Activity of Calpain in the Guinea Pig Brain under Conditions of Experimental Allergic Encephalomyelitis

We studied the activity of calpain in the brain tissue of guinea pigs at different stages of the development of experimental allergic encephalomyelitis (EAE). Eleven days after inoculation of a mixture containing myelin main protein into the experimental animals, we observed a drop in the calpain activity (on average, by 27% with respect to the control), whereas on the 20th and 27th days the activity of the enzyme under study exceeded the norm (by 12%). The calpain/calpastatin ratio also altered at the different stages of development of EAE: the amount of calpastatin increased significantly on the 11th and 27th days, while on the 20th day the level of calpastatin was close to that typical of the control animals. Therefore, we found that the state of calpain/calpastatin system in the guinea pig brain demonstrates some correlation with the dynamics of development of EAE.     

Factors reducing the superoxide dismutase activity of the liver of rats with Pliss lymphosarcoma

The procedures for isolation and purification of Cu/Zn superoxide dismutase (SOD) from small amounts of rat liver at different stages of Pliss lymphosarcoma growth were developed. Two stages of tumour growth were distinguished. At the first stage (4-5th day after reinoculation) the intensity of optical (680 nm) and EPR spectra of SOD was decreased, on the average, to 30%, while at the second stage--by 40% at 680 nm and by 32%, at 260 nm. The intensity of the EPR spectra was also diminished by 40% as compared to the control. It was assumed that at initial stages of tumour growth the decrease of the SOD activity is mainly due to the reduction of enzyme Cu, while at the second stage--to the decrease of the protein content. In all probability, the observed changes are induced by the activation of lipid peroxidation involving active O2 species. The second stage is associated with the superoxide-induced impairment of the function of nucleic acids involved in protein biosynthesis.     

Developmental pattern of poly (ADP-ribose) synthetase and NAD glycohydrolase in the brain of the fetal and neonatal rat

Poly (ADP-ribose) synthetase and NAD glycohydrolase were examined in nuclear fractions from rat brain at sequential times during late fetal and the first two weeks of neonatal life. In whole brain, both enzymes were demonstrable at all stages of development, but followed separate patterns. Activity of the synthetase which was greatest in fetal life, fell steadily with fetal maturation from 3.90±0.06 nmol/mg DNA at 16 days, to reach a nadir of 1.36±0.09 nmol/mg DNA on the 4th postnatal day. Subsequently it underwent a non sustained neonatal rise reaching a peak of 2.46±0.07 nmol/mg DNA on the 8th day. By contrast, NAD glycohydrolase activity increased steadily throughout late fetal and during the first two weeks of neonatal life, from 12.77±0.40 nmol/mg DNA on day 16 of gestation to 25.80±.95 nmol/mg DNA on neonatal day 12. In neonatal cerebellum the activity of poly (ADP-ribose) synthetase was greater at 8 than at 4 days, could be stimulated with graded concentrations of sonicated DNA up to 100 g, but was inhibited by higher concentrations of DNA and by all concentrations of exogenous histone. In an in vitro culture system of fetal rat brain cells, the activity of poly (ADP-ribose) synthetase increased steadily over six days. Cycloheximide 10^–3 M completely inhibited the activity of this enzyme. NAD glycohydrolase activity increased progressively in vitro, and after 6 days in cycloheximide (10^–3 M), the cultures contained significantly greater levels of enzyme activity. It is suggested that changing activities of poly (ADP-ribose) synthetase and NAD glycohydrolase could both provide potential markers for brain cell differentiation in this system.     

