Evaluation of carcinogenic/co-carcinogenic activity of chikusaku-eki, a bamboo charcoal by-product used as a folk remedy, in BALB/c 3T3 cells.
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Chikusaku-eki is an acidic dark brown liquid obtained as a by-product from bamboo charcoal burners. The solution diluted with water is gaining widespread popularity in Japan as a folk medicine for skin diseases such as scabies, eczema, and atopic dermatitis. In this study, the carcinogenic and tumor-promoting potential of chikusaku-eki was determined using the BALB/c 3T3 A31-1-1 cell transformation system. Carcinogenic activity was tested by treating A31-1-1 cells for 24 h with 0.06% solution, a dose resulting in 35% clonogenic cell survival. In both 2-O-tetradecanoylphorbol-13-acetate (TPA)-treated and non-treated groups, chikusaku-eki did not initiate carsinogenesis. Following initiation with 3-methylcholanthrene (3-MCA), A31-1-1 cells were chronically treated with a non-toxic concentration range of chikusaku-eki (< or = 0.01%), but chikusaku-eki did not act as a tumor promoter. Thus, chikusaku-eki was not carcinogenic/co-carcinogenic in the in vitro cell transformation assay examined in this study after being diluted more than 10(4)-fold with water. (+info)
Structural and mutational analyses of cis-acting sequences in the 5'-untranslated region of satellite RNA of bamboo mosaic potexvirus.
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The satellite RNA of Bamboo mosaic virus (satBaMV) contains on open reading frame for a 20-kDa protein that is flanked by a 5'-untranslated region (UTR) of 159 nucleotides (nt) and a 3'-UTR of 129 nt. A secondary structure was predicted for the 5'-UTR of satBaMV RNA, which folds into a large stem-loop (LSL) and a small stem-loop. Enzymatic probing confirmed the existence of LSL (nt 8-138) in the 5'-UTR. The essential cis-acting sequences in the 5'-UTR required for satBaMV RNA replication were determined by deletion and substitution mutagenesis. Their replication efficiencies were analyzed in Nicotiana benthamiana protoplasts and Chenopodium quinoa plants coinoculated with helper BaMV RNA. All deletion mutants abolished the replication of satBaMV RNA, whereas mutations introduced in most of the loop regions and stems showed either no replication or a decreased replication efficiency. Mutations that affected the positive-strand satBaMV RNA accumulation also affected the accumulation of negative-strand RNA; however, the accumulation of genomic and subgenomic RNAs of BaMV were not affected. Moreover, covariation analyses of natural satBaMV variants provide substantial evidence that the secondary structure in the 5'-UTR of satBaMV is necessary for efficient replication. (+info)
Functional analysis of the cloverleaf-like structure in the 3' untranslated region of bamboo mosaic potexvirus RNA revealed dual roles in viral RNA replication and long distance movement.
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The 3' untranslated region (UTR) of bamboo mosaic potexvirus (BaMV) RNA was identified to fold into a tertiary structure comprising a cloverleaf-like structure designated ABC domain followed by a major stem-loop D, which in turn is followed by a pseudoknot E and a poly(A) tail. The coat protein accumulation level of the mutant, BaMV40A/DeltaABC, lacking ABC domain was just 15% that of wild-type when inoculated into protoplasts of Nicotiana benthamiana. This suggested that ABC domain might play an important role in BaMV RNA replication. To define the precise role of each of the three stem-loops of ABC domain in RNA replication, three mutants BaMV40A/DeltaA, -/DeltaB, and -/DeltaC each lacking stem-loop A, B, and C, respectively, were created. Our results showed that accumulation of viral products of mutants BaMV40A/DeltaB and -/DeltaC were not as efficient as wild-type. On the contrary, level of accumulation of viral products of BaMV/DeltaA was similar to that of wild-type in protoplasts and inoculated leaves. Interestingly, the accumulation of viral products was not as efficient as that of wild-type in systemic leaves, implying that stem-loop A is dispensable for replication, but signifies a role in systemic accumulation. Using UV cross-linking and competition experiments, it was demonstrated that the E. coli expressed helicase domain of BaMV ORF1 can preferentially interact with the ABC domain. (+info)
Arg-16 and Arg-21 in the N-terminal region of the triple-gene-block protein 1 of Bamboo mosaic virus are essential for virus movement.
