Assessing hydrophobic regions of the plasma membrane H(+)-ATPase from Saccharomyces cerevisiae. The hydrophobic, photoactivatable probe TID [3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine] was used to label the plasma membrane H(+)-ATPase from Saccharomyces cerevisiae. The H(+)-ATPase accounted for 43% of the total label associated with plasma membrane protein and incorporated 0.3 mol of [125I]TID per mol of 100 kDa polypeptide. The H(+)-ATPase was purified by octyl glucoside extraction and glycerol gradient centrifugation, and was cleaved by either cyanogen bromide digestion or limited tryptic proteolysis to isolate labeled fragments. Cyanogen bromide digestion resulted in numerous labeled fragments of mass less than 21 kDa. Seven fragments suitable for microsequence analysis were obtained by electrotransfer to poly(vinylidene difluoride) membranes. Five different regions of amino-acid sequence were identified, including fragments predicted to encompass both membrane-spanning and cytoplasmic protein structure domains. Most of the labeling of the cytoplasmic domain was concentrated in a region comprising amino acids 347 to 529. This catalytic region contains the site of phosphorylation and was previously suggested to be hydrophobic in character (Goffeau, A. and De Meis, L. (1990) J. Biol. 265, 15503-15505). Complementary labeling information was obtained from an analysis of limited tryptic fragments enriched for hydrophobic character. Six principal labeled fragments, of 29.6, 20.6, 16, 13.1, 11.4 and 9.7 kDa, were obtained. These fragments were found to comprise most of the putative transmembrane region and a portion of the cytoplasmic region that overlapped with the highly labeled active site-containing cyanogen bromide fragment. Overall, the extensive labeling of protein structure domains known to lie outside the bilayer suggests that [125I]TID labeling patterns cannot be unambiguously interpreted for the purpose of discerning membrane-embedded protein structure domains. It is proposed that caution should be applied in the interpretation of [125I]TID labeling patterns of the yeast plasma membrane H(+)-ATPase and that new and diverse approaches should be developed to provide a more definitive topology model.


Several nongenotoxic carcinogens uncouple mitochondrial oxidative phosphorylation. A number of plasticizers and lipid-lowering drugs induce peroxisomes and cause hepatocellular carcinoma in rodents by mechanisms which remain unknown. In this study, seven structurally dissimilar peroxisome proliferating agents were shown to uncouple oxidative phosphorylation in isolated rat liver mitochondria. For example, perfluorooctanoate (0.5 mM) increased succinate-induced (state 4) mitochondrial respiration by over 50% while stimulation of state 3 respiration with ADP was minimal (i.e., uncoupling occurred). Interestingly, compounds which are potent carcinogens in vivo (e.g., Wy-14,643 and perfluorooctanoate) were more powerful uncouplers of oxidative phosphorylation in vitro than weak tumor-causing agents (e.g., valproate). Uncoupling also occurred in vivo. Basal rates of oxygen uptake in perfused livers from chronically treated rats were increased from 137 +/- 7 mumol g-1/h in pair-fed controls to 153 +/- 5 mumol g-1/h after 2.5 months of feeding Wy-14,643 (0.1% w/v in diet). Concomitantly, rates of urea synthesis from ammonia, a process highly dependent on ATP supply, were reduced almost completely from 104 +/- 10 mumol g-1/h to 13 +/- 6 mumol g-1/h. Bile flow, another energy-dependent process, was also reduced significantly by treatment with Wy-14,643 in vivo for 24 h. Taken together, these data indicate that energy supply for cellular processes such as urea synthesis and bile flow was disrupted in vivo due to uncoupling of oxidative phosphorylation by Wy-14,643. It is proposed that peroxisomal proliferators accumulate in the liver where they uncouple mitochondrial oxidative phosphorylation and interfere with cellular energetics.


Reactivity patterns for redox reactions of monomer forms of myoglobin, hemocyanin and hemerythrin. Electron-transfer reactions of myoglobin, hemocyanin and hemerythrin with the inorganic complexes [Fe(CN)6]3- (oxidant) and [Co(sep)]2+ (reductant) are considered. Rate constants kFe (25 degrees C) have been determined for the [Fe(CN)6]3- (410 mV) oxidation of horse deoxyMb, I = 0.100 M (NaCl). From the decrease in kFe over the range pH 5.5 to 9.0 a pKa of less than 6.2 is obtained, most likely due to the involvement of the heme propionate(s). At the higher pH values the rate constant is 1.2 x 10(6) M-1 s-1. Rate constants kCo (25 degrees C) for the [Co(sep)]2+ (-260 mV) reduction of metMb are also pH-dependent, pKa = 8.82, corresponding to acid dissociation of the H2O axially coordinated to the Fe(III). The rate constant for the aqua-met form is 2.8 x 10(3) M-1 s-1 at pH values less than 7.0. In contrast, no reaction is observed for the deoxy and met forms of P. interruptus hemocyanin monomer subunit a with the same two complexes (k less than 10(2) M-1 s-1). Comparisons are made with rate constants for hemerythrin, also as the monomer, which have been determined previously. Rate constants for the reactions of deoxy forms with the neutral small molecules, here O2 and H2O2, are also considered. Whereas the reactions of [Fe(CN)6]3- and [Co(sep)]2+ are at the protein surface, those of O2 and H2O2 are at the active site. Hemocyanin with the more buried (approximately 20 A) active site compared with myoglobin (3.8 A) and hemerythrin (6.3 A), does not readily undergo electron transfer with reagents at the surface. However, with the small molecules O2 and H2O2 penetration of the surrounding peptide occurs, with reaction at the active site. Rate constants for the three proteins are now of similar magnitude, and in the range (2.3-7.8) x 10(7) M-1 s-1 for O2, and 10.9 to 3600 M-1 s-1 for H2O2.


Kinetics of transport systems dependent on periplasmic binding proteins. Rate equations are derived for a transport model involving a water-soluble binding protein outside the plasma membrane. On addition of the substrate, the conformation of the binding protein changes; the complex then combines with the membrane carrier, transferring the substrate to the carrier site. The free binding protein leaves and the carrier shifts inward, releasing the substrate inside the cell. Exit follows the reverse path. The predicted behaviour is as follows. (i) Uptake does not necessarily conform to Michaelis-Menten kinetics. (ii) In both the energized and de-energized states, the maximum rate of exit is far lower than that of entry; the asymmetry is determined by the conformational change in the binding protein, which is independent of the energy state of the system. (iii) Exchange transport is inhibited by external substrate and is extremely slow; consequently counter-transport is not expected. (iv) The half-saturation constant in uptake can differ from the dissociation constant of the binding protein. (v) The maximum rate of uptake depends on the intrinsic substrate affinity of the membrane carrier relative to that of the binding protein. (vi) The maximum rate of uptake and the substrate half-saturation constant depend on the concentration of the binding protein.


Phloretin keto-enol tautomerism and inhibition of glucose transport in human erythrocytes (including effects of phloretin on anion transport). Under various pH conditions phloretin demonstrates keto-enol tautomerism with a pK value of 7.26 +/- 0.06. As Wilbrandt has shown ((1950) Arch. Exp. Pathol. Pharmacol. 212, 9-29) phloretin added to erythrocytes inhibits glucose efflux, but not glucose influx. At pH 6.5 a Ki value of 0.36 and at pH 9 of 22.7 microM was measured; only the ketonic form of phloretin contributes to the inhibition of glucose efflux. This was also the case for inhibition of galactose efflux and anion exchange. The geometry optimization of a large number of conformations of the ketonic and enolic forms of phloretin demonstrates different shapes of the molecules. Only the ketonic form shows several overlapping structures with beta-D-glucopyranose. Considering surplus binding of phloretin under glucose efflux conditions as being equivalent to the number of glucose transporters, a number of about 200,000 molecules was determined. By two independent methods 210,000 and 171,000 molecules per cell were calculated. This result is in close agreement with the number of glucose transporter sites of the erythrocyte.


Testing models for transport systems dependent on periplasmic binding proteins. A carrier model in which transport across the cytoplasmic membrane is mediated by a periplasmic binding protein (Krupka, R.M. (1992) Biochim. Biophys. Acta 1110, 1-10) is shown to account for many of the properties of these systems: (i) Michaelis-Menten kinetics; (ii) seemingly irreversible uptake; (iii) the absence of exchange transport and counter-transport; (iv) substrate half-saturation constants that in different systems may be lower or higher than the dissociation constant of the binding protein; (v) the high concentration of the binding protein in the periplasm and its weak association with the membrane component. The binding protein appears to function as a valve or rectifier that permits the substrate to enter the cell, but blocks exit in both the energized and de-energized states. The asymmetry depends on both the abruptness and the extent of the conformational change in the binding protein. Characteristically, these systems build up steep gradients across the membrane, circumstances in which such a valve might be important. In agreement with the mechanism, (a) the binding protein is missing in members of the same family of transporters that function in export of the substrate rather than import; and (b) in Gram-positive organisms, which have no periplasmic space, binding proteins function while anchored to the cytoplasmic membrane.


Reaction mechanism and asymmetric orientation of the reconstituted chloroplast phosphate translocator. Proteoliposomes loaded with varying levels of internal substrates were used in bisubstrate initial velocity studies to gain insight into the transport mechanism of the reconstituted chloroplast phosphate translocator. The kinetic response to trans substrates clearly indicated that the one-to-one exchange mediated by this translocator proceeds via a ping-pong type, and excluded a sequential type of reaction mechanism. It is also shown that reconstitution of the protein leads to an unidirectional orientation of the protein within the liposomes being orientated right-side-out with respect to chloroplasts. Different transport affinities were observed on either side of the membrane and only the outward-facing transport site of the translocator is able to bind inhibitors i.e. pyridoxal 5'-phosphate (PLP) and 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS).


Hexose-specific inhibition in vitro of human red cell Ca(2+)-ATPase activity. In a concentration-dependent manner (5.5-27.5 mmol/l), D-glucose incubated in vitro with human erythrocyte membranes at 37 degrees C for 1 h inhibited membrane Ca(2+)-ATPase activity by up to 75%. The IC50 was 11 mmol/l. L-Glucose was ineffective, as were 3-O-methylglucose, 2-deoxyglucose, sorbitol and myo-inositol. In contrast, D-fructose decreased Ca(2+)-ATPase activity nearly as effectively as D-glucose and mannose and galactose at 11 mmol/l were less than 50% as effective as D-glucose. Tunicamycin (12 pmol/l), but not 10 mmol/l aminoguanidine, progressively antagonized in vitro the D-glucose effect on the enzyme. Erythrocyte membrane Ca(2+)-ATPase activity may be regulated by glycosylation, rather than nonenzymatic glycation.


