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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.32, #1)

Biologically active D-amino acids by G. H. Fisher (pp. 1-1).

Biochemistry of D-aspartate in mammalian cells by H. Homma (pp. 3-11).

Recent investigations have shown that D-aspartate (D-Asp) plays an important physiological role(s) in the mammalian body. Here, several recent studies of free D-Asp metabolism in mammals, focusing on cellular localization in tissues, intracellular localization, biosynthesis, efflux, uptake and degradation are reviewed.D-Asp in mammalian tissues is present in specific cells, indicating the existence of specific molecular components that regulate D-Asp levels and localization in tissues. In the rat pheochromocytoma cell line (PC12) and its subclones, D-Asp is synthesized intracellularly, most likely by Asp racemase(s). Endogenous D-Asp apparently has two different intracellular localization patterns: cytoplasmic and vesicular. In PC12 cells, D-Asp release can occur through three distinct pathways: 1) spontaneous, continuous release of cytoplasmic D-Asp, which is not associated with a specific stimulus; 2) release of cytoplasmic D-Asp via a volume-sensitive organic anion channel that connects the cytoplasm and extracellular space; 3) exocytotic discharge of vesicular D-Asp. Under certain conditions, D-Asp can be released via a mechanism that involves the L-Glu transporter. D-Asp is thus apparently in dynamic flux at the cellular level to carry out its physiological function(s) in mammals.

Keywords: Keywords: D-Aspartate – Localization – Biosynthesis – Efflux – Uptake – Degradation

Alterations in D-amino acid levels in the brains of mice and rats after the administration of D-amino acids by A. Morikawa; K. Hamase; T. Inoue; R. Konno; K. Zaitsu (pp. 13-20).

To mutant ddY/DAO⁻ mice lacking D-amino-acid oxidase activity and normal ddY/DAO⁺ mice, five D-amino acids (D-Asp, D-Ser, D-Ala, D-Leu and D-Pro) were orally administered for two weeks, and the D-amino acid levels were examined in seven brain regions. The levels of D-Asp markedly increased in the pituitary and pineal glands in both strains. In the ddY/DAO⁺ mice, the levels of the other D-amino acids did not significantly change in most of the brain regions. While in the ddY/DAO⁻ mice the levels of D-Ser significantly increased in most of the brain regions except for the cerebrum and hippocampus. The levels of D-Ala and D-Leu increased in all regions but the levels of D-Pro did not significantly change. The same five D-amino acids were intravenously injected into Wistar rats and the D-amino acid levels in their brains were examined for 60 min after the administration. The levels of D-Asp markedly increased in the pineal gland 3 min after the administration, while the levels of D-Ser, D-Ala, and D-Pro increased both in the pineal and pituitary glands, the levels of D-Leu increased in all brain regions. These results are useful for the elucidation of the origins and regulation of D-amino acids in the mammalian body.

Keywords: Keywords: D-Amino acid – Brain distribution – D-Amino-acid oxidase – HPLC

D-Aspartic acid and L-amino acids in the neural system of the amphioxus Branchiostoma lanceolatum by S. D’Aniello; J. Garcia-Fernàndez (pp. 21-26).

The lancelet (amphioxus), a cephalochordate, is the closest invertebrate relative to vertebrates, with a simple vertebrate-like body plan and a prototypical genome. We have determined D-aspartic acid (D-Asp) and major free L-amino acids (L-AAs) content in the nervous system (neural tube) of the European amphioxus Branchiostoma lanceolatum, and have compared these values with those of molluscs and human brain. The B. lanceolatum neural tube contains relatively high amounts of L-Glu, L-Asp, L-Ala and L-Gly. Thus, the amphioxus neural tube has in common with the molluscan and human nervous systems the presence of appreciable amounts of L-Glu and L-Asp, which suggests that they are the most common neurotransmitters among these phylogenetically distant animal groups. The relatively high concentration of L-Ala in amphioxus is consistent with that found in molluscs and the low concentration of taurine is consistent with that described in the human brain.The D-Asp concentration, very high in the molluscan nervous system, was rather low in amphioxus, although a little higher than the extremely low amounts observed in the human brain. Our data on free amino acids composition is in agreement with the intermediate phylogenetic position of cephalochordates, in terms of the evolutionary transition from simple to complex neural systems.

