Contents of sulfur amino acids, and cystathionine beta-synthase and gamma-lyase activities in various tissues from agkistroden blomhoffi (mamushi)

The concentrations of sulfur-containing amino acids, taurine, cystathionine, methionine and cystine, as well as cystathionine beta-synthase and gamma-lyase activities in various tissues of Agkistrodon blomhoffi (mamushi) were measured. The concentration of taurine in examined tissues was greater than the concentration of other sulfur-containing amino acids. The concentration of cystathionine in various tissues was also much higher than those of methionine and cystine, but the concentration of cystathionine in the brain was lower than that of methionine. In all tissues examined in this study, cystathionine beta-synthase activity was much higher than that of cystathionine gamma-lyase. The ratios of cystathionine beta-synthase to gamma-lyase activities in various tissues were 5.6 to approximately 85.6. The concentration of sulfur-containing amino acids in muscle and skin divided into eight portions of the body were also determined. The concentrations of methionine and cystine in each portion of muscle and skin were almost the same, but the concentrations of taurine and cystathionine in each portion of the body were varied.     

Cystathionine in rat brain: Catabolism in vivo

The content of cystathionine was measured in 35 rat brains; the range was 10–120 nmol/g wet weight and thus the variability of cystathionine content in rat brain was emphasized. The regional distribution of cystathionine was also determined: the highest level was found in cerebellum; the lowest level was observed in the white and gray matter of the hemispheres. These results are different from those obtained in other species. The radioactive metabolites formed froml-[³⁵S]cystathionine injected intracisternally were measured in brains of rats killed at the following times after injection: 0.25, 1, 2, 4, 6, 9, 16, and 27 hr. The radioactivity was found both in the proteins and in the acid-soluble fraction. In the acid-soluble fraction the radioactivity was found in various ninhydrin-reacting compounds: (cysteic + cysteine sulfinic) acid, taurine, reduced and oxidized glutathione, cystine, cystathionine, and a compound tentatively identified as the mixed disulfide of cysteine and glutathione. The radioactivity of cystathionine decreased exponentially between the 1st and the 27th hour after injection and its half-life was estimated to be about 5 hr. The radioactivity in the other ninhydrin-reacting compounds increased until the 9th hour after injection, then decreased. Half of this radioactivity was present in reduced glutathione, the rest being shared equally between: (cysteic + cysteine sulfinic) acid, taurine, and the mixed disulfide. It is worthwhile to note that the radioactivity in the cystine fraction was always very low.     

Cystathionine Accumulation in Various Regions of Brain of DL-Propargylglycine-Treated Rats

The contents of cystathionine and taurine, as well as cystathionine beta-synthase activity in various regions of the brains of normal and DL-propargylglycine-treated rats, were measured. The content of cystathionine in each region of brain increased gradually from 0.5 mg to 20 mg/200 g body weight in relation to the dose of DL-propargylglycine. Cystathionine was found to be unevenly distributed in brains of both normal and DL-propargylglycine-treated rats. On the other hand, the activity of cystathionine beta-synthase was evenly distributed in various regions of normal rat brain, and was unaltered following treatment of rats with DL-propargylglycine. The concentration of taurine was similarly unaffected by DL-propargylglycine injection.     

REGIONAL DISTRIBUTION OF AMINO ACIDS IN HUMAN BRAIN OBTAINED AT AUTOPSY

Abstract— Contents (μmol/g wet wt.) of 35 free amino acids and related compounds were measured in 12 different regions of each of five human brains. Specimens were obtained at autopsy from patients who died suddenly without previous brain disease. These data may serve for later comparison with contents of amino compounds in similar regions of the brains of patients dying with various neurological or psychiatric disorders.
There were marked and consistent differences in the regional distribution of the following eight compounds: γ-aminobutyric acid, homocarnosine, glutamic acid, aspartic acid, taurine, cystathionine, glycerophosphoethanolamine, and phosphoethanolamine. These differences suggest that some of these compounds may have special physiological roles, including the possible mediation of synaptic transmission.
Human brain contains two previously unreported compounds, the mixed disulphide of cysteine and glutathione and α-(γ-aminobutyryl)-lysine. The latter dipeptide occurs in much higher concentrations in human brain than in the brains of lower mammals.     

