Investigating ß-N-Methylamino-l-alanine Misincorporation in Human Cell Cultures: A Comparative Study with Known Amino Acid Analogues.

Misincorporation of ß-N-methylamino-l-alanine (BMAA) into proteins has been proposed to be a mechanism of toxicity to explain the role of BMAA in neurodegenerative disease development. However, studies have shown that all detectable BMAA can be removed from proteins by SDS-PAGE purification and that the toxicity of l-canavanine cannot be reproduced in prokaryotes or in a rat pheochromocytoma cell line, strongly indicating that the misincorporation hypothesis of BMAA should be re-investigated. The aim of this study was therefore to determine if BMAA misincorporates into proteins in cells of human origin with subsequent misincorporation-type toxicity. Almost complete loss of viability in response to exposure to l-4-fluorophenylalanine and l-m-tyrosine was observed in all of the cell lines, corresponding to a concentration-dependent increase of the analogues in protein extracts from exposed cells. In contrast, BMAA exposure resulted in slight toxicity in one of the cell lines but the observed toxicity was not the result of misincorporation of BMAA into proteins, as no BMAA was detected in any of the SDS-PAGE purified protein extracts that were obtained from the cells following BMAA exposure. The results show that BMAA is not misincorporated into human proteins and that misincorporation is not a valid mechanism of toxicity.

Loss of heterozygosity of individual loci in Arabidopsis thaliana regenerants cultured with para-fluorophenylalanine.

Precise chromosome segregation is vital for speciation and hybrid formation. The aim of the work was to study the chromosomes behaviour and inheritance of maternal and paternal genomes in Arabidopsis regenerants de-rived from in vitro cultured cells on the medium with PFFA. The Arabidopsis thaliana model hybrid between Columbia and Landsberg erecta ecotypes was developed, which chromosomes were easy to distinguish using the 12 SSLP selected markers. Also, the influence of PFFA on the callus formation and regeneration of plants was analysed. 20 regenerated plants cultured with PFFA were derived, three of which were shown to loss the heterozygosity in six loci by DNA markers analysis. Different models are certainly required to understand how and when the mechanisms leading to proper chromosome segregation are established in species and hybrids.

Characterization of antinociceptive potency of endomorphin-2 derivatives with unnatural amino acids in rats.

This study reports on the in vivo effects of four endomorphin-2 (EM-2) derivatives (EMD1-4) containing unnatural amino acids, i.e. 2-aminocyclohexanecarboxylic acid (Achc2), para-fluorophenylalanine (pFPhe4), ß-methylphenylalanine (ßMePhe4) and/or 2',6'-dimethyltyrosine (Dmt1). After induction of osteoarthritis by monosodium iodoacetate into the ankle joint of male Wistar rats, a chronic intrathecal catheter was inserted for spinal drug delivery. The mechanical threshold was assessed by a dynamic aesthesiometer. Intrathecal injection of the original EM-2 and the ligands (0.3-10 µg) caused dose-dependent antiallodynic effects. The comparison of the different substances revealed that EMD3 and EMD4 showed more prolonged antinociception than EM-2, and the effects of the highest dose of EMD4 were comparable to morphine, while EMD3 caused paralysis at this dose. The potency of the different ligands did not differ from EM-2. The results show that the derivatives of EM-2 have similar in vivo potency to the original ligand, but their effects were more prolonged suggesting that these structural modifications may play a role in the development of novel endomorphin analogues with increased therapeutic potential.

Protein tyrosine kinase p56lck-deficiency confers hypersusceptibility to rho-fluorophenylalanine (pFPhe)-induced apoptosis by augmenting mitochondrial apoptotic pathway in human Jurkat T cells.

Phenylalanine analog, rho-fluorophenylalanine (pFPhe)-mediated cytotoxicity and several apoptotic events including mitochondrial cytochrome c release, activation of caspase-9, -3, and -8, Bid cleavage, degradation of PARP and PLCgamma-1, and DNA fragmentation were more significant in p56(lck)-deficient Jurkat T cells (JCaM1.6) than in wild-type Jurkat T cells (E6.1). The susceptibility of JCaM1.6 toward apoptogenic activity of pFPhe decreased after acquisition of p56(lck) by transfection. The p56(lck) kinase activity increased 1.6-fold at 15-30 min after pFPhe treatment. The pan-caspase inhibitor (z-VAD-fmk) completely blocked the pFPhe-mediated apoptotic changes except caspase-9 activation. The caspase-8 inhibitor (z-IETD-fmk), which failed to influence pFPhe-induced caspase-9 activation, completely blocked caspase-8 activation and PLCgamma-1 degradation with a marked reduction in caspase-3 activation and PARP degradation, indicating pFPhe-induced caspase-8 activation as a downstream event of mitochondria-dependent activation of caspase-9. These results indicate that the deficiency of p56(lck) augments pFPhe-induced mitochondrial cytochrome c release and resultant apoptotic cell death in Jurkat T cells.

