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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.27, #3-4)

Editorial: Is immunochemistry sufficient for protein detection, characterisation and identification? by G. Lubec (pp. 229-229).

Marfey’s reagent for chiral amino acid analysis: A review by R. Bhushan; H. Brückner (pp. 231-247).

The present paper describes characteristics and application of Marfey’s reagent (MR) including general protocols for synthesis of the reagent and diastereomers along with advantages, disadvantages and the required precautions. Applications, and comparison with other derivatizing agents, for the resolution of complex mixtures of DL-amino acids, amines and non-proteinogenic amino acids, peptides/amino acids from microorganisms, cysteine residues in peptides, and evaluation of racemizing characteristics have been discussed. Separation mechanisms of resolution of amino acid diastereomers and replacement of Ala–NH₂ by suitable chiral moieties providing structural analogs and different chiral variants and their application as a derivatizing agent to examine the efficiency, and reactivity of the reagent have been focussed. Use of MR for preparing CSPs for direct enantiomeric resolution has also been included.

Keywords: Keywords: Marfey’s reagent (1-Fluoro-2,4-dinitrophenyl-5-L-alanine amide, FDAA) – Pre-column derivatization – Enantiomeric resolution – Amino acids, proteinogenic, non-proteinogenic – HPLC – Chiral variants – Separation mechanisms

Depletion of the high-abundance plasma proteins by M. Fountoulakis; J.-F. Juranville; L. Jiang; D. Avila; D. Röder; P. Jakob; P. Berndt; S. Evers; H. Langen (pp. 249-259).

Body fluids, like plasma and urine, are comparatively easy to obtain and are useful for the detection of novel diagnostic markers by applying new technologies, like proteomics. However, in plasma, several high-abundance proteins are dominant and repress the signals of the lower-abundance proteins, which then become undetectable either by two-dimensional gels or chromatography. Therefore, depletion of the abundant proteins is a prerequisite for the detection of the low-abundance components. We applied affinity chromatography on blue matrix and Protein G and removed the most abundant human plasma proteins, albumin and the immunoglobulin chains. The plasma proteins, prior to albumin and immunoglobulin depletion, as well the eluates from the two chromatography steps were analyzed by two-dimensional electrophoresis and the proteins were identified by MALDI-TOF-MS. The analysis resulted in the identification of 83 different gene products in the untreated plasma. Removal of the high-abundance proteins resulted in the visualization of new protein signals. In the eluate of the two affinity steps, mostly albumin and immunoglobulin spots were detected but also spots representing several other abundant plasma proteins. The methodology is easy to perform and is useful as a first step in the detection of diagnostic markers in body fluids by applying proteomics technologies.

Keywords: Keywords: Affinity chromatography – Albumin depletion – Antibody chains depletion – Diagnostic markers – High-abundance proteins – Mass spectrometry – Plasma – Proteomics

Characteristics of basal taurine release in the rat striatum measured by microdialysis by S. Molchanova; S. S. Oja; P. Saransaari (pp. 261-268).

Taurine is a sulfur-containing amino acid thought to be an osmoregulator, neurotransmitter or neuromodulator in the brain. Our objective was to establish how much taurine is released in the striatum and examine the mechanisms controlling extracellular taurine concentrations under resting conditions. The experiments were made on rats by microdialysis in vivo. Changes in taurine were compared with those in glutamate, glycine and the non-neuroactive amino acid threonine. Using the zero net flux approach we showed the extracellular concentration of taurine to be 25.2 ± 5.1 μM. Glutamate was increased by tetrodotoxin and decreased by Ca²⁺ omission, glycine and threonine were not affected and both treatments increased extracellular taurine. The basal taurine release was increased by the taurine transport inhibitor guanidinoethanesulfonate and reduced by the anion channel blocker 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid.

Keywords: Keywords: Taurine – Glutamate – Glycine – Threonine – Zero net flux – Synaptic transmission

Aberrant cytosolic acyl-CoA thioester hydrolase in hippocampus of patients with mesial temporal lobe epilepsy by J. W. Yang; T. Czech; J. Yamada; E. Csaszar; C. Baumgartner; I. Slavc; G. Lubec (pp. 269-275).

