Amino Acids (v.34, #3)

The effect of glutamine supplementation and physical exercise on neutrophil function by C. J. Lagranha; A. C. Levada-Pires; D. F. Sellitti; J. Procopio; R. Curi; T. C. Pithon-Curi (337-346).
Glutamine is the most abundant free amino acid in the body. Its primary source is skeletal muscle, from where it is released into the bloodstream and transported to a variety of tissues. Several studies have shown that glutamine is important for rat and human neutrophil function and that these cells utilize glutamine at high rates. Physical exercise has also been shown to induce considerable changes in neutrophil metabolism and function. As neutrophils represent 50–60% of the total circulating leukocyte pool and play a key role in inflammation, both physical exercise and glutamine might be expected to regulate the inflammatory process. In this review, the changes in neutrophil function induced by physical exercise and glutamine supplementation are compared.
Keywords: Keywords: Neutrophil – Glutamine – Physical exercise – Phagocytosis – Apoptosis

SVCT1 and SVCT2: key proteins for vitamin C uptake by I. Savini; A. Rossi; C. Pierro; L. Avigliano; M. V. Catani (347-355).
Vitamin C is accumulated in mammalian cells by two types of proteins: sodium-ascorbate co-transporters (SVCTs) and hexose transporters (GLUTs); in particular, SVCTs actively import ascorbate, the reduced form of this vitamin.SVCTs are surface glycoproteins encoded by two different genes, very similar in structure. They show distinct tissue distribution and functional characteristics, which indicate different physiological roles. SVCT1 is involved in whole-body homeostasis of vitamin C, while SVCT2 protects metabolically active cells against oxidative stress. Regulation at mRNA or protein level may serve for preferential accumulation of ascorbic acid at sites where it is needed.This review will summarize the present knowledge on structure, function and regulation of the SVCT transporters. Understanding the physiological role of SVCT1 and SVCT2 may lead to develop new therapeutic strategies to control intracellular vitamin C content or to promote tissue-specific delivery of vitamin C-drug conjugates.
Keywords: Keywords: Ascorbic acid transport – Self-regulation – Vitamin C deficiency – Pharmacological applications

The focus has been on the development of methodology for stereoselective preparation of spiroannulated intermediates of the Schöllkopf chiron and further transformations to cyclic α-amino acids. The spiroannulations are effected by Ru(II)-catalysed ring-closing metathesis reactions, by Ru(II)- and Pd(0)-catalysed cycloisomerisations, by Rh(II)-carbenoid cyclisation reactions and by intramolecular aldol condensations. Hydrolytic reactions of the spirane intermediates have provided several groups of highly novel and functionalised five-, six- and seven-membered cyclic α-quaternary-α-amino acid derivatives as well as alicyclic derivatives. The novel cyclic amino acid derivatives can be regarded as cyclic constrained analogues of corresponding common amino acids, or in some cases as intermediates for further preparation of such amino acids. Some emphasis has been on the preparation of cyclic serine analogues. Major efforts have been on the preparation of cyclic α-quaternary bis(α-amino acid) derivatives as conformationally constrained dicarba-analogues of cystine.
Keywords: Keywords: Schöllkopf chiron – Stereoselective gem-dialkenylations – Transition metal promoted cyclisations – α-quaternary-α-amino acids – Spiroannulation

Inducible expression of maize polyamine oxidase in the nucleus of MCF-7 human breast cancer cells confers sensitivity to etoposide by L. Marcocci; M. Casadei; C. Faso; A. Antoccia; P. Stano; S. Leone; B. Mondovì; R. Federico; P. Tavladoraki (403-412).
In this study, polyamine oxidase from maize (MPAO), which is involved in the terminal catabolism of spermidine and spermine to produce an aminoaldehyde, 1,3-diaminopropane and H2O2, has been conditionally expressed at high levels in the nucleus of MCF-7 human breast cancer cells, with the aim to interfere with polyamine homeostasis and cell proliferation. Recombinant MPAO expression induced accumulation of a high amount of 1,3-diaminopropane, an increase of putrescine levels and no alteration in the cellular content of spermine and spermidine. Furthermore, recombinant MPAO expression did not interfere with cell growth of MCF-7 cells under normal conditions but it did confer higher growth sensitivity to etoposide, a DNA topoisomerase II inhibitor widely used as antineoplastic drug. These data suggest polyamine oxidases as a potential tool to improve the efficiency of antiproliferative agents despite the difficulty to interfere with cellular homeostasis of spermine and spermidine.
Keywords: Keywords: Polyamines – Polyamine oxidase – Hydrogen peroxide – Aminoaldehydes – Etoposide – Human breast cancer cells – Terminal catabolism

