Lipase-catalyzed regioselective synthesis of steryl (6′-<Emphasis Type="Italic">O</Emphasis>-acyl)glucosides

The regioselective acylation of cholesteryl β-d-glucoside, at the C-6 of the glucose moiety, was achieved using microbial lipases in organic solvents. With palmitic acid as an acyl donor 81 or 63% conversions of cholesteryl glucoside to its 6′-O-palmitoyl derivative were obtained using Candida antarctica or Rhizomucor miehei enzymes, respectively. High yields (64–92%) were also obtained with fatty acids 6:0–22:0 and 16:1 (n-7). The synthesis of cholesteryl (6′-O-palmitoyl)glucoside was also achieved via transesterification, using mono-, di- and tri-palmitoylglycerols or methyl and ethyl palmitate as acyl sources. With R. miehei lipase transesterification between methyl palmitate (80 mM) and cholesteryl glucoside (1 mM) proceeded after 24 h with a nearly quantitative yield (97%).     

Product solubility control in cellooligosaccharide production by coupled cellobiose and cellodextrin phosphorylase

Soluble cellodextrins (linear β-1,4-d -gluco-oligosaccharides) have interesting applications as ingredients for human and animal nutrition. Their bottom-up synthesis from glucose is promising for bulk production, but to ensure a completely water-soluble product via degree of polymerization (DP) control (DP ≤ 6) is challenging. Here, we show biocatalytic production of cellodextrins with DP centered at 3 to 6 (~96 wt.% of total product) using coupled cellobiose and cellodextrin phosphorylase. The cascade reaction, wherein glucose was elongated sequentially from α-d -glucose 1-phosphate (αGlc1-P), required optimization and control at two main points. First, kinetic and thermodynamic restrictions upon αGlc1-P utilization (200 mM; 45°C, pH 7.0) were effectively overcome (53% → ≥90% conversion after 10 hrs of reaction) by in situ removal of the phosphate released via precipitation with Mg²⁺. Second, the product DP was controlled by the molar ratio of glucose/αGlc1-P (∼0.25; 50 mM glucose) used in the reaction. In optimized conversion, soluble cellodextrins in a total product concentration of 36 g/L were obtained through efficient utilization of the substrates used (glucose: 98%; αGlc1-P: ∼80%) after 1 hr of reaction. We also showed that, by keeping the glucose concentration low (i.e., 1–10 mM; 200 mM αGlc1-P), the reaction was shifted completely towards insoluble product formation (DP ∼9–10). In summary, this study provides the basis for an efficient and product DP-controlled biocatalytic synthesis of cellodextrins from expedient substrates.     

Biosynthesis of a water solubility-enhanced succinyl glucoside derivative of luteolin and its neuroprotective effect

The natural flavonoids luteolin and luteoloside have anti-bacterial, anti-inflammatory, anti-oxidant, anti-tumour, hypolipidemic, cholesterol lowering and neuroprotective effects, but their poor water solubility limits their application in industrial production and the pharmaceutical industry. In this study, luteolin-7-O-β-(6″-O-succinyl)-d -glucoside, a new compound that was prepared by succinyl glycosylation of luteolin by the organic solvent tolerant bacterium Bacillus amyloliquefaciens FJ18 in an 8.0% DMSO (v/v) system, was obtained and identified. Its greater water solubility (2293 times that of luteolin and 12 232 times that of luteoloside) provides the solution to the application problems of luteolin and luteoloside. The conversion rate of luteolin (1.0 g l⁻¹) was almost 100% at 24 h, while the yield of luteolin-7-O-β-(6″-O-succinyl)-d -glucoside reached 76.2%. In experiments involving the oxygen glucose deprivation/reoxygenation injury model of mouse hippocampal neuron cells, the cell viability was significantly improved with luteolin-7-O-β-(6″-O-succinyl)-d -glucoside dosing, and the expressions of the anti-oxidant enzyme HO-1 in the nucleus increased, providing a neuroprotective effect for ischemic cerebral cells. The availability of biosynthetic luteolin-7-O-β-(6″-O-succinyl)-d -glucoside, which is expected to replace luteolin and luteoloside, would effectively expand the clinical application value of luteolin derivatives.     

