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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.37, #4)

Visfatin in pregnancy: proposed mechanism of peptide delivery by Laxmansa C. Katwa; Edward R. Seidel (pp. 555-558).

Visfatin is a newly identified 52 kD adipocytokine that appears to have insulinomimetic properties. We examined visfatin expression in visceral fat from lean and pregnant women. Visfatin gene expression was seven times higher in omental fat of pregnant women than in lean women. Both immunohistochemistry and immunoblot confirmed that visfatin protein was much higher in pregnant women than in nonpregnant women. However, serum visfatin was 20.8 ± 7.7 ng/ml (n = 7) in lean women as compared to only a slight increase to 40.3 ng/ml in pregnant women (n = 4). We measured visfatin mRNA content of human placenta and found that placenta expresses high levels of visfatin mRNA and protein. At a concentration of 2 nM, visfatin and insulin produced nearly identical increase in glucose transport. The discrepancy between the elevated visfatin expression and tissue visfatin compared to only a small increase in serum visfatin is a matter of controversy. The data on serum visfatin concentrations are replete with contradictory data. Taken together, we suggest that visfatin is not a hormone. Instead, we propose that visfatin acts in either a paracrine or autocrine mode. This hypothesis would explain what various laboratories have found widely discrepant values for serum visfatin. Since visfatin potently and efficaciously induced glucose transport in a cell culture model, any hypothetical role for visfatin in pregnancy should include the possibility that it may play a role in maternal/fetal glucose metabolism or distribution and that it may do so by acting locally.

Side-chain oxidative damage to cysteine on a glassy carbon electrode by Wansong Zong; Rutao Liu; Lingzi Zhao; Yanmin Tian; Dong Yuan; Canzhu Gao (pp. 559-564).

In this paper, oxidative damage to the cysteine (CySH) side-chain on a glassy carbon electrode (GCE) was investigated. Voltammetric studies show that there are three anodic peaks for the oxidation of CySH, which arise from (1) the oxidation of the –SH side-chain, forming cystine (0.71 V, vs. SCE) and (2) CySO xH, x = 2, 3 (0.98 V vs. SCE), and (3) the oxidation of the amino acid carboxyl group (around 1.51 V vs. SCE). The influence of dissolved oxygen, pH, scan rate, scan time, temperature and CySH concentration were investigated and the oxidative mechanism proposed. The peaks near 0.71 and 0.98 V are the promising candidates for measuring the oxidation of CySH on the GCE. This paper provides a new strategy for researching oxidative damage of amino acids, sulfur-containing peptides and proteins.

Keywords: Cycle volt-ampere; Glassy carbon electrode; Cysteine; Oxidative damage

The level of sulfane sulfur in the fungus Aspergillus nidulans wild type and mutant strains by Maria Wróbel; Irmina Lewandowska; Patrycja Bronowicka-Adamska; Andrzej Paszewski (pp. 565-571).

The interdependence of the sulfane sulfur metabolism and sulfur amino acid metabolism was studied in the fungus Aspergillus nidulans wild type strain and in mutants impaired in genes encoding enzymes involved in the synthesis of cysteine (a precursor of sulfane sulfur) or in regulatory genes of the sulfur metabolite repression system. It was found that a low concentration of cellular cysteine leads to elevation of two sulfane sulfurtransferases, rhodanase and cystathionine γ-lyase, while the level of 3-mercaptopyruvate sulfurtransferase remains largely unaffected. In spite of drastic differences in the levels of biosynthetic enzymes and of sulfur amino acids due to mutations or sulfur supplementation of cultures, the level of total sulfane sulfur is fairly stable. This stability confirms the crucial role of sulfane sulfur as a fine-tuning regulator of cellular metabolism.

Keywords: Sulfane sulfur; Aspergillus nidulans

Dietary supplementation with Chinese herbal powder enhances ileal digestibilities and serum concentrations of amino acids in young pigs by X. F. Kong; Y. L. Yin; Q. H. He; F. G. Yin; H. J. Liu; T. J. Li; R. L. Huang; M. M. Geng; Z. Ruan; Z. Y. Deng; M. Y. Xie; G. Wu (pp. 573-582).

