Utilization of endogenous diacylglycerol for the synthesis of triacylglycerol, phosphatidylcholine and phosphatidylethanolamine by lipid particles from baker's yeast (Saccharomyces cerevisiae). The activity of the enzymes diacylglycerol acyltransferase (EC 2.3.1.20), cholinephosphotransferase (EC 2.7.8.2) and ethanolaminephosphotransferase (EC 2.7.8.1) have been measured in a lipid particle preparation from baker's yeast (Saccharomyces cerevisiae) with endogenous 1,2-diacylglycerol as substrate. For all three enzymes the rate of diacylglycerol utilization was established with respect to substrate and Mg2+ concentration. Neither of the enzyme activities was stimulated significantly by addition of diacylglycerols. The conversion of diacylglycerol into triacylglycerol in the presence of CDP-choline and CDPethanolamine, and the synthesis of phospholipids in the presence of acyl-CoA either added or generated in situ were studied. Neither CDPcholine nor CDPethanolamine had an effect on triacylglycerol synthesis. Exogenous acyl-CoA had no effect on either choline- or ethanolaminephosphotransferase activity. However, when the necessary substrates for formation of acyl-CoAs in situ (ATP, CoA, Mg2+ and free fatty acids) were added a decrease in both cholinephosphotransferase and ethanolaminephosphotransferase activity was observed. This inhibition was shown to be due to ATP and might explained as a result of chelation of the Mg2+, a necessary activator of both the choline- and the ethanolaminephosphotransferase.


Low density lipoprotein receptors and catabolism in primary cultures of rabbit hepatocytes. Rabbit 125I-labelled low density lipoproteins (LDL) were incubated with primary monolayer cultures of rabbit hepatocytes in studies designed to assess the role of liver in LDL catabolism at the cellular level. After hepatocytes were preincubated for 20 h in lipoprotein-free medium, they exhibited time- and concentration-dependent interaction with 125I-labelled DLD at concentrations to 1 mg LDL protein/ml and times to 24 h. After a 3 h (37 degrees C) incubation with 50 microgram LDL protein/ml, hepatocytes bound 400 ng (LDL protein)/mg (cell protein), internalized 280 ng/mg, and degraded 660 ng/mg. Internalization and degradation may be greater than indicated by these values since pulse studies suggested the presence of a deiodinase which attacks cell associated 125I-labelled LDL. The amounts of LDL bound to hepatocytes after 3 h (37 degrees C) were similar to amounts for fibroblasts, but DLD internalization and degradation were considerably less. Rabbit hyperlipidemic 125I-labelled DLD showed the same amount of binding but 1.39 times more internalization and degradation than normolipidemic 125I-labelled LDL. Binding of both control and hyperlipidemic LDL was 3-fold greater at 24 and 42 h than at O or 3 h but addition of a 50-fold molar excess of high density lipoproteins (HDL) prevented increased LDL binding with time. Induction of specific high affinity receptors for binding LDL was shown to occur by preincubation of hepatocytes for increasing periods in lipoprotein-free medium and then measuring 125I-labelled LDL binding at 4 degrees C in the presence and absence of excess unlabelled LDL. Finally, hepatocytes took up 40 times more LDL than sucrose or dextran over a 24-h period, an indication that the uptake of LDL occurs via some mechanism other than simple bulk fluid endocytosis.


Uptake and degradation of 125I-labelled high density lipoproteins in rat liver cells in vivo and in vitro. 1. The uptake of 125I-labelled high density lipoproteins (HDL) in various organs of the rat was determined after an intravenous injection. The uptake of 125I-labelled polyvinylpyrrolidone in the same organs was determined in order to assess uptake by fluid endocytosis. The uptake/organ was highest for the liver. The adrenals showed the highest uptake/unit weight of the organs studied. The liver, the kidneys and the spleen showed comparable values for uptake/g of tissue. The uptake of 125I-labelled HDL exceeded by far that of 125I-labelled polyvinylpyrrolidone in the liver, the kidneys, the spleen and the adrenals, indicating that the uptake of 125I-labelled HDL was mediated by adsorptive endocytosis. 2. The in vivo uptake of 125I-labelled HDL was determined in purified hepatocytes and non-parenchymal cells prepared by collagenase perfusion of livers from animals after intravenous injections of 125I-labelled HDL. When expressed per cell, the hepatocytes and the non-parenchymal liver cells took up about the same amount of 125I-labelled HDL. 3. The in vitro uptake and degradation of 125I-labelled HDL in isolated rat hepatocytes was studied. The uptake at increasing concentrations of 125I-labelled HDL was saturable indicating uptake mediated through binding sites. 125I-labelled HDL were easily degraded by contaminating proteases from the perfusate. 4. Subcellular fractionation by isopycnic centrifugation indicated that the accumulation of 125I-labelled HDL did not take place in the lysosomes, but rather on the plasma membrane and possibly in the endosomes (phagosomes). 5. 125I-labelled HDL were internalized into the cells and degraded in the lysosomes. Leupetin and chloroquine, inhibitors of the lysosomal function effectively inhibited the formation of 125I-labelled acid-soluble radioactivity by the cells. Chloroquine, but not the protease inhibitor leupeptin, reduced the hydrolysis of the cholesteryl ester moiety of HDL.


Circular dichroism studies on cytochrome c peroxidase and cytochrome c-551 of Pseudomonas aeruginosa. Circular dichroism (CD) spectra of ferric, ferrous and ferrous-carbonyl forms of Pseudomonas cytochrome c peroxidase have been recorded in the wave length range 200 to 650 nm. CD spectra in the Soret region show that in the oxidized enzyme the two hemes are degenerate, whereas in the reduced form the hemes are perturbed differently and one of the hemes appears to be non-degenerate. Changes in optical activity upon formation of the carbonylderivative suggest a spin-state conversion and indicate the presence of one high-spin and a low-spin heme. A histidine residue is proposed for the axial ligand of the heme iron. The alpha-helical content of the enzyme is estimated to be 34%. Ligand binding or changes in the oxidation state of the heme iron do not alter the conformation of the protein backbone. The dichroic spectra of oxidized and reduced cytochrome c-551 (P. aeruginosa) are included for comparison. In the visible region the cytochrome exhibits CD spectra similar to those of the peroxidase, whereas in the Soret region the dichroic spectra of the cytochrome are simpler. CD spectra in the far-ultraviolet region show the cytochrome to have a high alpha-helix content.


Protected natural peptides as intermediates for preparing semisynthetic peptides and protein analogs. Selective acylation of epsilon-amino groups in cyanogen bromide peptides of cytochrome c using azidoformates. Treatment of CNBr peptides 66--80, 81--104 and 66--104 from cytochrome c with t-butyloxycarbonyl azide leads to selective acylation of the epsilon-amino groups of lysine residues and the phenolic hydroxyl groups of tyrosine residues with less than 25% acylation of the alpha-amino groups. Similar selectivity was obtained for reactions of benzyloxycarbonyl azide, p-nitrobenzyloxycarbonyl azide and p-methoxybenzyloxycarbonyl azide with peptide 81--104. All of these protective groups can be removed under mild conditions, and thus, the partially protected natural peptides are desirable intermediates for the preparation of semisynthetic peptides. Model condensation reactions of N alpha t-butyloxycarbonyl methionine N-hydroxysuccinimide ester with Z-protected peptide 81--104 produced a peptide corresponding to residues 80 to 104 of cytochrome c in 94% yield.


Semisynthetic analogs of cytochrome c reconstructed from natural and synthetic peptides. A biologically active semisynthetic hybrid of horse heart cytochrome c has been prepared by combining the heme peptide 1 through 65 (HP 1-65), prepared by CNBr cleavage of natural cytochrome c, with a semisynthetic peptide corresponding to positions 66 through 104. A fully protected synthetic peptide 66--79 was prepared by a modified solid phase peptide synthesis procedure and was converted to its N-hydroxysuccinimide ester. A peptide corresponding to residues 81--104 of cytochrome c was also isolated from the CNBr cleavage mixture and its epsilon-amino groups and tyrosyl hydroxyl group were protected selectively with the t-butyloxycarbonyl group. This partially protected peptide was reacted with t-butyloxycarbonyl methionine N-hydroxysuccinimide ester to give a derivative having methionine at position 80. This product was deprotected, purified and then t-butyloxycarbonyl groups were again introduced specifically on the epsilon-amino groups to give the peptide, Boc(Lys,Tyr)80--104. A semisynthetic peptide corresponding to residues 66 through 104 of cytochrome c was prepared by condensing the synthetic peptide 66--79 N-hydroxysuccinimide ester with t-butyloxycarbonyl (Lys,Tyr)80--104. The semisynthetic product was deprotected, purified and combined under anaerobic conditions with a heme peptide, HP 1-65, that was isolated from the products of CNBr cleavage of native cytochrome c. The reconstituted semisynthetic cytochrome c was purified by ion exchange chromatography and was shown to have the same oxygen uptake as native cytochrome c when assayed in the succinate oxidase system.


The iron-sulphur centres of soluble hydrogenase from Alcaligenes eutrophus. The soluble hydrogenase (hydrogen:NAD+ oxidoreductase (EC 1.12.1.2) from Alcaligenes eutrophus has been purified to homogeneity by an improved procedure, which includes preparative electrophoresis as final step. The specific activity of 57 mumol H2 oxidized/min per mg protein was achieved and the yield of pure enzyme from 200 g cells (wet weight) was about 16 mg/purification. After removal of non-functional iron, analysis of iron and acid-labile sulphur yielded average values of 11.5 and 12.9 atoms/molecule of enzyme, respectively. p-Chloromercuribenzoate was a strong inhibitor of hydrogenase and apparently competed with NAD not with H2. Chelating agents, CO and O2 failed to inhibit enzyme activity. The oxidized hydrogenase showed an EPR spectrum with a small signal at g = 2.02. On reduction the appearance of a high temperature (50--77 K) signal at g = 2.04, 1.95 and a more complex low temperature (less than 30 K) spectrum at g = 2.04, 2.0, 1.95, 1.93, 1.86 was observed. The pronounced temperature dependence and characteristic lineshape of the signals obtained with hydrogenase in 80--85% dimethylsulphoxide demonstrated that iron-sulphur centres of both the [2Fe-2S] and [4Fe-4S] types are present in the enzyme. Quantitation of the EPR signals indicated the existence of two identical centres each of the [4Fe-4S] and of the [2Fe-2S] type. The midpoint redox potentials of the [4Fe-4S] and the [2Fe-2S] centres were determined to be -445 mV and -325 mV, respectively. Spin coupling between two centres, indicated by the split feature of the low temperature spectrum of the native hydrogenase around g = 1.95, 1.93, has been established by power saturation studies. On reduction of the [Fe-4S] centres, the electron spin relaxation rate of the [2Fe-2S] centres was considerably increased. Treatment of hydrogenase with CO caused no change in EPR spectra.


