Effect of alcoholic intoxication of female mice prior to pregnancy on the ultrastructure of neurons in the sensorimotor cortex of the progeny

Sensorimotor cortex in the offspring of female rats alcoholized in the prepregnancy period revealed signs of delayed neuronal development and dystrophic changes in the neurons especially on the 14th day after birth. In 21-day-old animals the reparative changes increased, but normalization of neuronal ultrastructure was not observed. The dystrophic changes suggest that prenatal brain hypoxia plays an important role in the pathogenesis of alcohol-induced neuronal lesions in the offspring.     

Effect of antenatal ethanol exposures on the function of the hemato-encephalic barrier in animals

Blood-brain barrier permeability for P32, H3-valine, H3-tyrosine, S35-methionine was investigated in rat offsprings, 20 and 60 days old, that had been antenatally exposed to ethanol. In experimental 20-day-old rat offsprings P32 incorporation in brain structures was lower than in the controls. In mature rats that had been exposed to antenatal ethanol, a different degree decrease in labelled amino acid penetration and alterations in P32 penetration into different brain structures were found. Significant differences in stress-induced changes of blood-brain barrier permeability were observed in experimental and control animals.     

Study of some enzyme activities in cultured chick embryo brain nerve cells treated by chick embryo brain extracts

Brain extracts from 8-day-old chick embryos have been shown to influence morphological development of dissociated brain cells from 7-day-old chick embryos in culture. Stimulatory, effects on size of the neuronal somas and on growth of long processes were observed by adding the cytosol of the brain extract or the dialysate of the cytosol. These morphological changes parallel modifications of various enzyme activities according to the age of the cultures. Adenyl cyclase, (Na⁺, K⁺)- and Mg²⁺-ATPase, 5-nucleotidase, choline acetyltransferase, and acetylcholinesterase activities were studied between 5 and 14 days of culture. Adenyl cyclase activity was strongly stimulated at 8 days by both extracts. (Na⁺, K⁺)-and Mg²⁺-ATPase activities were stimulated in 8-day-old cultures only by the dialysate. 5-Nucleotidase activity was stimulated in 8-day-old cultures by the dialysate and in 11-day-old cultures by both extracts. Choline acetyltransferase activity was stimulated by the cytosol in 8-day-old cultures and by the dialysate in 11-day-old cultures. The total acetylcholinesterase activity was higher in 8-, 11-, and 14-day-old cultures treated with the cytosol. When the cells were treated with the dialysate, the activity was only higher in 14-day-old cultures. We also found that following the addition of brain extracts, the specific activity of the enzymes we studied was enhanced and became close to the values found in vivo during embryogenesis. Thus in parallel to the morphological modifications observed in nerve cell cultures treated by embryo brain extracts, biochemical variations especially involved in synaptogenesis and membrane development could be measured.     

ACETYLCHOLINESTERASE IN DEVELOPING CHICK EMBRYO BRAIN

–Acetylcholinesterase has been assayed at different stages of development to see whether changes in the activity of this enzyme are correlated in any way with the ontogenesis of electrical activity in the brain of growing chick embryo. The specific activity of the enzyme was highest in the synaptosomal fraction of the brain. The activity of the enzyme increased progressively with the age of the embryo. There were three isozymic forms of the enzyme in the 6-day-old embryo brain. A new isozyme appeared around the 9th day. The K_m values of the enzyme for acetylthiocholine from 6- and 20-day-old embryo brains were 6.5 ± 10^-5m and 3.3 ± 10^-5m respectively. Enzyme preparations from 6-day-old embryos were found to lose 50 per cent of their activity when heated at 50°C for 10 min. Under similar conditions the loss in activity in 18-day-old embryo brain enzyme was 22 per cent.     

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).     

Mechanism of action of ethanol on enkephalinase A activity in the rat brain

The influence of ethanol, its metabolites and some opiates on enkephalinase A activity was studied in rat experiments in vitro after acute and chronic administration of ethanol. It was demonstrated that addition of ethanol to the reaction mixture activated enkephalinase A of the midbrain and hypothalamus of intact rats, the maximal effect being attained at an ethanol concentration of 10(-3) M. Multiple washings with buffer of the ethanol-preincubated membranous fraction of these brain structures in the control rats did not lead to a significant reduction in the activating effect of ethanol on enkephalinase A. No activation was recorded upon the use of an enzymatic preparation of the brain from chronically alcoholized animals. Morphine, naltrexon, beta-carbolines, salsolinol (10(-4) M) and acetaldehyde (10(-8)-10(-2) M) did not activate the enzyme. It is suggested that enkephalinase A activation in rats given ethanol is determined by a direct action of ethanol on the enzyme.     

