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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.21, #2)

Sensitivity of taurine uptake to oxygen-derived reactive substances in MDR and non-MDR cells by C. Wersinger; I. H. Lelong-Rebel; G. Rebel (pp. 91-117).

In human KB and LoVo cell lines, high affinity taurine uptake was strongly reduced in both a time and dose-dependent manner by cumene hydroperoxide (CH) and to a lesser extent by hydrogen peroxide (H₂O₂). Uptake-inhibition was greater in multidrug resistant (MDR) cells than in their non-MDR counterparts. Basal taurine efflux was unaffected by the oxidants. Lipid peroxidation levels closely correlated with the uptake inhibition levels, and were greater in MDR cells than in their non-MDR counterparts. The two oxidants reduced the Vmax and, to a lesser extent, the affinity of the transporter for taurine. They also reduced low affinity taurine uptake and, to a lesser extent, taurine diffusion. The composition of the medium used for cell treatment, especially its pyruvate content, greatly affected the H₂O₂ effect. H₂O₂- or CH-induced reduction of the high affinity taurine uptake was unaffected by protein kinase C (PKC) inhibitors and by the calmodulin antagonist W-13, ruling out the involvement of PKC and perhaps of calmodulin kinases in their effect.

Keywords: Keywords: Amino acids; Taurine uptake; MDR cells; Hydrogen per-oxide; Cumene hydroperoxide; Malondialdehyde; Lipid peroxidation; Pyruvate; PKC; Calmodulin kinases

Regulation of the expression of low affinity GABA_A receptors in rat cerebellar granule cells by C. Luccardini; B. Barilà; A. Cupello; M. Robello; P. Mainardi (pp. 119-128).

GABA_A receptors of cerebellar granule cells obtained from neonatal rats and kept in culture were studied by labelled muscimol binding.The data show that, according to the maturational state of those cells in vivo, one or two binding components appear. The low affinity component seems to be the one appearing later. The expression of this component seems to be regulated by protein tyrosine phosphorylation. In fact, its expression is down regulated by the protein tyrosine kinase (PTK) inhibitor, genistein. Viceversa, its expression is upregulated by insulin like growth factor I (IGF-I), most probably via PTK activation.A possible interpretation of the data is that in vivo IGF-I is one of the endogenous messages leading to the expression of this component during development. Another endogenous factor involved may be GABA itself.Low affinity GABA_A receptors appear to be the ones involved in inhibitory synaptic transmission at glomeruli. Whereas the high affinity ones probably correspond to extrasynaptic GABA_A receptors mediating the tonic form of inhibition in cerebellar granules.

Keywords: Key words: Amino acids; Cerebellar granules; Development; Cells cultures; GABAA receptors; Muscimol binding; PTK

Determination of amino acid tissue concentrations by microdialysis: method evaluation and relation to plasma values by B. Rolinski; F. A. M. Baumeister; A. A. Roscher (pp. 129-138).

Microdialysis is an in vivo technique to monitor tissue concentrations of low molecular weight substances by means of a continuously perfused artificial capillary with a semipermeable membrane placed into the region of interest. The suitability of microdialysis to determine tissue concentrations of amino acids was evaluated in vitro by placing the catheter into Ringer buffer or into a plasma protein (50 g/l) solution containing 32 different amino acids (150 μmol/l each). All amino acids tested crossed freely the microdialysis membrane with recoveries close to 100%. Microdialysis fluid was sampled from subcutaneous tissue of five newborns and amino acid content analysed. Total and non protein bound amino acids were determined in the patients plasma by acid precipitation or ultrafiltration, respectively. Mean subcutaneous tissue concentrations were lower as compared to plasma for taurine, serine, alanine, aspartate, glutamate and ornithine and higher for valine, isoleucine, leucine, methionine, phenylalanine, tyrosine and arginine, indicating net uptake or release of amino acids from subcutaneous tissue. Thus, microdialysis offers a convenient and minimal invasive way to study tissue amino acid composition and appears to be a promising analytical tool for the study of amino acid metabolism in vivo.

Keywords: Keywords: Amino acids; Microdialysis; Ultrafiltration; Protein binding; Subcutaneous tissue; Newborn

The effects of Red Bull Energy Drink on human performance and mood by C. Alford; H. Cox; R. Wescott (pp. 139-150).

