Subependymal glycosaminoglycan networks in adult and developing rat brain

Histochemical studies of normal adult rat brain indicate two types of glycosaminoglycans in the subependymal region of the lateral ventricle. One network is characterized by an affinity for the cationic dyes alcian blue, aldehyde fuchsin and colloidal iron. These reactions occur at pH 1.0 and at 0.5-0.3 M concentration of MgCl2, which suggests that this material is chondroitin sulfate. The other system is identified by metachromasia with toluidine blue and a loss of PAS staining following sulfation. These findings are consistent with non-sulfated and non-anionic acid mucopolysaccharides. In developing rat brain the differential development of these networks enhances their separate identity. The metachromatic network is present at least by the 10th postnatal day but the polyanionic electrolytes cannot be identified until the 16th to the 22nd days. The possible functional importance of these systems is discussed.     

Generation of hydrogen peroxide in the developing rat heart: the role of elastin metabolism

Reports describing production of reactive oxygen species in neonatal heart are missing. As lysyl oxidase is potentially important source of H₂O₂, we studied its role during ontogenic development of rat heart. H₂O₂ was detected in thin sections of developing rat heart by fluorescence microscopy with the use of fluorescence probe 2??-7??-dichlorofluorescin. The experimental design comprised foetuses 21 days after conception, and then the animals sampled on the 1st, 4th, 7th, 10th, 15th, 30th and 60th day after birth. We also used 7-month-old animals as an example of ageing effects. Since the day 4 on, H₂O₂ was produced only extracellularly up to the day 15, between days 30 and 60 intracellular production was detected as well, and in 7-month-old animals only extracellular production was observed. The specific inhibitors of lysyl oxidase almost completely quenched the H₂O₂-dependent fluorescence. Starting from day 7, blue autofluorescence specific to oxidized proteins developed in the vessel wall. Intracellular blue autofluorescence specific to autoxidation products developed after day 30. Chloroform extraction diminished the intracellular blue fluorescence, leaving the extracellular fluorescence intact. This confirmed the protein nature of the fluorophores. Lysyl oxidase is significant source of H₂O₂ in the heart vessel wall during development and H₂O₂ oxidatively modifies elastin producing protein blue autofluorescence.     

On the nature of the metachromatic cells of pancreatic islets

Summary Islet cells with cytoplasmic granules metachromatically stained by toluidine blue are found in the pancreas of dog, cat, pig, rabbit, teleostian fish, monkey and man, but not in the rat, mouse and guinea pig; dubious results are obtained on duck and ox islets. These cells do not react with the staining methods for the demonstration of A- and B-cells; conversely, they are silver-impregnated by a modification of Davenport's method, and, at least in dog islets, can be readily identified with dark-field microscopy and stained blue with the Mallory-Heidenhain trichrome stain. No obvious changes of this cell type are found either in synthalintreated dogs or in diabetic men.The Authors suggest that the islet argyrophil-metachromatic cells (A₁-cells of Swedish Authors) are identical with D-cells and very likely represent a morphologically and functionally indipendent cell type.     