Oxidation-reduction reactions of copper-thiolate centres in Cu-thionein. Cu-thionein from yeast was investigated by EPR spectroscopy to probe the oxidation state of copper, and the effects on it of oxidizing and reducing agents. At pH 0.2 the copper was released, but no EPR signal from Cu(II) was observed, unless air was present. Optical experiments did not detect any disulphide groups which might have been formed during anaerobic release of copper. The mercurial, p-hydroxymercuribenzoate caused the release of EPR-detectable copper only under aerobic conditions, and EDTA caused release of Cu(II) on heating. No reduction of the copper-thiolate units in Cu-thionein by ascorbate was detected. Potentiometric titrations with hexachloroiridate(IV) or hexacyanoferrate(III) produced several different Cu(II) EPR signals at various stages of oxidation. The former oxidizing agent required a lower oxidation-reduction potential (+350 mV) to oxidize the copper, than the latter (+410 mV) and neither titration was fully reversible. The EPR signal from Cu(II) oxidized by hexachloroiridate(IV) resembled that produced by p-hydroxy-mercuribenzoate in air, suggesting that the copper was released from its thiolate ligands. It is concluded that the EPR non-detectable copper in the native protein is Cu(I). Oxidation-reduction of the copper-thiolate clusters of Cu-thionein is proposed to be decisive for controlling storage and transport of cellular copper.


(Na+ + K+)-ATPase: effects of detergents on the cross-linking of subunits in the presence of Cu2+ and o-phenanthroline. When (Na+ + K+)-ATPase is reacted with Cu2+ or Cu2+-phenanthroline, cross-linking of the two subunits (alpha and beta) occurs. The major products are alpha,beta- and alpha,alpha-dimers. The alpha,beta-dimer is unstable in the presence of EDTA, but becomes stable when it is first exposed to digitonin or Triton X-100. Conversion of alpha-CU2+-beta to alpha-S-S-beta is suggested. If the enzyme that is pretreated with these detergents is used, only the stable alpha,beta-dimer is obtained, and the formation of alpha,alpha-dimer is inhibited. The data are consistent with alpha 2 beta 2 quaternary structure of the enzyme.


In vitro characterization of skeletal muscle beta-adrenergic receptors coupled to adenylate cyclase. [3H]Dihydroalprenolol, a potent beta-adrenergic antagonist, was used to identify the adenylate cyclase-coupled beta-adrenoceptors in isolated membranes of rat skeletal muscle. The receptor sites, as revealed by [3H]dihydroalprenolol binding, were predominantly localized in plasmalemmal fraction. That skeletal muscle fraction may also contain the plasmalemma of other intramuscular cells, especially that of blood vessels. Hence, the [3H]dihydroalprenolol binding observed in that fraction may be due partly to its binding to the plasmalemma of blood vessels. Small but consistent binding was also observed in sarcoplasmic reticulum and mitochondria. The level of [3H]dihydroalprenolol binding in different subcellular fractions closely correlated with the level of adenylate cyclase present in those fractions. The binding of [3H]dihydroalprenolol to plasmalemma exhibited saturation kinetics. The binding was rapid, reaching equilibrium within 5 min, and it was readily dissociable. From the kinetics of binding, association (K1) and dissociation (K2) rate constants of 2.21 . 10(7) M-1 . min-1 and 3.21 . 10(-1) min-1, respectively, were obtained. The dissociation constant (Kd) of 15 mM for [3H]dihydroalprenolol obtained from saturation binding data closely agreed with the Kd derived from the ratio of dissociation and association rate constants (K2/K1). Several beta-adrenergic agents known to be active on intact skeletal muscle also competed for [3H]dihydroalprenolol binding sites in isolated plasmalemma with essentially similar selectivity and stereospecificity. Catecholamines competed for [3H]dihydroalprenolol binding sites with a potency of isoproterenol greater than epinephrine greater than norepinephrine. A similar order of potency was noted for catecholamines in the activation of adenylate cyclase. Effects of catecholamines were stereospecific, (-)-isomers being more potent than (+)-isomers. Phenylephrine, an alpha-adrenergic agonist, showed no effect either on [3H]dihydroalprenolol binding or on adenylate cyclase. Known beta-adrenergic antagonists, propranolol and alprenolol, stereospecifically inhibited the [3H]dihydroalprenolol binding and the isoproterenol-stimulated adenylate cyclase. The Ki values for the antagonists determined from inhibition of [3H]dihydroalprenolol binding agreed closely with the Ki values obtained from the inhibition of adenylate cyclase. The data suggest that the binding of [3H]dihydroalprenolol in skeletal muscle membranes possess the characteristics of a substance binding to the beta-adrenergic receptor.


Phosphoprotein phosphatase activity at the outer surface of intact normal and transformed 3T3 fibroblasts. Using 32P-labeled phosphocasein or phosphohistones as exogenous substrates it was possible to detect a phosphoprotein phosphatase activity on the outer surface of intact normal and transformed 3T3 fibroblasts. Incubation of monolayers of intact cells in buffered salt solution with the radioactively labeled substrate resulted in the release of alkali-labile 32P counts into the surrounding medium. The reaction was: (a) linear with time (at least up to 20 min); (b) proportional to the cell density; (c) dependent on the temperature and pH of the incubation medium; (d) stimulated by K+; and (e) inhibited by sodium fluoride, inorganic pyrophosphate, zinc chloride and relatively impermeant sulfhydryl reagents. Less than 2% of the externally located phosphoprotein phosphatase activity was detectable in pooled cell-free washings of the intact cell monolayer. Phosphocasein did not cause any detectable leakage of intracellular lactate dehydrogenase or soluble phosphoprotein phosphatase activity into the external medium; incubation of the cells with phosphohistones, on the other hand, resulted in appreciable leakage of both these cytoplasmic activities. Neoplastic transformation was associated with a nearly two-fold decrease in the activity of the surface phosphoprotein phosphatase. Addition of serum to either non-transformed 3T3 or spontaneously transformed 3T6 cells resulted in a rapid and remarkeable drop in the cell surface dephosphorylating activity. Acrylamide gel electrophoresis of the dephosphorylated casein or histone substrate revealed no proteolytic degradation or change in electrophoretic mobility. The intact cells showed no damage upon microscopic examination as a result of exposure to phosphocasein or phosphohistones.


Location of rat liver iodothyronine deiodinating enzymes in the endoplasmic reticulum. The iodothyronine-deiodinating enzymes (iodothyronine-5- and 5'-deiodinase) of rat liver were found to be located in the parenchymal cells. Differential centrifugation of rat liver homogenate revealed that the deiodinases resided mainly in the microsomal fraction. The subcellular distribution pattern of these enzymes correlated best with glucose-6-phosphatase, a marker enzyme of the endoplasmic reticulum. Plasma membranes, prepared by discontinuous sucrose gradient centrifugation, were found to contain very little deiodinating activity. Analysis of fractions obtained during the course of plasma membrane isolation showed that the deiodinases correlated positively with glucose-6-phosphatase (r larger than or equal to 0.98) and negatively with the plasma membrane marker 5'-nucleotidase (r ranging between -0.88 and -0.97). It is concluded that the iodothyronine-deiodinating enzymes of rat liver are associated with the endoplasmic reticulum.


Rat liver glycogen metabolism in the perinatal period. The correlation between blood glucose levels, the concentration of glycogen, the activities of glycogen synthase and phosphorylase and their respective kinases and phosphatases was examined in liver of rat fetuses between day 18 of gestation and one day after birth. Between day 18 and 21 there is a rapid increase in the concentration of glycogen and in the activity of synthase a and a much slower increase in the activity of phosphorylase a. The activity of the respective kinases increased rapidly during this period and reached maximum on day 21. The activity of synthase phosphatase and phosphorylase phosphatase increased after day 18, to reach a maximum on day 19 and 20, respectively, but decreased again towards day 21. The possibility that the changes in glycogen concentration and enzyme activities were related to an effect of glucose or AMP on the respective phosphatases was considered. It was found that the Km of phosphorylase phosphatase for glucose in the prenatal period was 5--7 mM, as in the adult. Since the level of blood glucose during this period was constant (2.8 mM), an effect of glucose on phosphatase activity seems unlikely. AMP concentration increased between day 18 and 21 from 6--15 nmol/g. In view of the low level of phosphorylase a activity during this period, the increase in AMP concentration is not considered to be important in the regulation of glycogen breakdown at this time. Immediately after birth blood glucose levels dropped to 5 mg/dl. This was accompanied by a rapid decrease in glycogen concentration and in the activity of glycogen synthase and a rise in phosphorylase activity. Blood glucose levels returned to the initial level within 1 h after birth, whereas the changes in glycogen concentration and enzyme activities continued for at least 3 h after birth. On day 22 all parameters examined had reached the level found in adult rat liver. It is suggested that the rapid changes observed immediately after birth are due to an effect of gypoglycemia mediated by hormones and cannot be ascribed to direct effects of metabolites on the enzyme systems involved.


Adenosine-induced cyclic AMP increase in pig lymphocytes is not related to adenylate cyclase stimulation. Adenosine-cyclic AMP relationships have been studied in pig mesenteric lymph node lymphocytes. The early 2--3-fold increase in cyclic AMP accumulation elicited by adenosine and 2-chloroadenosine, an adenosine deaminase-resistant analogue, could not be correlated to similar effects on the adenylate cyclase activity of disrupted cell preparations, but rather to the competitive inhibition of the low Km (0.17 muM) cyclic AMP phosphodiesterase. The existence of adenosine receptors coupled to lymphocyte adenylate cyclase, which had been proposed by several authors, could not be confirmed by this study Adenosine-cyclic AMP relationships do not appear to be involved in concanavalin A stimulation of pig lymphocytes.


Relationship of prolactin receptors to concanavalin A binding. Concanavalin A, which binds to specific carbohydrate determinants on the cell surface, was used to investigate the binding of prolactin to its receptors in liver membranes from female rats. The binding of 125I-labeled ovine prolactin to receptors was sharply inhibited by concanavalin A. This effect was reversed by the competitive sugar alpha-methyl-D-mannopyranoside and thus required the presence of specifically bound lectin. Concentrations of concanavalin A of up to 50 mu/ml caused a progressive decrease in the apparent affinity of the prolactin receptor for hormone. When higher concentrations were used, the number of available binding sites decreased. Concanavalin A-resistant receptors, about 30% of the total, had the same dissociation constant (Kd) as the controls. The binding of 125I-labeled concanavalin A in the same membrane preparations showed the presence of two distinct types of concanavalin A binding. At low concentrations, the lectin bound with high affinity (Kd approximately equal to 6.6 . 10(-8) M. At high lectin concentrations, low affinity (Kd approximately equal to 6.7 . 10(-5) M) binding predominated. Since high affinity concanavalin A binding was saturated at 50 microgram/ml, this class of binding most likely alters the affinity of the prolactin receptor for hormone; low affinity concanavalin A binding may mask prolactin receptors, making them inaccessible to the hormone. Binding sites for concanavalin A and prolactin appear to be independent but closely related since (i) concanavalin A did not displace bound prolactin from its receptor, and (ii) detergent-solubilized 125I-labeled prolactin-receptor complexes bound to concanavalin A-Sepharose and were eluted by alpha-methyl-D-mannopyranoside.


