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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.24, #1-2)

Extending the scope of the journal by G. Lubec (pp. i-i).

Homocysteine in the context of cobalamin metabolism and deficiency states by A. Briddon (pp. 1-12).

It is becoming increasingly clear that serum vitamin B12 (cobalamin) concentration is a dubious indicator of functional B12 status and, in contrast to long-standing convention, correlates poorly with haematological indices. This, in turn, has led to poorly defined reference intervals for serum B12. Patients presenting with neurological disturbance due to B12 deficiency are at risk of not being diagnosed if total reliance is placed on serum B12 levels and haematological parameters. Plasma homocysteine remethylation is uniquely placed at the metabolic end-point of B12 metabolism such that plasma total homocysteine is proving to be a sensitive marker of functional B12 status. Studies also show that plasma homocysteine correlates better with holotranscobalamin than serum B12. It is suggested that clinicians should cease to be guided by surrogate haematological markers when more specific tests of B12 deficiency, such as holotranscobalamin and total homocysteine, exist. These tests demand greater prevalence in routine diagnostic use.

Keywords: Keywords: Vitamin B12; Transcobalamin; Homocysteine; Cobalamin

New perspectives on the role of amine oxidases in physiopathology by S. Nocera; L. Marcocci; P. Pietrangeli; B. Mondovì (pp. 13-17).

In the paper here presented we summarize some results obtained in our laboratory in the last few years on new structural and functional aspects of some amine oxidases (AOs), which have to be taken into consideration in defining new strategies of controlling the cellular physiopathology.In particular, the ability of Cu-AO purified from vegetal sources or from bovine serum to bind different cellular targets inducing in them conformational as well as chemical modifications are described and the consequences of this interaction on cellular functions are discussed. This is the case of the protective effect of Cu-AO against the damage induced by free radicals, cell enrichment with Cu-AO, induction of cataract and the leukocyte-endothelia interaction.The role of Cu and FAD-amine oxidases related as to the protection or damage of cells is also discussed. In this context the involvement of MAOs in the modulation of the mitochondrial functions and in the induction of apoptosis is described and some aspects of the molecular mechanism of AO inhibition by H₂O₂ and metronidazole analyzed.

Keywords: Keywords: Amine oxidase; Hydrogen peroxide; Polyamine; Aldehyde; Apoptosis; Histamine

Proteomic analysis of the cell envelope fraction of Escherichia coli by M. Fountoulakis; R. Gasser (pp. 19-41).

We applied proteomics technologies to analyze a membrane preparation of Escherichia coli, wild type strain and of transformants expressing human cytochrome P450s. The proteins were analyzed by two-dimensional electrophoresis and identified by matrix-assisted laser desorption ionization mass spectrometry. The membrane proteins were solubilized with both mild detergents such as CHAPS and strong detergents, such as sodium and lithium dodecyl sulfate, sodium cholate and sodium deoxycholate. In the E. coli membrane fraction, 394 different gene products were identified. Approximately 28% of them were predicted to be integral membrane proteins, of which 100 proteins have been predicted to carry one transmembrane region, ten proteins to carry two, and two proteins to include three transmembrane domains. The remaining are probably membrane-associated and cytosolic proteins. Cytochrome P450s did not enter the immobilized pH gradient strips but were efficiently analyzed in a two-dimensional, two-detergent system. Use of strong solubilizing agents resulted in the detection of about 20 membrane proteins, which were not detected following extraction with mild detergents and chaotropes. The present database is one of the largest for membrane proteins.

Keywords: Keywords: Proteomics; Membrane proteins; Escherichia coli; Matrix-assisted laser desorption ionization mass spectrometry; Hydrophobicity

Chromatographic determination of L- and D-amino acids in plants by H. Brückner; T. Westhauser (pp. 43-55).

Quantities of free L- and D-amino acids (L- and D-AAs) in plants (leaves of coniferous and decidious trees, fleshy fruits, leaf blades of fodder grasses, and seeds and seedlings of edible legumes) were determined. Amino acid (AA) enantiomers were converted into diastereomers using pre-column derivatization with o-phthaldialdehyde together with N-isobutyryl-L(or D)-cysteine followed by separation of the resulting fluorescent isoindol derivatives on an octadecylsilyl stationary phase using high-performance liquid chromatography. Relative amounts of D-AAs were also determined by enantioselective gas chromatography-mass spectrometry on Chirasil-L-Val^®. Free D-AAs acids in the range of about 0.2% up to 8% relative to the corresponding L-AAs acids were found in plants. D-Asp, D-Asn, D-Glu, D-Gln, D-Ser and D-Ala could be detected in most of the plants, and D-Pro, D-Val, D-Leu and D-Lys in certain plants. As D-AAs were detected in gymnosperms as well as mono- and dicotyledonous angiosperms of major plant families it is concluded that free D-AAs in the low percentage range are principle constituents of plants.

