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Bacterial resistance to mercury compounds (mercurials) is mediated by proteins encoded by mercury resistance (mer) operons. Six merE variants with site-directed mutations were constructed to investigate the roles of the cysteine and histidine residues in MerE protein during mercurial transport. By comparison of mercurial uptake by the cell with intact and/or variant MerE, we showed that the cysteine pair in the first transmembrane domain was critical for the transport of both Hg(II) and CH 3Hg(I). Also, the histidine residue located near to the cysteine pair was critical for Hg(II) transport, whereas the histidine residue located on the periplasmic side was critical for CH 3Hg(I) transport. Thus, enhanced mercurial uptake mediated by MerE may be a promising strategy for the design of new biomass for use in the bioremediation of mercurials in the environment.

MerC, encoded by merC in the transposon Tn21 mer operon, is a heavy metal transporter with potential applications for phytoremediation of heavy metals such as mercuric ion and cadmium. In this study, we demonstrate that MerC also acts as a transporter for methylmercury. When MerC was expressed in Escherichia coli XL1-Blue, cells became hypersensitive to CH3Hg(I) and the uptake of CH3Hg(I) by these cells was higher than that by cells of the isogenic strain. Moreover, transgenic Arabidopsis plants expressing bacterial MerC or MerC fused to plant soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) accumulated CH3Hg(I) effectively and their growth was comparable to the wild-type plants. These results demonstrate that when the bacterium-derived merC gene is ectopically introduced in genetically modified plants, MerC expression in the transgenic plants promotes the transport and sequestration of methylmercury. Thus, our results show that the expression of merC in Arabidopsis results in transgenic plants that could be used for the phytoremediation and elimination of toxic methylmercury from the environment.

Some methylmercury (MeHg) is converted to inorganic mercury (Hg2+) after incorporation into human and animal tissues, where it can remain for a long time. To determine the overall toxicity of MeHg in tissues, studies should evaluate low concentrations of Hg2+. Although demethylation is involved, the participating enzymes or underlying mechanisms are unknown; in addition, the low cell membrane permeability of Hg2+ makes these analyses challenging. We established model cell lines to assess toxicities of low concentrations of Hg2+ using bacterial organomercury lyase (MerB). We engineered MerB-expressing HEK293 and HeLa cell lines that catalyze MeHg demethylation. These cells were significantly more sensitive to MeHg exposure compared to the parental cells. MeHg treatment remarkably induced metallothioneins (MTs) and hemeoxygenase-1 (HMOX-1) mRNAs and modest expression of superoxide dismutase 1, whereas catalase and glutathione peroxidase 1 mRNAs were not up-regulated. merB knockdown using small interfering RNA supported the induction of MT and HMOX-1 mRNA by MerB enzymatic activity. Pretreatment with Trolox, a water-soluble vitamin E analog, did not inhibit MeHg-induced elevation of MT-Ix and HMOX-1 mRNAs in MerB-expressing cells, suggesting that Hg2+ works independently of reactive oxygen species generation. Similar results were obtained in cells expressing MerB, suggesting that high MTs and HMOX-1 induction and cytotoxicity are common cellular responses to low intracellular Hg2+ concentrations. This is the first study to establish cell lines that demethylate intracellular MeHg to Hg2+ using bacterial MerB for overcoming the low membrane permeability of Hg2+ and exploring the intracellular responses and toxicities of low Hg2+ concentrations.

Intravital (in vivo) imaging of lymphatic vessels and lymph node is clinically necessary during diagnosis or treatment of many conditions and diseases, including lymphedema and cancer metastasis. Cancers are complex diseases, and the cancer microenvironment, including lymphatic vessels and blood vessels, play important roles throughout cancer development, from carcinogenesis to malignancy. Efforts aimed at elucidating the pathology of complex cancers and developing novel therapeutic agents for cancer treatment are limited by the exclusive use of in vitro analysis of conventional cultured cells and tissue sections. Therefore, it is necessary to analyze cancer cells and their microenvironments spatiotemporally in vivo.