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The protein encoded by the first gene of the triple gene block (TGBp1) of potexviruses is required for movement of the viruses. It has been reported that single Arg-->Ala substitutions at position 11, 16 or 21 of TGBp1 of Bamboo mosaic virus (BaMV) eliminate its RNA-binding activity, while substitutions at position 16 or 21 only affect its NTPase activity (Liou et al., Virology 277, 336-344, 2000). However, it remains unclear whether these Arg-->Ala substitutions also affect the movement of BaMV in plants. To address this question, six mutants of BaMV, each containing either a single- or a double-alanine substitution at Arg-11, Arg-16 and Arg-21 of TGBp1, were constructed and used to infect Chenopodium quinoa and Nicotiana benthamiana. We found that all of the BaMV mutants were able to replicate in protoplasts of N. benthamiana. However, only the mutant with an Arg-11-->Ala substitution in TGBp1 remained capable of movement from cell to cell in plants. Mutants with Arg-16, Arg-21 or both Arg-16 and Arg-21 of TGBp1 replaced with alanine were defective in virus movement. This defect was suppressed when a wild-type TGBp1 allele was co-introduced into the cells using a novel satellite replicon. The ability to trans-complement the movement defect by the wild-type TGBp1 strongly suggests that the Arg-->Ala substitution at position 16 or 21 of TGBp1, which diminishes the RNA-binding and NTPase activities of TGBp1, also eliminates the capability of BaMV to move from cell to cell in host plants. (+info)
Critical residues for GTP methylation and formation of the covalent m7GMP-enzyme intermediate in the capping enzyme domain of bamboo mosaic virus.
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Open reading frame 1 of Bamboo mosaic virus (BaMV), a Potexvirus in the alphavirus-like superfamily, encodes a 155-kDa replicase responsible for the formation of the 5' cap structure and replication of the viral RNA genome. The N-terminal domain of the viral replicase functions as an mRNA capping enzyme, which exhibits both GTP methyltransferase and S-adenosylmethionine (AdoMet)-dependent guanylyltransferase activities. We mutated each of the four conserved amino acids among the capping enzymes of members within alphavirus-like superfamily and a dozen of other residues to gain insight into the structure-function relationship of the viral enzyme. The mutant enzymes were purified and subsequently characterized. H68A, the mutant enzyme bearing a substitution at the conserved histidine residue, has an approximately 10-fold increase in GTP methyltransferase activity but completely loses the ability to form the covalent m(7)GMP-enzyme intermediate. High-pressure liquid chromatography analysis confirmed the production of m(7)GTP by the GTP methyltransferase activity of H68A. Furthermore, the produced m(7)GTP sustained the formation of the m(7)GMP-enzyme intermediate for the wild-type enzyme in the presence of S-adenosylhomocysteine (AdoHcy), suggesting that the previously observed AdoMet-dependent guanylation of the enzyme using GTP results from reactions of GTP methylation and subsequently guanylation of the enzyme using m(7)GTP. Mutations occurred at the other three conserved residues (D122, R125, and Y213), and H66 resulted in abolition of activities for both GTP methylation and formation of the covalent m(7)GMP-enzyme intermediate. Mutations of amino acids such as K121, C234, D310, W312, R316, K344, W406, and K409 decreased both activities by various degrees, and the extents of mutational effects follow similar trends. The affinity to AdoMet of the various BaMV capping enzymes, except H68A, was found in good correlations with not only the magnitude of GTP methyltransferase activity but also the capability of forming the m(7)GMP-enzyme intermediate. Taken together with the AdoHcy dependence of guanylation of the enzyme using m(7)GTP, a basic working mechanism, with the contents of critical roles played by the binding of AdoMet/AdoHcy, of the BaMV capping enzyme is proposed and discussed. (+info)
Silica deposition in relation to ageing of leaf tissues in Sasa veitchii (Carriere) Rehder (Poaceae: Bambusoideae).
