Effect of cadmium on enzymatic digestion and sugar transport in the small intestine of rabbit

Cadmium compounds are found widely in our environment: for example, in food, water, soil, and ambient air. The most important exposure route of animals to cadmium in the general environment is via oral exposure. In oral cadmium intoxication, the immediate target organ is the gastrointestinal tract. The aim of the present work was to determine how cadmium acts on the intestinal absorption of sugars and on the sucrase activity through rabbit jejunum, after in vitro administration and/or oral administration of CdCl₂ in drinking water. Results obtained show that cadmium decreasesD-galactose accumulation in the jejunum tissue. This effect seems to be the result of an action mainly located on Na⁺-dependent sugar transport of the mucosal border of the intestinal epithelium, because cadmium seemnnot to modify the sugar diffusion across the intestinal epithelium. Cadmium has also been shown to inhibit the (Na⁺-K⁺)-ATPase activity of the enterocyte, which might explain the inhibition of theD-galactose Na⁺-dependent transport. Nevertheless, a direct action of the cadmium molecule on the Na⁺-dependent carrier cannot be discarded. Cadmium altered the sucrose activity when it was administered in the drinking water for 4 d.     

Origin ofd-serine present in urine of mutant mice lackingd-amino-acid oxidase activity

Summary Urine of ddY/DAO mice lackingd-amino-acid oxidase contained 5.7 times more serine than that of normal ddY/DAO⁺ mice. Most of the serine wasd-isomer. The origin of this^d-serine was examined. Oral administration of 0.02% amoxicillin and 0.004% minocycline to the ddY/ DAO^- mice for 7 days did not reduce the urinaryd-serine, indicating that thed-serine was not of intestinal bacterial origin. When the mouse diet was changed to one with different compositions, the urinaryd-serine was considerably reduced. Furthermore, starvation of the ddY/DAO^- mice for 24 hours reduced the urinaryd-serine to 33% of the original level. These results indicate that most of the urinaryd-serine comes from the diet. However, the urine of the starved ddY/DAO^- mice still contained 4.6 times mored-serine than that of the ddY/DAO⁺ mice, suggesting a part of the D-serine have an endogenous origin.     

d-Aspartyl residue in a peptide can be liberated and metabolized by pig kidney enzymes

Summary The presence of an enzyme activity which hydrolyzes glycyl-d-aspartate was found in the homogenates of pig kidney cortex. The activity was inhibited by metal chelating agents and cilastatin, suggesting that the enzyme was a cilastatin-sensitive metallo-peptidase. Of the two hydrolysis products,d-aspartate was found to be less accumulated than glycine. The fate ofd-aspartate was, therefore, examined and the amino acid was found to be converted tol-aspartate,l-alanine and pyruvate, in the presence ofl-glutamate. Experiments with enzyme inhibitors suggested that the conversion involvedd-aspartate oxidase, aspartate aminotransferase and alanine aminotransferase as well as decarboxylation of oxaloacetate produced fromd-aspartate. All the results indicate that the enzymes in the pig kidney can liberate thed-aspartyl residue in the peptide and convert it to the compounds readily utilizable. The finding suggests a probable metabolic pathway of thed-aspartate-containing peptide.     

Biochemical and genetic characterization of l-glutamate transport and utilization in Salmonella typhimurium LT-2 mutants

Two systems for l-glutamate transport were found in Salmonella typhimurium LT-2 GltU⁺ (glutamate utilization) mutants. The first one is similar to the glt system previously described in Escherichia coli; by transductional analysis the structural gene, gltS, coding for the transport protein was located at minute 80 of the chromosome as part of the operon gltC-gltS, and its regulator, the gltR gene, near minute 90; the gltS gene product transports both l-glutamate and l-aspartate, is sodium independent, and is -hydroxyaspartate sensitive. The second transport system, whose structural gene was called gltF and is located at minute 0, was l-glutamate specific, sodium independent, and -methylglutamate sensitive. Two aspartase activities occurred in S. typhimurium LT-2: the first one was present only in the GltU⁺ mutants, had a pH 6.4 optimum, was essential for both l-glutamate and l-aspartate metabolism, and mapped at minute 94, close to the ampC gene. The second one had a pH 7.2 optimum, could be induced by several amino acids, and thus may have a general role in nitrogen metabolism.     