Keywords: Keywords: Amphioxus – D-aspartic acid – L-amino acids – Nervous system – GABA – Chordates – Evolution

Is there a correlation between age and D-aspartic acid in human knee cartilage? by G. Fisher; S. Lopez; K. Peterson; T. Goff; I. Philip; R. Gaviria; N. Lorenzo; M. Tsesarskaia (pp. 27-30).

L-Aspartic acid (L-Asp) is one of the fastest racemizing amino acids such that the abnormal D-form (D-Asp) has been found in stable biological human tissues such as dentin in teeth, eye lens and brain. Earlier reports showed that there was a linear correlation between age and D-Asp in teeth. We have previously reported that significant levels of D-Asp were found in normal and osteoarthritic knee cartilage. Since cartilage is a slow regenerating tissue, we hypothesized that D-Asp should accumulate in knee cartilage and that there might be a correlation between the age of the person and the amount of D-Asp found in cartilage. Our analysis of approximately 100 samples of normal knee cartilage showed that there are detectable amounts of D-Asp (2–4% of total Asp) in knee cartilage. However, there was only a slight correlation (r = 0.35) between the age of the person and the amount of D-Asp (nmoles/g). Surprisingly, there was a better correlation between age and the amount of D-Asp in the male subjects (r = 0.57) than in the female subjects (r = 0.21).

Keywords: Keywords: D-Aspartic acid – D-Asp – Knee cartilage

Effects of D-amino acids on lipoperoxidation in rat liver and kidney mitochondria by C. Cortés-Rojo; M. Clemente-Guerrero; A. Saavedra-Molina (pp. 31-37).

The effects of the amino acids D-ser, D-asp, and D-ala on lipoperoxidation under conditions of hypertension, alcoholism, and ammonemia in rat liver and kidney mitochondria were studied. Under normal conditions, D-alanine increased in 54% free radicals production in liver mitochondria (p < 0.05). The D-amino acids had no effect on kidney mitochondria. D-ser and D-ala increased lipoperoxidation in spontaneously hypertensive rats (SHR) as compared with their normotensive genetic control Wistar-Kyoto (WKY) rats (p < 0.05). During hypertension and in oxidative stress in the presence of calcium, only D-ala produced 46% and 29% free radicals in liver and kidney mitochondria (p < 0.05), respectively. During chronic alcoholism, D-ser increased lipoperoxidation in 80% in kidney mitochondria (p < 0.05), as compared to control. During ammonemia, D-ser produced 41% free radicals.

Keywords: Keywords: Lipoperoxidation – D-amino acids – Hypertension – Oxidative stress – Chronic alcoholism – Liver mitochondria – Kidney mitochondria

The influence of manufacture on the free D-amino acid content of Cheddar cheese by J. Csapó; É. Varga-Visi; K. Lóki; Cs. Albert (pp. 39-43).

The changes in the concentration and those of composition of alanine, aspartic acid and glutamic acid enantiomers were investigated during manufacture of Cheddar cheese. The amount of D-alanine increased continuously during ripening following the liberation of L-alanine originated from the proteolysis of milk proteins. There was slightly more D-aspartic and D-glutamic acid in the dry matter of curd after pressing than before pressurization. The D-amino acid content and the ratio of the D-enantiomers related to the total amount of free amino acids differed significantly among cheeses produced with different single-strain starters. The D-amino acid composition changed during manufacture, but the influence of the strain selection was not significant on the D-amino acid pattern.

Keywords: Keywords: D-alanine – D-aspartic acid – D-glutamic acid – Cheddar cheese manufacture – Autolysis

Involvement of D-Asp in P450 aromatase activity and estrogen receptors in boar testis by C. Lamanna; L. Assisi; V. Botte; M. M. Di Fiore (pp. 45-51).

