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Objective : Although deep vein thrombosis (DVT) followed by pulmonary thromboembolism (PE) is a critical complication during pregnancy, there have been few reports about its intrapartum management. We evaluated intrapartum management by using a temporary inferior vena cava filter (IVCF) in pregnant women with PE/DVT. Materials and Methods : Eleven women with PE/DVT during pregnancy between January 2004 and December 2016 were included. The patients were hospitalized for intravenous unfractionated heparin infusion after acute PE/DVT onset. Seven patients were discharged and continued treatment with subcutaneous injection of heparin at the outpatient unit. IVCF was implanted 1-3 days before delivery in 10 patients. Anticoagulant therapy was discontinued 6-12 h before delivery. We retrospectively analyzed rates of maternal or perinatal death, and recurrence of symptomatic PE/DVT. Results : One patient was diagnosed as having PE/DVT and 10 had DVT alone. One patient suffered hemorrhagic shock during delivery; however, maternal or perinatal death and recurrence of symptomatic PE/DVT did not occur in any patient. Conclusion : Maternal or perinatal death and recurrence of symptomatic PE/DVT was not seen in women diagnosed as having PE/DVT during pregnancy and treated with anticoagulant therapy and IVCF.

The older dialysis population is growing, and malnutrition and wasting syndrome are great concerns in this population. The management of these syndromes includes appropriate nutritional intake and physical activity. However, whether management in the form of an increase in protein intake has a beneficial effect on muscle mass has not been demonstrated. In this study, we investigated an association between changes of normalized protein catabolic rate (nPCR), as a proxy for protein intake and percent creatinine generation rate (%CGR), as a proxy for muscle mass in patients receiving hemodialysis. Multiple linear regression models were employed, and we included several sensitivity analyses. The results showed that increases in nPCR were associated with increases in %CGR. The association was stronger in patients with baseline %CGR levels below 100%. This was the first study to demonstrate that an increase in dietary protein intake might increase the muscle mass, but this study had certain limitations. Future interventional studies will be needed to investigate whether increases in protein intake have a beneficial effect on sarcopenia, protein-energy wasting, and frailty., (© 2018 S. Karger AG, Basel.)

Background: Serum ferritin is one of the most important and widely used markers of iron metabolism, and is a recommended standard index of iron dynamics in guidelines for renal anemia in many countries. However, serum ferritin measurement has several disadvantages. For example, it is prone to being influenced by underlying disease, and there is considerable systematic bias among different methods of measurement., Summary: Systematic bias affects not only the measurement results, but also the therapeutic strategy to be used in accordance with the relevant guidelines, and consequently the cost of medical treatment. Key Messages: Manufacturers should reduce variability in their serum ferritin assay kits. Researchers should take into account propagation of error in the analysis of measurement values, and clinicians should evaluate laboratory data with care., (© 2018 S. Karger AG, Basel.)

Objective : To assess the safety and efficacy of edoxaban for the treatment of venous thromboembolism (VTE) secondary to active malignancy. Materials and Methods : We enrolled 48 patients with newly diagnosed VTE secondary to active malignancy that was treated with oral edoxaban for 1 year between September 2014 and August 2015. We retrospectively examined the presence or absence of recurrent symptomatic VTE, VTE-related mortality, and bleeding events. Results : No recurrent symptomatic VTE or VTE-related deaths were recorded, enabling efficient assessment. Treatment safety was determined based on the reports of bleeding. Bleeding was reported in two patients, with serious bleeding in one of them. Conclusion : Edoxaban is safe and effective for the treatment of VTE secondary to active malignancy.

The extracellular egg-coating envelope that comprises a meshwork of filaments polymerized by glycoproteins plays a pivotal role in species-selective sperm recognition and subsequent fertilization; however, the structural and rheological properties conferring fertilization competence to the egg-coating envelope remain poorly unveiled. Here we show several nanoscale-structural and viscoelastic properties of the egg-coat using the transmission electron microscopy and the quartz crystal microbalance experiments, following clamp of the egg-coat at either fertilization-competent or -incompetent statuses by short-term pretreatment with synthetic peptides. Individual filament of approximately 4.8 nm diameter crossed one another, forming several types of intersections. Higher competence-inducing treatment changed the proportion of V-, Y-, and T-type intersections, and induced more randomly deflected angles at intersections. Incompetence-inducing treatment increased the median of a Gaussian distribution of filament lengths that had a peak of 10-20 nm under control conditions; furthermore, this treatment created bumps in the 30-40 and 50-60 nm windows. Quartz crystal microbalance study revealed that viscoelasticity of the competent VE suspension was lower than that of incompetent VE, indicating that viscoelastic property required for successful fertilization resides within a specific range. These findings indicated that the architecture of the egg-coat is capable of rapid and dynamic remodeling, which determines fertilization efficiency.

We report a unique case of recurrent thromboembolism and its histological findings that were revealed in an autopsy. Although it is well known that repeating thrombosis presents organization and recanalization, we also observed rare findings of thickening of the bronchial artery and fixation of the valve of the lower extremity vein in this case., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: The prognosis of super-elderly patients (age≥85 years) who undergo bradycardia pacemaker (PM) implantation remains unknown., Methods: We retrospectively enrolled 868 patients (men 49.0%, 76.6±10.6 years) who could walk unassisted and whose expected life expectancy was more than 1 year, receiving their first bradycardia PM implantation between January 1, 2006, and June 30, 2013. Clinical outcomes were compared between super-elderly patients (n=201, mean age 88.6±3.2 years) and younger patients (n=667, 73.0±9.3 years)., Results: At the end of a median 1285-day follow-up, 128 patients (14.7%) died, of which 54 were cardiac deaths (42.2%). Mortality rates were similar between the groups (16.4% vs. 14.2%, log-rank p=0.56) and across different indications for implantation (atrio-ventricular conduction disturbance or sick sinus syndrome, p=0.59), initial rhythms (sinus rhythm or persistent atrial fibrillation, p=0.62), pacing modes (dual chamber pacing or VVI pacing, p=0.26), and ventricular lead positions (septum or apex, p=0.52). On Cox proportional hazard model analysis, hypertension [hazard ratio (HR)=1.74, 95% confidence interval (CI)=1.19-2.54, p=0.004], diabetes mellitus (HR=2.18, 95% CI=1.51-3.14, p<0.001), history of myocardial infarction (HR=3.59, 95% CI=2.49-5.16, p<0.001), and history of stroke (HR=2.26, 95% CI=1.51-3.37, p<0.001) were independent predictors for mortality., Conclusions: The mortality rate of super-elderly patients who had no critical illnesses and were healthy enough to walk unassisted at the time of PM implantation was not inferior to that of younger patients. Prognosis was determined by comorbidities, but not by age, PM indication, initial rhythm, pacing leads, or mode., (Copyright © 2016 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.)

In the last decade, many studies have shown that hydrogen gas or hydrogen water can reduce the levels of reactive oxygen species in the living body. Molecular hydrogen has antioxidant and antiapoptotic effects and a preventive effect on oxidative stress-induced cell death. In the present study, we investigated solidified hydrogen-occluding-silica (H 2 -silica) that can release molecular hydrogen into cell culture medium because the use of hydrogen gas has strict handling limitations in hospital and medical facilities and laboratories, owing to its physicochemical characteristics. Human esophageal squamous cell carcinoma (KYSE-70) cells and normal human esophageal epithelial cells (HEEpiCs) were used to investigate the effects of H 2 -silica on cell viability and proliferation. Cell migration was examined with wound healing and culture-insert migration assays. The intracellular levels of reactive oxygen species were evaluated with a nitroblue tetrazolium assay. To assess the apoptotic status of the cells, the Bax/Bcl-2 ratio and cleaved caspase-3 were analyzed by western blot. The results showed that KYSE-70 cells and HEEpiCs were generally inhibited by H 2 -silica administration, and there was a significant proliferation-inhibitory effect in an H 2 -silica concentration-dependent manner compared with the control group ( P < 0.05) in KYSE-70. Apoptosis-inducing effect on KYSE-70 cells was observed in 10, 300, 600, and 1,200 ppm H 2 -silica, and only 1,200 ppm H 2 -silica caused a 2.4-fold increase in apoptosis in HEEpiCs compared with the control group as the index of Bax/Bcl-2. H 2 silica inhibited cell migration in KYSE-70 cells, and high concentrations had a cytotoxic effect on normal cells. These findings should provide insights into the mechanism of inhibition of H 2 -silica on human cancer cells in vitro ., Competing Interests: Conflicts of interest The authors declare no conflict of interest.