An expedient synthesis of l-ribulose and derivatives

A significant improvement in the production of l-ribulose from inexpensive and commercially available starting materials, l-arabinose and sodium aluminate, is demonstrated. This has facilitated expeditious access to gram-scale quantities of l-ribulofuranoside derivatives.     

d-Aminoaciduria in mutant mice lackingd-amino-acid oxidase activity

Summary Urine of mutant ddY/DAO^– mice lackingd-amino-acid oxidase activity contained more serine and proline than that of normal ddY/DAO⁺ mice.d-Amino-acid oxidase treatment of urinary amino acids decreased the serine and proline, suggesting that they containedd-isomers. An HPLC analysis confirmed the presence ofd-serine. Urinary serine and proline contents were not decreased when the ddY/DAO^– mice were fed a diet which did not contain supplementaryd-methionine or when they were given water containing antibiotics. These results suggest that thed-serine andd-proline do not derive from thed-methionine supplemented in the diet or from intestinal bacteria. In urine of the ddY/DAO^– mice, a substance which seemed to bed-methionine sulfoxide and/ord-methionine sulfone was present. It is probably a metabolite of thed-methionine supplemented in the diet. Thed-aminoaciduria in the mutant mice lackingd-amino-acid oxidase activity indicates that this enzyme is involved in the metabolism of thed-amino acids in normal mice.     

Crystal structures of rare disaccharides, α-d-glucopyranosyl β-d-psicofuranoside, and α-d-galactopyranosyl β-d-psicofuranoside

The crystal structures of α-d-glucopyranosyl β-d-psicofuranoside and α-d-galactopyranosyl β-d-psicofuranoside were determined by a single-crystal X-ray diffraction analysis, refined to R₁ = 0.0307 and 0.0438, respectively. Both disaccharides have a similar molecular structure, in which psicofuranose rings adopt an intermediate form between ⁴E and ⁴T₃. Unique molecular packing of the disaccharides was found in crystals, with the molecules forming a layered structure stacked along the y-axis.     

Determination of [d-Ala, d-Leu]enkephalin and the metabolites containing C-terminal d-leucine by high-performance liquid chromatography

A high-performance liquid chromatographic procedure has been developed for the determination of [d-Ala², d-Leu⁵]enkephalin (DADLE) and the fragments containing d-leucine in rat blood. The procedure was applied to the determination of blood levels of [³H-d-Leu⁵]DADLE and the C-terminal fragments after intravenous administration of [³H-d-Leu⁵]DADLE to a rat. Unlabelled DADLE and the C-terminal fragments were spiked as carriers to rat blood samples and the blood samples were extracted with 1% trifluoroacetic acid in methanol. The recoveries from rat blood were quantitative for all compounds. DADLE and the C-terminal four fragments were well separated on a reversed-phase column with gradient elution using a mobile phase composed of 0.14% HClO₄ and acetonitrile.     

Characterization of <Emphasis Type="SmallCaps">d</Emphasis>-tagatose-3-epimerase from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> that converts <Emphasis Type="SmallCaps">d</Emphasis>-fructose into <Emphasis Type="SmallCaps">d-</Emphasis>psicose

