Synthesis and biological evaluation of molecular probes based on the 9-methylstreptimidone derivative DTCM-glutarimide

Molecular probes based on 3-[(dodecylthiocarbonyl)methyl]glutarimide (DTCM-glutarimide) were synthesized and assessed for inhibitory activity against LPS-induced NO production. Among the probes examined, several derivatives exhibited potential for use in determining the target proteins of DTCM-glutarimide.     

β-d-2'-α-F-2'-β-C-Methyl-6-O-substituted 3',5'-cyclic phosphate nucleotide prodrugs as inhibitors of hepatitis C virus replication: A structure-activity relationship study

The 3',5'-cyclic phosphate prodrug 9-[β-d-2'-deoxy-2'-α-fluoro-2'-β-C-methylribofuranosyl]-2-amino-6-ethoxypurine, PSI-352938 1, has demonstrated promising anti-HCV efficacy in vitro and in human clinical trials. A structure-activity relationship study of the nucleoside 3',5'-cyclic phosphate series of β-d-2'-deoxy-2'-α-fluoro-2'-β-C-methylribofuranosyl nucleoside prodrugs was undertaken and the anti-HCV activity and in vitro safety profile were assessed. Cycloalkyl 3',5'-cyclic phosphate prodrugs were shown to be significantly more potent as inhibitors of HCV replication than branched and straight chain alkyl 3',5'-cyclic phosphate prodrugs. No cytotoxicity and mitochondrial toxicity for prodrugs 12, 13 and 19 were observed at concentrations up to 100μm in vitro. Cycloalkyl esters of 3',5'-cyclic phosphate nucleotide prodrugs demonstrated the ability to produce high levels of active triphosphate in clone-A cells and primary human hepatocytes. Compounds 12, 13 and 19 also demonstrated the ability to effectively deliver in vivo high levels of active nucleoside phosphates to rat liver.     

Hypoxic stress activates chaperone-mediated autophagy and modulates neuronal cell survival

Autophagy is a conserved mechanism responsible for the continuous clearance of unnecessary organelles or misfolded proteins in lysosomes. Three types of autophagy have been reported in the difference of substrate delivery to lysosome: macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA). Among these types, CMA is a unique autophagy system that selectively degrades substrates detected by heat shock cognate protein 70 (HSC70). Recently, autophagic cell death has been reported to be involved in neuronal death following brain ischemia; however, the contribution of CMA to neuronal death/survival after ischemic stress has not been addressed. In the present study, we determined whether quantitative alterations in LAMP-2A, which is the key molecule in CMA, would modulate neuronal cell survival under hypoxic conditions. Incubation of Neuro2A cells in a hypoxic chamber (1% O(2), 5% CO(2)) increased the level of LAMP-2A and induced accumulation of LAMP-2A-positive lysosomes in the perinuclear area, which is a hallmark of CMA activation. The activation of CMA in response to hypoxia was also confirmed by the GAPDH-HaloTag CMA indicator system at the single cell level. Next, we asked whether CMA was involved in cell survival during hypoxia. Blocking LAMP-2A expression with siRNA increased the level of cleaved caspase-3 and the number of propidium iodide-positive cells after hypoxic stress regardless of whether macroautophagy could occur, whereas the administration of mycophenolic acid, a potent CMA activator, rescued hypoxia-mediated cell death. Finally, we asked whether CMA was activated in the neurons after middle cerebral artery occlusion in vivo. The expression of LAMP-2A was significantly increased in the ischemic hemisphere seven days after brain ischemia. These results indicate that CMA is activated during hypoxia and contributes to the survival of cells under these conditions.     

