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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.36, #2)

Possible antioxidant role of SPA therapy with chlorine–sulphur–bicarbonate mineral water by M. Costantino; G. Giuberti; M. Caraglia; A. Lombardi; G. Misso; A. Abbruzzese; F. Ciani; E. Lampa (pp. 161-165).

The aim of our research was to analyze the antioxidant role and efficacy of thermal or salus per aquam (spa) therapy with chlorine–sulphur–bicarbonate mineral water. The study has been performed on 30 rats. The animals were randomized in three groups, each of them composed by ten animals, denominated A, B and C. The A group was the control group and was not subjected to any specific treatment (placebo); the B group has been treated with a standard cycle of hydropinics treatment with mineral water of Therme of Stabia in Castellammare (Naples, Italy) denominated STABIA; the C group was treated with a standard cycle of hydropinic treatment with mineral water of Therme of Stabia in Castellammare (Naples, Italy) denominated SULFUREA. After two weeks of treatment all the rats were sacrificed and blood was collected for the plasmatic determination of reactive oxygen species (ROS). The results demonstrated a significant (P < 0.05) reduction of ROS in B (374 Carr. U. ±73) and C group (399 carr. U. ±62) treated with mineral waters if compared with control group (571 + 69 Carr. U.). In conclusion this study suggests a possible antioxidant effect of chlorine–sulphur–bicarbonate spa hydropinic treatment with a consequent suitable intestinal physiology, with reduction of the functional and organic modifications that can lead to pathological disorders of the gastroenteric diseases in whose pathogenesis the oxidative stress can develop an important role.

Keywords: Spa therapy; Sulphur mineral water; Reactive radicals of the oxygen; Free radicals; Oxidative stress; Polyamines

An ensemble of reduced alphabets with protein encoding based on grouped weight for predicting DNA-binding proteins by Loris Nanni; Alessandra Lumini (pp. 167-175).

It is well known in the literature that an ensemble of classifiers obtains good performance with respect to that obtained by a stand-alone method. Hence, it is very important to develop ensemble methods well suited for bioinformatics data. In this work, we propose to combine the feature extraction method based on grouped weight with a set of amino-acid alphabets obtained by a Genetic Algorithm. The proposed method is applied for predicting DNA-binding proteins. As classifiers, the linear support vector machine and the radial basis function support vector machine are tested. As performance indicators, the accuracy and Matthews’s correlation coefficient are reported. Matthews’s correlation coefficient obtained by our ensemble method is ≈0.97 when the jackknife cross-validation is used. This result outperforms the performance obtained in the literature using the same dataset where the features are extracted directly from the amino-acid sequence.

Keywords: Multi-classifier; Amino-acid alphabets; Support vector machine; DNA-binding proteins; Ensemble classifier

How does fish metamorphosis affect aromatic amino acid metabolism? by Wilson Pinto; Luís Figueira; Maria Teresa Dinis; Cláudia Aragão (pp. 177-183).

Aromatic amino acids (AAs, phenylalanine and tyrosine) may be specifically required during fish metamorphosis, since they are the precursors of thyroid hormones which regulate this process. This project attempted to evaluate aromatic AA metabolism during the ontogenesis of fish species with a marked (Senegalese sole; Solea senegalensis) and a less accentuated metamorphosis (gilthead seabream; Sparus aurata). Fish were tube-fed with three l-[U-¹⁴C] AA solutions at pre-metamorphic, metamorphic and post-metamorphic stages of development: controlled AA mixture (Mix), phenylalanine (Phe) and tyrosine (Tyr). Results showed a preferential aromatic AA retention during the metamorphosis of Senegalese sole, rather than in gilthead seabream. Senegalese sole’s highly accentuated metamorphosis seems to increase aromatic AA physiological requirements, possibly for thyroid hormone production. Thus, Senegalese sole seems to be especially susceptible to dietary aromatic AA deficiencies during the metamorphosis period, and these findings may be important for physiologists, fish nutritionists and the flatfish aquaculture industry.

Keywords: Solea senegalensis ; Sparus aurata ; Metamorphosis; Aromatic amino acids; Amino acid metabolism; Tyrosine

Synthesis, spectroscopic and structural elucidation of sympathomimetic amine, tyraminium dihydrogenphosphate by Tsonko M. Kolev; Bojidarka B. Koleva; Michael Spiteller; William S. Sheldrick; Heike Mayer-Figge (pp. 185-193).

