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.     

Differentiation of Langerhans Cells from Interdigitating Cells Using CD1a and S-100 Protein Antibodies

The present study shows that Langerhans cells can be differentiated from Interdigitating cells at the light microscopic level. Superficial lymph nodes and skin taken from necropsies and the lymph nodes of dermatopathic lymphadenopathy (DPL) were used for this experiment. Sections of lymph node and skin were embedded using the acetone, methyl benzoate and xylene (AMeX) method and dendritic cells were immunostained with anti S-100 protein antibody (S-100, and OKT-6 (CD1a) using the restaining method. Langerhans cells in the skin were positive for both CD1a and S-100. Dendritic cells positive for both CD1a and S-100, and dendritic cells positive for S-100, but not for CD1a were observed in superficial lymph nodes. In normal superficial lymph nodes, there were more interdigitating cells than Langerhans cells. The majority of the dendritic cells in the DPL were Langerhans cells. We conclude that the S-100 and CD1a positive cells are Langerhans cells, and the S-100 positive-CD1a negative cells are interdigitating cells.     

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.     

Taq I-generated HLA-DQ αpolymorphism in Japanese patients with narcolepsy

Taq I-generated HLA-DQrestriction fragment length polymorphism was examined in Japanese patients with narcolepsy. All patients were DR2 positive and shared a 6.0 kb fragment, although this fragment was found only in 54 % of the healthy DR2-positive Japanese. This finding added the DQ gene to the list of candidates for the possible narcolepsy-susceptibility gene. In contrast, there was no complete association between narcolepsy and DXrestriction fragment length polymorphism. These findings suggest that a narcolepsy-susceptibility gene is located closer to the DQ locus than to the DX locus.     

Effect of various factors on serotonin-induced Ca2+ response in human platelets

Serotonin (5-HT)-stimulated intracellular Ca2+ concentration change was studied in the platelets of healthy subjects, using fluorescent Ca indicator fura-2. 5-HT increased the Ca2+ response in a concentration-dependent manner. 10 microM of 5-HT induced the maximal response and its EC50 value was 0.4 microM. This response was potently inhibited by selective 5-HT2 antagonists, suggesting that 5-HT-induced Ca2+ mobilization in human platelets is mediated by 5-HT2 receptors. This 5-HT2-mediated Ca2+ response was not significantly affected by the time of blood sampling, gender, meal or exercise. However, this response declined with time after blood drawing, suggesting that it must be measured as soon as possible after sampling. These results indicate that 5-HT-stimulated Ca2+ response in human platelets is a stable parameter and that it will be suitable for assessing 5-HT2 receptor function in depressed patients.     

Genetic mapping of a gene causing hypertension in the stroke-prone spontaneously hypertensive rat.

The stroke-prone spontaneously hypertensive rat (SHRSP) is a well-characterized model for primary hypertension in humans. High blood pressure in SHRSP shows polygenic inheritance, but none of the loci responsible have previously been identified. To locate genes controlling this quantitative trait, we mapped a large collection of DNA polymorphisms in a cross between SHRSP and the normotensive WKY strain. Here we report strong genetic evidence that a gene, Bp1, having a major effect on blood pressure maps to rat chromosome 10 with a LOD score of 5.10 and is closely linked to the rat gene encoding angiotensin-converting enzyme (ACE), an enzyme that plays a major role in blood pressure homeostasis and is an important target of anti-hypertensive drugs. We also find significant, albeit weaker, linkage to a locus, Bp2, on chromosome 18. We discuss the implications of genetic dissection of quantitative disease-related phenotypes in mammals.     

Interaction of glycylglycine and Na+ at the mucosal border of Guinea-pig small intestine: A non-mutual stimulation of transport

Sodium-dependence of glycylglycine (Gly-Gly) influx and stimulation of Na⁺ transport by Gly-Gly were studied in everted sacs, sheet preparations and brush-border membrane vesicles isolated from guinea-pig ileum. Gly-Gly influx was found to be independent of the presence of Na⁺, while Na⁺ transport was stimulated by Gly-Gly as evidenced by increases in transmural potential difference (PD_t), short-circuit current (I_sc) and Na⁺ influx. The change in PD_t (ΔPD_t) induced by Gly-Gly was a saturable function of Gly-Gly concentration, showing a Michaelis-Menten type relationship. The half-saturation concentration for Gly-Gly estimated from the electrical data was nearly identical with that estimated from influx data. At a constant Gly-Gly concentration the relationship between I_sc and Na⁺ concentration was sigmoid, and the Hill coefficient was 1.5. Kinetic analysis according to Garay Garrahan indicates that each Gly-Gly carrier has two equivalent non-interacting binding sites for Na⁺, and that translocation of Na⁺ occurs when the two Na⁺ sites on the carrier loaded with Gly-Gly are occupied by Na⁺. However, our results indicate that the resultant Na⁺ flow is not capable of stimulating Gly-Gly translocation.     

3-Aminobenzamide,a potent inhibitor of poly (ADP-ribose) polymerase,causes a rapid death of HL-60 cells cultured in serum-free medium

HL-60 cells transferred from serum-supplemented to serum-free culture medium initially bound to culture plate tightly and then released from the plate on increasing the culture time and resumed exponential growth after about 8 h lag. At the initial stage of the culture, the cells became extremely sensitive to 3-aminobenzamide, a potent inhibitor of poly (ADP-ribose) polymerase, and, at 1 mM, 80 to 90% of the cells were lysed within 20 h, whereas the inhibitor was totally ineffective on the cell growth in serum-supplemented medium at the concentration. Non-inhibitory analogs of the inhibitor were ineffective. Assay of poly(ADP-ribose) polymerase activity in permeable cells indicated that a transient activation of the enzyme occurred during the culture in serum-free medium (the maximum activation was observed at 8 h of the culture). The cells conditioned in serum-free medium for 24 h acquired significant resistancy to the inhibitor. A low concentration of fibronectin (5 to 10/ml) and a relatively high concentration of bovine serum albumin (0.5 to 1 mg/ml) effectively blocked the cell attachment to plate and also the 3-aminobenzamide-induced cell lysis. These results suggest that poly(ADP-ribose) polymerase is involved in a process essential for HL-60 cells to adapt to a serumdeprived growth condition.