Effects of poly(L-lysine) on the structural and thermotropic properties of dipalmitoylphosphatidylglycerol bilayers. The effects of poly(L-lysine) on the structural and thermotropic properties of dipalmitoylphosphatidylglycerol (DPPG) bilayers were studied with differential scanning calorimetry (DSC), X-ray diffraction and freeze-fracture electron microscopy. For thermal behavior, in the DPPG/poly(L-lysine) system the main transition temperature rises to 45.7 degrees C and the pretransition disappears in opposition to pure DPPG vesicles. An additional transition appears approximately at 36 degrees C for the DPPG/poly(L-lysine) system after incubation at 4 degrees C for two months. The incubated sample gives a X-ray diffraction pattern having several additional reflections in the range of 0.2-0.9 nm at 15 degrees C. These results suggest that even in the presence of poly(L-lysine) the DPPG bilayers form the subgel (Lc) phase after the long incubation at a low temperature. The X-ray diffraction measurements indicate that the structure of the Lc phase for DPPG/poly(L-lysine) system is different from that of pure DPPG bilayers. On the other hand, in the gel (L beta') phase, the wide-angle X-ray diffraction pattern suggests that the presence of poly(L-lysine) hardly affects the packing of hydrocarbon chains in the DPPG bilayers. The small-angle X-ray diffraction and freeze-fracture electron microscopy exhibit that the DPPG/poly(L-lysine) system forms a tightly packed multilamellar structure in which the poly(L-lysine) is intercalated between the subsequent DPPG bilayers.


Monensin intercalation in liposomes: effect on cytotoxicities of ricin, Pseudomonas exotoxin A and diphtheria toxin in CHO cells. Monensin, a carboxylic ionophore was intercalated in liposomes (liposomal monensin) and its effect on cytotoxicities of ricin, Pseudomonas exotoxin A and diphtheria toxin in CHO cells was studied. Intercalation of monensin in liposomal bilayer is found to have no effect on its stability and interaction with cells. Liposomal monensin (1 nM) substantially enhance the cytotoxicities of ricin (62-fold) and Pseudomonas exotoxin A (11.5-fold) while it has no effect on diphtheria toxin. This observed effect is highly dependent on the liposomal lipid composition. The potentiating ability of monensin (1 nM) in neutral vesicles is significantly higher (2.2-fold) as compared to negatively charges vesicles. This ability is drastically reduced by incorporation of stearylamine in liposomes and is found to be dependent on the density of stearylamine as well as on the concentration of serum in the medium. Monensin in liposomes containing 24 mol% stearylamine has a very marginal effect on the cytotoxicity of ricin (7.5-fold) which is further reduced (1.5-fold) in the presence of 20% serum. The uptake of 125I-gelonin from neutral vesicles is significantly higher (approximately 2.0-fold) than that from the negative vesicles. The uptake from positive vesicles is highly dependent on the concentration of stearylamine. The reduction in the lag period (30 min) of ricin action by monensin in neutral and negative vesicle is comparable with free monensin. However, monensin in positive vesicle has no effect on it. These studies have suggested that liposomes could be used as a delivery vehicle for monensin for selective elimination of tumor cells in combination with hybrid toxins.


Lipid mixing is mediated by the hydrophobic surfactant protein SP-B but not by SP-C. Pulmonary surfactant contains two families of hydrophobic proteins, SP-B and SP-C. Both proteins are thought to promote the formation of the phospholipid monolayer at the air/fluid interface of the lung. The excimer/monomer ratio of pyrene-labeled PC fluorescence intensities was used to investigate the capacity of the hydrophobic surfactant proteins, SP-B and SP-C, to induce lipid mixing between protein-containing small unilamellar vesicles and pyrene-PC-labeled small unilamellar vesicles. At 37 degrees C SP-B induced lipid mixing between protein-containing vesicles and pyrene-PC-labeled vesicles. In the presence of negatively charged phospholipids (PG or PI) the SP-B-induced lipid mixing was enhanced, and dependent on the presence of (divalent) cations. The extent of lipid mixing was maximal at a protein concentration of 0.2 mol%. SP-C was not capable of inducing lipid mixing at 37 degrees C not even at protein concentrations of 1 mol%. The SP-B-induced lipid mixing may occur during the Ca(2+)-dependent transformation of lamellar bodies into tubular myelin and the subsequent formation of the phospholipid monolayer.


Nucleoside transport-deficient mutants of PK-15 pig kidney cell line. Previous studies indicated that PK-15 pig kidney cells express solely a nitrobenzylthioinosine-sensitive, equilibrative nucleoside transporter. In the present study, PK-15 cells were mutagenized by treatment with ICR-170 and nucleoside transport-deficient mutants selected in a single step in growth medium containing tubercidin and cytosine arabinoside at a frequency of about 2 x 10(6). The mutants were simultaneously at least 100-times more resistant to tubercidin, cytosine arabinoside and 5-fluorodeoxyuridine than the wild-type parent cells. The mutants failed to transport thymidine and uridine and had lost all high affinity nitrobenzylthioinosine binding sites. Residual low level uptake of thymidine by the mutants was shown to be due to nonmediated permeation (passive diffusion), which explains the sensitivity of the mutants to growth inhibition by high concentrations of the nucleoside drugs. Passive diffusion of thymidine at a concentration of 16 microM was not rapid enough to support the growth of nucleoside transport-deficient mutant cells that had been made thymidine-dependent by treatment with methotrexate, whereas wild-type cells grew normally under these conditions. The nucleoside transport-deficient mutants exhibited about the same growth rate and plating efficiency (60-80%) as wild-type cells, but formed larger colonies than wild-type cells because of a more extensive spread of the cells on the surface of culture dishes. PK-15 cells adhere very strongly to the surface of culture dishes and have been transformed with high efficiency with plasmid DNA either via lipofection or electroporation.


Enrichment of saturated fatty acid containing phospholipids in sheep brain serotonin receptor preparations: use of microwave irradiation for rapid transesterification of phospholipids. During enrichment of the 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT)-binding serotonin 5-HT1A receptors from sheep brain gray matter (membrane isolation, detergent solubilization and reconstitution into vesicles) a consistent and striking increase in the composition of saturated fatty acids was observed in phospholipids which were coisolated with the receptors. A rapid procedure has been developed for the methylation of free and phospholipid linked fatty acids which were thus analyzed by gas chromatography-mass spectrometry (GC/MS). Esterification of free fatty acids and transesterification of phospholipid linked fatty acids were achieved with 14% boron trifluoride in methanol (BF3-CH3OH) in 20 s and 50 s, respectively, under low power microwave irradiation (60 W) with a post-reaction cooling of less than 5 min. This is in contrast to the conventional method of heating in a boiling water bath for 10-15 min with BF3-CH3OH which is inevitably preceded by time-consuming and inconvenient clamping of vials and followed by cooling for 10 min before the vials can be safely opened. Analysis of fatty acid profiles in phosphatidylethanolamine (PE) and phosphatidylcholine (PC) from egg yolk, phosphatidylinositol (PI) from bovine liver and phosphatidylserine (PS) from bovine brain by both techniques showed comparable results. During detergent solubilization of sheep brain gray matter, the overall proportion of saturated fatty acids in PE (major lipid), PI, PC (major lipid) and PS increased from 50-60% in sheep brain phospholipids to 70-75% in 1.5% CHAPS solubilized, reconstituted and biologically active serotonin 5-HT1A preparations. In sharp contrast, the proportions of saturated fatty acids in 1.5% Triton X-100 solubilized PE (48.1%) (major lipid), PI (63.6%), PC (60.6%) (major lipid) and PS (62.2%) were not significantly different from those in the original sheep brain membranes. Strikingly, this was coupled with the occurrence of very low levels of 5-HT1A receptor activity in the Triton X-100 solubilized preparations. The abundance of 5-HT1A sites in the enriched vesicles obtained only from the CHAPS-solubilized preparations was further confirmed by specific radiolabeling of a 58-kDa polypeptide by the 5-HT1A specific ligand p-aminophenylethyl-m-trifluoromethylphenylpiparazine (PAPP) which was coupled to a 125I-labeled, photoreactive, heterobifunctional cross-linker, sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3'-dithiopropiona te (SASD). Thus CHAPS-solubilized 5-HT1A receptor preparations are depleted in the more rigid lipids such as sphingolipids and cholesterol, (Banerjee et al. (1990) Biochim. Biophys. Acta 1044, 305-314), but are enriched in vesicle-stabilizing, phospholipid-linked saturated fatty acids which in turn probably stabilize the heptahelical, membrane bound 5-HT1A receptor.


Inhibition of anion transport in the human red blood cell membrane with para- and meta-methoxyphenylglyoxal. The positional isomers para-methoxyphenylglyoxal and meta-methoxyphenylglyoxal were newly synthesized and found to be potent inhibitors of sulfate exchange in the red blood cell membrane. The rate of inactivation of the transport system with both reagents obeys pseudo-first-order kinetics and increases with increasing pH and reagent concentration. The degree of inhibition of the transport system with the meta-isomer exceeds the inhibition caused by the para-isomer. At 2 mM 3-methoxyphenylglyoxal (3-MOPG) and 37 degrees C the half-lifetime of the anion transporter is 5.4 min at pH 8.0. Under the same experimental conditions the half-lifetime of the transporter at 2 mM 4-methoxyphenylglyoxal (4-MOPG) is found to be 24.7 min. The binding site of these reagents is found to be the same as binding site of the reversibly acting phenylglyoxal derivative 4-hydroxy-3-nitrophenylglyoxal (HNPG). Chloride ions are able to protect the transporter against inhibition with both reagents. Anion transport inhibitors like 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) and flufenemate, which are known to act on band 3 protein, are able to interact with the binding of the newly synthesised reagents. Phloretin and phloridzin show no interaction.


Liposome shrinkage and swelling under osmotic-diffusional stress: evaluation of kinetic parameters from spectrophotometric measurements. Analyses of spectrophotometric measurements of shrinkage and swelling of liposomes under osmotic-diffusional stress tacitly assume an 'empirical' inverse relationship between the absorbance (A) and the liposomal volume (v). In this paper, we have proposed an alternate more explicit relationship: A = EcL e - alpha Lv/1-Lv where L is liposomal concentration, Ec the extinction coefficient and alpha a dimensionless parameter. The exponential term, in essence, defines the partitioning of aqueous volume into intra- and extra-liposomal compartments. Experimental data obtained with glycerol as model compound are used to test the validity and internal consistency of the proposed formalism.


Kinetics of melittin induced pore formation in the membrane of lipid vesicles. We have investigated the permeabilization of POPC unilamellar vesicle bilayers upon the addition of melittin. This process was measured in an early time range of a few minutes by means of monitoring the release of an entrapped marker, the self-quenching fluorescent dye carboxyfluorescein. Pore formation is indicated by an apparent 'all-or-none' efflux out of individual vesicles and a higher than linear dependence on melittin concentration. Applying a recently developed evaluation procedure, the data are readily converted into the gross number of pores per vesicle formed within the elapsed measuring time t. The results can be generally described in terms of a fast initial rate of pore formation that slows down to a much lower value after a period of about 1 to 2 minutes, following a single exponential time course. The three rate parameters involved are shown to be power functions of the concentration of melittin that is actually associated with the vesicle membrane. These findings are in excellent quantitative agreement with a proposed scheme of reaction steps where the formation of lipid associated peptide dimers becomes rate determining once an initial fast deposit is exhausted.


