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.

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.

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.

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.

Chromosome condensing ability of mitotic proteins diminished by the substitution of phenylalanine with parafluorophenylalanine.

One of the objectives of this study was to develop a method for the reversible arrest of HeLa cells in G2 phase by using p-fluorophenylalanine (FPA), an analog of phenylalanine. Addition of 0.5 mM of FPA to synchronized HeLa cells during the middle of S phase, i.e., 4 h after reversal of the double thymidine block, arrested 93% of the cells in G2 phase. After the drug was removed from the medium, these G2 cells proceeded into mitosis synchronously. The results of the study also indicate that incorporation of FPA into mitotic proteins results in the inhibition of chromosome condensation as assayed by their ability to induce premature chromosome condensation in G1 HeLa cells and meiotic maturation in amphibian oocytes.

A novel cloning vector for the direct selection of recombinant DNA in E. coli.

A new plasmid cloning vector (pHE3) is described carrying dominant p-fluorophenylalanine-sensitivity (pheS) and chloramphenicol-resistance markers. This vector is used in combination with a p-fluorophenylalanine-resistant Escherichia coli recipient (strain RR28). Foreign DNA can be cloned into pHE3 leading to insertional inactivation of pheS. Transformation of RR28, and plating on minimal medium with chloramphenicol plus p-fluorophenylalanine (pfp) directly selects for colonies containing recombinant DNA.

Evidence for an increase in presumed somatic mutation during the ageing of human cells in culture.

A method has been developed for the direct measurement of genetic variants in mammalian cells in culture which does not require cell division and is therefore suitable for ageing studies. Previous evidence suggests that these variants are mutations. The variant frequency was measured during the lifespan of populations of MRC-5 human embryo lung fibroblasts. The frequency increased substantially during the lifespan of the culture and regression analysis shows that the increase is exponential. No increase invariant frequency was found in a immortal transformed line cultured for over 100 passages. Evidence is presented that the variants are regulatory mutations. This is the first direct evidence for the involvement of presumed somatic mutations in the ageing of mammalian cells. 5-Fluorouracil but not p-fluorophenylalanine shortened the life of the cells and increase the variant frequency. The data lend support to a model in which mutations are an expected consequence of errors in protein synthesis.

The influence of fluoro-phenyl-alanine on the synthesis of simian virus 40 DNA and T antigens.

Treatment of Simian Virus 40 (SV40) infected monkey cells with fluorophenylalanine (FPA) resulted in increased uptake of thymidine by the cells, and progressive inhibition of both viral and cellular DNA synthesis. Viral DNA synthesis was more sensitive to inhibition by FPA than cell DNA synthesis. Synthesis of SV40 T antigens was however unaffected by FPA, as judged from immunofluorescence assays. The M.W. of the major polypetides immunoprecipitated from cell extracts by antibodies from tumor bearing hamster sera was similarly unaffected. It is suggested that T antigen synthesized in the presence of FPA is non functional.

Meiotic aneuploidy: its origins and induction following chemical treatment in Sordaria brevicollis.

A system suitable for the detection of meiotic aneuploidy is described in which various different origins of the aneuploidy can be distinguished. Aneuploid meiotic products are detected as black disomic spores held in asci containing all the products of a single meiosis. Aneuploidy may result from nondisjunction or from a meiosis in which an extra replica of one of the chromosomes has been generated in some other way, e.g., extra replication. By using this system it has been shown that pFPA treatment increase aneuploidy, primarily through an effect on nondisjunction. Preliminary results with trifluralin have indicated that this compound, too, may increase aneuploidy. There is a good possibility that the system can be further developed to permit a more rapid screening using a random plating method; this will allow a more efficient two-part analysis of the effects of compounds under test.

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.

Mutational synergism between p-fluorophenylalanine and UV in Coprinus lagopus.

