Difference in the Electromyographic Behavior of the Masticatory and Swallowing Muscles During Cued Versus Spontaneous Swallowing.

The risk of dysphagia and/or aspiration is determined using screening tests, such as the repeated saliva swallowing test and modified water swallowing test, which evaluate cued swallowing. However, humans masticate and swallow foods with various consistencies, forms, and amounts, without conscious awareness. Therefore, this study aimed to examine the difference in the behavior of masticatory and swallowing muscles during spontaneous versus cued swallowing through a series of mastication and swallowing processes by evaluating surface electromyogram (sEMG) signals. The effect of the consistency and amount of food on the behavior of these muscles was also investigated. The sEMG recordings of the masseter muscles and anterior belly of the digastric muscle for 12 subjects, and genioglossus muscle for 5 subjects were obtained. The genioglossus activity was recorded using custom-made ball electrodes. The test foods were cookies and tofu, in amounts of 2 g and 4 g. The normalized muscle activity (integrated EMG), duration of the muscle activity, initial activation timepoint of each muscle, and total duration of swallowing were compared among four conditions. The activity of each muscle was significantly higher during the swallowing of cookies than tofu, for 4 g vs 2 g, and for cued versus spontaneous swallowing. The duration of each muscle activity, initial activation timepoint, and total duration of swallowing were significantly longer for cookies versus tofu, for 4 g vs 2 g, and for spontaneous versus cued swallowing. These results suggest that the behavior of the masticatory and swallowing muscles is affected by cued swallowing and by the consistency and amount of food.

The pseudophosphatase phogrin enables glucose-stimulated insulin signaling in pancreatic ß cells.

Autocrine insulin signaling is critical for pancreatic ß-cell growth and activity and is at least partially controlled by protein-tyrosine phosphatases (PTPs) that act on insulin receptors (IRs). The receptor-type PTP phogrin primarily localizes on insulin secretory granules in pancreatic ß cells. We recently reported that phogrin knockdown decreases the protein levels of insulin receptor substrate 2 (IRS2), whereas high-glucose stimulation promotes formation of a phogrin-IR complex that stabilizes IRS2. However, the underlying molecular mechanisms by which phogrin affects IRS2 levels are unclear. Here, we found that relative to wildtype mice, IRS2 levels in phogrin-knockout mice islets decreased by 44%. When phogrin was silenced by shRNA in pancreatic ß-cell lines, glucose-induced insulin signaling led to proteasomal degradation of IRS2 via a negative feedback mechanism. Phogrin overexpression in a murine hepatocyte cell line consistently prevented chronic insulin treatment-induced IRS2 degradation. In vitro, phogrin directly bound the IR without the assistance of other proteins and protected recombinant PTP1B from oxidation to potentiate its activity toward the IR. Furthermore, phogrin expression suppressed insulin-induced local generation of hydrogen peroxide and subsequent PTP1B oxidation, which allowed progression of IR dephosphorylation. Together, these results suggest that a transient interaction of phogrin with the IR enables glucose-stimulated autocrine insulin signaling through the regulation of PTP1B activity, which is essential for suppressing feedback-mediated IRS2 degradation in pancreatic ß cells.

Novel approach to HER2 quantification using phosphor-integrated dots in human breast invasive cancer microarray.

HER2 expression in breast cancer is evaluated to select patients for anti-HER2 therapy. With the advent of newly approved HER2-targeted drugs for low HER2 expression breast cancer, more solid evidence on the whole spectrum of HER2 expression is needed. In this study, we quantitatively assessed HER2 expression from the whole core by combining high-intensity phosphor-integrated dot (PID) immunostaining and whole slide imaging (WSI) analysis. Two types of staining were performed using a 170-core tissue microarray of invasive breast cancer. First, HER2 was stained by immunohistochemistry (IHC), and IHC scores were determined by two practicing pathologists according to the ASCO/CAP HER2 guideline. Second, HER2 was stained with PID, and tentative PID scores were determined by quantitative analysis. The results show that PID can numerically classify HER2 expression status into scores 3+, 2+, 1+, and 0. The HER2 value quantified by PID strongly correlated with the 3, 3'-diaminobenzidine (DAB) IHC score determined by pathologists (R2 = 0.93). PID IHC score 1+ cases included both DAB IHC score 1+ and 0 cases, and low HER2 expression cases appeared to be often evaluated as DAB IHC score 0. Therefore, digital image analysis by PID and WSI can help stratify HER2 IHC. It may also help classify low HER2 expression.