Fatty acid synthesis in isolated leucoplasts of developing seeds of <Emphasis Type="Italic">Brassica campestris</Emphasis> L

Under in vitro conditions, the fatty acid synthesis from labelled substrates was studied in the leucoplasts isolated from developing seeds of Brassica campestris L. The rate of fatty acid synthesis with Na-(1-¹⁴C) acetate was higher at lower concentrations (up to 1 mM). However, with ¹⁴C(U)-D-glucose, the rate was higher at higher concentrations (3–4 mM) at all the three stages of seed development. ATP and NAD(P)H were absolutely required in acetate utilization. Even for glucose utilization, the exogenous supply of ATP and NAD(P)H was required. At the early stage of seed development, the maximum reduction in labelled glucose and acetate utilization for fatty acid synthesis was observed with pyruvate and glucose, respectively. However, at mid-early and mid-late stages, maximum reduction in their utilization for fatty acid synthesis was observed with glc-6-P. This suggests a shift in the utilization of substrates for fatty acid synthesis during the development of seeds probably via different translocators activated at different stages.     

NONDIALYZABLE SULFATED SIALOGLYCOPEPTIDE FRACTIONS DERIVED FROM BOVINE HEIFER BRAIN GLYCOPROTEINS. ISOLATION,CHARACTERIZATTON AND CARBOHYDRATE-PEPTIDE LINKAGE STUDIES1

The nondialyzable sialoglycopeptides dcrived from defatted, protease digested whole boyine heifer brain tissue were isolated and characterized. The partially purified bovine heifer brain tissue were isolated and characterized. The partially purified sialoglycopeptide mixtures contained 7-8% ester sulfate. The amount of nonsulfated material present was of the order of 15%. The total mixture was fractionated by anion exchange chromatography on AG1-X2(CI^-) using a sodium chloride gradient to yield twelve fractions all of which contained ester sulfate to varying degrees (5.6-21.9%). The molecular size of the fractions ranged from molecular weights of 10,400-12,500. The molar sulfate/galactose plus glucosamine ratios ranged from 0.3 to 1.1. Carbohydrate peptide linkage studies of the fractions by treatment with 0.2 M-NaOH-O.3 M-NaBH₄ led to the destruction of a portion of the threonine, serine and galactosamine and the appearance in acid hydrolysates of α-amino-n-butyric acid and galactosaminitol with an increase in alanine. Partial acid hydrolysis of the most abundant sialoglycopeptide liberated detectable amounts of 2-acetarnido-1-(L-4-aspartamido-)-1,2-dideoxy-β-D- glucose. These results indicated that the GalNac at the reducing end of the alkali labile carbohydrate prosthetic groups were linked O-glycosidically to thc hydroxyl groups of threonine and serine and the alkali-stable region of the principal sialoglycopeptide involves N-acetylglycosaminyl-asparagine linkages. Mild acid hydrolysis of the alkali-stable portion of the sulfated fractions indicated that the ester sulfate was confined to the alkali-stable region and exists as galactose 6-O-sulfate and N-acetylglucosa- mine 6-O-sulfate, structural fcatures similar to those present in rat brain glycopcptides (Markgolis & Margolis , 1970; Margolis et al.; 1972). Fucose and ester sulfate were confined to the alkali-stable region while mannose was located in both the alkali-labilc and alkali-stable regions of the sialoglyco-peptide fractions.     

