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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.36, #3)

Human S100A12: a novel key player in inflammation? by Jens Pietzsch; Susan Hoppmann (pp. 381-389).

S100A12 is a member of the S100 family of EF-hand calcium-binding proteins. Human S100A12 is predominantly expressed and secreted by neutrophil granulocytes and, therefore, has been assigned to the S100 protein subfamily of calgranulins or myeloid-related proteins. Intracellular S100A12 exists as an anti-parallel homodimer and upon calcium-dependent activation interacts with target proteins to regulate cellular functions. Extracellular S100A12 exists majorily as homodimer and hexamer, respectively, and shows cytokine-like characteristics. It is part of the innate immune response and linked to certain autoimmune reactions. Human S100A12 is markedly overexpressed in inflammatory compartments, and elevated serum levels of S100A12 are found in patients suffering from various inflammatory, neurodegenerative, metabolic, and neoplastic disorders. In this regard, interaction of calcium-activated S100A12 with the multiligand receptor for advanced glycation endproducts (RAGE) and its soluble form (sRAGE) plays a central pathogenetic role. Recent clinical evidence suggests a high potential of S100A12 as a sensitive and specific diagnostic marker of localized inflammatory processes.

Keywords: Calcium-binding proteins; Canonical EF-hand; Copper-binding proteins; Damage-associated molecular pattern molecules; Inflammatory diseases; Pseudo EF-hand; Receptor for advanced glycation endproducts; Soluble RAGE

Kinetic determination of tryptophan by using the B-Z oscillating chemical system by Jinzhang Gao; Jie Qu; Wu Yang; Xiaoxia Wei; Hongxia Dai; Dongyu Lv; Jie Ren; Hua Chen (pp. 391-397).

A simple and rapid method was devised for determination of tryptophan, based on the Belousov-Zhabotinskii (B-Z) oscillating chemical system. Changes in oscillating period and amplitude were linearly proportional to the negative logarithm of l-tryptophan concentration over the range of 6.44 × 10⁻⁷–2.55 × 10⁻⁴M, with the regression coefficients of near unity and a lower detection limit of 6.5 × 10⁻⁸M. d-tryptophan was also examined although it is rarely found in most biological fluids, and perhaps not at all in natural proteins. The change of period against to negative logarithm of d-tryptophan concentration over the range of 4.9 × 10⁻⁵–8.24 × 10⁻⁴M is linear. Because the optimum conditions for determination of l- and d-tryptophan are not the same, a little amount of d-tryptophan does not affect the determination of l-tryptophan. Various influences were studied and a possible mechanism of perturbation to the B-Z oscillator by tryptophan was also discussed. Spectrophotometry and fluorescence spectrophotofluorimetry were used for comparision and confirmation of the results.

Keywords: L-tryptophan; d-tryptophan; B-Z oscillating chemical reaction; Kinetic determination

Binding of brilliant red compound to lysozyme: insights into the enzyme toxicity of water-soluble aromatic chemicals by Fang-Fang Chen; Yi-Nan Tang; Shi-Long Wang; Hong-Wen Gao (pp. 399-407).

The non-covalent interaction of brilliant red (BR) with lysozyme was investigated by the UV spectrometry, circular dichroism (CD) and isothermal titration calorimetry (ITC). The thermodynamic characterization of the interaction was performed and the assembly complexes were formed: lysozyme(BR)₁₇ at pH 2.03, lysozyme(BR)₁₅ at pH 3.25 and lysozyme(BR)₁₂ at pH 4.35, which corresponded to the physiological acidities. The ionic interaction induces a combination of multiple non-covalent bonds including hydrogen bond, hydrophobic interaction and van der Waals force. The two-step binding model of BR was found, in which one or two BR molecules entered the hydrophobic intracavity of lysozyme and the others bound to the hydrophilic outer surface of lysozyme. Moreover, BR binding resulted in change of the lysozyme conformation and inhibition of the lysozyme activity. The possible binding site and type of BR and the conformational transition of lysozyme were speculated and illustrated. This work provided a useful approach for study on enzyme toxicity of aromatic azo chemicals.

Keywords: Aromatic azo compound; Lysozyme; Non-covalent binding; Enzyme toxicity

Using ensemble of classifiers for predicting HIV protease cleavage sites in proteins by Loris Nanni; Alessandra Lumini (pp. 409-416).