Lipids and caplain in guinea pig tissues in the process of development of experimental autoimmune encephalomyelitis

The character of some lipids level change--cholesterol and phospholipids--as basic lipid components of cell membranes in the guinea-pig brain and liver tissue, and in serum in conditions of development of experimental autoimmune encephalomyelitis (EAE) have been investigated on the 11th, 21st, 27th day after inoculation. It has been detected, that the level of the investigated lipids changes wavely and indifferent-direction in the brain tissue on the 21st day of EAE. Similar variability observed in the activity of proteolytic ferment calpain, which is authentically reduced in the brain tissue by the 11th hour and increases up to the test objective level in the subsequent periods of EAE development. In the liver the level of alpha-tocopherol is reduced, while the content of studied lipids does not change. The investigated parameters can be attributed to the factors, which play an essential role in structural stability of cell membranes and their variability in conditions of EAE development is related to the processes of nervous cells demyelinisation and, hence to occurrence of such pathology as multiple sclerosis in people.     

Enzymes and microemulsions. Activity and kinetic properties of liver alcohol dehydrogenase in ionic water-in-oil microemulsions

The activity and the kinetic properties of horse liver alcohol dehydrogenase have been studied in water-in-oil microemulsions containing sodium dodecyl sulfate (SDS) or hexadecyl trimethylammonium bromide (CTAB), 1-butanol or 1-pentanol or 1-hexanol or t-butanol, water and cyclohexane alone or with octane. In the anionic microemulsions (i.e. containing sodium dodecyl sulfate), the enzyme quickly lost its activity, but was efficiently protected by the coenzyme and some adenine nucleotides. In the cationic microemulsions (i.e. containing hexadecyl trimethylammonium bromide), the enzyme activity was more stable and with higher alcohols was stable for at least 20 min. The Michaelis constant of NAD+ calculated with respect to the water content was nearly constant and higher than in water. The maximum velocity in anionic microemulsions depends on the water content whereas in cationic microemulsions, the maximum velocity did not show a clear dependence on the water content and was close to the maximum velocity found in water. The pH dependence of Km and Vmax in these microemulsions was similar to that observed in water. The kinetic data for a hydrophobic substrate, cinnamyl alcohol, showed that this alcohol partitions between the pseudo-phases and thus the apparent Michaelis constant and the concentration at which substrate-excess inhibition appeared were increased. The catalytic properties of the enzyme in microemulsions were illustrated by the preparative reduction of cinnamaldehyde with cofactor recycling. The rate determination of NAD+ reduction and of 1-butanol/cinnamaldehyde redox reaction showed that at low water content (2.8%), the NAD+ reduction rate was close to zero whereas the redox reaction rate was about half of the rate at higher water content. Probably at low water content the coenzyme binding-dissociation rates are reduced much more than the binding-dissociation rates of the substrates and the rates of the ternary complex interconversion. The cationic microemulsions seemed to be very favorable medium for enzyme activity, the tetraalkyl ammonium surfactant causing less denaturation than the anionic detergent dodecyl sulfate.     

State of the antioxidant system during induction in rats of primary generalized epileptic activity

Antioxidation system in the brain and blood of rats with generalized bemegride-induced epileptic activity was studied. Antioxidation enzyme activity (superoxide dismutase, glutathione peroxidase and glutathione reductase) and alpha-tocopherol content were determined at an early convulsive stage, immediately after generalized seizures and 10-15 min after seizure. Antioxidation enzyme activity and alpha-tocopherol level in the brain homogenate and blood remained unchanged at any stages of investigation. It is suggested that the increased level of lipid peroxidation products in the brain and blood of rats upon the development of bemegride-induced epileptic activity is not related to the decrease in antioxidation system activity. The effect is mediated by the activation of the reaction initiating free radical brain lipid transformations.     

Lipid peroxidation and antioxidant defence enzyme activity in multiple sclerosis

Changes of state of lipid peroxidation and activity of antioxidant defence enzymes katalase, superoxide dismutase, glutathione peroxidase and glutathione reductase - in the brain and liver tissue of guinea-pig in conditions of different stages of experimental autoimmune encephalomyelitis (EAE; 11th, 21st, 27th day after inoculation) and in blood of multiple sclerosis (MS) patients with different types, degrees of severity and length of disease and blood level of reduced glutathione have been investigated. We have found, that the development of oxidative stress in animal organism during the disease development is progressive and intensive lipid peroxide oxidation without compensation by antioxidant mechanisms have been shown in the late period (27th day) of the experiment. In MS conditions this state was accompanied with high activity of demyelination process, severe degree of neuronal injury and length of disease above 5 years. In addition reduced glutathione level was increased in many patient groups: remitting type, light (II) degree of severity and among the patients with strongly disturbed neurological functions and long course of the disease. The obtained data allow us to suppose that the development of oxidative stress under demyelination conditions is a result of strong metabolic disorders and decrease of antioxidant defence in the patients during the disease development. The necessity of individual approaches for antioxidant therapy of patients with MS is discussed.     

