Glycine,Serine, and Leucine Metabolism in Different Regions of Rat Central Nervous System

We have investigated the glycine, serine and leucine metabolism in slices of various rat brain regions of 14-day-old or adult rats, using [1-¹⁴C]glycine, [2-¹⁴C]glycine, L-[3-¹⁴C]serine and L-[U-¹⁴C]leucine. We showed that the [1-¹⁴C]glycine oxidation to CO₂ in all regions studied occurs almost exclusively through its cleavage system (GCS) in brains of both 14-day-old and adults rats. In 14-day-old rats, the highest oxidation of [1-¹⁴C]glycine was in cerebellum and the lowest in medulla oblongata. In these animals, the L-[U-¹⁴C]leucine oxidation was lower than the [1-¹⁴C]glycine oxidation, except in medulla oblongata where both oxidations were the same. Serine was the amino acid that showed lowest oxidation to CO₂ in all structure studied. In adult rats brains, the highest oxidation of [1-¹⁴C]glycine was in cerebral cortex and the lowest in medulla oblongata. We have not seen difference in the lipid synthesis from both glycine labeled, neither in 14-day-old rats nor in adult ones, indicating that the lipids formed from glycine were not neutral. Lipid synthesis from serine was significantly high than lipid synthesis and from all other amino acids studied in all studied structures. Protein synthesis from L-[U-¹⁴C]leucine was significantly higher than that from glycine in all regions and ages studied.     

Nitroxyl anion regulation of the NMDA receptor

Nitric oxide (NO) is an important regulator of NMDA channel function in the CNS. Recent findings suggest that nitroxyl anion (NO(-)) may also be generated by nitric oxide synthase, which catalyzes production of NO. Using recombinant NMDA receptors (NMDA-r) transfected into human embryonic kidney cells, our data demonstrate that the nitroxyl anion donor, Angeli's salt (AS; Na(2)N(2)O(3)) dramatically blocked glycine-independent desensitization in NMDA-r containing NR1-NR2A subunits. AS did not affect glycine-dependent desensitization, calcium dependent inactivation or glutamate affinity for the NMDA-r. This effect could be mimicked by treatment with DPTA, a metal chelator and was not evident under hypoxic conditions. In contrast, receptors containing the NR1-NR2B subunits demonstrated an approximate 25% reduction in whole cell currents in the presence of AS with no apparent change in desensitization. Our data suggest that the regulation of NMDA-r function by nitroxyl anion is distinctly different from NO and may result in different cellular outcomes compared with NO.     

Shoot inversion-induced ethylene in pharbitis nil induces the release of apical dominance by restricting shoot elongation

Shoot inversion induces outgrowth of the highest lateral bud (HLB) adjacent to the bend in the stem in Pharbitis nil. In order to determine whether or not ethylene produced by shoot inversion plays a direct role in promoting or inhibiting bud outgrowth, comparisons were made of endogenous levels of ethylene in the HLB and HLB node of plants with and without inverted shoots. That no changes were found suggests that the control of apical dominance does not involve the direct action of ethylene. This conclusion is further supported by evidence that the direct application of ethylene inhibitors or ethrel to inactive or induced lateral buds has no significant effect on bud outgrowth. The hypothesis that ethylene evolved during shoot inversion indirectly promotes the outgrowth of the highest lateral bud (HLB) by restricting terminal bud (TB) growth is found to be supported by the following observations: (1) the restriction of TB growth appears to occur before the beginning of HLB outgrowth; (2) the treatment of the inverted portion of the shoot with AgNO₃, an inhibitor of ethylene action, dramatically eliminates both the restriction of TB growth and the promotion of HLB outgrowth which usually accompany shoot inversion; and (3) the treatment of the upper shoot of an upright plant with ethrel mimics shoot inversion by retarding upper shoot growth and inducing outgrowth of the lateral bud basipetal to the treated region.     