Dual specificity of sterol-mediated glycoalkaloid induced membrane disruption. In this study the effects of the glycoalkaloids alpha-solanine, alpha-chaconine, alpha-tomatine and the aglycone solanidine on model membranes composed of PC in the absence and presence of sterols have been analysed via permeability measurements and different biophysical methods. The main result is that glycoalkaloids are able to interact strongly with sterol containing membranes thereby causing membrane disruption in a way which is specific for the type of glycoalkaloid and sterol. For this dual specificity both the sugar moiety of the glycoalkaloid and the side-chain of the sterol on position 24 turned out to be of major importance for the membrane disrupting activity. The order of potency of the glycoalkaloids was alpha-tomatine > alpha-chaconine > alpha-solanine. The plant sterols beta-sitosterol and fucosterol showed higher affinity for glycoalkaloids as compared to cholesterol and ergosterol. The mode of action of the glycoalkaloids is proposed to consist of three main steps: (1) Insertion of the aglycone part in the bilayer. (2) Complex formation of the glycoalkaloid with the sterols present. (3) Rearrangement of the membrane caused by the formation of a network of sterol-glycoalkaloid complexes resulting in a transient disruption of the bilayer during which leakage occurs.


Separation and inhibitor specificity of a second unidirectional efflux route for methotrexate in L1210 cells. L1210 cells mediate the unidirectional and energy-dependent efflux of methotrexate. Efflux occurs primarily via a system which has a high sensitivity to prostaglandin A1, vincristine, reserpine, verapamil, and bromosulfophthalein, but evidence has also been obtained for a second efflux component with a lower response to these inhibitors. Pretreatment of L1210 cells with low concentrations of vincristine reduces methotrexate efflux by three fold and uncovers a second efflux component with an inhibitor specificity which is distinctly different from the primary efflux route. Vincristine treatment increased by 8-20-fold the concentration required for half-maximal efflux inhibition by prostaglandin A1, reserpine, bromosulfophthalein, and verapamil but had no effect on inhibition by probenecid, quinidine, or carbonylcyanide m-chlorophenylhydrazone. A selective block in the primary efflux system and retention of the second component was also achieved in cells exposed to low concentrations of prostaglandin A1 or bromosulfophthalein. These results support prior conclusions that L1210 cells contain both a primary and secondary unidirectional efflux route for methotrexate. The second system has been difficult to detect and quantitate since it comprises only 25% of total unidirectional efflux and shows a relatively low response to various efflux inhibitors.


Labelling of liposomes with intercalating perylene fluorescent dyes. The high fluorescent potential and the exceptional photostability of lipophilic derivatives of perylene-3,4:9,10-bis(dicarboximides) are utilized for the fluorescence-labelling of liposomes. The preparation of the liposomes is effected by supersonic starting from a lipid mixture consisting of the matrix lipids soy lecithin, cholesterol, alpha-tocopherol and the perylene dyes. From a multitude of perylene derivatives investigated only those are optimally incorporated into the bilayer membrane of unilamellar liposomes which are substituted at both nitrogen atoms by one or two linear hydrocarbon groups. In order to attain an optimal fluorescent quantum yield, about 200 to 300 dye molecules can be incorporated per liposome. The liposomes thus obtained have a diameter of about 70 to 80 nm, are homogeneous and may be stored for more than seven months. Neither the fluorescent properties nor the stability of these liposomes are influenced by the additional incorporation of various ara C-derivatives and lipophilic anchor groups which subsequently enable the coupling of antibodies to the liposomes. As the water-insoluble perylene dyes are incorporated into the bilayer membrane, the aqueous inner volume of the liposomes remains available for a further utilization.


Dextran sulfate inhibits fusion of influenza virus and cells expressing influenza hemagglutinin with red blood cells. The influence of dextran sulfate with molecular weights of 500,000 and 8000 on binding and fusion of influenza virus (X31 strain) and of cells expressing influenza hemagglutinin (GP4F) with red blood cells (RBC) was investigated by spectrofluorimetry using virus and RBC labeled with the fluorescent dye octadecyl rhodamine B (R18). There was no significant inhibition of binding of virus and GP4F cells to red blood cells by dextran sulfate, but the polymer strongly inhibited the low pH induced fusion. Virus-RBC fusion was completely blocked by the high molecular weight dextran sulfate at concentrations as low as 0.5 mg/ml. Inhibition of RBC-GP4F cell fusion by dextran sulfate in the same concentration range was not as pronounced but the effect was potentiated by Ca2+. The polymer was only inhibitory when added at early steps of the fusion reaction, but the pH-induced conformational change of the hemagglutinin was not affected by dextran sulfate as measured by its susceptibility to proteolytic digestion. Removal of dextran sulfate after low pH-requiring steps allowed the system to fuse at neutral pH indicating that the inhibitory effect requires the continuous presence of dextran sulfate during the fusion reaction.


Characterization of the TRH-induced activation of Na+/H(+)-exchange in pituitary GH4C1 cells. In the present study in GH4C1 cells, the dependence of TRH-induced activation of Na+/H(+)-exchange on extracellular Na+ and Ca2+ was examined. Furthermore, the effects of both extracellular and intracellular H+ on Na+/H(+)-exchange were investigated. The buffering capacity was 63 +/- 11.8 mM (pH unit)-1 at basal intracellular pH (pHi) of 7.02 +/- 0.02. The initial rate of alkalinization in cells acidified with nigericin increased with increasing concentrations of extracellular Na+ according to simple Michaelis-Menten kinetics. The apparent Km-value for Na+ was 53 +/- 17.5 mM and the Vmax value was 28 +/- 4.5 mM H+/min. Addition of Na+ together with TRH increased Vmax to 56 +/- 6.4 mM H+/min (P < 0.05), while no difference was observed in Km. Decreasing extracellular pH (pHo) decreased the rate of alkalinization of acid-loaded cells, despite a large inward Na+ gradient. Furthermore, a decrease in pHi was necessary to obtain activation of Na+/H+ exchange. At pHi-values close to basal pHi no activation of Na+/H(+)-exchange was obtained. In addition, the results showed that extracellular Ca2+ was necessary for TRH-induced activation of Na+/H+ exchange. Blocking influx of extracellular Ca2+ with Ni2+ abolished the effect of TRH, suggesting that the TRH-induced activation of Na+/H(+)-exchange in GH4C1 cells is dependent on influx of extracellular Ca2+.


Archaebacterial lipid models: highly salt-tolerant membranes from 1,2-diphytanylglycero-3-phosphocholine. 1,2-Di(3RS,7R,11R-phytanyl)-sn-glycero-3-phosphocholine and its glycerol epimers were synthesized as model lipids of archaebacterial halophiles. These amphiphiles, upon sonication of aqueous suspensions, gave rise to small unilamellar vesicles (SUV) of 300-800 A in diameter and about 80 A in the membrane thickness. The liposomes were very stable for at least a month even in a highly concentrated suspension or 5 M aqueous NaCl. The vesicles could retain Na+ and Cl- ions as well as 5(6)-carboxyfluorescein in the aqueous interior at temperature as high as 70 degrees C. The liposomes of ordinary diester lipids such as 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and egg-yolk lecithin were less stable and more permeable than those of the diphytanyl lipids.


Nuclear magnetic relaxation dispersion and 31P-NMR studies of the effect of covalent modification of membrane surfaces with poly(ethylene glycol). Covalent attachment of methoxypoly(ethylene glycol) (MPEG) 5000 to the surface of unilamellar liposomes composed of egg phosphatidylcholine and dioleoylphosphatidylethanolamine (DOPE) (8:2) containing paramagnetic chelates, either entrapped within the interior volume of the liposomes, or associated with the membrane surface, had no effect upon the measured spin-lattice relaxation rates (1/T1) for water in these systems. 31P-NMR studies indicate no destabilization of dioleoylphosphatidylcholine (DOPC)/(DOPE) (1:1) vesicles following attachment of MPEG. However, in DOPC/DOPE (1:3) mixtures, covalent modification with MPEG results in a destabilization of multilamellar vesicles into smaller vesicular structures. These results indicate that covalent attachment of poly(ethylene glycol) to liposomal magnetic resonance agents may prove a useful method for increasing their utility as vascular MR agents by extending their lifetime in the circulation, without decreasing the relaxivity of paramagnetic species associated with the liposome, but that the presence of PEG covalently attached to the membrane surface may modify the polymorphic phase behavior of the lipid system to which it is covalently linked.


Activation of the Na+/K+/Cl- cotransport system by reorganization of the actin filaments in Ehrlich ascites tumor cells. Reorganization (disassembly) of the actin filaments in Ehrlich ascites tumor cells, either by hypotonic treatment in the presence of Ca2+ or by addition of cytochalasin B, results in activation of the Na+/K+/Cl- cotransport system. However, other regulatory processes, some of which may be dependent on an intact filament system, are responsible for the activation of the Na+/K+/Cl- cotransport system after cell shrinkage.


The fusion of artificial lipid membranes induced by the synthetic arenavirus 'fusion peptide'. The fusing activity of the synthetic 23 amino-acid fragment (fusion peptide, FP) of the fusion protein of the Lassa arenavirus membrane was tested in a model liposomal system. The resonance energy transfer between two fluorescent phospholipid probes was monitored in order to detect dioleoylphosphatidylcholine liposome fusion induced by the peptide. Fusion rates were compared at different pH values, ionic strength and calcium concentrations. FP demonstrated fusing activity at pH 4.5-5.5, indicating that the protonated form of the FP is the active one. A transmembrane proton-gradient induced by acidification was not relevant to the fusion process, since its elimination with nigericin did not affect the FP-mediated fusion. Both Ca2+ (8 mM) and the increase of the ionic strength (1 M NaCl) inhibited liposome fusion. The efficacy of liposome fusion depended also on the lipid-to-lipid ratio. Non-linear dependence was observed at a saturation ratio of 10 mol lipid per mol peptide. A model of 'side insertion' is suggested, describing FP interaction with the membrane.


Polarity of transport of 2-deoxy-D-glucose and D-glucose by cultured renal epithelia (LLC-PK1). At least two types of glucose transporter exist in cultured renal epithelial cells, a Na(+)-glucose cotransporter (SGLT), capable of interacting with D-glucose but not 2-deoxy-D-glucose (2dglc) and a facilitated transporter (GLUT) capable of interacting with both D-glucose and 2dglc. In order to examine the polarity of transport in cultured renal epithelia, 2dglc and D-glucose uptakes were measured in confluent cultures of LLC-PK1 cells grown on collagen-coated filters that permitted access of medium to both sides of the monolayer. The rates of basolateral uptake of both 1 mM glucose (Km 3.6 mM) and 1 mM 2dglc (Km 1.5 mM) were greater than apical uptake rates and the (apical-to-basolateral)/(basolateral-to-apical) flux ratio was high for glucose (9.4) and low for 2dglc (0.8), thus, confirming the lack of interaction of 2dglc with the apical SGLT. Specific glucose transport inhibitor studies using phlorizin, phloretin and cytochalasin B confirmed the polarised distribution of SGLT and GLUT in LLC-PK1 cells. Basolateral sugar uptake could be altered by addition of insulin (1 mU/ml) which increased 2dglc uptake by 72% and glucose uptake by 50% and by addition of 20 mM glucose to the medium during cell culture which decreased 2dglc uptake capacity at confluence by 30%. During growth to confluence, 2dglc uptake increased to a maximum, then decreased at the time of confluence, coincident with a rise in uptake capacity for alpha-methyl-D-glucoside, a hexose that interacts only with the apical SGLT. It was concluded that the non-metabolisable sugar 2dglc was a useful, specific probe for GLUT in LLC-PK1 cells and that GLUT was localised at the basolateral membrane after confluence.