[Study of sarcoplasmic reticulum Ca-ATPase by the paramagnetic label--paramagnetic probe method]. ESR spectra of nitroxyl derivative labelling with parachlormercury benzoate bound with SH-groups of Ca, Mg-dependent ATPase in the presence of Mn2+ ions are studied. It has been concluded from the saturation curves of ESR spectra that Mn2+ is localized at the distance approximately 40 A from the SH-group of the enzyme active centre. Platinum compound (K2PtCl4) changes the spectrum of ESR 1 due to the displacement of the label from the enzyme SH-groups and disintegration of the sarcoplasmic reticulum structure. Palladium compound (Pd(NH3)4Cl2) produced no effect on the ESR 1 spectrum.


[Establishment of the site of electron transport chain disruption in mutant Chlamydomonas chloroplasts with an inactive photosystem 2]. It has been shown in the studies of 6 strains of non-synthesizing Chlamydomonas reinhardi mutants with a damaged electron-transport chain (ETC) in the region of the Photosystem 2 (PS 2) that the damage is localized on the oxidizing side of PS 2. The ESR studies of the mutants have shown that signal 2 is absent in all the mutants, the width of signal 1 in some mutants is lower than in the control, which is, probably, concerned with the differences in the reaction centre structures. The ETC region from electron inlet from the donor--diphenylcarbaside to P700 is capable of functioning. It is suggested that in all the mutants studied the complex responsible for photodissociation of water is damaged.


New trends in cryoenzymology : II. - Aqueous solutions of enzymes in apolar solvents. In order to set up new procedures to investigate enzyme systems at subzero temperatures in pure aqueous media, we used micromicellar solutions which are homogeneous, optically transparent and of low viscosity in that range of temperatures. The preparation and the main properties of such solutions are described along with the behavior of enzyme-substrate intermediates. A critical discussion of results permits to examine advantages as well as limitations of this very promising procedure.


Combined dynamics of EEG and evoked potentials. I. Studies of simultaneously recorded EEG-EPograms in the auditory pathway, reticular formation, and hippocampus of the cat brain during the waking stage. This study is carried out on single (not averaged) recordings combining the spontaneous activity preceding the stimulus onset and the EP recorded upon acoustical stimulation. These recordings, which we call EEG-EPograms, are measured simultaneously from different subdural brain structures, such as the auditory cortex, medial geniculate nucleus, inferior colliculus, reticular formation and the hippocampus of awake cats. Using a combined analysis procedure (C.A.P.), the relevant frequency components of spontaneous EEG and EPs, recorded simultaneously from these brain nuclei, are analyzed according to the consistent selectivity bands depicted by the determined amplitude-frequency characteristics. These analyses provide us the following information: (1) there is an important congruency in the time courses of simultaneous response components in common frequency bands, especially in the alpha and beta frequency ranges; (2) there exist significant coupling and synchrony between the evoked amplitude enhancements in the simultaneously recorded single response components; (3) the inter-nuclei coherency in the brain's electrical activity is enormously increased upon stimulation; (4) the evoked response magnitude can be predicted, with reasonable accuracy, from the spontaneous activity preceding the stimulation. The strong dependence of the response magnitude on the stimulus-preceding EEG is explained by means of a model network consisting of a population of relaxation oscillators, which can be brought to different states of synchrony and asynchrony. Some suggestions and comments are also made for investigators working toward theories of signal transmission in the brain.


Pattern separability in a random neural net with inhibitory connections. Some interesting properties on pattern separation have been shown through researches by neural models of cerebellar cortex. It seems to us that those results are a part of the properties of pattern separation. A two layer random nerve net with inhibitory connections is given as a model of the cerebellar cortex. The model is composed of threshold elements there. A more general theory of pattern separation than those studied earlier is given, and the pattern separability of the model is considered. It is revealed that the standard deviation of threshold values of threshold elements has a great effect on the pattern separability and the control of the firing rate. The present study is also intended to investigate the pattern separability in such a case that the firing rate of input patterns are not equal, and a pattern includes the other pattern. It is assumed there that the standard deviation is small. Some properties of the degree of pattern separation are cleaned up.


Information transmission in multi-input-output stochastic neuron models. The Shannon's information theory in multi-way channels (Shannon, 1961) is applied to multi-input-output relations of the stochastic automaton models for interaction of excitatory and inhibitory impulse sequences proposed in the previous papers (Tsukada et al., 1977). In these models, the output spike train depends upon several statistical characteristics (mean frequency, standard deviation, form, order-dependence or order-independence, etc.) of the excitatory and inhibitory input spike trains. By the use of the multiple-access channel in information theory, some stochastic properties of temporal pattern discrimination in neurons are analyzed and discussed with biological systems.


[Lipids as possible proton carriers from the respiratory chain to ATP-synthetase and the mechanism of oxidative phosphorylation]. A scheme of oxidative phosphorylation is suggested according to which at the first stage due to the functioning of the electron-transport chain (ETC) in the points of coupling during relaxation of protein non-equilibrium conformation thermodynamically unfavourable transfer of H+ from H2O into the membrane takes place. Athe 2nd stage H+memb is carried by lipids from ETC-protein to ATP-synthetase. At the 3rd stage ATPase with ATP already contained in the active center seizes 2H+. In the course of subsequent protein relaxation the ATP interaction with the active center is disturbed, and ATP with protons transfers to H2O. In terms of the scheme proposed it proves possible to explain the respiratory control and formation of transmembrane potential difference, as well as the action mechanism of uncouplers and inhibitors of oxidative phosphorylation.


Analysis of "integrate-to-threshold" neural coding schemes. Methods of analysis for some deterministic and stochastic variants of the integrate-to-threshold neural coding scheme are presented. Adaptation phenomena are modeled by means of feedforward and feedback adaptive threshold control. Simulations of sinusoidal and step responses reproduce satisfactorily the qualitative characteristics of adaptation as compared with physiological data. It is postulated that such adaptive threshold control may be accomplished by the release, or conformation change, of molecules involved in the control of excitable-channel dynamics.


Synergism and antagonism of neurons caused by an electrical synapse. To investigate the role of electrical junction, a model system consisting of two electrically coupled neurons was studied. It was revealed that the model system generates both the in-phase pattern and the anti-phase pattern stably. Physiological condierations revealed the following. The in-phase discharge pattern (synergism) is a unique output of a electrically coupled pacemaker neurons. However, both the in-phase (synergism) and the anti-phase (antagonism) discharge patterns are possible for electrically coupled bursting neurons. We may expect other discharge patterns, such as a random discharge pattern (chaos), than the inphase and the anti-phase pattern in electrically coupled neurons. Random firing patterns in the inferior olive may be attributed to electrical synapses. Until now, we have assumed that excitation periods of two neurons were almost the same. When two periods are greatly different, various phenomena are expected. Determination of the stability of the anti-phase solution by the analytical methods, when diffusion constants are very small, is one of our future problems.


Plasma renin activity and adrenal angiotensin II receptors in fetal, newborn, adult and pregnant rabbits. Plasma renin activity (PRA) and adrenal angiotensin II receptors have been studied simultaneously in the rabbit at various stages of development. In the fetus both parameters show very low values but increase rapidly during the last 4 days of gestation. PRA reaches a maximal level in the early post-natal period but the concentration of adrenal angiotensin II receptors continues to increase further up to the adult state. In adult females, pregnancy results in an initial rise in PRA and adrenocortical angiotensin II receptors. However, PRA remains at a high level throughout the gestation period whereas the number of adrenocortical angiotensin II receptors decreases progressively as pregnancy progresses. The role of circulating angiotensin in the regulation of the concentration and affinity of its receptor sites is discussed.


Effect of cyclic nucleotides on weight of gastrocnemius and creatine kinase activity after denervation of muscle in young rats. Denervation of the gastrocnemius muscle at various stages of development reduces muscle weight and creatine kinase activity. An inverse relationship between the muscle-specific enzyme, creatine kinase, and the lysosomal enzyme, acid phosphatase, was shown. An increased percentage of the BB isoenzyme of creatine kinase is observed after long-term denervation. Apparently the muscle tissue has the ability to regenerate and presumptive myoblasts are formed from satellite cells. When the denervated muscle is treated with dibutyryl-cyclic-GMP administration new muscle tissue has been formed. Similar effects could not be demonstrated with either cyclic AMP or succinylcholine. The higher percentage of the BB isoenzyme after dibutyryl-cyclic-GMP administration supports the theory that presumptive myoblasts are derived from satellite cells. Succinylcholine also causes an increase of the B-type of creatine kinase. It can be concluded that cyclic GMP, generated via the nerve, has an important role in maintaining muscle weight.


Bimodal distribution of REM sleep latencies in depression. The REM sleep latency of endogenously depressed patients was investigated by analyzing 90 polysomnograms of six patients during depression and 58 polysomnograms of four of these patients after remission. During depression the REM sleep latencies are distributed bimodally with peaks at sleep onset (sleep onset REM phases, SOREMPs) and 60 min later. During the follow-up examinations some time after remission, the occurrence of SOREMPs is very rare. A model is proposed according to which the occurrence of SOREMPs in the sleep of these patients is caused by a reduced amplitude of the circadian rhythm of the arousal system.


Surface chemistry and cytotoxic properties of silica. Aluminium inhibits the harmful effect of quartz on the lungs. Current interest in such protective phenomena has lead us to consider a possible method of bonding aluminium to the crystal lattice, and to see if other atoms might have the same effect. A study of the structure of natural and artificial alumino-silicic compounds shows a surface state specific to quartz, namely the existence of vacancies in the three-dimensional SiO4 tetrahedral structure. Aluminium is a known inhibitor of the cytotoxic action of quartz, and can replace the missing silicon atoms in the crystal lattice. The result is the generation of finite numbers of heat-resistant acid sites. Thus it is not a question of a simple zeolite surface layer, but of the substitution of aluminium atoms for silicon atoms in the remaining crystal lattice. It can be shown that other atoms, such as sulphur and specially phosphorus, are capable of behaving in the same way as aluminium. There is reason to believe that the cytotoxic properties of quartz may be related to the crystal capturing atoms from the cell membrane. The large number of thermally stable acid sites detected after contact with alumina would then be a factor for assessing the cytotoxic properties of the various forms of quartz.


