The gs3 allele from a large-grain rice cultivar, Akita 63, increases yield and improves nitrogen-use efficiency.

The Green Revolution allowed a large amount of nitrogen (N) fertilization to increase crop yield but has led to severe environmental pollution. Therefore, increasing the crop grain yield must be achieved without such considerable input of N fertilization. A large-grain japonica rice cultivar, Akita 63, significantly increased grain yield and improved N-use efficiency (NUE) for yield per amount of N absorbed by plants. This study found that the nonsense mutated GS3 gene, the gs3 allele of Akita 63, has a superior yield production with enlarged grain size. The gs3 allele increased the yield with improvements in harvest index and NUE for yields per plant N content by analyzing the near-isogenic line of rice plants with a large grain (LG-Notohikari), which was developed by introducing the gs3 allele of Akita 63 into normal-grain japonica cultivar, Notohikari. Thus, the gs3 allele would be promising for further yield increase without additional large input of N fertilization in non-gs3-allele rice varieties.

Loss of the fragile X syndrome protein FMRP results in misregulation of nonsense-mediated mRNA decay.

Loss of the fragile X protein FMRP is a leading cause of intellectual disability and autism1,2, but the underlying mechanism remains poorly understood. We report that FMRP deficiency results in hyperactivated nonsense-mediated mRNA decay (NMD)3,4 in human SH-SY5Y neuroblastoma cells and fragile X syndrome (FXS) fibroblast-derived induced pluripotent stem cells (iPSCs). We examined the underlying mechanism and found that the key NMD factor UPF1 binds directly to FMRP, promoting FMRP binding to NMD targets. Our data indicate that FMRP acts as an NMD repressor. In the absence of FMRP, NMD targets are relieved from FMRP-mediated translational repression so that their half-lives are decreased and, for those NMD targets encoding NMD factors, increased translation produces abnormally high factor levels despite their hyperactivated NMD. Transcriptome-wide alterations caused by NMD hyperactivation have a role in the FXS phenotype. Consistent with this, small-molecule-mediated inhibition of hyperactivated NMD, which typifies iPSCs derived from patients with FXS, restores a number of neurodifferentiation markers, including those not deriving from NMD targets. Our mechanistic studies reveal that many molecular abnormalities in FMRP-deficient cells are attributable-either directly or indirectly-to misregulated NMD.

Growth inhibition of imatinib-resistant CML cells with the T315I mutation and hypoxia-adaptation by AV65--a novel Wnt/ß-catenin signaling inhibitor.

We investigated the effect of a novel Wnt/ß-catenin signaling inhibitor, AV65 on imatinib mesylate (IM)-sensitive and -resistant human chronic myeloid leukemia (CML) cells in vitro. AV65 inhibited the proliferation of various CML cell lines including T315I mutation-harboring cells. AV65 reduced the expression of ß-catenin in CML cells, resulting in the induction of apoptosis. Moreover, AV65 inhibited the proliferation of hypoxia-adapted primitive CML cells that overexpress ß-catenin. The combination of AV65 with IM had a synergistic inhibitory effect on the proliferation of CML cells. These findings suggest that AV65 could be a novel therapeutic agent for the treatment of CML.

Rapid automated detection of ABL kinase domain mutations in imatinib-resistant patients.

ABL tyrosine kinase inhibitor (TKI), imatinib is used for BCR-ABL(+) leukemias. We developed an automatic method utilizing guanine-quenching probes (QP) to detect 17 kinds of mutations frequently observed in imatinib-resistance. Results were obtained from 100µL of whole blood within 90min by this method. Detected mutations were almost identical between QP method and direct sequencing. Furthermore, the mutation-biased PCR (MBP) was added to the QP method to increase sensitivity, resulting earlier detection of T315I mutation which was insensitive to any ABL TKIs. Thus, the QP and MBP-QP may become useful methods for the management of ABL TKI-treated patients.

Rakicidin A effectively induces apoptosis in hypoxia adapted Bcr-Abl positive leukemic cells.

