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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.20, #2)

The role of eukaryotic initiation factor 5A in the control of cell proliferation and apoptosis by M. Caraglia; M. Marra; G. Giuberti; A. M. D'Alessandro; A. Budillon; S. del Prete; A. Lentini; S. Beninati; A. Abbruzzese (pp. 91-104).

In the past years, the attention of scientists has mainly focused on the study of the genetic information and alterations that regulate eukaryotic cell proliferation and that lead to neoplastic transformation. An increasing series of data are emerging about the involvement of the initiation phase of translational processes in the control of cell proliferation. In this paper we review the novel insights on the biochemical and molecular events leading to the initiation and its involvement in cell proliferation and tumourigenesis. We describe the structure, regulation and proposed functions of the eukaryotic initiation factor 5A (eIF-5A) focusing the attention on its involvement in the regulation of apoptosis and cell proliferation. Moreover, we describe the modulation of its activity (through the reduction of hypusine synthesis) in apoptosis induced either by tissue transglutaminase or interferon α. Finally, we propose eIF-5A as an additional target of anti-cancer strategies.

Keywords: Keywords: Amino acids; eIF-5A; Hypusine; tTGase; Apoptosis; IFNα; Tumour cells

Importance of direct determination of amino acid co- and counter-transport stoichiometries by L. J. Van Winkle (pp. 105-111).

The stoichiometry of amino acid transport with co- or counter-substrates of a given system has been found to vary with the amino acid species. This phenomenon has been studied directly in only a few cases, however, by measuring the fluxes of the substrates simultaneously. More frequently, the apparent transport stoichiometries of single amino acid species with co- or counter-substrates are estimated indirectly by thermodynamic criteria or cooperative kinetic effects. Unfortunately the latter indirect mea-sures of apparent stoichiometry often yield different results than direct measurement of simultaneous fluxes. These differences often cannot be explained by uncoupled transport of one of the co- or counter-substrates or by other characteristics of the transport process that would make the direct measurement of stoichiometry inaccurate. For these reasons, investigators are encouraged to measure the stoichiometry of transport directly by measuring simultaneous fluxes of co- and counter-substrates. Indirect measures of apparent stoichiometry may, however, reflect important details of a transport mechanism even if they are inconsistent with the actual stoichiometry of transport.

Keywords: Keywords: Amino acids; Kinetics; Thermodynamics; Transport; Transporters

Syntheses of glycine and L-serine by their interconversion in the posterior silkgland of the silkworm, Bombyx mori by M. Osanai; M. Okudaira (pp. 113-121).

It was observed by solution-state ¹³C NMR spectroscopy that a great portion of the ¹³C of [1-¹³C]L-serine fed to the 5th instar larvae of the silkworm, Bombyx mori was incorporated into C1 of glycine in silk fibroin. [1-¹³C]Glycine was detected along with [1-¹³C]serine in fibroin of the posterior silkgland cultured in a medium containing [1-¹³C]serine. This formation of [1-¹³C]glycine was inhibited by addition of aminopterin to the culture medium. These findings suggest that an active conversion from serine to glycine, which needs tetrahydrofolate, occurs in the posterior silkgland for fibroin synthesis. Moreover, the solid-state ¹³C CP/MAS spectrum of the fibroin prepared from cocoons spun by larvae fed with [¹³C]formate revealed that serine C3 was labelled specifically with ¹³C, suggesting that the reverse conversion from glycine to serine took place in the silkworm. The posterior silkgland has the ability to synthesize not only fibroin but also its major materials, glycine and serine.

Keywords: Keywords: Amino acids; Glycine-serine interconversion; Tetrahydrofolate; Aminopterin; Formate; Fibroin; Silkgland of Bombyx mori

Effects of vitamins on hepatic nuclear binding of L-tryptophan by H. Sidransky; E. Verney (pp. 123-134).

This study investigated the in vitro effects of selected vitamins on nuclear L-tryptophan receptor binding of rat liver. Our results revealed that some fat-soluble vitamins, β-carotene, retinyl acetate, calciferol, α-tocopherol, and Trolox, as well as some water-soluble vitamins, thiamine and riboflavin, acted to inhibit in vitro ³H-tryptophan binding to hepatic nuclei. On the other hand, pyridoxine had little or no effect. The addition of dithiothreitol, a protective agent for sulfhydryl groups, along with each vitamin decreased the vitamin's inhibitory effect on in vitro ³H-tryptophan binding to nuclei, with the exception of riboflavin and calciferol. The addition of L-leucine, which alone had no inhibitory effect on in vitro ³H-tryptophan binding to hepatic nuclei but when added with unlabeled L-tryptophan negated the effect of unlabeled L-tryptophan, caused a markedly diminished inhibitory binding effect due to each of the following vitamins, thiamine, β-carotene, retinyl acetate, and α-tocopherol and Trolox, but no effect on riboflavin and calciferol.

