The anticlastogenic effect of various combinations of cysteamine,AET, HCT,and amino acids on chromosome damage by Trenimon and bleomycin in human lymphocytes in vitro

Summary The protective activity of the combined application of anticlastogens against the chromosome-damaging action of Trenimon and bleomycin was studied by analyzing more than 32000 metaphases from cultures of human peripheral lymphocytes. Screening tests with the combinations cysteine/cysteamine/AET, cysteine/methionine/asparagine, cysteine/serine/HCT, and AET/HCT, with Trenimon as clastogen, in no case revealed a hyperadditive (synergistic) effect. From the results of detailed analyses of the action of the combination AET/HCT it was concluded that the (nonsynergistic) anticlastogenic effects observed were induced due to intracellular biologic mechanism, and not due to a reaction in the culture fluid between clastogens and anticlastogens. Although the observations gained with Trenimon or bleomycin differed in some respects, the anticlastogens apparently act via a mechanism at least common to AET and HCT, i.e., they manifest their effect in lymphocyte cultures by a limited interaction with the process of aberration formation, rather than by influencing repair processes, which are blocked by caffeine.Dedicated to Professor Dr. med. Gerhard Koch on the occasion of his 65th birthday     

A comparison of alternative distributions of postimplantation death in the dominant lethal assay

The action of beta-aminoethylisothiouronium bromide hydrobromide (AET) and sodium fluoride (NaF) on the clastogenic activity of Trenimon, cyclophosphamide, and bleomycin was tested on cultures of human peripheral lymphocytes with and without the addition of rat liver S9 mix. In addition, the influence of both anticlastogens on the SCE-inducing activity of Trenimon and cyclophosphamide was examined under the same conditions. In the absence of S9 mix both substances displayed the known anticlastogenic action when TR was the standard clastogen but acted coclastogenically in the experiments with BM. Under the influence of rat-liver S9 mix this action on TR-induced chromosome damage was decreased and only a slight anticlastogenic effect was observed in the experiments with activated cyclophosphamide. S9-activated BM lost some of its strong chromosome-damaging effect and AET proved clearly anticlastogenic under these test conditions. AET displayed a slight decreasing effect on SCE induced by TR, but had no effect on CP-induced SCE. No anti-SCE effect at all was found in the experiments with NaF. Detailed analyses revealed different actions of both anticlastogens on the different types of structural chromosome damage.     

Untersuchungen über die Beeinflussung der chromosomenschädigenden Aktivität von Trenimon® an menschlichen Lymphocyten in vitro durch Aminosäuren

Zusammenfassung Der Einfluß der Aminosäuren L-Alanin, L-Arginin, L-Asparagin, L-Glutaminsäure, L-Histidin und L-Methionin auf die cytogenetische Wirkung von Trenimon^® (2,3,5-Tris-äthylenimino-benzochinon-1,4) wurde an menschlichen Lymphocyten in vitro untersucht. Unter dem Einfluß von L-Asparagin und L-Methionin im Überschuß wurde (bei gleichzeitiger Einwirkung mit dem Aberrationsinduktor) die chromosomenschädigende Wirkung des Trenimon deutlich reduziert. Zwischen Protektordosis und Protektoreffekt bestand dabei keine klare Korrelation. Die beiden Aminosäuren entfalten eine unterschiedliche Effektivität gegenüber den verschiedenen Defekttypen. Untersuchungen zur Zellcyclussperzifität der Protektorwirkung von Methionin erbrachten ein unterschiedliches Effektivitätsmuster in verschiedenen Zellcyclusphasen.Für die Aminosäuren L-Alanin, L-Arginin, L-Glutaminsäure und L-Histidin ließ sich eine mehr (Ala,Arg) oder weniger (Glu,His) deutliche Verstärkung des chromosomenschädigenden Effektes von Trenimon (bei gleichzeitiger Einwirkung) nachweisen. Dabei fiel insbesondere die Zunahme Trenimon-induzierter Interchanges unter dem Einfluß der genannten Aminosäuren auf.