Methylmercury (MeHg) is a widespread environmental pollutant and causes a serious hazard to health worldwide. However, molecular mechanisms underlying MeHg toxicity remain elusive. We show that MeHg reduced mouse embryonic fibroblast (MEF) viability in a dose-dependent manner. Furthermore, MeHg treatment increased levels of autophagy markers LC3-II and p62, possibly by acting on the MAPKs signaling pathway in several cell types. MeHg exposure elevated the number of LC3 puncta in stable GFP-LC3 MEFs and the number of autophagic vacuoles. The accumulation of LC3-II and p62 increased further when complementing MeHg with autophagy inhibitor, chloroquine. Moreover, we found that autophagy-related gene 5-deficient (Atg5-/-) MEFs exhibited higher sensitivity and higher levels of p62 compared to their wild-type counterparts following MeHg exposure. This suggested that p62 was upregulated at the transcription level by MeHg and degraded by Atg5-dependent autophagy. Our data demonstrate that MeHg exposure promotes autophagy, and Atg5-dependent autophagy serves to protect cells from MeHg cytotoxicity.

Fish consumption has both the risk of methylmercury (MeHg) poisoning and the benefit of obtaining n-3 polyunsaturated fatty acids (n-3 PUFAs), particularly docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). However, the cellular interaction between MeHg and PUFAs remains unknown. Therefore, the aim of this study was to investigate the effects of MeHg and n-3 PUFA exposure on mouse embryonic fibroblasts (MEFs). The results showed that EPA had a negligible effect on MeHg-induced cell death, whereas DHA promoted it. Thiobarbituric acid reactive substance (TBARS) concentrations in cells exposed to DHA and MeHg were higher than in those exposed to EPA and MeHg. Treatment with DHA and MeHg markedly induced the expression of endoplasmic reticulum (ER) stress (CHOP and DNAJB9) and Nrf2 target gene (p62 and HMOX-1) mRNA levels. Unexpectedly, EPA supplementation in addition to DHA and MeHg attenuated DHA- and MeHg-induced cell death and suppressed ER stress and expression of Nrf2 target genes. Our results revealed a differential impact of DHA and EPA on MeHg-induced cell death, and combined treatment with DHA and EPA along with MeHg attenuated MeHg-induced toxicity.

Bone mineral density (BMD) is a commonly used diagnostic indicator for bone fracture risk in osteoporosis. Along with low BMD, bone fragility accounts for reduced bone quality in addition to low BMD, but there is no diagnostic method to directly assess the bone quality. In this study, we investigated changes in bone quality using the Raman spectroscopic technique. Sciatic neurectomy (NX) was performed in male C57/BL6J mice (NX group) as a model of disuse osteoporosis, and sham surgery was used as an experimental control (Sham group). Eight months after surgery, we acquired Raman spectral data from the anterior cortical surface of the proximal tibia. We also performed a BMD measurement and micro-CT measurement to investigate the pathogenesis of osteoporosis. Quantitative analysis based on the Raman peak intensities showed that the carbonate/phosphate ratio and the mineral/matrix ratio were significantly higher in the NX group than in the Sham group. There was direct evidence of alterations in the mineral content associated with mechanical properties of bone. To fully understand the spectral changes, we performed principal component analysis of the spectral dataset, focusing on the matrix content. In conclusion, Raman spectroscopy provides reliable information on chemical changes in both mineral and matrix contents, and it also identifies possible mechanisms of disuse osteoporosis.