Luminal interaction of phogrin with carboxypeptidase E for effective targeting to secretory granules.

Phogrin, a receptor tyrosine phosphatase-like protein, is localized to dense-core secretory granules (SGs) in various neuroendocrine cells. A previous report showed that the N-terminal luminal domain mediates targeting of this protein to SGs in AtT-20 cells. Here, we show that the luminal domain specifically interacts with carboxypeptidase E (CPE), one of the key proteins involved in peptide hormone sorting, in a weakly acidic condition. The luminal domain consists of pro-sequence domain (pro) and subsequent N-side mature domain and the pro domain was preferentially required for phogrin interaction with CPE and for its targeting to SGs. Small interfering RNA-directed reduction of the CPE protein level resulted in an improper accumulation of phogrin at the trans-Golgi network in AtT-20 cells. This finding indicates that CPE is involved in the sorting process of phogrin to SGs. However, SG localization of CPE was hindered by overexpression of the phogrin mutants that lack the transport motif of binding to clathrin adaptor complexes. Phogrin-depleted AtT-20 cells also exhibited reduced CPE targeting and increased CPE degradation. Our results suggest that the luminal interaction between phogrin and CPE contributes to their targeting to SGs in a cooperative manner in neuroendocrine cells.

CBS9106 is a novel reversible oral CRM1 inhibitor with CRM1 degrading activity.

CRM1 plays an important role in the nuclear export of cargo proteins bearing nuclear exporting signal sequences. Leptomycin B (LMB), a well-known CRM1 inhibitor, possesses strong antitumor properties. However, its toxicity prevents it from being clinically useful. In this study, we demonstrate that a novel compound, CBS9106, inhibits CRM1-dependent nuclear export, causing arrest of the cell cycle and inducing apoptosis in a time- and dose-dependent manner for a broad spectrum of cancer cells, including multiple myeloma cells. CBS9106 reduces CRM1 protein levels significantly without affecting CRM1 mRNA expression. This effect could be reversed by adding bortezomib or LMB. Moreover, CBS9106-biotin allows capture of CRM1 protein by streptavidin beads in a competitive manner with LMB and vice versa. Mass spectrometric analysis shows that CBS9106 reacts with a synthetic CRM1 peptide that contains Cys528 but not with a Cys528 mutant peptide. Oral administration of CBS9106 significantly suppresses tumor growth and prolongs survival in mice bearing tumor xenograft without a significant loss in body weight. A reduced level of CRM1 protein is also observed in tumor xenografts isolated from mice treated with CBS9106. Taken together, these results indicate that CBS9106 is a novel reversible CRM1 inhibitor and a promising clinical candidate.

Reactive oxygen species-mediated pancreatic beta-cell death is regulated by interactions between stress-activated protein kinases, p38 and c-Jun N-terminal kinase, and mitogen-activated protein kinase phosphatases.

Pancreatic beta-cells are susceptible to reactive oxygen species (ROS), which are known to be generated by high or low glucose (LG), hypoxic, or cytokine-producing conditions. When we cultured mouse beta-cell-derived MIN6 cells in a LG condition, we detected a significant generation of ROS, including hydrogen peroxide, which was comparable to the ROS production in hypoxic or cytokine-treated conditions. ROS accumulation induced by the LG culture led to cell death, which was prevented by the ROS scavengers N-acetylcysteine and manganese(III)tetrakis(4-benzoic acid) porphyrin. We next investigated the mechanism of stress-activated protein kinases (SAPKs), c-jun N-terminal kinase (JNK) and p38, in ROS-induced MIN6 cell death. Activation of p38 occurred immediately after the LG culture, whereas JNK activation increased slowly 8 h later. Adenoviral p38 expression decreased MIN6 cell death, whereas the JNK expression increased it. Consistently, blocking p38 activation by inhibitors increased beta-cell death, whereas JNK inhibitors decreased it. We then examined the role of MAPK phosphatases (MKPs) specific for stress-activated protein kinases in beta-cell death. We found that MKP-1 presented an increase in its oxidized product after the LG culture. ROS scavengers prevented the appearance of this oxidized product and JNK activation. Thus, ROS-induced MKP inactivation causes sustained activation of JNK, which contributes to beta-cell death. Adenoviral overexpression of MKP-1 and MKP-7 prevented the phosphorylation of JNK at 36 h after the LG culture, and decreased MIN6 beta-cell death. We suggest that beta-cell death is regulated by interactions between JNK and its specific MKPs.