Formation of non-bilayer structures induced by M13 coat protein depends on the conformation of the protein. A comparison is made of the interaction of the coat protein of bacteriophage M13 in a predominant alpha-helix conformation and in a predominant beta-sheet conformation. To perform a systematic study of the interaction between the protein in these two different forms of the surrounding lipid matrix, NMR spectra of 2H-nuclei of specific labelled phospholipid systems are measured. In addition 31P-NMR is employed to provide information about the morphological structure adopted by the reconstituted lipid/protein systems. From the 2H-NMR studies on specific headgroup and chain deuterium labelled phospholipids it is found that the protein in the predominant beta-sheet conformation causes a fraction of lipids to be trapped. By combining the results from the headgroup and acyl chains of the phospholipids, it is concluded that the trapped lipids are arranged in a non-bilayer structure, probably caused by a misfitting of the hydrophobic core of the protein and the membrane bilayer. The protein in the predominant alpha-helix conformation perfectly fits in the lipid bilayer and has only minor influences on the surrounding lipid matrix. A new model is proposed to explain the presence of the trapped lipids in the lipid/protein systems.


Alcohols dehydrate lipid membranes: an infrared study on hydrogen bonding. The effects of alcohols (methanol, ethanol, and n-butanol) on the hydrogen bonding of dipalmitoylphosphatidylcholine (DPPC) were studied by Fourier-transform infrared spectroscopy (FTIR) in water-in-oil (carbon tetrachloride) reversed micelles. The bound O-H stretching mode of water, bonded to DPPC, appeared as a broad band at around 3400 cm-1. The O-H bending mode of this complex appeared as a weak broad band at 1644 cm-1. No free O-H signal was observed. When alcohols were added, a part of DPPC-bound water was replaced by the alcohols. The released 'free' water appeared at 3680 cm-1. This free O-H stretching band represents water-alcohol complex. A new broad band of O-H stretching appeared at 3235 cm-1, which represents the alcohol molecules bound to the phosphate moiety of DPPC. When the alcohol concentration was increased, the intensities of the free O-H stretching and bending bands increased. The P = O- antisymmetric stretching band at 1238 cm-1 became broader and shifted to lower frequencies. This means that alcohols interacted with the phosphate moiety and replaced the bound water. In the deconvoluted spectra of the C = O stretching mode, the ratio between the free sn-2 and the hydrogen-bonded sn-2 bands increased; a part of the bound water at the sn-2 carbon in the glycerol skeleton is also released and the free sn-2 signal increased. From the change in the intensity of the P = O- stretching band, the partition coefficients of alcohols between the phosphate region of DPPC and water were estimated: methanol 7.8, ethanol 16.7 at 22.0 degrees C in mole fraction bases. In molality, these values translates into methanol 0.21 and ethanol 0.45. These results indicate that short-chain alcohols interact with lipid membranes at the phosphate moiety at the hydrophilic head, weaken the membrane-water interaction, and destabilize membranes.


Osmotic swelling and membrane conductances in A6 cells. Hyposmotic basolateral perturbations (-30 mosmol/kg) in cultured renal layers (A6) increased basolateral membrane conductance more than 2-fold within 10 min; the increase was partly due to upregulation of K+ conductance, but other conductive pathways were also activated. The raise in apical membrane amiloride-sensitive Na+ conductance was less pronounced; it appears to be due to secondary effects.


Evidence that the scavenger receptor is not involved in the uptake of negatively charged liposomes by cells. Scavenger receptors have a broad ligand specificity, ranging from modified low-density lipoproteins to a variety of high-molecular-weight poly-anions. A recent report by Nishikawa et al. (J. Biol. Chem. (1990) 265, 5226-5231) suggested that this receptor is also involved in the binding and endocytosis of liposomes containing negatively charged phospholipids. The mechanism by which liposomes are taken up by cells is of interest because liposomes are promising versatile carriers for macromolecules and drugs both in vitro and in vivo. In this report, we re-examine the role of the scavenger receptor in the uptake of liposomes using both Chinese hamster ovary cells transfected with the type I or type II bovine scavenger receptor, and smooth muscle cells induced to increase scavenger receptor expression by phorbol ester treatment. Expression of both types of scavenger receptors by Chinese hamster ovary cells induced an increase in the uptake of chemically modified low-density lipoproteins, but not the uptake of negatively charged liposomes. In smooth muscle cells treated with phorbol ester, scavenger receptor expression was upregulated and the uptake of chemically modified low-density lipoproteins was enhanced dramatically, but there was no effect on the uptake of negatively charged liposomes. We conclude that the existing evidence does not support the suggestion that the scavenger receptor is involved in the uptake of anionic liposomes by cells.


Effects of membrane lipids and -proteins and cytoskeletal proteins on the kinetics of cholesterol exchange between high density lipoprotein and human red blood cells, ghosts and microvesicles. To better understand the effects of plasma membrane lipids and proteins and the cytoskeleton on the kinetics of cellular cholesterol efflux, the effects of (1), selectively depleting either sphingomyelin (SM) or phosphatidylcholine (PC); (2), cross-linking the cytoskeleton, and (3), removing certain cytoskeletal and integral membrane proteins on radiolabelled cholesterol efflux from red blood cells (RBC) have been studied. When RBC were treated with either phospholipase A2 or sphingomyelinase C to hydrolyze either 30-40% of the PC or 40-50% of the SM, respectively, the halftimes (t1/2) for cholesterol efflux to excess HDL3 were not significantly altered, with the values being 4.4 +/- 0.8 h or 3.7 +/- 0.4 h, respectively, compared to 4.6 +/- 0.6 h for control RBC. To investigate the effects of the cytoskeleton on the rate of free cholesterol (FC) desorption from the plasma membrane, the cytoskeletal proteins were cross-linked by either heat-treatment or exposure to diamide and cholesterol efflux from ghosts of these cells was measured. Cross-linking the cytoskeletal proteins by diamide treatment resulted in no significant change in t1/2 for treated (3.6 +/- 0.6 h) compared to control (4.2 +/- 0.4 h) ghosts: this suggests that the cytoskeleton does not play a large role in modulating cholesterol efflux. To investigate the effects of membrane proteins on cholesterol efflux, RBC microvesicles, containing mainly band 3 and 4 proteins and little of the cytoskeletal proteins, such as spectrin (bands 1,2) or actin (band 5), were obtained by incubation with the ionophore A23187. With excess HDL3 present, microvesicles exhibited a t1/2 of 4.2 +/- 1.9 h (compared to the t1/2 of 4.2 +/- 0.4 h for control ghosts). The results described in this paper suggest that neither changing the SM/PC ratio in the membrane nor cross-linking the cytoskeletal proteins nor removing the cytoskeleton changes the t1/2 for cholesterol efflux to excess HDL3. Presumably, the cholesterol-phospholipid interactions are insensitive to these perturbations in membrane structure.


Na+/K+/Cl- cotransport in resealed ghosts from erythrocytes of the Milan hypertensive rats. The erythrocytes (RBC) of the Milan hypertensive rats (MHS) have a smaller volume and faster Na+/K+/Cl- cotransport than RBC from normotensive controls (MNS). The difference in Na+/K+/Cl- cotransport is no longer present in inside-out Vesicles (IOV) of RBC membrane. To differentiate between cytoplasmic or membrane skeleton abnormalities as possible causes of these differences. Resealed ghosts (RG) were used to measure ion transport systems. The following results have been obtained: (1) RG from MHS have a smaller volume than MNS (mean +/- S.E. 20.7 +/- 0.45 vs. 22.09 +/- 0.42 fl, P < 0.05). (2) RG showed a bumetanide-sensitive Na efflux that retains the characteristics of the Na+/K+/Cl- cotransport of the original RBC: it is K(+)- and Cl(-)-sensitive and dependent on the intracellular Na+ concentration. (3) The Na+/K+/Cl- cotransport was faster in RG from MHS than in those from MNS (mean +/- S.E. 0.095 +/- 0.01 vs. 0.066 +/- 0.01 rate constant h-1, P < 0.01). These results, together with those of IOV, support the hypothesis that an abnormality in the membrane skeletal proteins may play a role in the different Na+/K+/Cl- cotransport modulation between MHS and MNS erythrocytes.


Transport of phospholipids between subcellular membranes of wild-type yeast cells and of the phosphatidylinositol transfer protein-deficient strain Saccharomyces cerevisiae sec 14. The transfer of glycerophospholipids between microsomes and mitochondria, and from internal membranes to the plasma membrane of Saccharomyces cerevisiae was characterized. Cellular energy production was found to be essential for intracellular translocation of phospholipids, but neither a membrane potential nor an intact cytoskeleton are required for this process. Using the temperature-sensitive mutant strain Saccharomyces cerevisiae sec 14, which is defective in the phosphatidylinositol transfer protein, it could be demonstrated that this protein is not involved in the transport of phosphatidylinositol and phosphatidylcholine from internal membranes to the plasma membrane. Our results also confirm earlier findings that phosphatidylinositol and phosphatidylcholine can be delivered to the plasma membrane in a process independent of the flux of vesicles competent for protein secretion.


The influence of cholesterol 3-sulphate on phase behaviour and hydrocarbon order in model membrane systems. Cholesterol 3-sulphate (CS) is a component of the intercellular lipid found in the uppermost layer of human epidermis (the 'stratum corneum') and is thought to play an important role in tissue cohesion. In this investigation we have compared the influence of cholesterol (CH) and CS on the gel-to-liquid crystalline phase behaviour, the polymorphic phase behaviour, and the hydrocarbon order profile in selected model membranes. It is shown that in sphingomyelin (SPM) systems, the presence of equimolar amounts of either CH or CS eliminates the gel-to-liquid crystalline transition as detected by calorimetry. Similarly, in 1-palmitoyl,2-oleoyl-phosphatidylethanolamine (POPE) dispersions containing a perdeuterated palmitoyl chain (POPE-d31), it is shown that both CH and CS exert an ordering effect as determined by 2H-NMR techniques, however, CS is less potent at temperatures both above and below that of the main transition for the native phospholipid. Alternatively, in mixed systems containing dioleoylphosphatidylethanolamine (DOPE) and SPM (DOPE/SPM, 6:1 mol/mol) CH promotes thermotropic L alpha-->HII phase transitions, whereas CS stabilizes the bilayer organization. These bilayer stabilization effects can be diminished by addition of Ca2+. These effects are consistent with a larger area per molecule of CS as compared to CH, presumably related to the presence of the negatively charged sulphate moiety of CS.


Incorporation of carotenoids in aqueous systems: uptake by cultured rat hepatocytes. The in vitro investigations about carotenoid functions and metabolism are hindered by their hydrophobicity. In order to mimic as close as possible the physiological events, we prepared by rapid and easy methods sterile liposomes and emulsions containing carotenoids which are absorbed by cultured rat hepatocytes as a function of time and temperature. The lipid composition of the vesicles was shown to influence the carotenoid encapsulation.


Substrate-specific differences in the rate of bile acid carrier reorientation: studies on human placental basal vesicles. The initial rate of transport of the bile acid glycocholic acid (GCA) has been measured in influx and efflux across placental basal membrane vesicles, and the mechanism of inhibition of its transport by the analogue taurochenodeoxycholic acid (TCDCA) analysed kinetically. This analogue, although trans-stimulating GCA efflux, inhibits influx in a way which does not depend upon substrate concentration; moreover, its potency as an inhibitor is markedly influenced by whether it is placed on one or on both sides of the vesicles membrane. These findings can be accounted for by postulating that both GCA and TCDCA are translocated through the carrier, but that the rate of loaded carrier reorientation is higher than that of the free carrier only when loaded with TCDCA and not with GCA.