A standardized battery of quantitative sensory tests developed by the German Research Network on Neuropathic Pain (DFNS) was used to assess the association between somatosensory dysfunction and disease duration in patients with burning mouth syndrome (BMS). The 28 female participants with BMS were classified according to disease duration: ≤ 6 months (subchronic BMS, n = 15) and > 6 months (chronic BMS, n = 13); 29 age- and sex-matched healthy volunteers (control group) were recruited from staff of a dental hospital. The DFNS quantitative sensory testing protocol was applied at the ulnar surface of the right forearm and the tip of the tongue. Values for BMS patients and controls were compared and analyzed. The mechanical detection threshold (MDT) was significantly higher (i.e., loss of sensation) at the tongue tip in the chronic BMS group than in the control group (p = 0.011), whereas mechanical pain sensitivity (MPS) at the forearm was significantly higher (i.e., gain of sensation) in the chronic BMS group than in the control group (Z score = − 2.13 and 1.99, respectively). Multivariate analyses revealed that BMS patients could be discriminated from controls by using pressure pain threshold at the tongue (79.3%) (in the subchronic BMS group) and by MDT and MPS at the tongue tip and MPS at the forearm (96.6 and 89.7%, respectively) (in the chronic BMS group). In BMS patients with long disease duration, MDT showed loss of sensation. Increased MPS suggests that a neuropathic mechanism in the peripheral and central nervous systems is involved in BMS development.

The demand for low transmission loss materials applicable for printed circuit boards (PCBs) used in high-speed high-frequency devices has been increasing. Transmission loss consists of dielectric loss and conductor loss, and the reduction of both losses is necessary to achieve the demand of PCB materials. From our simulation results, the higher the frequency, the higher the ratio of dielectric loss in transmission loss. The impact of dielectric loss increases in higher frequency. Under such a situation, we developed the new thermosetting resin with low dielectric constant (Dk of 2.4 at 10 GHz), dissipation factor (Df of 0.0016 at 10 GHz) resin. Then we developed the new low loss film by applying this new resin to smooth surface roughness copper foil. Transmission loss of the new low loss film (-0.58 dB/cm at 76 GHz) is 23 % lower than the current Ultra-Low-Loss (ULL) grade material (-0.75 dB/cm at 76 GHz). This material has good processability which can make laser IVH and electroplating without special treatment such as plasma desmear. The connection resistance value of laser IVH did not decrease after 3,000 cycles of thermal test at 65 °C (15 min) ? 125 °C (15 min). The insulation reliability is also good with no insulation deterioration after thermal test of 2000 h at 85 oC /85 %RH. In addition, this material is applicable for multi-layer construction without other adhesive sheets. Therefore, we can propose new PCBs design as multi-layer antenna for ultra-wide band, contributing to increase the latitude of the design of PCBs.

To detect the bone quality loss in osteoporosis, we performed Raman spectroscopic analysis of sciatic nerve resection (NX) mice. Eight months after surgery, lower limbs were collected from the mice and fixed with 70% ethanol. Raman spectra of anterior cortical surface of the proximal tibia at 5 points in each bone were measured by RENISHAW inVia Raman Microscope. Excitation wave length was 785 nm. We also performed DXA and micro CT measurement to confirm the bone mineral density and bone microstructure in the osteoporotic model induced by sciatic nerve resection. In the result of Raman spectroscopy, we detected changes of Raman peak intensity ratio in carbonate/phosphate, mineral/combined proline and hydroxyproline and mineral/phenylalanine. In addition, in the result of micro CT, we found significant changes in VOX BV/TV, Trabecular number, thickness, cancellous bone mineral density, cortical thickness and cortical bone mineral density. The results suggest that not only the bone mineral density but also bone quality reduced in the NX mice. We conclude that Raman spectroscopy is a useful for bone quality assessment as a complementary technique for conventional diagnostics.