A series of enzyme alterations has been shown to be associated with several forms of epilepsy, in mesial temporal lobe epilepsy (MTLE), however, information is limited. It was therefore the aim of the study to determine brain enzyme protein expression using a proteomic screening approach. Hippocampi of controls and patients with drug-resistant MTLE were used for evaluation of protein expression. We applied two-dimensional electrophoresis (2-DE) with mass spectrometrical identification and immunoblotting. 2-DE revealed a remarkably decreased spot identified as cytosolic acyl-CoA thioester hydrolase (BACH; EC 3.1.2.2) in patients with MTLE. Western blotting showed absence of bands at 37 kDa in MTLEs using an antibody against mouse BACH and at 140 kDa in MTLEs using anti-rat BACH. This study demonstrates that BACHs were deranged in hippocampus of MTLE patients. This finding may well contribute to the understanding of the still elusive pathomechanisms involved in MTLE.

Keywords: Keywords: Acyl-CoA thioester hydrolase – Epilepsy – Hippocampus – Proteomics

The HPLC resolution of N-2,4-dinitrophenylated amino acids and peptides stereoisomers on naphthylethylcarbamate-β-cyclodextrin bonded phase using the acetonitrile-based mobile phase: evidence for the chiral recognition pattern by S. Chen (pp. 277-284).

A facile method of enantioresolving a variety of α-amino acids and peptides on naphthylethylcarbamate-β-cyclodextrin bonded phases (i.e., SN- and RN-β-CDs) under the elution of acetonitrile-based mobile phase makes use of 2,4-dinitrofluorobenzene (DNFB) as the tagging reagent, which undergoes nucleophilic substitution by the free amino group in alkaline medium to give a N-2,4-dinitrophenyl (DNP) derivative. The resolution is better obtained on RN-β-CD phase and fails to reproduce on the intact β-cyclodextrin bonded phase under the same chromatographic conditions, which strongly suggests that the observed resolution should be due to the interaction of analyte with naphthylethylcarbamate moiety, not with the residual secondary hydroxyl groups on the β-cyclodextrin.

Keywords: Keywords: 2,4-Dinitrofluorobenzene – Amino acid – Peptide – Enantioresolution – Chiral recognition pattern

Hexafluoroacetone as protecting and activating reagent: Site-selective functionalization of iminodiacetic acid by T. Rühl; C. Böttcher; L. Hennig; K. Burger (pp. 285-290).

Hexafluoroacetone was applied as a bidentate protecting and activating agent for the syntheses of RGD-peptide mimetics starting from iminodiacetic acid in solution and on solid phase.

Keywords: Keywords: Iminodiacetic acid – Hexafluoroacetone – Integrins – Site selective functionalization – Solid phase synthesis – Wang resin – PEGA resin – Affinity labeling

Optimal and effective oral dose of taurine to prolong exercise performance in rat by T. Miyazaki; Y. Matsuzaki; T. Ikegami; S. Miyakawa; M. Doy; N. Tanaka; B. Bouscarel (pp. 291-298).

The aim of this study was to determine the effective and optimum dose of taurine for exercise performance and to maintain tissue taurine concentration. Rats received a respective daily dose of 0, 20, 100, and 500 mg/kg body weight of taurine (EC and ET-1, -2, -3 groups, respectively) for two weeks, and then, were subjected to treadmill until exhaustion. The running time to exhaustion was significantly prolonged by 25% and 50% in the ET-2 and -3 groups, respectively, compared to that in the EC group accompanied with maintenance of taurine tissue concentrations. Furthermore, the oxidative glutathione per total glutathione ratio in tissues was inhibited in the ET-2 and -3 groups whereas it was higher in the EC group than in both the no exercise and taurine-administered groups. Therefore the effective and optimal doses of oral taurine administration for two weeks on a transient exercise performance were between 100 and 500 mg/kg/day.

Keywords: Keywords: Taurine – Exercise – Glutathione – Oxidative stress – Lipid hydroperoxide – Administration

The enthalpies of interactions of some L-α-amino acids with urea molecule in aqueous solutions at 298.15 K by B. Pałecz (pp. 299-303).

Dissolution enthalpies of L-α-aminobutyric acid, L-α-isoleucine, L-α-phenylalanine, L-α-methionine, L-α-serine, L-α-threonine, L-α-cysteine, L-α-asparagine and L-α-glutamine in aqueous solutions of urea have been measured by calorimetry at 298.15 K. The obtained results were used to calculate the enthalpic interaction coefficients between the zwitterions of the L-α-amino acids and a molecule of urea in water. These values were interpreted in terms of the hydrophobic or hydrophilic effects of the side chains of amino acids on their interactions with a polar molecule of urea in water.