The focus of this work is the use of ensembles of classifiers for predicting HIV protease cleavage sites in proteins. Due to the complex relationships in the biological data, several recent works show that often ensembles of learning algorithms outperform stand-alone methods. We show that the fusion of approaches based on different encoding models can be useful for improving the performance of this classification problem. In particular, in this work four different feature encodings for peptides are described and tested. An extensive evaluation on a large dataset according to a blind testing protocol is reported which demonstrates how different feature extraction methods and classifiers can be combined for obtaining a robust and reliable system. The comparison with other stand-alone approaches allows quantifying the performance improvement obtained by the ensembles proposed in this work.

Keywords: Machine learning; Ensembles of classifiers; HIV-1 protease prediction

Taurine plays a beneficial role against cadmium-induced oxidative renal dysfunction by Prasenjit Manna; Mahua Sinha; Parames C. Sil (pp. 417-428).

The present study has been carried out to investigate the role of taurine (2-aminoethanesulfonic acid), a conditionally essential amino acid, in ameliorating cadmium-induced renal dysfunctions in mice. Cadmium chloride (CdCl₂) has been selected as the source of cadmium. Intraperitoneal administration of CdCl₂(at a dose of 4 mg/kg body weight for 3 days) caused significant accumulation of cadmium in renal tissues and lessened kidney weight to body weight ratio. Cadmium administration reduced intracellular ferric reducing/antioxidant power (FRAP) of renal tissues. Levels of serum marker enzymes related to renal damage, creatinine and urea nitrogen (UN) have been elevated due to cadmium toxicity. Cadmium exposure diminished the activities of enzymatic antioxidants, superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GPx) and glucose-6-phosphate dehydrogenase (G6PD) as well as non-enzymatic antioxidant, reduced glutathione (GSH) and total thiols. On the other hand, the levels of oxidized glutathione (GSSG), lipid peroxidation, protein carbonylation, DNA fragmentation, concentration of superoxide radicals and activities of cytochrome P450 enzymes (CYP P450s) have been found to increase due to cadmium intoxication. Treatment with taurine (at a dose of 100 mg/kg body weight for 5 days) before cadmium intoxication prevented the toxin-induced oxidative impairments in renal tissues. The beneficial role of taurine against cadmium-induced renal damage was supported from histological examination of renal segments. Vitamin C, a well-established antioxidant was used as the positive control in the study. Experimental evidence suggests that both taurine and vitamin C provide antioxidant defense against cadmium-induced renal oxidative injury. Combining all, results suggest that taurine protects murine kidneys against cadmium-induced oxidative impairments, probably via its antioxidative property.

Keywords: Cadmium; Oxidative impairment; Renal dysfunction; Taurine; Antioxidant; Renoprotective effect

Synthesis of bis-amino acid derivatives by Suzuki cross-coupling, Michael addition and substitution reactions by P. M. T. Ferreira; L. S. Monteiro; M.-J. R. P. Queiroz; G. Pereira (pp. 429-436).

Several bis-amino acids were prepared using a bis-Suzuki coupling (compounds 4–8, 10), a sequential Michael addition and bis-Suzuki coupling (compounds 12, 13) and a Michael addition followed by a substitution reaction (compounds 18, 19). Thus, the pure stereoisomer of the methyl esters of N-(tert-butoxycarbonyl)-β-bromodehydroaminobutyric acid and dehydrophenylalanine and of N-benzyloxycarbonyl-β-bromodehydroaminobutyric acid were reacted with 1,4-phenylene-bis-boronic acid or 9,9-dioctyl-9H-fluorene-2,7-bis-boronic acid using modified Suzuki coupling conditions. The corresponding bis-dehydroamino acid derivatives were obtained in good to high yields maintaining the stereochemistry of the starting materials. This reaction was also applied successfully to a brominated dehydrodipeptide and 1,4-phenylene-bis-boronic acid showing that it could be used to create cross-links in peptide chains. An N,N-diacyldehydroalanine derivative was used in a sequential Michael addition and bis-Suzuki coupling giving a p-terphenyl bis-amino acid and a fluorenyl bis-amino acid in good yields. Two bis-α,β-diamino acids were obtained by a Michael addition of 1,2,4-triazole to the methyl esters of N-(4-toluenesulfonyl), N-(tert-butoxycarbonyl) dehydroamino acids followed by treatment with ethylenediamine.