Intracellular uptake and inhibitory activity of aromatic fluorinated amino acids in human breast cancer cells.

Nonproteinogenic amino acids that either occur naturally or are synthesized chemically are becoming important tools in modern drug discovery. In this context, fluorinated amino acids have great potential in the development of novel pharmaceuticals and drugs. To assess whether different fluorinated aromatic amino acid analogues of phenylalanine, tyrosine, and tryptophan are potentially interesting as therapeutic drugs, we examined their cytostatic and cytotoxic effects on the growth of the human breast cancer cell line MCF-7. Of all the tested analogues L-4-fluorotryptophan, L-6-fluorotryptophan and L-p-fluorophenylalanine effectively and irreversibly inhibited cell growth with IC(50) values in the low micromolar range (3-15 microM). Additionally, using L-4-[14C]fluorotryptophan, and L-6-[14C]fluorotryptophan, we discovered that the cellular uptake of these fluorinated amino acids occurs through active transport with a 70-fold excess of intracellular over extracellular concentrations. We identified system L as the responsible amino acid transporter. Our findings fully support the idea that fluorinated aromatic amino acid analogues are promising chemotherapeutics with the potential for use in combination with classical cancer therapy, and as new cytotoxic drugs for certain tumor types such as melanoma.

A novel transport and delivery mechanism underpins the effectiveness of prolyl-m-sarcolysyl-p-fluorophenylalanine (PSF) in a human melanoma xenograft nude-mouse model.

The alkylating peptide PSF shows very promising results in vitro on different cancer cells but its efficacy in animals has not been assessed. Here we evaluate the efficacy of PSF in human melanoma-bearing nude mice and examine the underlying mechanism. In melanoma-bearing nude mice, escalating doses of PSF showed dose-dependent responses and reached tumor regression with an optimal dose of 20 mg/kg for 1 month. A comparison of PSF with its free moiety m-sarcolysin and melphalan showed a highly significant advantage of PSF. Furthermore, dose fractionation yielded an even better control of tumor regrowth. In vitro studies unraveled an original delivery mechanism based on the rapid binding of PSF mainly due to red blood cells to form a pro-drug complex and the subsequent release of active metabolites by tumor-associated proteolytic enzymes. Blood kinetics showed one major metabolite partially released over time, while in the presence of melanoma cells three additional metabolites are generated. Interestingly, tumor-shed proteases also induce the production of these metabolites and varying combinations of enzyme inhibitors indicate the involvement of metallo- and other families of proteases in the delivery process. This particular transport and delivery of such an alkylating agent may have several benefits, mainly lowering the drug-free moiety in plasma and at the same time increasing its concentration in protease rich areas such as tumors.

Cloning and characterization of Fpmtr1, an amino acid transporter gene of Fusarium proliferatum (Gibberella intermedia).

Fpmtr1, an amino acid transporter gene from Fusarium proliferatum was strongly expressed during conidial germination and repressed in late stationary phase. To identify the specific function of this gene, DeltaFpmtr1 knock-out mutants were generated by gene replacement. Vegetative growth of the DeltaFpmtr1 mutants was normal both in liquid and on solid media, but conidial germination was delayed. The DeltaFpmtr1 mutants and the wild type were equally fertile when used as males in sexual crosses, however if the mutants were used as the female parent then the fertility of the cross decreased dramatically. Inactivation of Fpmtr1 abolished vegetative self-incompatibility in strain ITEM 2287 of F. proliferatum, but the DeltaFpmtr1 mutants were still vegetatively incompatible with the other strains of the fungus. Endophytic colonization capability of the mutants, assessed on maize seedlings also was adversely affected. These data suggest that Fpmtr1 is involved in multiple developmental processes related to both sexual and parasexual events in F. proliferatum. Furthermore, the fungus might have problems in adapting to a less than optimal environment if this otherwise dispensable transporter has been inactivated.

The rolB gene-induced overproduction of resveratrol in Vitis amurensis transformed cells.