Mammalian testis contains D-aspartic acid (D-Asp), which enhances testosterone production. D-Asp, on other hand, also stimulates 17β-estradiol synthesis in the ovary of some lower vertebrates. We studied boar testis in order to determine if D-Asp intervenes in 17β-estradiol synthesis in the testis of those mammals which produce significant amounts of estrogens as well as testosterone. The boar testis contains D-Asp (40 ± 3.6 nmol/g tissue) which, according to immunohistological techniques, is localized mainly in Leydig cells, and, to a lesser extent, in sustentacular (Sertoli), peritubular and some germ cells. The enzyme P450aromatase is present in Leydig cells and few germ cells. In vitro experiments showed that the addition of D-Asp to testicular tissue extracts induced a significant increase of aromatase activity, as evaluated by testosterone conversion into 17β-estradiol. The enzyme’s K_m was not affected by D-Asp (about 25 nM in both control and D-Asp added tests). On the basis of these results we suggest that, as in the ovary, D-Asp is involved in the local control of aromatase activity of boar testis and, therefore, it intervenes in the 17β-estradiol production. In the testis, the D-Asp targets are presumably the Leydig cells, which having also a nuclear estrogen receptor are, in turn, one of the putative targets of the 17β-estradiol that they produce (autocrine effect).

Keywords: Keywords: P450arom – D-Asp – Estrogen receptors – 17β-estradiol synthesis – Boar testis

Modulation of an AMPA-like glutamate receptor (SqGluR) gating by L- and D-aspartic acids by E. R. Brown; S. Piscopo; J.-T. Chun; M. Francone; I. Mirabile; A. D’Aniello (pp. 53-57).

L- and D-aspartic acids (L-Asp and D-Asp) are present in the majority of nervous systems. In phylogeny, significant levels have been reported in mollusc brains, particularly cephalopods. To examine the role of L- and D-Asp on a cephalopod receptor, we studied ligand gating of a squid glutamate receptor (SqGluR) expressed in HEK 239 (human embryonic kidney) cells. Under voltage clamp, application of L-glutamate (L-Glu; 1–30 mM), but not D-glutamate (D-Glu), or L- or D-Asp, evoked an inward current of 0.1 nA. L- or D-Asp (200 µM) applied with 20 mM L-Glu, slowed the time course of activation and inactivation of the L-Glu gated current (time constant increased from 1 s (L-Glu alone) to 3 s (D-Asp and L-Glu) and to 19 s (L-Asp and L-Glu)). Our results suggest that in molluscan systems, aspartic acid could act as a neuromodulator during glutamatergic transmission and could significantly alter synaptic integration by slowing glutamate receptor gating.

Keywords: Keywords: Glutamate receptor – SqGluR – Heterologous expression – L- and D-aspartic acid – Neuromodulation

Alanine racemase from the green alga Chlamydomonas reinhardtii by K. Nishimura; Y. Tomoda; Y. Nakamoto; T. Kawada; Y. Ishii; Y. Nagata (pp. 59-62).

Chlamydomonas reinhardtii, a unicellular green microalga, could grow to a stationary phase having optical density of 2.0–2.5 at 750 nm in Tris-acetate-phosphate (TAP) medium containing 0.1% D-alanine. D-alanine has no inhibitory effect on growth and induced alanine racemase activity 130-fold more than without D-alanine in the green alga. Although C. reinhardtii cultured in the TAP medium showed alanine racemase activity, the content of free D-alanine was only 0.14%. The enzyme was partially purified by ammonium sulfate fractionation followed by three kinds of liquid chromatography using DEAE Toyopearl, Phenyl Sepharose, and TSK G3000 SWXL columns. The specific activity for L-alanine of the partially purified alanine racemase was 3.8 µmol/min/mg. The molecular weight of the enzyme was determined to be approximately 72,000 by gel filtration. The enzyme showed a maximum activity at 45 °C and pH 8.4 and requires pyridoxal 5′-phosphate as a coenzyme.

Keywords: Keywords: Alanine racemase – D-Amino acid – D-Alanine, Chlamydomonas reinhardtii – Green alga

Characterization and isolation of L-to-D-amino-acid-residue isomerase from platypus venom by A. M. Torres; M. Tsampazi; E. C. Kennett; K. Belov; D. P. Geraghty; P. S. Bansal; P. F. Alewood; P. W. Kuchel (pp. 63-68).

