Interaction of α2-macroglobulin with alkaline proteinase from an alkalophilicBacillus sp. grown in an extraordinarily alkaline environment

The interaction of ₂-macroglobulin (₂M) with an alkaline serine proteinase (ALPase I) from alkalophilicBacillus sp. grown in an extraordinarily alkaline environment was investigated. Stoichiometry of the reaction showed that ALPase I bound to ₂M in a molar ratio of about 21. The ₂M-ALPase I complex showed about 80% of the proteinase activity shown by ALPase I in the hydrolysis of succinyl-l-alanyl-l-alanyl-l-prolyl-l-phenylalanyl-4-methyl-coumaryl-7-amide (Suc-Ala-Ala-Pro-Phe-MCA) and casein. The conformational changes in the ₂M molecule caused by the complex formation at pH 7.5 were determined from electron micrographs and difference spectra. The antigenic activity of the ₂M-ALPase I complex with the anti-ALPase I antiserum was found to be completely abolished. Immunoelectrophoresis of the complex incubated at pH 7.5 after 48 h showed no appreciable change, and the complex was recognized as exhibiting enhanced stability at pH 7.5.     

Structure-guided design and synthesis of P1' position 1-phenylcycloalkylamine-derived pentapeptidic BACE1 inhibitors

Previously, we reported potent pentapeptidic BACE1 inhibitors with the hydroxymethylcarbonyl isostere as a substrate transition-state mimic. To improve the in vitro potency, we further reported pentapeptidic inhibitors with carboxylic acid bioisosteres at the P(4) and P1' positions. In the current study, we screened new P1' position 1-phenylcycloalkylamine analogs to find non-acidic inhibitors that possess double-digit nanomolar range IC(50) values. An extensive structure-activity relationship study was performed with various amine derivatives at the P1' position. The most potent inhibitor of this pentapeptide series, KMI-1830, possessing 1-phenylcyclopentylamine at the P1' position had an IC(50) value of 11.6 nM against BACE1 in vitro enzymatic assay.     

Hsp105α suppresses Adriamycin-induced cell death via nuclear localization signal-dependent nuclear accumulation

Heat shock protein 105 (Hsp105) is a molecular chaperone, and the isoforms Hsp105α and Hsp105β exhibit distinct functions with different subcellular localizations. Hsp105β localizes in the nucleus and induces the expression of the major heat shock protein Hsp70, whereas cytoplasmic Hsp105α is less effective in inducing Hsp70 expression. Hsp105 shuttles between the cytoplasm and the nucleus; the subcellular localization is governed by the relative activities of the nuclear localization signal (NLS) and nuclear export signal (NES). Here, we show that nuclear accumulation of Hsp105α but not Hsp105β is involved in Adriamycin (ADR) sensitivity. Knockdown of Hsp105α induces cell death at low ADR concentration, at which ADR is less effective in inducing cell death in the presence of Hsp105α. Of note, Hsp105 is localized in the nucleus under these conditions, even though Hsp105β is not expressed, indicating that Hsp105α accumulates in the nucleus in response to ADR treatment. The exogenously expressed Hsp105α but not its NLS mutant localizes in the nucleus of ADR-treated cells. In addition, the expression level of the nuclear export protein chromosomal maintenance 1 (CRM1) was decreased by ADR treatment of cells, and CRM1 knockdown caused nuclear accumulation of Hsp105α both in the presence and absence of ADR. These results indicating that Hsp105α accumulates in the nucleus in a manner dependent on the NLS activity via the suppression of nuclear export. Our findings suggest a role of nuclear Hsp105α in the sensitivity against DNA-damaging agents in tumor cells.     

Establishment and characterization of a novel ovarian clear cell carcinoma cell line,TU-OC-2, with loss of ARID1A expression

A new cell line of human ovarian clear cell carcinoma (CCC), TU-OC-2, was established and characterized. The cells were polygonal in shape, grew in monolayers without contact inhibition and were arranged in islands like pieces of a jigsaw puzzle. The chromosome numbers ranged from 41 to 96. A low rate of proliferation was observed and the doubling time was 37.5 h. The IC₅₀ values of cisplatin, 7-ethyl-10-hydroxycamptothecin (SN38), which is an active metabolite of camptothecin, and paclitaxel were 7.7 μM, 17.7 nM and 301 nM, respectively. The drug sensitivity assay indicated that TU-OC-2 was sensitive to SN38, but resistant to cisplatin and paclitaxel. Mutational analysis revealed that TU-OC-2 cells have no mutations of PIK3CA in exons 9 and 20 and of TP53 in exons 4–9. We observed the loss of ARID1A protein expression in TU-OC-2 cells by western blot analysis and in the original tumor tissue by immunohistochemistry. This cell line may be useful for studying the chemoresistant mechanisms of CCC and exploring novel therapeutic targets such as the ARID1A-related signaling pathway.     

