The differential effect of thyrotropin on the electrical responses of thyroid cells in monolayer cultures of varying duration. The acute effects of thyrotropin on the membrane potential of thyroid cells maintained in the presence or absence of thyrotropin (0.2 U/ml) in the culture medium was determined. Monolayer cultures were prepared from porcine thyroid glands and cultured for 4--17 days after which the culture medium was exchanged for a buffered salt solution for intracellular measurements of the membrane potential. Cells were serially impaled with a microelectrode, first in the absence and then in the presence of 10 mU/ml thyrotropin. Cells cultured for 4--9 days depolarized from --29.6 +/- 1.7 (mean +/- S.E.) to --19.3 +/- 1.3 mV within 10 min after acute addition of 10 mU/ml thyrotropin. From 11 to 17 days of culture, basal membrane potentials were lower and, in most instances, cell hyperpolarization occurred within 30 min in response to thyrotropin. There was no difference in the electrical response of cells maintained in culture with or without thyrotropin. However, cells cultured with thyrotropin formed follicle-like structures in contrast to the monolayer formation of cells cultured without thyrotropin. The changes in the basal and stimulated electrical responses occur within a time frame similar to that reported for changes in the biosynthetic capacity of thyroid cells in culture. The data further emphasize the possible regulatory role of the cell membrane in stimulus-secretion coupling in the thyroid.


The modulation of membrane fluidity by hydrogenation processes. III. The hydrogenation of biomembranes of spinach chloroplasts and a study of the effect of this on photosynthetic electron transport. A method is reported for the in situ modification of the lipids of isolated spinach chloroplast membranes. The technique is based on a direct hydrogenation of the lipid double bonds in the presence of the catalyst, chlorotris(triphenylphosphine)rhodium (I). The pattern of hydrogenation achieved suggests that the catalyst distributes amongst all of the membranes. The polyunsaturated lipids within the membranes are hydrogenated at a faster rate and at an earlier stage than are the monoenoic lipids. Whilst addition of the catalyst to the chloroplast causes an initial 10--20% decrease in Hill activity, saturation of up to 40% of the double bonds present can be accomplished without causing further significant alterations in photosynthetic electron transport processes or marked morphological changes of the chloroplast structure as observed in the electron microscope.


Thermotropic phase transitions of phosphatidylcholines with odd-numbered n-acyl chains. Diacyl phosphatidylcholines with n-C13, -C15, -C17 and -C19 saturated acyl chains have been synthesized and their phase transitions in the presence of excess water monitored by differential thermal analysis. The C15-, C17- and C19-diacyl species show gel to liquid-crystalline transitions and pretransitions like those of the even-chain phosphatidylcholines. A plot of the main phase transition temperature, Tc, vs. acyl chain length is a smooth curve on which the Tc values of both odd- and even-chain species fall, while a similar plot for the pretransition temperature, Tp, shows significant alternation of Tp between odd- and even-chain species. Consideration of these results in terms of the physical basis of the odd-even alternation of phase transition temperatures in homologous series of paraffinic compounds suggests that the acyl chains of disaturated phosphatidylcholines are tilted with respect to the bilayer normal below Tp but become perpendicular to the bilayer surface above the pretransition temperature.


Physicochemical characterization of 1,2-diphytanoyl-sn-glycero-3-phosphocholine in model membrane systems. We report here on a series of studies aimed at characterization of the structural and dynamical properties of the synthetic lipid diphytanoyl phosphatidylcholine, in multilamellar dispersions and vesicle suspensions. The lipid exhibits no detectable gel to liquid crystalline phase transition over a large temperature range (-120 degrees C to +120 degrees C). Examination of proton nuclear magnetic resonance (NMR) free induction decays obtained from multilayer dispersions of diphytanoyl phosphatidylcholine provided an estimate of the methylene proton order parameter. The estimated magnitude of 0.21 is comparable to those determined for other phospholipids. Sonication of aqueous dispersions of diphytanoyl phosphatidylcholine led to formation of bilayer vesicles as determined by the measurement of the outer/inner choline methyl proton resonances, vesicle sizes in electron micrographs, and comparison of proton NMR linewidths between multilayer and sonicated dispersions. Ultracentrifugation studies of diphytanoyl phosphatidylcholine vesicles in H2O and 2H2O media yielded a value of 1.013 +/- 0.026 ml/g for the partial specific volume of this lipid. We have measured spin lattice relaxation rates for the methyl and methylenemethyne protons of the hydrocarbon chains of diphytanoyl phosphatidylcholine in bilayer vesicles over a range of temperatures and at two NMR frequencies (100 and 220 MHz). The observed relaxation rates for the methylene protons in this system were approximately twice those previously reported for dipalmitoyl phosphatidylcholine at comparable temperatures and resonance frequencies, whereas the relaxation rates measured for the methyl protons were greater than those of the straight chain lipid by an order of magnitude. Measurement of the spin lattice relaxation rates of the hydrocarbon protons of the diphytanoyl phosphatidylcholine in a 10 mol% mixture of the branched-chain lipid in a deuterated host lipid, diperdeuteropalmitoyl phosphatidylcholine, showed a discontinuity in the temperature dependence of the proton NMR longitudinal relaxation rates of the branched-chain lipid in the region of the gel to liquid crystalline phase transition temperature of the deuterated dipalmitoyl phosphatidylcholine host lipid. This result may be taken as evidence of lateral phase separation of a liquid cyrstalline phase enriched in diphytanoyl phosphatidylcholine from a gel phase enriched in diperdeuteropalmitoyl phosphatidylcholine at temperatures below the phase transition temperature of deuterated host lipid. This conclusion is supported by the observation of an abrupt change in the hydrocarbon methylene linewidth (at 100 MHz) of 10 mol% diphytanoyl phosphatidylcholine in diperdeuteropalmitoyl phosphatidylcholine over the temperature range where lateral phase separation is taking place according to differential thermograms.


Negatively charged phospholipids and their position in the cholesterol affinity sequence. The effects of low concentrations of cholesterol in mixtures of a negatively charged phospholipid (phosphatidylserine or phosphatidylglycerol) and another phospholipid (phosphatidylcholine, sphingomyelin or phosphatidylethanolamine) have been studied by differential scanning calorimetry. Only mixtures which showed a gel phase miscibility gap have been employed. It was demonstrated that in mixtures with phosphatidylethanolamine, cholesterol was preferentially associated with the negatively charged phospholipid, regardless whether this species represented the component with the high or with the low transition temperature in the mixture. In mixtures of a negatively charged phospholipid and phosphatidylcholine, cholesterol associated with the negatively charged phospholipid; when the phosphatidylcholine was the species with the low transition temperature, cholesterol had an affinity for the phosphatidylcholine and for the negatively charged phospholipid as well. Cholesterol, in a mixture of sphingomyeline with a high and phosphatidylserine with a low transition temperature, was preferentially associated with sphingomyelin. From these experiments it is concluded that phospholipids show a decrease in affinity for cholesterol in the following order: sphingomyelin greater than phosphatidylserine, phosphatidylglycerol greater than phosphatidylcholine greater than phosphatidylethanolamine.


The lateral distribution of intramembrane particles in the erythrocyte membrane and recombinant vesicles. Triton X-100 (in concentrations which did not cause a significant solubilization of membrane material) caused aggregation of the intramembrane particles of human erythrocyte ghosts. Ghosts from which the extrinsic proteins had been removed by alkali treatment showed a temperature-induced aggregation of the particles. With virtually no spectrin present, the particles in these stripped ghosts could still be aggregated by manipulations with ionic strength and pH, or by the addition of calcium. Recombinant vesicles were made from a Triton X-100 extract and a mixture of phospholipids with a composition which resembled that of the inner monolayer of erythrocyte membrane. In these recombinants the same manipulations with ionic strength and pH and the addition of calcium caused a rearrangement of the particles, resulting in the appearance of particle-free areas. In recombinants prepared from a Triton X-100 extract and egg phosphatidylcholine the lateral distribution of the particles was not altered by these manipulations. It is concluded that in the erythrocyte membrane the intramembrane particles can be aggregated by effects of external agents on lipid components. In this light the role of spectrin in stabilizing the membrane by interactions with lipids in the inner monolayer is discussed.


Inside-outside distribution and diffusion of phosphatidylcholine in rat sarcoplasmic reticulum as determined by 13C NMR and phosphatidylcholine exchange protein. 1. The transverse distribution of phosphatidylcholine in rat sarcoplasmic reticulum was investigated employing 13C NMR in conjunction with the shift reagent DyCl3. 2. Sarcoplasmic reticulum phosphatidylcholine was enriched with 13C by feeding rats a diet containing [N-Me3-13C]choline. Up to 32% of the sarcoplasmic reticulum phosphatidyl-[N-Me3-12C]choline was replaced by phosphatidyl-[N-Me3-13C]choline. 3. Titration of 13C-enriched sarcoplasmic reticulum with Dy3+ indicates that 40% of the phosphatidyl-[N-Me3-13C]choline is exposed to the external medium, whereas 60% is shielded from interaction with Dy3+. 4. Incubation of 32P-labelled sarcoplasmic reticulum with excess mitochondria and phosphatidylcholine exchange protein results in a fast transport of approx. 80% of [32P]phosphatydylcholine to the mitochondria indicating that part of the phosphatidylcholine pool is involved in a rapid transbilayer movement.


The occurrence of lipidic particles in lipid bilayers as seen by 31P NMR and freeze-fracture electron-microscopy. A new type of lipid organization is observed in mixtures of phosphatidyl-choline with cardiolipin (in the presence of Ca2+), monoglycosyldiglyceride and phosphatidylethanolamine (in the presence of cholesterol). This phase is characterised by an isotropic 31P NMR signal and is visualised by freeze-fracturing as particles and pits on the fracture faces of the lipid bilayer. As the most favourable model for this phase we propose the inverted micelle sandwiched in between the two monolayers of the lipid bilayer.


Transverse distribution and movement of lysophosphatidylcholine in sarcoplasmic reticulum membranes as determined by 13C NMR and lysophospholipase. 1. The transverse distribution of 1-palmitoyl-sn-glycero-3-phospho-N-[Me-13C]-choline in vitro incorporated in sarcoplasmic reticulum has been measured by means of 13C NMR and DyCl3 as an impermeable shift reagent. 2. Lysophosphatidylcholine added to the membranes equilibrates within 30 min at 20 degrees C between outer and inner membrane leaflet so that 42% is located in the inner leaflet. 3. Lysophosphatidylcholine diffuses back from the inner leaflet to the outer upon lysophospholipase action on the outer lysophosphatidylcholine pool.


