPS as the effects of LPS could be blocked by inclusion of polymyxin B sulfate in the culture medium. However, neither tumoricidal function nor protein modulation could be readily induced in C3H/HeJ proteose peptone-elicited or resident macrophages. These results identify biochemical responses to stimuli which may be requisite to acquisition or execution of cytolytic activity.     

Cyclic AMP-stimulated protein kinases at brain synaptic junctions.

We have examined endogenous cyclic AMP-stimulated phosphorylation of subcellular fractions of rat brain enriched in synaptic plasma membranes (SPM), purified synaptic junctions (SJ), and postsynaptic densities (PSD). The analyses of these fractions are essential to provide direct evidence for cyclic AMP-dependent endogenous phosphorylation at discrete synaptic junctional loci. Protein kinase activity was measured in subcellular fractions using both endogenous and exogenous (histones) proteins as substrates. The SJ fraction possessed the highest kinase activity toward endogenous protein substrates, 5-fold greater than SPM and approximately 120-fold greater than PSD fractions. Although the kinase activity as measured with histones as substrates was only slightly higher in SJ than SPM fractions, there was a marked preference of kinase activity toward endogenous compared to exogenous substrates in SJ fractions but in SPM fractions. Although overall phosphorylation in SJ fractions was increased only 36% by 5 micron cyclic AMP, there were discrete proteins of Mr = 85,000, 82,000, 78,000, and 55,000 which incorporated 2- to 3-fold more radioactive phosphate in the presence of cyclic AMP. Most, if not all, of the cyclic AMP-independent kinase activity is probably catalyzed by catalytic subunit derived from cyclic AMP-dependent kinase, since the phosphorylation of both exogenous and endogenous proteins was greatly decreased in the presence of a heat-stable inhibitor protein prepared from the soluble fraction of rat brain. The specific retention of SJ protein kinase(s) activity during purification and their resistance to detergent solubilization was achieved by chemical treatments which produce interprotein cross-linking via disulfide bridges. Two SJ polypeptides of Mr = 55,000 and 49,000 were photoaffinity-labeled with [32P]8-N3-cyclic AMP and probably represent the regulatory subunits of the type I and II cyclic AMP-dependent protein kinases. The protein of Mr = 55,000 was phosphorylated in a cyclic AMP-stimulated manner suggesting autophosphorylation as previously observed in other systems.     

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.     

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.     

Circadian organization of a subarctic rodent, the northern red-backed vole (Clethrionomys rutilus)

Arctic and subarctic environments are exposed to extreme light: dark (LD) regimes, including periods of constant light (LL) and constant dark (DD) and large daily changes in day length, but very little is known about circadian rhythms of mammals at high latitudes. The authors investigated the circadian rhythms of a subarctic population of northern red-backed voles (Clethrionomys rutilus). Both wild-caught and third-generation laboratory-bred animals showed predominantly nocturnal patterns of wheel running when exposed to a 16:8 LD cycle. In LL and DD conditions, animals displayed large phenotypic variation in circadian rhythms. Compared to wheel-running rhythms under a 16:8 LD cycle, the robustness of circadian activity rhythms decreased among all animals tested in LL and DD (i.e., decreased chi-squared periodogram waveform amplitude). A large segment of the population became noncircadian (60% in DD, 72% in LL) within 8 weeks of exposure to constant lighting conditions, of which the majority became ultradian, with a few individuals becoming arrhythmic, indicating highly labile circadian organization. Wild-caught and laboratory-bred animals that remained circadian in wheel running displayed free-running periods between 23.3 and 24.8 h. A phase-response curve to light pulses in DD showed significant phase delays at circadian times 12 and 15, indicating the capacity to entrain to rapidly changing day lengths at high latitudes. Whether this phenotypic variation in circadian organization, with circadian, ultradian, and arrhythmic wheel-running activity patterns in constant lighting conditions, is a novel adaptation to life in the arctic remains to be elucidated.