Arabino-Galactan Proteins from Pistacia lentiscus var. chia: isolation, characterization and biological function by F. Kottakis; F. Lamari; Ch. Matragkou; G. Zachariadis; N. Karamanos; T. Choli-Papadopoulou (413-420).
Arabino-Galactan Proteins (AGPs) were isolated from Chios mastic gum (CMG) by using a buffer containing 0.1 M NaCl, 20 mM Tris–HCl, pH 7.5. Protein analytical methods, combined with specific procedures for carbohydrate characterization, indicated the presence of highly glycosylated protein backbone. In particular, staining by Yariv reagent of the electrophoretically separated molecules revealed the existence of arabinose and galactose and such a modification is characteristic for AGPs.After experiments involving extensive dialysis of the isolated extracts against water and atomic absorption, there was evidence of the existence of zinc ions that are probably covalently bound to the AGPs. By using anion-exchange chromatography, capillary electrophoresis, colorimetric methods and GC-MS, it was found that the extracts were separated into three major populations (A, B, and C), which were consistent with their respective negative charge content namely, uronic acid. The characterization of neutral sugars that was investigated with GC-MS showed the existence of arabinose and galactose in different amounts for each group.Experiments concerning the inhibition of growth of Helicobacter pylori in the presence of AGPs, as is shown for other CMG constituents, showed that the extracts of at least 1.4 g CMG affected the viability of the bacterium. There is no evidence as to whether the AGPs provoke abnormal morphologies of H. pylori, as is reported for the total CMG, or for O-glycans that possess terminal α1, 4-linked N-acetylglucosamine and are expressed in the human gastric mucosa; this has to be further investigated.
Keywords: Keywords: Arabino-Galactan Proteins (AGPs) – Chios mastic gum (CMG) – Biological function

Ornithine decarboxylase (ODC) has a very fast turnover in mammalian cells, but is a stable enzyme in T. brucei and other trypanosmatid parasites like Leishmania donovani. However, Crithidia fasciculata, which is a phylogenetically closely related trypanosomatid to L. donovani, has an ODC with a rapid turnover. Interestingly, C. fasciculata ODC, but not L. donovani ODC, is rapidly degraded also in mammalian systems. In order to obtain information on what sequences are important for the rapid degradation of C. fasciculata ODC, we produced a variety of C. fasciculata/L. donovani ODC hybrid proteins and characterized their turnover using two different mammalian expression systems. The results obtained indicate that C. fasciculata ODC contains several sequence elements essential for the rapid turnover of the protein and that these regions are mainly located in the central part of the enzyme.
Keywords: Keywords: Polyamines – Ornithine decarboxylase – Protein turnover – Crithidia fasciculataLeishmania donovani

