3Development-Dependent Regulation of N-Acetyl-β-d-Hexosaminidase of Cerebellum and Cerebrum of Normal and Staggerer Mutant Mice

Distinctive activities of various glycosidases were expressed in the cerebellum and cerebral cortex of mice during their development. In particular, N-acetyl-beta-D-hexosaminidase (EC 3.2.1.30) appeared to be developmentally regulated. A transient peak of enzyme activity at postnatal day 7 was characteristic for the cerebellum, whereas the activity in the cerebral cortex gradually increased through the 1st postnatal month and was maintained at a high level of activity throughout adulthood. The regulation of N-acetylhexosaminidase activity in the developing cerebellum of the staggerer mouse deviated clearly from enzyme activities in the wild-type, whereas the activity pattern in the staggerer cerebral cortex remained unaffected. In experiments mixing wild-type and staggerer cerebellum homogenates, the specific activity was additive. Thus, involvement of inhibitors or activating molecules can be excluded. This developmentally controlled regulation or disregulation in staggerer appears to be enzyme specific, sine beta-glucosidase, alpha-glucosidase, and beta-galactosidase did not exhibit such a pattern in either normal or staggerer mice. In the mutation weaver that, like staggerer, loses the majority of its cerebellar granule cells, N-acetyl-beta-hexosaminidase activity of the cerebellum was not elevated, indicating a specific defect in staggerer rather than a general effect on lysosomal enzymes due to cell death.     

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.     

Brain nucleic acids and protein in various neurological mutant mice

A study was made to compare alterations in the cerebral contents of nucleic acids and protein of several mouse strains affected by different neurological mutations: jimpy, msd, quaking, reeler, weaver, and dwarf. In normal and affected jimpy and msd mice the brain components analyzed were very similar. On the other hand, the cerebral hemispheres of quaking mice showed significant decreases in total RNA and DNA, when compared with those of normal littermates. In the affected reeler and weaver mice, total protein, RNA, and DNA in the cerebellum differed markedly from controls. Protein decreased slightly, whereas nucleic acids showed no significant variation in the cerebral hemispheres of the same mutants. The cerebella and cerebral hemispheres of affected dwarf mice had wet weights and total protein contents that were about 20% lower than those of their controls; DNA did not vary significantly in the various brain regions analyzed. The decrease of DNA we report in reeler and weaver mutant cerebellum in toto quantifies the lack of cell number, in contrast to histological studies which give only semiquantitative information.     

Mitochondrial respiratory chain and creatine kinase activities following trauma brain injury in brain of mice preconditioned with N-methyl-d-aspartate

Traumatic brain injury (TBI) induces glutamatergic excitotoxicity through N-methyl-d-aspartate (NMDA) receptors, affecting the integrity of the mitochondrial membrane. Studies have pointed to mitochondria as the master organelle in the preconditioning-triggered endogenous neuroprotective response. The present study is aimed at understanding energy metabolism in the brains of mice after preconditioning with NMDA and TBI. For this purpose, male albino CF-1 mice were pre-treated with NMDA (75 mg/kg) and subjected to brain trauma. Mitochondrial respiratory chain and creatine kinase activities were assessed at 6 or 24 h after trauma. The mice preconditioned and subjected to TBI exhibited augmented activities of complexes II and IV in the cerebral cortex and/or cerebellum. Creatine kinase activity was also augmented in the cerebral cortex after 24 h. We suggest that even though NMDA preconditioning and TBI have similar effects on enzyme activities, each manage their response via opposite mechanisms because the protective effects of preconditioning are unambiguous. In conclusion, NMDA preconditioning induces protection via an increase of enzymes in the mitochondria.     

Several Galactosyltransferase Activities Are Associated with Mouse Chromosome 17

Indirect evidence suggests that some major histocompatibility complex (MHC) proteins are glycosyltransferases. No sequence or mapping information is available for transferases, although ganglioside variations in mice are linked to the H-2 complex on chromosome 17, and one galactosyltransferase activity on mouse sperm varies with T/t complex genotypes, also on chromosome 17. In the present experiments, diploid and trisomy 17 mouse embryos were assayed for four different galactosyltransferase activities. The same preparations were assayed for isocitrate dehydrogenase (Id-1, chromosome 1) and glyoxalase-1 (Glo-1, chromosome 17). Galactosyltransferase specific activities in trisomy 17 embryos are almost 1.5 times higher than in diploid embryos. The correlation between galactosyltransferase activities and chromosome 17 dosage indicates that the structural or regulatory gene for these enzymes are located on chromosome 17.     

