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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.43, #4)

Paraoxonase 1 and homocysteine metabolism by Joanna Perła-Kaján; Hieronim Jakubowski (pp. 1405-1417).

Paraoxonase 1 (PON1), a component of high-density lipoprotein (HDL), is a calcium-dependent multifunctional enzyme that connects metabolisms of lipoproteins and homocysteine (Hcy). Both PON1 and Hcy have been implicated in human diseases, including atherosclerosis and neurodegeneration. The involvement of Hcy in disease could be mediated through its interactions with PON1. Due to its ability to reduce oxidative stress, PON1 contributes to atheroprotective functions of HDL in mice and humans. Although PON1 has the ability to hydrolyze a variety of substrates, only one of them—Hcy-thiolactone—is known to occur naturally. In humans and mice, Hcy-thiolactonase activity of PON1 protects against N-homocysteinylation, which is detrimental to protein structure and function. PON1 also protects against neurotoxicity associated with hyperhomocysteinemia in mouse models. The links between PON1 and Hcy in relation to pathological states such as coronary artery disease, stroke, diabetic mellitus, kidney failure and Alzheimer’s disease that emerge from recent studies are the topics of this review.

Keywords: Paraoxonase 1; Homocysteine; N-homocysteinylation; Thiolactonase; Atherosclerosis; Alzheimer’s disease

Interactions of acidic pharmaceuticals with human serum albumin: insights into the molecular toxicity of emerging pollutants by Jiabin Chen; Xuefei Zhou; Yalei Zhang; Yajie Qian; Haiping Gao (pp. 1419-1429).

Acidic pharmaceuticals such as diclofenac (DCF), clofibric acid (CA) and ketoprofen (KTP) have been detected frequently in environmental media. In order to reveal the toxicity of such emerging pollutants, their interactions with human serum albumin (HSA) were investigated by capillary electrophoresis, molecular spectrometry, and equilibrium dialysis. The binding constants and sites of these acidic pharmaceuticals with HSA were obtained. The thermodynamic parameters, e.g. enthalpy change and entropy change of these interactions were calculated to characterize that all the reactions resulted from hydrophobic and electrostatic interactions. The static quenching of the fluorescence of HSA was observed when interacted with acidic pharmaceuticals, indicating acidic pharmaceuticals bound to Tryptophan residue of HSA. The 3D fluorescence and circular dichroism confirmed that the secondary conformation of HSA changed after the interactions with the pharmaceuticals. At physiological condition, only 0.12 mM acidic pharmaceuticals reduced the binding of vitamin B₂ to HSA by 37, 30 and 21% for DCF, KTP and CA, respectively. This work provides an insight into non-covalent interactions between emerging contaminants and biomolecule, and is helpful for clarifying the toxic mechanism of such emerging contaminants.

Keywords: HSA; Non-covalent interaction; Acidic pharmaceuticals; Fluorescence; Capillary electrophoresis

Site-selective radiolabeling of peptides by ¹⁸F-fluorobenzoylation with [¹⁸F]SFB in solution and on solid phase: a comparative study by Manuela Kuchar; Marc Pretze; Torsten Kniess; Jörg Steinbach; Jens Pietzsch; Reik Löser (pp. 1431-1443).

Peptides labeled with short-lived positron-emitting radionuclides are of outstanding interest as probes for molecular imaging by positron emission tomography (PET). Herein, the site-selective incorporation of fluorine-18 into lysine-containing peptides using the prosthetic labeling agent N-succinimidyl 4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB) is described. The reaction of [¹⁸F]SFB with four biologically relevant resin-bound peptides was studied and optimized. For comparison, each peptide was ¹⁸F-fluorobenzoylated in solution under different conditions and the product distribution was analyzed confirming the advantages of the solid-phase approach. The method’s feasibility for selective radiolabeling either at the N-terminus or at the lysine side chain was demonstrated. Labeling on solid phase with [¹⁸F]SFB resulted in crude ¹⁸F-fluorobenzoylpeptides whose radiochemical purities were typically greater than 90% and that could be prepared in synthesis times from 65 to 76 min.

Keywords: Fluorine-18; Solid-phase synthesis; Regioselectivity; Positron emission tomography

Cytotoxic activity of new racemic and optically active N-phosphonoalkyl bicyclic β-amino acids against human malignant cell lines by Petar T. Todorov; Diana W. Wesselinova; Nikola D. Pavlov; Jean Martinez; Monique Calmes; Emilia D. Naydenova (pp. 1445-1450).

The cytotoxic effects of novel racemic and optically active constrained N-phosphonoalkyl bicyclic β-amino acids were tested against a panel of human tumor cell lines. All of the compounds investigated exhibited different concentration-dependent antiproliferative effects against the HT-29, MDA-MB-231, HepG2 and HeLa cell lines after 24 h treatment. The most sensitive cells were the HeLa cells at various concentrations of the four compounds tested. The aminophosphonate 3 exerted the most pronounced antiproliferative effect against the HeLa cells (inhibition of the cell vitality up to 70% at 0.5 mg/ml) and was not toxic to the normal Lep3 cells at lower concentration. Furthermore, the N-phosphonophenyl derivatives 1 and 2 displayed antiproliferative effect against mainly the MDA-MB-231 tumour cells at higher concentration.

Keywords: Cytotoxic activity; Human tumor cell lines; Aminophosphonates; N-phosphonoalkyl β-amino acids

Insilico study of the A_2AR–D₂R kinetics and interfacial contact surface for heteromerization by Amresh Prakash; Pratibha Mehta Luthra (pp. 1451-1464).

G-protein-coupled receptors (GPCRs) are cell surface receptors. The dynamic property of receptor–receptor interactions in GPCRs modulates the kinetics of G-protein signaling and stability. In the present work, the structural and dynamic study of A_2AR–D₂R interactions was carried to acquire the understanding of the A_2AR–D₂R receptor activation and deactivation process, facilitating the design of novel drugs and therapeutic target for Parkinson’s disease. The structure-based features (Alpha, Beta, SurfAlpha, and SurfBeta; GapIndex, Leakiness and Gap Volume) and slow mode model (ENM) facilitated the prediction of kinetics (K _off, K _on, and K _d) of A_2AR–D₂R interactions. The results demonstrated the correlation coefficient 0.294 for K _d and K _on and the correlation coefficient 0.635 for K _d and K _off, and indicated stable interfacial contacts in the formation of heterodimer. The coulombic interaction involving the C-terminal tails of the A_2AR and intracellular loops (ICLs) of D₂R led to the formation of interfacial contacts between A_2AR–D₂R. The properties of structural dynamics, ENM and KFC server-based hot-spot analysis illustrated the stoichiometry of A_2AR–D₂R contact interfaces as dimer. The propensity of amino acid residues involved in A_2AR–D₂R interaction revealed the presence of positively (R, H and K) and negatively (E and D) charged structural motif of TMs and ICL3 of A_2AR and D₂R at interface of dimer contact. Essentially, in silico structural and dynamic study of A_2AR–D₂R interactions will provide the basic understanding of the A_2AR–D₂R interfacial contact surface for activation and deactivation processes, and could be used as constructive model to recognize the protein–protein interactions in receptor assimilations.

Keywords: A_2AR; D₂R; Kinetic constant; Dimer; Parkinson’s disease

Synthesis and aggregation properties of a novel enzymatically resistant nucleoamino acid by Giovanni N. Roviello; Anna Mottola; Domenica Musumeci; Enrico M. Bucci; Carlo Pedone (pp. 1465-1470).

