Showing papers in "Journal of Neurochemistry in 1956"

The quantitative estimation of cerebrosides in nervous tissue.

Abstract: THE extensive research work in the physiological and pathological processes in which lipids are involved has increased the necessity for accurate micromethods for their quantitative estimation. BRANTE (1949) reviewed modern micromethods for the determination of the lipids in nervous tissue, and made a thorough investigation of the various factors which may influence them. Like most other investigators, BRANTE determined the cerebroside content only by estimating the reducing substances in a lipid extract before and after hydrolysis. From the important work of KLENK and collaborators (KLENK, 1941, 1942; KLENK and LANGERBEINS, 1941 ; SCHUWIRTH. 1940) we know that cerebrosides are not the only lipids containing carbohydrates in the central nervous system. They have isolated gangliosides from the brain and determined their amount in different nervous tissues. Besides these two glycolipids, a third has been described by ARSOVE, FOLCH, and MEATH (1951a). They named the new lipid strandin, but my preparations like DAWN'S (1952), showed that strandin consisted of gangliosides in a different physico-chemical state contaminated with low-molecular substances and mucopolysaccharides. CHATAGNON and CHATAGNON (1953, 1954) have suggested that sphingomyelin is also a part of the complex. BRANTE (1949) discussed the interference of gangliosides on the cerebroside values, and we (BRANTE and SVENNERHOLM, 1949, 1951) determined both total glycolipids and gangliosides in some foetuses. Other investigators (JOHNSON, MCNABB, and ROSSITER, 1950; CUMINGS, 1953; BLACKWOOD and CUMINGS, 1954) have neglected the gangliosides. Therefore the values for cerebrosides found in the current literature include gangliosides. EDGAR (1955) who has thoroughly discussed the problem, used the term glyco-sphingosides instead of cerebrosides. He has also made some estimations of both gangliosides and cerebrosides. Consequently, the carbohydrates in a lipid extract are derived from at least two lipid sources. But lipid extracts are contaminated to some extent with low-molecular substances, partly of carbohydrate nature. It is impossible to remove them completely by the methods generally used, i.e., precipitation of the lipids before extraction or re-extraction of a primary lipid extract. Generally, the error introduced by the contaminants is negligible i n the determination of glycolipids in adult nervous tissue, but this is not so in foetal tissue, where the amount of glycolipids is low and that of the contaminants is both relatively and absolutely higher. FOLCH, ASCOLI, LEES. MEATH, and LEBARON (1951b), however, succeeded in removing these substanccs by partition dialysis. By this procedure the gangliosides too are separated from the other lipids. It therefore appeared that it might be possible to use this method to separate cerebrosides from other substances containing carbohydrates.

Depression by reserpine of the noradrenaline concentration in the hypothalamus of the cat.

Abstract: THE observation that reserpine causes a 'severe loss of 5-hydroxytryptamine from brain tissue (PLETSCHER, SHORE, and BRODIE, 1956; PAASONEN and VOGT, 1956) prompted the investigation of the effect of this drug on the noradrenaline content of brain. The region analysed was the hypothalamus, as its noradrenaline content is high and as the effect of reserpine on the 5-hydroxytryptamine of brain had been demonstrated on this region (PAASONEN and VOGT, 1956). Cats were used in which the left adrenal gland had been denervated in an aseptic operation under ether between twelve and twenty days before the experiment. This procedure allowed simultaneous observation of the noradrenaline content of the hypothalamus and of any central stimulation of the sympathetic system produced by the drug; such stimulation would appear as a difference in the amount of medullary amines (adrenaline and noradrenaline) found in the innervated and the denervated gland. The methods used have all been reported (VOGT, 1954) with the exception of the use of pithed rats (SHIPLEY and TILDEN. 1947) for some of the assays of noradrenaline and adrenaline; this preparation gives a steadier baseline than the anaesthetized rat treated with hexamethonium. Control experiments showed that small doses of reserpine, such as might be present in the brain extracts, did not interfere with the assays. The reserpine (Serpasil Ciba, ampoules of 2.5 mg/ml) was given i.p., and doses of the drug and duration of the experiments are shown in Table 1. They were the same as those found to release most of the 5-hydroxytryptamine from the hypothalamus of the dog. The results are presented in Table 1. Cats 1-3 belonged to a litter aged 44 months, cats 4 8 to a second litter of 14 months. Motility and posture were more seriously affected in the young than i n the adult mimals. The figures for the noradrenaline content of the hypothalamus are recorded in column 6: in column 7 they are expressed as percentages o f a mean of 1.4 pg/g fresh tissue (range 0.90-2.08 pg) obtained previously for twenty normal cats of all ages. Column 8 expresses the findings in litter 2 as percentages of the noradrenaline concentration found in cat 8, an uninjected control of the same litter. These figures are probably more reliable than those of column 7, as the variation within litters has previously been found to be small (VOGT, 1954). Irrespective of which calculation is chosen, the results leave no doubt that the injection of reserpine caused a loss in hypothalamic noradrenaline; the loss was very severe in the young kittens, less so in the adult animals. It was at least as great as that produced by large doses of morphine (VOGT, 1954). The effect of reserpine on the adrenal glands appears, howeker, to differ from that of morphine. After morphine.

