Effect of In Vivo Administration of Naloxone on ATP-ase's Enzyme Systems of Synaptic Plasma Membranes from Rat Cerebral Cortex

Naloxone is a specific competitive antagonist of morphine, acting on opiate receptors, located on neuronal membranes. The effects of in vivo administration of naloxone on energy-consuming non-mitochondrial ATP-ases were studied in two different types of synaptic plasma membranes from rat cerebral cortex, known to contain a high density of opiate receptors. The enzyme activities of Na⁺, K⁺-ATP-ase, Ca²⁺, Mg²⁺-ATP-ase and Mg²⁺-ATP-ase and of acetylcholinesterase (AChE) were evaluated on synaptic plasma membranes obtained from control and treated animals with effective dose of naloxone (12g · kg^–1 i.m. 30 minutes). In control (vehicle-treated) animals specific enzyme activities assayed on these two types of synaptic plasma membranes are different, being higher on synaptic plasma membranes of II type than of I type, because the first fraction is more enriched in synaptic plasma membranes. The acute treatment with naloxone produced a significant decrease in Ca²⁺,Mg²⁺-ATP-ase activity and an increase in AChE activity, only in synaptic plasma membranes of II type. The decrease of Ca²⁺,Mg²⁺-ATP-ase enzymatic activity and the increased AChE activity are related to the interference of the drug on Ca²⁺ homeostasis in synaptosoplasm, that leads to the activation of calcium-dependent processes, i.e. the extrusion of neurotransmitter. These findings give further evidence that pharmacodynamic characteristics of naloxone are also related to increase [Ca²⁺] _i , interfering with enzyme systems (Ca²⁺,Mg²⁺-ATP-ase) and that this drug increases acetylcholine catabolism in synaptic plasma membranes of cerebral cortex.     

Biotechnological production of <Emphasis Type="SmallCaps">l</Emphasis>-ribose from <Emphasis Type="SmallCaps">l</Emphasis>-arabinose

l-Ribose is a rare and expensive sugar that can be used as a precursor for the production of l-nucleoside analogues, which are used as antiviral drugs. In this work, we describe a novel way of producing l-ribose from the readily available raw material l-arabinose. This was achieved by introducing l-ribose isomerase activity into l-ribulokinase-deficient Escherichia coli UP1110 and Lactobacillus plantarum BPT197 strains. The process for l-ribose production by resting cells was investigated. The initial l-ribose production rates at 39°C and pH 8 were 0.46 ± 0.01 g g⁻¹ h⁻¹ (1.84 ± 0.03 g l⁻¹ h⁻¹) and 0.27 ± 0.01 g g⁻¹ h⁻¹ (1.91 ± 0.1 g l⁻¹ h⁻¹) for E. coli and for L. plantarum, respectively. Conversions were around 20% at their highest in the experiments. Also partially purified protein precipitates having both l-arabinose isomerase and l-ribose isomerase activity were successfully used for converting l-arabinose to l-ribose.     

Spectroscopic,theoretical and structural characterization of hydrogensquarates of <Emphasis Type="SmallCaps">l</Emphasis>-threonyl-<Emphasis Type="SmallCaps">l</Emphasis>-serine and <Emphasis Type="SmallCaps">l</Emphasis>-serine

Summary. Hydrogensquarates of dipeptide l-threonyl-l-serine (H-Thr-Ser-OH) and l-serine (HSq × Ser) have been synthesized, isolated and spectroscopic characterized by solid-state linear-polarized IR-spectroscopy, ¹H- and ¹³C-NMR, ESI-MS and HPLC with tandem masspectrometry (MS-MS) methods. The structures of the salts and neutral dipeptide have been predicted theoretically by ab initio calculations. In the case of H-Thr-Ser-OH the theoretical data are supported by IR-LD ones. The hydrogensquarates consist in positive charged dipeptide or amino acid moiety and negative hydrogensquarate anion (HSq) stabilizing by strong intermolecular hydrogen bonds. The data about the l-serine hydrogensquarate are compared with known crystallographic data thus indicating a good correlation between the theoretical predicted structures and experimentally obtained by single crystal X-ray diffraction.     