THE RELATIVE SIGNIFICANCE OF CO2-FIXING ENZYMES IN THE METABOLISM OF RAT BRAIN

To evaluate the relative significance of CO₂-fixing enzymes in the metabolism of rat brain, the subcellular distribution of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, NADP-isocitrate dehydrogenase and NADP-malate dehydrogenase, as well as the fixation of H¹⁴CO₃^? by the cytosol and the mitochondria was investigated. Pyruvate carboxylase and phosphoenol-pyruvate carboxykinase are mainly localized in the mitochondria whereas NADP-isocitrate dehydrogenase and NADP-malate dehydrogenase are present in both the cytosol and the mitochondria. In the presence of pyruvate rat brain mitochondria fixed H¹⁴CO₃^? at a rate of about 170 nmol/g of tissue/min whereas these organelles fixed negligible amounts of H¹⁴CO₃^? in the presence of α-ketoglutarate or phosphoenolpyruvate. Rat brain cortex slices fixed H¹⁴CO₃^? at a rate of about 7 nmol/g of tissue/min and it was increased by two-fold when pyruvate was added to the incubation medium. The carboxylation of α-ketoglutarate and pyruvate by the reversal of the cytosolic NADP-isocitrate dehydrogenase and NADP-malate dehydrogenase respectively was very low as compared to that by pyruvate carboxylase. The rate of carboxylation reaction of both NADP-isocitrate dehydrogenase and NADP-malate dehydrogenase was only about 1/10th of that of decarboxylation reaction of the same enzyme. It is suggested that under physiological conditions these two enzymes do not play a significant role in CO₂-fixation in the brain. In rat brain cytosol, citrate is largely metabolized to α-ketoglutarate by a sequential action of aconitate hydratase and NADP-isocitrate dehydrogenase. The operation of the citrate-cleavage pathway in rat brain cytosol is demonstrated. The data show that among four CO₂-fixing enzymes, pyruvate carboxylase, an anaplerotic enzyme, plays the major role in CO₂-fixation in the brain.     

Expression of Cytochrome P450 Side-Chain Cleavage Enzyme and 3β-Hydroxysteroid Dehydrogenase in the Rat Central Nervous System: A Study by Polymerase Chain Reaction and In Situ Hybridization

Abstract: In examining steroid synthesis in the CNS, expression of the mRNAs encoding for cytochrome P450 side-chain cleavage enzyme (P450_SCC) and 3β-hydroxysteroid dehydrogenase/Δ⁵-Δ⁴ isomerase (3β-HSD) has been studied in the rat brain. P450_SCC transforms cholesterol into pregnenolone and 3β-HSD transforms pregnenolone into progesterone. PCR was used to amplify cDNA sequences from total RNA extracts. Classical steroidogenic tissues, like adrenal and testis, as well as the non-steroidogenic tissue lung have been used as controls. The expression of P450_SCC and 3β-HSD have been demonstrated by PCR in cortex, cerebellum, and spinal cord. In addition, primary cultures of rat cerebellar glial cells and rat cerebellar granule cells were found to express P450_SCC and 3β-HSD at comparable levels. Furthermore, three of the four known isoenzymes of 3β-HSD were identified, as determined using selective PCR primers coupled with discriminative restriction enzymes and sequencing analysis of the amplified brain products. Using RNA probes, in situ hybridization indicated that P450_SCC and 3β-HSD are expressed throughout the brain at a low level and mainly in white matter. Enrichment of glial cell cultures in oligodendrocytes, however, does not increase the relative abundance of P450_SCC and 3β-HSD mRNA detected by PCR. This discrepancy suggests that the developmental state of cultured cells and their intercellular environment may be critical for regulating the expression of these enzymes. These findings support the proposal that the brain apparently has the capacity to synthesize progesterone from cholesterol, through pregnenolone, but that the expression of these enzymes appears to be quite low. Furthermore, the identification of these messages in cerebellar granule cell cultures implies that certain neurons, in addition to glial cells, may express these steroidogenic enzymes.     

Oxygen regime in rat brain tissues under conditions of acute nitrite methemoglobinemia

Oxygen regime in rat brain tissues was studied under conditions of nitrite hypoxia. The local brain circulation (LCC) and pO₂ were recorded by polargraphic method in microareas of the brain cortex one hour after a subcutaneous injection of NaNO₂ (3 mg/100 g body mass). A LCC decrease by 55% was shown by the 30th min of the nitrite intoxication, with its restoration to 85% of the initial blood-flow by the 60th min. In some brain microareas, a pO₂ increase by 40.7% was observed by the 45th min of the action and its decrease by 32.2% by the 60th min, whereas in other microareas, a pO₂ decrease by 24.5% with its subsequent increase to 78.7% of the initial level, respectively. A statistically significant correlation is revealed between changes of the oxygen pressure in the process of development of nitrite hypoxia and the LCC dynamics. It is concluded that the circulatory disturbances developing at the first moments of the nitrite action lead to an increase of the degree of the pO₂ differentiated distribution in the brain: to hyperoxygenation in some microareas and to severe hypoxia in others, what can be the cause of functional brain disturbances under the effect of nitrogen oxides.     

