Rad9, Rad17, TopBP1 and Claspin Play Essential Roles in Heat-Induced Activation of ATR Kinase and Heat Tolerance

Hyperthermia is widely used to treat patients with cancer, especially in combination with other treatments such as radiation therapy. Heat treatment per se activates DNA damage responses mediated by the ATR-Chk1 and ATM-Chk2 pathways but it is not fully understood how these DNA damage responses are activated and affect heat tolerance. By performing a genetic analysis of human HeLa cells and chicken B lymphoma DT40 cells, we found that heat-induced Chk1 Ser345 phosphorylation by ATR was largely dependent on Rad9, Rad17, TopBP1 and Claspin. Activation of the ATR-Chk1 pathway by heat, however, was not associated with FancD2 monoubiquitination or RPA32 phosphorylation, which are known as downstream events of ATR kinase activation when replication forks are stalled. Downregulation of ATR, Rad9, Rad17, TopBP1 or Claspin drastically reduced clonogenic cell viability upon hyperthermia, while gene knockout or inhibition of ATM kinase reduced clonogenic viability only modestly. Suppression of the ATR-Chk1 pathway activation enhanced heat-induced phosphorylation of Chk2 Thr68 and simultaneous inhibition of ATR and ATM kinases rendered severe heat cytotoxicity. These data indicate that essential factors for activation of the ATR-Chk1 pathway at stalled replication forks are also required for heat-induced activation of ATR kinase, which predominantly contributes to heat tolerance in a non-overlapping manner with ATM kinase.     

Ethanol production from high cellulose concentration by the basidiomycete fungus Flammulina velutipes

Ethanol production by Flammulina velutipes from high substrate concentrations was evaluated. F. velutipes produces approximately 40–60 g l⁻¹ ethanol from 15 % (w/v) d-glucose, d-fructose, d-mannose, sucrose, maltose, and cellobiose, with the highest conversion rate of 83 % observed using cellobiose as a carbon source. We also attempted to assess direct ethanol fermentation from sugarcane bagasse cellulose (SCBC) by F. velutipes. The hydrolysis rate of 15 % (w/v) SCBC with commercial cellulase was approximately 20 %. In contrast, F. velutipes was able to produce a significant amount of ethanol from 15 % SCBC with the production of β-glucosidase, cellobohydrolase, and cellulase, although the addition of a small amount of commercial cellulase to the culture was required for the conversion. When 9 mg g⁻¹ biomass of commercial cellulase was added to cultures, 0.36 g of ethanol was produced from 1 g of cellulose, corresponding to an ethanol conversion rate of 69.6 %. These results indicate that F. velutipes would be useful for consolidated bioprocessing of lignocellulosic biomass to bioethanol.     

Transmural and apicobasal gradients in repolarization contribute to T-wave genesis in human surface ECG

The cellular basis of the T-wave morphology of surface ECG remains controversial in clinical cardiology. We examined the effect of action potential duration (APD) distribution on T-wave morphology using a realistic model of the human ventricle and torso. We developed a finite-element model of the ventricle consisting of ～26 million elements, including the conduction system, each implemented with the ion current model of cardiomyocytes. This model was embedded in a torso model with distinct organ structures to obtain the standard ECG leads. The APD distribution was changed in the transmural direction by locating the M cells in either the endocardial or epicardial region. We also introduced apicobasal gradients by modifying the ion channel parameters. Both the transmural gradient (with M cells on the endocardial side) and the apicobasal gradient produced positive T waves, although a very large gradient was required for the apicobasal gradient. By contrast, T waves obtained with the transmural gradient were highly symmetric and, therefore, did not represent the true physiological state. Only combination of the transmural and the moderate apicobasal gradients produced physiological T waves in surface ECG. Positive T waves in surface ECG mainly originated from the transmural distribution of APD with M cells on the endocardial side, although the apicobasal gradient was also required to attain the physiological waveform.     

Effect of lime pretreatment of brown midrib sorghums

The effect of lime pretreatment of brown midrib sorghums on enzymatic saccharification was investigated. Under most of the pretreatment conditions, the saccharification yields of bmrs were higher than those of the normal counterparts. This result suggests that bmr is useful to reduce pretreatment costs, because the amount of lime necessary for the pretreatment of biomass can reduced by using bmr mutants.     