FREE AMINO ACIDS AND RELATED COMPOUNDS IN BIOPSIES OF HUMAN BRAIN

Abstract— Contents (μmol/g wet wt.) of 35 free amino acids and related compounds were measured in biopsies of human brain from ten patients. Brain specimens were frozen in liquid nitrogen within 10 sec of their removal at neurosurgery; thus, the values found should approximate those which occur in living brain.
Levels in free pools of biopsied cerebral cortex of most of the amino acids that are constituents of proteins were only 20-50 per cent of those found in autopsied cortex. The content of cystine and ethanolamine was much lower in biopsied than in autopsied cortex. Concentrations of GABA in biopsied cortex were only 20 per cent as high as those found in autopsied cortex, and levels of γ-aminobutyryl dipeptides were also significantly lower in biopsied cortex. Amounts of cystathionine in biopsied cortex varied markedly, but averaged much higher than in autopsied cortex; a single biopsy specimen of cerebellar grey matter had a cystathionine content 36-fold greater than the mean found in autopsied cerebellum.
Appreciable variability in contents among cortical biopsies was found for glycerophosphoethanolamine, phosphoethanolamine, ethanolamine, taurine, aspartic acid, glutamic acid, glutamine, and GABA, as well as for cystathionine. Whether this variability occurred between different subjects, or between different cortical areas, was not clear, although the former possibility was suggested by findings in multiple cortical biopsies from one patient.     

METHYLATED AMINO ACID RESIDUES OF PROTEINS OF BRAIN AND OTHER ORGANS

—Methods for the determination of methyl-lysine, methyllarginine and methylhistidine residues of tissue proteins are described. They consist of preliminary purification of basic amino acids, enzymic removal of lysine, arginine and histidine followed by amino acid analysis. Recovery rates and specificities of the method were satisfactory. The contents of methylamino acids in proteins of mammalian organs were determined. The distribution of proteins containing the methylamino acids in human brain showed that the concentrations of methyl-lysine and N^G,N′^G-dimethylarginine were highest in the gray matter of the cerebellar cortex and relatively high in regions rich in gray matter, while those of N^G-mono- and N^G,N′^G-dimethylarginine were highest in the white matter. The following findings suggest that most of the N^G-mono- and N^G,N′^G-dimethylarginine was associated with the myelin basic protein. The distribution of the methylarginine residues of acid-soluble proteins in bovine brains coincided with the cerebroside pattern. The concentrations of the amino acids in acid-soluble proteins of rat brain increased concomitantly with the increase of cerebroside. The methylamino acid content in proteins increased during the purification of the myelin basic protein from the white matter of human and bovine brains. Proteins containing N^G,N^G-dimethyiarginine and di- and trimethyl-lysine are concentrated in cell nuclei. The first amino acid was found mainly in nucleoplasmic proteins and the other two were found in histones. The concentration of 3-methylhistidine residue, highest in muscular proteins, is low in cerebral proteins and is probably derived from proteins of walls of blood vessels in the brain.     

Sequence of cDNA for rat cystathionine gamma-lyase and comparison of deduced amino acid sequence with related Escherichia coli enzymes.

