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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.27, #1)

Needle in a haystack: microdissecting the proteome of a tissue by H. J. Ball; N. H. Hunt (pp. 1-7).

Laser-assisted microdissection is a recent technology that enables cells to be harvested from tissue sections. Proteins can be extracted from the dissected cells for molecular analysis. This enables the analysis of proteins in specific cell types in an in vivo system. Although quantities of protein obtained from the dissected material can be small, it is possible to use established methods such as Western Blotting and 2D-PAGE, as well as newer technologies such as SELDI-MS, to analyse the proteins. This review describes the applications and technical considerations for using laser-assisted dissected cells in proteomics research.

Keywords: Keywords: Laser capture microdissection – Laser-assisted microdissection – Proteomics

N-Terminal pyrazinones: a new class of peptide-bound advanced glycation end-products by R. Krause; J. Kühn; I. Penndorf; K. Knoll; T. Henle (pp. 9-18).

The reaction of peptide Gly-Ala-Phe with the α-dicarbonyl compounds glyoxal and methylglyoxal was studied under physiological conditions (pH=7.4, 37°C). Using HPLC with UV and fluorescence detection, a rapid derivatization of the peptide and the concomitant formation of well-defined products were observed. The products, which showed characteristic UV absorbance (λ_max=320 to 340 nm) and fluorescence (λ_ex=330 to 340 nm, λ_em=395 to 405 nm), were identified by ESI-MS and NMR spectroscopic analysis as the N-terminally pyrazinone-modified peptides I (N-[2-(2-oxo-2H-pyrazin-1-yl)-propyl]-phenylalanine) and II (N-[2-(5-methyl-2-oxo-2H-pyrazin-1-yl)-propionyl]-phenylalanine). Model experiments revealed that the reactivity of the N-termini of peptides towards a derivatization by glyoxal is in the same order of magnitude as that of arginine, which generally is attributed as main target for α-dicarbonyl compounds in proteins. Incubation of insulin with glyoxal proved the protein-bound formation of pyrazinones, with the N-terminus of the B-chain as the main target. According to these results, we conclude that N-terminal pyrazinones represent a new type of advanced glycation end-products (AGEs) with significance for biological systems and foods.

Keywords: Keywords: Glycation – Maillard reaction – Pyrazinone – Advanced glycation end-product (AGE) – Glyoxal – Methylglyoxal – Insulin

Synthesis and application of methyleneoxy pseudodipeptide building blocks in biologically active peptides by J. Hlaváček; J. Mařík; J. Konvalinka; B. Bennettová; R. Tykva (pp. 19-27).

Pseudodipeptides H-Pheψ[CH₂O]Phe-OH, H-Tyrψ[CH₂O] Asp-OH and H-Proψ[CH₂O]-D-Thr-OH were synthesized using the intramolecular Williamson reaction via substituted morpholin-3-one ring with the nitrogen atom protected with bulky Boc group. This protection and the substituent at C₅ position induced the stereospecific alkylation at the C₂ position introducing the side chain of the C-terminal amino acid mimetic. In the first pseudodipeptide a quenching of the enolate with benzaldehyde was followed by dehydration and corresponding double bond was hydrogenated with high stereospecific purity. In the other pseudodipeptides, this alkylation was carried out directly by tert-butyl 2-bromoacetate or acetaldehyde. However, in the latter reaction an R configuration of C₃ substituent in conjugated lactame ring was determined using a NOE NMR. Consequently, after opening this ring by acidic hydrolysis, the C-terminal part of corresponding pseudodipeptide possessed the side-chain of D-Thr mimetic, contrary to former one. Synthesized pseudodipeptides were introduced into HIV protease inhibitors and into peptides with oostatic activity.

Keywords: Keywords: Methyleneoxy isoster–HIV protease inhibitor–Oostatic activity–Peptide synthesis

Taurine chloramine and taurine bromamine induce heme oxygenase-1 in resting and LPS-stimulated J774.2 macrophages by R. Olszanecki; J. Marcinkiewicz (pp. 29-35).