Diagnosis of Niemann-Pick disease by analysis of hair-roots. A micromethod for the measurement of sphingomyelinase in hair root lysates was described. With this method, 22 normal individuals, 3 children affected with Niemann-Pick disease and 2 carriers have been studied. It seems clear that Niemann-Pick homozygotes can be diagnosed definitively, but more extensive studies arenecessary to decide if carrier detection with hair-roots is possible. The particular interest of using hair roots is that the material can be sampled easily and mailed without special precautions.


Specific passive immunotherapy of experimental lymphoma. Effects of macrophage toxic drugs. Sub-lethal irradiation experiments have indicated that undamaged host cells are necessary for efficiency of specific passive tumour immunotherapy. To further indentify these host cells, the effect of known macrophage toxic agents (silica, carrageenan, trypan blue) were tested on passive immunotherapy of a Gross virus induced C57BL/6 mouse lymphoma with specific W/Fu rat anti-Gross cell surface antigens serum. Median survival time of serum treated mice was significantly decreased when toxic drug was injected three hours before tumour cell injection and seven hours before the beginning of passive immunotherapy, only if silica (5 mg i.p./mouse) or carrageenan (2.5 mg i.p./mouse) but not if trypan blue (4 mg i.p./mouse) was used. Pretreatment by PVNO, a macrophage protecting agent, 24 hours before, inhibited silica effect but not carrageenan effect on the passive immunotherapy. These results indicate that host's phagocytic cells are likely to be involved in the efficiency of passive antibody mediated tumour cell destruction, possibly through opsonization. However, more than one phagocyte population may participate in such a mechanism.


The growth-hormone in long-term steroid-treated patients with chronic active hepatitis. To determine whether the hypothalamo-pituitary axis (HPA) became suppressed in patients with chronic active hepatitis (CAH) treated with prednisone or long-activity synthetic corticotrophin (L.A.S.C.) for at least 12 months 36 patients with CAH were assessed for growth hormone release following insulin hypoglycemia test (IHT). Sixteen out of the 36 patients were treated with steroids, 11 with L.A.S.C. and 9 were untreated. Sixteen patients were less than 12 years old of whom 8 were treated with prednisone and 5 with L.A.S.C. and 3 untreated. Suppression of HPA was observed only in two patients of whom one was treated with prednisolone for 24 months and the other one received L.A.S.C. for 23 months. Normal HPA was observed in all the other 34 patients. Two patients aged 6 and 8 years were treated with steroids for 3 and 6 years respectively and showed impairment of the growth in height and remained permanently stunted even when steroids had been discontinued. Our data suggest that both steroids and L.A.S.C. treatment does not impair HPA in patients with CAH when administered for less than two years.


Infantile gastroenteritis caused by rotavirus: comparison of enzyme-linked immunosorbent assay and electron microscopy for rapid diagnosis. ELISA and negative contrast EM have used for the detection of rotavirus in one hundred stools from children, less than two years old, hospitalized for acute gastro-enteritis during the winter 1977-78. Samples obtained from the same children some months after hospitalization were also tested. A good correlation was found between the results given by EM and ELISA, but the later technique turned out to be more sensitive (12% more positive using ELISA). A rotavirus infection could be demonstrated in 73% of the patients. In the stools of 3 children we found a second virus in association with the rotavirus, and in two cases a pathogenic bacterium. When a second serum specimen was available from children previously infected by rotavirus it was always possible to detect a significant increase in CF antibodies. Several months after hospitalization a 2nd survey indicated that the rotavirus was no longer present but calicivirus, echovirus, coxsackievirus and adenovirus could be detected in those asymptomatic children.


Cholesterol synthesis by human fibroblasts in the presence of LDL and anti-LDL IgA. LDL interact with fibroblasts through specific membrane sites. This reaction is the first step of a mechanism which leads to the regulation of intracellular cholesterol synthesis. When LDL form a complex with an IgA from a myeloma serum with mixed hyperlipidemia and xanthomatosis, they no longer function as a regulator, and the result is excess production of free intracellular cholesterol. No excess cholesterol production is observed when myeloma IgA is replaced by control IgG and IgA.


Thermodynamic compensation process in interacting protein systems: definition of thermodynamic compensatory temperature, (Tc). In our thermodynamic analysis of the non-linear Van't Hoff expression as applied to several self-associating systems -- specifically in the cases of bovine liver L-glutamate dehydrogenase (GDH), glucagon and S-carboxymethylated apo A-II protein from human high density lipoprotein -- we have examined the interrelationships of a number of thermodynamic temperatures as they affect the association process. We found the principal determinants of the linear thermodynamic compensation process to be delta S0(T)/delta C0p(T) = (delta T'C)/(Texp), where (delta T'C) = Texp). We have defined the unique compensatory temperature, (TC), for any interacting system, at which the contributions of enthalpy and entropy to the association process are balanced.


The water proton spin-lattice relaxation times in virus-infected cells. The water proton spin-lattice relaxation times in HEp-2 cell cultures were determined immediately after 1 h of polio-virus adsorption. The shortening of the water T1 was closely related to the multiplicity of infection, allowing direct inspections of the virus--cell interaction since the first steps of the infectious cycle. Virus-induced structural and conformational changes of cell constituents were suggested to be detectable by NMR investigation of cell water.


Potentiometric method for substrate analysis using immobilized NAD + -dependent oxidoreductase enzymes. Two coenzyme-dependent oxidoreductases, glucose dehydrogenase and alcohol dehydrogenase, were immobilized in polyacrylamide gel over a platinum grid matrix and used as enzyme electrodes to measure their substrate concentrations in buffered aqueous solutions. The immobilized enzymes were used to oxidize their substrates in the presence of NAD +. Ferricyanide was used as the redox mediator and electroactive species. The determinations of glucose and ethanol were utilized to demonstrate and evaluate the performance of the system. The described methodology should be readily applicable to the analysis of numerous other substrates of coenzyme-dependent oxidoreductases.


Cholangiocarcinoma.

Cholangiocarcinoma. Cholangiocarcinoma may arise in the small bile ducts within the liver or in the large ducts in the hilum or the extrahepatic biliary tree. Curative surgical excision is being increasingly attempted for tumours at all sites and the best results are achieved with cancer at the lower end of the common bile duct. Palliative bypass surgery to drain the biliary tree gives good symptomatic relief and may provide prolonged survival because these tumours are often slow growing. Radiotherapy deserves more extensive trials since about 50 per cent of patients derive benefit. The results of treatment with cytotoxic drugs are so far disappointing.


Incidence of carcinoma in situ of germ cells in contralateral testis of men with testicular tumours. Biopsy specimens from the contralateral testicle in 50 consecutive patients with germinal testicular cancer were examined for carcinoma in situ. Three out of 21 men with seminomas and one out of 29 with other types of germinal cancer (8%) had carcinoma in situ in the contralateral testicle without any clinical signs. One of these men developed early invasive germ-cell cancer 46 months after carcinoma in situ was first diagnosed. The others have been followed up for less than a year without signs of tumour growth. If these results are confirmed routine biopsy of the contralateral testicle in patients with germinal cancer may be indicated.


Static and dynamic fusimotor interaction and the possibility of multiple pace-makers operating in the cat muscle spindle. The interaction of static and dynamic fusimotor activation on the firing of primary muscle spindle afferents has been studied in the cat soleus muscle at constant length and during sinusoidal stretching. Cycle histogram analysis revealed summation of static and dynamic action during the peak of the afferent response to sinusoidal stretching, while static action completely occluded the dynamic effect during the trough of the response. Occlusion was complete as long as, for single fusimotor activation, the static-induced trough response exceeded the dynamic-induced one by about 25%. The investigation of inter-spike interval distributions obtained at constant muscle length revealed occlusion of dynamic by static action in 8 out of 13 cases. A model of multiple spike generation in primary spindle afferents is considered which is based on two or more pacemakers arranged in parallel, with a common pacemaker in series.


Pharmacological characterization of GABA receptors mediating vasodilation of verebral arteries in vitro. GABA (gamma-aminobutyric acid) produced a dose-dependent dilation of isolated cat and dog cerebral artery segments which had been given an active, tonic contraction by either prostaglandin F2 alpha or serotonin. No effect of GABA on extracranial blood vessels was observed. The GABA-induced dilation could be blocked in a dose-dependent manner by either bicuculline or picrotoxin. The latter agent appeared to act as a competitive antagonist. GABA agonists muscimol, imidazoleacetic acid, delta-aminovaleric acid, (+/-)gamma-amino-beta-hydroxybutyric acid, and beta-alanine also relaxed actively contracted cerebral arteries dose-dependently. The relative potency of these agonists was consistent with that established for GABA receptors on neurons and invertebrate striated muscle. GABA was also tested on two human cerebral arteries and found to cause a small dilation. The results support the existence of a cerebrovascular GABA receptor which may mediate an interaction between GABA and the cerebral circulatory system.


Two different and compatible intraneuronal labels for ultrastructural study of synaptically related cells. To examine the structural interactions between synaptically connected neurons in Aplysia, we have developed a method for simultaneously labeling two identified cell with different and compatible intraneuronal marking agents (horseradish peroxidase and [3H]N-acetyl-D-galactosamine) visible in both the light and electron microscopes. Combining these two agents within a single cell yields a third label.


Circulation of cortical spreading depression around electrically stimulated areas and epileptic foci in the neocortex of rats. Spreading depression (SD) elicited in the cerebral cortex of anaesthetized rats does not penetrate into epileptic foci (penicillin) or cortical regions subjected to repetitive electrical stimulation (6--10 Hz, 0.05--0.01 msec). The extent of the SD block, monitored by the absence of the slow potential change and preservation of spontaneous and evoked EEG activity, can be varied in certain limits by changing the stimulus parameters. When the diameter of the block in the parietal cortex is reduced at a time when its temporal boundary is circumvented by an SD wave, propagating in the rostral direction, a strip of cortical tissue between the block and sagittal sulcus is opened for SD which spreads through this route from the frontal to the occipital cortex and starts reverberating around the block. Other methods for eliciting SD reverberation employ removal of an additional block in a section of the circular pathway and directionally biased propagation around the stimulation point. SD reverberation lasting for at least 3 cycles was observed 109 times (median number of completed cycles 5.2, average cycle duration 4.4 +/- 0.1 min). It is suggested that SD reverberation around an epileptic focus can account for slow oscillations of ictal and interictal discharge and for recurrent seizures.