Production of cellulases and xylanases under catabolic repression conditions from mutant PR-22 of Cellulomonas flavigena

Derepressed mutant PR-22 was obtained by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) mutagenic treatment of Cellulomonas flavigena PN-120. This mutant improved its xylanolytic activity from 26.9 to 40 U mg⁻¹ and cellulolytic activity from 1.9 to 4 U mg⁻¹; this represented rates almost 2 and 1.5 times higher, respectively, compared to its parent strain growing in sugarcane bagasse. Either glucose or cellobiose was added to cultures of C. flavigena PN-120 and mutant PR-22 induced with sugarcane bagasse in batch culture. The inhibitory effect of glucose on xylanase activity was more noticeable for parent strain PN-120 than for mutant PR-22. When 20 mM glucose was added, the xylanolytic activity decreased 41% compared to the culture grown without glucose in mutant PR-22, whereas in the PN-120 strain the xylanolytic activity decreased by 49% at the same conditions compared to its own control. Addition of 10 and 15 mM of glucose did not adversely affect CMCase activity in PR-22, but glucose at 20 mM inhibited the enzymatic activity by 28%. The CMCase activity of the PN-120 strain was more sensitive to glucose than PR-22, with a reduction of CMCase activity in the range of 20–32%. Cellobiose had a more significant effect on xylanase and CMCase activities than glucose did in the mutant PR-22 and parent strain. Nevertheless, the activities under both conditions were always higher in the mutant PR-22 than in the PN-120 strain. Enzymatic saccharification experiments showed that it is possible to accumulate up to 10 g l⁻¹ of total soluble sugars from pretreated sugarcane bagasse with the concentrated enzymatic crude extract from mutant PR-22.     

The effects of phenylpyruvate and hyperphenylalaninemia on incorporation of (6- 3 H)glucose into macromolecules of slices of rat cerebral cortex

The effects of phenylpyruvate and hyperphenylalaninemia on the incorporation of [6-³H]glucose into lipids, proteins and nucleic acids were examined in differentiating and adult rat brain. Foetal brain was most sensitive to inhibition by phenylpyruvate in vitro, with significant effects occurring at 2·5 mM for labelling of lipids and proteins and at 5 mM for labelling RNA and DNA. Older age groups were less affected, and cortical slices from adult brain were slightly or not at all affected by phenylpyruvate. The inhibition by phenylpyruvate of incorporation of [6-³H]glucose into nucleic acids, proteins, and lipids could be further distinguished by the reversibility of the effect on nucleic acid and protein synthesis at high levels of glucose and the irreversibility of the effect on lipid synthesis. Lipid synthesis was most sensitive to inhibition by phenylpyruvate at the stage of fatty acid synthesis, with lesser effect on the formation of glyceride glycerol. Exposure in utero of the foetal brain to maternal hyperphenylalaninemia resulted in reduction of 26–38 per cent in the subsequent incorporation in vitro of [6-³H]glucose into lipids, proteins, RNA and DNA of brain slices from foetal animals. Feeding hyperphenylalaninemic pregnant rats a high-glucose diet significantly protected the foetal brain from the neurotoxicity accompanying the hyperphenylalanemia.     

Controlling selectivity and enhancing yield of flavonoid glycosides in recombinant yeast

Flavonoid glycosides are known for their medicinal properties and potential use as natural sweeteners. In this study, Saccharomyces cerevisiae expressing a flavonoid glucosyltransferase from Dianthus caryophyllus was used as a whole-cell biocatalyst. The yeast system’s performance was characterized using the flavanone naringenin as a model substrate for the production of naringenin glycosides. It was found that final naringenin glycoside yields increased in a dose-dependent manner with increasing initial naringenin substrate concentrations. However, naringenin concentrations >0.5 mM did not give further enhancements in glycoside yield. In addition, a method for controlling overall selectivity was discovered where the glucose content in the culture medium could be altered to control the selectivity, making either naringenin-7-O-glucoside (N7O) or naringenin-4′-O-glucoside (N4O) the major products. The highest yields achieved were 87 mg/L of N7O and 82 mg/L of N4O using 40MSGI and 2xMSGI media, respectively. The effects of two intermediates involved in UDP-glucose biosynthesis, uridine 5′-monophosphate (UMP) and orotic acid, on glycoside yields were also determined. Addition of UMP to the culture medium significantly decreased glycoside yield. In contrast, addition of orotic acid to the culture medium significantly enhanced the glycoside yield and shifted the selectivity toward N7O. The highest naringenin glycoside yield achieved using 10 mM orotic acid in the 40MSGI media was 155 mg/L, a 71% conversion of substrate to product.     