Taurine chloramine (TauCl) and taurine bromamine (TauBr) are products of activated neutrophils and eosinophils, respectively. It has been reported that TauCl, has strong anti-inflammatory properties. In a number of separate studies it has been shown that heme oxygenase-1 (HO-1), a stress inducible protein, exerts similar anti-inflammatory effects. In this study we investigated the influence of HO-1 on TauCl/TauBr mediated suppression of NO generation in J774.2 macrophages. Expression of HO-1 and inducible nitric oxide synthase (NOS-2) in LPS stimulated J774.2 cells provides an opportunity for determining these interactions. TauCl and TauBr, at non-cytotoxic concentrations, in a similar, dose-dependent manner, inhibited the expression of NOS-2, as evidenced by western blotting technique. Surprisingly, TauCl and TauBr induced expression of HO-1 in both non-activated and LPS-activated macrophages. Importantly, the fall in NOS-2 protein level was associated with a concomitant, dose-dependent induction of HO-1. In addition, an inhibitor of HO-1 activity, chromium III mesoporhyrin (CrMP), attenuated the inhibitory activity of TauBr but not that of TauCl, as measured by nitrite accumulation. These results suggest that at a site of inflammation, TauCl and TauBr may provide a link between taurine-dependent and HO-1-dependent cytoprotective mechanisms.

Keywords: Keywords: Taurine chloramine – N-Chlorotaurine – Taurine bromamine – Heme oxygenase – Macrophages – Nitric oxide

Taurine protected myocardial mitochondria injury induced by hyperhomocysteinemia in rats by L. Chang; J. Xu; F. Yu; J. Zhao; X. Tang; C. Tang (pp. 37-48).

Taurine can protect against cardiovascular diseases, whereas elevated levels of plasma homocysteine are associated with atherosclerotic and thromboembolic cardiovascular diseases. To illustrate the effects of taurine on hyperhomocysteinemia, we observed the myocardial mitochondria dysfunction in the rats with hyperhomocysteinemia induced by diet methionine loading, and the therapeutic effect of taurine. A methionine diet increased plasma homocysteine concentration (133.51 ± 27.91 μmol/L vs 12.31 ± 2.58 μmol/L in control, P < 0.01), stimulated the production of reactive oxygen species (ROS) in the myocardial mitochondria, and inhibited the activities of mitochondrial Mn-superoxide dismutase and catalase. The ⁴⁵Ca uptake and Ca²⁺-ATPase activity in the myocardial mitochondria were significantly lowered in rats with hyperhomocysteinemia. Taurine supplements effectively attenuated the hyperhomocysteinemia-induced ROS production and inhibition of Mn-superoxide dismutase and catalase activities in the myocardial mitochondria, and increased its ⁴⁵Ca uptake and Ca²⁺-ATPase activity. Thus, taurine antagonizes the oxidative stress injury in the myocardial mitochondria induced by the hyperhomocysteinemia.

Keywords: Keywords: Hyperhomocysteine – Taurine – Heart – Mitochondria – Rat

Use of capillary (zone) electrophoresis for determining felinine and it’s application to investigate the stability of felinine by S. M. Rutherfurd; F. Zhang; D. R. K. Harding; A. D. Woolhouse; W. H. Hendriks (pp. 49-55).

A rapid capillary electrophoresis method was established to quantify felinine (2-amino-7-hydroxy-5,5-dimethyl-4-thiaheptanoic acid) in cat urine and used to investigate felinine stability. Synthetic felinine was stable in the presence of oxygen while 11% of the natural felinine in urine disappeared after 4 days exposure to air. Both synthetic felinine and the natural felinine (in urine) were stable for up to 3 months when stored at −5°C and 20°C. Thirty percent of the synthetic felinine was lost after 5 hours at 100°C while 95% of the natural felinine disappeared after only 2 hours at the same temperature. The recovery of felinine under certain conditions was greater than 100%. It is possible that acetyl-felinine may be present in the urine and that it is deacetylated during incubation. Overall synthetic felinine was found to be stable but the felinine in cat urine much less so. Other compounds present in the urine may contribute to the decomposition of felinine.

Keywords: Keywords: Felinine – Capillary electrophoresis – Cats – Urine

Ischemia and reoxygenation induced amino acid release release and tissue damage in the slices of rat corpus striatum by R. L. Büyükuysal (pp. 57-67).