Proteins associated with tubulin.

The distribution of proteins on SDS-urea polyacrylamide (7.5%) disc gel electrophoresis is studied from rat brain tubulin purified by three different procedures, including ammonium sulfate precipitation followed by DEAE cellulose chromotography, three cycles of polymerization-depolymerization and colchicinecontaining agarose affinity columns. Three tubulin-associated proteins other than the principal tubulin dimer are identified and characterized with respect to molecular weight, behavior on gel filtration chromatography and method of tubulin purification. One of these proteins (H₁) is released from the tubulin complex when colchicine is irreversibly bound to tubulin. These proteins may participate in processes related to microtubule assembly or function.     

DEVELOPMENT OF LIPOGENESIS IN RAT BRAIN CORTEX: THE DIFFERENTIAL INCORPORATION OF GLUCOSE AND ACETATE INTO BRAIN LIPIDS IN VITRO

—The oxidation to CO₂ and the incorporation of [U-¹⁴C]glucose and [U-¹⁴C]acetate into lipids by cortex slices from rat brain during the postnatal period were investigated. The oxidation of [U-¹⁴C]glucose was low in 2-day-old rat brain, and increased by about two-fold during the 2nd and 3rd postnatal weeks. The oxidation of [U-¹⁴C]acetate was increased markedly in the second postnatal week, but decreased to rates observed in 2-day-old rat brain at the time of weaning. Both labeled substrates were readily incorporated into non-saponifiable lipids and fatty acids by brain slices from 2-day-old rat. Their rates of incorporation and the days on which maximum rates occurred were different, however, maximum incorporation of [U-¹⁴C]glucose and [U-¹⁴]acetate into lipid fractions being observed on about the 7th and 12th postanatal days, respectively. The metabolic compartmentation in the utilization of these substrates for lipogenesis is suggested. The activities of glucose-6-phosphate dehydrogenase, cytosolic NADP-malate dehydrogenase, cytosolic NADP-isocitrate dehydrogenase, ATP-citrate lyase and acetyl CoA carboxylase were measured in rat brain during the postnatal period. All enzymes followed somewhat different courses of development; the activity of acetyl CoA carboxylase was, however, the lowest among other key enzymes in the biosynthetic pathway, and its developmental pattern paralleled closely the fatty acid synthesis from [U-¹⁴C]glucose. It is suggested that acetyl CoA carboxylase is a rate-limiting step in the synthesis de novo of fatty acids in developing rat brain.     

THE BIOSYNTHESIS OF 3-METHOXY-4-HYDROXYPHENYLGLYCOL SULPHATE BY LIVER AND BRAIN

—3-Methoxy-4-hydroxyphenylglycol (MHPG) formed a sulphate conjugate when incubated with ATP, Mg²⁺ ions, Na₂³⁵SO₄ and the high-speed supernatant preparations of rabbit or rat brain. The same reactions could be catalysed by similar enzyme preparations from liver. The sulphated product was separated and identified by paper chromatography. On acid hydrolysis, it released both Na₂³⁵SO₄ and the free glycol. The measurement of this labelled sulphate was used as a specific assay procedure for determining the overall sulphoconjugatory process. The pH optimum of the reaction is 7.8. For rabbit brain, the K_m for Na₂SO₄ determined for the activating system is 3.6 × 10⁻⁴m , and that for MHPG for the sulphotransferase reaction is 1.05 × 10⁻⁴m . The specific enzyme activity, expressed as nmol ³⁵SO₄ incorporated/h/mg protein for a 30-min assay is as follows: rat brain, 2.8; rabbit brain, 1.6; rat liver, 33.4and rabbit liver, 15.0. Dithiothreitol at 3 mm concentration had no significant effect on the sulphation of MHPG in all these preparations.     