Glycosaminoglycans from earthworms (<Emphasis Type="Italic">Eisenia andrei</Emphasis>)

The whole tissue of the earthworm (Eisenia andrei) was lyophilized and extracted to purify glycosaminoglycans. Fractions, eluting from an anion-exchange column at 1.0 M and 2.0 M NaCl, showed the presence of acidic polysaccharides on agarose gel electrophoresis. Monosaccharide compositional analysis showed that galactose and glucose were most abundant monosaccharides in both fractions. Depolymerization of the polysaccharide mixture with glycosaminoglycan-degrading enzymes confirmed the presence of chondroitin sulfate/dermatan sulfate and heparan sulfate in the 2.0 M NaCl fraction. The content of GAGs (uronic acid containing polysaccharide) in the 2.0 M NaCl fraction determined by carbazole assay was 2%. Disaccharide compositional analysis using liquid chromatography–electrospray ionization mass spectrometry (LC–ESI–MS) analysis after chondroitinase digestion (ABC and ACII), showed that the chondroitin sulfate/dermatan sulfate contained a 4-O-sulfo (76%), 2,4-di-O-sulfo (15%), 6-O-sulfo (6%), and unsulfated (4%) uronic acid linked N-acetylgalactosamine residues. LC–ESI–MS analysis of heparin lyase I/II/III digests demonstrated the presence of N-sulfo (69%), N-sulfo-6-O-sulfo (25%) and 2-O-sulfo-N-sulfo-6-O-sulfo (5%) uronic acid linked N-acetylglucosamine residues.     

Isolation and properties of two sialate-<Emphasis Type="Italic">O</Emphasis>-acetylesterases from horse liver with 4- and 9-<Emphasis Type="Italic">O</Emphasis>-acetyl specificities

Sialate-O-acetylesterase was purified almost 900-fold from particle-free supernatants of horse liver by gel filtration, ion-exchange chromatography and isoelectric focussing. The native enzyme on gel filtration exhibits a molecular weight of 54,000 Da. It was separated by isoelectric focussing into two forms with pI values of 4.8 and 5.7, respectively. The esterase with a lower pI hydrolyses only 9-O-acetyl groups from sialic acids (K_M 1.1 mM), while that with the higher pI esterifies both 4- and 9-O-acetylated monosaccharides at similar rates (K_M 0.3 M and 1.3 mM, respectively). Both forms are inactive with 7-O-acetylated N-acetylneuraminic acid. Enzyme assays were carried out at the pH optimum (pH 8.4–8.6) using free O-acetylated sialic acids followed by direct analysis of the reaction products by isocratic anion-exchange HPLC. Glycosidically bound sialic acids can also be de-O-acetylated. Horse liver esterase seems to be an essential enzyme for the catabolism of 4-O-acetylated sialoglycoconjugates, since sialidase from this tissue cannot act on 4-O-acetylated sialic acids.     

Permeabilization of <Emphasis Type="Italic">Microbacterium oxylans</Emphasis> shifts the conversion of puerarin from puerarin-7-<Emphasis Type="Italic">O</Emphasis>-glucoside to puerarin-7-<Emphasis Type="Italic">O</Emphasis>-fructoside