Along with the advancement in miniaturizing of mobile devices, typified by smart phones and tablet PCs, the semiconductor PKG substrate installed in these devices is demanded to be thinner and higher in density. As one of the most innovative solutions, the PoP (package on package) technology, which has the three-dimensional construction, has been expanding rapidly in recent years. However, thinner PKG such as PoP tends to warp at the assembly process and cause the decrease in the connection reliability. Therefore ultra low CTE (coefficient of thermal expansion) core materials have been needed as a key solution for the reduction of the warpage for PoP. We have already developed and mass-produced ultra low CTE core material named E-770G (CTE value: 1.8 ppm/°C) for this application. However, the demand for lower warpage is still stronger especially for thinner PKG applications. To meet this requirement, we confirmed that not only low CTE but also lower resin shrinkage control gives big impact to warpage. In this paper, we would like to introduce new ultra low CTE and low shrinkage core material, named E-777G for next generation thin CSP. At the same time, we would like to explain the relationship between the warpage and material properties (CTE and resin shrinkage). E-777G has ultra low CTE and low shrinkage. These properties were achieved by applying our original resin system designed by hard segments and soft segments. Hard segments have a stack structure of aromatic ring and the strong intermolecular force between them, which lead to the ultra low CTE and low shrinkage. Soft segments of low elastic modulus can follow approximately the thermal behavior of glass fabric when heated and cooled. Consequently, the material can show the ultra low CTE which is similar to that of glass fabric itself. As a result, E-777G has achieved the ultra low CTE of 0.7 ppm/°C by TMA method which leads to significant reduction of the warpage. In the cooling process after the reflow, the bottom PKG of PoP shows a convex warpage. It is caused by resin shrinkage of substrate in the cooling process. As the method of decreasing resin shrinkage in the cooling process, we focused on decreasing free volume of the resin. By using molecular dynamics simulation, we designed the most suitable resin composition. Confirming the warpage property, we evaluated the warpage behavior of the bottom PKG before/after assembly process. The bottom PKG using E-777G material showed lower warpage than E-770G. We confirmed that not only the ultra low CTE but also low shrinkage of material effectively contributes to the decrease of the warpage.

text, 紀要論文 / Departmental Bulletin Paper

Autophagy is a cell survival process that represents a therapeutic target in cancer treatment. Many types of cytochalasins have been identified and some of them have been reported to interfere with the formation of the autophagosome, although only limited data are available to assess their potential effects. Therefore, in this study, we examined the effects of cytochalasins and structurally related compounds on cell survival and the regulation of autophagy in human lung A549 adenocarcinoma cells. Cytochalasin D (CD) and cytochalasin E (CE) prominently inhibited the growth of A549 cells in a dose-dependent manner. Following treatment with CE, F-actin filaments were disrupted, and the proportion of binucleated cells increased, whereas no such effects were observed with the seven other cytochalasins tested. We found that cytochalasin H (CH), CD, and especially CE could induce the up-regulation of autophagy-related protein (LC3-II) and SQSTM1/p62. Using bafilomycin A1, we demonstrated that CD, CE, and CH inhibited autophagosome turnover, resulting in a dysfunctional autophagic process. The results of this study reveal that CE is the most potent cytochalasin in terms of its ability to induce cell death and inhibit autophagy. CE may therefore be an effective therapeutic agent against lung cancer.

Bacterial resistance to mercury compounds (mercurials) is mediated by proteins encoded by mercury resistance (mer) operons. Six merE variants with site‐directed mutations were constructed to investigate the roles of the cysteine and histidine residues in MerE protein during mercurial transport. By comparison of mercurial uptake by the cell with intact and/or variant MerE, we showed that the cysteine pair in the first transmembrane domain was critical for the transport of both Hg(II) and CH 3Hg(I). Also, the histidine residue located near to the cysteine pair was critical for Hg(II) transport, whereas the histidine residue located on the periplasmic side was critical for CH 3Hg(I) transport. Thus, enhanced mercurial uptake mediated by MerE may be a promising strategy for the design of new biomass for use in the bioremediation of mercurials in the environment.