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Studies on the influence of some amino acids on the chromosome-damaging activity of trenimon^® in human lymphocytes in vitro

Summary The influence of the amino acids l-alanine, l-arginine, l-asparagine, l-glutamic acid, l-histidine and l-methionine on the cytogenetic action of Trenimon^® (2,3,5-triethylene-imine—1,4-benzoquinone) was tested in human lymphocytes in vitro. An excess of l-asparagine and l-methionine added to the cultures simultaneously with the inductor of aberrations distinctly reduced the chromosome-damaging effect of Trenimon. There was no clear correlation between protector dose and protector effects. The two amino acids exerted different effects on the various types of aberrations. Studies on the specificity of the protector activity of l-methionine during the cell cycle showed different patterns of sensibility for the different phases of the cell cycle.In contrast to these results the effect of Trenimon was increased quite markedly by l-alanine and l-arginine, and to a lesser extent by l-glutamic acid and l-histidine. Under the influence of these amino acids, the number of interchanges induced by Trenimon was strikingly increased.

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Der Firma Bayer, Leverkusen, danke ich für die Überlassung von Trenimon^®.     

Asparagine transport in Lactobacillus plantarum and Streptococcus faecalis

The properties of het asparagine transport systems in Lactobacillus plantarum and Streptococcus faecalis are described. In both organisms the uptake of isotopically labeled l-asparagine was markedly stimulated by glucose. Kinetic studies yielded curvilinear Lineweaver-Burk plots in both organisms. These data were most consistently accounted for in both organisms by assuming the operation of two catalytic uptake components in addition to a diffusion component. The occasional limitation of kinetic studies in distinguishing between single or multiple catalytic components is illustrated. A large selection of structurally related amino acids and other substances were tested as competitors in initial rate studies. In L. plantarum the most effective competitors. structurally related dicarboxylic acid amide derivatives were only moderately effective competitors. In contrast, the most effective competitors of l-arparagine uptake in S. faecalis were relatively small neutral amino acids such as l-alanine, l-serine. l-αaminobutyric acid, l-cysteine and l-methionine, suggesting that asparagine enters this organism by reaction with a catalyst having relatively unspecific structural discrimination among neutral amino acids. Both organisms rapidly converted a large proportion of the transported asparagine to aspartic acid. In S. faecalis, the deamidation of l-asparagine was shown to be relatively insensitive to inhibition by those amino acids which were most effective in reducing the asparagine entry rate.     

l-Cysteine-induced up-regulation of the low-density lipoprotein receptor is mediated via a transforming growth factor-alpha signalling pathway

Sulphur-containing amino acids regulate plasma cholesterol levels in animals and humans. However, their mechanism of action remains unclear. Low-density lipoprotein receptor (LDLR) plays an important role in cholesterol metabolism. We therefore investigated the effects of sulphur-containing amino acids on the expression of LDLR in hepatocytes. HepG2 cells were cultured in Dulbecco’s Modified Eagle’s Medium with or without sulphur-containing amino acids and cysteine-containing compounds. We found that l-cysteine increased LDLR mRNA and enhanced LDLR gene promoter activity through the extracellular-signal-related kinase and p38 mitogen-activated protein kinase signalling pathways in HepG2 cells. Moreover, we observed that l-cysteine stimulated the release of transforming growth factor-alpha (TGF-α) and that TGF-α increased the LDLR mRNA levels. This study provides a report of the l-cysteine mediated up-regulation of the LDLR expression via TGF-α signalling pathway. Our findings provide insights into cholesterol homeostasis and amino acid signalling.     

The clastogenic effect of 5-methoxypsoralen plus UV-A in human lymphocytes in vitro and its modification by the anticlastogen β-aminoethylisothiouronium

Summary The treatment of human lymphocytes in vitro with 5-methoxypsoralen (5-MOP) plus UV-A induces a dose-dependent increase in the SCE rate and in structural chromosome aberrations. We carried out tests to see whether the clastogenic effect of 5-MOP plus UV-A could be reduced by the anticlastogen -aminoethylisothiouronium (AET). The occurrence of a protective effect proved to be dependent upon the conditions of treatment. When AET was present over a long period of time (22h) in cultures with 5-MOP, the number of breaks was reduced compared with such cultures without AET (reduction factor 0.5–0.6). On the other hand, a short period of action by AET (1.5 h) in the presence of 5-MOP produced no reduction of breaks. Posttreatment with AET (20 h) yielded an obvious protective effect (reduction factor 0.2–0.4). The possible mechanisms of the protective effect of AET are discussed.     

The protective effect of energy substrates, vitamins, coenzymes and their complexes in the action on the body of the factors of an enclosed space

Experiments on mice were performed to study a protective action of amino acids and other oxidation substrates (L-aspartic acid, pyruvate, succinate, GABA, alpha-ketoglutarate), metabolites (pyridoxal-5'-phosphate) as well as vitamin-coenzyme complexes in combination with oxidation substrate while being under closed space conditions. GABA, aspartate, glutamate possessed the highest protective effect as against alpha-ketoglutarate and succinate.     