In this work, we describe the synthesis, evaluation of some biological properties, such as DNA- and RNA-binding ability and in sero stability, as well as the supramolecular assembly of a novel nucleoamino acid based on l-spinacine. More particularly, a thymine-containing l-spinacine derivative was synthesized in liquid phase by a simple peptide-coupling procedure. Subsequently, nucleic acid and Cu²⁺-binding ability, as well as self-assembly properties of the novel nucleoamino acid, were investigated by spectroscopy (CD and UV) and laser light scattering which furnished interesting information on the assembly of supramolecular networks based on the peptidyl nucleoside analog. Finally, nucleoamino acid enzymatic stability was studied and a half life of about 7 days was found in the presence of fresh human serum.

Keywords: Spinacine; Nucleobase; Supramolecular; DNA

The α-defensin salt-bridge induces backbone stability to facilitate folding and confer proteolytic resistance by Håkan S. Andersson; Sharel M. Figueredo; Linda M. Haugaard-Kedström; Elina Bengtsson; Norelle L. Daly; Xiaoqing Qu; David J. Craik; André J. Ouellette; K. Johan Rosengren (pp. 1471-1483).

Salt-bridge interactions between acidic and basic amino acids contribute to the structural stability of proteins and to protein–protein interactions. A conserved salt-bridge is a canonical feature of the α-defensin antimicrobial peptide family, but the role of this common structural element has not been fully elucidated. We have investigated mouse Paneth cell α-defensin cryptdin-4 (Crp4) and peptide variants with mutations at Arg⁷ or Glu¹⁵ residue positions to disrupt the salt-bridge and assess the consequences on Crp4 structure, function, and stability. NMR analyses showed that both (R7G)-Crp4 and (E15G)-Crp4 adopt native-like structures, evidence of fold plasticity that allows peptides to reshuffle side chains and stabilize the structure in the absence of the salt-bridge. In contrast, introduction of a large hydrophobic side chain at position 15, as in (E15L)-Crp4 cannot be accommodated in the context of the Crp4 primary structure. Regardless of which side of the salt-bridge was mutated, salt-bridge variants retained bactericidal peptide activity with differential microbicidal effects against certain bacterial cell targets, confirming that the salt-bridge does not determine bactericidal activity per se. The increased structural flexibility induced by salt-bridge disruption enhanced peptide sensitivity to proteolysis. Although sensitivity to proteolysis by MMP7 was unaffected by most Arg⁷ and Glu¹⁵ substitutions, every salt-bridge variant was degraded extensively by trypsin. Moreover, the salt-bridge facilitates adoption of the characteristic α-defensin fold as shown by the impaired in vitro refolding of (E15D)-proCrp4, the most conservative salt-bridge disrupting replacement. In Crp4, therefore, the canonical α-defensin salt-bridge facilitates adoption of the characteristic α-defensin fold, which decreases structural flexibility and confers resistance to degradation by proteinases.

Keywords: Defensin; Cryptdin-4; Crp4; Salt-bridge; Structure; Folding; Proteolytic stability

Decreased glutamate, glutamine and citrulline concentrations in plasma and muscle in endotoxemia cannot be reversed by glutamate or glutamine supplementation: a primary intestinal defect? by Claire Boutry; Hideki Matsumoto; Cécile Bos; Christophe Moinard; Luc Cynober; Yulong Yin; Daniel Tomé; François Blachier (pp. 1485-1498).

Endotoxemia affects intestinal physiology. A decrease of circulating citrulline concentration is considered as a reflection of the intestinal function. Citrulline can be produced in enterocytes notably from glutamate and glutamine. The aim of this work was to determine if glutamate, glutamine and citrulline concentrations in blood, intestine and muscle are decreased by endotoxemia, and if supplementation with glutamate or glutamine can restore normal concentrations. We induced endotoxemia in rats by an intraperitoneal injection of 0.3 mg kg⁻¹ lipopolysaccharide (LPS). This led to a rapid anorexia, negative nitrogen balance and a transient increase of the circulating level of IL-6 and TNF-α. When compared with the values measured in pair fed (PF) animals, almost all circulating amino acids (AA) including citrulline decreased, suggesting a decrease of intestinal function. However, at D2 after LPS injection, most circulating AA concentrations were closed to the values recorded in the PF group. At that time, among AA, only glutamate, glutamine and citrulline were decreased in gastrocnemius muscle without change in intestinal mucosa. A supplementation with 4% monosodium glutamate (MSG) or an isomolar amount of glutamine failed to restore glutamate, glutamine and citrulline concentrations in plasma and muscle. However, MSG supplementation led to an accumulation of glutamate in the intestinal mucosa. In conclusion, endotoxemia rapidly but transiently decreased the circulating concentrations of almost all AA and more durably of glutamate, glutamine and citrulline in muscle. Supplementation with glutamate or glutamine failed to restore glutamate, glutamine and citrulline concentrations in plasma and muscles. The implication of a loss of the intestinal capacity for AA absorption and/or metabolism in endotoxemia (as judged from decreased citrulline plasma concentration) for explaining such results are discussed.

Keywords: Endotoxemia; Citrulline; Monosodium glutamate; Glutamine; Intestine

Oxidative stress improvement is associated with increased levels of taurine in CKD patients undergoing lipid-lowering therapy by Angelo Zinellu; Salvatore Sotgia; Giacomina Loriga; Luca Deiana; Andrea Ercole Satta; Ciriaco Carru (pp. 1499-1507).

Lipid-lowering therapy has been reported to reduce several oxidative stress (OS) markers in hypercholesterolemia. Since OS is frequently associated with renal dysfunction, we aimed to investigate the effect of hypolipidemic drugs on oxidative stress and plasma taurine (Tau), a sulfur amino acid with a marked antioxidant effect, in chronic kidney disease (CKD). We enrolled 30 CKD randomized to receive three different hypolipidemic regimens for 12 months: simvastatin alone (40 mg/day) or ezetimibe/simvastatin combined therapy (10/20 or 10/40 mg/day). Low molecular weight (LMW) thiols including homocysteine, cysteine, cysteinylglycine, glutathione, and glutamylcysteine in their reduced and total form and oxidative stress indices as malondialdehyde (MDA) and allantoin/uric acid (All/UA) ratio were also evaluated. Tau concentration significantly increased throughout the therapy. The rise of taurine was more striking for the group with the concomitant administration of ezetimibe/simvastatin 10/40 mg/day (+31.6% after 1 year of therapy). A significant decrease of both MDA and All/UA ratio was observed during therapy for all patients (−19% for both MDA and All/UA ratio) with a more pronounced effect in patients treated with ezetimibe/simvastatin 10/40 mg/day (−26% for MDA and −28% for All/UA ratio). Besides, an increase of thiols reduced forms was found (+20.7% of LMW thiols redox status) with a greater effect in subjects treated with ezetimibe/simvastatin 10/40 mg/day (+24.7%). Moreover, we demonstrated that oxidative stress improvement during therapy was correlated with increased taurine levels. We hypothesize that taurine may be responsible for the oxidative stress improvement observed during lipid-lowering treatment through the reduction of superoxide anion production at the respiratory chain activity level.

Keywords: Chronic kidney disease; Taurine; LDL; Oxidative stress

Taurine ameliorates alloxan-induced diabetic renal injury, oxidative stress-related signaling pathways and apoptosis in rats by Joydeep Das; Parames C. Sil (pp. 1509-1523).