Keywords: Dehydroamino acids; Bis-amino acids; Bis-Suzuki coupling; Michael addition; Substitution reaction

A comparative study of the chemical reactivity of pyridoxamine, Ac-Phe-Lys and Ac-Cys with various glycating carbonyl compounds by Miquel Adrover; Bartolomé Vilanova; Juan Frau; Francisco Muñoz; Josefa Donoso (pp. 437-448).

Pyridoxamine (PM) has long been known to inhibit protein glycation via various mechanisms of action. One such mechanism involves the scavenging of carbonyl compounds with glycating ability. Despite the abundant literature on this topic, few quantitative kinetic studies on the processes involved have been reported. In this work, we conducted a comparative kinetic study under physiological pH and temperature conditions of the reactions of PM, Ac-Phe-Lys and Ac-Cys with various glycating carbonyl compounds (viz. aldehydes, α-oxoaldehydes and ketones). The microscopic formation rate constants for Schiff bases of PM and various carbonyl compounds, k ₁, are of the same order of magnitude as those for the Schiff bases of Ac-Phe-Lys. However, because PM exhibits a higher proportion of reactive form at physiological pH, its observed second-order rate constant is ca. five times greater than that for Ac-Phe-Lys. That could explain PM ability to compete with amino residues in protein glycation. On the other hand, the observed formation rate constant for thiohemiacetals is four orders of magnitude greater than the formation constants for the Schiff bases of PM, which excludes PM as a competitive inhibitor of Cys residues in protein glycation.

Keywords: Pyridoxamine (PM); Carbonyl compounds; Kinetics; Shiff base; Protein Glycation

Ca²⁺-independent effects of spermine on pyruvate dehydrogenase complex activity in energized rat liver mitochondria incubated in the absence of exogenous Ca²⁺ and Mg² ⁺ by E. Pezzato; V. Battaglia; A. M. Brunati; E. Agostinelli; A. Toninello (pp. 449-456).

In the absence of exogenous Ca²⁺ and Mg²⁺ and in the presence of EGTA, which favours the release of endogenous Ca²⁺, the polyamine spermine is able to stimulate the activity of pyruvate dehydrogenase complex (PDC) of energized rat liver mitochondria (RLM). This stimulation exhibits a gradual concentration-dependent trend, which is maximum, about 140%, at 0.5 mM concentration, after 30 min of incubation. At concentrations higher than 0.5 mM, spermine still stimulates PDC, when compared with the control, but shows a slight dose-dependent decrease. Changes in PDC stimulation are very close to the phosphorylation level of the E_1α subunit of PDC, which regulates the activity of the complex, but it is also the target of spermine. In other words, progressive dephosphorylation gradually enhances the stimulation of RLM and progressive phosphorylation slightly decreases it. These results provide the first evidence that, when transported in RLM, spermine can interact in various ways with PDC, showing dose-dependent behaviour. The interaction most probably takes place directly on a specific site for spermine on one of the regulatory enzymes of PDC, i.e. pyruvate dehydrogenase phosphatase (PDP). The interaction of spermine with PDC may also involve activation of another regulatory enzyme, pyruvate dehydrogenase kinase (PDK), resulting in an increase in E_1α phosphorylation and consequently reduced stimulation of PDC at high polyamine concentrations. The different effects of spermine in RLM are discussed, considering the different activities of PDP and PDK isoenzymes. It is suggested that the polyamine at low concentrations stimulates the isoenzyme PDP₂ and at high concentrations it stimulates PDK₂.

Keywords: Pyruvate dehydrogenase; Mitochondria; Spermine; E_1α phosphorylation; Pyruvate dehydrogenase kinase; Pyruvate dehydrogenase phosphatase

Effect of taurine on alcoholic liver disease in rats by Gaofeng Wu; Jiancheng Yang; Changmian Sun; Xinhong Luan; Jiao Shi; Jianmin Hu (pp. 457-464).

To investigate the effect of taurine on alcoholic liver disease in rats, male Wistar rats were administered alcohol intragastrically for 3 months. The effect of β-alanine-mediated taurine depletion and taurine administration on the development of alcoholic liver disease was examined. It was found that taurine administration produced lower levels of aspartate aminotransferase and alkaline aminotransferase than that of the untreated group. In addition, the levels of hepatic total protein, glutathione and superoxide dismutase were higher in the taurine treated groups than those in the untreated control or the taurine depleted groups, while hepatic malondialdehyde content exhibited the negative effect. Moreover, the concentrations of hepatic hydroxyproline, serum hyaluronic acid, interleukin-2, interleukin-6, tumor necrosis factor-α and laminin were all decreased in the taurine treated groups. The pathological changes showed that the percentage of fatty degeneration and inflammation in the taurine groups were lower than that of the control, taurine depleted and automatic recovery groups. These in vivo findings demonstrate that hepatic disease caused by chronic alcohol consumption can be prevented and cured by administration of taurine.