Keywords: Keywords: L- and D-amino acids; Chirality; High performance liquid chromatography; Gas chromatography; Mass spectrometry; Plant amino acids; Plant living fossils

Effects of human growth hormone (hrGH) treatment on amine metabolism in rats subjected to extensive small bowel resection by W. A. Fogel; K. Sasiak; J. Socha; W. Andrzejewski (pp. 57-62).

The effect of human recombinant growth hormone (hrGH) on intestinal adaptation in rats subjected to massive small bowel resection has been followed by monitoring changes in the tissue polyamine system and in red blood cell (RBC) polyamine levels. In parallel, the activities of monoamine oxidase A and B and diamine oxidase, the enzymes that catalyse one of the major routes of biogenic amine metabolism, oxidative deamination, were also examined.The results suggest that whilst hrGH treatment accelerates adaptive intestinal hyperplasia evoked by the resection, it has no significant effect on RBC polyamine level or gut mucosal DNA concentration as measured 3 weeks post surgery. hrGH treated operated rats exhibited significantly lower amine oxidase activities which implies that GH may alter biogenic amine systems.

Keywords: Key words: Growth hormone; Intestinal adaptation; Amine metabolism; Erythrocyte polyamines

Polyamine metabolism in primary human colon adenocarcinoma cells (SW480) and their lymph node metastatic derivatives (SW620) by B. Duranton; V. Holl; Y. Schneider; S. Carnesecchi; F. Gossé; F. Raul; N. Seiler (pp. 63-72).

The natural polyamines are multifunctional constituents of all eucaryotic cells. The objective of this work was to compare aspects of polyamine metabolism in two related cell lines with the idea to investigate whether metabolic differences can be attributed to functional differences of the cells. The human colon carcinoma-derived cell lines SW480 and SW620 were chosen as models. SW480 cells were isolated from the primary tumour, SW620 cells from a lymph node of the same patient. SW620 cells grow faster, and the key regulatory enzymes of polyamine biosynthesis (ODC and AdoMetDC) are more active in the metastatic cells. Moreover, their ability to accumulate polyamines from the environment is more important than of SW480 cells. Likewise polyamine concentrations were markedly higher in SW620 cells, although they are much smaller than SW480 cells, and have a particularly small cytoplasmic space. Both cell lines show a striking diminution of ODC and AdoMetDC activities and changes in the polyamine patterns at the transition from exponential to non-exponential growth – most probably as a consequence of high cell density. Depletion of putrescine and spermidine due to inactivation of ODC by DFMO causes accumulation of cells in G1, and a proportional decrease of S-phase cells in both cell lines. Based on morphologic and other criteria SW480 and SW620 cells were typified as poorly differentiated. In agreement with their low grade of differentiation they exhibit a low alkaline phosphatase activity. However, the time-dependent decrease of alkaline phosphatase is not typical of differentiation patterns of other adenocarcinoma-derived cell lines or of normal enterocytes. The high capacity of de novo polyamine biosynthesis and of polyamine uptake is presumably a prerequisite for the rapid growth and invasiveness. The fact that these properties were more accentuated in the case of SW620 cells and paralleled enhanced metastatic properties indicate relationships between basic parameters of polyamine metabolism and malignancy.

Keywords: Keywords: Polyamines; Metabolism; Uptake; Human colon carcinoma cells; SW480; SW620

Degradation of tryptophan and related indolic compounds by ruminal bacteria, protozoa and their mixture in vitro by N. Mohammed; R. Onodera; M. M. Or-Rashid (pp. 73-80).

In vitro experiments were conducted to examine the degradation of d- and l-isomers of tryptophan (Trp) and 10 related indolic compounds by mixed rumen bacteria (B), protozoa (P) and a combination of the two (BP). The analyses were carried out by HPLC. d-Trp (1.0 mM) was not degraded by rumen microorganisms during the 24-h incubation period. The net degradation of 1 mM l-Trp was 46.5%, 8.7% and 80.0% by B, P and BP suspensions, respectively. Trp was degraded into indoleacetic acid, indolelactic acid and indole by rumen bacteria and protozoa, and into skatole, p-cresol and indolepropionic acid by rumen bacteria only. Of them, indoleacetic acid was the major product of Trp found in B (15.4%) and P (3.1%), and skatole in BP (43.2%). This is the first report of the production of indolelactic acid and p-cresol from Trp by rumen microbes. Starch, d-glucose, salinomycin and monensin inhibited the production of skatole and indole from Trp, and skatole from indoleacetic acid by rumen bacteria.

