ATR-dependent activation of the kinase Chk1 is the initial step in signal transduction in the DNA replication checkpoint, which allows a cell to enter mitosis only after the completion of DNA replication. TopBP1-related proteins in higher eukaryotes are implicated in the replication checkpoint, but their exact role remains elusive because of their requirements for replication initiation. Here we report that the initiation function of Xenopus Cut5/TopBP1 could be entirely separated from its checkpoint function: the N-terminal half fragment, a region of Cut5 conserved through evolution, is sufficient for initiation, but is incapable of activating the checkpoint; the C-terminal half fragment, which is unique in metazoan species, is by itself capable of activating the checkpoint response without initiating replication. Upon the activation of Chk1, the Ser1131 within the C-terminal region of Cut5 is phosphorylated, and this phosphorylation is critical for the checkpoint response. Furthermore, Cut5 directly stimulated Chk1 phosphorylation in the in vitro kinase assay reconstituted with recombinant proteins and ATR immunoprecipitated from extracts. On the basis of replication protein A (RPA)-dependent loading of Cut5 on to replicating and replication-arrested chromatin, we propose that Cut5 plays a crucial role in the initial amplification step of the ATR-Chk1 signaling pathway at the stalled replication fork.

Cohesin-mediated sister chromatid cohesion is established during the S-phase, and recent studies demonstrate that a cohesin protein ring concatenates sister DNA molecules. However, little is known about how DNA replication is linked to the establishment of sister chromatid cohesion. Here, we used Xenopus egg extracts to show that AND-1 and Tim1-Tipin, homologues of Saccharomyces cerevisiae Ctf4 and Tof1-Csm3, respectively, are associated with the replisome and are required for proper establishment of the cohesion observed in the M-phase extracts. Immunodepletion of both AND-1 and Tim1-Tipin from the extracts leads to aberrant sister chromatid cohesion, which is similarly induced by the depletion of cohesin. These results demonstrate that AND-1 and Tim1-Tipin are key factors linking DNA replication and establishment of sister chromatid cohesion. On the basis of the physical interactions between AND-1 and DNA polymerases, we discuss a model to describe how replisome progression complex establishes sister chromatid cohesion.

The c-kit proto-oncogene encodes a receptor tyrosine kinase that is crucial to hematopoiesis, melanogenesis, and gametogeneis. Although the enzymatic activity of the c-kit product (KIT) is regulated by its ligand, both the Val559-->Gly (G559) mutation in the juxtamembrane domain and the Asp814-->Val (V814) mutation in the phosphotransferase domain lead to constitutive activation of KIT. By retroviral infection of hematopoietic progenitor cells with KIT(G559) or KIT(V814), KIT(G559) induced development of granulocyte/macrophage and mast-cell colonies in vitro without the addition of exogenous growth factors. KIT(V814) induced factor-independent growth of various types of hematopoietic progenitor cells, resulting in the development of mixed erythroid/myeloid colonies in addition to granulocyte/macrophage and mast-cell colonies. Furthermore, transplantation of KIT(G559) and KIT(V814)-infected bone marrow cells led to development of acute leukemia in one of 10 and six of 10 transplanted mice, respectively. No mice developed hematologic malignancies after transplantation of wild- type KIT-infected cells. Furthermore, transgenic mice expressing KIT(V814) developed acute leukemia or malignant lymphoma. These results demonstrate a direct role of the mutant KITs, particularly KIT(V814), in tumorigenesis of hematopoietic cells and suggest that similar mutations may contribute to the development of human hematologic malignancies.

