Brain ischemic stroke is one of the leading causes of death in developed countries, including Japan. Controlling the neuroinflammation in the penumbra region with mild ischemia is crucial for treating ischemic stroke. C-C chemokine receptor 5 (CCR5), noted for its functions in the progression of neuroinflammation, is considered a promising drug target. We recently found that three CCR5-regulated matrix metalloproteinases (MMPs) are detected in various cell types, including neurons, microglia, and blood vessel endothelial cells, in the ischemic brain of the photothrombosis model mouse. However, it is still unclear whether CCR5 is expressed in these cell types. This study examined the presence of CCR5 in the photothrombotic brain. Preceding the analysis, we evaluated and improved the photothrombosis induction protocol to obtain equable results with lower toxicity. Rose bengal, used to induce thrombosis to cause an infarction, is radicalized by laser and exhibits pancreatic toxicity. Therefore, we changed the administration route from the abdomen to the jugular vein and reduced the required dose of rose bengal. With this improved protocol, we found that the level of CCR5 protein was increased in neurons, microglia, and blood vessels in the ischemic core, in the infarct brain. The increase in CCR5 levels was sensitive to NSAIDs, especially to cyclooxygenase-2-selective etodolac. CD4, a collaborative membrane receptor for CCR5, was also detected in the migrating microglia. These results suggest that CCR5 is dynamically regulated and play diverse roles during ischemic stroke.

Green tea contains catechins, possessing anti-obesity and anti-oxidative effects, and has been consumed for hundreds of years. Our previous pilot study reported that Kosen-cha improves obesity and the parameters of metabolic syndromes in obese patients, however, the effect of Kosen-cha on obesity is still unclear in pre-obese subjects. The aim of this study was to investigate the effect of Kosen-cha on obesity and related clinical parameters including blood lipid and liver functions in a randomized placebo-controlled, double-blinded study. In total, 54 subjects with body mass index (BMI) of 25–30 were enrolled and randomized to receive either Kosen-cha or a placebo. The subjects drank Kosen-cha or the placebo thrice-daily for 12 weeks. Thereafter, we examined the effect of Kosen-cha on obesity (body weight, BMI, body fat, waist circumference, and visceral fat), lipid metabolism (triglyceride and high- and low-density lipoprotein cholesterol), and serum liver enzymes (aspartate aminotransferase, alanine aminotransferase (ALT), and γ-glutamyl transpeptidase). None of the subjects reported adverse effects from drinking Kosen-cha. Body weight, BMI, body fat, waist circumference, and visceral fat area remained unchanged in both groups. However, the change ratio of ALT significantly reduced between placebo and Kosen-cha groups after 12 weeks (Kosen-cha: −11.1 ± 32.7% vs. placebo: 8.46 ± 23.4%, p = 0.019). These results show that the consumption of Kosen-cha did not significantly improve obesity and may reduce liver enzyme levels in pre-obese Japanese subjects.

Inappropriate polypharmacy increases the risks of adverse drug reactions and hospitalization. Thus, it is important to evaluate the appropriateness of prescriptions in polypharmacy. We designed a checklist based on previous studies and guidelines for pharmacists in our hospital to evaluate the appropriateness of a prescription of multiple medications. The efficacy of checklist-based standardization was evaluated by investigating inpatient medical records and prescriptions. We designed a checklist for pharmacists in our hospital to evaluate the appropriateness of a prescription and reduce the prescription of medications with multiple administrations for all age groups. When patients using more than six medications were admitted, pharmacists assessed the prescriptions of these patients using the checklist. We examined 729 inpatients over the course of 4 months before and after the standardization. The research protocol was approved by the Human Ethical Committee of Showa University, School of Pharmacy. For prescriptions with six or more medications, the total number of suggestions for all patients significantly increased upon implementation of the checklist (50 vs. 21, P < 0.05). Additionally, the number of changes in prescriptions by doctors increased while using the checklist (44 vs. 17, P < 0.05), whereas the rate of changes per suggestions did not change. The most common reason for the increase in prescription suggestions after the standardization was a medication was prescribed to patients despite the absence of symptoms. Our checklist was effective in reducing the prescription of inappropriate medications in patients of all ages.