The synthesis, isolation, spectroscopic and structural elucidation of sympathomimetic amine, tyramine dihydrogenphosphate are of interest due to its biological activity and the establishing correlation between spectroscopic properties and structure. The complex approach for investigation included single crystal X-ray diffraction, new technique in linear-polarized IR-spectroscopy in solid state and quantum chemical calculations with a view to predict the electronic structure and vibrational data of interacting species in entitled compound, the correlation structure–spectroscopic properties as well as the influence of intermolecular interaction on IR-characteristic bands are carried out.

Keywords: Tyramine dihydrogenphosphate; Single crystal X-ray diffraction; Solid-state IR-LD spectroscopy; Quantum chemical calculations; Vibrational analysis

Synthesis, spectroscopic and structural elucidation of tyrosinamide hydrogensquarate monohydrate by Tsonko M. Kolev; Bojidarka B. Koleva; Michael Spiteller; William S. Sheldrick; Heike Mayer-Figge (pp. 195-201).

Synthesis, isolation, spectroscopic and structural elucidation of tyrosinamide hydrogensquarate monohydrate (I) is reported on the basis of quantum chemical DFT calculations, vibrational analysis and experimental linear-polarized IR-spectroscopy in solid state. These data are compared with those obtained using single crystal X-ray diffraction, which show that the molecules of (I) in the unit cell formed 3D network through moderate intermolecular _(Tyr)OH···O = C_(Sq) (2.727 Å), O=C–NH₂···OH_(Tyr) (2.991 Å), O=C–NH₂···OH_(Sq) (2.988 Å), O=C–NH₂···O=C–NH₂ (3.068 Å), N⁺H₃···O=C_(Sq) (2.737, 2.953, 2.954 Å), OH₂···O=C_(Sq) (2.839 Å) and _(Sq)OH···OH₂ (2.607 Å) hydrogen bonds. The relationship between the structure and spectroscopic properties is studied.

Keywords: Tyrosinamide hydrogensquarate monohydrate; IR-LD spectroscopy; DFT calculations; Vibrational analysis; Single crystal X-ray diffraction; FAB-MS

Solid phase and solution synthesis of NvocLys(CO(CH₂)₅NH–NBD)OCH₂CN, a trifunctional fluorescent lysine derivative by Kshitij A. Patkar; W. Edward Highsmith; Jane V. Aldrich (pp. 203-207).

Herein, we describe a general strategy for the facile synthesis of a multifunctional amino acid derivative bearing both fluorescent and photolabile groups such as the lysine derivative NvocLys(CO(CH₂)₅NH–NBD)OCH₂CN (1) that can be used as a biophysical tool for studying protein structure. The synthetic strategy involves functionalization of the amine groups while the amino acid is attached to a solid support, followed by esterification of the carboxylic acid in solution. The solid support protects the caboxylic acid, preventing a side reaction associated with the synthesis in solution and obviating the need for chromatographic purification of several intermediates. This synthetic strategy can be used for the preparation of a variety of amino acid derivatives with unusual α-amine and side chain functionalities.

Keywords: Fluorescent amino acid; NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl); Photolabile protecting group; Nvoc (nitroveratryloxycarbonyl); Lysine; Solid phase synthesis

Plasma catecholamine and nephrine responses to brief intermittent maximal intensity exercise by Richard M. Bracken; Denise M. Linnane; Stephen Brooks (pp. 209-217).

Catecholamines (noradrenaline, NA; adrenaline, AD; dopamine, DA) influence the metabolic and cardiovascular responses to exercise. However, changes in catecholamine metabolism during exercise are unclear. Plasma normetanephrine (NMET), metanephrine (MET) and catecholamine responses to a laboratory-based model of games-type exercise were examined. Twelve healthy men completed a resting control trial and a trial consisting of ten 6 s cycle ergometer sprints interspersed with 30 s recovery, in randomised order. Resting and post-sprint venous blood samples were taken. Plasma NA and AD increased after each sprint but DA was unaltered. Plasma nephrines increased significantly from sprint 4 onwards with peak NMET increasing 60% to 0.76 ± 0.19 nmol l⁻¹ and MET 230% to 0.37 ± 0.16 nmol l⁻¹ from resting values (P < 0.05). The results demonstrate increased catecholamine metabolism via elevated catechol-O-methyl transferase activity during intermittent sprinting. The results may aid regulation of the metabolic and cardiovascular responses to exercise by maintaining tissue adrenoceptor sensitivity to circulating catecholamines.

