Check out our New Publishers' Select for Free Articles

Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.28, #3)

Redox options in two-dimensional electrophoresis by R. Wait; S. Begum; D. Brambilla; A. M. Carabelli; F. Conserva; A. Rocco Guerini; I. Eberini; R. Ballerio; M. Gemeiner; I. Miller; E. Gianazza (pp. 239-272).

Two-dimensional electrophoresis is usually run on fully reduced samples. Under these conditions even covalently bound oligomers are dissociated and individual polypeptide chains may be fully unfolded by both, urea and SDS, which maximizes the number of resolved components and allows their pI and M_r to be most accurately evaluated. However, various electrophoretic protocols for protein structure investigation require a combination of steps under varying redox conditions. We review here some of the applications of these procedures. We also present some original data about a few related samples – serum from four species: Homo sapiens, Mus musculus, Rattus norvegicus, Bos taurus – which we run under fully unreduced and fully reduced conditions as well as with reduction between first and second dimension. We demonstrate that in many cases the unreduced proteins migrate with a better resolution than reduced proteins, mostly in the crowded ‘α-globulin’ area of pI 4.5–6 and M_r 50–70 kDa.

Keywords: Keywords: Cysteine – Cystine – Reduction – Alkylation – Oxidation – Two-dimensional electrophoresis – Serum

Long-chain acyl-CoA hydrolase in the brain by J. Yamada (pp. 273-278).

Long-chain acyl-CoA hydrolases are a group of enzymes that cleave acyl-CoAs into fatty acids and coenzyme A (CoA-SH). Because acyl-CoAs participate in numerous reactions encompassing lipid synthesis, energy metabolism and regulation, modulating intracellular levels of acyl-CoAs would affect cellular functions. Therefore, acyl-CoA synthetases have been intensively studied. In contrast, acyl-CoA hydrolases have been less investigated, especially in the brain despite the fact that its long-chain acyl-CoA hydrolyzing activity is much higher than that in any other organ in the body. However, recent studies have dissected the multiplicity of this class of enzymes on a genomic basis, and have allowed us to discuss their function. Here, we describe a cytosolic long-chain acyl-CoA hydrolase (referred to as BACH) that is constitutively expressed in the brain, comparing it with other acyl-CoA hydrolases found in peripheral organs that have a role in fatty acid oxidation.

Keywords: Keywords: Acyl-CoA thioesterase – Long-chain fatty acyl-CoA – Lipid metabolism – Neuron

Expression profiling using human tissues in combination with RNA amplification and microarray analysis: assessment of Langerhans cell histiocytosis by K. L. McClain; Y.-H. Cai; J. Hicks; L. E. Peterson; X.-T. Yan; S. Che; S. D. Ginsberg (pp. 279-290).

Advances in molecular genetics have led to sequencing of the human genome, and expression data is becoming available for many diverse tissues throughout the body, allowing for exciting hypothesis testing of critical concepts such as development, differentiation, homeostasis, and ultimately, disease pathogenesis. At present, an optimal methodology to assess gene expression is to evaluate single cells, either identified physiologically in living preparations, or by immunocytochemical or histochemical procedures in fixed cells in vitro or in vivo. Unfortunately, the quantity of RNA harvested from a single cell is not sufficient for standard RNA extraction methods. Therefore, exponential polymerase-chain reaction (PCR) based analyses, and linear RNA amplification including amplified antisense (aRNA) RNA amplification and a newly developed terminal continuation (TC) RNA amplification methodology have been used in combination with microdissection procedures such as laser capture microdissection (LCM) to enable the use of microarray platforms within individual populations of cells obtained from a variety of human tissue sources such as biopsy-derived samples {including Langerhans cell histiocytosis (LCH)} as well as postmortem brain samples for high throughput expression profiling and related downstream genetic analyses.

Keywords: Keywords: cDNA microarray – Postmortem human brain – Cancer genomics – Translational neuroscience – Molecular fingerprint

Effects of hypoxic and osmotic stress on the free D-aspartate level in the muscle of blood shell Scapharca broughtonii by T. Watanabe; K. Shibata; Y. Kera; S. Takahashi; R. Yamada (pp. 291-296).