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BACKGROUND AND AIMS: Silica deposition is one of the important characteristics of plants in the family Poaceae. There have been many investigations into the distribution, deposition and physiological functions of silica in this family. Two hypotheses on silica deposition have been proposed based on these studies. First, that silica deposition occurs passively as a result of water uptake by plants, and second, that silica deposition is controlled positively by plants. To test these two apparently contradictory hypotheses, silica deposition in relation to the ageing of leaf tissues in Sasa veitchii was investigated. METHODS: Tissues were examined using a light microscope and a scanning electron microscope equipped with an energy dispersive X-ray microanalyser. KEY RESULTS: The deposition process differed depending on cell type. In mesophyll tissue, fusoid cells deposited large amounts of silica depending on leaf age after maturation, while chlorenchyma cells deposited little. In epidermal tissue, comprised of eight cell types, only silica cells deposited large amounts of silica during the leaf's developmental process and none after maturation. Bulliform cells, micro-hairs and prickle hairs deposited silica densely and continuously after leaf maturation. Cork cells, guard cells, long cells and subsidiary cells deposited silica at low levels. CONCLUSIONS: The significance of these observations is discussed in relation to the two hypotheses proposed for silica deposition in Poaceae. The results of the present study clearly indicate that both hypotheses are compatible with each other dependent on cell types. (+info)
Sasa health exerts a protective effect on Her2/NeuN mammary tumorigenesis.
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Bamboo grass leaves of different Sasa species have been widely used in food and medicine in Eastern Asia for hundreds of years. Of special interest are Kumazasa (Sasa senanensis rehder) leaves used to prepare an alkaline extract known as Sasa Health. This extract was reported to inhibit both the development and growth of mammary tumors in a mammary tumor strain of virgin SHN mice (1). We found that Sasa Health exerts a significant protective effect on spontaneous mammary tumorigenesis in another mouse model of human breast cancer, the transgenic FVB-Her2/NeuN mouse model. Two cohorts of Her2/NeuN female mice of different age (eleven-week-old and twenty-four-week-old) chronically treated with Sasa Health in drinking water showed both a delay in the development of tumors and reduced tumor multiplicity. Sasa Health also induced inhibition of mammary duct branching and side bud development in association with reduced angiogenesis. Altogether these findings indicate that Sasa Health contains phytochemicals that can effectively retard spontaneous mammary tumorigenesis. (+info)
A conserved secondary structure in the hypervariable region at the 5' end of Bamboo mosaic virus satellite RNA is functionally interchangeable.
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Satellite RNA (satRNA) associated with Bamboo mosaic virus (BaMV) is dependent on BaMV for replication and encapsidation. Molecular analyses of total RNA extracted from bamboo species collected worldwide revealed that 26 out of 61 BaMV isolates harbored satBaMV. Among them, two phylogenetically distinguishable groups, A and B, with a genetic diversity of 6.9 +/- 0.7% were identified. Greatest sequence diversity occurred in the 5' untranslated region (UTR) that contained one hypervariable region with variations of up to 20.7%. Concurrent covariations in the 5' hypervariable sequences support the existence of a conserved apical hairpin stem-loop structure, which was earlier mapped by enzymatic probings and functional analyses [Annamalai, P., Hsu, Y.H., Liu, Y.P., Tsai, C.H., Lin, N.S., 2003. Structural and mutational analyses of cis-acting sequences in the 5'-untranslated region of satellite RNA of bamboo mosaic potexvirus. Virology 311 (1), 229-239]. Furthermore, chimeric satBaMVs generated by interchanging the hypervariable region between groups A and B demonstrated the replication competence of satBaMV isolates in Nicotiana benthamiana protoplasts co-inoculated with BaMV RNA. The results suggest that an evolutionarily conserved secondary structure exists in the hypervariable region of 5' UTR of satBaMV. (+info)