Regulatory mechanism of histidine-tagged homocitrate synthase from Saccharomyces cerevisiae. I. Kinetic studies

Homocitrate synthase (HCS) catalyzes one of the regulated steps of the alpha-aminoadipate pathway for lysine biosynthesis in fungi. The kinetic mechanism of regulation of HCS from Saccharomyces cerevisiae by Na+ and the feedback inhibitor lysine was studied by measuring the initial rate in the absence and presence of the effectors. The data suggest that Na+ is an activator at low concentrations and an inhibitor at high concentrations and that these effects occur as a result of the monovalent ion binding to two different sites in the free enzyme. Inhibition and activation by Na+ can occur simultaneously, with the net rate of the enzyme determined by Na+/K(iNa+) and Na+/K(act), where K(iNa+) and K(act) are the inhibition and activation constants, respectively. The inhibition by Na+ was eliminated at high concentrations of acetyl-CoA, the second substrate bound, but the activation remained. Fluorescence binding studies indicated that lysine bound with high affinity to its binding site as an inhibitor. The inhibition by lysine was competitive versus alpha-ketoglutarate and linear in the physiological range of lysine concentrations up to 5 mm. The effects of Na+ and lysine were independent of one another. A model is developed for regulation of HCS that takes into account all of the effects discussed above.     

Mitochondrial genomes suggest that hexapods and crustaceans are mutually paraphyletic

For over a century the relationships between the four major groups of the phylum Arthropoda (Chelicerata, Crustacea, Hexapoda and Myriapoda) have been debated. Recent molecular evidence has confirmed a close relationship between the Crustacea and the Hexapoda, and has included the suggestion of a paraphyletic Hexapoda. To test this hypothesis we have sequenced the complete or near-complete mitochondrial genomes of three crustaceans (Parhyale hawaiensis, Squilla mantis and Triops longicaudatus), two collembolans (Onychiurus orientalis and Podura aquatica) and the insect Thermobia domestica. We observed rearrangement of transfer RNA genes only in O. orientalis, P. aquatica and P. hawaiensis. Of these, only the rearrangement in O. orientalis, an apparent autapomorphy for the collembolan family Onychiuridae, was phylogenetically informative.We aligned the nuclear and amino acid sequences from the mitochondrial protein-encoding genes of these taxa with their homologues from other arthropod taxa for phylogenetic analysis. Our dataset contains many more Crustacea than previous molecular phylogenetic analyses of the arthropods. Neighbour-joining, maximum-likelihood and Bayesian posterior probabilities all suggest that crustaceans and hexapods are mutually paraphyletic. A crustacean clade of Malacostraca and Branchiopoda emerges as sister to the Insecta sensu stricto and the Collembola group with the maxillopod crustaceans. Some, but not all, analyses strongly support this mutual paraphyly but statistical tests do not reject the null hypotheses of a monophyletic Hexapoda or a monophyletic Crustacea. The dual monophyly of the Hexapoda and Crustacea has rarely been questioned in recent years but the idea of both groups' paraphyly dates back to the nineteenth century. We suggest that the mutual paraphyly of both groups should seriously be considered.     

E-learning: is there anything special about the "E"?

E-learning has been widely utilized in medical education and suggested by some proponents to represent a fundamental advance in educational methodology. We challenge this conclusion by examining e-learning in the context of broader learning theories, specifically as they relate to instructional design and methods. Core tenets of educational design are applied to e-learning in a unified model for instructional design, and examples of e-learning technologies are examined in the context of medical education, with reflections on research questions generated by these new modalities. Throughout, we argue that e-learning is a tool that, when designed appropriately, can be used to meet worthy educational goals.     

THE EFFECT OF LOW PRESSURES ON CELL OXIDATION

1. A method is described for measuring tissue oxidation under reduced barometric pressure. 2. The oxygen uptake of yeast is diminished by low barometric pressures to a greater extent than by a reduction of the partial pressure of oxygen, to a corresponding degree, at atmospheric pressure. 3. This effect of low pressure is not observed with certain in vitro oxidation systems. 4. The anaerobic respiration (carbon dioxide production) of yeast is not at all affected by low pressures. 5. The inhibition of tissue oxidation caused by carbon monoxide is removed by lowering the pressure.     