Increased permeability of vascular endothelial cells in the brain is an underlying cause of stroke, which is associated with high mortality rates worldwide. Vascular permeability is regulated by tight junctions (TJs) formed by claudin family and occludin proteins. In particular, increased vascular permeability is associated with decreased claudin domain-containing 1 (CLDND1) expression, which belongs to the TJs family. We previously reported that myeloid zinc finger 1 (MZF1) acts as an activator of CLDND1 expression by binding to its first intron. Several transcription factors regulate transcription by acting on the promoter regions of target genes. However, transcription factors acting on the promoter of CLDND1 are not completely elucidated. Thus, we focused on the promoter region of human CLDND1 to identify factors that could regulate its transcription. Reporter analysis of CLDND1 promoter region revealed an enhancer in the -742/-734 region with MZF1 and specificity protein 1 (SP1) binding sites. Chromatin immunoprecipitation assays confirmed that both MZF1 and SP1 could bind to CLDND1 enhancer region. MZF1 overexpression significantly increased CLDND1 expression, whereas overexpression of SP1 had no effect. Moreover, the identified enhancer region exhibited stronger transcriptional and binding capacity than the first intron. Thus, CLDND1 expression is more strongly regulated by competitive action of MZF1 and SP1 binding to the promoter-enhancer region than the first intron silencer region. These results provide novel insights for the development of potential therapies and preventive strategies for stroke in the future.

Moesin is a member of the ezrin, radixin and moesin proteins that are involved in the formation and/or maintenance of cortical actin organization through their cross-linking activity between actin filaments and proteins located on the plasma membranes as well as through regulation of small GTPase activities. Microglia are immune cells in the central nervous system. They show dynamic reorganization of the actin cytoskeleton in their process elongation and retraction as well as phagocytosis and migration. They also secrete proinflammatory cytokines such as tumor necrosis factor−α (TNF−α). Moesin is the predominant ezrin, radixin and moesin family protein in microglia. Recently, we found that moesin is involved in microglial activation accompanying morphological changes and reorganization of actin cytoskeleton by using moesin knockout mice in vivo and ex vivo. Here we studied the effects of a small molecule inhibitor specific for ezrin and moesin, NSC305787, on the moesin phosphorylation, phagocytosis, migration, and TNF−α secretion of the primary microglia. NSC305787 at the concentration of 10 μM inhibited the moesin phosphorylation, UDP-induced phagocytosis, ADP-induced migration and lipopolysaccharide-induced TNF−α secretion without effect on cell viability. These results in combination with the previous results using moesin knockout mice suggest the functional importance of moesin in microglial activities.

Lysophosphatidic acid (LPA), a small ubiquitous lipid found in vertebrate and non-vertebrate organisms, mediates diverse biological actions. LPA activates mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase, and low-molecular-weight G-proteins by binding to multiple LPA receptors (LPA_1–6, and GPR87). We previously demonstrated that colonies of A431 cells, a human epidermoid carcinoma cell line, were dispersed by LPA1 and GPR87 activation. This LPA-induced A431 cell dispersal is accompanied by epithelial-mesenchymal transition (EMT) and is believed to contribute to tumor progression. Endoplasmic reticulum (ER) stress has been implicated in tumor progression and growth. A recent study found that activation of the inositol-requiring enzyme 1α/X-Box binding protein 1 pathway promotes tumor progression and EMT in colorectal carcinoma. In addition, another report indicated that ER stress preconditioning using stress inducers promotes transforming growth factor β1-induced EMT and apoptosis in human peritoneal mesothelial cells. To investigate the effect of ER stress preconditioning on LPA-induced cell dispersal, we analyze the crosstalk between LPA-induced and ER stress-induced cellular responses using A431 cells. Interestingly, preconditioning via tunicamycin, an ER stress inducer, inhibited LPA-induced A431 cell dispersal, whereas thapsigargin, another inducer, promoted cell dispersal. Furthermore, western blot analysis illustrated that LPA-induced p38 MAPK phosphorylation was enhanced by thapsigargin pretreatment but not by tunicamycin. These results indicate that ER stress inducers differentially alter LPA-induced A431 cell dispersal by modifying LPA-related signals.