The aim of the present study is to investigate protective effects of hydrogen-rich water (HW) against reactive oxygen species (ROS)-induced cellular harmful events and cell death in human gingival fibroblasts (HGF) and three-dimensional (3D-) gingival tissue equivalents. HW was prepared with a magnesium stick in 600-mL double distilled water (DDW) overnight. Dissolved hydrogen was about 1460 ± 50 μg/L versus approximately 1600 μg/L for the saturated hydrogen. Under cell-free conditions, HW, dose-dependently, significantly scavenged peroxyl radicals (ROO·) derived from 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). Extract from HW-treated HGF cells scavenged ROO· more markedly than that from DDW-treated cells, suggesting that HW can increase the intracellular antioxidant capacity. Hydrogen peroxide dose-dependently increased the intracellular ROS generation, which was significantly repressed by HW, both in the cytoplasm and nuclei. LIVE/DEAD staining and our original cell viability dye-extraction assay showed that HW significantly protected HGF cells from hydrogen peroxide-induced cell death. Hydrogen peroxide also diminished the contents of intracellular glutathione, which were appreciably relieved by HW-pretreatment. Additionally, HW noticeably prevented cumene hydroperoxide-induced generation of cellular ROS in epidermis parts of 3D-gingival equivalents. The in vitro scratch assay showed that HW was able to diminish physical injury-induced ROS generation and promote wound healing in HGF cell monolayer sheets. In summary, HW was able to increase intracellular antioxidative capacity and to protect cells and tissue from oxidative damage. Thus, HW might be used for prevention/treatment of oxidative stress-related diseases.

The aim of this study was to investigate preventive effects of the lipophilic vitamin C derivative, 6-O-palmitoylascorbate (PlmtVC) against X-ray radiation-induced harmful events. Free radical scavenging activity tests showed that both fresh and old (being kept at 37°C for 72 h) solutions of PlmtVC showed significantly higher abilities for scavenging both DPPH and peroxyl radical (ROO·) radicals than L-ascorbic acid (L-AA) under the same conditions, suggesting that PlmtVC is an antioxidant more efficient and stable than L-AA. Irradiation with X-ray (15 Gy) increased intracellular ROS production, lipid peroxidation and protein carbonylation, in human keratinocytes HaCaT, all of which were repressed, especially for intracellular ROS more markedly, by PlmtVC than by L-AA. After X-ray (15 Gy)-irradiation, caspase 3/7 activation and TUNEL-detected DNA-strand-breakages characteristic of apoptosis obviously increased in HaCaT cells or 3D-skin tissue equivalents, respectively, both of which were prevented more appreciably by PlmtVC than by L-AA. PlmtVC also noticeably prevented cumene hydroperoxide-induced generation of cellular ROS in epidermis parts of 3D-skin equivalents. Thus, PlmtVC prevents X-ray-induced diverse harmful effects, through its antioxidant activity and the palmitoyl moiety-based lipophilicity, more efficiently than L-AA. J. Cell. Biochem. 118: 318-329, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Acetaldehyde is a detrimental substance produced in alcoholic liquor aging. We assessed an ability of hydrogen-storing microporous silica 'Microcluster' (MC+) to reduce acetaldehyde, as compared with autoclave-dehydrogenated MC+ (MC-). Acetaldehyde was quantified spectrophotometrically by an enzymatic method. Authentic acetaldehyde was treated by MC+ for 20min, and decreased from 43.4ppm to 10.9ppm, but maintained at 49.3ppm by MC-. On the other hand, acetaldehyde contained in a distilled spirit was decreased from 29.5ppm to 3.1ppm at 20min by MC+, but not decreased by MC-. Addition of MC+ or MC- to distilled water without acetaldehyde showed no seeming effect on the quantification used. Accordingly acetaldehyde in a distilled spirit is reduced to ethanol by hydrogen contained in MC+, but not by the silica moiety of MC+. Hydrogen gas of 1.2mL was released for 20min from MC+ of 0.59g in water, resulting in dissolved hydrogen of 1.09ppm and an oxidation- reduction potential of -687.0mV indicative of a marked reducing ability. Thus, MC+ has an ability to reduce acetaldehyde in a distilled spirit due to dissolved hydrogen released from MC+., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Our previous study demonstrated that platinum nanocolloid (Pt-nc), combined with lower-dose gamma irradiation at 3, 5, and 7 Gy significantly decreased proliferation and accelerated apoptosis of the human esophageal squamous cell carcinoma-derived cell line KYSE-70. The aim of the present study was to determine, under the same conditions as our previous study where gamma rays combined with Pt-nc were carcinostatic to KYSE-70 cells, if we could induce a radioprotective or the radiation-sensitizing effect on the human normal esophageal epithelial cells (HEEpiC). HEEpiC were treated with various Pt-nc concentrations and then irradiated with various gamma-ray doses. The proliferative status of HEEpiC was evaluated using trypan blue dye-exclusion and WST-8 assays. The cellular and nucleic morphological features were determined using crystal violet and Hoechst 33342 stainings, respectively. The intracellular level of reactive oxygen species (ROS) in HEEpiC was evaluated with a nitro blue tetrazolium (NBT) assay. The apoptotic status was detected with caspase-3, Bax, and Bcl-2 by Western blotting. Either Pt-nc or gamma irradiation could inhibit the growth of HEEpiC; however, their combined use exerted a significant proliferation-inhibitory effect in a Pt-nc dose-dependent manner than gamma irradiation alone. Pt-nc resulted in radiation sensitization rather than radiation protection on HEEpiC in vitro similar to KYSE-70 cells, when Pt-nc was administrated alone or combined with gamma irradiation. Thus, Pt-nc has an inhibitory effect on cell proliferation, a facilitative effect on apoptosis, and a certain degree of toxicity against HEEpiC.

In our previous study, we demonstrated that combined treatment with hydrogen (H2) and platinum nanocolloid (Pt-nc) exerted markedly antiproliferative effects on cancer cells compared with each treatment alone. However, because the related mechanisms remain unclear, we investigated carcinostatic mechanisms of the combined treatment with H2 + Pt-nc. Significant suppression of cell proliferation was confirmed at 52 h following combined treatment, and the similar effect was also observed by the 30- or 40-min transient treatment with H2 + Pt-nc. The transient treatments led to changes in cell size and morphology, loss of microvilli, and apoptosis-like cell death at 120 h after treatment. Moreover, transient combined treatment with H2 + Pt-nc induced cell-cycle arrest, as reflected by decreased proportions of G1-phase cells and accumulation of G2/M-phase cells. In contrast, intracellular peroxide levels were temporarily and significantly increased immediately after H2 + Pt-nc treatment but not after treatment with H2 or Pt-nc alone. Additionally, combined treatment-induced carcinostatic effects were significantly diminished in the presence of catalase, and marked hydrogen peroxide (H2O2) generation was confirmed after mixing Pt-nc into cell culture media containing a high concentration of H2. These changes are in agreement with the results that carcinostatic effects were induced after only 40 min of treatment with H2 + Pt-nc. Thus, transient and marked generation of H2O2 is responsible for the carcinostatic effects of combined treatment with H2 + Pt-nc.

Fertilization is a highly coordinated process whereby sperm interact with the egg-coating envelope (called the zona pellucida, ZP) in a taxon-restricted manner, Fertilization triggers the resumption of the cell cycle of the egg, ultimately leading to generation of a new organism that contains hereditary information of the parents. The complete sperm-ZP interaction comprises sperm recognition of the ZP, the acrosome reaction, penetration of the ZP, and fusion with the egg. Recent evidence suggests that these processes involve oligosaccharides associated with a ZP constituent (termed ZP protein), the polypeptide backbone of a ZP protein, and/or the proper three-dimensional filamentous structure of the ZP. However, a detailed description of the molecular mechanisms involved in sperm-ZP interaction remains elusive. Recently, I found that dicalcin, a novel ZP protein-associated protein, suppresses fertilization through its association with gp41, the frog counterpart of the mammalian ZPC protein. This review focuses on molecular aspects of sperm-ZP interaction and describes the fertilization-suppressive function of dicalcin and associated molecular mechanisms. The amount of dicalcin in the ZP significantly correlates with alteration of the lectin-staining pattern within the ZP and the orientation pattern of ZP filaments, which may assist in elucidating the complex molecular mechanisms that underlie sperm-ZP interaction.