A cDNA clone for cystathionine gamma-lyase was isolated from a rat cDNA library in lambda gt11 by screening with a monospecific antiserum. The identity of this clone, containing 600 bp proximal to the 3'-end of the gene, was confirmed by positive hybridization selection. Northern-blot hybridization showed the expected higher abundance of the corresponding mRNA in liver than in brain. Two further cDNA clones from a plasmid pcD library were isolated by colony hybridization with the first clone and were found to contain inserts of 1600 and 1850 bp. One of these was confirmed as encoding cystathionine gamma-lyase by hybridization with two independent pools of oligodeoxynucleotides corresponding to partial amino acid sequence information for cystathionine gamma-lyase. The other clone (estimated to represent all but 8% of the 5'-end of the mRNA) was sequenced and its deduced amino acid sequence showed similarity to those of the Escherichia coli enzymes cystathionine beta-lyase and cystathionine gamma-synthase throughout its length, especially to that of the latter.     

Lipids and proteins in multiple sclerosis white matter

Abstract— Quantitative analyses of white matter from four brains of patients with multiple sclerosis (MS) and four control brains were carried out for total and soluble proteins, individual lipid fractions, and their corresponding fatty acids. In three specimens from two of the MS brains there were reductions of cerebrosides and of the C_20:1 acid in the ethanolamine glycerophosphatide (EGP) fraction and a slight increase of tetraenes and trienes, while all other components were present in concentrations similar to those in the controls. In three other samples from two of the MS brains, galactolipids were deficient to a greater extent than cholesterol, EGP or CGP (choline glycerophosphatide), while proteins were within the control range. In samples where thinning of myelin was observed in Luxol-blue stained sections, there were proportional decreases of all components. The percentage of C_20:1 acid in the EGP fraction was reduced in two of three myelin preparations from corresponding samples of MS white matter, and that of C_24:1 acid in the cerebroside fraction was reduced in all three MS myelin preparations when compared with the two controls. The data suggest that inadequacy of the fatty acid elongation process together with deficits of cerebrosides represent one of the early biochemical lesions in the white matter of the MS brain.     

General Control Nonderepressible 2 (GCN2) Kinase Protects Oligodendrocytes and White Matter during Branched-chain Amino Acid Deficiency in Mice

Branched-chain amino acid (BCAA) catabolism is regulated by branched-chain α-keto acid dehydrogenase, an enzyme complex that is inhibited when phosphorylated by its kinase (BDK). Loss of BDK function in mice and humans causes BCAA deficiency and epilepsy with autistic features. In response to amino acid deficiency, phosphorylation of eukaryotic initiation factor 2α (eIF2∼P) by general control nonderepressible 2 (GCN2) activates the amino acid stress response. We hypothesized that GCN2 functions to protect the brain during chronic BCAA deficiency. To test this idea, we generated mice lacking both Gcn2 and Bdk (GBDK) and examined the development of progeny. GBDK mice appeared normal at birth, but they soon stopped growing, developed severe ataxia, tremor, and anorexia, and died by postnatal day 15. BCAA levels in brain were diminished in both Bdk^−/− and GBDK pups. Brains from Bdk^−/− pups exhibited robust eIF2∼P and amino acid stress response induction, whereas these responses were absent in GBDK mouse brains. Instead, myelin deficiency and diminished expression of myelin basic protein were noted in GBDK brains. Genetic markers of oligodendrocytes and astrocytes were also reduced in GBDK brains in association with apoptotic cell death in white matter regions of the brain. GBDK brains further demonstrated reduced Sod2 and Cat mRNA and increased Tnfα mRNA expression. The data are consistent with the idea that loss of GCN2 during BCAA deficiency compromises glial cell defenses to oxidative and inflammatory stress. We conclude that GCN2 protects the brain from developing a lethal leukodystrophy in response to amino acid deficiencies.     

