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Gene silencing by RNA interference （RNAi） is widely used for assessing gene function. An important advance in the RNAi field was the discovery that plasmid-based RNAi can substitute for synthetic small interfering RNA in vitro and in vivo. However, constitutive and ubiquitous knockdown of gene expression by RNAi in mice can limit the scope of experiments because this process can lead to embryonic lethality, or result in compensatory overexpression of other genes such that no phenotypic abnormalities occur. Either way, analyses of the physiological roles of the gene of interest in adult mice are not possible. To overcome these limitations, we previously constructed a double-conditional short-hairpin RNA （shRNA） expression vector that can regulate shRNA expression in a spatio-temporal manner with a tetracycline-inducible floxed stuffer sequence selectively excised by application of Cre recombinase. In this study, we aimed to modify this vector to create an all-in-one vector that produces double-conditional transgenic mice through a single round of gene transfer to fertilized eggs. We added a coding region for nuclear localizing Cre （NCre） recombinase with a multi-cloning site for a cell-specific promoter into the double-conditional short-hairpin RNA （shRNA） expression vector that we previously constructed. Using Escherichia coli, we confirmed successful construction of the vector. First, we confirmed isopropyl-β-D-thiogalactopyranoside-induced expression of NCre recombinase through the lac operon as a specific promoter by western blotting. Second, we confirmed functional recombination of the floxed sequence of loxP-like TATA-lox by analysing restriction enzyme-digested fragments. This all-in-one double-conditional shRNA expression vector will be useful for reversible in vitro and in vivo knockdown of target gene expression, in target cells via promoter-specific expression of NCre, and at specific times by tetracycline application.

Triple-negative breast cancer （TNBC） accounts for 10-15％ of all breast cancer cases and shows a poor prognosis with 30％ distant metastasis. With few specific target molecules and ineffective hormonal and anti-HER2 treatment, an alternative therapeutic method for TNBC is urgently required. Recently, a non-taxane inhibitor of microtubule dynamics called eribulin was developed for breast cancer therapy. Eribulin induces irreversible mitotic mass formation in cancer cells during the G2-M phase, initiating apoptosis; however, the mechanism underlying this eribulin activity remains unclear. We reported previously that exposing non-basal-like （NBL） TNBC cells to eribulin increases miR-195 expression, which in turn decreases the expression of targeted Wnt3a. The present study sought to further clarify the mechanism of this antitumor effect by exploring how eribulin affects Wnt/β - catenin signaling based on miRNA expression changes in TNBC. In an NBL type of human breast cancer cell line （MDA-MB-231 cells）, we compared the expression levels of Wnt/β catenin signaling pathway proteins in cells exposed to an miR-195 mimic （cells transfected with miR-195 and in which Wnt3a expression was suppressed） and in cells exposed to eribulin. Expression levels of Wnt3a, β -catenin, and GSK-3β were measured by ELISA and observed by fluorescence immunostaining. Wnt3a and β -catenin expression was significantly lower and GSK-3β expression was significantly higher in the cells exposed to eribulin and transfected with miR-195 mimic than in the untreated controls, suggesting that eribulin inactivates the Wnt/β -catenin signaling pathway. Therefore, a novel antitumor mechanism of eribulin was determined, whereby eribulin induces high expression of miR-195 to inactivate the Wnt/β -catenin signaling pathway in NBL-type TNBC.