Cytidine and uridine requirement of the brain.

Abstract: THE nutritional requirements of the brain were investigated previously in perfusion experiments on cats. It has been shown that the carbohydrate metabolism of the brain, and its survival, is dependent on substances which are produced or released by the liver into the blood circulation. In the absence of the liver, aerobic glycolysis developed in the brain, followed later by impermeability to glucose, reducing the survival time of the perfused brain to about one hour. Insertion of the liver into the circulation corrected the faulty carbohydrate metabolism of the brain and prolonged its survival by several hours. (GEIGER, MAGNFS, TAYJ,OR, and VFXALLI, 1954). It was found in the present experiments that, by adding small amounts of cytidine and uridme to the perfusion fluid, it was possible to maintain the perfused brain in good functional condition with a normal carbohydrate metabolism for four hours or longer without the liver. These experiments indicate the necessity of these nucleosides for the maintenance of normal brain functions and metabolism. An attempt is also made to elucidate the mechanism of action of these substances on brain metabolism.

Quantitative histochemical changes during the development of the rat cerebral cortex

Abstract: SUGITA (1917) described in detail the postnatal changes of the albino rat brain. He pointed out its immaturity and the lack of differentiation at birth when only four layers of cortex are present (Fig. 1). SUGITA divided the subsequent development into three phases: Phase I, starting at birth and lasting 10 days, is a period of rapid growth during which brain weight increases from 250 mg to 960 mg and the thickness of the cortex from 0-74 mm to 1.7 mm. During this phase cells migrate from the ventricles, multiply, and become organized, with the result that all fundamental cortical layers are present at 10 days of age, and all the major areas of the adult cortex are recognizable. Phase I1 extends from the 10th to the 20th day after birth. It is characterized by cellular enlargement and the development of axons, but few new cells make their appearance. Myelinization begins in the latter half of this phase. Brain weight increases from 960 mg to 2.0 g and cortical thickness increases to within 4 per cent of the final adult average. By the 20th day the cortex is completely organized. Phase I11 extends from the 20th to the 90th day and is the period in which myelinization is completed. The rat brain is peculiar in that it continues to increase in weight throughout life; however, there is no appreciable increase in cortical thickness or cellular arrangement after the 20th day. Comparable physiological changes follow this anatomical develop ment. Electrical activity recorded from the cerebral cortex is irregular and of low amplitude from 0 to 7 days of age, but by 10 days it is essentially similar to that in the adult ( C m , 1952). From a blind, poorly integrated, dependent animal at birth, the rat becomes, by 20 days, an active and autonomous unit. Recently FLJXNER and his co-workers (1948, 1950, 1951, 1953) have investigated the biochemical changes during the “critical period” as it occurs (in utero) in the guinea-pig. They demonsfrated rapid rises in ATPase, succinic dehydrogenase and cytochrome oxidase activity in whole brain homogenates. The levels of phosphocreatine and ATP were also studied throughout gestation. SUGITA (1917) in his investigation of the preand post-natal cellular changes of the rat cerebral cortex, found a rapid increase in the average cell and nuclear diameter of the pyramidal cells of layers I11 and V during the first 10 days after birth. These findings were similar to those of and FLEXNEX (1950) for the guinea-pig. P o r n (1945) measured

