Amino Acids (v.40, #2)

Two intracellular helices of G-protein coupling receptors could generally support oligomerization and coupling with transducers by Michael S. Parker; Edwards A. Park; Floyd R. Sallee; Steven L. Parker (261-268).
For many G-protein coupling receptors (GPCRs), the upkeep of receptor dimers could depend on association with functional Gi α subunits. This is known for Y1, Y2 and Y4 neuropeptide Y receptors [presented in the companion paper (Estes et al., Amino Acids, doi: 10.1007/s00726-010-0642-z , 2010)]. Interactions with transducers use mainly intracellular domains of the receptors. Intracellular loops 1 and 2 in GPCRs are short and lack extensive helicity that could support transducer anchoring. Interaction with G-proteins is known to use the juxtamembrane Helix 8 in the fourth intracellular domain, for which we document a helix-stabilizing n/(n + 4) pattern of large hydrophobic sidechains. Another intracellular helix located in the C-terminal portion of the third intracellular loop does not display a strong stabilizing pattern, and is found in many studies to serve dynamically in association and activation of transducers and effectors. We show that these tracts share features across metazoan phyla not only in opsins and opsin-like receptors (including the Y receptors), but also in Taste-2 and Frizzled receptors. Similarities of these helices across GPCR groups could have both phylogenetic and functional roots.
Keywords: G-protein coupling receptor; Neuropeptide Y; Receptor dimer; Receptor heteropentamer; Y1 receptor; Y2 receptor; Y4 receptor

Polyamine metabolism in Leishmania: from arginine to trypanothione by Gianni Colotti; Andrea Ilari (269-285).
Polyamines (PAs) are essential metabolites in eukaryotes, participating in a variety of proliferative processes, and in trypanosomatid protozoa play an additional role in the synthesis of the critical thiol trypanothione. The PAs are synthesized by a metabolic process which involves arginase (ARG), which catalyzes the enzymatic hydrolysis of l-arginine (l-Arg) to l-ornithine and urea, and ornithine decarboxylase (ODC), which catalyzes the enzymatic decarboxylation of l-ornithine in putrescine. The S-adenosylmethionine decarboxylase (AdoMetDC) catalyzes the irreversible decarboxylation of S-adenosylmethionine (AdoMet), generating the decarboxylated S-adenosylmethionine (dAdoMet), which is a substrate, together with putrescine, for spermidine synthase (SpdS). Leishmania parasites and all the other members of the trypanosomatid family depend on spermidine for growth and survival. They can synthesize PAs and polyamine precursors, and also scavenge them from the microenvironment, using specific transporters. In addition, Trypanosomatids have a unique thiol-based metabolism, in which trypanothione (N1-N8-bis(glutathionyl)spermidine, T(SH)2) and trypanothione reductase (TR) replace many of the antioxidant and metabolic functions of the glutathione/glutathione reductase (GR) and thioredoxin/thioredoxin reductase (TrxR) systems present in the host. Trypanothione synthetase (TryS) and TR are necessary for the protozoa survival. Consequently, enzymes involved in spermidine synthesis and its utilization, i.e. ARG, ODC, AdoMetDC, SpdS and, in particular, TryS and TR, are promising targets for drug development.
Keywords: Polyamines; Leishmania ; Arginine; Trypanothione; Redox metabolism; Drug targets

An overview of the therapeutic effects of leucine supplementation on skeletal muscle under atrophic conditions by Humberto Nicastro; Guilherme Giannini Artioli; André dos Santos Costa; Marina Yazigi Solis; Claudia Ribeiro da Luz; François Blachier; Antonio Herbert Lancha Jr (287-300).
The characterization of the mechanisms underlying skeletal muscle atrophy under different conditions has been a constant focus of research. Among anti-atrophic therapies, amino acid supplementation, particularly with leucine, has received a lot of attention. Supplementation has been shown to have remarkable effects on muscle remodeling through protein turnover modulation. This may then impact physiological parameters related to muscle function, and even quality of life. In light of this, leucine supplementation could be a useful therapy for mitigating the atrophic effects of catabolic conditions. The purpose of this review is to present the major results of human studies evaluating the effects of leucine supplementation on structure and function of skeletal muscle in atrophic conditions such as muscle disuse, sarcopenia, and cancer.
Keywords: Leucine; Branched-chain amino acids; Skeletal muscle; Atrophy; Disuse

The size of information that can be stored in nucleic acids, proteins, and carbohydrates was calculated. The number of hexamers for peptides is 64,000,000 (206) and seems to be impressive in comparison with 4,096 (46) hexanucleotides, but the number of isomers of hexasaccharides is 1.44 × 1015. Carbohydrates are therefore the best high-density coding system. This language has been named glycocode resp. sugar code. In comparison with peptide dendrimers, the amount of information carried by glycopeptide dendrimers or glycodendrimers is therefore much higher. This is reflected by the variability of structures and functions (activities). This review is about the broad area of peptide and glycopeptide dendrimers. The dendrimeric state and physicochemical properties and general consequences are described, together with a cluster effect. The impact of cluster effect to biological, chemical, and physical properties is discussed. Synthesis of dendrimers by convergent and divergent approaches, “Lego” chemistry, ligation strategies, and click chemistry is given with many examples. Purification and characterization of dendrimers by chromatographic methods, electromigration methods, and mass spectrometry are briefly mentioned. Different types of dendrimers with cyclic core, i.e. RAFTs, TASPs and analogous cyclic structures, carbopeptides, carboproteins, octopus glycosides, inositol-based dendrimers, cyclodextrins, calix[4]arenes, resorcarenes, cavitands, and porphyrins are given. Dendrimers can be used for creation of libraries, catalysts, and solubilizing agents. Biocompatibility and toxicity of dendrimers is discussed, as well as their applications in nanoscience, nanotechnology, drug delivery, and gene delivery. Carbohydrate interactions of glycopeptide dendrimers (bacteria, viruses, and cancer) are described. Examples of dendrimers as anti-prion agents are given. Dendrimers represent a fast developing area which partly overlaps with nanoparticles and nanotechnologies.
Keywords: Calixarene dendrimers; Calix[4]resorcarene dendrimers; Carbopeptide dendrimers; Contrast agents; Cyclodextrin dendrimers; Dendrimers; Drug delivery; Glycobiology; Glycocluster; Glycoconjugates; Glycodendrimers; Glycopeptide dendrimers; Glycopeptide libraries; Glycopeptides; Glycotope; Lectin; Imaging agents; Ligation chemistry; Multiple antigen glycopeptide (MAG)

Maintenance of Y receptor dimers in epithelial cells depends on interaction with G-protein heterotrimers by Anne-Marie Estes; Kathleen McAllen; Michael S. Parker; Renu Sah; Trevor Sweatman; Edwards A. Park; Ambikaipakan Balasubramaniam; Floyd R. Sallee; Mary W. Walker; Steven L. Parker (371-380).
Treatment of CHO cells expressing human Y receptors (Y1, Y2 or Y4 subtype) with pertussis toxin results in a large decrease in functional receptors, with a preferential loss of heteropentameric assemblies of receptor dimers and G-protein trimers. This occurs in parallel to inactivation of the nucleotide site of Gi α subunits, with a half period of about 4 h. The loss could be mainly due to proteolysis at the level of recycling/perinuclear endosomes, and of receptor completion in the ER, since it is reduced by co-treatment with ammonium chloride, an inhibitor of particulate proteinases. Antagonists do not strongly decrease the heteropentameric fraction. These findings indicate that the upkeep of Y receptor dimers in epithelial cell lines depends on the association of receptor oligomers with functional Gi α subunits. This interaction could use the juxtamembrane helix 8 in the fourth intracellular domain, and could also be supported by the C-terminal helix of the third intracellular loop, as outlined in the companion review (Parker et al., Amino Acids, doi: 10.1007/s00726-010-0616-1 , 2010).
Keywords: G-protein coupling receptor; Neuropeptide Y; Receptor dimer; Receptor heteropentamer; Y1 receptor; Y2 receptor; Y4 receptor