Exacerbation of Brain Pathology After Partial Restraint in Hypertensive Rats Following SiO2 Nanoparticles Exposure at High Ambient Temperature

This investigation examines the possibility that exposure to silica dust of hypertensive individuals may exacerbate brain pathology and sensory motor dysfunction at high environmental temperature. Hypertension was produced in rats (200–250 g) by two-kidney one clip (2K1C) method, and in these animals, SiO₂ nanoparticles (NPs; 50 to 60 nm) were administered at 50 mg/kg, i.p. daily for 1 week. On the 8th day, these rats were subjected to partial restraint in a Perspex box for 4 h either at room temperature (21 °C) or at 33 °C in a biological oxygen demand incubator (wind velocity, 2.6 cm/s; relative humidity, 65 to 67 %). In these animals, behavioral functions, blood–brain barrier (BBB) permeability to Evans blue albumin (EBA) and radioiodine (^[131]-Iodine), brain water content and neuronal injuries were determined. Hypertensive rats subjected to 4 h restraint at room temperature did not exhibit BBB dysfunction, brain edema, neural injury, or alterations in rotarod or inclined plane angle performances. However, when these hypertensive rats were subjected to restraint at 33 °C, breakdown of the cortical BBB (EBA, +38 %; radioiodine, +56 %), brain water (+0.88 %), neuronal damages (+18 %), and behavioral impairment were exacerbated. Interestingly, SiO₂ exposure to these rats further exacerbated BBB breakdown (EBA, 280 %; radioiodine, 350 %), brain edema (4 %), and neural injury (30 %) after identical restraint depending on the ambient temperature. SiO₂ treatment also induced brain pathology and alteration in behavioral functions in normotensive rats after restraint at high temperature. These observations clearly show that hypertension significantly enhances restraint-induced brain pathology, and behavioral anomalies particularly at high ambient temperature and SiO₂ intoxication further exacerbated these brain pathologies and cognitive dysfunctions.     

Phospholipase C β2 in vascular smooth muscle

Receptor-mediated inositol 1,4,5-trisphosphate formation in most tissues is dependent on a variety of phospholipase C isoforms. To determine which phospholipase C isoforms were present in vascular smooth muscle compared to brain, liver, and spleen, we extracted proteins from these tissues and separated and identified the phospholipase C isoforms by immunoblotting. Aliquots of rat tail artery were examined by this procedure, together with aliquots of rat liver, spleen, cerebral cortex, hippocampus, cerebellum, aorta, and mesenteric artery. Phospholipase C γ1 was shown to be present in all of these tissues, while phospholipase C β1 was shown to be limited to fractions from brain. Phospholipase C Δ1 was detected in rat tail artery, mesenteric artery, aorta, and brain. Phospholipase C β2 was found in rat tail artery, liver, and brain. This is the first report of phospholipase C β2 in tissues other than HL60 cells. Since G proteins activate IP₃ production via stimulation of phospholipase Cβ isoforms in many tissues, and agonist-stimulated IP₃ production in smooth muscle requires G protein activation, phospholipase C β2 may be required for agonist-stimulated force production in vascular smooth muscle. © 1996 Wiley-Liss, Inc.     

Assessment of Structural Connectivity in the Preterm Brain at Term Equivalent Age Using Diffusion MRI and T2 Relaxometry: A Network-Based Analysis

Preterm birth is associated with a high prevalence of adverse neurodevelopmental outcome. Non-invasive techniques which can probe the neural correlates underpinning these deficits are required. This can be achieved by measuring the structural network of connections within the preterm infant''s brain using diffusion MRI and tractography. We used diffusion MRI and T₂ relaxometry to identify connections with altered white matter properties in preterm infants compared to term infants. Diffusion and T₂ data were obtained from 9 term neonates and 18 preterm-born infants (born <32 weeks gestational age) at term equivalent age. Probabilistic tractography incorporating multiple fibre orientations was used in combination with the Johns Hopkins neonatal brain atlas to calculate the structural network of connections. Connections of altered diffusivity or T₂, as well as their relationship with gestational age at birth and postmenstrual age at the time of MRI, were identified using the network based statistic framework. A total of 433 connections were assessed. FA was significantly reduced in 17, and T₂ significantly increased in 18 connections in preterm infants, following correction for multiple comparisons. Cortical networks associated with affected connections mainly involved left frontal and temporal cortical areas: regions which are associated with working memory, verbal comprehension and higher cognitive function – deficits which are often observed later in children and adults born preterm. Gestational age at birth correlated with T₂, but not diffusion in several connections. We found no association between diffusion or T₂ and postmenstrual age at the time of MRI in preterm infants. This study demonstrates that alterations in the structural network of connections can be identified in preterm infants at term equivalent age, and that incorporation of non-diffusion measures such as T₂ in the connectome framework provides complementary information for the assessment of brain development.     