Keywords: Keywords: Tryptophan degradation; Skatole; P-cresol; Indoleacetic acid; Rumen bacteria; Rumen protozoa

Branched chain amino acids as source of specific branched chain volatile fatty acids during the fermentation process of fish sauce by N. G. Sanceda; E. Suzuki; T. Kurata (pp. 81-87).

The source of the formation of branched chain volatile fatty acids (VFA) in fish sauce was investigated. Certain branched VFA were derived from the degradation of specific amino acids as iso-butyric acid from valine and iso-valeric acid from leucine. Short and long straight chain VFA were significantly higher in the linoleic acid added sample than in the control but did not significantly bring changes to the branched chain VFA. It is suggested that straight chain VFA developed from fish fats. Alanine and isoleucine did not have a clear influence on the production of volatile fatty acids.

Keywords: Keywords: Fish sauce; Volatile fatty acids; Straight chain volatile fatty acids; Branched chain volatile fatty acids; Aerobic fermentation; Anaerobic fementation; Microbial activity

Co-variation of plasma sodium, taurine and other amino acid levels in critical illness by C. Chiarla; I. Giovannini; J. H. Siegel (pp. 89-93).

This study investigates the relationship between changes in plasma sodium and changes in amino acid levels in a patient with post-traumatic sepsis and prolonged critical illness. Ninety-two consecutive measurements were performed at regular intervals over a period of many weeks; these consisted in the determination of full amino-acidograms, plasma sodium and complementary variables. A unique, highly significant inverse correlation between taurine and plasma sodium was found (r² = 0.48, p < 0.001). All other amino acids were unrelated, or much more weakly related, to sodium. Taurine was also strongly and directly related to phosphoethanolamine, glutamate and aspartate. Changes in sodium and in levels of these amino acids explained up to 86% of the variability of taurine. Besides, levels of these amino acids maintained a high degree of co-variation, remaining reciprocally related one to each other, directly, with r² ranging between 0.33 and 0.59 (p < 0.001 for all). There were similar findings for β-alanine, which however was measured inconsistently. These data provide gross clinical evidence of a specific link binding plasma sodium and taurine levels, and may be consistent with occurrence of opposite and interdependent shifts of sodium and taurine between intravascular and extravascular space, to maintain osmoregulation. Co-variation of taurine with the other amino acids may be related to the same phenomenon, and/or to similarities in transport systems and chemical structure, or true metabolic interactions.

Keywords: Keywords: Amino acids; Osmoregulation; Sepsis; Acute-phase response; Taurine; Phosphoethanolamine

Mitochondrial nitric oxide inhibits ATP synthesis Effect of free calcium in rat heart by A. Saavedra-Molina; J. Ramírez-Emiliano; M. Clemente-Guerrero; V. Pérez-Vázquez; L. Aguilera-Aguirre; J. C. González-Hernández (pp. 95-102).

Nitric oxide is a small potentially toxic molecule and a diatomic free radical. We report the interaction of L-arginine, oxygen and calcium with the synthesis of nitric oxide in heart mitochondria. Nitric oxide synthesis is increased in broken rat heart mitochondria compared with intact and permeabilized mitochondria. Intact mitochondria subjected to hypoxia-reoxygenation conditions accumulated nitric oxide that inhibits oxygen consumption and ATP synthesis. ATPase activity is not affected during this augment of nitric oxide. Physiological free calcium concentrations protected mitochondria from the damage caused by the accumulation of nitric oxide. Higher concentrations of the divalent cation increase the damage exerted by nitric oxide.

Keywords: Keywords: Heart; Mitochondria; Nitric oxide; Hypoxia-reoxygenation; Calcium; ATP synthesis

Alterations in hepatic metabolism of sulfur-containing amino acids induced by ethanol in rats by S. K. Kim; J. M. Seo; Y. S. Jung; H. E. Kwak; Y. C. Kim (pp. 103-110).

