Phospholipid dependence of the anion transport system of the human erythrocyte membrane. Studies on reconstituted band 3/lipid vesicles

Band 3 protein extracted from human erythrocyte membranes by Triton X-100 was recombined with the major classes of phospholipid occurring in the erythrocyte membrane. The resulting vesicle systems were characterized with respect to recoveries, phospholipid composition, protein content and vesicle size as well as capacity and activation energy of sulfate transport. Transport was classified into band-3-specific fluxes and unspecific permeability by inhibitors. Transport numbers (sulfate ions per band 3 per minute) served as a measure of functional recovery after reconstitution. The transport properties of band 3 proved to be insensitive to replacement of phosphatidylcholine by phosphatidylethanolamine, while sphingomyelin and phosphatidylserine gradually inactivated band-3-specific anion transport when present at mole fractions exceeding 30 mol%. The activation energy of transport remained unaltered in spite of the decrease in transport numbers. The results, which are discussed in terms of requirements of band 3 protein function with respect to the fluidity and surface charge of its lipid environment, provide a new piece of evidence that the transport function of band 3 protein depends on the properties of its lipid environment just as the catalytic properties of some other membrane enzymes. The well-established species differences in anion transport (Gruber, W. and Deuticke, B. (1973) J. Membrane Biol. 13, 19–36) may to some extent reflect this lipid dependence.     

Effects of Organic Solvents on the Coupling Between ATP Hydrolysis and Sliding of Microtubules in Axonemes from Chlamydomonas Flagella

ABSTRACT. The effects of organic solvents on the ATPase activity and the sliding disintegration of axonemes from Chlamydomonas were investigated. The axonemal ATPase was markedly activated by methanol accompanying with marked inhibition of the sliding disintegration of axonemes. On the contrary, glycerol inhibited the ATPase activity without serious inhibition of the sliding disintegration. As far as the axonemes are not irreversibly denatured by extremely high concentration of solvents, the effects of solvents both on the ATPase and the ability of sliding are reversible. Therefore, the inhibition of sliding accompanied by the activation of ATPase is probably due to an inability to couple the hydrolysis of ATP to sliding between dynein and microtubule in the presence of methanol. The axonemal ATPase was less sensitive to vanadate inhibition after exposure to methanol. This indicates that methanol makes the dyneinADP.Pi complex unstable and increases product release. On the other hand, glycerol and ethylene glycol seem to stabilize the force generation responsible for the sliding through stabilizing the dynein.ADP.Pi complex.     

Detection of cellular responses to toxicants by dielectrophoresis

The dielectrophoretic (DEP) crossover method has been applied to the detection of cell responses to toxicants. Time and dose responses of the human cultured leukemia (HL-60) line were measured for paraquat, styrene oxide (SO), N-nitroso-N-methylurea (NMU) and puromycin. These toxicants were chosen because of their different predominant mechanisms of action, namely membrane free radical attack, simultaneous membrane and nucleic acid attack, nucleic acid alkylation, and protein synthesis inhibition, respectively. For all treatments, the specific membrane capacitance (C_mem) of the cells decreased while the specific membrane conductance (G_mem) increased in dose- and time-dependent manners. The DEP responses correlated sensitively with alterations in cell surface morphology, especially folds, microvilli, and blebs, observed by scanning electron microscopy. The DEP method was more sensitive to agents that had a direct action on the membrane than to agents for which membrane alterations were secondary. The responses to paraquat and SO, which directly damaged the cell membrane, could be detected 15 min after exposure, while those for puromycin and NMU, which acted on intracellular targets, could be detected after 30 min. The detection times and dose sensitivity results showed that the DEP method is much faster and more sensitive than conventional cell and higher organism viability testing techniques. The feasibility of producing small instruments for toxicity detection and screening based on cellular dielectric responses is discussed.     

Dictyosome vesicle production and plasma membrane turnover in auxin-stimulated outer epidermal cells of coleoptile segments fromAvena sativa (L.)