Keywords: Alcoholic liver disease; Effect; Protection; Rat; Taurine

Enolization and racemization reactions of glucose and fructose on heating with amino-acid enantiomers and the formation of melanoidins as a result of the Maillard reaction by Ji-Sang Kim; Young-Soon Lee (pp. 465-474).

This study investigated the enolization and racemization reactions of glucose and fructose on heating with amino acid enantiomers and the formation of melanoidins as a result of the Maillard reaction. The study measured reducing sugars and L- and D- amino acids using HPLC as an index for the amount of enolization of the sugars and isomerization of the amino acids. Additionally, the absorption of melanoidins was measured at different wavelengths (420, 450, 470, 490 nm); the UV–Vis spectra and the extinction coefficient were determined for the formation of melanoidins. Melanoidins were, rather arbitrarily, defined by a high-molecular-weight (HMW) if it was above a lower limit of 12.4 kDa, which was the nominal cut-off value in the dialysis system used. A remarkable enolization reaction of the sugars was observed in the course of the Maillard reaction. Especially, in the Fru/D-Asn model system, the degree of sugar enolization was more than in the other model systems. All of the FDAA (1-fluoro-2, 4-dinitrophenyl-5-L-alanine amide) amino acids were separated by TLC. The racemization of the amino acids was higher in the fructose-amino acids systems. Isomer formation was the highest in the Fru/D-Asn system. The L- and D- isomers showed different absorptions in the UV–Vis spectra, although these had similar shapes. The absorption of the melanoidins formed from glucose was higher than that formed from fructose. In particular, the sugar–asparagine system showed different characteristics according to the L- and D-isomers. The differences in the extinction coefficients of the melanoidins was significant (P < 0.05), except for the sugar–lysine system.

Keywords: Enolization; Extinction coefficient; Maillard reaction; Melanoidins; Racemization

Ultra performance liquid chromatography-mass spectrometric determination of the site specificity of modification of β-casein by glucose and methylglyoxal by Maria Lima; Catherine Moloney; Jennifer M. Ames (pp. 475-481).

Modification of protein by carbonyl compounds under in vitro physiological conditions is site-directed. There are few reports of the site specificity of glycation of proteins using heating conditions of relevance to food processing. The aim of this study was to determine the site specificity of modification of β-casein (βCN) by glucose and methylglyoxal (MGO). βCN (1.33 M, 3.2%) was heated with either glucose (1.345 M, 4.6%) or MGO (1 mM) at 95°C for up to 4 h. Tryptic digests were prepared and analysed by ultra performance liquid chromatography electrospray ionisation mass spectrometry (UPLC-ES/MS). The sites of formation of the Amadori product, N ε-(fructosyl)lysine (FL), and the advanced glycation end-products, N ε-(carboxymethyl)lysine (CML), MGO-derived dihydroxyimidazolidine (MG-DH) and MGO-derived hydroimidazolone (MG-HI), were located. FL and CML were detected at K107 and K176 residues in βCN/glucose incubations. Indigenous N ε-(lactulosyl)lysine was detected at K107 only. MG-DH and MG-HI were detected at R202 and possibly R183 residues in both βCN/glucose and βCN/MGO incubations. Glycation of βCN by glucose and MGO resulted in similar site specificity for MG-DH and MG-HI formation.

Keywords: Advanced glycation end-products; N ε-(fructosyl)lysine; N ε-(carboxymethyl)lysine; MGO-derived dihydroxyimidazolidine; MGO-derived hydroimidazolone; Ultra performance liquid chromatography-mass spectrometry

Binding specificity of the l-arginine transport systems in mouse macrophages and human cells overexpressing the cationic amino acid transporter hCAT-1 by Dániel Erős; László Őrfi; Ildikó Csuka; György Kéri; András Hrabák (pp. 483-492).