Nitric oxide (NO) has been shown to regulate neurotransmitter release in the brain; both inhibitory and excitatory effects have been seen. Taurine is essential for the development and survival of neural cells and protects them under cell-damaging conditions. In the brain stem, it regulates many vital functions such as cardiovascular control and arterial blood pressure. Now we studied the effects of the NO-generating compounds hydroxylamine (HA), S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SNP) on the release of preloaded [3H]taurine under normal and ischemic conditions in slices prepared from the mouse brain stem from developing (7-day-old) to young adult (3-month-old) mice. In general, the effects of NO on the release were somewhat complex and difficult to explain, as expected from the multifunctional role of NO in the central nervous system. The basal initial release under normal conditions was enhanced by the NO donors 5 mM HA and 1.0 mM SNAP at both ages, but SNP was inhibitory in developing mice. The release was markedly enhanced by K+ stimulation. The effects of HA, SNAP and SNP on the basal release were not antagonized by the NO synthase inhibitor NG-nitro-L-arginine (L-NNA, 1.0 mM), demonstrating that mechanisms other than NO synthesis are involved. Taurine release in developing mice in the presence of SNP was reduced by the inhibitor of soluble guanylate cyclase, 1H-(1,2,3)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), indicating the possible involvement of cGMP. In normoxia, N-methyl-D-aspartate (NMDA, 1.0 mM) enhanced the SNAP- and HA-evoked taurine release in developing mice and the HA-evoked release in adults. In ischemia, both K+ stimulation and NMDA potentiated the NO-induced release, particularly in the immature mice, probably without the involvement of the NO synthase or cGMP. The substantial release of taurine in the developing brain stem evoked by NO donors together with NMDA might represent signs of important mechanisms against excitotoxicity which protect the brain stem under cell-damaging conditions.
Keywords: Keywords: Taurine release – Nitric oxide – Ischemia – Brain stem – Tissue slices – Adult and developing mouse

A DING phosphatase in Thermus thermophilus by A. A. Pantazaki; G. P. Tsolkas; D. A. Kyriakidis (437-448).
Phosphate transport in bacteria occurs via a phosphate specific transporter system (PSTS) that belongs to the ABC family of transporters, a multisubunit system, containing an alkaline phosphatase. DING proteins were characterized due to the N-terminal amino acid sequence DINGG GATL, which is highly conserved in animal and plant isolates, but more variable in microbes. Most prokaryotic homologues of the DING proteins often have some structural homology to phosphatases or periplasmic phosphate-binding proteins. In E. coli, the product of the inducible gene DinG, possesses ATP hydrolyzing helicase enzymic activity. An alkaline phosphorolytic enzyme of the PSTS system was purified to homogeneity from the thermophilic bacterium Thermus thermophilus. N-terminal sequence analysis of this protein revealed the same high degree of similarity to DING proteins especially to the human synovial stimulatory protein P205, the steroidogenesis-inducing protein and to the phosphate ABC transporter, periplasmic phosphate-binding protein, putative (P. fluorescens Pf-5). The enzyme had a molecular mass of 40 kDa on SDS/PAGE, exhibiting optimal phosphatase activity at pH 12.3 and 70 °C. The enzyme possessed characteristics of a DING protein, such as ATPase, ds endonuclease and 3′ phosphodiesterase (3′-exonuclease) activities and binding to linear dsDNA, displaying helicase activity on supercoiled DNA. Purification and biochemical characterization of a T. thermophilus DING protein was achieved. The biochemical properties, N-terminal sequence similarities of this protein implied that the enzyme belongs to the PSTS family and might be involved in the DNA repair mechanism of this microorganism.
Keywords: Keywords: Thermus thermophilus – DING protein – Alkaline phosphatase – Phosphate specific transporter system (PSTS) – ATPase – Type II phosphodiesterase (3′-exonuclease)

Novel synthesis of α-PNA monomers by U-4CR by P.-C. Huang; G.-J. Hsu; B.-R. Zhuang; K. Sung (449-453).
A novel synthesis of α-PNA monomers was carried out by U-4CR, followed by photochemical cleavage of the 2-nitrobenzyl group and selective hydrolysis in the presence of 10% HCl in THF. Three of four functional components in the U-4CR were specially protected: cyclohexenyl isocyanide, Boc for protecting the amino group of glycine, and 2-nitrobenzyl group as a photocage (photoremovable protecting group) for ammonia. The amino group of aldehyde-containing adenine is too weak to interfere with the U-4CR, so that it is not necessary to be protected.
Keywords: Keywords: PNA – Multi-component reaction – Peptide