The amplitude of post-tetanic potentiation of the EPSP RC1-R15 in Aplysia is modulated by environmental parameters. Evidence is presented that the EPSP called RC1-R15, which is recorded from cell R15 of the abdominal ganglion of Aplysia californica upon appropriate stimulation of the right connective, is endogenously active. In previous studies we showed that after repetitive stimulation the amplitude of this EPSP increases and then slowly decays over many minutes, a phenomenon called post-tetanic potentiation (PTP). The rate of endogenous firing of this EPSP varies with time of day and tonicity of the animal's external environment. It is shown that this endogenous firing increases the magnitude of the PTP component of the amplitude of the EPSP. The degree of this potentiation varies with the endogenous firing rate. In general, daytime or a hypertonic external environment increases the firing rate of the EPSP and its degree of potentiation. Since the degree of this PTP reflects time of day and environmental tonicity, it is inferred that it conveys physiologically significant information to the postsynaptic cell R15.


Neocortical pyramidal cells: a model with dendritic calcium conductance reproduces repetitive firing and epileptic behavior. A computer model of a neocortical pyramidal cell has been constructed using ideas similar to those used for hippocampal pyramidal cells. This model has been applied to the study of (a) repetitive firing, and (b) the paroxysmal depolarizing shift (PDS), an important intracellular event during seizures. Although calcium spikes have not been demonstrated directly in neocortical cells, we have postulated (by analogy with hippocampal pyramidal cells) a dendritic calcium conductance and a 'slow potassium' conductance modulated by intracellular calcium ion. With these dendritic ionic conductances, the model is able to reproduce the following experimental features of neocortical pyramidal cells: the afterdepolarization and succeeding afterhyperpolarization after an antidromic spike, and the f-I (firing rate-injected current) curve. Some of the differences between 'fast' and 'slow' pyramidal tract neurons (PTNs) -- narrower spikes and a steeper f-I curve in the fast PTNs -- may be explained by differences in Hodgkin-Huxley potassium kinetics between the two kinds of cell. The same model which faithfully reproduces repetitive firing behavior also reproduces (given appropriate synaptic inputs) the following intracellular events recording during epileptic seizures: (a) a burst of action potentials superimposed on and followed by a PDS, and (b) rapid repetitive firing succeeded by an IPSP. Thus, a single set of parameters can reporduce both normal physiological behavior and 'epileptic' behavior: the particular behavior seen depending on how the cell is stimulated. This overall result is the same as for our model of the CA1 hippocampal cell. It suggests that certain acutely acting epileptogenic agents, e.g. penicillin, may act by increasing synaptic input (perhaps both excitatory and inhibitory) to pyramidal cells, rather than by altering their membrane properties. As in our CA1 hippocampal cell model, bursting seems to be a phenomenon generated by the apical dendrite.


Some aspects of the organization of the lateral geniculate nucleus in Galago senegalensis revealed by using horseradish peroxidase to label relay neurons. Following injection of horseradish peroxidase into area 17 of the prosimian Galago senegalensis, columns of labeled neurons are seen in the dorsal lateral geniculate nucleus extending through all cell layers. Individual counts of the number of labeled and unlabeled neurons reveal that from 91 to 98% of all neurons within these densely labeled columns are labeled. These results indicate that most of the neurons within the LGN of the bushbaby project to striate cortex. Average diameter measurements of labeled and unlabeled cells within the labeled columns were used to determine whether cell layers could be separated into two types (parvocellular and magnocellular) or three types (small, medium, and large) on the basis of cell body size. These measurements indicate that the LGN of the bushbaby is composed of two layers each of small (layers 4 and 5), medium (layers 3 and 6), and large (layers 1 and 2) relay neurons. These observations are consistent with the conclusion that layers 1 and 2 in the LGN of Galago are homologous with the magnocellular layers, and layers 3 and 6 homologous with the parvocellular layers, identified in the LGN of New and Old World monkeys.


Subcellular localization of luteinizing hormone releasing hormone degrading activity in the hypothalamus. The in vitro degradation of endogenous as well as exogenous luteinizing hormone releasing hormone (LHRH) by subcellular fractions of rat hypothalamic tissue was studied. Endogenous LHRH, localized an isolated nerve terminals (synaptosomes), was found to be resistant to enzymatic degradation (60 min, 37 degrees C) as long as the synaptosomal membrane remained intact. Endogenous LHRH was rapidly degraded by the 900 x g supernatant fluid and cytosol but not by myelin/microsomes, intact synaptosomes, or mitochondria. Lysed synaptosomes rapidly degraded exogenous LHRH. The LHRH degrading activity of synaptosomes was highly concentrated in the 'synaptosol', i.e., the cytosol of the nerve terminal. These data suggest that the LHRH degrading activity of the rat hypothalamus is a readily solubilized component of neurons, and possibly of non-neuronal cells.


Morphine derivatives with diminished opiate receptor potency show enhanced central excitatory activity. Central excitatory potency of morphine administered by cerebroventricular infusion in enhanced in derivatives substituted at the 3-position (phenolic group) and/or 6-position (alcoholic group). Morphine-3-glucuronide is several hundred times more potent than morphine in evoking dose-related hyperactive motor behavior which can progress to lethal convulsions. Excitatory potencies in decreasing order are: (1) 3-glucuronide; (2) 3-SO4; (3) 3-OAc, 6-OAc (heroin); (4) 6-OAc; (5) 3-OAc; (6) 3-OH, 6-OH (morphine); (7) 3-OCH3 (codeine); (8) 3-OCH3, 6-OCH3 (thebaine). Levorphanol, lacking a 6-OH group, is devoid of excitatory actions. In this series of substituted morphines, there is an inverse relationship between opiate receptor binding potency and central excitatory potency, but codeine and thebaine behave anomalously. These findings are compatible with the hypothesis that morphine acts upon a species of receptor which mediates behavioral and EEG excitation and is distinct from the recognized opiate receptor mediating sedation and analgesia.


Cocaine and D-amphetamine induce changes in central beta-adrenoceptor sensitivity: effects of acute and chronic drug treatment. The effects of acute and chronic treatment with psychomotor stimulants on specific binding of [3H]dihydroalprenolol to beta-adrenoceptors in rat brain were examined. At a dose of 10 mg/kg both acute and chronic treatment with cocaine and chronic treatment with D-amphetamine (10 mg/kg) caused increased binding of [3H]dihydroalprenolol. The molecular mechanism for this enhanced binding appears to be augmentation of the density of beta-adrenoceptors in rat brain. At a lower dose (5 mg/kg), however, chronic administration of D-amphetamine caused a decrease in the density of beta-adrenoceptors in rat brain. Chronic treatment with either D-amphetamine (10 mg/kg) or cocaine induced a marked increase in the magnitude of cyclic AMP accumulation in rat brain slices elicited by norepinephrine. Acute as well as chronic administration of D-amphetamine in vivo inhibited the temperature-dependent uptake of [3H]norepinephrine in rat brain synaptosomal homogenates, but no such inhibition was observed after chronic or acute treatment with cocaine. The results suggest that psychomotor stimulants induce beta-adrenoceptor supersensitivity which may be involved in the phenomenon of reverse tolerance and possibly psychosis in humans. The development of beta-adrenoceptor supersensitivity does not appear to be mediated through alterations in norepinephrine transport at the presynaptic sites.


Suppression of retinal spike discharge by dipropylacetate (Depakene): a possible involvement of GABA. Effects of dipropylacetate (DPA) were studied on spike discharges in the isolated carp retina. (1) DPA, applied electrophoretically or by pressure-microinjection in the vicinity of the recording electrode at the inner plexiform layer, consistently activated spike discharges. (2) DPA introduced into the perfusate produced complex actions; some units responded to DPA with an initial activation followed by suppression of spike discharges. (3) When gamma-aminobutyric acid (GABA) was applied electrophoretically together with perfusion of DPA, the depressing effect was greater than that obtained with each of these compounds alone. Bicuculline in the perfusate markedly attenuated the depressing actions of GABA or DPA singly, or in combination. (4) It is concluded that the delayed suppression of spike discharges by perfused DPA appears to be mediated by GABA-receptors on ganglion cells in the carp retina.


Locus coeruleus and substantia nigra: involvement in morphine-induced behavior. Cats pretreated with morphine (5 mg/kg, IP) received naloxone into the area of the locus coeruleus (LC) or the area of the substantia nigra (SN). The LC-treated animals stopped the morphine-induced stereotyped behavior and showed normal but hyperactive behavior. The SN-treated animals, however, ceased their movements of the head and the forelegs, adopted a rigid posture with extended forelegs and became hypoactive. It is concluded that both the LC area, which contains noradrenergic cell bodies, and the SN area, which contains dopaminergic cell bodies, are sites of action of morphine on behavior.


Intra- and interganglionic synaptic connections in the CNS of Aplysia. Synaptic connections were found between two groups of neurons in the CNS of Aplysia, the cerebral ganglion A and B cluster neurons which are involved in the control of pedal and parapodial movements and neurons in the pleural ganglion which has been shown to modulate locomotion. The A and B neurons made synaptic connections in both the cerebral and pleural ganglia. Pleural neurons had synaptic connections among themselves and with A and B neurons. The A neurons made excitatory monosynaptic connections with the B neurons and a minimum of 6 pleural neurons including the left giant cell (LGC). All of the A neuron synapses found were excitatory. The B neurons received excitatory synaptic input from two other groups of neurons in the cerebral ganglion and both excitatory and inhibitory input from pleural neurons. The latter were identified on the basis of their synaptic connections with the LGC and A neurons. The B neurons and LGC had several common synaptic inputs. The A neurons received monosynaptic input from only 2 pleural neurons. Complex synaptic circuits between A and B neurons and pleural neurons were found. These included recurrent inhibition of B neurons by A neurons via a pleural interneuron, feedforward summation of A neuron synaptic input to the LGC, and reciprocal excitatory synaptic connections between B and the pleural neurons. The activity of the B neurons was modulated by direct inhibitory and excitatory synaptic connections from pleural neurons. The A neurons were modulated primarily by a polysynaptic pathway through the B neurons. The modulation of cerebral A and B neurons by pleural neurons is consistent with behavioral results obtained studying locomotion.


Role of interganglionic synaptic connections in the control of pedal and parapodial movements in Aplysia. Locomotion in Aplysia can be elicited by food chemosensory, tactile, proprioceptive and nociceptive stimuli. The effects of these stimuli on cerebral B neurons, pleural neurons and motor activity in the foot were examined and the behavioral roles of identified synaptic connections among these neurons investigated in semi-intact preparations. The motor effects of intracellular stimulation of pleural neurons were determined. Sensory stimulation elicited spiking in the left giant cell (LGC) in quiescent preparations. When the LGC was spontaneously bursting, sensory stimulation caused decreased interburst intervals and increased burst durations. Following sensory stimulation, LGC firing was correlated with motor activity in the foot. Intracellular stimulation of the LGC evoked contractions in the foot and parapodia. Pleural neurons which produced EPSPs in the LGC and/or B neurons were excited by sensory stimuli that elicit locomotion and when driven caused contractions in the foot. Pleural neurons which inhibited the B neurons were excited by nociceptive stimuli that inhibit normal locomotion, and were inhibited by tactile and chemosensory stimulation of the tentacles which excited the B neurons and can elicit normal locomotion. Intracellular stimulation of the neurons which produced IPSPs in the B neurons evoked contractions in the posterior foot suggesting a motor function in nociceptive induced withdrawal. The synaptic connections between pleural and cerebral neurons are consistent with the proposed modulatory role of the pleural ganglion in locomotion, and may account for changes in locomotion with result from cerebro-pleural connective lesions.