Calcium transport by pigeon erythrocyte membrane vesicles. Membrane vesicles from pigeon erythrocytes show a rapid, ATP-dependent accumulation of 45Ca2+.Ca2+ accumulation ratios greater than or approximately equal to 10(4) are readily attained. For ATP-dependent Ca2+ uptake, V is 1.5 mmol.1(-1).min(-1) at 27 degrees C (approx. 0.9 nmol.mg-1 protein.min-1), [Ca2+]1/2 is 0.18 microM, [ATP]1/2 is 30--60 microM, the Ca2+ uptake rate depends on [Ca2+]2 and the dependence of uptake rate on ATP concentration implies strong ATP-ATP cooperativity. The Arrhenius activation energy is 19.1 +/- 1.4 kcal/mol and the pH optimum is approx. 6.9.


Molecular and biosynthetic heterogeneity of fucosyl glycoproteins associated with rat brain synaptic functions. The composition and biosynthesis of fucosyl glycoproteins present in rat brain synaptic membranes and synaptic junctions were investigated. Reaction with 125I-labelled fucose-binding protein (Lotus tetragonolobus) following sodium dodecyl sulphate gel electrophoresis identified 6--8 fucosyl glycoproteins in synaptic membranes but only three major high molecular classes (Mr = 180 000, 130 000 and 110 000) in synaptic junctions. Affinity chromatography on concanavalin A-Sepharose resolved each of the synaptic junctional fucosyl glycoproteins into concanavalin A-positive and negative components indicating the presence of at least six high molecular weight fucosyl glycoproteins in synaptic junctions. Following the administration of [3H]fucose synaptic membranes, synaptic junctions and post-synaptic densities incorporated isotope, the order of relative specific activities being synaptic membranes greater than synaptic junctions greater than post-synaptic densities. Fractionation of [3H]fucose-labelled synaptic junctions on concanavalin A-Sepharose revealed a time-dependent increase in the percentage of isotope associated with the concanavalin A-positive glycoproteins. The results demonstrate both molecular and biosynthetic heterogeneity of fucosyl glycoproteins associated with synaptic junctions.


Active Ca2+ transport by vesicles reconstituted from Triton X-100-solubilized pigeon erythrocyte membrane. Pigeon erythrocyte membrane was solubilized partially, but relatively unselectively by Triton X-100. Vesicles were reconstituted from mixtures of Triton-solubilized membrane and lipid (phosphatidylcholine plus phosphatidyl-ethanolamine plus cholesterol) by addition of bovine high-density lipoprotein. This efficiently removed the Triton X-100. Sodium dodecyl sulfate-polyacrylamide gel electropherograms of reconstituted vesicles showed band patterns resembling those of the original membrane. The reconstituted vesicles showed ATP-dependent active accumulation of 45Ca2+. ATP-dependent 45Ca2+ uptake by the reconstituted vesicles resembled the corresponding activity of the original membrane vesicles; in both preparations the Ca2+ uptake rate depended on the square of the Ca2+ concentration and had similar [Ca2+]1/2 values, 0.16 microM and 0.18 microM, respectively.


Effect of cholesterol on the valinomycin-mediated uptake of rubidium into erythrocytes and phospholipid vesicles. Human erythrocytes have been treated with lipid vesicles in order to alter the cholesterol content of the cell membrane. Erythrocytes have been produced with cholesterol concentrations between 33 and 66 mol% of total lipid. The rate of valinomycin-mediated uptake of rubidium into the red cells at 37 degrees C was lowered by increasing the cholesterol concentration of the cell membrane. Cholesterol increased the permeability to valinomycin at 20 degrees C of small (less than 50 nm), unilamellar egg phosphatidylcholine vesicles formed by sonication. Cholesterol decreased the permeability to valinomycin at 20 degrees C of large (up to 200 nm) unilamellar egg phosphatidylcholine vesicles formed by freeze-thaw plus brief sonication. It is concluded that cholesterol increases the permeability of small membrane vesicles to hydrophobic penetrating substances while above the transition temperature but has the opposite effect on large membrane vesicles and on the membranes of even larger cells.


Exchange and interactions between lipid layers at the surface of a liposome solution. Lipid organization and lipid transport processes occurring at the air-water interface of a liposome (lipid vesicle) solution are studied by conventional surface pressure-area measurements and interpreted by an adequate theory. At the interface of a dioleoyl phosphatidylcholine vesicle solution, used for demonstration, a well defined two layer structure selfassembles: vesicles disintegrate at the interface forming a surface-adsorbed lipid monolayer, which prevents further disintegration beyond about 1 dyne/cm surface pressure. A layer of vesicles now assembles in close association with the monolayer. This layer is in vesicle diffusion exchange with the solution and in lipid exchange with the monolayer. The lipid exchange occurs exclusively between the monolayer and the outer lipid layer of the vesicles; it is absent between outer and inner vesicle layers. Equilibration of the lipid density in the monolayer with that in the vesicle outer layer provides a coherent and quantitative explanation of the observed hysteresis effects and equilibrium states. The correspondence between monolayer and vesicle outer layer is traced down to equilibrium constants and rate constants and their dependences on surface pressure, vesicle size and concentration. Other alternate realizations of surface structure and exchange, including induced lipid flip-flop within vesicles or vesicle monolayer adhesion or fusion are potential applications of the proposed analysis.


Sodium influx rate and ouabain-sensitive rubidium uptake in isolated guinea pig atria. 1. Ouabain-sensitive 86Rb+ uptake by tissue preparations has been used as an estimate of Na+ pump activity. This uptake, however, may be a measure of the Na+ influx rate, rather than capacity of the Na+ pump, since intracellular Na+ concentration is a determinant of the active Na+/Rb+ exchange reaction under certain conditions. This aspect was examined by studying the effect of altered Na+ influx rate on ouabain-sensitive 86Rb+ uptake in atrial preparations of guinea pig hearts. 2. Electrical stimulation markedly enhanced ouabain-sensitive 86Rb+ uptake without affecting nonspecific, ouabain-insensitive uptake. Paired-pulse stimulation studies indicate that the stimulation-induced enhancement of 86Rb+ uptake is due to membrane depolarizations, and hence related to the rate of Na+ influx. 3. Alterations in the extracellular Ca2+ concentration failed to affect the 86Rb+ uptake indicating that the force of contraction does not influence 86Rb+ uptake. 4. Reduced Na+ influx by low extracellular Na+ concentration decreased 86Rb+ uptake, and an increased Na+ influx by a Na+-specific ionophore, monensin, enhanced 86Rb+ uptake in quiescent atria. 5. Grayanotoxins, agents that increase transmembrane Na+ influx, and high concentrations of monensin appear to have inhibitory effects on ouabain-sensitive 86Rb+ uptake in electrically stimulated and in quiescent atria. 6. Electrical stimulation or monensin enhanced ouabain binding to (Na+ + K+)-ATPase and also increased the potency of ouabain to inhibit 86Rb+ uptake indicating that the intracellular Na+ available to the Na+ pump is increased under these conditions. 7. The ouabain-sensitive 86Rb+ uptake in electrically stimulated atria was less sensitive to alterations in the extracellular Na+ concentration, temperature and monensin than that in quiescent atria. 8. These results indicate that the rate of Na+ influx is the primary determinant of ouabain-sensitive 86Rb+ uptake in isolated atria. Electrical stimulation most effectively increases the Na+ available to the Na+ pump system. The ouabain-sensitive 86Rb+ uptake by atrial preparations under electrical stimulation at a relatively high frequency seems to represent the maximal capacity of the Na+ pump in this tissue.


A high affinity site for sugar transport at the inner face of the human erythrocyte membrane? A disagreement centering on a method of analysis as to the existence of a high affinity site for glucose transport at the inner face of the human red cell membrane is resolved by using direct fitting methods to confirm the original parameter estimates.


Hypoglycemic drugs increase liver plasma membrane microviscosity in vitro. The effect of hypoglycemic drugs chlorpropamide and phenformin has been tested on isolated liver plasma membranes as to their microviscosity parameters. Results reported suggest that both drugs are able to increase in vitro plasma membrane microviscosity in a dose-dependent way.


The structure of melittin in lipid bilayer membranes. 0.15 M inorganic phosphate dramatically increased the alpha-helix content of melittin in aqueous solution. When melittin interacted with egg yolk phosphatidylcholine liposomes in the absence of inorganic phosphate, it was converted to an alpha-helix rich form, as postulated by Dawson et al. (Dawson, C.R., Drake, A.F. Helliwell, J. and Hider, R.C. (1978) Biochim. Biophys. Acta 510, 75--86).


Sodium dependence of neutral amino acid uptake into rabbit ileum. Alanine uses two mediated pathways to enter the rabbit ileal mucosa. Present results suggest that one of them (Km = 4.1 mM) is fully dependent on sodium in the mucosal medium, while the other (Km = 91 mM) is sodium-independent. Similar results are obtained for methionine and serine. Reinterpretation of previous alanine/sodium coupling coefficients suggests that two sodium ions per alanine molecule are transported via the high affinity system.


Complex phase mixing of phosphatidylcholine and phosphatidylserine in multilamellar membrane vesicles. The phase mixing in dipalmitoyl phosphatidylcholine and bovine brain phosphatidylserine mixtures suspended in aqueous salt solutions was studied by differential scanning calorimetry, freeze-fracture electron microscopy and X-ray and electron diffraction. The pure dipalmitoyl phosphatidylcholine has two well-defined solidus phases P beta' and L beta' and a liquidus phase L alpha while the pure phosphatidylserine has a broad transition from L beta to L alpha. The mixture exhibits some dipalmitoyl phosphatidylcholine characteristics up to 30% phosphatidylserine when pure P beta' phase no longer exists. Phase mixing is observed at all compositions. An addition of 3% phosphatidylserine is found to be sufficient to destroy the sharp L beta'--P beta' transition observed in pure dipalmitoyl phosphatidylcholine. The transition between L beta' and P beta' in these mixtures is displaced to lower temperatures and becomes gradual as depicted by all three techniques, but freeze-fracture gives more definite information of the continuous transition. The most striking observation on the morphology of the mixed membranes (3--30% phosphatidylserine in dipalmitoyl phosphatidylcholine) is the presence of banded patterns (P beta') at temperatures well below the main transition peak as detected by differential scanning calorimetry.


Stimulation of monovalent cation fluxes by electron donors in the human red cell membrane. When human red cells are incubated at 37 degrees C with the artificial electron donor system ascorbate + phenazine methosulphate the fluxes of Rb+ (K+) through the cell membrane are increased. The effect of this donor system is much stronger in energy-depleted than in normal cells. The same effects are produced by HS-glutathione, NADH or NADPH loaded into resealed ghosts, but these electron donors were ineffective when added to the incubation medium. The Rb+ (K+) fluxes induced by electron donors resemble closely those induced by an increase of intracellular Ca2+ (Gardos effect). The electron donors require the presence of intracellular Ca2+ to be effective, but at levels that do not stimulate by themselves the fluxes of K+. Flavoenzyme inhibitors (atebrin and chlorpromazine), oligomycin and quinine prevented the effects of both electron donors and Ca2+ alone; antimycin, upcouplers and ethacrynic acid inhibited them partially; ouabain, furosemide, and rotenone had no effect. The results could be explained if the effect of electron donors is to bring about a change in the redox state of some membrane component(s) that makes intracellular Ca2+ more effective to elicit rapid K+ movements. Plasma membrane oxidoreductase activities could be engaged in this change.