The main product of the conversion of puerarin by unpermeabilized cells of bacterium Microbacterium oxydans CGMCC 1788 was puerarin-7-O-glucoside (241 ± 31.9 μM). Permeabilization with 40% ethanol could not increase conversion yield, whereas it resulted in change of main product; a previous trace product became a main product (213 ± 48.0 μM) which was identified as a novel puerarin-7-O-fructoside by electrospray ionization time-of-flight MS, ¹³C NMR, ¹H NMR, and GC-MS analysis of sugar composition, and puerarin-7-O-glucoside became a trace product (14.8 ± 5.4 μM). However, the extract from cells of M. oxydans CGMCC 1788 permeabilized with ethanol converted puerarin to form 113.9 ± 27.7 μM puerarin-7-O-glucoside and 187.8 ± 29.5 μM puerarin-7-O-fructoside under the same conditions. When unpermeabilized intact cells were recovered and used repeatedly for the conversion of puerarin, with increase of reuse times, the yield of puerarin-7-O-glucoside gradually decreased, whereas the yield of puerarin-7-O-fructoside increased gradually in the conversion mixture. The main product of the conversion of puerarin by the tenth recycled unpremerbilized cells was puerarin-7-O-fructoside (288.4 ± 24.0 μM). Therefore, the change of permeability of cell membrane of bacterium M. oxydans CGMCC 1788 contributed to the change of conversion of the product’s composition.     

Regioselective synthesis of kojic acid esters by Bacillus subtilis protease

The lipophilicity of kojic acid [5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one] was improved by esterifying kojic acid with either divinyl adipate, vinyl hexanoate, vinyl octanoate or vinyl decanoate using protease from Bacillus subtilis for 7 d. ¹H-NMR and ¹³C-NMR showed that the primary hydroxyl group at the C-7 position of kojic acid was regioselectively esterified to afford 7-O-vinyl adipoyl kojic acid, 7-O-hexanoyl kojic acid, 7-O-octanoyl kojic acid and 7-O-decanoyl kojic acid (13–27% yield). The kojic acid esters had radical scavenging activities, inhibited tyrosinase activity and was biodegradable.     

Cobalt-chelated magnetic particles for one-step purification and immobilization of His6-tagged Escherichia coli γ-glutamyltranspeptidase

Cobalt-chelated magnetic (Fe₃O₄-AA-ANTA-Co^{2 +}) particles were prepared and one-step purification and immobilization of His₆-tagged Escherichia coli γ-glutamyltranspeptidase (His₆-EcGGT) using these particles were evaluated. The optimal conditions for the adsorption of His₆-EcGGT to Fe₃O₄-AA-ANTA-Co^{2 +} particles were found to be 24.7 U g^–1 adsorbent, pH 6.5, 300 mM NaCl and 30 min incubation at 4°C, while the elution solution was optimized to be 50 mM phosphate buffer (pH 6.5) containing 150 mM imidazole and 300 mM NaCl. The immobilized His₆-EcGGT was recycled five times without significant loss of GGT activity. The average yield rate for the synthesis l-theanine from glutamine and ethylamine reached 56.7%. These results indicate that one-step affinity purification and immobilization of His₆-EcGGT by Fe₃O₄-AA-ANTA-Co^{2 +} particles might serve as an effective process for industrial application.     

Acidic heteroxylans from medicinal plants and their anti-ulcer activity

Xylans are the main hemicelluloses found in higher plants, and are often present in phytotherapic medicines. An acidic heteroxylan was obtained from Maytenus ilicifolia leaves by hot 10% aqueous KOH extraction. This was subjected to freeze-thawing process, giving insoluble and soluble fractions and the latter treated with Fehling solution. Its insoluble fraction (MI-HX) was further examined. The acidic heteroxylan gave xylose, galactose, glucose, and 4-O-methylglucuronic acid in a 76:6:9:9 molar ratio and methylation analyses and ¹³C NMR spectroscopy showed its main chain consists of 4-O-linked β-d-Xylp units. This polysaccharide and another acidic heteroxylan from Phyllanthus niruri had anti-ulcer activity and were able to reduce gastric lesions induced by ethanol by 65% and 78%, with ED₅₀ = 40.0 and 20.4 mg/kg, each respectively. These results suggest that this class of polysaccharide has a protective anti-ulcer effect, and that there is a relation between its chemical structure and biological function.     