Keywords: Metanephrine; Normetanephrine; Dopamine; Intermittent exercise

Regulatory mechanisms of SNAT2, an amino acid transporter, in L6 rat skeletal muscle cells by insulin, osmotic shock and amino acid deprivation by Hitoshi Kashiwagi; Kojiro Yamazaki; Yoh Takekuma; Vadivel Ganapathy; Mitsuru Sugawara (pp. 219-230).

Several studies have demonstrated that the activity of system A is upregulated by insulin, osmotic shock and amino acid deprivation. However, the mechanisms are not clear. We carried out studies using L6 rat skeletal muscle cells to clarify the mechanisms of upregulation of system A activity by insulin, osmotic shock and amino acid deprivation. The upregulation was found to be due to an increase in V _max, not K _m. Chloroquine and wortmannin inhibited the upregulation induced by insulin stimulation and amino acid deprivation but not that induced by osmotic shock. On the other hand, cycloheximide and actinomycin D inhibited the upregulation by each stimulation. Moreover, PD98059 and SP600125 inhibited only amino acid deprivation-induced upregulation and SB202190 inhibited only insulin-induced upregulation. Our findings indicate that the mechanisms of upregulation of system A activity by insulin, osmotic shock and amino acid deprivation are different in L6 cells. Western blot and RT-PCR analysis showed an increase in system A at the protein and mRNA levels with each stimulation.

Keywords: SNAT2; Insulin stimulation; Osmotic shock; Amino acid deprivation; Transporter recruitment; de novo protein synthesis

Biosynthesis of ¹⁵NL-phenylalanine by phenylalanine ammonia-lyase from Rhodotorula glutinis by Wenya Wang; Haiyan Yue; Qipeng Yuan; Wenchuan Wang (pp. 231-233).

Catalyzed by phenylalanine ammonia-lyase from Rhodotorula glutinis, 2% trans-cinnamic acid and 0.5 mol/l (¹⁵NH₄)₂SO₄ was bioconverted to ¹⁵NL-phenylalanine. The yield and the purity of ¹⁵NL-phenylalanine reached 71 and 99.3%, respectively. The results showed that 96% of ¹⁵N was labeled on the l-phenylalanine and 88% of (¹⁵NH₄)₂SO₄ was recovered. The present paper provides a new and economic way for biosynthesis of ¹⁵NL-phenylalanine.

Keywords: ¹⁵NL-phenylalanine; Phenylalanine ammonia-lyase; (¹⁵NH₄)₂SO₄ ; Rhodotorula glutinis

Searching distant homologs of the regulatory ACT domain in phenylalanine hydroxylase by Jessica Siltberg-Liberles; Aurora Martinez (pp. 235-249).

High sequence divergence, evolutionary mobility, and superfold topology characterize the ACT domain. Frequently found in multidomain proteins, these domains induce allosteric effects by binding a regulatory ligand usually to an ACT domain dimer interface. In mammalian phenylalanine hydroxylase (PAH), no contacts are formed between ACT domains, and the domain promotes an allosteric effect despite the apparent lack of ligand binding. The increased functional scenario of this abundant domain encouraged us to search for distant homologs, aiming to enhance the understanding of the ACT domain in general and the ACT domain of PAH in particular. The PDB was searched using the FATCAT server with the ACT domain of PAH as a query. The hits that were confirmed by the SSAP algorithm were divided into known ACT domains (KADs) and potential ACT domains (PADs). The FATCAT/SSAP procedure recognized most of the established KADs, as well 18 so far unrecognized non-redundant PADs with extremely low sequence identities and high divergence in functionality and oligomerization. However, analysis of the structural similarity provides remarkable clustering of the proteins according to similarities in ligand binding. Despite enormous sequence divergence and high functional variability, there is a common regulatory theme among these domains. The results reveal the close relationships of the ACT domain of PAH with amino acid binding and metallobinding ACT domains and with acylphosphatase.