Identification and characterization of Xenopus kctd15, an ectodermal gene repressed by the FGF pathway

The FGF pathway regulates a variety of developmental processes in animals through activation and/or repression of numerous target genes. Here we have identified a Xenopus homolog of potassium channel tetramerization domain containing 15 (KCTD15) as an FGF-repressed gene. Kctd15 expression is first detected at the gastrula stage and gradually increases until the tadpole stage. Whole-mount in situ hybridization reveals that the spatial expression of kctd15 is tightly regulated during early embryogenesis. While kctd15 is uniformly expressed throughout the presumptive ectoderm at the early gastrula stage, its expression becomes restricted to the non-neural ectoderm and is excluded from the neural plate at the early neurula stage. At the mid-neurula stage, kctd15 shows a more restricted distribution pattern in regions that are located at the anterior, lateral or medial edge of the neural fold, including the preplacodal ectoderm, the craniofacial neural crest and the prospective roof plate. At the tailbud stage, kctd15 expression is mainly detected in neural crest- or placode-derived tissues that are located around the eye, including the mandibular arch, trigeminal ganglia and the olfactory placode. FGF represses kctd15 expression in ectodermal explants, and the inhibition of FGF receptor with a chemical compound dramatically expands the region expressing kctd15 in whole embryos. Dorsal depletion of kctd15 in Xenopus embryos leads to bent axes with reduced head structures, defective eyes and abnormal somites, while ventral depletion causes defects in ventral and caudal morphologies. These results suggest that kctd15 is an FGF-repressed ectodermal gene required for both dorsal and ventral development.     

Centaurin-alpha1 is a phosphatidylinositol 3-kinase-dependent activator of ERK1/2 mitogen-activated protein kinases

Centaurin-alpha1 is known to be a phosphatidylinositol 3,4,5-triphosphate (PIP3)-binding protein that has two pleckstrin homology domains and a putative ADP ribosylation factor GTPase-activating protein domain. However, the physiological function of centaurin-alpha1 is still not understood. Here we have shown that transient expression of centaurin-alpha1 in COS-7 cells results in specific activation of ERK, and the activation is inhibited by co-expression of a dominant negative form of Ras. We have also found that a mutant form of centaurin-alpha1 that is unable to bind PIP3 fails to induce ERK activation and that a phosphatidylinositol 3-kinase inhibitor LY294002 inhibits centaurin-alpha1-dependent ERK activation. Furthermore, transient knockdown of centaurin-alpha1 by small interfering RNAs results in reduced ERK activation after epidermal growth factor stimulation in T-REx 293 cells. These results suggest that centaurin-alpha1 contributes to ERK activation in growth factor signaling, linking the PI3K pathway to the ERK mitogen-activated protein kinase pathway through its ability to interact with PIP3.     

Dynamics of the Ras/ERK MAPK cascade as monitored by fluorescent probes

To comprehend the Ras/ERK MAPK cascade, which comprises Ras, Raf, MEK, and ERK, several kinetic simulation models have been developed. However, a large number of parameters that are essential for the development of these models are still missing and need to be set arbitrarily. Here, we aimed at collecting these missing parameters using fluorescent probes. First, the levels of the signaling molecules were quantitated. Second, to monitor both the activation and nuclear translocation of ERK, we developed probes based on the principle of fluorescence resonance energy transfer. Third, the dissociation constants of Ras.Raf, Raf.MEK, and MEK.ERK complexes were estimated using a fluorescent tag that can be highlighted very rapidly. Finally, the same fluorescent tag was used to measure the nucleocytoplasmic shuttling rates of ERK and MEK. Using these parameters, we developed a kinetic simulation model consisting of the minimum essential members of the Ras/ERK MAPK cascade. This simple model reproduced essential features of the observed activation and nuclear translocation of ERK. In this model, the concentration of Raf significantly affected the levels of phospho-MEK and phospho-ERK upon stimulation. This prediction was confirmed experimentally by decreasing the level of Raf using the small interfering RNA technique. This observation verified the usefulness of the parameters collected in this study.     

