Spin-label studies on phosphatidylcholine-cholesterol membranes: effects of alkyl chain length and unsaturation in the fluid phase

Dynamic properties of phosphatidylcholine-cholesterol membranes in the fluid phase and water accessibility to the membranes have been studied as a function of phospholipid alkyl chain length, saturation, mole fraction of cholesterol, and temperature by using spin and fluorescence labelling methods. The results are the following: (1) The effect of cholesterol on motional freedom of 5-doxyl stearic acid spin label (5-SASL) and 16-doxyl stearic acid spin label (16-SASL) in saturated phosphatidylcholine membrane is significantly larger than the effects of alkyl chain length and introduction of unsaturation in the alkyl chain. (2) Variation of alkyl chain length of saturated phospholipids does not alter the effects of cholesterol except in the case of dilauroylphosphatidylcholine, which possesses the shortest alkyl chains (12 carbons) used in this work. (3) Unsaturation of the alkyl chains greatly reduces the ordering effect of cholesterol at C-5 and C-16 positions although unsaturation alone gives only minor fluidizing effects. (4) Introduction of 30 mol% cholesterol to dimyristoylphosphatidylcholine membranes decreases the lateral diffusion constants of lipids by a factor of four, while it causes only a slight decrease of lateral diffusion in dioleoylphosphatidylcholine membranes. (5) If compared at the same temperature, 5-SASL mobilities plotted as a function of mole fraction of cholesterol in the fluid phases of dimyristoylphosphatidylcholine-, dipalmitoylphosphatidylcholine- and distearoylphosphatidylcholine-cholesterol membranes are similar in wide ranges of temperature (45-82 degrees C) and cholesterol mole fraction (0-50%). (6) In isothermal experiments with saturated phosphatidylcholine membranes, 5-SASL is maximally immobilized at the phase boundary between Regions I and III reported by other workers (Recktenwald, D.J. and McConnell, H.M. (1981) Biochemistry 20, 4505-4510) and becomes more mobile away from the boundary in Regions I and III. (7) 5-SASL in unsaturated phosphatidylcholine membranes showed a gradual monotonic immobilization with increase of cholesterol mole fraction without showing any maximum in the range of cholesterol fractions studied. (8) By rigorously determining rigid-limit magnetic parameters of cholestane spin labels in membranes from Q-band second-derivative ESR spectra to monitor the dielectric environment around the nitroxide radical, it is concluded that cholesterol incorporation increases water accessibility in the hydrophilic loci of the membrane. In contrast, 12-(9-anthroyloxy)stearic acid fluorescence showed that water accessibility is decreased in the hydrophobic loci of the membrane.     

Dynamic fluorescence quenching studies on lipid mobilities in phosphatidylcholine-cholesterol membranes

Bimolecular collision rate of 8-anilinonaphthalene-1-sulfonic acid (ANS) and the nitroxide doxyl group attached to various carbons on stearic acid spin labels (n-SASL) in phosphatidylcholine-cholesterol membranes in the fluid phase was studied by observing dynamic quenching of ANS fluorescence by n-SASL's. The excited-state lifetime of ANS and its reduction by the n-SASL doxyl group were directly measured by the time-correlated single photon counting technique to observe only dynamic quenching separately from static quenching and were analyzed by using Stern-Volmer relations. The collision rate of ANS with the n-SASL doxyl group ranges between 1 X 10(7) and 6 X 10(7), and the extent of dynamic quenching by n-SASL is in the order of 5-much much greater than 6- greater than 7- less than 9- less than 10- less than 12- less than 16-SASL (less than 5-SASL) in dimyristoylphosphatidylcholine (DMPC) membranes. Collision rate of 16-SASL is only 10% less than that of 5-SASL. Since the naphthalene ring of ANS is located in the near-surface region of the membrane, these results indicate that the methyl terminal of SASL appears in the near surface area frequently, probably due to extensive gauche-trans isomerism of the methylene chain. The presence of 30 mol% cholesterol decreases the collision rate of ANS with 12- and 16-SASL doxyl groups but not with the 5-SASL doxyl group in DMPC membranes. On the other hand, in egg-yolk phosphatidylcholine membranes, inclusion of 30 mol% cholesterol does not affect the collision of ANS with either 5-SASL or 16-SASL doxyl groups, in agreement with our previous observation that alkyl chain unsaturation moderates cholesterol effects on lipid motion in the membrane (Kusumi et al., Biochim. Biophys. Acta 854, 307-317). It is suggested that dynamic quenching of ANS fluorescence by lipid-type spin labels is a useful new monitor of membrane fluidity that reports on various lipid mobilities in the membrane; a class of motion can be preferentially observed over others by selecting a proper spin label, i.e., rotational diffusion of lipid about its long axis and translational diffusion by using 5-SASL, wobbling motion of the lipid long axis by using 7-SASL or androstane spin label, and gauche-trans isomerism by using 16-SASL.     