Raphe projections to the locus coeruleus in the rat. Afferent projections to the locus coeruleus from the various raphe nuclei, particularly of the midbrain (nuclei raphe dorsalis and medianus) and pons (nuclei raphe pontis and magnus), have been studied in the rat by retrograde transport methods using horseradish peroxidase (HRP). The locus coeruleus, in both its dorsomedial and ventrolateral divisions, and in its various anterior-posterior components, were injected with 0.05 microliters of horseradish peroxidase following which various structures of the brainstem, particularly the raphe nuclei, were examined for HRP reactive cells. It was found that injections in most components of the locus coeruleus were associated with HRP positive cells in varying degrees of density in the nuclei raphe dorsalis, medianus, pontis, and magnus, with considerably sparser labelling in the anterior aspects of the medullary raphe nuclei pallidus and obscurus. Labelled cells were also seen in the nuclei of the solitary tract, contralateral locus coeruleus. lateral reticular areas of the pons and midbrain, nuclei pontis oralis and caudalis, vestibular nuclei, mesencephalic nucleus of the trigeminal nerve, fastigial nuclei of cerebellum and medial parabrachial nuclei. These data, showing widespread innervation of the locus coeruleus from all raphe nuclei, as well as many other brainstem areas, in the rat support the general view of heavy innervation of the locus coeruleus from both extra-raphe and raphe nulcei. These latter raphe projections, probably serotonergic in nature, provide anatomical support for the various experiments indicating considerable regulation of locus coeruleus activities, such as phasic events of REM sleep, among other, by widespread projections from most raphe nuclei was well as several other regions of the brainstem.


[Modification of a method to study cell fusion induced in vitro by viruses]. A studying method of viruses induced cell-fusion is described, using monolayers of Vero monkey cells. Nature and density of cells and infection multiplicity are studied. The sensibility is equal to 4 hemagglutining units of Sendaï and accuracy about +/- 3 % of polykaryocytosis. Quantification. comparison and kinetics study of cell-fusion are allowed by this method which may lead to systematic and routine exploration of cell-fusion ability of biological extracts from slow degenerative diseases of the human central nervous system.


[In vitro fusion ability of agents causing slow diseases of the central nervous system]. The authors study cell fusion in vitro induced by two kinds of slow viruses : visna virus of sheep and scrapie of mouse and Creutzfeldt-Jakob disease of man agents. Cell-fusion induced by visna virus is either slow and late occuring (fusion from within) or early and rapid (fusion from without) according to experimental conditions. Whereas, cell-fusion induced by spongiform encephalopathies agents is only of slow appearance, looking an endogenous phenomenon.


[Nuclear inclusions in the posterior epithelium of the rat pituitary cleft]. Microfilamentous nuclear inculsions have been found at the ultrastructural level in the posterior epithelium of the rat pituitary cleft. They appear strictly located in ciliate cells of this epithelium. The microfilaments (7-8 nm in diameter) are gathered as a bundle. Their length is up to several microns and their average diameter is 0,35 micron. They can only be observed in adult rats.


Fornix transection: discrimination between neuropeptide effects on attention and memory. Transection of the fornix and the stria terminalis completely blocks the inhibitory action of ACTH 4-10 on extinction of a conditioned avoidance response (CAR), whereas this effect of the vasopressin analogue des-glycinamide-lysine-vasopressin (DG-LVP) is not affected. These data indicate that the behavioral effect of DG-LVP may be localized to certain anatomical substrates, while ACTH 4-10 needs an intact limbic system as a functional substrate for its effect on avoidance behavior. This differential effect of fornicotomy may also be interpreted as a discrimination between the effects of these neuropeptides on attention or on memory consolidation. Additionally, transection of the fornix and the stria terminalis induces an increase in motor responsiveness to an electric footshock (EFS) and a facilitation of acquisition of a CAR.


The identification of alpha-adrenergic receptors in adipose tissue by [3H]dihydroergocryptine binding. The 3H-labelled alpha-adrenergic antagonist dihydroergocryptine has been found to bind specifically to hamster white adipocyte membranes. Binding is rapid and is inhibited by alpha but not beta antagonists. In addition, adrenergic agonists compete for this binding site in order of affinities equal to their activity as alpha-adrenergic agonists. The (-) stereoisomer of adrenaline is 10 times more potent than the (+) stereoisomer in inhibiting binding.


Influence of choline and ethanolamine administration on choline and ethanolamine phosphorylating activities of mouse liver and kidney. The administration of ethanolamine to adult male mice resulted in a significant increase in ethanolamine kinase activity in liver and kidney. Similarly, choline administration resulted in a significant increase in choline kinase activity in liver and kidney. The administration of ethanolamine resulted in enhancement of choline kinase activity concomitantly with ethanolamine kinase activity in liver and kidney. The administration of choline, however, did not result in any significant increase in ethanolamine kinase activity in liver or kidney. Cycloheximide administration along with choline-ethanolamine prevented the increase in kinase activity in liver and kidney. The results obtained have been discussed in relation to the regulatory role of choline kinase and ethanolamine kinase by de novo synthesis in response to enhanced substrate concentration, the secondary nature of choline kinase induction on ethanolamine administration, and possible distinction between choline kinase and ethanolamine kinase.


The indirect fluorescent antibody technique as a method for detecting antibodies in aborted fetuses. In this investigation the indirect fluorescent antibody technique was used to titrate antibodies in bovine sera to parainfluenza 3, infectious bovine rhinotracheitis virus and bovine viral diarrhea virus. These results were compared to those determined on the same samples by hemagglutination inhibition for parainfluenza 3 virus and serum neutralization for bovine virus diarrhea and infectious bovine rhinotracheitis virus. The results of the serological methods agreed closely. The indirect fluorescent antibody technique is a rapid and sensitive method for detecting antibodies and the procedure lends itself to use in diagnostic laboratories. In addition to the above viruses the presence or absence of antibodies to bovine coronavirus and bovine adenovirus 3 were determined by the indirect fluorescent antibody technique in thoracic fluids from 100 aborted fetuses and 50 nonaborted fetuses. Results on these samples were not compared to hemagglutination inhibition or serum neutralization as the condition of fluid samples from aborted fetuses renders interpretation of such tests unreliable. Antibodies to one or more viruses were detected in 30 of the 100 aborted fetuses and in seven of the 50 nonaborted fetuses. Antibodies to more than one agent were detected in eleven of the 100 aborted and in one of the 50 nonaborted fetuses. Reasons for this occurrence and application of the test in determination of causes of abortion are discussed.


Evaluation of the agar-gel immunodiffusion test for the detection of precipitating antibodies against progressive pneumonia virus of sheep. Various cell cultures were evaluated for their ability to support progressive pneumonia virus infection in vitro. Ovine trachea cells supported progressive pneumonia virus infection for an extended time,were extremely durable and could be passaged up until 30 passages. Progressive pneumonia virus infected ovine trachea cells were then used for the production of antigen for agar-gel immunodiffusion. A method for concentrating antigen, diafiltration, was compared to dialysis against polyethylene glycol. Using diafiltration, the concentrated virus was easily quantitated, less viscous (and therefore easier to apply) and only produced one precipitation line. Agar-gel immunodiffusion was used to survey 401 animals from two sheep flocks. One flock (96 sheep) was free of progressive pneumonia while the other flock had 111 of 305 total animals positive for precipitating antibodies. The incidence of precipitating antibodies in sheep ranged from 23% for yearling ewes to 80% in ewes seven years old.


Immune response of cattle to antigens obtained from bovine herpesvirus 1-infected tissue culture. The ability of two antigens, termed EV and CM, derived from bovine herpesvirus 1 infected cultures to produce serum-virus neutralizing antibodies has been studied in cattle. Both EV and CM when inoculated with adjuvant induced significant increases in serum-virus neutralizing antibody titers. Control groups inoculated in a similar manner failed to induce significant increases in serum-virus neutralizing antibody. Some of the animals were vaccinated, then were bred, challenged with a virulent strain of bovine herpesvirus 1 and held until calving was completed. During this 18-month period titers declined slowly in the vaccinated animals. Proportionally there were fewer live calves born to the control cattle than to the CM vaccinated group but reduction was not large enough to conclude that this vaccine had protected the cattle against the abortigenic activity of bovine herpesvirus 1. Further challenge studies should be made to determine whether the administration of these antigens can prevent the subsequent onset of the clinical signs associated with bovine herpesvirus 1.


Aerosol stability of bovine parainfluenza type 3 virus. Aerosols of bovine parainfluenza type 3 virus were generated with a Devilbiss 40 nebulizer from Eagle's minimum essential medium and nasal secretion from a non-infected calf and stored in a rotating drum at temperatures of 6 degrees C or 32 degrees C and relative humidities of 30% or 90%. The aerosols were sampled at seven minutes, one, two and three hours after the start of generation with an all glass impinger (AGI-30) and titrated for infectivity in cell cultures. Physical decay was determined by a rhodamine tracer technique. Media, temperature or relative humidity had little effect on the survival of parainfluenza type 3 virus during spraying (zero to seven minutes). During aging of aerosols at 32 degrees C and 30% relative humidity, parainfluenza type 3 virus was less stable in Eagle's minimum essential medium than in nasal secretion from a noninfected calf, but at 6 degrees C and 30% relative humidity, the virus was more stable in Eagle's minimum essential medium. At 32 degrees C, the virus was less stable during aging at 90% relative humidity than at 30% relative humidity. The virus was consistently more stable during aging of aerosols at 6 degrees C than at 32 degrees C.


Aerosol stability of bovine adenovirus type 3. The WBR-1 strain of bovine adenovirus type 3 was suspended in Eagle's medium or bovine nasal secretion and atomized into a rotating drum at temperatures of 6 degrees C or 32 degrees C and relative humidities of 30% or 90%. Impinger samples of the aerosols were collected seven minutes, one, two and three hours postgeneration, and titrated for infectivity in embryonic bovine kidney cell cultures. Under certain conditions of temperature and relative humidity, the virus was more stable in aerosols of Eagle's medium than in nasal secretion. The bovine adenovirus was usually inactivated more rapidly at 30% relative humidity than at 90% relative humidity and during aging of the aerosols the virus was inactivated more rapidly at 32 degrees C than at 6 degrees C.