Provision of continuous maturation signaling to dendritic cells by RIG-I-stimulating cytosolic RNA synthesis of Sendai virus

Dendritic cell (DC)-based immunotherapy has potential for treating infections and malignant tumors, but the functional capacity of DC must be assessed in detail, especially maturation and Ag-specific CTL priming. Recent reports suggest that DC that are provided with continuous maturation signals in vivo after transfer into patients are required to elicit the full DC functions. We demonstrate in this study that the rSendai virus vector (SeV) is a novel and ideal stimulant, providing DC with a continuous maturation signal via viral RNA synthesis in the cytosol, resulting in full maturation of monocyte-derived DC(s). Both RIG-I-dependent cytokine production and CD4 T cell responses to SeV-derived helper Ags are indispensable for overcoming regulatory T cell suppression to prime melanoma Ag recognized by T cell-1-specific CTL in the regulatory T cell abundant setting. DC stimulated via cytokine receptors, or TLRs, do not show these functional features. Therefore, SeV-infected DC have the potential for DC-directed immunotherapy.     

Autoregulator protein PhaR for biosynthesis of polyhydroxybutyrate [P(3HB)] possibly has two separate domains that bind to the target DNA and P(3HB): Functional mapping of amino acid residues responsible for DNA binding

PhaR from Paracoccus denitrificans functions as a repressor or autoregulator of the expression of genes encoding phasin protein (PhaP) and PhaR itself, both of which are components of polyhydroxyalkanoate (PHA) granules (A. Maehara, S. Taguchi, T. Nishiyama, T. Yamane, and Y. Doi, J. Bacteriol. 184:3992-4002, 2002). PhaR is a unique regulatory protein in that it also has the ability to bind tightly to an effector molecule, PHA polyester. In this study, by using a quartz crystal microbalance, we obtained direct evidence that PhaR binds to the target DNA and poly[(R)-3-hydroxybutyrate] [P(3HB)], one of the PHAs, at the same time. To identify the PhaR amino acid residues responsible for DNA binding, deletion and PCR-mediated random point mutation experiments were carried out with the gene encoding the PhaR protein. PhaR point mutants with decreased DNA-binding abilities were efficiently screened by an in vivo monitoring assay system coupled with gene expression of green fluorescent protein in Escherichia coli. DNA-binding abilities of the wild-type and mutants of recombinant PhaR expressed in E. coli were evaluated using a gel shift assay and a surface plasmon resonance analysis. These experiments revealed that basic amino acids and a tyrosine in the N-terminal region, which is highly conserved among PhaR homologs, are responsible for DNA binding. However, most of the mutants with decreased DNA-binding abilities were unaffected in their ability to bind P(3HB), strongly suggesting that PhaR has two separate domains capable of binding to the target DNA and P(3HB).     

Spreds are essential for embryonic lymphangiogenesis by regulating vascular endothelial growth factor receptor 3 signaling

Spred/Sprouty family proteins negatively regulate growth factor-induced ERK activation. Although the individual physiological roles of Spred-1 and Spred-2 have been investigated using gene-disrupted mice, the overlapping functions of Spred-1 and Spred-2 have not been clarified. Here, we demonstrate that the deletion of both Spred-1 and Spred-2 resulted in embryonic lethality at embryonic days 12.5 to 15.5 with marked subcutaneous hemorrhage, edema, and dilated lymphatic vessels filled with erythrocytes. This phenotype resembled that of Syk^−/− and SLP-76^−/− mice with defects in the separation of lymphatic vessels from blood vessels. The number of LYVE-1-positive lymphatic vessels and lymphatic endothelial cells increased markedly in Spred-1/2-deficient embryos compared with WT embryos, while the number of blood vessels was not different. Ex vivo colony assay revealed that Spred-1/2 suppressed lymphatic endothelial cell proliferation and/or differentiation. In cultured cells, the overexpression of Spred-1 or Spred-2 strongly suppressed vascular endothelial growth factor-C (VEGF-C)/VEGF receptor (VEGFR)-3-mediated ERK activation, while Spred-1/2-deficient cells were extremely sensitive to VEGFR-3 signaling. These data suggest that Spreds play an important role in lymphatic vessel development by negatively regulating VEGF-C/VEGFR-3 signaling.     

A novel extracellular protease of Vibrio mimicus that mediates maturation of an endogenous hemolysin

Vibrio mimicus, a human pathogen that causes gastroenteritis, produces an enterotoxic hemolysin as a virulence factor. The hemolysin is secreted extracellularly as an inactive protoxin and converted to a mature toxin through removal of the N‐terminal propeptide, which comprises 151 amino acid residues. In this study, a novel protease having the trypsin‐like substrate specificity was purified from the bacterial culture supernatant. The N‐terminal amino acid sequence of the purified protein was identical with putative trypsin VMD27150 of V. mimicus strain VM573. The purified protease was found to cause maturation of the protoxin by cleavage of the Arg¹⁵¹? Ser¹⁵² bond. Deletion of the protease gene resulted in increased amounts of the protoxin in the culture supernatant. In addition, expression of the hemolysin and protease genes was detected during the logarithmic growth phase. These findings indicate that the protease purified may mediate maturation of the hemolysin.