Downregulation of CYP1A2, CYP2B6, and CYP3A4 in Human Hepatocytes by Prolyl Hydroxylase Domain 2 Inhibitors via Hypoxia-Inducible Factor-α Stabilization.

Hypoxia-inducible factor (HIF) is associated with the expression of CYP, but the underlying mechanism remains uncertain. In this study, we investigated the effect of HIF-α stabilization caused by novel prolyl hydroxylase domain (PHD) 2 inhibitors, which are HIF-α stabilizers that mimic hypoxia, on the expressions of CYP1A2, CYP2B6, and CYP3A4 in human hepatocytes. An mRNA expression analysis of human hepatocytes treated with PHD2 inhibitors for 72 hours showed the downregulation of genes encoding CYP1A2, CYP2B6, and CYP3A4. The mRNA repressions were accompanied with an increase in erythropoietin protein, a marker of HIF-α stabilization, indicating that HIF-α stabilization was involved in the downregulation of the CYP isoforms. To understand the underlying mechanisms, we assessed the relationship between the expressions of the CYP isoforms and those of their regulating transcription factors [aryl hydrocarbon receptor (AhR), AhR nuclear translocator (ARNT), constitutive androstane receptor (CAR), pregnane X receptor (PXR), and retinoid X receptor (RXR)] in human hepatocytes treated with the HIF-α stabilizers. As a result, the mRNA level of AhR did not decrease, although ARNT expression was repressed. On the other hand, the mRNA expression levels of CAR, PXR, and RXR were repressed and closely associated with those of CYP2B6 and CYP3A4. Although the underlying mechanism of the downregulation for CYP1A2 remains unclear, the presently reported results suggest that the downregulation of CYP2B6 and CYP3A4 via HIF-α stabilization is caused by a decrease in the expressions of CAR, PXR, and RXR. SIGNIFICANCE STATEMENT: We showed that hypoxia-inducible factor (HIF)-α stabilization downregulates CYP1A2, CYP2B6, and CYP3A4 using prolyl hydroxylase domain 2 inhibitors, which are HIF-α stabilizers, as a new tool to mimic hypoxia in human hepatocytes. To understand the underlying mechanisms, we assessed the relationship between the expressions of the CYP isoforms and those of their regulating transcription factors. Our findings would contribute to a better understanding of the hypoxia-triggered regulatory mechanism of drug-metabolizing enzymes in human hepatocytes.

[Relevance of the five-repetition sit-to-stand test for diagnosing sarcopenia in elderly inpatients with cardiac disease].

PURPOSE: The aim of this study was to determine the reference values for diagnosing sarcopenia using the five-repetition sit-to-stand test in elderly inpatients with cardiac disease. METHODS: We studied 71 inpatients with cardiac disease ≥65 years of age (mean age 78.0±7.9 years, 42.3% women) who were admitted between April 2015 and March 2016. Patients were assessed for sarcopenia, and we performed the five-repetition sit-to-stand test. We defined sarcopenia using the Asian Working Group for Sarcopenia-suggested diagnostic algorithm. A logistic regression analysis was performed to estimate the odds ratios (ORs) and 95% confidence intervals (CIs) of the relationship between sarcopenia and the five-repetition sit-to-stand test. A multivariate analysis showed that the age, admission diagnosis, the New York Heart Association classification, the Charlson comorbidity index, and the ratio of extracellular to total body water were relevant covariates. The cut-off value of the five-repetition sit-to-stand test to diagnose sarcopenia was determined using a receiver operating characteristic curve. RESULTS: Sarcopenia was diagnosed in 25 patients (35.2%). A multivariate logistic regression analysis showed that the five-repetition sit-to-stand test was significantly associated with sarcopenia (p=0.024), and the OR (95% CI) was 1.31 (1.04-1.65). The cut-off value of the five-repetition sit-to-stand test to diagnose sarcopenia was 10.9 s (sensitivity 80.0%, specificity 70.0%, area under the curve 0.83). CONCLUSIONS: The five-repetition sit-to-stand test is a useful screening tool for sarcopenia in elderly inpatients with cardiac disease. The cut-off value to diagnose sarcopenia was 10.9 s in this study.

[The isometric knee extension strength threshold for maintaining walking speed and step length in older male inpatients].