Alterations in hepatic metabolism of S-amino acids were monitored over one week in male rats treated with a single dose of ethanol (3 g/kg, ip). Methionine and S-adenosylhomocysteine concentrations were increased rapidly, but S-adenosylmethionine, cysteine, and glutathione (GSH) decreased following ethanol administration. Activities of methionine adenosyltransferase, cystathionine γ-lyase and cystathionine β-synthase were all inhibited. γ-Glutamylcysteine synthetase activity was increased from t = 8 hr, but GSH level did not return to control for 24 hr. Hepatic hypotaurine and taurine levels were elevated immediately, but reduced below control in 18 hr. Changes in serum and urinary taurine levels were consistent with results observed in liver. Cysteine dioxygenase activity was increased rapidly, but declined from t = 24 hr. The results show that a single dose of ethanol induces profound changes in hepatic S-amino acid metabolism, some of which persist for several days. Ethanol not only inhibits the cysteine synthesis but suppresses the cysteine availability further by enhancing its irreversible catabolism to taurine, which would play a significant role in the depletion of hepatic GSH.

Keywords: Keywords: Ethanol; Transsulfuration pathway; Cysteine; Glutathione; Taurine; Hypotaurine

Protein levels of genes encoded on chromosome 21 in fetal Down syndrome brain: Challenging the gene dosage effect hypothesis (Part I) by M. S. Cheon; S. H. Kim; M.-L. Yaspo; F. Blasi; Y. Aoki; K. Melen; G. Lubec (pp. 111-117).

Down syndrome (DS) is the most significant genetic disorder with mental retardation and is caused by trisomy 21. The phenotype of DS is thought to result from overexpression of a gene(s) located on the triplicated chromosome (region). An increasing body of evidence that challenge this “gene dosage effect” hypothesis, however, has been reported indicating that this hypothesis still remains to be elucidated. The availability of the complete sequence of genes on chromosome 21 could have an immediate impact on DS research, but no conclusions can be drawn from nucleic acid levels. This made us evaluate protein levels of six proteins, gene products, encoded on chromosome 21 (T-cell lymphoma invasion and metastasis inducing Tiam1 protein, holocarboxylase synthetase, human interferon-regulated resistance GTP-binding protein MxA, Pbx regulating protein 1, autoimmune regulator, and pericentrin) in fetal cortex from DS and controls at 18–19 weeks of gestational age using Western blot technique. None of the investigated proteins showed overexpression in DS compared to controls. Our present data showing unaltered expression of six proteins on chromosome 21 in fetal DS brain suggest that the existence of the trisomic state is not involved in abnormal development of fetal DS brain and that the gene dosage effect hypothesis is not sufficient to fully explain the DS phenotype. We are in the process of quantifying all gene products of chromosome 21 and our first results do not support the gene dosage hypothesis.

Keywords: Keywords: AIRE; Chromosome 21; Down syndrome; HCS; MxA; Pericentrin; Prep1; protein expression; Tiam1

Protein levels of genes encoded on chromosome 21 in fetal Down syndrome brain: Challenging the gene dosage effect hypothesis (Part II) by M. S. Cheon; M. Bajo; S. H. Kim; J. O. Claudio; A. K. Stewart; D. Patterson; W. D. Kruger; H. Kondoh; G. Lubec (pp. 119-125).

Down syndrome (DS) is the most common genetic cause of mental retardation. To explain the impact of extra chromosome 21 in the pathology of DS, gene dosage effect hypothesis has been proposed, but several investigators including our group have challenged this hypothesis. Although analysis of the sequence of chromosome 21 has been essentially completed, the molecular and biochemical mechanisms underlying the pathology are still unknown. We therefore investigated expression levels of six proteins encoded on chromosome 21 (HACS1, DYRK1A, αA-crystallin, FTCD, GARS-AIRS-GART, and CBS) in fetal cerebral cortex from DS and controls at 18–19 weeks of gestational age using Western blot analysis. Protein expression of HACS1 was significantly and remarkably decreased in DS, and the expression levels of five proteins were comparable between DS and controls suggesting that the gene dosage effect hypothesis is not sufficient to fully explain the DS phenotype. We are continuing to quantify proteins whose genes are encoded on chromosome 21 in order to provide a better understanding of the pathobiochemistry of DS at the protein level.

Keywords: Keywords: Chromosome 21; Down syndrome; HACS1; DYRK1A; alphaA-crystallin; FTCD; GARS-AIRS-GART; CBS

Protein levels of genes encoded on chromosome 21 in fetal Down syndrome brain: Challenging the gene dosage effect hypothesis (Part III) by M. S. Cheon; S. H. Kim; V. Ovod; N. Kopitar Jerala; J. I. Morgan; Y. Hatefi; T. Ijuin; T. Takenawa; G. Lubec (pp. 127-134).