Summary Dark grown coleoptile segments were floated on solutions of IAA alone and of IAA and the secretion inhibitors cytochalasin and monensin. The secretion inhibitors prevented normal elongation of the tissue segments, the monensin inhibition being virtually complete while cytochalasin gave a 40% reduction over the first six hours with little further further elongation in the following 18 hours. Vesicle production was assessed in outer epidermal cells after 6 hours of IAA-stimulated elongation using the vesicle accumulation method following a cytochalasin-block of vesicle transport. The results were compared with the area of plasma membrane required to enable cell elongation to proceed at the observed rate. The area of vesicle membrane delivered to the cell surface exceeded this requirement to such an extent that at least 65% of the delivered membrane must be recycled back into the cytoplasm. Expressed in terms of the whole cell, the plasma membrane turnover rate was found to be once every 200 minutes. It is concluded that limitation of elongation by secretion inhibitors is more likely to reflect a requirement for the vesicle contents than the vesicle membrane. These results are compared with those obtained from other secretory systems using a similar approach.Abbreviations IAA indole acetic acid - DMSO dimethyl sulphoxide - D dictyosome - ER endoplasmic reticulum - V vesicle     

The Treatment of Missing Values in Logistic Regression

The efficiencies of the estimators in the linear logistic regression model are examined using simulations under six missing value treatments. These treatments use either the maximum likelihood or the discriminant function approach in the estimation of the regression coefficients. Missing values are assumed to occur at random. The cases of multivariate normal and dichotomous independent variables are both considered. We found that in general, there is no uniformly best method. However, mean substitution and discriminant function estimation using existing pairs of values for correlations turn out to be favourable for the cases considered.     

The Monofactorial Model for Recurrence Risks with Generalized Penetrance

Recurrence risks are derived explicitly in terms of gene frequencies and penetrance coefficients for the general case in which all genotypes have incomplete penetrance. Maximum likelihood estimation of recurrence risks is achieved through the use of the semi-symmetric intraclass contingency table. The resulting formulas and estimation procedure can be useful for the analysis of population and family data, and in genetic counselling.     

Fluorescence anisotropy from diphenylhexatriene in rat liver plasma membranes

Rat livers were fractionated to obtain intracellular membrane preparations and a highly purified preparation of bile canaliculi. The fraction containing bile canaliculi was homogenized and subfractionated to give fractions representing fragments of contiguous membrane and of canalicular microvilli. The relative purity and extent of contamination of each preparation was determined. When the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene was incorporated into aliquots of each fraction at the same probe: lipid ratio and the steady-state anisotropy of its fluorescence measured, it was found that the plasma membrane preparations were much more ordered than the intracellular membrane preparations. Of the plasma membrane preparations, that containing the canalicular microvilli was the most ordered, even allowing for any contribution of contaminants. Thus the microvillus membrane of the bile canaliculus appears to be the most ordered domain of the plasma membrane of the hepatocyte. The high order in this domain may be a factor in reducing the susceptibility to bile salt damage during bile secretion, since it is this region which is exposed to high concentrations of bile salts in vivo.     

Describing Ordinal Odds Ratios for Stratified r × c Tables

For an r × ctable with ordinal responses, odds ratios are commonly used to describe the relationship between the row and column variables. This article shows two types of ordinal odds ratios where local‐global odds ratios are used to compare several groups on a c‐category ordinal response and a global odds ratio is used to measure the global association between a pair of ordinal responses. When there is a stratification factor, we consider Mantel‐Haenszel (MH) type estimators of these odds ratios to summarize the association from several strata. Like the ordinary MH estimator of the common odds ratio for several 2 × 2 contingency tables, the estimators are used when the association is not expected to vary drastically among the strata. Also, the estimators are consistent under the ordinary asymptotic framework in which the number of strata is fixed and also under sparse asymptotics in which the number of strata grows with the sample size. Compared to the maximum likelihood estimators, simulations find that the MH type estimators perform better especially when each stratum has few observations. This article provides variances and covariances formulae for the local‐global odds ratios estimators and applies the bootstrap method to obtain a standard error for the global odds ratio estimator. At the end, we discuss possible ways of testing the homogeneity assumption.