Differing responses of Nijmegen breakage syndrome and ataxia telangiectasia cells to ionizing radiation

Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder. Originally thought to be a variant of ataxia telangiectasia (AT), the cellular phenotype of NBS has been described as almost indistinguishable from that of AT. Since the gene involved in NBS has been cloned and its functions studied, we sought to further characterize its cellular phenotype by examining the response of density-inhibited, confluent cultures of human diploid fibroblasts to irradiation in the G(0)/G(1) phase of the cell cycle. Both NBS and AT cells were markedly sensitive to the cytotoxic effects of radiation. NBS cells, however, were proficient in recovery from potentially lethal damage and exhibited a pronounced radiation-induced G(1)-phase arrest. Irradiated AT cells showed no potentially lethal damage and no G(1)-phase arrest. Both cell types were hypersensitive to the induction of chromosomal aberrations, whereas the distribution of aberrations in irradiated NBS cells was similar to that of normal controls, AT cells showed a high frequency of chromatid-type aberrations. TP53 and CDKN1A (also known as p21(Waf1)) expression was attenuated in irradiated NBS cells, but maximal induction occurred 2 h postirradiation, as was observed in normal controls. The similarities and differences in cellular phenotype between irradiated NBS and AT cells are discussed in terms of the functional properties of the signaling pathways downstream of AT involving the NBS1 and TP53 proteins.     

Quantitative Measurements of <Emphasis Type="Italic">Xanthomonas Oryzae</Emphasis> pv. <Emphasis Type="Italic">Oryzae</Emphasis> Distribution in Rice Using Fluorescent-Labeling

The rice host sensor, XA21, confers robust resistance to most strains of Xanthomonas oryzae pv. oryzae (Xoo), the casual agent of bacterial blight disease. Using in planta fluorescence imaging of Xoo strain PXO99Az expressing a green fluorescent protein (Xoo-gfp) we show that XA21 restricts Xoo spread at the point of infection. This noninvasive and quantitative method to measure spatial distribution of Xoo populations in planta facilitates detailed assessment of plant disease resistance.     

Radiation sensitivity of primary fibroblasts from hereditary retinoblastoma family members and some apparently normal controls: colony formation ability during continuous low-dose-rate gamma irradiation

We previously described an enhanced sensitivity for cell killing and G(1)-phase cell cycle arrest after acute gamma irradiation in primary fibroblast strains derived from 14 hereditary-type retinoblastoma family members (both affected RB1(+/-) probands and unaffected RB1(+/+) parents) as well as distinctive gene expression profiles in unirradiated cultures by microarray analyses. In the present study, we measured the colony formation ability of these cells after exposure to continuous low-dose-rate (0.5-8.4 cGy/h) (137)Cs gamma radiation for a 2-week growth period. Fibroblasts from all RB family members (irrespective of RB1 genotype) and from 5 of 18 apparently normal Coriell cell bank controls were significantly more radiosensitive than the remaining apparently normal controls. The average dose rates required to reduce relative survival to 10% and 1% were approximately 3.1 and 4.7 cGy/h for the Coriell control strains with normal radiosensitivity and approximately 1.4 and 2.5 cGy/h for the radiosensitive RB family member and remaining apparently normal Coriell control strains. The finding that a significant proportion of fibroblast strains derived from apparently normal individuals are sensitive to chronic low-dose-rate irradiation indicates such individuals may harbor hypomorphic genetic variants in genomic maintenance and/or DNA repair genes that may likewise predispose them or their children to cancer.     

Intravacuolar Spherical Bodies in Polygonum cuspidatum

The vacuoles in the epidermal cells of light-grown seedlingsof Polygonum cuspidatum were found to contain intensely pigmentedspherical bodies (anthocyanoplasts). Dark-grown seedlings containedsimilar spherical bodies that were unpigmented. The unpigmentedspherical bodies accumulated anthocyanin and turned into anthocyanoplastswhen dark-grown seedlings were irradiated. The unpigmented sphericalbodies of dark-grown seedlings were heavily stained upon treatmentof seedlings with neutral red. The absorption spectra of sphericalbodies and vacuoles showed that the contents of the sphericalbody were different from those of the vacuole. The sphericalbody was stable in darkness or at low temperature but was unstableat 25°C in the light. There was no correlation between theamount of anthocyanin and the percentage of cells that containedspherical bodies, suggesting that, in P. cuspidatum, the sphericalbody is not the main site of anthocyanin synthesis. ²Present address: Department of Applied Biology, Faculty ofTextile Science and Technology, Shinshu University, Ueda, 386Japan     

Glycopeptidolipid of Mycobacterium smegmatis J15cs Affects Morphology and Survival in Host Cells