Keywords: Keywords: Amino acids; Vitamins; L-Tryptophan; Hepatic nuclear binding; Rats

Novel N-quinonyl amino acids and their transformation to 3-substituted p-isoxazinones by S. Gorohovsky; S. Bittner (pp. 135-144).

Quinonyl amino acids are building blocks in the preparation of peptides which target the quinonic drug to cancer damaged area. Novel N-(3-chloro-1,4-dihydro-1,4-dioxonaphthalen-2-yl)-α-amino acids 1a–f were prepared by direct substitution of 2,3-dichloro-1,4-naphthoquinone. The quinonic moiety was reduced by NaBH₄ to yield the corresponding hydroquinones 2a–f, which in acidic conditions underwent internal cyclization to yield the 3,4-dihydro-2H-naphth[1,2-b]-1,4-oxazine-2-ones (six-membered azlactones) 3a–f.

Keywords: Keywords: Amino acids; Quinones; Hydroquinones; Isoxazinones; Azlactones

Specific peptide inhibitors of trypanothione reductase with backbone structures unrelated to that of substrate: potential rational drug design lead frameworks by J. H. McKie; J. Garforth; R. Jaouhari; C. Chan; H. Yin; T. Besheya; A. H. Fairlamb; K. T. Douglas (pp. 145-153).

By introducing cationic charge sites novel peptide lead inhibitor structures for trypanothione reductase have been designed using molecular modelling methods. The inhibitors showed reversible, linear competitive inhibition and the strongest peptide inhibitor to date was found to be N-benzyloxycarbonyl-Ala-Arg-Arg-4-methoxy-β-naphthylamide with a K_i value of 2.4 μM and a selectivity for parasitic enzyme (trypanothione reductase) over the host enzyme (human glutathione reductase) of over 3 orders of magnitude.

Keywords: Keywords: Amino acids; Drug leads; Glutathione; Antiparasitics; Trypanosomiasis; Leishmaniasis

Analysis of the disease course of HIV-1 by entropic chaos degree by K. Sato; M. Ohya (pp. 155-162).

When a V3 sequence obtained on the n-th year after infection with human immunodeficiency virus type 1 (HIV-1) was supposed to change into a V3 sequence on the n + 1-th year, the variation between the above two sequences was analyzed by means of entropic chaos degree. The entropic chaos degree measures chaotic aspects of the dynamics causing the variation of sequence. If it is large, then the dynamics produces the large complexity, in other words, the variation of sequences becomes large.As a result, the chaos degree for the dynamics changing the V3 region showed the specific variation patterns throughout from the early stages of infection to death. That is, the variation patterns indicated that the entropic chaos degree is useful to measure the stage of disease progression after HIV-1 infection.

Keywords: Keywords: Amino acids; HIV-1; Entropic chaos degree

A fast and sensitive method for measuring picomole levels of total free amino acids in very small amounts of biological tissues by G. H. Fisher; I. Arias; I. Quesada; S. D'Aniello; F. Errico; M. M. Di Fiore; A. D'Aniello (pp. 163-173).

In the present study we describe a simple and fast method to measure the concentration of total free amino acids in very small amounts of biological tissues. The procedure described here is based on the reaction of free amino acids with o-phthaldialdehyde (OPA) in the presence of a reducing agent, β-mercaptoethanol (MET), to give a complex which can be measured by fluorescence. It is a very rapid process and has the same reliability as the conventional ninhydrin method of Moore and Stein but is about 500 times more sensitive. The sensitivity of the new protocol is such to permit the determination with high reliability of very small amounts of free amino acids at picomole levels, either in a standard amino acid mixture or in biological tissues, without chromatographic separation of the amino acids. It is particularly useful when the amount of the sample is very low, e.g. on a single pituitary or pineal gland of small animals or on single cells, such as oocytes or eggs, as well as single ganglions or axons of marine invertebrates.

Keywords: Keywords: Amino acids; Fluorometric method; OPA-β-Mercaptoethanol; Ninhydrin

Establishment of a rat hepatoma-derived cell line proliferating in d-phenylalanine medium and expressing d-amino-acid oxidase by N. Yoda; R. Konno; S. Nagashima (pp. 175-185).