AIM: To clarify the minimum knee extension muscle strength needed to maintain walking speed and step length in older male inpatients. METHOD: The participants were 786 male inpatients of ≥65 years of age without cerebrovascular disorder, orthopedic disease, malignancy, or dementia. We investigated the participants' isometric knee extension muscle force (kgf/kg), maximum walking speed (m/s) and step length, based on their medical records. The relationship of walking speed and step length to isometric knee extension muscle force was fitted to linear and nonlinear models, and the respective R2 values were compared. Next, the muscle force data were divided into two groups, and two linear functions were calculated. Then, the muscle force value that minimized the sum of the residual sum of squares of the two linear function expressions was obtained. RESULTS: The R2 values of each equation in the nonlinear model were higher than those in the linear model. Among all participants, the muscle force values that minimized the sum of the residual sum of squares for walking speed and step length were 0.33 kgf/kg and 0.43 kgf/kg, respectively. Among participants of ≤74 years of age, the muscle force value that minimized the sum of the residual sum of squares was 0.30 kgf/kg for both walking speed and step length, whereas the values were 0.32 kgf/kg and 0.43 kgf/kg, respectively, in participants of ≥75 years of age. CONCLUSION: Walking speed and step length were significantly decreased in male inpatients of 65-74 years of age when the isometric knee extension force values for both were <0.30 kgf/kg. In contrast, among male inpatients of ≥75 years of age, these values were significantly decreased when the respective isometric knee extension muscle force values were <0.32 kgf/kg and <0.43 kgf/kg.

Differences in physical performance based on the Geriatric Nutritional Risk Index in elderly female cardiac patients.

BACKGROUND AND AIMS: Little is known about differences in the risk of poor nutritional status as assessed by the Geriatric Nutritional Risk Index (GNRI) in relation to physical performance in elderly female cardiac inpatients. The present study aimed to determine both differences in physical performance based on the GNRI and physical performance cut-off values according to the GNRI in elderly female cardiac inpatients. METHODS: We enrolled 105 consecutive female Japanese inpatients aged ≥65 years (mean age, 74.6 years) with cardiac disease in this cross-sectional study. We divided the patients into two groups according to GNRI: high-GNRI group (≥92 points) (n = 71) and low-GNRI group (<92 points) (n = 34). Handgrip strength (HG), knee extensor muscle strength (KEMS), gait speed (GS), and one-leg standing time (OLST) were assessed as indices of hospital physical performance and compared between the two groups to determine cut-off values of physical performance. RESULTS: After adjustment for age and left ventricular ejection fraction, HG, KEMS, GS, and OLST were significantly lower in the low-GNRI versus high-GNRI group. Cut-off values by ROC curve analysis were 16.2 kgf (AUC = 0.66; p < 0.001) for HG, 34.3 % of body weight (AUC = 0.62; p = 0.04) for KEMS, 1.24 m/s (AUC = 0.72; p < 0.01) for GS, and 8.28 s (AUC = 0.62; p = 0.04) for OLST. CONCLUSION: The risk of poor nutrition, as indicated by a low GNRI, might be a predictor of lower physical performance. Cut-off values determined in this study might be minimum target goals for physical performance that can be attained by elderly female cardiac inpatients.

Alleviation effect of arbutin on oxidative stress generated through tyrosinase reaction with L-tyrosine and L-DOPA.

BACKGROUND: Hydroxyl radical that has the highest reactivity among reactive oxygen species (ROS) is generated through L-tyrosine-tyrosinase reaction. Thus, the melanogenesis might induce oxidative stress in the skin. Arbutin (p-hydroxyphenyl-ß-D-glucopyranoside), a well-known tyrosinase inhibitor has been widely used for the purpose of skin whitening. The aim of the present study was to examine if arbutin could suppress the hydroxyl radical generation via tyrosinase reaction with its substrates, L-tyrosine and L-DOPA. RESULTS: The hydroxyl radical, which was determined by an electron spin resonance-spin trapping technique, was generated by the addition of not only L-tyrosine but L-DOPA to tyrosinase in a concentration dependent manner. Arbutin could inhibit the hydroxyl radical generation in the both reactions. CONCLUSION: It is presumed that arbutin could alleviate oxidative stress derived from the melanogenic pathway in the skin in addition to its function as a whitening agent in cosmetics.