The isolation from brain tissue of a trypsin‐resistant protein fraction containing combined inositol, and its relation to neurokeratin*

Abstract: INTRODUCTION IT has been well established that meso-inositol exists in brain in both free and combined states. More than fifty years ago, THUDICHUM (1901) reported the presence of free inositol, and this observation has since been amply confirmed. More recent work (FOLCH and WOOLLEY, 1942) demonstrated the OcCuITence of inositol combined in brain lipides, and resulted in the isolation from brain of diphosphoinositide (FOLCH, 1949), a phosphatide which contains inositol metadiphosphate as a constituent. Results obtained in this laboratory in the course of work on the nature of neurokeratin have established the presence in brain tissue of a second form of combined inositol, namely, that of a phosphoinositide combined to protein. This paper reports these findings and some other observations bearing on the nature of neurokeratin. Preliminary reports of this work have already appeared (FOLCH and LEBARON, 195 1 , 1953). Neurokeratin is a fraction of brain proteins first isolated by EWALD and K u m (1877) which can be summarily described as a proteolytic enzyme-resistant protein residue obtained primarily from nerve tissue myelin, and which has traditionally been considered to be associated with the protein network of the myelin sheath. The classical procedure for preparing neurokeratin consists essentially of two major operations; namely, the quantitative removal of lipides with organic solvents, and the subsequent treatment of the proteins thus obtained, with digestive enzymes. The procedure takes several months, and involves Such drastic steps as continuous extraction with hot ethanol for four weeks, digestion with pepsin in dilute hydrochloric acid for three weeks, and, in some of its versions, treatment with hot alkali. The product is a protein residue insoluble in water and in dilute acids and bases, rich in sulphur, and resistant to the action of proteolytic enzymes. The preparations obtained by various workers differed somewhat in composition; and BLOCK (1951) concluded that neurokeratin is most likely an artifact, derived from a definite group of substances specific to nervous tissue. The work which led to the isolation of the protein fraction containing combined inositol was an attempt to develop a method of preparation of neurokeratin less

Histochemistry and characterization of hyaluronic acid in axons of peripheral nerve.

Abstract: CATIONIC adsorption, or accumulation, within tissues has been attributed to many substances, including proteins (see LING (1955) for discussion), phospholipids (FOLCH, 1952), soluble constituents, such as polyphosphates (STEINBACH, 1950), chondroitin sulphate (MEYER and RAPPORT, 1951), and to ground substance colloids (JOSEPH, ENGEL, and CATCHPOLE, 1952). In view of certain di!liculties concerning the hypothesis that the internal potassium concentration exists in a free form and is maintained by a “sodium pump mechanism,” LING (1955) has postulated that “fixed charges” within the nerve are responsible for the selective accumulation of potassium. Studies of the energy requirements of nervous tissue during excitation have suggested that “high-energy phosphates” are primarily concerned with the maintenance of structural relationships essential for activity and, presumably, are not speci6dy involved in a sodium pumping mechanism (AWOD and GOLDMAN, 1956). In an effort to determine whether nucleic acids, or the microsomes of which they are an integral part, are involved in ionic accumulation (ABOOD and ROMANCHEK, 1955), the suggestion offered itself that mucopolysaccharides may play a role, since they are implicated in ionic transfer elsewhere in the organism. ’The present communication reports the histochemical identification of hyaluronic acid (HA): in nerve, its isolation and chemical characterization, as well as certain cationic-exchange studies pointing to its possible role in nerve conduction.

The quantitative histochemistry of the cerebral cortex. I. Architectonic distribution of ten chemical constituents in the motor and visual cortices.