Vasopressin stimulation of vanadate-sensitive Na+ transport by liver plasma membrane vesicles. Evidence for regulation via phospholipase C and protein kinase C activities. The rate of vanadate-sensitive 22Na+ uptake by isolated liver membrane vesicles, reflecting transport by Na+/K(+)-ATPase, was measured to study the role played by phospholipase C and protein kinase C in the regulation of this process by vasopressin. Na+ uptake was enhanced 2-3-fold by 100 nM [Arg8]vasopressin and the hormone effect was mimicked by 0.1 microM inositol 1,4,5-trisphosphate as well as by 1.0 microM myo-inositol. The stimulation by vasopressin was potentiated by phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis (5-10 mU/ml). No effect of the bacterial enzyme was observed in the absence of the hormone. Phorbol myristate acetate (0.5-1 microM) suppressed the stimulation by vasopressin but had no effect in the absence of the hormone. High concentrations of bacterial phosphatidylinositol-specific phospholipase C (50-100 mU/ml) also antagonized the hormone stimulation. Staurosporine (50-100 nM) prevented the antagonistic effect of bacterial phospholipase C (50 mU/ml) and EGTA (1 mM) partially protected the hormonal stimulation in the presence of phorbol myristate acetate. Our results suggest that the stimulatory effect of vasopressin on Na+ transport is mediated by phospholipase C and products derived from the inositol moiety of membrane phospholipids. Membrane-associated protein kinase C appears to be at least partially responsible for the desensitization to stimulation by vasopressin.


Alcohols produce reversible and irreversible acceleration of phospholipid flip-flop in the human erythrocyte membrane. The slow, non-mediated transmembrane movement of the lipid probes lysophosphatidylcholine, NBD-phosphatidylcholine and NBD-phosphatidylserine in human erythrocytes becomes highly enhanced in the presence of 1-alkanols (C2-C8) and 1,2-alkane diols (C4-C8). Above a threshold concentration characteristic for each alcohol, flip rates increase exponentially with the alcohol concentration. The equieffective concentrations of the alcohols decrease about 3-fold per methylene added. All 1-alkanols studied are equieffective at comparable calculated membrane concentrations. This is also observed or the 1,2-alkane diols, albeit at a 5-fold lower membrane concentration. At low alcohol concentrations, flip enhancement is reversible to a major extent upon removal of the alcohol. In contrast, a residual irreversible flip acceleration is observed following removal of the alcohol after a treatment at higher concentrations. The threshold concentrations to produce irreversible flip acceleration by 1-alkanols and 1,2-alkane diols are 1.5- and 3-fold higher than those for flip acceleration in the presence of the corresponding alcohols. A causal role in reversible flip-acceleration of a global increase of membrane fluidity or membrane polarity seems to be unlikely. Alcohols may act by increasing the probability of formation of transient structural defects in the hydrophobic barrier that already occur in the native membrane. Membrane defects responsible for irreversible flip-acceleration may result from alterations of membrane skeletal proteins by alcohols.


Experimental evidence for the involvement of the cytoskeleton in mammalian cell electropermeabilization. Chinese hamster ovary (CHO) cells and human erythrocytes were pulsed by using square-wave electric-field pulses. This treatment induced their permeabilization. This phenomenon appears to be a three-step process of creation, expansion and annihilation of permeated structures. Altering the cell cytoskeleton, either with drugs, such as colchicine, known to depolymerise the microtubules in CHO cells, or by high temperature shock to affect the spectrin-actin network in erythrocytes, induced no modification on the first two steps of the electropermeabilization process, but was associated with a dramatic decrease in the stability of the electro-induced permeated structures. These experimental observations support the hypothesis of an implication of cytoskeleton in electropermeabilization in agreement with thermodynamic conclusions.


Endoplasmic reticulum: the major contributor to the PDE peak in hepatic 31P-NMR spectra at low magnetic field strengths. 31P-NMR spectra of liver in vivo, subcellular fractions and model systems were acquired in order to characterise further the hepatic phosphodiester peak seen at low magnetic field strengths previously shown to be predominantly due to phospholipid bilayers. The data obtained in this study in vitro suggested that the phospholipid membranes of the endoplasmic reticulum provide the dominant contribution to this phosphodiester peak. Support for this hypothesis was provided by experiments on rats. Phenobarbitone, which is known to induce proliferation of the endoplasmic reticulum produced a considerable increase in intensity of the phosphodiester peak in liver spectra in vivo.


Developmental changes in hepatic basolateral membrane lipid composition and fluidity. Membrane fluidity and lipid composition influence the activity of a variety of membrane proteins. Decreased rates of hepatic ion clearance are associated with the neonatal period. We postulated that hepatic basolateral membranes derived from suckling animals might be less fluid than those from adult animals. Basolateral membrane vesicles were prepared from the livers of 1-week-old (SBLMV) and adult (ABLMV) rats by a Percoll gradient method. Na+/K(+)-ATPase activities were similar in the two groups. Double bond index, cholesterol and cholesterol/phosphorus ratios were significantly higher in SBLMV compared with ABLMV, while lipid phosphorus and relative percentages of phospholipid subclasses did not differ. Fluorescence anisotropy measured using diphenylhexatriene as well as 2-(9-anthroyloxy)stearate was significantly greater in SBLMV compared with ABLMV, while measurements made with 12-(9-anthroyloxy)stearate were similar in both age groups. Mean excited state lifetimes, lifetime distributions, and rotational correlation times were similar in both groups. These data suggest that hepatic basolateral membranes derived from suckling rats are less fluid than those from adult animals and further suggest that this difference may be due to increased cholesterol in hepatic basolateral membranes derived from suckling animals.


Effect of the sulfhydryl reagent thimerosal on cytosolic free Ca2+ and membrane potential of thymocytes. The sulfhydryl reagent thimerosal at concentrations 5-100 microM has been found to induce a variety of changes in ion transport in rat thymocytes. In particular, [Ca2+]i increases about 10-fold from the basal level. The [Ca2+]i response to thimerosal displays a two-stage time course, with the main [Ca2+]i rise during the second stage. Evidence has been obtained for the depletion of intracellular Ca2+ pools in thimerosal-treated cells, however, Ca2+ mobilization from intracellular stores does not contribute significantly into [Ca2+]i rise. Thimerosal elicits permeability not only for Ca2+, but also for Mn2+ and Ni2+, which is Ca(2+)-dependent. We failed to get any evidence on thimerosal-induced inhibition of the plasma membrane Ca(2+)-ATPase. The induction of Ca2+ influx, rather than inhibition of Ca(2+)-ATPase, accounts for the disturbance of [Ca2+]i homeostasis in thimerosal-treated cells. Thimerosal also elicits changes in monovalent ion fluxes resulting in marked depolarization. The latter seems unrelated to the changes in [Ca2+]i and is suggested to be mediated both by increased permeability for Na+ and a decreased one for K+. Thimerosal significantly stimulates AA release from thymocytes. Evidence has been presented that AA metabolite(s), probably, LO product(s), may mediate the changes in the transport of mono- and divalent cations elicited by the sulfhydryl reagent. Prolonged treatment of thymocytes with thimerosal resulted in cell death.


Characterization and chemical modification of the Na(+)-dependent bile-acid transport system in brush-border membrane vesicles from rabbit ileum. The Na(+)-dependent uptake system for bile acids in the ileum from rabbit small intestine was characterized using brush-border membrane vesicles. The uptake of [3H]taurocholate into vesicles prepared from the terminal ileum showed an overshoot uptake in the presence of an inwardly-directed Na(+)-gradient ([Na+]out > [Na+]in), in contrast to vesicles prepared from the jejunum. The Na(+)-dependent [3H]taurocholate uptake was cis-inhibited by natural bile acid derivatives, whereas cholephilic organic compounds, such as phalloidin, bromosulphophthalein, bilirubin, indocyanine green or DIDS - all interfering with hepatic bile-acid uptake - did not show a significant inhibitory effect. Photoaffinity labeling of ileal membrane vesicles with 3,3-azo- and 7,7-azo-derivatives of taurocholate resulted in specific labeling of a membrane polypeptide with apparent molecular mass 90 kDa. Bile-acid derivatives inhibiting [3H]taurocholate uptake by ileal vesicles also inhibited labeling of the 90 kDa polypeptide, whereas compounds with no inhibitory effect on ileal bile-acid transport failed to show a significant effect on the labeling of the 90 kDa polypeptide. The involvement of functional amino-acid side-chains in Na(+)-dependent taurocholate uptake was investigated by chemical modification of ileal brush-border membrane vesicles with a variety of group-specific agents. It was found that (vicinal) thiol groups and amino groups are involved in active ileal bile-acid uptake, whereas carboxyl- and hydroxyl-containing amino acids, as well as tyrosine, histidine or arginine are not essential for Na(+)-dependent bile-acid transport activity. The irreversible inhibition of [3H]taurocholate transport by DTNB or NBD-chloride could be partially reversed by thiols like 2-mercaptoethanol or DTT. Furthermore, increasing concentrations of taurocholate during chemical modification with NBD-chloride were able to protect the ileal bile-acid transporter from inactivation. These findings suggest that a membrane polypeptide of apparent M(r) 90,000 is a component of the active Na(+)-dependent bile-acid reabsorption system in the terminal ileum from rabbit small intestine. Vicinal thiol groups and amino groups of the transport system are involved in Na(+)-dependent transport activity, whereas other functional amino acids are not essential for transport activity.


Membrane lipids of human peripheral nerve and spinal cord. Major membrane lipids were determined in specimens of human peripheral nerve (cauda equina) and spinal cord of 10 subjects aged 20-70 years. The same lipids were also assayed in myelin from the same tissues isolated with two different procedures and in myelin of cauda equina from 3 subjects aged 17-91 years isolated with a third method. The concentrations (mean and standard deviation) of phospholipids were 90 +/- 11 and 96 +/- 9 nmol/g fresh weight; of cholesterol 70 +/- 15 and 101 +/- 16; of cerebroside 19 +/- 3 and 41 +/- 7; of sulfatide 10 +/- 1 and 11 +/- l; and of gangliosides 0.80 +/- 0.08 and 0.40 +/- 0.05 N in cauda equina and spinal cord, respectively. The proportion of ethanolamine phosphoglyceride was lower and that of sphingomyelin higher in cauda equina than in spinal cord. The myelin of peripheral nerve and spinal cord contained almost the same proportions of lipids as the whole tissue. The protein-bound sialic acid content was 3-fold higher than the lipid-bound sialic acid content in cauda myelin. The fatty acid patterns of choline, ethanolamine, inositol and serine phosphoglycerides of spinal cord and its myelin, were very similar to those of cerebral white matter, while the phosphoglycerides of cauda equina had higher proportions of monoenoic acids and lower proportions of polyunsaturated fatty acids. The fatty acid patterns of sphingomyelin, cerebroside and sulfatide of spinal cord were similar to those of cerebral white matter, while those of cauda equina contained significantly more saturated fatty acids. This suggests that the lipid and fatty acid compositions of peripheral nerve are particularly suitable for the formation of a tightly packed myelin membrane which can be a powerful shield against infections and other injuries.


