Suppression of autophagy induces senescence in the heart.

Aging is a critical risk factor for heart disease, including ischemic heart disease and heart failure. Cellular senescence, characterized by DNA damage, resistance to apoptosis and the senescence-associated secretory phenotype (SASP), occurs in many cell types, including cardiomyocytes. Senescence precipitates the aging process in surrounding cells and the organ through paracrine mechanisms. Generalized autophagy, which degrades cytosolic materials in a non-selective manner, is decreased during aging in the heart. This decrease causes deterioration of cellular quality control mechanisms, facilitates aging and negatively affects lifespan in animals, including mice. Although suppression of generalized autophagy could promote senescence, it remains unclear whether the suppression of autophagy directly stimulates senescence in cardiomyocytes, which, in turn, promotes myocardial dysfunction in the heart. We addressed this question using mouse models with a loss of autophagy function. Suppression of general autophagy in cardiac-specific Atg7 knockout (Atg7cKO) mice caused accumulation of senescent cardiomyocytes. Induction of senescence via downregulation of Atg7 was also observed in chimeric Atg7 cardiac-specific KO mice and cultured cardiomyocytes in vitro, suggesting that the effect of autophagy suppression upon induction of senescence is cell autonomous. ABT-263, a senolytic agent, reduced the number of senescent myocytes and improved cardiac function in Atg7cKO mice. Suppression of autophagy and induction of senescence were also observed in doxorubicin-treated hearts, where reactivation of autophagy alleviated senescence in cardiomyocytes and cardiac dysfunction. These results suggest that suppression of general autophagy directly induces senescence in cardiomyocytes, which in turn promotes cardiac dysfunction.

Synthesis of Isotopic Atropisomers Based on 12C/13C Discrimination.

Quinazolin-4-one derivatives possessing an isotopic atropisomerism (isotopic N-C axial chirality) based on ortho-12CH3/13CH3 discrimination were prepared. The diastereomeric quinazolin-4-ones bearing an asymmetric carbon as well as an isotopic atropisomerism were clearly discriminated by 1H and 13C NMR spectra and revealed to possess high rotational stability and stereochemical purity.

Contribution of periodontal health in cardiovascular secondary prevention: Analyses on hospitalized patients in cardiology units.

AIM: To investigate the association between dental health and incident cardiovascular disease (CVD) in secondary prevention setting. MATERIALS AND METHODS: A total of 888 patients with known CVD hospitalized in the cardiology unit were prospectively enrolled. We assessed the association between missing teeth and three variables of periodontitis and major adverse cardiovascular events (MACE), defined as a composite of cardiac death, acute myocardial infarction, stroke and hospital re-admission for congestive heart failure. RESULTS: During a median (Q1, Q3) follow-up of 4.6 (1.4, 6.7) years, an additional missing tooth was associated with a 3% (95% confidence interval [CI]: 1%-5%) higher hazard of MACE (p = .004). Compared with patients with 0 to ≤4 missing teeth, periods free from MACE (95% CI) by 5 years of follow-up were, on average, shorter by 0.17 (-0.04 to 0.37) years, 0.26 (0.04-0.49) years and 0.59 (0.34-0.85) years in patients with 5 to ≤7, 8 to ≤13 and >13 missing teeth, respectively. No significant associations were observed between periodontal measures and MACE incidence. CONCLUSIONS: In hospitalized patients with existing CVD, the total number of missing teeth was associated with incident MACE.

Effect of Periodontal Disease on Long-Term Outcomes After Percutaneous Coronary Intervention for De Novo Coronary Lesions in Non-Smokers.

BACKGROUND: This study aimed to investigate the effect of periodontal disease (PD) on the outcomes of patients with coronary artery disease (CAD) treated with percutaneous coronary intervention (PCI).Methods and Results: The study included 77 consecutive non-smoking patients with de novo coronary lesions treated with a drug-eluting stent (DES). Periodontal measurements, including the community periodontal index (CPI), were performed by independent periodontists. A CPI score of ≥3 was used to define PD. The occurrence of major adverse cardiac events (MACEs), which were defined as a composite of cardiovascular death, non-fatal myocardial infarction, target lesion revascularization, or non-target lesion revascularization, was compared between patients with and without PD. Of the 77 patients, 49 (63.6%) exhibited a CPI score of 3 or 4 and were assigned to the PD group. The remaining 28 patients (36.4%) were assigned to the non-PD group. Baseline clinical characteristics and angiographic findings were comparable between the 2 groups. MACEs occurred in 13 (26.5%) of the PD patients and 2 (7.1%) of the non-PD patients. Kaplan-Meier analysis showed a significantly lower MACE-free survival rate in the PD group than for the non-PD group (P=0.034). CONCLUSIONS: PD at baseline was associated with an increased risk of MACEs in CAD patients who were treated with a DES for de novo coronary lesions.