The importance of preventing infectious disease for public health continues to increase, and effective disinfectants are needed to inactive pathogenic microorganisms. Chlorine dioxide (ClO₂) is known as one of the most efficient disinfectants. We studied the inhibitory effect of a novel disinfectant, MA-T, for three species of bacteria (Escherichia coli, Staphylococcus aureus, and Aggregatibacter actinomycetemcomitans). We found that NADH:O₂ oxidoreductase activity (NADH oxidase activity) was markedly decreased in all three species, corresponding to the decrease in colony-forming units following treatment with MA-T. In E. coli, NADH:ubiquinone-1(Q₁) oxidoreductase (NADH-Q₁ dehydrogenase; NDH) activity was decreased following MA-T exposure, indicating that both the NDH-1 and NDH-2 enzymes were targets of this disinfectant. The activity of ubiquinol-1 (Q₁H₂): O₂ oxidoreductase (Q₁H₂ oxidase) also was decreased, indicating that cytochromes bo3 and bd were damaged by MA-T. In S. aureus, NADH-ferricyanide dehydrogenase activity and Q₁H₂ oxidase activity were strongly decreased, suggesting that NDH-2, cytochrome bd, and cytochrome aa3 were targets of MA-T in this species. In A. actinomycetemcomitans, only Q₁H₂ oxidase activity was decreased, indicating that in this species, only cytochrome bd was impaired by MA-T treatment. NADH oxidase activity in membrane vesicles prepared from untreated E. coli was not markedly affected by treatment with MA-T, suggesting that MA-T may attack components of the respiratory chain only in live bacteria (i.e., those possessing a membrane potential), because the membrane vesicles cannot produce the membrane potential.

The performance of hexavalent chromium (Cr(VI)) adsorption by activated carbon (AC) prepared from coconut shell (AC1) and modified with silver nanoparticles (AC2), titanium oxide (AC3), and magnetic powder (AC4) was evaluated in this study. The interaction between AC surface properties and Cr(VI) was also assessed via elemental distribution and binding energy analyses. More Cr(VI) was adsorbed onto AC1 than onto any other AC, indicating that the specific surface area and surface functional groups are key factors for Cr(VI) adsorption from the aqueous phase (with correlation coefficients of 0.988 and 0.868–0.949, respectively). Activation of the coconut shell with silver nanoparticles, titanium oxide, and magnetic powder did not increase Cr(VI) adsorption. Cr atoms were detected on the AC1 surface by electron probe microanalysis only after adsorption. Moreover, the binding energies of Cr (2s, 2p, 3s, and 3p) and O (1s) were confirmed after absorption. These results indicate that AC surface properties were strongly related to adsorption performance. Finally, this study reveals the optimal pH conditions for the removal of Cr(VI) from the aqueous phase of approximately pH 2–3 (acidic conditions). In conclusion, this study elucidates the Cr(VI) adsorption mechanisms of coconut shell-derived AC.

Metal responsive transcription factor-1 (MTF-1) is ubiquitously expressed in various tissues, and is thought to be an intracellular zinc sensor that regulates transcriptional activation of zinc responsive genes. We investigated the distribution of MTF-1 in the mouse testis, where zinc plays an essential role in spermatogenesis. By performing immunohistochemical analysis of the ddY mouse testis using the anti-MTF-1 antibody, we observed a donut-shaped staining pattern in the seminiferous tubules in the region proximal to the lumen, where spermatocytes primarily localize. A similar staining pattern was obtained using in situ hybridization to detect MTF-1 mRNA. Furthermore, we confirmed that MTF-1 could not be detected at both the protein and mRNA levels in the premature 20-d-old mouse testis, where spermatocytes are thought not to have been formed in the seminiferous tubules yet. These lines of evidence strongly indicated the selective expression of MTF-1 in mouse spermatocytes and suggested that MTF-1 played a role in a certain stage of spermatogenesis.

