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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.16, #1)

Plasma amino acid levels after carbon tetrachloride induced acute liver damage. A dose-response and time-response study in rats by H. Skalská; J. Mráz; Dr. M. Holeček (pp. 1-11).

The aims of the present study were to assess the changes of individual plasma amino acid levels in relation (1) to the severity of liver damage and (2) to the process of liver recovery. Acute liver injury was induced by an intragastric administration of CCl₄ diluted in olive oil in doses of 2, 4 and /or 6 g of CCl₄ per kg b.w. The control rats received olive oil only. Animals were sacrificed at 16, 24, 48 and 96 hours after treatment. The severity of liver injury was assessed by histological examination, by changes in ALT and AST in the blood plasma and by changes in liver weight. Statistical analysis was carried by ANOVA, p < 0.05 was considered significant. The Spearman rank correlation coefficient was used as a measure of the degree of linear relationship between variable and dose. In the period of the development of acute liver damage, i.e. at 16 and 24 hours after treatment, an increase in blood plasma amino acid levels and positive correlations with the dose of CCl₄ were observed for most individual amino acids. The only exception was arginine which decreased in a dose dependent manner. At a phase of liver recovery, i.e. at 48 and 96 hours after CCl₄ treatment, the concentrations of some individual amino acids decreased below the control values. The negative correlation with the dose of CCl₄ occurred for taurine and isoleucine (at 48 hours) and taurine, threonine, valine, methionine, isoleucine and leucine (at 96 hours).

Keywords: Amino acids; Liver; Hepatic damage; Carbon tetrachloride

Determination of the protooncogene ets-2 gene transcript in human brain at the atto-gram-level by the use of competitive RT/PCR by D. Schatzmann-Turhani; S. Greber-Platzer; N. Cairns; .Prof. Dr. G. Lubec (pp. 13-19).

Protooncogenes (PO) play a crucial role for brain biology and pathology. Only the concerted action of protooncogenes enables normal brain development.The reliable and sensitive quantification of brain PO is still holding centre stage in neurobiological research. The aim of our study was therefore the determination of PO in minute amounts of brain areas. For this purpose we decided to apply the most sensitive detection principle of competitive reverse transcriptase polymerase chain reaction using capillary electrophoresis and laser-induced fluorescence detection.We selected the PO ets-2 for our studies as this transcription factor was shown to be involved in neurodegenerative disease. As little as 10ng of total RNA each were extracted from 5 different regions of human postmortem brain and used in the assay system. Our results revealed that the ets-2 gene transcript was detectable at the atto-gram level in the brain (54.5 ± 17.7 ag/ 10 ng RNA in the occipital lobe, 34.2 ± 7.5 in temporal lobe, 40.2 ± 15.6 in the frontal lobe, 31.4 ± 15.7 in the cerebellum, and undetectably low in the parietal lobe).This is the first report at this sensitivity level providing neurobiology with a powerful analytical tool.

Keywords: Amino acids; RT/PCR; Protooncogene; ets-2

Unusual amino acids VIII. Asymmetric hydrogenation of some heteroaryl-N-CBZ and N-BOC aminocinnamic acid derivatives by H. -J. Kreuzfeld; M. Michalik; .Dr. Chr Döbler (pp. 21-27).

(Z)-α-[(Benzyloxy)- or (tert.-butyloxy)carbonylamino]-β (thienyl)-or (furyl)-acrylic acids and their esters were prepared by known methods and hydrogenated to the corresponding optically active alanine derivatives with optical yields in the range of 58–93% ee using the cationic rhodium complex of “PROPRAPHOS”.

Keywords: Amino acids; Non-proteinogenic optically active amino acids; Dehydroamino acids; Chiral rhodium catalysts; Asymmetric hydrogenation

Contribution of serum albumin to the transport of orally administered L-tryptophan into liver of rats with L-tryptophan depletion by E. Sasaki; Y. Ohta; R. Shinohara; I. Ishiguro (pp. 29-39).