Hyperglycemia-induced oxidative stress plays a vital role in the progression of diabetic nephropathy. The renoprotective nature of taurine has also been reported earlier; but little is known about the mechanism of this beneficial action. The present study has, therefore, been carried out to explore in detail the mechanism of the renoprotective effect of taurine under diabetic conditions. Diabetes was induced in rats by alloxan (single i.p. dose of 120 mg/kg body weight) administration. Taurine was administered orally for 3 weeks (1% w/v in drinking water) either from the day on which alloxan was injected or after the onset of diabetes. Alloxan-induced diabetic rats showed a significant increase in plasma glucose, enhanced the levels of renal damage markers, plasma creatinine, urea nitrogen and urinary albumin. Diabetic renal injury was associated with increased kidney weight to body weight ratio and glomerular hypertrophy. Moreover, it increased the productions of reactive oxygen species, enhanced lipid peroxidation and protein carbonylation in association with decreased intracellular antioxidant defense in the kidney tissue. In addition, hyperglycemia enhanced the levels of proinflammatory cytokins (TNF-α, IL-6, IL-1β) and Na⁺–K⁺-ATPase activity with a concomitant reduction in NO content and eNOS expression in diabetic kidney. Investigation of the oxidative stress-responsive signaling cascades showed the upregulation of PKCα, PKCβ, PKCε and MAPkinases in the renal tissue of the diabetic animals. However, taurine administration decreased the elevated blood glucose and proinflammatory cytokine levels, reduced renal oxidative stress (via decrease in xanthine oxidase activity, AGEs formation and inhibition of p47phox/CYP2E1 pathways), improved renal function and protected renal tissue from alloxan-induced apoptosis via the regulation of Bcl-2 family and caspase-9/3 proteins. Taurine supplementation in regular diet could, therefore, be beneficial to regulate diabetes-associated renal complications.

Keywords: Alloxan; Apoptotis; Hyperglycemia; Kidney; Mitochondria; Renoprotection; Taurine

Antidepressant effect of taurine in diabetic rats by Greice Caletti; Danielly B. Olguins; Elis F. Pedrollo; Helena M. T. Barros; Rosane Gomez (pp. 1525-1533).

Clinical and preclinical studies have shown that diabetic individuals present more depressive behaviors than non-diabetic individuals. Taurine, one of the most abundant free amino acids in the central nervous system, modulates a variety of biological functions and acts as an agonist at GABA_A receptors. Our objective was to assess the antidepressant effect of taurine in diabetic rats. Additionally, we studied the effect of taurine on weight gain, water and food intake, and blood glucose levels in diabetic and non-diabetic rats. Male Wistar rats were divided into control (CTR) and streptozotocin-induced diabetic (STZ) groups and were administered daily 0, 25, 50 or 100 mg/kg of taurine (n = 10 per subgroup) intraperitoneally. After 28 days of treatment, the animals were exposed to the forced swimming test, and their behaviors were recorded. Weight gain, water and food intake, and blood glucose levels were measured weekly. Our results showed that STZ rats had a higher immobility duration than CTR rats, and taurine decreased this depressive-like behavior in STZ rats at doses of 25 and 100 mg/kg. Both of these doses of taurine also decreased water intake and improved weight gain in STZ rats. All doses of taurine decreased the water intake in CTR rats. Taurine, at a dose of 100 mg/kg, decreased food intake and blood glucose levels in STZ rats. Because taurine is a GABA agonist and both amino acids are lower in the plasma of diabetic and depressive individuals, we hypothesize that taurine may represent a new adjuvant drug for the treatment of depression in diabetic individuals.

Keywords: Water intake; Food intake; GABA agonist; Depression; Glycemia

Non-canonical interactions of porphyrins in porphyrin-containing proteins by Srđan Đ. Stojanović; Esma R. Isenović; Božidarka L. Zarić (pp. 1535-1546).

In this study we have described the non-canonical interactions between the porphyrin ring and the protein part of porphyrin-containing proteins to better understand their stabilizing role. The analysis reported in this study shows that the predominant type of non-canonical interactions at porphyrins are CH···O and CH···N interactions, with a small percentage of CH···π and non-canonical interactions involving sulfur atoms. The majority of non-canonical interactions are formed from side-chains of charged and polar amino acids, whereas backbone groups are not frequently involved. The main-chain non-canonical interactions might be slightly more linear than the side-chain interactions, and they have somewhat shorter median distances. The analysis, performed in this study, shows that about 44% of the total interactions in the dataset are involved in the formation of multiple (furcated) non-canonical interactions. The high number of porphyrin–water interactions show importance of the inclusion of solvent in protein–ligand interaction studies. Furthermore, in the present study we have observed that stabilization centers are composed predominantly from nonpolar amino acid residues. Amino acids deployed in the environment of porphyrin rings are deposited in helices and coils. The results from this study might be used for structure-based porphyrin protein prediction and as scaffolds for future porphyrin-containing protein design.

Keywords: Non-canonical interactions; Proteins; Porphyrins; Stabilization centers

Induction of NAD(P)H:quinone oxidoreductase 1 expression by cysteine via Nrf2 activation in human intestinal epithelial LS180 cells by Hideo Satsu; Emi Chidachi; Yuto Hiura; Haru Ogiwara; Yusuke Gondo; Makoto Shimizu (pp. 1547-1555).

In this study, we examined the effects of 20 amino acids on the expression level of NAD(P)H:quinone oxidoreductase 1 (NQO1) in human intestinal LS180 cells. Five amino acids were associated with significant increases in NQO1 mRNA expression; the most substantial increase was induced by cysteine, which markedly increased the NQO1 mRNA level in a time- and dose-dependent manner. Cysteine also increased the protein level of NQO1 and its enzymatic activity in LS180 cells. Furthermore, cysteine significantly up-regulated NQO1 promoter activity, and this induction was completely abolished by mutation of the antioxidant response element, a binding site of the nuclear factor erythroid 2-related factor 2 (Nrf2). Knockdown experiment using siRNA against Nrf2 showed the involvement of Nrf2 on cysteine-induced increase in NQO1 mRNA expression. Further, cysteine treatment increased the amount of Nrf2 protein in the nucleus and decreased the amount of Kelch-like ECH-associated protein 1 (a suppressor protein of Nrf2) in the cytosol, suggesting that Nrf2 was activated by cysteine. Oral administration of cysteine to mice significantly increased NQO1 mRNA levels in the mouse intestinal mucosa. These findings show that cysteine induces NQO1 expression in both in vitro and in vivo systems and also suggest that Nrf2 activation is involved in this induction.

Keywords: Cysteine; NAD(P)H:quinone oxidoreductase 1 (NQO1); Nuclear factor erythroid 2-related factor 2 (Nrf2); Intestinal epithelial cell

Metabolic stability of long-acting luteinizing hormone-releasing hormone antagonists by Jin-Feng Yao; Ning Zhou; Yu-Jian Lv; Ruifeng Zhang; Ke-Liang Liu; Ming Xue (pp. 1557-1566).

Long-acting luteinizing hormone-releasing hormone (LHRH) antagonists designed to be protease resistant consisted of a series of novel decapeptides structurally similar to LHRH. The aim of this study was to evaluate the in vitro metabolic stability of the LHRH decapeptides using pancreatin and homogenates models and identify the metabolites in rat liver homogenate for the purpose of illustrating the metabolic features of the decapeptides. The major metabolites in rat liver homogenate were identified by LC–ESI-MSⁿ. The half-lives of the 11 LHRH decapeptides were from 44 to 330 min in the pancreatin model. The half-lives of the five decapeptides in rat liver, kidney and lung homogenates were between 8 and 462 min. The most stable decapeptides were the LY616 and LY608 peptides with half-lives of 36 min in liver homogenate. Two major cleavage sites were found by analysing the metabolites of the LY618 peptide in rat liver homogenate, between the Pal³-Ser⁴ and the Leu⁷-Ilys⁸ peptide bonds. The major metabolites were produced via cleavages of peptide bonds at these sites, and further metabolic reactions such as hydroxylation, oxidative dechlorination, alcohol dehydration and isopropyl dealkylation were also observed.