Changes of malic enzyme activity in the developing rat brain are due to both the increase of mitochondrial protein content and the increase of specific activity.

1. The pattern of NADP-linked malic enzyme activity estimated in the whole brain homogenate did not parallel that found in liver of developing rat. 2. Studies on intracellular distribution of malic enzyme in brain showed that the mitochondrial enzyme increased about three-fold between 10th and 40th day of life. Thereafter, a slow gradual increase to the adult level was observed. 3. The extramitochondrial malic enzyme from brain, like the liver enzyme, increased at the time of weaning, although to a lesser extent. At day 5 the brain malic enzyme was equally distributed between mitochondria and cytosol. 4. During the postnatal development, the contribution of the mitochondrial malic enzyme in the total activity was increasing, reaching the value approx. 80% at day 150 after birth. 5. The increase with age of the malic enzyme specific activity was observed in both synaptosomal and non-synaptosomal mitochondria, the changes in the last fraction being more pronounced. 6. The activity of citrate synthase developed markedly between 10-40 postnatal days, increasing about five-fold, while the specific activity of the enzyme did change neither in the synaptosomal nor in non-synaptosomal mitochondria at this period. 7. We conclude that the changes in malic enzyme activity in the developing rat brain are mainly due both to the increase of mitochondrial protein content and to the increase of specific activity of the mitochondrial malic enzyme.     

Developmental changes of L-lysine-ketoglutarate reductase in rat brain and liver.

1. Developmental aspects of L-lysine-ketoglutarate reductase, the first enzyme in saccharopine pathway of L-lysine degradation in rat liver and brain tissues were studied. 2. Although the adult rat brain shows negligible activity, the enzyme activity was shown to be highly active during the early stages of development. 3. The enzyme activity gradually decreased through development in the brain, whereas it gradually increased in the liver, establishing the fact that the saccharopine pathway is the major pathway in liver. 4. Our results also show that glucagon stimulated the induction of this enzyme by 2-3-fold in both adult liver and brain tissues.     

Purification and properties of the NAD(P)-dependent malic enzyme from human placental mitochondria

The NAD(P)-dependent malic enzyme from human term placental mitochondria was purified 108-fold with a final yield of 72% and specific activity of about 2 mumol per minute per milligram protein. The final preparation was completely free of fumarase, malic, and lactic dehydrogenases. Divalent cations were required for NAD(P)-dependent malic enzyme activity, Mn2+ and Co2+ were by far more effective activators than Mg2+ and Ni2+, whereas the reaction did not proceed in the presence of Ca2+. The optimum pH with NAD and NADP as coenzymes was at around 7.1 and 6.4, respectively. The ratio of the rate of NAD:NADP reduction was 7.4 and 1.3 at pH 7.1 and 6.4, respectively. The enzyme is activated by succinate and fumarate and inhibited by ATP. In the absence of fumarate the Michaelis constants for L-malate and NAD were 2.82 and 0.33 mM; and in the presence of fumarate 1.18 and 0.22 mM, respectively. This study presents the first report showing the purification and kinetic properties of NAD(P)-dependent malic enzyme from human tissue.     

ADP-ribosyltransferase in isolated nuclei from sea-urchin embryos.