The uptake of l-arginine into mouse peritoneal macrophages can be inhibited by numerous amino acids and derivatives. Kinetic studies showed an almost entirely competitive inhibition for both cationic and neutral amino acids and derivatives suggesting that the comparison of their binding specificity by using a quantitative structure-activity relationship (QSAR) study is reasonable. The properties of the most efficient inhibitors were the following: the length of the aliphatic side chain, a general structural similarity to l-arginine (>0.79), cationic character, l-configuration, the presence of an α-amino group (with a mean pK_a of 9.41), the van der Waals volume (mean 225 Å³) and a low logP value (mean: −2.99). The significance of four other descriptors (neutral character, presence and the pK_a of an α-carboxyl group, and the presence of a modified guanidino group) is much lower. Similar results were obtained for the hCAT-1 cell line, but the significance of the descriptors was slightly different. The l-configuration, van der Waals volume, the low logP value and the length of aliphatic side chain were the most significant, while the pK_a value of the side chain (mean pK_a = 11.6) was found to be more important than that of the α-amino group. In addition, the general similarity to l-arginine, the presence of an amino group in the terminal position of the side chain (Orn, Lys) and the basic character were significant descriptors, while the significance of the acidity is negligibly low. As a final conclusion, the following descriptors were found to be important generally for the cationic transporters: the van der Waals volume, hydrophobicity (log P); l-configuration; the size of the side chain; the general similarity to l-arginine; the presence of an α-amino group; the general basicity of the molecule; the pK_a values of the α-amino group (in macrophages) or that of the side chain (in CAT-1 cells). These descriptors can be regarded as the general structurally important binding characteristics of the cationic amino transporters.

Keywords: Arginine; Cationic amino acid transporter; Binding specificity; Inhibition; Quantitative structure–activity relationship

Novel deprotection method of Fmoc group under neutral hydrogenation conditions by Tomohiro Maegawa; Yuta Fujiwara; Takashi Ikawa; Hideo Hisashi; Yasunari Monguchi; Hironao Sajiki (pp. 493-499).

Novel deprotection method of the Fmoc (9-flurorenylmethoxycarbonyl) protective group under Pd/C-catalyzed hydrogenation conditions at room temperature was developed. The addition of CH₃CN accelerated the deprotection of the Fmoc group, and also the application of H₂ pressure (3 atm) shows notable rate enhancement.

Keywords: Pd/C; Hydrogenation; Fmoc; Deprotection

Effects of 2-hydroxy-4-methylthiobutyrate on portal plasma flow and net portal appearance of amino acids in piglets by Z. F. Fang; J. Luo; Z. L. Qi; F. R. Huang; S. J. Zhao; M. Y. Liu; S. W. Jiang; J. Peng (pp. 501-509).

To determine whether portal plasma flow (PPF) and net portal appearance of amino acids (AA) could be affected by 2-hydroxy-4-methylthiobutyrate (HMB), six barrows (35-day-old, 8.6 ± 1.4 kg), implanted with arterial, portal and mesenteric catheters, were fed a dl-methionine (as the control) or HMB-supplemented diet once hourly and infused intramesenterically with 1% p-amino hippurate. PPF was numerically 9% higher (P = 0.09) in HMB-fed pigs than in controls over a 4–6 h period. Compared with controls, pigs fed the HMB diet had increased (P < 0.05) net portal balance and/or appearance of leucine, isoleucine, histidine, arginine and alanine, but had decreased (P < 0.05) portal appearance of glutamate over a 6-h period. The concentration of acetate in the lumen of the distal small intestine was higher (P = 0.01) in HMB-fed pigs than in controls (25.14 vs. 7.64 mmol/kg). mRNA levels for proglucagon and endothelial nitric-oxide synthase (eNOS) in stomach and proximal small intestine, and mRNA levels for GLP-2 receptor (GLP-2R) in stomach were higher (P < 0.05) in HMB-fed pigs compared with those in controls. Collectively, HMB supplementation increased concentrations of short-chain fatty acids in intestinal lumen, expression of proglucagon, GLP-2R, and eNOS genes, and net portal absorption of AA. These novel findings from the study with pigs may also have important implications for intestinal nutrition and health in humans.

Keywords: Amino acids; Portal plasma flow; Proglucagon; Short-chain fatty acids; 2-Hydroxy-4-methylthiobutyrate

Synthesis and characterization of new optically active poly(azo-ester-imide)s via interfacial polycondensation by Abdol R. Hajipour; Saeed Zahmatkesh; Parniyan Roosta; Arnold E. Ruoho (pp. 511-518).

