Effects of kyotorphin (l-tyrosyl-l-arginine) on [H]N-nitro-l-arginine binding to neuronal nitric oxide synthase in rat brain

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-Tyrosyl-

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-arginine (kyotorphin) is known as an endogenous analgesic neuropeptide. We examined whether kyotorphin and other arginine-containing neuropeptides were endogenous substrates for neuronal nitric oxide synthase (NOS) in the rat brain. Cytosol fractions of the rat cerebellum contained higher concentrations of neuronal NOS (nNOS) than endothelial NOS. In rat cerebellar cytosol, the binding activity of [³H]N^G-nitro-

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-arginine (NNA) was inhibited equally by

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-arginine (

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-Arg), kyotorphin, and

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-leucyl-

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-Arg (a kyotorphin receptor antagonist). Binding activities were inhibited to lesser degrees by fibronectin active fragments, bradykinin, and dynorphin A, but were not inhibited by

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-tyrosyl-

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-Arg or substance P. Interestingly, the inhibition of [³H]NNA binding by kyotorphin was attenuated by inhibitors of kyotorphin-hydrolyzing peptidases (KTPases) such as bestatin and arphamenine B. These results suggest that kyotorphin is degraded to

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-Arg by KTPases, which in turn may act as substrate for nNOS.     

Recognition of specific patterns of amino acid inhibition of growth in higher plants, uncomplicated by glutamine-reversible `general amino acid inhibition'

The complexity of the regulatory mechanisms that govern amino acid biosynthesis, particularly in multibranched pathways, frequently results in sensitivity to growth inhibition by exogenous amino acids. Usually the inhibition caused by a given amino acid(s) is relieved by another amino acid(s), thus indicating the cause of inhibition to be a specific interference with endogenous formation of the latter amino acid(s). We recently summarized the evidence that Nicotiana silvestris (and probably most higher plants), in suspension culture, exhibits a separate phenomenon of amino acid mediated growth inhibition called general amino acid inhibition. Every amino acid provokes general amino acid inhibition except for

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-glutamine. In fact,

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-glutamine completely overcomes general amino acid inhibition. We have now demonstrated that specific amino acid inhibition can be recognized and characterized at the level of growth inhibition without interference caused by general amino acid inhibition by the simple provision of exogenous

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-glutamine. Several examples of specific amino acid inhibition of growth were demonstrated in N. silvestris. In one case,

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-threonine inhibits growth partially in the presence of

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-glutamine. The residual amino acid inhibition was overcome by the additional presence of

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-lysine and

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-methionine, indicating that exogenous

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-threonine specifically inhibits the biosynthesis of both

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-lysine and

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-methionine. As a second example, the

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-valine-mediated inhibition of growth that persisted in the presence of

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-glutamine was overcome by

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-isoleucine, indicating that exogenous

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-valine inhibits

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-isoleucine biosynthesis. The use of amino acid analogs as experimental tools for biochemical-genetic studies in higher plants is also complicated by general amino acid inhibition. Conditions were demonstrated under which p-fluorophenylalanine and m-fluorotyrosine could be used as specific antimetabolites of

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-phenylalanine and

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-tyrosine biosynthesis without interference from general amino acid inhibition. We thus present a rigorous basis for recognition of specific relationships between metabolic branches that can guide detailed enzymological analyses.     

Enzymatic synthesis of mannobioses and mannotrioses by reverse hydrolysis using α-mannosidase from Aspergillus niger

Various manno-oligosaccharides including and

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were formed when a highly concentrated mannose solution was incubated in the presence of α-mannosidase from Aspergillus niger. and

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were isolated by activated carbon chromatography followed by high performance liquid chromatography using an amino-silica column. In addition to the above oligosaccharides,

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, and

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were also isolated.     