Platypus venom contains an isomerase that reversibly interconverts the second amino-acid residue in some peptides between the L-form and the D-form. The enzyme acts on the natriuretic peptides OvCNPa and OvCNPb, and on the defensin-like peptides DLP-2 and DLP-4, but it does not act on DLP-1. While the isomerization of DLP-2 to DLP-4 is inhibited by the amino-peptidase inhibitor amastatin, it is not affected by the leucine amino-peptidase inhibitor bestatin. The enzyme, that is only present in minute quantities in an extract of the venom gland, is thermally stable up to 55 °C, and it was found by anion-exchange chromatography to be acidic. Isolation of the isomerase was carried out by combined ion-exchange chromatography and reverse-phase high performance liquid chromatography (HPLC).

Keywords: Keywords: DLP – Peptide isomerase – Platypus venom peptides

Molecular cloning of a cDNA encoding mouse D-aspartate oxidase and functional characterization of its recombinant proteins by site-directed mutagenesis by M. Katane; T. Furuchi; M. Sekine; H. Homma (pp. 69-78).

The cDNA encoding D-aspartate oxidase (DASPO) was cloned from mouse kidney RNA by RT–PCR. Sequence analysis showed that it contained a 1023-bp open reading frame encoding a protein of 341 amino acid residues. The protein was expressed in Escherichia coli with or without an N-terminal His-tag and had functional DASPO activity that was highly specific for D-aspartate and N-methyl-D-aspartate. To investigate the roles of the Arg-216 and Arg-237 residues of the mouse DASPO (mDASPO), we generated clones with several single amino acid substitutions of these residues in an N-terminally His-tagged mDASPO. These substitutions significantly reduced the activity of the recombinant enzyme against acidic D-amino acids and did not confer any additional specificity to other amino acids. These results suggest that the Arg-216 and Arg-237 residues of mDASPO are catalytically important for full enzyme activity.

Keywords: Keywords: D-aspartate oxidase – Flavoprotein – D-aspartate – Mouse – cDNA cloning – Site-directed mutagenesis

Isolation and characterization of mammalian D-aspartyl endopeptidase by T. Kinouchi; H. Nishio; Y. Nishiuchi; M. Tsunemi; K. Takada; T. Hamamoto; Y. Kagawa; N. Fujii (pp. 79-85).

The accumulation of D-isomers of aspartic acid (D-Asp) in proteins during aging has been implicated in the pathogenesis of Alzheimer’s disease (AD), cataracts and arteriosclerosis. Here, we identified a specific lactacystin-sensitive endopeptidase that cleaves the D-Asp-containing protein and named it D-aspartyl endopeptidase (DAEP). DAEP has a multi-complex structure (MW: 600 kDa) and is localized in the inner mitochondrial membrane. However, DAEP activity was not detected in E. coli, S. cerevisiae, and C. elegans. A specific inhibitor for DAEP, i-DAEP: (benzoyl-L-Arg-L-His-[D-Asp]-CH₂Cl; MW: 563.01), was newly synthesized and inhibited DAEP activity (IC₅₀, 3 µM), a factor of ten greater than lactacystin on DAEP. On the other hand, i-DAEP did not inhibit either the 20S or 26S proteasome. And we identified succinate dehydrogenase and glutamate dehydrogenase 1 as components of DAEP by affinity label using biotinylated i-DAEP. In the long life span of mammals, DAEP may serve as a scavenger against accumulation of racemized proteins in aging. Insights into DAEP will provide the foundation for developing treatments of diseases, such as AD, in which accumulation of D-Asp-containing proteins are implicated.

Keywords: Keywords: D-Aspartate – Protease – Mitochondria – Alzheimer’s disease – Amyloid

Age-related changes of alpha-crystallin aggregate in human lens by N. Fujii; Y. Shimmyo; M. Sakai; Y. Sadakane; T. Nakamura; Y. Morimoto; T. Kinouchi; Y. Goto; K. Lampi (pp. 87-94).