Evaluation of Electroencephalogram Using Exact Low-Resolution Electromagnetic Tomography During Photic Driving Response in Patients with Migraine.

BACKGROUND: Photophobia is a common feature of migraine, which may involve abnormal cortical information processing. In electroencephalograms (EEG), photic driving is known as a reaction to visual stimulation. Both photophobia and photic driving response are present during light stimulation. We hypothesized that cortical response to photic stimulation would differ between migraine patients with and without aura. METHODS: We recruited 50 migraine patients (migraine with aura [MWA] = 21; migraine without aura [MWOA] = 29). Spontaneous eyes-closed resting EEG from 20 electrodes on the scalp during the interictal phase was recorded. After recording, each photic stimulation was separately selected. We analyzed EEG by fast Fourier transform and observed the spectrum frequency peaks and topographies in response to photic stimulation. Exact low-resolution electromagnetic tomography (eLORETA) was used to compute the 3-dimensional intracerebral distribution of EEG activity. RESULTS: Photic stimulation at frequencies 5, 8, 15, and 20 Hz showed significant differences between migraine patients with and without aura. MWOA patients consistently had a stronger response to photic stimulation than MWA patients. In all patients, the differential response was located in the visual cortex, except for the stimulation at 20 Hz, where the difference at subharmonic 10 Hz was located in the parietal cortex (Brodmann Area 7). CONCLUSION: We confirmed high incidences of photic hypersensitivity and photic driving responses in migraine patients. We suggest that repeated occurrences of cortical spreading depression in MWA may suppress cortical function, thus contributing to a weaker visual cortical response to photic stimulation in MWA patients compared with MWOA patients.

The origin of ß-strand bending in globular proteins.

BACKGROUND: Many ß-strands are not flat but bend and/or twist. However, although almost all ß-strands have a twist, not all have a bend, suggesting that the underlying force(s) driving ß-strand bending is distinct from that for the twist. We, therefore, investigated the physical origin(s) of ß-strand bends. METHODS: We calculated rotation, twist and bend angles for a four-residue short frame. Fixed-length fragments consisting of six residues found in three consecutive short frames were used to evaluate the twist and bend angles of full-length ß-strands. RESULTS: We calculated and statistically analyzed the twist and bend angles of ß-strands found in globular proteins with known three-dimensional structures. The results show that full-length ß-strand bend angles are related to the nearby aromatic residue content, whereas local bend angles are related to the nearby aliphatic residue content. Furthermore, it appears that ß-strands bend to maximize their hydrophobic contacts with an abutting hydrophobic surface or to form a hydrophobic side-chain cluster when an abutting hydrophobic surface is absent. CONCLUSIONS: We conclude that the dominant driving force for full-length ß-strand bends is the hydrophobic interaction involving aromatic residues, whereas that for local ß-strand bends is the hydrophobic interaction involving aliphatic residues.

Topographic Analysis of Electroencephalographic Changes during Photic Driving Responses in Patients with Migraine.

OBJECTIVE: Migraineurs demonstrate abnormal information processing such as photic hypersensitivity. The photic driving response to photic stimulation (PS) is a reaction to the visual stimulation of electroencephalography (EEG). Both the photic driving response and photic hypersensitivity appear during light stimulation. We considered that evaluation of a migraineur's photic driving response may help to elucidate the mechanism of the migraineur's sensitive condition. Our study aimed to investigate EEG photic driving responses with a source-localizing method. METHODS: We recorded spontaneous resting EEG with eyes closed from 20 electrodes on the scalp during the interictal phase. After recording, each PS was separately selected. We also analyzed EEG by fast Fourier transform and observed the spectrum frequency peaks and topographies in response to PS. RESULTS: The photic driving response could be observed at a flashing rate of >15 Hz. Patients with photic hypersensitivity tended to show more photic driving regardless of the migraine subtype. Moreover, in topographies, their activated areas were shifted anteriorly from fundamental driving to harmonic driving for each photic frequency. This anterior shift was more evident with a longer duration of illness, although no significant differences were seen between migraine subtypes. The global field power value had a positive correlation with the duration of illness. CONCLUSION: Photic hypersensitivity and photic driving responses were increased in migraineurs. Photic hypersensitivity might result in sensitization of the limbic system. © 2015 S. Karger AG, Basel.