Myocardial carnitine palmitoyltransferase of the mitochondrial outer membrane is not altered by fasting. The regulation of heart carnitine palmitoyltransferase was studied during the transition to the fasting state. Using decanoyl-CoA or palmitoyl-CoA as substrates, we found no differences in carnitine palmitoyltransferase activity or in its sensitivity to inhibition by malonyl-CoA between fed and fasted states. No cooperativity was seen with either substrate, and the malonyl-CoA-induced shift to sigmoid kinetics normally observed with liver mitochondria was not obvious with heart mitochondria. Analysis of malonyl-CoA inhibition data revealed that mitochondria from rat heart exhibited incomplete maximum inhibition of carnitine palmitoyltransferase (partial inhibition). Homogenization of intact liver mitochondria resulted in a similar pattern of incomplete inhibition and suggested that the malonyl-CoA-insensitive carnitine palmitoyltransferase of the inner membrane was also being assayed. Carnitine palmitoyltransferase in mitochondrial outer membranes, isolated from the heart, proved to be extremely sensitive to malonyl-CoA inhibition and had maximum inhibition values of 90-100% with either decanoyl-CoA or palmitoyl-CoA as substrates, but fasting had no effect. Fasting produced no change in the Ki for malonyl-CoA (0.10 +/- 0.04 and 0.14 +/- 0.02 microM for the fed and fasted groups, respectively). Acyl-CoA chain length specificity was C10 greater than C16 greater than C14 greater than C12 greater than C18 = C8 for carnitine palmitoyltransferase in heart mitochondrial outer membranes. It is concluded that the regulation of carnitine palmitoyltransferase of heart mitochondrial outer membranes differs from regulation of the liver enzyme in three characteristics--the heart enzyme (a) has greater sensitivity to malonyl-CoA inhibition, (b) is resistant to the effects of fasting and (c) has somewhat different acyl-CoA substrate specificity.


12-Lipoxygenase from rat basophilic leukemia cells, an oxygenase with leukotriene A4-synthase activity. Rat basophilic leukemia cells exhibit 12-lipoxygenase activity only upon cell disruption. 12-Lipoxygenase may also possess 15-lipoxygenase activity, as is indicated by the formation of low amounts of 15(S)-HETE, in addition to the predominant product 12(S)-HETE, upon incubation of partially purified 12-lipoxygenase with arachidonic acid. With 5(S)-HPETE as substrate not only 5(S), 12(S)-diHETE and 5(S), 15(S)-diHETE are formed, but also LTA4, as was indicated by the presence of LTA4-derived LTB4-isomers. 12-Lipoxygenase from rat basophilic leukemia cells has many features in common with 12-lipoxygenase from bovine leukocytes. As was suggested for the latter enzyme, 12-lipoxygenase from rat basophilic leukemia cells may represent the remaining LTA4-synthase activity of 5-lipoxygenase, of which the 5-dioxygenase activity has disappeared upon cell disruption. Such a possible shift from 5-lipoxygenase activity to 12-lipoxygenase activity could not simply be induced by interaction of cytosolic 5-lipoxygenase with a membrane fraction after cell disruption, but may involve release of membrane-associated 5-lipoxygenase upon disruption of activated rat basophilic leukemia cells.


Spontaneous hypercholesterolemia in cynomolgus monkeys: evidence for defective low-density lipoprotein catabolism. Spontaneously hypercholesterolemic (SH) cynomolgus monkeys were identified that have average plasma cholesterol of 202 mg/dl, while that in normal monkeys is 119 mg/dl. The LDL from these SH monkeys have lower affinity for fibroblast LDL receptors in vitro. The amount of LDL2 (1.030 mean value of d 1.063 g/ml) required to displace 50% of [125I]LDL was 3.8 micrograms/ml for normal LDL2 and 6.6 micrograms/ml for SH-LDL2. The binding affinity of LDL1 (1.019 mean value of d 1.030 g/ml) was the same in normal and SH animals. LDL turnover experiments showed that the SH monkeys were comprised of two populations. Normal LDL2 was cleared much slower in two of the SH monkeys than in normocholesterolemic animals, suggesting that these two animals have an LDL receptor defect. However, LDL2 isolated from these two SH monkeys was cleared normally in normal monkeys. LDL2 isolated from two other SH monkeys is cleared slower than is normal LDL2 in normal animals, suggesting that these animals have an LDL defect. Thus, the hypercholesterolemia of these SH monkeys is associated with defective LDL catabolism; two animals appear to have functionally defective LDL receptors, and two animals appear to have functionally defective LDL.


Changes in fatty acid composition in rat blood and organs after infusion of eicosapentaenoic acid ethyl ester. An infusible emulsion of 10% eicosapentaenoic acid ethyl ester (EPA-EE, 99.2% pure) was prepared. Three ml of the emulsion was infused into tail veins of 20 Wistar rats weighing approx. 300 g. They were killed 1 h, 6 h, 24 h, 3 d and 7 d after the infusion, and fatty acid composition of various organs and plasma was analyzed along with that of control rats. There was no lipidosis in any organs of any rats. It was estimated that not less than 98% of infused EPA was cleared from plasma during the first hour after the infusion. EPA concentrations reached their peaks within 6 h after EPA infusion in plasma lipid fractions and in the phospholipid fraction of liver, heart, lung, kidney and spleen. The fatty acid composition of the phospholipid fraction of heart was very stable, and was not altered by EPA infusion except for a very slight increase in EPA at 1 h after the infusion (0.18% at 0 h to 0.56%). However, EPA concentrations increased markedly in the free fatty acid fraction of heart at 1 h after the infusion (0.15% at 0 h to 4.23%). Arachidonic acid concentrations decreased significantly within 24 h in the phospholipid fraction of organs except for heart. EPA emulsion might be useful for patients in whom a rapid increment in EPA in tissues is beneficial.


Prolonged circulation time in vivo of large unilamellar liposomes composed of distearoyl phosphatidylcholine and cholesterol containing amphipathic poly(ethylene glycol). The effect of poly(ethylene glycol) (PEG) on the circulation time of liposomes in mice was examined by employing amphipathic PEGs (phosphatidylethanolamine (PE) derivatives of PEG) with average molecular weights of 1000, 2000, 5000 and 12,000. The activity of dioleoyl phosphatidylethanolamine-PEG (DOPE-PEG) in prolonging the circulation time of egg phosphatidylcholine/cholesterol large unilamellar liposomes (ePC/CH LUVs) (200 nm) was proportional to the molecular weight of PEG, i.e., 12000 = 5000 greater than 2000 greater than 1000. On the other hand, inclusion of distearoylphosphatidylethanolamine-PEG (DSPE-PEG) or dipalmitoyl-phosphatidylethanolamine-PEG (DPPE-PEG) of low molecular weight such as 1000 and 2000 in distearoylphosphatidylcholine (DSPC)/CH LUVs or dipalmitoyl phosphatidylcholine (DPPC)/CH LUVs effectively increased their blood circulation time. At least 3 mol% of amphipathic PEG in liposomes was required for activity. Addition of CH, which has a bilayer-tightening effect, to DSPC/CH/DSPE-PEG2000 LUVs further increased the blood residence time. A size of less than 300 nm was essential for prolonging the residence time of amphipathic PEG-containing liposomes in blood. DSPC/CH/DSPE-PEG2000 LUVs (1:1:0.13, m/m) containing 6 mol% of PEG and 200 nm in diameter remained in the circulation for over 24 h after injection and may be clinically useful for sustained release of an entrapped drug in the bloodstream and for drug accumulation in solid tumors.


The influence of medium components on Cu(2+)-dependent oxidation of low-density lipoproteins and its sensitivity to superoxide dismutase. The extent of in vitro Cu(2+)-dependent oxidation of low-density lipoproteins (LDL) has been reported to vary widely depending upon reaction conditions. In this study, the effect of proteins and amino acids on Cu(2+)-induced LDL oxidation was examined. Treatment of LDL with 5 microM CuSO4 for 18 h in either phosphate-buffered saline (PBS) or Ham's F-10 medium resulted in extensive oxidation as determined by the content of thiobarbituric acid reactive substances (TBARS) and by increased lipoprotein electronegativity. In PBS, oxidation was entirely blocked by histidine and the tripeptide, gly-his-lys (GHK). Oxidation was also prevented by bovine serum albumin, but superoxide dismutase (SOD) provided only 20% protection. Both proteins bound similar amounts of Cu2+, but albumin appeared to be a more effective peroxyl radical trap as evidenced by its ability to prevent LDL oxidation induced by 2,2'-azo-bis(2-amidinopropane hydrochloride). In F-10 medium, SOD had marked inhibitory effects, in contrast to PBS. The addition of disulfides to PBS markedly enhanced the ability of SOD to inhibit oxidation. These results indicate that medium components which affect Cu2+ availability influence LDL oxidation and suggest that albumin is ideally suited as a plasma antioxidant to prevent oxidative modification of LDL. Furthermore, in certain instances, the inhibitory effects of SOD may be attributable to effects such as Cu2+ binding rather than dismutation of superoxide.


7 alpha-hydroxylation of 27-hydroxycholesterol in human liver microsomes. Human liver microsomes were found to catalyze 7 alpha-hydroxylation of 27-hydroxycholesterol at a rate of up to about 0.2 nmol/mg protein per min. The product of the reaction, 5-cholestene-3 beta, 7 alpha, 27-triol, was identified by means of combined gas chromatography-mass spectrometry. Liver microsomes from two patients with an upregulated cholesterol 7 alpha-hydroxylase, did not have higher 7 alpha-hydroxylase activity towards 27-hydroxycholesterol than those from untreated patients, suggesting that the 7 alpha-hydroxylase active towards 27-hydroxycholesterol is not the same as that active towards cholesterol. The mitochondrial fraction of liver from untreated patients and patients treated with cholestyramine, had negligible 7 alpha-hydroxylase activity towards 27-hydroxycholesterol less than 0.01 nmol/mg protein per min). The results are in accord with the possibility that there is a pathway to bile acids in human liver in which the first step is a 27-hydroxylation of cholesterol.


Altered positional specificity of human plasma lecithin-cholesterol acyltransferase in the presence of sn-2 arachidonoyl phosphatidyl cholines. Mechanism of formation of saturated cholesteryl esters. The positional specificity of purified human lecithin-cholesterol acyltransferase (LCAT) was studied by analyzing the labeled cholesteryl ester (CE) species formed in the presence of proteoliposome substrates containing mixed chain phosphatidylcholine (PC) species, labeled cholesterol and apoprotein A-I. Whereas over 90% of the acyl groups used for CE synthesis were derived from the sn-2 position of most of the naturally occurring PC substrates, about 75% of the CE species formed in the presence of sn-1-myristoyl 2-arachidonoyl PC, sn-1-palmitoyl-2-arachidonoyl (PAPC) and sn-1-palmitoyl 2-docosahexaenoyl PC were derived from the sn-1-position. On the other hand, rat LCAT utilized mostly sn-2-acyl group from either PAPC or from sn-1-palmitoyl 2-linoleoyl PC. The positional specificity of the human enzyme was not affected by the alteration in the matrix fluidity, type of the apoprotein activator used, or by the free cholesterol/PC ratio in the substrate. These results show that the positional specificity of human plasma LCAT is altered in the presence of sn-2-arachidonoyl PC, or sn-2-docosahexaenoyl PC, probably due to steric restrictions at the active site, and this may account for the formation of disproportionately high concentrations of saturated CE, and low concentrations of long-chain polyunsaturated CE in human plasma, relative to the composition of sn-2-acyl groups in plasma PC.