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The Ws mutant allele of rats represents a 12-base deletion at the tyrosine kinase domain of the c-kit gene. Although homozygous Ws/Ws rats were deficient in both connective tissue-type mast cells (CTMC) and mucosal-type mast cells (MMC), mast cells did develop when bone marrow cells of Ws/Ws rats were cultured in the presence of concanavalin A-stimulated spleen cell conditioned medium (ConA-SCM). Although the proliferative response of rat cultured mast cells (RCMC) derived from Ws/Ws rats to ConA-SCM was comparable to that of RCMC derived from control normal (+/+) rats, the proliferative response of Ws/Ws RCMC to rat recombinant stem cell factor (rrSCF; a ligand for the c-kit receptor tyrosine kinase) was much lower than that of +/+ RCMC. However, a slight c-kit kinase activity was detectable in Ws/Ws RCMC, and the proliferation of Ws/Ws RCMC was accelerated when rrSCF was added to ConA-SCM. Because CTMC contain rat mast cell protease-I (RMCP- I) and MMC contain RMCP-II, the phenotype of +/+ and Ws/Ws RCMC in various culture conditions was evaluated by immunohistochemistry of RMCPs. Both +/+ and Ws/Ws RCMC showed the MMC-like phenotype (RMCP-I- /II+) when they were cultured with ConA-SCM alone. Most +/+ RCMC and about half of Ws/Ws RCMC acquired a novel protease (RMCP-I+/II+) phenotype when they were cultured with rrSCF alone. However, because the number of Ws/Ws RCMC dropped to one-tenth in the medium containing rrSCF alone, the absolute number of Ws/Ws RCMC with the RMCP-I+/II+ phenotype did not increase significantly. The effect of rrSCF in inducing the novel phenotype was suppressed when ConA-SCM was added to rrSCF. In contrast, +/+ and Ws/Ws RCMC cocultured with +/+ fibroblasts showed the RMCP-I+/II+ phenotype even in the presence of ConA-SCM. Moreover, a fibroblast cell line derived from SI/SI mouse embryos that did not produce SCF did not support the survival of both +/+ and Ws/Ws RCMC but did induce the RMCP-I+/II+ phenotype in about half of +/+ and Ws/Ws RCMC when their survival was supported by the addition of ConA- SCM. The normal signal transduction through the c-kit receptor did not appear to be prerequisite for the acquisition of the RMCP-I+/II+ phenotype.

One-sentence summary:This study shows a novel function of GAPDH in homeostatic control of the heart, which is disturbed and results in cardiac hypertrophy with pathological stressors. Abstract:Pathological stressors disrupt cellular and organ homeostasis, causing various diseases. We discovered a novel role for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the pathological growth response of the heart, independent of its functions in glycolysis and cell death. In a cellular model for cardiac hypertrophy, endothelin-1 elicited nuclear translocation of GAPDH and activation of p300 histone acetyl-transferase (HAT), followed by activation of myocyte enhancer factor 2 (MEF2). GAPDH nuclear translocation and p300 HAT activation was also identified in rodent pathological hypertrophied hearts. The hypertrophy was markedly ameliorated by molecular and pharmacological interventions that antagonize the nuclear GAPDH pathway, including a novel antagonist selective to its nuclear function. This pathway may be the key to stress response/homeostatic control, and thus the potential therapeutic target for stressassociated diseases. Main Text:Besides glycolytic function, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) translocate to the nuclei in response to stress where it has been described to mainly regulate cell death (1-4).The nuclear GAPDH pathway is triggered by a specific oxidation/S-nitrosylation of GAPDH at cysteine-150, which enables the interaction of the pool of GAPDH with Siah1 (5, 6). The protein complex translocates to the nucleus where GAPDH can affect p300, p53 and p53 upregulated modulator of apoptosis (PUMA) resulting in cell death; in this cascade, only a small fraction of GAPDH is converted to a signaling molecule, and the overall change in cytosolic and glycolytic GAPDH is negligible (5, 7). Despite it is involvement in cell death, it remains unclear whether and how this cascade plays general and diverse roles.The heart is among the organs with the highest expression of GAPDH (8), which has been simply thought to play a "house keeping" glycolytic role in this organ. The heart develops hypertrophy or abnormal growth in response to pathological stress, which can ultimately result in the organ failure (9). Indeed, cardiac hypertrophy and failure is a leading cause of death worldwide, imposing an enormous burden on society (10). Although p300 has been implicated in this pathophysiology (11,12), the overall significance and its regulatory mechanism remain elusive. We hypothesize that nuclear GAPDH-p300 signaling may mediate this stress response, which disrupts critical homeostasis of cardiac myocytes and causes hypertrophic growth.To address this question, we used a mouse model of pressure-overload cardiac hypertrophy induced by transverse aortic constriction (TAC) (13). In this model, cardiac hypertrophy is developed at 7 to 10 days after TAC, transitioning to failure in 63 days after TAC.The histone acetyl transferase (HAT) activity of p300 was markedly increased in TAC hearts at Reagents and RR compound...

