Polyamine synthesis in plants. Purification and properties of amidinotransferase from soybean (Glycine max) axes

Three-day-old soybean (Glycine max) seedlings were exposed to 0.4 M sorbitol solution for 4 h to induce amidinotransferase activity, with the corresponding enzyme being purified to homogeneity by chromatographic separation on DEAE-Sephacel, Sephacryl S-300 and L-arginine Sepharose 4B. The purified enzyme used L-arginine and L-glycine as the major donor/acceptor of the amidino group, respectively, with formation of guanidinoacetic acid and ornithine products being confirmed by ESI-MS. The enzyme is a tetrameric protein having a molecular mass of 240,000 Da, whose thiol group is needed for enzymatic activity. The K(M)s for arginine and glycine were 3.8 and 0.89 mM, respectively, with optimal temperature and pH being 37 degrees C and 9.5, respectively. The soybean amidinotransferase could be indirectly involved in nitrogen metabolism, as suggested by the observation that arginine:glycine amidinotransferase in soybean axes is indirectly involved in putrescine biosynthesis and displays feedback control at high levels of an endogenous regulator, putrescine.     

Expression of the Bovine Growth Hormone Alters the Root Morphology in Transgenic Tobacco Plants

The bovine growth hormone (bGH) is a natural peptide hormone that controls the differentiation, growth and metabolism, and is produced in the pituitary gland of cows. For the production of bGH from plants, two different bgh clones, of which the pGAbGH1 contaions only mature peptide sequences and the pGAbGH15 contains signal sequences and the first intron, as well as mature peptide sequences, were used. Those bghs under the control of the CaMV 35S promoter and NOS terminator were introduced to tobacco plants via Agrobacterium tumefaciens-mediated transformation. By PCR analyses using bgh and nptII specific primers, 17 and 21 putative transformants were respectively selected from pGAbGH1- and pGAbGH15-transformed tobacco plants. Northern blot analysis showed that the most of the transgenic lines expressed the bgh mRNA. Western blot analysis revealed that the pGAbGH1-transformed tobaccos produced recombinant bGH, but pGAbGH15-transformed ones did not produce the protein. Interestingly, some morphological changes were observed in the roots of transgenic tobacco plants. The transgenic tobacco plants had thick and short roots containing few root hairs in contrast to the non-transformed wild type plants.     

Autotaxin promotes motility via G protein-coupled phosphoinositide 3-kinase gamma in human melanoma cells

Autotaxin (ATX), an exo-nucleotide pyrophosphatase and phosphodiesterase, stimulates tumor cell motility at sub-nanomolar levels and augments invasiveness and angiogenesis. We investigated the role of G protein-coupled phosphoinositide 3-kinase gamma (PI3Kgamma) in ATX-mediated tumor cell motility stimulation. Pretreatment of human melanoma cell line A2058 with wortmannin or LY294002 inhibited ATX-induced motility. ATX increased the PI3K activity in p110gamma, but not p85, immunoprecipitates. This effect was abrogated by PI3K inhibitors or inhibited by pertussis toxin. Furthermore, stimulation of tumor cell motility by ATX was inhibited by catalytically inactive form of PI3Kgamma, strongly indicating the crucial role of PI3Kgamma for ATX-mediated motility in human melanoma cells     

Different role of functional domains of hTR in DNA binding to telomere and telomerase reconstruction

Even if template sequence of hTR played an essential role in telomere binding, a 326 nucleotide fragment of hTR containing template, pseudoknot, and CR4-5 domains is critical for both binding with telomeric DNA and reconstitution of telomerase activity. A functional study with antisense oligonucleotides suggested that targeted disruption of the template region efficiently abrogated both telomeric DNA binding and telomerase activity, whereas disruption of the CR4-5 region induced only loss of telomerase activity. hTR interacts with telomeric DNA via structural region composed of the template, pseudoknot, and CR4-5 domains, however, each structural domain plays a distinct role in telomere binding and telomerase activity reconstitution.     