In Japan, the number of patients receiving dialysis is 314,180 at the end of 2013 and 97% are treated with hemodialysis. And the mean age of patients and the percentage of diabetes have been increasing. For this reason, preparations of a new vascular access (VA) and its long-term maintenance have become difficult. In the guidelines by the Japanese Society for Dialysis Therapy (JSDT), endovascular treatment (ET) is positioned as the first line for VA stenosis. The procedure of ET itself is very simple. The revision of Japanese health insurance set an expensive technical fee for ET in 2012. It also added a restriction by which the claims for both technical and material fees would be denied, if the treatment was performed within 3 months after a previous treatment. This makes determination of best treatment timing more important. The functional evaluation using ultrasonography (US) is a useful monitoring index for determination of the ET timing for patients with stenosis. We investigated the cumulative relative frequency of flow volume (FV) and resistant index (RI) of brachial artery in arteriovenous graft (AVG) and arteriovenous fistula (AVF) cases with access failures. As a result, the cut-off values of FV and RI in AVG were 480 mL/min and 0.57, and in AVF were 354 mL/min and 0.61, respectively. Therefore we determine the treatment timing based on these results. Since 2012, active monitoring using US could have decreased the number of treatment patients by 100 per year. This meant that objective evaluation by US enabled treatments at a more suitable time to promote the proper use of medical expenses for EV treatment.

β-Catenin signaling plays a pivotal role in the genesis of a variety of malignant tumors, but its role in breast cancer has not been fully elucidated. Here, we examined whether deregulation of β-catenin signaling is related to the aggressive characteristics of certain types of breast cancers. Analysis of cytokine levels in MDA-MB-231 cells overexpressing a constitutively active form of β-catenin (CAβ-catenin) revealed a higher level of CCL5 expression. Cells transfected with CAβ-catenin or stimulated with recombinant CCL5 exhibited increased cell invasion activity and spheroid formation in vitro. Furthermore, CAβ-catenin-transfected MDA-MB-231 cells formed larger tumor masses that contained more Ki-67-positive cells and infiltrating lymphocytes than did the control cells. An inhibitor of CCR5 and a pan-CXCR neutralizing antibody dramatically reduced CAβ-catenin-promoted activities. In addition to CCL5, 6-BIO, a chemical activator of β-catenin, induced cell invasion and spheroid formation in MDA-MB-231 cells. Furthermore, high levels of nuclear β-catenin accumulation were detected in breast cancer in patients with metastasis but not in those without metastasis. Nuclear β-catenin localization is related to increased CCL5 production in breast cancer. These findings suggest that β-catenin expression enhances tumor progression via chemokine production in breast cancers and that β-catenin signaling is a critical regulator of the aggressive traits of breast cancers., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Fertilization begins with species-restricted interaction of sperm and the egg-coating envelope, which includes a three-dimensional meshwork of filaments composed of glycoproteins (called ZP proteins). Growing evidence has unveiled the molecular nature of ZP proteins; however, the structural property conferring fertilization competence to the egg-coating envelope remains unknown. Here, we show the molecular mechanism that mediates direct interaction between dicalcin, a novel fertilization-suppressive ZP protein-associated protein, and gp41, a Xenopus laevis ortholog of mammalian ZP3, and subsequently demonstrate the structural basis of the envelope for fertilization competence. The interactive regions between dicalcin and gp41 comprised five and nine amino acid residues within dicalcin and twenty-three within gp41 [corrected]. Synthetic peptides corresponding to these regions dramatically affected fertilization: treatment with dicalcin- or gp41-derived peptides decreased or increased fertilization rates, respectively. Prior application of these peptides caused distinct alterations in the in vivo lectin-staining pattern of the envelope as well. Transmission electron microscopy analysis revealed that the dicalcin-derived peptide induced the formation of a well-organized meshwork, whereas the gp41-derived peptide caused the formation of a significantly disorganized meshwork. These findings indicated that the fertilization competence of the egg-coating envelope is crucially regulated by the direct interaction between dicalcin and gp41.

We prepared nano-bubble hydrogen-dissolved water (nano-H water) which contained hydrogen nano-bubbles of <717-nm diameter for 54% of total bubbles. In the DMPO-spin trap electron spin resonance (ESR) method, the DMPO-OH:MnO ratio, being attributed to amounts of hydroxyl radicals (OH), was 2.78 for pure water (dissolved hydrogen [DH]≤0.01 ppm, oxidation-reduction potential [ORP]=+324 mV), 2.73 for tap water (0.01 ppm, +286 mV), 2.93 for commercially available hydrogen water (0.075 ppm, +49 mV), and 2.66 for manufactured hydrogen water (0.788 ppm, -614 mV), whereas the nano-H water (0.678 ppm, -644 mV) exhibited 2.05, showing the superiority of nano-H water to other types of hydrogen water in terms of OH-scavenging activity. Then, the reduction activity of nano-H water was assessed spectrophotometrically by the 2,2'-bipyridyl method. Differential absorbance at 530 nm was in the order: 0.018 for pure water, 0.055 for tap water, 0.079 for nano-H water, 0.085 for commercially available hydrogen water, and 0.090 for manufactured hydrogen water, indicating a prominent reduction activity of hydrogen water and nano-H water against oxidation in ascorbate-coupled ferric ion-bipyridyl reaction. Thus, nano-H water has an improved antioxidant activity as compared to hydrogen water of similar DH-level, indicating the more marked importance of nano-bubbles rather than the concentration of hydrogen in terms of OH-scavenging., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Herpes simplex virus 2 caused a genital ulcer, and a secondary herpetic whitlow appeared during acyclovir therapy. The secondary and recurrent whitlow isolates were acyclovir-resistant and temperature-sensitive in contrast to a genital isolate. We identified the ribonucleotide reductase mutation responsible for temperature-sensitivity by deep-sequencing analysis.

Aim: In this study, we evaluated the carcinostatic effects of combined ascorbic acid (AsA) and a capacitive-resistive electric transfer (CRet) hyperthermic apparatus-induced hyperthermic treatment on Ehrlich ascites tumor (EAT) cells., Materials and Methods: EAT cells were exposed to various AsA (0-10 mM) concentrations for 1 h; they subsequently underwent CRet treatment for 15 min at 42 °C. Cell viability was assessed by the WST-8 assay 24 h after the combined treatment. Reactive oxygen species involvement was evaluated using catalase and tempol; caspase-3/7 activation was determined by their fluorescent substrates; cell proliferation were estimated by time-lapse observation. The effect on the cell cycle was analyzed by flow cytometry., Results: Combined AsA and CRet treatment synergistically suppressed cell viability compared with either treatment alone, and these synergistically carcinostatic effects were evident even at noncytotoxic concentrations of AsA alone (≤ 2 mM). The carcinostatic effects of combined AsA and CRet treatment were attenuated in a dose-dependent manner by catalase addition, but not by the superoxide anion radical scavenger tempol. Time-lapse observation revealed that combined AsA and CRet treatment activated caspase-3/7 at 10-24 h after treatment, accompanied by significant cell growth suppression. Cell cycle analysis revealed that the rate of sub-G1-phase (apoptotic) cells was drastically increased at 12 h and 24 h, and that the G2/M-phase cells gradually increased at 6-24 h after treatment., Conclusion: These results indicate that combined AsA and CRet treatment synergistically inhibits EAT cell growth through G2/M arrest and apoptosis induction via H2O2 generation at lower AsA concentrations; this carcinostatic effect cannot be exerted by AsA alone.