α-N-Acetylglucosaminidase from Bifidobacterium bifidum specifically hydrolyzes α-linked N-acetylglucosamine at nonreducing terminus of O-glycan on gastric mucin.

α-Linked N-acetylglucosamine is one of the major glyco-epitopes in O-glycan of gastroduodenal mucin. Here, we identified glycoside hydrolase (GH) family 89 α-N-acetylglucosaminidase, termed AgnB, from Bifidobacterium bifidum JCM 1254, which is essentially specific to GlcNAcα1-4Gal structure. AgnB is a membrane-anchored extracellular enzyme consisting of a GH89 domain and four carbohydrate-binding module (CBM) 32 domains. Among four CBM32 domains, three tandem ones at C-terminus showed to bind porcine gastric mucin, suggesting that these domains enhance the enzyme activity by increasing affinity for multivalent substrates. AgnB might be important for assimilation of gastroduodenal mucin by B. bifidum and also applicable to production of prebiotic oligosaccharides from porcine gastric mucin.

Local sequence of protein ß-strands influences twist and bend angles.

ß-Sheet twisting is thought to be mainly determined by interstrand hydrogen bonds with little contribution from side chains, but some proteins have large, flat ß-sheets, suggesting that side chains influence ß-structures. We therefore investigated the relationship between amino acid composition and twists or bends of ß-strands. We calculated and statistically analyzed the twist and bend angles of short frames of ß-strands in known protein structures. The most frequent twist angles were strongly negatively correlated with the proportion of hydrophilic amino acid residues. The majority of hydrophilic residues (except serine and threonine) were found in the edge regions of ß-strands, suggesting that the side chains of these residues likely do not affect ß-strand structure. In contrast, the majority of serine, threonine, and asparagine side-chains in ß-strands made contacts with a nitrogen atom of the main chain, suggesting that these residues suppress ß-strand twisting.

Ubiquitin-negative, eosinophilic neuronal cytoplasmic inclusions associated with stress granules and autophagy: an immunohistochemical investigation of two cases.

Identification of the proteinaceous components of the pathological inclusions is an important step in understanding the associated disease mechanisms. We immunohistochemically examined two previously reported cases with eosinophilic neuronal cytoplasmic inclusions (NCIs)(case 1, Mori et al. Neuropathology 2010; 30: 648­53; case 2, Kojima et al. Acta Pathol Jpn 1990; 40: 785­91) using 67 antibodies against proteins related to cytoskeletal constituents, ubiquitin-proteasome system, autophagy-lysosome pathway and stress granule formation. Regional distribution pattern of eosinophilic NCIs in case 1 was substantially different from that in case 2. However, NCIs in both cases were immunonegative for ubiquitin and p62 and were immunopositive for stress granule markers as well as autophagy-related proteins, including valosin-containing protein. Considering that eukaryotic stress granules are cleared by autophagy and valosin-containing protein function, our findings suggest that eosinophilic NCIs in the present two cases may represent the process of autophagic clearance of stress granules.

Suppression of autophagy induces senescence in the heart.

Aging is a critical risk factor for heart disease, including ischemic heart disease and heart failure. Cellular senescence, characterized by DNA damage, resistance to apoptosis and the senescence-associated secretory phenotype (SASP), occurs in many cell types, including cardiomyocytes. Senescence precipitates the aging process in surrounding cells and the organ through paracrine mechanisms. Generalized autophagy, which degrades cytosolic materials in a non-selective manner, is decreased during aging in the heart. This decrease causes deterioration of cellular quality control mechanisms, facilitates aging and negatively affects lifespan in animals, including mice. Although suppression of generalized autophagy could promote senescence, it remains unclear whether the suppression of autophagy directly stimulates senescence in cardiomyocytes, which, in turn, promotes myocardial dysfunction in the heart. We addressed this question using mouse models with a loss of autophagy function. Suppression of general autophagy in cardiac-specific Atg7 knockout ( Atg7 cKO) mice caused accumulation of senescent cardiomyocytes. Induction of senescence via downregulation of Atg7 was also observed in chimeric Atg7 cardiac-specific KO mice and cultured cardiomyocytes in vitro , suggesting that the effect of autophagy suppression upon induction of senescence is cell autonomous. ABT-263, a senolytic agent, reduced the number of senescent myocytes and improved cardiac function in Atg7 cKO mice. Suppression of autophagy and induction of senescence were also observed in doxorubicin-treated hearts, where activation of autophagy alleviated senescence in cardiomyocytes and cardiac dysfunction. These results suggest that suppression of general autophagy directly induces senescence in cardiomyocytes, which in turn promotes cardiac dysfunction.