A non-characterized gene, previously proposed as the d-tagatose-3-epimerase gene from Rhodobacter sphaeroides, was cloned and expressed in Escherichia coli. Its molecular mass was estimated to be 64 kDa with two identical subunits. The enzyme specificity was highest with d-fructose and decreased for other substrates in the order: d-tagatose, d-psicose, d-ribulose, d-xylulose and d-sorbose. Its activity was maximal at pH 9 and 40°C while being enhanced by Mn²⁺. At pH 9 and 40°C, 118 g d-psicose l⁻¹ was produced from 700 g d-fructose l⁻¹ after 3 h. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Antigen 85C-mediated acyl-transfer between synthetic acyl donors and fragments of the arabinan

Antigen 85 (ag85) is a complex of acyltransferases (ag85A–C) known to play a role in the mycolation of the d-arabino-d-galactan (AG) component of the mycobacterial cell wall. In order to better understand the chemistry and substrate specificity of ag85, a trehalose monomycolate mimic p-nitrophenyl 6-O-octanoyl-β-d-glucopyranoside (1) containing an octanoyl moiety in lieu of a mycolyl moiety was synthesized as an acyl donor. Arabinofuranoside acceptors, methyl α-d-arabinofuranoside (2), methyl β-d-arabinofuranoside (3), and methyl 2-O-β-d-arabinofuranosyl-α-d-arabinofuranoside (9) were synthesized to mimic the terminal saccharides found on the AG. The acyl transfer reaction between acyl donor 1 and acceptors 2, 3, and 9 in the presence of ag85C from Mycobacterium tuberculosis (M. tuberculosis) resulted in the formation of esters, methyl 2, 5-di-O-octanoyl-α-d-arabinofuranoside (10), methyl 5-O-octanoyl-β-d-arabinofuranoside (11), and methyl 2-O-(5-O-octanoyl-β-d-arabinofuranosyl)-5-O-octanoyl-α-d-arabinofuranoside (12) in 2 h, 2 h and 8 h, respectively. The initial velocities of the reactions were determined with a newly developed assay for acyltransferases. As expected, the regioselectivity corresponds to mycolylation patterns found at the terminus of the AG in M. tuberculosis. The study shows that d-arabinose-based derivatives are capable of acting as substrates for ag85C-mediated acyl-transfer and the acyl glycoside 1 can be used in lieu of TMM extracted from bacteria to study ag85-mediated acyl-transfer and inhibition leading to the better understanding of the ag85 protein class. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Physiological comparison of d-cysteine desulfhydrase of Escherichia coli with 3-chloro-d-alanine dehydrochlorinase of Pseudomonas putida CR 1-1

d-Cysteine desulfhydrase of Escherichia coli W3110 trpED102/F trpED102 was physiologically characterized. It was found to be located in the cytosolic fraction, as 3-chloro-d-alanine dehydrochlorinase is. d-Cysteine desulfhydrase catalyzed not only the ,-elimination reaction of O-acetyl-d-serine to form pyruvate, acetic acid and ammonia, but also the -replacement reaction of O-acetyl-d-serine with sulfide to form d-cysteine. However, these reactions appeared not to proceed in vivo. No other activity of d-cysteine synthesis from O-acetyl-d-serine and sulfide was detected in a crude cell extract of E. coli which was immunotitrated with antibodies raised against the purified d-cysteine desulfhydrase. Although d-cysteine desulfhydrase catalyzes the degradation (,-elimination reaction) of 3-chloro-d-alanine, which is an effective antibacterial agent, E. coli W3110 trpED102/F trpED102 did not show resistance against 3-chloro-d-alanine. Therefore, d-cysteine desulfhydrase does not contribute to 3-chloro-d-alanine detoxification in vivo.     