Resveratrol is a stilbene, which prevents carcinogenesis at stages of tumor initiation, promotion and progression. In the present investigation, we developed cell cultures of wild-growing grape (Vitis amurensis Rupr.). The cultures produced low levels of resveratrol, up to 0.026% dry wt., i.e., comparable to levels reported for other plant cell cultures previously established. Different methods commonly used to increase secondary metabolite production (cell selection, elicitor treatments and addition of a biosynthetic precursor) only slightly enhanced cell productivity. Transformation of V. amurensis V2 callus culture by the rolB gene of Agrobacterium rhizogenes resulted in more than a 100-fold increase in resveratrol production in transformed calli. The rolB-transformed calli are capable of producing up to 3.15% dry wt. of resveratrol. We show that the capability to resveratrol biosynthesis is tightly correlated with the abundance of rolB mRNA transcripts. Tyrosine phosphatase inhibitors abolished the rolB-gene-mediated stimulatory effect, thus documenting for the first time the involvement of tyrosine phosphorylation in plant secondary metabolism.

Comparison of two alternative dominant selectable markers for wine yeast transformation.

Genetic improvement of industrial yeast strains is restricted by the availability of selectable transformation markers. Antibiotic resistance markers have to be avoided for public health reasons, while auxotrophy markers are generally not useful for wine yeast strain transformation because most industrial Saccharomyces cerevisiae strains are prototrophic. For this work, we performed a comparative study of the usefulness of two alternative dominant selectable markers in both episomic and centromeric plasmids. Even though the selection for sulfite resistance conferred by FZF1-4 resulted in a larger number of transformants for a laboratory strain, the p-fluoro-DL-phenylalanine resistance conferred by ARO4-OFP resulted in a more suitable selection marker for all industrial strains tested. Both episomic and centromeric constructions carrying this marker resulted in transformation frequencies close to or above 10(3) transformants per microg of DNA for the three wine yeast strains tested.

Construction of a Bacillus subtilis (natto) with high productivity of vitamin K2 (menaquinone-7) by analog resistance.

To invent a functional natto promoting bone formation, the construction of a strain with high productivity of vitamin K2 (menaquinone-7: MK-7), which is important in the carboxylation of a kind of bone protein participating in bone formation, osteocalcin, was investigated. To screen for a strain appropriate to making natto (a Japanese traditional fermented soybean food) with high productivity of MK-7, a combination of analog resistance to the compounds on the biosynthetic pathway of menaquinones with mutation was done. Consequently, strain OUV23481, with 2-fold higher productivity (1,719 microg/100 g natto) of MK-7 than that of a commercial strain, was constructed as a mutant with analog resistance to 1-hydroxy-2-naphthoic acid (HNA), p-fluoro-D,L-phenylalanine (pFP), m-fluoro-D,L-phenylalanine (mFP), and beta-2-thienylalanine (betaTA). This strain was classified as Bacillus subtilis (natto). The natto made using this strain was evaluated to have a good quality as natto in all the viewpoints of appearance, flavor, taste, texture, and stringiness.

Shikonin production by p-fluorophenylalanine resistant cells of Lithospermum erythrorhizon.

Studies were conducted with a BK-39 callus culture of Lithospermum erythrorhizon, which produced seven shikonin derivatives (acetylshikonin, propionylshikonin, isobutyrylshikonin, beta,beta-dimethylacrylshikonin, isovalerylshikonin, beta-hydroxyisovalerylshikonin and alpha-methyl-n-butyrylshikonin). A selection of cell aggregates of BK-39 culture on a medium containing p-fluorophenylalanine (PFP) yields a cell line possessing a higher resistance to the inhibitor than the initial culture. Selected BK-39F cultures produced almost the same profile of shikonin naphthoquinones as the initial culture. The shikonin derivative content of PFP-resistant culture was approximately two times higher than that of the control, reaching 12.6% of DW cell biomass.

Phenylalanine ammonia lyase production by gamma irradiated and analog-resistant mutants of Rhodotorula glutinis.

Mutants resistant to phenylalanine analogs (L-tyrosine, p-fluoro-D, L-phenylalanine (PFP) and trans-cinnamic acid) were isolated from a wild type strain of Rhodotorula glutinis A-97 by mutagenic treatment with gamma radiation and screened for phenylalanine ammonia lyase (PAL) production. One such mutant, gammaT11 (resistant to L-tyrosine), exhibited four times the PAL activity of the parent wild strain A-97. Mutant isolate gammaTFP5.6 which was selected as L-tyrosine and PFP resistant isolate, produced inducible PAL activity at levels 5.94-fold higher than the wild-type A-97 and 2.66-fold higher than its parent mutant isolate gammaT5 which was resistant to L-tyrosine. The mutant isolate gammaTC5d which was resistant to L-tyrosine and trans-cinnamic acid, exhibited 3.48 and 1.56-fold increase in PAL activity compared to the parent wild strain A-97 and its parent mutant isolate gammaT5, respectively. Different media have been examined for the induction of PAL.

The genetic activity of 6-N-hydroxylaminopurine in Aspergillus nidulans.