A high-pressure liquid chromatographic method for measuring 3', 5'-cyclic AMP in brain.

A method is described for the estimation of adenosine 3′,5′-monophosphate (3′,5′-cyclic AMP) in rat brain by high-pressure liquid chromatography (HPLC). The nucleotide is purified initially by being passed through two columns, alumina and AG-1X2. The peak in HPLC was identified by a number of methods. Optimum parameters for HPLC were obtained by using 1 mm KH₂PO₄ buffer, pH 4.8, at a flow rate of 57 ml/hr at room temperature. Using this technique the concentration of 3′,5′-cyclic AMP in rat brain was found to be 2.53 ± 0.40 nmol/g (mean ± SD, n = 5).     

Stereoselective inhibition of rat brain cyclooxygenase by dexketoprofen

Although it has been assumed that the effects of nonsteroidal antiinflammatory drugs (NSAIDs) are mainly the result of their action on local synthesis of prostaglandins, there is growing evidence to suggest that they may also exert a central analgesic action. Some authors have suggested that inhibition of prostaglandin synthesis in the brain could contribute to the analgesic action. The effect of dexketoprofen trometamol (tromethamine salt of the enantiomer (+)-S-ketoprofen) on prostaglandin synthesis was investigated in rat brain fragments and in cyclooxygenase preparations from rat brain microsomes. Effects of the (-)-R-enantiomer and the racemic mixture were also evaluated. Significant levels of prostaglandin F_2α (PGF_2α) were synthesized in rat brain fragments after 10 min of incubation at 37°C. Dexketoprofen was found to be a potent inhibitor of this PGF_2α production in rat brain (IC₅₀ = 6.2 nM), and it completely suppressed PGF_2α production at 1 μM concentration. In addition, inhibition of PGF_2α synthesis by dexketoprofen was highly stereoselective since the enantiomer (-)-R-ketoprofen was significantly less potent (IC₅₀ = 294 nM); with this enantiomer, even at high concentrations such as 1 μM, less than 60% inhibition was achieved. These results correlated with those obtained in the study of racemic ketoprofen and its enantiomers on cyclooxygenase activity of rat brain microsomes, where dexketoprofen also inhibited enzymatic activity stereoselectively. IC₅₀ values obtained for dexketoprofen, (-)-R-ketoprofen, and rac-ketoprofen were 3.5 μM, 45.3 μM, and 5.8 μM, respectively. The above results could be related to the potent analgesic effect of dexketoprofen observed in vivo, which was also stereoselective. Taken together, these findings suggest that prostaglandin synthesis inhibition in rat brain by dexketoprofen could be associated, at least in part, with the analgesic effect of this NSAID. Chirality 9:281–285, 1997. © 1997 Wiley-Liss, Inc.     

