The Sodium/Proton Exchanger NHE8 Regulates Late Endosomal Morphology and Function

The pH and lumenal environment of intracellular organelles is considered essential for protein sorting and trafficking through the cell. We provide the first evidence that a mammalian NHE sodium (potassium)/proton exchanger, NHE8, plays a key role in the control of protein trafficking and endosome morphology. At steady state, the majority of epitope-tagged NHE8 was found in the trans-Golgi network of HeLa M-cells, but a proportion was also localized to multivesicular bodies (MVBs). Depletion of NHE8 in HeLa M-cells with siRNA resulted in the perturbation of MVB protein sorting, as shown by an increase in epidermal growth factor degradation. Additionally, NHE8-depleted cells displayed striking perinuclear clustering of endosomes and lysosomes, and there was a ninefold increase in the cellular volume taken up by LAMP1/LBPA-positive, dense MVBs. Our data points to a role for the ion exchange activity of NHE8 being required to maintain endosome morphology, as overexpression of a nonfunctional point mutant protein (NHE8 E225Q) resulted in phenotypes similar to those seen after siRNA depletion of endogenous NHE8. Interestingly, we found that depletion of NHE8, despite its function as a sodium (potassium)/proton antiporter, did not affect the overall pH inside dense MVBs.     

Free and polymerized tubulin in cultured bone cells and Chinese hamster ovary cells: the influence of cold and hormones

A low pH method of liposome-membrane fusion (Schneider et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 77:442) was used to enrich the mitochondrial inner membrane lipid bilayer 30-700% with exogenous phospholipid and cholesterol. By varying the phospholipid-to- cholesterol ratio of the liposomes it was possible to incorporate specific amounts of cholesterol (up to 44 mol %) into the inner membrane bilayer in a controlled fashion. The membrane surface area increased proportionally to the increase in total membrane bilayer lipid. Inner membrane enriched with phospholipid only, or with phospholipid plus cholesterol up to 20 mol %, showed randomly distributed intramembrane particles (integral proteins) in the membrane plane, and the average distance between intramembrane particles increased proportionally to the amount of newly incorporated lipid. Membranes containing between 20 and 27 mol % cholesterol exhibited small clusters of intramembrane particles while cholesterol contents above 27 mol % resulted in larger aggregations of intramembrane particles. In phospholipid-enriched membranes with randomly dispersed intramembrane particles, electron transfer activities from NADH- and succinate-dehydrogenase to cytochrome c decreased proportionally to the increase in distance between the particles. In contrast, these electron- transfer activities increased with decreasing distances between intramembrane particles brought about by cholesterol incorporation. These results indicate that (a) catalytically interacting redox components in the mitochondrial inner membrane such as the dehydrogenase complexes, ubiquinone, and heme proteins are independent, laterally diffusible components; (b) the average distance between these redox components is effected by the available surface area of the membrane lipid bilayer; and (c) the distance over which redox components diffuse before collision and electron transfer mediates the rate of such transfer.     

Experimental analyses of competition between two species of bumble bees (Hymenoptera: apidae)

Summary Dynamical aspects of flower usage and forager body size in sympatric and experimentally-induced allopatric populations of Bombus flaviforns and Bombus rufocinctus were studied in 10 discrete subalpine meadows and over the last half of one summer. Results indicate that there is a high degree of asymmetry in the competitive effects and that B. flavifrons is the clear competitive and numerical dominant. When occurring alone, B. rufocinctus used the same spectrum of flowers in similar frequencies to that of B. flavifrons whose flower use was invariant over all meadows and treatments. When sympatric with B. flavifrons, B. rufocinctus was relegated to secondary, less preferred flowers. Shifts in flower use by B. rufocinctus were accompanied by changes in forager body-size: body weights were greater in allopatric populations of B. rufocinctus and smaller in those sympatric with B. flavifrons. Competitive dominance may be related to differences in species phenologies. Bombus flavifrons initiated colonies in the spring three weeks before B. rufocinctus and maintained higher densities over the summer; by virtue of greater numbers of foragers, it may control exploitation of the most profitable flowers.     

Analysis of folate cofactor levels in tissues using high-performance liquid chromatography

A method for the isolation and concentration of the monoglutamate forms of folate cofactors from tissues and for their subsequent separation and quantitation using HPLC coupled with uv detection at 284 nm is described. A chromatographic procedure utilizing Dowex 50 has been developed for the separation of the folate monoglutamates from a large portion of the nonfolate-related material following digestion of the polyglutamated froms with a highly purified preparation of rat liver conjugase. This chromatographic procedure combined with concentration of the Dowex eluate by lyophilization eliminates uv-absorbing material, which interferes with the detection and quantitation of the folate cofactors and makes possible uv measurement of the individual folates. Reverse-phase paired-ion chromatography on μBondapak C₁₈ coupled with uv detection allows direct quantitation of the folates in the nanogram range.     

Preharvest aflatoxin contamination: effect of moisture and substrate variation in developing cottonseed and corn kernels.

Variations in moisture and substrate in preharvest corn kernels and cottonseed were linked with the ability of Aspergillus parasiticus to infect the seed and produce aflatoxin. Osmotic pressures and moisture content (MC) levels of developing starch-rich corn kernels and lipid-rich cottonseed were determined. For in vivo studies, corn kernels and cottonseed were inoculated with A. parasiticus conidia and retained on plants through maturation. For in vitro studies, samples of corn kernels and cottonseed were collected at various stages, sterilized, inoculated, incubated for 2 weeks, and assayed for toxin. Aflatoxin levels were highest in corn kernels inoculated at 28 days postflowering (52% MC) in both the in vivo and in vitro tests. Toxin concentrations in cottonseed were greatest with inoculation at 35 days postflowering (70% MC) in seed retained on the plant, but toxin accumulation continued to increase with the maturity of the seed inoculated in cottonseed used in the in vitro trials. Moisture and substrate conditions in the midrange of seed development provided optimum conditions for fungal development and toxin production in seed retained on the plant.     