Hpa1Xoo (harpin) is a type III secreted protein of the rice blight bacterial pathogen Xanthomonas oryzae pv. oryzae that elicits a hypersensitive response (HR) in nonhost tobacco. Hpa1Xoo is predicted to contain two potential coiled-coil (CC) regions, one at the N-terminus with a high probability of formation, and one at the C-terminus with a lower probability of formation. We constructed several CC-equivalent peptides by a chemosynthetic method, and investigated the structure–function of the predicted Hpa1Xoo CC regions, using biophysical and biochemical approaches. Both peptides elicited an HR in tobacco. Mutant versions of the N- and C-terminal peptides that were predicted to disrupt or favor CC formation were generated. The resulting altered HR activity and oligomerization indicated that the N-terminal CC region is essential for eliciting HR, but the C-terminus is not. The results also indicate that a 14-residue fragment (LDQLLCQLISALLQ) within the N-terminal CC region is a minimal and independent functional element for HR-induction in tobacco leaves. We propose that HR-induction requires a specific oligomerization of the CC regions of Hpa1Xoo.
Keywords: Xanthomonas oryzae ; Hpa1; Coiled-coil; Oligomerization; α-helix; Hypersensitive response

Daily hypoxia increases basal monocyte HSP72 expression in healthy human subjects by Lee Taylor; Adrian W. Midgley; Bryna Chrismas; Angela R. Hilman; Leigh A. Madden; Rebecca V. Vince; Lars R. McNaughton (393-401).
Heat shock protein 72 (HSP72) performs vital roles within the body at rest and during periods of stress. In vitro, research demonstrates HSP72 induction in response to hypoxia. Recently, in vivo, an acute hypoxic exposure (75 min at 2,980 m) was sufficient to induce significant increases in monocyte expressed HSP72 (mHSP72) and a marker of oxidative stress in healthy human subjects. The purpose of the current study was to identify the impact of 10 consecutive days of hypoxic exposures (75 min at 2,980 m) on mHSP72 and erythropoietin (EPO) expression, markers of oxidative stress, and maximal oxygen consumption in graded incremental aerobic exercise. Eight male subjects were exposed to daily normobaric hypoxic exposures for 75 min at 2,980 m for 10 consecutive days, commencing and ceasing at 0930 and 1045, respectively. This stressor was sufficient to induce significant increases in mHSP72, which was significantly elevated from day 2 of the hypoxic exposures until 48 h post-final exposure. Notably, this increase had an initial rapid (30% day on day compared to baseline) and final slow phase (16% day on day compared to baseline) of expression. The authors postulate that 7-day hypoxic exposure in this manner would be sufficient to induce near maximum hypoxia-mediated basal mHSP72 expression. Elevated levels of mHSP72 are associated with acquired thermotolerance and provide cross tolerance to non-related stressors in vivo, the protocol used here may provide a useful tool for elevating mHSP72 in vivo. Aside from these major findings, significant transient daily elevations were seen in a marker of oxidative stress, alongside sustained increases in EPO expression. However, no physiologically significant changes were seen in maximal oxygen consumption or time to exhaustion.
Keywords: Stress protein; Oxidative stress; EPO; TBARS

Two new chiral monochloro-s-triazines (MCT) were synthesized [viz N-(4-chloro-6-piperidinyl-[1,3,5]-triazine-2-yl)-l-leucine amide and N-(4-chloro-6-piperidinyl-[1,3,5]-triazine-2-yl)-l-leucine) (CDR 1 and 2, respectively)] by the nucleophilic displacement of chlorine atoms in s-triazine moiety. One of the Cl atoms was replaced with piperidine, and the second Cl atom in the 6-piperidinyl derivative was replaced with amino acid amide (viz l-Leu–NH2) and amino acid (l-Leu). These reagents were characterized and used as CDRs for chiral separation of protein and non-protein amino acids, and were separated on a reversed-phase C18 column. The reaction conditions were optimized for the synthesis of diastereomers using one MCT reagent. The separation method was validated for limit of detection, linearity, accuracy, precision, and recovery.
Keywords: Cyanuric chloride; Amino acids; Reversed-phase liquid chromatography; Indirect resolution

Towards non-peptide ANG II AT1 receptor antagonists based on urocanic acid: rational design, synthesis and biological evaluation by George Agelis; Amalia Resvani; Minos-Timotheos Matsoukas; Theodore Tselios; Konstantinos Kelaidonis; Dimitra Kalavrizioti; Demetrios Vlahakos; John Matsoukas (411-420).
A series of o-, m- and p-benzyl tetrazole derivatives 11ac has been designed, synthesized and evaluated as potential Angiotensin II AT1 receptor antagonists, based on urocanic acid. Compound 11b with tetrazole moiety at the m-position showed moderate, however, higher activity compared to the o- and p-counterpart analogues. Molecular modelling techniques were performed in order to extract their putative bioactive conformations and explore their binding modes.
Keywords: AT1 receptor; Urocanic acid; Pharmacophore; Molecular modelling

Activation of the AtoSC two-component system in the absence of the AtoC N-terminal receiver domain in E. coli by Evaggelos C. Theodorou; Marina C. Theodorou; Margarita N. Samali; Dimitrios A. Kyriakidis (421-430).
The AtoSC two-component system in E. coli consists of the AtoS sensor kinase and the AtoC response regulator. It regulates positively the transcriptional activation of atoDAEB operon, encoding enzymes involved in short-chain fatty acid catabolism upon acetoacetate-mediated induction. AtoSC acting on atoDAEB operon, regulates the biosynthesis and the intracellular distribution of short-chain poly-(R)-3-hydroxybutyrate (cPHB). A phosphorylation-incompetent AtoC form was constructed lacking its N-terminal receiver domain, trAtoC, to study the effects of AtoC domains on cPHB biosynthesis and atoDAEB operon regulation. Both cPHB biosynthesis and atoDAEB gene expression were regulated positively by trAtoC in the absence of any inducer in E. coli of both atoSC + and ΔatoSC genotypes. The presence of acetoacetate or spermidine further promoted these trAtoC actions. Competitive regulatory functions between the full length AtoC and trAtoC were observed referring to atoDAEB and cPHB targets as well as growth of trAtoC-overproducing atoSC + cells on butyrate as the sole carbon source. trAtoC in contrast to the wild-type AtoC presented different modes of cPHB and atoDAEB regulation in the presence of compounds involved in fatty acid metabolism including CoA-SH, acetyl-CoA, sodium acetate or 3-hydroxybutyryl-CoA. These data provide evidence for a role of the AtoC N-terminal receiver domain in regulating the biological activities of AtoSC as well as additional mechanisms of interactions between the AtoSC constituents including their established inducers or new effectors towards the accomplishment of the AtoSC TCS signal transduction.
Keywords: AtoSC; Truncated AtoC; atoDAEB ; Two-component system; Poly-(R)-3-hydroxybutyrate

TI2BioP: Topological Indices to BioPolymers. Its practical use to unravel cryptic bacteriocin-like domains by Guillermín Agüero-Chapin; Gisselle Pérez-Machado; Reinaldo Molina-Ruiz; Yunierkis Pérez-Castillo; Aliuska Morales-Helguera; Vítor Vasconcelos; Agostinho Antunes (431-442).
Bacteriocins are proteinaceous toxins produced and exported by both gram-negative and gram-positive bacteria as a defense mechanism. The bacteriocin protein family is highly diverse, which complicates the identification of bacteriocin-like sequences using alignment approaches. The use of topological indices (TIs) irrespective of sequence similarity can be a promising alternative to predict proteinaceous bacteriocins. Thus, we present Topological Indices to BioPolymers (TI2BioP) as an alignment-free approach inspired in both the Topological Substructural Molecular Design (TOPS-MODE) and Markov Chain Invariants for Network Selection and Design (MARCH-INSIDE) methodology. TI2BioP allows the calculation of the spectral moments as simple TIs to seek quantitative sequence-function relationships (QSFR) models. Since hydrophobicity and basicity are major criteria for the bactericide activity of bacteriocins, the spectral moments (HPμ k ) were derived for the first time from protein artificial secondary structures based on amino acid clustering into a Cartesian system of hydrophobicity and polarity. Several orders of HPμ k characterized numerically 196 bacteriocin-like sequences and a control group made up of 200 representative CATH domains. Subsequently, they were used to develop an alignment-free QSFR model allowing a 76.92% discrimination of bacteriocin proteins from other domains, a relevant result considering the high sequence diversity among the members of both groups. The model showed a prediction overall performance of 72.16%, detecting specifically 66.7% of proteinaceous bacteriocins whereas the InterProScan retrieved just 60.2%. As a practical validation, the model also predicted successfully the cryptic bactericide function of the Cry 1Ab C-terminal domain from Bacillus thuringiensis’s endotoxin, which has not been detected by classical alignment methods.
Keywords: Bacteriocin; Topological indices; Spectral moments; Alignment methods; Artificial secondary structure