Protein-carbohydrate interaction regulates multiple important processes during fertilization, an essential biological event where individual gametes undergo intercellular recognition to fuse and generate a zygote. In the mammalian female reproductive tract, sperm temporarily adhere to the oviductal epithelium via the complementary interaction between carbohydrate-binding proteins on the sperm membrane and carbohydrates on the oviductal cells. After detachment from the oviductal epithelium at the appropriate time point following ovulation, sperm migrate and occasionally bind to the extracellular matrix, called the zona pellucida (ZP), which surrounds the egg, thereafter undergoing the exocytotic acrosomal reaction to penetrate the envelope and to reach the egg plasma membrane. This sperm-ZP interaction also involves the direct interaction between sperm carbohydrate-binding proteins and carbohydrates within the ZP, most of which have been conserved across divergent species from mammals to amphibians and echinoderms. This review focuses on the carbohydrate-mediated interaction of sperm with the female reproductive tract, mainly the interaction between sperm and the ZP, and introduces the fertilization-suppressive action of dicalcin, a Xenopus laevis ZP protein-associated protein. The action of dicalcin correlates significantly with a dicalcin-dependent change in the lectin-staining pattern within the ZP, suggesting a unique role of dicalcin as an inherent protein that is capable of regulating the affinity between the lectin and oligosaccharides attached on its target glycoprotein.

Aims: To explore the carcinostatic effects of platinum nanocolloid (Pt-nc) combined with gamma rays on human esophageal squamous cell carcinoma (ESCC)., Main Methods: ESCC-derived KYSE-70 cells were treated with various concentrations of Pt-nc and/or gamma irradiation, and subsequently cultured in phenol red free DMEM with 10% FBS for 48 h. The proliferative status of the KYSE-70 cells was evaluated using trypan blue dye exclusion and WST-8 assays. Cellular and nucleic morphological aspects were evaluated using crystal violet and Hoechst 33342 stainings, respectively. Radiosensitivity was quantified by a cell viability assay, and the activated form of caspase-3, a characteristic apoptosis-related protein, was detected by Western blotting., Key Findings: Although single treatment with either Pt-nc or gamma irradiation could slightly inhibit the growth of the KYSE-70 cells, their combination exerted remarkable carcinostatic effects in a manner dependent on either Pt-nc concentrations or gamma ray doses, compared with the effect of each treatment alone (p<0.05). By fluorescence micrographic observation, the KYSE-70 cells that were treated with Pt-nc and subsequently irradiated with gamma rays, were shown to undergo distinct apoptotic morphological changes. The carcinostatic effect of gamma rays at 7 Gy without Pt-nc was approximately equal to that when 3-Gy irradiation was combined with 100 ppm Pt-nc or that 5-Gy irradiation was combined with 50 ppm Pt-nc., Significance: Pt-nc in combination with gamma rays may exert a cooperative effect through platinum- or gamma ray-induced apoptosis resulting in the inhibition of growth of cancer cells, while concurrently enabling the lowering of the radiative dose., (Copyright © 2015 Elsevier Inc. All rights reserved.)

We assessed the repression of lipid-droplet formation in mouse mesenchymal stromal preadipocytes OP9 by specified oat extracts (Hatomugi, Coix lacryma-jobi var. ma-yuen) named "SPH" which were proteolytically and glucosyl-transferredly prepared from finely-milled oat whole-grain. Stimulation of OP9 preadipocytes with insulin-containing serum-replacement promoted differentiation to adipocytes, concurrently with an increase in the intracellular lipid droplets by 51.5%, which were repressed by SPH-bulk or SPH-water-extract at 840ppm, to 33.5% or 46.9%, respectively, but not by SPH-ethanol-extract at the same dose, showing the hydrophilic property of the anti-adipogenetic ingredients. The intracellular lipid droplets were scanty for intact preadipocytes, small-sized but abundant for the SPH-unadministered adipocytes, and large-sized but few for SPH-bulk-administered adipocytes being coexistent with many lipid-droplet-lacking viable cells, suggesting "the all-or-none rule" for lipid-droplet generation in cell-to-cell. Hydrogen-peroxide-induced cell death in human epidermal keratinocytes HaCaT was prevented by SPH-bulk at 100 or 150ppm by 5.6-8.1%, being consistent with higher viabilities of SPH-bulk-administered OP9 cells, together with repressions of both cell shrinkage and cell detachment from the culture substratum. In three-dimensional subcutaneous adipose tissue models reconstructed with HaCaT-keratinocytes and OP9-preadipocytes, lipid droplets were accumulated in dermal OP9-cell-parts, and repressed to 43.5% by SPH-bulk at 840ppm concurrently with marked diminishment of huge aggregates of lipid droplets. Thus SPH-bulk suppresses adipogenesis-associated lipid-droplet accumulation during differentiation of OP9 preadipocytes together with lowered cytotoxicity to either HaCaT keratinocytes or the preadipocytes., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Ataxia telangiectasia (AT) is caused by a mutation in the ataxia-telangiectasia-mutated (ATM) gene; the condition is associated with hyper-radiosensitivity, abnormal cell-cycle checkpoints, and genomic instability. AT patients also show cerebellar ataxia, possibly due to reactive oxygen species (ROS) sensitivity in neural cells. The ATM protein is a key regulator of the DNA damage response. Recently, several AT-like disorders have been reported. The genes responsible for them are predicted to encode proteins that interact with ATM in the DNA-damage response. Ataxia with oculomotor apraxia types 1-3 (AOA1, 2, and 3) result in a neurodegenerative and cellular phenotype similar to AT; however, the basis of this phenotypic similarity is unclear. Here, we show that the cells of AOA3 patients display aberrant ATM-dependent phosphorylation and apoptosis following γ-irradiation. The ATM-dependent response to H2O2 treatment was abrogated in AOA3 cells. Furthermore, AOA3 cells had reduced ATM activity. Our results suggest that the attenuated ATM-related response is caused by an increase in endogenous ROS in AOA3 cells. Pretreatment of cells with pyocyanin, which induces endogenous ROS production, abolished the ATM-dependent response. Moreover, AOA3 cells had decreased homologous recombination (HR) activity, and pyocyanin pretreatment reduced HR activity in HeLa cells. These results indicate that excess endogenous ROS represses the ATM-dependent cellular response and HR repair in AOA3 cells. Since the ATM-dependent cell-cycle checkpoint is an important block to carcinogenesis, such inactivation of ATM may lead to tumorigenesis as well as neurodegeneration., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The synthetic thymidine analogue, 5-bromo-2'-deoxy-uridine (BrdU) was encapsulated in cationic liposome composed of dipalmitoylphosphatidylcholine, cholesterol and stearylamine (molar ratio = 1/1/0.2; diameter = 120 nm), and the radiosensitization of cationic liposomal BrdU was assessed on human melanoma cells HMV-II, with comparing to anionic or nonionic liposomal BrdU and free-BrdU. HMV-II cells were pretreated by cationic liposomal BrdU or free-BrdU and then exposed to X-ray, followed by WST-8 assay to examine cell proliferation. The radiation-induced change of nuclei was defined with Hoechst33342 staining. The rates of thymidine replacement by BrdU and DNA double-strand breaks on HMV-II cells were determined with an anti-BrdU antibody and anti-53BP1 antibody, respectively. On 7th day after X-ray irradiation at 3 Gy, cell proliferation was suppressed more markedly in the administration of cationic liposomal BrdU than free-BrdU at equivalent BrdU doses. Giant polykaryocytes were observed in cationic liposomal BrdU-treated HMV-II cells. Radiosensitization was enhanced dose-dependently along with BrdU doses of 0.1-0.8 μM in the order: cationic liposomal BrdU > anionic liposomal BrdU > nonionic liposomal BrdU (see symbol) free-BrdU. Similarly, the cationic liposomal BrdU augmented the rate of thymidine-moiety replacement by BrdU and DNA double-strand breaks more appreciably than free-BrdU. Therefore, the cationic liposome-encapsulation of BrdU would be one of favorable drug deliveries for facilitating the X-ray therapy against cancer.