The glutathione dependence of inorganic sulfate formation from l- or d-cysteine in isolated rat hepatocytes

The GSH dependence of the metabolic pathways involved in the conversion of cysteine to sulfate in intact cells has been investigated. It was found that hepatocyte-catalysed sulfate formation from added

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-cysteine did not occur if hepatocyte GSH was depleted beforehand, but was restored when GSH levels recovered. Furthermore, sulfate formation did not recover in GSH-depleted hepatocytes if GSH synthesis was prevented with buthionine sulfoximine. Thiosulfate formation was, however, markedly enhanced in GSH-depleted hepatocytes. These results suggest that thiosulfate is an intermediate in the formation of inorganic sulfate from

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-cysteine and that GSH was required for the conversion of thiosulfate to inorganic sulfate. Much less sulfate was formed if the cysteine was replaced with cysteinesulfinate. Furthermore, sulfate formation from

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-cysteine was markedly inhibited by the addition of the transaminase inhibitor

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-cycloserine or the γ-cystathionase inhibitor

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-propargylglycine. The major routes of sulfate formation from

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-cysteine therefore seems to involve pathways that do not involve

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-cysteinesulfinate. Similar amounts of sulfate were formed from

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-cysteine as

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-cysteine. Thiosulfate instead of sulfate was also formed in GSH-depleted hepatocytes. However, sulfate formation from

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-cysteine differed from

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-cysteine in that it was inhibited by the

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-aminoacid oxidase inhibitor sodium benzoate and was not affected by transaminase or γ-cystathionase inhibitors. These results suggest that thiosulfate is an intermediate in sulfate formation from

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-cysteine and involves the oxidation of

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-cysteine by

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-amino acid oxidase to form β-mercaptopyruvate.     

Synthesis of methyl

Mutacins are bactericidal substances of proteinaceous nature produced by Streptococcus mutans. Lantibiotics are antibacterial substances containing post-translationally modified amino acids such as lanthionine. Mutacin B-Ny266 was purified from the cell pellet of S. mutans strain Ny266 by ethanol extraction at pH 2.0 followed by reversed-phase chromatography (Sep-Pak® cartridge) and by HPLC on a C₁₈ column. The mean purification factor was 3240±81 and the mean yield was 1.0±0.1%. Molecular mass of mutacin B-Ny266 as determined by mass spectroscopy is 2270.29±0.21 Da. The amino acid sequence of the purified active fraction was obtained by Edman degradation after treatment with alkaline ethanethiol. Twenty-one amino acids were detected in this analysis. Mutacin B-Ny266 belongs to the type A lantibiotics. The proposed sequence is: F–K–

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–W–U–F–

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–

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–P–G–

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–A–K–O–G–

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–F–N–

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–Y–

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. The molecule differs from that of epidermin/staphylococcin 1580 and gallidermin at positions 1, 2, 4, 5 and 6.     

Purification and structure of mutacin B-Ny266: a new lantibiotic produced by Streptococcus mutans

Mutacins are bactericidal substances of proteinaceous nature produced by Streptococcus mutans. Lantibiotics are antibacterial substances containing post-translationally modified amino acids such as lanthionine. Mutacin B-Ny266 was purified from the cell pellet of S. mutans strain Ny266 by ethanol extraction at pH 2.0 followed by reversed-phase chromatography (Sep-Pak® cartridge) and by HPLC on a C₁₈ column. The mean purification factor was 3240±81 and the mean yield was 1.0±0.1%. Molecular mass of mutacin B-Ny266 as determined by mass spectroscopy is 2270.29±0.21 Da. The amino acid sequence of the purified active fraction was obtained by Edman degradation after treatment with alkaline ethanethiol. Twenty-one amino acids were detected in this analysis. Mutacin B-Ny266 belongs to the type A lantibiotics. The proposed sequence is: F–K–

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–W–U–F–

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–

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–P–G–

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–A–K–O–G–

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–F–N–

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–Y–

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. The molecule differs from that of epidermin/staphylococcin 1580 and gallidermin at positions 1, 2, 4, 5 and 6.     