Keywords: ACT domain; Aromatic amino acid hydroxylases; Phenylalanine hydroxylase; Structural homology; Sequence divergence

Non-covalent binding of azo compound to peptide chain: interactions of biebrich scarlet and naphthochrome green with four model proteins by Hong-Wen Gao; Xiang-Hu Liu; Zhi Qiu; Lu Tan (pp. 251-260).

We studied the non-specific interactions of two azo compounds: biebrich scarlet (BS) and naphthochrome green (NG), with four model proteins: bovine serum albumin, ovalbumin, poly-l-lysine and hemoglobin by UV-VIS spectrometry, fluorophotometry and circular dichroism melting technique. The optimal acidities of NG and BS for binding to proteins correspond to the physiological pHs of skin and gastro tissues. The saturation binding numbers of BS and NG on peptide chains were determined and the effects of electrolytes and temperature were investigated. These interactions were fitted by the Temkin absorption model and their thermodynamic parameters were calculated. The different bindings of BS and NG to proteins were compared from their molecular structures. We inferred that an ion-pair electrostatic interaction first fixes azo compounds to basic amino acid residues and subsequent binding involves the collective action of other non-covalent bonds: hydrogen bond, van der Waals force, and hydrophobic interaction. This combination of bonds caused a change of secondary conformation of protein from β-sheet to helix and the possible process was illustrated. The potential protein toxicity resulting from such a non-specific binding was analyzed. Besides, the interaction of BS with peptide chains was applied to protein assay.

Keywords: Protein–ligand interaction; Non-covalent binding; Azo compound; Conformational change; Protein toxicity; Molecular spectrometry

Organisms can essentially be classified according to two codon patterns by T. Okayasu; K. Sorimachi (pp. 261-271).

We recently classified 23 bacteria into two types based on their complete genomes; “S-type” as represented by Staphylococcus aureus and “E-type” as represented by Escherichia coli. Classification was characterized by concentrations of Arg, Ala or Lys in the amino acid composition calculated from the complete genome. Based on these previous classifications, not only prokaryotic but also eukaryotic genome structures were investigated by amino acid compositions and nucleotide contents. Organisms consisting of 112 bacteria, 15 archaea and 18 eukaryotes were classified into two major groups by cluster analysis using GC contents at the three codon positions calculated from complete genomes. The 145 organisms were classified into “AT-type” and “GC-type” represented by high A or T (low G or C) and high G or C (low A or T) contents, respectively, at every third codon position. Reciprocal changes between G or C and A or T contents at the third codon position occurred almost synchronously in every codon among the organisms. Correlations between amino acid concentrations (Ala, Ile and Lys) and the nucleotide contents at the codon position were obtained in both “AT-type” and “GC-type” organisms, but with different regression coefficients. In certain correlations of amino acid concentrations with GC contents, eukaryotes, archaea and bacteria showed different behaviors; thus these kingdoms evolved differently. All organisms are basically classifiable into two groups having characteristic codon patterns; organisms with low GC and high AT contents at the third codon position and their derivatives, and organisms with an inverse relationship.

Keywords: Classification; Prokaryotes; Eukaryotes; Amino acid compositions; Codon usage; GC content; Cluster analysis

Polyamine synthesis inhibition induces S phase cell cycle arrest in vascular smooth muscle cells by M. Odenlund; B. Holmqvist; B. Baldetorp; P. Hellstrand; Bengt-Olof Nilsson (pp. 273-282).

Polyamines are important for cell growth and proliferation and they are formed from arginine and ornithine via arginase and ornithine decarboxylase (ODC). Arginine may alternatively be metabolised to NO via NO synthase. Here we study if vascular smooth muscle cell proliferation can be reversed by polyamine synthesis inhibitors and investigate their mechanism of action. Cell proliferation was assessed in cultured vascular smooth muscle A7r5 cells and in endothelium-denuded rat arterial rings by measuring [³H]-thymidine incorporation and by cell counting. Cell cycle phase distribution was determined by flow cytometry and polyamines by HPLC. Protein expression was determined by Western blotting. The ODC inhibitor DFMO (1–10 mM) reduced polyamine concentration and attenuated proliferation in A7r5 cells and rat tail artery. DFMO accumulated cells in S phase of the cell cycle and reduced cyclin A expression. DFMO had no effect on cell viability and apoptosis as assessed by fluorescence microscopy. Polyamine concentration and cellular proliferation were not affected by the arginase inhibitor NOHA (100–200 μM) and the NO synthase inhibitor l-NAME (100 μM). Lack of effect of NOHA was reflected by absence of arginase expression. Polyamine synthesis inhibition attenuates vascular smooth muscle cell proliferation by reducing DNA synthesis and accumulation of cells in S phase, and may be a useful approach to prevent vascular smooth muscle cell proliferation in cardiovascular diseases.