Deamination of amino acids by Bacillus pasteurii

Resting cells of Bacillus pasteurii as employed in the treatment of distillery waste showed deamination of amino acids. The deamination of l-glutamic acid and dl-aspartic acid was found to be oxidative while that of dl-serine, dl-threonine and l-asparagine was non-oxidative. dl-Alanine and glycine were not deaminated when present individually but when incubated together showed oxidative deamination. NAD stimulated the oxidation of l-glutamic acid and α,α′-dipyridyl completely inhibited it.     

Comparative studies on the specificity of anticlastogenic action in human lymphocytes in culture

Summary Comparative studies on human lymphocyte cultures yielded a certain specificity of the anticlastogenic action of the SH compounds 1-cysteine, cysteamine, and -aminoethylisothiouronium (AET) as well as of the amide 1-asparagine and the amino acid 1-methionine. This specific anticlastogenic activity manifested itself in specific changes of the spectrum of aberration types induced by the clastogens and of the pattern of intercellular distribution of the induced aberrations. It was clearly dependent on the concentration of the anticlastogens but was also influenced by the used clastogen. The use of different culture media yielded some quantitative influences on the anticlastogenic activity, but fundamental changes in the spectrum of anticlastogenic action have not been observed except with cysteamine. The patterns of activity ascertained for the different anticlastogens specifically differed from those changes in the spectrum and pattern of distribution of aberrations induced by a mere reduction of the concentration for instance of Trenimon. Therefore a direct reaction between the protectors and the clastogen Trenimon as the cause of the observed anticlastogenic action was again excluded. The presented data are also discussed under the aspects of the hypotheses of aberration induction as well as of their importance for further antimutagen research.Some parts of this paper have been supported by grants of Deutsche Forschungsgemeinschaft.     

Nitrogen metabolism in plant cell suspension cultures: I. Effect of amino acids on growth

Certain amino acids inhibit growth of tobacco (Nicotiana tabacum L. var. xanthi), tomato (Lycopersicon esculentum) carrot (Daucus carota), and soybean (Glycerine max L. co. Mandarin) cell cultures when nitrate or urea are the nitrogen sources but not when ammonia is the nitrogen source. These amino acids also inhibit development of nitrate reductase activity (NADH:nitrate oxidoreductase EC 1.6.6.1) in tobacco and tomato cultures. Threonine, the most inhibitory amino acid, also inhibits nitrate uptake in tobacco cells. Arginine, and some other amino acids, abolish the inhibition effects caused by other amino acids. We suggest that amino acids inhibit assimilation of intracellular ammonium into amino acids in cells grown on nitrate or urea.     

Alteration of Carbon Metabolism by a Base Analog

The study of carbon metabolism by cultures of the yeast C. utilis exposed to 5-fluorouracil revealed that the growth rate and synthesis of macromolecules was altered. The amino acid composition of the metabolic pool of amino acids was vastly altered, but the protein composition was unchanged. It is suggested that the analog may exert a selective action on certain amino acids, and that this action may be related to a template-like mechanism.     

Die Schutzwirkung von Cysteamin und β-Aminoäthylisothiouronium (AET) gegen die chromosomenschädigende Aktivität von Trenimon® in menschlichen Lymphocyten in vitro

Zusammenfassung Die als Cytostatikum verwendete alkylierende Substanz Trenimon^® (2.3.5-Tris-äthylenimino-benzochinon-1,4) verursacht an menschlichen Chromosomen in hohem Maße sekundäre Strukturaberrationen. Durch Zugabe von Cysteamin und AET in Kulturen menschlicher Lymphocyten in vitro war es möglich, die aberrationsauslösende Wirkung des Trenimon^® zu reduzieren.Der Schutzeffekt beider Substanzen erstreckte sich auf verschiedene Aberrationsparameter wie Gaps, Brüche und Chromosomenumbaufiguren (Exchanges). Eine Abhängigkeit des Schutzeffektes von der Protektorkonzentration konnte für beide Substanzen nachgewiesen werden. Die cytostatische Wirkung des Trenimon^® wurde sowohl durch AET als auch Cysteamin nicht beeinträchtigt.