A method for the synthesis of C-(2-deoxy-β-glycosyl) arenes

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(2) was converted by a Wittig reaction into a mixture of (

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(4,5). Selective deprotection of the 5,6-O-isopropylidene group in compounds 4 and 5 followed by selective silylation at position 6 afforded the separate

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8a–d and the corresponding E-isomers (9a–d). Iodonium-ion-induced cyclization of compounds 8c and 9a-c furnished stereoselectively the

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10a–c. Full deprotection of compounds 10a–c and the O-acetylation led to compounds 11a–c, which on treatment with tributyltin hydride-azobisisobutyromnitrile yielded and the title compounds (12a–c).     

Mass spectrometric quantification of the mu opioid receptor agonist Tyr-

A rapid and sensitive assay method for the determination of glycine carboxypeptidase activity has been reported. This method is based on the monitoring of the absorption at 460 nm of 4-dimethylaminoazobenzene-4′-sulfonyl-Gly-

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-Phe, enzymatically formed from the substrate 4-dimethylaminoazobenzene-4′-sulfonyl-Gly-

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-Phe-Gly, after separation by high-performance liquid chromatography (HPLC) using a TSK gel ODS-80TM reversed-phase column by isocratic elution. This method is sensitive enough to measure 4-dimethylaminoazobenzene-4′-sulfonyl-Gly-

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-Phe at concentrations as low as 1 pmol and yield highly reproducible results and requires less than 7.5 min per sample for separation and quantitation. The pH optimum for glycine carboxypeptidase activity was 4.8 to 5.4. The K_m and V_max values were respectively 21.1 μmol and 3.73 pmol/μg/h with the use of enzyme extract obtained from bovine pituitary. Glycine carboxypeptidase activity was strongly inhibited by Ag⁺, Cu²⁺ and p-chloromercuriphenylsulfonic acid. Among the organs examined in a mouse, the highest specific activity of the enzyme was found in testis. The sensitivity and selectivity of this method will aid in efforts to examine the physiological role of this peptidase.     

High-performance liquid chromatographic–colorimetric assay for glycine carboxypeptidase activity

A rapid and sensitive assay method for the determination of glycine carboxypeptidase activity has been reported. This method is based on the monitoring of the absorption at 460 nm of 4-dimethylaminoazobenzene-4′-sulfonyl-Gly-

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-Phe, enzymatically formed from the substrate 4-dimethylaminoazobenzene-4′-sulfonyl-Gly-

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-Phe-Gly, after separation by high-performance liquid chromatography (HPLC) using a TSK gel ODS-80TM reversed-phase column by isocratic elution. This method is sensitive enough to measure 4-dimethylaminoazobenzene-4′-sulfonyl-Gly-

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-Phe at concentrations as low as 1 pmol and yield highly reproducible results and requires less than 7.5 min per sample for separation and quantitation. The pH optimum for glycine carboxypeptidase activity was 4.8 to 5.4. The K_m and V_max values were respectively 21.1 μmol and 3.73 pmol/μg/h with the use of enzyme extract obtained from bovine pituitary. Glycine carboxypeptidase activity was strongly inhibited by Ag⁺, Cu²⁺ and p-chloromercuriphenylsulfonic acid. Among the organs examined in a mouse, the highest specific activity of the enzyme was found in testis. The sensitivity and selectivity of this method will aid in efforts to examine the physiological role of this peptidase.     

