Characteristics of ouabain binding to isolated trout hepatocytes

Summary Na⁺, K⁺ exchanges were studied in isolated hepatocytes of the rainbow trout, Salmo gairdneri. Ouabain at 10^–4 M produced maximal inhibition (95%) of K⁺ uptake and enhanced intracellular Na⁺ accumulation, showing that active fluxes account for a very large proportion of Na⁺ and K⁺ exchanges. Inhibition of the Na–K pump by ouabain was significant at low concentrations (10^–8 M). When external K⁺ concentration was reduced from 7 mM to 0.5 mM, half maximum inhibition (IC₅₀) of K⁺ uptake was obtained at a 22-fold lower concentration of ouabain confirming that ouabain and potassium compete at the same pump site. Time-course analysis of [³H]ouabain binding indicated a two-component kinetics: one component saturable and dependent on K⁺ concentration in the medium, the other linear and independent of external K⁺. The ouabain binding site number, determined by Scatchard plots, remained constant (ca. 2.5·10⁵ per cell) and independent of the external K⁺ concentration (7, 0.5 or 0 mM), while the dissociation constant (K_D) decreased from 4.2 M to 7.3 nM when K⁺ was removed from the Hank's medium. These ouabain binding sites are characterized by an exceptionally low turnover rate (400 min^–1), as estimated from ouabain-sensitive K⁺ flux, in comparison to those described in other cell types of higher vertebrates. At each external K⁺ concentration studied, the inhibition of K⁺ uptake and ouabain binding measured as a function of ouabain concentration indicated a strict correlation between the degree of K pump inhibition and the amount of bound glycoside.     

Linkage disequilibrium and extended haplotypes in the HLA-A to D6S105 region: implications for mapping the hemochromatosis gene (HFE)

The hemochromatosis gene (HFE) maps to 6p21.3, in close linkage with the HLA Class I genes. Linkage disequilibrium (LD) studies were designed to narrow down the most likely candidate region for HFE, as an alternative to traditional linkage analysis. However, both the HLA-A and D6S105 subregions, which are situated 2–3 cM and approximately 3 Mb apart, have been suggested to contain HFE. The present report extends our previous study based upon the analysis of a large number of HFE and normal chromosomes from 66families of Breton ancestry. In addition to the previously used RFLP markers spanning the 400-kb surrounding HLA-A, we examined three microsatellites: D6S510, HLA-F, and D6S105. Our combined data not only confirm a peak of LD at D6S105, but also reveal a complex pattern of LD over the i82 to D6S105 interval. Within our ethnically well-defined population of Brittany, the association of HFE with D6S105 is as great as that with HLA-A, while the internal markers display a lower LD. Fine haplotype analysis enabled us to identify two categories of haplotypes segregating with HFE. In contrast to the vast majority of normal haplotypes, 50% of HFE haplotypes are completely conserved over the HLA-A to D6S105 interval. These haplotypes could have been conserved through recombination suppression, selective forces and/or other evolutionary factors. This particular haplotypic configuration might account for the apparent inconsistencies between genetic linkage and LD data, and additionally greatly complicates positional cloning of HFE through disequilibrium mapping.The authors contributed equally to this work     

Impacts of introduced common wasps (Vespula vulgaris) on experimentally placed mealworms in a New Zealand beech forest

An introduced social wasp Vespula vulgaris may compete with native birds for honeydew and invertebrates in New Zealand forests. Experimentally hidden mealworms (Tenebrio molitor) persisted longer at two sites following wasp poisoning that at two sites where wasps were not poisoned. Mealworms persisted longer in the morning than in the afternoon within all study sites. An unusually low mealworm removal rate during a morning trial before wasp poisoning heavily influences the results of this experiment but we have no ecological reason to ignore it. Wasps may therefore be having a heavy impact on invertebrate abundance on very short time scales (within a day following dawn emergence). They may also remove cached food items that would otherwise be retrieved by the South Island robin (Petroica australis australis) during cold or dark feeding conditions.     