Lysoplasmalogenase–A Microsomal Enzyme from Rat Brain

Abstract: An enzymic activity of rat brain that liberates radioactive free aldehydes from 1-[1-¹⁴C]alk-1'-enyl- sn -glycero-3-phosphoethanolamine (lysoplasmalogen) is described. It was present mainly in microsomal fractions (crude) of brains of rats of different ages. The highest specific enzyme activity was found in 21-day-old animals. The formation of free aldehyde was dependent on the amount of enzyme protein as well as the amount of substrate added, and was linear to the incubation time up to 60 min. The pH optimum was between 7.1 and 7.3. Bivalent cations (Mg²⁺, Ca²⁺) and detergents inhibited the reaction. However, the same cell fractions as well as extracts of acetone-dried powder of brain from young or old rats possessed no enzyme activity for liberating the aldehyde from the acylated substrates: 1-[1-¹⁴C]alk-1'-enyl-2-acyl- sn -glycero-3-phosphoethanolamine (plasmalogen) or plasmalogen of ox corpus callosum.     

Hormonal modulation of neuronal cells behaviour in vitro

In this study we have investigated the effect of insulin and/or of nerve growth factor (NGF) on enzyme activities of cholinergic neurotransmission, in cultured embryonic rat mesencephali. Our data show that choline-O-acetyltransferase (ChAT) and acetylcholinesterase (AChE) activity display a prominent change in the embryonic brain tissues as a function of time in vitro. The change depends on the age of embryos from which the brain cell cultures have been set up. Namely, ChAT activity increases in the cultures taken from 13-17-day-old embryos as a function of time in vitro. AChE activity shows a striking decrease if the cultures have been set up from the older embryos (17-day-old), while AChE activity increases in the cultures prepared from 13-day-old embryos continuously. Insulin (amount ranging 10-27 micrograms/ml) causes a significant inhibition in the ChAT activity in comparison with the increased enzyme activity measured in control cultures (insulin ranging from 1 to 100 ng). AChE activity of 13-day-old embryos was not influenced by insulin (20-27 micrograms/ml) but the same amount of insulin prevents the decrease of AChE activity in cultured brain cells originating from 17-day-old-embryos. Biochemical studies of NGF treated cultures (30 ng/ml) revealed that nerve growth factor resulted in 5-12-fold increase in specific activity of the cholinergic enzyme, choline acetyltransferase (ChAT). NGF did not influence the AChE activity in cultured brain cells (13-17-day-old).     

Studies on the hydrolysis of 1-alk-1'-enyl-sn-glycero-3-phosphoethanolamine by microsomes from myelinating rat brain.

Microsomal fractions of 14-day-old rat brain were incubated at pH 7.1 with 1-[1'-14C]-alk-1'-enyl-sn-glycero-3-phosphoethanolamine (lysoplasmalogen). 1-[1'-14C]alkenylglycerol was produced by hydrolyzing enzyme activities, which were stimulated by Mg2 and inhibited by SH-group reagents. Hydrolysis of 1-[1'-14C]alkyl-sn-glycero-3-phosphoethanolamine is very similar in this respect, but the Km value is higher in the former case. The 1-alkyl compound acts as a non-competitive inhibitor of the hydrolyzing enzyme activity described, whereas the hydrolysis of the 1-alkyl derivative is not inhibited by the 1-alkenyl compound.     

Unilateral injury of sensorimotor cortex of the dominant or subdominant hemispheres of rats-mothers and emotional behavior of their offsprings in the "open field"

Neurohumoral disorders in mothers caused by brain injury, infection, hypoxia, and other pathological factors result in motor and psychoemotional disorders in children. Emotional behavior of 30-day-old offsprings of female rats with unilateral sensorimotor brain injury was studied in the "open field". Individual behavior was estimated (the probability of certain acts and significant transfers between them). Behavioral disorders in rat offsprings depended on the side (left of right) of mother's brain injury and "handedness". The right-side mother's injury turned to be more deleterious. Behavioral alterations were stronger in offsprings of ambidextral and left-handed females than in those born by right-handed males with the same sensorimotor injury.     