Abstract: THE development of quantitative methods for the measurement of chemical and enzymatic tissue constituents in samples of tissue weighing less than 10 p g has made possible the study of the amount and localization of these constituents at rather precise locations in the brain (ROBINS and SMITH, 1953; LOWRY, ROBERTS, LEINER, Wu, FARR, and ALBERS, 1954; POPE, 1952). By utilization of these methods, it is possible to study the chemical and enzymatic architectonics of the cerebral cortex. In this report, the results of a study of the distribution of dry weight per unit volume, protein, total lipide, cephalins, lecithins, sphingomyelins, total sphingolipides. non-phosphorus-containing sphingolipides, total phospholipides, cholesterol, and number of cells per unit volume in each histologically defined layer and sublayer of the motor cortex and visual cortex of the monkey are presented. In thc accompanying papers, studies of some of the enzymatic constituents of these layers ( R O B I ~ S , MITH, EYDT, and MCCAMAN, 1956) and of analyses at 50 p intervals compared with those by architectonic layers throughout the depth of the two cortices (ROBINS. MITH, and EYDT, 1956) are reported.

Enzymic development and maturation of the hypothalamus.

Abstract: FLFXNER (1955) has shown that the cytological changes occurring during the development of nervous tissues are associated with concurrent changes in the pattern of enzymic activity. Changes have been reported in the activity of various enzymes in a number of localized regions of the brain (NACHMANSOHN, 1939, 1940; YOUNGSTROM, 1941; H~MWICH and APRXSON, 1955), but little systematic information is available, however, about the enzymes of the hypothalamus. The hypothalamic centres are of interest in view of their role in relation to the chief autonomic and endocrine functions of the body, and since they may also influence the activity of the cerebral cortex (LE GROS CLARK and MEYER, 1950). The vegetative functions of the hypothalamus are particularly important in the 'early life of the animal. The development of biochemical activity in the hypothalamo-neurohypophysial system was studied by HELLW and ZAIMIS (1949), who found a lower concentration of antidiuretic hormone in the neurohypophysis of the newborn than of the adult rat. DAWSON (1953) observed the appearance of neurosecretory substance in the hypothalamus and posterior pituitary of the rat within a few days after birth. In the present investigation the maturation of the hypothalamus of the rat was studied by following the changes with age in the activities of the enzymes phosphomonoesterase I and I1 (nonspecific alkaline and acid phosphatase) and acetylcholinesterase. The values obtained for the hypothalamus were compared with values obtained concurrently for the cerebral cortex and the thalamus. In addition, histochemical techniques were applied to study the localization of the two phosphomonoesterases in the different hypothalamic nuclei. More information was thus obtained about the distribution of these enzymes than could be obtained by biochemical or histochemical methods alone.

The occurrence and distribution of 5-hydroxytryptamine (enteramine) in the central nervous system of vertebrates.

Abstract: IT is now hell established that the central nervous system of mammals as well as certain ganglia and peripheral nerves of invertebrates contain detectable quantities of 5-hydroxytryptamine (5-HT). AMIN, CRAWFORD, and GADDUM (1954) first found that acetone extracts from various parts of the central nervous system of the dog stimulated the atropinized estrous uterus of the rat. The contraction produced (spasmogenic effect) corresponded, per gram of fresh tissue, to that provoked by 0-01-0.35 p g of 5-HT. The area richest in 5-HT was the hypothalamus, followed by the area postrema, the mid-brain, and other areas of grey matter. 5-HT was not found in the cerebellum, the white matter, or peripheral nerves. Similar results were obtained by TWAROG and PAGE (1953), ZETLER and SCHLOSSER (1954) and, quite recently, by GARVEN (1955). The first investigators found that the 5-HT content of the whole brain of the dog is 0.1-0.36 pg/g and that of the rat 0.24 ,~ ig /g. The isolated heart of Venus tnercenaria (which is five to ten-fold more sensitive to bufotenine and twice as sensitive to N-methyl-5-HT as to 5-HT itself) was used as a test-object. ZETLER and SCHLOSSER (1954), also using a molluscan heart preparation, the isolated heart of Helix pomatia, estimated the 5-HT content of ala cinerea and trigonurn hypoglossi of the ox brain as 0.07 pg/g. Finally, GARVEN (1955), working on the rat uterus preparation, gives the following 5-HT values for the rabbit brain: hypothalamus 0.40 pg/g, mid-brain 0.37 pg/g, olfactory bulbs 0 17 pg/g , cerebellum 0.03 ,ug/g. In the invertebrates, FLOREY and FLOREY (1953, 1954) found a substance biologically indistinguishable from 5-HT in aqueous extracts of cuttle-fish and crab ganglia (20-82 p g per gram of dry weight) as well as in extracts of the nerves of crab legs (30-66 pg/g). WELSH (1953, 1955) has confirmed these data, working with extracts of molluscan ganglia and heart. In ganglia of Venus mercenaria (pooled cerebro-pleural, visceral, and pedal ganglia) the ratio of 5-HT (about 15 pg/g fresh tissue) to acetylcholine was found to be 4 : 1 , in ganglia of Busycon canalicularurn about 1 : 1. The occurrence of 5-HT in ganglia and nerves of molluscs has been again established by ERSPAMER (1955), both by bioassay and by paper chromatography (Octopus culgaris: 0.3-2.2 pg 5-HT per gram fresh tissue). This investigator found, in contrast, unexpectedly low or even doubtful values in the nervous tissue of crustaceans (Palinurus idgaris: (0.05 pg/g, both in ganglia and in leg nerves) and failed to detect 5-HT in the cerebral ganglia of an insect, Locusra nii