The activity of ADP-ribosyltransferase in nuclei isolated from sea-urchin embryos was estimated by the incorporation of [adenosine-14C]NAD+ into the acid-insoluble fraction. Hydrolysis of this acid-insoluble product by snake venom phosphodiesterase yielded radioactive 5'-AMP and phosphoribosyl-AMP. The incorporation of [14C]-NAD+ was inhibited by 3-aminobenzamide and nicotinamide, potent inhibitors of ADP-ribosyltransferase. [14C]NAD+ incorporation into the acid-insoluble fraction results from the reaction of ADP-ribosyltransferase. The optimum pH for the enzyme in isolated nuclei was 7.5. The enzyme, in 50 mM-Tris/HCl buffer, pH 7.5, containing 0.5 mM-NAD+ and 0.5 mM-dithiothreitol, exhibited the highest activity at 18 degrees C in the presence of 14 mM-MgCl2. The apparent Km value for NAD+ was 25 microM. The activity of the enzyme was measured in nuclei isolated from the embryos at several stages during early development. The activity was maximum at the 16-32-cell stage and then decreased to a minimum at the mesenchyme blastula stage. Thereafter its activity slightly increased at the onset of gastrulation and decreased again at the prism stage.     

Specific cellular expression of monoamine oxidase B during early stages of quail embryogenesis

Monoamine oxidase-B (MAO-B) is one of two distinct molecular forms of MAO that in part regulate the cellular levels of biogenic amines. In order to determine whether discrete cell populations known to express aminergic properties in the vertebrate embryo also express MAO-B, the distribution of MAO-B-immunoreactive cells was examined in early developing quail embryos. Two major patterns of staining emerge. First, tissues known to express several aminergic characteristics are initially MAO-B positive at early stages of development and continue to express immunoreactivity through Zacchei stage 20, the oldest stage examined. These include cells of the sympathetic and some cranial and trunk sensory ganglia (beginning at stage 13), the pancreas (stage 14), and the brain stem raphe (stage 14). Second, other structures that contain or accumulate biogenic amines are transiently MAO-B immunopositive during early stages of development. These tissues include extraembryonic yolk sac and presumptive gut endoderm (with most intense staining between stages 8 and 13), the ventral trunk neural tube (stages 14 and 16), and the notochord (stages 8–10). MAO-B immunoreactivity disappears from these regions at different stages, and none are MAO-B positive by stage 19–20. Other structures without known aminergic properties during early development also stain; these include liver (stage 20), mesenchymal cells that surround the Wolffian duct and lung buds (stages 14 and 18), and endothelial cells surrounding the dorsal aorta (stages 14 and 20). In general, however, MAO-B appears to be distributed in embryonic tissues that can use this enzyme to regulate biogenic amine levels either transiently or during initial phenotypic expression of aminergic traits.     

Studies on sex-organ development. Oestrogenic effect on ornithine decarboxylase activity in the differentiating Müllerian ducts and other organs of the chick embryo.

The activity of ornithine decarboxylase in the differentiating left and right Müllerian ducts was assayed and compared with that in other embryonic organs, i.e. the liver and the brain throughout the stages of development. In general the enzyme activity was high in the early stages and decreased extensively in the late stages of development. Specifically, in the left and righ Müllerian ducts, the enzyme activity was high from day 8 to day 9 of incubation. In the right duct the enzyme activity started to decline on day 9 and then continuously decreased to an almost undetectable value on day 18 of incubation. In the left duct the enzyme activity also decreased slightly from day 9 to day 12; however, it increased from day 13 to day 15 and finally decreased to a constant value from day 18 until hatching. The alteration in enzyme activity in the Müllerian duct as assayed in vitro during development is not due to the effect of the size of the endogenous ornithine pool. When the enzyme activity was subjected to oestrogen stimulation, an increase of 5--10-fold for the left duct and of 5--3-fold for the right duct was observed during the course of development. No such stimulation was observed with the treatment of progesterone. Testosterone consistently caused a 25--30% inhibition of the enzyme activity in the Müllerian duct. Oestrogen slightly stimulated the enzyme activity in the developing liver but inhibited that of the brain. The concentration of the three polyamines measured in the Müllerian duct corresponds to the activity of the enzyme determined.     