Developmental and Regional Studies of the Metabolism of Inositol 1,4,5-Trisphosphate in Rat Brain

Coupling of CNS receptors to phosphoinositide turnover has previously been found to vary with both age and brain region. To determine whether the metabolism of the second messenger inositol 1,4,5-trisphosphate also displays such variations, activities of inositol 1,4,5-trisphosphate 5'-phosphatase and 3'-kinase were measured in developing rat cerebral cortex and adult rat brain regions. The 5'-phosphatase activity was relatively high at birth (approximately 50% of adult values) and increased to adult levels by 2 weeks postnatal. In contrast, the 3'-kinase activity was low at birth and reached approximately 50% of adult levels by 2 weeks postnatal. In the adult rat, activities of the 3'-kinase were comparable in the cerebral cortex, hippocampus, and cerebellum, whereas much lower activities were found in hypothalamus and pons/medulla. The 5'-phosphatase activities were similar in cerebral cortex, hippocampus, hypothalamus, and pons/medulla, whereas 5- to 10-fold higher activity was present in the cerebellum. The cerebellum is estimated to contain 50-60% of the total inositol 1,4,5-trisphosphate 5'-phosphatase activity present in whole adult rat brain. The localization of the enriched 5'-phosphatase activity within the cerebellum was examined. Application of a histochemical lead-trapping technique for phosphatase indicated a concentration of inositol 1,4,5-trisphosphate 5'-phosphatase activity in the cerebellar molecular layer. Further support for this conclusion was obtained from studies of Purkinje cell-deficient mutant mice, in which a marked decrement of cerebellar 5'-phosphatase was observed. These results suggest that the metabolic fate of inositol 1,4,5-trisphosphate depends on both brain region and stage of development.     

小鼠脑内NO/NOS-cGMP信号系统与吗啡依赖形成的机制

本文观察了吗啡依赖小鼠脑组织ｃＧＭＰ含量,钙依赖性及非钙依赖性ＮＯＳ活性的变化,蛋白激酶Ａ对ＮＯＳ活性的磷酸化调节以及一氧化氮合酶（ＮＯＳ）抑制剂对吗啡依赖形成的影响。结果发现：（１）小脑,纹状体,海马及大脑皮质ｃＧＭＰ含量明显下降;（２）纹状体及大脑皮质钙依赖性ＮＯＳ活性明显升高,而ＩＰ２０（ＰＫＡ抑制剂）可抑制比变化,小脑及海马依赖性ＮＯＳ活性及以上各脑区非钙依赖性ＮＯＳ活性无明显变化;（３）     

Galactosyltransferase, pyrophosphatase and phosphatase activities in luminal plasma of the cauda epididymidis and in the rete testis fluid of some mammals

Galactosyltransferase activity was measured in the luminal plasma of the cauda epididymidis of mice, rats, rabbits, rams and boars, and in the rete testis fluid of rams and boars. The activities of nucleotide pyrophosphatase and alkaline phosphatase, which compete with galactosyltransferase for substrate, were also determined. In these species, galactosyltransferase activity in the luminal plasma of the cauda epididymidis was similar when the inhibitory effect of pyrophosphatase and phosphatase was minimized by assay conditions. However, under assay conditions that did not minimize the effect of these enzymes, the galactosyltransferase activities of these species were very different and were inversely correlated with the activities of pyrophosphatase and phosphatase. The ratio of galactosyltransferase activity to pyrophosphatase and phosphatase activity was much higher in the rete testis fluid than in the luminal plasma of the cauda epididymidis in both rams and boars. In rams, galactosyltransferase in the luminal plasma of the cauda epididymidis was more heat resistant than that in serum. These results suggest that there is a species difference in the availability of galactosyltransferase activity in the luminal plasma of the cauda epididymidis and that in some species, galactosyltransferase in the luminal fluid is unlikely to have any function. The results are also discussed with respect to the possible function of galactosyltransferase, pyrophosphatase and phosphatase in epididymal luminal plasma and rete testis fluid.     

Effects of ammonia on monoamine oxidase and enzymes of GABA metabolism in mouse brain.