Maedi-visna in Canadian sheep.

Maedi-visna in Canadian sheep. Lesions of maedi-visna were seen in sheep from the institutional research flock of the Animal Research Institute, Research Branch, Agriculture Canada in Ottawa. Viral particles demonstrated by electron microscopy in tissue culture cells and serological results confirm the diagnosis of maedi-visna. The extent of the problem in this flock will be described in a future paper.


Use of electron microscopy and an enzyme-linked immunosorbent assay for the detection of rotaviruses in neonatal calf diarrhea. Electron microscopy and an enzyme-linked immunosorbent assay were compared for the diagnosis of rotaviruses associated with neonatal calf diarrhea. One hundred percent correlation was observed when 125 samples were tested by both techniques. Both techniques were equally efficient in detecting rotaviruses. The enzyme-linked immunosorbent assay was more sensitive but being specific could not detect other viruses.


Cyclic nucleotides, calcium, bicarbonate, and protein secretion in canine pancreatic juice in response to cholecystokinin-pancreozymin. The secretion of cyclic AMP, cyclic GMP, protein, calcium, and bicarbonate in the pancreatic juice of three nonanesthetized dogs with chronic gastric and duodenal Thomas cannulae has been studied. Intravenous infusions of increasing doses of cholecystokinin-pancreozymin (CCK) (1.5, 3, 6, 12, 24 Crick Harper-Raper (CHR) U kg-1 h-1) were administered together with a continuous submaximal dose of secretin (1 clinical unit (CU) kg-1 h-1). Doubling CCK doses every 45 min induced a parallel increase in the output of both cyclic nucleotides. Cyclic AMP output peaked at between 15 and 30 min for 3 and 6 U kg-1 h-1 of CCK and later for 12 and 24 U kg-1 h-1 of CCK whereas cyclic GMP output increased more constantly. Calcium output followed a pattern similar to that of cyclic GMP secretion. Flow rate and protein output attained their peaks at between 30 and 45 min. A strong linear correlation was found between the quantities of cyclic AMP, cyclic GMP, and the quantities of protein secreted in response to each CCK dose. This study demonstrates the presence of cyclic GMP in the canine pancreatic juice and the dose-dependent stimulation of the secretion of cyclic GMP and cyclic AMP by CCK in the presence of secretin.


Patterns of early and late discharges in somatotopically identified precentral neurons in awake monkeys in response to somatic inputs. Extracellular recordings were made of single neurons in precentral cortex of awake monkeys. These neurons were somatotopically identified with respect to their responses to inputs from single joints or their somatic surround. Many of these neurons exhibited early (less than 50 ms) and late (greater than 50 ms) discharges in response to flexion or extension torques delivered about the wrist. With the monkey in a mode requiring opposition to the injected torque, all responsive neurons showed a parallel excitatory or inhibitory modification in the early and late discharges. This was true both for cells identified as wrist (flexion-extension) neurons and those identified as nonwrist (flexion-extension) neurons. These findings indicate that the reflex and voluntary components of percentral discharge invariably show a congruent functional response to a torque disturbance, for this particular instruction set.


Structure-activity studies on the inhibition of gamma-aminobutyric acid uptake in brain slices by compounds related to nipecotic acid. Various N-methyl derivatives of nipecotic acid and related compounds were tested as inhibitors of gamma-aminobutyric acid (GABA) uptake into mini slices. N-Methylnipecotic acid, N,N-dimethylnipecotic acid, N-methylguvacine, and N-methylnicotinic acid were effective inhibitors. None of them, however, were as potent as nipecotic acid itself. All the effective inhibitors, including nipecotic acid, also inhibited the uptake of L-proline, but to a much lesser extent. Four of the test compounds produced a depressant action on cerebral cortical neurons, but even N-methylisoguvacine, the most potent in this respect, was considerably less active than GABA. None of the test compounds caused any clearly discernible changes in the gross behaviour or appearance of mice in the 1-h period following intramuscular injection. It was concluded that methylation of the N atom of nipecotic acid and its derivatives was unlikely to lead to the development of agents with greater experimental or therapeutic potential than that of nipecotic acid itself, if the action of the agent was dependent on its effects on GABA uptake.


The effects of cold-induced muscle spindle secondary activity on monosynaptic and stretch reflexes in the decerebrate cat. The effects of muscle spindle secondary ending activity on the stretch reflex were studied in unanesthetized decerebrate cats. Activation of secondary endings was accomplished by reducing the muscle temperature. This has been shown to cause a sustained asynchronous discharge from secondary endings. Cooling of the medial gastrocnemius or lateral gastrocnemius-soleus muscles caused an increase in the phasic and tonic components of their stretch reflexes. Cooling of the relaxed medial gastrocnemius muscle caused similar increases in the components of the stretch reflex of the synergistic lateral gastrocnemius-soleus muscle and an increase in its monosynaptic reflex. It was concluded that the facilitatory autogenetic and synergistic effects of muscle cooling on the stretch and monosynaptic reflexes were brought about by activity in group II afferents from muscle spindle secondary endings and could not be ascribed to any other type of muscle receptor. These results support the concept of an excitatory role for the secondary endings of the muscle spindle in the stretch reflex of the decerebrate cat.


Neck muscle and trigeminal input to the upper cervical cord and lower medulla of the cat. Experiments on chloralose-anaesthetized cats have shown that low-threshold neck muscle afferents project to laminae IV and V in the dorsal horn of the upper cervical cord, to lamina VI including the region which encompasses the central cervical nucleus, as well as to extensive regions of the ventral horn. At posterior medullary levels projections also exist to laminae IV, V, and VI of the spinal nucleus of V (although those to lamina IV are circumscribed), to the deep layers and lateral margin of the cuneate nucleus, and to the inferior olive. These projections are both from low- and high-threshold afferents. Evidence of a functional relationship between the trigeminal and neck muscle afferent system was found both in the upper cervical cord and lower medulla. About 40% of units in both regions receive a convergent input and when convergence could not be demonstrated, prior stimulation of one modality in some instances affected the responsiveness of the unit to the other modality. A motor role was found for some trigeminal afferent projections to the upper cervical cord. Trigeminal afferents consistently activated antidromically identified motoneurons of splenius, biventer cervicis, and complexus.


The effect of ambient temperature on beta-adrenergic responsiveness in rats. The responses of tail skin and colonic temperatures of female rats to ambient temperatures of 20, 22, 24, 26, 28, and 30 degrees C were measured. Within this range, colonic temperature was stable while tail skin temperature increased linearly with increasing ambient temperature. Administration of the beta-adrenergic agonist, d,l-isoproterenol, at 10.0, 25.0, and 62.5 micrograms/kg, sc, at each ambient temperature was accompanied by increases in tail skin and colonic temperatures that were dependent on both the dose of isoproterenol administered and the ambient temperature. The integrated responses of tail skin temperature following administration of the three doses of isoproterenol were maximal at an ambient temperature of 26 degrees C while the integrated responses of colonic temperature were maximal at 30 degrees C. The results suggest that tests of beta-adrenergic responsiveness using this technique should be performed at an ambient temperature of 26 degrees C for maximal sensitivity.


The Syrian golden hamster: a model for the cardiomyopathy of Friedreich's ataxia. In light of the available information on the cardiomyopathy of Friedreich's ataxia, the cardiomyopathic Syrian hamster may be an appropriate laboratory model. Cardiomyopathy in these animals is a result of calcium accumulation. We analyzed the atria and right and left ventricles from cardiomyopathic (CM) and random bred (RB) animals for calcium, magnesium, and iron concentrations at 30-40 and 60-70 days of age (age of maximum lesioning). There are no significant differences in the concentration of iron or magnesium among age-matched groups. The concentration of calcium in the left ventricles of the CM animals at 60 days old is 14 fold higher than that of RB animals. Although there is a significant difference in the concentration of calcium in the left ventricles of younger animals, it is not as pronounced as the difference in older animals. Analysis of the taurine concentration in 30-40 day old animals revealed that the CM animals show slightly higher taurine concentrations than RB in the whole heart. In 60 day old CM hamsters in the beta-adrenergic receptor density of the ventricles is unchanged. This indicates that calcium overload is not due to adrenergic supersensitivity.


Brain neurotransmitter receptors in Friedreich's ataxia. The binding of 3H-quinuclidinyl benzilate, a muscarinic cholinergic antagonist, of 3H-dihydroalprenolol, a beta adrenergic antagonist, and of 3H-flunitrazepam, a ligand which labels benzodiazepine receptors, was examined in several regions of control and Friedreich's ataxia (FA) brains. 3H-Quinuclidinyl benzilate binding appeared to increase in the inferior olivory nucleus, anterior and posterior cerebellar vermi but was unaltered in the dentate nucleus and cerebellar hemisphere of FA brain. The binding of 3H-dihydroalprenolol seemed to increase in the inferior olivary nucleus yet was not different from controls in the dentate nucleus, cerebellar hemisphere, anterior and posterior cerebellar vermi of FA brains. 3H-Flunitrazepam binding was slightly lowered in the inferior olivary and dentate nuclei but was unchanged in the other FA brain regions examined. The present study suggests possible trends in neurotransmitter receptor alterations in post-mortem brain tissue of FA patients.


Fatty acid profile of major lipid classes in plasma lipoproteins of patients with Friedreich's ataxia-demonstration of a low linoleic acid content most evident in the cholesterol-ester fraction. Studies were undertaken to further characterize plasma lipids and lipoprotein abnormalities in Friedreich's ataxia. The high density lipoprotein (HDL) apo AI/AII ratio was quantitated by densitometry and found to be normal. The free to esterified cholesterol ratio in HDL was lower in Friedreich's ataxia because of a reduction in the amount of free cholesterol in this lipoprotein class. The fatty acid profile of the cholesteryl-ester (CE) fraction was markedly deficient in linoleic acid (18:2) in both total plasma and HDL. There was a compensatory increase in saturated acids. The HDL phospholipid (PL) fraction also showed a reduction in the proportion of 18:2 with a concomitant increase in stearic (18:0) and oleic acid (18:1) while the HDL triglyceride (TG) fraction showed only an increase in palmitoleic (16:1) and oleic acids. Feeding of soya lecithin rich in 18:2 failed to increase significantly the 18:2 content of HDL-CE and HDL-PL but lowered the percentage of 16:1 and 18:1 in all 3 lipid classes of HDL. Although the total plasma CE fatty acid profile was perturbed in Friedreich's Ataxia, total plasma PL and TG fatty acid patterns were unaffected. Among the plasma lipoprotein fatty acid profiles, that of the low density lipoprotein (LDL) was most affected, then that of the HDL. The very low density lipoprotein (VLDL) fatty acid composition showed an increase in 16:1 and a decrease in 18:2 which were entirely corrected by lecithin feeding. These results suggest the existence of a metabolic defect in the incorporation of 18:2 into chylomicron phospholipids within the intestinal mucosa.