This study was designed to determine the effect of ultra-fine Chinese herbal powder as a dietary additive on serum concentrations and apparent ileal digestibilities (AID) of amino acids (AA) in young pigs. In Experiment 1, 60 Duroc × Landrace × Yorkshire piglets weaned at 21 days of age were randomly assigned to one of three treatments, representing supplementation with 0 or 2 g/kg of the powder, or 0.2 g/kg of colistin (an antibiotic) to corn- and soybean meal-based diets (n = 20 per group). Blood samples from five piglets per group were collected on days 7, 14, and 28 to determine serum AA concentrations. In Experiment 2, 12 barrows with an average initial body weight of 7.64 kg were randomly assigned to one of the three dietary treatments, followed by surgical placement of a simple T-cannula at the terminal ileum. All of the diets contained 0.1% titanium oxide as a digestibility marker. The samples of terminal ileal digesta were collected on day 7 for determining AID of AA. Results show that dietary supplementation with the herbal powder increased (P < 0.05) serum concentrations and AID of most AA by 10–50% and 10–16%, respectively. As an indicator of improved intestinal function, AID values of calcium were also enhanced in piglets supplemented with the herbal powder. Dietary supplementation of colistin increased serum concentrations and AID values of some AA by 8–44% and 10–15%, respectively, in comparison with the non-supplemented group. These novel findings demonstrate that the herbal powder can enhance the digestibility of dietary protein and the intestinal absorption of AA into the systemic circulation in post-weaning pigs, therefore providing a new mechanism for its growth- and immunity-promoting efficacy.

Keywords: Ultra-fine Chinese herbal powder; Amino acids; Digestion; Piglets

Using multidimensional patterns of amino acid attributes for QSAR analysis of peptides by G. Liang; L. Yang; L. Kang; H. Mei; Z. Li (pp. 583-591).

On the basis of exploratory factor analysis, six multidimensional patterns of 516 amino acid attributes, namely, factor analysis scales of generalized amino acid information (FASGAI) involving hydrophobicity, alpha and turn propensities, bulky properties, compositional characteristics, local flexibility and electronic properties, are proposed to represent structures of 48 bitter-tasting dipeptides and 58 angiotensin-converting enzyme inhibitors. Characteristic parameters related to bioactivities of the peptides studied are selected by genetic algorithm, and quantitative structure–activity relationship (QSAR) models are constructed by partial least square (PLS). Our results by a leave-one-out cross validation are compared with the previously known structure representation method and are shown to give slightly superior or comparative performance. Further, two data sets are divided into training sets and test sets to validate the characterization repertoire of FASGAI. Performance of the PLS models developed by training samples by a leave-one-out cross validation and external validation for test samples are satisfying. These results demonstrate that FASGAI is an effective representation technique of peptide structures, and that FASGAI vectors have many preponderant characteristics such as straightforward physicochemical information, high characterization competence and easy manipulation. They can be further applied to investigate the relationship between structures and functions of various peptides, even proteins.

Keywords: Peptide; Factor analysis scales of generalized amino acid information; Quantitative structure–activity relationship; Partial least squares; Genetic algorithm-partial least square

Molecular cloning, distribution and ontogenetic expression of the oligopeptide transporter PepT1 mRNA in Tibetan suckling piglets by Wence Wang; Changyou Shi; Jianshe Zhang; Wanting Gu; Tiejun Li; Meimei Gen; Wuying Chu; Ruilin Huang; Yulan Liu; Yongqing Hou; Peng Li; Yulong Yin (pp. 593-601).

The gene encoding the oligopeptide transporter PepT1 (HGMW-approved gene symbol SLC15A1) from Tibetan porcine intestine was cloned. The open reading frame of this cDNA encodes 708 deduced amino acid residues that show high sequence similarity with its ovine and bovine counterparts. The putative protein has 12 putative transmembrane domains, including many structural features that are highly conserved among the vertebrate orthologs. PepT1 mRNA expression can be detected in duodenum, jejunum and ileum from Tibetan pigs at 28 days by RT-PCR. Real-time PCR analysis indicated that the jejunum had the highest expression of PepT1 when compared with the duodenum and ileum. PepT1 mRNA expression in the duodenum and proximal jejunum increases continuously from day 1 to day 14: expression was highest at day14 (P < 0.01) and then decreased gradually from day 21 to day 35. Our findings show that PepT1 mRNA expression in the distal jejunum increased gradually with age in suckling Tibetan piglet, and this may have important implications for amino acid and protein nutrition in young animals.

