The primary acid product of DPNH

Analysis of the proton magnetic resonance spectra obtained at 220 MHz confirms the axial conformation of the C-6 hydroxyl in the model primary acid product 1-n-(2,6-dichlorobenzyl)-6-hydroxy-1,4,5,6-tetrahydronicotinamide. In the primary acid product of DPNH however the reaction occurs stereospecifically with the substitution at the C-6 position equatorial and on the B-side of the pyridine ring and the C-4A proton axial. A cyclic structure α,O^2′-6B cyclotetrahydronicotinamide is proposed for the primary acid product of DPNH, formed by epimerization of βDPNH to the α configuration followed by protonation at C-5 and subsequent attack of the ribose C-2′-OH on the C-6 position forming a new five membered ring.     

Occurrence of a galactofuranose disaccharide in immunoadjuvant fractions of Mycobacterium tubercolosis (cell walls and wax D)

By partial acid hydrolysis a digalactoside was obtained from the arabinogalactan of the cell walls and Wax D of Mycobacterium tuberculosis. By using instrumental and chemical analysis methods (gas-liquid chromatography, mass spectrometry, PMR spectroscopy, periodate oxidation, permethylation) the saccharide was identified as 6-O-β-d-galactofuranosyl-d-galactose     

Novel epoxides formed during the liver cytochrome P-450 oxidation of arachidonic acid

Arachidonic acid is enzymatically oxidized by the rat liver microsomal mixed-function oxidase system, in the presence of NADPH and oxygen, to a wide variety of products. We report here, the identification of the major organic-soluble metabolites. They are the 5,6-,8,9-,11,12-, and 14,15-epoxy-eicosatrienoic acid derivatives of arachidonic acid.     

Finding of a galactose-oxidation-product in lens of galactose-fed guinea pig

From studies on polyols in lens of galactose-fed guinea pigs, r-galactono-1,4-lactone was found, which proves the presence of galactonic acid as a product of galactose oxidation, by gas liquid chromatography and mass spectrometry. The content of this component was one tenth of that of galactitol. In vitro culture of rat lens in 30 mM galactose-loaded media demonstrated the formation of the lactone. The significance of the lactone was discussed with respect to the galactose metabolism in lens.     

Distribution of saccharopine and 2-aminoadipic acid in higher plants

Saccharopine and 2-aminoadipic acid have been identified in eleven plant species belonging to nine families. The amino acids have been isolated from green parts of the plants using ion-exchange chromatography and preparative high voltage electrophoresis, and in three cases the identification was supported by mass spectroscopy. Mild conditions were used during the isolation to avoid lactamization, and the contents of saccharopine and 2-aminoadipic acid have been determined semiquantitatively. The significance of the occurrence of the two amino acids with regard to lysine metabolism is briefly discussed.     

2-C-Methylaldotetronic acid,a new lactone-forming acid present in plants

2-C-Methylaldotetronic acid (probably the erythro form) was found in considerable amounts in Cannabis sativa, Cereus forbesii, C. peruvianus, Lophophora williamsii, Trichocereus santiaguensis, T. spachianus and T. strigosus. In addition, the acid was present in minor amounts in another five species, all from the Cactaceae. In total, this new plant acid was detected in 12 of 19 investigated species.     

Effect of trophic conditions on asparagine transamination in wheat plants

In dialyzed extracts from winter wheat plants transamination reactions occurred between asparagine and α-ketoglutaric acid (L-asparagine+2-oxoacid=2-oxosuccinamate+ +amino acid; 2. 6. 1. 14). Reactions with pyruvate exhibited a very low activity. Besides transamination products,i. e. glutamate and alanine, aspartic acid was formed in both reactions. Deamidation was more intensive in the weak reaction asparagine-alanine and less intensive in the asparagine-glutamate reaction. When calculated per dry weight unit the activity was the same in plants of all variants (three experimental variants—Knop, potassium humate, water). A higher, activity was found in root dialysates; however, a highly significant difference could be observed only between shoots and roots of Knop variant. When evaluating results in terms of protein content we found a significant difference between mineral variant (Knop—the lowest activity) and both deficient variants (potassium humate, water—the highest activity). Thus the highest growth activity was in connection with the lowest transamination activity and vice versa, which was the same as in transaminations of aspartic acid. In the case of asparagine, too, one can consider the possibility of its utilization via transamination for biosynthesis of glutamic acid in plants which have, for reasons of nutrition, a low level of this metabolically important amino acid.     