The changes with age in the protein composition of the rat brain.

Abstract: THE proportion of protein in the rat brain on a dry-weight basis remains relatively constant during the period of foetal and subsequent growth when the brain is most rapidly increasing in size. It is not known whether the chemical composition of the protein remains approximately constant during growth, or whether the ratios of the individual amino acids in the proteins vary significantly with the age of the animaI. However, such parameters as enzymic activity and nucleoprotein content show that differentiation of the protein must occur to some extent. In this connexion a report of B ~ K (193%) that the concentration of the basic amino acids arginine and histidine is lower in young than in adult rat brain is of interest. BLOCK’S data suggest also that owing to a relatively lower concentration of histidine in young animals, the argininahistidine ratio (A/H) is increased. In the present investigation a study was made of the protein composition of the rat brain in relation to the age of the animal. The arginine and histidine content of the proteins were determined in the brain of animals of different ages, and in particular regions of the brain representing different stages of ontogenetic development. Simultaneous determinations in muscle were carried out to aid in recognizing general growth phenomena. Measurements of the body weight, brain weight, and water content of the brain in rats of different ages were obtained to provide reference standards for these experiments.

The quantitative histochemistry of the cerebral cortex. II. Architectonic distribution of nine enzymes in the motor and visual cortices.

Abstract: IN Paper I (ROBINS, SMITH, and EYDT, 1956a) the architectonic distribution of protein. lipides, and deoxyribonucleic acid was described in the individual layers of motor and visual cortices of the monkey (Mucaca mulatra). In this paper the distribution of nine enzymes-acid phosphatase, alkaline phosphatase, adenosinetriphosphatase (ATPase). purine nucleoside phosphorylase, glutamic dehydrogenase, fumarase, malic dehydrogenase, aldolase, and lactic dehydrogenase-in the architectonic layers of these same cortical areas is described.

On the participation of the glutamic acid-glutamine system in metabolic processes in the rat brain during physical exercise.