Keywords: Amino acid transporter; PepT1; Tibetan pig; Expression

Facile syntheses of l-β-haloalanine derivatives from l-cysteine or l-cystine by Feiyan Tao; Yinggang Luo; Qingchun Huang; Yan Liu; Bogang Li; Guolin Zhang (pp. 603-607).

l-β-Haloalanines are physiologically active unnatural amino acids and they are useful intermediates for the synthesis of natural and unnatural amino acids, S-linked glycopeptides, and lanthionines. In general l-β-haloalanines were prepared predominantly from l-serine via functional group transformation. Here we reported an alternative approach for the preparation of l-β-haloalanines via halogenation of protected l-cysteine esters which was obtained from l-cysteine or l-cystine, respectively. The mercapto group of protected l-cysteine esters was efficiently transformed to halo groups by triphenylphosphine/N-halosuccinimides. It has been proved to be a versatile desulfurization strategy via this functional group transformation.

Keywords: l-β-Haloalanine; Cysteine; Cystine; Halogenation; Desulfurization; Functional group transformation

Regulation of muscle protein degradation, not synthesis, by dietary leucine in rats fed a protein-deficient diet by Takayuki Sugawara; Yoshiaki Ito; Naoyuki Nishizawa; Takashi Nagasawa (pp. 609-616).

The aim of this study was to elucidate the effects of long-term intake of leucine in dietary protein malnutrition on muscle protein synthesis and degradation. A reduction in muscle mass was suppressed by leucine-supplementation (1.5% leucine) in rats fed protein-free diet for 7 days. Furthermore, the rate of muscle protein degradation was decreased without an increase in muscle protein synthesis. In addition, to elucidate the mechanism involved in the suppressive effect of leucine, we measured the activities of degradation systems in muscle. Proteinase activity (calpain and proteasome) and ubiquitin ligase mRNA (Atrogin-1 and MuRF1) expression were not suppressed in animals fed a leucine-supplemented diet, whereas the autophagy marker, protein light chain 3 active form (LC3-II), expression was significantly decreased. These results suggest that the protein-free diet supplemented with leucine suppresses muscle protein degradation through inhibition of autophagy rather than protein synthesis.

Keywords: Skeletal muscle; Protein synthesis; Protein degradation; Leucine; Sarcopenia; Autophagy

Chronic methionine load-induced hyperhomocysteinemia impairs the relaxation induced by bradykinin in the isolated rat carotid by Daniella Bonaventura; Carlos R. Tirapelli; Ana Maria de Oliveira (pp. 617-627).

This study investigates the effects of chronic methionine intake on bradykinin (BK)-relaxation. Vascular reactivity experiments were performed on carotid rings from male Wistar rats. Treatment with methionine (0.1, 1 or 2 g kg⁻¹ per day) for 8 and 16 weeks, but not for 2 and 4 weeks, reduced the relaxation induced by BK. Indomethacin, a non-selective cyclooxygenase (COX) inhibitor, and SQ29548, a selective thromboxane A₂ (TXA₂)/prostaglandin H₂ (PGH₂) receptor antagonist prevented the reduction in BK-relaxation observed in the carotid from methionine-treated rats. Conversely, AH6809, a selective prostaglandin F_2α (PGF_2α) receptor antagonist did not alter BK-relaxation in the carotid from methionine-treated rats. The nitric oxide synthase (NOS) inhibitors L-NAME, L-NNA and 7-nitroindazole reduced the relaxation induced by BK in carotids from control and methionine-treated rats. In summary, we found that chronic methionine intake impairs the endothelium-dependent relaxation induced by BK and this effect is due to an increased production of endothelial vasoconstrictor prostanoids (possibly TXA₂) that counteracts the relaxant action displayed by the peptide.

Keywords: Methionine; Homocysteine; Bradykinin; Relaxation; Prostanoids

Changes in amino acid concentration in plasma and type I and type II fibres during resistance exercise and recovery in human subjects by Eva Blomstrand; Birgitta Essén-Gustavsson (pp. 629-636).