Keywords: Arginase; Cell cycle distribution; DNA synthesis; Ornitihine decarboxylase; Polyamines; Vascular smooth muscle cells

Systematic comparison of two novel, thiol-reactive prosthetic groups for ¹⁸F labeling of peptides and proteins with the acylation agent succinimidyl-4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB) by Frank Wuest; Lena Köhler; Mathias Berndt; Jens Pietzsch (pp. 283-295).

A systematic comparison of 4-[¹⁸F]fluorobenzaldehyde-O-(2-{2-[2-(pyrrol-2,5-dione-1-yl)ethoxy]-ethoxy}-ethyl)oxime ([¹⁸F]FBOM) and 4-[¹⁸F]fluorobenzaldehyde-O-[6-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-hexyl]oxime ([¹⁸F]FBAM) as prosthetic groups for the mild and efficient ¹⁸F labeling of cysteine-containing peptides and proteins with the amine-group reactive acylation agent, succinimidyl-4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB), is described. All three prosthetic groups were prepared in a remotely controlled synthesis module. Synthesis of [¹⁸F]FBOM and [¹⁸F]FBAM was accomplished via oxime formation through reaction of appropriate aminooxy-functionalized labeling precursors with 4-[¹⁸F]fluorobenzaldehyde. The obtained radiochemical yields were 19% ([¹⁸F]FBOM) and 29% ([¹⁸F]FBAM), respectively. Radiolabeling involving [¹⁸F]FBAM and [¹⁸F]FBOM was exemplified by the reaction with cysteine-containing tripeptide glutathione (GSH), a cysteine-containing dimeric neurotensin derivative, and human native low-density lipoprotein (nLDL) as model compounds. Radiolabeling with the acylation agent [¹⁸F]SFB was carried out using a dimeric neurotensin derivative and nLDL. Both thiol-group reactive prosthetic groups show significantly better labeling efficiencies for the peptides in comparison with the acylation agent [¹⁸F]SFB. The obtained results demonstrate that [¹⁸F]FBOM is especially suited for the labeling of hydrophilic cysteine-containing peptides, whereas [¹⁸F]FBAM shows superior labeling performance for higher molecular weight compounds as exemplified for nLDL apolipoprotein constituents. However, the acylation agent [¹⁸F]SFB is the preferred prosthetic group for labeling nLDL under physiological conditions.

Keywords: ¹⁸F-labeled prosthetic groups; Peptides; LDL; Apolipoproteins; Positron emission tomography (PET)

Tuberin, p27 and mTOR in different cells by S. Burgstaller; M. Rosner; C. Lindengrün; M. Hanneder; N. Siegel; A. Valli; C. Fuchs; M. Hengstschläger (pp. 297-302).

Mutations in the genes TSC1 or TSC2 cause the autosomal dominantly inherited tumor suppressor syndrome tuberous sclerosis, which is characterized by the development of tumors, named hamartomas, in different organs. The TSC gene products, hamartin and tuberin, form a complex, of which tuberin is assumed to be the functional component. Both, hamartin and tuberin have been implicated in the control of the cell cycle by activating the cyclin-dependent kinase inhibitor p27 and in cell size regulation by inhibiting the mammalian target of rapamycin (mTOR) a regulator of the p70 ribosomal protein S6 kinase (p70S6K) and its target the ribosomal protein S6. The tuberin/hamartin complex was shown to protect p27 from protein degradation. Within the mTOR signaling pathway tuberin harbors GTPase activating (GAP) potential toward Rheb, which is a potent regulator of mTOR. In this study, we have analyzed the protein levels of tuberin, p27, cyclin D1, mTOR and phospho mTOR Ser2448 (activated mTOR), S6 and phospho S6 Ser240/244 (activated S6) and as controls α-tubulin and topoisomerase IIβ, in ten different cells, including primary normal cells, immortalized and transformed cell lines.