Previous studies have shown that the amino acid analogue p-fluorophenylalanine (PFP) is mutagenic to Coprinus lagopus due to its incorporation into proteins [32]. Spontaneous mutations, PFP and UV mutagenesis and PFP/UV synergism have been studied in a UV resistant strain and in two complementing UV sensitive mutant strains. By comparison to the UV resistant strain, one UV sensitive strain shows normal spontaneous mutations, 1.4% PFP-induced mutations and 50-fold UV mutagenesis. The second UV sensitive strain has 19-fold spontaneous mutation frequency, 8% PFP induced mutations and slightly elevated UV mutagenesis. In all 3 strains the PFP/UV synergism is comparable (4--5 times the arithmetic expected). The results indicate that PFP mutagenesis is due to the incorporation of PFP into enzymes normally functioning in the organism but which also participate in UV repair mechanisms. A model is proposed for UV repair which is based on a PFP sensitive excision repair system of at least two enzymes, and alternative "error proof" pathway which is not suscetible to PFP and an "error prone" pathway which is responsible for UV mutagenesis and is susceptible to PFP as shown by the PFP/UV synergism. Because PFP is given before UV treatment, this implies a UV inducible cofactor and a PFP sensitive enzyme which only functions after UV activation.

Testing for the chemical induction of aneuploidy in the male mouse.

Dyad scores of metaphase II spermatocytes in the mouse have been used as an end point to assess the aneuploidy-inducing potential of three different chemicals; p-fluorophyalanine, phenylalanine and 6-mercaptopurine. The sensitivities of three different spermatogenic stages have been tested; pre-leptotene, zygotene and metaphase I. No effect was found at any treated stage for 6-mercaptopurine and phenylalanine. p-Fluorophenylalanine, when compared to control treatments, did, however, induce non-disjunction when applied at metaphase I. It also caused a delay to spermatogenesis when applied at this stage. The potential of mammalian test systems for the routine screening of chemicals as non-disjunction inducers, is discussed.

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.

Biosynthesis of lasalocid A. Biochemical alteration of polyether antibiotic production.

The effect of known precursors, their fluorinated analogs, and biochemical inhibitors on the production of the polyether antibiotic, lasalocid A (1), by resting cells of Streptomyces lasaliensis was determined to study the biochemistry and regulation of antibiotic biosynthesis in vivo.

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.

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.

[Effect of treatment with p-fluorophenylalanine on the mating capacity of industrial polyploid yeasts]. / Efecto del tratamiento con p-fluorfenilalanina sobre la capacidad de conjugar levaduras poliploides industriales.

Brewing yeast strains of the genus Saccharomyces uvarum (carlsbergensis) were grown on media containing p-fluorophenylalanine (p-FPA). After the treatment, non-sporulation colonies were selected, and these were mated with haploid strains of flocculent and amylolytic yeasts of the genus Saccharomyces. The selected hybrids, which carried the greater part of the parental genetic markers and produced asci containing 2,3 and 4 spores per ascus, were placed on sporulation medium. Some aspects of the probable action of p-FPA are discussed.

[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.

[Disulfide maturation of the glycoproteins of influenza virus hemagglutinin and neuraminidase in infected cells]. / Disul'fidnoe sozrevanie glikoproteidov gemaggliutinina i neiraminidazy virusa grippa v zarazhennykh kletkakh.

Fractionation by polyacrylamide gel electrophoresis (PAGE) demonstrated that in the infected cells the newly synthesized influenza virus glycoproteins, hemagglutinin (HA) and neuraminidase (NA), differ from mature proteins of virus particles. After some time of life in the cells the differences are levelled. Since this phenomenon was demonstrable only in an analysis under the conditions favourable for the retention of disulphide bonds, it was designated as "disulphide maturation" of glycoproteins. Two causes of disulphide maturation of HA are considered: posttranslational folding of molecules conducive to drawing closer of the oxidizable thiol groups, and gradual loss of sensitivity to endogenous reducing agents. As for NA, the observed maturation here is the result of disulphide dimerization of monomers. Some factors affecting disulphide maturation of glycoproteins have been studied.

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.