Ischemic incubation significantly increased amino acid release from rat striatal slices. Reoxygenation (REO) of the ischemic slices, however, enhanced only taurine and citrulline levels in the medium. Ischemia-induced increases in glutamate, taurine and GABA outputs were accompanied with a similar amount of decline in their tissue levels. Tissue final aspartic acid level, however, was doubled by ischemia. Lactate dehydrogenase (LDH) leakage was not altered by ischemia, but enhanced during REO. Presence of tetrodotoxine (TTX) during ischemic period caused significant decline in ischemia-induced glutamate output, but not altered REO-induced LDH leakage. Although omission of extracellular calcium ions from the medium during ischemic period protected the slices against REO-induced LDH leakage, this treatment failed to alter ischemia-induced glutamate and GABA outputs. The release of other amino acids, however, declined 50% in calcium-free medium. Blockade of the glutamate uptake transporter by L-trans-PDC, on the other hand, doubled ischemia induced glutamate and aspartic acid outputs. These results indicate that more than one mechanisms probably support the ischemia-evoked accumulation of glutamate and other amino acids in the extracellular space. Although LDH leakage enhanced during REO, processes involved in this increment were found to be dependent on extracellular calcium ions during ischemia but not REO period.

Keywords: Keywords: Ischemia – Reoxygenation – Glutamate release – Amino acids – LDH

Stability of immobilized D-hydantoinase from Vigna angularis and repeated cycles of highly enantioenriched production of N-carbamoyl-D-phenylglycines by M. B. Arcuri; O. A. C. Antunes; S. P. Machado; C. H. F. Almeida; E. G. Oestreicher (pp. 69-74).

D-hydantoinase from Vigna angularis was immobilized by covalent linkage to aminopropyl glass beads. Thermal stability, resistance to storage at different pH values and temperatures of this biocatalyst were studied. This enzyme preparation was used as a catalyst to prepare enantioenriched N-carbamoyl-D-phenylglycine, N-carbamyl-D-p-fluorophenylglycine and N-carbamoyl-D-p-trifluoromethylphenylglycine, using a stirred batch reactor. Reactions were conducted during eight repeated reaction cycles, without loss of enzymatic activity or variation of the enantiomeric excess of the respective product (>98%).

Keywords: Keywords: D-hydantoinase – Vigna angularis – Biocatalysis – N-carbamoyl-D-amino acids – Immobilized enzyme

Interaction of aluminium ions with some amino acids present in human blood by D. Bohrer; P. C. do Nascimento; J. K. A. Mendonça; V. G. Polli; L. M. de Carvalho (pp. 75-83).

The interaction of aluminium with some amino acids present in human blood was studied combining ion-chromatography (IC), atomic absorption spectrometry (AAS) and ultrafiltration (UF) techniques. An IC system for simultaneous determination of ornithine, lysine, glutamic acid, aspartic acid and tyrosine was developed. By adding aluminium to standard solutions of the amino acids and keeping the pH at 6 and 7 it was possible to verify that aluminium caused a reduction on the amino acid chromatographic signals. Similar experiment, carried out for copper showed the same behaviour (with different percentage of signal reductions) and validated the results for aluminium, considering that the interaction Cu-amino acid is well-established. The AAS analysis of sample fractions (500 μl) after the IC separation showed that aluminium (as copper as well) is not present in the fractions in which the amino acid peaks appear in the chromatogram. These approaches carried out with serum samples after UF showed that part of the “free” fraction of serum aluminium is distributed, besides other ligands, among these amino acids. It was found that in serum the affinity for aluminium followed the sequence Lys>Orn>Tyr>Glu ≈ Asp.

Keywords: Keywords: Aluminium – Serum – HPLC – Metal-binding – Amino acids

The effect of sugar, amino acid, metal ion, and NaCl on model Maillard reaction under pH control by E.-J. Kwak; S.-I. Lim (pp. 85-90).

The color intensities was determined of Maillard reaction products (MRPs) prepared by heating each of five sugars (maltose, fructose, glucose, arabinose, and xylose) with each of 12 amino acids (aspartic acid, glutamic acid, alanine, leucine, isoleucine, valine, proline, serine, cysteine, phenylalanine, arginine, and lysine). The remaining percentages of glucose and rate of change of color intensity due to the addition of a metal ion and NaCl were monitored for nine MRPs that had been formed between glucose and each of nine amino acids (aspartic acid, glutamic acid, alanine, valine, serine, cysteine, phenylalanine, arginine, and lysine). Model MRPs were prepared in a block heater at 100°C for 1–12 h with the pH value controlled at 6.5. The resulting color intensity of each MRPs formed from the basic amino acids was greater due to the higher reactivity than those from the acidic amino acids. The remaining percentage of glucose in each MRPs from the basic amino acids was lower than those from the acidic amino acids. The MRPs from the nonpolar amino acids showed an intermediate color intensity and remaining percentages of glucose between those formed from the basic and acidic amino acids. Browning tended to be accelerated in the presence of metal ions, especially Fe²⁺ and Cu²⁺, although it was affected by the property of the amino acid and heating time as well as by the type of metal ion. On the other hand, browning was greatly inhibited by a high concentration of NaCl.