Keywords: Tuberin; p27; mTOR; S6

Changes in free amino acids in the brain during embryonic development in layer and broiler chickens by M. Sato; S. Tomonaga; D. M. Denbow; M. Furuse (pp. 303-308).

Developmental changes in the levels of the excitatory amino acids l-glutamate (Glu) and l-Aspartate (Asp) and inhibitory amino acids glycine (Gly) and γ-amino butyric acid (GABA), as well as taurine and its related amino acids l-methionine (Met), l-cysteine (Cys) and l-serine (Ser) in the brain and pectoralis muscle at various embryonic stages and hatch in broiler and layer type chickens were determined. Brain concentrations of Asp, GABA and taurine were higher than those in the muscle, but the difference in the two types was small. The concentrations of the precursors of taurine including Met, Cys and Ser were lower than that of taurine. In conclusion, the synthesis of some amino acids and their metabolites such as Asp, GABA and taurine in the chick embryo is very high in order to support brain development.

Keywords: Brain; Chicken; Embryo; Free amino acid; Skeletal muscle; Taurine

Solid phase synthesis of peptides containing novel amino acids, substituted 3-benzimidazolealanines by Małgorzata Koprowska-Ratajska; Alicja Kluczyk; Piotr Stefanowicz; Hubert Bartosz-Bechowski; Zbigniew Szewczuk (pp. 309-315).

A direct solid-phase synthesis of a series of substituted benzimidazole-containing peptides is described. The method involves on-resin formation of new amino acids containing benzimidazole derivatives in the side chain. The heterocycle conjugates were obtained by reaction between aldehydes and peptides containing β-(3,4-diaminophenyl)alanine residue, immobilized on a polymeric solid support.

Keywords: Solid phase peptide synthesis; Unnatural amino acids; 4-Amino-3-nitrophenylalanine; Benzimidazoles; Combinatorial synthesis

Determination of N ^ɛ-(carboxymethyl)lysine in food systems by ultra performance liquid chromatography-mass spectrometry by Shima H. Assar; Catherine Moloney; Maria Lima; Ronald Magee; Jennifer M. Ames (pp. 317-326).

We report the use of ultra pressure liquid chromatography (UPLC), coupled to a tandem mass spectrometer operated in multiple reaction monitoring mode to determine the advanced glycation endproduct, N ^ɛ-(carboxymethyl)lysine (CML). The procedure was applied to acid hydrolyzates of protein isolated from a range of foods (milks processed at different temperatures, butter, cheese, infant formulae, bread, raw and cooked minced beef and olive oil). Highest levels of CML were determined in white bread crust (15.2 ± 0.63 mmol/mol Lys), wholemeal bread crust (13.1 ± 0.61 mmol/mol Lys) and evaporated full-fat milk (4.86 ± 0.77 mmol/mol Lys). Lowest levels of CML were measured in raw minced beef beef (0.03 ± 002 mmol/mol Lys), raw full-fat cow’s milk (0.08 ± 0.03 mmol/mol Lys) and pasteurized skimmed cow’s milk (0.09 ± 0.002 mmol/mol Lys). CML could not be detected in olive oil.

Keywords: N ^ɛ-(carboxymethyl)lysine; Maillard reaction; Advanced glycation endproducts; Ultra performance liquid chromatography-mass spectrometry; Dietary advanced glycation endproducts

Hypotransferrinemia and changes in plasma lipid and metabolic patterns in sepsis by Carlo Chiarla; Ivo Giovannini; John H. Siegel (pp. 327-331).