An <Emphasis Type="SmallCaps">l</Emphasis>-Glutamine Transporter Isoform for Neurogenesis Facilitated by <Emphasis Type="SmallCaps">l</Emphasis>-Theanine

l-Theanine (=γ-glutamylethylamide) is an amino acid ingredient in green tea with a structural analogy to l-glutamine (l-GLN) rather than l-glutamic acid (l-GLU), with regards to the absence of a free carboxylic acid moiety from the gamma carbon position. l-theanine markedly inhibits [³H]l-GLN uptake without affecting [³H]l-GLU uptake in cultured neurons and astroglia. In neural progenitor cells with sustained exposure to l-theanine, upregulation of the l-GLN transporter isoform Slc38a1 expression and promotion of both proliferation and neuronal commitment are seen along with marked acceleration of the phosphorylation of mammalian target of rapamycin (mTOR) and relevant downstream proteins. Stable overexpression of Slc38a1 leads to promotion of cellular growth with facilitated neuronal commitment in pluripotent embryonic carcinoma P19 cells. In P19 cells stably overexpressing Slc38a1, marked phosphorylation is seen with mTOR and downstream proteins in a fashion insensitive to the additional stimulation by l-theanine. The green tea amino acid l-theanine could thus elicit pharmacological actions to up-regulate Slc38a1 expression for activation of the mTOR signaling pathway required for cell growth together with accelerated neurogenesis after sustained exposure in undifferentiated neural progenitor cells. In this review, I summarize a novel pharmacological property of the green tea amino acid l-theanine for embryonic and adult neurogenesis with a focus on the endogenous amino acid analog l-GLN. A possible translational strategy is also discussed on the development of dietary supplements and nutraceuticals enriched of l-theanine for the prophylaxis of a variety of untoward impairments and malfunctions seen in patients with different neurodegenerative and/or neuropsychiatric disorders.     

Removal of <Emphasis Type="SmallCaps">l</Emphasis>-alanine from the production of <Emphasis Type="SmallCaps">l</Emphasis>-2-aminobutyric acid by introduction of alanine racemase and <Emphasis Type="SmallCaps">d</Emphasis>-amino acid oxidase

l-2-Aminobutyric acid can be synthesized in a transamination reaction from l-threonine and l-aspartic acid as substrates by the action of threonine deaminase and aromatic aminotransferase, but the by-product l-alanine was produced simultaneously. A small amount of l-alanine increased the complexity of the l-2-aminobutyric acid recovery process because of their extreme similarity in physical and chemical properties. Acetolactate synthase has been introduced to remove the pyruvate intermediate for reducing the l-alanine concentration partially. To eliminate the remnant l-alanine, alanine racemase of Bacillus subtilis in combination with d-amino acid oxidase of Rhodotorula gracilis or Trigonopsis variabilis respectively was introduced into the reaction system for the l-2-aminobutyric acid synthesis. l-Alanine could be completely removed by the action of alanine racemase of B. subtilis and d-amino acid oxidase of R. gracilis; thereby, high-purity l-2-aminobutyric acid was achieved. The results revealed that alanine racemase could discriminate effectively between l-alanine and l-2-aminobutyric acid, and selectively catalyzed l-alanine to d-alanine reversibly. d-Amino acid oxidase then catalyzed d-alanine to pyruvate stereoselectively. Furthermore, this method was also successfully used to remove the by-product l-alanine in the production of other neutral amino acids such as l-tertiary leucine and l-valine, suggesting that multienzymatic whole-cell catalysis can be employed to provide high purity products.     

Substrate specificity of a peptidyl-aminoacyl-<Emphasis Type="SmallCaps">l</Emphasis>/<Emphasis Type="SmallCaps">d</Emphasis>-isomerase from frog skin

In the skin of fire-bellied toads (Bombina species), an aminoacyl-l/d-isomerase activity is present which catalyses the post-translational isomerization of the l- to the d-form of the second residue of its substrate peptides. Previously, this new type of enzyme was studied in some detail and genes potentially coding for similar polypeptides were found to exist in several vertebrate species including man. Here, we present our studies to the substrate specificity of this isomerase using fluorescence-labeled variants of the natural substrate bombinin H with different amino acids at positions 1, 2 or 3. Surprisingly, this enzyme has a rather low selectivity for residues at position 2 where the change of chirality at the alpha-carbon takes place. In contrast, a hydrophobic amino acid at position 1 and a small one at position 3 of the substrate are essential. Interestingly, some peptides containing a Phe at position 3 also were substrates. Furthermore, we investigated the role of the amino-terminus for substrate recognition. In view of the rather broad specificity of the frog isomerase, we made a databank search for potential substrates of such an enzyme. Indeed, numerous peptides of amphibia and mammals were found which fulfill the requirements determined in this study. Expression of isomerases with similar characteristics in other species can therefore be expected to catalyze the formation of peptides containing d-amino acids.     