Structure of polymerizable lipid bilayers. V. Synthesis, bilayer structure and properties of diacetylenic ether and ester lipids. Four diacetylenic phosphatidylcholines (PC's) have been synthesized and the structures of bilayers of these lipids have been determined at low resolution by low-angle X-ray diffraction. The PC's all have 18-carbon chains but differ with respect to the ether/ester linkage at the sn-1 and sn-2 positions and the relative position of the diacetylene moiety: diester-PC (1): 1,2-bis(octadeca-4',6'-diynoyl)-sn-glycero-3-phosphocholine diester-PC (2): 1-(octadeca-4',6'-diynoyl)-2-(octadeca-5',7'-diynoy l)-sn- glycero-3-phosphocholine diester-PC (3): 1,2-bis(octadeca-8',10'-diynoyl)-sn-glycerol-3-phosphocholin e diether-PC (4): 1-O-(octadeca-4',6'-diynyl)-2-O-(octadeca-5",7"-din yl)-sn- glycero-3-phosphocholine Only (1) exhibits the typical bilayer profile, whereas (2), (3) and (4) show evidence of interdigitation and/or significant disorder. Only (1) polymerized effectively upon illumination with 254 nm light, turning deep blue in seconds, indicating the formation of long, well-ordered polydiacetylenic structures. Liposomes of these derivatives were tested for permeability by osmotic swelling. Polymerized liposomes of (1) underwent osmotic swelling with urea, glycerol, and acetamide more rapidly than did liposomes of stearoyl-oleoyl-PC, but the initial rates of osmotic swelling of polymerized liposomes of (1) were 3-10-times lower than those of unpolymerized liposomes of (1). Blue polymerized multilayer samples of (1) exhibited an irreversible thermochromic transition to red at approx. 40 degrees C. Differential scanning calorimetry with liposome suspensions of (1) revealed an endotherm at 28.3 degrees C with a transition enthalpy of 40 J/g. PC (1) is a potentially useful diacetylenic lipid which exhibits facile, complete polymerization and a bilayer thickness comparable to that of biomembrane lipids.


Molecular, functional and structural properties of an archaebacterial elongation factor 2. The elongation factor 2 (aEF-2) from the extreme thermo-acidophilic archaebacterium Sulfolobus solfataricus, is the only cytosolic target protein which is ADP-ribosylated by diphtheria toxin in presence of NAD. Once ADP-ribosylated, aEF-2 is no longer able to sustain poly(Phe) synthesis in vitro. aEF-2 displays a great thermoresistance: at the growth temperature of the archaebacterium, 87 degrees C, its half-life is 3 h. The amino acid sequence of the N-terminal region of aEF-2 has been determined up to residue 22. In the first 15 positions such a sequence is identical to that of EF-2 from Sulfolobus acidocaldarius and very similar to that of EF-2 from other archaebacteria or eukaryotes. The same is true for the primary structure of the peptide containing the ADP-ribosylation site. The fact that the primary structure of EF-2 at the ADP-ribosylation site is highly conserved ensures either the correct recognition of the histidine residue by the enzymes involved in its modification to diphthamide, or the proper interaction with the diphtheria toxin.


The bovine protamine 2 gene: evidence for alternative splicing. Protamine 2 (PRM2) is a low molecular weight arginine-rich protein which is present in haploid spermatogenic cells of human and mouse. Although the bull PRM2 gene is translated and transcribed at low levels, the protein could not be detected. The gene was isolated from a cosmid library and was found to consist of two exons (298 and 50 bp, respectively) interrupted by an intron of 142 bp. As compared to the PRM2 genes of man, mouse and rat the bovine gene lacks a highly conserved sequence coding for the amino acids RLHRIH. Furthermore, primer extension experiments on bull PRM2 mRNA and sequencing of junction fragments revealed alternative splicing of mRNA resulting in two putative isoforms of the protein. The most abundant transcript is spliced at the conserved splice donor site found in exon 1 at position 236 giving rise to an in-frame deletion of 63 bp as compared to the cDNA sequence (Maier et al. (1990) Nucleic Acids Res. 18, 1249-1254). The less abundant longer mRNA was not detectable by radioactive primer extension. The corresponding cDNA was obtained by performing PCR with reverse transcribed bull testis RNA or with a spermatid specific cDNA library. Alternative splicing should result in an addition of 21 nonpolar amino acids in the derived polypeptide and an altered protein conformation and function.


Magnetic field-induced changes in specific gene transcription. Magnetic fields are physical, environmental agents that have been shown to produce a variety of responses in cellular and animal studies, including general changes in gene transcription. In this study, the nuclear run-off assay has been employed to assess alterations in specific gene transcription in CEM-CM3 T-lymphoblastoid cells exposed for 15-120 min to a 1 gauss sinusoidal magnetic field at 60 Hz. Time-dependent and cell density-dependent changes in the transcription of c-fos, c-jun, c-myc and protein kinase C (beta-form) have been observed and quantitated. Additionally, changes in transcript levels, assessed by slot-blot analysis, have been found to parallel the changes in gene transcription. These data suggest an important role for magnetic field exposure in altering cellular processes.


Readthrough of the Bacillus subtilis stop codon produces an extended enzyme displaying a higher polymerase activity. It has been generally accepted that the structural sacB gene of Bacillus subtilis levansucrase encodes a 50,000 Da extracellular protein. However, examination of the DNA sequence of the sacB flanking regions shows a putative open reading frame coding for a 20 amino acid peptide downstream immediately following the terminal TAA stop codon. By site-directed mutagenesis we have changed this stop codon to a glutamine codon. This stop codon readthrough leads to the synthesis and secretion by B. subtilis of a levansucrase possessing an extended polypeptide chain. The extended levansucrase has a molecular weight of 53,000 with a new carboxyl-terminus, rich in basic and hydrophobic amino acids and possessing one cysteine residue. This enzyme synthesizes fructosyl polymer levan of higher molecular weight than the shorter levansucrase. The increase in molecular weight was achieved by increasing the number of branches. These results suggest that the C-terminal part of the enzyme plays a specific role in the degree of branching of the synthesized polymer. Moreover, the extended enzyme is able to form an active dimer from two polypeptide chains linked by an S-S bridge.


Structure and expression of gene coding for a pupal cuticle protein of Bombyx mori. A specific protein termed as PCP accumulates in the newly synthesized pupal cuticle of the silkworm, Bombyx mori. We have cloned the genomic sequence encoding PCP and analyzed its structure. The PCP gene comprises two exons interspersed by a single intron approx. 5.8 kb in length. Transcription initiation sites of the PCP gene were located at nucleotide level. The 5' flanking region of the gene contains a sequence homologous to the Pit-1 DNA recognition element of the rat prolactin and growth hormone genes. The developmental profile of the PCP precursor RNA in epidermal cells showed that the biosynthesis of PCP is regulated at the transcriptional level in a stage- and tissue-specific fashion during post-embryonic development. Administration of 20-hydroxyecdysone to the isolated abdomens prepared from the early fifth instar larvae provoked the accumulation of PCP mRNA in epidermis, suggesting that the molting hormone triggers the expression of PCP gene.


Recombinant mouse tumor necrosis factor expressed in mammalian cells: effect of glycosylation on cytotoxic activity. A mouse tumor necrosis factor-alpha (TNF) expression vector, pTNFNeo, was constructed by inserting a 1.3 kb cDNA coding for a full structural region of mouse TNF into an expression plasmid BCMGSNeo. COS7 cells were transfected with the pTNFNeo and a G418-resistant transfectant, BK-2, which stably secreted lytic activity to L929 cells was cloned. The lytic activity in the BK-2 culture spent medium reached up to 6000 U/ml, and was completely and specifically inhibited with antiserum to mouse TNF. Gel filtration chromatography and Western blot analysis indicated that the recombinant TNF in the medium existed in associated forms composed of a mixture of 22 kDa and 17.5 kDa components. Glycopeptidase F digestion indicated that the 22 kDa species was an N-glycosylated form of the 17.5 kDa species. Specific activities of the 22 kDa and the 17.5 kDa species isolated were 6.9 x 10(5) U/mg and 8.1 x 10(6) U/mg, respectively, suggesting that carbohydrate moiety impaired the lytic activity.


Yeast ribosomal proteins: XIV. Complete nucleotide sequences of the two genes encoding Saccharomyces cerevisiae YL16. We isolated and sequenced YL16A and YL16B encoding ribosomal protein YL16 of Saccharomyces cerevisiae. The two nucleotide sequences within coding regions retain 91.1% identity, and their predicted sequences of 176 amino acids show 93.8% identity. Out of the ribosomal protein sequences from various organisms currently available, no counterpart to YL16 could be found.


Molecular cloning and characterization of genes encoding rat pancreatic cholecystokinin (CCK)-releasing peptide (monitor peptide) and pancreatic secretory trypsin inhibitor (PSTI). The genes encoding a rat pancreatic cholecystokinin (CCK)-releasing peptide (monitor peptide) and its structurally related peptide, rat pancreatic secretory trypsin inhibitor (PSTI), have been isolated and sequenced. The two genes share extremely high sequence similarity in the 5' flanking regions, suggesting that these regions may be responsible for the characteristic coordinate expression of the two peptides.


Isolation and nucleotide sequence of cecropin B cDNA clones from the silkworm, Bombyx mori. Two cDNA clones encoding cecropin B, an antibacterial protein, were isolated from a fat body cDNA library of the silkworm, Bombyx mori. Amino acid sequences of these clones, deduced from nucleotide sequences, were identical, including signal peptide regions. However, the nucleotide sequences were different at 30 positions. Deduced amino acid sequences of Bombyx mori cecropin B showed higher homology with cecropins from Lepidoptera than with those from Diptera.


Isolation and characterization of the rat gene encoding ornithine aminotransferase. Herein we describe the isolation and characterization of the rat gene encoding ornithine aminotransferase (rOAT). Six unique genomic clones were characterized and assigned to two nonoverlapping contigs representing approx. 33 kb of the rat genome. The 5' contig contains the rOAT promoter, exons 1 and 3, and a portion of exon 4; an exon corresponding to exon 2 of the human OAT gene (hOAT) was not identified. The rOAT promoter contains several putative regulatory elements in positions similar to hOAT. The 3' contig contains exons 7 through 11 in their entirety. Data presented and discussed herein suggest that approx. 3.0 kb of uncloned genomic DNA, containing the remainder of exon 4 and all of exons 5 and 6, separate the two contigs. Together, these data suggest that rOAT extends over approx. 20 kb and is organized into at least 10 exons, thereby closely resembling hOAT in size and exon/intron organization. Isolation of rOAT provides an important tool for examining the molecular mechanisms through which estrogen and thyroid hormone regulate transcription of this gene in a cell-type specific manner.


cDNA sequence for rat dermatan sulfate proteoglycan-II (decorin). A cDNA clone for dermatan sulfate proteoglycan-II, or decorin, has been isolated from a rat uterus library and sequenced. The cDNA and deduced amino acid sequences are 79 and 77% identical to the previously reported human and bovine sequences, respectively. The rat protein contains potential attachment sites for two glycosaminoglycan chains and four N-linked oligosaccharides, six conserved cysteine residues and multiple repeats of a leucine-rich sequence, LXXLXLXXNXL/I. Overlapping the C-end of one of these repeats is an NKISK sequence, which has been implicated in binding to fibronectin.


