Enolase Activity and Isoenzyme Distribution in Human Brain Regions and Tumors

Abstract: The distribution of enolase (EC 4.2.1.11) activity and isoenzymes in various regions of human brain at different ages (from 23 weeks of gestation to 95 years) and in brain tumors has been determined. Total enolase activity increased in all regions with age. No significant differences were found in the relative proportions of αα-, αγ-, and γγ-enolase isoenzymes in the various brain regions, determined by agarose gel electrophoresis. Type αα-enolase was the predominant isoenzyme, and αγ-enolase represented a substantial proportion of the total enolase activity. Astrocytomas, anaplastic astrocytomas, glioblastomas, and meningiomas possessed lower enolase activity than normal brain. Among astrocytic tumors, total enolase activity correlated with malignancy. Astrocytomas possessed the lowest and glioblastomas the highest enolase activity. All tumors possessed a higher proportion of αα-enolase and a lower proportion of γγ-enolase than the normal human brain. Among astrocytic tumors, glioblastomas were the tumors with the highest proportion of αα-enolase and lowest proportion of γγ-enolase.     

Determination of Brain Enolase Isozymes with an Enzyme Immunoassay at the Level of Single Neurons

Abstract Ultrasensitive enzyme immunoassay systems for the assay of rat brain enolase isozymes ( αα , αγ , and γγ forms) were prepared by use of β- d -galactosidase from Escherichia coli as label and the purified rabbit antibodies to αα and γγ enolases. The antibodies were purified from the immunoglobulin G (IgG) fractions of antisera by immunoaffinity chromatography with a column of the corresponding antigen-coupled Sepharose. Sandwich-type immunoassay systems with the galactosidase-labeled antibody Fab'fragments and the antibody F(abapos;)₂-immobilized polystyrene beads could determine amounts as small as 1 amol (10⁻¹⁸ mol) of each isozyme. Purkinje cell bodies picked up from the bulk-separated fraction by means of a nylon loop were subjected to the assay at the level of single cells. In contrast to previous report, this neuron contained not only the γγ but also the αγ and αα enolases at a level of amol per cell body, although the concentration of γγ was the highest. Immunohistochemical experiments on the cerebellum with the peroxidase-labeled antirabbit IgG antibody and the unlabeled antibody method confirmed the above results, and indicated that both α and γ subunits of the enolase were stained intensely in axons.     

Dolichol in Human Brain: Regional and Developmental Aspects

Distinct regional differences in dolichol content were defined in human brain from 15 to 76 years of age. Concerning the regional distribution of dolichol, levels were: higher in cortical gray matter than in subcortical white matter, highest among cortical regions in temporal gray matter, highest among all brain regions in thalamus, and lowest among all brain regions in lower brain stem and spinal cord. The developmental changes in the contents of dolichol were found to be different among brain regions. For example, among regions with the highest levels of dolichol, in thalamus there was a six to sevenfold increase, but in parietal gray matter, only a 2.5-fold increase. Regional and developmental changes in the proportions of the individual molecular species (isoprenologues) of dolichol were also observed. The findings indicate that the metabolism of dolichol is not uniform among regions of developing and aging human brain and may have implications for the role of dolichol in normal and diseased human brain.     

Developmental Changes in the NGF Content in the Brain of Young,Growing, Low-Birth-Weight Rats

The NGF content in each region of the brain of four-week-old rats was ranked in the decreasing order of cerebral cortex, hippocampus, cerebellum, midbrain/diencephalon, and pons/medulla ob-longata, and the NGF concentration, in the decreasing order of hippocampus, cerebral cortex, cerebellum, midbrain/diencephalon, and pons/medulla oblongata in both AFD and SFD groups. The NGF content and concentration in the cerebral cortex were about the same value at each age between those in the AFD and SFD groups. Those in the hippocampus were a little higher in the SFD group than in the AFD group at the ages of three and four weeks, unlike those in the other regions, where the values for the cerebellum, midbrain/diencephalon and pons/medulla oblongata tended to be somewhat higher in the AFD group than in the SFD group. The NGF concentrations in the hippocampus and cerebral cortex increased with growth: the concentration in the hippocampus at four weeks of age was about 4-fold of that at one week in the AFD group and about 5.7-fold of that at one week in the SFD group; and likewise the concentration in the cerebral cortex at four weeks of age was about 5.3-fold in the AFD group and about 7-fold in the SFD group. The NGF concentrations in the cerebellum decreased, and those in midbrain/diencephalon and pons/medulla oblongata hardly changed with growth in either AFD or SFD group. From these results NGF may have stronger implications for the neuronal growth in the hippocampus compared with those in the lower brain regions of the SFD rats.     

