Action of the Protease from Streptomyces cellulosae on l-Leu-Gly-Gly

We introduced efficient incorporation of unsaturated fatty acids into volicitin [N-(17-hydroxylinolenoyl)-L-glutamine] and N-linolenoyl-L-glutamine, insect-derived elicitors of plant volatiles, in the common cutworms Spodoptera litura by the incubation of larval gut tissues with unsaturated (linolenic, linoleic, and oleic acids) or saturated fatty acids (palmitic and stearic acids) sodium salt, and L-[α-¹⁵N]glutamine.     

Identification of Volicitin-related Compounds from the Regurgitant of Lepidopteran Caterpillars

Volicitin-related compounds were found in the oral secretion of the three noctuid species, Helicoverpa armigera, Mythimna separata and Spodoptera litura, and one sphingid species, Agrius convolvuli. Volicitin [N-(17-hydroxylinolenoyl)-L-glutamine], N-(17-hydroxy-linoleoyl)-glutamine, N-linolenoylglutamine and N-linoleoylglutamine were identified in the secretion from the noctuid larvae. In secretions from the sphingid larvae, N-linolenoylglutamine and N-linoleoylglutamine were the main components. Furthermore, there were significant differences in the amounts of the N-acylamino acid conjugates in the secretions from the three noctuid species. These results suggest that the proportion of volicitin-related compounds in the regurgitant was species-specific.     

New Amides from Buckwheat Seeds (Fagopyrum esculentum Moench)

Two new amino acid amides which yield in acid hydrolysis isomeric hydroxybenzylamines and amino acids have been isolated from the achenes of Fagopyrum esculentum Moench. One of them called BN-II is composed of salicylamine and allo-4-hydroxy-l-glutamic acid, and the other, BN-III, p-hydroxybenzylamine and l-glutamic acid. These coupled compounds link one another to form an amide respectively. Finally the structures of BN-II and BN-III were determined to be N⁵-(2′-hydroxybenzyl)-allo-4-hydroxy-l-glutamine and N⁵-(4′-hydroxybenzyl)-l-glutamine respectively from their chemical and spectrometry properties.     

Specific Incorporation of L-Glutamine into Volicitin in the Regurgitant of Spodoptera litura

Volicitin, [N-(17-hydroxylinolenoyl)-L-glutamine], was identified as an elicitor of plant volatiles from a Spodoptera exigua regurgitant. It has been proposed that gut microbes synthesize volicitin from glutamine, a predominant amino acid component in the insect gut. However, we found that glutamine was not a major component in the regurgitant of Spodoptera litura, although L-glutamine was exclusively incorporated into volicitin by S. litura fed on diets enriched with various amino acids. This selectivity of glutamine as a substrate was not due to a dominant occurrence in the insect gut.     

Helix–coil stability constants for the naturally occurring amino acids in water. XXI. Glutamine parameters from random poly(hydroxypropylglutamine-co-L-glutamine) and poly(hydroxybutylglutamine-co-L-glutamine)

Water-soluble, random copolymers containing L -glutamine and either N⁵-(3-hydroxypropyl)-L -glutamine or N⁵-(4-hydroxybutyl)-L -glutamine were synthesized, fractionated, and characterized. The thermally induced helix–coil transitions of these copolymers were studied in water. A short-range interaction theory was used to deduce the Zimm-Bragg parameters σ and s for the helix–coil transition in poly(L -glutamine) in water from an analysis of the melting curves of the copolymers in the manner described in earlier papers. The computed values of s indicate that L -glutamine is helix-indifferent at low temperature and a helix-destabilizing residue at high temperature in water. At all temperatures in the range of 0–70°C, the glutamine residue promotes helix–coil boundaries since the computed value of σ is large.     

Isolation of N-acetylglutamine, pyroglutamic acid and the butyl ester of pyroglutamic acid from human brain

N-acetyl-l -glutamine, pyroglutamic acid, and the butyl ester of pyroglutamic acid were isolated in pure form from an aqueous extract of human brain. These compounds were isolated by combination of paper and ion exchange chromatography. The isolated substance identified as N-acetyl-l -glutamine did not react with the ninhydrin reagent but yielded glutamic acid and ammonia upon acid hydrolysis. An acetyl hydrazide was identified by paper chromatography from hydrazinolysates of the isolated substance. The glutamic acid liberated by hydrolysis had the l -configuration. The results of elementary analysis of the isolated compound were in full accord with the analysis calculated for synthetic N-acetyl-l -glutamine. A large amount of pyroglutamic acid and a substance identical with the butyl ester of pyroglutamic acid were isolated in pure form. The results of our studies suggest that pyroglutamic and the butyl ester derivative were artifacts formed during the isolation and purification procedures.     

Dansylcadaverine specific staining for transamidating enzymes.