Eight male subjects performed leg press exercise, 4 × 10 repetitions at 80% of their maximum. Venous blood samples were taken before, during exercise and repeatedly during 2 h of recovery. From four subjects, biopsies were taken from the vastus lateralis muscle prior to, immediately after and following one and 2 h of recovery. Samples were freeze-dried, individual muscle fibres were dissected out and identified as type I or type II. Resistance exercise led to pronounced reductions in the glutamate concentration in both type I (32%) and type II fibres (70%). Alanine concentration was elevated 60–75% in both fibre types and 29% in plasma. Glutamine concentration remained unchanged after exercise; although 2 h later the concentrations in both types of fibres were reduced 30–35%. Two hours after exercise, the plasma levels of glutamate and six of the essential amino acids, including the branched-chain amino acids were reduced 5–30%. The data suggest that glutamate acts as an important intermediate in muscle energy metabolism during resistance exercise, especially in type II fibres.

Keywords: Amino acids; Muscle fibre types; Resistance exercise

High concentration of kynurenic acid in bile and pancreatic juice by Piotr Paluszkiewicz; Wojciech Zgrajka; Tomasz Saran; Janusz Schabowski; Jose L. Valverde Piedra; Olexandr Fedkiv; Sofia Rengman; Stefan G. Pierzynowski; Waldemar A. Turski (pp. 637-641).

Kynurenic acid (KYNA) is an agonist of the G-protein-coupled receptor GPR35, which is predominantly expressed in gastrointestinal tissues. The aim of this study was to determine the content of KYNA in gastric juice, bile and pancreatic juice and intestinal content. KYNA was determined by means of high performance liquid chromatography. The mean concentrations of KYNA in human gastric juice is 9.91 ± 0.71 nM in contrast to human bile (832.5 ± 204.1 and 306.8 ± 35.2 nM) obtained from patients with cholecystolithiasis and obstructive jaundice, respectively. In pigs, the KYNA levels in bile and pancreatic juice are 1,113.3 ± 63.34 and 757.0 ± 394.4 nM, respectively. The KYNA concentration increases along the digestive system, reaching 1,638 nM in the colon content. We suggest that the liver and pancreas affect the content of kynurenic acid in the lumen of the digestive tract.

Keywords: Kynurenic acid; Bile; Gastric juice; Pancreatic juice

Dietary l-arginine supplementation alleviates immunosuppression induced by cyclophosphamide in weaned pigs by J. Han; Y. L. Liu; W. Fan; J. Chao; Y. Q. Hou; Y. L. Yin; H. L. Zhu; G. Q. Meng; Z. Q. Che (pp. 643-651).

A study was conducted to investigate the effects of l-arginine (Arg) on performance and immune function in cyclophosphamide (CY) immunosuppressed weaned pigs. The weaned pigs were allotted randomly into one of three treatments, including: (1) non-challenged control; (2) CY-challenged group; and (3) CY + 0.5% Arg. On days 14 and 21 of the trial, pigs were injected with CY or sterile saline. Blood samples were obtained on days 21 and 28 of the trial for further analysis. On day 28, delayed-type hypersensitivity reaction was evaluated. Arg alleviated the decrease of average daily gain (P < 0.05) induced by CY challenge from days 21 to 28. Arg mitigated the CY-induced decrease of total white blood cell numbers (P < 0.05) on day 28 and improved the lymphocyte percentage on day 21 (P < 0.05). Arg increased the delayed-type hypersensitivity reaction (P < 0.05), and attenuated the decrease of bovine serum albumin antibody level caused by CY treatment (P < 0.05) on day 28. In addition, Arg elevated the levels of serum interleukin-2 and interferon-γ (P < 0.05) on day 28, and mitigated the decrease of serum interferon-γ level on day 21 (P < 0.05). These results indicate that Arg supplementation has beneficial effects in attenuating the immunosuppressive effects of CY challenge, therefore improving growth performance of young pigs.

Keywords: Arginine; Cyclophosphamide; Immunosuppression; Weaned pigs

d-Aspartate affects secretory activity in rat Harderian gland: molecular mechanism and functional significance by Rossella Monteforte; Alessandra Santillo; Marcello Di Giovanni; Antimo D’Aniello; Antimo Di Maro; Gabriella Chieffi Baccari (pp. 653-664).