Down syndrome (DS) is the most frequent genetic disorder with mental retardation and caused by trisomy 21. Although the gene dosage effect hypothesis has been proposed to explain the impact of extra chromosome 21 on the pathology of DS, a series of evidence that challenge this hypothesis has been reported. The availability of the complete sequences of genes on chromosome 21 serves now as starting point to find functional information of the gene products, but information on gene products is limited so far. We therefore evaluated expression levels of six proteins whose genes are encoded on chromosome 21 (synaptojanin-1, chromosome 21 open reading frame 2, oligomycin sensitivity confering protein, peptide 19, cystatin B and adenosine deaminase RNA-specific 2) in fetal cerebral cortex from DS and controls at 18–19 weeks of gestational age using Western blot analysis. Synaptojanin-1 and C21orf2 were increased in DS, but others were comparable between DS and controls, suggesting that the DS phenotype cannot be simply explained by gene dosage effects. We are systematically quantifying all proteins whose genes are encoded on chromosome 21 in order to provide a better understanding of the pathobiochemistry of DS at the protein level. These studies are of significance as they show for the first time protein levels that are carrying out specific function in human fetal brain with DS.

Keywords: Keywords: Adenosine deaminase RNA-specific 2; chromosome 21; Chromosome 21 open reading frame 2; Cystatin B; Down syndrome; Oligomycin sensitivity conferring protein; Peptide 19; Synaptojanin-1

Studies on the utilization of methionine sulfoxide and methionine sulfone by rumen microorganisms in vitro by M. M. Or-Rashid; R. Onodera; S. Wadud (pp. 135-139).

An in vitro experiment was conducted to test the ability of mixed rumen bacteria (B), protozoa (P), and their mixture (BP) to utilize the oxidized forms of methionine (Met) e.g., methionine sulfoxide (MSO), methionine sulfone (MSO₂). Rumen contents were collected from fistulated goats to prepare the microbial suspensions and were anaerobically incubated at 39°C for 12 h with or without MSO (1 mM) or MSO₂ (1 mM) as a substrate. Met and other related compounds produced in both the supernatants and hydrolyzates of the incubation were analyzed by HPLC. During 6- and 12-h incubation periods, MSO disappeared by 28.3 and 42.0%, 0.0 and 0.0%, and 40.6 and 62.4% in B, P, and BP suspensions, respectively. Rumen bacteria and the mixture of rumen bacteria and protozoa were capable to reduce MSO to Met, and the production of Met from MSO in BP (156.6 and 196.1 μmol/g MN) was about 17.3 and 14.1% higher than that in B alone (133.5 and 171.9 μmol/g MN) during 6- and 12-h incubations, respectively. On the other hand, mixed rumen protozoa were unable to utilize MSO. Other metabolites produced from MSO were found to be MSO₂ and 2-aminobutyric acid (2AB) in B and BP. MSO₂ as a substrate remained without diminution in all-microbial suspensions. It was concluded that B, P, and BP cannot utilize MSO₂; but MSO can be utilized by B and BP for producing Met.

Keywords: Keywords: Methionine; Methionine sulfoxide; Methionine sulfone; Rumen microorganisms

Preservation of amino acids during long term ischemia and subsequent reflow with supplementation of L-arginine, the nitric oxide precursor, in the rat heart by M. Desrois; M. Sciaky; C. Lan; P. J. Cozzone; M. Bernard (pp. 141-148).

We investigated whether L-arginine, used in heart preservation to limit endothelial damage, may influence the pool of amino acids during long term ischemia and reflow. Isolated isovolumic rat hearts (n = 23) were submitted to 8 h of hypothermic ischemia after cardioplegic arrest with the Centre de Résonance Magnétique Biologique et Médicale (CRMBM) solution with or without L-arginine (Arg and No Arg groups respectively). Hearts were freeze-clamped after ischemia (n = 11) or submitted to 60 min of reflow (n = 12) and freeze-clamped. Eight hearts were perfused aerobically for 20 min and freeze-clamped (No ischemia group). Addition of L-arginine to the CRMBM solution limited aspartate depletion and decreased lysine level at the end of ischemia. After reflow, L-arginine supplementation increased the pool of glutamate and arginine and limited the depletion of serine, asparagine, glycine and taurine. We conclude that adding L-arginine to the CRMBM cardioplegic solution during long term ischemia preserved the amino acids pool.

