Structural and functional studies of cinnamomin, a new type II ribosome-inactivating protein isolated from the seeds of the camphor tree.

Cinnamomin is a new type II ribosome-inactivating protein (RIP). Its A-chain exhibits RNA N-glycosidase activity to inactivate the ribosome and thus inhibit protein synthesis, whereas the glycosylated B-chain is a lectin. The primary structure of cinnamomin, which exhibits approximately 55% identity with those of ricin and abrin, was deduced from the nucleotide sequences of cDNAs of cinnamomin A- and B-chains. It is composed of a total of 549 amino-acid residues: 271 residues in the A-chain, a 14-residue linker and 264 residues in the B-chain. To explore its biological function, the cinnamomin A-chain was expressed in Escherichia coli with a yield of 100 mg per L of culture, and purified through two-step column chromatography. After renaturation, the recovery of the enzyme activity of the expressed A-chain was 80% of that of native A-chain. Based on the modeling of the three-dimensional structure of the A-chain, the functional roles of five amino acids and the only cysteine residues were investigated by site-directed mutagenesis or chemical modification. The conserved single mutation of the five amino-acid residues led to 8-50-fold losses of enzymatic activity, suggesting that these residues were crucial for maintaining the RNA N-glycosidase activity of the A-chain. Most interestingly, the strong electric charge introduced at the position of the single cysteine in A-chain seemed to play a role in enzyme/substrate binding.

Involvement of ethylene in the maturation of black spruce embryogenic cell lines with different maturation capacities.

To examine the possible relationship between ethylene and the capacity of embryogenic cell lines to produce mature somatic embryos of black spruce (Picea mariana (Mill.) B.S.P.), two embryogenic cell lines which exhibit different maturation capacities were used to analyse ethylene biosynthesis and that of its immediate precursor, 1-aminocyclopropane-1- carboxylic acid (ACC). Several compounds known to alter ethylene metabolism were also evaluated for their effect on the number of mature somatic embryos produced. The results showed that in the high capacity cell line, ethylene production and endogenous ACC pools were less than in the low capacity cell line. It was also demonstrated that limiting ethylene biosynthesis by adding inhibitors of ethylene biosynthesis or its physiological action to the maturation medium promoted somatic embryo production for the low capacity cell line. Conversely, lowering ethylene biosynthesis reduced the number of somatic embryos in the high capacity cell line. These results were further substantiated by the finding that the effects of amino-oxyacetic acid (AOA), an inhibitor of ethylene biosynthesis, were partially reversed by adding ethylene to both embryogenic cell lines. It is concluded that ethylene is implicated in somatic embryogenesis of black spruce and that the low capacity cell line had excess, i.e. supraoptimal, ethylene production, whereas the high capacity cell line had nearly optimal ethylene production. The relationship between ethylene and other phytohormones, and the possible effects of the interaction between ethylene and polyamines on the maturation of the somatic embryos are discussed.

Two-dimensional electrophoretic analysis of proteins associated with somatic embryogenesis development in Cupressus sempervirens L.

Two-dimensional gel electrophoretic analysis and histological studies were performed on somatic embryos in cypress. Embryogenic cultures were obtained from in vitro culture of immature seeds. On a modified Murashige and Skoog (MS) medium they showed an intense and repetitive cleavage polyembryogenesis phenomenon which maintained them in a continuous proliferating status instead of undergoing a complete embryogenic development. Only the addition of bovine serum albumin to the culture allowed somatic embryo development and maturation. Major histological differences were noticed between developing and nondeveloping embryogenic cultures. Attempts to find proteins that could be associated with developmental stages of somatic embryos have been achieved. Proteins were extracted and analyzed by two-dimensional electrophoresis from nondeveloping embryogenic cultures (S0) and from embryogenic cultures at three different stages of somatic embryo development: small size and rounded shape embryos (S1), increased size embryos with a well-developed suspensor (S2) and embryos with two well-separated cotyledons (S3). The results revealed some qualitative and quantitative protein variations between the two cultures. Some could be connected with the induction of pro-embryo differentiation whereas others should be more related to the mechanisms involved in somatic embryo development and maturation. Specific polypeptides associated with the presence of bovine serum albumin (BSA) in the medium have been detected.

[Radiation-induced changes in the genome of pine seeds, forming under conditions of chronic irradiation in the Chernobyl exclusion zone]. / Radiatsionno-indutsirovannye izmeneniia v genome semian sosny, sformirovabshikhsiia v usloviiakh khronicheskogo oblucheniia v zone otchuzhdeniia ChAES.

Comparative study of long-term existing genome damages in the chronically irradiated pine seeds from different parts of Chernobyl zone have been performed by a pulsed field gel electrophoresis and hydroxyapatite chromatography. Some differences were established for DNA degradation kinetic from protoplasts with various accumulated doses of irradiation. Results obtained could testify the existence of long-living changes in the experimental plants genome.

The effect of heat shock on the formaldehyde cycle in germinating acorns of European Turkey oak.

The effect of heat shock (40 degrees C) on the formaldehyde cycle has been studied in European Turkey oak (Quercus cerris L.) acorns germinated to a 10% increase in mass. Hydroxy-methyl groups bonded to sulfur, oxygen and nitrogen atoms were made to react with dimedone and the derivative obtained (formaldemethone), which represented the endogenous formaldehyde level, was determined by high performance liquid chromatography. Qualitative alterations of methyl donors and acceptors in the response of acorns to the heat shock have been mapped by MALDI (matrix assisted laser desorption ionization) mass analysis. In the first experiment the acorns were prevented from withering by wrapping them in aluminium foil and in the second they were not. The relatively high temperature of the acorns wrapped in aluminium foil was the dominant stress effect and the role of withering was subsidiary. Alteration of the endogenous formaldehyde level in the seed-leaves reflected the phases of the stress syndrome. If the withering were not hindered, two local minima in the alteration of endogenous formaldehyde level were found. First, the increase in temperature decreased the endogenous formaldehyde level and after a local maximum a repeated local minimum was observed as a delayed response. It is presumed that the second minimum was induced by the decreasing water amount becoming more and more significant in the seed-leaves.

Asimilobin and cis- and trans-murisolinones, novel bioactive Annonaceous acetogenins from the seeds of Asimina triloba.

Three new bioactive Annonaceous acetogenins, asimilobin [1], cis-murisolinone [2], and trans-murisolinone [3], have been isolated from an ethanolic extract of the seeds of Asimina triloba by directing the fractionation with brine shrimp lethality. The structure were elucidated based on spectroscopic and chemical methods, In addition, cis- and trans-bullatacinone, which are known compounds, were obtained. Asimilobin [1] has adjacent bis-THF rings, located at C-10 to C-17 and having only one flanking hydroxyl group at C-18. Compounds 1-3 showed cytotoxicity values comparable with adriamycin against six human solid tumor cell lines.