Mycobacterium smegmatis has been widely used as a mycobacterial infection model. Unlike the M. smegmatis mc²155 strain, M. smegmatis J15cs strain has the advantage of surviving for one week in murine macrophages. In our previous report, we clarified that the J15cs strain has deleted apolar glycopeptidolipids (GPLs) in the cell wall, which may affect its morphology and survival in host cells. In this study, the gene causing the GPL deletion in the J15cs strain was identified. The mps1-2 gene (MSMEG_0400-0402) correlated with GPL biosynthesis. The J15cs strain had 18 bps deleted in the mps1 gene compared to that of the mc²155 strain. The mps1-complemented J15cs mutant restored the expression of GPLs. Although the J15cs strain produces a rough and dry colony, the colony morphology of this mps1-complement was smooth like the mc²155 strain. The length in the mps1-complemented J15cs mutant was shortened by the expression of GPLs. In addition, the GPL-restored J15cs mutant did not survive as long as the parent J15cs strain in the murine macrophage cell line J774.1 cells. The results are direct evidence that the deletion of GPLs in the J15cs strain affects bacterial size, morphology, and survival in host cells.     

Detection and Characterization of a Vacuolar Protein (VP24) in Anthocyanin-Producing Cells of Sweet Potato in Suspension Culture

Vacuoles containing large amounts of protein and anthocyaninwere isolated and purified from anthocyanin-producing culturedcells of sweet potato (Ipomoea batatas). A 24 kDa protein (VP24),identified as a major protein in the isolated vacuoles, wasdetected by SDS-polyacrylamide gel electrophoresis. NeitherVP24 nor anthocyanin was detectable in dark-cultured cells,but VP24 appeared three days after the start of irradiationwith light and the level of VP24 increased for up to a week.High concentrations of 2,4-D markedly inhibited the accumulationof both VP24 and anthocyanin. These results indicated that theexpression of VP24, which was induced by exposure to light,was closely accompanied by the accumulation of anthocyanin inthe vacuoles. VP24 was recovered as an insoluble reddish precipitateafter ultracentrifugation of a lysate of anthocyanin-containingvacuoles. Immunoblot analysis indicated that this vacuolar proteinwas distinct from sporamin, a major storage protein of sweetpotato tuberous root. These results suggest that VP24 is notan integral protein of the vacuolar membrane but tends to formaggregates via interactions with anthocyanin during extraction.VP24 may be involved in the formation of intravacuolar pigmentedstructures that develop in vivo in the anthocyanin-containingvacuoles of sweet potato cells in culture. ³Present address: Department of Applied Biology, Faculty ofTextile Science and Technology, Shinshu University, Tokida 3-15-1,Ueda, 386 Japan     

A host-vector system for molecular study of the intracellular growth of Mycobacterium tuberculosis in phagocytic cells

The mechanisms by which Mycobacterium tuberculosis survives and persists in phagocytic cells remain poorly understood. To study the question, a convenient and safe host-vector system is indispensable. In this study it has been shown that, in contrast with M . smegmatis strain mc²155 which has been widely used for molecular analysis, M. smegmatis strain J15cs is able to survive even at day 6 post-infection in a murine macrophage cell line, J774. The survivability of J15cs was found to depend on the culture medium used for the bacteria prior to infection. Bacteria precultured on nutrient agar medium showed a high survivability and a characteristic cell wall ultrastructure. A plasmid vector, pYT923hyg, was developed from an Escherichia coli - mycobacterium shuttle vector pYT923 (previously constructed in our laboratory) to obtain three drug resistant genes (amp-, hyg- and km-resistant gene) and cloning sites in the km resistant gene. The vector pYT923hyg exerted no influence on in vitro growth of J15cs and intracellular survival in J774 cells, and was stably retained in J15cs after serial subculturing (three subcultures) in Luria-Bertani broth and at day 5 post-infection into J774 cells. Furthermore, using this system, the possibility of a relationship between some seemingly essential genes of M. tuberculosis and intracellular growth was demonstrated.
In this study, M. smegmatis strain J15cs and pYT923hyg were found to be capable of serving as an appropriate host-vector system for molecular study of the intracellular growth of M . tuberculosis in phagocytic cells; this system may be useful as a screening tool for M . tuberculosis genes.     

Diurnal regulation of plant growth

Life occurs in an ever-changing environment. Some of the most striking and predictable changes are the daily rhythms of light and temperature. To cope with these rhythmic changes, plants use an endogenous circadian clock to adjust their growth and physiology to anticipate daily environmental changes. Most studies of circadian functions in plants have been performed under continuous conditions. However, in the natural environment, diurnal outputs result from complex interactions of endogenous circadian rhythms and external cues. Accumulated studies using the hypocotyl as a model for plant growth have shown that both light signalling and circadian clock mutants have growth defects, suggesting strong interactions between hypocotyl elongation, light signalling and the circadian clock. Here, we review evidence suggesting that light, plant hormones and the circadian clock all interact to control diurnal patterns of plant growth.