Adenovirus E1A oncogene expression in tumor cells enhances killing by TNF-related apoptosis-inducing ligand (TRAIL)

Expression of the adenovirus serotype 5 (Ad5) E1A oncogene sensitizes cells to apoptosis by TNF-alpha and Fas-ligand. Because TNF-related apoptosis-inducing ligand (TRAIL) kills cells in a similar manner as TNF-alpha and Fas ligand, we asked whether E1A expression might sensitize cells to lysis by TRAIL. To test this hypothesis, we examined TRAIL-induced killing of human melanoma (A2058) or fibrosarcoma (H4) cells that expressed E1A following either infection with Ad5 or stable transfection with Ad5-E1A. E1A-transfected A2058 (A2058-E1A) or H4 (H4-E1A) cells were highly sensitive to TRAIL-induced killing, but Ad5-infected cells expressing equally high levels of E1A protein remained resistant to TRAIL. Infection of A2058-E1A cells with Ad5 reduced their sensitivity to TRAIL-dependent killing. Therefore, viral gene products expressed following infection with Ad5 inhibited the sensitivity to TRAIL-induced killing conferred by transfection with E1A. E1B and E3 gene products have been shown to inhibit TNF-alpha- and Fas-dependent killing. The effect of these gene products on TRAIL-dependent killing was examined by using Ad5-mutants that did not express either the E3 (H5dl327) or E1B-19K (H5dl250) coding regions. A2058 cells infected with H5dl327 were susceptible to TRAIL-dependent killing. Furthermore, TRAIL-dependent killing of A2058-E1A cells was not inhibited by infection with H5dl327. Infection with H5dl250 sensitized A2058 cells to TRAIL-induced killing, but considerably less than H5dl327-infection. In summary, expression of Ad5-E1A gene products sensitizes cells to TRAIL-dependent killing, whereas E3 gene products, and to a lesser extent E1B-19K, inhibit this effect.     

Eastward Ho: phylogeographical perspectives on colonization of hosts and parasites across the Beringian nexus

The response of Arctic organisms and their parasites to dramatic fluctuations in climate during the Pleistocene has direct implications for predicting the impact of current climate change in the North. An increasing number of phylogeographical studies in the Arctic have laid a framework for testing hypotheses concerning the impact of shifting environmental conditions on transcontinental movement. We review 35 phylogeographical studies of trans-Beringian terrestrial and freshwater taxa, both hosts and parasites, to identify generalized patterns regarding the number, direction and timing of trans-continental colonizations. We found that colonization across Beringia was primarily from Asia to North America, with many events occurring in the Quaternary period. The 35 molecular studies of trans-Beringian organisms we examined focused primarily on the role of glacial cycles and refugia in promoting diversification. We address the value of establishing testable hypotheses related to high-latitude biogeography. We then discuss future prospects in Beringia related to coalescent theory, palaeoecology, ancient DNA and synthetic studies of arctic host–parasite assemblages highlighting their cryptic diversity, biogeography and response to climate variation.     

Survival of Campylobacter jejuni and Escherichia coli in groundwater during prolonged starvation at low temperatures

AIMS: To evaluate the survival of Campylobacter jejuni relative to that of Escherichia coli in groundwater microcosms varying in nutrient composition. METHODS AND RESULTS: Studies were conducted in groundwater and deionized water incubated for up to 470 days at 4 degrees C. Samples were taken for culturable and total cell counts, nutrient and molecular analysis. Die-off in groundwater microcosms was between 2.5 and 13 times faster for C. jejuni than for E. coli. Campylobacter jejuni had the lowest decay rate and longest culturability in microcosms with higher dissolved organic carbon (4 mg l(-1)). Escherichia coli survival was the greatest when the total dissolved nitrogen (12.0 mg l(-1)) was high. The transition of C. jejuni to the coccoid stage was independent of culturability. CONCLUSION: The differences in the duration of survival and response to water nutrient composition between the two organisms suggest that E. coli may be present in the waters much longer and respond to water composition much differently than C. jejuni. SIGNIFICANCE AND IMPACT OF THE STUDY: The data from these studies would aid in the evaluation of the utility of E. coli as an indicator of C. jejuni. This study also provided new information about the effect of nutrient composition on C. jejuni viability.     

Myoblast proliferation and syncytial fusion both depend on connexin43 function in transfected skeletal muscle primary cultures

Muscles are formed by fusion of individual postmitotic myoblasts to form multinucleated syncytial myotubes. The process requires a well-coordinated transition from proliferation, through migratory alignment and cycle exit, to breakdown of apposed membranes. Connexin43 protein and cell-cycle inhibitor levels are correlated, and gap junction blockers can delay muscle regeneration, so a coordinating role for gap junctions has been proposed. Here, wild-type and dominant-negative connexin43 variants (wtCx43, dnCx43) were introduced into rat myoblasts in primary culture through pIRES-eGFP constructs that made transfected cells fluoresce. GFP-positive cells and vitally-stained nuclei were counted on successive days to reveal differences in proliferation, and myotubes were counted to reveal differences in fusion. Individual transfected cells were injected with Cascade Blue, which permeates gap junctions, mixed with FITC-dextran, which requires cytoplasmic continuity to enter neighbouring cells. Myoblasts transfected with wtCx43 showed more gap-junctional coupling than GFP-only controls, began fusion sooner as judged by the incidence of cytoplasmic coupling, and formed more myotubes. Myoblasts transfected with dnCx43 remained proliferative for longer than either GFP-only or wtCx43 myoblasts, showed less coupling, and underwent little fusion into myotubes. These results highlight the critical role of gap-junctional coupling in myotube formation.