Oxidative damage and antioxidant enzyme activities in experimental hypothyroidism

Free radicals are now well known to damage cellular components. To investigate whether age and thyroid level affect peroxidation speed, we examined the levels of malondialdehyde and antioxidant enzyme activities in different age groups of hypothyroid rats. Hypothyroidism was induced in 30- and 60-day-old Wistar Albino rats by the i.p. administration of propylthiouracil (10 mg kg(-1) body weight) for 15 days. While malondialdehyde levels of 30- or 60-day-old hypothyroid rats were increased in liver, they were decreased in the tissues of the heart and thyroid. While glucose-6-phosphate dehydrogenase activity levels did not change in heart, brain and liver tissues of 30-day-old rats, they increased in brain and heart tissues of 60-day-old experimental groups, but decreased in the liver. Catalase activities decreased in the liver and heart of rats with hypothyroidism, but increased in erythrocytes. In control groups while malondialdehyde levels increased in brain, heart and thymus with regard to age, they decreased in plasma. Glucose-6-phosphate dehydrogenase and catalase activities were not affected by age in tissues of the thymus, thyroid and brain, but they were decreased in the heart tissue. The changes in the levels of lipid peroxidation and antioxidant enzyme activities which were determined in different tissues of hypothyroid rats indicate a cause for functional disorder of these tissues. Moreover, there may be changes depending on age at lipid peroxidation and antioxidant enzyme activity levels.     

Regional Development of Glutamate Dehydrogenase in the at Brain

The development of glutamate dehydrogenase enzyme activity in rat brain regions has been followed from the late foetal stage to the adult and through to the aged (greater than 2 years) adult. In the adult brain the enzyme activity was greatest in the medulla oblongata and pons greater than midbrain = hypothalamus greater than cerebellum = striatum = cortex. In the aged adult brain, glutamate dehydrogenase activity was significantly lower in the medulla oblongata and pons when compared to the 90-day-old adult value, but not in other regions. The enzyme-specific activity of nonsynaptic (free) mitochondria purified from the medulla oblongata and pons of 90-day-old animals was about twice that of mitochondria purified from the striatum and the cortex. The specific activity of the enzyme in synaptic mitochondria purified from the above three brain regions, however, remained almost constant.     

ADENYLATE CYCLASE ACTIVITY IN MOUSE VAGINAL EPITHELIUM. AGE RELATED CHANGES IN BASAL ENZYME ACTIVITY AND ESTRADIOL SENSITIVITY

Using neonatal and immature female NMRI mice, age related variations in histochemically demonstrable adenylate cyclase activity were studied in the vaginal epithelium. Basal enzyme activity increased from day 3 to 6 after birth, only later to decrease. Almost no activity was seen on day 14. An estradiol induced increase in enzyme activity occurred in 3- and 6-day-old animals but not in 14-day-old animals. In spite of this difference both neonatal and 14-day-old animals respond to estradiol with an increased amount of a cellular product with antigenic properties specific for the cervicovaginal epithelium. The results are discussed in relation to estrogen induced irreversible effects in the neonatal cervicovaginal epithelium.     

Udp-galactose: glycoprotein galactosyltransferase activity in a clonal line of rat brain.

1. UDPgalactose:glycoprotein galactosyltransferase (EC 2.4.1.-) activity was demonstrated in homogenates from whole rat brain, isolated neuromal perikarya, enriched glial cell fractions, and cultured rat glial tumor cells (clone C6). 2. Galactosyltransferase activity was enriched 3-9-fold in neuronal perikarya and 1.4--1.8-fold in the glial cell fraction over the activity in whole brains from 19- and 40-day-old rats. The activity of galactosyltransferase in neuronal perikarya decreased with age. Extensive contamination of the glial cell fraction with membranous fragments appeared to obscure the precise specific activity of this fraction. 3. The specific activity of the enzyme in glial tumor cells was 4--8-fold higher than in brain tissue when the enzyme was assayed under identical conditions using endogenous and different exogenous acceptors. 4. Galactosyltransferase activities from adult brain and glial tumor cells had similar properties. They both required Mn-2 plus and Triton, and exhibited pH optima between 5 and 7. The apparent Km of the enzyme for UDPgalactose was 1.3-10-minus 4 M for brain tissue and 2.2-10-minus 4 M for glial tumor cells. 5. The high galactosyltransferase activity in glial tumor cells and in neuronal perikarya of younger rats is compatible with the possibility of a role of this enzyme in developing brain.     