Keywords: Keywords: L-α-amino acids – Enthalpies of solution – Aqueous solutions of urea

Proteomic identification of collagens and related proteins in human fibroblasts by J. E. Oh; K. Krapfenbauer; G. Lubec (pp. 305-311).

Fibroblasts are used for diagnosis of a series of metabolic diseases and are particularly suitable for the diagnosis of collagen disorders. We aimed to generate a skin fibroblast map that would be suitable for the concomitant determination of collagen and collagen-related proteins.A human skin fibroblast cell line was cultivated, homogenised, proteins extracted and subject to two-dimensional gel electrophoresis with subsequent in-gel-digestion of protein spots and mass spectrometrical identification (MALDI-TOF).Collagen alpha1 (I) chain precursor, collagen alpha1 (III) chain precursor, collagen alpha2 (VI) precursor and collagen modifying enzymes prolyl 4-hydroxylase alpha-2-subunit precursor, procollagen-lysine 2-oxoglutarate 5-dioxygenase 1 and 2, protein disulfide isomerase ER-60 precursor and peptidyl-prolyl cis-trans isomerase were among the abundant proteins.The finding of collagen and collagen-related structures as well as the identification of other metabolic enzyme systems on one 2D gel may propose the use of this proteomic method for further characterization of collagen and collagen-related proteins or for preliminary screening of metabolic disorders.

Keywords: Keywords: Fibroblast – Collagen – Collagen alpha1 chain – Procollagen-lysine – Prolyl 4-hydroxylase – Protein disulfide isomerase

Effects of ornithine on neutrophil (PMN) free amino acid and α-keto acid profiles and immune functions in vitro by J. Mühling; M. Fuchs; M. Campos; J. Gonter; A. Sablotzki; J. Engel; I. D. Welters; M. Wolff; R. Matejec; M. G. Dehne; T. Menges; M. Krüll; G. Hempelmann (pp. 313-319).

The objective of this study was to determine the effects of ornithine on polymorphonuclear leucocyte (PMN) free amino- and α-keto acid profiles, superoxide anion (O₂⁻) generation, hydrogen peroxide (H₂O₂) formation and released myeloperoxidase acitivity (MPO). Exogenous ornithine significantly increased PMN asparagine, glutamine, asparatate, glutamate, arginine, citrulline, alanine, α-ketoglutarate and pyruvate as intracellular ornithine increased. Concerning PMN immune function markers ornithine increased H₂O₂-generation and MPO acitivity while O₂⁻-formation was decreased. We believe therefore that ornithine is important for affecting PMN “susceptible free amino- and α-keto acid pool” although the mechanisms are not yet clear. This may be one of the determinants in PMN nutrition considerably influencing and modulating PMN host defense capability.

Keywords: Keywords: Ornithine – Neutrophil – Amino acids – α-Keto acids – Immune function

The effect of EGF application in gel form on histamine content of experimentally induced wound in mice by A. Babül; B. Gönül; S. Dinçer; D. Erdoğan; C. Özoğul (pp. 321-326).

The factors participating to the wound healing are complex and still obscure. Among these factors, epidermal growth factor (EGF) and histamine by increasing reepithelization and reparation tissue strength via enhancing collagen deposition to the wound site have a beneficial effect. This study was performed to investigate the effect of EGF dosage forms on the histamine content of the experimentally induced wound and some wound healing criters in the mice.Histological investigation of reepithelization, wound tensile strength for healing and collagen maturation, and histamine levels were assessed in the present study. Thirty two mice were divided into control, and EGF treated groups. Controls included three subgroups; untreated (n=5), 0.9% NaCl applied (n=5), and gel applied (n=5). Experimental groups were treated with two forms of EGF; EGF, solution form in 0.9% NaCl (n=5) and the gel form in 0.2% w/w in carbopol 940 (n=7). The discrepancy between these forms were evaluated. This evaluation was done by the application of two forms of EGF for 15 days on experimentally induced wound healing.Gel form of EGF by sustained release from bioadhesive polymer is found to be more effective than the soluble form, on the healing of the wound, by acceleration of reepithelization and increment of wound tensile strength. The tensile strength of the wound indicates the rate of repair and collagen maturation. It has been observed that when physiological saline and carbopol 940 exposed to incision without EGF causes a significant increase in tissue histamine content.According to the results of the present investigation; the histamine content is found to be decreased by EGF gel dosage form treatment, therefore preventing abnormal collagen formation has a beneficial effect on wound healing.