Keywords: Keywords: L-arginine; Amino acids; Heart; Nitric oxide; Ischemia; Reperfusion

Determination of the standard Gibbs energies of transfer of cations and anions of amino acids and small peptides across the water nitrobenzene interface by R. Gulaboski; V. Mirčeski; F. Scholz (pp. 149-154).

The standard Gibbs energies of transfer of anions and cations of amino acids and small peptides across the water nitrobenzene interface were determined with the help of a novel electrochemical technique using three-phase electrodes. This is the first time that reliable data are reported for the anions of amino acids. The main result is that the standard Gibbs energies of transfer of the anion and cation of an amino acid are almost the same.

Keywords: Keywords: Amino acids; Voltammetry; Ion transfer; Liphophilicity

Taurine-evoked chloride current and its potentiation by intracellular Ca²⁺ in immature rat hippocampal CA1 neurons by Z.-Y. Wu; T.-L. Xu (pp. 155-161).

Taurine is one of the most abundant free amino acids in the immature mammalian central nervous system. In the present study, whole-cell patch-clamp recordings were made to examine taurine-evoked currents (I _Tau) in acutely dissociated immature rat hippocampal CA1 neurons. Taurine at low concentrations (≤1 mM) activated glycine receptors while at high concentrations (≥3 mM) activated both glycine and GABA_A receptors. Moreover, elevation of intracellular Ca²⁺ via non-NMDA receptor activation enhanced I _Tau reversibly.The results indicate that taurine may act as a native ligand of glycine receptors and modulate neurotransmissions in the immature hippocampus, and under certain conditions it can also activate GABA_A receptors. The potentiation of I _Tau by intracellular Ca²⁺ may contribute to the protection effect of taurine under some cell-damaging conditions.

Keywords: Keywords: Taurine; Glycine receptors; GABAA receptors; Intracellular Ca2+

Effect of D-amino acids on some mitochondrial functions in rat liver by J. C. González-Hernández; L. Aguilera-Aguirre; V. Pérez-Vázquez; J. Ramírez; M. Clemente-Guerrero; C. Cortés-Rojo; A. Saavedra-Molina (pp. 163-169).

We studied the role of the D-amino acids (D-aa) D-serine, D-alanine, D-methionine, D-aspartate, D-tyrosine and D-arginine on rat liver mitochondria. The stability of D-amino acids, mitochondrial swelling, transmembrane potential and oxygen consumption were studied under oxidative stress conditions in rat liver mitochondria. In the presence of glutamate-malate all D-aas salts increased mitochondrial swelling, while in the presence of succinate plus rotenone only D-ala, D-arg and D-ser, induced mitochondrial swelling. The transmembrane potential (ΔΨ) was decreased in the presence of 1 μM Ca²⁺. The D-aas inhibited oxygen consumption in state 3. The D-aa studied exerted effects on mitochondria via an increase of free radicals production.

Keywords: Keywords: Mitochondria; Liver; D-Amino acids; Membrane potential; Mitochondrial Swelling; Free radicals

Volatile constituents of glutathione – ribose model system and its antioxidant activity by K. El-massry; A. Farouk; A. El-Ghorab (pp. 171-177).

Reaction between glutathione and ribose was carried out to study the volatiles formed via Maillard reaction and their antioxidant activity as well as their role in inhibition of LDL oxidation. The simultaneous distillation – extraction technique was used for trapping the volatile components followed by GC – MS analysis. Thirty six compounds were identified with the predominance of carbonyls and sulfur – containing compounds in the volatiles of this model system. Sensory evaluation was performed for the model system product according to the International Standard Methods (ISO). The results showed a high decrease in roasted and burnt attributes and remarkable increase in the like – boiled and roasted meat attributes. The sensory results of the model system product were confirmed by the presence of high concentrations of some volatile compounds having meat – like aroma such as 2-methyl-3-furanthiol and 2-furylmethanethiol. The radical scavenging activity of glutathione – ribose model system was quantified spectrophotometrically, using DPPH radical. The activity of the model system product was found to be slightly lower than that of gallic acid and BHA, but it was much higher than that of cinnamic acid (200 ppm. for each). A highly antioxidative activity was recorded by the model system product during the inhibition of LDL – oxidation in comparison with L-ascorbic acid as well as reduced glutathione (as a concentration of 0.5 μmol/L, for each) which may be due to the presence of some compounds such as 2-furylmethanethiol, 2-acetyl thiazole, 4-hydroxy-5-methyl-3(2H)-furanone.