Chromosome condensation outside of mitosis: mechanisms and new tools

A basic principle of cell physiology is that chromosomes condense during mitosis. However, condensation can be uncoupled from mitotic events under certain circumstances. This phenomenon is known as "premature chromosome condensation (PCC)." PCC provides insights in the mechanisms of chromosome condensation, thus helping clarifying the key molecular events leading to the mitosis. Besides, PCC has proved to be an useful tool for analyzing chromosomes in interphase. For example, using PCC we can visualize genetic damage shortly after the exposure to clastogenic agents. More than 30 years ago, the first report of PCC in interphase cells fused to mitotic cells using Sendai virus was described (virus-mediated PCC). The method paved the way to a great number of fundamental discoveries in cytogenetics, radiation biology, and related fields, but it has been hampered by technical difficulties. The novel drug-induced PCC method was introduced about 10 years ago. While fusion-induced PCC exploits the action of external maturation/mitosis promoting factor (MPF), migrating from the inducer mitotic cell to the interphase recipient, drug-induced PCC exploits protein phosphatase inhibitors, which can activate endogenous intracellular MPF. This method is much simpler than fusion-induced PCC, and has already proven useful in different fields.     

Augmentation of innate immunity by low-dose irradiation

The effect of low-dose irradiation on the immune system was investigated in mice. When a 0.2 Gy dose of X-ray irradiation was administered every other day for a total of four times, the number of lymphocytes yielded by the liver, spleen and thymus decreased at the initial stage (around day 10). At this stage, NK cells, extrathymic T cells and NKT cells were found to be radioresistant. In other words, conventional lymphocytes were radiosensitive, even in the case of low-dose irradiation. However, the number of lymphocytes in all tested immune organs increased beyond the control level at the recovery stage (around day 28). Enumeration of the absolute number of lymphocyte subsets showed that the most prominently expanding populations were NK cells, extrathymic T cells and NKT cells, especially in the liver where primordial lymphocytes are primarily present. Functional and phenotypic activation of these populations also occurred at the recovery stage. It raised a possibility that an initial activation of macrophages by low-dose irradiation then mediated the present phenomenon. These results suggest that low-dose irradiation eventually has the potential to induce a hormesis effect on the immune system.     

Regulation of nuclear translocation of extracellular signal-regulated kinase 5 by active nuclear import and export mechanisms

Extracellular signal-regulated kinase 5 (ERK5), a member of the mitogen-activated protein kinase family, plays an important role in growth factor signaling to the nucleus. However, molecular mechanisms regulating subcellular localization of ERK5 have remained unclear. Here, we show that nucleocytoplasmic shuttling of ERK5 is regulated by a bipartite nuclear localization signal-dependent nuclear import mechanism and a CRM1-dependent nuclear export mechanism. Our results show that the N-terminal half of ERK5 binds to the C-terminal half and that this binding is necessary for nuclear export of ERK5. They further show that the activating phosphorylation of ERK5 by MEK5 results in the dissociation of the binding between the N- and C-terminal halves and thus inhibits nuclear export of ERK5, causing its nuclear import. These results reveal the mechanism by which the activating phosphorylation of ERK5 induces its nuclear import and suggest a novel example of a phosphorylation-dependent control mechanism for nucleocytoplasmic shuttling of proteins.     

ErbB and HB-EGF signaling in heart development and function

The epidermal growth factor (EGF)-ErbB signaling network is composed of multiple ligands of the EGF family and four tyrosine kinase receptors of the ErbB family. In higher vertebrates, these four receptors bind a multitude of ligands. Ligand binding induces the formation of various homo- and heterodimers of ErbB, potentially providing for a high degree of signal diversity. ErbB receptors and their ligands are expressed in a variety of tissues throughout development. Recent advances in gene targeting strategies in mice have revealed that the EGF-ErbB signaling network has fundamental roles in development, proliferation, differentiation, and homeostasis in mammals. The heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that binds to and activates the EGF receptor (EGFR/ErbB1) and ErbB4. Recent studies using several mutant mice lacking HB-EGF expression have revealed that HB-EGF has a critical role in normal heart function and in normal cardiac valve formation in conjunction with ErbB receptors. HB-EGF signaling through ErbB2 is essential for the maintenance of homeostasis in the adult heart, whereas HB-EGF signaling through EGFR is required during cardiac valve development. In this review, we introduce and discuss the role of ErbB receptors in heart function and development, focusing on the physiological function of HB-EGF in these processes.