Keywords: Luteinizing hormone-releasing hormone; Antagonists; Degradation; Kinetics; Cleavage site; Mass spectrometry

Metal ion dependency of serine racemase from Dictyostelium discoideum by Tomokazu Ito; Hirotaka Murase; Motoki Maekawa; Masaru Goto; Shuhei Hayashi; Hajime Saito; Masatoshi Maki; Hisashi Hemmi; Tohru Yoshimura (pp. 1567-1576).

d-Serine is known to act as an endogenous co-agonist of the N-methyl-d-aspartate receptor in the mammalian brain and is endogenously synthesized from l-serine by a pyridoxal 5′-phosphate-dependent enzyme, serine racemase. Though the soil-living mycetozoa Dictyostelium discoideum possesses no genes homologous to that of NMDA receptor, it contains genes encoding putative proteins relating to the d-serine metabolism, such as serine racemase, d-amino acid oxidase, and d-serine dehydratase. D. discoideum is an attractive target for the elucidation of the unknown functions of d-serine such as a role in cell development. As part of the elucidation of the role of d-serine in D. discoideum, we cloned, overexpressed, and examined the properties of the putative serine racemase exhibiting 46% amino acid sequence similarity with the human enzyme. The enzyme is unique in its stimulation by monovalent cations such as Na⁺ in addition to Mg²⁺ and Ca²⁺, which are well-known activators for the mammalian serine racemase. Mg²⁺ or Na⁺ binding caused two- to ninefold enhancement of the rates of both racemization and dehydration. The half-maximal activation concentrations of Mg²⁺ and Na⁺ were determined to be 1.2 μM and 2.2 mM, respectively. In the l-serine dehydrase reaction, Mg²⁺ and Na⁺ enhanced the k _cat value without changing the K _m value. Alanine mutation of the residues E207 and D213, which correspond to the Mg²⁺-binding site of Schizosaccharomyces pombe serine racemase, abolished the Mg²⁺- and Na⁺-dependent stimulation. These results suggest that Mg²⁺ and Na⁺ share the common metal ion-binding site.

Keywords: d-Serine; Serine racemase; Dictyostelium discoideum ; Metal ion activation

2-Benzamido-N-(1H-benzo[d]imidazol-2-yl)thiazole-4-carboxamide derivatives as potent inhibitors of CK1δ/ε by Joachim Bischof; Johann Leban; Mirko Zaja; Arnhild Grothey; Barbara Radunsky; Olaf Othersen; Stefan Strobl; Daniel Vitt; Uwe Knippschild (pp. 1577-1591).

In this study we identified two heterocyclic compounds (5 and 6) as potent and specific inhibitors of CK1δ (IC₅₀ = 0.040 and 0.042 μM, respectively). Whereas compound 5 exhibited fivefold higher affinity towards CK1δ than to CK1ε (IC₅₀ CK1ε = 0.199 μM), compound 6 also inhibited CK1ε (IC₅₀ = 0.0326 μM) in the same range as CK1δ. Selected compound 5 was screened over 442 kinases identifying 5 as a highly potent and selective inhibitor of CK1δ. X-ray analysis of 5 bound to CK1δ demonstrated its binding mode. In addition, characterization of 5 and 6 in a cell biological approach revealed the ability of both compounds to inhibit proliferation of tumor cell lines in a dose and cell line specific manner. In summary, our optimizations lead to the development of new highly selective CK1δ and ε specific inhibitors with biological activity.

Keywords: CK1δ; CK1ε; Phosphorylation; Small molecule inhibitor; Crystal structure; MTT; FACS

Arginine nutrition and fetal brown adipose tissue development in diet-induced obese sheep by M. Carey Satterfield; Kathrin A. Dunlap; Duane H. Keisler; Fuller W. Bazer; Guoyao Wu (pp. 1593-1603).

The global incidence of human obesity has more than doubled over the past three decades. An ovine model of obesity was developed to determine effects of maternal obesity and arginine supplementation on maternal, placental, and fetal parameters of growth, health, and well being. One-hundred-twenty days prior to embryo transfer, ewes were fed either ad libitum (n = 10) to induce obesity or 100% National Research Council-recommended nutrient requirements (n = 10) as controls. Embryos from superovulated ewes with normal body condition were transferred to the uterus of control-fed and obese ewes on day 5.5 post-estrus to generate genetically similar singleton pregnancies. Beginning on day 100 of gestation, obese ewes received intravenous administration of saline or l-arginine-HCl three times daily (81 mg arginine/kg body weight/day) to day 125, whereas control-fed ewes received saline. Fetal growth was assessed at necropsy on day 125. Maternal obesity increased (1) percentages of maternal and fetal carcass lipids and (2) concentrations of leptin, insulin, glucose, glutamate, leucine, lysine and threonine in maternal plasma while reducing (1) concentrations of progesterone, glycine and serine in maternal plasma and (2) amniotic and allantoic fluid volumes. Administration of l-arginine to obese ewes increased arginine and ornithine concentrations in maternal and fetal plasma, amniotic fluid volume, protein content in maternal carcass, and fetal brown adipose tissue (+60%), while reducing maternal lipid content and circulating leptin levels. Fetal or placental weight did not differ among treatments. Results indicate that arginine treatment beneficially reduces maternal adiposity and enhances fetal brown adipose tissue development in obese ewes.

Keywords: Adipose tissue; Pregnancy; Fetus; Arginine; Sheep; Obese

Novel nanostructure amino acid-based poly(amide–imide)s enclosing benzimidazole pendant group in green medium: fabrication and characterization by Shadpour Mallakpour; Mohammad Dinari (pp. 1605-1613).

In the present work, several novel optically active nanostructure poly(amide–imide)s (PAI)s were synthesized via step-growth polymerization reaction of chiral diacids based on pyromellitic dianhydride-derived dicarboxylic acids containing different natural amino acids such as l-alanine, S-valine, l-leucine, l-isoleucine, l-methionine, and l-phenylalanine with 2-(3,5-diaminophenyl)-benzimidazole under green conditions using molten tetrabutylammonium bromide. The new optically active PAIs were achieved in good yields and moderate inherent viscosity up to 0.41 dL/g. The synthesized polymers were characterized with FT-IR, ¹H-NMR, X-ray diffraction, field emission scanning electron microscopy (FE-SEM), elemental and thermogravimetric analysis (TGA) techniques. These polymers show high solubility in organic polar solvents due to the presence of amino acid and benzimidazole pendant group at room temperature. FE-SEM results show that, these chiral nanostructured PAIs have spherical shapes and the particle size is around 20–80 nm. On the basis of TGA data, such PAIs are thermally stable and can be classified as self-extinguishing polymers. In addition due to the existence of amino acids in the polymer backbones, these macromolecules are not only optically active but also could be biodegradable and thus may well be classified under environmentally friendly materials.

Keywords: α-Amino acids; Chiral nanostructure polymers; Benzimidazole; Poly(amide–imide); Green chemistry

Dakin–West reaction on 1-thyminyl acetic acid for the synthesis of 1,3-bis(1-thyminyl)-2-propanone, a heteroaromatic compound with nucleopeptide-binding properties by Giovanni N. Roviello; Giuseppina Roviello; Domenica Musumeci; Enrico M. Bucci; Carlo Pedone (pp. 1615-1623).