The activity of a base analog (6-N-hydroxylaminopurine, HAP) has been tested on Aspergillus nidulans. In germinating haploid conidia HAP is a strong mutagen, while it does not have any activity in resting conidia. Moreover, HAP does not increase the frequency of recombination in germinating conidia. The mutagenic activity of this base analog has also been tested in diploid conidia of A. nidulans; in fact, it has been shown (Pavlov et al., 1991) that the HAP-induced frequency of heteroallelic recessive mutations in diploid cells of the yeast S. cerevisiae is higher than expected. In A. nidulans, we did not observe any increase in the frequency of recessive homozygous fpaA/fpaA (p-fluorophenylalanine-resistant) mutants over the expected one, which has been calculated on the basis of the observed mutation frequency in the haploid strain.

Genetic transformation of industrial yeasts using an amino acid analog resistance gene as a directly selectable marker.

Prototrophic and often polyploid yeasts of industrial use require some dominant genes as directly selective markers for the transformation. We examined the applicability of a dominant gene, ARO4-OFP, which causes the resistance to PFP plus tyrosine, to direct selection of the transformants from 2 laboratory and 6 industrial strains, including bakers', distillers', winery, and saké yeasts. Although the transformation rates were low and seemed different among strains, the ARO4-OFP gene was applicable to all strains tested for direct selection of the transformants.

[Preparation and analysis of mutants of rhizospheric pseudomonads, resistant to toxic analogs of tryptophan and phenylalanine]. / Poluchenie i analiz mutantov rizosfernykh psevdomonad, ustoichivykh k toksichnym analogam triptofana i fenilalanina.

The absence of plasmids in strains of fluorescent pseudomonads characterized by high level of synthesis of phytohormone indole-3-acetic acid (IAA) as well as invariability of this feature in plasmid and non-plasmid variants of strain BSP8 suggests chromosomal control of IAA synthesis by the rhizosphere bacteria tested. Using toxic analogues of aromatic amino acids -5-fluorine-tryptophan and 5-methyl-tryptophan variants were obtained which synthesized and secreted only anthranilic acid. Mutants with resistance to p-fluorine-phenylalanine and capable of secreting tryptophan and/or phenylalanine were found. Testing of the secreting variants failed to reveal any differences between the levels of IAA biosynthesis in comparison with the wild-type strains.

Isolation and genetic study of p-fluoro-DL-phenylalanine-resistant mutants overproducing beta-phenethyl-alcohol in Saccharomyces cerevisiae.

p-Fluoro-DL-phenylalanine (PFP)-resistant mutants which produce a large amount of beta-phenethyl-alcohol, a rose-like flavor component, were isolated from the isogenic strains X2180-1A and X2180-1B of Saccharomyces cerevisiae. Cells of these mutants accumulated phenylalanine and tryptophan more than 3-fold times that of wild-type cells, while they accumulated less than half the tyrosine. The activity of prephenate dehydrogenase (PDG) (EC 1.3.1.12) was markedly decreased while that of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (EC 4.1.2.15) was increased. Genetic analysis revealed that the mutation occurred at the TYR1 locus, encoding PDG, and that the mutated TYR1 gene, try1-pfp, caused both PFP resistance and beta-phenethyl-alcohol overproduction. This was supported by molecular genetic studies with cloned tyr1-pfp DNA.

p-fluoro-phenylalanine resistance in Aspergillus nidulans diploid cells: evidence that dominant, lethal mutations are involved.

An unexpectedly large number of p-fluoro-phenylalanine (FPA)-resistant mutants have been recovered after UV-irradiation of wild type diploid conidia of Aspergillus nidulans. At least five different classes of mutants, possibly corresponding to five different loci, have been identified. Two of them may be the dominant loci which have already been described but the others (a minimum of three loci) are completely different. Mutations in these loci confer high level FPA resistance in the heterozygous diploids, being lethal in the haploids; one mutation has been preliminarily mapped to chromosome I and another to chromosome III.

Frequency of spontaneous and induced recessive mutations in a diploid strain of Aspergillus nidulans.

The spontaneous and UV-induced frequencies of recessive mutations have been studied in a diploid strain of Aspergillus nidulans, by the p-fluoro-phenylalanine (FPA) and 8-azaguanine (8-AZA) resistance tests, on either resting or germinating conidia. Observed frequencies are in the order of magnitude of those expected, which have been calculated considering the observed mutation frequencies in the haploid strain as well as the mitotic recombination frequencies. We also review some papers which claim to have found higher rates of recessive mutations in mammalian cell lines; in some cases no really higher rates are evident and the authors' conclusions often rest on misinterpretation of their own data.