Vincenzo Bonavita

NEUROCHEMICAL STUDIES ON THE INHERITED RETINAL DEGENERATION OF THE RAT. I. LACTATE DEHYDROGENASE IN THE DEVELOPING RETINA.

Abstract Lactate dehydrogenase activity has been shown to increase in the normal retina and to decrease progressively in the retina of rats with inherited retinal degeneration after the third week of age. In contrast with the quantitative measurements of lactate dehydrogenase in the whole organ, an investigation of the electrophoretic and catalytic features of the enzyme has allowed a very early discrimination between normal and affected rats.

NEUROCHEMICAL STUDIES WITH l‐CYCLOSERINE, A CENTRAL DEPRESSANT AGENT

CENTRAL inhibition has received less experimental attention than other inhibitory phenomena in nervous activity, for quite a long time. Theories, however, have not been lacking, as seen in the physiological literature since 1872, when HERING first proposed that inhibition acted by its regulation of basic metabolic processes in the nerve cell. While KUFFLER and EDWARDS (1958) have given conclusive evidence that GABA simulates the action of physiological inhibitory processes in the stretch receptor of the crayfish, the same direct evidence is still to be provided for single cells in the central nervous system, before GABA or related compounds can be seriously considered as physiological inhibitory substances. Whereas this type of evidence has not been produced up till now, considerable neurochemical and pharmacological data have accumulated on the homeostatic mechanisms regulating the level of GABA in the central nervous system, and on the relevance of its variations to the stability of neuronal membranes (ROBERTS et ul. 1960). Most recently, FERRARI and ARNOLD (1961) have carried out studies on the effect of agents which act on the central nervous system on rat brain GABA level. Although they tested several compounds, none produced dramatic changes in the GABA content of the brain. Furthermore depressant compounds such as diphenylhydantoin, trimethadione and mephobarbital did, in fact, cause a decrease of the amino acid level, which did not support the hypothesis of central depression mediated by a tissue increase in GABA. As far as we know, amino-oxyacetic acid and hydroxylamine have been found to determine the greater elevation of GABA in the brain of various mammals (WALLACH, 1960; BAXTER and ROBERTS, 1961). The two compounds are powerful inhibitors in cico of ABAT without affecting the activity of GDC* in the intact brain, while they inhibit both the enzymes at low concentrations in vitro. This is not SO with 0-methylhydroxylamine, which inhibits the transaminase in citro, but has no effect i l l ciuo upon the level of GABA in rat brain (BAXTER and ROBERTS, 1961). Since L-cycloserine, a central depressant agent (MONACO, 1959), is a cyclic derivative of 0-substituted hydroxylamine, one would assume that it would not elevate brain GABA when injected into mammals. However, that the compound may cause an increase in the level of GABA is easily supposed, when considering that L-cycloserine is a strong antagonist of convulsions induced by isoniazid, which is known to be responsible for a decrease of GABA in the brain (MONACO, 1959; KILLAM and BAIN, 1957).

Nicotinamide adenine dinucleotides in the developing rat brain

Summary Measurements of oxidized and reduced nicotinamide adenine dinucleotides (NAD, NADH, NADP, NADPH) in the developing rat brain have been carried out at various time intervals after birth. The concentration of the two oxidized coenzymes (μg/g wet weight) increased in the developing brain, while the level of the reduced coenzymes underwent a progressive decline. The relevance of these results to the understanding of the metabolic organization of the nervous tissue is briefly discussed.

PURINE AND PYRIMIDINE NUCLEOTIDES IN THE BRAIN OF NORMAL AND CONVULSANT RATS

Abstract— Purine and pyrimidine nucleotides were measured in the brain of normal and electroshocked rats after chromatographic separation on ion‐exchange resin of mono‐, di‐ and tri‐phosphorylated derivatives.

Aspartate aminotransferase in the brain of the developing rat.