Nicotinamide coenzyme content in the normal and regenerating liver of rats

The content of NADH and NADPH was measured in the intact and regenerating rat liver. In the intact rat liver, the content of NAD+, NADH, NADP+ and NADPH was 235 +/- 6.4, 66.6 +/- 4.3, 73.3 +/- 2.5 and 148.0 +/- 4.6 micrograms/g crude liver weight, respectively. Seasonal alterations in the rat liver content of coenzymes were established. No changes were found in the content of nicotinamide coenzymes in the regenerating liver 4 and 18 h after operation. Twenty-four hours after operation, a 25.6% increase in the content of NAD+ and a 57.8% reduction in the NADH content were recorded in the liver of hepatectomized animals. At the same time the total content of NAD+ plus NADH changed but insignificantly (14.7%). The total content of NADP+ plus NADPH dropped by 29.8% (within the above period). Thirty-two hours after operation the content of all the nicotinamide coenzymes returned to the initial level.     

Enzymes of glycogen metabolism in chick embryo liver]

At all stages of ontogenesis glycogen phosphorylase (EC 2.4.1.1) from liver chick embryos in represented by an isoenzyme whose properties are close to those of isoenzyme IL or F. Total enzyme activity (a+b forms) from the 8th day of development up to hatching gradually increases 1.5-fold, a practically complete activation of enzyme being observed by the end of embryogenesis. Phosphorylase b possesses high catalytic activity in the presence of 1 mM AMP and it activated by protamine and 0.2 M Na2SO4. Glycogen synthetase (EC 2.4.1.11) has a constant Km(UDFG) value during ontogenesis. This value is about 5.10(-4) M in the presence of 10 mM glucose-6-phosphate, both for I- and D-forms of enzyme. The total enzyme activity reaches its maximum on the 17th postembryonic day and is decreased more than 6-fold thereafter. In the course of embryogenesis the I/D ratio is increased from 0.2 on the 8th day of development up to 0,45 during extensive accumulation of glycogen and falls down to 0.33 before hatching. Glycogen biosynthesis in embryonic liver is wellcorrelated with the increase in the I/D ratio, i.e. the increase of the active form of enzyme. The proportion of granular glycogen in embryonic liver is increased from 15% up to 90% of total glycogen content between the 8th and 14th days of development. The activity of glycogen synthetase contained in granular glycogen is increased from 40% in the 8-day-old embryos up to 90% in the 18-day-old ones. The activity of phosphorylase is found in granular glycogen only on the 12th day of embryogenesis and reaches its maximum (80% of total enzyme activity) only on the 19th days of development. It is concluded that in the adult chicken liver the embronic enzymes--glycogen phosphorylase and glycogen synthetase--are retained.     

Discovery and characterization of a Coenzyme A disulfide reductase from Pyrococcus horikoshii. Implications for this disulfide metabolism of anaerobic hyperthermophiles

We have cloned NADH oxidase homologues from Pyrococcus horikoshii and P. furiosus, and purified the recombinant form of the P. horikoshii enzyme to homogeneity from Escherichia coli. Both enzymes (previously referred to as NOX2) have been shown to act as a coenzyme A disulfide reductases (CoADR: CoA-S-S-CoA + NAD(P)H + H+-->2CoA-SH + NAD(P)+). The P. horikoshii enzyme shows a kcat app of 7.2 s(-1) with NADPH at 75 degrees C. While the enzyme shows a preference for NADPH, it is able to use both NADPH and NADH efficiently, with both giving roughly equal kcats, while the Km for NADPH is roughly eightfold lower than that for NADH. The enzyme is specific for the CoA disulfide, and does not show significant reductase activity with other disulfides, including dephospho-CoA. Anaerobic reductive titration of the enzyme with NAD(P)H proceeds in two stages, with an apparent initial reduction of a nonflavin redox center with the first reduction resulting in what appears to be an EH2 form of the enzyme. Addition of a second of NADPH results in the formation of an apparent FAD-NAD(P)H complex. The behavior of this enzyme is quite different from the mesophilic staphylococcal version of the enzyme. This is only the second enzyme with this activity discovered, and the first from a strict anaerobe, an Archaea, or hyperthermophilic source. P. furiosus cells were assayed for small molecular mass thiols and found to contain 0.64 micromol CoA.g dry weight(-1) (corresponding to 210 microM CoA in the cell) consistent with CoA acting as a pool of disulfide reducing equivalents.     