The aim of this study was to investigate protective effects of the lipophilic vitamin C derivative, 6-o-palmitoylascorbate (PlmtVC), against X-ray radiation-induced damages including cell death, DNA double-strand breaks (DSBs), lipid peroxidation, and protein carbonylation in human lymphocytes HEV0082, and the stability of PlmtVC under cell-cultured or cell-free condition. Irradiation with X-ray (1.5 Gy) diminished the cell viability and induced apoptosis, both of which were protected by pre-irradiational administration with PlmtVC. Gamma-H2A.X foci as a hallmark of DSBs were markedly enhanced in the irradiated cells. PlmtVC prevented X-ray-induced DSBs more appreciably than L-ascorbic acid (L-AA). Intracellular ROS production, lipid peroxidation, and protein carbonylation in HEV0082 cells were increased by X-ray at 1.5 Gy, all of which were significantly repressed by PlmtVC. PlmtVC also elevated endogenous reduced glutathione (GSH) in HEV0082 cells, and prevented X-ray-induced GSH depletion that are more appreciably over L-AA. Thus, PlmtVC prevents X-ray-induced cell death through its antioxidative activity. Stability tests showed that after being kept under physiological conditions (pH 7.4, 37 °C) for 14 days, vitamin C residual rates in PlmtVC solutions (62.2-82.0 %) were significantly higher than those in L-AA solutions (20.5-28.7 %). When PlmtVC or L-AA was added to HEV0082 lymphocytes, intracellular vitamin C in L-AA-treated cells was not detectable after 24 h, whereas PlmtVC-treated cells could keep a high level of intracellular vitamin C, suggesting an excellent stability of PlmtVC. Thus, X-ray-induced diverse harmful effects could be prevented by PlmtVC, which was suggested to ensue intrinsically from the persistent enrichment of intracellular vitamin C, resulting in relief to X-ray-caused oxidative stress.

Microcorpuscular titanium dioxide (TiO2), a useful sunscreen agent, photocatalyzes generation of reactive oxygen species (ROS). We assessed protective effects of fullerene-C60 derivatives or microcolloidal platinum (Pt) against ultraviolet ray (UV)-irradiation in the presence of TiO2 in vitro. UV-irradiation (8 J/cm2, mixed UVA and UVB) in the presence of 15 ppm TiO2 on HaCaT keratinocytes decreased cell viability as quantified by WST-1 assay, and increased both intracellular ROS and cell-membrane-lipid peroxidation, as quantified by nitroblue-tetrazolium (NBT) assay and diphenyl-1-pyrenylphosphine (DPPP) assay, respectively, whereas all of three phototoxicity-related symptoms were appreciably repressed almost to UV-unirradiational levels by pretreatment with polyvinylpyrrolidone-entrapped fullerene-C60 (C60/PVP) or fullerene-C60 dissolved in squalane (C60/Sqn) in a dose-dependent manner of C60, but scarcely by PVP alone or Sqn alone. In contrast, Pt repressed intracellular ROS generation, but did not prevent either peroxidation of cell-membrane-lipid or cell mortality. Then in the epidermis of 3-dimensional human skin tissue model, UV-irradiation in the presence of TiO2 extensively induced two symptoms such as ROS-generation around perinuclear regions and membrane-lipid peroxidation, both of which were repressed by C60/PVP or C60/Sqn, whereas Pt did not prevent membrane-lipid peroxidation adequately. Thus the advantageous application of the lipophilic antioxidant fullerene-C60 which effectively protects cell membrane against peroxidation. In conclusion, fullerene-C60 can be expected to serve as an antioxidant for scavenging of TiO2-photocatalyzed ROS in the skin surface, and therefore provide a functional improvement of TiO2-containing sunscreens.

A water-soluble complex of fullerene [C60]:polyethylene glycol (PEG) (1:350 wt/wt) (C60-PEG), but not PEG alone, was found in the present study by ESR/DMPO spin-trap method to generate hydroxyl radicals 6.5-fold as abundant as the non-irradiation level, when irradiated with visible light (400-600 nm, 140 J/cm(2): 450-fold as intense as in average outdoor), but not to generate without irradiation. At 3 h after irradiation with C60-PEG, human fibrosarcoma cells HT1080 were obviously degenerated together with diminished microvilli, cell shrinkage and cell fragmentation as observed by SEM and were shown either for increased cytotoxicity by dual stains with calcein-AM and propidium iodide or for nuclear condensation and fragmentation by Hoechst 33342 stain, any of which were, in contrast, scarcely changed in normal human fibroblastic cells DUMS16 derived from the same connective tissue type as HT1080 cells. Under the conditions, the maximum intracellular uptake amount was more abundant for HT1080 cells than for DUMS16 cells, either by immunostain/fluorography using polyclonal antibody against fullerene [C60], or by HPLC method indicating the 2.4-fold preferential uptake of C60-PEG into HT1080 cells, suggested to greater phagocytotic ability characteristic of cancer cells, over DUMS16 cells being non-macrophage-like normal cells. Thus, C60-PEG is expected as a photosensitizer for photodynamic therapy with scarce side effects to normal cells and preferential reactive oxygen species generation in cancer cells.

Although fatty acids are important components of biological membranes, energy sources, and signal transducers or precursors of lipid mediators, excess intake of fatty acids and their accumulation cause obesity and metabolic syndrome. Thus, fatty acid quantity is known to be an important factor for obesity-related diseases, but the effects of different types of fatty acids (i.e., fatty acid quality) on human health are not completely understood. We here focused on the relationship between fatty acid quality and oxidative stress by investigating whether resistibility to tert-butyl hydrperoxide (t-BuOOH)-induced oxidative stress in 3T3-L1 cells varied according to the fatty acid type. Among eight fatty acids (both saturated and unsaturated) tested, oleic acid (OA) exerted the most pronounced cytoprotective effects, with efficacy over a wide range of concentrations. OA treatment markedly enhanced the intracellular levels of lipid peroxidation markers, including N(ε)-(hexanoyl)lysine, 4-hydroxy-2-nonenal, and acrolein. The levels of these markers in OA-treated cells were decreased after t-BuOOH exposure, whereas the levels in untreated control cells were notably increased after t-BuOOH exposure. Our results suggested that unsaturated fatty acids, particularly OA, could promote an adaptive response and enhance cell tolerance through increased cellular antioxidative capacity via OA-induced mild lipid peroxidation (lipohormesis), and thus protect cells against subsequent oxidative stress-related injury.

Background: Pressure ulcer (PU) is common in immobile elderly patients, and there are some research works to investigate a preventive and curative method, but not to find sufficient effectiveness. The aim of this study is to clarify the clinical effectiveness on wound healing in patients with PU by hydrogen-dissolved water (HW) intake via tube-feeding (TF). Furthermore, normal human dermal fibroblasts OUMS-36 and normal human epidermis-derived cell line HaCaT keratinocytes were examined in vitro to explore the mechanisms relating to whether hydrogen plays a role in wound-healing at the cellular level., Methods: Twenty-two severely hospitalized elderly Japanese patients with PU were recruited in the present study, and their ages ranged from 71.0 to 101.0 (86.7 ± 8.2) years old, 12 male and 10 female patients, all suffering from eating disorder and bedridden syndrome as the secondary results of various underlying diseases. All patients received routine care treatments for PU in combination with HW intake via TF for 600 mL per day, in place of partial moisture replenishment. On the other hand, HW was prepared with a hydrogen-bubbling apparatus which produces HW with 0.8-1.3 ppm of dissolved hydrogen concentration (DH) and -602 mV to -583 mV of oxidation-reduction potential (ORP), in contrast to reversed osmotic ultra-pure water (RW), as the reference, with DH of < 0.018 ppm and ORP of +184 mV for use in the in vitro experimental research. In in vitro experiments, OUMS-36 fibroblasts and HaCaT keratinocytes were respectively cultured in medium prepared with HW and/or RW. Immunostain was used for detecting type-I collagen reconstruction in OUMS-36 cells. And intracellular reactive oxygen species (ROS) were quantified by NBT assay, and cell viability of HaCaT cells was examined by WST-1 assay, respectively., Results: Twenty-two patients were retrospectively divided into an effective group (EG, n = 12) and a less effective group (LG, n = 10) according to the outcomes of endpoint evaluation and the healing criteria. PU hospitalized days in EG were significantly shorter than in LG (113.3 days vs. 155.4 days, p < 0.05), and the shortening rate was approximately 28.1%. Either in EG or in LG, the reducing changes (EG: 91.4%; LG: 48.6%) of wound size represented statistically significant difference versus before HW intake (p < 0.05, p < 0.001). The in vitro data demonstrate that intracellular ROS as quantified by NBT assay was diminished by HW, but not by RW, in ultraviolet-A (UVA)-irradiated HaCaT cells. Nuclear condensation and fragmentation had occurred for UVA-irradiated HaCaT cells in RW, but scarcely occurred in HW as demonstrated by Hoechst 33342 staining. Besides, under UVA-irradiation, either the mitochondrial reducing ability of HaCaT cells or the type-I collagen construction in OUMS-36 cells deteriorated in RW-prepared culture medium, but was retained in HW-prepared culture medium as shown by WST-1 assay or immunostain, respectively., Conclusions: HW intake via TF was demonstrated, for severely hospitalized elderly patients with PU, to execute wound size reduction and early recovery, which potently ensue from either type-I collagen construction in dermal fibroblasts or the promoted mitochondrial reducing ability and ROS repression in epidermal keratinocytes as shown by immunostain or NBT and WST-1 assays, respectively.