Keywords: Keywords: Glutathione; Meat-like aroma; Maillard reaction; Sensory evaluation; LDL; DPPH

Isolation of the bifunctional enzyme lysine 2-oxoglutarate reductase-saccharopine dehydrogenase from Phaseolus vulgaris by S. T. Cunha Lima; R. A. Azevedo; L. G. Santoro; S. A. Gaziola; P. J. Lea (pp. 179-186).

Lysine is catabolyzed by the bifunctional enzyme lysine 2-oxoglutarate reductase-saccharopine dehydrogenase (LOR-SDH) in both animals and plants. LOR condenses lysine and 2-oxoglutarate into saccharopine, using NADPH as cofactor and SDH converts saccharopine into α-aminoadipate δ-semialdehyde and glutamic acid, using NAD as cofactor. The distribution pattern of LOR and SDH among different tissues of Phaseolus vulgaris was determined. The hypocotyl contained the highest specific activity, whereas in seeds the activities of LOR and SDH were below the limit of detection. Precipitation of hypocotyl proteins with increasing concentrations of PEG 8000 revealed one broad peak of SDH activity, indicating that two isoforms may be present, a bifunctional LOR-SDH and possibly a monofunctional SDH. During the purification of the hypocotyl enzyme, the LOR activity proved to be very unstable, following ion-exchange chromatography. Depending on the purification procedure, the protein eluted as a monomer of 91–94 kDa containing only SDH activity, or as a dimer of 190 kDa with both, LOR and SDH activities, eluting together.

Keywords: Keywords: Phaseolus vulgaris; Catabolism; Lysine; Lysine 2-oxoglutarate reductase; Saccharopine dehydrogenase

Polyamine oxidase activity in rats treated with mitoguazone: Specific and permanent decrease in thymus by M. E. Ferioli; A. Armanni (pp. 187-194).

To extend the knowledge on the role of polyamine oxidase in thymus physiology, we evaluated the in vivo effect of the polyamine biosynthetic pathway inhibitor mitoguazone. The drug markedly and permanently decreased the enzyme activity in the organ, in which the level of putrescine also decreased at the later times observed. A byproduct of the reaction catalyzed by polyamine oxidase is hydrogen peroxide, a well known inducer of apoptosis. The decrease in polyamine oxidase activity, with the consequent decrease in hydrogen peroxide production, is correlated with a positive effect on thymus physiology. Since mitoguazone has been successfully employed in patients with AIDS-related diseases, in which the reconstitution of the immune function is a favorable prognostic index, we hypothesized that mitoguazone may have the thymus as target organ, and that the decrease in polyamine oxidase activity may have a role in the positive effect of the drug.

Keywords: Keywords: Polyamine oxidase; Spermidine/spermine N1-acetyltransferase; Mitoguazone; Thymus; Rat

Production of cysteine for bacterial and plant biotechnology: Application of cysteine feedback-insensitive isoforms of serine acetyltransferase by M. Wirtz; R. Hell (pp. 195-203).

The first step of cysteine biosynthesis in bacteria and plants consists in the formation of O-acetylserine catalyzed by serine acetyltransferase (SAT). SAT is highly sensitive to feedback inhibition by cysteine as part of the regulatory circuit of cysteine biosynthesis und thus hampers over-expression and fermentation of cysteine in biotechnological production processes. Since plants contain multiple SAT isoforms with different cysteine feedback sensitivity, this resource was exploited to demonstrate the suitability of plant SATs for the production of cysteine in both bacteria and plants. Three new cDNAs encoding SATs were isolated from Nicotiana tabacum. The catalytic activity of SAT4 was insensitive up to 0.6 mM cysteine. Expression of SAT4 in a newly constructed Escherichia coli host strain without endogenous SAT activity yielded a significant accumulation of cysteine in the culture medium compared to expression of cysteine sensitive SATs in the same strain. The application of a similarly insensitive SAT isoform from A. thaliana demonstrated the suitability of this approach to increase cysteine levels in transgenic tobacco plants.

Keywords: Keywords: Plant biotechnology; Bacterial fermentation; Sulfur amino acids; Gene cloning

Free amino acids in the haemolymph of honey bee queens(Apis mellifera L.) by N. Hrassnigg; B. Leonhard; K. Crailsheim (pp. 205-212).