METABOLIC PARAMETERS OF ONTOGENESIS OF ELECTRICAL ACTIVITY IN THE BRAIN. SODIUM-POTASSIUM ACTIVATED ADENOSINE TRIPHOSPHATASE IN DEVELOPING CHICK EMBRYO BRAIN

—(1) The activity of the Na-K ATPase in the particulate fraction of the chick embryo brain has been assayed at different stages of development with the objective of finding whether or not changes in the activity of this enzyme bear any relation to the maturation of spontaneous and evoked electrical activity of the growing chick brain. (2) The specific activity of the enzyme is low on day 6 and it rises rapidly between days 10 and 12, at which time it attains a plateau and remains essentially unchanged from day 12 until day 20. Experimental evidence rules out the possible presence of an inhibitor of the enzyme in 8-day-old brain homogenates, suggesting that these developmental changes in the activity of the enzyme may represent new synthesis of enzyme rather than its activation. The period between days 10 and 12 does not represent a unique stage of general protein synthesis. (3) The chick brain particulate enzyme has an optimum activity at pH 7·4 and at 37°. It is optimally activated by a Na⁺ concentration of 100mm and K⁺ concentration of 20 mm . The enzyme is inhibited by ouabain and Ca²⁺. (4) The results have been discussed.     

Transglutaminase levels in brain and reticuloendothelial cells during allergic encephalomyelitis determined by a radiochemical method

A sensitive method for the assay of transglutaminase based on incorporation of [¹⁴C]methylamine into carbobenzoxy l -glutaminyl l -valine methyl ester is described. Transglutaminase levels were measured in the livers, brains and white cells obtained from peritoneal exudates of two strains of mice during the development of experimental allergic encephalomyelitis (EAE). The sensitive (BSVS) strain developed the disease, while the resistant (BRVR) mice did not succumb to EAE. The liver enzyme in the sensitive mice increased to significantly higher levels during the early stages of the disease. Low values were recorded in the same animals during paralysis. Brain transglutaminase increased to 300 per cent of normal values in the sensitive mice during paralytic stages of EAE. In the resistant strain of mice transglutaminase levels doubled in the mixture of cells obtained from the peritoneal wash. In the sensitive mice, normal values were recorded in those cells. It is suggested that transglutaminase is actively involved in immunological reactions taking place in the brain in EAE sensitive mice and in the reticuloendothelial system of EAE resistant mice.     

The effect of ethanol exposure in pregnancy on maturation of monoaminergic systems in the developing rat bran

Simultaneous study of the main neurotransmitter of monoaminergic system of the brain, its metabolites, activity of catechol-O-methyltransferase (COMT) and the state of different subtypes of dopamine (DA) receptors in the developing brain of offspring from mothers alcoholized in gestation and feeding periods revealed a decrease in activity of all monoaminergic systems studied with reduction of noradrenaline and DA level in alcoholized fetus as well as of mPNA of COMT, an enzyme of catecholamine metabolism, in the structures of the forebrain on the 17th day but not on 13th day of prenatal development. In parallel experiments, an increase of the contents of both long and short splice variants of D2 DA receptor was registered. In postnatal period (days 4, 10, 17), further decrease of the DA system activity was observed, particularly a reduction of DOPAC level and DOPAC/DA ratio in rat litter, mothers of whom took alcohol in the gestation period with withdrawal it after birth of offspring. The serotonin system activity was also reduced in alcoholized litter in the postnatal period and was registered in the early stages (on the 4th day of life). Therefore, the serotonin system activity is changing at early stages of development (the 4th day), whereas inhibition of the DA system activity is registered at later stages (the 10th day of life).