N-acetyl-l-glutamate in brain: Assay,levels, and regional and subcellular distribution

N-Acetyl-L-glutamate (NAG), the activator of mitochondrial carbamoyl phosphate synthetase (CPS), is demonstrated by several methods, including a new HPLC assay, in the brain of mammals and of chicken. The brain levels of NAG are 200–300 times lower than the levels of N-acetyl-l-aspartate (NAA), and are similar to the levels of NAG in rat liver. The NAG levels in chicken liver are very low. Although NAG is mitochondrial in the liver, it is cytosolic in brain. Using enzyme activity and immuno assays we did not detect CPS in brain (detection limit, 12.5 g/g brain), excluding that brain NAG is involved in citrullinogenesis. The regional distribution of brain NAG differs from that of NAA and resembles that of N-acetyl-l-aspartyl-l-glutamate (NAAG), suggesting that NAG and NAAG are related. NAG might be involved in the modulation of NAAG degradation.Special issue dedicated to Dr. Santiago Grisolía     

Purification, enzymatic properties of a recombinant d-hydantoinase and its dissociation by zinc ion

Summary A d-hydantoinase was expressed in the soluble form by a recombinant E. coli strain, pE-HDT/E. coli BL21 in LB medium. The enzymatic activity of cultured cells reached 5.2–6.5 IU/ml culture at a cell turbidity of 10 at 600 nm. The expressed enzyme was efficiently purified by three steps, ammonium sulfate fractionation, Phenyl-Sepharose hydrophobic interaction chromatography and Sephacryl S-200 size-exclusion chromatography. With the above purification process, the enzyme was purified to more than 95% purity as estimated by SDS-PAGE. The overall recovery of enzymatic activity was 54.4% and the specific activity for substrate dl-hydantoin achieved 48 U/mg. The purified enzyme appeared as a dimer with a molecular mass of 103 kDa, as measured by size-exclusion chromatography. The enzyme was stable from pH 6 to 12 with an optimum pH at 9.5 The optimum temperature of the enzyme was 45 °C and it activity was rapidly lost over 55 °C. Divalent metal ions, including Co²⁺, Mn²⁺ and Ni ²⁺ ions obviously enhanced the enzymatic activity, while Zn²⁺ ion had a slight inhibitory effect. In addition, the dissociation of purified enzyme into its subunits occurred in the presence of 1 mM Zn²⁺ ion. The effect of different metal ions on the d-hydantoinase activation/attenuation was discussed.     

Visible wavelength spectrophotometric assays of l-aspartate and d-aspartate using hyperthermophilic enzyme systems

Methods with which to simply and rapidly assay l-aspartate (l-Asp) and d-aspartate (d-Asp) would be highly useful for physiological research and for nutritional and clinical analyses. Levels of l- and d-Asp in food and cell extracts are currently determined using high-performance liquid chromatography. However, this method is time-consuming and expensive. Here we describe a simple and specific method for using an l-aspartate dehydrogenase (l-AspDH) system to colorimetrically assay l-Asp and a system of three hyperthermophilic enzymes—aspartate racemase (AspR), l-AspDH, and l-aspartate oxidase (l-AO)—to assay d-Asp. In the former, the reaction rate of nicotinamide adenine dinucleotide (NAD⁺)-dependent l-AspDH was measured based on increases in the absorbance at 438 nm, reflecting formation of formazan from water-soluble tetrazolium-1 (WST-1), using 1-methoxy-5-methylphenazinum methyl sulfate (mPMS) as a redox mediator. In the latter, d-Asp was measured after first removing l-Asp in the sample solution with l-AO. The remaining d-Asp was then changed to l-Asp using racemase, and the newly formed l-Asp was assayed calorimetrically using NAD⁺-dependent aspartate dehydrogenase as described above. This method enables simple and rapid spectrophotometric determination of 1 to 100 μM l- and d-Asp in the assay systems. In addition, methods were applicable to the l- and d-Asp determinations in some living cells and foods.     