The biosynthesis of plasmalogens by rat brain: involvement of the microsomal electron transport system.

The biosynthesis of 1-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine (ethanolamine plasmalogens) was studied using 1-[1-14C]hexadecyl-sn-glycero-3-phosphoethanolamine as the substrate and EDTA-washed microsomes from brains of 14-day-old rats. It was found that the 1-E11-14C]hexadecyl-sn-glycero-3-phosphoethanolamine was first acylated to form 1-[1-14C]hexadecyl-2-acyl-sn-glycero-3-phosphoethanolamine, then was desaturated to form 1-[1-14C]hexadec-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine. The desaturation required O2 and NADH or NADPH and was inhibited by KCN but not by CO. The data indicated that the desaturation is carried out by a mixed-function oxidase system similar to that involved in the desaturation of fatty acids and that the pathway for the biosynthesis of plasmalogens in brain is similar to that previously found in other tissues. The desaturase was not stimulated by ATP and Mg2plus nor inhibited by EDTA. The specific activity of microsomes from brains of rats of different ages was determined; the activity decreased with age until in adults the activity was only 15% that of the 12--14-day-old rats.     

Proteolytic and peptidase activities of the jejunum and ileum of the rat during postnatal development

1. Proteolytic (substrate nitrocasein), tripeptidase (substrate glycylglycylgly-cine) and aminopeptidase (substrate leucyl-beta-naphthylamide) activities were studied in homogenates of jejunal and ileal mucosa of 7-, 10-, 14-, 21-, 35- and 60-day-old rats. 2. Proteolytic activity was practically the same in jejunum of 7-, 10-, 14- and 21-day-old rats, but after day 21 a significant increase was observed. The activity of the ileum changed very little during postnatal development and was always higher than that of the jejunum. 3. Tripeptidase activity was low in the jejunum of 7- and 14-day-old rats, an increase was observed between day 14 and 21, but later no substantial changes were found. There were no changes in the ileum. The activity in the jejunum of 7- and 14-day-old rats was lower than in the ileum, but later the jejunum was more active than the ileum. 4. Aminopeptidase activity had a similar developmental pattern to tripeptidase activity. A low activity was found in the jejunum of 7- and 14-day-old rats, the maximum was in 21-day-old rats and then a decrease was observed, though values for 60-day-old rats were still higher than for 7- and 10-day-old rats. The activity in the ileum was practically the same in all age groups studied except in 14- and 21-day-old rats, where a transient peak was observed.     

PROTEIN SYNTHESIS BY HIGHLY AGGREGATED AND PURIFIED POLYSOMES FROM YOUNG AND ADULT RAT BRAIN

The state of aggregation and the activity of polyribosomes as well as the activity of the pH 5 enzyme fraction were studied at two stages of postnatal brain development, 9 and 50 days after birth. When the polyribosomes were prepared at 0°C in the presence of 5 mm -Mg²⁺, more than 85 per cent of the polyribosome material exhibited a sedimentation coefficient higher than 110 S. High Mg²⁺ concentrations are, therefore, unnecessary to obtain highly aggregated brain polyribosomes. The basal amino acid incorporating activity of both 9- and 50-day-old rat brain preparations is at least equal to that of rat liver. When prepared by the same procedure as above, 9-day-old rat brain polyribosomes seem to be more active (20 per cent) than those of adult brain. However, this difference in activity depends on the presence of a non-ribosomal inactive contaminant which is always present in higher amounts in adult brain preparations. When purified from this contaminant, the preparations do not differ in activity. High Mg²⁺ concentrations are also not necessary for optimal protein synthetic activity and, in fact, are inhibitory. When assayed with both types of highly aggregated polyribosomes, the pH 5 enzyme fraction from adult brain is clearly less active than that of 9-day-old rats. These results suggest that the loss of brain protein synthesis during development does not depend on the stability of the messenger RNA-ribosome complex but only on the soluble pH 5 enzyme fraction.     