Many studies have suggested that there is a close correlation among declines in internal ascorbic acid (AsA) levels, various disorders, and senescence. To clarify the relationships between age-associated changes in intracellular AsA levels and the effects of AsA administration on intracellular reactive oxygen species (ROS) levels, we investigated aging-related changes in AsA uptake, ROS levels, and the effects of AsA administration on intracellular ROS levels in young and old (senescent) human fibroblasts. Our results demonstrated that AsA uptake was increased in old cells compared with young cells, although mRNA and protein expression of sodium-dependent vitamin C transporter 2 was barely altered between the young and old cells. We also demonstrated that the intracellular superoxide anion level was higher in young cells, whereas the level of intracellular peroxides was significantly increased in old cells under both normal and oxidative stress conditions. Moreover, AsA administration markedly decreased the augmentation of intracellular peroxides in old cells, whereas there was no effect of AsA treatment in young cells under both normal and oxidative stress conditions. Therefore, our results also indicate that AsA could play an important role in regulating the intracellular ROS levels in senescent cells and that the need for AsA is enhanced by cellular senescence.

The effect of protein-glutaminase (PG) on the heat-induced conformational changes in whey protein isolate (WPI) and its relation to gel properties was investigated. The structural properties of WPI treated with PG were examined by several analytical methods. The analysis of the fluorescence spectrum and the binding capacity of a fluorescent probe demonstrated that deamidation prevented the increase in the fluorescence intensity caused by subsequent heat treatment. Measurements of the molecular weight distribution of WPI showed that PG-treated WPI was not likely to polymerize even after heating. This is thought to be due to an increase in electrostatic repulsion between carboxylic acid groups and a decrease in the formation of disulfide bonds, which results in the decrease in heat-induced aggregation. The properties of heat-induced WPI gels were modified by deamidation. PG-treated WPI gels had a soft texture and a high water-holding capacity in the presence of salts.

The effect of washing in Vibrio parahaemolyticus contaminated and hygienic seawater on fish, and the frequency and level of natural V. parahaemolyticus contamination in fish were investigated. In the first experiment, live horse mackerel was experimentally kept in seawater artificially contaminated with V. parahaemolyticus. After washing in contaminated and hygienic seawater, the contamination in fish was quantitatively analyzed. Washing fish in the seawater contaminated with V. parahaemolyticus increases the contamination level on the surface and in the gills of the fish. Washing in hygienic seawater was effective in reducing the contamination in fish and cutting board surfaces, but not in the gills or viscera. In the second experiment, natural V. parahaemolyticus contamination in various fish caught by us was analyzed. V. parahaemolyticus was detected in 6 of 28 gill samples and 10 of 28 viscera samples of naturally contaminated fish. The means of V. parahaemolyticus level on gills were 3.3 and 3.9 log cfu/g, and those in viscera were 2.6 and 4.4 log cfu/g by culture method and a real-time PCR assay, respectively. These results indicate that the gills and viscera are able to spread the pathogens to fish meat as well as fish surface contamination by washing in the contaminated seawater. Washing with hygienic seawater and control of contamination from gills and viscera are critically important to prevent V. parahaemolyticus infections.

Purpose: The aim of this study was to evaluate inhibitory effects of L-ascorbic acid-2-O-phosphate-Na(2) (APS), a pro-vitamin C, combined with hyperthermia on adipogenic differentiation of mouse stromal cells, OP9., Materials and Methods: OP9 preadipocytes were differentiated with serum replacement, administered with APS, and simultaneously treated with hyperthermia using a capacitive-resistive electric transfer (CRet) apparatus, which was conducted repeatedly twice a day. After 2 days, intracellular lipid droplets were stained with Oil Red O, then observed by microscopy and assessed spectrophotometrically., Results: After stimulation by serum replacement for 2 days, lipid droplets were accumulated surrounding nucleus of OP9 cells. When APS of 0.15-0.6 mM was administered without hyperthermia, the amount of lipid droplets was markedly suppressed to 50.5%∼-11.3% versus the undifferentiated control, and diminished huge aggregates of lipid droplets. In OP9 cells treated by hyperthermia at 42°C for 0.5 min, 1 min or 3 min in the absence of APS, adipogenesis was suppressed abruptly in a time-dependent manner to 95.4%, 18.7% or -5.5%, respectively. Whereas, the percentage of adipogenesis was 96.8% in OP9 cells treated by mild hyperthermia alone at 41°C for 1 min. The simultaneous application of APS and hyperthermia at 41°C for 1 min markedly suppressed the accumulation of lipid droplets to 25.7%∼-66.2%. By scanning electron microscopy (SEM) observation, the surface of OP9 cells treated with APS and hyperthermia appeared to have the morphological property of undifferentiated OP9 cells., Conclusion: Combined treatment of APS and mild hyperthermia suppresses adipogenesis in OP9 cells, particularly in lipid droplets accumulation during spontaneous differentiation of OP9 preadipocytes.

Objective: Clinically, the ankle-brachial blood pressure index (ABI) and skin perfusion pressure (SPP) are used to screen for subclinical peripheral artery disease. However, the association between the SPP and mortality in hemodialysis patients has not been previously reported. We investigated these factors and compared the ABI and SPP in patients receiving hemodialysis., Methods: A total of 102 patients receiving maintenance hemodialysis were enrolled in this study. The ABI was determined using an ABI-form (Colin, Japan). The SPP was measured using a SensiLase(TM) PAD3000 (Kaneka, Osaka, Japan)., Results: The mean follow-up period was 3.2 ± 1.4 years. A multivariate Cox analysis identified a low ABI (p=0.019) and a low SPP (p=0.047) as being independent predictors of mortality. A receiver operating characteristic (ROC) analysis of the ABI revealed a cutoff point of 1.1 and an area under the curve (AUC) of 0.79, with a sensitivity of 90% and a specificity of 62%. A ROC analysis of the SPP revealed a cutoff point of 54.0 mmHg and an AUC of 0.71, with a sensitivity of 55% and a specificity of 84%., Conclusion: Both low ABI and SPP values were found to be independent risk factors for mortality among hemodialysis patients. The cutoff point for ABI as a predictor of mortality was 1.1, while that for SPP was 54.0 mmHg.

The survival and recovery of Salmonella Enteritidis inoculated on stainless steel surfaces with different metal contents and surface finishes were examined. Two S. Enteritidis strains possessing different levels of biofilm productivity were inoculated with tryptone soya broth (TSB) and egg yolk emulsion (EY) on the surface of stainless steel squares (1 cm × 1 cm) and stored at 22℃ under a dry condition. After storage, cells were recovered from the stainless steel surfaces by swabbing with a cotton swab. The numbers of cells recovered by swabbing and the cells remaining on the stainless steel squares were counted. The survival ratio of the strain possessing high biofilm productivity was greater than that of the strain possessing low biofilm productivity. The survival ratio of S. Enteritidis suspended in TSB was often higher than that in EY. There were no significant differences in the survival and recovery ratios of S. Enteritidis based on stainless steel composition or surface finish. From all except one sample, more than 98% of viable cells of S. Enteritidis were recovered by swabbing with a cotton swab.