High-resolution proton and carbon-13 NMR of membranes: why sonicate?

We have obtained high-field (11.7-T) proton and carbon-13 Fourier transform (FT) nuclear magnetic resonance (NMR) spectra of egg lecithin and egg lecithin-cholesterol (1:1) multibilayers, using "magic-angle" sample spinning (MASS) techniques, and sonicated egg lecithin and egg lecithin-cholesterol (1:1) vesicles, using conventional FT NMR methods. Resolution of the proton and carbon-13 MASS NMR spectra of the pure egg lecithin samples is essentially identical with that of sonicated samples, but spectra of the unsonicated lipid, using MASS, can be obtained very much faster than with the more dilute, sonicated systems. With the 1:1 lecithin-cholesterol systems, proton MASS NMR spectra are virtually identical with conventional FT spectra of sonicated samples, while with 13C NMR, we demonstrate that most 13C nuclei in the cholesterol moiety can be monitored, even though these same nuclei are essentially invisible, i.e., are severely broadened, in the corresponding sonicated systems. In addition, 13C MASS NMR, spectra can again be recorded much faster than with sonicated samples, due to concentration effects. Taken together, these results strongly suggest there will seldom be need in the future to resort to ultrasonic disruption of lipid bilayer membranes in order to obtain high-resolution proton or carbon-13 NMR spectra.     

The ovulated ovum of the horse: cytology of nonfertilized ova to pronuclear stage ova

Fertilization and early development in the horse were studied by recovering oviductal ova at various times after postovulatory mating. Ova collected between 7 and 22 h post coitum (pc) were examined for evidence of fertilizing sperm, cellular changes accompanying fertilization, and pronuclear development. Five ova collected between 7 and 9 h pc contained a marginal metaphase plate, but had no indication of sperm components; three of these, however, showed reduced numbers of cortical granules. Two activated ova (10 and 14 h pc) were in telophase of the second meiotic division, following incorporation of the fertilizing sperm. The fertilizing sperm was situated in a slight elevation; the nucleus was expanding but lacked a nuclear envelope. The pronuclear stage in the horse began as early as 12 h pc, and lasted at least until 21 h pc. Sperm tail remnants were seen in 5 of 7 pronuclear-stage ova, although the crowding of the cytoplasm with clusters of lipid and vacuoles made discerning sperm tail remnants difficult. The spindles of the metaphase stage of the second meiotic division were oriented radially, that is, at right angles to the cell surface, in all but one ovum, so this orientation is not a response to fertilization.     

The population dynamics of pike,Esox lucius,and perch,Perca fluviatilis,in a simple predator-prey system

Synopsis The population dynamics and predator-prey relationship of pike, Esox lucius, and perch, Perca fluviatilis, were examined in simple fish communities in two adjacent shallow lakes, Lochs Kinord and Davan, Deeside, Scotland. Few perch survive to age 3 but Z is low for fish > 3 years and perch live up to 17 years. Population fecundity remained relatively high and constant in perch because of the multi-age spawning stock and the presence of older more fecund perch. Growth rates of perch in both lochs are relatively high as a consequence of low stock abundance. The N, B, and P of adult perch were unusually low. The age range of pike, and N, B, P, and growth were in the range of values reported elsewhere. There was little variation in the strength of pike year classes and the importance of cannibalism and low occurrence of alternative prey in the lochs suggest that the populations were self-regulating. Cannibalism by adults was responsible for most of mortality in perch larvae, and predation by pike and adult perch was responsible for the majority of juvenile losses. This conclusion is supported by the high biomass ratios of pike:juvenile perch of 1.0–30.8. While the number of adult fish was almost equal, the biomass of adult pike was 2–3 × that of perch in Kinord and 6 × in Davan. In L. Kinord, where year class strength was stable, high predation pressure from perch and pike reduced perch abundance rather than eliminated year classes. Perch year classes fluctuated in abundance in L. Davan and were eliminated in the first summer in two sampling years. The pike, and particularly the perch populations, have features characteristic of fish communities in unperturbed ecosystems: namely, a wide range of age classes, stability in biomass with variation dampened by longevity, and low production.     

Indoxyl-beta-D-glucuronide, a novel chromogenic reagent for the specific detection and enumeration of Escherichia coli in environmental samples

About 97% of Escherichia coli strains produce beta-glucuronidase, but almost all other Enterobacteriaceae lack this enzyme. A D-glucopyranosiduronic acid (glucuronide) possessing a readily detectable beta-linked aglycone should, therefore, constitute a specific reagent for the detection of this organism. For this purpose, the title compound has been synthesized for the first time. The synthesis proceeds in eight steps from readily available D-glucuronolactone, anthranilic acid, and chloroacetic acid and can be carried out on a large scale. The compound has the predicted properties: when included in the standard membrane filter test for the analysis of water, indoxyl-beta-D-glucuronide allows specific detection of E. coli through the formation of blue colonies that are the result of rapid conversion of the liberated aglycone to indigo. The recovery of E. coli is easily measured and almost quantitative.