Action of acetylxylan esterase from Trichoderma reesei on acetylated methyl glycosides

Substrate specificity of purified acetylxylan esterase (AcXE) from Trichoderma reesei was investigated on partially and fully acetylated methyl glycopyranosides. Methyl 2,3,4-tri-O-acetyl-β-

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-xylopyranoside was deacetylated at positions 2 and 3, yielding methyl 4-O-acetyl-β-

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-xylopyranoside in almost 90% yield. Methyl 2,3-di-O-acetyl β-

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-xylopyranoside was deacetylated at a rate similar to the fully acetylated derivative. The other two diacetates (2,4- and 3,4-), which have a free hydroxyl group at either position 3 or 2, were deacetylated one order of magnitude more rapidly. Thus the second acetyl group is rapidly released from position 3 or 2 after the first acetyl group is removed from position 2 or 3. The results strongly imply that in degradation of partially acetylated β-1,4-linked xylans, the enzyme deacetylates monoacetylated xylopyranosyl residues more readily than di-O-acetylated residues. The T. reesei AcXE attacked acetylated methyl β-

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-glucopyranosides and β-

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-mannopyranosides in a manner similar to the xylopyranosides.     

Catalytic mechanism of aldose reductase studied by the combined potentials of quantum mechanics and molecular mechanics

The catalytic reduction of

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-glyceraldehyde to glycerol by aldose reductase has been investigated with the combined potentials of quantum mechanics (QM) and molecular mechanics (MM) to resolve the question of whether Tyr48 or His110 serves as the proton donor during catalysis. Site directed mutagenesis studies favor Tyr48 as the proton donor while the presence of a water channel linking the Nδ1 of His110 to the bulk solvent suggests that His110 is the proton donor. Utilizing the combined potentials of QM and MM, the binding mode of substrate

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-glyceraldehyde was investigated by optimizing the local geometry of Asp43, Lys77, Tyr48, His110 and NADPH at the active site of aldose reductase. Reaction pathways for the reduction of

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-glyceraldehyde to glycerol were then constructed by treating both Tyr48 and His110 as proton donors. Comparison of energetics obtained from the reaction pathways suggests His110 to be the proton donor. Based on these findings, a reduction mechanism of

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-glyceraldehyde to glycerol is described.     

Molecular cloning and characterization of FHL2, a novel LIM domain protein preferentially expressed in human heart

A full-length cDNA clone encoding a novel LIM-only protein was isolated and sequenced from a human fetal heart cDNA library. This full-length clone consists of 1416 base pairs and has a predicted open reading frame (ORF) encoding 279 amino acids. The ORF of this polypeptide codes for the human heart-specific

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our and a

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alf

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IM-only protein

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(FHL2). It possesses an extra zinc finger that is a half LIM domain and four repeats of LIM domain. When the human FHL2 cDNA probe was used to hybridize with poly-A RNA of various human tissues, a very strong signal could be seen in heart tissues, and only moderately low signals could be detected in placenta, skeletal muscle and ovary. Virtually no signal could be detected in brain, lung, liver, kidney, pancreas, spleen, thymus, prostate, testis, small intestine, colon or peripheral blood leukocyte. FHL2 was mapped to chromosome 2q12–q13 by fluorescent in-situ hybridization (FISH).     

Direct enantioseparation of some β-adrenergic blocking agents using impregnated thin-layer chromatography

The resolution of (±)-atenolol, (±)-propranolol and (±)-metoprolol into their enantiomers was achieved by TLC on silica-gel plates impregnated with optically pure

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-lysine (0.5%) and

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-arginine (0.5%) as the chiral selectors. In all cases, different combinations of acetonitrile–methanol solvent systems were found to be successful in resolving these compounds. Spots were detected using iodine vapour. The detection limit for both (±)-atenolol and (±)-propranolol was 2.6 μg and for (±)-metoprolol, it was 0.26 μg.     

Reaction of FcCCo3(CO)9 with 2,3-bis(diphenylphosphino)maleic anhydride (bma). X-ray diffraction structure and redox properties of

Kynurenine 3-monooxygenase, an NADPH-dependent flavin monooxygenase, catalyses the hydroxylation of

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-kynurenine to

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-3-hydroxykynurenine. By hybridization screening using a cDNA probe encoding the entire exon 2 of Drosophila melanogaster kynurenine 3-monooxygenase, we isolated a 2.0 kb cDNA clone coding for the corresponding human liver enzyme. The deduced amino acid sequence of the human protein consists of 486 amino acids with a predicted molecular mass of 55 762 Da. Transfection of the human cDNA in HEK-293 cells resulted in the functional expression of the enzyme with kinetic properties similar to those found for the native human protein. RNA blot analysis of human tissues revealed the presence of a major mRNA species of 2.0 kb in liver, placenta and kidney.     