Versatile fluorescent staining methodologies, based on the incorporation of dansylcadaverine[N-(5-aminopentyl)-5-dimethylamino-l-naphthalenesulfonamide] into N,N-dimethylcasein, are described for the detection of transamidating enzymes of the endo-γ-glutamine:ε-lysine transferase type. Activity staining was employed for comparing the electrophoretic behaviors of such transamidating enzymes derived from human and guinea pig tissues. Two enzymatically active forms of guinea pig liver transglutaminase were found.     

Molecular Cloning,Sequencing, and Expression of a <Emphasis Type="SmallCaps">l</Emphasis>-Glutamine <Emphasis Type="SmallCaps">d</Emphasis>-Fructose 6-Phosphate Amidotransferase Gene from <Emphasis Type="Italic">Volvariella volvacea</Emphasis>

Using 3′-RACE and 5′-RACE, we have cloned and sequenced the genomic gene and complete cDNA encoding l-glutamine d-fructose 6-phosphate amidotransferase (GFAT) from the edible straw mushroom, Volvariella volvacea. Gfat contains five introns, and encodes a predicted protein of 697 amino acids that is homologous to other reported GFAT sequences. Southern hybridization indicated that a single gfat gene locus exists in the V. volvacea genome. Recombinant native V. volvacea GFAT enzyme, over-expressed using Escherichia coli and partially purified, had an estimated molecular mass of 306 kDa and consisted of four equal-sized subunits of 77 kD. Reciprocal plots revealed K _m values of 0.55 and 0.75 mM for fructose 6-phosphate and l-glutamine, respectively. V. volvacea GFAT activity was inhibited by the end-product of the hexosamine pathway, UDP-GlcNAc, and by the glutamine analogues N ³-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid and 2-amino-2-deoxy-d-glucitol-6-phosphate.     

Helix-coil stability constants for the naturally occurring amino acids in water. XIII. The presence of by-products in amino-acid analysis of copolymers and their effect on the guest parameters; recomputed values of σ and s for L-serine

Previously used procedures for processing the amino acids from 6N hydrochloric acid hydrolysis of poly[N⁵-(4-hydroxybutyl)-L -glutamine], poly[N⁵-(3-hydroxypropyl)-L -glutamine], and several random copolymers derived from these, led to the formation of spurious products. These have been isolated and characterized as the γ-ester of glutamic acid and the hydroxyalkyl amine, and chloro-alkyl amine hydrochloride. The former reduces the observed values for glutamic acid, but the latter has no effect on them. A method is used to avoid formation of these artifacts in the amino-acid analysis. Of all the copolymers studied previously in this series, the compositions of only those containing L -serine are in error as a result of the formation of the γ-ester. A redetermination of the amino-acid compositions of the copolymer fractions studied earlier leads to slightly revised values for the Zimm-Bragg parameters σ and s of serine.     

Elicitors and priming agents initiate plant defense responses

Biotic elicitors produced by plant pathogens or herbivore pests rapidly activate a range of plant chemical defenses when translocated to plant tissue. The fatty acid conjugate volicitin has proven to be a robust elicitor model for studying herbivore-induced plant defense responses. Here we review the role of insect-derived volicitin (N-[17-hydroxylinolenoyl]-L-glutamine) as an authentic elicitor of defense responses, specifically as an activator of signal volatiles that attract natural enemies of herbivore pests. Comparisons are drawn between volicitin as an elicitor of plant defenses and two other classes of signaling molecules, C₆ green-leaf volatiles and C₄ bacterial volatiles that appear to prime plant defenses thereby enhancing the capacity to mobilize cellular defense responses when a plant is faced with herbivore or pathogen attack.     

Cloning and primarily function study of two novel putative N5-glutamine methyltransferase (Hemk) splice variants from mouse stem cells

Methylation is one of epigenetic mechanisms regulating gene expression. The methylation pattern is determined during embryogenesis and passed over to differentiating cells and tissues. Beginning with the ESTs which were highly expressed in undifferentiated human ES cells and using homology research in mouse dbEST database, we cloned two novel putative (N ⁵)-glutamine methyltransferase (Hemk) splice variants termed mHemk1 and mHemk2 (Genbank accession number AY456393 and AY583759). Sequence analysis revealed that mHemk1 and mHemk2 cDNAs are 1,792 bp and 1,696 bp in length respectively. The deduced proteins have 214 amino acid residues (mHemk1) and 138 residues (mHemk2) in length and both share significant homology with (N ⁵)-glutamine methyltransferase (Hemk proteins) in database. Northern blot and RT-PCR analysis showed that mHemk mRNAs were abundantly expressed in undifferentiated ES cells, testis and brain, weakly expressed in differentiated ES cells and kidney, and not expressed in muscle, heart, placenta, pancreas, lung and stomach. Immunohistochemical analysis further revealed that the protein was most abundant in undifferentiated ES cells. The green fluorescent protein produced by pEGFP-C3/mHemk1 was detected mainly in the nucleus of COS7 cell lines after 24 h post-transfection. RNA interference (RNAi)-mediated knock-down method was established. Cell cycle analysis suggests that the cell proliferation decreases after RNAi with mHemk1. In vitro bioactivity assay showed that no evidence for a DNA adenine-methyltransferase activity was detected. The accumulating functional information from Hemk homology proteins in bacteria and yeast suggests that it may be an uncharacterized new mammalian N ⁵-glutamine methyltransferase.     