Keywords: Keywords: Maillard Reaction Products (MRP) – Browning – Melanoidin

Taurine restores ethanol-induced depletion of antioxidants and attenuates oxidative stress in rat tissues by G. Pushpakiran; K. Mahalakshmi; C. V. Anuradha (pp. 91-96).

Ethanol by its property of generating free radicals during the course of its metabolism causes damage to cell structure and function. The study investigates the protective effects of the antioxidant aminoacid taurine on ethanol-induced lipid peroxidation and antioxidant status. Male Wistar rats of body weight 170–190 g were divided into 4 groups and maintained for 28 days as follows: a control group and taurine-supplemented control group, taurine supplemented and unsupplemented ethanol-fed group. Ethanol was administered to rats at a dosage of 3 g/kg body weight twice daily and taurine was provided in the diet (10 g/kg diet). Lipid peroxidation products and antioxidant potential were quantitated in plasma and in following tissues liver, brain, kidney and heart.Increased levels of thiobarbituric acid substances (TBARS) and lipid hydroperoxides (LHP) in plasma and tissues, decreased activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were observed in hemolysate and tissues of ethanol-fed rats. The contents of reduced glutathione (GSH), α-tocopherol and ascorbic acid in plasma and tissues were significantly reduced in these animals as compared to control animals. Simultaneous administration of taurine along with ethanol attenuated the lipid peroxidation process and restored the levels of enzymatic and non-enzymatic antioxidants. We propose that taurine may have a bioprotective effect on ethanol-induced oxidative stress.

Keywords: Keywords: Ethanol – Taurine – Lipid peroxidation – Antioxidants – Tissues – Oxidative stress

The relationship between plasma cholesterol, amino acids and acute phase proteins in sepsis by C. Chiarla; I. Giovannini; J. H. Siegel (pp. 97-100).

The purpose of the study was to correlate degree of hypocholesterolemia to changes in plasma levels of amino acids and other metabolic variables in severely injured septic patients. Measurements included plasma cholesterol, full amino-acidograms, acute phase proteins, complementary variables and blood cell counts. The Fischer plasma molar amino acid ratio (leucine+isoleucine+valine)/(phenylalanine+tyrosine) was calculated. Plasma cholesterol for all measurements (n=145) was 3.1±1.1 mmol/L and, upon entry in the study, it was correlated inversely with sepsis severity score (p<0.05). Along the clinical course, changes in cholesterol were clearly paralleled by opposite changes in C-reactive protein, which was the best correlate of cholesterol (r²=0.70, p<0.0001). Furthermore cholesterol was inversely related to phenylalanine, fibrinogen, lactate and white blood cell count, and directly to the Fischer molar amino acid ratio, cystathionine, methionine, glycine and transferrin (r² between 0.36 and 0.15, p<0.0001 for all). Within this pattern of correlations, cholesterol was also directly related to alkaline phosphatase, which accounted for the effect of cholestasis, when present. For any given value of the other variables, cholesterol increased significantly with increase in alkaline phosphatase (p<0.0001). C-reactive protein (CRP, mg/dl) and alkaline phosphatase (ALKPH, U/L) together in the same regression explained 79% of the variability of cholesterol (CHOL, mmol/L): CHOL=5.90–0.74[Log_eCRP]+0.004[ALKPH]; multiple r²=0.79, p<0.0001. Inclusion in this regression of other variables did not increase the r². By using only amino acid variables, the best fit was provided by a regression including the Fischer ratio and cystathionine, which explained 55% of the variability of cholesterol (multiple r²=0.55 p<0.0001), and this result was not improved by the inclusion of other amino acids. These data show that severity of hypocholesterolemia in sepsis is quantifiably related to changes in plasma amino acids, and to severity of acute phase response and metabolic decompensation. More study is needed to understand whether hypocholesterolemia in sepsis has only diagnostic or prognostic implications, or that it may also contribute actively to worsening of the disease.