Postnatal Developmental Changes in Fucosyltransferase Activity in Rat Brain

Abstract: GDP-fucose:asialofetuin fucosyltransferase activity was studied during the postnatal development of rat brain. The enzymatic activity was very low during the first days of life and reached a maximum level around 21 days. This increase in enzymatic activity was characterized by two periods of rapid change. A rapid increase occurred between 3 and 7 days after birth, followed by a slow increase from 7 to 17 days, then a new rapid change from 17 to 21 days. Stimulation of the enzymatic activity by Triton X-100 increased with age. The developmental profiles of GDP-fucose pyrophosphatase and fucosidase did not change during this period.     

Selective postmortem degradation of inducible heat shock protein 70 (hsp70) mRNAs in rat Brain

Summary 1. Altered mRNA levels in postmortem brain tissue from persons with Alzheimer's disease (AD) or other neurological diseases are usually presumed to be characteristic of the disease state, even though both agonal state (the physiological state immediately premortem) and postmortem interval (PMI) (the time between death and harvesting the tissue) have the potential to affect levels of mRNAs measured in postmortem tissue. Although the possible effect of postmortem interval on mRNA levels has been more carefully evaluated than that of agonal state, many studies assume that all mRNAs have similar rates of degradation postmortem.2. To determine the postmortem stability of inducible heat shock protein 70 (hsp70) mRNAs, themselves unstablein vivo at normal body temperature, rats were heat shocked in order to induce synthesis of the hsp70 mRNAs. hsp70 mRNA levels in cerebellum and cortex were then compared to those of their heat shock cognate 70 (hsc70) mRNAs, as well as to levels of 18S rRNAs, at 0 and at 24 hr postmortem.3. Quantiation of northern blots after hybridization with an hsp70 mRNA-specific oligo probe indicated a massive loss of hsp70 mRNA signal in RNAs isolated from 24-hr postmortem brains; quantitation by slot-blot hybridization was 5- to 15-fold more efficient. Even using the latter technique, hsp70 mRNA levels were reduced by 59% in 24-hr-postmortem cerebellum and by 78% in cortex compared to mRNA levels in the same region of 0-hr-postmortem brain. There was little reduction postmortem in levels of the hsp70 mRNAs or of 18S rRNAs in either brain region.4.In situ hybridization analysis indicated that hsp70 mRNAs were less abundant in all major classes of cerebellar cells after 24 hr postmortem and mRNAs had degraded severalfold more rapidly in neurons than in glia. There was no corresponding loss of intracellular 18S rRNA in any cell type.5. We conclude from these results that the effect of postmortem interval on mRNA degradation must be carefully evaluated when analyzing levels of inducible hsp70 mRNAs, and perhaps other short-lived mRNAs, in human brain.     

Root Hypoxia-lnduced Changes in the Pattern of Translatable mRNAs In Poplar Leaves

Shoot responses to root stress may be modulated by hormonestransported from root tissues. In this study, the pattern ofin vitro translatable mRNAs from leaves on plants with oxygen-deprived(hypoxic) roots was used as a molecular phenotype to test forthe action and specificity of putative root-to-shoot signalmolecules. Root hypoxia caused a decrease in the abundance ofa group of leaf mRNAs which translated to give low molecularweight (LMW) proteins (12 to 28 kDa). Root excision induceda similar reduction in a subset of the same LMW leaf mRNAs.This suggests that the effects of root hypoxia on leaves arephysiologically related to the effects of root excision. Feedingabscisic acid to leaves did not mimic the effects of root aerationor hypoxia. Applications of zeatin riboside (ZR) to leaves ofplants with hypoxic roots duplicated the effects of root aerationon the LMW translation products. The specificity of the leafresponse to ZR was tested by feeding KC1 and sucrose to leavesof root excised plants. Applications of either KC1 or sucrosealso increased the abundance of the LMW translation products.Although the results reported in this paper are consistent withthe concept that shoot responses to root stress may be modulatedby substances moving in the xylem, a thorough understandingof how hormones control cellular responses needs to be resolvedbefore the mechanism of root-shoot integration can be understood. Key words: ABA, cytokinins, hypoxia, poplar     