The role of serum albumin in the transport of orally administered L-tryptophan (Trp) into rat tissues was examined using analbuminemic and Sprague-Dawley (SD) rats with and without a-methyl-DL-tryptophan (AMT)-induced Trp depletion. Trp was orally administered to rats 16h after AMT or 0.85% NaCl administration, when liver tryptophan 2,3-dioxygenase and protein synthetic activities in AMT-treated rats were similar to those of 0.85% NaCl-treated rats. After oral Trp administration, regardless of the presence or absence of Trp depletion, free serum Trp concentrations were similar in the analbuminemic and SD rats, while total serum Trp concentrations were lower in analbuminemic rats than in SD rats. Although liver, brain, and muscle Trp concentrations after oral Trp administration under Trp depletion were lower in analbuminemic rats than in SD rats, the ratio of the liver Trp concentration in analbuminemic rats to that in SD rats was smaller than that of the brain or muscle Trp concentration. These results suggest that variations in serum albumin levels could affect the transport of orally administered Trp into the liver of rats with Trp depletion.

Keywords: Amino acids; L-Tryptophan; Serum albumin; Transport; L-Tryptophan depletion; α-Methyl-DL-tryptophan; Analbuminemic rat

Fatty acids and glycerol or lactate are required to induce gluconeogenesis from alanine in isolated rabbit renal cortical tubules by T. Lietz; J. Rybka; Dr. J. Bryla (pp. 41-58).

In isolated rabbit renal cortical tubules, glucose synthesis from 1 mM alanine is negligible, while the amino acid is metabolized to glutamine and glutamate. The addition of 0.5 mM octanoate plus 2 mM glycerol induces incorporation of [U-¹⁴C]Alnine into glucose and decreases glutamine synthesis, whereas oleate and palmitate in the presence of glycerol are less potent than octanoate. Gluconeogenesis is also significantly accelerated when glycerol is substituted by lactate. In view of an increase in¹⁴CO₂ fixation and elevation of both cytosolic and mitochondrial NADH/NAD⁺ ratios, the activation of glucose formation from alanine upon the addition of glycerol and octanoate is likely due to (i) stimulation of pyruvate carboxylation, (ii) increased availability of NADH for glyceraldehyde-3-phosphate dehydrogenase and (iii) elevation of mitochondrial redox state causing a diminished provision of ammonium for glutamine synthesis. The induction of gluconeogenesis in the presence of alanine, glycerol and octanoate is not related to cell volume changes. The results presented in this paper show the importance of free fatty acids and glycerol for regulation of renal gluconeogenesis from alanine. The possible physiological significance of the data is discussed.

Keywords: Amino acids; Alanine; Fatty acids; Glutamine and glutamate synthesis; Glycerol and lactate metabolism; Malate-aspartate shuttle; Rabbit kidney-cortex tubules; Renal gluconeogenesis

Terfenadine induces toxicity in cultured cerebellar neurons: A role for glutamate receptors by R. Díaz-Trelles; A. Solana-López; J. R. Fernández-González; Antonello Novelli; María Teresa Fernández-Sánchez (pp. 59-70).

Exposure of cultured cerebellar neurons to the histamine H1 receptor antagonist terfenadine resulted in neuronal degeneration and death. Terfenadine neurotoxicity was dependent upon concentration and time of exposure. After 2h exposure, 20µM terfenadine reduced the number of surviving neurons by 75%, and as low as 10nM terfenadine induced significant neurotoxicity after 5 days of exposure. Neuronal sensitivity to terfenadine changed with age in culture, and at 25 days in culture neurons appeared to be much less sensitive than at 5 or 9–17 days in culture. Neurotoxicity by terfenadine could not be prevented by high concentrations of histamine (5 mM), but it was significantly delayed by blocking NMDA or non-NMDA glutamate receptors with MK-801 or CNQX respectively, suggesting the involvement of excitatory transmission mediated by glutamate in the neurotoxicity induced by terfenadine in these neurons. We also found that the presence of terfenadine (5,µM) unveiled the potential excitotoxicity of the non-NMDA receptor agonist AMPA (100µM), and reduced the concentration of glutamate necessary to induce excitotoxicity, compared to untreated cultures. These results suggest a role for terfenadine in the modulation of the excitotoxic response mediated in cerebellar neurons through ionotropic glutamate receptors.