Artificial intelligence systems based on texture descriptors for vaccine development by Loris Nanni; Sheryl Brahnam; Alessandra Lumini (443-451).
The aim of this work is to analyze and compare several feature extraction methods for peptide classification that are based on the calculation of texture descriptors starting from a matrix representation of the peptide. This texture-based representation of the peptide is then used to train a support vector machine classifier. In our experiments, the best results are obtained using local binary patterns variants and the discrete cosine transform with selected coefficients. These results are better than those previously reported that employed texture descriptors for peptide representation. In addition, we perform experiments that combine standard approaches based on amino acid sequence. The experimental section reports several tests performed on a vaccine dataset for the prediction of peptides that bind human leukocyte antigens and on a human immunodeficiency virus (HIV-1). Experimental results confirm the usefulness of our novel descriptors. The matlab implementation of our approaches is available at .
Keywords: Peptide classification; Vaccine development; HIV-1 protease prediction; Locally binary patterns; Discrete cosine transform; Support vector machine

The agmatine-degrading enzyme agmatinase: a key to agmatine signaling in rat and human brain? by H.-G. Bernstein; C. Derst; C. Stich; H. Prüss; D. Peters; M. Krauss; B. Bogerts; R. W. Veh; G. Laube (453-465).
Agmatinase, an ureohydrolase belonging to the arginase family, is widely expressed in mammalian tissues including the brain. Here, it may serve two different functions, the inactivation of the arginine derivative agmatine, a putative neurotransmitter, and the formation of the diamine putrescine. In order to identify the cellular sources of agmatinase expression in the brain, we generated a polyclonal monospecific antibody against recombinant rat agmatinase. With immunocytochemistry, selected areas of rat and human brain were screened. Clearly, in both species agmatinase-like immunoreactivity was predominantly detected in distinct populations of neurons, especially cortical interneurons. Also, principal neurons in limbic regions like the habenula and in the cerebellum robustly expressed agmatinase protein. When comparing the overall agmatinase expression with immunocytochemical data available for agmatine and polyamine biosynthetic enzymes, the observed pattern may argue in favor of an agmatine inactivating function rather than fueling the alternative pathway of polyamine synthesis. The putative neurotransmitter agmatine is seemingly involved with mental disorders. Therefore, agmatinase may be similarly important for pathogenesis. The normal expression profile of the protein as described here may therefore be altered under pathological conditions.
Keywords: Agmatine; Calretinin; Polyamines; Habenula; Epithalamus; Depression

Role of the active site residues arginine-216 and arginine-237 in the substrate specificity of mammalian d-aspartate oxidase by Masumi Katane; Yasuaki Saitoh; Kazuhiro Maeda; Toshihiko Hanai; Masae Sekine; Takemitsu Furuchi; Hiroshi Homma (467-476).
d-Aspartate oxidase (DDO) and d-amino acid oxidase (DAO) are flavin adenine dinucleotide-containing flavoproteins that catalyze the oxidative deamination of d-amino acids. Unlike DAO, which acts on several neutral and basic d-amino acids, DDO is highly specific for acidic d-amino acids. Based on molecular modeling and simulated annealing docking analyses, a recombinant mouse DDO carrying two substitutions (Arg-216 to Leu and Arg-237 to Tyr) was generated (R216L-R237Y variant). This variant and two previously constructed single-point mutants of mouse DDO (R216L and R237Y variants) were characterized to investigate the role of Arg-216 and Arg-237 in the substrate specificity of mouse DDO. The R216L-R237Y and R216L variants acquired a broad specificity for several neutral and basic d-amino acids, and showed a considerable decrease in activity against acidic d-amino acids. The R237Y variant, however, did not show any additional specificity for neutral or basic d-amino acids and its activity against acidic d-amino acids was greatly reduced. The kinetic properties of these variants indicated that the Arg-216 residue is important for the catalytic activity and substrate specificity of mouse DDO. However, Arg-237 is, apparently, only marginally involved in substrate recognition, but is important for catalytic activity. Notably, the substrate specificity of the R216L-R237Y variant differed significantly from that of the R216L variant, suggesting that Arg-237 has subsidiary effects on substrate specificity. Additional experiments using several DDO and DAO inhibitors also suggested the involvement of Arg-216 in the substrate specificity and catalytic activity of mouse DDO and that Arg-237 is possibly involved in substrate recognition by this enzyme. Collectively, these results indicate that Arg-216 and Arg-237 play crucial and subsidiary role(s), respectively, in the substrate specificity of mouse DDO.
Keywords: d-Aspartate oxidase; d-Amino acid oxidase; Flavoprotein; Site-directed mutagenesis; Substrate specificity

Memantine reduces consumption of highly palatable food in a rat model of binge eating by Piotr Popik; Tomasz Kos; Yulei Zhang; Adam Bisaga (477-485).
Excessive consumption of highly palatable food has been linked to the development of eating disorders and obesity, and can be modeled in non-food-deprived rats by offering them a limited (2-h daily) access to an optional dietary fat. Since the glutamatergic system has recently emerged as a viable target for binge-eating medication development, we compared the effects of subchronic treatment with glutamatergic receptor antagonists to the effects of a reference appetite-suppressing agent sibutramine on highly palatable food (lard) and normal chow intake. In three separate experiments, the consumption of a standard laboratory chow and lard were measured during 12 days of medication treatment and for 6 days afterwards. Generalized estimating equations analysis demonstrated that sibutramine (7.5 mg/kg, PO) significantly decreased lard consumption, with a concurrent increase in chow consumption. Sibutramine effects disappeared after treatment discontinuation. The NMDA receptor antagonist memantine (5 mg/kg, IP) significantly decreased lard consumption and increased chow consumption, comparable to effects of sibutramine; however, memantine’s effects persisted after treatment discontinuation. The effects of the mGluR5 antagonist MTEP (7.5 mg/kg, IP) on food consumption were in the same direction as seen with memantine, but the observed differences were not significant. In an additional control experiment, sibutramine and memantine reduced unlimited (24 h) chow intake during the treatment phase. Present results provide evidence that glutamatergic neurotransmission might be involved in the regulation of excessive consumption of highly palatable foods, and suggest that NMDA receptor may be an attractive target for developing obesity and disordered eating pharmacotherapies.
Keywords: Binge eating; Compulsive behavior; Generalized estimating equations; Memantine; mGluR5 antagonist; Sibutramine

In this study, an optically active diamine, N,N′-(pyromellitoyl)-bis{N-[4(4-aminophenoxy)phenyl]-2-(4-methyl)pentanamide} (1) containing amino acid l-leucine was prepared in three steps. The step-growth polymerization of this chiral diamine with several diisocyanates in room temperature ionic liquid (IL), 1,3-dipropylimidazolium bromide as an environmentally friendly solvent and in a volatile organic solvent, is investigated. The polymerization yields and inherent viscosities of the resulting poly(amide-ether-imide-urea)s are compared in both solvents. The results show that the IL to be the superior polymerization media. All of the obtained polymers exhibited good solubility in some polar aprotic organic solvents such as N,N-dimethyacetamide, N,N-dimethyformamide, dimethyl sulfoxide while thermal stability was not disturbed based on thermogravimetric analysis and differential scanning calorimetry experiments. X-ray diffraction analysis of polymers shows that they are amorphous. The observation of optical rotation confirms the optical activity of prepared polymers.
Keywords: Chiral diamine; l-Leucine; Polyurea; Optically active polymers; Imidazolium ionic liquid; Green chemistry