Treatment with Abl tyrosine kinase inhibitors (TKI) drastically improves the prognosis of chronic myelogenous leukemia (CML) patients. However, quiescent CML cells are insensitive to TKI and can lead to relapse of the disease. Thus, research is needed to elucidate the properties of these quiescent CML cells, including their microenvironment, in order to effectively target them. Hypoxia is known to be a common feature of solid tumors that contributes to therapeutic resistance. Leukemic cells are also able to survive and proliferate in severely hypoxic environments. The hypoxic conditions in the bone marrow (BM) allow leukemic cells that reside there to become insensitive to cell death stimuli. To target leukemic cells in hypoxic conditions, we focused on the hypoxia-selective cytotoxin, Rakicidin A. A previous report showed that Rakicidin A, a natural product produced by the Micromonospora strain, induced hypoxia-selective cytotoxicity in solid tumors. Here, we describe Rakicidin A-induced cell death in hypoxia-adapted (HA)-CML cells with stem cell-like characteristics. Interestingly, apoptosis was induced via caspase-dependent and -independent pathways. In addition, treatment with Rakicidin A in combination with the TKI, imatinib, resulted in synergistic cytotoxicity against HA-CML cells. In conclusion, Rakicidin A is a promising compound for targeting TKI-resistant quiescent CML stem cells in the hypoxic BM environment.

Galectin-9 ameliorates acute GVH disease through the induction of T-cell apoptosis.

Galectins comprise a family of animal lectins that differ in their affinity for ß-galactosides. Galectin-9 (Gal-9) is a tandem-repeat-type galectin that was recently shown to function as a ligand for T-cell immunoglobin domain and mucin domain-3 (Tim-3) expressed on terminally differentiated CD4(+) Th1 cells. Gal-9 modulates immune reactions, including the induction of apoptosis in Th1 cells. In this study, we investigated the effects of Gal-9 in murine models of acute GVH disease (aGVHD). First, we demonstrated that recombinant human Gal-9 inhibit MLR in a dose-dependent manner, involving both Ca(2+) influx and apoptosis in T cells. Next, we revealed that recombinant human Gal-9 significantly inhibit the progression of aGVHD in murine BM transplantation models. In conclusion, Gal-9 ameliorates aGVHD, possibly by inducing T-cell apoptosis, suggesting that gal-9 may be an attractive candidate for the treatment of aGVHD.

Activity of the multitargeted kinase inhibitor, AT9283, in imatinib-resistant BCR-ABL-positive leukemic cells.

Despite promising clinical results from imatinib mesylate and second-generation ABL tyrosine kinase inhibitors (TKIs) for most BCR-ABL(+) leukemia, BCR-ABL harboring the mutation of threonine 315 to isoleucine (BCR-ABL/T315I) is not targeted by any of these agents. We describe the in vitro and in vivo effects of AT9283 (1-cyclopropyl-3[5-morpholin-4yl methyl-1H-benzomidazol-2-yl]-urea), a potent inhibitor of several protein kinases, including Aurora A, Aurora B, Janus kinase 2 (JAK2), JAK3, and ABL on diverse imatinib-resistant BCR-ABL(+) cells. AT9283 showed potent antiproliferative activity on cells transformed by wild-type BCR-ABL and BCR-ABL/T315I. AT9283 inhibited proliferation in a panel of BaF3 and human BCR-ABL(+) cell lines both sensitive and resistant to imatinib because of a variety of mechanisms. In BCR-ABL(+) cells, we confirmed inhibition of substrates of both BCR-ABL (signal transducer and activator of transcription-5) and Aurora B (histone H3) at physiologically achievable concentrations. The in vivo effects of AT9283 were examined in several mouse models engrafted either subcutaneously or intravenously with BaF3/BCR-ABL, human BCR-ABL(+) cell lines, or primary patient samples expressing BCR-ABL/T315I or glutamic acid 255 to lysine, another imatinib-resistant mutation. These data together support further clinical investigation of AT9283 in patients with imatinib- and second-generation ABL TKI-resistant BCR-ABL(+) cells, including T315I.

A combination of a DNA-chimera siRNA against PLK-1 and zoledronic acid suppresses the growth of malignant mesothelioma cells in vitro.