In queen honey bees the free amino acid content in the haemolymph clearly depends on the physiological function and social environment of the individual. While in drones and workers the content of free amino acids increases after emergence until it reaches a peak in 5-day-old animals and decreases afterwards, the amino acid content in queens reaches its highest level (>60 nmol/μl haemolymph) with the onset of egg laying (10 d of age). This level is about 2.5 times more than the highest level found in workers. Queens maintain this high level also when they are older (>30 d) and continue to lay eggs in average colonies. As in drones and workers, in queens the predominant amino acid is proline, which accounts for more than 50% of the total content of free amino acids in egg-laying individuals. When 10-day-old queens are prevented from mating and do not lay eggs, their amino acid content is significantly lower compared to laying queens of the same age. Also the social environment influences the contents of free amino acids in queens. When virgin queens were kept for 6 days with 20 worker bees and sufficient honey and pollen in an incubator, they had significantly lower concentrations of amino acids than virgin queens living for the same period with about 8000 workers in a colony. Most probably, the high amino acid concentration in the haemolymph is the basis for the high protein synthesis activity of laying queens.

Keywords: Keywords: Amino acids; Queen; Apis mellifera; Proline; Egg-production; Social environment

Characterization of N-methyl-D-aspartate-evoked taurine release in the developing and adult mouse hippocampus by P. Saransaari; S. S. Oja (pp. 213-221).

Taurine is an inhibitory amino acid acting as an osmoregulator and neuroromodulator in the brain, with neuroprotective properties. The ionotropic glutamate receptor agonist N-methyl-D-aspartate (NMDA) greatly potentiates taurine release from brain preparations in both normal and ischemic conditions, the effect being particularly marked in the developing hippocampus. We now characterized the regulation of NMDA-stimulated taurine release from hippocampal slices from adult (3-month-old) and developing (7-day-old) mouse using a superfusion system. The NMDA-stimulated taurine release was receptor-mediated in both adult and developing mouse hippocampus. In adults, only NO-generating compounds, sodium nitroprusside, S-nitroso-N-acetylpenicillamine and hydroxylamine reduced the release, as did also NO synthase inhibitors, 7-nitroindazole and nitroarginine, indicating that the release is mediated by the NO/cGMP pathway. On the other hand, the regulation of the NMDA-evoked taurine release proved to be somewhat complex in the immature hippocampus. It was not affected by the NOergic compounds, but enhanced by the protein kinase C activator 4β-phorbol 12-myristate 13-acetate and adenosine receptor A₁ agonists, N⁶-cyclohexyladenosine and R(−)N⁶-(2-phenylisopropyl)adenosine in a receptor-mediated manner. The activation of both ionotropic 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors and metabotropic glutamate group I receptors also enhanced the evoked release. The NMDA-receptor-stimulated taurine release could be a part of the neuroprotective properties of taurine, being important particularly under cell-damaging conditions in the developing hippocampus and hence preventing excitotoxicity.

Keywords: Keywords: Taurine release; N-Methyl-D-aspartate receptors; Hippocampal slices; Adult; Developing mouse

Hamster d-amino-acid oxidase cDNA: rodents lack the 27th amino acid residue in d-amino-acid oxidase by M. Tsuchiya; A. Kurabayashi; R. Konno (pp. 223-226).

The nucleotide sequence of cDNA that encodes hamster d-amino-acid oxidase (DAO) was determined. The cDNA consisted of 1,590 nucleotides and a poly(A) tail. It had an open reading frame for a protein consisting of 346 amino acid residues. The number of the amino acid residues is the same as that of the rat DAO. However, the hamster DAO has one residue more than mouse DAO and one residue less than human, pig, rabbit, and guinea pig DAOs. Amino acid sequence of the hamster DAO was highly similar to those of mouse and rat DAOs: 89% and 88% of the amino acid residues were identical between the hamster and mouse DAOs and between the hamster and rat DAOs, respectively. The homology was slightly less between the hamster DAO and the human (81%), pig (78%), rabbit (78%), or guinea pig DAO (82%). It has been proposed that the mouse and rat DAOs lack an amino acid residue corresponding to the 25th residue of the DAOs of other mammals. However, a detailed comparison of the amino acid sequences as well as the underlying nucleotide sequences by inclusion of the hamster ones revealed that the rodent DAOs does not lack the 25th, but the 27th residue.

Keywords: Keywords: d-Amino-acid oxidase; Hamster; Rodents; Amino acid sequence; Nucleotide sequence; Deletion

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.24, #1-2)