Periplasmic oligopeptide-binding protein (OppA) is the initial receptor in the ATP-binding cassette (ABC) system of bacteria, which exhibits a broad specificity in binding oligopeptides without regard to sequence. Here, we present a computational study on the structural properties and energetic landscapes of OppA protein interacting with its cognate ligands on the basis of 28 structure/affinity-known OppA–tripeptide complexes. By employing a well-designed protocol that couples the hybrid quantum mechanical/molecular mechanical (QM/MM) scheme and the sophisticated Poisson–Boltzmann/surface area (PB/SA) solvent model together to analyze and decompose the energy components associated with the OppA–peptide binding, we demonstrate that the broad specificity of OppA-recognizing peptides is originated from a series of exquisite balances between the free energy contributions from, for example, the direct nonbonded interactions and indirect desolvation effects, the main chains and side chains, and the different residue positions of the tripeptide ligands. We also show that, in a framework of structure-based quantitative structure–activity relationship (SB-QSAR) methodology, the QM/MM–PB/SA-derived energy terms could be used as a good descriptor to characterize the interaction profile of OppA with peptides and correlate pretty well with the experimentally measured affinities of the binding.
Keywords: OppA protein; Peptide; QM/MM; PB/SA; SB-QSAR

Immunomodulatory activities of a new pentapeptide (Bursopentin) from the chicken bursa of Fabricius by D. Y. Li; Z. R. Geng; H. F. Zhu; C. Wang; D. N. Miao; P. Y. Chen (505-515).
The bursa of Fabricius (BF) is a central immune organ in birds, and some peptides from chicken BF have demonstrated important immune functions. Here, a new 626.27 Da pentapeptide, Bursopentin (BP5, Cys-Lys-Arg-Val-Tyr) was isolated and purified by reverse-phase high-performance liquid chromatography. In this study, we examined the effects of BP5 on antigen-specific immune response in BALB/c mice sensitized with inactivated avian influenza virus (AIV) [A/Duck/Jiangsu/NJ08/05 (AIV H9N2 subtype)]. The results suggested that BP5 enhanced anti-hemagglutinin antibody (IgG, the isotypes IgG1 and IgG2a) production, induced both of Th1- (IL-2 and IFN-γ) and Th2-type (IL-4 and -10) cytokines, increased proliferations of splenic lymphocyte subsets CD4+ T cells (CD3+CD4+), CD8+ T cells (CD3+CD8+) and B cells, and enhanced cytotoxic T-lymphocyte activity of the activated splenocytes against NIH3T3 cells. The effects of BP5 on the proliferation of isolated T- and/or B-cell populations of BALB/c mice were assessed, and the data suggested that BP5 promoted spleen lymphocyte proliferation by activating B cells directly and T cells indirectly. Further analysis revealed that B-lymphocyte proliferation induced by BP5 is mediated by reactive oxygen species generated from thiol auto-oxidation of BP5. Furthermore, our data indicated that protein kinase C, mitogen-activated protein kinase, and nuclear factor kappa B are involved in the signal transductions during the BP5-induced B lymphocyte proliferation. This study indicates that BP5 could be a potential immunomodulator for future immuno-pharmacological use.
Keywords: Peptide; Immunomodulation; Lymphocyte proliferation; Reactive oxygen species

Intracellular cysteine availability is an important rate-limiting factor governing glutathione synthesis in the heart. This is also dependent on the magnitude and rate of cysteine uptake into cardiomyocytes, which has been little studied. This study investigated the hypothesis that changes to cysteine transporter expression and activity during oxidative stress influence cardiomyocyte glutathione levels. The uptake of 0–3 mM l-[35S]cysteine into ventricular cardiomyocytes isolated from adult male Wistar rats was measured using oil filtration. Cysteine transporter expression was investigated by conventional and real-time quantitative reverse-transcription polymerase chain reaction and Western blotting. Glutathione levels were measured enzymatically. Oxidative stress was induced via 0–6 h incubation with 0.05 mM H2O2. Cysteine uptake was greatest in sodium-containing media and was inhibited by glutamine, 2-(methylamino)-isobutyric acid (αMeAIB), serine or alanine. The K m and V max of the αMeAIB insensitive and sensitive portions were 0.133 ± 0.01 mM and 468.11 ± 9.04 pmol/μl cell vol/min, and 0.557 ± 0.096 mM and 279.87 ± 16.06 pmol/μl cell vol/min, respectively. Cardiomyocytes expressed ASCT2, SNAT1 and SNAT2 but not ASCT1. Oxidative stress significantly enhanced cysteine uptake, which was attenuated by αMeAIB. This was accompanied by significantly enhanced SNAT1 expression, whilst SNAT2 and ASCT2 were unaffected. Incubation with cysteine significantly reduced the oxidative-stress-induced decline in cardiomyocyte glutathione as compared to cells incubated without cysteine or cells incubated with cysteine and αMeAIB. In conclusion, under control conditions SNAT transporters aid in the delivery of cysteine for cardiomyocyte GSH synthesis, whilst oxidative stress increases cardiomyocyte cysteine uptake and stimulates cardiomyocyte SNAT1 expression.
Keywords: SNAT1; Oxidative stress; Isolated cardiomyocytes; Glutathione

A new chiral derivatizing agent for α-amino acids is described which leads to diastereomers that can be separated by reverse-phase HPLC with direct detection by a diode array detector. The main advantage of the presented procedure is the fact that an excess of the derivatizing reagent can be employed as the product exhibits an absorption maximum at 360 nm, while the reagent has its absorption maximum at 260 nm. Therefore, it is possible to suppress the reagent signal by a detection wavelength of 400 nm leading to an easy and general method for the enantioseparation of a mixture of dl-amino acids and the determination of the enantiomeric purity of α-amino acid as exemplified by 16 different α-amino acids.
Keywords: Sanger reagent; Marfey reagent; Chiral derivatization agent (CDA); Amino acids; Enantiomeric purity determination

N,N′-Pyromelliticdiimido-di-l-alanine (1), N,N′-Pyromelliticdiimido-di-l-phenylalanine (2), and N,N′-Pyromelliticdiimido-di-l-leucine (3) were prepared from the reaction of Pyromellitic dianhydride with corresponding l-amino acids in a mixture of glacial acetic acid and pyridine solution (3/2 ratio) under refluxing conditions. A series of poly (amide-imide)s containing l-amino acids were prepared from the synthesized dicarboxylic acids with two synthetic aromatic diamines in an ionic liquid (IL) as a green, safe and eco-friendly medium and also reactions catalysis agent. Evaluation of data shows that IL is the better polyamidation medium than the reported method and the catalysis stand on the higher inherent viscosities of the obtained PAIs and the rate of polymerizations beyond the greener reaction conditions and deletion of some essential reagents in conventional manners. Characterization were performs by means of IR, MS and 1H NMR spectroscopy, elemental analysis, specific rotation, thermogravimetric analysis and differential scanning calorimetric techniques. Molecular weights of the obtained polymers were evaluated viscometrically, and the measured inherent viscosities were in the range 0.43–0.85 dL/g. These polymers were readily soluble in many organic solvents. These polymers still kept good thermal stability with glass transition temperatures in the range of 94–154°C, and the decomposition temperature under the nitrogen atmosphere for 10% weight-loss temperatures in excess of 308°C.
Keywords: Biodegradable; Amino acid; Green chemistry; Poly (amide-imide); Ionic liquid