In this paper, the role of d-aspartate in the rat Harderian gland (HG) was investigated by histochemical, ultrastructural, and biochemical analyses. In this gland, substantial amounts of endogenous d-Asp were detected, along with aspartate racemases that convert d-Asp to l-Asp and vice versa. We found that the gland was capable of uptaking and accumulating exogenously administered d-Asp. d-Asp acute treatment markedly increased lipid and porphyrin secretion and induced a powerful hyperaemia in inter-acinar interstitial tissue. Since d-Asp is known to be recognized by NMDA receptors, the expression of such receptors in rat HG led us to the hypothesis that d-Asp acute treatment induced the activation of the extracellular signal-regulated protein kinase (ERK) and nitric oxide synthase (NOS) pathways mediated by NMDA. Interestingly, as a result of enhanced oxidative stress due to increased porphyrin secretion, the revealed activation of the stress-activated protein kinase/c-jun N-terminal kinase (SAPK/JNK) pro-apoptotic pathway was probably triggered by the gland itself to preserve its cellular integrity.

Keywords: Harderian gland; d-Aspartate; d-Amino acids; Exocrine gland

Microwave-induced synthesis of new optically active and soluble polyamides containing pendent 4-(2-phthalimidiylpropanoylamino)benzoylamino-groups by Shadpour Mallakpour; Zahra Rafiee (pp. 665-672).

An aromatic chiral diacid monomer, 5-[4-(2-phthalimidiylpropanoylamino)-benzoylamino]isophthalic acid was synthesized in five steps under conventional heating in high yield and purity. A series of soluble, thermally stable and optically active polyamides (PA)s containing pendent groups made of phthalimide, flexible l-alanine and benzamide sequence have been successfully synthesized under microwave irradiation. Excellent yields and very short reaction time were the main characteristics of this method. The same polymerization reactions were also carried out by conventional thermal heating and the results are compared. The resulting PAs had inherent viscosity in the range of 0.50–0.79 dL g⁻¹. All of the these polymers are readily dissolved in various solvents such as N-methyl-2-pyrrolidinone, N,N-dimethylacetamide and N,N-dimethylformamide and showed glass-transition temperature above 200°C. Thermogravimetric analysis demonstrated that the 10% weight-loss temperatures in nitrogen were 372 and 422°C for selected two PAs. All of these polymers showed optical rotation which is due to successful insertion of l-alanine in the structure of chiral diacid monomer.

Keywords: Direct polycondensation; Microwave; 5-[4-(2-Phthalimidiylpropanoylamino)benzoyl-amino]isophthalic acid; Polyamide

The effects of alanine ingestion on metabolic responses to exercise in cyclists by Janet Klein; William L. Nyhan; Mark Kern (pp. 673-680).

The influence of alanine on plasma amino acid concentrations and fuel substrates as well as cycling performance was examined. Four solutions [6% alanine (ALA); 6% sucrose (CHO); 6% alanine and 6% sucrose (ALA–CHO); an artificially sweetened placebo (PLC)] were tested using a double-blind, randomised, cross-over design. During each trial, ten cyclists ingested 500 mL of test solution 30 min before exercise and 250 mL after 15, 30, and 45 min of exercise. Participants cycled for 45 min at 75% VO₂max followed by a 15-min performance trial. Blood was collected before beverage consumption and prior to the performance trial. Alanine concentration was increased (p < 0.05) by approximately tenfold for ALA and ALA–CHO and less than twofold for CHO and PLC. Alanine ingestion increased concentrations of most gluconeogenic amino acids. Overall, alanine supplementation tended to produce favourable metabolic effects, but did not influence performance.

Keywords: Amino acids; Carbohydrate; Cycling; Athletes; Endurance

The heme-binding protein HbpS regulates the activity of the Streptomyces reticuli iron-sensing histidine kinase SenS in a redox-dependent manner by Gabriele Bogel; Hildgund Schrempf; Darío Ortiz de Orué Lucana (pp. 681-691).