Cloning and functional expression of human kynurenine 3-monooxygenase

Kynurenine 3-monooxygenase, an NADPH-dependent flavin monooxygenase, catalyses the hydroxylation of

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-kynurenine to

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-3-hydroxykynurenine. By hybridization screening using a cDNA probe encoding the entire exon 2 of Drosophila melanogaster kynurenine 3-monooxygenase, we isolated a 2.0 kb cDNA clone coding for the corresponding human liver enzyme. The deduced amino acid sequence of the human protein consists of 486 amino acids with a predicted molecular mass of 55 762 Da. Transfection of the human cDNA in HEK-293 cells resulted in the functional expression of the enzyme with kinetic properties similar to those found for the native human protein. RNA blot analysis of human tissues revealed the presence of a major mRNA species of 2.0 kb in liver, placenta and kidney.     

Effect of pasteurization and storage on some components of pooled human milk

Pooled human milk was subjected to Holder pasteurization and storage at −20°C up to 90 days and examined for its content of fat and

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-lactate and for lipid composition. This treatment reduced fats by 6% and

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-lactate by at least 7%. In addition, pasteurization and storage induced triglyceride hydrolysis. The absolute amount of free fatty acids (FFAs) which was 0.5% after collection, doubled after pasteurization and rose even more after storage. Different FFA compositions were found by several authors using the same analytical method even for milk samples subjected to the same treatment. More detailed information on procedures must be given to explain the different results.     

Determination of urinary mercapturic acids of styrene in man by high-performance liquid chromatography with fluorescence detection

A method for the determination of urinary

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(M1) and

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(M2) in man was developed. Clean-up of urine samples was obtained by a chromatographic technique using a short reversed-phase precolumn; purified samples were then deacetylated with porcine acylase I for 16 h at 37°C and deproteinized by centrifugal ultrafiltration. Derivatization was performed with o-phthaldialdehyde and 2-mercaptoethanol and the fluorescent derivatives were separated on a reversed-phase analytical column with a gradient mobile phase consisting of 50 mM acetate buffer (pH 6.5) and methanol. The retention times of the diastereoisomers of M1 (M1-“S” and M1-“R”) were 52.8 and 73.7 min, respectively; M2 diastereoisomers eluted as a single peak at 70.5 min. The fluorescence detector was set at 330 nm (excitation) and 440 nm (emission). The detection limit (at a signal-to-noise ratio of three) was about 7 μg/l. The method was applied to 25 urine samples from workers exposed to styrene. A relationship was found between urinary mandelic and phenylglyoxylic acids and mercapturic acids specific for styrene. Urine samples from ten non-exposed subjects showed no detectable amounts of analytes.     

Process for the isolation of preparative quantities of

A general and flexible synthetic approach to biologically important 5,6-unsaturated C₁₈-phytosphingosines was developed via olefin cross-metathesis employing truncated C₆-phytosphingosines as the key intermediates. These were efficiently prepared in high yields by zinc-mediated reductive opening of methyl 2-amino-2-deoxy-β-hexopyranosides.

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Efficient synthesis of d-xylo and d-ribo-phytosphingosines from methyl 2-amino-2-deoxy-β-d-hexopyranosides

A general and flexible synthetic approach to biologically important 5,6-unsaturated C₁₈-phytosphingosines was developed via olefin cross-metathesis employing truncated C₆-phytosphingosines as the key intermediates. These were efficiently prepared in high yields by zinc-mediated reductive opening of methyl 2-amino-2-deoxy-β-hexopyranosides.

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