Generation of one-carbon units in methionine-supplemented Euglena cells

The possible effect of L-methionine supplements on the folate metabolism of division-synchronized Euglena gracilis (strain Z) cells has been examined. Cells receiving 1 mM L-methionine for four cell cycles were examined for folate derivatives, prior to and during cell division. Before cell division, methionine-supplemented cells contained less formylfolate but more methylfolate than unsupplemented cells. During division, both types of folates were present in lower concentrations in the supplemented cells. Growth in methionine for 10 and 34 hr also increased the levels of free aspartate, threonine, serine, cysteine and methionine relative to the controls. Methionine-supplemented cells contained ca 50% of the 10-formyltetrahydrofolate synthetase (EC 6.3.4.3) activity per cell of unsupplemented control cultures and specific enzyme activity was reduced ca 90%. Supplemented cells contained almost twice as much serine hydroxymethyltransferase (EC 2.1.2.1) activity per cell but comparable levels of glycollate dehydrogenase. Growth in methionine also reduced the incorporation of formate-¹⁴C] into serine, RNA, DNA, adenine and protein methionine. In contrast, incorporation of glycine-[2-¹⁴C] and serine-[3-¹⁴C] into folate-related products was not greatly altered by this treatment. Levels of radioactivity in these products suggested that formate was a more important C₁ unit source than glycine or serine when growth occurred in unsupplemented medium. It is concluded that methionine reduces formylfolate production by an effect on the cellular levels of formyltetrahydrofolate synthetase.     

Algal metallothioneins: secondary metabolites and proteins

Metallothioneins, MT's, are low-molecular-weight, cysteine-rich, polypeptides that complex ‘soft’ metal ions in thiol clusters. They are structurally diverse. Some MT's are gene products, while others are secondary metabolites. Two of the three classes of MT have been identified in algae. Eukaryotic algae possess the secondary metabolites referred to as class III MT. There is no unequivocal evidence that MT genes occur in eukaryotic algae. However, the products of MT genes have been identified in cyanobacteria. These genes and their metal regulatory elements remain to be isolated and characterized. MT's have attracted interest from researchers involved in a wide range of disciplines including bioinorganic chemistry, biochemistry, molecular biology, physiology, toxicology, environmental science and medicine. Although, the precise physiological roles of these polypeptides remain undefined, a large number of functions have been speculated. These molecules chelate toxic trace metals, such as Cd, thereby reducing the concentration of cytotoxic, free-metal ions. Furthermore, some MT's are believed to be involved in zinc and copper homoeostasis. Future studies should reveal whether or not some of the diversity of MT structure reflects a diversity of function.     

Reduced Glycine Stimulation of [3H]MK-801 Binding in Alzheimer's Disease

The novel N-methyl-D-aspartate receptor channel ligand (+)-[3H]5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5, 10-imine maleate ([3H]MK-801) has been utilized to label this receptor in human brain tissue. Characteristics of [3H]MK-801 binding to well-washed membranes from 17 control subjects and 16 patients with Alzheimer's disease were determined in frontal, parietal, and temporal cerebral cortex and cerebellar cortex. In control tissue the pharmacological specificity of the binding of this substance is entirely consistent with the profile previously reported for rat brain. Binding could be stimulated by the addition of glutamic acid to the incubation medium; addition of glycine produced further enhancement which was not prevented by strychnine. The specificity of the effects of these and other amino acids on the binding was the same as in the rat. In Alzheimer's disease significantly less binding was observed in the frontal cortex under glutamate- and glycine-stimulated conditions. This appears to be associated with a reduced affinity of the site whereas the pharmacological specificity of the site remained unchanged. The effect did not appear to be due to differences in mode of death between Alzheimer's disease and control subjects and is unlikely to be related to factors for which the groups were matched. In contrast, binding was not altered in the absence of added amino acids and presence of glutamate alone. These results imply that in the cerebral cortex the agonist site and a site in the cation channel of the receptor are not selectively altered, but that their coupling to a strychnine-insensitive glycine recognition site is impaired.     

Comparative biochemical and immunological studies of the glycine betaine synthesis pathway in diverse families of dicotyledons

Members of the Chenopodiaceae can accumulate high levels (>100 mol·(g DW)^-1) of glycine betaine (betaine) in leaves when salinized. Chenopodiaceae synthesize betaine by a two-step oxidation of choline (cholinebetaine aldehyde betaine), with the second step catalyzed by betaine aldehyde dehydrogenase (BADH, EC 1.2.1.8). High betaine levels have also been reported in leaves of species from several distantly-related families of dicotyledons, raising the question of whether the same betaine-synthesis pathway is used in all cases.Fast atom bombardment mass spectrometry showed that betaine levels of >100 mol·(g DW)^-1 are present in Lycium ferocissimum Miers (Solanaceae), Helianthus annuus L. (Asteraceae), Convolvulus arvensis L. (Convolvulaceae), and Amaranthus caudatus L. (Amaranthaceae), that salinization promotes betaine accumulation in these plants, and that they can convert supplied choline to betaine aldehyde and betaine. Nicotiana tabacum L. and Lycopersicon lycopersicum (L.) Karst. ex Farw. (Solanaceae), Lactuca sativa L. (Asteraceae) and Ipomoea purpurea L. (Convolvulaceae) also contained betaine, but at a low level (0.1–0.5 mol·(g DW)^-1. Betaine aldehyde dehydrogenase activity assays, immunotitration and immunoblotting demonstrated that the betaine-accumulating species have a BADH enzyme recognized by antibodies raised against BADH from Spinacia oleracea L. (Chenopodiaceae), and that the M_r of the BADH monomer is in all cases close to 63 000. These data indicate that the cholinebetaine aldehydebetaine pathway may have evolved by vertical descent from an early angiosperm ancestor, and might be widespread (albeit not always strongly expressed) among flowering plants. Consistent with these suggestions, Magnolia x soulangiana was found to have a low level of betaine, and to express a protein of M_r 63 000 which cross-reacted with antibodies to BADH from Spinacia oleracea.Abbreviations BADH Betaine aldehyde dehydrogenase - DCIMS desorption chemical ionization mass spectrometry - FABMS fast atom bombardment mass spectrometry - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate - TLC thin-layer chromatography     