Lipid composition and microviscosity of subcellular fractions from rabbit thymocytes. Differences in the microviscosity of plasma membranes from subclasses of thymocytes. There are indications from freeze-fracture experiments that subclasses of rabbit thymocytes show different mobilities of plasma membrane components. Consequently, one would expect differences in the fluidity of the plasma membrane. For this reason, rabbit thymocytes were separated on a Ficoll/Metrizoate gradient yielding three subclasses representing various levels of cell differentiation. These thymocyte subclasses did not show any significant differences in the degree of fluorescence polarization using the probe 1,6-diphenyl-1,3,5-hexatriene. The fluorescence polarization of the plasma membrane may be overshadowed by the contribution of all cellular lipids due to penetration of the fluorescent probe into the cell. Therefore, plasma membranes were isolated from rabbit thymocytes using a cell-disrupting pump, differential centrifugation, and sucrose density gradient centrifugation. As shown by biochemical and electron microscopical analyses, plasma membranes with a high degree of purity were obtained. As expected the plasma membrane fractions showed a higher microviscosity than the other subcellular fractions. This was attributed to a higher cholesterol to phospholipid molar ratio and a higher degree of saturation of phospholipid fatty acid chains. Subsequently, the microviscosity was measured of plasma membrane preparations obtained from two main subclasses of thymocytes representing mature and immature lymphocytes. The immature thymocytes yielded two plasma membrane fractions with higher microviscosity than the mature cells. These finding is in line with earlier observed differences in the glycerol-induced clustering of intramembranous particles. Furthermore, the results of this study support the view that the fluorescence polarization technique applied to whole cells does not exclusively monitor the plasma membrane.


Sodium gradient dependence of proline and glycine uptake in rat renal brush-border membrane vesicles. The sodium-dependent entry of proline and glycine into rat renal brush-border membrane vesicles was examined. The high Km system for proline shows no sodium dependence. The low Km system for glycine entry is strictly dependent on a Na+ gradient but shows no evidence of the carrier system having any affinity for Na+. The low Km system for proline and high Km system for glycine transport appear to be shared. Both systems are stimulated by a Na+ gradient and appear to have an affinity for the Na+. The effect of decreasing the Na+ concentration in the ionic gradient is to alter the Km for amino acid entry and, at low Na+ concentrations, to inhibit the V for glycine entry.


The calculated glucose concentration profile in the intercellular spaces of everted jejunum of rat. On the basis of experimental data from isolated and everted rat jejunum and on extension of Diamond and Bossert's mathematical model, the glucose concentration in the intercellular spaces has been calculated and has been found higher (less than 2 mM) than that present in the serosal space.


Levels and distributions of phospholipids and cholesterol in the plasma membrane of neuroblastoma cells. Murine neuroblastoma cells (clone N-2A) grown in suspension (spinner cells) or attached on a plastic surface (monolayer cells) were used in studies of the phospholipid and cholesterol composition of whole cells, primary plasma membranes, plasma membranes internalized during phagocytosis of polystyrene latex beads, mitochondria and microsomes. Monolayer cells contained higher concentrations of total phospholipid, phosphatidylserine and phosphatidylcholine, and lower concentration of phosphatidylethanolamine than spinner cells. The cholesterol levels and the relative proportions of the various phospholipids were similar in both cell types except phosphatidylethanolamine and sphingomyelin whose proportions were lower in monolayer cells. The primary plasma membranes of the two cell types differed significantly in the relative proportions of all phospholipids, except sphingomyelin, and the phospholipid to protein and the cholesterol to protein ratios were all higher in the membranes of spinner cells. In contrast to these results, all the phospholipid to protein and the cholesterol to protein ratios of the internalized plasma membranes were higher in monolayer than in spinner cells, and the proportions of all phospholipids, except phosphatidylethanolamine, were similar in both cell types. The membrane distributions of individual phospholipids and cholesterol were inferred from comparison of the phospholipid and cholesterol compositions of primary plasma membranes and plasma membranes internalized during phagocytosis of polystyrene beads. The results are consistent with a non-random distribution of most phospholipids in both spinner and monolayer cells, but the patterns of these distributions were different in the two cell types. With regard to cholesterol the results are compatible with a random or a heterogeneous distribution. All the phospholipid to protein ratios of the mitochondrial fraction of both cell types were lower than those of the plasma membranes. However, these ratios of the microsomal fraction were higher than those of the plasma membranes of monolayer cells, whereas they were comparable, with a few exceptions, to those of spinner cell membranes. The cholesterol to phospholipid molar ratios of plasma membranes were 6.4 and 4.3 fold greater than those of the mitochondrial and microsomal fractions, respectively.


Synthesis and characterization of a new fluorescent phospholipid. A novel fluorescent phospholipid, whose structure was tentatively assigned as 1-(2'-thio-1'-hydroxyethyl)-2-(ethylphosphatidyl)isoindole), was synthesized by reacting O-phthalaldehyde and beta-mercaptoethanol with phosphatidylethanolamine. The fluorescent lipid product was purified by silicic acid chromatography. The purity was demonstrated by thin-layer chromatography. This fluorescent phospholipid could not form stable lipid vesicles. However, a mixture of phosphatidylcholine and this fluorescent phospholipid did form stable vesicles after sonication, as demonstrated by Sepharose 4B column chromatography and electron microscopy. The absorption and fluorescence properties of this lipid, both as aqueous micelles or incorporated into vesicles, have been determined. The potential usage of this new fluorescent phospholipid in membrane studies is discussed.


Characteristics of phospholipids in microvillar membranes of octopus photoreceptor cells. Characteristics of lipids in the microvillar membranes of octopus photoreceptor cells were studied in order to obtain some information on the membrane environment with rhodopsin in the invertebrate. (1) The membranes contain lipid and protein in almost equal proportion. The majority of lipids are phospholipids. Neutral lipids make up 16% of the total lipids, the major constituent of which is cholesterol. (2) Phosphatidylethanolamine and phosphatidylcholine are the major phospholipids. Phosphatidylserine, ceramide 2-aminoethylphosphonate and sphingomyelin occur as minor components. An unidentified alkaline and acid stable phospholipid was found. (3) The predominant fatty acids of phosphatidylethanolamine and phosphatidylcholine are highly unsaturated such as 22 : 6, 20 : 5 and 20 : 4. The 22 : 6 and 20 : 5 are exclusively linked at the 2-position, but the 20 : 4 is linked significantly at the 1-position of the phospholipids. (4) Major molecular species are 16 : 0/22 : 6 (48.4%) and 16 : 0/20 : 4 (19.6%) in phosphatidylcholine, and 20 : 4/22 : 6 (50.7%) and 16 : 0/22 : 6 (25.6%) in phosphatidylethanolamine.


A theory of the electric field-induced phase transition of phospholipid bilayers. Improving the statistical mechanical model of Jacobs et al. (Jacobs, R.E., Hudson, B. and Andersen, H.C. (1975) Proc. Natl. Acad. Sci. U.S. 72, 3993--3997) we have constructed a model which describes not only the temperature but also the external field dependence of the membrane structure of phospholipid bilayers. In addition to the interactions between head groups, between hydrocarbon chains, and the internal conformational energy of the chains (which were considered in Jacobs' model), our model includes the energy of deformation and the field energy as well. By the aid of this model we can explain the phenomenon of dielectric breakdown, the non-linearity of current-voltage characteristics, and the mechanism of membrane elasticity. The free energy of the membrane, the average number of the gauche conformations in the hydrocarbon interior and at the membrane surface, gauche distribution along the chain, the membrane thickness, area and volume are calculated at different temperatures and voltages. The calculation also gives the temperature dependence of Young's modulus and that of the linear thermal expansion coefficient.


Phosphonic and arsonic acids as inhibitors of human red cell acid phosphatase and their use in affinity chromatography. 1. In order to obtain an effective ligand for affinity chromatography of the low molecular weight acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) from human red cells nine phosphonic and two arsonic acid substrate analogues were investigated as potential inhibitors. The two forms of acid phosphatase type B (b1 and b2) were isolated and partially purified using conventional methods and the inhibitory action of the substrate analogs investigated. 2. Four of the phosphonic acids were relatively effective competitive inhibitors. It appears that certain structural and electronic requirements have to be fulfilled by the phosphonic acids in order to exhibit significant affinity for the enzyme. A high affinity appears to require the presence of a bulky, hydrophobic moiety which has to be separated from the phosphorus atom by the distance of one atom. 3. p-Aminobenzylphosphonic acid exerted the highest affinity for acid phosphatase with a pH optimum at 6.5. Ki values of 4 . 10(-4) and 6 . 10(-4) M were found for the b1 and b2 forms, respectively. 4. Coupling of p-aminobenzylphosphonic acid to Agarose yielded an effective and specific affinity medium. By means of affinity chromatography using this medium, acid phosphatase was purified 500-fold in a single step.


Post-proline cleaving enzyme. Synthesis of a new fluorogenic substrate and distribution of the endopeptidase in rat tissues and body fluids of man. Synthesis and application of the first fluorogenic substrate, N-carbobenzoxyglycylprolyl-4-methylcoumarinyl amide (Z-Gly-Pro-MeCouNH) for the determination of the post-proline cleaving enzyme (EC 3.4.21.-) were reported. Maximal activity of the enzyme purified from lamb kidney for the new substrate was observed at pH 7.0. This substrate showed a higher affinity (Km = 0.02 mM) for the enzyme than the proline containing substrates studied previously and allowed the detection of 10-50 ng post-proline cleaving enzyme activity per ml sample after a 1 min incubation period. Distribution of post-proline cleaving enzyme and other proline specific peptidases in rat tissues was studied using Z-Gly-Pro-MeCouNH and other proline-containing substrates. High post-proline cleaving enzyme activity was observed in testis, liver and skeletal muscle. Inhibition experiments indicated that post-proline cleaving enzyme activity was completely inactivated by 0.1 mM diisopropylphosphofluoridate and Z-Gly-Pro-chloromethylketone, as had been found in the case of the enzyme isolated from lamb kidney. Activity in human body fluids was also tested for levels of post-proline cleaving enzyme activity using Z-Gly-Pro-MeCouNH and semen was found to show the highest cleaving activity.