Lens alpha-crystallin, composed of two subunits alpha A- and alpha B-crystallin, forms large aggregates in the lens of the eye. The present study investigated the aggregate of human lens alpha-crystallin from elderly and young donors. Recombinant alpha A- and alpha B-crystallins in molar ratios of alpha A to alpha B at 1:1, corresponding to the aged sample, were also studied in detail. We found by ultra-centrifugation analysis that the alpha-crystallin aggregate from elderly donors was large and heterogeneous with an average sedimentation coefficient of 30 S and a range of 20–60 S at 37 °C. This was higher compared to the young samples that had an average sedimentation coefficient of 17 S. The sedimentation coefficients of recombinant alpha A- and alpha B-crystallins were approximately 12 S and 15 S, respectively. Even when recombinant alpha-crystallins were mixed in molar ratios equivalent to those found in vivo, similar S values as the native aged alpha-crystallin aggregates were not obtained.Changes in the self-association of alpha-crystallin aggregate were correlated to changes in chaperone activity. Alpha-crystallin from young donors, and recombinant alpha A- and alpha B-crystallin and their mixtures showed chaperone activity, which was markedly lost in samples from the aged alpha-crystallin aggregates.

Keywords: Keywords: Aging – Aggregation – Analytical ultracentrifugation – Lens alpha-crystallin

Enhancement of transglutaminase activity and polyamine depletion in B16-F10 melanoma cells by flavonoids naringenin and hesperitin correlate to reduction of the in vivo metastatic potential by A. Lentini; C. Forni; B. Provenzano; S. Beninati (pp. 95-100).

The in vitro and in vivo effects of two flavonons, naringenin (NG) and hesperitin (HP) on the proliferation rate of highly metastatic murine B16-F10 melanoma cell were investigated. NG or HP treatment of melanoma cells produced a remarkable reduction of cell proliferation, paralleled with both the lowering of the intracellular levels of polyamine, spermidine and spermine and the enhancement of transglutaminase (TGase, EC 2.3.2.13) activity. Orally administered NG or HP in C57BL6/N mice inoculated with B16-F10 cells affected the pulmonary invasion of melanoma cells in an in vivo metastatic assay. The number of lung metastases detected by a computerized image analyzer was reduced, compared to untreated animals, by about 69% in NG-treated mice and by about 36% in HP-treated mice. Survival studies showed that 50% of the NG-treated animals died 38 ± 3.1 days after tumor cell injection (control group: 18 ± 1.5 days) and HP-treated mice died 27 ± 2.3 days after cell inoculation. Taken together, these findings provide further evidences for the potential anticancer properties of dietary flavonoids as chemopreventive agents against malignant melanoma.

Keywords: Keywords: Flavonoids – Melanoma – Metastasis – Transglutaminase – Polyamines

Abnormal changes of plasma acute phase proteins in schizophrenia and the relation between schizophrenia and haptoglobin (Hp) gene by C. Wan; Y. La; H. Zhu; Y. Yang; L. Jiang; Y. Chen; G. Feng; H. Li; H. Sang; X. Hao; G. Zhang; L. He (pp. 101-108).

In this study we focused on detecting schizophrenia related changes of plasma proteins using proteomic technology and examining the relation between schizophrenia and haptoglobin (Hp) genotype. We investigated plasma proteins from schizophrenic subjects (n = 42) and healthy controls (n = 46) by two-dimensional gel electrophoresis (2-DE) in combination with mass spectrometry. To further reveal the genetic relationship between acute phase proteins (APPs) and schizophrenia disease, we tested Hp α1/Hp α2 (Hp 1/2) polymorphism and two single nucleotide polymorphisms (SNPs) of Hp, rs2070937 and rs5473, for associations with schizophrenia in the Chinese Han population. With the relatively high number of samples for 2-DE work, we found that four proteins in the family of positive APPs were all up-regulated in patients. In genetic association study, we found significant associations existing between schizophrenia and Hp polymorphisms, Hp 1/2 and rs2070937 variants. Schizophrenia is accompanied by both an altered expression of Hp protein and a different genotype distribution of Hp gene, demonstrating that Hp is associated with schizophrenia. The results from proteomic and genomic aspects both indicate that acute phase reaction is likely to be an aetiological agent in the pathophysiology of schizophrenia, but not just an accompanying symptom. The positive APPs are schizophrenic related proteins, with the highly concordant results on four positive APPs.