This work deals with the Dakin–West synthesis, starting from the nucleoamino acid 1-thyminyl acetic acid, as well the NMR, ESI MS, and X-ray characterization of a heteroaromatic compound denominated by us T₂CO, comprising two thymine moieties anchored to a 2-propanonic unit, the spectroscopic properties of which were studied by UV as a function of temperature and ionic strength. Preliminary binding-studies with molecules of biomedical interest such as nucleic acids and proteins, performed on samples containing T₂CO, suggested that this molecule is able to interact very weakly with double-stranded RNA, whereas it does not seem to bind other nucleic acids or proteins. Moreover, by studies with fresh human serum we found that T₂CO is resistant to enzymatic degradation till 24 h, whereas UV metal binding-studies, performed using solutions of copper (II) chloride dihydrate and nickel (II) chloride hexahydrate, revealed a certain ability of T₂CO to bind copper (II) cation. Finally, by CD spectroscopy we investigated the influence of T₂CO on the already described supramolecular networks based on l-serine-containing nucleopeptides. More particularly, we found that T₂CO is able to increase the level of structuration of the non-covalent supramolecular assembly of the chiral nucleopeptides, which is a feature of remarkable interest for the development of innovative drug delivery tools.

Keywords: Thymine; 2-Propanone; Nucleopeptide

Evaluation of fluorine-labeled gastrin-releasing peptide receptor (GRPR) agonists and antagonists by LC/MS by Ying Ma; Min Yang; Haokao Gao; Gang Niu; Yongjun Yan; Lixin Lang; Dale O. Kiesewetter; Xiaoyuan Chen (pp. 1625-1632).

An LC/MS method was used to evaluate 2-fluoropropionyl (FP) and 4-fluorobenzoyl (FB) modified bombsin peptides: GRPR agonist [Aca-QWAVGHLM-NH₂] and antagonist [fQWAVGHL-NHEt], and their hydrophilic linker modified counterparts with the attachment of GGGRDN sequence. This study developed strategies to evaluate the in vitro receptor mediated cell uptake and metabolic profile of the various GRPR agonists and antagonists. We identified the metabolites produced by rat hepatocytes and quantitatively analyzed the uptake and internalization of the ligands in PC-3 human prostate cancer cells. The major metabolites of both GRPR agonists and antagonists were the result of peptide bond hydrolysis between WA and AV. The agonists also formed a unique metabolite resulting from hydrolysis of the C-terminal amide. The antagonists showed significantly higher stability against metabolism compared to the agonists in rat hepatocytes. The directly modified agonists (FP-BBN and FB-BBN) had higher internalization with similar cell binding compared to the unmodified agonist (BBN), whereas the hydrophilic linker modified agonists (G-BBN and FG-BBN) had much lower total cell uptake. The labeled antagonists (FP-NBBN, FB-NBBN, G-NBBN and FP-G-NBBN) displayed lower internalization. The optimal imaging agent will depend on the interplay of ligand metabolism, cellular uptake, and internalization in vivo.

Keywords: LC/MS; Gastrin releasing peptide receptor (GRPR); Bombesin (BBN); Agonist; Antagonist; PET

Solid-phase route to Fmoc-protected cationic amino acid building blocks by Jacob Dahlqvist Clausen; Lars Linderoth; Hanne Mørck Nielsen; Henrik Franzyk (pp. 1633-1641).

Diamino acids are commonly found in bioactive compounds, yet only few are commercially available as building blocks for solid-phase peptide synthesis. In the present work a convenient, inexpensive route to multiple-charged amino acid building blocks with varying degree of hydrophobicity was developed. A versatile solid-phase protocol leading to selectively protected amino alcohol intermediates was followed by oxidation to yield the desired di- or polycationic amino acid building blocks in gram-scale amounts. The synthetic sequence comprises loading of (S)-1-(p-nosyl)aziridine-2-methanol onto a freshly prepared trityl bromide resin, followed by ring opening with an appropriate primary amine, on-resin N^β-Boc protection of the resulting secondary amine, exchange of the N^α-protecting group, cleavage from the resin, and finally oxidation in solution to yield the target γ-aza substituted building blocks having an Fmoc/Boc protection scheme. This strategy facilitates incorporation of multiple positive charges into the building blocks provided that the corresponding partially protected di- or polyamines are available. An array of compounds covering a wide variety of γ-aza substituted analogs of simple neutral amino acids as well as analogs displaying high bulkiness or polycationic side chains was prepared. Two building blocks were incorporated into peptide sequences using microwave-assisted solid-phase peptide synthesis confirming their general utility.

Keywords: Aziridine; Cationic amino acids; Hydrophobic amino acids; Peptides; Solid-phase synthesis

High yielding synthesis of N-ethyl dehydroamino acids by Luís S. Monteiro; Ana S. Suárez (pp. 1643-1652).

Recently we reported the use of a sequence of alkylation and dehydration methodologies to obtain N-ethyl-α, β-dehydroamino acid derivatives. The application of this N-alkylation procedure to several methyl esters of β,β-dibromo and β-bromo, β-substituted dehydroamino acids protected with standard amine protecting groups was subsequently reported. The corresponding N-ethyl, β-bromo dehydroamino acid derivatives were obtained in fair to high yields and some were used as substrates in Suzuki cross-coupling reactions to give N-ethyl, β,β-disubstituted dehydroalanine derivatives. Herein, we further explore N-ethylation of β-halo dehydroamino acid derivatives using triethyloxonium tetrafluoroborate as alkylating agent, but substituting N,N-diisopropylethylamine for potassium tert-butoxide as auxiliary base. In these conditions, for all β-halo dehydroamino acid derivatives, reactions were complete and the N-ethylated derivative could be isolated in high yield. This method was also applied for N-ethylation of non-halogenated dehydroamino acids. Again, with all compounds the reactions were complete and the N-ethyl dehydroamino acid derivatives could be isolated in high yields. Some of these N-ethyl dehydroamino acid methyl ester derivatives were converted in high yields to their corresponding acids and coupled to an amino acid methyl ester to give N-ethyl dehydrodipeptide derivatives in good yields. Thus, this method constitutes a general procedure for high yielding synthesis of N-ethylated dehydroamino acids, which can be further applied in peptide synthesis.

Keywords: Amino acids; Nonnatural amino acids; Alkylation; N-ethyl dehydroamino acids; Protecting groups

New approach for amino acid profiling in human plasma by selective fluorescence derivatization by Małgorzata Jaworska; Marta Stańczyk; Małgorzata Wilk; Gabriela Kłaczkow; Elżbieta Anuszewska; Justyna Barzał; Piotr Rzepecki (pp. 1653-1661).

A new approach for the separation of 6-aminoquinolyl-carbamyl (AQC)-derivatized amino acids has been proposed. The chromatography used ion-pairing mechanism to increase the method selectivity. Mobile phase was based on triethylamine buffer containing N,N-dimethyloctylamine as a modifier. A number of factors, buffer composition and pH, counterion concentration, temperature and acetonitrile gradient profile, were optimized to achieve final chromatographic conditions. With the presented analytical method, the separation and identification of 34 AQC-amino acids and amino compounds present in human plasma is possible. The results of validation proved the applicability of the method for quantification of 27 amino acids in biological samples. The ultrafiltration proposed as deproteinization procedure gave repeatable and reliable results for the amino acids under investigation. This method introduced in routine testing can be a suitable tool for amino acid profiling in plasma including all aspects of clinical application.

Keywords: Amino acids; Human plasma; Ultrafiltration; Derivatization; 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC); Ion-pair chromatography; N,N-dimethyloctylamine

Kynurenic acid in human renal cell carcinoma: its antiproliferative and antimigrative action on Caki-2 cells by Katarzyna Walczak; Małgorzata Żurawska; Jacek Kiś; Radosław Starownik; Wojciech Zgrajka; Krzysztof Bar; Waldemar A. Turski; Wojciech Rzeski (pp. 1663-1670).