Photokeratitis is a disease in which the ocular surface is directly affected by oxidative stress caused by exposure to ultraviolet （UV） light and oxygen. It is speculated that the production of free radicals and reactive oxygen species （ROS） is caused by UV-induced cytotoxicity. Recent studies have reported that catechins have antioxidant, antiallergic, antitumor, and antibacterial effects. The aim of our study was to investigate the mechanism of UV-induced cytotoxicity in cultured human corneal epithelial （HCE-T） cells and evaluate the protective effects of the catechins, （?）-epigallocatechin gallate （EGCG） and （?）-epigallocatechin 3-O-（3-O-methyl） gallate （EGCG3”Me）, on apoptosis. HCE-T cells were UV irradiated at 312nm （4.94mW/cm2, 296mJ/cm2）. EGCG and EGCG3”Me were dissolved in methanol and adjusted to 5, 10, or 20?M. Absorption was measured from 250 to 400nm. EGCG and EGCG3”Me were pre-incubated for 1 hr. After UV irradiation, membrane lipid peroxide, tumor necrosis factor （TNF）-α production, ROS generation, caspase-3 and -8 activities, mitochondrial membrane potential, and cytochrome c levels were measured. Both EGCG and EGCG3”Me had UV absorption, and increased with concentration dependently. The increases in the levels of membrane lipid hyperoxidation, activation of caspase-3 and -8, production of TNF-α and ROS were found, by UV irradiation, to be significant. But these levels were significantly decreased by pretreatment with EGCG and EGCG3”Me. There were no changes in mitochondrial membrane potential and cytoplasmic cytochrome c levels after UV irradiation. Oxidative stress occurs early near the cell membrane in response to UV irradiation. As a result, TNF-α is induced, leading to apoptosis mainly through caspase-8 activation. Conversely, EGCG and EGCG3”Me absorb UV light directly and inhibit lipid peroxidation in the cell membrane. Catechins inhibit the apoptosis cascade by inactivating caspase-3 and caspase-8.

A pterygium is a benign growth that develops on the conjunctiva and, in some cases, extends to the cornea and interferes with vision. Excessive exposure to ultraviolet (UV) light is one of the causes of pterygium development. We previously reported that UV-induced apoptosis is led by production of reactive oxygen species (ROS) that activate p38 mitogen-activated protein kinase (MAPK) in human conjunctival epithelial (HCE) cells. Also, ROS-dependent induction of interleukin-11 (IL-11) has been reported to upregulate MAPK pathways, which results in compensatory proliferation. In this study, we examined the effect of UV exposure on HCE cells, in terms of change in apoptosis, ROS generation, phosphorylation of c-Jun N-terminal kinase (JNK), levels of IL-11 (a key cytokine in tissue repair and compensatory proliferation), production of activator protein 1 (AP-1), and expression of c-myc, c-fos and c-jun (which provides evidence of healthy cell proliferation). Apoptosis in HCE cells was induced by UV light irradiation (312nm, 4.94mW/cm2). Apoptosis was measured using the Muse Annexin V and Dead Cell Assay Kit. ROS generation was measured by using 5-(and 6-) chloromethyl-2'7'-dichlorodihydrofluorescein diacetate, acetyl ester. JNK phosphorylation, IL-11 levels and AP-1 production were measured by enzyme-linked immunosorbent assays (ELISAs). Imnunocytochemical staining was used to measure c-myc, c-fos and c-jun expression. UV irradiation increased ROS generation, phosphorylation of JNK, and apoptotic cell count. IL-11 levels and AP-1 production were significantly increased by UV irradiation. The irradiated cells had increased expression of c-myc, c-fos and c-jun, and treatment of the cells with IL-11 significantly increased expression of c-myc, c-fos and c-jun. These results suggest that the release of IL-11 from UV-induced apoptotic HCE cells and surrounding healthy cells could promote proliferation to maintain homeostasis.