Cysteine fluxes across the portal-drained viscera of enterally fed minipigs: effect of an acute intestinal inflammation by Didier Rémond; Caroline Buffière; Corine Pouyet; Isabelle Papet; Dominique Dardevet; Isabelle Savary-Auzeloux; Gary Williamson; Magali Faure; Denis Breuillé (543-552).
Cysteine is considered as a conditionally indispensable amino acid. Its dietary supply should thus be increased when endogenous synthesis cannot meet metabolic need, such as during inflammatory diseases. However, studies in animal models suggest a high first-pass extraction of dietary cysteine by the intestine, limiting the interest for an oral supplementation. We investigated here unidirectional fluxes of cysteine across the portal-drained viscera (PDV) of multi-catheterized minipigs, using simultaneous intragastric l-[15N] cysteine and intravenous l-[3,3D2] cysteine continuous infusions. We showed that in minipigs fed with an elemental enteral solution, cysteine first-pass extraction by the intestine is about 60% of the dietary supply, and that the PDV does not capture arterial cysteine. Beside dietary cysteine, the PDV release non-dietary cysteine (20% of the total cysteine release), which originates either from tissue metabolism or from reabsorption of endogenous secretion, such as glutathione (GSH) biliary excretion. Experimental ileitis induced by local administration of trinitrobenzene sulfonic acid, increased liver and ileal GSH fractional synthesis rate during the acute phase of inflammation, and increased whole body flux of cysteine. However, cysteine uptake and release by the PDV were not affected by ileitis, suggesting an adaptation of the intestinal sulfur amino acid metabolism in order to cover the additional requirement of cysteine linked to the increased GSH synthesis. We conclude that the small intestine sequesters large amounts of dietary cysteine during absorption, limiting its release into the bloodstream, and that the other tissues of the PDV (colon, stomach, pancreas, spleen) preferentially use circulating methionine or cysteine-containing peptides to cover their cysteine requirement.
Keywords: Cysteine; Glutathione; Portal-drained viscera; Ileitis

Uptake and conversion of d-amino acids in Arabidopsis thaliana by Dirk Gördes; Üner Kolukisaoglu; Kerstin Thurow (553-563).
The d-enantiomers of proteinogenic amino acids fulfill essential functions in bacteria, fungi and animals. Just in the plant kingdom, the metabolism and role of d-amino acids (d-AAs) still remains unclear, although plants have to cope with significant amounts of these compounds from microbial decay in the rhizosphere. To fill this gap of knowledge, we tested the inhibitory effects of d-AAs on plant growth and established a method to quantitate 16 out of 19 proteinogenic amino acids and their d-enantiomers in plant tissue extracts. Therefore, the amino acids in the extracts were derivatized with Marfey’s reagent and separated by HPLC–MS. We used two ecotypes (Col-0 and C24) and a mutant (lht1) of the model plant Arabidopsis thaliana to determine the influence and fate of exogenously applied d-AAs. All of them were found in high concentrations in the plant extracts after application, even in lht1, which points to additional transporters facilitating the import of d-AAs. The addition of particular amino acids (d-Trp, d-Phe, d-Met and d-His) led to the accumulation of the corresponding l-amino acid. In almost all cases, the application of a d-AA resulted in the accumulation of d-Ala and d-Glu. The presented results indicate that soil borne d-AAs can actively be taken up and metabolized via central metabolic routes.
Keywords: Arabidopsis thaliana ; l- and d-Amino acids; Chiral separation; High performance liquid chromatography; Mass spectrometry; d-Amino acids in soil and plants

Exogenous proline effects on water relations and ions contents in leaves and roots of young olive by Ch. Ben Ahmed; S. Magdich; B. Ben Rouina; S. Sensoy; M. Boukhris; F. Ben Abdullah (565-573).
The ability of exogenous compatible solutes, such as proline, to counteract salt inhibitory effects was investigated in 2-year-old olive trees (Olea europaea L. cv. Chemlali) subjected to different saline water irrigation levels supplied or not with exogenous proline. Leaf water relations [relative water content (RWC), water potential], photosynthetic activity, leaf chlorophyll content, and starch contents were measured in young and old leaves. Salt ions (Na+, K+, and Ca2+), proline and soluble sugars contents were determined in leaf and root tissues. Supplementary proline significantly mitigated the adverse effects of salinity via the improvement of photosynthetic activity (Pn), RWC, chlorophyll and carotenoid, and starch contents. Pn of young leaves in the presence of 25 mM proline was at 1.18 and 1.38 times higher than the values recorded under moderate (SS1) and high salinity (SS2) treatments, respectively. Further, the proline supply seems to have a more important relaxing effect on the photosynthetic chain in young than in old leaves of salt-stressed olive plants. The differential pattern of proline content between young and old leaves suggests that there would be a difference between these tissues in distinguishing between the proline taken from the growing media and that produced as a result of salinity stress. Besides, the large reduction in Na+ accumulation in leaves and roots in the presence of proline could be due to its interference in osmotic adjustment process and/or its dilution by proline supply. Moreover, the lower accumulation of Na+ in proline-treated plants, compared to their corresponding salinity treatment, displayed the improved effect of proline on the ability of roots to exclude the salt ions from the xylem sap flowing to the shoot, and thus better growth rates.
Keywords: Olea europaea L.; Salinity stress; Proline supplement; Photosynthetic activity; Ions contents

Hyperammonemia is considered to be the main cause of decreased levels of the branched-chain amino acids (BCAA), valine, leucine, and isoleucine, in liver cirrhosis. In this study we investigated whether the decrease in BCAA is caused by the direct effect of ammonia on BCAA metabolism and the effect of ammonia on BCAA and protein metabolism in different types of skeletal muscle. M. soleus (SOL, slow-twitch, red muscle) and m. extensor digitorum longus (EDL, fast-twitch, white muscle) of white rat were isolated and incubated in a medium with or without 500 μM ammonia. We measured the exchange of amino acids between the muscle and the medium, amino acid concentrations in the muscle, release of branched-chain keto acids (BCKA), leucine oxidation, total and myofibrillar proteolysis, and protein synthesis. Hyperammonemia inhibited the BCAA release (81% in SOL and 60% in EDL vs. controls), increased the release of BCKA (133% in SOL and 161% in EDL vs. controls) and glutamine (138% in SOL and 145% in EDL vs. controls), and increased the leucine oxidation in EDL (174% of controls). Ammonia also induced a significant increase in glutamine concentration in skeletal muscle. The effect of ammonia on intracellular BCAA concentration, protein synthesis and on total and myofibrillar proteolysis was insignificant. The data indicates that hyperammonemia directly affects the BCAA metabolism in skeletal muscle which results in decreased levels of BCAA in the extracellular fluid. The effect is associated with activated synthesis of glutamine, increased BCAA oxidation, decreased release of BCAA, and enhanced release of BCKA. These metabolic changes are not directly associated with marked changes in protein turnover. The effect of ammonia is more pronounced in muscles with high content of white fibres.
Keywords: Ammonia; Branched-chain amino acids; Leucine metabolism; Protein metabolism; Encephalopathy

Glutamine prevents myostatin hyperexpression and protein hypercatabolism induced in C2C12 myotubes by tumor necrosis factor-α by Andrea Bonetto; Fabio Penna; Valerio G. Minero; Patrizia Reffo; Domiziana Costamagna; Gabriella Bonelli; Francesco M. Baccino; Paola Costelli (585-594).
Depletion of skeletal muscle protein mainly results from enhanced protein breakdown, caused by activation of proteolytic systems such as the Ca2+-dependent and the ATP-ubiquitin-dependent ones. In the last few years, enhanced expression and bioactivity of myostatin have been reported in several pathologies characterized by marked skeletal muscle depletion. More recently, high myostatin levels have been associated with glucocorticoid-induced hypercatabolism. The search for therapeutical strategies aimed at preventing/correcting protein hypercatabolism has been directed to inhibit humoral mediators known for their pro-catabolic action, such as TNFα. The present study has been aimed to investigate the involvement of TNFα in the regulation of both myostatin expression and intracellular protein catabolism, and the possibility to interfere with such modulations by means of amino acid supplementation. For this purpose, C2C12 myotubes exposed to TNFα in the presence or in the absence of amino acid (glutamine or leucine) supplementation have been used. Myotube treatment with TNFα leads to both hyperexpression of the muscle-specific ubiquitin ligase atrogin-1, and enhanced activity of the Ca2+-dependent proteolytic system. These changes are associated with increased myostatin expression. Glutamine supplementation effectively prevents TNFα-induced muscle protein loss and restores normal myostatin levels. The results shown in the present study indicate a direct involvement of TNFα in the onset of myotube protein loss and in the perturbation of myostatin-dependent signaling. In addition, the protective effect exerted by glutamine suggests that amino acid supplementation could represent a possible strategy to improve muscle mass.
Keywords: Myostatin; TNFα; Muscle wasting; Amino acids; Protein breakdown

