HEAT repeats mediate plasma membrane localization of Tor2p in yeast

The subcellular distribution of Tor1p and Tor2p, two phosphatidylinositol kinase homologs and targets of the immunosuppressive drug rapamycin in Saccharomyces cerevisiae, was analyzed. We found that Tor protein is peripherally associated with membranes. Subcellular fractionation and immunofluorescence studies showed that Tor1p and Tor2p associate with the plasma membrane and a second fraction that is distinct from Golgi, vacuoles, mitochondria, and nucleus and may represent vesicular structures. Pulse-chase experiments showed that association of Tor protein with plasma membrane and the second compartment is fast, does not appear to involve components of endocytic, secretory, or Golgi to vacuole transport pathways, and is not affected by the immunosuppressive drug rapamycin. Deletion analysis reveals that two domains within Tor2p independently mediate localization to both compartments. These domains are composed of HEAT repeats that are thought to act as protein-protein interaction surfaces. Our studies therefore place Tor proteins at the site of action of their known downstream effectors and suggest that they may be part of a multiprotein complex.     

Biophysical and lipofection studies of DOTAP analogs

In order to investigate the relationship between lipid structure and liposome-mediated gene transfer, we have studied biophysical parameters and transfection properties of monocationic DOTAP analogs, systematically modified in their non-polar hydrocarbon chains. Stability, size and (by means of anisotropy profiles) membrane fluidity of liposomes and lipoplexes were determined, and lipofection efficiency was tested in a luciferase reporter gene assay. DOTAP analogs were used as single components or combined with a helper lipid, either DOPE or cholesterol. Stability of liposomes was a precondition for formation of temporarily stable lipoplexes. Addition of DOPE or cholesterol improved liposome and lipoplex stability. Transfection efficiencies of lipoplexes based on pure DOTAP analogs could be correlated with stability data and membrane fluidity at transfection temperature. Inclusion of DOPE led to rather uniform transfection and anisotropy profiles, corresponding to lipoplex stability. Cholesterol-containing lipoplexes were generally stable, showing high transfection efficiency at low relative fluidity. Our results demonstrate that the efficiency of gene transfer mediated by monocationic lipids is greatly influenced by lipoplex biophysics due to lipid composition. The measurement of fluorescence anisotropy is an appropriate method to characterize membrane fluidity within a defined system of liposomes or lipoplexes and may be helpful to elucidate structure-activity relationships.     

Intraspecific mating system evolution and its effect on complex male secondary sexual traits: Does male–male competition increase selection on size or shape?

Sexual selection is generally held responsible for the exceptional diversity in secondary sexual traits in animals. Mating system evolution is therefore expected to profoundly affect the covariation between secondary sexual traits and mating success. Whereas there is such evidence at the interspecific level, data within species remain scarce. We here investigate sexual selection acting on the exaggerated male fore femur and the male wing in the common and widespread dung flies Sepsis punctum and S. neocynipsea (Diptera: Sepsidae). Both species exhibit intraspecific differences in mating systems and variation in sexual size dimorphism (SSD) across continents that correlates with the extent of male–male competition. We predicted that populations subject to increased male–male competition will experience stronger directional selection on the sexually dimorphic male foreleg. Our results suggest that fore femur size, width and shape were indeed positively associated with mating success in populations with male‐biased SSD in both species, which was not evident in conspecific populations with female‐biased SSD. However, this was also the case for wing size and shape, a trait often assumed to be primarily under natural selection. After correcting for selection on overall body size by accounting for allometric scaling, we found little evidence for independent selection on any of these size or shape traits in legs or wings, irrespective of the mating system. Sexual dimorphism and (foreleg) trait exaggeration is therefore unlikely to be driven by direct precopulatory sexual selection, but more so by selection on overall size or possibly selection on allometric scaling.     

The binding of pyridoxal 5'-phosphate to human serum albumin

Most of the pyridoxal 5'-phosphate (PLP) in plasma is bound to protein, primarily albumin. Binding to protein is probably important in transporting PLP in the circulation and in regulating its metabolism. The binding of PLP to human serum albumin (HSA) was studied using absorption spectral analysis, equilibrium dialysis, and inhibition studies. The kinetics of the changes in the spectrum of PLP when mixed with an equimolar concentration of HSA at pH 7.4 followed a model for two-step consecutive binding with rate constants of 7.72 mM-1 min-1 and 0.088 min-1. The resulting PLP-HSA complex had absorption peaks at 338 and 414 nm and was reduced by potassium borohydride. The 414-nm peak is probably due to a protonated aldimine formed between PLP and HSA. The binding of PLP to bovine serum albumin (BSA) at equimolar concentrations at pH 7.4 occurred at about 10% the rate of its binding to HSA. The final PLP-BSA complex absorbed maximally at 334 nm and did not appear to be reduced with borohydride. Equilibrium dialysis of PLP and HSA indicated that there were more than one class of binding sites of HSA for PLP. There was one high affinity site with a dissociation constant of 8.7 microM and two or more other sites with dissociation constants of 90 microM or greater. PLP binding to HSA was inhibited by pyridoxal and 4-pyridoxic acid. It was not inhibited appreciably by inorganic phosphate or phosphorylated compounds. The binding of PLP to BSA was inhibited more than its binding to HSA by several compounds containing anionic groups. It is concluded that PLP binds differently to HSA than it does to BSA.     