We investigated the anti-melanogenetic efficacy of hydrogen-occluding silica microcluster (H2-Silica), which is a silsesquioxane-based compound with hydrogen interstitially embedded in a matrix of caged silica, against melanogenesis in HMV-II human melanoma cells and L-DOPA-tyrosinase reaction [EC1.14.18.1]. HMV-II cells were subjected to oxidative stress by ultraviolet ray-A (UVA) exposure of 3-times of 0.65 J/cm2 summed up to 1.95 J/cm2. After UVA irradiation, HMV-II cells were stimulated to produce melanin by 2.72-fold more abundantly than unirradiated control. When HMV-II cells were treated with H2-Silica of 20 ppm or kojic acid of 28.4 ppm before and after UVA-irradiation, the amount of melanin was repressed to 12.2% or 14.5% as compared to that of UVA-irradiated control, respectively. That is, H2-Silica exhibited a comparable efficacy to the whitening agent kojic acid. The H2-Silica could prevent melanogenesis in HMV-II cells by low-level doses at 1-10 ppm, and cell viability and apoptosis event did not change even by high-level doses at 100-1000 ppm. On the contrary, kojic acid was cytotoxic at the concentration of 14-28 ppm or more. By microscopic observation, H2-Silica suppressed such properties indicative of melanin-rich cells as cellular hypertrophy, cell process formation, and melanogenesis around the outside of nuclei. The enzymatic assay using L-DOPA and mushroom tyrosinase demonstrated that H2-Silica restrained UVA-mediated melanin formation owing to down-regulation of tyrosinase activity, which could be attributed to scavenging of free radicals and inhibition of L-DOPA-to-dopachrome oxidation by hydrogen released from H2-Silica. Thus H2-Silica has a potential to prevent melanin production against UVA and serves as a skin-lightening ingredient for supplements or cosmetics.

To determine the prevalence and antimicrobial susceptibility profiles of Campylobacter, Salmonella, Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), and vancomycin-resistant enterococci (VRE) in food-producing animals and retail raw meats in Japan, raw meat samples as well as food-producing animal feces, cutaneous swabs, and nasal swabs collected from 2004 to 2006 were analyzed. Isolation rates of Campylobacter jejuni and Campylobacter coli, Salmonella, and S. aureus were 34.6% (363 of 1,050), 2.7% (28 of 1,050), and 32.8% (238 of 725), respectively. MRSA was isolated from 3% (9 of 300) of meat samples. No VRE were isolated in this study. Antibiotic resistance in C. coli was higher than that in C. jejuni. Three C. jejuni isolates from a patient with diarrhea in a hospital of Shizuoka Prefecture and two chicken samples that exhibited resistance to ciprofloxacin had identical pulsed-field gel electrophoresis patterns, suggesting that ciprofloxacin-resistant C. jejuni could have been distributed in meat. S. aureus isolates showed the highest level of resistance to ampicillin and tetracycline. Resistance to tetracycline in S. aureus isolates from beef was lower than that seen in isolates from chicken and pork (P < 0.01). This study revealed that the prevalence of MRSA and VRE were low in food-producing animals and retail domestic meats in Japan, although Campylobacter isolates resistant to fluoroquinolone and erythromycin were detected. The occurrence of antimicrobial-resistant pathogens should be monitored continuously to improve the management of the risks associated with antimicrobial drug resistance transferred from food-producing animals to humans.

Reactive oxygen species has been suggested to be one of the key factors associated with the development of obesity. During spontaneous differentiation of mouse stromal preadipocytes OP9 into adipocytes, intracellular superoxide anion radicals (O (2) (-.) ) level markedly increases and is accompanied by a significant elevation of intracellular lipid accumulation. This differentiation-dependent increase in intracellular O (2) (-.) level positively correlated with the intracellular augmentation of the lipid level. Super-highly hydroxylated fullerene (SHH-F; C(60)(OH)(44)), a novel polyhydroxylated fullerene derivative, quenched intracellular O (2) (-.) , and lipid accumulation to 38.7 and 42.7 % of that in the control, respectively. By thin-layer chromatographic analysis of extracted cellular lipid components, SHH-F clearly decreased the triglycerides ratio in the whole lipid droplet fraction, but scarcely influenced other lipids components. PPARγ2 expression, which plays a key role in regulating adipogenic differentiation, was significantly suppressed by SHH-F at the late stage of differentiation, with unaltered PPARγ1 expression. The intracellular superoxide anion radical augmentation preceded expression of PPARγ2, strongly suggesting that the primary O (2) (-.) generation was closely associated with lipid accumulation and subsequent PPARγ2 induction. These results indicate that SHH-F suppresses intracellular lipid accumulation, particularly in lipid droplets, and decreases O (2) (-.) level and subsequent PPARγ2 upregulation during spontaneous differentiation of OP9 preadipocytes into adipocytes.

The nitroimidazole-related hypoxic radiosensitizer, pimonidazole (Pmz) was encapsulated in liposome composed of dipalmitoylphosphatidylcholine, cholesterol and dipalmitoylphosphatidylglycerol (molar ratio = 1:1:0.2; diameter = 112.9 nm), and the radiosensitization was evaluated in human melanoma cells HMV-II. Cell proliferation was examined by WST-8 assay after X-ray irradiation in the presence of liposomal Pmz or free-Pmz under hypoxic conditions. On 7th day after X-ray irradiation of 5 Gy, cell proliferation decreased more markedly in the administration of liposomal Pmz than free-Pmz at equivalent Pmz doses. Chromatin fragmentation or nuclear condensation was observed in liposomal Pmz-treated HMV-II cells. Radiosensitization was enhanced dose-dependently along with Pmz amounts of 250-2000 microM contained in liposomal Pmz. Intracellular uptake was more abundant for liposomal Pmz for 60-240 min than for free-Pmz. Thus liposomal Pmz has a potential to overcome radiation resistance in hypoxia, owing to enhanced intracellular uptake by melanoma cells.

In this study, using human tongue squamous carcinoma cells (HSC-4) carcinostatic activity was compared for diverse L-ascorbic acid (Asc) derivatives, including the 'straight-C(16)-chain types', 6-O-palmitoyl-Asc (A6-P) and Asc-2-phosphate-6-O-palmitate sodium salt (APPS), as well as the 'branched-C(16)-chain types', Asc-2-phosphate-6-O-(2'-hexyl)decanoate (APHD), an isomer of APPS, and Asc-2,3,5,6-O-tetra-(2'-hexyl)decanoate (VCIP). The order of magnitude of the carcinostatic effects at 37°C was: APPS>A6-P = APHD>VCIP and at 42°C was APPS = A6-P>APHD>VCIP. Therefore, the two straight-C(16)-chain derivatives, APPS and A6-P, had a greater effect compared to the two branched-C(16)-chain Asc derivatives, which are considered to have more difficulty with 'orientation along cell-membrane-glycerolipid direction'. APPS-treated HCS-4 cells were observed for a decrease in cell number, cell shrinkage, pycnosis indicative of apoptosis and cell deformation. The order of cytotoxicity for the normal human dermal fibroblasts (OUMS-36) at 37°C was: A6-P (50% inhibitory concentration: 150-300 μM)>APHD (450-600 μM)>>Asc = APPS (800-1000 μM). Accordingly, APHD was more cytotoxic than APPS, since the straight-C(16)-chain type, which was eliminated after the enzymatic esterolysis of APPS, is metabolized via the 'fatty acid β-oxidation cycle' more efficiently in normal cells. Thus, APPS had a greater advantage over APHD, A6-P and VCIP in terms of carcinostatic effects at 37°C, carcinostasis promotion at 42°C and a decrease of cytotoxicity to normal cells. This observation suggests a marked potential for aliphatic chain-moiety structures as anticancer agents, due to their cancer-selective carcinostasis and combined efficacy with hyperthermia, without causing side effects.