Background: Approved anticancer drug combinations are classified into the following groups: broader than (broad label), the same as (same label), or smaller than (narrow label) the series of combination regimens investigated in clinical studies. The present research attempted to elucidate the characteristics of the broad/narrow label to clarify what types of combination regimens are given these labels. Methods: All anticancer drugs approved in Japan between April 2006 and March 2020 and their review reports were selected from the Pharmaceuticals and Medical Devices Agency (PMDA) website. The differences in the number of regimens in clinical trials given each label were investigated using Tukey’s test. Multinomial logistic regression analysis was also conducted to examine the factors influencing each category. Results and Discussion: There were significant differences in the numbers of regimens among the labels. The factors that significantly contributed to labeling could not be identified. However, key features were identified. If there were multiple clinically comparable regimens and a clinical trial was conducted to evaluate the clinical benefit of adding new anticancer agents to one of the major regimens, there was a high probability of receiving a broad label. A narrow label may be granted if a regimen considered clinically comparable despite possessing a different mechanism of action does not exhibit clinical benefits in phase III studies. Conclusion: The present study revealed the PMDA stance for reviewing the clinical data of anticancer combination therapies submitted by sponsors in their totality to allow physicians to provide patient-centric, evidence-based, optimized cancer care to patients.

The incidence rate of bacterial meningitis caused by methicillin-resistant staphylococci is lower than that of bacterial meningitis caused by other gram-positive bacteria. However, considering the high mortality rate of staphylococcal infections, empirical vancomycin (VCM) therapy is often used. On the other hand, VCM is known to affect renal function. Therefore, it is necessary to understand the risks of empirical VCM therapy in patients with suspected bacterial meningitis. We aimed to investigate the risk of acute kidney injury (AKI) associated with empirical VCM therapy in patients with suspected bacterial meningitis. We reviewed the records of 35 suspected bacterial meningitis patients treated with empirical VCM therapy at Fukuoka University Hospital between 2011 and 2017. The incidence rate of AKI associated with empirical VCM therapy was evaluated based on Kidney Disease: Improving Global Outcome criteria. The patients were aged 65.0 (44.0-75.0) years, and had various underlying diseases such as subarachnoid hemorrhage, cerebral hemorrhage, other diseases of the head and community-acquired infection. Methicillin-resistant staphylococci were detected in only 3 patients. In 4 patients with negative culture results, empirical VCM therapy was continued for more than 1 week; this resulted in AKI. The incidence rate of AKI associated with empirical VCM therapy was 11.4%. For patients with suspected bacterial meningitis requiring empirical VCM therapy, it is important to check the necessity of VCM by bacterial culture tests and ensure the safety by monitoring blood concentrations in order to avoid the risk of AKI.

Clostridium perfringens iota-toxin consists of an enzymatic Ia component and binding Ib component. Ib binds to membrane receptors, forms heptamers in lipid rafts, and associates with Ia. The toxin complex is internalized into cells. In this study, we evaluated the inhibitory effects of cholera toxin B subunit (CTB), which binds to lipid rafts, on iota-toxin-mediated cytotoxicity. We examined the effect of CTB on iota-toxin-induced cell rounding activity against MDCK (Madin-Darby canine kidney) cells and Ib-induced cell death in A431 (human epithelial) cells. The presence of CTB inhibited both the cell rounding activity of iota-toxin (MDCK cells) and Ib-induced cell death (A431 cells). Moreover, CTB blocked binding of the Ib monomer to both cells at 4°C. We found by immunofluorescence microscopy that, after loading with Ib and CTB at 4°C, Ib and CTB bound to distinct regions in the plasma membranes. In MDCK cells at 37°C, Ib internalized and partially coexisted with CTB in cytoplasmic vesicles. These findings demonstrated that CTB blocks the binding of iota-toxin to target cells. CTB may provide a protective effect against infection.