Non-invasive visualization of physiological changes of insects during metamorphosis based on biophoton emission imaging.

Spontaneous ultra-weak photon emission from living organisms, designated as biophoton emission, is a generally observed phenomenon irrespective of the organism species. Biophoton emission is attributed to the production of excited molecules in a metabolic biochemical reaction, especially in processes involving reactive oxygen species (ROS). Although many plant and mammal subjects have reportedly been used to study its application to biological measurements, biophoton emission properties of insects remain unclear. For this study, we strove to measure the variation of two-dimensional images of biophoton emission during the metamorphosis of lepidopterous insects as a moving picture to elucidate the physiological changes underlying the mechanism of drastic changes of morphological and ecological characteristics of the insects. We used our developed biophoton imaging system incorporating a cooled charge-coupled device (CCD) camera and a specially designed lens system to elucidate the spatiotemporal dynamics of biophoton emission during metamorphosis, larval-pupal ecdysis/pupation of Papilio protenor, suggesting its applicability for in vivo observation of physiological changes during the regulation of metamorphosis.

Development of biodegradable scaffolds based on patient-specific arterial configuration.

Biodegradable scaffolds are of great value in tissue engineering. We have developed a method for fabricating patient-specific vascular scaffolds from a biocompatible and biodegradable polymer, poly(L-lactide-co-epsilon-caprolactone). This method's usefulness is due to flexibility in the choice of materials and vascular configurations. Here, we present a way to fabricate scaffolds of human carotid artery by combining processes of rapid prototyping, lost wax, dip coating, selective dissolution, and salt leaching. The result was the successful development of porous biodegradable scaffolds, with mechanical strength covering the range of human blood vessels (1-3 MPa). Human umbilical vein endothelial cells were also cultured on the scaffolds and their biocompatibility was confirmed by cell growth. The Young's modulus of scaffolds could be controlled by changing polymer concentration and porosity. The wall thickness of the tubular scaffold was also controllable by adjusting polymer concentration and pull-up velocity during dip coating. We believe that this fabrication technique can be applied to patient-specific regeneration of blood vessels.

Early diagnosis of cancer by detecting the chemiluminescence of hematoporphyrins in peripheral blood lymphocytes.

Early detection and optimal treatment are the most effective means to improve cancer mortality. Mass screening for cancer has yielded a marked reduction of cancer mortality in the United States. Simple and effective methods are expected for screening of malignancy. Hematoporphyrin derivatives (HPDs) are known to accumulate in cancer cells; thus, HPD has been used for local diagnosis and photodynamic therapy of cancer. The lymphocytes of cancer patients also demonstrate the active uptake of HPD and this phenomenon has been applied for the diagnosis of cancer. In the present study, we have developed a novel method for measurement of the chemiluminescence of HPD in peripheral blood lymphocytes. HPD is composed of hematoporphyrin and its oligomers. Seven cancer patients and seven controls were recruited for this study. The primary cancers included two prostate cancers (one without metastasis and the other with lung metastasis), a renal cancer, a lung adenocarcinoma with systemic metastasis, two gallbladder cancers with lung metastasis, and a colon cancer with liver metastasis. HPD in lymphocytes was measured using a highly sensitive chemiluminescence analyzer with laser light irradiation to detect photoemission by (1)O(2) from HPD. The intensity of chemiluminescence exhibited a linear correlation with the concentrations of HPD. In addition, the level of HPD in lymphocytes was significantly higher in cancer patients than that in healthy volunteers (p < 0.05). These results suggest that detection of the chemiluminescence of HPD in lymphocytes could be a sensitive and simple method for cancer diagnosis and screening.

Antimicrobial Intervention by Photoirradiation of Grape Pomace Extracts via Hydroxyl Radical Generation.