Dream-enacting behaviour is associated with impaired sleep and severe headache-related disability in migraine patients.

BACKGROUND: Sleep disorders, nightmares and visual hallucinations have been reported in migraine patients, which may suggest the involvement of rapid eye movement (REM) sleep regulation in migraine. However, the relationship between migraine and REM sleep behaviour disorder (RBD) remains unclear. METHODS: To investigate the clinical correlates of dream-enacting behaviours (DEB) in migraine patients, we assessed episodic migraine patients ( N = 161, mean age 33.1 years) and headache-free control subjects ( N = 140, mean age 33.1 years) under 50 years of age in a cross-sectional, case-control study. The Japanese version of the RBD screening questionnaire was used, and subjects scoring 5 or higher were defined as having DEB. RESULTS: A significantly increased frequency of DEB was observed in migraine patients compared to controls (24.2% vs. 14.3%). Migraine patients with DEB presented higher scores on the Migraine Disability Assessment and Pittsburgh Sleep Quality Index and an increased rate of smoking compared to those without DEB. Duration of migraine and headache frequency and intensity were not different between migraine patients with or without DEB. CONCLUSION: DEB was associated with impaired sleep and severe headache-related disability in migraine patients and may reflect brainstem dysfunction and increased brain excitability in migraine patients.

Probable rapid eye movement sleep behavior disorder, nocturnal disturbances and quality of life in patients with Parkinson's disease: a case-controlled study using the rapid eye movement sleep behavior disorder screening questionnaire.

BACKGROUND: Increasing evidence provides a clear association between rapid eye movement sleep behavior disorders (RBD) and Parkinson's disease (PD), but the clinical features that determine the co-morbidity of RBD and PD are not yet fully understood. METHODS: We evaluated the characteristics of nocturnal disturbances and other motor and non-motor features related to RBD in patients with PD and the impact of RBD on their quality of life. Probable RBD (pRBD) was evaluated using the Japanese version of the RBD screening questionnaire (RBDSQ-J). RESULTS: A significantly higher frequency of pRBD was observed in PD patients than in the controls (RBDSQ-J ≥ 5 or ≥ 6: 29.0% vs. 8.6%; 17.2% vs. 2.2%, respectively). After excluding restless legs syndrome and snorers in the PD patients, the pRBD group (RBDSQ-J≥5) showed higher scores compared with the non-pRBD group on the Parkinson's disease sleep scale-2 (PDSS-2) total and three-domain scores. Early morning dystonia was more frequent in the pRBD group. The Parkinson's Disease Questionnaire (PDQ-39) domain scores for cognition and emotional well-being were higher in the patients with pRBD than in the patients without pRBD. There were no differences between these two groups with respect to the clinical subtype, disease severity or motor function. When using a cut-off of RBDSQ-J = 6, a similar trend was observed for the PDSS-2 and PDQ-39 scores. Patients with PD and pRBD had frequent sleep onset insomnia, distressing dreams and hallucinations. The stepwise linear regression analysis showed that the PDSS-2 domain "motor symptoms at night", particularly the PDSS sub-item 6 "distressing dreams", was the only predictor of RBDSQ-J in PD. CONCLUSION: Our results indicate a significant impact of RBD co-morbidity on night-time disturbances and quality of life in PD, particularly on cognition and emotional well-being. RBDSQ may be a useful tool for not only screening RBD in PD patients but also predicting diffuse and complex clinical PD phenotypes associated with RBD, cognitive impairment and hallucinations.

Factors associated with spontaneous reporting of adverse drug reactions in Japan.