Exposure to cadmium, an environmental pollutant, has been associated with adverse health effects such as atherosclerosis. Nucleolin is a multifunctional protein present in the nucleus, cytoplasm, and cell surface and involved in the angiogenesis and repair of the vascular endothelium following injury. Nucleolin dysfunction has been suggested to cause the development of vascular lesions. In the present study, we assessed the effect of cadmium on nucleolin expression in cultured bovine aortic vascular endothelial cells. After incubation with cadmium (0–5 μM), cellular NCL (nucleolin) mRNA levels and nucleolin protein levels were determined by real-time RT-PCR and western blotting, respectively. Cadmium did not alter nucleolin levels in vascular endothelial cells. We then evaluated whether nucleolin modulates endothelial cadmium-induced cytotoxicity by lactate dehydrogenase leakage. In nucleolin-knockdown vascular endothelial cells, cadmium-induced cytotoxicity was significantly higher than in control cells. Furthermore, nucleolin knockdown did not decrease the mRNA levels of metallothionein 1A and 2A, suggesting that nucleolin protects vascular endothelial cells against cadmium-induced cytotoxicity via metallothionein-independent pathways.

Polymethoxyflavones (PMFs) are flavone compounds that contain more than two methoxyl groups and are almost exclusively found in citrus peel. In this study, we examined and compared the effects of the PMFs nobiletin, heptamethoxyflavone and natsudaidain on antigen-specific T cell activation in vitro and in vivo. The three PMFs suppressed proliferation responses in an in vitro study. The PMFs also suppressed IFN-γ, IL-2 and IL-10 production from splenocytes in a dose-dependent manner. Interestingly, nobiletin and heptamethoxyflavone, but not natsudaidain, enhanced IL-4 production from splenocytes. In an in vivo model, antigen-specific T cell proliferation responses were reduced in mice treated with nobiletin, heptamethoxyflavone and natsudaidain compared with responses in control mice. The results suggest that citrus PMFs suppress T cell activation and that nobiletin and heptamethoxyflavone enhance IL-4 production.

Spinal muscular atrophy (SMA) is a progressive neuromuscular disease, associated with motoneuron loss and muscle wasting. Numerous SMA-causative mutations have been reported in survival motor neuron (SMN) gene(s); however, the pathogenic mechanism underlying SMA remains unclear. In the present study, we showed that SMN modulates the expression of transcription factor EB (TFEB), a master regulator of lysosomal genes that plays a key role in lysosome function, autophagy, and the mammalian target of rapamycin (mTOR) signaling pathway. The transfection of small interfering RNA (siRNA) targeting SMN caused a reduction in TFEB expression in the motoneuron-like NSC-34 cell line. In differentiated NSC-34 cells, either SMN or TFEB knockdown resulted in reduced lysosomes at neurites and the atypical accumulation of swollen and enlarged lysosomes in cell bodies. SMN knockdown caused the reduced expression of lysosome-related genes, resulting in the decline of lysosomal degradation and increased autophagic flux. These SMN-depletion-induced aberrations in lysosomes and autophagy could be rescued by the exogenous expression of TFEB. Furthermore, SMN depletion in NSC-34 cells resulted in the decreased phosphorylation of mTOR and its downstream signals. Finally, SMA transgenic mice exhibited reduced TFEB and lysosomal protein expression and the inactivation of mTOR signaling in the lumbar spinal cord at postnatal day 11, compared with their counterparts. These findings indicated that SMN regulates lysosomal gene expression and functions by altering TFEB expression in motoneurons. The targeting of lysosomes might represent a new strategy for the treatment of SMA.