Comparisons of brain weight and neocortical thickness in 21-day-old fetuses and 1-day-old rats with the histophysiological indices of the ovaries and adrenal glands of their mothers (a correlational and cluster analysis)]

The correlation and cluster analysis revealed that the brain weight of the rat 21-day-old foetus obviously depended on 3 beta-hydroxysteroiddehydrogenase activity in adrenal cortex. No such dependence was found in 1-day-old foetus. Thickness of the brain cortex in 21-day-old foetus and in 1-day-old rat correlated with parameters of the enzyme activity in the corpus luteum, corpus atretic, and theca folliculi of mother's ovaries.     

Purification, characterization, and ontogeny of acetyl-CoA carboxylase isozyme of chick embryo brain.

Acetyl-CoA carboxylase catalyzes the first committed step in the synthesis of fatty acids. Because fatty acids are required during myelination in the developing brain, it was proposed that the level of acetyl-CoA carboxylase may be highest in embryonic brain. The presence of acetyl-CoA carboxylase activity was detected in chick embryo brain. Its activity varied with age, showing a peak in the 17-18-day-old embryo and decreasing thereafter. The enzyme, affinity-purified from 18-day-old chick embryo brain, appeared as a major protein band on polyacrylamide electrophoresis gels in the presence of sodium dodecyl sulfate (Mr 265,000), indistinguishable from the 265 kDa isozyme of liver acetyl-CoA carboxylase. It had significant activity (Sp act = 1.1 mumol/min per mg protein) in the absence of citrate. There was a maximum stimulation of only 25% in the presence of citrate. Dephosphorylation using [acetyl-CoA carboxylase] phosphatase 2 did not result in activation of the enzyme. Palmitoyl-CoA (0.1 mM) and malonyl-CoA (1 mM) inhibited the activity to 95% and 71%, respectively. Palmitoylcarnitine, however, did not show significant inhibition. The enzyme was inhibited (greater than 95%) by avidin; however, avidin did not show significant inhibition in the presence of excess biotin. The enzyme was also inhibited (greater than 90%) by antibodies against liver acetyl-CoA carboxylase. An immunoblot or avidin-blot detected only one protein band (Mr 265,000) in preparations from chick embryo brain or adult liver. These observations suggest that acetyl-CoA carboxylase is present in embryonic brain and that the enzyme appears to be similar to the 265 kDa isozyme of liver.     

Inhibition by the branched-chain 2-oxo acids of the 2-oxoglutarate dehydrogenase complex in developing rat and human brain

1. The effect of the branched-chain amino acids, namely leucine, isoleucine and valine and their corresponding 2-oxo acids on the metabolism of 2-oxoglutarate by developing rat and human brain preparations was investigated. 2. The decarboxylation of 2-oxo[1-(14)C]glutarate to (14)CO(2) by mitochondria from adult rat brain was inhibited by the branched-chain 2-oxo acids whereas the branched-chain amino acids had no inhibitory effect on this process. 3. The activity of 2-oxoglutarate dehydrogenase complex was about 0.2unit/g of brain from 2-day-old rats and increased by about fourfold reaching an adult value by the end of the third postnatal week. 4. The K(m) value for 2-oxoglutarate of the 2-oxoglutarate dehydrogenase complex in rat and human brain was 100 and 83mum respectively. 5. The branched-chain 2-oxo acids competitively inhibited this enzyme from suckling and adult rats brains as well as from foetal and adult human brains, whereas the branched-chain amino acids had no effect on this enzyme. 6. Approximate K(i) values for the branched-chain 2-oxo acids found for this enzyme were in the range found for these 2-oxo acids in plasma from patients with maple-syrup-urine disease. 7. The possible significance of the inhibition by the branched-chain 2-oxo acids of the 2-oxoglutarate dehydrogenase complex in brains of untreated patients with maple-syrup-urine disease is discussed in relation to the energy metabolism and the biosynthesis of lipids from ketone bodies.