Keywords: Keywords: Schizophrenia – Acute phase proteins – Proteomics – Plasma – Haptoglobin

Could activated tissue remodeling be considered as early marker for progressive valve degeneration? Comparative analysis of checkpoint and ECM remodeling gene expression in native degenerating aortic valves and after bioprosthetic replacement by K. Yeghiazaryan; D. Skowasch; G. Bauriedel; H. Schild; O. Golubnitschaja (pp. 109-114).

Objectives. Aortic stenosis is the leading cause of heart valve disease in elderly. Little is known about molecular mechanisms leading to altered left ventricular geometry generally and, particularly, to remodeling of degenerating aortic valve. Alterations in native degenerating aortic valves and valvular tissue after replacement might result from a stage specific tissue remodeling protein core induced by stress responsible factors. Here we were looking for a possible stage specificity of tissue remodeling and stress responsive checkpoint gene activation in native degenerating human aortic valves and bioprosthetic valvular tissue after replacement. Materials and methods. Specimens of native degenerating aortic valves as well as bioprosthetic valves after replacement were tested for their morphological properties. Native degenerating valves were selected for two groups: non-calcified (7 samples) and calcified (5 samples) one; the third group (5 samples) was consisting of bioprosthetic valve samples after replacement. Individual mRNA-pools were isolated from each tissue sample, and semi-quantitative RT-PCR was performed. Target transcripts of p21^waf1/cip1, MT1-MMP, MMP-2, MMP-9 and TIMP-1 were measured. The specificity was controlled by restriction analysis of PCR products. Results and conclusions. According to the abundant expression of p21^waf1/cip1, a highly activated stress response was found in non-calcified native degenerating aortic valves, whereas no stress response was monitored in valvular tissue after replacement. Whereas MT1-MMP expression was almost equally induced in all three groups investigated, MMP-9 was higher expressed in non-calcified versus calcified native valves, and was not expressed after replacement. An induced expression of MMP-2 was detected in non-calcified native degenerating aortic valves only. An abundant expression of tissue inhibitor of metalloproteinases TIMP-1 was observed in all three groups tested. Apparently, the ECM degradation potential is specifically enhanced in non-calcified native degenerating aortic valves e.g. at the early degeneration stages. In contrast, the replaced valves were found to be actively resorbing tissue with no detectable stress response, where both MT1-MMP and TIMP-1 might play the key role in geometry remodeling.

Keywords: Keywords: Degenerating aortic valves – Bioprosthetic valvular replacement – Stress responsive checkpoints – Extracellular matrix remodeling – Metalloproteinases – Diagnostic molecular marker

Decreases in taurine levels induced by β-alanine treatment did not affect the susceptibility of tissues to lipid peroxidation by H. Parıldar-Karpuzoğlu; S. Doğru-Abbasoğlu; J. Balkan; G. Aykaç-Toker; M. Uysal (pp. 115-119).

We aimed to investigate the effect of decreased taurine levels on endogenous and induced lipid peroxide levels in liver, brain, heart and erythrocytes as well as prooxidant and antioxidant balance in the liver of rats administered β-alanine (3%, w/v) in drinking water for 1 month to decrease taurine levels of tissues. This treatment caused significant decreases in taurine levels of liver (86%), brain (36%) and heart (15%). We found that endogenous and ascorbic acid-, NADPH- and cumene hydroperoxide-induced malondialdehyde (MDA) levels did not change in the liver, brain and heart homogenates following β-alanine treatment. Also, H₂O₂-induced MDA levels remained unchanged in erythrocytes. In addition, we did not observe any changes in levels of MDA, diene conjugates, glutathione, α-tocopherol, ascorbic acid and the activities of superoxide dismutase, glutathione peroxidase and glutathione transferase in the liver. According to this, buffering or sequestering capacity of tissues to exogenous stimuli was not influenced by reduced taurine levels in tissues of rats.

Keywords: Keywords: Taurine – β-Alanine – Lipid peroxidation – Antioxidants – Rats

Influence of pegylated interferon-α therapy on plasma levels of citrulline and arginine in melanoma patients by D. Fekkes; M. Bannink; W. H. J. Kruit; A. R. Van Gool; P. G. H. Mulder; S. Sleijfer; A. M. M. Eggermont; G. Stoter (pp. 121-126).