Different cytoplasmic/nuclear distribution of S6 protein phosphorylated at S240/244 and S235/236 by M. Rosner; C. Fuchs; H. Dolznig; M. Hengstschläger (595-600).
Higher eukaryotic ribosome biogenesis takes place in the nucleolus and requires the import of ribosomal proteins from the cytoplasm. The ribosomal protein S6 is essential for the formation of ribosome subunits, and in mice S6 heterozygosity triggers embryonal lethality. Downstream of the mTOR (mammalian target of rapamycin) and MAPK (mitogen-activated protein kinase) signalling pathways S6 protein is phosphorylated at clustered residues S235/236 and S240/244 upon numerous physiological and pathological stimuli. Here, we show that S240/244-phosphorylated S6 is predominantly nuclear but also detectable in the cytoplasm, whereas S235/236-phosphorylated S6 is almost exclusively localized to the nucleus of primary human cells and virtually undetectable in the cytoplasm. However, in transformed cells the latter can also be detected in the cytoplasm. Experiments with the mTOR inhibitor rapamycin revealed that neither blocking the phosphorylation of S6 at S235/236 and S240/244 nor arresting the cell cycle affects the cytoplasmic/nuclear localization of S6 protein. Our findings provide new insights into the regulation of S6 phosphorylation and S6 protein localization in mammalian cells.
Keywords: S6; Posphorylation; Localization; mTOR; S6Kinase

Beta-amyloid (Aβ) is considered to be responsible for the pathogenesis of Alzheimer’s disease (AD), and accumulation and aggregation of Aβ peptide in the brains of AD patients result in activation of glial cells which, in turn, initiates neuroinflammatory responses that involve reactive oxygen intermediates and release of inflammatory cytokines. In the present study, the protective effects of S-propargyl-cysteine (SPRC), also named as ZYZ-802, a sulphur-containing amino acid, on cognitive impairment and neuronal ultrastructure damage induced by Aβ were examined in rats, and the possible mechanisms were explored. These data showed that SPRC administration at the doses of 40, 80 mg/kg by intraperitoneal injection (i.p.) may inhibit cognitive impairment and neuronal ultrastructure damage induced by intracerebroventricular (i.c.v.) injection of 10 μg of Aβ25–35 in rats. Subsequently, SPRC inhibited the expressions of tumor necrosis factor (TNF)-α, cyclooxygenase-2 (COX-2) mRNA, and protein in rat hippocampus. SPRC afforded a beneficial action on inhibitions of extracellular signal-regulated kinase (ERK1/2), as well as inhibitions of IκB-α degradation and activation of transcription factors of the nuclear factor κB (NF-κB) produced by Aβ. These findings suggested that SPRC might be a potential agent for treatment of AD.
Keywords: Alzheimer’s disease; S-propargyl-cysteine; A sulphur-containing amino acid; Beta-amyloid; Cognitive impairment

In this investigation N,N′-(pyromellitoyl)-bis-l-tyrosine dimethyl ester (7) as a chiral bioactive diphenolic monomer was prepared in three steps. The aim of this work was to obtain novel optically and biologically active pseudo-poly(amino acid)s (PAA)s that are more soluble in common organic solvents while maintaining their high thermal stability. Thus, several new, highly soluble, thermally stable, optically active and biodegradable PAAs containing different amino acid moieties in the main chain were prepared with moderate molecular weights via direct polycondensation using tosyl chloride, pyridine and N,N′-dimethylformamide as a condensing agent. The resulting novel polymers were characterized with FT-IR, 1H-NMR, elemental and thermogravimetric analysis techniques. In addition, in vitro toxicity and biodegradability behavior of the diphenolic monomer 7, different synthetic diacids (3a3e) and obtained PAAs, which were investigated in culture media, showed that the synthesized compounds and polymers derived from them are biologically active and biodegradable under a natural environment.
Keywords: Pseudo-poly(amino acid)s; Nontoxic aromatic diol; l-tyrosine; Biodegradable

In this study, we investigated the activating role of Mcm1p at ARG1 during arginine starvation. Our results showed that two Mcm1p binding sites positively contribute to ARG1 transcription and cell growth. Especially, we provide strong evidence that the Mcm1p binding sites play a positive role in ARG1 transcription to overcome arginine starvation in the absence of Gcn4p. In addition, we found that the Mcm1p binding sites are not only regulated by the presence or absence of arginine but also in the presence or absence of other amino acids. These findings suggest that the ARG1 promoter utilizes different DNA elements to control transcription, depending on which amino acids are detected in the medium.
Keywords: Gcn4p; Mcm1p; ARG1 ; SD and SC media; Arginine starvation

An efficient route towards a new branched tetrahydrofurane δ-sugar amino acid from a pyrolysis product of cellulose by Andrea Defant; Ines Mancini; Cristian Torri; Danilo Malferrari; Daniele Fabbri (633-640).
(1R,5S)-1-Hydroxy-3,6-dioxa-bicyclo[3.2.1]octan-2-one, is a bicyclic lactone obtained in gram-scale by catalytic pyrolysis of the renewable source cellulose. Now it has been used as a chiral building block in the preparation of the new δ-sugar amino acid, (3R,5S)-5-(aminoethyl)-3-hydroxytetrahydrofurane-3-carboxylic acid, by an efficient synthesis in five steps with a 67% overall yield. The structure of this tetrahydrofurane amino acid, isolated in protonated form, was assigned by extensive mono- and bidimensional 1H- and 13C-NMR analysis and mass spectrometry, including measurements by electrospray and matrix-assisted laser desorption ionization techniques, the latter one for high-resolution experiments. This amino acid is an isoster of dipeptide glycine-alanine (H-Gly-Ala-OH), with a potential use in the access of new peptidomimetics with conformationally restricted structures due to the presence of tetrahydrofurane ring. As a preliminary study in order to disclose this effect, density functional theory calculation performed in water using polar continuum model was applied to the new amino acid and H-Gly-Ala-OH dipeptide, so that to evaluate and compare the relative torsional angles for the energy-minimized structures.
Keywords: Sugar amino acids; Chiral building block; Dipeptide isoster; DFT calculation; Hydroxylactone; Cellulose pyrolysis

A proteomic analysis of PKCε targets in astrocytes: implications for astrogliosis by Miguel Burgos; Noelia Fradejas; Soledad Calvo; Sung Ung Kang; Pedro Tranque; Gert Lubec (641-651).
Astrocytes are glial cells in the central nervous system (CNS) that play key roles in brain physiology, controlling processes, such as neurogenesis, brain energy metabolism and synaptic transmission. Recently, immune functions have also been demonstrated in astrocytes, influencing neuronal survival in the course of neuroinflammatory pathologies. In this regard, PKCepsilon (PKCε) is a protein kinase with an outstanding role in inflammation. Our previous findings indicating that PKCε regulates voltage-dependent calcium channels as well as morphological stellation imply that this kinase controls multiple signalling pathways within astrocytes, including those implicated in activation of immune functions. The present study applies proteomics to investigate new protein targets of PKCε in astrocytes. Primary astrocyte cultures infected with an adenovirus that expresses constitutively active PKCε were compared with infection controls. Two-dimensional gel electrophoresis clearly detected 549 spots in cultured astrocytes, and analysis of differential protein expression revealed 18 spots regulated by PKCε. Protein identification by mass spectrometry (nano-LC–ESI-MS/MS) showed that PKCε targets molecules with heterogeneous functions, including chaperones, cytoskeletal components and proteins implicated in metabolism and signalling. These results support the notion that PKCε is involved in astrocyte activation; also suggesting that multiple astrocyte-dependent processes are regulated by PKCε, including those associated to neuroinflammation.
Keywords: Neuroinflammation; Glia; Reactive astrocytes; Chaperone; α-B-crystallin; HSP60; Protein kinase