Portable and noise-tolerant magnetic field generation system.

We have successfully developed a portable pulsed magnetic field generation system incorporating a number of techniques to avoid the effects of noise, including shielding, a self-power capability, and a high-capability semiconductor switch. The system fits into a cubical box less than 0.5 m in linear dimensions and can easily be installed in experimental facilities, including noisy environments such as high-power laser facilities. The system can generate a magnetic field of several tesla sustainable for several tens of microseconds over a spatial scale of several centimeters. In a high-power laser experiment with Gekko-XII, the system operated stably despite being subjected to a high level of electrical noise from laser shots of 600 J.

CBP501 suppresses macrophage induced cancer stem cell like features and metastases.

CBP501 is an anti-cancer drug candidate which has been shown to increase cis-diamminedichloro-platinum (II) (CDDP) uptake into cancer cell through calmodulin (CaM) inhibition. However, the effects of CBP501 on the cells in the tumor microenvironment have not been addressed. Here, we investigated new aspects of the potential anti-tumor mechanism of action of CBP501 by examining its effects on the macrophages. Macrophages contribute to cancer-related inflammation and sequential production of cytokines such as IL-6 and TNF-α which cause various biological processes that promote tumor initiation, growth and metastasis (1). These processes include the epithelial to mesenchymal transition (EMT) and cancer stem cell (CSC) formation, which are well-known, key events for metastasis. The present work demonstrates that CBP501 suppresses lipopolysaccharide (LPS)-induced production of IL-6, IL-10 and TNF-α by macrophages. CBP501 also suppressed formation of the tumor spheroids by culturing with conditioned medium from the LPS-stimulated macrophage cell line RAW264.7. Moreover, CBP501 suppressed expression of ABCG2, a marker for CSCs, by inhibiting the interaction between cancer cells expressing VCAM-1 and macrophages expressing VLA-4. Consistently with these results, CBP501 in vivo suppressed metastases of a tumor cell line, 4T1, one which is insensitive to combination treatment of CBP501 and CDDP in vitro. Taken together, these results offer potential new, unanticipated advantages of CBP501 treatment in anti-tumor therapy through a mechanism that entails the suppression of interactions between macrophages and cancer cells with suppression of sequential CSC-like cell formation in the tumor microenvironment.

CBP501 inhibits EGF-dependent cell migration, invasion and epithelial-to-mesenchymal transition of non-small cell lung cancer cells by blocking KRas to calmodulin binding.

The anti-cancer agent CBP501 binds to calmodulin (CaM). Recent studies showed that migration and metastasis are inhibited by several CaM antagonists. However, there is no available evidence that CBP501 has similar effects. Here we found that CBP501 inhibits migration of non-small cell lung cancer (NSCLC) cells in vitro, even in the presence of migration inducing factors such as WNT, IL-6, and several growth factors. CBP501 also inhibited epidermal growth factor (EGF) enhanced invasion and the epithelial-to-mesenchymal transition (EMT), and this inhibition was accompanied by (i) suppression of Akt and ERK1/2 phosphorylation, and (ii) suppression of expression of transcription factor Zeb1 and the mesenchymal marker Vimentin. A pull down analysis performed using sepharose-immobilized CaM showed that CBP501 blocks the interaction between CaM and KRas. Furthermore, EGF induced Akt activation and cell migration was effectively suppressed by KRas down-regulation in NSCLC cells. Stable knockdown of KRas also made cells insensitive to CBP501's inhibition of growth factor-induced migration. Taken together, these results indicate that CBP501 inhibits binding of CaM with KRas and thereby suppresses the PI3K/AKT pathway, migration, invasion and EMT. These findings have identified a previously unrecognized effect of CBP501 on downstream KRas signaling mechanisms involving EMT and invasion, and provide support for the further clinical development of this agent.

Control of unsteady laser-produced plasma-flow with a multiple-coil magnetic nozzle.

We report an experimental demonstration of controlling plasma flow direction with a magnetic nozzle consisting of multiple coils. Four coils are controlled separately to form an asymmetric magnetic field to change the direction of laser-produced plasma flow. The ablation plasma deforms the topology of the external magnetic field, forming a magnetic cavity inside and compressing the field outside. The compressed magnetic field pushes the plasma via the Lorentz force on a diamagnetic current: j × B in a certain direction, depending on the magnetic field configuration. Plasma and magnetic field structure formations depending on the initial magnetic field were simultaneously measured with a self-emission gated optical imager and B-dot probe, respectively, and the probe measurement clearly shows the difference of plasma expansion direction between symmetric and asymmetric initial magnetic fields. The combination of two-dimensional radiation hydrodynamic and three-dimensional hybrid simulations shows the control of the deflection angle with different number of coils, forming a plasma structure similar to that observed in the experiment.