Methylmercury (MeHg) is a widely distributed environmental pollutant that causes a series of cytotoxic effects. However, molecular mechanisms underlying MeHg toxicity are not fully understood. Here, we report that sequestosome1/p62 protects mouse embryonic fibroblasts (MEFs) against low-dose MeHg cytotoxicity via clearance of MeHg-induced ubiquitinated proteins. p62 mRNA and protein expression in MEFs were temporally induced by MeHg exposure p62-deficient MEFs exhibited higher sensitivity to MeHg exposure compared to their wild-type (WT) counterparts. An earlier and higher level of accumulation of ubiquitinated proteins was detected in p62-deficient cells compared with WT MEFs. Confocal microscopy revealed that p62 and ubiquitinated proteins co-localized in the perinuclear region of MEFs following MeHg treatment. Further analysis of MEFs revealed that ubiquitinated proteins co-localized with LC3-positive puncta upon co-treatment with MeHg and chloroquine, an autophagy inhibitor. In contrast, there was minimal co-localization in p62-deficient MEFs. The present study, for the first time, examined the expression and distribution of p62 and ubiquitinated proteins in cells exposed to low-dose MeHg. Our findings suggest that p62 is crucial for cytoprotection against MeHg-induced toxicity and is required for MeHg-induced ubiquitinated protein clearance.

To evaluate the bone quality in the osteoporosis, we generated sciatic nerve resection (NX) mice as an osteoporosis model and analyzed by Raman spectroscopy. Raman spectra were measured in anterior cortical surface of the proximal tibia at 5 points in each bone. After that, the samples were fixed with 70% ethanol. We then performed DXA and μCT measurement. Raman peak intensity ratios were significantly different between NX and Control. Those changes in the Raman peak intensity ratios may reflect loss of bone quality in the osteoporosis model. Raman spectroscopy is a promising technique for measuring the bone quality and bone strength.

The efficacy of endothelin (ET) receptor antagonist bosentan in patients with severe pulmonary arterial hypertension (PAH) remains limited, partly because its higher doses for potential blockade of ET receptors have never been tested due to liver dysfunction. We hypothesized that rigorous blockade of ET receptors using the novel dual ET receptor antagonist macitentan would be effective in treating severe PAH without major side effects in a preclinical model appropriately representing the human disorder. In normal rats, 30 mg·kg·d of macitentan completely abolished big ET-1-induced increases in right ventricle (RV) systolic pressure. Adult male rats were injected with SU5416, a vascular endothelial growth factor blocker, and exposed to hypoxia for 3 weeks and then to normoxia for an additional 5 weeks (total 8 weeks). In intrapulmonary arterial rings isolated from rats with severe PAH, macitentan concentration dependently inhibited ET-1-induced contraction. Long-term treatment with macitentan (30 mg·kg·d, from week 3 to 8) reversed the high RV systolic pressure with preserved cardiac output. Development of RV hypertrophy, luminal occlusive lesions and medial wall thickening were also significantly improved without increasing serum levels of liver enzymes by macitentan. In conclusion, efficacious blockade of ET receptors with macitentan would reverse severe PAH without major adverse effects.

Cellular differentiation proceeds along complicated pathways, even when it is induced by extracellular signaling molecules. One of the major reasons for this complexity is the highly multidimensional internal dynamics of cells, which sometimes causes apparently stochastic responses in individual cells to extracellular stimuli. Therefore, to understand cell differentiation, it is necessary to monitor the internal dynamics of cells at single-cell resolution. Here, we used a Raman and autofluorescence spectrum analysis of single cells to detect dynamic changes in intracellular molecular components. MCF-7 cells are a human cancer-derived cell line that can be induced to differentiate into mammary-gland-like cells with the addition of heregulin (HRG) to the culture medium. We measured the spectra in the cytoplasm of MCF-7 cells during 12 days of HRG stimulation. The Raman scattering spectrum, which was the major component of the signal, changed with time. A multicomponent analysis of the Raman spectrum revealed that the dynamics of the major components of the intracellular molecules, including proteins and lipids, changed cyclically along the differentiation pathway. The background autofluorescence signals of Raman scattering also provided information about the differentiation process. Using the total information from the Raman and autofluorescence spectra, we were able to visualize the pathway of cell differentiation in the multicomponent phase space.