Background/Aims: Chronic kidney disease (CKD) is the common cause of end-stage renal disease. Antihypertensive agents are clinically used to inhibit the progression of CKD. However, these agents cannot completely prevent progression to renal failure. We have previously reported that 5-chloro-2-{(1E)-3-[2-(4-methoxybenzoyl)-4-methyl-1H- pyrrol-1-yl]prop-1en-1-yl}-N-(methylsulfonyl)benzamide (SMP-534) improves renal disease and prevents the production of extracellular matrix in vitro. Additionally, SMP-534 inhibits glomerular fibrosis and provides renoprotection in vivo. In the present study, we investigated the effect of SMP-534 on renal dysfunction in a 5/6 nephrectomized (5/6Nx) rat model. Method: Five groups of rats were studied: sham operated, 5/6Nx + vehicle, 5/6Nx + SMP-534 30 mg/kg, 5/6Nx + SMP-534 60 mg/kg and 5/6Nx + SMP-534 90 mg/kg. Treatment with SMP-534 began 13 weeks after surgery, when hypertension and renal insufficiency had developed. Serum creatinine, blood urea nitrogen levels, creatinine clearance and urinary albumin were measured at specific time points. Results: Serum creatinine and blood urea nitrogen levels were significantly reduced in SMP-534-treated groups. In addition, SMP-534 dose-dependently suppressed the increase in urinary albumin excretion observed in 5/6Nx rats. Moreover, survival rates were improved in SMP-534-treated groups. Conclusion: We have shown in this study that chronic oral administration of SMP-534 improves renal dysfunction in 5/6Nx rats. These findings indicate that SMP-534 may be a new therapeutic agent for the treatment of CKD.

The Wsh is a mutant allele at the W (c-kit) locus of mice. Mice of Wsh/Wsh genotype have white hairs and black eyes. Although adult C57BL/6-Wsh/Wsh mice were not anemic, they showed a remarkable depletion of mast cells. Most homozygous or double heterozygous mutant mice at the W (c-kit) locus, of which mast-cell depletion was comparable to that of Wsh/Wsh mice, are deficient in germ cells. However, male and female Wsh/Wsh mice have an appreciable number of germ cells in their gonads. We investigated the mechanism of specific depletion of mast cells in Wsh/Wsh mice. Cultured mast cells (CMC) derived from the spleen of Wsh/Wsh mice neither attached to normal (+/+) fibroblasts nor survived in the coculture with +/+ fibroblasts. The c-kit messenger RNA (mRNA) was strongly expressed in +/+ CMC, but not detectable in Wsh/Wsh CMC. Despite the lack of c-kit mRNA in Wsh/Wsh CMC, the c-kit mRNA was normally detectable in the cerebellum and weakly detectable in the testis and spleen of Wsh/Wsh mice. No significant changes were found in the nucleotide sequence of the c-kit transcripts obtained from the cerebellum of Wsh/Wsh mice. Development of mast cells, erythrocytes, and germ cells in Wsh/Wsh mice appeared to be parallel with the magnitude of the c-kit gene expression in each cell type.