FREE AMINO ACIDS AND RELATED COMPOUNDS IN FIVE REGIONS OF BIOPSIED CAT BRAIN

Abstract— Contents (μmol/g wet wt.) of 34 free amino acids and related compounds were measured in grey matter from three areas of cerebral cortex, from the cerebellum, and from the caudate nucleus in unanaesthetized cats with classical cerveau isolé preparations. Brain specimens were frozen in liquid nitrogen within 10 s of removal; thus, the values found were expected to approximate those which occur in living cat brain. Levels of most of the compounds measured were lower than those previously reported for the cat. In the case of GABA, alanine, and ethanolamine, the lower values found seemed attributable to the rapid freezing of brain tissue, and may more closely approximate levels occurring in living cat brain. On the other hand, the relatively low levels of aspartic and glutamic acids found may have resulted from use of the cerveau isolé preparation. Little difference in levels of amino compounds was found among the three cerebral cortical areas examined. However, there were significant differences in the contents of a number of amino acids between cerebral cortex and the cerebellum or caudate nucleus. These differences resembled those previously observed in autopsied human brain. The content of GABA was two-fold higher in biopsied cat cortex than in biopsied human cortex, whereas the content of cystathionine was only 10 per cent of that in human cortex. Homocarnosine and α-(γ-aminobutyryl)-lysine, two GABA-containing dipeptides found in relatively large amounts in human brain, were not detectable in cat brain. Living cat brain contained two amino acids not previously reported for this species:putreanine and ɛ- N -methyllysine.     

TRANS-SULPHURATION IN PRIMATE BRAIN: REGIONAL DISTRIBUTION OF CYSTATHIONINE SYNTHASE, CYSTATHIONINE AND TAURINE IN THE BRAIN OF THE RHESUS MONKEY AT VARIOUS STAGES OF DEVELOPMENT

—The regional distributions of cystathionine synthase, cystathionine and taurine in the brain of the Rhesus monkey were determined at various stages of foetal and postnatal development. Activity of cystathionine synthase was highest in cerebellum, cortical grey areas and globus pallidus, and lowest in subcortical white matter and corpus callosum. There was no marked change in activity in any area during development from the first-trimester foetus to the juvenile animal. In the brain of the juvenile monkey concentrations of cystathionine were highest in subcortical white matter, corpus callosum, and globus pallidus, and lowest in cortical grey matter. There was a sharp increase in concentration between late foetal life and the first 2 weeks of postnatal life and a subsequent more gradual increase during the next 2 years. Concentrations of taurine were highest in lateral cerebellum and neostriatum and lowest in brain stem areas and spinal cord. During the first 6 months of postnatal life, there was a marked decrease in concentration as the brain matured. The regional distribution of cystathionine in brain suggests that this compound may be synthesized in the perikaryon of the nerve cell and transported down axons into white matter. The changes during development suggest the further possibility that cystathionine may have some relationship to myelin and/or myelination.     

Human brain GABA transaminase is immunologically distinct from those of other mammalian brains

Monoclonal antibodies (mAbs) to bovine brain γ-aminobutyric acid (GABA) transaminase were characterized by epitope mapping analysis, and used as probes to compare the epitopes of the enzymes from several mammalian brains including man. From the epitope mapping analysis, three subgroups of mAbs recognizing different peptide fragments were identified. In the immunoblots probed with the mAbs, only one out of the three subgroups of mAbs reacted with a protein band of 50 kDa from human brain; the two other mAbs failed to detect any signal on the blots. In contrast, all of the mAbs did recognize a GABA-T protein band on immunoblots of all other mammalian brains tested. The results suggest that human brain GABA transaminase is immunologically distinct from those of other mammalian brains.     

Studies on the neurochemical properties of cystathionine

Following the intracerebral administration of [³⁵S]cystathionine, the synaptosome fraction of rat brain was labelled, the greatest uptake of amino acid being associated with hypothalamus.The uptake of [³⁵S]cystathionine by synaptosome preparations isolated from different regions of brain, was typical of that exhibited by amino acids which are not neurotransmitters.Depolarization of the synaptic membrane had no effect on the efflux of [³⁵S]cystathionine from preloaded synaptosomes.The intracerebral administration of cystathionine resulted in an elevation of the levels of brain cyclic AMP, the effect being particularly evident in the cerebellum. Attempts to reproduce this effect in vitro were unsuccessful.     