The Roles of Striatal Serotonin and <Emphasis Type="SmallCaps">l</Emphasis>-Amino-acid Decarboxylase on <Emphasis Type="SmallCaps">l</Emphasis>-DOPA-induced Dyskinesia in a Hemiparkinsonian Rat Model

The administration of l-DOPA is the standard treatment for Parkinson’s disease (PD). However, the symptomatic relief provided by long-term administration may be compromised by l-DOPA-induced dyskinesia (LID) that presents as adverse fluctuations in motor responsiveness and progressive loss of motor control. In the later stages of PD, raphestriatal serotonin neurons compensate for the loss of nigrostriatal dopamine (DA) neurons by converting and releasing DA derived from exogenous l-DOPA. Since the serotonin system does not have an autoregulatory mechanism for DA, raphe-mediated striatal DA release may fluctuate dramatically and precede the development of LID. The 6-hydroxydopamine lesioned rats were treated with l-DOPA (6 mg/kg) and benserazide (15 mg/kg) daily for 3 weeks to allow for the development of abnormal involuntary movement score (AIMs). In rats with LID, chronic treatment with l-DOPA increased striatal DA levels compared with control rats. We also observed a relative increase in the expression of striatal l-amino-acid decarboxylase (AADC) in LID rats, even though tyrosine hydroxylase (TH) expression did not increase. The administration of l-DOPA also increased striatal serotonin immunoreactivity in LID rats compared to control rats. Striatal DA and 5-hydroxytryptamine (5-HT) levels were negatively correlated in l-DOPA-treated rats. These results of this study reveal that 5-HT contributes to LID. Striatal DA positively influences LID, while 5-HT is negatively associated with LID. Finally, we suggest that by strategic modification of the serotonin system it may be possible to attenuate the adverse effects of chronic l-DOPA therapy in PD patients.     

Metabolic engineering of <Emphasis Type="Italic">Escherichia coli</Emphasis> for improving <Emphasis Type="SmallCaps">l</Emphasis>-3,4-dihydroxyphenylalanine (<Emphasis Type="SmallCaps">l</Emphasis>-DOPA) synthesis from glucose

l-3,4-dihydroxyphenylalanine (l-DOPA) is an aromatic compound employed for the treatment of Parkinson's disease. Metabolic engineering was applied to generate Escherichia coli strains for the production of l-DOPA from glucose by modifying the phosphoenolpyruvate:sugar phosphotransferase system (PTS) and aromatic biosynthetic pathways. Carbon flow was directed to the biosynthesis of l-tyrosine (l-Tyr), an l-DOPA precursor, by transforming strains with compatible plasmids carrying genes encoding a feedback-inhibition resistant version of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase, transketolase, the chorismate mutase domain from chorismate mutase-prephenate dehydratase from E. coli and cyclohexadienyl dehydrogenase from Zymomonas mobilis. The effects on l-Tyr production of PTS inactivation (PTS⁻ gluc⁺ phenotype), as well as inactivation of the regulatory protein TyrR, were evaluated. PTS inactivation caused a threefold increase in the specific rate of l-Tyr production (q _l-Tyr), whereas inactivation of TyrR caused 1.7- and 1.9-fold increases in q _l-Tyr in the PTS⁺ and the PTS⁻ gluc⁺ strains, respectively. An 8.6-fold increase in l-Tyr yield from glucose was observed in the PTS⁻ gluc⁺ tyrR ⁻ strain. Expression of hpaBC genes encoding the enzyme 4-hydroxyphenylacetate 3-hydroxylase from E. coli W in the strains modified for l-Tyr production caused the synthesis of l-DOPA. One of such strains, having the PTS⁻ gluc⁺ tyrR ⁻ phenotype, displayed the best production parameters in minimal medium, with a specific rate of l-DOPA production of 13.6 mg/g/h, l-DOPA yield from glucose of 51.7 mg/g and a final l-DOPA titer of 320 mg/l. In a batch fermentor culture in rich medium this strain produced 1.51 g/l of l-DOPA in 50 h.     