The aim of this study was to evaluate the effect of pegylated interferon-alpha (PEG-IFN-α) on the plasma citrulline/arginine ratio, regarded as an index of nitric oxide (NO) synthesis, in patients with high-risk melanoma. Forty patients were randomly assigned to either PEG-IFN-α treatment (n = 22) or to observation only (control group, n = 18). The treatment group received 6 µg PEG-IFN-α/kg once a week during 8 weeks, followed by a maintenance dose of 3 µg/kg/wk. Blood was collected at different time points, plasma concentrations of citrulline and arginine were measured and the ratio of citrulline/arginine was calculated. Patients treated with PEG-IFN-α showed a significant decrease in the concentrations of citrulline and in the citrulline/arginine ratio during the whole study period, both compared to baseline values and to the control group. The data suggest that therapy with PEG-IFN-α results in a marked decrease in the synthesis of NO in melanoma patients.

Keywords: Keywords: Interferon – Nitric oxide – Melanoma – Citrulline – Arginine

The role of L-arginine in toxic liver failure: interrelation of arginase, polyamine catabolic enzymes and nitric oxide synthase by J. Nikolic; I. Stojanovic; R. Pavlovic; D. Sokolovic; G. Bjelakovic; S. Beninati (pp. 127-131).

The existing interrelation in metabolic pathways of L-arginine to polyamines, nitric oxide (NO) and urea synthesis could be affected in sepsis, inflammation, intoxication and other conditions. The role of polyamines and NO in the toxic effect of mercury chloride on rat liver function was studied. Administration of mercury chloride for 24 h led to significantly elevated plasma activities of Alanine transaminase (ALT) and Aspartate transaminase (AST). Malondyaldehyde (MDA) levels were unaffected (p > 0.05) and arginase activity was significantly decreased (p < 0.05) while nitrate/nitrite production was significantly elevated (p < 0.001) in liver tissue. Polyamine oxidase (PAO) and diamine oxidase (DAO) activities, enzymes involved in catabolism of polyamines, were decreased. L-arginine supplementation to intoxicated rats potentiated the effect of mercury chloride on NO production and it was ineffective on arginase activity.Results obtained in this study show that mercury chloride-induced toxicity leads to abnormally high levels of ALT and AST that may indicate liver damage with the involvement of polyamine catabolic enzymes and NO.

Keywords: Keywords: Arginase – Nitric oxide synthase – Polyamine oxidase – Diamine oxidase – Mercury chloride – Liver

The biological properties of lysine-derived surfactants by L. Sánchez; M. Mitjans; M. R. Infante; M. T. García; M. A. Manresa; M. P. Vinardell (pp. 133-136).

We examine the effects of aquatic toxicity on Daphnia magna, the antimicrobial activity of new anionic lysine-derivative surfactants, and the influence of different-sized counterions associated with the surfactants. Surfactants with Tris and Lithium had less of a toxic effect on Daphnia, while all surfactants proved highly active against yeasts and the gram-negative bacteria Bordetella bronchiseptica. Counterion size was found to have no effect on aquatic toxicity or antimicrobial activity.

Keywords: Keywords: Surfactants – Lysine – CMC – Aquatic toxicity – Antimicrobial activity

High concentrations of D-amino acids in human gastric juice by Y. Nagata; T. Sato; N. Enomoto; Y. Ishii; K. Sasaki; T. Yamada (pp. 137-140).

The concentrations of free D- and L-amino acids were determined in the gastric juice from four groups: patients suffering from early gastric carcinoma with or without Helicobacter pylori infection, and patients without carcinoma but with peptic ulcers, duodenal ulcers or chronic gastritis with or without H. pylori infection. H. pylori is a bacterium associated with gastric inflammation and peptic ulcers and is a risk factor for stomach cancer. The highest D-amino acid ratios (free D-amino acid concentration to the total corresponding free D- and L-amino acid concentration) were 29%, 26%, 18%, 4% and 1% for proline, alanine, serine, aspartate and glutamate, respectively. The gastric juice levels of L-alanine, L-serine, L-proline, L-glutamate and D-alanine in the samples obtained from subjects bearing early gastric carcinoma and H. pylori were significantly higher than in the samples from the other three groups. Except for D-alanine, there was no correlation between the D-amino acid concentrations and presence of carcinoma or H. pylori.