Brain hydrogen sulfide is severely decreased in Alzheimer's disease

Although hydrogen sulfide (H2S) is generally thought of in terms of a poisonous gas, it is endogenously produced in the brain from cysteine by cystathionine beta-synthase (CBS). H2S functions as a neuromodulator as well as a smooth muscle relaxant. Here we show that the levels of H2S are severely decreased in the brains of Alzheimer's disease (AD) patients compared with the brains of the age matched normal individuals. In addition to H2S production CBS also catalyzes another metabolic pathway in which cystathionine is produced from the substrate homocysteine. Previous findings, which showed that S-adenosyl-l-methionine (SAM), a CBS activator, is much reduced in AD brain and that homocysteine accumulates in the serum of AD patients, were confirmed. These observations suggest that CBS activity is reduced in AD brains and the decrease in H2S may be involved in some aspects of the cognitive decline in AD.     

Metabolism of ethanol by human brain to fatty acid ethyl esters

Although the most prominent acute and chronic effect of alcohol ingestion in man is alteration of brain function, metabolism of ethanol by human brain has not been documented. This study was designed to detect and localize a new family of nonoxidative ethanol metabolites, fatty acid ethyl esters, in human brain and characterize their synthetic pathways. Fatty acid ethyl ester synthase activity was present in 10 different locations in human brain, with gray matter containing more activity than white matter (0.53 nmol of ethyl oleate/mg of protein/h and 0.25 nmol of ethyl oleate/mg of protein/h, respectively). Two forms of this synthase, present in cytosol or loosely bound to membrane fractions, were isolated from human gray and white matter and then partially purified by ion-exchange chromatography. Both were active at low ethanol concentrations easily attained in vivo in man. Importantly, fatty acid ethyl esters were also detected in brains of individuals dying while intoxicated; only small amounts were present in control subjects at autopsy. Thus, alcohol metabolism in human brain has been documented for the first time by identifying both fatty acid ethyl esters and their synthases in this important target-organ of alcohol abuse.     

THE INTRASPINAL DISTRIBUTION OF SOME DEPRESSANT AMINO ACIDS

Abstract— —Six known depressant amino acids were found in extracts of feline spinal grey matter: α-alanine, cystathionine, GABA, glycine, serine and taurine.
Of these, glycine has the distribution within the nervous system anticipated of a sub-stance whose main function is as a spinal inhibitory transmitter at strychnine-sensitive synapses, GABA being a likely transmitter candidate at any strychnine-insensitive spinal inhibitory synapses.
The intraspinal distributions of α-alanine, cystathionine, serine and taurine reveal little to indicate that a synaptic role is a major function of these amino acids.     

Evidence of Nitrosative Damage in the Brain White Matter of Patients with Multiple Sclerosis

Nitric oxide (NO) has been implicated in the pathophysiology of both experimental autoimmune encephalomyelitis and multiple sclerosis (MS). NO-mediated protein damage in MS appears to be confined to large plaques where 3-nitrotyrosine has been detected. To determine whether nitrosative damage takes place beyond visible MS plaques, the occurrence of various NO-triggered protein modifications in normal-appearing white matter (NAWM) of eight MS brains was assessed and compared to that in white matter (WM) of four control brains. As determined by amino acid analysis and western blotting, no evidence of tyrosine nitration was found in the MS samples studied, suggesting that they did not contain appreciable amounts of plaque-derived material. The amino acid composition of total myelin proteins and proteolipid protein (PLP) was also unaltered in the diseased tissue, as was the fatty acid composition of PLP. In addition, we detected no changes in the number of protein free thiols suggesting that oxidation do not occur to any appreciable extent. However, the levels of nitrite in MS-NAWM were higher than those in control WM, while in the MS-gray matter (GM) the concentration of this ion was unaltered. Furthermore, five of the MS samples analyzed, and the same as those with high levels of glial fibrilary acidic protein, showed increased amounts of protein nitrosothiols as determined by the biotin switch method. S-nitrosation of GM proteins was again normal. There was no indication of N-nitrosation of tryptophan and N-terminal amino groups in both control and MS tissue. Overall, the data suggests that WM, but not GM, from MS brains is subjected to considerable nitrosative stress. This is the first report to present direct evidence of increased protein S-nitrosation and nitrite content in the brain parenchyma of MS patients.     

