Self-association of Calcium-binding Protein S100A4 and Metastasis

Elevated levels of the calcium-binding protein S100A4 promote metastasis and in carcinoma cells are associated with reduced survival of cancer patients. S100A4 interacts with target proteins that affect a number of activities associated with metastatic cells. However, it is not known how many of these interactions are required for S100A4-promoted metastasis, thus hampering the design of specific inhibitors of S100A4-induced metastasis. Intracellular S100A4 exists as a homodimer through previously identified, well conserved, predominantly hydrophobic key contacts between the subunits. Here it is shown that mutating just one key residue, phenylalanine 72, to alanine is sufficient to reduce the metastasis-promoting activity of S100A4 to 50% that of the wild type protein, and just 2 or 3 specific mutations reduces the metastasis-promoting activity of S100A4 to less than 20% that of the wild type protein. These mutations inhibit the self-association of S100A4 in vivo and reduce markedly the affinity of S100A4 for at least two of its protein targets, a recombinant fragment of non-muscle myosin heavy chain isoform A, and p53. Inhibition of the self-association of S100 proteins might be a novel means of inhibiting their metastasis-promoting activities.     

High-level tryptophan accumulation in seeds of transgenic rice and its limited effects on agronomic traits and seed metabolite profile

Metabolic manipulation of plants to improve their nutritional quality is an important goal of plant biotechnology. Expression in rice (Oryza sativa L.) of a transgene (OASA1D) encoding a feedback-insensitive alpha subunit of rice anthranilate synthase results in the accumulation of tryptophan (Trp) in calli and leaves. It is shown here that the amount of free Trp in the seeds of such plants is increased by about two orders of magnitude compared with that in the seeds of wild-type plants. The total Trp content in the seeds of the transgenic plants was also increased. Two homozygous lines, HW1 and HW5, of OASA1D transgenic rice were generated for characterization of agronomic traits and aromatic metabolite profiling of seeds. The marked overproduction of Trp was stable in these lines under field conditions, although spikelet fertility and yield, as well as seed germination ability, were reduced compared with the wild type. These differences in agronomic traits were small, however, in HW5. In spite of the high Trp content in the seeds of the HW lines, metabolic profiling revealed no substantial changes in the amounts of other phenolic compounds. The amount of indole acetic acid was increased about 2-fold in the seeds of the transgenic lines. The establishment and characterization of these OASA1D transgenic lines have thus demonstrated the feasibility of increasing the Trp content in the seeds of rice (or of other crops) as a means of improving its nutritional value for human consumption or animal feed.     

Chk1 phosphorylation at Ser286 and Ser301 occurs with both stalled DNA replication and damage checkpoint stimulation

We previously reported Chk1 to be phosphorylated at Ser286 and Ser301 by cyclin-dependent kinase (Cdk) 1 during mitosis [T. Shiromizu et al., Genes Cells 11 (2006) 477-485]. Here, we demonstrated that Chk1-Ser286 and -Ser301 phosphorylation also occurs in hydroxyurea (HU)-treated or ultraviolet (UV)-irradiated cells. Unlike the mitosis case, however, Chk1 was phosphorylated not only at Ser286 and Ser301 but also at Ser317 and Ser345 in the checkpoint response. Treatment with Cdk inhibitors diminished Chk1 phosphorylation at Ser286 and Ser301 but not at Ser317 and Ser345 with the latter. In vitro analyses revealed Ser286 and Ser301 on Chk1 to serve as two major phosphorylation sites for Cdk2. Immunoprecipitation analyses further demonstrated that Ser286/Ser301 and Ser317/Ser345 phosphorylation occur in the same Chk1 molecule during the checkpoint response. In addition, Ser286/Ser301 phosphorylation by Cdk2 was observed in Chk1 mutated to Ala at Ser317 and Ser345 (S317A/S345A), as well as Ser317/Ser345 phosphorylation by ATR was in S286A/S301A. Therefore, Chk1 phosphorylation in the checkpoint response is regulated not only by ATR but also by Cdk2.     

(-)-Epicatechin gallate accumulates in foamy macrophages in human atherosclerotic aorta: implication in the anti-atherosclerotic actions of tea catechins

The localization and target sites of tea catechins underlying their biological activity including anti-atherosclerotic activity have not yet been fully understood. To identify the target sites of catechins in vivo, we have developed a novel monoclonal antibody (mAb5A3) specific for (−)-epicatechin-3-gallate (ECg), one of the major tea catechins. The immunoreactive materials with mAb5A3 were detected in the human atherosclerotic lesions but not in the normal aorta, and were specifically localized in the macrophage-derived foam cells. In vitro experiments using macrophage-like cell lines also showed the significant accumulation of ECg in the cells. We also demonstrated that ECg could suppress the gene expression of a scavenger receptor CD36, a key molecule for foam cell formation, in macrophage cells. These results, for the first time, showed the target site of a tea component ECg in the aorta and might provide a mechanism for the anti-atherosclerotic actions of the catechins.     