N,N′-Pyromelliticdiimido-di-l-amino acids (1a–1d) were prepared from the reaction of pyromellitic dianhydride with the corresponding l-amino acids in a solution of glacial acetic acid/pyridine (3:2) at refluxing temperature. 4,4′-sulfonyl bis(4,1-phenylene) bis(diazene-2,1-diyl) diphenol, 4,4′-oxy bis(4,1-phenylene) bis(diazene-2,1-diyl) diphenol and 4,4′-methylene bis(4,1-phenylene) bis(diazene-2,1-diyl) diphenol, were prepared from 4,4′-diamino diphenyl sulfone, 4,4′-diamino diphenyl ether, 4,4′-diamino diphenyl methane, sodium nitrite and phenol following the general procedure of diazo coupling. Interfacial polycondensation method was used to prepare the corresponding poly(azo-ester-imid)s (PAEI 1–12) in biphasic solution of water/dichloromethane. The resulting polymers (PAEIs) have been obtained in high yields having good inherent viscosities (0.32–0.57 dl g⁻¹), optical activities and thermal stabilities.

Keywords: Poly(azo-ester-imide); Interfacial polycondensation; Optically active; Thermally stable; Azo compound; Diazo coupling

Perioperative application of l-alanyl-l-glutamine in cardiac surgery: effect on the polarized T cell cytokine expression by J. M. Engel; S. Ruhs; J. Mühling; C. Katzer; M. Müller; T. Menges; T. Langefeld; G. Hempelmann (pp. 519-527).

At risk patients undergoing cardiac surgery with cardiopulmonary bypass have increased rates of postoperative infectious morbidity. Postoperatively, after cardiac surgery, an immunosuppression in the form of a polarization of T helper (Th) cells with a decreased Th1 response (IL-2 and IFN-γ) and an increased Th2 response (IL-4 and IL-10) is recognized. Therapeutic strategies to modulate the immunological response include special key nutrients such as the amino acid glutamine favoring the Th2 response. There is no information available concerning its effect in patients undergoing cardiac surgery. The aim of this clinical study was to evaluate the effects of a perioperative infusion of glutamine on the polarized lymphocyte T cell cytokine expression and on infectious morbidity in cardiac surgery patients at risk of infection. Seventy-eight patients were included in the study undergoing elective cardiac surgery with a lymphopenia less than 1.2 giga/l. One or more of the following criteria had to be met: age older than 70 years, ejection fraction less than 40%, or mitral valve replacement. We randomly assigned patients to receive infusions of either high-dose l-alanyl-l-glutamine dipeptide [0.5 g/(kg day) glutamine] dissolved in an amino acid solution or an isonitrogeneous, isocaloric, isovolemic nutritional solution. An additional group with normal saline served as control to eliminate any nonspecific nutritional effect. We started the infusion after induction of anesthesia with 1,000 ml/24 h and continued it for 3 days. The primary endpoint was intracellular T cell cytokine expression (including the description in tertiles) on the first postoperative day (pod 1). Secondary endpoints were postoperative infection rate, mortality rate, cardiovascular circulation ventilation time, and renal function. A high-dose perioperative glutamine application leading to mean plasma levels of 1,177 μM had only a minor influence on the polarized intracellular T cell cytokine expression. On pod 1 there was a polarization of T cells, i.e., an augmented Th2 response with an increased number of IL-6 and IL-10 producing cells. On the other side the Th1 response with IL-2 and TNF-α declined on pods 1 and 2. Only the intracellular IL-2 response in the lower tertile of IL-2 production was improved with glutamine indicating a small influence. We did not observe any effects on the numbers of postoperative infections; on mortality rate; on cardiovascular circulation; on ventilation time or on renal function. The elevation of glutamine plasma levels by a perioperative intravenous infusion of l-alanyl-l-glutamine influenced the intracellular expression of IL-2 in the lower tertile only slightly. However, mean glutamine values in the other groups remained above or close 500 μM, thus suggesting that glutamine supply to the immune cells was still adequate in most patients, and that glutamine deficiency, if it occurred, was marginal. In the event of a severe glutamine deficiency the observed effect on cytokine production could be more pronounced. Furthermore, we could not observe any obvious clinical advantage in this at risk cardiac surgical patient population. A glutamine supplementation for patients undergoing cardiac surgery without a clear glutamine deficiency is not recommended.