Acute and chronic ammonia toxicity was produced in the mice by intraperitoneal injection of ammonium chloride (200 mg/kg) and by exposure of mice to ammonia vapours (5% v/v) continuously for 2 days and 5 days respectively. The ammonia content was elevated in the cerebellum, cerebral cortex and brain stem and in liver. In acute ammonia intoxication there was a decrease in the monoamine oxidase (MAO) activity in all the three regions of brain. In chronic ammonia toxicity (2 days of exposure) a significant increase in the activity of MAO was observed in the cerebral cortex while in cerebellum and brain stem there was a significant decrease. In cerebral cortex and cerebellum there was a rise in the activity of MAO as a result of exposure to ammonia vapours for 5 days. A significant decrease was observed in the activity of glutamate decarboxylase (GAD) in all the three regions of the brain both in acute and chronic ammonia toxicity (2 days). There was a decrease in the activity of this enzyme only in the cerebral cortex in the animals exposed to ammonia for 5 days. The activity of GABA-aminotransferase (GABA-T) showed a significant rise in cerebellum and a fall in the brain stem in acute ammonia toxicity. In chronic ammonia toxicity GABA-T showed a rise in all the three regions of brain. Chronic ammonia toxicity produced a significant decrease in the content of glutamate in all the three regions without a significant change in the content of aspartate. GABA and glutamine. The content of alanine increased in all the three regions of brain under these experimental conditions. The ratio of glutamate + aspartate/GABA and glutamate/glutamine showed a decrease in all the three regions as a result of ammonia toxicity.     

Mosaic Expression of the Reeler and Normal Phenotypes in the Cerebral Cortex in Reeler-Normal Chimeras at a Late Embryonic Stage

The cerebral cortex of reeler -normal chimera embryos was studied by hematoxylin-eosin staining and fractographic scanning electron microscopy in comparison with the cortices of normal and reeler mutant mice. The cerebral cortex of normal mice had a plexiform layer, which was composed of a fine meshwork of matrix cell processes. Spindle shaped neuroblasts formed a radial lining columnar structure, which was formed by attachment of migrating neuroblasts to the radial bundles. The cerebral cortex of reeler mutants did not show a plexiform layer and the cells were round with no radially columnar structures, and no radial bundles. In reeler -normal chimera embryos, the thickness of the plexiform layer varied in different parts of the cerebral cortex. In parts where the plexiform layer was present, neuroblasts were spindle-shaped and had a radially oriented columnar structure (normal type). But where the plexiform layer was absent, the neuroblasts were round with a radial architecture ( reeler type). Intermediates between the reeler and control types were also observed. Since mosaic expression of the two phenotypes, was observed in chimeras, the reeler abnormality is apparently not caused by humoral factors. The possible mechanism of cell migration is discussed.     

Acute metabolic effects of taurine on the enzymes metabolizing glutamate and gaba

100 mg of taurine per kg body weight had been administered intraperitoneally and 30 min after the administration the animals were sacrificed. Glutamate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, glutaminase, glutamine synthetase, glutamate decarboxylase and GABA aminotransferase along with the content of glutamate and GABA in cerebral cortex, cerebellum and brain stem were studied and compared with the same obtained in the rats treated with normal saline in place of taurine. The results indicated a significant decrease in the activity of glutamate dehydrogenase in cerebral cortex and cerebellum and a significant increase in brain stem. Glutaminase and glutamine synthetase were found to increase significantly both in cerebral cortex and cerebellum. The activities of glutamate decarboxylase was found to increase in all the three regions along with a significant decrease in GABA aminotransferase while the content of glutamate showed a decrease in all the three brain regions, the content of GABA was observed to increase significantly. The above effects of taurine on the metabolism of glutamate and GABA are discussed in relation to the functional role of GABA and glutamate. The results indicate that taurine administration would result in a state of inhibition in brain.     

Activity of pyruvate- and ketoglutarate dehydrogenase complexes on various regions of the rat brain

The activity of pyruvate dehydrogenase and ketoglutarate dehydrogenase complexes (PDC; EC 1.2.4.1 and KDC; EC 1.2.4.2, respectively) was studied in extracts and lysates of mitochondria isolated from the cortex, cerebellum and stem of the rat brain. In all the mentioned cerebral areas the PDC activity calculating per 1 mg protein noticeably increased that for KDC. Under conditions of solubilization the activity of KDC lowered to a greater extent than that of PDC. The studied brain areas are arranged according to the activity of PDC and KDC in lysates in the following order: stem much much greater than cortex greater than cerebellum, however, the highest stock of the PDC activity manifesting with activation of endogenous phosphatase and dephosphorylation of the complex is observed in cerebellum. When calculating per mitochondria isolated from 1 g of tissue, the value of the PDC activity/KDC activity ratio in all areas of the brain is more than 1, and in cerebellum allowing for the complete PDC activity it exceeds 3. The data obtained emphasize the biochemical originality of the cerebellum and the presence of specific peculiarities in regulation of the pyruvate dehydrogenase complex activity in this cerebral area.     