The annual production of grape worldwide amounts to almost 70 million tons, and around 80% is used for winemaking. The two major wastes from winemaking process, pomace and lees account for 20 and 7% of the grapes, respectively. They have been expected as a valuable resource to be recycled because they are rich in polyphenols. Polyphenols possess prooxidatve activity as well as antioxidative one just like a two sides of a coin. A typical example of the prooxidative activity is antibacterial activity of catechins. The activity is exerted through oxidation of phenolic hydroxyl moiety coulpled with reduction of dissolved oxygen leading to hydrogen peroxide (H2O2) generation. In addition, once the oxidation of phenolic hydroxyl moiety is augmented by photoirradiation, highly reactive hydroxyl radical (·OH) is generated. Accordingly, there have been several reports showing that photoirardiation of polyphenols exerts bactericidal activity via ·OH generation. This review focuses mainly on antimicrobial intervention by photoirradiation of grape pomace extract in relation to ·OH generation analyzed by an electron spin resonance-spin trapping method.

Polychromatic spectral pattern analysis of ultra-weak photon emissions from a human body.

Ultra-weak photon emission (UPE), often designated as biophoton emission, is generally observed in a wide range of living organisms, including human beings. This phenomenon is closely associated with reactive oxygen species (ROS) generated during normal metabolic processes and pathological states induced by oxidative stress. Application of UPE extracting the pathophysiological information has long been anticipated because of its potential non-invasiveness, facilitating its diagnostic use. Nevertheless, its weak intensity and UPE mechanism complexity hinder its use for practical applications. Spectroscopy is crucially important for UPE analysis. However, filter-type spectroscopy technique, used as a conventional method for UPE analysis, intrinsically limits its performance because of its monochromatic scheme. To overcome the shortcomings of conventional methods, the authors developed a polychromatic spectroscopy system for UPE spectral pattern analysis. It is based on a highly efficient lens systems and a transmission-type diffraction grating with a highly sensitive, cooled, charge-coupled-device (CCD) camera. Spectral pattern analysis of the human body was done for a fingertip using the developed system. The UPE spectrum covers the spectral range of 450-750nm, with a dominant emission region of 570-670nm. The primary peak is located in the 600-650nm region. Furthermore, application of UPE source exploration was demonstrated with the chemiluminescence spectrum of melanin and coexistence with oxidized linoleic acid.

Bactericidal Action of Photo-Irradiated Aqueous Extracts from the Residue of Crushed Grapes from Winemaking.

Our previous studies revealed that photo-irradiation of polyphenols could exert bactericidal action via reactive oxygen species (ROS). In the present study, the photo-irradiation-induced bactericidal activity of the aqueous extract from the residue of crushed grapes from winemaking was investigated in relation to ROS formation. Staphylococcus aureus suspended in the extract was irradiated with LED light at 400 nm. This solution killed the bacteria, and a 3-4 log and a >5-log reduction of the viable counts were observed within 10 and 20 min, respectively. LED light irradiation alone also killed the bacteria, but the viable counts were 2-4 log higher than those of the photo-irradiated extract. In contrast, almost no change occurred in the suspension without LED irradiation. When hydroxyl radical scavengers were added to the suspension, the bactericidal effect of the photo-irradiated extract was attenuated. Furthermore, electron spin resonance analysis demonstrated that hydroxyl radicals were generated by the photo-irradiation of the extract. The present study suggests that polyphenolic compounds in the extract exert bactericidal activity via hydroxyl radical formation upon photo-irradiation.

Simultaneous Real-Time Monitoring of Oxygen Consumption and Hydrogen Peroxide Production in Cells Using Our Newly Developed Chip-Type Biosensor Device.

All living organisms bear its defense mechanism. Immune cells during invasion by foreign body undergoes phagocytosis during which monocyte and neutrophil produces reactive oxygen species (ROS). The ROS generated in animal cells are known to be involved in several diseases and ailments, when generated in excess. Therefore, if the ROS generated in cells can be measured and analyzed precisely, it can be employed in immune function evaluation and disease detection. The aim of the current study is to introduce our newly developed chip-type biosensor device with high specificity and sensitivity. It comprises of counter electrode and working electrodes I and II. The counter electrode is a platinum plate while the working electrodes I and II are platinum microelectrode and osmium-horseradish peroxidase modified gold electrode, respectively which acts as oxygen and hydrogen peroxide (H2O2) detection sensors. Simultaneous measurement of oxygen consumption and H2O2 generation were measured in animal cells under the effect of exogenous addition of differentiation inducer, phorbol 12-myristate 13-acetate. The results obtained showed considerable changes in reduction currents in the absence and presence of inducer. Our newly developed chip-type biosensor device is claimed to be a useful tool for real-time monitoring of the respiratory activity and precise detection of H2O2 in cells. It can thus be widely applied in biomedical research and in clinical trials being an advancement over other H2O2 detection techniques.