Visible light-induced highly selective transformation of olefin to ketone by 2,4,6-triphenylpyrylium cation encapsulated within zeolite Y.

2,4,6-Triphenylpyrylium cation encapsulated within zeolite Y promotes highly selective transformation of olefins to ketones with molecular oxygen, under visible light (lambda > 400 nm) irradiation at room temperature.

CBS9106-induced CRM1 degradation is mediated by cullin ring ligase activity and the neddylation pathway.

Chromosome region maintenance 1 (CRM1) mediates the nuclear export of proteins and mRNAs, and is overexpressed in various cancers. Recent studies have also reported that CRM1 protein expression is a negative prognostic factor in patients with cancer. Therefore, CRM1 is considered a potential target for anticancer therapy. Our previous study demonstrated that CBS9106, a synthetic small-molecular inhibitor of CRM1, decreases CRM1 protein through proteasomal degradation without affecting CRM1 mRNA levels. However, the mechanism by which CRM1 is degraded is not well understood. Here, we demonstrate a novel signaling pathway that plays an important role in CBS9106-induced CRM1 degradation. We found that MLN4924, a selective inhibitor of NEDD8-activating enzyme (NAE), effectively inhibits cullin neddylation and attenuates CBS9106-induced CRM1 degradation in a time- and dose-dependent manner. MLN4924 also attenuated CBS9106-induced nuclear accumulation of Ran-binding protein 1 (RanBP1), cell growth inhibition, and apoptosis. Furthermore, RNAi-mediated knockdown of neddylation pathway proteins (NEDD8 and UBA3) or cullin ring ligase (CRL) component protein (Rbx1) attenuated CRM1 protein degradation and G1 phase cell-cycle arrest by CBS9106. Knockdown of CSN5 or CAND1 also partially inhibited CBS9106-induced CRM1 degradation. These findings demonstrate that CBS9106-induced CRM1 degradation is conferred by CRL activity involving the neddylation pathway, and that this response to CBS9106 leads to cell growth inhibition and apoptosis.

CBP501-calmodulin binding contributes to sensitizing tumor cells to cisplatin and bleomycin.

CBP501 is an anticancer drug currently in randomized phase II clinical trials for patients with non-small cell lung cancer and malignant pleural mesothelioma. CBP501 was originally described as a unique G(2) checkpoint-directed agent that binds to 14-3-3, inhibiting the actions of Chk1, Chk2, mitogen-activated protein kinase-activated protein kinase 2, and C-Tak1. However, unlike a G(2) checkpoint inhibitor, CBP501 clearly enhances the accumulation of tumor cells at G(2)-M phase that is induced by cisplatin or bleomycin at low doses and short exposure. By contrast, CBP501 does not similarly affect the accumulation of tumor cells at G(2)-M that is induced by radiation, doxorubicin, or 5-fluorouracil treatment. Our recent findings point to an additional mechanism of action for CBP501. The enhanced accumulation of tumor cells at G(2)-M upon combined treatment with cisplatin and CBP501 results from an increase in intracellular platinum concentrations, which leads to increased binding of platinum to DNA. The observed CBP501-enhanced platinum accumulation is negated in the presence of excess Ca(2+). Some calmodulin inhibitors behave similarly to, although less potently than, CBP501. Furthermore, analysis by surface plasmon resonance reveals a direct, high-affinity molecular interaction between CBP501 and CaM (K(d) = 4.62 × 10(-8) mol/L) that is reversed by Ca(2+), whereas the K(d) for the complex between CBP501 and 14-3-3 is approximately 10-fold weaker and is Ca(2+) independent. We conclude that CaM inhibition contributes to CBP501's activity in sensitizing cancer cells to cisplatin or bleomycin. This article presents an additional mechanism of action which might explain the clinical activity of the CBP501-cisplatin combination.

Gene silencing of phogrin unveils its essential role in glucose-responsive pancreatic beta-cell growth.