Rescue of ataxia and preplate splitting by ectopic expression of Reelin in reeler mice

The gene mutated in reeler (reelin) encodes a protein secreted by neurons in the developing brain that controls laminar positioning of migrating cells in the CNS by an unknown mechanism. To investigate Reelin function, we used the nestin promoter to express Reelin ectopically in the ventricular zone and other brain regions in transgenic mice. In the presence of the endogenous protein, ectopic Reelin did not alter cell migration in the neocortex or the cerebellum. However, in the reeler background, ectopic Reelin induced tyrosine phosphorylation of Dab-1 in the ventricular zone and rescued some, but not all, of the neuroanatomic and behavioral abnormalities characteristic of reeler. These results indicate that Reelin does not function simply as a positional signal. Rather, it appears to participate in multiple events critical for neuronal migration and cell positioning.     

Changes in galactosyltransferase activity in chick pectoral muscle during embryonic development.

The two major vertebrate galactosyltransferases have been investigated in developing chick muscle in ovo and in vitro, and in cultured chick fibroblasts. The two enzymes were UDP-galactose-N-acetylglucosamine galactosyltransferase (galactosyltransferase I) and UDP-galactose-N-acetylgalactosamine galactosyltransferase (galactosyltransferase II). Both activities fell during muscle development in ovo. Galactosyltransferase I activity was constant from day 7 to day 16, after which it declined 5-fold, whereas galactosyltransferase II activity fell markedly from day 9 to 13 and 16 to 20, displaying an overall 8-fold decrease. In primary muscle cultures, galactosyltransferase I activity fell slightly during 7 days in culture, whereas galactosyltransferase II increased 2-fold during the same period. No significant change in activity of either galactosyltransferase was observed during intercellular recognition and fusion. Analysis of muscle cultures treated with cytosine arabinoside and of fibroblast cultures revealed that the majority of galactosyltransferase I activity in primary muscle cultures is associated with fibroblasts, whereas the majority of galactosyltransferase II activity is muscle-associated. The addition of 5-bromodeoxyuridine to primary muscle cultures resulted in a 3-fold rise in activities of both transferases.     

Age and Strain Comparisons of Neurotransmitter Synthetic Enzyme Activities in the Mouse

Activities of the neurotransmitter synthetic enzymes, choline acetyltransferase (EC 2.3.1.6; ChAT), glutamic acid decarboxylase (EC 4.1.1.15; GAD), and tyrosine hydroxylase (EC 1.14.3.2; TH), were assayed in four brain regions of A/J and C57BL/6J mice at three ages (4, 18, and 24 months). The brain regions assayed were the fronto-parietal cortex, hippocampus, striatum, and cerebellum. Strain effects: In some brain regions, at several ages, ChAT activity did not differ among the two strains. However, ChAT was higher in the C57BL/6J strain in the cortex at 18 months, the hippocampus at 18 and 24 months, the striatum at 24 months, and the cerebellum at 4 months. The reverse was true in the cerebellum at 24 months, where ChAT was higher in A/J mice. GAD activity in C57BL/6J mice compared to that of A/J mice was higher in the striatum and cortex, and lower in the hippocampus and cerebellum. TH activities in all four regions were generally higher in C57BL/6J mice than in A/J mice. Age effects: Age differences in enzyme activities varied with the genetic strain. ChAT activity generally was higher in brain regions of older mice of both strains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calpastatin, an endogenous calpain-inhibitor protein, regulates the cleavage of the Cdk5 activator p35 to p25