Continuous 2-keto-<Emphasis Type="SmallCaps">l</Emphasis>-gulonic acid fermentation from <Emphasis Type="SmallCaps">l</Emphasis>-sorbose by <Emphasis Type="Italic">Ketogulonigenium vulgare</Emphasis> DSM 4025

A single-stage continuous fermentation process for the production of 2-keto-l-gulonic acid (2KGA) from l-sorbose using Ketogulonigenium vulgare DSM 4025 was developed. The chemostat culture with the dilution rate that was calculated based on the relationship between the 2KGA production rate and the 2KGA concentration was feasible for production with high concentration of 2KGA. In this system, 112.2 g/L of 2KGA on the average was continuously produced from 114 g/L of l-sorbose. A steady state of the fermentation was maintained for the duration of more than 110 h. The dilution rate was kept in the range of 0.035 and 0.043 h⁻¹, and the 2KGA productivity was 3.90 to 4.80 g/L/h. The average molar conversion yield of 2KGA from l-sorbose was 91.3%. Under the optimal conditions, l-sorbose concentration was kept at 0 g/L. Meanwhile, the dissolved oxygen level was changing in response to the dilution rate and 2KGA concentration. In the dissolved oxygen (DO) range of 16% to 58%, it was revealed that the relationship between DO and D possessed high degree of positive correlation under the l-sorbose limiting condition (complete consumption of l-sorbose). Increasing D closer to the critical value for washing out point of the continuous fermentation, DO value tended to be gradually increased up to 58%. In conclusion, an efficient and reproducible continuous fermentation process for 2KGA production by K. vulgare DSM 4025 could be developed using a medium containing baker’s yeast without using a second helper microorganism.     

Central <Emphasis Type="SmallCaps">l</Emphasis>-arginine reduced stress responses are mediated by <Emphasis Type="SmallCaps">l</Emphasis>-ornithine in neonatal chicks

Recently, we observed that central administration of L-arginine attenuated stress responses in neonatal chicks, but the contribution of nitric oxide (NO) to this response was minimal. The sedative and hypnotic effects of L-arginine may be due to L-arginine itself and/or its metabolites, excluding NO. To clarify the mechanism, the effect of intracerebroventricular (i.c.v.) injection of L-arginine metabolites on behavior under social separation stress was investigated. The i.c.v. injection of agmatine, a guanidino metabolite of L-arginine, had no effect during a 10 min behavioral test. In contrast, the i.c.v. injection of L-ornithine clearly attenuated the stress response in a dose-dependent manner, and induced sleep-like behavior. The L-ornithine concentration in the telencephalon and diencephalon increased following the i.c.v. injection of L-arginine. In addition, several free amino acids including L-alanine, glycine, L-proline and L-glutamic acid concentrations increased in the telencephalon. In conclusion, it appears that L-ornithine, produced by arginase from L-arginine in the brain, plays an important role in the sedative and hypnotic effects of L-arginine observed during a stress response. In addition, several other amino acids having a sedative effect might partly participate in the sedative and hypnotic effects of L-arginine.     

A mutant leucine aminopeptidase from <Emphasis Type="Italic">Streptomyces cinnamoneus</Emphasis> with enhanced <Emphasis Type="SmallCaps">l</Emphasis>-aspartyl <Emphasis Type="SmallCaps">l</Emphasis>-amino acid methyl ester synthetic activity

l-Aspartyl l-amino acid methyl ester was synthesized using a mutant of a thermostable leucine aminopeptidase from Streptomyces cinnamoneus, D198 K SSAP, obtained in previously. A peptide of high-intensity sweetener, l-aspartyl-l-phenylalanine methyl ester, was selected as a model for demonstrating the synthesis of l-aspartyl l-amino acid methyl ester. The hydrolytic activities of D198 K SSAP toward l-aspartyl-l-phenylalanine and its methyl ester were, respectively, 74-fold and fourfold higher than those of wild type. Similarly, the initial rate of the enzyme for l-aspartyl-l-phenylalanine methyl ester synthesis was over fivefold higher than that of wild-type SSAP in 90% methanol (v/v) in a one-pot reaction. Furthermore, other l-aspartyl l-amino acid methyl esters were synthesized efficiently using D198 K SSAP. Results show that the substitution of Asp198 of SSAP with Lys is effective for synthesizing l-aspartyl l-amino acid methyl ester.     