The SenS/SenR system of Streptomyces reticuli regulates the expression of the redox regulator FurS, the catalase-peroxidase CpeB and the heme-binding protein HbpS. SenS/SenR is also proposed to participate in sensing redox changes, mediated by HbpS. Here, we show in vitro that heme-free HbpS represses the autokinase activity of SenS; whereas hemin-treated HbpS considerably enhances SenS autophosphorylation under redox conditions using either H₂O₂ or DTT. The presence of iron ions alone or in combination with H₂O₂ or DTT also leads to significantly increased phosphorylation levels of SenS. Further comparative physiological studies using the S. reticuli WT, a S. reticuli hbpS mutant and a S. reticuli senS-senR mutant corroborates the importance of HbpS and the SenS/SenR system for resistance against high concentrations of iron ions and hemin in vivo. Hence SenS/SenR and HbpS act in concert as a novel three-component system which detects redox stress, mediated by iron ions and heme.

Keywords: Streptomyces ; Two-component system SenS/SenR; Heme-binding protein HbpS; Redox stress

l-Leucinamide hydrogensquarate: spectroscopic and structural elucidation by Tsonko Kolev; Sonya Zareva; Heike Mayer-Figge; Michael Spiteller; William S. Sheldrick; Bojidarka B. Koleva (pp. 693-701).

The hydrogensquarate [LeuNH₂] (HSq) of l-leucinamide has been synthesized and its structure has been determined by single crystal X-ray diffraction. A three dimensional network is formed by hydrogen bonds with participation of the O=C–NH₂ function, the hydrogensquarate ion and the N⁺H₃ group [NH₂⋯O=C_(Sq) (2.840 and 2.749 Å), _(Sq)OH⋯O=C(NH₂) (2.618 Å), NH₃ ⁺⋯O=C_(Sq) (3.246, 2.804 and 2.823 Å)], respectively. A theoretical approximation of the electronic structure was carried out by means of ab initio UMP2 and MP2 level of theory at the 6-311++G** basis set. The IR-spectroscopic assignment in the solid-phase was obtained by linear-polarized IR-spectroscopy of oriented samples as colloid suspensions in a nematic host and application of the reducing-difference procedure for the interpretation of polarized IR-spectra.

Keywords: l-Leucinamide hydrogensquarate; Crystal structure; Solid-state IR-LD spectroscopy; ab initio calculations; ¹H-NMR data

Nitric oxide production and monoamine oxidase activity in cancer patients during interferon-α therapy by Durk Fekkes; Arthur R. Van Gool; Marjolein Bannink; Stefan Sleijfer; Wim H. J. Kruit; Bronno van der Holt; Alexander M. M. Eggermont; Michiel W. Hengeveld; Gerrit Stoter (pp. 703-708).

Both increased and decreased nitric oxide (NO) synthesis have been reported in patients treated with interferon-α (IFN-α). Animal studies showed that IFN-α administration results in increased levels of biogenic amines, subsequent activation of monoamine oxidases (MAOs), and finally in a change in NO production due to the H₂O₂ generated by MAOs. We examined the potential relationship between NO production in plasma and MAO-B activity in platelets of 43 cancer patients during 8 weeks of treatment with IFN-α. NO synthesis was quantitated by measuring both the ratio of citrulline and arginine (CIT/ARG-ratio) and total nitrite/nitrate (NOx) levels. Compared to baseline, MAO activity and NOx increased, while the CIT/ARG-ratio decreased. No associations were found between NOx, MAO and CIT/ARG-ratio. Only few associations were observed between changes in the biochemical parameters and changes in psychopathology induced by IFN-α, of which the association between changes in CIT and lassitude was the most consistent. The results suggest that peripheral NO production and MAO activity are unrelated to each other, and that peripheral changes in these biochemical parameters induced by IFN-α are unlikely to contribute to definite psychiatric disturbance.

Keywords: Citrulline; Depression; Interferon-α; Nitric oxide; Monoamine oxidase; Cancer

Dietary supplementation with cholesterol and docosahexaenoic acid affects concentrations of amino acids in tissues of young pigs by Peng Li; Sung Woo Kim; Xilong Li; Sujay Datta; Wilson G. Pond; Guoyao Wu (pp. 709-716).