Although novel agents effective against malignant mesothelioma (MM) have been developed, the prognosis of patients with MM is still poor. We generated a DNA-chimeric siRNA against polo-like kinase-1 (PLK-1), which was more stable in human serum than the non-chimeric siRNA. The chimeric PLK-1 siRNA inhibited MM cell proliferation through the induction of apoptosis. Next, we investigated the effects of zoledronic acid (ZOL) on MM cells, and found that ZOL also induced apoptosis in MM cells. Furthermore, ZOL augmented the inhibitory effects of the PLK-1 siRNA. In conclusion, combining a PLK-1 siRNA with ZOL treatment is an attractive strategy against MM.

Osteoclasts are involved in the maintenance of dormant leukemic cells.

Osteoclasts (OCs) are specialized cells for the resorption of bone matrix that have also been recently reported to be involved in the mobilization of hematopoietic progenitor cells. When Ba/F3 cells expressing wild-type bcr-abl were co-cultured with osteoblasts (OBs), OCs, and bone slices, their proliferation was significantly suppressed, and the Ki-67 negative population, which is believed to be in G(0) phase, was increased. The results of our in vitro experiments suggest that OCs could be involved in the maintenance of dormant leukemic cells in the bone marrow (BM) microenvironment through the release of soluble factors, one of which could be TGF-beta.

Production of short-chain-length/medium-chain-length polyhydroxyalkanoate (PHA) copolymer in the plastid of Arabidopsis thaliana using an engineered 3-ketoacyl-acyl carrier protein synthase III.

Short-chain-length/medium-chain-length (SCL/MCL) polyhydroxyalkanoate (PHA) was produced in the plastids of Arabidopsis thaliana. Phe87Thr (F87T) mutated 3-ketoacyl-acyl carrier protein (ACP) synthase III (FabH) from Escherichia coli , and Ser325Thr/Gln481Lys (ST/QK) mutated polyhydroxyalkanoate (PHA) synthase (PhaC1) from Pseudomonas sp. 61-3, along with the beta-ketothiolase (PhaA) and acetoacetyl-CoA reductase (PhaB) from Ralstonia eutropha (Cupriavidus necator) genes were introduced into Arabidopsis. The transgenic Arabidopsis produced PHA copolymers composed of monomers consisting of 4-14 carbons. The introduction of the engineered PHA synthase resulted in a 10-fold increase in PHA content compared to plants expressing the wild-type PHA synthase. In addition, expression of the engineered fabH gene in the plastid led to an increase in the amount of the SCL monomer, 3-hydroxybutyrate, incorporated into PHA, and contributed to supply of MCL monomers for PHA production.

A refined two-hybrid system reveals that SCF(Cdc4)-dependent degradation of Swi5 contributes to the regulatory mechanism of S-phase entry.

Ubiquitin-dependent degradation is implicated in various cellular regulatory mechanisms. The SCF(Cdc4) (Skp1, Cullin/Cdc53, and the F-box protein Cdc4) complex is an ubiquitin ligase complex that acts as a regulator of cell cycle, signal transduction, and transcription. These regulatory mechanisms are not well defined because of the difficulty in identifying the interaction between ubiquitin ligases and their substrates. To identify substrates of the yeast SCF(Cdc4) ubiquitin ligase complex, we refined the yeast two-hybrid system to allow screening Cdc4-substrate interactions under conditions of substrate stabilization, and identified Swi5 as a substrate of the SCF(Cdc4) complex. Swi5 is the transcriptional activator of Sic1, the inhibitor of S phase cyclin-dependent kinases (CDKs). We showed that Swi5 is indeed ubiquitinated and degraded through the SCF(Cdc4) complex. Furthermore, the SCF(Cdc4)-dependent degradation of Swi5 was required to terminate SIC1 transcription at early G(1) phase, which ensured efficient entry into S phase: Hyperaccumulation of Sic1 was noted in cells expressing stabilized Swi5, and expression of stabilized Swi5 delayed S phase entry, which was dominantly suppressed by SIC1 deletion. These findings indicate that the SCF(Cdc4) complex regulates S phase entry not only through degradation of Sic1, but also through degradation of Swi5.

Oxidative stress-induced ubiquitination of RCAN1 mediated by SCFbeta-TrCP ubiquitin ligase.