Oxygen dependence of tyrosine hydroxylase by M. Rostrup; A. Fossbakk; A. Hauge; R. Kleppe; E. Gnaiger; J. Haavik (455-464).
The effects of dioxygen on tyrosine hydroxylase (TH) activity was studied, measuring the formation of DOPA from tyrosine, 3H2O from 3,5-3H-tyrosine, or by direct oxygraphic determination of oxygen consumption. A high enzyme activity was observed during the initial 1–2 min of the reactions, followed by a decline in activity, possibly related to a turnover dependent substoichiometrical oxidation of enzyme bound Fe(II) to the inactive Fe(III) state. During the initial reaction phase, apparent K m-values of 29–45 µM for dioxygen were determined for all human TH isoforms, i.e. 2–40 times higher than previously reported for TH isolated from animal tissues. After 8 min incubation, the K m (O2)-values had declined to an average of 20 ± 4 µM. Thus, TH activity may be severely limited by oxygen availability even at moderate hypoxic conditions, and the enzyme is rapidly and turnover dependent inactivated at the experimental conditions commonly employed to measure in vitro activities.
Keywords: Keywords: Catecholamines – Human – Hypoxia – Oxygen – Tyrosine hydroxylase

Solid-phase peptide synthesis and circular dichroism study of chiral β-peptoid homooligomers by C. A. Olsen; M. Lambert; M. Witt; H. Franzyk; J. W. Jaroszewski (465-471).
N-alkyl-β-alanine oligomers (β-peptoids) with α-chiral side chains [(R)- or (S)-1-(phenylethyl)amino groups] were synthesized and analyzed by CD spectroscopy. These chiral β-peptoid homomers exhibited chain-length-dependent and solvent-dependent ellipticity, strongly indicating the presence of a secondary structure in solution. The CD behaviour was only slightly temperature-dependent upon heating, as also previously observed for stable α-peptoid helices containing the same type of side chains. Thus, the data presented here comprise the first evidence for a chain length-dependent secondary folding of compounds with this novel peptidomimetic backbone design. In addition, applicability of a novel hyphenated technique, HPLC-SPE-NMR/MS, for analysis of crude SPPS reaction products was demonstrated.
Keywords: Keywords: Peptidomimetics – Chiral β-peptoids – Solid-phase synthesis – Circular dichroism – Secondary folding

Production of γ-aminobutyric acid by Streptococcus salivarius subsp. thermophilus Y2 under submerged fermentation by S.-Y. Yang; F.-X. Lü; Z.-X. Lu; X.-M. Bie; Y. Jiao; L.-J. Sun; B. Yu (473-478).
γ-Aminobutyric acid (GABA), a major inhibitory neurotransmitter in the central nervous system, has several well-known physiological functions and has been applied to the production of many drugs and functional foods. The technology of GABA production via submerged fermentation by Streptococcus salivarius subsp. thermophilus Y2 was investigated in this paper. It indicated that the GABA production was related to the biochemical characteristics of glutamate decarboxylase (GAD) of S. salivarius subsp. thermophilus Y2. After 24 h of fermentation at 37 °C, which is the suitable culture conditions for GAD-production, then the culture condition were adjusted to the optimal temperature (40 °C) and pH (4.5) for the GAD reaction activity in biotransformation of cells and pyridoxal 5′-phosphate (0.02 mmol/l) were added to the broth at the 48 h, the GABA production was increased up to 1.76-fold, reaching 7984.75 ± 293.33 mg/l. The strain shows great potential use as a starter for GABA-containing yoghurt, cheese and other functional fermented food productions.
Keywords: Keywords: γ-Aminobutyric acid – Glutamate decarboxylase – Streptococcus salivarius subsp. thermophilus

Sub-epitopic dissection of HCV E1315–328HRMAWDMMMNWSPT sequence by similarity analysis by L. Polimeno; A. Mittelman; L. Gennero; A. Ponzetto; G. Lucchese; A. Stufano; A. Kusalik; D. Kanduc (479-484).
Our labs are focused on identifying amino acid sequences having the ability to react specifically with the functional binding site of a complementary antibody. Our epitopic definition is based on the analysis of the similarity level of antigenic amino acid sequences to the host proteome. Here, the similarity profile to the human proteome of an HCV E1 immunodominant epitope, i.e. the HCV E1315–328HRMAWDMMMNWSPT sequence, led to i) characterizing the immunoreactive HCV E1 315–328 region as a sequence endowed with a low level of similarity to human proteins; ii) defining 2 contiguous immunodominant linear determinants respectively located at the NH2 and COOH terminus of the conserved viral antigenic sequence. This study supports the hypothesis that low sequence similarity to the host’s proteome modulates the pool of epitopic amino acid sequences in a viral antigen, and appears of potential value in defining immunogenic viral peptide sequences to be used in immunotherapeutic approaches for HCV treatment.
Keywords: Keywords: Epitope mapping – Similarity level – Amino acid sequence uniqueness – Computational biology – Anti-HCV peptide vaccines