Abstract: IT is known that increased functional activity of the brain is associated with ammonia formation (BUDANOVA, 1950; PRAVDIE-NEMINSKIJ, 1933 : RICHTER and DAWSON, 1948; ROSCH and TEKAMP, 1928; TASHIRO, 1922; VLADIMIROVA, 1953, 1954; VRBA, 1954b; WINTERSTEIN and HIRSCHBERG. 1925). Ammonia is toxic to nervous tissue (RICHTER and DAWSON. 1948: VLADIMIROVA, 1953) and produces a neuromuscular block even in small concentrations (GOODMANN and GILLMAN, 1941). For this reason the system which detoxicates the ammonia produced during activity is important for maintaining the working capacity of the tissue. This paper deals with the mechanism of detoxication of ammonia in the brain and the participation of glutamic acid in this process. Swimming served as a method of producing increased activity of the brain in the experimental animals. In our previous experiments we found that during physical exercise the concentration of free ammonia in the brain of the experimental animals was not increased (VRBA, 1954b). The explanation of this apparent disagreement seems to be that ammonia produced in the brain during diffuse neuromuscular activity lasting for several hours is chemically bound to glutamic acid, and glutamine is produced. There is considerable evidence (ELLIOT, 1951 ; SPECK. 1949; WEILMALHERBE, 1950, 1952) of the activity in brain tissue of the enzymic system which forms glutamine. If this system is active in rice, the ammonia produced is bound to glutamic acid, and, instead of an increased ammonia concentration, an increased concentration of free glutamine should occur. In previous experiments we found that this is actually the case, and we could demonstrate a significant increase of free glutamine in the brain during diffuse neuromu~cular activity (VRBA, 1954b). If the increased concentration of free glutamine in the brain is the result of an increased rate of amidation of glutamic acid, we should expect a simultaneous decrease in the concentration of free glutamic acid. This assumption was tested experimentally, and the results are given in the present paper. A preliminary account of these experiments has already been published (VRBA, 1955~).

The content and the concentration of ribonucleic acid in motor anterior horn cells from the rabbit.

Abstract: THE basophilic material of the nerve cell has been the object of a large number of investigations. The striking alterations found in it in various physiological and pathological states have attracted much attention and led to much speculation about its function (for literature, see HYDBN, 1943). The following facts make it likely that the basophilic and ultra-violet-absorbing component is ribonucleic acid (RNA): (a) the affinity for basic stains has been shown to be a property of nucleic acids in in vitro experiments (MICHAELIS, 1947); (b) ultra-violet microphotometry has shown it to have an absorption curve practically identical with that of RNA (HYDBN et al., 1941): (c) it is extracted by ribonuclease (BRACHET, 1940; GERSH and BODIAN, 1943); and (d) finally it has been found to contain the four mononucleotides characteristic of RNA (EDSTROM and HYD~N, 1954; EDSTR~M, 1956). The motor anterior horn cells belong to the neurons rich in basophilic material. Preliminary estimations have been made of the amount and concentration of RNA in these cells in the rabbit (EDSTROM, 1953a,b). In the earlier of the reports cited, where freshly fixed cells were used, they were found to contain about 220 pg of RNA (1 pg = 10-l2 g) and to have a mean concentration of about 0.7% (w/v). In the latter report, 550 pg and 2.4% are the values given for cells first freeze-dried and afterwards fixed. The discrepancy between the values obtained after the two different treatments is evident and calls for a reinvestigation. A further object of the present investigation was to obtain a picture of the distribution of RNA content and volume of the nerve cell body. It is well known that the mean value for the cell volume varies at different segmental levels in the spinal cord. The question is whether this applies also to RNA. It has been suggested that there may be a correlation between the amount of basophilic substance and the plasma volume (HEIDENHEIN, 1911). The present study was undertaken partly with the object of investigating whether there is any relation between the amount of RNA in a nerve cell body and its volume or some other function of the cell body.

Chloride content and metabolism of cerebral tissues in fluids low in chlorides

Abstract: FUNCTIONAL activity in nervous tissues involves ion movements, and several metabolic characteristics of cerebral tissues are affected by the ionic composition of the media in which they are immersed. A previous study (GORE and MCILWAIN, 1952) surveyed effects of cations on the respiration and glycolysis of separated cerebral tissues, and on their response to stimulating agents. The anions of the tissue and surrounding medium have now been examined from this point of view. In this study, attention has been directed especially towards chloride ions. because these normally preponderate and appear to be related to the marked central effects of bromides. Also, chloride ions may be specifically involved in the transmission of inhibitory effects in the central nervous system which is disturbed by strychnine (COOMBS et a]., 1953).

The depression of phospholipid turnover in brain tissue by chlorpromaztne

Abstract: IT has been previously reported that the turnover of the phospholipid phosphorus of the brain is affected by changes in the physiological state (DAWSON and RICHTER, 1950). However, little information is available about changes in the different phospholipid fractions. In the course of studies on the effect of various experimental conditions on the metabolism of phospholipids in uiuo, the effect of the tranquillizing drug, chlorpromazine (Largactil), was tested. The present paper reports the effect of this drug on the turnover of total rat brain phospholipid and of the cholineand ethanolamine-containing phospholipids.