A change in the protein level of RCAN1 (DSCR1/MCIP/Adapt78/CSP1) has been implicated in oxidative stress-induced cell death in neurons and in the pathogenesis of Alzheimer's disease. The pathogenic processes in neurodegenerative diseases are closely related to oxidative stress and the ubiquitin proteasome system (UPS). Therefore, we investigated whether oxidative stress induces a change in the protein level of RCAN1 through the UPS. H2O2 induced ubiquitination of RCAN1 at the same concentrations as those causing a decrease in RCAN1 in HEK293T cells. beta-TrCP, the F-box protein component of SCF ubiquitin ligase, interacted with RCAN1 in response to H2O2 stimulation. Although FBW4, another F-box protein, interacted with RCAN1, its interaction was independent of H2O2 stimulation. In vitro ubiquitination assay showed that SCFbeta-TrCP but not SCFFBW4 increased ubiquitination of RCAN1, dependent on H2O2 stimulation. In addition, knockdown of beta-TrCP by siRNA abolished the H2O2-induced decrease in RCAN1 in HEK293T cells. We further examined whether RCAN1 undergoes ubiquitination by H2O2 in primary neurons, similarly to that in HEK293T cells. An H2O2-induced decrease in RCAN1 was exhibited also in hippocampal and cortical neurons. Ubiquitination of RCAN1 was induced by 500 muM H2O2, the concentration at which H2O2 induced a decrease in RCAN1 in primary neurons. These results suggest that H2O2 induces SCF beta-TrCP-mediated ubiquitination of RCAN1, leading to a decrease in the protein level of RCAN1.

The SCFCdc4 ubiquitin ligase regulates calcineurin signaling through degradation of phosphorylated Rcn1, an inhibitor of calcineurin.

The highly conserved RCN family of proteins regulates the serine/threonine protein phosphatase calcineurin, which is required for the expression of genes involved in Ca(2+)-dependent processes, such as the control of memory, apoptosis, T cell activation, cell cycle, Ca(2+)-homeostasis, and skeletal and cardiac muscle growth and differentiation. However, RCNs regulate calcineurin through two paradoxical actions: they act as feedback inhibitors of calcineurin, whereas their phosphorylation stimulates calcineurin. Here we show that phosphorylation of yeast RCN, Rcn1, triggers degradation through the SCF(Cdc4) ubiquitin ligase complex. Degradation of phosphorylated Rcn1 is required to mitigate inhibition of calcineurin by Rcn1 and results in activation of calcineurin activity in response to Ca(2+) as well as in reactivation of calcineurin in response to changes in Ca(2+) concentration. The SCF(Cdc4)-dependent degradation required phosphorylation of Rcn1 by Mck1, a member of the GSK3 family of protein kinases, and was promoted by Ca(2+). However, such degradation was counteracted by dephosphorylation of Rcn1, which was promoted by Ca(2+)-stimulated calcineurin. Thus, calcineurin activity is fine-tuned to Ca(2+) signals by mechanisms that have opposite functions. Our results identify the molecular mechanism of Rcn1 phosphorylation-induced stimulation of the phosphatase activity of calcineurin. The results provide insight into the mechanism involved in maintaining proper responses to Ca(2+) signals.

Enhancement of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production in the transgenic Arabidopsis thaliana by the in vitro evolved highly active mutants of polyhydroxyalkanoate (PHA) synthase from Aeromonas caviae.

In this study, the enhancement of photosynthetic PHA production was achieved using the highly active mutants of PHA synthase created by the in vitro evolutionally techniques. The wild-type and mutated PHA synthase genes from Aeromonas caviae were introduced into Arabidopsis thaliana together with the NADPH-dependent acetoacetyl-CoA reductase gene from Ralstonia eutropha. Expression of the highly active mutated PHA synthase genes, N149S and D171G, led to an 8-10-fold increase in PHA content in the T1 transgenic Arabidopsis, compared to plants harboring the wild-type PHA synthase gene. In homozygous T2 progenies, PHA content was further increased up to 6.1 mg/g cell dry weight. GC/MS analysis of the purified PHA from the transformants revealed that these PHAs were poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] copolymers consisting of 0.2-0.8 mol % 3HV. The monomer composition of the P(3HB-co-3HV) copolymers synthesized by the wild-type and mutated PHA synthases reflected the substrate specificities observed in Escherichia coli. These results indicate that in vitro evolved PHA synthases can enhance the productivity of PHA and regulate the monomer composition in transgenic plants.