Keywords: Keywords: EGF – Carbomer – Histamine – Wound healing

Effects of taurine in glucose and taurine administration by B. Kaplan; G. Karabay; R. D. Zağyapan; ç. Özer; H. Sayan; İ. Duyar (pp. 327-333).

Taurine has several biological processes such as hypoglycemic action, antioxidation, detoxification, etc. To assess the effect of taurine administration on the guinea pigs with hyperglycemia, blood glucose, C-peptide levels together with morphologic alterations in the pancreatic ultrastructure were investigated in terms of hypoglycemic action and malondialdehyde and total sulfhydryl group levels with regard to oxidation-antioxidation relation. Animals were divided into four groups of six. Glucose supplementation group was administrated a single dose of glucose (400 mg/kg, i.p.) injection. Glucose and taurine supplementation group was administrated glucose treatment (a single dose, 400 mg/kg, i.p.) following taurine (a single dose, 200 mg/kg, i.p.). Taurine and glucose supplementation group was administered taurine treatment (a single dose, 200 mg/kg, i.p.) following glucose treatment (a single dose, 400 mg/kg, i.p.). Control animals received no treatment. Blood samples were collected at the end of the experiments for the determination of glucose, C-peptide (indicator of insulin secretion), lipid peroxidation (thiobarbituric acid reactive substances), and total sulfhydryl groups levels. Pancreatic tissue samples were then collected and processed for transmission electron microscopy. The findings showed that glucose supplementation following taurine administration significantly decreased blood glucose level by increasing C-peptide level and the pancreatic secretion stimulated morphologically and insignificantly changed thiobarbituric acid reactive substances and total sulfhydryl group levels. These observations suggest that taurine administration may be useful in hyperglycemia because of its hypoglycemic and protective effects.

Keywords: Keywords: Taurine – Glucose – Lipid peroxidation – Pancreas

Differential protein expression profile in gastrointestinal stromal tumors by B. Landuyt; H. Prenen; M. Debiec-Rychter; R. Sciot; E. A de Bruijn; A. T. van Oosterom (pp. 335-337).

Gastrointestinal stromal tumors (GISTs) arise from the interstitial cells of Cajal through gain of function mutations of the oncogene KIT. Imatinib offers the first effective treatment for patients with GISTs, but the therapeutic outcome strongly depends on the type of KIT mutation. We used ProteinChip technology to investigate whether GISTs with different KIT mutations express different proteins. In total, 154 proteins were significantly differentially expressed in GISTs with exon 9 KIT mutation compared to GISTs with exon 11 KIT mutation.

Keywords: Keywords: GIST – KIT – Imatinib – SELDI TOF MS – ProteinChip

Mass spectrometrical analysis of phosphoprotein enriched in astrocytes of 15 kDa in mouse hippocampi by J.-H. Shin; J.-M. Delabar; G. Lubec (pp. 339-344).

Phosphoprotein enriched in astrocytes of 15 kDa (PEA-15) is a small protein that was first identified as an abundant phosphoprotein in brain. PEA-15 was characterised so far at the immunochemical level and by a microsequencing attempt. In order to update characterisation of this important structure by advanced methodology unambiguously identifying proteins independent of antibody availability and specificity, we used a proteomic method for this purpose: Performing protein profiling in mouse hippocampi using two dimensional gel electrophoresis with subsequent mass spectrometrical (MS/MS) identification we detected this protein and demonstrate proteomic characterisation of PEA-15 (Q62048). This study enables further specific and unambiguous determination serving as an analytical tool.

Keywords: Keywords: Astrocytic phosphoprotein PEA-15 – Two-dimensional gel electrophoresis – Hippocampus – Mouse

Introduction: Amine oxidases and polyamines in biological systems by E. Agostinelli (pp. 345-346).

The biological functions of polyamine oxidation products by amine oxidases: Perspectives of clinical applications by E. Agostinelli; G. Arancia; L. Dalla Vedova; F. Belli; M. Marra; M. Salvi; A. Toninello (pp. 347-358).

