Sucrose protectable inhibition of the sucrose transporter by N-ethylmaleimide

To understand contradictory data published in the literature,the sensitivity of sucrose and of valine uptake to N-ethylmaleimide(NEM) was reinvestigated in detail with plasma membrane vesiclespurified by phase partitioning from mature sugar beet (Betavulgaris) leaves. Uptake in the vesicles was energized by anartificial proton-motive force combining a pH gradient and anelectrical gradient. Three main parameters were varied in theexperiments: the presence of a reducing agent, dlthiothreitol(DTT) In the medium used to store the vesicles, the temperatureof pretreatment with NEM (12 or 23°C) and the temperatureof incubation with the labelled substrate (12 or 23°C).Sensitivity of sucrose uptake to NEM only appeared with vesiclesthat had been stored in the presence of DTT, and if the pretreatmentwas run at 23°C. The temperature of incubation with labelledsucrose did not affect NEM sensitivity. The NEM sensitivityof valine uptake was not affected in the same way as sucroseuptake by the temperature of preincubation, showing that theeffects observed were specific for a given transporter. Underconditions which normally inhibit sucrose uptake, addition ofsucrose during NEM pretreatment protected the sucrose transporteragainst NEM inhibition. Key words: Sugar transport, plasma membrane, differential labelling, thiol reagents     

Intracellular Cl regulates Na-K-Cl cotransport activity in human trabecular meshwork cells

The trabecular meshwork (TM) of the eye plays a central role inmodulating intraocular pressure by regulating aqueous humor outflow,although the mechanisms are largely unknown. We and others have shownpreviously that aqueous humor outflow facility is modulated byconditions that alter TM cell volume. We have also shown that theNa-K-Cl cotransport system is a primary regulator of TM cell volume andthat its activity appears to be coordinated with net efflux pathways tomaintain steady-state volume. However, the cellular mechanisms thatregulate cotransport activity and cell volume in TM cells have yet tobe elucidated. The present study was conducted to investigate thehypothesis that intracellular Cl concentration([Cl]_i) acts toregulate TM cell Na-K-Cl cotransport activity, as has been shownpreviously for some other cell types. We demonstrate here that thehuman TM cell Na-K-Cl cotransporter is highly sensitive to changes in[Cl]_i. Our findingsreveal a marked stimulation of Na-K-Cl cotransport activity, assessedas ouabain-insensitive, bumetanide-sensitive K influx, in TM cells following preincubation of cells with Cl-free medium as a means ofreducing [Cl]_i. Incontrast, preincubation of cells with media containing elevated Kconcentrations as a means of increasing [Cl]_i results ininhibition of Na-K-Cl cotransport activity. The effects of reducing[Cl]_i, as well aselevating [Cl]_i, onNa-K-Cl cotransport activity are concentration dependent. Furthermore, the stimulatory effect of reduced[Cl]_i is additive withcell-shrinkage-induced stimulation of the cotransporter. Our studiesalso show that TM cell Na-K-Cl cotransport activity is altered by avariety of Cl channel modulators, presumably through changes in[Cl]_i. These findingssupport the hypothesis that regulation of Na-K-Cl cotransport activity,and thus cell volume, by[Cl]_i may participatein modulating outflow facility across the TM.

     

New markers and map sequences inNeurospora crassa,with a description of mapping by duplication coverage,and of multiple translocation stocks for testing linkage

Thirty previously unmapped markers have been located; 13 are at newly designated loci. Numerous sequences for previously mapped genes have also been determined. A revised map of linkage group I is presented. The order from conventional mapping has been confirmed by testing recessive markers in IL for coverage by duplications. Assignment of new mutants to linkage groups is greatly facilitated by using gene-tagged multiple translocation strains for linkage detection; these “alcoy” tester strains and procedures for using them are described. Recent mapping data of other workers are compiled. Distal markers are now known for all but one of the 14 chromosome arms, but extensive map segments are still devoid of markers.     