Importance of the l-galactonolactone pool for enhancing the ascorbate content revealed by l -galactonolactone dehydrogenase-overexpressing tobacco plants

l-Galactono-1,4-lactone (GalL) dehydrogenase (GLDH) is an enzyme that catalyzes the last step of l-ascorbate (AsA) biosynthesis in plants. To re-evaluate the importance of the enzyme and the possibility of manipulating the AsA content in plants, a cDNA encoding GLDH from sweet potato was introduced into tobacco plants by Agrobacterium-mediated transformation under the control of a CaMV 35S promoter. Protein blot analysis revealed the elevation of GLDH protein contents in three GLDH-transformed lines. Furthermore, these transgenic lines showed 6- to 10-fold higher GLDH activities in the roots than the non-transformed plants, SR1. Despite the elevated GLDH activity, the AsA content in the leaves did not change in all lines; i.e., the AsA content in GLDH-transformed lines was 3–7 μmol g⁻¹ FW, comparable to that in the non-transformed plants. Incubation of leaf discs in a GalL solution led to a rapid 2- to 3-fold increase in the AsA content in both GLDH-transformed and non-transformed plants in the same manner. These results suggest that the supply of GalL is a crucial factor for determining the AsA pool size and that the upstream genes in the AsA biosynthetic pathway are responsible for enhancing the AsA content in plants.     

Development of a high-throughput method for the determination of pharmacological levels of plasma d-serine

d-Serine administration has been shown to be effective for the treatment of schizophrenia symptoms. However, d-serine must be administered at high doses to observe clinical effects. This is due in large part to d-serine undergoing oxidation by d-amino acid oxidase (DAAO) before it reaches the brain. Consequently, coadministration of d-serine with a DAAO inhibitor has been suggested as a way to lower the dose of d-serine required to treat schizophrenia. During the characterization of DAAO inhibitors as potential drugs, inhibitors are evaluated in rodents for their ability to increase plasma d-serine levels after oral coadministration. Current high-performance liquid chromatography (HPLC)-based methodologies to measure d-serine in plasma are time-consuming and are not amenable to concomitant analysis of multiple samples. We report the characterization of a 96-well format assay to monitor d-serine in plasma that greatly expedites analysis time. The assay involves the use of strong cation exchange solid phase extraction (SPE) to isolate d-serine from plasma followed by quantitation of d-serine using the DAAO-catalyzed reaction. Plasma d-serine determination using this assay could also be used as pharmacodynamic marker and as biomarker.     

Organization and characterization of three genes involved in d-xylose catabolism in Lactobacillus pentosus

Summary A cluster of three genes involved in d-xylose catabolism (viz. xylose genes) in Lactobacillus pentosus has been cloned in Escherichia coli and characterized by nucleotide sequence analysis. The deduced gene products show considerable sequence similarity to a repressor protein involved in the regulation of expression of xylose genes in Bacillus subtilis (58%), to E. coli and B. subtilis d-xylose isomerase (68% and 77%, respectively), and to E. coli d-xylulose kinase (58%). The cloned genes represent functional xylose genes since they are able to complement the inability of a L. casei strain to ferment d-xylose. NMR analysis confirmed that ¹³C-xylose was converted into ¹³C-acetate in L. casei cells transformed with L. pentosus xylose genes but not in untransformed L. casei cells. Comparison with the aligned amino acid sequences of d-xylose isomerases of different bacteria suggests that L. pentosus d-xylose isomerase belongs to the same similarity group as B. subtilis and E. coli d-xylose isomerase and not to a second similarity group comprising d-xylose isomerases of Streptomyces violaceoniger, Ampullariella sp. and Actinoplanes. The organization of the L. pentosus xylose genes, 5-xylR (1167 bp, repressor) — xylA (1350 bp, D-xylose isomerase) — xylB (1506 bp, d-xylulose kinase) — 3 is similar to that in B. subtilis. In contrast to B. subtilis xylR, L. pentosus xylR is transcribed in the same direction as xylA and xylB.     