Identification and functional analysis of novel (p)ppGpp synthetase genes in Bacillus subtilis

Bacterial alarmone (p)ppGpp, is a global regulator responsible for the stringent control. Two homologous (p)ppGpp synthetases, RelA and SpoT, have been identified and characterized in Escherichia coli, whereas Gram-positive bacteria such as Bacillus subtilis have been thought to possess only a single RelA-SpoT enzyme. We have now identified two genes, yjbM and ywaC, in B. subtilis that encode a novel type of alarmone synthetase. The predicted products of these genes are relatively small proteins ( approximately 25 kDa) that correspond to the (p)ppGpp synthetase domain of RelA-SpoT family members. A database survey revealed that genes homologous to yjbM and ywaC are conserved in certain bacteria belonging to Firmicutes or Actinobacteria phyla but not in other phyla such as Proteobacteria. We designated the proteins as small alarmone synthetases (SASs) to distinguish them from RelA-SpoT proteins. The (p)ppGpp synthetase function of YjbM and YwaC was confirmed by genetic complementation analysis and by in vitro assay of enzyme activity. Molecular genetic analysis also revealed that ywaC is induced by alkaline shock, resulting in the transient accumulation of ppGpp. The SAS proteins thus likely function in the biosynthesis of alarmone with a mode of action distinct from that of RelA-SpoT homologues.     

New monoclonal antibodies that define multiple epitopes and a human-specific marker on the interleukin 2 receptor molecules of primates

Twelve hybridoma cell lines producing monoclonal antibodies to the human interleukin 2 (IL-2) receptor (IL-2R) molecule were prepared. These antibodies were characterized by competitive antibody-binding assay and sequential immunoprecipitation assay with four known monoclonal antibodies to the human IL-2R molecule. The twelve new monoclonal antibodies were divided among the four known antibody types, the HIEI-, H-A26-, H-31-, and anti-Tac-type, and an additional new type, the H-48-type. The H-48 antibody did not compete with any other antibodies in the competitive binding assay. The binding of 125I-IL-2 to MT-2 cells and the IL-2-dependent growth of normal activated T-cells were both strongly inhibited by all the H-31- and anti-Tac-type antibodies, and partially or slightly inhibited by HIEI- and H-A26-type antibodies, but were not inhibited by the H-48 antibody. Thus, the same type of monoclonal antibodies had a similar effect on the function of IL-2R. These results suggest that epitopes for the same type of antibodies could be single identical epitopes or epitopes closely associated with each other. On the other hand, these antibodies also reacted variously with a panel of various human and simian lymphoid cell lines immortalized with human T-cell leukemia virus type-I (HTLV-I): the H-45 antibody reacted only with the human cell lines, the H-C1 and H-44 and H-47 antibodies reacted with human and ape cell lines, and the other antibodies reacted with cell lines of humans, apes and Old and New World monkeys. These differences in the reactivity of the antibodies with the primate cell lines suggest that the antigenic structure of the IL-2R molecule changed during evolutionary divergence of the primates.     

Essential role of Duox in stabilization of Drosophila wing

NADPH oxidase produces reactive oxygen species (ROS). Drosophila melanogaster has two homologs of NADPH oxidase, dNox and dDuox, with functions that remain unclear in vivo. To clarify these functions, two independent transgenic fly lines expressing dsRNA targeted for different portions of dDuox mRNA were used. In both flies, en-GAL4> UAS-dDuoxIR(976-1145) and en-GAL4> UAS-dDuoxIR(370-518), in which dDuox was knocked down selectively in the posterior area of the wing disc, the posterior compartment of the adult wings became paler and more fragile with wing veins that were indistinct by comparison with the anterior one. Fluorescence staining of the en-GAL4> UAS-dDuoxIR(976-1145) adult wings revealed that the ROS concentration in the posterior compartment was significantly lower than that in the anterior compartment. Moreover, in these flies, the posterior compartment of the wing imaginal disc showed a greater number of apoptotic cells detected by immunostaining with anti-cleaved caspase-3 antibody than those in the anterior compartment. Respective knockdown of tyrosine hydroxylase or dopa-decarboxylase showed paler wing blades in the posterior compartment similar to the phenotype of dDuox-knockdown files. Along with this observation, analysis of the catecholic and dityrosine components in the wings of adult flies proved that dDuox plays important roles in the stabilization of the cuticle structure of the wings via tyrosine cross-linking, the sclerotization and melanization processes possibly through ROS production. These dDuox-knockdown fly lines would be useful tools for further studying dDuox functions during the development of Drosophila.