OBJECTIVE: Phogrin and IA-2, autoantigens in insulin-dependent diabetes, have been shown to be involved in insulin secretion in pancreatic beta-cells; however, implications at a molecular level are confusing from experiment to experiment. We analyzed biological functions of phogrin in beta-cells by an RNA interference technique. RESEARCH DESIGN AND METHODS: Adenovirus-mediated expression of short hairpin RNA specific for phogrin (shPhogrin) was conducted using cultured beta-cell lines and mouse islets. Both glucose-stimulated insulin secretion and cell proliferation rate were determined in the phogrin-knockdown cells. Furthermore, protein expression was profiled in these cells. To see the binding partner of phogrin in beta-cells, coimmunoprecipitation analysis was carried out. RESULTS: Adenoviral expression of shPhogrin efficiently decreased its endogenous expression in pancreatic beta-cells. Silencing of phogrin in beta-cells abrogated the glucose-mediated mitogenic effect, which was accompanied by a reduction in the level of insulin receptor substrate 2 (IRS2) protein, without any changes in insulin secretion. Phogrin formed a complex with insulin receptor at the plasma membrane, and their interaction was promoted by high-glucose stimulation that in turn led to stabilization of IRS2 protein. Corroboratively, phogrin knockdown had no additional effect on the proliferation of beta-cell line derived from the insulin receptor-knockout mouse. CONCLUSIONS: Phogrin is involved in beta-cell growth via regulating stability of IRS2 protein by the molecular interaction with insulin receptor. We propose that phogrin and IA-2 function as an essential regulator of autocrine insulin action in pancreatic beta-cells.

Evaluation of gastric cancer by high-resolution three-dimensional CISS MR imaging in vitro.

OBJECTIVE: The objective of this study was to demonstrate the usefulness of high-resolution three-dimensional (3D) constructive interference in steady-state (CISS) MR imaging for evaluating mural invasion and the morphologic features of gastric cancers in vitro. MATERIALS AND METHODS: High-resolution 3D-CISS MR images were obtained in three surgical specimens containing three different gastric cancers. RESULTS: In early carcinoma, advanced carcinoma, and leiomyosarcoma, the depth of mural invasion at 3D-CISS MR imaging correlated well with the histopathologic stage. CONCLUSION: High-resolution 3D-CISS MR imaging is a useful method for evaluating mural invasion and the macroscopic features of gastric cancers in vitro.

Adsorption-driven photocatalytic activity of mesoporous titanium dioxide.

Titanium dioxide with a mesoporous structure, when photoactivated in water, demonstrates an unprecedented photocatalytic activity, driven strongly by an adsorption degree of molecules onto the catalyst surface, which promotes a preferential conversion of a well-adsorbed molecule. This catalyzes a selective transformation of a well-adsorbed molecule into a less-adsorbed molecule, so-labeled "stick-and-leave" transformation, which promotes a direct hydroxylation of benzene to phenol, one of the most difficult synthetic reactions, with very high selectivity (>80%) and using water as a source of oxidant.

Titanosilicate molecular sieve for size-screening photocatalytic conversion.

Titanosilicate molecular sieves, when activated by ultraviolet light irradiation in water in the presence of molecular oxygen, catalyze a conversion of molecules having a size close to the pore of the catalysts but are inactive for molecules having much larger or smaller size. This unprecedented size-screening photocatalytic activity is triggered by a combination of H2O-induced shortened lifetime of active species (charge-transfer excited state of tetrahedrally coordinated titanium oxide) and restricted diffusion of a molecule inside the pore. This catalytic property demonstrates a potential utility of the catalyst for selective transformation of molecules that is associated with a size reduction of molecules, so-labeled "molecular shave" transformation.

Cytoplasmic transport signal is involved in phogrin targeting and localization to secretory granules.

Phogrin is an integral glycoprotein primarily expressed in neuroendocrine cells. The predominant localization of phogrin is on dense-core secretory granules, and the lumenal domain has been shown to be involved in its efficient sorting to the regulated secretory pathway. Here, we present data showing that a leucine-based sorting signal [EExxxIL] within the cytoplasmic tail contributes its steady-state localization to secretory granules. Deletion mutants in the tail region failed to represent granular distribution in pancreatic beta-cell line, MIN6, and anterior pituitary cell line, AtT-20. A sorting signal mutant with two glutamic acids substituted into alanines (EE/AA) is primarily accumulated in the Golgi area instead of secretory granules, and another mutant (IL/AA) is trapped at the plasma membrane due to a defect in endocytosis. We further demonstrate that the leucine-based sorting signal of phogrin specifically interacts with both adaptor protein (AP)-1 and AP-2 clathrin adaptor complexes in vitro. These observations, along with previous studies, suggest that distinct domains of phogrin mediate proper localization of this transmembrane protein on secretory granules.