Keywords: Keywords: Amino acids – Plasma cholesterol – Acute phase proteins – C-reactive protein – Sepsis

Total enzymatic synthesis of cholecystokinin CCK-5 by H. Xiang; G. Y. Xiang; Z. M. Lu; L. Guo; H. Eckstein (pp. 101-105).

This paper describes the enzymatic synthesis of the C-terminal fragment H-Gly-Trp-Met-Asp-Phe-NH₂ of cholecystokinin. Immobilized enzymes were used for the formation of all peptide bonds except thermolysin. Beginning the synthesis with phenylacetyl (PhAc) glycine carboxamidomethyl ester (OCam) and H-Trp-OMe by using immobilized papain as biocatalyst in buffered ethyl acetate, the dipeptide methyl ester was then coupled directly with Met-OEt·HCl by α-chymotrypsin/Celite 545 in a solvent free system. For the 3+2 coupling PhAc-Gly-Trp-Met-OEt had to be converted into its OCam ester.The other fragment H-Asp(OMe)-Phe-NH₂ resulted from the coupling of Cbo-Asp(OMe)-OH with H-Phe-NH₂·HCl and thermolysin as catalyst, followed by catalytic hydrogenation.Finally PhAc-Gly-Trp-Met-Asp-Phe-NH₂ was obtained in a smooth reaction from PhAc-Gly-Trp-Met-OCam and H-Asp(OMe)-Phe-NH₂ with α-chymotrypsin/Celite 545 in acetonitrile, followed by basic hydrolysis of the β-methyl ester. The PhAc-group is removed with penicillin G amidase and CCK-5 is obtained in an overall isolated yield of 19.6%.

Keywords: Keywords: Enzymatic peptide synthesis – Immobilized enzymes – Organic solvent system – Solvent free system – CCK-5 – Pentagastrin

Validity of elevated interstitial levels of taurine as a predictor of myocardial ischemic injury by M. Kavianipour; G. Wikström; G. Ronquist; A. Waldenström (pp. 107-111).

The microdialysis (MD) technique allows for continuous in vivo monitoring of dynamic changes in the interstitial levels of energy-related metabolites. The release of taurine from the myocyte has been suggested as a marker of ischemic injury. The relationship between (interstitial) taurine release and the degree of myocardial ischemic injury was evaluated following a 40 min long ischemia in a porcine heart-infarct-model. Different protocols of ischemia and reperfusion were used in order to achieve a graded level of myocardial injury. Both interstitial peak levels and the area under curve of taurine obtained during ischemia and reperfusion correlated with the degree of ischemic injury (assessed by developed infarct size estimation). The release of taurine in the myocardium measured by the MD-technique correlated with the degree of ischemic injury during ongoing ischemic insult. Hence, taurine determination in the MD-setting represents a powerful tool to follow the development of myocardial ischemic injury over time.

Keywords: Keywords: Microdialysis – Ischemia – Taurine – Infarct size – Myocardium – Pig

A review of adsorption of amino acids on minerals: Was it important for origin of life? by D. A. M. Zaia (pp. 113-118).

Minerals more readily adsorb amino acids with charged R groups than uncharged R groups, so that the incorporation of amino acids with charged R groups into peptides would be more frequent than for amino acids with uncharged R groups. However, 74% of the amino acids in the proteins of modern organisms contain uncharged R groups. Thus, what could have been the mechanisms that produced peptides/proteins with more amino acids with uncharged R groups than precursors with charged R groups? Should we expect the composition of amino acids adsorbed on minerals to be similar to those of present proteins? Was the adsorption of amino acids on minerals important for the origin of life? The lipid world offers an alternative view of origin of life. Liposomes contributed to elongation of peptides as well as select hydrophobic amino acids and peptides. These experiments could be showing the mechanism, which hydrophobic amino acids have been selected. However, liposomes have no influence on the stereoselectivity in the oligomerization of amino acids. In the present paper, several other mechanisms are also discussed that could produce peptides with a greater proportion of amino acids with uncharged R groups.

Keywords: Keywords: Adsorption – Minerals – Amino acids – Proteins – Side chain

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.27, #1)