This study was performed to obtain a characterization of the changes in plasma transferrin (Tf, g/L) in sepsis. More than four hundred determinations of Tf, and of a large series of simultaneously collected blood and hemodynamic variables, were obtained in 17 patients with post-traumatic sepsis. Tf during sepsis was consistently low (mean ± SD = 1.46 ± 0.46) however fluctuated markedly according to changes in metabolic and hemodynamic patterns. Regression analysis showed that decreases in Tf were simultaneously correlated with the plasma lipid pattern (in particular with decreasing cholesterol and increasing triglycerides), with decreases in albumin and peripheral O₂ extraction, and with increasing cardiac index (p < 0.001 for all). Decreases in Tf were moderated by increasing the parenteral amino acid dose (p < 0.001). Combinations of these variables in multiple regressions explained nearly 80% of the variability of Tf. There were no similar correlations for other acute phase proteins except ceruloplasmin, which showed opposite changes compared to those of Tf. These results show that within the hypotransferrinemia which characterizes sepsis, Tf may oscillate remaining strongly correlated with changes in metabolic and hemodynamic patterns, which may account for nearly 80% of the variability of Tf.

Keywords: Sepsis; Plasma transferrin; Hypocholesterolemia; Hypertriglyceridemia; Peripheral oxygen extraction; Ceruloplasmin; Hyperdynamic cardiovascular state

Protease-catalysed coupling of N-protected amino acids and peptides with 4-aminoantipyrine by Alexander Lang; Catharina Hatscher; Charline Wiegert; Peter Kuhl (pp. 333-340).

The enzymatic synthesis of N-protected l-aminoacyl- and l-peptidyl-antipyrine amides was accomplished by proteases from different classes. Serine and cysteine proteases proved to be suitable tools for the production of amino acids and peptides conjugated to 4-aminoantipyrine, whereas metalloproteases do not seem to be very qualified for accepting this nucleophile. The product yields were optimised by applying ample opportunities of medium engineering, e.g. aqueous-organic, biphasic, suspension and solid-to-solid reaction systems. Thus, yields up to 100% could be obtained. The products were purified and characterised by polarimetry and NMR spectroscopy. These results broaden the common knowledge of the catalytic potential of proteases, in particular with regard to the suitability of a special heterocyclic 1,2-amino ketone as a nucleophile for the biocatalytic amidation of amino acids and peptides.

Keywords: Amidation; Amino acids; 4-aminoantipyrine; Enzymatic synthesis; Peptides; Proteases

Process improvement in amino acid N-carboxyanhydride synthesis by N-carbamoyl amino acid nitrosation by Olivier Lagrille; Grégoire Danger; Laurent Boiteau; Jean-Christophe Rossi; Jacques Taillades (pp. 341-347).

Amino acid N-carboxyanhydrides (NCA), convenient monomer for polypeptide synthesis, are easily prepared in high purity as the result of N-carbamoyl amino acids (CAA) nitrosation by gaseous NOx (4:1 NO + O₂ mixture, or NOCl) in toluene. Removal of polar side products is then efficiently carried out during subsequent work-up and crystallisation so that the resulting NCA obtained in good yield is suitable for controlled, primary amine-initiated polymerisation.

Keywords: N-carboxyanhydrides; Nitrosation; Polypeptides; N-carbamoylamino acids

Mice transgenic for reduced folate carrier: an animal model of Down syndrome? by Joachim Höger; David Patterson; Harald Höger; Ki-Shuk Shim; Hermann Bubna-Littitz; Gert Lubec (pp. 349-357).

In a previous publication we observed aberrant levels of the human reduced folate carrier (hRFC) in cortex from fetal Down syndrome (DS) subjects. Immunoreactivity for hRFC was increased as the only chromosome 21 gene product studied. We, therefore, analyzed mice transgenic for hRFC (TghRFC1) and wild-type (WT) mice for cognitive functions, behavior and in an observational neurological battery (FOB). Cognitive functions were evaluated by the Morris water maze (MWM), the open field (OF) was used for exploratory behavior, locomotor activity and anxiety-related behavior. The elevated plus maze (EPM) was used to confirm findings in the OF testing anxiety-related behavior and the rota rod (RR) to evaluate motor function. In the MWM TghRFC1 mice performed significantly worse (P < 0.0003) on the probe trial than WT mice. In the FOB visual placing was significantly reduced inTghRFC1 mice. In the OF TghRFC1 mice crossed twice as often (P < 0.029) and in the EPM individuals from this group showed a reduced number of exits from the closed arm (P < 0.044) compared to WT mice. TghRFC1 mice showed impaired performance on the RR, spending one-fourth of the time of WT on the revolving rod (P < 0.0003). Cognitive impairment is an obligatory symptom of DS and this deficiency corresponds to findings in the MWM of mice transgenic for hRFC. Findings of visual placing and failure on the RR may reflect impaired motor performance including muscular hypotonia in DS subjects. Increased crossings in the OF may indicate modulated anxiety-related behavior observed in patients with DS.