A purification and some properties of two proteases from papaya latex. Two proteases, one of which is papaya peptidase A and the other a previously unknown enzyme in papaya latex have been purified to homogeneity in a simple two stage process. Both are markedly less reactive than papain or chymopapain. Each has a molecular weight of 24,000, N-terminal sequences commencing Leu-Pro-Glu, and contains no carbohydrate. Their amino acid compositions differ for several residues. The essential -SH groups of the enzymes examined appear to be 'masked' in the native state.


Isolation of completely inactive plasma prorenin and its activation by kallikreins. A possible new link between renin and kallikrein. Existing views on prorenin are conflicting and its physiological activation mechanism is not clear. In an attempt to obtain clearcut views on the molecular properties of prorenin in human plasma, the renin zymogen (prorenin) was separated from active renin by two steps of affinity chromatography and it was demonstrated that prorenin is a completely inactivate zymogen contrary to the existing information. Inactive prorenin has an apparent molecular of 56,000 contrary to 46,000-43,000 of partially active prorenin. Isolated and acid-treated human prorenin was shown to be activated by kallikreins from human urine and plasma. This activation was completely blocked by Trasylol. Hog pancreatic kallikrein also activated human prorenin. The kallikrein mediated activation of prorenin indicates the existence of a new link between the vasoconstricting renin-angiotensin system and the vasodilating kallikreinkinin system.


Fluoride inhibition of inorganic pyrophosphatase. IV. Evidence for metal participation in the active center and a four-site model of metal effect on catalysis. Atomic spectroscopy of native yeast inorganic pyrophosphatase (pyrophosphate phosphohydrolase, EC 3.6.1.1) after gel filtration showed that it only binds activating Mg2% in an easily dissociable manner. Formation of a covalent intermediate between the enzyme and an entire substrate molecular in the presence of fluoride, however, dramatically strengthened the binding of two Mg2+ per subunit and eliminated at neutral pH the effect of added metals on protein fluorescence but not on the absorption spectrum, suggesting that different mental binding sites influence the two spectra. This conclusion was confirmed by spectra studied on native enzyme. A third, low-affinity site for Mg2+ was found on the enzyme pH greater than 8. A model of enzyme-substrate-metal interactions was proposed, according to which the fluorescence-controlling site belongs to the active center and substrate can only be bound to it as a 1 : 1 complex with metals.


Mechanism of action of iodothyronine-5'-deiodinase. Production of 3,3'-di-iodothyronine (3,3'-T2) from 3,3',5'-tri-iodothyronine (reverse T3, rT3) as catalysed by rat liver microsomal fraction was measured with a specific radioimmunoassay. The effect of the addition of 2-thiouracil and of varying concentrations of cofactor (dithiothreito) on the kinetic parameters of this reaction were studied. It was found that thiouracil is an uncompetitive inhibitor with respect to substrate and a competitive inhibitor with respect to cofactor. The effect of a decrease in the concentration of cofactor was similar to the effect of addition of thiouracil, i.e. a proportional decrease in Km and V. The results strongly suggest that enzymatic 5'-deiodination of iodothyronines follows a ping-pong mechanisms, which may be envisaged as a transiodination and the subsequent reduction of the iodo-enzyme complex by cofactor. The intermediate is probably a sulfenyl iodide form of the enzyme, which reacts with thiouracil to yield a mixed disulfide.


A study of the reaction catalysed by alginate lyase VI from the sea mollusc, Littorina sp. The molecular weight of polymeric alginic acid digested by alginate lyase (poly(1,4-beta-D-mannuronide) lyase, EC 4.2.2.3) was determined at various stages of the lysis. Low molecular weigh fragments were detected only after 60-100% lysis. Some high molecular weight fragments remained intact even after addition of a fresh aliquot of enzyme to the digest. The enzyme showed maximal activity at pH 5.6 in 0.05 M salt. Enzyme activity was stimulated by addition of 7.5 mM CaCl2 and 0.2 M NaCl, when the pH optimum was between 8 and 8.5. Only mannuronic acid was detected at the reducing end of fragments after exhausive enzymolysis, reduction and hydrolysis. On studying the reaction products by NMR, a double-bound signal (sigma = 5.98 ppm) was observed. A considerable decrease in intensity of the D-mannuronic acid residue signal was detected after hydrolysis of alginate lyase VI on poly-(ManUA-GulUA), but not poly(GulUA). The results suggest that alginate lyase VI may be an endoalginate lyase that splits glycoside bonds only between two mannuronic acid residues.


The oxidation and decarboxylation of retinoic acid by horseradish peroxidase. The decarboxylation of retinoic acid by horseradish peroxidase was investigated. A marked increase in the yield of products was obtained. However, the data indicated the reaction was a nonenzymatic, heme catalyzed peroxidation. Previously reported requirements for phosphate, oxygen and ferrous ion were eliminated when hydrogen peroxide was provided. Peroxide also eliminated the EDTA and cyanide induced inhibition of the phosphate dependent system. In the presence of hydrogen peroxide, horseradish peroxidase was not essential to the reaction; heme equivalent amounts of hemoglobin decarboxylated retinoic acid with equal facility. However, hemoglobin was ineffective in the absence of hydrogen peroxide. Attainment of 50--60% decarboxylation represented complete utilization of the available retinoic acid. Thus the products of the reaction can be divided into two groups, products of retinoic acid oxidation and products of an oxidative decarboxylation of retinoic acid.


Purification and properties of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase. 3-Hydroxy-3-methylglutaryl coenzyme A reductase has been purified from rat liver microsomes with a recovery of approx. 25%. The enzyme was homogeneous on gel electrophoresis and enzyme activity comigrated  with the single protein band. The molecular weight of the reductase determined by gel filtration on Sephadex G-200 was 200,000. SDS-polyacrylamide gel electrophoresis gave a subunit molecular weight of 52,000 +/- 2000, suggesting that the enzyme was a tetramer. The specific activities of the purified enzyme obtained from rats fed diets containing 0% or 5% cholestyramine were 11,303 and 19,584 nmol NADPH oxidized/min per mg protein, respectively. The reductase showed unique binding properties to Cibacron Blue Sepharose; the enzyme was bound to the Cibacron Blue via the binding sites for both substrates, NADPH and (S)-3-hydroxy-3-methylglutaryl coenzyme A. Antibodies prepared against purified reductase inactivated 100% of the soluble and at least 91% of the microsomal enzyme activity. Immunotitrations of solubilized enzyme obtained from normal and cholestyramine-fed rats indicated that cholestyramine feeding both increased the amount of enzyme protein and resulted in enzyme activation. Administration of increasing amounts of mevalonolactone to rats decreased the equivalence point obtained from immunotitration studies with solubilized enzyme. These data indicate that the antibody cross-reacts with the inactive enzyme formed after mevalonolactone treatment.


Lysozyme sensitivity of pantothenate-deficient Lactobacillus plantarum grown with exogenous fatty acids. A pantothenic acid deficiency in Lactobacillus plantarum reduces lipid synthesis, prevents normal uptake and retention of extracellular amino acids and markedly increases sensitivity of these cells to lysozyme induced lysis. Pantothenate-deficient cells provided with exogenous fatty acids synthesize additional lipids and express nearly normal solute transport activities. The present study has shown that such cells retain a heightened sensitivity to lysozyme induced lysis. These observations indicate that the lysozyme sensitivity of pantothenate-deficient cells is not produced as in indirect effect of membrane lipid depletion, but represents an independent consequence of pantothenate insufficiency.


Factors regulating the elongation of palmitic and stearic acid by rat liver microsomes. Analysis of the rates of overall chain elongation and condensation of malonyl-CoA with palmitoyl-CoA and stearoyl-CoA as primers demonstrated that for each primer, the rate of the overall metabolic process was similar to the initial condensation. The specific activity for condensation with palmitoyl-CoA was eleven times greater than for stearoyl-CoA. The specific activities of both the beta-hydroxyacyl-CoA dehydrase and 2-trans-enoyl-CoA reductase reactions were much higher than for either condensation or chain elongation, although these rates were somewhat greater with the intermediates required in chain elongating palmitoyl-CoA than for stearoyl-CoA. Both substrates were incorporated into phospholipids at low rates and there was a time-dependent hydrolytic cleavage of the acyl-CoA primers which was partially prevented by bovine serum albumin. These findings demonstrate that there was no selective removal of either primer which could result in specific substrate depletion and an apparent reduction in the rate of condensation. These combined results firmly establish the rate-limiting nature and high degree of substrate specificity exhibited during the initial condensation step in fatty acid elongation.


Origin of the arachidonic acid released post-mortem in rat forebrain. To determine the origins of the arachidonic acid released post-mortem in brain tissue, [3H]arachidonic acid was injected by the intracerebro-ventricular route and radioactivity monitored in complex lipids and free arachidonic acid at various times after decapitation. The specific activity of the released arachidonic acid was close to that in the total phospholipid fraction and much lower than that of the neutral lipids. The phospholipid with the closest specific activity to the free arachidonic acid recovered at the end of the post-mortem period was phosphatidylinositol. Phosphatidylcholine showed a small but significant decrease in radioactivity post-mortem and could contribute 37% of the arachidonic acid released to the free fatty acid fraction. Arachidonic acid released in rat forebrain after decapitation thus comes from a mixture of phospholipids with phosphatidylinositol and phosphatidylcholine being the major source. Phosphatidylserine and phosphatidic acid did not make important contributions to the free arachidonic acid. In the microsomal fraction, the specific activity of the free arachidonic acid was very close to that in phosphatidylinositol.


Metabolism of prostaglandin E1 and of glutathione conjugate of prostaglandin A1 (GSH-prostaglandin A1) by prostaglandin 9-ketoreductase from rabbit kidney. Rabbit kidney prostaglandin 9-ketoreductase was found to metabolize the glutathione conjugate of prostaglandin A1 (GSH-prostaglandin A1). Apparent Km (GSH-prostaglandin A1) 13 microM and apparent Km (prostaglandin E1) 200 microM. The cytosolic preparation was subjected to gelfiltration and isoelectric focusing, which revealed that metabolism of prostaglandin E1 and GSH-prostaglandin A1 occurs by means of the same fractions. Furthermore, prostaglandin E1 and GSH-prostaglandin A1 are competitive inhibitors of the enzyme, when GSH-prostaglandin A1 and prostaglandin E1 are tested as substrates, respectively. It si concluded, that GSH-prostaglandin A1 is a much better substrate for prostaglandin 9-ketoreductase from rabbit kidney than is prostaglandin E1.