Cholesterol and docosahexaenoic acid (DHA) are important nutrients for neural development of infants. However, little is known about the effect of cholesterol or DHA on concentrations of amino acids (AA) in neonatal tissues. This study was conducted with the piglet (an established model for studying human infant nutrition) to test the hypothesis that dietary supplementation with the lipids may modulate AA availability in tissues. Sixteen newborn pigs were nursed by sows for 24 h and then assigned to one of four treatment groups, representing supplementation with 0.0% (control), 0.2% cholesterol, 0.2% DHA, or cholesterol plus DHA to the basal milk-formula. All piglets were euthanized at 49 days of age. In brain, cholesterol supplementation reduced (P < 0.05) concentrations of glutamate, serine, glutamine, threonine, β-alanine, alanine, methionine, isoleucine, leucine, and γ-aminobutyrate but increased (P < 0.05) concentrations of glycine and lysine, whereas DHA supplementation similarly affected (P < 0.05) concentrations of the same AA (except for isoleucine and lysine) and taurine. In addition, concentrations of most AA in liver, muscle and plasma were substantially altered by dietary supplementation of cholesterol and DHA in a tissue-dependent manner. Further, DHA reduced concentrations of carnosine in skeletal muscle, as well as ammonia in both plasma and brain. The results reveal that cholesterol and DHA can regulate AA metabolism and availability in various tissues of piglets. These novel findings have important implications for designing the next generation of infant formula to optimize neonatal growth and development.

Keywords: Cholesterol; Docosahexaenoic acid; Amino acids; Pigs

Methods for ¹⁸F-labeling of RGD peptides: comparison of aminooxy [¹⁸F]fluorobenzaldehyde condensation with ‘click labeling’ using 2-[¹⁸F]fluoroethylazide, and S-alkylation with [¹⁸F]fluoropropanethiol by Matthias Glaser; Magne Solbakken; David R. Turton; Roger Pettitt; Jon Barnett; Joseph Arukwe; Hege Karlsen; Alan Cuthbertson; Sajinder K. Luthra; Erik Årstad (pp. 717-724).

Three strategies for chemoselective labeling of RGD peptides with ¹⁸F have been compared. Aminooxy [¹⁸F]fluorobenzaldehyde conjugation provided 40 ± 12% decay-corrected radiochemical yield using a fully automated method. An one-pot protocol for ‘click labeling’ of the RGD scaffold with 2-[¹⁸F]fluoroethylazide afforded 47 ± 8% decay-corrected radiochemical yield. Attempted conjugation with 3-[¹⁸F]fluoropropanethiol led to extensive decomposition and was therefore found unsuitable for labeling of the RGD peptide investigated. The results suggest that ‘click labeling’ of RGD peptides provides an attractive alternative to aminooxy aldehyde condensation, however, 2-[¹⁸F]-fluoroethylazide may be too small to allow separation of large ¹⁸F-labeled RGD peptides from their precursors.

Keywords: PET; RGD; Angiogenesis; Fluorine-18; Automation

Source of oseltamivir resistance in avian influenza H5N1 virus with the H274Y mutation by Maturos Malaisree; Thanyada Rungrotmongkol; Nadtanet Nunthaboot; Ornjira Aruksakunwong; Pathumwadee Intharathep; Panita Decha; Pornthep Sompornpisut; Supot Hannongbua (pp. 725-732).

Molecular dynamics simulations were carried out for the mutant oseltamivir-NA complex, to provide detailed information on the oseltamivir-resistance resulting from the H274Y mutation in neuraminidase (NA) of avian influenza H5N1 viruses. In contrast with a previous proposal, the H274Y mutation does not prevent E276 and R224 from forming the hydrophobic pocket for the oseltamivir bulky group. Instead, reduction of the hydrophobicity and size of pocket in the area around an ethyl moiety at this bulky group were found to be the source of the oseltamivir-resistance. These changes were primarily due to the dramatic rotation of the hydrophilic –COO⁻ group of E276 toward the ethyl moiety. In addition, hydrogen-bonding interactions with N1 residues at the -NH₃ ⁺ and -NHAc groups of oseltamivir were replaced by a water molecule. The calculated binding affinity of oseltamivir to NA was significantly reduced from −14.6 kcal mol⁻¹ in the wild-type to −9.9 kcal mol⁻¹ in the mutant-type.

Keywords: Oseltamivir resistance; H274Y mutation; Neuraminidase; H5N1

N-Methyl-d-aspartate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors involved in the induction of sedative effects under an acute stress in neonatal chicks by H. Yamane; Y. Tsuneyoshi; D. M. Denbow; M. Furuse (pp. 733-739).