Keywords: Down syndrome; Mouse; Reduced folate carrier

Comparison of the carnosine and taurine contents of vastus lateralis of elderly Korean males, with impaired glucose tolerance, and young elite Korean swimmers by Hyo-Jeong Kim (pp. 359-363).

The carnosine and taurine contents of the vastus lateralis of two diverse groups of Korean male subjects (elderly and impaired glucose-tolerant (IGT) subjects and young elite swimmers at a national sport university) having a similar national diet, were examined. Despite marked differences in age, fitness and clinical status the two groups showed almost identical muscle carnosine and taurine contents. In the case of carnosine, the results suggest a similar contribution to intracellular buffering capacity in the two groups of subjects, with no evidence of a reduction of this in elderly IGT subjects. In addition, both groups showed the same inverse relationship between the muscle carnosine and taurine contents; the spread of values between subjects, within-groups, most likely reflect variations in the type I (low carnosine, high taurine) or type II (high carnosine, low taurine) composition of the vastus lateralis. The relationship is consistent with a role of taurine in osmoregulation, compensating for variations between fibre types in the carnosine content.

Keywords: Carnosine; Taurine; Korean males; Swimmers; Impaired glucose tolerance; Ageing

Induction of apoptosis by l-carnitine through regulation of two main pathways in Hepa1c1c 7 cells by Jiang Ping Fan; Han Seop Kim; Gi Dong Han (pp. 365-372).

This study shows the effects of l-carnitine treatment on cell proliferation with hepa1c1c7 mouse cancer cells and NCTC 1469 normal cells. In an MTT assay, l-carnitine increased the number of dead hepa1c1c7 cells, while there was no difference in the number of NCTC 1469 cells. mRNA and protein levels of TNF-α, Fas, and caspase-8, which are closely related to cell apoptosis by a death ligand/receptor-dependent apoptosis pathway, were increased by l-carnitine treatment. In addition, l-carnitine treatment regulated mitochondria-dependent apoptosis pathways by inducing the up-regulation of caspase-9 and caspase-3 and the down-regulation of Bcl-2 in hepa1c1c 7 cells. Taken together, the findings of this study have demonstrated that l-carnitine could induce apoptosis in hepa1c1c7 cells by regulating Fas ligands and inhibiting the expression of Bcl-2. These results suggest that l-carnitine treatment could be related to both a mitochondrion-dependent and a death ligand/receptor-dependent apoptosis pathway in hepa1c1c7 cells. Our results could give information for understanding the l-carnitine-induced apoptosis mechanism in some cancer cells.

Keywords: l-carnitine; Apoptosis; Hepa1c1c 7 cell; TNF-α; Bcl-2; Caspase-3

The protonation equilibria of selected glycine dipeptides in ethanol–water mixture: solvent composition effect by Alev Doğan; Ayça Demirel Özel; Esma Kılıç (pp. 373-379).

Knowledge of the protonation constants of small dipeptide is important, interesting and necessary for complete understanding of the physiochemical behavior of dipeptide. In this study, the protonation constants of some aliphatic dipeptides (Gly–Gly, Gly–Val, Gly–Leu, Gly–Thr, Gly–Phe and Gly–Met) were studied in water and ethanol–water mixtures [20% ethanol–80% water, 40% ethanol–60% water, 60% ethanol–40% water, (v/v)] at 25 ± 0.1°C under nitrogen atmosphere and ionic strength at 0.10 mol dm⁻³ by potentiometry. The constants of the systems were calculated by using BEST computer program, and distribution species diagrams were produced using the SPE computer program. The protonation constants were influenced by changes in solvent composition, and their variations were discussed in terms of solvent and structural properties. The concentration distribution of the various species in ethanol–water mixtures was evaluated.

Keywords: Dipeptides; Protonation constants; Ethanol–water mixture; Potentiometry

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.36, #2)

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.36, #2)