Plasma nitroproteome of kidney disease patients by Marta Piroddi; Angelo Palmese; Francesca Pilolli; Angela Amoresano; Piero Pucci; Claudio Ronco; Francesco Galli (653-667).
3′-nitrotyrosine (3NT) is a post-translational modification (PTM) of body fluids and tissues that is sustained by chronic inflammation and oxidative stress, two main clinical traits of chronic kidney disease (CKD). Despite this background, protein targets and their differential susceptibility to in vivo nitration remain almost completely unexplored in CKD. This study reports a first investigation of plasma nitroproteome in these patients, carried out by both immunorecognition and LC-MS/MS techniques. Plasma proteins of chronic and end-stage KD patients showed a higher burden of nitration than in healthy controls, but main nitration targets appeared to be the same in these populations. Immunoblotting data showed that uremic albumin is largely represented in the uremic nitroproteome together with fibrinogen chains (A, B and C), transferrin, α1-antitrypsin, complement factor D, haptoglobin, and IgG light and heavy chains. However, immunopurification and affinity chromatography experiments demonstrated that the relative content of 3NT on the albumin molecule was very low when compared with that of the remaining plasma proteins. The uremic nitroproteome was investigated using also plasma proteins obtained by in vivo ultrafiltration from patients treated with protein leaking or standard high-flux hemodialyzers. The study of these samples revealed the possibility to selectively remove protein nitration products during hemodialysis. Identification of intramolecular sites of nitration was preliminarily obtained in IgG chains isolated by 2D PAGE and assessed by bidimensional tandem mass spectrometry after chemoselective tagging. Further studies are needed to confirm at the molecular level the presence of nitrated Tyr residues in other proteins tentatively identified as nitration targets in this study and to explore the biological meaning of such a selective modification of plasma proteins by reactive nitrogen species in uremia and dialysis patients.
Keywords: 3′-nitrotyrosine; Protein nitration; Plasma proteins; Uremia; Hemodialysis; Chronic kidney disease; Uremic toxins; Proteomics; Nitroproteome; Mass spectrometry

LC/MS evaluation of metabolism and membrane transport of bombesin peptides by Dongyu Gu; Ying Ma; Gang Niu; Yongjun Yan; Lixin Lang; Haji Akber Aisaand; Haokao Gao; Dale O. Kiesewetter; Xiaoyuan Chen (669-675).
Two bombsin peptides, GRPR agonist [Aca-QWAVGHLM-NH2] and antagonist [fQWAVGHL-NHEthyl] were evaluated. We employed the highly sensitive Waters Q-Tof Premier MS coupled with a UPLC system to identify the metabolites produced by rat hepatocytes or PC-3 human prostate cancer cells; and we utilized the AB/MDS 4000 Q-Trap LC/MS/MS system with highly sensitive quantitative and qualitative performance, to quantitatively analyze the internalization of GRPR agonist and antagonist in PC-3 cells. The major metabolites of both GRPR agonist and antagonist were the result of peptide bond hydrolysis between W and A which was demonstrated by observation of the N-terminal fragment m/z 446 (Aca-QW-OH) for agonist and m/z 480 (fQW-OH) for antagonist. Both peptides were also hydrolyzed between A and V which formed peaks m/z 517 [Aca-QWA-OH] and m/z 555 (VGHLM-NH2) for the agonist and m/z 551 [fQWA-OH] and m/z 452 (VGHL-NHEthyl) for the antagonist. The peptide agonist also formed a unique metabolite that resulted from hydrolysis of the C-terminal amide. The antagonist showed significantly slower metabolism as compared to the agonist in both rat hepatocytes and PC-3 cells. The antagonist also showed significantly lower PC-3 cell internalization rate than that of the agonist. In conclusion, the metabolism profiles of both GRPR agonist and antagonist peptides were identified by LC/MS. The antagonist peptide was more stable than the agonist peptide in rat hepatocyte incubation. One major factor could be the hydrolysis-resistant C-terminal L-NHEthyl group compared with the unsubstituted amide of the agonist. Another factor could be different amino acid sequences of the agonist and antagonist that may also influence the enzymatic hydrolysis. The antagonist ligand is potentially more useful for receptor-targeted imaging due primarily to its higher metabolic stability.
Keywords: LC/MS/MS; Gastrin-releasing peptide receptor (GRPR); Bombesin (BBN); Agonist; Antagonist

Erratum to: LC/MS evaluation of metabolism and membrane transport of bombesin peptides by Dongyu Gu; Ying Ma; Gang Niu; Yongjun Yan; Lixin Lang; Haji Akber Aisa; Haokao Gao; Dale O. Kiesewetter; Xiaoyuan Chen (677-677).

Calcium and zinc complexes of pyrroglutamate analogs detected by electrospray ionization mass spectrometry by Joséphine Beck; Laetitia Maton; Jean-Louis Habib Jiwan; Jacqueline Marchand-Brynaert (679-687).
The complexation of calcium and zinc cations by pyrroglutamate analogs has been studied in the gas phase by means of electrospray ionization mass spectrometry (ESI–MS). Complexes were obtained from the solutions of calcium perchlorate and zinc perchlorate in acetonitrile. The complexes with calcium are singly and doubly charged with various stoichiometries while zinc complexes are singly charged except for one ligand. Solvation with acetonitrile and presence of perchlorate counter-ions are observed when the complexes are in the gas phase. The complexes formed with both metals are mainly L2M and LM species. All tested compounds are better complexing agents for calcium than for zinc.
Keywords: Non natural aminoacids; Aminophosphonates; Aminobisphosphonates; Zinc complexes; Calcium complexes; Electrospray ionization mass spectrometry

Plasma free amino acid kinetics in rainbow trout (Oncorhynchus mykiss) using a bolus injection of 15N-labeled amino acids by Jacob William Robinson; Dan Yanke; Jeff Mirza; James Stuart Ballantyne (689-696).
To gain insight into the metabolic design of the amino acid carrier systems in fish, we injected a bolus of 15N amino acids into the dorsal aorta in mature rainbow trout (Oncorhynchus mykiss). The plasma kinetic parameters including concentration, pool size, rate of disappearance (R d), half-life and turnover rate were determined for 15 amino acids. When corrected for metabolic rate, the R d values obtained for trout for most amino acids were largely comparable to human values, with the exception of glutamine (which was lower) and threonine (which was higher). R d values ranged from 0.9 μmol 100 g−1 h−1 (lysine) to 22.1 μmol 100 g−1 h−1 (threonine) with most values falling between 2 and 6 μmol 100 g−1 h−1. There was a significant correlation between R d and the molar proportion of amino acids in rainbow trout whole body protein hydrolysate. Other kinetic parameters did not correlate significantly with whole body amino acid composition. This indicates that an important design feature of the plasma-free amino acids system involves proportional delivery of amino acids to tissues for protein synthesis.
Keywords: Oncorhynchus mykiss ; Plasma amino acid kinetics; Amino acid turnover; Bolus injection; 15N; Fish

Observed peptide pI and retention time shifts as a result of post-translational modifications in multidimensional separations using narrow-range IPG-IEF by Johan Lengqvist; Hanna Eriksson; Marcus Gry; Kristina Uhlén; Christina Björklund; Bengt Bjellqvist; Per-Johan Jakobsson; Janne Lehtiö (697-711).
Modified peptides constitute a sub-population among the tryptic peptides analyzed in LC–MS based shotgun proteomics experiments. For larger proteomes including the human proteome, the tryptic peptide pool is very large, which necessitates some form of sample fractionation. By carefully choosing the sample fractionation and separation methods applied as shown here for the combination of narrow-range immobilized pH gradient isoelectric focusing (IPG-IEF) and nanoUPLC–MS, significantly increased information content can be achieved. Relatively low standard deviations were obtained for such multidimensional separations in terms of peptide pI (<0.05 pI units) and retention time (<0.3 min for a 350 min gradient) for a selection of highly complex proteomics samples. Using narrow-range IPG-IEF, experimental and predicted pI were in relative good agreement. However, based on our data, retention time prediction algorithms need further improvements in accuracy to match state-of-the-art reversed-phase chromatography performance. General trends of peptide pI shifts induced by common modifications including deamidations and N-terminal modifications are described. Deamidations of glutamine and asparagines shift peptide pI by approximately 1.5 pI units, making the peptides more acidic. Additionally, a novel pI shift (+~0.4 pI units) was found associated with dethiomethyl Met modifications. Further, the effects of these modifications as well as methionine oxidation were investigated in terms of experimentally observed retention time shifts in the chromatographic separation step. Clearly, post-translational modification-induced influences on peptide pI and retention time can be accurately and reproducibly measured using narrow-range IPG-IEF and high-performance nanoLC–MS. Even at modest mass accuracy (±50 ppm), the inclusion of peptide pI (±0.2 pI units) and/or retention time (±20 min) criteria are highly informative for human proteome analyses. The applications of using this information to identify post-translationally modified peptides and improve data analysis workflows are discussed.
Keywords: Isoelectric focusing; Post-translational modifications; Retention time