Kidneys possess a complex enzyme system which plays a major role in tryptophan metabolism. Taking into account a considerably high concentration of one of the tryptophan metabolites, kynurenic acid (KYNA) in this organ and previously reported antiproliferative activity against colon cancer cells in vitro, we measured its content in human normal and tumour kidney tissue. KYNA concentration was considerably higher in normal renal tissue (379.7 ± 39.7 pmol/g wet weight) than in renal cell carcinomas (115.5 ± 20.8 pmol/g wet weight). In in vitro experiments, KYNA in higher micro- and millimolar concentrations significantly inhibited proliferation, DNA synthesis and migration of renal cancer Caki-2 cells. Our results suggest that KYNA may affect cell cycle regulators and signalling pathways through overexpression of p21 Waf1/Cip1 and inhibition of phosphorylation of Rb protein and p38 MAPK. In conclusion, KYNA may be suggested as an endogenous agent, controlling the growth of tumour, or a chemopreventive agent.

Keywords: Kynurenic acid; Renal cell carcinoma; Proliferation; Migration

Proteomics analysis of human umbilical vein endothelial cells treated with resveratrol by Bin Shao; Mei Tang; Ziqiang Li; Rui Zhou; Yaqi Deng; Chunlai Nie; Zhu Yuan; Liangxue Zhou; Minghai Tang; Aiping Tong; Yuquan Wei (pp. 1671-1678).

In the past decade, the small polyphenol resveratrol has received widespread attention as either a potential therapy or as a preventive agent for numerous age-related chronic diseases, including cardiovascular atherosclerosis, cancer, hypertension, and diabetes, but the biological processes and molecular pathways by which resveratrol induces these beneficial effects, as well as its safety and toxicology remain largely undefined. To explore the molecular mechanisms of resveratrol involved in the amelioration of endothelial dysfunction and vascular disease, in the present study the protein profile changes of human umbilical vein endothelial cells in response to resveratrol treatment were investigated using proteomics approaches (2-DE combined with MS/MS). As a result, four down-regulated protein species named elongation factor 2 (EEF2), carboxymethyl-cofilin-1 (cofilin-1), acetyl-eukaryotic translation initiation factor 5A-1 (acetyl-EIF5A) and barrier-to-autointegration factor, and five up-regulated protein species named heat shock protein beta-1 (HSP27), phospho-HSP27, phospho-stathmin, Nicotinate-nucleotide pyrophosphorylase and 1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase were identified. Among them, two translation-related protein species (EEF2 and acetyl-EIF5A) were the most significantly changed (over tenfold). Phospho-EEF2 was further verified to be dramatically up-regulated by immunoblot assays. It is notable that in the present study several protein species with post-transcriptional modification (carboxymethyl-, acetyl-, and phospho-) were found to be altered following exposure to resveratrol. These findings may improve our understanding of the molecular mechanisms underlying the pleiotropic effects of resveratrol on endothelial cells.

Keywords: Resveratrol; HUVECs; 2-DE; Proteomics

AW00179 potentiates TRAIL-mediated death of human lung cancer H1299 cells through ROS-JNK-c-Jun-mediated up-regulation of DR5 and down-regulation of anti-apoptotic molecules by Mi-Kyung Hwang; Byung Jun Ryu; Seong Hwan Kim (pp. 1679-1687).

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers apoptosis in tumor cells, but when used alone, it is not effective at treating TRAIL-resistant tumors. This resistance is challenging for TRAIL-based anti-cancer therapies. In this study, we found that 1-(4-trifluoromethoxy-phenyl)-3-[4-(5-trifluoromethyl-2,5-dihydro-pyrazol-1-yl)-phenyl]-urea (AW00179) sensitized human lung cancer H1299 cells to TRAIL-mediated apoptosis. Even in the absence of TRAIL, AW00179 strongly induced DR5 expression and decreased the expression of anti-apoptotic proteins, suggesting that the sensitizing effect of AW00179 on TRAIL-mediated apoptosis is due to increased levels of DR5 protein and decreased anti-apoptotic molecules. AW00179 also induced the activation of c-Jun and ERK; however, a pharmacologic inhibition study revealed that JNK-c-Jun signaling is involved in the induction of DR5 expression. In addition, reactive oxygen species (ROS) appear to be involved in AW00179 activity. In conclusion, AW00179 has the potential to sensitize H1299 cells to TRAIL-mediated apoptosis through two distinct mechanisms: ROS-JNK-c-Jun-mediated up-regulation of DR5, and down-regulation of anti-apoptotic molecules.

Keywords: TRAIL; Sensitization; DR5; ROS; c-Jun

Expression, purification and antimicrobial activity of puroindoline A protein and its mutants by Yingjie Miao; Ling Chen; Cheng Wang; Yajuan Wang; Qian Zheng; Chunbao Gao; Guangxiao Yang; Guangyuan He (pp. 1689-1696).

Wheat puroindoline proteins, PINA and PINB, play key roles in determining wheat grain hardness as well as in defending the plant against pathogens. PINA has much greater membrane-binding property and antimicrobial activity because it contains more tryptophan residues in the unique tryptophan-rich domain (TRD). In order to obtain proteins with higher antimicrobial activity, mutants of PINA containing two or three copies of TRD, designated ABBC and ABBBC, respectively, were constructed and expressed in E. coli Rosetta-gami (DE3). Metal affinity chromatography was used to purify the soluble affinity-tagged recombinant proteins. The secondary structures of the recombinant proteins were predicted by the online program Protein Homology/analog Y Recognition Engine v2.0 and experimentally assessed using circular dichroism. Minimum inhibition concentration tests and fluorescence microscope analyses were employed to evaluate the antimicrobial activities of the mutants. The results showed that the purified recombinant ABBC was correctly folded and presented significantly higher antimicrobial activities against E. coli and S. aureus than wild-type PINA, suggesting its potential use as an antimicrobial agent. The results also confirmed that TRD is a determinant of the antimicrobial activity of PINA and demonstrated that it is feasible to enhance the antimicrobial activity of PINA by adding one copy of TRD.

Keywords: Puroindoline A; Artificial mutants; Tryptophan-rich domain; Antimicrobial activity; Circular dichroism; Minimum inhibitory concentration

Taurine suppresses osteoblastic differentiation of aortic valve interstitial cells induced by beta-glycerophosphate disodium, dexamethasone and ascorbic acid via the ERK pathway by Xiang Feng; Jian-ming Li; Xiao-bo Liao; Ye-rong Hu; Bao-peng Shang; Zhi-yuan Zhang; Ling-qing Yuan; Hui Xie; Zhi-feng Sheng; Hao Tang; Wei Zhang; Lu Gu; Xin-min Zhou (pp. 1697-1704).

Aortic valve calcification (AVC) is an active process characterized by osteoblastic differentiation of the aortic valve interstitial cells (AVICs). Taurine is a free β-amino acid and plays important physiological roles including protective effect of cardiovascular events. To evaluate the possible role of taurine in AVC, we isolated human AVICs from patients with type A dissection without leaflet disease. We demonstrated that the cultured AVICs express SM α-actin, vimentin and taurine transporter (TAUT), but not CD31, SM-myosin or desmin. We also established the osteoblastic differentiation model of the AVICs induced by pro-calcific medium (PCM) containing β-glycerophosphate disodium, dexamethasone and ascorbic acid in vitro. The results showed that taurine attenuated the PCM-induced osteoblastic differentiation of AVICs by decreasing the alkaline phosphate (ALP) activity/expression and the expression of the core binding factor α1 (Cbfα1) in a dose-dependent manner (reaching the maximum protective effect at 10 mM), and taurine (10 mM) inhibited the mineralization level of AVICs in the form of calcium content significantly. Furthermore, taurine activated the extracellular signal-regulated protein kinase (ERK) pathway via TAUT, and the inhibitor of ERK (PD98059) abolished the effect of taurine on both ALP activity/expression and Cbfα1 expression. These results suggested that taurine could inhibit osteoblastic differentiation of AVIC via the ERK pathway.