Delivery of DNA into mammalian cells by receptor-mediated endocytosis and gene therapy

The correction of genetically based disorders by the introduction of a therapeutic genetic construct into the appropriate cell type (“gene therapy”), has become a distinct possibility in recent years. In order for gene therapy to be a practical alternative to more conventional pharmaceutical approaches to treatment, it must be administrable in vivo. This demands that a system be developed that can specifically target the DNA to the desired cell type once introduced into the patient. Among the procedures that are currently being pursued, the delivery of DNA to cells by receptor mediated endocytosis (RME), comes closest to fulfilling this crucial requirement. The natural physiological process of RME can be exploited to deliver genetic material to cells. An antibody or ligand to a cell surface receptor that is known to undergo endocytosis, is complexed with DNA through a covalently linked polycationic adjunct (e.g., polylysine, protamines). Such complexes retain their binding specificity to the cell surface and are taken up into the cell where they enter the endosomal compartment via normal endocytotic processes. In addition, steps must be taken to avoid degradation of the DNA within the endosome-lysosome. Cells can be treated with the lysosomatropic agent chloroquine during the transfection procedure. Alternatively, the components of viruses that enter cells by endocysis and possess an endosomal “break out” capacity can be used. Replication defective adenovirus coupled to the ligand-DNA complex gives transfection efficiencies of virtually 100% on tissue culture cells in vitro. Synthetic peptides that mimic the membrane fusing region of influenza virus hemagglutinin, have also been successfully used as part of the ligand-DNA complex to bring about endosomal escape. Preliminary studies have demonstrated the potential of this method to specifically target DNA to the cell type of choice in vivo. Delivery of genes by receptor-mediated endocytosis offers the greatest hope that gene therapy can be an inexpensive, easily applicable, widespread technology.     

Comparative limnology,species diversity and biomass relationship of zooplankton and phytoplankton in five freshwater lakes in Kenya

Comparative studies on the limnology, species diversity and standing stock biomass of phytoplankton and zooplankton in five freshwater lakes, Naivasha and Oloidien, Ruiru, Masinga and Nairobi reservoirs, were undertaken. Phytoplankton chlorophyll a, dissolved oxygen and temperature were also measured. Thermocyclops oblongatus (Copepoda) was dominant in all the lakes. Ceriodaphnia cornuta and Diaphanosoma excisum (Cladocera) dominated in lakes Naivasha and Oloiden, whereas in Ruiru, Masinga and Nairobi reservoirs, Brachionus angularis and Hexarthra mira (Rotifera) were the dominant zooplankters. Phytoplankton biomass as chlorophyll a was lowest in Ruiru dam 5.64 ± 4.0 µg l^-1 and highest in the eutrophic Nairobi dam 71.5 ± 12.02 µg l^-1. The endorheic lakes Naivasha and Oloidien showed medium values of 24.5 ± 4.0 µg l^-1.     

Cell physiology and antibiotic production of Streptomyces coelicolor grown on solid medium

Summary In order to examine the physiology ofStreptomyces coelicolor when growing on solid media, we have employed a membrane overlay technique and used a new approach to extract substrate and product compounds from the agar. Comparisons made with liquid grown cultures indicate a change from non-growth associated productivity of actinorhodin in liquid culture, to growth associated production on agar plates. In contrast, the temporal control of methylenomycin production was virtually identical under both culture conditions. Considerable extracellular protein production was observed during growth on agar.     

Differential expression of several E2-type ubiquitin carrier protein genes at different developmental stages inArabidopsis thaliana andNicotiana sylvestris

We characterized three genes encoding different E2-type ubiquitin carrier proteins involved in the ubiquitin-mediated proteolytic pathway:UbcAt3 shows homologies to the yeastCDC34 gene andUbcAt4a andUbcAt4b are two different genes homologous to theUbc1/4/5 subfamily in yeast. Their accumulation was analysed and compared with that of the different families encoding polyubiquitins, as well as the monoubiquitin fusion protein, which is considered as a marker for cell division, during various developmental stages including GO/S transition and senescence of higher plant cells. Our results imply that theseUbc genes are under the control of complex mechanisms, and are differentially regulated, but not necessarily co-regulated with ubiquitin genes. Even the closely relatedUbcAt4a andUbcAt4b genes of the same multigene subfamily are controlled by distinct regulatory mechanisms.