Keywords: Keywords: D-Amino acids – Gastric juice – Gastric carcinoma – H. pylori

Manifold decrease of sialic acid synthase in fetal Down syndrome brain by T. Gulesserian; E. Engidawork; M. Fountoulakis; G. Lubec (pp. 141-144).

Background: Down syndrome (DS, trisomy 21) is the most common genetic cause of mental retardation. A large series of biochemical defects have been observed in fetal and adult DS brain that help in unraveling the molecular mechanisms underlying mental retardation. Aims: As sialylation of glycoconjugates plays an important role in brain development, this study aimed to look at the sialic acid metabolism by measuring sialic acid synthase (SAS; N-acetylneuraminate synthase) in early second trimester fetal control and DS brain. Results: In this regard, protein profiling was performed by two-dimensional gel electrophoresis coupled to matrix-assisted laser desorption/ionization mass-spectrometry followed by database search and subsequent quantification of spot using specific software. SAS, the enzyme catalyzing synthesis of N-acetyl-neuraminic acid (syn: sialic acid) was represented as a single spot and found to be significantly and manifold reduced (P < 0.01) in cortex of fetuses with DS (control vs. DS, 0.052 ± 0.025 vs. 0.012 ± 0.006). Conclusion: The intriguing finding of the manifold decrease of SAS in DS fetal cerebral cortex as early as in the second trimester of pregnancy may help to explain the brain deficit observed in DS. Decreased SAS may well lead to altered sialic acid metabolism, required for brain development and, more specifically, for sialylation of key brain proteins, including neuronal cell adhesion molecule and myelin associated glycoprotein.

Keywords: Keywords: Sialic acid synthase – Sialic acid – Two-dimensional gel electrophoresis – Down syndrome – Fetal brain

Focus on phosphohistidine by P. V. Attwood; M. J. Piggott; X. L. Zu; P. G. Besant (pp. 145-156).

Phosphohistidine has been identified as an enzymic intermediate in numerous biochemical reactions and plays a functional role in many regulatory pathways. Unlike the phosphoester bond of its cousins (phosphoserine, phosphothreonine and phosphotyrosine), the phosphoramidate (P–N) bond of phosphohistidine has a high ΔG° of hydrolysis and is unstable under acidic conditions. This acid-lability has meant that the study of protein histidine phosphorylation and the associated protein kinases has been slower to progress than other protein phosphorylation studies.Histidine phosphorylation is a crucial component of cell signalling in prokaryotes and lower eukaryotes. It is also now becoming widely reported in mammalian signalling pathways and implicated in certain human disease states. This review covers the chemistry of phosphohistidine in terms of its isomeric forms and chemical derivatives, how they can be synthesized, purified, identified and the relative stabilities of each of these forms. Furthermore, we highlight how this chemistry relates to the role of phosphohistidine in its various biological functions.

Keywords: Keywords: Phosphohistidine – Histidine kinase – Phosphoramidate

Adrenomedullin: molecular mechanisms and its role in cardiac disease by B. Yanagawa; N. Nagaya (pp. 157-164).

Adrenomedullin (AM) is a potent, long-lasting vasoactive peptide originally isolated from human pheochromocytoma. Since its discovery, serum and tissue AM expression have been shown to be increased in experimental models and in patients with cardiac hypertrophy, myocardial infarction and end-stage heart failure with several beneficial effects. Considerable evidence exists for a wide range of autocrine, paracrine and endocrine mechanisms for AM which include vasodilatory, anti-apoptotic, angiogenic, anti-fibrotic, natriuretic, diuretic and positive inotropic. Thus, through regulation of body fluid or direct cardiac mechanisms, AM has additive and beneficial effects in the context of heart disease. Notable molecular mechanisms of AM include cyclic adenosine monophosphate, guanosine-3′,5′-monophosphate, PI3K/Akt and MAPK-ERK-mediated cascades. Given the endogenous and multifunctional nature of AM, we consider this molecule to have great potential in the treatment of cardiovascular diseases. In agreement, early experimental and preliminary clinical studies suggest that AM is a new and promising therapy for cardiovascular diseases.

Keywords: Keywords: Adrenomedullin – Heart disease – Signaling

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.32, #1)

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.32, #1)