Preliminary kinetic characterization of a copper amine oxidase from rat liver mitochondria matrix by Roberto Stevanato; Sara Cardillo; Michele Braga; Angela De Iuliis; Valentina Battaglia; Antonio Toninello; Enzo Agostinelli; Fabio Vianello (713-720).
Kinetic measurements of a novel copper-dependent amine oxidase, purified from rat liver mitochondria matrix, were carried out using various substrates in a large pH (5.6–10.2) and ionic strength range (5–200 mM), in order to study the docking of substrates to the enzyme and, as a consequence, to verify the physicochemical characteristics of the active site. Relatively small changes of V max values (approx. 2.5-folds) over the substrates tested, suggest that the rate determining step of the catalysis is only slightly affected by amine chemical structure. In contrast, the strong change of K M and k c/K M values (approx. two orders of magnitude) indicates electrostatic control of the docking process, since the changes of K M and k c/K M values appear due to the presence of positively charged groups in the substrate molecules. These results suggest the presence in the enzyme active site of two negatively charged amino acid residues which seem to interact with positively charged groups of the substrate molecules. Analogies and differences with bovine serum amine oxidase are also described.
Keywords: Amine oxidases; Mitochondrion; Enzyme function; Ionic strength; Polyamines

In this paper, we report on a catanionic vesicles-based strategy to reduce the cytotoxicity of the diacyl glycerol arginine-based synthetic surfactants 1,2-dimyristoyl-rac-glycero-3-O-(N α-acetyl-l-arginine) hydrochloride (1414RAc) and 1,2-dilauroyl-rac-glycero-3-O-(N α-acetyl-l-arginine) hydrochloride (1212RAc). The behavior of these surfactants was studied either as pure components or after their formulation as pseudo-tetra-chain catanionic mixtures with phosphatidylglycerol (PG) and as cationic mixtures with 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) used as control. The antimicrobial activity of the negatively charged formulations against Acinetobacter baumannii was maintained with respect to the surfactant alone, while a significant improvement of the antimicrobial activity against Staphylococcus aureus was observed, together with a strong decrease of hemolytic activity. The influence of the net charge of the catanionic vesicles on membrane selectivity was studied using model membranes. The dynamics of surface tension changes induced by the addition of 1414RAc/PG aqueous dispersions into phospholipid monolayers composed of zwitterionic DPPC as model system for mammalian membranes and of negatively charged PG mimicking cytoplasmic membrane of Gram-positive bacteria was followed by tensiometry. Our results constitute a proof of principle that tuning formulation can reduce the cytotoxicity of many surfactants, opening their possible biological applications.
Keywords: Amino acid-based surfactant; Antimicrobial agent; Phosphatidylglycerol; Pseudo-tetra-chain catanionic mixtures; Hemolysis; Dynamic surface tension

A peptide mimic of the chemotaxis inhibitory protein of Staphylococcus aureus: towards the development of novel anti-inflammatory compounds by Anton Bunschoten; Johannes H. Ippel; John A. W. Kruijtzer; Louris Feitsma; Carla J. C. de Haas; Rob M. J. Liskamp; Johan Kemmink (731-740).
Complement factor C5a is one of the most powerful pro-inflammatory agents involved in recruitment of leukocytes, activation of phagocytes and other inflammatory responses. C5a triggers inflammatory responses by binding to its G-protein-coupled C5a-receptor (C5aR). Excessive or erroneous activation of the C5aR has been implicated in numerous inflammatory diseases. The C5aR is therefore a key target in the development of specific anti-inflammatory compounds. A very potent natural inhibitor of the C5aR is the 121-residue chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS). Although CHIPS effectively blocks C5aR activation by binding tightly to its extra-cellular N terminus, it is not suitable as a potential anti-inflammatory drug due to its immunogenic properties. As a first step in the development of an improved CHIPS mimic, we designed and synthesized a substantially shorter 50-residue adapted peptide, designated CHOPS. This peptide included all residues important for receptor binding as based on the recent structure of CHIPS in complex with the C5aR N terminus. Using isothermal titration calorimetry we demonstrate that CHOPS has micromolar affinity for a model peptide comprising residues 728 of the C5aR N terminus including two O-sulfated tyrosine residues at positions 11 and 14. CD and NMR spectroscopy showed that CHOPS is unstructured free in solution. Upon addition of the doubly sulfated model peptide, however, the NMR and CD spectra reveal the formation of structural elements in CHOPS reminiscent of native CHIPS.
Keywords: C5a receptor; CHIPS; Inflammation; Anaphylatoxin

Characterization of calmodulin binding domains in TRPV2 and TRPV5 C-tails by Blanka Holakovska; Lenka Grycova; Jan Bily; Jan Teisinger (741-748).
The transient receptor potential channels TRPV2 and TRPV5 belong to the vanilloid TRP subfamily. TRPV2 is highly similar to TRPV1 and shares many common properties with it. TRPV5 (and also its homolog TRPV6) is a rather distinct member of the TRPV subfamily. It is distant for being strictly Ca2+-selective and features quite different properties from the rest of the TRPV subfamily. It is known that TRP channels are regulated by calmodulin in a calcium-dependent manner. In our study we identified a calmodulin binding site on the C-termini of TRPV2 (654–683) and TRPV5 (587–616) corresponding to the consensus CaM binding motif 1-5-10. The R679 and K681 single mutants of TRPV2 caused a 50% decrease in binding affinity and a double mutation of K661/K664 of the same peptide lowered the binding affinity by up to 75%. A double mutation of R606/K607 and triple mutation of R594/R606/R610 in TRPV5 C-terminal peptide resulted in the total loss of binding affinity to calmodulin. These results demonstrate that the TRPV2 C-tail and TRPV5 C-tail contain calmodulin binding sites and that the basic residues are strongly involved in TRP channel binding to calmodulin.
Keywords: TRP channel; Steady-state fluorescence anisotropy; Binding site; Calmodulin; Calcium

Membrane-interaction and assembly of a leucine zipper peptide (LZP), and its single (SASA) and double (DASA) alanine-substituted analog onto mammalian, hRBCs and 3T3 cells and bacteria, Escherichia coli and Staphylococcus aureus were studied as a model system to understand the plausible role of assembly on their contrasting cytotoxic but similar bactericidal activities. Peptides’ ability to depolarize and damage the membrane organization of hRBC and 3T3 cells decreased from LZP to SASA and to DASA which may be related to their decrease in assembly onto these mammalian live cells and oligomerization states in the presence of these cell membranes or zwitterionic PC/Chol lipid vesicles. However, LZP and its analogs showed appreciable similarities in damaging or depolarizing the E. coli or S. aureus cells, which further matched with their comparable assembly and oligomerization either onto these live cells or the cell membranes or in the presence of negatively charged PC/PG lipid vesicles.
Keywords: Antimicrobial peptides, bactericidal and cytotoxic activity; Cellular interactions; Peptide-induced depolarization and damage of membrane organization of bacteria and mammalian cells; Leucine zipper motif; Assembly and oligomerization of antibacterial peptides

β-Lactam antibiotic produces a sustained reduction in extracellular glutamate in the nucleus accumbens of rats by Bruce A. Rasmussen; David A. Baron; Jae K. Kim; Ellen M. Unterwald; Scott M. Rawls (761-764).
We investigated the short- and long-term effects of ceftriaxone on glutamate transporter subtype 1 (GLT-1) transporter activity and extracellular glutamate in the rat nucleus accumbens. Repeated ceftriaxone administration (50, 100 or 200 mg/kg, i.p.) produced a dose-dependent reduction in glutamate levels that persisted for 20 days following discontinuation of drug exposure. The ceftriaxone effect was prevented by the GLT-1 transporter inhibitor dihydrokainate (1 μM, intra-accumbal). These results suggest that β-lactam antibiotics produce an enduring reduction in glutamatergic transmission in the brain reward center.
Keywords: Ceftriaxone; Glutamate; Nucleus accumbens; β-Lactam antibiotic; Microdialysis; GLT-1