Blood shell, Scapharca broughtonii, contains large quantities of free D-aspartate comparable to free L-aspartate in its tissues. When the shell was reared in hypoxic seawater, D-aspartate as well as L-aspartate in the foot muscle decreased rapidly, and their total level became about one-fourth within 24 hr. None of the other amino acids examined showed a similar behavior, but many of them rather increased during the same period. The increase in L-alanine was especially remarkable and was almost equal to the sum of the decrease in aspartate enantiomers. When the shell that had been acclimated to hypoxic seawater for 96 hr was transferred to normoxic seawater, all the amino acid levels mostly returned to the control levels within 96 hr. In contrast to these effects of hypoxic stress, hyperosmotic stress of 150% seawater had no effect on the D- and L-aspartate levels in the same tissue. These results suggest that D-aspartate is involved in anaerobic energy metabolism of this bivalve as well as L-aspartate, whose vital role in anoxia-tolerant bivalves is well known.

Keywords: Keywords: Anaerobic metabolism – Scapharca broughtonii – D-Aspartate – Aspartate racemase – Hypoxic stress – Osmotic stress

Effect of adding dietary L-lysine, L-threonine and L-methionine to a low gluten diet on urea synthesis in rats by K. Tujioka; S. Lyou; Y. Fukaya; A. Sano; K. Hayase; H. Yokogoshi (pp. 297-303).

We have shown that urinary urea excretion increased in rats fed a low quality protein. The purpose of present study was to determine whether an addition of dietary limiting amino acids affected urea synthesis in rats fed a low gluten diet. Experiments were done on three groups of rats given diets containing 10% gluten, 10% gluten+0.5% L-lysine or 10% gluten+0.5% L-lysine, 0.2% L-threonine and 0.2% L-methionine for 10 d. The urinary excretion of urea, and the liver concentrations of serine and ornithine decreased with the addition of dietary L-lysine, L-threonine and L-methionine. The fractional and absolute rates of protein synthesis in tissues increased with the treatment of limiting amino acids. The activities of hepatic urea-cycle enzymes was not related to the urea excretion. These results suggest that the addition of limiting amino acids for the low gluten diet controls the protein synthesis in tissues and hepatic ornithine and decline urea synthesis.

Keywords: Keywords: Dietary limiting amino acids – Urea synthesis – Protein synthesis – Amino acids – Ornithine – Rats

Effect of hyperprolinemia on acetylcholinesterase and butyrylcholinesterase activities in rat by D. Delwing; F. Chiarani; C. M. D. Wannmacher; M. Wajner; A. T. S. Wyse (pp. 305-308).

We observed here that acute proline (Pro) administration provoked a decrease (32%) of acetylcholinesterase (AChE) activity in cerebral cortex and an increase (22%) of butyrylcholinesterase (BuChE) activity in the serum of 29-day-old rats. In contrast, chronic administration of Pro did not alter AChE or BuChE activities. Furthermore, pretreatment of rats with vitamins E and C combined or alone, N^ϖ-nitro-L-arginine methyl ester or melatonin prevented the reduction of AChE activity caused by acute Pro administration, suggesting the participation of oxidative stress in such effects.

Keywords: Keywords: Hyperprolinemia type II Proline Acetylcholinesterase Butyrylcholinesterase Experimental model

Repeated-testing of place preference expression for evaluation of anti-craving-drug effects by V. Herzig; W. J. Schmidt (pp. 309-317).

In addiction research, the conditioned place preference (CPP) paradigm is a widely used animal model of conditioned reward. Usually, CPP development is studied, while only few studies examine CPP expression. In the present study, the suitability of a schedule allowing repeated testing of CPP expression was evaluated. Two groups of rats were either conditioned with cocaine or morphine then the repeated-testing-schedule was applied. This schedule consisted of four repeated applications of a sequence of drug- (i.e. cocaine or morphine), saline- and anti-craving-drug- (i.e. acamprosate, naloxone, their joint administration or saline as internal control) tests. Methodologically, the repeated-testing-schedule produced stable CPP expression in both groups over 12 subsequent tests. In conclusion, it is suggested as a useful method to study effects of anti-craving-drugs on CPP expression, thereby reducing the overall number of experimental animals. The evaluation of the anti-craving-drug effects revealed that neither acamprosate and naloxone given separately nor their combined administration significantly reduced cocaine- or morphine-CPP expression. Thus, we suggest that these anti-craving-drugs are unlikely to be effective for relapse prevention in cocaine- or morphine-addicts.