Plasma membrane lipids of human diploid fibroblasts from normal individuals and patients with cystic fibrosis. The lipid composition of isolated plasma membranes of human skin fibroblasts is described for the first time. Plasma membranes from a number of strains of fibroblasts from patients with cystic fibrosis and matched normals were isolated by a recently described procedure and analysed for major phospholipid classes, cholesterol and fatty acids. No differences in the quantities of these compounds were detected between cells of the two different origins. The fetal calf serum used to supplement the growth medium contained relatively more palmitoleate and oleate but less stearate than the membranes. There were also no consistent differences between cystic fibrosis and normal membranes in terms of the fatty acid compositions of their individual phospholipid classes. Consistent with this lack of chemical change in the lipids of membranes of cystic fibrosis cells, the degree of fluorescence polarization of diphenylhexatriene, an index of fluidity, was also unchanged.


Studies on molecular species of choline glycerophospholipids of developing rat brain. The chronological changes in molecular species of choline glycerophospholipids were studied for cerebra of 17-, 19- and 21-day-old rat fetuses, and 3-, 6-, 12-, 24- and 90-day-old rats. The molecular species found by gas chromatography-mass spectrometry and selected ion retrieval technique were phosphatidylcholines of '30 : 0, 32 : 0, 32 : 1, 34 : 0, 34 : 1, 34 : 2, 36 : 0, 36 : 1, 36 : 2, 36 : 3, and 36 : 4' where the larger number indicates the sum of chain lengths on positions C-1 and C-2; the smaller number is the total number of double bonds. Of these molecular species, '32 : 0' (mainly 16 : 0/16 : 0, dipalmitoyl glycerophosphorylcholine), '34 : 1' (mainly 16 : 0/18 : 1, palmitoyloleoyl glycerophosphorylcholine), '34 : 0' (16 : 0/18 : 0, palmitoylstearoyl glycerophosphorylcholine), '32 : 1' (mainly 16 : 0/16 : 1, palmitoylpalmitoleoyl glycerophosphorylcholine and '30 : 0' (14 : 0/16 : 0, myristoylpalmitoyl glycerophosphorylcholine) were main species. The '32 : 0' species increased to about 44% at around the 10th day and thereafter remained nearly constant. '34 : 1' and '34 : 0' decreased to about 17 and 6% at that time and then increased to about 30 and 14%, respectively. '30 : 0' increased from last stage of gestation to the 6th day and then decreased. '32 : 1' was about 16% for 17-day-old fetus and decreased grandually. '36 : 1' (18 : 0/18 : 1, stearoyloleoyl glycerophosphorylcholine) increased at the latter part of development.


A facile permethylation of cord factor.

A facile permethylation of cord factor. Small quantities of cord factor (trehalose-6,6'-dimycolates) can be readily and almost quantitatively permethylated in anhydrous diethyl ether/dimethylformamide mixtures with CH3I and NaH-oil dispersion in the presence of molecular sieve. Hydrolysis of the permethylated products from cord factor "Peurois" and 'P3-Aoyama-b' prove these to be 6,6'-dimycolates. Experiments with a 4-palmitoyl glucose suggest that acyl migration in this system may not occur.


Positional distribution of exogenous and endogenous fatty acids in triacylglycerols formed by rat adipocytes in vitro. Rat adipocytes were used in vitro to compare the positional distributions of fatty acids of intra- and extra-cellular origin in triacyl-sn-glycerols. Fatty acids of extracellular origin were esterified to each position in similar, but not identical, proportions to the natural distributions. A high proportion of the oleic acid synthesised in the tissue by desaturation of exogenous stearic acid was found in position sn-3. When palmitic acid was the only fatty acid added, tripalmitoylglycerol was synthesised by the adipocytes. The rate and pattern of fatty acids synthesised de novo from acetate was dependent on the age of the donor rat and the concentration of acetate and presence or absence of long-chain fatty acids in the medium. The newly synthesised fatty acids were esterified in very different proportions from the natural distributions and thus from those of extracellular fatty acids. The results are discussed in terms of esterification of the fatty acids from the two sources in different compartments of the cell.


Isolation and structure of diglycosylsterols and triglycosylsterols in rice bran. Novel oligoglycosylsterols, a series of sterylglycosides, were detected in the glycolipids of rice bran, and di- and triglycosylsterols were isolated and their structures were investigated. The principal sterol components were sitosterol, campesterol and stigmasterol; glucose was the only component sugar. The major species of diglycosylsterol and triglycosylsterol were characterized as D-glucopyranosyl-(beta 1 leads to 4)-D-glucopyranosyl-(beta 1 leads to 3')-sitosterol, i.e. cellobiosylsitosterol, and D-glucopyranosyl-(beta 1 leads to 4)-D-glucopyranosyl-(beta 1 leads to 4)-D-glucopyranosyl-(beta 1 leads to 3')-sitosterol, i.e. cellotriosylsitosterol, respectively.


Pyruvate cycling involving possible oxaloacetate decarboxylase activity. With high concentrations of pyruvate as substrate for hepatocytes from fasted rats, high rates of cycling between pyruvate and the dicarboxylic acids occur, as shown isotopically. This rate of cycling is adequate to account for the hydrogen translocation from the mitochondria to the cytosol to furnish NADH for lactate formation. Addition of sufficiently high concentrations of mercaptopicolinate to block almost completely glucose formation from pyruvate, depresses isotopic cycling and lactate formation by only about 50-75%. Under some conditions, when the normal phosphoenolpyruvate carboxykinase activity is inhibited, cytosolic oxaloacetate may be decarboxylated directly to pyruvate, possibly via the decarboxylase activity of phosphoenolpyruvate carboxykinase.


Quantitative assay of types I and III collagen synthesized by keloid biopsyes and fibroblasts. Molecular sieve column chromatography was used to determine the amount of type I and III collagen synthesized by normal dermis and keloid biopsies and fibroblasts derived from these tissues. After incubation with radioactive proline, the collagen was extracted and separated into types I and III and then quantitated. There was no significant difference in the percent type III collagen synthesized by fresh keloid biopsies compared to normal dermis. Likewise, there was no significant difference in the percent type III collagen synthesized by keloid fibroblasts compared to normal dermal fibroblasts. However, fibroblasts from both keloid and normal dermis synthesized a lower percentage of type III collagen in cell culture compared to the original biopsies. These findings demonstrate that keloid collagen has the same type distribution as normal dermis and suggest that increased collagen synthesis in these lesions is not related to altered collagen types.


Age-related effects on the incorporation of acetate into rat liver histones. Incorporation of sodium [3H]acetate into histones of rats was examined as a function of age. Incorporation was observed to decline with age up to 24 months, at which time a levelling occurred. Controls indicated that this decrease in histone acetylation could not be attributed to variability in isotope delivery to the liver or to alterations in intracellular 'pools' available for acetylation. Polyacrylamide gel electrophoresis established that, in all cases, acetate was incorporated primarily into histone fractions H3 and H4 and the pattern of incorporation exhibited age-dependent phenomena. H4 was predominantly labelled in 2 months animals, while in 12, 16, and 24 months animals H3 was more highly labelled; at 27 months the two fractions were labelled equally. Assessment of histone acetylase and deacetylase activities indicates that deacetylase activity increased with age.


Partition behaviour of a cultured mouse mammary cancer cell line in aqueous two-phase polymer systems. Cell surface-associated changes in behaviour of cultured cells on partition in an aqueous two-phase polymer system were studied using FM3A cell line (a cultured mammary cancer of mouse) with respect to aging. The aqueous polymer system consisted of dextran, polyethyleneglycol and sodium phosphate, equilibrated at 6 degrees C to separate into two phases. Enzyme treatment of cells with neuraminidase reduced cell electrophoretic mobility, as well as the cell partition ratio. Hyaluronidase produced no observable effects on partition and cell electrophoretic mobility, suggesting that the partition is related to culture time was similar for both cell electrophoretic mobility and cell partition, showing a rise and fall of charge-associated cell surface change during cell growth, the maxium occurring at the beginning of exponential growth. This change was reflected in the pattern of countercurrent distribution of the cells in respective stages of growth. Countercurrent distribution with our two-phase system is expected to be capable of fractionating cell populations according to cell surface properties.


Secretion and uptake of beta-N-acetylglucosaminidase by fibroblasts. Effect of chloroquine and mannose 6-phosphate. The effects of chloroquine and mannose 6-hosphate on the secretion and uptake of the lysosomal enzyme, beta-N-acetylglucosaminidase (EC 3.2.1.30), by human fibroblasts have been compared. There was a reciprocal relationship between intracellular depletion, and extracellular accumulation, of enzyme at chloroquine concentrations ranging from 5 micrometers to 100 micrometers. A loss of enzyme activity from the system (intra- plus extracellular activity) with increasing concentrations of chloroquine was due to inhibition of the beta-N-acetylglucosaminidase. At a concentration of 50 micrometers, chloroquine elicited a three fold increase in the extracellular accumulation of beta-N-acetylglucosaminidase in 24 h whereas the addition of 5 micrometers mannose 6-phosphate (a competitive inhibitor of receptor-mediated uptake) resulted in only a 13% increase. Uptake of beta-N-acetylglucosaminidase by enzyme-deficient fibroblasts was completely inhibited by 5 micrometers mannose 6-phosphate. In the presence of chloroquine there was also no uptake of enzyme, however ther was a marked decrease in the residual activity of the cells. The results suggest that the effect of chloroquine on fibroblasts is to stimulate secretion rather than to inhibit uptake as previously reported. The isoenzyme pattern of the beta-N-acetylglucosaminidase from normal culture medium was compared with that accumulating in the medium following exposure of the cells to 50 micrometers chloroquine. In the presence of chloroquine, there was an increase in the A isoenzyme, however the activity was eluted in a broad peak which probably represents several closely related forms of the enzyme. There was an almost total loss of the A isoenzyme of beta-N-acetylglucosaminidase from fibroblasts cultured in the presence of chloroquine. A small peak of activity eluting at a similar position to the secreted, As, isoenzyme was present in extracts of chloroquine-treated fibroblasts, suggesting that the As isoenzyme is formed and/or stored at a site distinct from the intracellular isoenzyme.


Stimulation of de novo synthesis of L-phenylalanine ammonia-lyase in relation to phytoalexin accumulation in Colletotrichum lindemuthianum elicitor-treated cell suspension cultures of french bean (Phaseolus vulgaris). (1) The regulation of the accumulation of the isoflavonoid-derived phytoalexin phaseollin in cell suspension cultures of Dwarf French Bean (Phaseolus vulgaris/ has been investigated. (2) An elicitor preparation from cell walls of Colletotrichum lindemuthianum, the causal agent of anthracnose disease of French bean, caused a marked accumulation of phaseollin in the cultures. The elicitor induced phaseollin accumulation to a level of 60% that obtained with the artificial elicitor autoclaved ribonuclease A and was maximally active at a concentration (weight basis) of at least 50 times lower than required for maximal response to ribonuclease. (3) Elicitor preparations from cell walls of Phytophthora megasperma var. sojae, a fungal pathogen of soybean, and Botrytis cinerea, the common grey mould, were much less effective than the C. lindemuthianum wall-released elicitor. (4) There was a marked but transient increase in the extractable activity of phenylalanine ammonia-lyase, the enzyme catalysing the first reaction in the biosynthesis of phaseollin from L-phenylalanine, in response to the elicitor from C. lindemuthianum. (5) Comparative density labelling with 2H from 2H2O indicated that the elicitor stimulates de novo synthesis of phenylalanine ammonie findings provide the basis of a scheme for elicitor induction of phytoalexin accumulation.