Abstract The biochemical immaturity of the rat brain at birth has been largely investigated in recent years (see for references, Sperry, 1955, 1962; Himwich, 1962). It has been tempting to relate neurochemical data to older findings on the physiological maturation of the organ.

LACTATE DEHYDROGENASE ISOENZYMES IN THE NERVOUS TISSUE. V. THE EFFECT OF X- RAYS ON THE ENZYME OF THE DEVELOPING AND ADULT RAT BRAIN

DEVELOPING neurons and proliferating glial cells are highly susceptible to ionizing radiations (BAILEY, 1962). The brain of the new-born rat would appear, therefore, to be an exceptional target to ionizing radiations. The immaturity of rat brain at birth is reflected in three main morphogenetic processes: (i) the development of dendrites and axons from neurons which have attained their final number; (ii) the active proliferation of glial cells; (iii) the myelinisation (SUGITA, 19 17). Parallel to these phenomena are several biochemical changes among which the molecular evolution of lactate dehydrogenase (LDH) has been recently investigated in this laboratory (BONAVITA, PONTE and AMORE, 1962, 19646). The present article describes an attempt to modify the maturation curve of LDH by ionizing radiations. It has been supposed that the block of glial proliferation (BAILEY, 1962) would have been responsible for an apparent acceleration of the maturation process previously reported. The experimental evidence has been in keeping with the hypothesis. It has been found, however, that there is also a delayed effect of radiation on some kinetic properties of the enzyme.

LACTATE‐DEHYDROGENASE ISOENZYMES IN NERVOUS TISSUE—III

THE BIOCHEMICAL properties of single nervous structures have not been the topic of many systematic investigations. With reference to the enzyme composition, regional neurochemistry was first studied by Lowry and coworkers who also collected data on LDHt (STROMINGER and LOWRY, 1955; ROBERTS et al., 1958). Using a different approach, BAILEY and HEALD (1961) have recently shown startling differences in the protein composition of various nervous structures by means of starch-gel electrophoresis of tissue extracts. According to these authors (BAILEY and HEALD, 1961), cytoplasmic proteins exhibit electrophoretic patterns which are peculiar to the species and to the brain structure. Clearly, differences in the electrophoretic patterns given by the pool of cytoplasmic proteins do not imply functional differences at a biochemical level per se, although the hypothesis is quite likely to be true. BONAVITA and GUARNERI (1962) have already reported significant catalytic differences among LDH isoenzymes from ox brain as separated by agar-gel electrophoresis according to GRABAR and WILLIAMS (1955). Though the electrophoretic migration on agar gel causes great conformational changes of ox brain isoenzymes, satisfactory evidence has been obtained to suggest that LDH isoenzymes of mammalian brain are catalytically different also in their native condition. Experiments with pyridine analogues of DPN support this view (FLEXNER et al., 1960; BONAVITA and GUARNERI, 1962). Starting from this observation, the possibility of a differential isoenzymic composition of various brain structures has now been examined. This paper summarizes electrophoretic and catalytic findings concerning ox brain LDH’s. Regional differences in the distribution of single isoenzymes and in the kinetic properties of unresolved LDH are not so great as those observed in the comparative analysis of vertebrates (BONAVITA and GUARNERJ, 1963). Regional data, however, are certainly much more relevant than comparative observations to the molecular basis of systemic nervous pathology.

Lactic dehydrogenase isozymes in the nervous tissue I. The reaction of isozymes with diphosphopyridine nucleotide analogues and their inhibition by sodium metabisulfite