Syntheses of dopa glycosides using glucosidases

Syntheses of l-dopa 1a glucoside 10a,b and dl-dopa 1b glycosides 10–18 with d-glucose 2, d-galactose 3, d-mannose 4, d-fructose 5, d-arabinose 6, lactose 7, d-sorbitol 8 and d-mannitol 9 were carried out using amyloglucosidase from Rhizopus mold, β-glucosidase isolated from sweet almond and immobilized β-glucosidase. Invariably, l-dopa and dl-dopa gave low to good yields of glycosides 10–18 at 12–49% range and only mono glycosylated products were detected through glycosylation/arylation at the third or fourth OH positions of l-dopa 1a and dl-dopa 1b. Amyloglucosidase showed selectivity with d-mannose 4 to give 4-O-C1β and d-sorbitol 8 to give 4-O-C6-O-arylated product. β-Glucosidase exhibited selectivity with d-mannose 4 to give 4-O-C1β and lactose 7 to give 4-O-C1β product. Immobilized β-glucosidase did not show any selectivity. Antioxidant and angiotensin converting enzyme inhibition (ACE) activities of the glycosides were evaluated glycosides, out of which l-3-hydroxy-4-O-(β-d-galactopyranosyl-(1′→4)β-d-glucopyranosyl) phenylalanine 16 at 0.9 ± 0.05 mM and dl-3-hydroxy-4-O-(β-d-glucopyranosyl) phenylalanine 11b,c at 0.98 ± 0.05 mM showed the best IC₅₀ values for antioxidant activity and dl-3-hydroxy-4-O-(6-d-sorbitol)phenylalanine 17 at 0.56 ± 0.03 mM, l-dopa-d-glucoside 10a,b at 1.1 ± 0.06 mM and dl-3-hydroxy-4-O-(d-glucopyranosyl)phenylalanine 11a-d at 1.2 ± 0.06 mM exhibited the best IC₅₀ values for ACE inhibition. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Glutamate and Schizophrenia: Beyond the Dopamine Hypothesis

1. After 50 years of antipsychotic drug development focused on the dopamine D2 receptor, schizophrenia remains a chronic, disabling disorder for most affected individuals.2. Studies over the last decade demonstrate that administration of low doses of NMDA receptor antagonists can cause in normal subjects the negative symptoms, cognitive impairments and physiologic disturbances observed in schizophrenia.3. Furthermore, a number of recently identified risk genes for schizophrenia affect NMDA receptor function or glutamatergic neurotransmission.4. Placebo-controlled trials with agents that directly or indirectly activate the glycine modulatory site on the NMDA receptor have shown reduction in negative symptoms, improvement in cognition and in some cases reduction in positive symptoms in schizophrenic patients receiving concurrent antipsychotic medications.5. Thus, hypofunction of the NMDA receptor, possibly on critical GABAergic inter-neurons, may contribute to the pathophysiology of schizophrenia.     

Fermentation of <Emphasis Type="SmallCaps">d</Emphasis>-glucose and <Emphasis Type="SmallCaps">d</Emphasis>-xylose mixtures by <Emphasis Type="Italic">Candida tropicalis</Emphasis> NBRC 0618 for xylitol production

The fermentation of d-glucose and d-xylose mixtures by the yeast Candida tropicalis NBRC 0618 has been studied under the most favourable operation conditions for the culture, determining the most adequate initial proportion in these sugars for xylitol production. In all the experiments a synthetic culture medium was used, with an initial total substrate concentration of 25 g L⁻¹, a constant pH of 5.0 and a temperature of 30 °C. From the experimental results, it was deduced that the highest values of specific rates of production and of overall yield in xylitol were achieved for the mixtures with the highest percentage of d-xylose, specifically in the culture with the initial d-glucose and d-xylose concentrations of 1 and 24 g L⁻¹, respectively, with an overall xylitol yield of 0.28 g g⁻¹. In addition, the specific rates of xylitol production declined over the time course of the culture and the formation of this bioproduct was favoured by the presence of small quantities of d-glucose. The sum of the overall yield values in xylitol and ethanol for all the experiments ranged from 0.26 to 0.56 g bioproduct/g total substrate.