text, 紀要論文 / Departmental Bulletin Paper

text, 紀要論文 / Departmental Bulletin Paper

Methylmercury (MeHg) is one of the most toxic environmental pollutants and presents a serious hazard to health worldwide. Although the adverse effects of MeHg, including neurotoxicity, have been studied, its effects on immune function, in particular the immune response, remain unclear. This study examined the effects of low-dose MeHg on immune responses in mice. Mice were orally immunized with ovalbumin (OVA) or subcutaneously injected with mite extract to induce a T-helper 2 (Th2) allergic response. They were then exposed to MeHg (0, 0.02, 1.0, or 5.0 mg·kg(-1)·d(-1)). Immunization with oral OVA or subcutaneous mite extract increased serum levels of OVA-specific immunoglobulin (Ig) E (OVA-IgE), OVA-IgG1, interleukin (IL)-4, and IL-13, and total IgE, total IgG, and IL-13 when compared with levels in non-immunized mice. However, no interferon (IFN)-γ was detected. By contrast, serum levels of OVA-IgE, OVA-IgG1, IL-4, and IL-13, or total IgE, total IgG, and IL-13 in Th2 allergy model mice subsequently treated with MeHg were no higher than those in MeHg-untreated mice. These results suggest that MeHg exposure has no adverse effects on Th2 immune responses in antigen-immunized mice.

To reduce warpage of thinner package application such as POP (package on package), the ultra-low CTE (coefficient of thermal expansion) core materials has been developed. The resin system was originally designed by the concept of the combination of the hard and the soft segments. The hard segments composed of a stack structure of aromatic rings and the strong intermolecular force between them, which are the origin of the ultra-low CTE. The soft segments can follow the thermal motion of the glass fabric well due to the low elastic modulus. The CTE of the newly developed core material was as low as 0.7 ppm/°C. Then, it showed the ability to reduce warpage of PoP before/after assembly process. Our fundamental study revealed that the amount of the warpage is affected by not only the CTE but also the shrinkage of resin component in the core.

A compressor was developed using R32 which was a low GWP R32 refrigerant for coping with global warming. However, there is a tendency for vibrations of a compressor to also increase because the circulation of refrigerant increases according to specification for using R32 refrigerant. Since large vibrations of a compressor causes outdoor units to generate solid propagation sound, there is a need for a technology that can reduce vibration of a compressor. However, a vibration-measuring method to analyze such vibration had not been fully developed and it was difficult to specify the cause. Accordingly, a new vibration-measuring method was developed specifically for compressors. The use of this measuring method allowed to discover that one of the unresolved problems with sound of outdoor units was the vertical vibration of the accumulator housed in the compressor. Moreover, it was found that the accumulator vibrates vertically due to the acoustic resonance inside the casing, which led to develop a new (accumulator) design with a focus on resonance. There is a type of spatial resonance inside an accumulator that has a phase difference with the antinode of sound pressure appearing at the upper and lower ends of the space. The vertical force caused by the difference in the sound pressure becomes excitation force, which then causes vertical vibration of the accumulator. Therefore, preventing this resonance from occurring can help suppress the vertical vibration. In order to prevent the generation of resonance, a plate-shaped reflection member was placed inside the accumulator, and it proved to be effective in suppressing the vertical vibration.

text, 紀要論文 / Departmental Bulletin Paper

text, 紀要論文 / Departmental Bulletin Paper

text, 紀要論文 / Departmental Bulletin Paper

The torsional vibration between metal rolling rolls and a rolling mill motor, may occur in recent days, as a result of higher speed response adjustment for variable speed rolling mill motor drive system. Issues in this paper are focused on excess acceleration value, in tangential direction of the mill motor rotor, which is caused by the motor shaft torsional resonance at the white noise signal superposition to the speed reference signal of the motor drive system for the online transfer function analysis. As a result of the acceleration analysis, the acceleration values in “G” (Relative acceleration value on the basis of Gravity) can be plotted on “Bode-Diagram”, which is namely frequency response for the speed signal amplitude transmission ratio. In addition, relation between the white noise amplitude reduction and the transfer function analysis accuracy deterioration is also examined, in this paper. As the amplitude of the white noise decreases, the analysis error increases because of the reduction in the resolution when the amplitude of the white noise signal is small.