Purification and characterization of <Emphasis Type="SmallCaps">l</Emphasis>-arabinose isomerase from <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> producing <Emphasis Type="SmallCaps">d</Emphasis>-tagatose

l-arabinose isomerase (EC5.3.1.4. AI) mediates the isomerization of d-galactose into d-tagatose as well as the conversion of l-arabinose into l-ribulose. The AI from Lactobacillus plantarum SK-2 was purified to an apparent homogeneity giving a single band on SDS–PAGE with a molecular mass of 59.6 kDa. Optimum activity was observed at 50°C and pH 7.0. The enzyme was stable at 50°C for 2 h and held between pH 4.5 and 8.5 for 1 h. AI activity was stimulated by Mn²⁺, Fe³⁺, Fe²⁺, Ca²⁺ and inhibited by Cu²⁺, Ag⁺, Hg²⁺, Pb²⁺. d-galactose and l-arabinose as substrates were isomerized with high activity. l-arabitol was the strongest competitive inhibitor of AI. The apparent Michaelis–Menten constant (K _m), for galactose, was 119 mM. The first ten N-terminal amino acids of the enzyme were determined as MLSVPDYEFW, which is identical to L. plantarum (Q88S84). Using the purified AI, 390 mg tagatose could be converted from 1,000 mg galactose in 96 h, and this production corresponds to a 39% equilibrium.     

<Emphasis Type="SmallCaps">l</Emphasis>-Proline nutrition and catabolism in <Emphasis Type="Italic">Staphylococcus saprophyticus</Emphasis>

Staphylococcus saprophyticus strains ATCC 15305, ATCC 35552, and ATCC 49907 were found to require l-proline but not l-arginine for growth in a defined culture medium. All three strains could utilize l-ornithine as a proline source and contained l-ornithine aminotransferase and Δ¹-pyrroline-5-carboxylate reductase activities; strains ATCC 35552 and ATCC 49907 could use l-arginine as a proline source and had l-arginase activity. The proline requirement also could be met by l-prolinamide, l-proline methyl ester, and the dipeptides l-alanyl-l-proline and l-leucyl-l-proline. The bacteria exhibited l-proline degradative activity as measured by the formation of Δ¹-pyrroline-5-carboxylate. The specific activity of proline degradation was not affected by addition of l-proline or NaCl but was highest in strain ATCC 49907 after growth in Mueller–Hinton broth. A membrane fraction from this strain had l-proline dehydrogenase activity as detected both by reaction of Δ¹-pyrroline-5-carboxylate with 2-aminobenzaldehyde (0.79 nmol min⁻¹ mg⁻¹) and by the proline-dependent reduction of p-iodonitrotetrazolium (20.1 nmol min⁻¹ mg⁻¹). A soluble fraction from this strain had Δ¹-pyrroline-5-carboxylate dehydrogenase activity (88.8 nmol min⁻¹ mg⁻¹) as determined by the NAD⁺-dependent oxidation of dl-Δ¹-pyrroline-5-carboxylate. Addition of l-proline to several culture media did not increase the growth rate or final yield of bacteria but did stimulate growth during osmotic stress. When grown with l-ornithine as the proline source, S. saprophyticus was most susceptible to the proline analogues L-azetidine-2-carboylate, 3,4-dehydro-dl-proline, dl-thiazolidine-2-carboxylate, and l-thiazolidine-4-carboxylate. These results indicate that proline uptake and metabolism may be a potential target of antimicrobial therapy for this organism.     