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The protective effect of cysteamine and -aminoethylisothiouronium (AET) on the chromosome-damaging activity of Trenimon^® Dose-effect-ratios

Summary The cytostatic alkylating substance Trenimon^® (2,3,5-tristhyleneimine-1,4-benzoquinone) is extremely detrimental to human chromosomes to a high degree. Addition of cysteamine or aminoethylisothiouronium (AET) made reduction of this aberration-inducing activity of Trenimon possible. The protective effect of both cysteamine and AET was demonstrated in different types of aberrations (gaps, breaks, and interchanges). It was dependent on the concentration of the protective agent in each case. The cytostatic activity of Trenimon^® was affected by neither AET nor cysteamine.

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Herrn Professor Dr. G. Koch zum 60. Geburtstag gewidmet.

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Die Arbeit enthält die Ergebnisse einer Dissertation von R. Becher, die 1972 der Medizinischen Fakultät der Universität Erlangen vorgelegt wurde.     

Identification of Bacillus thuringiensis Delta-Endotoxin Cry1C Domain III Amino Acid Residues Involved in Insect Specificity

Cry1C domain III amino acid residues involved in specificity for beet armyworm (Spodoptera exigua) were identified. For this purpose, intradomain III hybrids between Cry1E (nontoxic) and Cry1E-Cry1C hybrid G27 (toxic) were made. Crossover points of these hybrids defined six sequence blocks containing between 1 and 19 of the amino acid differences between Cry1E and G27. Blocks B, C, D, and E of G27 were shown to be required for optimal activity against S. exigua. Block E was also required for optimal activity against the tobacco hornworm (Manduca sexta), whereas block D had a negative effect on toxicity for this insect. The mutagenesis of individual amino acids in block B identified Trp-476 as the only amino acid in this block essential, although not sufficient by itself, for full S. exigua activity. In block D, we identified a seven-amino-acid insertion in G27 that was not in Cry1E. The deletion of either one of two groups of four consecutive amino acids in this insertion completely abolished activity against S. exigua but resulted in higher activity against M. sexta. Alanine substitutions of the first group had little effect on toxicity, whereas alanine substitutions of the second group had the same effect as its deletion. These results identify groups of amino acids as well as some individual residues in Cry1C domain III, which are strongly involved in S. exigua-specific activity as well as sometimes involved in M. sexta-specific activity.     

NEUROTOXICITY OF A NON-METABOLIZABLE AMINO ACID, 1-AMINOCYCLOPENTANE-1-CARBOXYLIC ACID: ANTAGONISM BY AMINO ACIDS IN CULTURES OF CEREBELLUM

Abstract— The non-metabolizable amino acid 1-aminocyclopentane-1-carboxylic acid (ACPC) induced degeneration of myelinated axons but spared nerve cell bodies in well myelinated organotypic cultures of cerebellum. The ACPC concentrations used were comparable to those which induce axonal degeneration in vivo. Developing unmyelinated cultures were more sensitive to ACPC than mature cultures and newly myelinating axons appeared to be particularly affected. Supplementing the medium with amino acids, but not with vitamins, prevented toxicity at the lower concentrations of ACPC and afforded considerable protection against the highest concentrations. The protective effect of amino acids could not be accounted for by inhibition of intracellular ACPC transport. These results are considered in terms of other evidence indicating defective protein metabolism in ACPC-treated mice.     

Involvement of phospholipase A2 in the release of silymarin to the culture medium of Silybum marianum cell suspensions

In suspension cell cultures of Silybum marianum, methyl jasmonate (MJ) stimulated the accumulation and release of silymarin (Sm) to the culture medium. This study shows that phospholipase A2 (PLA2) plays a role in the release of Sm in elicited cultures. PLA2 activity increased in cell suspensions treated with MJ. Addition of aristolochic acid (AA) or bromoenol lactone (BEL) compounds that inhibit PLA2 activity impeded silymarin release. The addition of linoleic or linolenic acid reversed the inhibitory action of AA. Fatty acids (FAs) stimulated Sm release when added alone to control cultures. By contrast, oleic acid and saturated FA were ineffective in emulating MJ action.     