The effects of Red Bull Energy Drink, which includes taurine, glucuronolactone, and caffeine amongst the ingredients, were examined over 3 studies in a total of 36 volunteers. Assessments included psychomotor performance (reaction time, concentration, memory), subjective alertness and physical endurance. When compared with control drinks, Red Bull Energy Drink significantly (P < 0.05) improved aerobic endurance (maintaining 65–75% max. heart rate) and anaerobic performance (maintaining max. speed) on cycle ergometers. Significant improvements in mental performance included choice reaction time, concentration (number cancellation) and memory (immediate recall), which reflected increased subjective alertness. These consistent and wide ranging improvements in performance are interpreted as reflecting the effects of the combination of ingredients.

Keywords: Keywords: Amino acids; Red Bull Energy Drink; Caffeine; Glucuronolactone; Taurine; Physical endurance; Psychomotor performance

Regulation of taurine transport in murine macrophages by L. Romio; O. Zegarra-Moran; L. Varesio; L. J. V. Galietta (pp. 151-160).

We studied the regulation of taurine transport in ANA1 murine macrophage cell line. Taurine uptake was upregulated by hypertonicity and downregulated by bacterial lypopolysaccharide (LPS) and other stimuli leading to macrophage activation. However combined stimulation with LPS plus hypertonic shock evoked an increase of taurine uptake that was even higher than with hypertonic shock alone. Taurine transport was not modified by LPS in GG2EE macrophages derived from C3H/Hej mouse strain, which harbour a mutated Toll-like receptor 4 (TLR4) and thus are not activated by LPS. The extracellular signal-regulated kinase (ERK) inhibitor PD98059 abrogates the effect of both LPS and hyperosmotic shock on ANA1 taurine uptake, while the p38 inhibitor SB203580 reduces the taurine uptake in control conditions and impairs only the response to hypertonicity. These results suggest that the effect of LPS on taurine transport depends on ERK pathway and can be influenced by environmental conditions.

Keywords: Keywords: Amino acids; Cell volume regulation; Hypertonic shock; Bacterial lypolysaccharide; Organic osmolytes; Endotoxin resistance

Substrate recognition by proline permease in Salmonella by M.-K. Liao; S. Maloy (pp. 161-174).

Proline transport is required for catabolism of proline as a carbon, nitrogen, and energy source, and for accumulation of proline during adaptation to osmotic stress. These physiological processes are widespread in nature, and play essential roles in the virulence of both prokaryotic and eukaryotic pathogens. In enteric bacteria, the major proline permease is encoded by the putP gene. To identify the structural features required for substrate recognition by PutP, we assayed the transport and toxicity of a variety of natural and synthetic derivatives of proline. The results indicate that the substrate binding site of proline permease consists of a hydrophobic pocket that accommodates C3, C4, and C5 of the pyrrolidine ring. Both 4- and 5-membered rings fit into the substrate binding pocket, but 6-membered rings are excluded. Analogs with substituents on the C4 position are also excluded. In addition, the binding site includes a hydrophilic region that recognizes the imino and carbonyl groups. A free carboxyl group is not required. Taken together, these results may be used to design new synthetic inhibitors of proline transport that can effectively block proline uptake by microbial pathogens.

Keywords: Keywords: Amino acids; Proline permease substrate specificity

Chum salmon trypsin-catalyzed preferential formation of peptides containing d-amino acid by H. Sekizaki; K. Itoh; E. Toyota; K. Tanizawa (pp. 175-184).

Chum salmon trypsin-catalyzed peptide synthesis has been studied by using nine series of "inverse substrates," i.e., p-amidinophenyl, p- and m-guanidinophenyl, p- and m-(guanidinomethyl)phenyl, and four position isomers of guanidinonaphthyl esters derived from N α-(tert-butyloxycarbonyl)amino acid as acyl donor components. They were found to couple with an acyl acceptor such as l-alanine p-nitroanilide to produce dipeptide in the presence of trypsin. All substrates tested in this study undergo less enantioselective coupling reaction, and the coupling product was the favorably obtained d-series rather than l-series (in the present case; N α-Boc-d-Ala and N α-Boc-l-Ala). The optimum condition for the coupling reaction was studied by changing the organic solvent, buffer solution, pH, and acyl acceptor concentration. It was found that the enzymatic hydrolysis of the resulting product was negligible.

Keywords: Keywords: Amino acids; Inverse substrate; Chum salmon trypsin; Enzymatic peptide synthesis; d-Amino acid; Amidinophenyl ester; Guanidinophenyl ester

Interactions of surfactants with living cells.Induction of apoptosis by detergents containing a β-lactam moiety by A. Perani; C. Gérardin; G. Stacey; M.-R. Infante; P. Vinardell; L. Rodehüser; C. Selve; M. Maugras (pp. 185-194).