The dipeptidyl carboxypeptidase of <Emphasis Type="Italic">Escherichia coli</Emphasis> novablue: overproduction and molecular characterization of the recombinant enzyme

To express Escherichia coli novablue dipeptidyl carboxypeptidase (EcDCP), the gene was amplified by PCR and cloned into the expression plasmid pQE-31 to yield pQE-EcDCP. His₆-tagged EcDCP (His₆-EcDCP) was over-expressed in E. coli M15 (pQE-EcDCP) as a soluble and active form under 0.05 mM IPTG induction at 26°C for 12 h. The recombinant enzyme was purified to homogeneity by Ni²⁺-NTA resin and had a molecular mass of approximately 75 kDa. The temperature and pH optima for His₆-EcDCP were 37°C and 7.0, respectively. In the presence of 200 mM NaCl, His₆-EcDCP was stimulated by 1.5 fold. The K _M and k _cat values of the enzyme for N-benzoyl-l-glycyl-l-histidyl-l-leucine were 1.83 mM and 168.3 s⁻¹, respectively. His₆-EcDCP activity was dramatically inhibited by 10 mM EDTA, 0.25 mM 1.10-phenanthroline, and 2.5 mM DEPC, but it was not affected by Ser, Asp, Lys, and Trp protease inhibitors. Analysis of His₆-EcDCP by circular dichroism revealed that the secondary structures of the enzyme in 30 mM universal buffer (pH 7.0) were 17% α-helix, 35% β-sheet and 47% random coil. Mid point of thermal transition was calculated to be 55°C for the recombinant enzyme.     

Characterization of cytosolic phosphoglucoisomerase from immature wheat (Triticum aestivum L.) endosperm

Phosphoglucoisomerase from cytosol of immature wheat endosperm was purified 650-fold by ammonium sulphate fractionation, isopropyl alcohol precipitation, DEAE-cellulose chromatography and gel filtration through Sepharose CL-6B. The enzyme, with a molecular weight of about 130,000, exhibited maximum activity at pH 8.1. It showed typical hyperbolic kinetics with both fructose 6-P and glucose 6-P withK _m of 0.18 mM and 0.44mM respectively. On either side of the optimum pH, the enzyme had lower affinity for the substrates. Using glucose 6-P as the substrate, the equilibrium was reached at 27% fructose 6-P and 73% glucose 6-P with an equilibrium constant of 2.7. The ΔF calculated from the apparent equilibrium constant was +597 cal mol^-1. The activation energy calculated from the Arrhenius plot was 5500 cal mol^-1. The enzyme was completely inhibited by ribose 5-P, ribulose 5-P and 6-phosphogluconate, withK _i values of 0.17, 0.25 and 0.14 mM respectively. The probable role of the enzyme in starch biosynthesis is discussed.     

Sucrose and starch metabolism in carrot (Daucus carota L.) cell suspensions analysed by 13-labelling: indications for a cytosol and a plastid-localized oxidative pentose phosphate pathway

Cells were grown in batch culture on a mixture of 50 mM glucose and fructose as the carbon source; either the glucose or the fructose was [1-¹³C]-labelled. In order to investigate the uptake and conversion of glucose and fructose during long-term labelling experiments in cell suspensions of Daucus carota L., samples were taken every 2 d during a 2 week culture period and sucrose and starch were assayed by means of HPLC and ¹³C-nuclear magnetic resonance. The fructose moieties of sucrose had a lower labelling percentage than the glucose moieties. Oxidative pentose phosphate pathway activity in the cytosol is suggested to be responsible for this loss of label of especially C-1 carbons. A combination of oxidative pentose phosphate pathway activity, a relatively high activity of pathway to sucrose synthesis and a slow equilibration between glucose-6-phosphate and fructose-6-phosphate could explain these results. Starch contained glucose units with a much lower labelling percentage than glucose moieties of sucrose: it was concluded that a second, plastid-localized, oxidative pentose phosphate pathway was responsible for removal of C-1 carbons of the glucosyl units used for synthesis of starch. Redistribution of label from [1-¹³C]-hexoses to [6-¹³C]-hexoses also occurred: 18-45% of the label was found at the C-6 carbons. This is a consequence of cycling between hexose phosphates and those phosphates in the cytosol catalysed by PFP. The results indicate that independent (oxidative pentose phosphate pathway mediated) sugar converting cycles exist in the cytosol and plastid.Key words: Daucus carotaL., cell suspensions, carbon-13 nuclear magnetic resonance, ¹³C-NMR, carbohydrate cycling, oxidative pentose phosphate pathway, plastid.      