Separation and Characterization of Four Forms of β-N-Acetylhexosaminidase from Chicken Brain

Chicken brain beta-N-acetylhexosaminidases from embryos (16 and 21 days old), newborns (1 and 4 days old), and adults (3 1/2 months and 2 years old) were separated into four different forms by ion exchange chromatography on diethylaminoethyl-cellulose. Three of these forms were "acid" hexosaminidases (I, IIA, and IIB), and the fourth was a "neutral" form. Throughout development of the chicken, forms IIA and III maintained the same activity ratio, whereas that for form I decreased and that for form IIB showed an increase.     

Brain Levels of Neuron-Specific and Nonneuronal Enolase in Huntington's Disease

Abstract— Levels of the cell-specific brain isoenzymes of enolase were determined in basal ganglia and cerebral cortical tissue of Huntington's disease and age- and sex-matched control brain. Neuron-specific enolase (NSE) levels are decreased an average of 45% in basal ganglia from patients with Huntington's disease whereas the glial-specific form of enolase, nonneuronal enolase (NNE), is not significantly altered. In contrast, levels of NSE in cerebral cortical tissue from Huntington's disease patients remains unchanged in comparison with controls whereas NNE levels are significantly increased. NNE and NSE levels appear to be specific biochemical indicators of glial and neuronal cell number and viability. Levels of these cell-specific isoenzymes may therefore prove useful in quantitating neuropathological changes in various neurological disorders.     

Developmental Changes in Expression of a 60-kDa Somatostatin Receptor Immunoreactivity in the Rat Brain

Abstract: The neuropeptide somatostatin (SRIF) exerts several important physiological actions in the adult CNS through interactions with membrane-bound receptors. SRIF expression is developmentally regulated and this regulation is most apparent in the cerebellum, where SRIF immunoreactivity is expressed at early postnatal ages and then disappears toward adulthood. The transitory nature of SRIF expression at a time of major changes in cerebellum suggests that this peptide may have a role in cerebellar development. To further investigate the role of the SRIF transmitter system during development, we have examined the levels of expression of SRIF receptors in the developing rat brain by immunoblotting using antiserum selective for a 60-kDa brain SRIF receptor. In whole rat brain, SRIF receptor immunoreactivity first appears at embryonic day 13 (E13), is elevated at E16. increases at birth, peaks at early postnatal ages, and then gradually declines with age. No apparent changes in size of the receptor occur with age. No consistent changes in levels of SRIF receptor immunoreactivity are detected from early postnatal ages to adulthood in the hippocampus, cerebral cortex, and striatum, but levels gradually decline in the hypothalamus. In contrast, SRIF receptor immunoreactivity is expressed transiently in cerebellum. SRIF receptor immunoreactivity is detectable in cerebellum at E16, increases in levels at birth, is apparent from postnatal day 3 to postnatal day 8, and then disappears. The transitory nature of SRIF receptor expression in cerebellum is unique and parallels the expression of SRIF immunoreactivity in this brain region. These findings support the hypothesis that SRIF has a role in cerebellar development.     

Developmental Changes in the Activity of Enzymes of Purine Metabolism in Rat Neuronal Cells in Culture and in Whole Brain