The primary structure of rat rig/ribosomal protein S15 gene. We have isolated rat rig/ribosomal protein S15 gene from a DNA library derived from a rat insulinoma and determined the complete nucleotide sequence. The rat rig/S15 gene is composed of four exons and three introns spanning 2 kbp and exhibits distinctive structural features unique for a ribosomal protein gene.


Platelet adhesion to collagen-coated wells: analysis of this complex process and a comparison with the adhesion to matrigel-coated wells. The mechanisms of platelet adhesion to collagen type III-coated wells and Matrigel-coated wells were analyzed. The adhesion of 51Cr-labeled platelets to collagen-coated wells showed a biphasic pattern. The early stage of adhesion was inhibited by antibodies against platelet glycoprotein(GP)s Ia/IIa and VI. The later stage of platelet adhesion was inhibited by an antibody against the GPIIb/IIIa complex and a concomitant release of 14C-labeled serotonin was observed. The percentage of adhered platelets was increased when a higher platelet concentration was added in the reaction medium. These results indicated that the adhesion assay of platelets to collagen-coated wells was composed of two reactions: the first one is the platelet-collagen interaction that depends on GPIa/IIa and GPVI on the platelet surface; and the second reaction is the platelet-platelet interaction, platelet aggregation, which depends on GPIIb/IIIa. The adhesion of platelets to Matrigel-coated wells was indicated to involve platelet-Matrigel interactions that were partly dependent on the laminin in the Matrigel solution.


Skeletal muscle Pi transport and cellular [Pi] studied in L6 myoblasts and rabbit muscle-membrane vesicles. In the rat skeletal myoblast line L6 and in a rabbit skeletal muscle sarcolemma/t-tubule vesicle preparation, [32P]Pi uptake was largely dependent on the transmembrane Na gradient. Na-dependent [32P]Pi uptake had a hyperbolic relationship to [Pi] and [Na], being half-maximal at 0.2-0.3 mM [Pi] and at 25-40 mM [Na]. In vesicles the Na-dependence suggests that approx. two Na are transported with each Pi, but the inhibition of [32P]Pi uptake at high pH suggests that the Pi monoanion is the transported form. Together these imply electrogenic transport and this is confirmed by the results of manipulating the vesicle membrane potential. Thus, electrogenic Na-Pi co-transport exploits both the sodium gradient and the cell membrane potential to maintain muscle cellular [Pi] against an unfavourable electrochemical gradient. The low [Pi] for half-maximal flux may partly explain the small effect of altered extracellular [Pi] on cellular [Pi]. In L6 myoblasts most 32P was first detectable in an organic phosphate pool rather than cellular Pi, while the specific activity of cell Pi rapidly reached 40% of that of extracellular Pi and was stable for at least 3 h. These results are discussed in terms of the organisation of cellular phosphate metabolism.


Antiproliferative effect of phorbol esters on MCF-7 human breast adenocarcinoma cells: relationship with enhanced expression of transforming growth-factor-beta 1. In human breast carcinoma MCF-7 cells, phorbol diesters inhibit proliferation and induce cell maturation. We have recently reported that exogenous TGF-beta 1 reverses the resistance of a breast adenocarcinoma MCF-7 subline (MCF-7:RPh-4) to these phorbol ester effects. Here, we investigated the involvement of TGF-beta 1 in the PKC-mediated inhibition of breast-cancer cell proliferation. Parental MCF-7-conditioned medium contained a 20-fold higher transforming activity on NRK-49F fibroblasts than the TPA-resistant subline. TPA increased TGF-beta activity in MCF-7 conditioned medium. MCF-7 cells also expressed more TGF-beta 1 mRNA than the resistant subline. TPA induced a dose-dependent increase in TGF-beta 1 mRNA levels that paralleled the inhibitory effect on MCF-7 proliferation. The lower level of TGF-beta mRNA expression in TPA resistant subline was not modified after addition of TPA, but was significantly increased in the presence of exogenous TGF-beta 1. These data argue in favor of a role of endogenous TGF-beta 1 in the maturation process induced by protein kinase C activation.


What dual-action agents reveal about acid secretion: a combined experimental and modeling analysis. A previously described simple mathematical model for gastric acid secretion is employed to characterize the acid secretion response to thiocyanate and omeprazole, two partially conservative inhibitors that inhibit both the formation and translocation of acid. In addition, the effect of dithiothreitol on the omeprazole inhibition was investigated. The model parameters were estimated from four data sets by a non-linear least squares procedure: a dynamic (time-dependent) set, made up of individual acid secretion rate curves; and three integral (time-independent) sets consisting of the curves of acid secreted and suppressed above and below baseline, respectively, and the index of conservation, all three as functions of agent exposure. At the translocation step the inhibition function was the same for the two inhibitors, and though similar at the formation step, they differ in that a Hill coefficient larger than unity is necessary in the thiocyanate inhibition function of the acid formation. Dithiothreitol protects only the formation step from inhibition by omeprazole. Hence, the binding sites for omeprazole are different for formation and translocation. This study exemplifies a non-invasive method for the investigation of the mechanism of gastric acid secretion following perturbation by external agents.


Interleukin-1 beta enhances the response of human articular chondrocytes to extracellular ATP. It has been observed that both interleukin-1 (IL-1) and extracellular ATP stimulate the production of prostaglandin E (PGE) by human articular chondrocytes in monolayer culture. The combined effects of recombinant human IL-1 beta and ATP were therefore studied using these cells. IL-1 beta rapidly enhanced the response to a maximally effective concentration of ATP (100 microM). On continuous exposure of the cells to the cytokine, its effect was greatest after approx. 24 h and tended to decline thereafter. The enhancement of the response to 100 microM ATP by IL-1 beta was dose-dependent. Removal of IL-1 beta prior to treating the cells with 100 microM ATP did not affect the degree of enhancement of the response. The effect of the cytokine on the response to suboptimal concentrations of extracellular ATP was also tested. IL-1 beta lowered the minimum concentration of ATP required to elicit an increase in the production of PGE by human articular chondrocytes. These findings are of interest, since they indicate a synergistic interaction between a cytokine and a purinergic agonist. Moreover, since both the sensitivity of the cells to extracellular ATP and the maximum response to this agent were enhanced, it is possible that IL-1 modulates more than one step in the process of P2-purinoceptor-mediated stimulation of PGE production. These observations may be relevant to the pathogenesis of some forms of arthritis.


A retrovirus encoding the v-fps protein-tyrosine kinase induces factor-independent growth and tumorigenicity in FDC-P1 cells. There is increasing evidence that protein-tyrosine kinases play pivotal roles in the response to growth-factor signals. The cytoplasmic tyrosine kinase c-fps/fes, due to its restricted expression in hematopoietic tissue, is likely to participate in hematopoietic growth-factor signalling. We have introduced a retrovirus containing an activated fps gene (encoding P130gag-fps) into the growth factor-dependent myeloid cell line FDC-P1. Clonal cell lines were derived by selection for a marker gene coding for G418 resistance in the absence or presence of the hematopoietic growth factor IL-3. G418 resistant clones expressed P130gag-fps and its associated protein-tyrosine kinase activity and displayed either a factor-independent or IL-3 hypersensitive phenotype and were tumorigenic in syngeneic recipients. Thus, introduction of the activated v-fps gene was able to circumvent the requirement for exogenous growth factors by FDC-P1 cells. Bioassay of conditioned medium from the various clones did not detect hematopoietic growth factor activity and PCR analysis for IL-3 transcripts were negative, suggesting that growth-factor independence was achieved by a mechanism other than autocrine production of a growth factor. We suggest that P130gag-fps is acting to directly stimulate a hematopoietic growth-factor signalling pathway, perhaps one that normally involves the endogenous c-fps/fes protein-tyrosine kinase of FDC-P1 cells.


The synthesis of p58cyclin A and the phosphorylation of p34cdc2 are inhibited in human lymphoid cells arrested in G1 by alpha-interferon. Daudi cells arrest in G1 in the presence of alpha-interferon. Such cells have little p58cyclin A, probably due to inhibition of p58cyclin A synthesis. The phosphorylation-associated migration shift of p34cdc2 is not seen in alpha-interferon-arrested cells. Cells arrested in late G1 by aphidicolin have abundant p58cyclin A and phosphorylated p34cdc2. Cell sorting showed that p58cyclin A increases in proliferating cells in late G1 and coincides with phosphorylation of p34cdc2.


Photoaffinity labeling of integrin alpha IIb beta 3 (glycoprotein IIb-IIIa) on intact platelets with 8-azido-[gamma-32P]ATP. The fibrinogen receptor GPIIb-IIIa plays a crucial role in platelet aggregation. Here we show that the adenine nucleotide, 8-azido-ATP, inhibits ADP-induced conformational change of the platelet fibrinogen receptor GPIIb-IIIa (integrin alpha IIb beta 3). Photoaffinity labeling of intact platelets with 8-azido-[gamma-32P]ATP exclusively modifies two plasma-membrane glycoproteins which are identical with both subunits of GPIIb-IIIa. The presence of adenine-nucleotide-binding sites on GPIIb-IIIa implies that the platelet fibrinogen receptor is directly regulated by extracellular adenine nucleotides.


Alpha 5 integrin subunit expression changes during myogenesis. Fibronectin and its cellular receptor, the alpha 5 beta 1 integrin, are involved in the transmembrane signalling events that control muscle cell differentiation. In this study, the expression of the alpha 5 integrin subunit was followed by reverse transcription-polymerase chain reaction (RT-PCR) to determine alterations during myogenesis. In studies of murine muscle, we found a 90% reduction in the level of the alpha 5 integrin subunit mRNA during early postnatal development. Concurrently, the fibronectin alternative splicing pattern changed markedly in the EIIIB and V exons. In-vitro analyses of these molecules during myoblast differentiation revealed changes that followed trends similar to those observed in vivo, although of lesser magnitude. These observations imply an important role of fibronectin and the alpha 5 integrin subunit in muscle development.


Comparative investigations on the amino-acid sequences of different isolectins from the sponge Axinella polypoides (Schmidt). The sponge Axinella polypoides contains four different D-galactose binding lectins and one, termed lectin IV, which is specific for hexuronic acids. Only the D-galactose binding lectins were investigated in this study. The complete amino-acid sequence of lectin I, the main component in the crude extract was determined. Lectin I is a homodimer and each subunit comprises 144 amino acids with a M(r) of 15,847 +/- 10, as calculated from the sequence data and determined by mass spectrometry. Each subunit contains one intrachain disulfide bridge between positions 4 and 46. Of lectin II, only the first 49 amino acids of the NH2-terminal end were analysed. This part has 29 amino acids in common with lectin I, including a cysteine residue at position 4, also suggesting an intrachain loop in a identical position as in lectin I. The molecular mass of its subunit is 16,235 +/- 10 Da. Only the first 15 NH2-terminal amino acids of lectins III and V could be sequenced. Lectin V was identical to lectin II in all positions, whereas lectin III showed only 5 residues identical to lectins I or II. Thus, lectins I, II and III are derived from three different genes, whereas lectin V may either be a proteolytic cleavage product, or result from different splicing events or may be derived also from a separate gene. Neither of the four lectins showed any similarity to known lectin sequences of animal or plant origin.