Gene cloning,expression, and characterization of a novel trehalose synthase from <Emphasis Type="Italic">Arthrobacter aurescens</Emphasis>

Trehalose synthase (TreS) is an intramolecular transglycosylase. It specially catalyzes the conversion of maltose and trehalose. In this study, a novel treS gene, which had a length of 1,797 bp and encoded 598 amino acids, was cloned from Arthrobacter aurescens CGMCC 1.1892 and expressed in Escherichia coli. Thin layer chromatography results indicated that it could catalyze the conversion between maltose and trehalose in one step. However, the ion chromatography results showed that, as a byproduct, about 13% glucose was also produced. The purified recombinant enzyme had a molecular weight of 68 kDa and showed its optimal activity at 35 °C and pH 6.5. This enzyme was not thermostable, and its activity was increased by 1 mM Mg²⁺, Mn²⁺, and Ca²⁺ while strongly inhibited by 5 mM Cu²⁺ and SDS.     

Contribution of Extracellular Glutamine as An Anaplerotic Substrate to Neuronal Metabolism: A Re-Evaluation by Multinuclear NMR Spectroscopy in Primary Cultured Neurons

Multinuclear NMR spectroscopy is used to investigate the effect of glutamine on neuronal glucose metabolism. Primary neurons were incubated with [1-¹³C]glucose in the absence or presence of glutamine (2 mM) and/or NH₄Cl (5 mM). After ammonia-treatment, the concentrations of high-energy phosphates decreased up to 84% of control, which was aggravated in glutamine-containing medium (up to 42% of control). These effects could not be attributed to changes in mitochondrial glucose oxidation. Withdrawal of glutamine decreased amino acid concentrations, e.g. of glutamate to 53%, but also considerably lessened the ¹³C enrichment in [4-¹³C]glutamate to 8.3% of control, and decreased the ¹³C-enrichment in acetyl-CoA entering the Krebs cycle (P<0.001). Thus, although glutamine is potent in replenishing neuronal glutamate stores, glutamate formation is mainly attributed to its de novo synthesis from glucose. Furthermore, mitochondrial glucose metabolism strongly depends on the supply of carbons from glutamine, indicating that exogenous glutamine is a well-suited substrate to replenish neuronal Krebs cycle intermediates.     

13C CP/MAS NMR Spectral Analysis of 6-O-Tosyl, 6-Deoxy-6-iodo,and 6-Deoxy Derivatives of N-Acetylchitosan in a Solid State

6-O-Tosyl (1, d.s. 0.94, 80% yield), 6-deoxy-6-iodo (2, d.s. 0.49, 86% yield) and 6-deoxy (3, d.s. 0.49, 50% yield) derivatives of N-acetylchitosan were prepared, and a ¹³C CP/MAS NMR spectral analysis was performed because no suitable solvent for 3 was found. The ¹³C signal for CH₃ at C-6 in 3 was detected at 18.9 ppm, and that for C-4 in 1–3 appeared at 72.2–72.7 ppm, which is in a higher magnetic field than those (82.5–86.0 ppm) in N-acetylchitosan, 6-O- (ethylthio), 6-O-(benzylthio)- and 6-O-(methylthio)-thiocarbonyl derivatives, chitosan, and chitin. This strongly suggests a different molecular conformation for 1–3.