The C-Terminal Part of Microcin B Is Crucial for DNA Gyrase Inhibition and Antibiotic Uptake by Sensitive Cells

Microcin B (McB) is a ribosomally synthesized antibacterial peptide. It contains up to nine oxazole and thiazole heterocycles that are introduced posttranslationally and are required for activity. McB inhibits the DNA gyrase, a validated drug target. Previous structure-activity analyses indicated that two fused heterocycles located in the central part of McB are important for antibacterial action and gyrase inhibition. Here, we used site-specific mutagenesis of the McB precursor gene to assess the functional significance of the C-terminal part of McB that is located past the second fused heterocycle and contains two single heterocycles as well as an unmodified four-amino-acid C-terminal tail. We found that removal of unmodified C-terminal amino acids of McB, while having no effect on fused heterocycles, has a very strong negative effect on activity in vivo and in vitro. In fact, even nonconservative point substitutions in the last McB amino acid have a very strong effect by simultaneously decreasing uptake and ability to inhibit the gyrase. The results highlight the importance of unmodified McB amino acids for function and open the way for creation of recombinant McB derivatives with an altered or expanded spectrum of antibacterial action.     

Growth of Escherichia coli cells in the presence of cysteine on sulphate-deficient media

Summary The behaviour of E. coli B culture grown on SO₄ ^2--free minimal glucose-salt medium was examined in the presence of exogenous cysteine at various concentrations. This was done by means of using the following parameters: length of lag, growth rate and total population. Up to a concentration of cysteine at 0.2mm the growth sets in without a lag phase, the growth rate is optimal (identical with that of cultures grown on media containing Na₂SO₄ as source of sulphur), only the size of total population being decreased by cysteine. At concentrations of 0.2mm and upwards, after a concentration-dependent lag-period, the cultures were found to increase at various lower growth rates.The toxic effect of cysteine was reduced by leucine itself, as well as by a mixture of leucine, isoleucine, valine and threonine. The anti-cysteine action of these amino acids showed itself in the shortening of the lag period and in the recovery of the growth rate which, however, failed to reach the original level.Cysteamine failed to provide sulphur for cultures of E. coli B grown on the above medium. Neither was the utilization of cysteamine affected by the application of amino acids possessing an anti-cysteine action.We have postulated that beside the inhibition of the biosynthesis of amino acids having an anti-cysteine effect, toxic concentrations of cysteine posses additional sites of action.     

Regulation of cell proliferation and morphogenesis by amino acids inBrassica tissue cultures and its correlation with threonine deaminase

The effect of amino acids has been investigated with respect to the capacity ofBrassica cultures to undergo proliferation and differentiation. Hormone medium without any amino acid resulted in 6% shoot formation. Addition of optimal concentrations of L-leucine and L-isoleucine enhanced shoot formation upto 30% and 60%, respectively. L-methionine, L-threonine and pyruvic acid supported only proliferation but no differentiation. Amino acids had a marked effect on the activity of enzyme threonine deaminase (TD), bothin vivo andin vitro. TD in proliferating callus cultures was 3-fold higher than in differentiating cultures. Amino acids which induced cell proliferation increased TD while those which supported differentiation repressed it. Amino acids which did not alter TD activity had no effect on morphogenesis. The results suggest that amino acids play a regulatory role inBrassica morphogenesis which can be correlated with the activity of threonine deaminase.     

The effect of L-lysine alpha-oxidase from Trichoderma cf. aureoviride Rifai VKM F-4268D on the rat pheochromocytoma PC12 cell line

L-Amino acid oxidases (L-AAO; EC 1.4.3.2) comprise a group of flavoproteins that catalyze oxidative deamination of L-alpha amino acids to corresponding alpha-keto acids, NH₃ and H₂O₂. Most of these enzymes are homodimers with molecular mass of 100–150 kDa that exhibit antiviral, antifungal, antibacterial, and anticancer activity. Among this group of enzymes L-lysine alpha-oxidase (LO) is especially important as its biological effects may differ from the effects of other L-AAO, because this enzyme preferentially oxidizes L-lysine, the essential amino acid for the human body, without any practical effect on other amino acids. Since molecular mechanisms of the cytotoxic action of LO still require better understanding, in this study we have investigated a possible mechanism of action of LO from Trichoderma cf. aureoviride Rifai VKMF-4268D. A rat pheochromocytoma PC12 cell culture was used as a model. Using flow cytometry a dose-dependent cell death induced by LO was shown. The increase in intracellular reactive oxygen species detected by the 2,7-dichlorodihydrofluorescein assay suggests that the oxidative pathway is one of mechanisms underlying the cytotoxic LO action; however, this does not rule out the involvement of other (previously demonstrated) mechanisms of LO effects on cell death.