The polyamines spermine, spermidine and putrescine are ubiquitous cell components. If they accumulate excessively within the cells, due either to very high extracellular concentrations or to deregulation of the systems which control polyamine homeostasis, they can induce toxic effects. These molecules are substrates of a class of enzymes that includes monoamine oxidases, diamine oxidases, polyamine oxidases and copper containing amine oxidases. Polyamine concentrations are high in growing tissues such as tumors. Amine oxidases are important because they contribute to regulate levels of mono- and polyamines. These enzymes catalyze the oxidative deamination of biogenic amines and polyamines to generate the reaction products H₂O₂ and aldehyde(s) that are able to induce cell death in several cultured human tumor cell lines. H₂O₂ generated by the oxidation reaction is able to cross the inner membrane of mitochondria and directly interact with endogenous molecules and structures, inducing an intense oxidative stress. Since amine oxidases are involved in many crucial physiopathological processes, investigations on their involvement in human diseases offer great opportunities to enter novel classes of therapeutic agents.

Keywords: Keywords: Polyamines – Amine oxidases – Multidrug resistance – Colon adenocarcinoma – Acrolein – Mitochondria

Signal transduction pathways linking polyamines to apoptosis by C. Pignatti; B. Tantini; C. Stefanelli; F. Flamigni (pp. 359-365).

Polyamines are important multifunctional cellular components and are classically considered as mediators of cell growth and division. Recently polyamines have been also implicated in cell death. Now it appears that polyamines are bivalent regulators of cellular functions, promoting proliferation or cell death depending on the cell type and on environmental signals. This review draws a picture about the role of polyamines in signalling pathways related to apoptotic cell death and the proposed molecular targets of these polycations at the level of the apoptotic cascade. Solid evidence indicates that polyamines may affect the mitochondrial and postmitochondrial phases of apoptosis, by modulating cytochrome c release from mitochondria and activation of caspases. Recently, polyamines have been also implicated in the regulation of the premitochondrial phase of apoptosis, during which upstream apoptotic signal transduction pathways are activated. The studies reviewed here suggest that polyamines may participate in loops involving interaction with signal transduction pathways and activation/expression of proteins that may control cell death or cell growth.

Keywords: Keywords: Spermine – Apoptosis – Spermidine – Mitochondria – MAPK – NF-κB

Polyamines biosynthesis and oxidation in free-living amoebae by P. Ruggeri; G. Laganà; E. Bellocco; C. Fabiano; R. Leonaldi; D. Forino (pp. 367-372).

In this paper we describe the polyamine biosynthesis and oxidation processes, giving an overview about recent results in free-living Amoebae.The protozoa polyamine levels are different in comparison with mammalian cells. Also, the polyamine levels in protozoa cells change if these species are pathological or not for the human beings. All the amoeba strains show high concentrations of 1,3-diaminopropane (DAP), spermidine and acetylspermidine while spermine is absent. In these amoeba a considerable polyamine oxidase activity has been found, which acts on N⁸-acetylspermidine, but not on free polyamines. This enzyme is responsible, together with polyamine acetylase, of DAP synthesis whose function is not well known.

Keywords: Keywords: Polyamines – Acetylpolyamines – Ornithine decarboxylase – S-adenosylmethionine decarboxylase – Polyamine oxydase – Free-living amoebae

Excitotoxic and post-ischemic neurodegeneration: Involvement of transglutaminases by D. Caccamo; A. Campisi; M. Currò; G. Li Volti; A. Vanella; R. Ientile (pp. 373-379).

Neurodegeneration induced by excitotoxicity is a common feature in various neurological disorders. This pathological condition is caused by prolonged stimulation of glutamate receptor subtypes, followed by both intracellular Ca²⁺ overload and activation of specific genes, resulting in synthesis of enzymes involved in cell stress response.Using experimental in vitro models of excitotoxicity, we demonstrated that glutamate exposure up-regulated tissue transglutaminase in primary cultures of both cerebellar granule cells and astrocytes. These changes were consequent to receptor-mediated Ca²⁺ influx, as demonstrated by the inhibition with selective antagonists, MK-801 and GYKI 52466. Early increases in different transglutaminase isoforms were also observed in global cerebral ischemia, which closely resembles neuronal damage caused by NMDA receptor activation.These findings agree with a postulated role for transglutaminases in molecular mechanisms of several neurodegenerative diseases. Indeed, increased cross-linking reactions could be of pathologic relevance, as part of biochemical changes observed in neurological disorders.

Keywords: Keywords: Transglutaminases – Excitotoxicity – Neurodegenerative diseases – Ischemia – Cerebellar granule neurons – Astroglial cells

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.27, #3-4)