DEVELOPMENT OF MITOCHONDRIAL PYRUVATE METABOLISM IN RAT BRAIN

The activities of a number of mitochondrial enzymes involved in the metabolism of pyruvate during development of the rat brain were investigated. The rates of decarboxylation of [1-¹⁴C]pyruvate to ¹⁴CO₂ via pyruvate dehydrogenase and the fixation of H¹⁴CO₃^? in the presence of pyruvate via pyruvate carboxylase by brain homogenates were very low in newborn rats. These rates increased markedly by about four-fold and 15-fold respectively during 10–35 postnatal days. The rates of the fixation of H¹⁴CO₃^? by cerebral homogenates were supported by the development of the activity of pyruvate carboxylase in rat brain. The activities of citrate synthase, aconitase, NAD-malate dehydrogenase, aspartate aminotransferase, alanine aminotransferase and phosphoenol-pyruvate carboxykinase were very low in the particulate fraction of the newborn rat brain. The activities of all these enzymes increased makedly by about three- to 10-fold during 10–35 days after birth. The activity of mitochondrial phosphoenolpyruvate carboxykinase from rat brain was not precipitated by an antibody prepared against rat liver cytosolic phosphoenolpyruvate carboxykinase suggesting that cerebral mitochondrial enzyme is immunologically different from that of the cytosolic form in hepatocytes. The significance of the development of the cerebral mitochondrial metabolism is discussed in relation to biochemical maturation of the brain.     

Expression of stress fibers in bullfrog mesothelial cells in situ

Summary Monoclonal antibodies (MABs) have been raised against acidic glycolipids extracted from the electric organ of Torpedo marmorata. One of these, designated L9, appears to recognize acidic glycolipids in adult T. marmorata electric organ, electromotor nerves and brain, adult rat sciatic nerve, and in embryonic and neonatal rat brain, starting at embryonic day (ED) 15 and disappearing by the 20th day of post-natal life. The epitope is present in growth cones isolated from 4-day-old rats; its proportion relative to total gangliosides is, however, no higher than that found in whole neonatal brain membranes. Desialidation of the acidic glycolipid fraction modifies neither the immunoreactivity nor the R_F value following thin-layer chromatography (TLC) of the antigen; it is concluded that the antigen is not a ganglioside. The MAB, HNK-1, recognizes the L9 antigen. Both HNK-1 and L9 recognize a sulphoglycolipid of the same R_F in TLC. The function of the L9 antigen is not known but its evolutionary conservation, presence in growth cones and its developmental regulation in the mammalian central nervous system indicate that it plays an important role in nervous system maturation.     

MEMBRANE AFFINITIES AND SUBCELLULAR DISTRIBUTION OF THE DIFFERENT MOLECULAR FORMS OF CHOLINE ACETYLTRANSFERASE FROM RAT

Choline acetyltransferase from rat brain is present in three different molecular forms with isoelectric points at pH 7·4-7.6, 7·7-7·9 and 8·3. The three forms were identified in a highly purified enzyme preparation, in a preparation of synaptosomes and in a cyto-plasmic preparation from disrupted axons and perikarya (fraction S₃). The three molecular forms differed in their affinities for synaptosome membranes and for a cation exchange resin (CM-Sephadex C-50). The positive surface charges of the different molecular forms and their affinities for membranes correlated well with their isoelectric points. The molecular form with jsoelectric point 8·3 had the largest positive surface charge and the highest membrane affinity. On isoelectric focusing of an extract from rat brain synaptosomes, the molecular form with isoelectric point 8·3 formed a complex with a negatively charged compound, presumably a protein. A method was developed to remove this compound by treatment with DEAE-Sephadex or by precipitation with vinblastine. These procedures are similar to methods known to remove the neurotubular protein. The complex formation did not occur in fraction S₃.     

THE DIFFERENTIATION OF PHOSPHOLIPASE A1 AND A2 IN RAT AND HUMAN NERVOUS TISSUES

—Lipid-free extracts of rat and human brain have been prepared and shown to contain phospholipase A₁ and A₂ activities and a lysophospholipase. The phospholipase Aj activity has pH optima of 4·2 and 4·6 in rat and human brain, respectively; it can be partially purified and isolated in high yields by dialysing the extracts at low pH. The purified preparations hydrolyse the ester bond at the 1-position in lecithin, phosphatidyl-ethanolamine and phosphatidylserine, but have little or no action on triglyceride or cholesterol ester. An assay system for the enzyme is described. Phospholipase A₂ activity is optimal at pH 5·5 in rat brain extracts and at pH 5·0 in extracts of human brain. The phospholipase A₂ activity of human cerebral cortex is largely unaffected by heating extracts at 70°C for 5 min, whereas this treatment substantially inactivates phospholipase A₁ and completely destroys lysophospholipase. Phospholipase A₁ is widely distributed in both grey and white matter of human brain and is also present in peripheral nerve. Phospholipase A₂ activity is lower than A₁ in all regions of the CNS examined so far, and is absent from peripheral nerve. Neither enzyme appears to require Ca²⁺ but both are inhibited by di-isopropylfluorophosphate (DFP, 2 × 10^?6 m) and thus differ from phospholipase A of pancreas. These studies confirm that the phospholipase A₁ and A₂ activities in brain are due to separate enzymes.     