Effects of High-Potassium-Induced Depolarization on Amino Acid Chemistry of the Dorsal Cochlear Nucleus in Rat Brain Slices

High K⁺ was used to depolarize glia and neurons in order to study the effects on amino acid release from and concentrations within the dorsal cochlear nucleus (DCN) of brain slices. The release of glutamate, -aminobutyrate (GABA) and glycine increased significantly during exposure to 50 mM K⁺, while glutamine and serine release decreased significantly during and/or after exposure, respectively. After 10 min of exposure to 50 mM K⁺, glutamine concentrations increased in all three layers of DCN slices, to more than 5 times the values in unexposed slices. In the presence of a glutamate uptake blocker, L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC), glutamine concentrations in all layers did not increase as much during 50 mM K⁺. Similar but smaller changes occurred for serine. Mean ATP concentrations were lower in 50 mM K⁺-exposed slices compared to control. The results suggest that depolarization, such as during increased neural activity, can greatly affect amino acid metabolism in the cochlear nucleus.     

Effect of Salinity on Photosynthesis and Biochemical Characteristics in Mulberry Genotypes

Mulberry genotypes were subjected to salinity (0–12 mS cm^–1) in pot culture experiment. Chlorophyll and total carotenoid contents were reduced considerably by salinity. At low salinity, photosynthetic CO₂ uptake increased over the control, but it decreased at higher salinity. Contents of soluble proteins, free amino acids, soluble sugars, sucrose, starch, and phenols increased at salinity of 1–2 mS cm^–1 and decreased at higher salinity (8–12 mS cm^–1). Glycine betaine accumulated more than proline, the maximum accumulation of both was at salinity of 2–4 mS cm^–1. Among the genotypes studied, BC2-59 followed by S-30 showed better salinity tolerance than M-5.     

Formation of glycine conjugate and (-)-(R)-enantiomer from (+)-(S)-2-phenylpropionic acid suggesting the formation of the CoA thioester intermediate of (+)-(S)-enantiomer in dogs.

It has been proposed that the chiral inversion of the 2-arylpropionic acids is due to the stereospecific formation of the (-)-R-profenyl-CoA thioesters which are putative intermediates in the inversion. Accordingly, amino acid conjugation, for which the CoA thioesters are obligate intermediates, should be restricted to those optical forms which give rise to the (-)-R-profenyl-CoA, i.e., the racemates and the (-)-(R)-isomers. We have examined this problem in dogs with respect to 2-phenylpropionic acid(2-PPA). Regardless of the optical configuration of 2-phenylpropionic acid administered, the glycine conjugate was the major urinary metabolite and this was shown to be exclusively the (+)-(S)-enantiomer by chiral HPLC. Both (-)-(R)- and (+)-(S)-2-phenylpropionic acid were present in plasma after the administration of either antipode, and further evidence of the chiral inversion of both enantiomers was provided by the presence of some 25% of the opposite enantiomer in the free 2-phenylpropionic acid and its glucuronide excreted in urine after administration of (-)-(R)- and (+)-(S)-2-phenylpropionic acid. The (+)-(S)-enantiomer underwent chiral inversion to the (-)-(R)-antipode when incubated with dog hepatocytes. These data suggests that both enantiomers of 2-phenylpropionic acid are substrates for canine hepatic acyl CoA ligase(s) and thus undergo chiral inversion, but that the CoA thioester of only (+)-(S)-2-phenylpropionic acid is a substrate for the glycine N-acyl transferase. These studies are presently being extended to the structure and species specificity of the reverse inversion and amino acid conjugation of profen NSAIDs.