Effect of hydrophobic side chains on the pressure-induced changes in the NMR spectra of water-soluble biopolymers

Measurements have been made of the high-resolution nmr spectra of the polyamino acids poly[N⁵-(2-hydroxyethyl)-L -glutamine] and poly[N⁵-(4-hydroxybutyl)-L -glutamine] in mixed deuterium oxide and water solvent at varying pressures from 1.03 to 3163.7 kg/cm². The results are compared with previously reported results for the polymer poly[N⁵-(3-hydroxypropyl)-L -glutamine] under similar conditions. The significance of the behaviour of the polymers is considered in terms of the effect of the presence of hydrophobic residues in their side chains.     

The synthesis of isomeric 4-prolinylamines and 4,4′-diprolinylamines

Starting from the previously describedtert-butyl esters of 4-epimericN-benzyloxycarbonyl-4-hydroxyprolines andN-benzyloxycarbonyl-4-trans- and 4-cis-trifluoroacetaminoprolinetert-butyl esters, the corresponding uprotected 4-aminoprolines and a number of their partially protected derivatives were synthesized via the intermediate 4-O-mesyl and 4-azide derivatives. The reductive amination ofN-benzyloxycarbonyl-4-oxoprolinetert-butyl ester with ammonium acetate led toN-benzyloxycarbonyl-4-cis-4′-cis- and 4-cis-4′-trans-diprolinylamines. The¹H NMR and CD spectra of the synthesized compounds are described.     

Thermal Stability of Immobilized Glucoamylase in the Presence of a Substrate

N-(1-Arylethenyl)-2-chloroacetamides were synthesized and their herbicidal activities were tested. Among them, both 2-chloro-N-(2-methoxyethyl)-N-(2-methyl-1-phenylpropen-1-yl)acetamide and 2-chloro-N(2-ethoxyethyl)-N-(2-methyl-1-phenylpropen-1-yl)acetamide were found to be highly active against upland weeds.     

Nuclear magnetic resonance study on the model compounds for poly(N-alkylglycine)s

Cis-trans isomerism was investigated with N-acetyl and N-propionyl, N-alkylglycine dimethylamides as model compounds for poly(N-alkylglycine dimethlamides as model compounds for (N-alkylglycine)s using n.m.r. spectroscopy. The population of the cis isomer measured in benzene and methylene chloride solutions did not show any marked dependence on the bulkiness of N-alkyl substituents. This contrasts with polyN-alkylglycine)s, whose cis isomer population increased with the introduction of bulky N-alkyl groups. Kinetics of the Cis-trans isomerization was also investigated with N-acetyldimethylamides of sacrosine, N-n-propylglycine, and N-isopropylglycine in tetrachloroethane solution. The δG? values for Cis-trans isomerization in these amides were 18 ～ 19 kcal/mole, which were virtually the same as that of polysacrosine.     

Structural Determination of Monosialyl Trisaccharides Obtained From Caprine Colostrum

Acidic oligosaccharides were separated by dialysis, ion-exchange, preparative paper and gel chromatography from caprine colostrum. Four sialyl trisaccharides were characterized by ¹H-NMR spectrometry as follows: α-N-acetylneuraminyl-(2,6)-β-d-galactopyranosyl-(1,4)-2-N-acetamido-2-deoxy-d-glucopyranose (Neu5Ac α 2-6Gal β 1-4GlcNAc), α-N-acetylneuraminyl-(2,3)-β-d-galactopyranosyl-(1,4)-d-glucopyranose (Neu5Ac α 2-3Gal β-1-4Glc), α-N-acetylneuraminyl-(2,6)-β-d-galactopyranosyl-(1,4)-d-glucopyranose (Neu5Ac α 2-6Gal β 1-4Glc) and α-N-glycolylneuraminyl-(2,6)-β-d-galactopyranosyl-(1,4)-d-glucopyranose (Neu5Gc α 2-6Gal β 1-4Glc).     