The activities (Vmax) of several enzymes of purine nucleotide metabolism were assayed in premature and mature primary rat neuronal cultures and in whole rat brains. In the neuronal cultures, representing 90% pure neurons, maturation (up to 14 days in culture) resulted in an increase in the activities of guanine deaminase (guanase), purine-nucleoside phosphorylase (PNP), IMP 5'-nucleotidase, adenine phosphoribosyltransferase (APRT), and AMP deaminase, but in no change in the activities of hypoxanthine-guanine phosphoribosyltransferase (HGPRT), adenosine deaminase, adenosine kinase, and AMP 5'-nucleotidase. In whole brains in vivo, maturation (from 18 days of gestation to 14 days post partum) was associated with an increase in the activities of guanase, PNP, IMP 5'-nucleotidase, AMP deaminase, and HGPRT, a decrease in the activities of adenosine deaminase and IMP dehydrogenase, and no change in the activities of APRT, AMP 5'-nucleotidase, and adenosine kinase. The profound changes in purine metabolism, which occur with maturation of the neuronal cells in primary cultures in vitro and in whole brains in vivo, create an advantage for AMP degradation by deamination, rather than by dephosphorylation, and for guanine degradation to xanthine over its reutilization for synthesis of GMP. The physiological meaning of the maturational increase in these two ammonia-producing enzymes in the brain is not yet clear. The striking similarity in the alterations of enzyme activities in the two systems indicates that the primary culture system may serve as an appropriate model for the study of purine metabolism in brain.     

衰老大鼠的某些脑区组织中游离氨基酸水平的改变

使用D 半乳糖建立衰老大鼠模型组与同龄、同饲的正常对照组大鼠的某些脑区游离氨基酸 (FAA)水平的比较发现 :( 1 )衰老模型组的海马、纹状体以及皮层等脑区中谷氨酸 (Glu)、天门冬氨酸 (Asp)水平明显降低 ;( 2 )γ 氨基丁酸 (GABA)水平在衰老模型组大鼠的海马 ,纹状体以及小脑等脑区中明显升高 ;( 3)衰老模型组的皮层、小脑、海马、纹状体等脑区的牛磺酸 (Tau)水平明显下降。以此探讨动物衰老与脑区游离氨基酸水平的关系     

Localization and Developmental Changes of τ Protein Kinase I/Glycogen Synthase Kinase-3β in Rat Brain

Abstract: τ protein kinase I (TPKI) purified from bovine brain extract has been shown to phosphorylate τ and to form paired helical filament (PHF) epitopes and was found recently to be identical to glycogen synthase kinase-3β (GSK-3β). Before elucidating a role of TPKI/GSK-3β in PHF formation, it is necessary to investigate the normal function of the enzyme. To study the distribution and developmental changes of the enzyme, specific polyclonal antibodies were prepared against TPKI and GSK-3α. Immunoblot analysis demonstrated that TPKI was nearly specifically localized in the brain of adult rats. The level of TPKI in the rat brain was high at gestational day 18, peaked on postnatal day 8, and then decreased rapidly to a low level, which was sustained up to 2 years. Immunohistochemistry indicated primarily neuronal localization of TPKI. Growing axons were stained most intensely in the developing cerebellum, but the immunoreactivity became restricted to the gray matter in the mature tissue. Parallel fibers had a high level of TPKI and also stained intensely for τ. These findings indicate that τ is one of the physiological substrates of TPKI and suggest that the enzyme plays an important role in the growth of axons during development of the brain.     

Changes in Free Amino Acid Levels in Developing Human Foetal Brain Regions

The levels of free amino acids were determined in human foetal brain regions during prenatal development. Variation in the distribution of amino acids and their rate of change in five segments of the CNS at different stages of ontogeny was observed. Striking developmental changes were found in the levels of aspartic acid in medulla-pons and spinal cord, glycine in the spinal cord, gamma-aminobutyric acid in the cerebral cortex, glutamic acid in the cerebral cortex, midbrain, and spinal cord, and taurine in the medulla-pons and spinal cord. At a late gestational period, glutamic acid was found most abundantly over all the brain regions, whereas the level of taurine was highest at an early gestational stage but not in spinal cord.     

Developmental and Regional Changes of mRNA for a Cerebellar Protein (Spot 35) in the Rat Brain

Spot 35 protein is a cerebellar Ca-binding protein. Because Southern blot analysis showed evidence for the nucleotide sequence of spot 35 protein cDNA in the rat genome, we applied cDNA to quantitate the mRNA of rat spot 35 protein. The size of this mRNA was about 1,900 nucleotides in length, and that of mRNA from bovine cerebellum was larger. We also examined the developmental changes and regional distribution of spot 35 protein mRNA in rat brains by dot-blot analysis using cDNA as a probe. During postnatal days 1-20, a rapid increase of mRNA levels was observed. Further, the level of mRNA for spot 35 protein was found in the cerebellum and was negligible in the cerebral cortex, striatum, and hippocampus.     