Absorption, serum levels and urinary excretion of inorganic sulfate after oral administration of sodium sulfate in the conscious rat. The absorption of inorganic sulfate after ingestion was investigated in rats. After oral administration of Na235SO4, 35S radioactivity was measurable in plasma already after 15 min and its plasma concentration reached a peak after about 1.5--2 h. The 35S-radioactivity excreted in urine during 24 h after ingestion of Na235SO4 together with varying amounts of unlabelled Na2SO4 (0.25--5.0 mmol Na2SO4 per rat) indicated an almost complete absorption of inorganic sulfate from the gastrointestinal tract. Determination of the inorganic sulfate concentration in rat serum 2 h after oral administration of 5.0 mmol Na2SO4 revealed a three-fold increase in serum sulfate concentration. The data suggest a rapid and almost complete absorption of inorganic sulfate after oral administration in the rat. Its importance in relation to the sulfate availability for sulfate conjugation of drugs is discussed.


Lipid carriers in the synthesis of high-mannose glycoproteins in algae. Particulate preparations from the chlorophyta Prototheca zopfii catalyze the incorporation of mannose and N-acetylglucosamine into glycolipids. These had been characterized as lipid monophosphate mannose, lipid pyrophosphate N,N'-diacetylchitobiose and various lipid-linked oligosaccharides containing two N-acetylglucosamine residues plus a variable number of mannose residues. The lipid moiety has the properties expected for dolichyl phosphate. The oligosacchride-linked lipids serve as precursors for the formation of a polymer sensible to pronase digestion. The oligosaccharide is linked by N-glycosidic linkage to an asparagine residue. In longer incubation periods, a polymer insensitive to pronase hydrolysis, but precipitable by copper salts such as cell wall mannans is formed. Polymer formation is inhibited by 1 mM bacitracin. The reactions leading to the formation of the mannoprotein were found associated to the rough endoplasmic reticulum. The synthesis of mannans was found to occur in the Golgi vesicles.


ADP-ribosylation of membrane proteins and activation of adenylate cyclase by cholera toxin in fat cell ghosts from euthyroid and hypothyroid rats. Incubation of fat cell ghosts with activated cholera toxin, nucleoside triphosphate, cytosol, and NAD results in increased adenylate cyclase activity and the transfer of ADP-ribose to membrane proteins. The major ADP-ribose protein comigrates on sodium dodecyl sulfate-polyacrylamide gels with the putative GTP-binding protein of pigeon erythrocyte membranes (Mr 42 000), which is also ADP-ribosylated by cholera toxin. The treatment with cholera toxin enhances the stimulation of the fat cell membrane adenylate cyclase by GTP, but the stimulation by guanyl-5'-yl imidodiphosphate is unaltered. Subsequent stimulation of fat cell adenylate cyclase by 10 micrometers epinephrine is not particularly affected. These changes were qualititatively the same for membranes isolated from fat cells of hypothyroid rats. Although the cyclase of these membranes has a reduced response to epinephrine, guanyl-5'-yl imidodiphosphate or GTP, as compared to euthyroid rat fat cell membranes, the defect is not rectified by toxin treatment and cannot be explained by a deficiency in the cholera toxin target.


Epinephrine and glucagon counteract inhibition of protein synthesis induced by D-galactosamine in isolated mouse hepatocytes. 10 mM D-galactosamine enhibited protein synthesis (1 h incubation time) by 67% in isolated mouse liver cells. Counteracting uridylate deficiency induced by D-galactosamine by preventive administration of 20 mM uridine did not decrease the extent of protein synthesis inhibition. 20 mM D-galactose reverted the inhibition of protein synthesis by D-galactosamine. 10(-5) M epinephrine and 10(-7) M glucagon decreased the incorporation of D-galactosamine into glycogen to 38% and 26% of the control value, respectively, after a 35 min incubation and reduced the inhibition of protein synthesis by D-galactosamine effectively. Experimental evidence supports the view that aminoglycogen formed after D-galactosamine treatment is responsible for the inhibition of protein synthesis.


Ultracentrifugation evidence for a somatostatin-binding protein in serum. Using a technique of high speed centrifugation of serum and a well validated immunoassay for the measurement of serum somatostatin-like immunoreactivity, we have demonstrated that somatostatin, unlike other peptide hormones, appears to sediment with large molecular weight proteins. When synthetic somatostatin of increasing concentration was incubated with serum prior to ultracentrifugation, a linear plot of concentration of somatostatin added against concentration sedimenting (or apparently bound to protein) revealed an association curve. These data provide further evidence for the existence of a serum-binding protein for somatostatin.


Interaction of lectins with human platelets. Effects on platelet stimulation by thrombin and ristocetin. Wheat germ agglutinin induced aggregation and secretion of fresh platelets. Aggregation, but not secretion of serotonin by platelets in plasma, by the lectin was inhibited by 5 mM EDTA. Further, the lectin-induced stimulation of fresh platelets was blocked by prostaglandin E1. Thus, this lectin stimulates platelets by a mechanism which closely mimics thrombin activation and is independent of intercellular crosslinking. Lentil lectin did not stimulate platelets. Each platelet contained about 6 . 10(-5) binding sites for the lectins with an apparent dissociation constant of 3.0 . 10(-7) M. Wheat germ agglutinin, which binds mainly to glycoprotein I (Mr 150 000), increased the subsequent binding of thrombin to fixed platelets while lentil lectin was without effect. It appears that thrombin and wheat germ agglutinin bind to independent but interacting sites. Wheat germ agglutinin, but neither thrombin nor lentil lectin, inhibited the agglutination of platelets by ristocetin. Further, rat platelets were not aggregated by either ristocetin or wheat germ agglutinin. It appears that the interaction sites of ristocetin and wheat germ agglutinin on platelets are overlapping.


Evidence for a plasma inhibitor of the heparin accelerated inhibition of factor Xa by antithrombin III. The ability of heparin fractions of different molecular weight to potentiate the action of antithrombin III against the coagulation factors thrombin and Xa has been examined in purified reaction mixtures and in plasma. Residual thrombin and Xa have been determined by their peptidase activities against the synthetic peptide substrates H-D-Phe-Pip-Arg-pNA and Bz-Ile-Gly-Arg-pNA. High molecular weight heparin fractions were found to have higher anticoagulant activities than low molecular weight heparin when studied with both thrombin and Xa incubation mixtures in purified mixtures and in plasma. The inhibition of thrombin by heparin fractions and antithrombin III was unaffected by other plasma components. However, normal human plasma contained a component that inhibited the heparin and antithrombin III inhibition of Xa particularly when the high molecular weight heparin fraction was used. Experiments using a purified preparation of platelet factor 4 suggested that the platelet-derived heparin-neutralizing protein was not responsible for the inhibition.


Estrogen-dependent and progesterone-arrested synthesis and secretion of sulfated glycoproteins in luminal epithelia of rat uteri. 1. 35SO4 administered intraperitoneally was specifically incorporated into a glycopeptide component separated by electrophoresis of the glycosaminoglycan fraction prepared from the uterine epithelia (luminal), as well as the uterine fluid of ovariectomized rats treated with estradiol-17 beta, in contrast to the rats without estrogen treatment. 2. The epithelial cells of uteri isolated from estrogen-treated ovariectomized rats incorporated 35SO4 in vitro into at least two macromolecular components. The larger molecular weight component (sodium dodecyl sulfate polyacryl-amide gel electrophoresis and/or gel filtration) labelled with 35S was observed in both the cytosol and particulate fractions, whereas the smaller molecular weight component labelled with 35S was found only in the particulate fraction. 35SO4 was also incorporated into two macromolecular components in the incubation medium, similarly to the particulate fraction. A 35 SO4-labelled glycopeptide similar to that from the epithelial particulate fraction and the incubation medium, and not from the epithelial cytosol fraction. 3. Progesterone, in contrast to estrogen, did not stimulate the sulfated blycoprotein synthesis. Moreover, progesterone administered together with or after estrogen-administration completely arrested the estrogen-dependent synthesis and secretion of the sulfated glycoproteins in the uterine epithelia.


Properties and affinity purification of the mixed-type putative acetylcholine receptor from wild and a mutant strain of hose flies. Binding of decamethonium to a soluble preparation from house fly head (either wild or a mutant strain) showed a single kind of binding with values for wild strain of Kd = 0.095 micrometers and Bmax = 0.22 nmol/mg protein. The mutant had a four-fold greater affinity and a seven-fold lesser amount. The binding was blocked by both nicotinic and muscarinic drugs. The decamethonium binding migrated in sucrose gradients as a single peak, with sedimentation coefficient s20,w = 12.5 S and therefore a molecular weight of 342 000. Purification by affinity chromatography was achieved with only partial loss of activity, andthe purified material demonstrated a single band on analytical disc gel electrophoresis. Electrophoresis in sodium dodecyl sulphate gels showed two subunits of molecular wegiths 94 000 and 64 000. Both subunits had an isoelectric point of 4.8.


Characterization of endogenous folate and incorporation of labelled folates into the brain of the South African fruit bat. Folate in the brain of the South African fruit bat consists of 10-formyltetrahydropteroyglutamic acid and the tri-, tetra- and penta- forms of 5-methyltetrahydropteroylglutamic acid. Following parenteral injection, only 5-[3H]-methyltetrahydropteroylglutamic acid was taken up by the brain, but none of a dose of 14C-labelled tetrahydropteroylglutamic acid was detectable. Only trace smounts of the 5-methyltetrahydropteroylglutamic acid were converted into the formyl compounds and a small amount of methyltetrahydropteroyltriglutamic acid appeared after 96 h. There was no significant difference in vitamin B-12-deficient animals.


Ionic partial molar volumes of density gradient salts at finite concentrations. A general method is presented for estimating concentration-dependent partial molar volumes for individual ions of heavy density gradient salts used in the isopycnic ultracentrifugation of charged macromolecules.


The increase in hexokinase activity in hereditary avian muscular dystrophy. Hexokinase activity was found to be increased in both the more severely affected red (thigh) muscle of dystrophic chickens. The increase in activity was largely associated with the particulate fraction. These findings may indicate early events in the pathogenesis of avian muscular dystrophy.