Keywords: Taurine; Aortic valve interstitial cells; Extracellular signal-regulated protein kinase

Taurine protects cerebellar neurons of the external granular layer against ethanol-induced apoptosis in 7-day-old mice by Andrey G. Taranukhin; Elena Y. Taranukhina; Pirjo Saransaari; Markku Pelto-Huikko; Irina M. Podkletnova; Simo S. Oja (pp. 1705-1711).

Acute alcohol administration is harmful especially for the developing nervous system, where it induces massive apoptotic neurodegeneration leading to alcohol-related disorders of newborn infants. Neuroprotection against ethanol-induced apoptosis may save neurons and reduce the consequences of maternal alcohol consumption. Previously we have shown that taurine protects immature cerebellar neurons in the internal granular layer of cerebellum from ethanol-induced apoptosis. Now we describe a similar protective action for taurine in the external layer of cerebellum of 7-day-old mice. The mice were divided into three groups: ethanol-treated, ethanol + taurine-treated and controls. Ethanol (20% solution) was administered subcutaneously at a total dose of 5 g/kg (2.5 g/kg at time 0 h and 2.5 g/kg at 2 h) to the ethanol and ethanol + taurine groups. The ethanol + taurine group also received subcutaneously two injections of taurine (1 g/kg each, 1 h before the first dose of ethanol and 1 h after the second dose of ethanol). To verify apoptosis, immunostaining for activated caspase-3 and TUNEL staining were made in the mid-sagittal sections containing lobules I–X of the cerebellar vermis at 8 h after the first ethanol injection. Ethanol induced apoptosis in the cerebellar external granular layer. Taurine treatment significantly reduced the number of activated caspase-3-immunoreactive and TUNEL-positive cells. Taurine has thus a neuroprotective antiapoptotic action in the external granular layer of the cerebellum, preserving a number of neurons from ethanol-induced apoptosis.

Keywords: Taurine neuroprotection; Ethanol-induced apoptosis; TUNEL staining; Activated caspase-3; External granular layer; Developing cerebellum

Lipid metabolism in pigs fed supplemental conjugated linoleic acid and/or dietary arginine by Gwangwoong Go; Guoyao Wu; David T. Silvey; Seongho Choi; Xilong Li; Stephen B. Smith (pp. 1713-1726).

We proposed that the combination of conjugated linoleic acid (CLA) and arginine would decrease adiposity by depressing lipid synthesis in liver and adipose tissues of growing pigs. Pigs were allotted to treatments in a 2 × 2 factorial design with two lipids (CLA or canola oil) and two amino acids [l-arginine or l-alanine (isonitrogenous control)]; supplements were provided from 80 to 110 kg body weight (approximately 4 weeks). Treatment groups (n = 4) were: control (2.05% l-alanine plus 1% canola oil); CLA (2.05% l-alanine plus 1% CLA); arginine (1.0% l-arginine plus 1.0% canola oil); arginine plus CLA (1.0% arginine plus 1.0% CLA). Arginine increased backfat thickness (P = 0.07) in the absence or presence of CLA, and arginine supplementation increased subcutaneous and retroperitoneal adipocyte volume, especially in combination with dietary CLA (interaction P = 0.001). Arginine increased palmitate incorporation into total lipids by over 60% in liver (P = 0.07). Dietary CLA increased palmitate incorporation into lipids in longissimus muscle by over 100% (P = 0.01), and CLA increased longissimus muscle lipid by nearly 20%. CLA increased glucose oxidation to CO₂ by over 80% in retroperitoneal and subcutaneous adipose tissues (P = 0.04), and doubled palmitate oxidation to CO₂ in intestinal duodenal mucosal cells (P = 0.07). Arginine supplementation decreased muscle pH at 45 min postmortem (P = 0.001), indicating elevated early postmortem glycolysis, and CLA and arginine independently increased PGC-1α gene expression in longissimus muscle. CLA but not arginine depressed mTOR gene expression in intestinal duodenal mucosal cells. CLA decreased serum insulin by 50% (P = 0.02) but increased serum triacylglycerols by over 40%. CLA supplementation increased (P ≤ 0.01) total saturated fatty acids in liver and adipose tissue. In conclusion, neither CLA nor arginine depressed tissue lipid synthesis in growing/finishing pigs, and in fact dietary CLA promoted elevated intramuscular lipid and arginine increased carcass adiposity.

Keywords: Arginine; Conjugated linoleic acid; Substrate oxidation; Lipogenesis; Gene expression

An in silico model of enterocytic glutamine to citrulline conversion pathway by J. Bensaci; E. Curis; I. Nicolis; J.-P. de Bandt; S. Bénazeth (pp. 1727-1737).

Enterocyte is one of the main sites of amino acids metabolism and particularly of the citrulline biosynthesis. Working at the cellular scale and applying ordinary differential equations (ODEs) formalism, we have built a mathematical model of the enterocytic glutamine to citrulline conversion in the fasting state. This model enables us to test different physiopathological scenarios of enzyme activity loss. Results from two different approaches were compared: a standard approach (KA) based on the Michaelis–Menten assumptions and an association–dissociation approach (VH) based on the kinetic mass action law. For both approaches, ODEs system was numerically solved using Mathematica™. In both cases, the model correctly predicts the physiological plasma citrulline steady-state, but the two approaches present clear differences for metabolites of enzymes having a complex mechanism, challenging the validity of the KA approach in such cases. When physiopathological scenarios of enzyme activity loss are simulated, both approaches predict a very sharp transition from the physiological citrulline plasma level to the lack of its production: the concentration profiles of these simulations show a clear threshold of which characteristics vary with the involved enzyme. Moreover, amongst all enzymes included in the model, the ornithine aminotransferase (OAT) shows the highest sensitivity in the system whatever the approach used. This model points out the limits of the Michaelis–Menten approach to model complex enzyme mechanisms. It highlights the key role of OAT in the studied citrulline synthesis pathway and also suggests an order of magnitude about the optimal ratio of enzyme concentrations in this pathway.

Keywords: Citrulline; Glutamine; Enterocyte; Mathematical modelling; Ordinary differential equations

Strain-independent global effect of hippocampal proteins in mice trained in the Morris water maze by Kongzhao Li; Iris Müller; Sudarshan Patil; Harald Höger; Arnold Pollak; Nina Russo-Schlaff; Gert Lubec; Lin Li (pp. 1739-1749).

A series of individual proteins have been linked to performance in the Morris water maze (MWM) but no global effects have been reported. It was therefore the aim of the study to show which proteins were strain-independent, global factors for training in the MWM. Strains C57BL/6J, apodemus sylvaticus and PWD/PhJ were used. MWM and gels from trained animals were from a previous own study and corresponding yoked groups were generated. Hippocampal proteins were extracted and run on two-dimensional gel electrophoresis. Spots with different expressional levels between trained and yoked groups were punched and identified by mass spectrometry (nano-LC-ESI-MS/MS, ion trap). Two-way ANOVA with two factors (strain and training) was carried out and a Bonferroni test was used to compare groups. 12 proteins from several pathways and cascades showed different levels in trained mice versus corresponding yoked animals in all strains tested. Four out of these proteins were verified by immunoblotting: beta-synuclein, profilin 2, nucleoside diphosphate kinase A (NME1) and isocitrate dehydrogenase 3. Four proteins verified by immunoblotting could be shown to be involved in training in the MWM as a global effect, independent of the strain tested.