A cell line (R-Y121B·DF) has been established from a cell line (R-Y121B) derived from a rat hepatoma line (H4-II-E). The R-Y121B·DF cells have been continuously cultured in a serum-free modified Eagle's minimum essential medium in which l-phenylalanine was replaced by d-phenylalanine. They had d-amino-acid oxidase (DAO) activity which is essential for the growth in the medium containing d-amino acids. The enzyme activity of the R-Y121B·DF cells was approximately one-fourth of that of the rat liver. Northern hybridization using a DAO cDNA probe detected a hybridizing signal in the R-Y121B·DF cells and the rat liver but not in the parental R-Y121B and H4-II-E cells. Reverse transcription-polymerase chain reaction using DAO-specific primers amplified a DNA fragment of the expected size in the R-Y121B·DF cells but not in the R-Y121B and H4-II-E cells. This fragment was confirmed to be DAO cDNA by nucleotide sequencing. Western blotting showed that DAO protein was present in the R-Y121B·DF cells and the rat liver but not in the R-Y121B and H4-II-E cells. Southern hybridization showed that the DAO gene structure was not different among the R-Y121B·DF cells, R-Y121B cells, H4-II-E cells, and the rat liver. These results indicate that the R-Y121B·DF is a unique cell line which proliferates in the medium containing d-phenylalanine and explicitly expresses DAO. This line is useful for the study of DAO in vitro.

Keywords: Keywords: Amino acids; Hepatoma cell line; d-Amino-acid oxidase; Rat; d-Phenylalanine; Gene expression

High-performance liquid chromatographic quantification of allysine as bis-p-cresol derivative in elastin by H. Umeda; K. Kawamorita; K. Suyama (pp. 187-199).

The first step in normal cross-linking in elastin is the formation of α-aminoadipic-δ-semialdehyde, allysine, through oxidative deamination of specific peptidyl lysine by the enzyme lysyl oxidase (EC 1.4.3.13). For the analysis of allysine, allysine was derivatized with p-cresol. The derivatization was carried out by acid hydrolysis (6N HCl containing 5% (w/v) p-cresol at 110°C for 48 h) accompanied with the hydrolysis of elastin. A bis-p-cresol derivative of allysine was isolated from bovine ligamentum nuchae elastin hydrolysates, and was characterized by UV, FAB-MS and NMR. This derivative was identified as 2-amino-6,6-bis(2-hydroxy-5-methylphenyl)hexanoic acid. A rapid, sensitive reverse-phase high-performance liquid chromatographic method with UV detection was developed for the quantitative determination of allysine as its bis-p-cresol derivative. The lower limit of detection of the bis-p-cresol derivative was 58 pmol in the standard sample with a 20-μl injection at a signal-to-noise ratio of 3. This method was applied to the determination of allysine in bovine ligamentum nuchae, aorta, lung, and rat aorta elastin. The allysine content in rat aorta elastin dramatically increased from 1 week to 2 weeks of age.

Keywords: Keywords: Amino acids; Allysine; Cross-link; Desmosine; Elastin; Isodesmosine

Renal amino acid transport in immature and adult rats during chromate and cisplatinum-induced nephrotoxicity by Ch. Fleck; I. Kretzschel; T. Sperschneider; D. Appenroth (pp. 201-215).

The effects of sodium dichromate (chromate; 1 mg/100 g b. wt. s.c.) and cisdiamminedichloroplatinum(II) (CP; 0.6 mg/100 g b. wt. i.p.) on renal amino acid excretion and plasma amino acid composition were investigated in 10- and 55-day-old anaesthetised rats. On the basis of diuresis experiments on conscious rats the mentioned doses and times (1^st day after chromate in both age groups and in 10-day-old rats after CP and 3^rd day after CP in adult rats) were found out to be optimal for the characterisation of amino acid transport after heavy metal poisoning. Interestingly, in conscious 10-day-old rats chromate nephrotoxicity is not detectable after 1 mg/100 g b. wt. whereas all of the other experimental groups showed nephrotoxic effects of chromate and CP in conscious rats. Urine volumes are lower, but not significantly, in anaesthetised immature rats, independently of the administered nephrotoxin. But GFR is significantly lower in 10-day-old rats, both in controls and after CP, whereas after chromate GFR is significantly reduced only in adult rats and age differences disappeared. In principle the renal fractional excretion (FE) of amino acids was distinctly higher in immature rats as a sign of lower amino acid reabsorption capacity. Nevertheless, the amino acid plasma concentrations were relatively high in immature rats. However, both chromate and CP did not distinctly influence amino acid plasma concentrations. But in both age groups the administration of chromate and CP significantly decreased amino acid reabsorption capacity (increase in FE) as a sign of nephrotoxicity, most pronounced in adult rats after CP. The investigation of renal amino acid handling confirms (1) that both CP and chromate are nephrotoxins, (2) that CP was more nephrotoxic in 55-day-old animals compared to immature rats as could be demonstrated before using other parameters for nephrotoxicity testing and showed (3) that determination of renal amino acid handling is a highly sensitive marker for nephrotoxicity testing, especially in immature rats.

Keywords: Keywords: Amino acids; Cisplatinum; Chromate; Nephrotoxicity; Amino acid transport; Kidney; Ontogeny; Rats

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.20, #2)