[Comparative study of the characteristics of the proton NMR spin echo decay of different globular proteins in water]. Spin-echo decays were studied for protons of 7 enzymes and 4 other globular proteins. Similar decay behaviour of the skin-echo decay curves was observed for all non-enzymatic proteins (motion of protein globules as whole units was taken into account). The spin-echo decay surves of 5 monomeric enzymes also exhibited very close decay patterns, although they decayed more slowly than non-enzymatic proteins. Greater slowing of the decay was observed for proton spin echoes in two subunit enzymes. Observed slowing of the decay is interpreted in terms of more intense inner motions within the macromolecules of enzymes, compared to non-enzymes. In subunit enzymes it occurs in addition because of the motion of separate units.


[Conformation of hydrocarbon chains in a lipid bilayer]. Based on the linear relationship between the entropy change deltaS during the phase transition and the length of he hydrocarbon chains of some lipids, the dependence of the probability of the trans--(pt) and gauche--(pg) conformation of the "averaged" methylene group of the chain on the increment deltas is deduced. Values of the probabilities for liquid n-alkanes, tri-glycerines and fatty acids are pt approximately equal to 0.6 and pg+ approximately equal to pg- approximately equal to 0.2, for liquid crystalline lecithin bilayers pt approximately equal to 0.8 and pg+ = pg- approximately equal to 0.1. From these data the average number of gaucheisomers per a plamitin chain in the dipalmitoylphosphatidylcholine molecule is estimated to be approximately 2.8-3.


[Study of the kinetics of chlorophyll photosensitized dye reduction by photopolarography]. It has been shown by photopolarographic method that under stationary illumination chlorophyll-photosensitized reduction of dyes with ascorbic acid proceeds according to the first kinetic order. Rate constants of photoreduction of vitamin K3, methelene red, safranin-T and methylviologen were found in the course of sensitization which proceeded by oxidative and reduction mechanisms. Similarity of kinetics at both sensitization mechanisms was shown. Results obtained by polarographic and spectroscopic methods were compared.


[Mechanisms of adaptive regulation of complex motor systems (regulation with respect to position and acceleration)]. Possible mechanisms of adaptive control over human and animal motor systems are described for the case when the information about position or movement acceleration of its parts is used. A model of the control system with the standard which functions in two modes of operations is treated. The first mode is distinguished by measured control signal leaps used for the identification of a system state and for a further movement correction and precise definition of active muscle composition. The second mode is characterized by the estimation of movement direction only, in case when a certain value of a regulated parameter is maintained or reached. Some problems of the control over a multilinked system are also discussed.


The effects of EMG-assisted relaxation training with hyperkinetic children: a behavioral alternative. Six developmentally hyperkinetic children, ages 6 to 9, participated in either an A-B-A-B-A or an A-B-A-BC-A research design to evaluate the effect of EMG-assisted relaxation training in the acquisition of self regulatory skills to decrease hyperkinetic behavior. Descriptive verbal feedback regarding subject performance was provided during the BC phase in addition to the continuous rate-proportional auditory feedback normally available. Posttraining psychological assessment yielded significant gains for the experimental group on various visual motor tasks at the .05 level. All six subjects significantly decreased EMG activity across the 15 sessions. In a follow-up, five of the six maintained such activity at or below levels reached at the conclusion of the study. Five out of six made significant behavioral improvements at home during training and were maintaining these at the follow up. One out of four made significant improvements in school behavior during training, with five out of six exhibiting such improvements at the follow-up during the next school year. Auditory feedback was effective at the .05 level; the benefit of the verbal feedback, however, was not demonstrated. Two subjects were maintained on stimulant medication, one with a slight dose reduction during training. According to the observations of their parents and teachers, four subjects for whom medication had been recommended prior to the training were subsequently judged not to need it at the time of the follow-up.


Alpha detection: some comments on Hardt and Kamiya, "Conflicting results in EEG alpha feedback studies". The problem of defining EEG alpha activity, and of detecting and measuring it, is outlined. Some advantages of a threshold method are presented and compared with an amplitude-integration method. Characteristics of percent-time scoring, based upon observable alpha spindles in the waking EEG, are discussed. A probability distribution of percent-time alpha as a function of detection threshold is derived and compared with empirical data.


Biofeedback control of skin potential level. This study explored the possibility of using biofeedback to arrest spontaneous declines in tonic electrodermal levels. For 3 days, 10 subjects (S) received 20 minutes of training in increasing skin potential level (SPL) negativity (arrested declines), followed by 20 minutes of training in decreasing SPL negativity (facilitating declines), with reverse order for half of the Ss. Simultaneous analogue and binary feedback of SPL were used. Training in the direction of SPL increases resulted in arrest of spontaneous declines in SPL negativity but did not result in large-magnitude increases above baseline. Training in the direction of SPL decreases facilitated declines in SPL. The absolute mean difference between the increase condition and the decrease condition on the 3rd day was 8.28 mV. Correlations suggested that considerable intraindividual variability may have been independently related to the Locus of Control personality dimension and to a Law of Initial Values limitation on SPL change. It was concluded that SPL was controllable to the extent of facilitating or arresting spontaneous declines.


[Conformational transitions of DNA in concentrated neutral salt solutions]. Salt anions can be arranged in lyotropic series of the action of conformational stability of DNA. This effect is universal for proteins and DNA. It is explained by the salt changes of transfer free energy of macromolecular inner groups to the solvent. Effect mechanism is the combination of anion direct interaction with the exposed inner groups and in indirect way through the changes of water structure. Individual features of the salt effect on DNA are the induced transitions in DNA duplex and the changes of differential stability of AT- and GC-base pairs. An important role in these phenomena plays the dehydration of DNA in the reduction of water activity in concentrated salt solutions. Local changes in medium polarity, hydration level of DNA molecule at different stages of their functioning can explain the regulatory role in intracellular processes, as well as selectivity and specificity of the action of individual ions in the cell.


[Successive phases in continuous work of human skeletal muscle during dentate tetanus functioning]. The sustained unfused tetanus of the human flexor digitorum sublimis was studied in isometrical conditions during electrical stimulation of the muscle. The frequency of stimulation was 8-12 p.p.s., train duration 20-60 s. 3 distinct successive phases of tetanus were revealed. These phases differ in the ratio of the mean tension to the amplitude of its oscillations, in relaxation rate and the efficiency of the duble pulse action. The duration of phases depends on muscle length, stimulation frequency and previous activity. It is supposed that phase II is adopted for the quick movements and muscle properties in phase III for the efficient mantaining of sustained tension.


[Dynamics of intramembrane particles during proteolysis of certain membranes]. Freeze-fracture electron microscopy has been used to study the ultrastructure of proteolytic enzymes treated of the bovine photoreceptor membranes, the rat liver microsome ghosts and the rabbit sarcoplasmic reticulum membranes. The observed increase in the intramembranous particle number in the inner fracture face suggests transmembrane dipping of amphipathic integral proteins affected by the partial proteolysis.


[Ionic balance during cultivation of cell populations in a spatially homogeneous medium]. In culture medium many ionic reactions proceed. Extracellular substances and the cell proper participate in these reactions. For the analysis of ionic processes proceeding in this system the classical approach of physical chemistry is broadened, the effect of flow and cells being taken into account. Fast and slow variables are divided. Ionic equilibria in culture medium are subjected to the same laws as in the absence of the flow and cells. Slow variables are common concentrations of substances of each type. An equation for the cell charge is derived. The set of equations derived describes the dependence of pH culture medium, concentration of the biomass, substrates, metabolic products, charge of these substances on flow rates and metabolism stechiometry.


[Photosensitized luminescence of singlet oxygen in aqueous solutions]. The photoluminescence of singlet oxygen has been observed in air saturated solutions of riboflavin in D2O and mixtures of D2O and H2O. The excitation spectrum coincides with the absorption spectrum of the pigment, the emission maximum lies at 1275 nm. In D2O the quantum yield is approximately 1,2 x 10(-7). H2O quenches the luminescence. Analysis of quenching has shown that the quantum yield in H2O is less than in D2O by the factor of 20.


Operant conditioning of brain steady potential shifts in man. Steady potential shifts (SPS) recorded from the scalp were conditioned operantly by visual and acoustical feedback. Three groups of seven subjects were each tested with a different response-reinforcement contingency: positive reinforcement for a positive SPS after a cue stimulus, positive reinforcement for a negative SPS after a cue stimulus, and noncontingent reinforcement. The steady potential shifts learned under these three conditions differed significantly. Negative shifts were associated with subjective feelings of activation, positive shifts with inactivation. Cortical genesis and possible artifacts are discussed.


Biofeedback as therapy in Raynaud's disease. The following dimensions of Raynaud's disease are reviewed: (a) etiological factors, particularly those of a psychological nature, (b) proposed biological mechanisms of vasospastic episodes, (c) efficacy of pharmaceutical and surgical interventions, and (d) use of biofeedback as therapy. Emotional stress appears to be wholly sufficient to induce vasopastic episodes in victims. Some authors further hypothesize that suppressed anger may be involved in the phenomenon. Vehicles proposed to account for the symptoms include local vascular pathology, sympathetic discharge, and biochemical phenomena. Sympathectomy does not produce permanent remission and vasodilator medications tend to have undesirable side effects even when successful. Documented success with feedback techniques is sparse but encouraging. Published outcome studies are often confounded by the use of additional treatment modes such as relaxation training and hypnosis. The study of Raynaud's disease has enormous potential for the understanding of psychosomatic illness in a holistic fashion. Stresses induce immediate physiological responses, and corrective conditioning is easily implemented and monitored.


Treatment of hypertonicity in muscles of lip retraction. An EMG biofeedback program was developed for a 56-year-old Parkinsonism patient who exhibited pathological lip hypertonia and retraction. The program was designed to achieve the following goals: (1) to demonstrate a reduction in postural lip hypertonicity and (2) to demonstrate a reduction in lip hypertonicity during a series of increasingly complex speech activities. To achieve the first goal, contrastive tasks of full contraction and relaxation were utilized. Each posture was sustained while voltage measurements were made at specific intervals. Procedures to modify lip retraction during speech included five tasks in which the patient was to monitor the audio feedback signal. The tasks involved: prolongation of a neutral vowel, consonant-vowel combinations, monosyllabic words, sentences, and a paragraph-reading task. Data collected over six biofeedback sessions are presented. Trend analyses showed consistent muscular reduction within each task. The following explanations for the decrease in the patient's hypertonicity were discussed: (1) reduction of anisometric contraction, (2) reduction of isometric contraction, (3) relearning of agonistic-antagonistic muscle balance.