A novel <Emphasis Type="SmallCaps">l</Emphasis>-aspartate dehydrogenase from the mesophilic bacterium <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PAO1: molecular characterization and application for <Emphasis Type="SmallCaps">l</Emphasis>-aspartate production

l-aspartate dehydrogenase (EC 1.4.1.21; l-AspDH) is a rare member of amino acid dehydrogenase superfamily and so far, two thermophilic enzymes have been reported. In our study, an ORF PA3505 encoding for a putative l-AspDH in the mesophilic bacterium Pseudomonas aeruginosa PAO1 was identified, cloned, and overexpressed in Escherichia coli. The homogeneously purified enzyme (PaeAspDH) was a dimeric protein with a molecular mass of about 28 kDa exhibiting a very high specific activity for l-aspartate (l-Asp) and oxaloacetate (OAA) of 127 and 147 U mg⁻¹, respectively. The enzyme was capable of utilizing both nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) as coenzyme. PaeAspDH showed a T _m value of 48°C for 20 min that was improved to approximately 60°C by the addition of 0.4 M NaCl or 30% glycerol. The apparent K _m values for OAA, NADH, and ammonia were 2.12, 0.045, and 10.1 mM, respectively; comparable results were observed with NADPH. The l-Asp production system B consisting of PaeAspDH, Bacillus subtilis malate dehydrogenase and E. coli fumarase, achieved a high level of l-Asp production (625 mM) from fumarate in fed-batch process with a molar conversion yield of 89.4%. Furthermore, the fermentative production system C released 33 mM of l-Asp after 50 h by using succinate as carbon source. This study represented an extensive characterization of the mesophilic AspDH and its potential applicability for efficient and attractive production of l-Asp. Our novel production systems are also hopeful for developing the new processes for other compounds production.     

Racemic Resolution of some <Emphasis Type="SmallCaps">dl</Emphasis>-Amino Acids using <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis><Emphasis Type="SmallCaps">l</Emphasis>-Amino Acid Oxidase

The ability of Aspergillus fumigatus l-amino acid oxidase (l-aao) to cause the resolution of racemic mixtures of dl-amino acids was investigated with dl-alanine, dl-phenylalanine, dl-tyrosine, and dl-aspartic acid. A chiral column, Crownpak CR+ was used for the analysis of the amino acids. The enzyme was able to cause the resolution of the three dl-amino acids resulting in the production of optically pure d-alanine (100% resolution), d-phenylalanine (80.2%), and d-tyrosine (84.1%), respectively. The optically pure d-amino acids have many uses and thus can be exploited industrially. This is the first report of the use of A. fumigatus l-amino acid oxidase for racemic resolution of dl-amino acids.     

Membrane-bound <Emphasis Type="SmallCaps">l</Emphasis>- and <Emphasis Type="SmallCaps">d</Emphasis>-lactate dehydrogenase activities of a newly isolated <Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis> strain

Pseudomonas stutzeri SDM was newly isolated from soil, and two stereospecific NAD-independent lactate dehydrogenase (iLDH) activities were detected in membrane of the cells cultured in a medium containing dl-lactate as the sole carbon source. Neither enzyme activities was constitutive, but both of them might be induced by either enantiomer of lactate. P. stutzeri SDM preferred to utilize lactate to growth, when both l-lactate and glucose were available, and the consumption of glucose was observed only after lactate had been exhausted. The Michaelis–Menten constant for l-lactate was higher than that for d-lactate. The l-iLDH activity was more stable at 55°C, while the d-iLDH activity was lost. Both enzymes exhibited different solubilization with different detergents and different oxidation rates with different electron acceptors. Combining activity staining and previous proteomic analysis, the results suggest that there are two separate enzymes in P. stutzeri SDM, which play an important role in converting lactate to pyruvate. Ma and Gao contributed equally to this work.     

Fungal arabinan and <Emphasis Type="SmallCaps">l</Emphasis>-arabinose metabolism

l-Arabinose is the second most abundant pentose beside d-xylose and is found in the plant polysaccharides, hemicellulose and pectin. The need to find renewable carbon and energy sources has accelerated research to investigate the potential of l-arabinose for the development and production of biofuels and other bioproducts. Fungi produce a number of extracellular arabinanases, including α-l-arabinofuranosidases and endo-arabinanases, to specifically release l-arabinose from the plant polymers. Following uptake of l-arabinose, its intracellular catabolism follows a four-step alternating reduction and oxidation path, which is concluded by a phosphorylation, resulting in d-xylulose 5-phosphate, an intermediate of the pentose phosphate pathway. The genes and encoding enzymes l-arabinose reductase, l-arabinitol dehydrogenase, l-xylulose reductase, xylitol dehydrogenase, and xylulokinase of this pathway were mainly characterized in the two biotechnological important fungi Aspergillus niger and Trichoderma reesei. Analysis of the components of the l-arabinose pathway revealed a number of specific adaptations in the enzymatic and regulatory machinery towards the utilization of l-arabinose. Further genetic and biochemical analysis provided evidence that l-arabinose and the interconnected d-xylose pathway are also involved in the oxidoreductive degradation of the hexose d-galactose.     