Reevaluation of Taurine Levels and Distribution of Cysteic Acid Decarboxylase in Developing Human Fetal Brain Regions

The possibilities of interference by glycerophosphoryl ethanolamine (GPE) in the estimation of taurine levels in cerebral cortex, midbrain, cerebellum, medullapons, and spinal cord of developing human fetal brain regions were eliminated by hydrolyzing tissue extracts with 6 M HCl. Cysteic acid thus produced was separated from taurine by ion-exchange chromatography using Biorad-AG resin. Fluorescamine was used as fluorogen. Data reveal that the estimation of taurine in human fetal brain regions is affected if GPE is present as a contaminant in the assay system. Cysteic acid decarboxylase activity was measured using cysteic acid as the substrate. Higher enzymic activity was recorded with increased fetal body weight, but the reverse was true for taurine level.     

Homologous Mammalian Brain Cell Lysate System for the Initiation and Translation of Exogenous mRNAs

Abstract: The rate of protein synthesis in mammalian brain tissue is affected by a variety of physiological conditions, both natural and induced. The process of initiation may be involved in some of the observed changes, although as yet the actual rates of initiation of natural mRNAs have not been directly measured in these circumstances. One approach to studying the regulation of protein synthesis in brain tissue would be to utilize a homologous cell-free system to examine in vitro the translation of various added mRNAs. The present report describes a micrococcal nuclease-treated cell-free lysate system derived from fetal mouse brain tissue which is capable of actively initiating and translating exogenously added mRNA. Sodium dodecyl sulfate-polyacrylamide slab gel electrophoretic analysis of the specific protein products of the reaction mixture allowed a qualitative and quantitative assessment of the translational process under a variety of experimental conditions. Optimal conditions for mRNA-dependent protein synthesis were the following: 30°C incubation temperature; 80–100 mM-KCl; 2.1 mM-Mg²⁺; 50 μM-spermhe; and 10 μg/ml poly A(+) mRNA. Incorporation of L-[³⁵S]methionine into proteins required ATP, GTP, and an energy regenerating system. The addition of saturating amounts of a homologous "initiation factors" fraction stimulated incorporation twofold during the first 20 min of incubation, while the patterns of inhibition observed upon the addition of 5 × 10^-5 M-aurin tricarboxylic acid at various periods during incubation demonstrated the occurrence of multiple rounds of initiation.     

Effect of Hypothyroidism on the Subcellular Distribution of Ca2+/Calmodulin-Stimulated Protein Kinase II in Chicken Brain During Posthatch Development

Abstract: In developing chicken brain Ca²⁺/calmodulin-stimulated protein kinase II (CaMPK-II) changes from being primarily cytosolic to being primarily particulate during the protracted maturation period. To investigate whether thyroid hormone levels may be involved in regulating this subcellular redistribution, we raised chickens from 1 day posthatching on food soaked in 0.15% (wt/vol) propylthiouracil (PTU) plus 0.05% (wt/vol) methimazole (MMI). This produced a mild hypothyroidism specifically during the maturation period and resulted in a 67% reduction in the levels of free triiodothyronine (T₃) at 42 days. The concentrations of α- and β-CaMPK-II in cytosol (S3) and crude synaptic membrane (P2M) fractions from forebrain were measured by three methods: Ca²⁺/calmodulin- or Zn²⁺-stimulated autophosphorylation or binding of biotinylated calmodulin. By all three methods hypothyroid animals showed a marked retardation of the redistribution of both subunits of CaMPK-II: an increase in the concentration of the enzyme in S3 and a corresponding decrease in P2M with no overall change in the total amount of enzyme and little apparent change in the concentration of other proteins. In both fractions, there was a parallel change in the Ca²⁺/calmodulin-stimulated phosphorylation of endogenous protein substrates but no change in the basal or cyclic AMP-stimulated protein phosphorylation. Supplementing the PTU/MMI-treated diet with thyroxine (0.5 ppm) prevented all of the observed changes.