Keywords: Glutamine; Cardiac surgery; T helper cells; Infection

Taurine in plasma and CSF: a study in healthy male volunteers by M. Samuelsson; M.-L. Dahl; R. C. Gupta; C. Nordin (pp. 529-533).

In order to explore the interrelationship between plasma and cerebrospinal fluid taurine concentrations, three consecutive 6-ml fractions of cerebrospinal fluid were drawn from 30 healthy male volunteers in the early morning after 8 h in the fasting condition. Repeated plasma samples were drawn over 24 h the day before lumbar puncture. Taurine in plasma and cerebrospinal fluid was determined by high performance liquid chromatography. The subjects were categorized as extensive or poor metabolizers with respect to the cytochrome P450 2D6 genotype. The taurine cerebrospinal fluid/plasma ratio at 8 a.m. was negatively influenced by the plasma taurine concentration at 4 p.m. the previous day. It was also negatively influenced by body mass index and positively by the intraspinal pressure. Three poor metabolizers of cytochrome P450 2D6 had higher plasma taurine areas under the curve than 27 extensive metabolizers. Hypothetically, cytochrome P450 2D6 influences the transport of taurine across the blood–brain barrier.

Keywords: Taurine; Cerebrospinal fluid; Plasma; Cytochrome P450 2D6; Genotype

In silico quantitative prediction of peptides binding affinity to human MHC molecule: an intuitive quantitative structure–activity relationship approach by F. Tian; L. Yang; F. Lv; Q. Yang; P. Zhou (pp. 535-554).

In this paper, we have handpicked 23 kinds of electronic properties, 37 kinds of steric properties, 54 kinds of hydrophobic properties and 5 kinds of hydrogen bond properties from thousands of amino acid structural and property parameters. Principal component analysis (PCA) was applied on these parameters and thus ten score vectors involving significant nonbonding properties of 20 coded amino acids were yielded, called the divided physicochemical property scores (DPPS) of amino acids. The DPPS descriptor was then used to characterize the structures of 152 HLA-A^*0201-restricted CTL epitopes, and significant variables being responsible for the binding affinities were selected by genetic algorithm, and a quantitative structure–activity relationship (QSAR) model by partial least square was established to predict the peptide-HLA-A*0201 molecule interactions. Statistical analysis on the resulted DPPS-based QSAR models were consistent well with experimental exhibits and molecular graphics display. Diversified properties of the different residues in binding peptides may contribute remarkable effect to the interactions between the HLA-A*0201 molecule and its peptide ligands. Particularly, hydrophobicity and hydrogen bond of anchor residues of peptides may have a significant contribution to the interactions. The results showed that DPPS can well represent the structural characteristics of the antigenic peptides and is a promising approach to predict the affinities of peptide binding to HLA-A*0201 in a efficient and intuitive way. We expect that this physical-principle based method can be applied to other protein–peptide interactions as well.

Keywords: Quantitative structure–activity relationship; Computer-aided vaccine design; Immunoinformatics; Divided physicochemical property scores of amino acids; HLA-A*0201; CTL epitope; Genetic algorithm-partial least square regression

Adaptational modification of serine and threonine metabolism in the liver to essential amino acid deficiency in rats by Kenji Nagao; Makoto Bannai; Shinobu Seki; Masato Mori; Michio Takahashi (pp. 555-562).

It is known that plasma serine and threonine concentrations are elevated in rats chronically fed an essential amino acid deficient diet, but the underlying mechanisms including related gene expressions or serine and threonine concentrations in liver remained to be elucidated. We fed rats lysine or valine deficient diet for 4 weeks and examined the mRNA expressions of serine synthesising (3-phosphoglycerate dehydrogenase, PHGDH) and serine/threonine degrading enzymes (serine dehydratase, SDS) in the liver. Dietary deficiency induced marked elevation of hepatic serine and threonine levels associated with enhancement of PHGDH mRNA expression and repression of SDS mRNA expression. Increases in plasma serine and threonine levels due to essential amino acid deficiency in diet were caused by marked increases in hepatic serine and threonine levels. Proteolytic responses to the amino acid deficiency may be lessened by storing amino radicals as serine and inducing anorexia through elevation of threonine.

Keywords: Amino acid deficiency; Serine; Threonine; PHGDH; SDS

Molecularly imprinted polymers for RGD selective recognition and separation by Emmanuel Papaioannou; Christos Koutsas; Maria Liakopoulou-Kyriakides (pp. 563-569).