Mitochondrial Aconitase is a Transglutaminase 2 Substrate: Transglutamination is a Probable Mechanism Contributing to High-Molecular-Weight Aggregates of Aconitase and Loss of Aconitase Activity in Huntington Disease Brain

Transglutaminase activity was found to be present in highly purified non-synaptosomal rat brain mitochondria. A 78-kDa protein in these organelles was shown to be a transglutaminase 2 substrate, and incubation of a non-synaptosomal mitochondrial lysate with transglutaminase 2 yielded high-M_r proteins. The 78-kDa protein was identified as mitochondrial aconitase by MALDI-TOF analysis. Aconitase activity was decreased in a dose-dependent manner when non-synaptosomal rat brain mitochondria were incubated with transglutaminase 2. Transglutaminase activity is increased about 2-fold in the mitochondrial fraction of HD caudate. Moreover, Western blotting of the mitochondrial fraction revealed that most of the mitochondrial aconitase in HD caudate is present as high-M_r aggregates. Aconitase activity was previously shown to be decreased in Huntington disease (HD) caudate (a region severely damaged by the disease). The present findings suggest that an increase of transglutaminase activity in HD caudate may contribute to mitochondrial dysfunction by incorporating aconitase into inactive polymers.     

Activites of Enzymes Synthesizing Diacyl, Alkylacyl, and Alkenylacyl Glycerophosphoethanolamine and Glycerophosphocholine During Development of Chicken Brain

Abstract: Ethanolamine and choline glycerophospholipids are the major phospholipids of brain membranes. During brain development, the accumulation of these phospholipids is most intense when myelination occurs. In order to gain knowledge about the regulatory mechanisms for synthesis of these lipids in relation to membrane synthesis, we investigated the activities of the 1,2-diradyl-sn-gIycerol: CDPethanolamine phosphoethanolarnine transferase and 1,2-diradyl-sri-glycerol:CDPcholine phosphocholine transferase during chicken brain development. Diacyl, alkenylacyl, and alkylacylglycerols are substrates for both enzymes. The specific activities of microsomal phospho-ethanolamine and phosphocholine transferases are constant between the 8th and 18th day of embryonic life. The specific activities of both enzymes double around hatching, which is the period of intense myelination and marked ac-cumulation of ethanolamine and choline glycerophospholipids in brain. At the same time, the amount of microsomes increases by 50%; thus the total activities increase threefold. Four days after hatching the specific activities of both enzymes are at adult values. Similar results were obtained in the presence of exogenous diacyl or alkylacylglycerols. During brain development the apparent K_m, value of rnicrosomal phosphoethanolamine transferase for CDP ethanolamine increases when assayed with diaclyglycerols or alkylacyl-glycerols a s lipid substrates. The apparent K_m, value of phosphocholine trans-ferase for CDP choline does not change during brain development in the presence of exogenous diacylglycerols, but increases in the presence of exogenous alkylacylglycerols. These changes in K_m, values may be due to the appearance of glial isoenzyme at the beginning of myelination. The apparent K_m, values of diacylglycerol phosphocholine, alklyacylglycerol phosphocholine, and diacyl-glycerol phosphoethanolamine transferases for their CDP bases are similar in adult brain microsomes and are threefold higher than the apparent K_m, value of alkylacylglycerolphosphoethanolamine transferase. The high affinity of alkylacylglycerolphosphoethanolamine transferase for CDPethanolamine may be responsible for the preferential synthesis of ethanolamine plasmalogens in brain.