The 'colloidal platinum' stabilized with polyvinylpyrrolidone (Pt/PVP-colloid) was dispersed in hydrogen-rich water (HW; hydrogen concentration, 0.82 ppm; oxidation-reduction potential, -583 mV) or regular water (RW; <0.01 ppm, +218 mV). And we evaluated the antioxidant activity of Pt/PVP-colloid in HW or RW on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and improvement of blood fluidity under 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH)-induced oxidative stress. When applied with the 0.25-0.5 ppm Pt/PVP-colloid in RW or HW, the level of DPPH radicals decreased to 77.5-59.6% or 16.1-5.6%, in contrast to the level as high as 81.3% for HW alone, respectively, as measured by an electron spin resonance method. The horse blood, which was subjected to AAPH-induced oxidative stress, was incubated for 24 hr with RW or HW, and thereafter required 13.7 sec (100%) or 5.7 sec (42.3%) for passing through the micro-channels in a rheology equipment. When treated with 0.5-1.0 ppm Pt/PVP-colloid in RW or HW, the blood passage time in the micro-channels decreased dose-dependently to 9.7-7.3 sec (71.6-53.8%) or 4.3-1.3 sec (32.8-10.3%), and the rate of micro-channels clogged with erythrocyte aggregates decreased to 23.8-21.0% or 15.8-9.8%, respectively, from 42.8% for no addition of Pt/PVP. By scanning electron microscopy, AAPH-treated erythrocytes lost intact surface morphology on the membrane together with protrusions and without hollows, being indicative of impaired transforming ability, and the rate of erythrocyte agglutination was increased to 46.2%. When treated the horse blood with HW alone significantly decreased the rate of erythrocyte agglutination to 29.6%, whereas 1.0 ppm Pt/PVP-colloid in RW or HW decreased it to 24.1% or 21.1%, respectively. Thus, DPPH-radical-scavenging and erythrocyte-protecting effects of Pt/PVP-colloid in HW were superior to those of Pt/PVP-colloid in RW or Pt/PVP-free HW. The results could be mainly attributed to the enhanced antioxidant activity of Pt/PVP in HW, which may be due to captured-hydrogen on platinum.

Cockayne syndrome (CS) and xeroderma pigmentosum (XP) are caused by deficient nucleotide excision repair. CS is characterized by cachectic dwarfism, mental disability, microcephaly and progeria features. Neuropathological examination of CS patients reveals dysmyelination and basal ganglia calcification. In addition, arteriosclerosis in the brain and subdural hemorrhage have been reported in a few CS cases. Herein, we performed elastica van Gieson (EVG) staining and immunohistochemistry for collagen type IV, CD34 and aquaporin 4 to evaluate the brain vessels in autopsy cases of CS, XP group A (XP-A) and controls. Small arteries without arteriosclerosis in the subarachnoid space had increased in CS cases but not in either XP-A cases or controls. In addition, string vessels (twisted capillaries) in the cerebral white matter and increased density of CD34-immunoreactive vessels were observed in CS cases. Immunohistochemistry findings for aquaporin 4 indicated no pathological changes in either CS or XP-A cases. Hence, the increased subarachnoid artery space may have caused subdural hemorrhage. Since such vascular changes were not observed in XP-A cases, the increased density of vessels in CS cases was not caused by brain atrophy. Hence, brain vascular changes may be involved in neurological disturbances in CS., (© 2011 Japanese Society of Neuropathology.)

S100 proteins comprise a large family of Ca(2+)-binding proteins and exhibit a variety of intra- and extracellular functions. Despite our growing knowledge about the biology of S100 proteins in some tissues such as brain and smooth muscle, little is known about S100 proteins in the normal mammalian reproductive tissue. In the present study, we investigated the distribution pattern of S100A6 (alternatively named calcyclin) in the murine ovary by immunohistochemical study using specific antibody. S100A6 was localized substantially in the cytoplasm of luteal cells, with concomitant expression of S100A11, another S100 protein, but not in the other type of cells such as oocytes, follicle epithelial cells (granulosa cells), and cells of stroma including theca interna cells in the murine ovary. S100A6-immunoreactive corpora lutea (CLs) were divided into two types: homogeneously and heterogeneously stained CLs, and possibly they may represent differentiating and mature CL, respectively. Our regression analysis revealed that expression level of S100A6 positively correlated with that of cytochrome P450 11A, a steroidogenic enzyme in the heterogeously stained CL. These results suggested that S100A6 may contribute to differentiation of steroidogenic activity of luteal cells in a synergistic manner with S100A11 by facilitating some shared functions.

Hydrogen-rich electrolyzed warm water (HW) was prepared at 41°C and exhibited dissolved hydrogen (DH) of 1.13 ppm and an oxidation-reduction potential (ORP) of -741 mV in contrast to below 0.01 ppm and+184 mV for regular warm water (RW). Fibroblasts OUMS-36 and keratinocytes HaCaT were used to examine effects of HW against UVA-ray irradiation. Type-I collagen was synthesized 1.85- to 2.03-fold more abundantly by HW application for 3-5 days than RW in OUMS-36 fibroblasts, and localized preferentially around the nuclei as shown by immunostain. HW application significantly prevented cell death and DNA damages such as nuclear condensation and fragmentation in UVA-irradiated HaCaT keratinocytes as estimated by WST-1 and Hoechst 33342 assays. HW significantly suppressed UVA-induced generation of intracellular superoxide anion radicals in both the cell lines according to NBT assay. Wrinkle repression was clinically assessed using a HW-bathing. Six Japanese subjects were enrolled in a trial of HW-bathing (DH, 0.2-0.4 ppm) every day for 3 months. HW-bathing significantly improved wrinkle in four subjects on the back of neck on 90th day as compared to 0 day. Thus, HW may serve as daily skin care to repress UVA-induced skin damages by ROS-scavenging and promotion of type-I collagen synthesis in dermis., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The antitumor and anti-invasive activities of the low-molecular-weight macrocyclic ketones (MCKs), such as musk secreted from the mammalian genital glands and musk released from relatively unkown plants, were investigated comparatively together with the enhancement of the effects in combination with hyperthermia. Ehrlich ascites tumor cells were treated with each MCK and cultured, followed by evaluation of the cell viability using the mitochondrial dehydrogenase-based WST-8 assay. The number of HT-1080 human fibrosarcoma cells cultured with the MCKs or invading through a reconstituted basement membrane was measured using microscopy. The order of the efficiency was as follows: (Z)-g-cycloheptadecen-1-one (Hp) (17:1, musk rats), 8-cyclohexadecen-1-one (16:1, musk ferns), cyclopentadecanone (15:0, musk rats) and 3-methylcyclopentadecanone (16:0, musk deer), having 15-17 carbon atoms with and without a double bond, which exhibited a carcinostatic effect either at 100 µM for 20-h culture or at 50 µM for 72-h culture. The effects were markedly enhanced by heat treatment at 42˚C. MCKs were not found in the cells by gas-liquid chromatographic determination, indicating that the carcinostatic effects were attributed to their surface activity on the cell membrane. Invasion of HT-1080 cells was inhibited by MCKs at doses scarcely diminishing the cell viability, indicating that the suppression of invasiveness did not ensue from the secondary action due to carcinostasis. The order of invasion-inhibitory efficacy of the MCKs was, however, similar to that of their carcinostatic effects. Hp17:1 also exhibited the highest anti-invasive activity in addition to the highest carcinostatic activity. The two inhibitory effects were promoted by combination with hyperthermia. MCKs with a double bond, particularly Hp17:1 rather than 8-Hx16:1, but not saturated-aliphatic MCKs, may be potent multi-applicable antitumor agents due to their dual inhibitory activities against tumor progression and invasion and in hyperthermia-combined therapy.

Protein-glutaminase (PG) is an enzyme that catalyzes the deamidation of protein-bound glutamine residues. We found that an enzyme labeling technique (ELT), which is a stable isotope labeling method based on transglutaminase (TGase) reaction, is applicable for PG. PG catalyzed incorporation of (15)N-labeled ammonium ions into reactive glutamine amide groups in α-lactalbumin similarly to TGase and deamidated the most reactive glutamine amide group once labeled with (15)N. Furthermore, we investigated the effect of ammonium ions on the PG activity by peptide mapping, and more reactive glutamine residues were detected than were detected by the ELT in the presence of ammonium ions. This is probably because ammonium ions are competitive inhibitors, causing decreased reactivity for glutamine residues. We propose the reaction scheme of PG in the presence of the (15)N-labeled ammonium ions and show that the ELT method with PG is useful for evaluating the activity of PG.