Cyclin-dependent kinase 5 (Cdk5) is a Ser/Thr kinase that is activated by binding to its regulatory subunit, p35. The calpain-mediated cleavage of p35 to p25 and the resulting aberrant activity and neurotoxicity of Cdk5 have been implicated in neurological disorders, such as Alzheimer's disease. To gain further insight into the molecular mechanisms underlying the pathological function of Cdk5, we investigated the role of the calpain inhibitor protein calpastatin (CAST), in controlling the aberrant production of p25. For this purpose, brain tissue from wild-type, CAST-over-expressing (transgenic), and CAST knockout mice were analyzed. Cleavage of p35 to p25 was increased in extracts from CAST knockout mice, compared with wild-type. Conversely, generation of p25 was not detected in brain lysates from CAST-over-expressing mice. CAST expression was 5-fold higher in mouse cerebellum than cerebral cortex. Accordingly, p25 production was lower in the cerebellum than the cerebral cortex. Furthermore, the Ca(2+) -dependent degradation of p35 by proteasome was evident when calpain was inhibited. Taken together, these results suggest that CAST is a crucial regulator of calpain activity, the production of p25, and, hence, the deregulation of Cdk5. Therefore, impairment of CAST expression and its associated mechanisms may contribute to the pathogenesis of neurodegenerative disorders.     

Effect of Acute Administration of 3-Butyl-1-Phenyl-2-(Phenyltelluro)Oct-En-1-One on Oxidative Stress in Cerebral Cortex,Hippocampus, and Cerebellum of Rats

Organotellurium compounds have been synthesized since 1840, but pharmacological and toxicological studies about them are still incipient. Therefore, the objective of this study was to verify the effect of acute administration of the organochalcogen 3-butyl-1-phenyl-2-(phenyltelluro)oct-en-1-one on some parameters of oxidative stress in the brain of 30-day-old rats. Animals were treated intraperitoneally with a single dose of the organotellurium (125, 250, or 500 μg/kg body weight) and sacrificed 60 min after the injection. The cerebral cortex, the hippocampus, and the cerebellum were dissected and homogenized in KCl. Afterward, thiobarbituric acid reactive substances (TBARS), carbonyl, sulfhydryl, catalase (CAT), superoxide dismutase (SOD), nitric oxide (NO) formation, and hydroxyl radical production were measured in the brain. The organotellurium enhanced TBARS in the cerebral cortex and the hippocampus, and increased protein damage (carbonyl) in the cerebral cortex and the cerebellum. In contrast, the compound provoked a reduced loss of thiol groups measured by the sulfhydryl assay in all the tissues studied. Furthermore, the activity of the antioxidant enzyme CAT was reduced by the organochalcogen in the cerebral cortex and the cerebellum, and the activity of SOD was inhibited in all the brain tissues. Moreover, NO production was increased in the cerebral cortex and the cerebellum by this organochalcogen, and hydroxyl radical formation was also enhanced in the cerebral cortex. Our findings indicate that this organotellurium compound induces oxidative stress in the brain of rats, corroborating that this tissue is a potential target for organochalcogen action.     

METABOLIC CONTROL MECHANISMS IN MAMMALIAN SYSTEMS

Abstract— The regulation by thyroid hormone of the activities of hexokinase (ATP: D-hexose 6-phosphotransferase; EC 2.7.1.1), phosphofructokinase (ATP: D-fructose-6- phosphate 1-phosphotransferase; EC 2.7.1.11) and pyruvate kinase (ATP: pyruvate phosphotransferase; EC 2.7.1.40) has been investigated in the soluble fractions of the cerebral cortex and cerebellum of the rat. Ontogenetic studies on these key glycolytic enzymes demonstrated marked increases in the normal cerebral cortex between 1 day and 1 yr of age; less pronounced increases in enzyme activities were noted in the normal cerebellum. Neonatal thyroidectomy, induced by treatment of 1-day-old rats with 100 μCi of ¹³¹I, ied to an impairment of body and brain growth and inhibited the developmental increases in hexokinase, phosphofructokinase and pyruvate kinase in both the cerebral cortex and cerebellum. Whereas 50 μCi of ¹³¹I had little or no effect on these brain enzymes, 200 μCi of the radioisotope markedly inhibited (35–65 per cent) the developmental increases of the various enzyme activities investigated. When administration of the radioisotope was delayed for 20 days after birth, little or no inhibition of the development of brain glycolytic enzymes was observed. Whereas treatment of normal neonatal animals with L-tri-iodothyronine had no significant effect on the activities of cerebro-cortical and cerebellar glycolytic enzymes, the hormone increased their activities in young cretinous rats. However, when the initiation of tri-iodothyronine treatment was delayed until neonatally thyroidectomized rats had reached adulthood, this hormone failed to produce any appreciable change in enzyme activity. Our results indicate that thyroid hormone exerts an important regulatory influence on the activities of hexokinase, phosphofructokinase and pyruvate kinase in the developing cerebral cortex and cerebellum.