Organ and cellular localization of asparagine synthetase in rice plants

DNA gel blot analysis suggested that asparagine synthetase (AS; EC 6.3.5.4) occurred as a single gene in rice. A fusion protein consisting of 17 kDa tagged-region from pET32a(+) expression plasmid and 42 kDa N-terminal region of rice AS was first expressed in Escherichia coli. The resulting polypeptide was purified and a mono-specific antibody for rice AS was prepared after affinity-purification with the antigen. Immunoblotting revealed a high content of AS protein in the leaf sheath at the second position from the fully expanded top leaf and in grains at the middle stage of ripening. Accumulation of mRNA for AS was also observed in these organs. During the ripening of the spikelets, the AS protein contents increased during the first 21 days after flowering, then declined rapidly. Immunolocalization analysis revealed signals for AS protein in the companion cells of vascular bundles of leaf sheath and phloem-parenchyma cells, nucellar projection, and nucellar epidermis of dorsal vascular bundles of grains.     

Hepatic metabolism of prostacyclin (PGI2) in the rabbit: Formation of a potent novel inhibitor of platelet aggregation

Metabolism of [9-³H]-PGI₂ was studied in the isolated Tyrode's perfused rabbit liver. Five products, four radioactive and one non-radioactive, were identified in the perfusate: 19-hydroxy-6-keto-PGF_1α, 6-keto-PGF_1α, dinor-6-keto-PGF_1α, pentanor PGF_1α and a 6-keto-PGE₁-like substance. The first two, 19-hydroxy-6-keto-PGF_1α and 6-keto-PGF_1α, represented 5% and 45% respectively, of the total radioactivity; the last two accounted for 39%. The presence of dinor and pentanor derivatives of 6-keto-PGF_1α indicated that β -oxidation and oxidative-decarboxylation occurs in the liver as the major metabolic pathway of PGI₂. One non-radioactive metabolite which co-migrated with authentic 6-keto-PGE₁ was found to inhibit platelet aggregation, having a potency similar to authentic 6-keto-PGE₁, and its effect can be eliminated by boiling and by alkali treatment. This metabolite, having similar Rf value on TLC and biological behavior as 6-keto-PGE₁, may arise from oxidation of 6-keto-PGF_1α via the 9-hydroxyprostaglandin dehydrogenase pathway, as suggested by recovery of tritiated water in the aqueous phase of the perfusate. This material, a potent inhibitor of platelet aggregation, may arise from PGI₂ or its hydrolysis product, 6-keto-PGF_1α.     

Three-dimensional structure of Escherichia coli asparagine synthetase B: a short journey from substrate to product

Asparagine synthetase B catalyzes the assembly of asparagine from aspartate, Mg(2+)ATP, and glutamine. Here, we describe the three-dimensional structure of the enzyme from Escherichia colidetermined and refined to 2.0 A resolution. Protein employed for this study was that of a site-directed mutant protein, Cys1Ala. Large crystals were grown in the presence of both glutamine and AMP. Each subunit of the dimeric protein folds into two distinct domains. The N-terminal region contains two layers of antiparallel beta-sheet with each layer containing six strands. Wedged between these layers of sheet is the active site responsible for the hydrolysis of glutamine. Key side chains employed for positioning the glutamine substrate within the binding pocket include Arg 49, Asn 74, Glu 76, and Asp 98. The C-terminal domain, responsible for the binding of both Mg(2+)ATP and aspartate, is dominated by a five-stranded parallel beta-sheet flanked on either side by alpha-helices. The AMP moiety is anchored to the protein via hydrogen bonds with O(gamma) of Ser 346 and the backbone carbonyl and amide groups of Val 272, Leu 232, and Gly 347. As observed for other amidotransferases, the two active sites are connected by a tunnel lined primarily with backbone atoms and hydrophobic and nonpolar amino acid residues. Strikingly, the three-dimensional architecture of the N-terminal domain of asparagine synthetase B is similar to that observed for glutamine phosphoribosylpyrophosphate amidotransferase while the molecular motif of the C-domain is reminiscent to that observed for GMP synthetase.     

Analysis of metabolic profiles of prostaglandins in urine using a lipophilic anion exchanger

A method is described for the fractionation of prostaglandins and their metabolites in urine. Following acidification and extraction on Amberlite XAD-2, samples were separated by chromatography on the lipophilic anion exchanger diethyl-aminohydroxypropyl Sephadex LH-20 into fractions containing neutral compounds, monocarboxylic, dicarboxylic and polycarboxylic acids. The compounds in resulting fractions were further separated by reversed phase partition chromatography. As an application, the metabolic profiles in urine of [9β-^3H]-labeled prostaglandin F_2α¹ and prostaglandin analogs 15-methyl-PGF_2α and 16,16-dimethyl-PGF_2α were investigated in the cynomolgus monkey. It was demonstrated that the resolution of individual prostaglandin metabolites by reversed phase partition chromatography was considerably simplified by initial group separation on the anion exchanger, and several metabolites were much purified. A glucuronic acid conjugate of the main metabolite of 15-methyl-PGF_2α (dinor-15-methyl-PGF_2α) was tentatively identified using computerized gas chromatography - mass spectrometry.     