Enzymatic studies of mitochondria and other constituents of lobster and squid nerve fibres.

Abstract: BEFORE it is possible to understand the energy coupling which underlies impulse propagation in peripheral nerve fibres, it is necessary to obtain information concerning not only the molecular and macromolecular structure of the nerve fibre (the structure of the machine), but also the source of the chemical energy. In this connection ABOOD and GERARD (1953) studied the particulates isolated from mammalian nerve, and to show that passage of current through suspensions of nerve mitochondria inhibited the phosphorylation of the mitochondria. MULLINS (1953) found that electrical stimulation inhibits the uptake of P3200, by frog sciatic-nerve fibres. It has also been shown by KOECHLIN (1950) and others (LEWIS, 1952; SCHMITT, BEAR, and SILBER, 1939; SILBER, 1941; SILBER and SCHMITT, 1940) that dicarboxylic acids of the KREBS cycle, such as malic, fumaric, and succinic, and compounds related to them metabolically, constitute an appreciable fraction of the total anions in invertebrate nerve. Since the general programme of research on nerve chemistry in this laboratory concerns primarily invertebrate nerves such as the giant fibre of the squid and peripheral nerves of lobster, it was desirable to determine whether mitochondria isolated from these fibre types behave in typical fashion chemically. Electron microscope investigations of the structure of lobster fibres in thin sections show not only that the mitochondria are clustered almost exclusively at the periphery of the axon just under the Schwann-cell-axon interface, but also that the Schwann cell itself appears to form mitochondria-like particulates by evaginations from the axon surface of the Schwann cell (GEREN and SCHMITT, 1954). Such observations, together with other facts, suggest that the SCHWANN cell may play a significant role in the chemical metabolism of the fibre and possibly in the energy coupling which underlies the process of impulse propagation itself. Using conventional extrusion methods, LIBET (1948) found the ATP-ase activity of the sheath to be about a hundred times higher than the axoplasm. Choline esterase is also said to be conccntrated i n the sheath (NACHMANSOHN, 1952). To get information about the chemical properties of the Schwann cell, new methods were developed which make possible the preparntion of the squid giant fibre sheath with a minimum of damage.

The action of chlorpromazine and reserpine on brain enzyme systems.

Abstract: STUDIES on the mechanism of action of chlorpromazine have indicated that the drug can act as an uncoupling agent of oxidative phosphorylation and also as an inhibitor of ATP-ase and cytochrome oxidase (ABOOD, 1955; BERNSOHN, NAMMUSKA, and COCHRANE, 1956a). The relationships that may exist between these mechanisms are imperfectly understood. The large variety of compounds which can depress the P : 0 ratio suggests that there is no specific correlation between this inhibitory action and physiological function. Also, there is no apparent explanation of how the inhibition of cytochrome oxidase and/or of ATP-ase can provide a basis for the action of chlorpromazine, since other cytochrome oxidase inhibitors, viz. cyanide, azide, and other ATP-ase inhibitors viz, fluoride, do not possess pharmacological properties akin to chlorpromazine. However, the kinetics of the inhibition, as well as the binding sites of the inhibitor to the enzyme and the accessibility of the target enzyme to the organic inhibitor, may vary sufficiently to impart properties to the drug which are different from those of other inhibitors of these enzymes. Since both reserpine and chlorpromazine can produce ataraxia, hypotension, and hypothermia, it is of interest to determine whether the two drugs have any similar mode of action. CENTURY and HORWITT (1956) have shown that a synergistic effect of reserpine on chlorpromazine occurs in inhibiting oxidative phosphorylation, though the molar concentrations of the drugs are in excess of that expected in vivo on a uniform distribution basis. On the other hand, BRODY, SHORE, and PLETSCHER (1956) have found that chlorpromazine does not act by releasing serotonin from brain tissue, whereas this has been postulated as a basis for the clinical effects of reserpine. While a common mechanism may be looked for, dissimilarity in behaviour is also of interest, since each drug has properties which are confined to only one of the compounds. For example, reserpine does not possess the barbiturate-potentiating property of chlorpromazine (STEPHAN, B ~ u R G E ~ I ~ G A v A R D I N , and MARTIN, 1955). In this work, further studies have been made on the effect of these drugs on the cytochrome oxidase and ATP-ase systems. M E T H O D S