Biotransformation of <Emphasis Type="SmallCaps">l</Emphasis>-Selenomethionine and Selenite in Rat Gut Contents

l-Selenomethionine (SeMet) and sodium selenite are widely used selenium nutritional supplements with potential benefit in preventing cancer. However, supplementation is not without risks of toxicity if intake is too high. The aim of the present study was to investigate SeMet and selenite metabolism in the gastrointestinal tract with particular focus on the formation of the volatile selenium excretion products, dimethylselenide (DMSe) and dimethyldiselenide (DMDSe). Adult male Wistar rats (n = 5) were euthanized, their intestinal tracts removed and the contents of jejunum, ileum, caecum and colon used to prepare 10% suspensions in saline. SeMet and selenite (0.5–0.6 mM) were then incubated with these suspensions at 37°C for 3 h. Caecum and colon contents were the most metabolically active towards SeMet with 30% and 15% metabolized over 3 h. DMDSe was the only volatile selenium metabolite detected accounting for 8.7 ± 1.3% of the selenium lost in caecum contents. Selenite was completely metabolized by caecum contents and 73% by colon contents under the same conditions forming DMSe (5.7 ± 0.9% of the selenium lost in caecum) and a precipitate of red amorphous elemental selenium. Based on previous literature and these results, we conclude that the gut microbiota contributes to the excretion of excess selenium through the production of methylated selenium compounds and elemental selenium.     

Production of 3,4-dihydroxy phenyl-<Emphasis Type="SmallCaps">l</Emphasis>-alanine (<Emphasis Type="SmallCaps">l</Emphasis>-DOPA) by Egyptian halophilic black yeast

Production of 3,4-dihydroxy phenyl-l-alanine (l-DOPA) using an Egyptian isolate of halophilic black yeast was studied. Optimum aeration level and incubation period for high yield production of l-DOPA were 50 ml medium/250 ml flask with 200 rpm and 36 h, respectively. Two new techniques (addition of aniline or NaCl to the medium) have been investigated to enhance the monophenolase activity and inhibit or reduce diphenolase activity of tyrosinase to form high yield of l-DOPA without more oxidation to melanin. Addition of aniline to tyrosine medium at 3 μl/ml medium enhanced l-DOPA production 2.9 fold, however, addition of NaCl at 20% showed the same amount of l-DOPA as the control. Peptone and ram horn hydrolysate were studied as nitrogen sources instead of tyrosine in the medium and they showed good nitrogen sources for l-DOPA production as tyrosine. Finally, addition of aniline (3 μl/ml) to ram horn hydrolysate was economically feasible and cost effective for l-DOPA production by Egyptian halophilic black yeast.     

Crystal Structure of the Protein <Emphasis Type="SmallCaps">l</Emphasis>-Isoaspartyl Methyltransferase from <Emphasis Type="Italic">Escherichia coli</Emphasis>

Among the known covalent damages that can occur spontaneously to proteins, the formation of isoaspartyl linkages through deamidation of asparagines and isomerization of aspartates may be one of the most rapid forms under conditions of physiological pH and temperature. The protein l-isoaspartyl methyltransferase (PIMT) is thought to recognize l-isoaspartyl residues and repair this kind of damaged proteins. Curiously, there is a potential functional difference between bacterial and mammalian PIMTs. Herein, we present the crystal structure of Escherichia coli PIMT (EcPIMT) at a resolution of 1.8 Å. The enzyme we investigated was able to remain bound to its product S-adenosylhomocysteine (SAH) during crystallization. Analysis indicates that the high affinity of EcPIMT for SAH might lead to the lower activity of the enzyme.