Cyclooxygenase inhibitors induce apoptosis in oral cavity cancer cells by increased expression of nonsteroidal anti-inflammatory drug-activated gene

We have investigated whether NAG-1 is induced in oral cavity cancer cells by various NSAIDs and if apoptosis induced by NSAIDs can be linked directly with the induction of NAG-1. NAG-1 expression was increased by diclofenac, aceclofenac, indomethacin, ibuprofen, and sulindac sulfide, in the order of NAG-1 induction, but not by acetaminophen, piroxicam or NS-398. Diclofenac was the most effective NAG-1 inducer. Incubation with diclofenac inhibited cell proliferation and induced apoptosis. The expression of NAG-1 was observed in advance of the induction of apoptosis. Conditioned medium from NAG-1-overexpressing Drosophila cells inhibited SCC 1483 cells proliferation and induced apoptosis. In summary, some NSAIDs induce NAG-1 expression in oral cavity cancer cells and the induced NAG-1 protein appears to mediate apoptosis. Therefore, NSAIDs may be considered as a possible chemopreventive agent against oral cavity cancer.     

Identification of fungal (Magnaporthe grisea) stress-induced genes in wild rice (Oryza minuta)

To identify fungal stress-related genes in wild rice, Oryza minuta, we constructed a subtracted library using suppression subtractive hybridization in combination with mirror orientation selection. DNA chips containing 960 randomly selected cDNA clones were applied by reverse Northern analysis to eliminate false positive clones from the library and to prescreen differentially expressed genes. In total, 377 cDNA clones were selected on the basis of their signal intensities and expression ratios. Sequence analyses of these 377 cDNA fragments revealed that 180 of them (47.7%) represented unique genes. Of these180 cDNAs, 89 clones (49.6%) showed significant homologies to previously known genes, while the remaining 91 did not match any known sequences. The putative functions of the 180 unique ESTs were categorized by aligning them with MIPS data. They were classified into seven different groups using microarray data-derived expression patterns and verified by Northern blotting.Abbreviations ER: Endoplasmic reticulum - EST: Expressed sequence tag - MIPS: Munich Information Center for Protein Sequences - MOS: Mirror orientation selection - NCBI: National Center for Biotechnology Information - omfi: Oryza minuta fungal-stress induced - PCD: Programmed cell death - PDI: Proteins disulfide isomerase - SSH: Suppression subtractive hybridization Communicated by I.S. ChungK.S. Shim and S.K. Cho contributed equally to this work.     

Rapid, large-scale generation of Ds transposant lines and analysis of the Ds insertion sites in rice

Rapid, large-scale generation of a Ds transposant population was achieved using a regeneration procedure involving tissue culture of seed-derived calli carrying Ac and inactive Ds elements. In the F(2) progeny from genetic crosses between the same Ds and Ac starter lines, most of the crosses produced an independent germinal transposition frequency of 10-20%. Also, many Ds elements underwent immobilization even though Ac was expressed. By comparison, in a callus-derived regenerated population, over 70% of plants carried independent Ds insertions, indicating transposition early in callus formation. In the remaining population, the majority of plants carried only Ac. Most of the new Ds insertions were stably transmitted to a subsequent generation. An exceptionally high proportion of independent transposants in the regenerated population means that selection markers for transposed Ds and continual monitoring of Ac/Ds activities may not necessarily be required. By analyzing 1297 Ds-flanking DNA sequences, a genetic map of 1072 Ds insertion sites was developed. The map showed that Ds elements were transposed onto all of the rice chromosomes, with preference not only near donor sites (36%) but also on certain physically unlinked arms. Populations from both genetic crossing and tissue culture showed the same distribution patterns of Ds insertion sites. The information of these mapped Ds insertion sites was deposited in GenBank. Among them, 55% of Ds elements were on predicted open-reading frame (ORF) regions. Thus, we propose an optimal strategy for the rapid generation of a large population of Ds transposants in rice.