PURPOSE: Spontaneous reported adverse drug reactions (ADRs) are currently the main source of pharmacovigilance activities in each country. The number of ADRs reported to the authority warns of safety risks to patients, but it also reflects the efficiency and limitations of the reporting system itself. This article explored how the accumulation of safety information, status in foreign countries (e.g., US approval), drug company attributes, and regulatory actions (e.g., early post-marketing phase vigilance; EPPV) were associated with the numbers of spontaneously reported ADRs in Japan. METHODS: All serious ADRs for drugs for which the active ingredients or route of administration were approved in Japan from 2000 through 2005 were collected from the national database. The numbers of serious ADRs within the first 2 and 3 years after launch were analyzed using the negative binominal distribution model. RESULTS: The launch lag and the presence of drugs with a similar mode of action were negatively associated with the ADR numbers, but the number of study subjects for the new drug application (NDA) showed no clear association. The number of sales representatives and the implementation of EPPV were positively associated with the ADR numbers. CONCLUSION: The accumulation of foreign post-market evidence seemed to be more important for drug safety in Japan than was the amount of pre-approval safety data. The positive impacts of sales representatives and EPPV suggested a critical role for drug companies in drug safety and also the importance of considering how best to collect information in local situations with unique regulatory requirements.

[Proposal for contrast-amplified image reconstruction methods by adjusting computed tomography value scale in three-dimensional image processing].

To create three-dimensional computed tomography angiography (3D-CTA) images of blood vessels, it is important to acquire a high-contrast original image of the blood vessels and the surrounding tissues. However, reexamination is likely to be needed if, due to the condition of the examinee, insufficient contrast enhancement is achieved. This prompted us to design a method of amplifying image contrast using software that simply adds the CT value to each corresponding pixel of the original image, i.e., the same image is added after the scan. This prevents spatial and time discontinuities with the original image. The CT value of the original image can also be increased arbitrarily to any value by adjusting the weighting level of the added image. The difference in CT values between the pixels can thus be amplified without changing the contours of the original image. Since this method enables the original image's contrast to be amplified by post-processing, i.e., without increasing radiation load exposure or employing additional contrast medium, it is a technique with high clinical utility.

Porphyromonas gingivalis, a periodontal pathogen, impairs post-infarcted myocardium by inhibiting autophagosome-lysosome fusion.

While several previous studies have indicated the link between periodontal disease (PD) and myocardial infarction (MI), the underlying mechanisms remain unclear. Autophagy, a cellular quality control process that is activated in several diseases, including heart failure, can be suppressed by Porphyromonas gingivalis (P.g.). However, it is uncertain whether autophagy impairment by periodontal pathogens stimulates the development of cardiac dysfunction after MI. Thus, this study aimed to investigate the relationship between PD and the development of MI while focusing on the role of autophagy. Neonatal rat cardiomyocytes (NRCMs) and MI model mice were inoculated with wild-type P.g. or gingipain-deficient P.g. to assess the effect of autophagy inhibition by P.g. Wild-type P.g.-inoculated NRCMs had lower cell viability than those inoculated with gingipain-deficient P.g. This study also revealed that gingipains can cleave vesicle-associated membrane protein 8 (VAMP8), a protein involved in lysosomal sensitive factor attachment protein receptors (SNAREs), at the 47th lysine residue, thereby inhibiting autophagy. Wild-type P.g.-inoculated MI model mice were more susceptible to cardiac rupture, with lower survival rates and autophagy activity than gingipain-deficient P.g.-inoculated MI model mice. After inoculating genetically modified MI model mice (VAMP8-K47A) with wild-type P.g., they exhibited significantly increased autophagy activation compared with the MI model mice inoculated with wild-type P.g., which suppressed cardiac rupture and enhanced overall survival rates. These findings suggest that gingipains, which are virulence factors of P.g., impair the infarcted myocardium by cleaving VAMP8 and disrupting autophagy. This study confirms the strong association between PD and MI and provides new insights into the potential role of autophagy in this relationship.

Endoplasmic Reticulum Selective Autophagy Alleviates Anthracycline-Induced Cardiotoxicity.