Abstract 1. 1. Brain l (+)lactic acid dehydrogenases from several species have been submitted to agar-gel electrophoresis. When the pH is 7.6, the beef-brain enzyme, which has been studied most extensively, may be resolved into three main components. 2. 2. Some kinetic properties of three l (+)lactic acid dehydrogenase isozymes from beef brain have been investigated by using several DPN analogues and the determination of other kinetic parameters ( K m for sodium pyruvate, excess substrate inhibition, inhibition by sodium metabisulfite). 3. 3. Sodium metabisulfite has been found to be a powerful competitive inhibitor of brain l (+)lactic acid dehydrogenase. Resolved isozymes are less sensitive to the inhibitor than the native enzyme. pH-activity curves in the presence of sodium metabisulfite show definite differences among the multiple forms of l (+)lactic acid dehydrogenase. 4. 4. Experiments with the pyridine analogues of DPN and sodium metabisulfite, and also the analysis of the enzyme affinity for pyruvate, have shown that the electrophoretic migration on agar gel causes a conformational change of the single l (+)lactic acid dehydrogenases. The nature of this modification has been discussed.

Diphosphopyridine nucleotide synthesis in brain following injection of various compounds

Abstract Diphosphopyridine nucleotide synthesis in brain has been investigated following intraperitoneal and/or intrathecal injections of nicotinamide, nicotinic acid, reserpine and azaserine. The findings described in this note give conclusive evidence that pyridine nucleotide synthesis is an active process in brain as well as in liver, although it is much easier to alter DPN turnover in liver than in brain.

ELECTROPHYSIOLOGTCAL AND NEUROCHEMICAL STUDIES FOLLOWING INJECTION OF MONONUCLEOTIDES AND THEIR DERlVATIVES

THE PERFUSED brain rapidly deteriorates if the liver is not included in the perfusion circuit. It has been found, however, by GEIGER and YAMASAKI (1956) that addition of cytidine and uridine to the perfusion fluid produces an effect similar to inclusion of the liver. The neurochemical significance of free nucleotides and their derivatives has been realized only recently (BRADY and TOWER, 1960), but also the peculiar position of ribonucleic acid in the biology of the neuron (HYD~N, 1960) stimulates investigation of the actions of purine and pyrimidine nucleotides in vivo. CAMERON (1958) reported on the use of ribonucleic acid in aged patients with memory impairment and claimed a considerable or complete restoration of retention memory when ribonucleic acid (RNA) was given orally, This observation led to an attempt to obtain a clear cut effect of an alkaline hydrolysate of RNA on the human brain (BONAVITA, SCARANO and ZITO, 1961, 1962). The structural similarity of uracil, cytosine and barbituric acid suggested an attempt to reveal a possible RNA effect by recording the pentothalactivated EEG, before and after treatment of an experimental group with the alkaline hydrolysate of yeast RNA. The activation or synchronization of the EEG with sodium pentothal has been used for several purposes, e.g. the localization of focal lesions (SCHNEIDER and THOMALSKE, 1956) and the analysis of drug effects in psychoses (GOLDMAN, 1960). BONAVITA, SCARANO and ZITO (1961, 1962) showed that the injection of sodium pentothal into subjects pre-treated with hydrolysed RNA led to deeper changes of stages C and D, as described in the pentothal-activated EEG by SCHNEIDER and THOMALSKE (1956). Moreover, BONAVITA, SCARANO and ZITO (1961, 1962) found that the pentothal-induced electrographic changes and sleep appear significantly earlier, and last longer, when the barbiturate injection follows treatment with RNA. This suggested the investigation of the EEG in the rat after intraperitoiieal injection of a mixture of mononucleotides, four mononucleotides (uridylic acid, cytidylic acid, adenylic acid, guanylic acid), four nucleosides (uridine, cytidine, adenosine, guanosine) and the corresponding heterocyclic bases. Clear cut changes in the EEG of human subjects could only be recorded after pentothal injection (BONAVITA, SCARANO and ZITO, 1961, 1962). In the present paper it is shown that the electroencephalogram of the rat undergoes conspicuous changes even when some rnononucleotides or nucleosides or bases are injected alone. Sensitive and accurate EEG readings are difficult in the rat but it is a convenient animal for biochemical tissue studies and shows deep electrographic modifications after injection of