The use of metrizamide for stabilizing against convection in sedimentation equilibrium

In meniscus depletion sedimentation equilibrium experiments, the solute concentration gradient in much of the cell is insufficient to stabilize against thermal convection. Experiments with spectrin at 35 degrees C led to sufficient convection that, particularly at low concentrations of solute, no apparent redistribution occurred. At low angular velocity, the density gradient provided by moderate concentrations of NaCl or additional sucrose is inadequate to overcome the convective disturbances. We have found that the use of low concentrations of metrizamide can overcome these problems. The higher molecular weight and lower partial specific volume of metrizamide vis-a-vis sucrose allow a significant and stabilizing density gradient even at angular velocities below 8000 rpm.     

Purification of a water-soluble Mg2+-ATPase from human erythrocyte membranes

A water-soluble Mg2+-ATPase previously reported (White, M.D. and Ralston, G.B. (1976) Biochim. Biophys. Acta 436, 567-576) has been purified from human erythrocyte membranes. The purified enzyme has a molecular weight of 575 000; the apparent minimum molecular weight was 100 000, corresponding to a soluble protein of the component 3 region. The Km value for ATP was 1 mM and apparent Km for Mg2+ was 3.6 mM. By means of histochemical activity staining in acrylamide gels it was shown that the purified ATPase preparation could be inhibited by Cd2+ and Zn2+ salts, p-chloromercuribenzoate and N-ethylmaleimide, known inhibitors of membrane endocytosis.     

Infection of naïve, free-living brushtail possums (Trichosurus vulpecula) with the nematode parasite Parastrongyloides trichosuri and its subsequent spread

Despite the importance of spatial processes in host-parasite interactions, parasite dispersal has been the subject of few experimental studies. Introduced marsupial common brushtail possums (Trichosurus vulpecula) are a major environmental and agricultural pest in New Zealand. Parastrongyloides trichosuri, an intestinal rhabdiasoid nematode parasite specific to possums, is being evaluated as a self-disseminating delivery system for engineered fertility control vaccines. This study addressed whether an artificial infection could be established in a na?ve, free-living possum population, by measuring the post-release dynamics of possum-parasite interactions at the release site, and by following the spread of the parasite into surrounding possum populations. Infection was established efficiently by applying infective larvae to the skin of possums on a single occasion. All experimentally infected possums recaptured 3 weeks after infection had parasite eggs in their faeces. Over the subsequent 2.5 years, infection spread steadily over an area of about 6000 ha. Infection persisted at the original release site for the 3.5 years of the study and at a nearby site infected by natural spread for more than 3 years. Seasonal changes in faecal egg counts were similar at the two sites. The rapid establishment of the parasite and its spread provide additional support for its ongoing development as a vaccine delivery system.     

Dystrophin is a microtubule-associated protein

Cytolinkers are giant proteins that can stabilize cells by linking actin filaments, intermediate filaments, and microtubules (MTs) to transmembrane complexes. Dystrophin is functionally similar to cytolinkers, as it links the multiple components of the cellular cytoskeleton to the transmembrane dystroglycan complex. Although no direct link between dystrophin and MTs has been documented, costamere-associated MTs are disrupted when dystrophin is absent. Using tissue-based cosedimentation assays on mice expressing endogenous dystrophin or truncated transgene products, we find that constructs harboring spectrinlike repeat 24 through the first third of the WW domain cosediment with MTs. Purified Dp260, a truncated isoform of dystrophin, bound MTs with a K_d of 0.66 µM, a stoichiometry of 1 Dp260/1.4 tubulin heterodimer at saturation, and stabilizes MTs from cold-induced depolymerization. Finally, α- and β-tubulin expression is increased ∼2.5-fold in mdx skeletal muscle without altering the tubulin–MT equilibrium. Collectively, these data suggest dystrophin directly organizes and/or stabilizes costameric MTs and classifies dystrophin as a cytolinker in skeletal muscle.     

Transmembrane partitioning of boron and other elements in RAW 264.7 and HL60 cell cultures

The trace element boron is essential for all higher plants and is beneficial or has been established as essential for several animal models of human nutrition. To help identify the biomolecules that require boron for function in humans, we determined whether intracellular boron is retained against a concentration gradient. Cells (Abelson leukemia virus BALB murine monocyte-macrophage RAW 264.7 [RAW] and HL60) and supplemented media (Dulbecco’s modified essential media [+10% fetal calf serum] and Iscove’s modified Dulbecco’s medium [+5% fetal calf serum], respectively) were analyzed for mineral concentrations after culture and subculture. Special corrections were made for trapped extracellular media in cell pellets and endocytosed media. For RAW cells, the partitioning coefficients (PC; intracellular/extracellular ratios) were, in rank order, as follows: Mn, 110; Fe, 67; P, 65; Zn, 32; K, 15; Cu, 7.1; Mg, 4.3; B, 1.7; Ca, 0.4; Na, 0.3. For HL60 cells, the partitioning coefficients were, in rank order, as follows: Mn, 212; Zn, 211; P, 123; K, 21; Fe, 16; Mg, 11; B, 1.7; Ca, 0.8; Na, 0.3. Trapped extracellular media was estimated to be 6.7±0.8%; trapped extracellular and endocytosed media together was 24.8±0.3% of the mass within the isolated cell pellets. The partitioning coefficients indicate a positive gradient for intracellular accumulation of boron, zinc, phosphorus, managanese, magnesium, potassium, iron, and copper in RAW264.7 and HL60 cells. Specifically, the data indicate the existence of a selective boron-binding molecular species within the cell or the existence of a boron-specific membrane transporter.