Modulation of K+ channels by hydrogen peroxide.

External application of hydrogen peroxide (H2O2) was found to inhibit the time-dependent fast inactivation process of three cloned voltage-gated K+ channels expressed in Xenopus oocytes: KShIIIC, KShIIID and HukII. As expected from kinetic models where some channels are still opening while a significant fraction of channels is already inactivated there was a large increase in current magnitude concomitant to inactivation block. The channels otherwise functioned normally. The effects of H2O2 were specific (other cloned voltage-gated K+ channels were not affected), and reversible, the currents returned to normal upon removal of the H2O2. H2O2 is produced during normal metabolism; it could act as a modulator of excitability through effects on K+ channels if effective local concentrations are reached in neuronal regions close to the channel. KShIIIC and KShIIID currents are very similar to an O2-sensitive K+ current present in type I cells of the carotid body which is believed to underlie the modulation of excitability of these cells by changes in arterial O2 pressure. H2O2 has been proposed as an intermediary between O2 and cellular response in the carotid body; our results provide support for this model.     

Characterization of the pH-mediated solubility of Bacillus thuringiensis var. san diego native delta-endotoxin crystals.

Native crystals of Bacillus thuringiensis var. san diego, a coleopteran-specific delta-endotoxin, were metabolically labelled with [35S]methionine. Specific activity was 82,000 CPM/micrograms (2.44 Ci/mmol). Using a universal buffer formulated with the same ionic strength at every pH, we determined that native crystals dissolve above pH 10 and below pH 4. At the acidic pH, the rate of solubilization was substantially slower than at the alkaline pH. Recrystallization rates for the toxin were similar regardless of solubilization conditions. The banding patterns in denatured polyacrylamide gel electrophoresis were unaffected by solubilization conditions. Toxicity was higher for soluble toxin compared to crystal toxin, but virtually identical for the acidic and alkaline produced solutions. Acid solubilization is significant because of the acidic midgut of susceptible Coleoptera.     

Bivariate flow cytometry of farm animal chromosomes: a potential tool for gene mapping

Bivariate flow karyotypes of chromosomes from sheep, cattle and pig lymphocytes and from a cattle-mouse somatic cell hybrid line were obtained using a dual laser fluorescence-activated cell sorter (FACS). Pig chromosomes were resolved into 19-20 peaks, indicating that most, if not all, pig chromosomes could be separated by this technique. Sheep chromosomes showed incomplete separation but three clear peaks, presumably representing the three large metacentric chromosomes, plus five other clusters were obtained. Cattle chromosomes showed poor separation but about four peaks could be distinguished, indicating that certain chromosomes could be sorted in this species. The use of cattle-mouse hybrids may enable other individual cattle chromosomes to be obtained. It is concluded that FACS separation will be a useful additional tool for gene mapping.     

The unique headpore of Monospilus dispar (Cladocera,Chydoridae) is not lonesome: new record of minute lateral pores

The headshield of Monospilus dispar (Cladocera, Chydoridae, Aloninae) was characterized by the presence of a unique headpore. Scanning Electron Microscopy shows the occurrence of minute pores close to the main headpore. The intimate structure of the main headpore is formed by concentric lamellae. This new information leads to new considerations about phylogeny.     

The relationship between water velocity,energetic costs,and microhabitat use in four North American stream fishes

Freshwater crayfish are key members of aquatic communities due to their large size and abundance. Although most commonly regarded as herbivores and detritivores, they are also selective predators. The crayfish plague fungus Aphanomyces astaci (Schikora) led to the elimination of a stock of white-clawed crayfish, Austropotamobius pallipes (Lereboullet) from Lough Lene, Co. Westmeath, in 1987. Samples taken of the flora and benthic communities of two Irish lakes, one (Lough Bane) formerly containing crayfish and the other (Lough Lene) immediately following a plague outbreak, were compared to similar samples taken a year later and ecological shifts were noted and compared to laboratory feeding results. Over time, Chara strands increased in mean length, and molluscs became more abundant.     

Primary sequence and functional expression of a novel ouabain-resistant Na,K-ATPase. The beta subunit modulates potassium activation of the Na,K-pump.

In order to understand the molecular mechanism of ouabain resistance in the toad Bufo marinus, Na,K-ATPase alpha and beta subunits have been cloned and their functional properties tested in the Xenopus laevis oocyte expression system. According to sequence comparison between species, alpha 1, beta 1, and beta 3 isoforms were identified in a clonal toad urinary bladder cell line (TBM 18-23). The sequence of the alpha 1 isoform is characterized by two positively charged amino acids (Arg, Lys) at the N-terminal border of the H1-H2 extracellular loop and no charged amino acid at the C terminus, a pattern distinct from the ouabain-resistant rat alpha 1 isoform. The coexpression of alpha 1 beta 1 or alpha 1 beta 3 TBM subunits in the Xenopus oocyte resulted in the expression of identical maximum Na,K-pump currents with identical inhibition constant for ouabain (Ki) (alpha 1 beta 1: 53 +/- 3 microM; n = 7 vs. alpha 1 beta 3: 57 +/- 3.0 microM; n = 8) but distinct potassium half activation constant (K1/2) (alpha 1 beta 1: 0.87 +/- 0.08 mM, n = 16; alpha 1 beta 3: 1.29 +/- 0.07 mM, n = 17; p less than 0.005). We conclude that (i) the TBM alpha 1 isoform is necessary and sufficient to confer the ouabain resistant phenotype; (ii) the beta 3 or beta 1 subunit can associate with the alpha 1 equally well without affecting the ouabain-resistant phenotype; (iii) some specific sequence of the beta subunit can modulate the activation of the Na,K-pump by extracellular potassium ions.     

Inhibition of glycosylphosphatidylinositol anchor formation by mannosamine.

Many eucaryotic cell surface proteins are anchored to the plasma membrane via a glycosylphosphatidylinositol (GPI), of which the core region is highly conserved from protozoa to mammalian cells. Previous studies (Lisanti, M. P., Field, M. C., Caras, I. W., Menon, A. K., and Rodiguez-Boulan, E. (1991) EMBO J. 10, 1969-1977) showed that mannosamine blocked the expression of a recombinant GPI-anchored protein in Madin-Darby canine kidney cells and converted this protein to an unpolarized secretory product. In the present study, we examined the effect of mannosamine on the formation of the glycan portion of the GPI anchor precursors. This amino sugar inhibited the incorporation of mannose into the glycan portion, and the inhibition was dose-dependent. Mannosamine was shown to be incorporated into the glycan as mannosamine, probably mostly in the second mannose position and thereby to block the further addition of mannose and other anchor components. The products formed in the presence of this drug were characterized by gel filtration and high resolution TLC both before and after deamination with nitrous acid and dephosphorylation by HF. Galactosamine and trehalosamine were inactive in this system, whereas glucosamine also inhibited mannose incorporation into GPI intermediates.