Keywords: Amino acids; Cerebellar granule cells; Neurotoxicity; Terfenadine; Excitatory amino acids; Histamine

Changes in plasma taurine levels after different endurance events by R. J. Ward; M. Francaux; C. Cuisinier; X. Sturbois; Prof. Ph. De Witte (pp. 71-77).

The sulphonated amino acid taurine increased significantly in the plasma of trained athletes after three endurance exercises of different duration and intensity, a 90 min run on a treadmill at 75% of an individual's VO₂ peak, a Marathon, 42.2km and a 100km run, by 19%, 77% and 36%, respectively. Such results indicated that the speed at which the exercise is per formed, referred to as the intensity, rather than the duration of the exercise, correlated with the elevated taurine levels possibly indicating its release from muscle fibres. The plasma amino acid pool decreased significantly in relationship with the duration of the exercise, caused by their utilisation for glucogenesis. The possible sources of the increased plasma taurine are discussed.

Keywords: Amino acids; Taurine; Marathon; Endurance exercise

Substituted 4-hydroxyproline di- and tri-peptides as cytotoxic agents by Dr. I. H. Hall; S. Y. Chen (pp. 79-89).

4-Hydroxyproline di- and tri-peptides and N-cbz-hydroxypropylglycinamides were observed to be potent cytotoxic agents against the growth of suspended single cells, L-1210, Tmolt₃, and HeLa-S³. The agents were not as potent against the growth of cultured solid tumor cells. Selected derivatives were investigated for their mode of action in Tmolt₃ leukemia cells. The compounds selectively inhibited DNA synthesis at 50 and 100smM. The target site of action of the agents appeared to be the purinede novo pathway with marked inhibition of the activities of the two regulatory enzymes of the pathway, i.e. PRPP amido-transferase and IMP dehydrogenase. d[NTP] pools were reduced by the agents consistent with their overall reduction of DNA synthesis. Other marginally inhibited targets of the agents were r-RNA polymerase and TMP-kinase activities. The DNA molecule itself did not appear to be a target of these agents.

Keywords: Amino acids; Anti-neoplastic amino acids; Purine and DNA synthesis inhibitors; PRPP amido transferase and IMP dehydrogenase inhibitors

Structural organization of the human eukaryotic initiation factor 5A precursor and its site-directed variant Lys50 → Arg by P. Stiuso; G. Colonna; R. Ragone; M. Caraglia; J. W. B. Hershey; S. Beninati; Dr. Alberto Abbruzzese (pp. 91-106).

The molecular properties of the human eukaryotic initiation factor 5A precursor and its site directed Lys50 → Arg variant have been investigated and compared. Structure perturbation methods were used to gain information about the protein architecture in solution. Intrinsic and extrinsic spectroscopic probes strategically located in the protein matrix detected the independent unfolding of two molecular regions. Three cystemes out of four were titrated in the native protein and the peculiar presence of a tyrosinate band at neutral pH was detected. At alkaline pH only two tyrosines out of three were titratable in the native protein, with an apparent pK of about 9.9. Native protein and its Lys50 → Arg variant reacted in a similar fashion to guanidine and to pH variation, but differently to thermal stress. The complex thermal unfolding of both proteins indicated the presence of intermediates. Spectroscopic data showed that these intermediates are differently structured. Consequently, the two proteins seem to have different unfolding pathways.

Keywords: Amino acids; eIF-5A; Hypusine; Protein folding; Posttranslational modification

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.16, #1)