The Lichen Connections of Black Fungi

Many black meristematic fungi persist on rock surfaces—hostile and exposed habitats where high doses of radiation and periods of desiccation alternate with rain and temperature extremes. To cope with these extremes, rock-inhabiting black fungi show phenotypic plasticity and produce melanin as cell wall pigments. The rather slow growth rate seems to be an additional prerequisite to oligotrophic conditions. At least some of these fungi can undergo facultative, lichen-like associations with photoautotrophs. Certain genera presenting different lifestyles are phylogenetic related among the superclass Dothideomyceta. In this paper, we focus on the genus Lichenothelia, which includes border-line lichens, that is, associations of melanised fungi with algae without forming proper lichen thalli. We provide a first phylogenetic hypothesis to show that Lichenothelia belongs to the superclass Dothideomyceta. Further, culture experiments revealed the presence of co-occurring fungi in Lichenothelia thalli. These fungi are related to plant pathogenic fungi (Mycosphaerellaceae) and to other rock-inhabiting lineages (Teratosphaeriaceae). The Lichenothelia thallus-forming fungi represent therefore consortia of different black fungal strains. Our results suggest a common link between rock-inhabiting meristematic and lichen-forming lifestyles of ascomycetous fungi.     

Immunological cross-reactions among gadidae parvalbumins

Antisera raised against apparently homogeneous whiting parvalbumin III have been found to recognize two non cross-reacting molecular species of parvalbumins. Aliquots of these antisera have been separately absorbed with two distinct parvalbumins from a near-related fish species, namely haddock parvalbumins II and III, and also with the homologous antigen. The immunochemical reactivities of absorbed and non-absorbed antisera toward parvalbumins from nine Gadidae species have been systematically explored by immunoelectrophoresis. The observed cross-reactions lead to distinguish two groups among Gadidae parvalbumins. So far this discrimination can be correlated with differences in amino-acid compositions, peptide maps and sequences which are known to characterize several protein members from each of the two groups. Using the same anti-whiting antisera, a tenuous common antigenic reactivity is shown between Gadidae and some Cyprinidae parvalbumins.     

Pseudomonas syringae isolated in lichens for the first time: Unveiling Peltigera genus as the exclusive host

Pseudomonas syringae is a bacterial complex that is widespread through a range of environments, typically associated with plants where it can be pathogenic, but also found in non-plant environments such as clouds, precipitation, and surface waters. Understanding its distribution within the environment, and the habitats it occupies, is important for examining its evolution and understanding behaviours. After a recent study found P. syringae living among a range of vascular plant species in Iceland, we questioned whether lichens could harbour P. syringae. Sixteen different species of lichens were sampled all over Iceland, but only one lichen genus, Peltigera, was found to consistently harbour P. syringae. Phylogenetic analyses of P. syringae from 10 sampling points where lichen, tracheophyte, and/or moss were simultaneously collected showed significant differences between sampling points, but not between different plants and lichens from the same point. Furthermore, while there were similarities in the P. syringae population in tracheophytes and Peltigera, the densities in Peltigera thalli were lower than in moss and tracheophyte samples. This discovery suggests P. syringae strains can localize and survive in organisms beyond higher plants, and thus reveals opportunities for studying their influence on P. syringae evolution.     

Kif26b controls endothelial cell polarity through the Dishevelled/Daam1-dependent planar cell polarity–signaling pathway

Angiogenesis involves the coordinated growth and migration of endothelial cells (ECs) toward a proangiogenic signal. The Wnt planar cell polarity (PCP) pathway, through the recruitment of Dishevelled (Dvl) and Dvl-associated activator of morphogenesis (Daam1), has been proposed to regulate cell actin cytoskeleton and microtubule (MT) reorganization for oriented cell migration. Here we report that Kif26b—a kinesin—and Daam1 cooperatively regulate initiation of EC sprouting and directional migration via MT reorganization. First, we find that Kif26b is recruited within the Dvl3/Daam1 complex. Using a three-dimensional in vitro angiogenesis assay, we show that Kif26b and Daam1 depletion impairs tip cell polarization and destabilizes extended vascular processes. Kif26b depletion specifically alters EC directional migration and mislocalized MT organizing center (MTOC)/Golgi and myosin IIB cell rear enrichment. Therefore the cell fails to establish a proper front–rear polarity. Of interest, Kif26b ectopic expression rescues the siDaam1 polarization defect phenotype. Finally, we show that Kif26b functions in MT stabilization, which is indispensable for asymmetrical cell structure reorganization. These data demonstrate that Kif26b, together with Dvl3/Daam1, initiates cell polarity through the control of PCP signaling pathway–dependent activation.