Hydrolysis of polyphosphates and permeability changes in response to osmotic shocks in cells of the halotolerant alga dunaliella

The effects of osmotic shocks on polyphosphates and on the vacuolar fluorescent indicator atebrin have been investigated to test whether acidic vacuoles in the halotolerant alga Dunaliella salina have a role in osmoregulation. Upshocks and downshocks induce different patterns of polyphosphate hydrolysis. Upshocks induce rapid formation of new components, tentatively identified as 5 or 6 linear polyphosphates, formed only after upshocks with NaCl and not with glycerol, indicative of compartmentation of Na⁺ into the vacuoles. Conversely, downshocks induce a slower transient accumulation of tripolyphosphates, indicating activation of a different hydrolytic process within the vacuoles. Osmotic shocks do not lead to release of atebrin from acidic vacuoles, indicating that they do not induce a major intravacuolar alkalinization. However, osmotic shocks induce transient permeability changes measured by amine-induced atebrin release from vacuoles. Hypoosmotic shocks transiently increase the permeability (up to 20-fold), whereas hyperosmotic shocks induce a rapid drop in permeability. Electron micrographs of osmotically shocked cells also reveal transient changes in the surface and internal organelles of D. salina cells. It is suggested that hyperosmotic and hypoosmotic shocks induce different changes within acidic vacuoles and in the organization and/or composition of the plasma membrane in Dunaliella.     

Current perspectives on plasmodesmata: structure and function

Recent studies on plasmodesmata have shown that these important intercellular passages for communication and transport are much more sophisticated in both structure and regulatory abilities than previously imagined. A complex, but not well understood, substructure has been revealed by a variety of increasingly reliable ultrastructural techniques. Proteinaceous particles are seen within the cytoplasmic sleeve surrounding the desmotubule. Dye-coupling studies have provided experimental evidence for the physical pathway of solute movement, supporting conclusions about substructural dimensions within plasmodesmata drawn from the ultrastructural studies. Calcium has been identified as a major factor in the regulation of intercellular communication via plasmodesmata. Evidence from studies on virus movement through plasmodesmata suggests a direct interaction between virallycoded movement proteins and plasmodesmata in the systemic spread of many viruses. There is increasing evidence, albeit indirect, that in some plant species phloem loading may involve transport of photoassimilate entirely within the symplast from mesophyll cells to the sieve element-companion cell complexes of minor veins.     

Structural and functional analysis of NS1 and NS2 proteins of H1N1 subtype

Influenza A virus (H1N1), a genetic reassortment of endemic strains of human, avian and swine flu, has crossed species barrier to human and apparently acquired the capability of human to human transmission. Some strains of H5N1 subtype are highly virulent because NS1 protein inhibits antiviral interferon α/β production. Another protein NS2 mediates export of viral ribonucleoprotein from nucleus to the cytoplasm through export signal. In this paper, we have studied structure-function relationships of these proteins of H1N1 subtype and have determined the cause of their pathogenicity. Our results showed that non-conservative mutations slightly stabilized or destabi- lized structural domains of NS1 or NS1-dsRNA complex, hence slightly increased or decreased the function of NS1 protein and consequently enhanced or reduced the pathogenicity of the H1N1 virus. NS2 protein of different strains carried non-conservative mutations in different domains, resulting in slight loss of function. These mutations slightly decreased the pathogenicity of the virus. Thus, the results confirm the structure-function relationships of these viral proteins.     

Downregulation of mu opioid receptor by RNA interference in the ventral tegmental area reduces ethanol consumption in mice

Pharmacological and genetic studies have implicated the mu opioid receptor (MOR) in the regulation of ethanol intake in animal models and humans. Non-specific antagonists of opioid receptors have been shown to affect ethanol consumption when infused directly into the ventral tegmental area (VTA) of rats. However, administration of MOR-selective antagonists into the VTA has yielded mixed results. We used RNA interference (RNAi) to specifically decrease levels of MOR messenger RNA in the VTA of mice and examined the effect on ethanol consumption in a two-bottle choice paradigm. Mice were injected in the VTA with lentivirus expressing either a small hairpin RNA (shRNA) targeting MOR or a control shRNA. One week after virus injection, mice were examined for ethanol consumption in a two-bottle choice experiment with increasing concentrations of ethanol over the course of 1 month. Expression of an shRNA targeting MOR in the VTA led to a significant reduction in ethanol consumption. These results strengthen the hypothesis that MOR in the VTA is one of the key brain substrates mediating alcohol consumption. The RNAi combined with lentiviral delivery can be used successfully in brain to effect a sustained reduction in expression of specific genes for behavioral analysis.     

Improved Luciferase Tagging System for Listeria monocytogenes Allows Real-Time Monitoring In Vivo and In Vitro

An improved system for luciferase tagging Listeria monocytogenes was developed by constructing a highly active, constitutive promoter. This construct gave 100-fold-higher activity in broth than any native promoter tested and allowed for imaging of lux-tagged L. monocytogenes in food products, during murine infections, and in tumor targeting studies.     

New taxa ofCosmarium Corda (Desmidiaceae) from Karnataka State,India

Six new taxa including two new species (Cosmarium bourrellyi Bongale sp. nov. andC. desikacharyi Bongale sp. nov.) and four new varieties (C. auriculatum var.protrusum Bongale var. nov.,C. pachydermum var.inflatum Bongale var. nov.,C. pseudoconnatum var.tuberculans Bongale var. nov. andC. quadrum var.rotundatum Bongale var. nov.) are reported. Algae were collected from a paddy field at Belgaum.