Assessment of Still and Moving Images in the Diagnosis of Gastric Lesions Using Magnifying Narrow-Band Imaging in a Prospective Multicenter Trial

Objectives

Magnifying narrow-band imaging (M-NBI) is more accurate than white-light imaging for diagnosing small gastric cancers. However, it is uncertain whether moving M-NBI images have additional effects in the diagnosis of gastric cancers compared with still images.

Design

A prospective multicenter cohort study.

Methods

To identify the additional benefits of moving M-NBI images by comparing the diagnostic accuracy of still images only with that of both still and moving images. Still and moving M-NBI images of 40 gastric lesions were obtained by an expert endoscopist prior to this prospective multicenter cohort study. Thirty-four endoscopists from ten different Japanese institutions participated in the prospective multicenter cohort study. Each study participant was first tested using only still M-NBI images (still image test), then tested 1 month later using both still and moving M-NBI images (moving image test). The main outcome was a difference in the diagnostic accuracy of cancerous versus noncancerous lesions between the still image test and the moving image test.

Results

Thirty-four endoscopists were analysed. There were no significant difference of cancerous versus noncancerous lesions between still and moving image tests in the diagnostic accuracy (59.9% versus 61.5%), sensitivity (53.4% versus 55.9%), and specificity (67.0% versus 67.6%). And there were no significant difference in the diagnostic accuracy between still and moving image tests of demarcation line (65.4% versus 65.5%), microvascular pattern (56.7% versus 56.9%), and microsurface pattern (48.1% versus 50.9%). Diagnostic accuracy showed no significant difference between the still and moving image tests in the subgroups of endoscopic findings of the lesions.

Conclusions

The addition of moving M-NBI images to still M-NBI images does not improve the diagnostic accuracy for gastric lesions. It is reasonable to concentrate on taking sharp still M-NBI images during endoscopic observation and use them for diagnosis.

Trial registration

Umin.ac.jp UMIN-CTR000008048     

Enhancement of oxidative stress-induced apoptosis by Hsp105alpha in mouse embryonal F9 cells.

Hsp105alpha is one of the major mammalian heat shock proteins that belongs to the HSP105/110 family, and is expressed at especially high levels in the brain as compared with other tissues in mammals. Previously, we showed that Hsp105alpha prevents stress-induced apoptosis in neuronal PC12 cells, and is a novel anti-apoptotic neuroprotective factor in the mammalian brain. On the other hand, we have also demonstrated that Hsp105alpha is expressed transiently at high levels during mouse embryogenesis and is found not only in various tissues but also in apoptotic cells. In the present study, to elucidate the role of Hsp105alpha during mouse embryogenesis, we established mouse embryonal F9 cell lines that constitutively over-express Hsp105alpha. Over-expression of Hsp105alpha enhanced hydrogen peroxide-induced apoptosis by enhancing the activation of caspase-3, poly(ADP-ribose)polymerase cleavage, cytochrome c release and activation of p38 mitogen-activated protein kinase (p38). Furthermore, oxidative stress-induced apoptosis was suppressed by SB202190, a potent inhibitor of p38, in F9 cells. These findings indicated that the activation of p38 is an essential step for apoptosis in F9 cells and that Hsp105alpha enhances activation of p38, release of cytochrome c and caspase activation. Hsp105alpha may play important roles in organogenesis, during which marked apoptosis occurs, by enhancing apoptosis during mouse embryogenesis.     

The tyrosine kinase v-Src modifies cytotoxicities of anticancer drugs targeting cell division

v-Src oncogene causes cell transformation through its strong tyrosine kinase activity. We have revealed that v-Src-mediated cell transformation occurs at a low frequency and it is attributed to mitotic abnormalities-mediated chromosome instability. v-Src directly phosphorylates Tyr-15 of cyclin-dependent kinase 1 (CDK1), thereby causing mitotic slippage and reduction in Eg5 inhibitor cytotoxicity. However, it is not clear whether v-Src modifies cytotoxicities of the other anticancer drugs targeting cell division. In this study, we found that v-Src restores cancer cell viability reduced by various microtubule-targeting agents (MTAs), although v-Src does not alter cytotoxicity of DNA-damaging anticancer drugs. v-Src causes mitotic slippage of MTAs-treated cells, consequently generating proliferating tetraploid cells. We further demonstrate that v-Src also restores cell viability reduced by a polo-like kinase 1 (PLK1) inhibitor. Interestingly, treatment with Aurora kinase inhibitor strongly induces cell death when cells express v-Src. These results suggest that the v-Src modifies cytotoxicities of anticancer drugs targeting cell division. Highly activated Src-induced resistance to MTAs through mitotic slippage might have a risk to enhance the malignancy of cancer cells through the increase in chromosome instability upon chemotherapy using MTAs.