Generation Mechanism of Deferoxamine Radical by Tyrosine-Tyrosinase Reaction.

Nitroxide radical formations of deferoxamine mesylate (DFX) that is used clinically to treat iron-overload patients was examined by a tyrosine-tyrosinase reaction system as models of the H-atom transfer or proton-coupled electron transfer. When DFX was exposed to the tyrosine-tyrosinase reaction, nine-line ESR spectrum (g = 2.0063, hfcc; aN = 0.78 mT, aH(2) = 0.63 mT) was detected, indicating that the oxidation of DFX leads to a nitroxide radical. The signal intensity of the DFX radical increased dependently on the concentrations of tyrosine and tyrosinase. The amounts of DMPO-OH spin adducts via the tyrosine-tyrosinase reaction declined with DFX. Furthermore, mass spectra of an extra removed from the tyrosine-tyrosinase reaction mixture showed that the enzyme reactions might not be degradations of DFX. Therefore, there might be two types of DFX reaction passways, which could be through an internal electron transfer from tyrosine and hydrogen absorptions by ·OH directly.

Detection of hydrogen peroxide in Photosystem II (PSII) using catalytic amperometric biosensor.

Hydrogen peroxide (H2O2) is known to be generated in Photosystem II (PSII) via enzymatic and non-enzymatic pathways. Detection of H2O2 by different spectroscopic techniques has been explored, however its sensitive detection has always been a challenge in photosynthetic research. During the recent past, fluorescence probes such as Amplex Red (AR) has been used but is known to either lack specificity or limitation with respect to the minimum detection limit of H2O2. We have employed an electrochemical biosensor for real time monitoring of H2O2 generation at the level of sub-cellular organelles. The electrochemical biosensor comprises of counter electrode and working electrodes. The counter electrode is a platinum plate, while the working electrode is a mediator based catalytic amperometric biosensor device developed by the coating of a carbon electrode with osmium-horseradish peroxidase which acts as H2O2 detection sensor. In the current study, generation and kinetic behavior of H2O2 in PSII membranes have been studied under light illumination. Electrochemical detection of H2O2 using the catalytic amperometric biosensor device is claimed to serve as a promising technique for detection of H2O2 in photosynthetic cells and subcellular structures including PSII or thylakoid membranes. It can also provide a precise information on qualitative determination of H2O2 and thus can be widely used in photosynthetic research.

Identification by an EPR technique of decreased mitochondrial reducing activity in puromycin aminonucleoside-induced nephrosis.

The temporal changes in the electron paramagnetic resonance (EPR) signal intensities of a nitroxide radical, 4-hydroxy 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), in the kidney in rat puromycin aminonucleoside (PAN) nephrosis were investigated in vivo and in vitro. The rats of the PAN nephrosis group received intraperitoneal injections of PAN at 75 mg/kg body weight while those of control group received saline. The in vivo renal half-lives of TEMPOL were calculated from the decay curve of EPR signal intensities after the intravenous injection of the TEMPOL solution. The mitochondrial half-lives were obtained from the decay curve of the EPR signals after mixing the mitochondrial fraction of the kidney and TEMPOL solution. The in vivo half-lives of TEMPOL of the kidney from 7 to 14 d after PAN administration were significantly longer than those of the controls. The mitochondrial half-lives of TEMPOL on the 9th day after the PAN administration prolonged remarkably compared to the controls (378 +/- 69 vs. 676 +/- 183 s, p <.01). These findings indicate that the in vivo and mitochondrial reducing activity in PAN treated rats decreased markedly, because the half-life of TEMPOL in the kidney reflects the renal reducing activity.

Solution-state dynamics of sugar-connected spin probes in sucrose solution as studied by multiband (L-, X-, and W-band) electron paramagnetic resonance.