[Study of structural transformations of the sugar-phosphate chain and nitrogen bases of DNA hydrate by IR-spectroscopy]. The IR-spectra (900-1800 cm-1) of films of native and denatured DNA were studied within the temperature range 4-100 degrees C at different relative humidity (r.h.). Two temperature intervals of S-like change of spectral parameters of absorption bands of sugar-phosphate chain of double-helical DNA are found on temperature relationships at high humidity. These two intervals point to two types of cooperative transitions of sugar-phosphate pattern. The band parameters of nitrous bases are changed only under high temperature transition. In the region of structural transitions of different subsystems of DNA hydrate hyperchromism was wound for the bands of sugar-phosphate chain and hydrate cover unlike hyperchromism specific of most nitrous bases bands. From the data obtained it is proposed to use the basic characteristics of temperature relationship of spectral parameters of the absorption bands of sugar-phosphate pattern and hydrate envelope in the region of low-temperature transition for the "in vivo" analysis of the DNA structure in complex biological systems.


[Heavy cation position in the structure of the crystalline B-form of DNA]. Coordinates of caesium ions in DNA crystalline B-form were obtained by X-ray study of CsDNA with the use of synchrotron radiation. In conformity with the model that well meets the obtained set of structure factor amplitudes, the cations are located rather close to diad axes lying between the planes of successive nucleotide pairs. The cations in the narrow groove touch DNA surface, while those in the wide groove are rather far from the DNA molecule.


[Structure of the B-form of DNA using the homopolymer poly(dA) . poly(dT) as an example. Refinement in accordance with x-ray and energy criteria]. The refinement of the B-form DNA structure was made using homopolymer poly(dA) (dT) as a model system. In the course of refinement 9 conformational and 22 independent experimental parameters (20 intensities of reflections and 2 helical parameters) were used. After the goal-function, including R-factor, helical and conformational parameters, was optimised, the structures with the values of potential energy and R-factor close correspondingly to the optimal values were selected. "The best" conformations obtained according to energy criteria have the value of an axial projection per one nucleotide pair of about 2,9 A that is equal to the corresponding value in the recently discovered relaxed form of DNA in solution. The most probable conformations according to combine energy and structural criteria have better value of the R-factor in comparison with those derived on the basis of X-ray only, although they are slightly less satisfactory according to the energy criteria. The conformation of DNA which have the values of an axial projection per one nucleotide pair--3,29 A; of the angle of rotation of neighboring nucleotide pair relative each other--36 degrees and of the angle of nucleotide pair with an axis of the helix -13,7 degrees seems to be the most accurate description of the doublehelical structure of poly(dA) . poly(dT). The atomic coordinates of the new model may be used in analysis of stereochemistry of the complexes of nucleic acids with polypeptide and proteins, that is in consideration of the problem of protein-nucleic acid recognition.


The use of temperature biofeedback in the treatment of chronic pain due to causalgia. A patient with chronic pain due to a reflex sympathetic dystrophy in his hand and arm was successfully treated with temperature biofeedback after several months of conservative standard medical care brought little relief. Over the 18 treatment sessions the patient learned to emit a reliable handwarming response of 1 to 1.5 degrees C. Coincident with his learning, the pain in his hand and arm decreased markedly and remained absent at 1-year follow-up.


Glycosylation of mouse embryonic hemoglobins in cultured yolk sac erythroblasts. Embryonic hemoglobins of yolk sac nucleated erythroblasts were glycosylated in vitro by (3H)-glucose and the rate of glycosylation was maximal within 24 h of incubation. Isoelectric focusing of the glycosylated products revealed that both EI and EII embryonic hemoglobins have glycosylated components, EIg, EIIg, with pI very close to the native hemoglobins.


Influence of progressive starvation upon brain and adrenal monoaminergic activity in developing rats of two different ages. Experiments show the influence of progressive starvation upon the synthetic and metabolic activity of monoaminergic function in the brain and the adrenal gland of young rats of two different ages. Brain and adrenal monoamine oxidase (MAO) showed a tendency to decline with the prolongation of the starvation interval. After 60 h of starvation, MAO activity was irreversibly decreased, even with 24 h of feeding, in the two age groups. Cerebral catechol-O-methyltransferase (COMT) activity was very slightly affected in response to the starvation in the older group, but the younger group showed an increased level of enzyme activity, and refeeding after 60 h of starvation of the young rats produced further increases. 60 h of starvation produced an increase in COMT activity of the adrenal gland of the older rats whereas the younger group did not show any marked change. Adrenal phenylethanolamine-N-methyltransferase (PNMT) declined after 24 and 48 h of starvation in the older rats, but the younger rats showed progressive increases after similar intervals of starvation. After 60 h of starvation, PNMT in the adrenal gland of the old rats increased significantly when compared to the control value, but the younger rats did not show any important change. Adrenal stores of adrenaline rose progressively up to 60 h of starvation in the old rats whereas the younger group responded in a contrary manner. Adrenal noradrenaline followed a similar pattern of evolution in both groups up to 60 h of starvation (when the results are expressed per milligram of adrenal protein), and refeeding had very little influence on the effects of starvation. The effects of starvation upon adrenal and cerebral MAO activity were verified with two different substrates. The results provide evidence that the metabolism of monoamines by oxidative deamination can be markedly affected by starvation, and this can be irreversible even after 24 h of feeding of starved rats. COMT activity augments when MAO activity declines.


Monoamine oxidase inhibition and recovery in the mouse brain at various ages after birth. Mice between the ages of 1 day postpartum and adulthood were exposed to the monoamine oxidase inhibitors tranylcypromine and pargyline. The mice were killed at seven different times following drug injection and assayed in vitro for monoamine oxidase activity in four brain regions. Inhibition and recovery of monoamine oxidase activity varied by age, and with the two inhibitors used. During the early maturational period, the potential for new monoamine oxidase synthesis was greater than normal maturational patterns of increase would suggest; and during later maturation either the proportions of various monoamine oxidase forms or their rates of synthesis are different.


Electrokinetic behaviour of cord blood platelets in acidosis: an in vitro study. The effect of acidosis on platelet surface potential was studied in vitro in samples obtained from newborn infants. It was found that acidosis induced by CO2 and lactic acid decreased the electrophoretic mobility of platelets in the presence of plasma. It is suggested that the changed electrokinetic properties of platelets in acidosis may be an additional factor contributing to bleeding manifestations and thrombotic complications often seen in babies with perinatal asphyxia.


Maternal and fetal plasma renin activity during ritodrine infusion to the mother. The effect of ritodrine to the mother on plasma renin activity (PRA) in the mother and fetus was examined in 10 mothers coming to elective cesarean section. 10 comparable mothers without ritodrine infusion served as controls. At the end of 2 h infusion of ritodrine, the mean maternal PRA was significantly (p less than 0.001) higher than in the control group. Also the mean PRA levels in the umbilical vein (p less than 0.001) and umbilical artery (p less than 0.01) were significantly higher after ridodrine infusion to the mother than the respective levels in the control group.


Correlations between neonatal body weight and adolescent brain development in rats. Correlations between neonatal body weight, and body weight and several brain parameters at 30 days of age were studied in normal rats. At 30 days ('adolescence') cortex has already reached its final thickness and the rat exhibits long-term memory. Brain parameters included cerebral weight, DNA, protein and cholesterol contents and densities, as well as cortical and cerebral dimensions (cerebral sections). As expected, most of these parameters in 30-day-old animals were significantly correlated with each other. Unexpectedly, neonatal body weight was also significantly correlated with cholesterol content and density at 30 days, as well as with cortical and cerebral dimensions at 30 days. Thus, statistically, neonatal body weight already predetermines the extent of neuronal (cerebral) development at adolescence (30 days). This finding also makes it possible to make at birth statistical predictions about future brain development without having to sacrifice neonatal animals.


[Spectral forms of chlorophyll and chloroplast structure of Chlamydomonas mutants with disortions in their light-gathering pigments]. A 2-3-fold reduction of chlorophyll content in pigment mutants of Chlamydomonas reinhardii is conditioned by the disturbances in light-harvesting pigments. When the activity of both photosystems is preserved, the reduction of short-wave forms of chlorophyll a (667, 678 nm) is accompanied by a fall of the fluorescence maximum at 685-690 nm; the disturbance of the long-wave forms with the adsorption maxima at 689, 698 an 703 nm results in a decrease of the chlorophyll fluorescence band at 707-718 nm. The long-wave forms of chlorophyll appertain to the pigment-protein complex of PS-1 which is not involved in the formation of contacts between the grand thylacoids.


Reincorporation of iodinated peptides in vivo and the comparative effectiveness of tungstic and trichloroacetic acids in peptide separation. The absorption of 125I-bovine IgG after oral administration to young rats has been followed by gel filtration and quantitatively by TCA precipitation. The wide spectrum of protein derivatives crossing the gut wall have been utilised to compare the relative effectiveness of 10% TCA, and 1% tungstic acid, as reagents for the quantitative assessment of radioactive proteins and peptides. Radioactive iodide was the chief radioactive metabolite when 125I-labelled bovine IgG was fed to young rats, there was no significant incorporation of small radioactive metabolites into protein during a second exposure to the gut wall.


Maintenance of central and peripheral monoamine oxidase activity in developing rats subjected to disturbed alimentary rhythms and undernutrition. Monoamine oxidase (MAO) activity was studied in whole brain, hypothalamus, adrenals and liver of developing rats subjected to disturbed feeding patterns or undernutrition for 3 weeks. The rats were divided into six groups: (1) normally fed controls; (2) rats starved for 24 h, fed for the following 8 h and killed after the last starvation period (PAS); (3) same treatment as in (2) but killed after the last feeding period (PAF); (4) rats starved for 16 h, fed for the following 8 h at a constant schedule, and killed after the last starvation period (PS); (5) same treatment as in (4) but killed after the last feeding period (PF), and (6) undernourished (U). Alteration of the feeding time resulted in significant decreases of MAO activity in the brain and the adrenal gland whereas the hypothalamus and the liver showed a slight increase in activity in the PAS group. In PS rats, MAO activity increased in the brain, adrenals and hypothalamus; in PF rats, the effects of the treatment were inverse. Both in the PS and PF rats, hepatic MAO activity was strongly decreased when assayed with kynuramine. In U rats, hepatic MAO activity was highly increased when assayed with kynuramine but the other tissues responded differently. The adrenaline and noradrenaline stocks of the adrenal gland were markedly increased in all the treated groups; the maximum increase in noradrenaline was observed in the PS rats. The results suggest that any disturbance in the feeding pattern affects the MAO activity in the central and peripheral regions of the young rat during postnatal development. The developing rat seems to get accustomed to new alimentary rhythms, and normal monoaminergic function is rapidly restored when the rat is given a compensatory diet. Increased adrenal catecholamines after a disturbance in the feeding patterns seem to be a response to stress.


Single injection clearance in the neonate. The single injection clearance of inulin was compared to the standard clearance in neonates aged 1--20 days. In the first 3 days of life, the single injection method overestimated glomerular filtration rate by approximately 30%. From the 2nd week of life, values obtained by the two techniques were not significantly different. We conclude that caution should be paid when interpreting results obtained by the single injection technique during the very first days of life.