Strain-dependent effects of cognitive enhancers in the mouse by B. Sunyer; S. Patil; C. Frischer; H. Hoeger; G. Lubec (485-495).
A series of cognitive enhancers (CEs) have been reported to increase spatial memory in rodents, information on behavioral effects, however, is limited. The aim of the study was therefore to examine the behavioral effects of three CEs in two well-documented inbred mouse strains.C57BL/6J and DBA/2 mice were administered intraperitonial. D-cycloserine (DCS; NMDA receptor agonist), 1-(4-Amino-5-chloro-2-methoxyphenyl)-3-[1-butyl-4-piperidinyl]-1-propanone hydrochloride (RS67333; 5HT4-receptor agonist), and (R)-4-{[2-(1-methyl-2-pyrrolidinyl)ethyl]thio}phenol hydrochloride (SIB-1553A; β-4-nicotinic receptor agonist) and tested in the open field (OF), elevated plus maze (EPM), neurological observational battery and rota-rod. Cognitive performance was tested in the Morris water maze.All compounds modified behavioral performance in the OF, DCS showed an anxiolytic effect in the EPM, and differences in the observational battery were observed i.e. vestibular drop was decreased by SIB-1553A and RS67333 treatment in C57BL/6J and increased with DCS treatment in DBA/2 mice. In the rota rod SIB-1553A improved motor performance.DCS effects on learning and memory was comparable to controls whereas the other compounds impaired performance in the Morris water maze.In conclusion, behavioral testing of CEs in the mouse revealed significant changes that may have to be taken into account for evaluation of CEs, interpretation of cognitive studies and warrant further neurotoxicological studies. Moreover, strain-dependent differences were observed that in turn may confound results obtained from behavioral and cognitive testing.
Keywords: Keywords: Inbred strains – D-cycloserine – RS67333 – SIB-1553A – Behavioral testing – Morris water maze

Cell-based signal chemical genomics can profile the signalling pathway for certain cellular events by using a target-known chemical library. To ascertain its usefulness, the receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis in mouse monocyte/macrophage cells RAW264.7 was used as an in vitro experimental model. Of 180 target-known inhibitors/activators formatted in a 384-well plate, 8 chemicals were shown to inhibit the osteoclast formation, but 4 chemicals enhanced this process. A variety of references support, or possibly lead one to expect the effects of these 12 chemicals on the cellular process of osteoclastogenesis in RAW264.7 cells, but several signalling pathways were newly found in this study; for example, CA-074 Me inhibiting cathepsin B and nitrendipine blocking the calcium channel could have the potential to inhibit the osteoclast formation as well as bone resorption. This is a simple but very fast and powerful method of profiling the signalling pathway of certain cellular events. Signal chemical genomics could provide invaluable information for the exploration of new target signalling processes and further target-based drug discovery strategies.
Keywords: Keywords: Drug discovery – Osteoclastogenesis – RAW264.7 cells – Receptor activator of NF-κB ligand (RANKL) – Signal chemical genomics

In plasma and serum, the presence of high-abundance proteins can overwhelm the signals of low-abundance proteins, which then become undetectable either by two-dimensional gels or chromatographic techniques. Therefore, depletion of abundant proteins is a prerequisite to detect low-abundance components. Furthermore, the regeneration of pre-purification tools could be money-saving. We applied an affinity chromatography kit to remove albumin and the immunoglobulin chains from plasma and propose a simple and effective technical procedure for the regeneration of these affinity columns.
Keywords: Keywords: Body fluids – HSA/IgG Removal kit – Proteomics – Two-dimensional electrophoresis