Metabolic properties of cerebral tissues modified by neoplasia and by freezing

Abstract: NEOPLASTIC cerebral tissues have received much less metabolic study than have those from several other parts of the body. Aspects of the resting metabolism of cerebral tumours have been reported by VICTOR and WOLF (1 937) and during the course of the present investigation by HELLER and ELLIO~T (1955). However, an especial interest of such tissues lies in their derivation from components of an excitable organ whose level of metabolism changes markedly with its level of functional activity. Techniques for studying metabolic responses to excitation in separated cerebral tissues have been developed (MCILWAIN, 1951, 1955) and are here applied to neoplasms obtained during neurosurgical operations. A preliminary report has been made (MCILWAIN, 1954). Results are also presented of a cognate study of cerebral tissues modified experimentally. Portions of the animal brain were exposed to low temperatures, and samples were examined metabolically after various survival periods, for each of which the corresponding histological appearances were known (BR~ERLEY, 1956). In each case comparison has been made with the metabolic behaviour of normal cerebral tissues from the same animal species.

The quantitative histochemistry of the cerebral cortex. III. Analyses at 50 intervals compared with analyses by architectonic layers in the motor and visual cortices.

Abstract: THE measurements of chemical constituents and enzymes in the motor and visual cortices of the monkey reported in the first two papers of this series (ROBINS, MITH, and EYDT, 1956: ROBINS, SMITH, EYDT, and MCCAMAN, 1956) were made at nine architectonic levels of the cortex and in the immediately subjacent white matter. In the motor cortex, which is approximately 4 mm thick from pial surface to subjacent white matter, this means that each measurement averaged approximately 400 p apart. In the visual cortex. which is approximately 2 mm in thickness, this means that each measurement averaged approximately 200 p apart. Whether changes in the amounts of any of these constituents occurred between the loci measured was a n important question. It was decided to measure protein, fumarase, and glutamic dehydrogenase at 50 ,u intervals throughout the depth of the cortex. Protein was chosen as a n example of a chemical constituent, fumarase as a n enzyme with a distribution in both cortices similar to the majority of the other enzymes studied, and glutamic dehydrogenase as an enzyme with an atypical distribution. Also, it was of interest to confirm the previously found increase in glutamic dehydrogenase activity in the deeper layers of both cortices.

Observations on the acetalphospholipids of brain tissue

Abstract: OH THANNHAUSER, BONCODDO, and SCHMIDT (1951 a) isolated an acetalphospholipid in low yield from brain tissue. A lipid of the same type as (I) (2-aminoethyl 2 : 3-0hexadecylidene-I-glycerophosphate) has recently been synthesized by EGERTON and MALKIN (1953). It has generally been assumed that the acetalphospholipids are stable to alkali, and both FEULCEN and BERSIN (1939), and more recently KLENK (1944), have used hot alkali to destroy ester phosphatides in lipid mixtures and leave acetalphospholipids intact, although it is known that hot alkali treatment leads also to the formation of some plasmalogenic acid (acetalphosphatidic acid) (11) (FEULGEN and BEKSIK, 1939). On the other hand, acetalphospholipids are generally considered to be very ttnsiable to acids, which decompose them into the free aldehyde and glycerylpiiosphoiyIethanolamine (GPE) (THANNHAUSER ef a/.. 1951 b). CH, ~ -0 HC*R CHO '

Purification and properties of sphingosine.

Abstract: IN the course of studies on the biochemistry of the complex lipids of nerve tissue, it has become necessary to have available analytically pure sphingosine for use as substrate in enzyme systems and for the organic synthesis of labelled lipids. We wish to describe a new method for the isolation of pure sphingosine base, a prccedure for the analysis of minute quantities of this material, and some of its chemical and physical properties.