Malignant hyperthermia (MH) is a potentially fatal pharmacogenetic disorder triggered by exposure to commonly used volatile anesthetics. Pharmacological and genetic analyses implicated the type 1 of ryanodine receptor (RyR1) /Ca2+ release channel as the main candidate gene for causing MH. Genetic diagnosis of MH was proposed to replace conventional methods using biopsied muscle samples that are painful for patients and require skillful diagnosticians to interpret. However, more than 250 RyR1 gene variants have now been reported in MH-susceptible patients, although most have yet to be associated with functional abnormalities using exogenous constructs of these mutants expressed in living cells. To directly compare the pharmacological characteristics of some of the MH-related RyR1 mutants, we have established doxycycline -inducible cell lines expressing two of the unconfirmed rabbit RyR1 mutants, Q156K or R534H (corresponding to the Q155K or R533H mutations in human RyR1 reported in MH patients in Japan) and a confirmed mutant, R164C RyR1 (corresponding to the R163C mutation in human). The caffeine sensitivity of Q156K-expressing cells was remarkably enhanced compared to wild-type RyR1 and similarly to previously reported levels for R164C-expressing cells, while that of the R534H mutants was not different from wild-type cells. The resting cytosolic Ca2+ concentrations of cell lines expressing Q156K or R164C were much higher than those expressing R534H or wild-type RyR1. These results indicated that the RyR1 gene mutation causing the Q156K phenotype (Q155K in human) is potentially susceptible to MH, and that screening for this mutation could be useful for the noninvasive genetic diagnosis of MH in humans.

Glioblastoma (GBM) is difficult to completely cure by surgical treatment alone, and it is generally treated with a combination of surgery, radiotherapy, and chemotherapy. However, GBM is resistant to radiotherapy and chemotherapy, and complete cure cannot be achieved. Cancer stem cells (CSC) and survivin, which inhibit apoptosis, are considered as factors underlying tumor recurrence and the radiation- and drug-resistance of these tumors. We analyzed CSC and survivin expression in surgically excised specimens of malignant brain tumors to establish the relationships between the grades and CSC and survivin expression and between MIB-1 (Ki-67) expression and resistance. No relationship was noted between the grades and CSC or survivin expression, or between MIB-1 and CSC expression or between Grade 3 and 4 MIB-1 and survivin expression, although a correlation was noted between MIB-1 and survivin expression in Grade II tumors. These findings suggested that CSC are consistently contained in tumor tissue at a specific rate regardless of the histological grade, and the apoptosis of cells with low-level proliferative and cell cycling activities does not occur because these cells do not respond to chemotherapy or radiation, being resistant to treatment.

Silencing of gene expression by RNA interference (RNAi) has become a widely used tool for assessing gene function in a fast and easy manner. An important advance in the RNAi field was the discovery that plasmid-based short hairpin RNA transcription can substitute for synthetic small interference RNAs both in vitro and in vivo. The constitutive knockdown of gene expression by RNAi can limit the scope of experiments, especially if the inhibition of genes leads to cell lethality, which prevents in vivo functional analysis. We have generated double-conditional short hairpin RNA from a single vector that can regulate its own transcription by a combination of tetracycline-inducible and Cre-loxP systems. This vector will be useful for the control of short hairpin RNA expression in a spatially-regulated manner by Cre recombinase as well as a temporally-regulated manner by tetracycline induction. These control features provide for the specific silencing of genes to facilitate functional genetic analysis in mammals in vivo.

The ryanodine receptor type 1 (RyR1) is capable of homotetrameric assembly to form a Ca2+ release channel at intracellular Ca2+ storage sites such as endoplasmic reticulum (ER). The mRNA transcript encoding full-length RyR1 is approximately 16kb and is mainly distributed in excitable cells. A 2.4-kb mRNA splice variant from the 3'-terminal region of the RyR1 gene coexists specifically in brain together with the full-length form, although the functions of this brain-specific splice variant remain unclear. To investigate the short form of RyR1 in intracellular Ca2+ signaling in brain at the cellular level, we established an experimental system whereby the green fluorescent protein (GFP) -tagged brain-specific variant of RyR1 is coexpressed with the full-length protein in the same cell. Both forms of RyR1 were localized in the ER. Caffeine-induced Ca2+-release activities in cells expressing both the brain-specific and full-length RyR1 were reduced compared to cells expressing only the full-length form of RyR1. These results suggested that coexpression of the brain-specific splice variant of RyR1 with its full-length counterpart modulates intracellular Ca2+ signaling by acting as a dominant-negative subunit of the Ca2+ release channel in a tissue-specific fashion.