Response of Glutamine Synthetase and Glutamate Synthase Isoforms to Nitrogen Sources in Rice Cell Cultures

As a model system with no photorespiration and no long distancetransport, rice cell cultures (Oryza saliva L. cv Sasanishiki)were used to investigate the effect of nitrogen sources on thelevels of isoforms of glutamine synthetase (GS) and glutamatesynthase (GOGAT). Isoforms of GS and GOGAT were analyzed byimmunoblotting methods and their activities in early growthphase of the cells. Cytosolic type GS (41 kDa subunit) and NADH-GOGATwere the major isoforms in the rice cells grown in normal R-2medium. However, contents of plastid type GS (44 kDa subunit)and Fd-GOGAT increased in response to NO_–³ supply. NADH-GOGATactivity also increased following the supply of NO_–³.In vitro translated products from poly(A)⁺RNA prepared fromthe cells showed that the precursor of plastid type GS (49 kDa)was detected at 48 h after the inoculation. Supply of NH₊⁴ resultedin an increase in NADH-GOGAT activity but had no effect on thelevels of Fd-GOGAT, of polypeptides of the plastid type GS orof the corresponding mRNAs. (Received May 30, 1990; Accepted August 23, 1990)     

Demand and supply of N in seed production of soybean (<Emphasis Type="Italic">Glycine max</Emphasis>) at different N fertilization levels after flowering

Nitrogen (N) has been suggested as a determinant of seed production especially in species with high seed N content. Assuming that seed yield was determined as the balance between N demand and supply for seed production, we studied the effect of N fertilization after flowering on soybean (Glycine max L. Merr.) yield. Seed N concentration was nearly constant irrespective of N fertilization, indicating that seed production was proportional to the amount of N available for seed growth. N demand for seed production was analyzed as the product of seed number, the rate of N filling in individual seeds, and the length of the reproductive period. N fertilization increased seed number and the reproductive period, but did not influence the N filling rate. Seed number was positively correlated with dry mass productivity after flowering. Three N sources were distinguished: mineral N uptake, symbiotic N₂ fixation and N remobilization from vegetative body. N fertilization increased N uptake and N remobilization, but lowered N₂ fixation. We concluded that N availability in the reproductive period determined seed yield directly through increasing N supply for seed growth and indirectly through increasing seed N demand with enhanced plant dry mass productivity.     

Mechanism of oxidative DNA damage induced by capsaicin, a principal ingredient of hot chili pepper

Although capsaicin exhibits antitumor activity, carcinogenic potential has also been reported. To clarify the mechanism for expression of potential carcinogenicity of capsaicin, we examined DNA damage induced by capsaicin in the presence of metal ion and various kinds of cytochrome P450 (CYP) using ³²P-5'-end-labeled DNA fragments. Capsaicin induced Cu(II)-mediated DNA damage efficiently in the presence of CYP1A2 and partially in the presence of 2D6. CYP1A2-treated capsaicin caused double-base lesions at 5'-TG-3', 5'-GC-3' and CG of the 5'-ACG-3' sequence complementary to codon 273, a hotspot of p53 gene. DNA damage was inhibited by catalase and bathocuproine, a Cu(I) chelator, suggesting that reactive species derived from the reaction of H₂O₂ with Cu(I) participate in DNA damage. Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine was significantly increased by CYP1A2-treated capsaicin in the presence of Cu(II). Therefore, we conclude that Cu(II)-mediated oxidative DNA damage by CYP-treated capsaicin seems to be relevant for the expression of its carcinogenicity.     

Genetic transformation of gentian using wild-type Agrobacterium rhizogenes

Leaf sections of greenhouse-grown Miscanthus x ogiformis Honda 'Giganteus' plants and leaf sections or shoot apices of in vitro shoot cultures were grown on Murashige and Skoog medium containing various concentrations of benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid. On leaf sections, the callus induction decreased with increasing BA concentration. The percentage of embryogenic callus was increased, the percentage of root-forming callus decreased, and a new shoot-forming callus type was formed by inclusion of BA during callus induction. A higher percentage of shoot-forming callus was formed on shoot apices compared with leaf sections of in vitro-grown shoots when cultured on 0.4 μM BA. The largest number of plants per callus piece was regenerated from shoot-forming callus, but maintenance of the high regeneration capacity proved difficult. This revised version was published online in June 2006 with corrections to the Cover Date.     

H2O2 accelerates cellular senescence by accumulation of acetylated p53 via decrease in the function of SIRT1 by NAD+ depletion.