A multiband (L-band, 0.7GHz; X-band, 9.4GHz; and W-band, 94GHz) electron paramagnetic resonance (EPR) study was performed for two glycosidated spin probes, 4-(alpha,beta-D-glucopyranosyloxy)-TEMPO (Glc-TEMPO) and 4-(alpha,beta-D-lactopyranosyloxy)-TEMPO (Lac-TEMPO), and one non-glycosylated spin probe, 4-hydroxy-TEMPO (TEMPOL), where TEMPO=2,2,6,6-tetramethyl-1-piperidinyloxyl, to characterize fundamental hydrodynamic properties of sugar-connected spin probes. The linewidths of these spin probes were investigated in various concentrations of sucrose solutions (0-50wt%). The multiband approach has allowed full characterization of the linewidth parameters, providing insights into the molecular shapes of the spin probes in sucrose solution. The analysis based on the fast-motional linewidth theory has yielded anisotropy parameters of rho(x) approximately 2.6 and rho(y) approximately 0.9 for Glc-TEMPO, and rho(x) approximately 4.2 and rho(y) approximately 0.9 for Lac-TEMPO. These values indicate that the glycosidated spin probes have a prolate-type molecular shape elongated along the x-axis (NO(rad) axis) with Lac-TEMPO elongated more remarkably, consistent with their molecular structures. The interaction parameters k (the ratios of the effective hydrodynamic volumes to the real ones) corrected for the difference in molecular shape have been estimated and found to have the relation k(TEMPOL)

Evaluation of antioxidative effects of sesamin on the in vivo hepatic reducing abilities by a radiofrequency ESR method.

Antioxidative effects of sesamin (a mixture of sesamin and episesamin) were evaluated in the liver, kidney and inferior vena cava of living rats using a radiofrequency ESR method. TEMPOL, 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl, was used as an in vivo redox probe, the half-life of which is believed to be correlated with the antioxidant status. The oral administration of sesamin (250 mg/kg rat weight) 3 h before ESR measurements shortened the half-life of TEMPOL in the liver by 10 - 15% as compared with the controls, but did not affect the other organs. This effect was maintained for at least 3 h after the administration, and then disappeared at 24 h, corresponding to the results of our preliminary pharmacokinetic studies. Changes in the reducing ability were observed only in the hepatic sites of the sesamin-treated rats. These findings suggest that sesamin exhibits effective antioxidant activity in the liver via modulation of the intracellular redox status related to TEMPOL reduction.

Scavenging or quenching effect of melanin on superoxide anion and singlet oxygen.

Although photoprotective properties of skin melanin have been well documented, a few studies on the effect of melanin on reactive oxygen species (ROS) generated by ultraviolet (UV) irradiation have been reported. To study the interaction of melanin with ROS, scavenging or quenching effect of melanin on O(2) (*-) and (1)O(2) was examined by electron spin resonance (ESR)-spin trapping methods and a spectrophotometric method, respectively. Melanin potently interacted with O(2) (*-) generated in a hypoxanthine (HPX)-xanthine oxidase (XOD) reaction, and with (1)O(2) generated from a peroxidase, H(2)O(2), and halide system. In the HPX-XOD reaction, it was proved that melanin doses not interfere with the enzyme reaction. It is confirmed that one of the mechanisms by which melanin protects UV-induced skin damage is likely scavenging or quenching activity against ROS such as O(2) (*-) and (1)O(2).

Generation mechanism of radical species by tyrosine-tyrosinase reaction.

Alleviated melanin formation in the skin through inhibition of tyrosine-tyrosinase reaction is one of the major targets of cosmetics for whitening ability. Since melanin has a pivotal role for photoprotection, there are pros and cons of inhibition of melanin formation. This study applying electron spin resonance (ESR)-spin trapping method revealed that (•)H and (•)OH are generated through tyrosine-tyrosinase reaction. When deuterium water was used instead of H(2)O, the signal of 5,5-dimethyl-1-pyrroline N-oxide (DMPO)-H (a spin adduct of DMPO and (•)H) greatly decreased, whilst DMPO-OH (a spin adduct of DMPO and (•)OH) did not. Thus, it is suggested that (•)H was derived from H(2)O, and (•)OH through oxidative catalytic process of tyrosine to dopaquinone. Our study suggests that tyrosinase inhibitors might contribute to alleviate the oxidative damage of the skin by inhibiting (•)OH generation via the enzyme reaction.