Glutamate, an excitatory amino acid, acts at several glutamate receptor subtypes. Recently, we reported that central administration of glutathione induced hypnosis under stressful conditions in neonatal chicks. Glutathione appears to bind to the N-methyl-d-aspartate (NMDA) receptor. To clarify the involvement of each glutamate receptor subtype during stressful conditions, intracerebroventricular (i.c.v.) injection of several glutamate receptor agonists was given to chicks under social separation stress. Glutamate dose-dependently induced a hypnotic effect. NMDA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and kainate are characterized as ionotropic glutamate receptors (iGluRs). Although NMDA also induced sleep-like behavior or sedative effects, the potency of NMDA was less than that of glutamate. AMPA tended to decrease distress vocalizations induced by acute stress and brought about a sedative effect. Kainate and (S)-3, 5-dehydroxyphenylglycine, which is a metabotropic glutamate receptor agonist, had no influence on chick behavior. Thus, it is suggested that the iGluRs, NMDA and AMPA, are important in inducing hypnosis and sedation under acute stress in chicks.

Keywords: Glutamate; N-methyl-d-aspartate; Intracerebroventricular injection; Social separation stress; Neonatal chick

Application of protein grey incidence degree measure to predict protein quaternary structural types by Xuan Xiao; Wei-Zhong Lin (pp. 741-749).

Many proteins are composed of two or more subunits, each associated with different polypeptide chains. The number and arrangement of subunits forming a protein are referred to as quaternary structure. It has been known for long that the functions of proteins are closely related to their quaternary structure. In this paper the grey incidence degree is introduced that can calculate the numerical relation between various components, expressed the similar or different degree between these components. We have demonstrated that introduction of the grey incidence degree can remarkably enhance the success rates in predicting the protein quaternary structural class. It is anticipated that the grey incidence degree can be also used to predict many other protein attributes, such as subcellular localization, membrane protein type, enzyme functional class, GPCR type, protease type, among many others.

Keywords: Protein sequence distance measure; Grey system; Grey incidence degree; Protein quaternary structural type; Nearest neighbor algorithm

Colonic luminal ammonia and portal blood l-glutamine and l-arginine concentrations: a possible link between colon mucosa and liver ureagenesis by Mamy Eklou-Lawson; Françoise Bernard; Nathalie Neveux; Catherine Chaumontet; Cécile Bos; Anne-Marie Davila-Gay; Daniel Tomé; Luc Cynober; François Blachier (pp. 751-760).

The highest ammonia concentration in the body is found in the colon lumen and although there is evidence that this metabolite can be absorbed through the colonic epithelium, there is little information on the capacity of the colonic mucosa to transfer and metabolize this compound. In the present study, we used a model of conscious pig with a canula implanted into the proximal colon to inject endoluminally increasing amounts of ammonium chloride and to measure during 5 h the kinetics of ammonia and amino acid concentration changes in the portal and arterial blood. By injecting as a single dose from 1 to 5 g ammonia into the colonic lumen, a dose-related increase in ammonia concentration in the portal blood was recorded. Ammonia concentration remained unchanged in the arterial blood except for the highest dose tested, i.e. 5 g which thus apparently exceeds the hepatic ureagenesis capacity. By calculating the apparent net ammonia absorption, it was determined that the pig colonic epithelium has the capacity to absorb 4 g ammonia. Ammonia absorption through the colonic epithelium was concomitant with increase of l-glutamine and l-arginine concentrations in the portal blood. This coincided with the expression of both glutamate dehydrogenase and glutamine synthetase in isolated colonic epithelial cells. Since l-glutamine and l-arginine are known to represent activators for liver ureagenesis, we propose that increased portal concentrations of these amino acids following increased ammonia colonic luminal concentration represent a metabolic link between colon mucosa and liver urea biosynthesis.

Keywords: Ammonia absorption; Pig colon mucosa

Addendum to 11th International Congress on Amino Acids, Peptides and Proteins (pp. 761-766).

N-Methyl-d-aspartate and α-amino-3-hydroxy-5-methyl-4- isoxazolepropionate receptors involved in the induction of sedative effects under an acute stress in neonatal chicks by H. Yamane; Y. Tsuneyoshi; D. M. Denbow; M. Furuse (pp. 767-767).

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.37, #4)