It has been reported that p53 acetylation, which promotes cellular senescence, can be regulated by the NAD(+)-dependent deacetylase SIRT1, the human homolog of yeast Sir2, a protein that modulates lifespan. To clarify the role of SIRT1 in cellular senescence induced by oxidative stress, we treated normal human diploid fibroblast TIG-3 cells with H(2)O(2) and examined DNA cleavage, depletion of intracellular NAD(+), expression of p21, SIRT1, and acetylated p53, cell cycle arrest, and senescence-associated beta-galactosidase (SA-beta-gal) activity. DNA cleavage was observed immediately in TIG-3 cells treated with H(2)O(2), though no cell death was observed. NAD(+) levels in TIG-3 cells treated with H(2)O(2) were also decreased significantly. Pre-incubation with the poly (ADP-ribose) polymerase (PARP) inhibitor resulted in preservation of intracellular NAD(+) levels. The amount of acetylated p53 was increased in TIG-3 cells at 4h after H(2)O(2) treatment, while there was little to no decrease in SIRT1 protein expression. The expression level of p21 was increased at 12h and continued to increase for up to 24h. Additionally, exposure of TIG-3 cells to H(2)O(2) induced cell cycle arrest at 24h and increased SA-beta-gal activity at 48h. This pathway likely plays an important role in the acceleration of cellular senescence by oxidative stress.     

The conformation of calreticulin is influenced by the endoplasmic reticulum luminal environment

In order to understand the dynamics of the endoplasmic reticulum (ER) luminal environment, we investigated the role of Ca(2+), Zn(2+), and ATP on conformational changes of calreticulin. Purified calreticulin was digested with trypsin in the presence or absence of Ca(2+), Zn(2+), and ATP. At low Ca(2+) concentration (<100 micrometer), calreticulin is rapidly and fully degraded by trypsin, indicating that under these conditions the protein is in a highly trypsin-susceptible conformation. Increasing Ca(2+) concentration up to 500 micrometer or 1 mm resulted in protection of the full-length calreticulin and in generation of the 27-kDa fragment highly resistant to trypsin digestion. The 27-kDa protease-resistant core of the protein represented the NH(2)-terminal half of calreticulin and was identified by its reactivity with specific antibodies and by NH(2)-terminal amino acid sequence analysis. Ca(2+)-dependent changes in calreticulin's sensitivity to proteolysis indicate that agonist-induced fluctuation in the free ER luminal Ca(2+) concentration may affect the protein conformation and function. Trypsin digestion of calreticulin in the presence of Zn(2+) resulted in the formation of a 17-kDa central protease-resistant core in the protein corresponding to the central region of the protein, indicating that under these conditions the N- and C-domains of the protein are in an extended conformation. Here we also show that calreticulin is an ATP-binding protein but that it does not contain detectable ATPase activity. Digestion of the protein with trypsin in the presence of Mg(2+)-ATP protects the full-length protein. These results indicate that calreticulin may undergo frequent, ion-induced conformation changes, which may affect its function and its ability to interact with other proteins in the lumen of the ER.     

Hyper‐reactive cloned mice generated by direct nuclear transfer of antigen‐specific CD4+ T cells

T‐cell receptor (TCR)‐transgenic mice have been employed for evaluating antigen‐response mechanisms, but their non‐endogenous TCR might induce immune response differently than the physiologically expressed TCR. Nuclear transfer cloning produces animals that retain the donor genotype in all tissues including germline and immune systems. Taking advantage of this feature, we generated cloned mice that carry endogenously rearranged TCR genes from antigen‐specific CD4⁺ T cells. We show that T cells of the cloned mice display distinct developmental pattern and antigen reactivity because of their endogenously pre‐rearranged TCRα (rTα) and TCRβ (rTβ) alleles. These alleles were transmitted to the offspring, allowing us to establish a set of mouse lines that show chronic‐type allergic phenotypes, that is, bronchial and nasal inflammation, upon local administrations of the corresponding antigens. Intriguingly, the existence of either rTα or rTβ is sufficient to induce in vivo hypersensitivity. These cloned mice expressing intrinsic promoter‐regulated antigen‐specific TCR are a unique animal model with allergic predisposition for investigating CD4⁺ T‐cell‐mediated pathogenesis and cellular commitment in immune diseases.     

Reduced DNase I sensitivity of the rearranged c-myc gene in somatic cell hybrids between murine plasmacytoma cells and fibroblasts

In mouse plasmacytoma (MPC) S194, the rearranged c-myc gene was much more sensitive to DNase I digestion than the nonrearranged gene. The sensitivity of the rearranged c-myc was markedly reduced to the same extent as that of the nonrearranged one in hybrids between the MPC cells and the fibroblasts, but not in a hybrid between the MPC and the spleen cells. These results suggest that trans-acting factors in fibroblasts alter the DNase I-sensitive structure of the rearranged c-myc gene.