Brain amino acid concentrations during diving and acid-base stress in turtles

To assess the role of brain amino acid neurotransmitters in the breath hold of diving animals, concentrations of free amino acids present in the brains of turtles immediately after 2 h of apneic diving (at 20 degrees C) were measured. Additionally, the same measurements were performed on four other groups of animals subjected to 2 h of hypercapnia (8% CO2 in air), anoxia (N2 breathing), anoxia plus hypercapnia (8% CO2-92% N2), or air breathing (control). Significant changes in the concentrations of the inhibitory amino acid neurotransmitters known to affect respiration [gamma-aminobutyric acid (GABA) and taurine] were seen. GABA increased significantly in those animals subjected to anoxia, whereas taurine decreased significantly in the diving animals and increased significantly in those subjected to anoxia plus hypercapnia. These results suggest that the attenuated central ventilatory drive during diving in these animals may be related to alterations in brain concentrations of GABA and taurine.     

Application of PIXE analysis to the study of the regional distribution of trace elements in normal human brain

A particle-induced X-ray emission (PIXE) analysis method is presented, which allows measurement of eight elements (i.e., K, Ca, Mn, Fe, Cu, Zn, Se, and Rb) in human brain samples of only a few mg dry weight. The precision and accuracy of the method were investigated by analyzing animal brain matter with both PIXE and instrumental neutron activation analysis (INAA). The method was applied to measure the 8 elements in 46 different regions of 3 human brains. The sections analyzed originated from either the left or the right cerebral hemisphere, brain stem, and cerebellum. For one of the brains, sections were also analyzed from 26 corresponding regions of both hemispheres. For all elements, similar concentrations were found in the corresponding areas of the left and right sides of the brain. The concentrations (in μg/g dry weight) of the elements K, Fe, Cu, Zn, Se, and Rb were consistently higher in cortical structures than in white matter. Deep nuclei and brain stem, which have a mixed composition, showed intermediate values for K, Zn, Se, and Rb. A hierarchical cluster analysis indicated that the various brain regions clustered into two large groups, one comprising gray and mixed matter regions and the other, white and mixed matter brain areas.     

CYSTATHIONINE SYNTHESIS AND DEGRADATION IN BRAIN, LIVER AND KIDNEY OF THE DEVELOPING MONKEY

Abstract— The concentration of cystathionine, along with the specific activities of the enzymes involved in its synthesis and degradation, cystathionine synthasc and cystathionase, respectively, have been measured in brain, liver and kidney of the developing Rhesus monkey from mid-gestation, through birth and neonatal life, to maturity. The concentration of cystathionine and the specific activity of cystathionine synthase are low in fetal brain. Both parameters increase slowly after birth and reach values found in adult brain at approx 3 months of postnatal age. The activity of cystathionase in brain is low throughout development.
Liver provides a direct contrast in that the concentration of cystathionine and the specific activity of cystathionine synthase are high in the fetus, decreasing rapidly after birth to values found in the adult by 2 weeks of postnatal age. Cystathionase activity is low in fetal liver and increases slowly after birth reaching values found in adult liver after 2–3 months. Kidney has no more than trace amounts of cystathionine throughout development, higher activity of cystathionine synthase in the fetus than in the adult and high, unchanged activity of cystathionase throughout the period of development studied.
These results indicate that the high concentrations of cystathionine found in primate brain are reached postnatally and suggest that this high concentration of cystathionine may be associated with the functioning of mature brain.