Preparation of 3-ketovalidoxylamine A C–N lyase substrate: <Emphasis Type="Italic">N</Emphasis>-<Emphasis Type="Italic">p</Emphasis>-nitrophenyl-3-ketovalidamine by <Emphasis Type="Italic">Stenotrophomonas maltrophilia</Emphasis> CCTCC M 204024

3-Ketovalidoxylamine A C–N lyase is one of three key enzymes in the production of valienamine, which is a potent glucosidase inhibitor from validamycin A. N-p-Nitrophenyl-3-ketovalidamine, used as the substrate of 3-ketovalidoxylamine A C–N lyase, was prepared from N-p-nitrophenylvalidamine with free cells of Stenotrophomonas maltrophilia CCTCC M 204024. The yield and selectivity of N-p-nitrophenyl-3-ketovalidamine from cells were improved by treatment with 10 mM ethylenediaminetetraacetic acid. The optimal pH and temperature for N-p-nitrophenyl-3-ketovalidamine formation was pH 6.0 and 30°C, respectively. N-p-Nitrophenyl-3-ketovalidamine was formed with a yield of 0.68 in the first batch.     

Cell surface-associated lysosomal enzymes in cultured human skin fibroblasts

Trypsin released from the surface of intact human skin fibroblasts β-N-acetylglucosaminidase. The amount of trypsin removable β-N-acetylglucosaminidase in 4 control and 14 mucopolysaccharidosis cell lines was equivalent to 1.5% (range 0.5–4.3%) of the intracellular activity. Cell surface-associated β-N-acetylglucosaminidase was absent in mucolipidosis II and III fibroblasts that form lysosomal enzymes defective in binding to the cell surface receptors of fibroblasts and in β-N-acetylglucosaminidase deficient fibroblasts (Sandhoff's disease). Indirect immunofiuorescence with monospecific antisera allowed the demonstration of β-N-acetylglucosaminidase, α-N-acetylglucosaminidase, α-mannosidase and β-glucuronidase on the cell surface of fibroblasts, whereas these enzymes were absent on the cell surface of mucolipidosis II and III fibroblasts. Simultaneous staining for β-glucuronidase and β-N-acetylglucosaminidase showed presence of both enzymes in almost identical areas of the same cell. Cross-reacting material was present on the cell surface of fibroblasts with a deficiency of β-N-acetylglycosaminidase, α-N-acetylglucosaminidase (mucopolysaccharidosis III B), α-mannosidase (mannosidosis) and β-glucuronidase (mucopolysaccharidosis VII). The demonstration of lysosomal enzymes on the cell surface is in agreement with the hypothesis that in fibroblasts transport of lysosomal enzymes to the lysosomal apparatus involves cycling of lysosomal enzymes via the cell surface.     

Synthesis of Glyceroyl β-N-Acetyllactosaminide and Its Derivatives through a Condensation Reaction by Cellulase

A condensation reaction between N-acetyllactosamine and glycerol was directly catalyzed by using a commercially available cellulase preparation from Trichoderma reesei. 1-O-β-N-Acetyllactosaminyl-(R, S)-glycerols (1) were readily synthesized in a 5% yield based on the N-acetyllactosamine added and conveniently isolated by two-step column chromatographies. The use of a partially purified enzyme increased 2.3-fold the yield of 1, compared to that of the crude enzyme containing β-D-galactosidase activity. When various alkanols (n:2-4) were used in the condensation reaction, the corresponding alkyl β-N-acetyllactosaminides were obtained in yields of 0.3-1.1% of the desired compounds.     

Experimental characterization of poly(hydroxyalkyl-L-glutamine) conformations in aqueous calcium chloride and sodium perchlorate solutions

Poly(hydroxyalkyl-L -glutamine) (alkyl = ethyl, propyl, butyl) solutions have been studied by CD as functions of temperature and activity of calcium chloride and sodium perchlorate. Helical content is altered by changes in salt activity and temperature. The helicity of poly(hydroxybutyl-L -glutamine) and poly(hydroxypropyl-L -glutamine) falls to zero in a monotonic fashion with increasing calcium chloride activity. A nonzero helicity reappears at activities in excess of 5–50 mol kg^?1. Poly(hydroxypropyl-L -glutamine) is much more sensitive to calcium chloride than is poly(hydroxybutyl-L -glutamine), and both polypeptides are more sensitive to calcium chloride than are typical proteins. Markedly different behavior is observed with sodium perchlorate. This salt acts as a helix stabilizer at low activities but becomes a destabilizer at activities higher than 0.3–1.0 mol kg^?1. In this respect the effect of sodium perchlorate on nonionic poly(hydroxyalkyl-L -glutamines) resembles that seen with cationic poly(L -lysine) and poly(L -arginine). Helix stabilization at low sodium perchlorate activity is moderate for poly(hydroxybutyl-L -glutamine) and large for poly(hydroxypropyl-L -glutamine) and poly(hydroxyethyl-L -glutamine).