Characterization of Triosephosphate Isomerase Mutants with Reduced Enzyme Activity in Mus Musculus

Four heterozygous triosephosphate isomerase (TPI) mutants with approximately 50% reduced activity in blood compared to wild type were detected in offspring of 1-ethyl-1-nitrosourea treated male mice. Breeding experiments displayed an autosomal, dominant mode of inheritance for the mutations. All mutations were found to be homozygous lethal at an early postimplantation stage of embryonic development, probably due to a total lack of TPI activity and consequently to the inability to utilize glucose as a source of metabolic energy. Although activity alteration was also found in liver, lung, kidney, spleen, heart, brain and muscle the TPI deficiency in heterozygotes has no influence on the following physiological traits: hematological parameters, plasma glucose, glucose consumption of blood cells, body weight and organo-somatic indices of liver, spleen, heart, kidney and lung. Biochemical investigations of TPI in the four mutant lines indicated no difference of physicochemical properties compared to the wild type. Results from immunoinactivation assays indicate that the decrease of enzyme activity corresponds to a decrease in the level of an immunologically active moiety. It is suggested that the mutations have affected the Tpi-1 structural locus and resulted in alleles which produce no detectable enzyme activity and no immunologically cross-reacting material. The study furthermore suggests one functional TPI gene per haploid genome in the erythrocyte and seven other tested organs of the mouse.     

Asparagine-linked oligosaccharides containing poly-N-acetyllactosamine chains are preferentially bound by immobilized calf heart agglutinin

We have investigated the carbohydrate-binding specificity of a mammalian lectin, calf heart agglutinin, by determining the interaction of the immobilized lectin with a variety of complex-type Asn-linked oligosaccharides. Our results demonstrate that calf-heart agglutinin binds with high affinity to oligosaccharides containing the repeating disaccharide (3Gal beta 1-4GlcNAc beta 1)n or poly-N-acetyllactosamine sequence and that the presence of terminal beta-linked galactosyl residues is neither sufficient nor necessary for high affinity interactions.     

Defining null expectations for animal site fidelity

Site fidelity—the tendency to return to previously visited locations—is widespread across taxa. Returns may be driven by several mechanisms, including memory, habitat selection, or chance; however, pattern-based definitions group different generating mechanisms under the same label of ‘site fidelity’, often assuming memory as the main driver. We propose an operational definition of site fidelity as patterns of return that deviate from a null expectation derived from a memory-free movement model. First, using agent-based simulations, we show that without memory, intrinsic movement characteristics and extrinsic landscape characteristics are key determinants of return patterns and that even random movements may generate substantial probabilities of return. Second, we illustrate how to implement our framework empirically to establish ecologically meaningful, system-specific null expectations for site fidelity. Our approach provides a conceptual and operational framework to test hypotheses on site fidelity across systems and scales.     

Hereditary Lactate Dehydrogenase a-Subunit Deficiency as Cause of Early Postimplantation Death of Homozygotes in Mus Musculus

Two ethylnitrosourea-induced heterozygous mouse mutants with approximately 58 and 50% of wild-type lactate dehydrogenase (LDH) activity and a gamma-ray-induced heterozygous mutant with 50% of wild-type LDH activity in blood, liver and spleen (expressing predominantly the Ldh-1 gene) were recovered in mutagenicity experiments following spermatogonial treatment. Physiological and genetic studies revealed no indications for differences in fertility as well as hematological or other physiological traits between heterozygotes of each mutant line and wild types. This suggests that neither the mutations in the heterozygous state per se nor the resulting approximate 42 to 50% LDH deficiency affect metabolism and fitness. Physicochemical and immunological studies clearly demonstrated that the two mutations with 50% deficiency in heterozygotes result from null alleles of the Ldh-1 structural locus, generating neither enzyme activity nor immunological cross-reacting material. In contrast, the heterozygous mutant with approximately 58% of normal blood LDH activity was shown to be due to a Ldh-1 allele creating protein subunits, which in random assortment with wild-type subunits in vivo exhibit a reduced specific activity and further alterations of kinetic and physicochemical characteristics. All the mutations in the homozygous state were found to be lethal at an early postimplantation stage of embryonic development, probably due to a block of glycolysis with the corresponding loss of the main source of metabolic energy during this ontogenetic stage. The distinct physiological consequences of the total absence of a functioning LDH-A subunit in mice and humans are discussed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gonadal hormones stimulate ultrasound production by female hamsters.

Two studies were conducted to examine the role of sex hormones in ultrasound production by female hamsters. Ovariectomized hamsters were treated with estradiol benzoate (EB), testosterone propionate (TP), progesterone (P), estradiol plus progesterone (EB + P), testosterone plus progesterone (TP+P), or oil vehicle. Rates of ultrasound production by these females were observed in response to brief male-female contact, and during exposure to synthetic ultrasounds. Maximal rates of ultrasound production required EB + P or TP+P replacement therapy. Intermediate call rates were stimulated by EB and TP individually, whereas P alone had no significant effect. These results support the hypothesis that ovarian estrogens and progesterone control proceptive as well as receptive behaviors in female hamsters.