Background: The administration of anthracycline drugs induces progressive and dose-related cardiac damage through several cytotoxic mechanisms, including endoplasmic reticulum (ER) stress. The unfolded protein response plays a crucial role for mitigating misfolded protein accumulation induced by excessive ER stress. Objectives: We aimed to clarify whether endoplasmic reticulum-selective autophagy machinery (ER-phagy) serves as an alternative system to protect cardiomyocytes from ER stress caused by anthracycline drugs. Methods: Primary cultured cardiomyocytes, H9c2 cell lines, and cardiomyocyte-specific transgenic mice, all expressing ss-RFP-GFP-KDEL proteins, were used as ER-phagy reporter models. We generated loss-of-function models using RNA interference or gene-trap mutagenesis techniques. We assessed phenotypes and molecular signaling pathways using immunoblotting, quantitative polymerase chain reaction, cell viability assays, immunocytochemical and histopathological analyses, and cardiac ultrasonography. Results: The administration of doxorubicin (Dox) activated ER-phagy in ss-RFP-GFP-KDEL-transduced cardiomyocytes. In addition, Dox-induced cardiomyopathy models of ER-phagy reporter mice showed marked activation of ER-phagy in the myocardium compared to those of saline-treated mice. Quantitative polymerase chain reaction analyses revealed that Dox enhanced the expression of cell-cycle progression gene 1 (CCPG1), one of the ER-phagy receptors, in H9c2 cells. Ablation of CCPG1 in H9c2 cells resulted in the reduced ER-phagy activity, accumulation of proapoptotic proteins, and deterioration of cell survival against Dox administration. CCPG1-hypomorphic mice developed more severe deterioration in systolic function in response to Dox compared to wild-type mice. Conclusions: Our findings highlight a compensatory role of CCPG1-driven ER-phagy in reducing Dox toxicity. With further study, ER-phagy may be a potential therapeutic target to mitigate Dox-induced cardiomyopathy.

Erratum to "Association between periodontal disease and pericardial adipose tissue in patients with cardiovascular disease" [American Heart Journal Plus: Cardiol. Res. and Pract. Volume 30, 2023, 100,298].

[This corrects the article DOI: 10.1016/j.ahjo.2023.100298.].

Association between periodontal disease and pericardial adipose tissue in patients with cardiovascular disease.

Background: Periodontal disease (PD) is associated with an increased risk of cardiovascular disease (CVD). Pericardial adipose tissue (PAT) is known as a marker of progressive CVD. This study sought to assess the association between PD and PAT in patients with CVD. Methods: We retrospectively investigated 135 patients admitted for CVD who underwent computed tomography coronary angiography (CTCA) and periodontal examinations. Periodontal assessment using the community periodontal index (CPI) was based on the probing pocket depth around teeth. Patients with CPI ≥3 were categorized as having PD. PAT volume was measured with a quantitative semi-automated procedure using CTCA images. Patients were divided into tertiles according to PAT volume. Baseline characteristics and PD findings were compared among the tertiles. Results: Eighty-six patients were diagnosed with PD (63.7 %). Mean PAT volume was 181.4 ml, and patients were categorized as small-PAT (PAT <148.9 ml), intermediate-PAT (148.9 ml ≤ PAT ≤204.6 ml), and large-PAT (PAT >204.6 ml). The prevalence of PD was significantly higher in large-PAT (38/46, 82.6 %) than in small-PAT (18/45, 40.0 %) and intermediate-PAT (30/44, 68.2 %) patients. Multivariate logistic regression analysis showed that body weight, history of hypertension, and the presence of PD were independent predictors for large-PAT (odds ratio [OR]: 1.12, P < 0.001, OR: 3.97, P = 0.017, and OR: 4.18, P = 0.0078, respectively). Conclusion: The presence and severity of PD were significantly correlated with PAT volume, which has been associated with progressive CVD. Further prospective studies are warranted to assess the impact of PD on the onset and outcomes of CVD.

Inhibitory effects of daiokanzoto (da-huang-gan-cao-tang) on p-glycoprotein.

We have studied the effects of various Kampo medicines on P-glycoprotein (P-gp), a drug transporter, in vitro. The present study focused on Daiokanzoto (Da-Huang-Gan-Cao-Tang), which shows the most potent inhibitory effects on P-gp among the 50 Kampo medicines studied, and investigated the P-gp inhibitory effects of Daiokanzoto herbal ingredients (rhubarb and licorice root) and their components by an ATPase assay using human P-gp membrane. Both rhubarb and licorice root significantly inhibited ATPase activity, and the effects of rhubarb were more potent than those of licorice root. The content of rhubarb in Daiokanzoto is double that in licorice root, and the inhibition patterns of Daiokanzoto and rhubarb involve both competitive and noncompetitive inhibition, suggesting that the inhibitory effects of Daiokanzoto are mainly due to rhubarb. Concerning the components of rhubarb, concentration-dependent inhibitory effects were observed for (-)-catechin gallate, (-)-epicatechin gallate, and (-)-epigallocatechin gallate. In conclusion, rhubarb may cause changes in the drug dispositions of P-gp substrates through the inhibition of P-gp. It appears that attention should be given to the interactions between these drugs and Kampo medicines containing rhubarb as an herbal ingredient.