Keywords: Keywords: Conditioned place preference (CPP) expression – Conditioned reward – Repeated testing – Morphine – Cocaine – Naloxone – Acamprosate

Characterization of developmental autolysis in myxobacterial fruiting body morphogenesis with profiling of amino acids using capillary electrophoresis method by H. Zhang; H. Dong; J. Zhao; W. Hu; Y.-Z. Li (pp. 319-325).

Capillary electrophoresis equipped with Laser-induced fluorescence (CE-LIF), combining with micro-culture technique was employed to determine extracellular amino acids in single myxobacterial fruiting body morphogenesis. The result showed that in the early aggregation stage, there was a remarkable increase of extracellular amino acids, which was produced by developmentally induced autolysis. The amino acids were then consumed by the vegetative cells in aggregation stage. In the following developmental events, the extracellular amino acids were kept at low level, which indicated that in the stages of fruiting body formation and myxospore development, there was no further cell autolysis. Using this novel method may provide detailed insight into the mechanisms of the developmental phenomena.

Keywords: Keywords: Capillary electrophoresis – Laser-induced fluorescence detection – Myxobacterium – Morphogenesis – Autolysis

Protein metabolism and strength performance after bovine colostrum supplementation by A. Mero; T. Nykänen; O. Keinänen; J. Knuutinen; K. Lahti; M. Alen; S. Rasi; J. Leppäluoto (pp. 327-335).

This study was designed to determine the responses of muscle protein, serum amino acids, and strength performance to bovine colostrum supplementation in physically active men. The rest (R) group (n=6) and the exercise (E) group (n=6) carried out twice a 2-week experiment randomly assigned in a double-blind fashion with either placebo (PLA; consuming daily 20 g maltodextrin) or bovine colostrum (COL; consuming daily 20 g colostrum supplement) treatment with one month between. On the test day after the treatment period the measurements were carried out in fasting conditions and E carried out a strength training session (STS). The methods involved the infusion of ring-²H₅-phenylalanine, femoral arterial and venous blood sampling, and biopsies from the vastus lateralis muscle. Serum concentration of essential amino acids during recovery was greater (p<0.05) in the COL groups compared with the PLA groups. Both muscle protein synthesis and breakdown increased (p<0.05) with COL. There were no differences in phenylalanine net balance or strength performance between the PLA and COL groups. It was concluded that a 2-week supplementation with bovine colostrum in physically active men increases serum concentration of essential amino acids but has no effect either on strength performance or protein net balance in fasting conditions during recovery after STS.

Keywords: Keywords: Amino acids – Protein synthesis – Protein breakdown – Physical training

Effect of a swim training on homocysteine and cysteine levels in rats by V. Gaume; H. Figard; F. Mougin; J. C. Guilland; J. M. Alberto; J. L. Gueant; D. Alber; C. Demougeot; A. Berthelot (pp. 337-342).

The purpose of this study was to investigate the effects of a 8-week of swim training on total plasma homocysteine and cysteine levels in 16 male Sprague-Dawley rats aged 17 weeks. We also evaluated the activity of hepatic cystathionine β-synthase (CBS), an enzyme involved in the metabolism of Hcy, the concentration of plasma glutathione, taurine, and a fraction of vitamin B6: the pyridoxal 5-phosphate (PLP). After one week of acclimatization, rats were randomly divided into two groups: 8 non-trained (NTR) and 8 trained rats (TR). Following the training period, body weight gain was lower in TR than in NTR. Plasma homocysteine did not differ among groups while significantly lower plasma cysteine and taurine levels were found in TR (157.83±8.6 μmol/L; 133.01±9.32 μmol/L; P<0.05) compared with data of NTR (176.19±4.9 μmol/L; 162.57±8.16 μmol/L; P<0.05). No significant changes in hepatic CBS activity were observed in TR compared with NTR. Moreover, values for plasma glutathione and PLP concentrations were not affected by training.These results indicate that training reduces plasma cysteine and taurine levels whereas it does not modify other studied parameters. Thus, physical training may regulate cysteine metabolism.

Keywords: Keywords: Cystathionine β synthase – Cysteine – Glutathione – Homocysteine – Pyridoxal 5′ phosphate taurine – Swim training

Sections

Personal tools

Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.28, #3)

Sections

Personal tools

Local resources

Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.28, #3)