Molecularly imprinted polymers that could recognize the tripeptide Arg-Gly-Asp have been produced with the use of two functional monomers and three different cross-linkers, respectively. Methacrylic acid and acrylamide were used as functional monomers and the role of the ethylene glycol dimethacrylate, trimethylpropane trimethacrylate and N,N′-methylene-bisacrylamide as crosslinking monomers, was investigated on their recognition capability. The % net rebinding and the imprinting factor values were obtained, giving for the methacrylic acid–trimethylpropane trimethacrylate polymer the highest values 12.3% and 2.44, respectively. In addition, this polymer presented lower dissociation constant (K _D) value and the higher B _max% of theoretical total binding sites than all the other polymers. Rebinding experiments with Lys-Gly-Asp, an analogue of Arg-Gly-Asp, and other different peptides, such as cholecystokinin C-terminal tri- and pentapeptide and gramicidin, further indicated the selectivity of methacrylic acid-trimethylpropane trimethacrylate copolymer for Arg-Gly-Asp giving specific selectivity factor values 1.27, 1.98, 1.31 and 1.67, respectively.

Keywords: Arg-Gly-Asp; RGD; Copolymerization; Molecular imprinting; Radical polymerization

Chromatographic separation of enantiomers of non-protein α-amino acids after derivatization with Marfey’s reagent and its four variants by R. Bhushan; Virender Kumar; Shivani Tanwar (pp. 571-579).

Some non-protein α-amino acids were derivatized with 1-fluoro-2,4-dinitrophenyl-5-l-alaninamide (Marfey’s reagent, MR, FDNP-l-Ala-NH₂,) and four of its structural variants FDNP-l-Phe-NH₂, FDNP-l-Val-NH₂, FDNP-l-Leu-NH₂ and FDNP-l-Pro-NH₂. The resultant diastereomers were separated by normal and reversed phase thin layer chromatography (TLC) and reversed phase HPLC. In normal phase TLC, best resolution was obtained with solvent combination of phenol-water (3:1) while in reversed phase TLC mixtures of acetonitrile with triethylammonium phosphate buffer were found successful for resolution of diastereomers. The separation behavior of diastereomers prepared with different reagents was compared. The diastereomers of most of the amino acids prepared with FDNP-l-Leu-NH₂ were best separated while those prepared with FDNP-l-Pro-NH₂ failed to separate in most of the cases. The diastereomers were also separated on a reversed phase C₈ column with gradient elution using mixture of aqueous-trifluoroacetic acid (TFA) and acetonitrile and with detection at 340 nm. The effects of TFA concentration, flow rate and run time on HPLC separation were studied.

Keywords: Chiral separation; Non-protein α-amino acids; FDNP-l-Ala-NH₂ ; FDNP-l-Phe-NH₂ ; FDNP-l-Val-NH₂ ; FDNP-l-Leu-NH₂ ; FDNP-l-Pro-NH₂ ; Normal and RPTLC; RPHPLC

Peptide substrate for caspase-3 with 2-aminoacridone as reporting group by Valentin Lozanov; Ivaylo P. Ivanov; Bistra Benkova; Vanio Mitev (pp. 581-586).

Synthesis and properties of a new fluorescent/fluorogenic substrate Ac-DEVD-AMAC for caspase-3 are reported. The substrate is obtained by conventional Fmoc-based solid phase peptide synthesis and its properties are investigated with regard to fluorescence, sensitivity, applicability and kinetic constants. A non-traditional approach to assay the proteases activity using 2-aminoacridone labeled peptides is proposed. This approach utilizes the decrease of fluorescence intensity of a sample as a measure for the enzyme activity.

Keywords: 2-Aminoacridone; Fluorogenic substrate; Fluorescent substrate; Caspase-3; Apoptosis

Localization and expression of serine racemase in Arabidopsis thaliana by Manabu Sugimoto; Wataru Sakamoto; Yoshiyuki Fujitani (pp. 587-590).

Arabidopsis plants transformed by promoter of A. thaliana serine racemase fused with β-glucuronidase (GUS) reporter gene showed strong GUS staining in elongating and developing cells such as tip regions of primary and lateral roots, developing leaves, and shoot meristems. RT-PCR and digital northern hybridization showed that expression of the serine racemase gene was not induced by l- and d-serine, light irradiation, biotic and abiotic stresses.

Keywords: Serine racemase; d-amino acid; Arabidopsis thaliana ; GUS; Promoter

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.36, #3)

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.36, #3)