Evidence for an altered physical state of membrane proteins in erythrocytes in Friedreich's ataxia. Electron spin resonance, scanning electron microscopic, and SDS-polyacrylamide gel electrophoretic studies of erythrocytes in Friedreich's ataxia have been performed. No alteration in the physical state of membrane lipids, in morphology, or in the staining profile of erythrocytes in Friedreich's ataxia could be demonstrated. An altered conformation and/or organization of proteins in erythrocyte membranes in this disorder was suggested by spin labeling studies (P less than 0.025), favoring the possibility of a generalized membrane abnormality in Friedreich's ataxia. These findings are discussed in relation to other inherited neurological diseases where similar studies have been performed.


Equine herpesvirus 1 infection in mares vaccinated with a live-virus rhinopneumonitis vaccine attenuated in cell culture. Vaccination, in July and again in either November or December 1976, of 55 pregnant Standardbred mares with a live-virus rhinopneumonitis vaccine attenuated in cell culture failed to protect some mares from infection with equine herpesvirus 1. From 1976-12-08 to 1977-03-08, 33 mares foaled healthy foals, 16 mares foaled dead foals or live foals which died usually within 48 hours and six mares aborted. Gross and histological examinations and virus isolation studies confirmed that equine herpesvirus 1 caused 18 of the 22 neonatal deaths, stillbirths or abortions.


Cells transformed by temperature-sensitive mutants of avian sarcoma virus cause tumors in vivo at permissive and nonpermissive temperatures. Chick embryo (CE) fibroblasts and normal rat kidney (NRK) cells transformed by temperature-sensitive (ts) mutants of avian sarcoma virus (NY68, LA23, LA24, LA25, LA29, LA31, GI201, GI202, GI251, GI253 induce tumors on the chorioallantoic membrane (CAM) of chick eggs at temperatures that correspond to the permissive and nonpermissive temperatures used to induce conditional expression of the "transformed" phenotype in these cells when cultured in vitro. Chick embryo cells infected with transformation-defective mutants of ASV (td101, td108) or RAV-50 were nontumorigenic under the same conditions, as were nontransformed CE and NRK cells. This indicates that the CAM is not an unusually susceptible substrate for cell growth and that the ability of tsASV-transformed cells to form tumors at nonpermissive temperatures reflects their true tumorigenicity. In contrast, a ts mutant chemically transformed rat liver cell line, ts-223, only formed tumors on the CAM under permissive conditions. The wild-type parent cells (W-8) of this mutant produced tumors at both permissive and nonpermissive temperatures. Direct implantation of microprobe thermometers into tumors caused by ts-ASV-transformed cells at nonpermissive temperatures confirmed that tumor formation occurred in a stable temperature environment and was not due to temperature fluctuations which might have created semi-permissive conditions for tumor growth. Cells isolated from tumors formed at nonpermissive temperatures and recultured in vitro displayed temperature-dependent hexose transport and colony formation in agar similar to the orginal parent cell inoculum. Similarly, virus recovered from tumors at nonpermissive temperatures retained the ts mutation.


Expression of the autonomous parvovirus H1 genome: evidence for a single transcriptional unit and multiple spliced polyadenylated transcripts. We identified viral transcripts in parvovirus H1-infected rodent cells using the S1 nuclease mapping technique of Berk and Sharp (1977, 1978). The most abundant viral transcript, present in both nucleus and cytoplasm, is approximately 2.8 kb long and represents about 56% of the viral genome. Less abundant viral transcripts of 3.0, 1.45 and 1.30 kb, and possibly other minor viral transcripts, are also detected in nuclear and cytoplasmic fractions. In contrast, a prominent 4.7 kb viral transcript which corresponds to 95% of the viral DNA is found only in the nucleus; this finding suggests that the parvovirus genome may function as a single transcription unit. Virus-infected cells pretreated with cycloheximide accumulate all these viral transcripts. Analyses of RNA-DNA hybrids (isolated from neutral agarose gels) by electrophoresis on alkaline agarose gels indicate that the 4.7, 3.0 and 2.8 kb viral transcripts are "spliced" RNAs. The nuclear-specific 4.7 kb transcript appears to be encoded by two noncontiguous DNA segments of 2.2 and 2.6 kb. The 3.0 and 2.8 kb transcripts are apparently encoded by.a 2.6 kb segment of DNA and one or more much smaller noncontiguous DNA segments, one of which is approximately 170 nucleotides long.


Defective interfering particles modulate VSV infection of dissociated neuron cultures. Infection of dissociated neuron cultures of mice with VSV and its defective particle DI-T was studied using fluorescent light microscopy as well as transmission and scanning electron microscopy. When cultures are infected with wild virus, VSV replicates selectively in neurons, producing cell death within 24-48 hr. Sensory and immature neurons express viral antigen most rapidly. Viral antigen and viral budding sites are detected along the neuron soma and dendrites. When large amounts of DI-T particles are added to the wild virus inoculum, viral growth is completely suppressed in mature neurons, the cell killing effects of VSV are considerably delayed and co-infected cultures survive for 5-16 days. Viral antigen accumulates in cytoplasmic inclusions and on the membrane of neuron cell somas and dendrites in the virtual absence of viral assembly. Identical modulation of VSV infection in mature neuron cultures is obtained when DI-T particles are added before or after the wild virus, but ultraviolet inactivation of DIs completely abolishes their protective effect. Immature neurons or Vero cells cannot be protected from acute cytopathic changes by an equivalent amount of DI particles. Thus DIs interfere with replication and assembly of the wild virus and attenuate cell killing effects in mature neurons in vitro.


[Effects of cyclic AMP treatment on the migration of primordial germ cells in the embryo of Xenopus laevis]. After a treatment of early embryos by AMPc, only 40% of the primordial germ cells are found in the gonads; the others are found in the dorsal mesentery and especially in the intestinal wall.


[Histochemical localization of lactate dehydrogenase (LDH) during cartilaginous cell differentiation in pleurodeles (Pleurodeles waltlii Michah]. As revealed in chondrogenetic cells the LDH activity that is very low during cellular migration, suddenly increases at the time of cytodifferentiation and reaches a peak at the onset of the first chondrocyte differentiation. A relationship is likely to exist between this increase in LDH-activity, chondroitin-sulfate synthesis and NAD metabolism.


[Suppressor effect induced by sodium periodate on the antibody response of mouse splenic cells in vitro : inhibition of this effect by sodium borohydride]. Mild oxidation of mouse spleen cells by sodium periodate induces blastogenesis. Concomitantly, the in vitro response of these cells to sheep red blood cells is inhibited. Exposure of spleen cells to sodium borohydride following periodate treatment reduces blastogenesis and restores the immune response.


Visualization of the distance between perfusion and anoxia along an ischemic border. The distance between perfusion and anoxia was measured on the border of an experimental ischemic area in the rabbit heart. Reduced nicotinamide adenine dinucleotide (NADH) fluorescence photography was used to detect myocardial anoxia. Fluorescein angiography marked areas of myocardial perfusion. The hearts were isolated, perfused with a hemoglobin-free solution and performed no external work. In all hearts there was a narrow band between areas of perfusion and anoxia that measured 329 +/- 42 mu (mean +/- SD). The transition from minimal to full NADH fluorescence was abrupt, less than 80 mu. We conclude that the normoxic/anoxic transition is sharp, and the gap between perfusion and anoxia is narrow along an ischemic border in the isolated heart performing no external work. These data suggest that in the vivo working heart the gap between perfusion and anoxia would be even narrower.


Evaluation of a rapid, sensitive and specific assay for the determination of collagenolytic activity in biological samples. Several methods for the determination of collagenolytic activity were compared from the point of view of sensitivity, selectivity, simplicity and practical value for large numbers of biological samples. A labelled collagen substrate was prepared using [3H]acetic anhydride. The specificity of the assay as well as conditions allowing an optimum detection limit were investigated. The influence of low temperatures, lyophilisation and salt concentration on Clostridium histolyticum collagenase have been investigated.


Serum high density lipoprotein cholesterol determination: a simple modification. A simple modification to the method of Burnstein et al. (Burnstein, M., Scholnick, H.R. and Morfin, R. (1970) J. Lip. Res. 11, 583) [1] for the measurement of serum high-density lipoprotein (HDL) cholesterol concentrations is reported. The modification obviates the need for a refrigerated centrifuge and makes the method suitable for routine use in almost any laboratory. Good correlation was obtained for values in 80 healthy adults using the method as originally described by Burnstein et al. [1] and then with the modification. The presented method was linear and precise and the study showed that it was not necessary to fast before giving a blood sample for the assay. It was found that HDL cholesterols were higher in women than in men, but did not change significantly with age in either group.


[A simple micromethod for sphingomyelinase determination (author's transl)]. A rapid and sensitive micromethod for routine determination of sphingomyelinase activity of leukocytes, fibroblasts and hair roots is described. This method makes use of the natural radioactive substrate of the enzyme. The product of the reaction is isolated by direct application of the incubation mixture on silica gel plates followed by ascending chromatography. The sensitivity of the method allows sphingomyelinase determination in less than 0.5 microgram of fibroblast proteins.


Combined hyperlipidemia and hypertriglyceridemia in renal allograft recipients. Comparison with non-renal combined hyperlipidemic or hypertriglyceridemic patients and normal subjects. In order to investigate the degree of similarity between renal transplant and non-renal combined hyperlipidemic and hypertriglyceridemic patients, serum and lipoprotein lipid compositions were compared in transplant and non-renal combined hyperlipidemic and in transplant and non-renal hypertriglyceridemic patients, and normal subjects. Although certain similarities were demonstrated, combined hyperlipidemia in transplants differed from that in non-renal patients in a number of respects: (1) LDL-triglyceride levels were increased to a greater extent in transplant than in non-renal patients in females, while LDL-phospholipid was elevated in male transplants only; (2) HDL-cholesterol levels were raised in transplants relative to non-renal patients in females, and relative to both non-renal patients and normal subjects in males; (3) a number of differences in lipoprotein-lipid ratios between transplant and non-renal patients were demonstrated for all three lipoprotein fractions. In hypertriglyceridemia, changes in lipoprotein-lipid levels were similar in transplant and non-renal patients with the exception of HDL-cholesterol levels, which were decreased in non-renal patients only. Furthermore, the ratio of esterified to free cholesterol in LDL and HDL was decreased in non-renal but not in transplant patients. The data presented demonstrate that, despite certain similarities, a number of the lipoprotein-lipid changes observed in transplant combined hyperlipidemia and in transplant hypertriglyceridemia differ from those observed in non-renal patients with similarly elevated serum lipids.