Keywords: Morris water maze; Hippocampal proteins; Beta-synuclein; Profilin-2; Nucleoside diphosphate dehydrogenase

Pharmacological characterization of the ghrelin receptor mediating its inhibitory action on inflammatory pain in rats by Valeria Sibilia; Francesca Pagani; Emanuela Mrak; Elisa Dieci; Giovanni Tulipano; Francesco Ferrucci (pp. 1751-1759).

Recent research suggests a role for ghrelin in the modulation of inflammatory disorders. However, the type of ghrelin receptor (GHS-R) involved in both the anti-inflammatory and anti-hyperalgesic actions of ghrelin remains to be characterized. In this study, we examined whether the inhibitory effect of ghrelin in the development of hyperalgesia and edema induced by intraplantar carrageenan administration depends on an interaction with GHS-R1a. Both central (1 nmol/rat, i.c.v.) and peripheral (40 nmol/kg, i.p.) administration of the selective GHS-R1a agonist EP1572 had no effect on carrageenan-induced hyperalgesia measured by Randall–Selitto test and paw edema. Furthermore, pre-treatment with the selective GHS-R1a antagonist, d-lys³-GHRP-6 (3 nmol/rat, i.c.v.) failed to prevent the anti-hyperalgesic and anti-inflammatory effects exerted by central ghrelin administration (1 nmol/rat), thus indicating that the type 1a GHS-R is not involved in these peptide activities. Accordingly, both central (1 and 2 nmol/rat, i.c.v.) and peripheral (40 and 80 nmol/kg, i.p.) administration of desacyl-ghrelin (DAG), which did not bind GHS-R1a, induced a significant reduction of the hyperalgesic and edematous activities of carrageenan. In conclusion, we have shown for the first time that DAG shares with ghrelin an inhibitory role in the development of hyperalgesia, as well as the paw edema induced by carrageenan and that a ghrelin receptor different from type 1a is involved in the anti-inflammatory activities of the peptide.

Keywords: Ghrelin receptors; Desacyl-ghrelin; Hyperalgesia; Edema; Randall and Selitto

Antimicrobial lipopeptaibol trichogin GA IV: role of the three Aib residues on conformation and bioactivity by Marta De Zotti; Barbara Biondi; Yoonkyung Park; Kyung-Soo Hahm; Marco Crisma; Claudio Toniolo; Fernando Formaggio (pp. 1761-1777).

The lipopeptaibol trichogin GA IV is a natural, non-ribosomally synthesized, antimicrobial peptide remarkably resistant to the action of hydrolytic enzymes. This feature may be connected to the multiple presence in its sequence of the non-coded residue α-aminoisobutyric acid (Aib), which is known to be responsible for the adoption of particularly stable helical structures already at the level of short peptides. To investigate the role of Aib residues on the 3D-structure and bioactivity of trichogin GA IV, we synthesized and fully characterized four analogs where one or two Aib residues are replaced by l-Leu. Our extensive conformational studies (including an X-ray diffraction analysis) and biological assays performed on these analogs showed that the Aib to l-Leu replacements do not affect the resistance to proteolysis, but modulate the bioactivity of trichogin GA IV in a 3D-structure related manner.

Keywords: Antibiotics; Bioactivity; Conformational analysis; Membranes; Peptides; C^α-Tetrasubstituted α-amino acids

Novel emissive bio-inspired non-proteinogenic coumarin-alanine amino acid: fluorescent probe for polyfunctional systems by Elisabete Oliveira; José Luis Capelo; João Carlos Lima; Carlos Lodeiro (pp. 1779-1790).

Two new bio-inspired non-proteinogenic compounds L1 and L2, containing coumarin and/or acridine chromophores and bearing as spacer an alanine amino acid were successfully synthesized and fully characterized by elemental analysis, ¹H and ¹³C NMR, infrared spectroscopy (KBr discs), melting point, ESI-TOF (electrospray ionization-time of flight-mass), UV–vis absorption and emission spectroscopy, fluorescence quantum yields and lifetime measurements. A relative fluorescence quantum yield of 0.02 was determined for both compounds. In L2 the presence of an intramolecular energy transfer from the coumarin to the acridine unit was observed. L1 and L2 are quite sensitive to the basicity of the environment. At alkaline values both compounds show a strong quenching in the fluorescence emission, attributed to the photoinduced electron transfer (PET). However, both deprotonated forms recover the emission with the addition of Zn²⁺, Cd²⁺ and Al³⁺ metal ions. As multifunctional emissive probes, the titration of L1 and L2 with lanthanides (III), Eu³⁺ and Tb³⁺ was also explored as new visible bio-probes in the absence and in the presence of liposomes. In a liposomal environment a lower energy transfer was observed.

Keywords: Alanine; Coumarin; Acridine; Liposomes; Lanthanide(III); Transition metals

Taurine supplementation prevents morpho-physiological alterations in high-fat diet mice pancreatic β-cells by Rosane Aparecida Ribeiro; Junia Carolina Santos-Silva; Jean Francisco Vettorazzi; Beatriz Borghi Cotrim; Daniela D. M. Mobiolli; Antonio Carlos Boschero; Everardo Magalhães Carneiro (pp. 1791-1801).

Taurine (Tau) is involved in beta (β)-cell function and insulin action regulation. Here, we verified the possible preventive effect of Tau in high-fat diet (HFD)-induced obesity and glucose intolerance and in the disruption of pancreatic β-cell morpho-physiology. Weaning Swiss mice were distributed into four groups: mice fed on HFD diet (36 % of saturated fat, HFD group); HTAU, mice fed on HFD diet and supplemented with 5 % Tau; control (CTL); and CTAU. After 19 weeks of diet and Tau treatments, glucose tolerance, insulin sensitivity and islet morpho-physiology were evaluated. HFD mice presented higher body weight and fat depots, and were hyperglycemic, hyperinsulinemic, glucose intolerant and insulin resistant. Their pancreatic islets secreted high levels of insulin in the presence of increasing glucose concentrations and 30 mM K⁺. Tau supplementation improved glucose tolerance and insulin sensitivity with a higher ratio of Akt phosphorylated (pAkt) related to Akt total protein content (pAkt/Akt) following insulin administration in the liver without altering body weight and fat deposition in HTAU mice. Isolated islets from HTAU mice released insulin similarly to CTL islets. HFD intake induced islet hypertrophy, increased β-cell/islet area and islet and β-cell mass content in the pancreas. Tau prevented islet and β-cell/islet area, and islet and β-cell mass alterations induced by HFD. The total insulin content in HFD islets was higher than that of CTL islets, and was not altered in HTAU islets. In conclusion, for the first time, we showed that Tau enhances liver Akt activation and prevents β-cell compensatory morpho-functional adaptations induced by HFD.

Keywords: Glucose homeostasis; High-fat diet mice; Insulin secretion; Islet morphometry; Taurine supplementation

Creatine-induced glucose uptake in type 2 diabetes: a role for AMPK-α? by Christiano Robles Rodrigues Alves; Júlio César Ferreira; Mário Alves de Siqueira-Filho; Carla Roberta Carvalho; Antonio Herbert Lancha Jr.; Bruno Gualano (pp. 1803-1807).

This study focused on understanding the signaling mechanisms leading to GLUT-4 translocation and increased skeletal-muscle glucose uptake that follow creatine (Cr) supplementation in type 2 diabetes (n = 10). AMPK-α protein content presented a tendency to be higher (p = 0.06) after Cr supplementation (5 g/d for 12w). The changes in AMPK-α protein content significantly related (p < 0.001) to the changes in GLUT-4 translocation (r = 0.78) and Hb1Ac levels (r = −0.68), suggesting that AMPK signaling may be implicated in the effects of supplementation on glucose uptake in type 2 diabetes.

Keywords: Creatine supplementation; Insulin resistance; Molecular pathways

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.43, #4)