The toxicity of new surfactants containing a β-lactam ring has been established by studying their interaction with a hybridoma cell line. An hour of contact is sufficient to generate an apoptotic signal after two days of culture. Under the experimental conditions chosen for the experiments, surfactants have been divided into three categories: i) biocompatible and non-apogenic; ii) surfactants triggering an apoptotic signal without inducing cell necrosis; iii) surfactants triggering an apoptotic signal at low concentrations and destroying the cells by necrosis at higher concentrations. The necrosis inducing surfactants also had haemolytic properties. These properties were related to the values of the hydrophilic-lipophilic balance of the molecules.

Keywords: Keywords: Amino acids; Surfactants; Toxicity; Apoptosis

Effect of proline on the production of singlet oxygen by Alia; P. Mohanty; J. Matysik (pp. 195-200).

Molecular oxygen in electronic singlet state is a very powerful oxidant. Its damaging action in a variety of biological processes has been well recognized. Here we report the singlet oxygen quenching action of proline. Singlet oxygen (¹O₂) was produced photochemically by irradiating a solution of sensitiser and detected by following the formation of stable nitroxide radical yielded in the reaction of ¹O₂ with the sterically hindered amine (2,2,6,6-tetramethylpiperidine, TEMP). Illumination of a sensitiser, toluidine blue led to a time dependent increase in singlet oxygen production as detected by the formation of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) by EPR spectrometry. Interestingly, the production of TEMPO was completely abolished by the presence of proline at concentration as low as 20 mM. These results show that proline is a very effective singlet oxygen quencher. Other singlet oxygen generating photosensitizer like hematopophyrin and fluorescein also produced identical results with proline. Since proline is one of the important solutes which accumulate in many organisms when they are exposed to environmental stresses, it is likely that proline accumulation is related to the protection of these organisms against singlet oxygen production during stress conditions. A possible mechanism of singlet oxygen quenching by proline is discussed.

Keywords: Keywords: Amino acids; Proline; Singlet oxygen; EPR

A modified spray reagent for the detection of amino acids on thin layer chromatography plates by S. Laskar; A. Sinhababu; K. M. Hazra (pp. 201-204).

Identification of amino acids is extremely important for the evaluation of protein structure. Thin layer chromatography is an important tool for detecting amino acids by variety of spray reagents. Among these ninhydrin is the most popular due to its high sensitivity. However, ninhydrin produces the same purple/violet color with most amino acids. A spray reagent with high sensitivity for easy and rapid identification of amino acids on thin-layer plates has been introduced.

Keywords: Keywords: Amino acids; Ninhydrin; N-Cyanoguanidine

L-arginine stimulates insulin secretion from the pancreas of normal and diabetic rats by E. Adeghate; A. S. Ponery; T. El-Sharkawy; H. Parvez (pp. 205-209).

Several reports have shown that nitric oxide (NO) stimulates glucose-induced insulin secretion in the pancreas of normal rat but the effect of L-arginine (a NO donor) on insulin secretion from the pancreas of diabetic pancreas is unknown. Fragments of pancreatic tissue from normal and diabetic rats were incubated for 45 min in Krebs solution containing 100 mM L-arginine. The supernatant was subsequently analyzed for the insulin content using radioimmunoassay technique. L-arginine evoked large increases in insulin secretion from the pancreas of diabetic rat. The insulin secreted from the pancreas of diabetic rat was numerically but not significantly lower compared to that of normal rat pancreas. In conclusion, L-arginine, a nitric oxide donor stimulates insulin secretion from the pancreas of diabetic rats.

Keywords: Keywords: Amino acids; Insulin secretion; Radioimmunoassay; L-Arginine; NO; Pancreas; Diabetes mellitus; Rat

Analysis of the genomic organization of the human cationic amino acid transporters CAT-1, CAT-2 and CAT-4 by R. Hammermann; G. Brunn; K. Racké (pp. 211-219).

By screening nucleotide databases, sequences containing the complete genes of the human cationic amino acid transporters (hCATs) 1, 2 and 4 were identified. Analysis of the genomic organization revealed that hCAT-2 consists of 12 translated exons and most likely of 2 untranslated exons. The splice variants hCAT-2A and hCAT-2B use exon 7 and 6, respectively. The hCAT-2 gene structure is closely related to the structure of hCAT-1, suggesting that they belong to a common gene family. hCAT-4 consists of only 4 translated exons and 3 short introns. Exons of identical size and highly homologous to exon 3 of hCAT-4 are present in hCAT-1 and hCAT-2.

Keywords: Keywords: Cationic amino acids; Human cationic amino acid transporter; Solute carrier family; Genomic organization; Exon-intron structure

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.21, #2)