Ophthalmic acid is a marker of oxidative stress in plants as in animals

Background

Ophthalmic acid (OPH), γ-glutamyl-L-2-aminobutyryl-glycine, a tripeptide analogue of glutathione (GSH), has recently captured considerable attention as a biomarker of oxidative stress in animals. The OPH and GSH biosynthesis, as well as some biochemical behaviors, are very similar. Here, we sought to investigate the presence of OPH in plants and its possible relationship with GSH, known to possess multiple functions in the plant development, growth and response to environmental changes.

N-Acylethanolamine accumulation in infarcted myocardium

Long-chain N-acylethanolamines were found at levels of 400–500 nmol per g tissue in the infarcted areas of canine myocardium 24 hours after coronary artery ligation. Peripheral infarct areas also contained substantial amounts (200 nmol/g) while apparently normal heart muscle contained very little (< 10 nmol/g). The amide linked fatty acids were mainly 16:0, 18:0, 18:1 and 18:2. Because of its anti-inflammatory activity, N-acylethanolamine may exert beneficial effects in the infarcted area and may be produced as a response to ischemic injury.     

Amino acid requirements of the mosquito Culex pipiens: asparagine essential

When any of the ten “rat essential” amino acids was omitted singly from a fully-defined synthetic dietary medium, newly-hatched Culex pipiens larvae were unable to develop to the second instar. With proline omitted, development was greatly retarded and survival to the adult stage reduced. Without aspargine (but not aspartic acid) growth and development ceased in most individuals before larval-pupal ecdysis, and no adults were obtained. These twelve amino acids are considered nutritionally essential for this mosquito. With glycine omitted singly, development was markedly retarded, but survival to the adult stage was not affected; thus this amino acid is required for good growth, but these experiments do not demonstrate it as essential. Single omission of alanine, aspartic acid, cysteine, glutamic acid or amide, serine or tyrosine had virtually no effect on development and they are therefore considered nutritionally non-essential. With diets containing the twelve culex-essential amino acids only, very little development occurred, but augmentation with either glycine or serine allowed growth and development almost as good as with the complete amino acid mixture. Augmentation of the essential twelve with alanine, cysteine, glutamic acid/amide, or tyrosine singly failed to improve development. The requirement for dietary asparagine shown by these studies appears to be unique among insects so far studied. In particular, another mosquito, Aedes aegypti, has no such requirement.     

Mass spectrometric differentiation of leucine and isoleucine in proteins derived from bacteria or cell culture

The differentiation of leucine and isoleucine is a well known difficulty in mass spectrometric peptide sequencing. A technique has been developed which allows these two amino acids to be distinguished by growing a bacterial or cell culture in a medium containing γ,δ-dl-dideuteroleucine. The isotopically labelled residue is incorporated into the cell's proteins, and the resulting mass spectra of leucine containing peptides exhibit sequence ions 2 amu higher than the corresponding isoleucine peptides.     

Haemoglobin Arya: alpha 2-47 (CD5), aspartic acid yields asparagine.

A new haemoglobin variant (haemoglobin Arya), is described from an Iranian female. The substitution is at residue 47 (CD5) of the alpha chain in which aspartic acid has been substituted by asparagine. The presence of haemoglobin Arya was not associated with clinical symptoms. This variant has normal stability at 50 degrees C, but is slightly unstable when tested at 55 degrees C.     

水杨酸在植物抗环境胁迫中的作用

水杨酸在植物的抗病方面发挥着重要作用。近年来的研究还表明 ,水杨酸在植物的抗冷、热、盐、干旱、臭氧、重金属等环境胁迫方面也有明显的作用。该文介绍近年来有关水杨酸在植物抗环境胁迫方面的研究进展 ,并探讨了其作用机理。     

OsSEND-1: a new RAD2 nuclease family member in higher plants

A novel endonuclease, a new member of the RAD2 nuclease family, has been identified from the higher plant, rice (Oryza sativa L. cv. Nipponbare), and designated as OsSEND-1. The open reading frame of the OsSEND-1 cDNA encoded a predicted product of 641 amino acid residues with a molecular weight of 69.9 kDa. The encoded protein showed a relatively high degree of sequence homology with the RAD2 nuclease family proteins, especially RAD2 nuclease, but it differed markedly from FEN-1, XPG or HEX1/EXO1. The N- and I-domains in the family were highly conserved in the OsSEND-1 sequence. The protein was much smaller than XPG, but larger than HEX1/EXO1 and FEN-1. The genome sequence was composed of 14 exons, and was localized at the almost terminal region of the short arm of chromosome 8. Northern blotting and in situ hybridization analyses demonstrated preferential expression of OsSEND-1 mRNA in proliferating tissues such as meristem. The mRNA level of OsSEND-1 was induced by UV and DNA-damaging agent such as MMS or H₂O₂, indicating that OsSEND-1 has some roles in the repair of many types of damaged DNA. The recombinant peptide showed endonuclease activity.