Melanocytes increase melanin production upon exposure to ultraviolet rays (UV) as a defense mechanism. Melanin synthesis in melanocytes is regulated by bioactive factors produced and secreted by keratinocytes. In addition, melanocytes transfer biosynthesized melanin to keratinocytes, causing skin pigmentation. During the pigmentation process, melanocytes communicate with surrounding cells. However, the molecules involved in this cell-to-cell communication, particularly in melanocytes, are poorly understood. In this study, we focused on changes in cell membrane protein expression following exposure of melanocytes to UV irradiation. After mouse melanoma B16 cells were exposed to UV irradiation, alterations in the expression of cell membrane proteins were analyzed using peptide mass fingerprinting. We identified the membrane protein vinculin from proteins with enhanced expression. Western blot results confirmed UV exposure increased vinculin in cell membranes. In contrast, there was no change in vinculin levels in whole cell extracts. Furthermore, we observed no variation in mRNA expression levels using real time PCR. Melanocytes exposed to UV enhanced vinculin migration to cell membranes without altering expression levels. We suggest vinculin involved in the cellular responses of melanocytes and keratinocytes.

Thiazolidinediones, such as troglitazone and rosiglitazone, are anti-diabetic insulin-sensitizing agents that bind to the peroxisome proliferator-activated receptor γ (PPARγ) and have potent adipogenic effects on 3T3-L1 preadipocytes. During 3T3-L1 preadipocyte differentiation, which was induced by isobutyl methylxanthine, dexamethasone, and insulin, troglitazone treatment increased lipid content and decreased PPARγ protein levels compared with DMSO-treated control cells. However, the level of CCAAT/enhancer binding protein α (C/EBPα) and C/EBPβ proteins did not decrease in troglitazone-treated cells compared with DMSO-treated cells. Real-time PCR analysis showed that PPARγ mRNA but not C/EBPα mRNA was downregulated in troglitazone-treated adipocytes, suggesting that PPARγ protein reduction occurred due to the decrease in its transcription level. Rosiglitazone treatment also increased lipid content but decreased PPARγ expression during 3T3-L1 preadipocyte differentiation. Both thiazolidinediones significantly increased the levels of adipokines such as adipocyte protein 2 (aP2) and adiponectin in 3T3-L1 adipocytes compared with that in DMSO-treated cells. We propose that thiazolidinediones are involved in adipogenic homeostasis rather than act as agonists of PPARγ during 3T3-L1 adipocyte differentiation.

Claudin family proteins play an important role in the formation of tight junctions in several tissues. Individual claudins display organ- and tissue-specific expression. Claudin domain containing 1 (CLDND1), also known as claudin 25 (CLDN25), is a homolog of the claudin family, and its expression was reported to be downregulated in a mouse model of cerebellar hemorrhage. We have also reported that the retinoic acid receptor-related orphan receptor α (RORα) is involved in the transcriptional activation of CLDND1 by binding to the RORα responsive element (RORE) in the CLDND1 promoter region. Cholesterol and its derivative oxysterol reportedly serve as ligands for the nuclear receptor RORα. However, the effect of cholesterols on CLDND1 expression is unclear. The present study aimed to evaluate the effect of inhibiting steroid synthesis via lovastatin on RORα-mediated CLDND1 transcriptional regulation. Chromatin immunoprecipitation and luciferase reporter assays revealed that RORα-mediated transcriptional regulation of CLDND1 was suppressed upon lovastatin treatment of HepG2 cells; however, this inhibitory effect was attenuated by supplementation with cholesterol. Furthermore, quantitative reverse transcription-PCR and immunoblotting analyses revealed the downregulated expression of CLDND1 mRNA and protein in HepG2 cells upon lovastatin treatment with no parallel changes in RORα mRNA and protein levels. These results confirm that cholesterol serve as ligands for RORα and are, therefore, involved in the activation of CLDND1 transcriptional regulation by RORα.

Cholesterol deficiency is believed to result in fragile plasma membranes. It remains unclear whether a reduction in the amounts of both mevalonate pyrophosphate decarboxylase (MPD), which is involved in cholesterol biosynthesis, and cholesterol content occurs in the cerebrum and brain stem (diencephalon and midbrain) in or near the sites of strokes in stroke-prone spontaneously hypertensive rats (SHRSP). In this study, we investigated whether a reduction in both the amounts of MPD and cholesterol content corresponded to the sites of strokes in the SHRSP brain. The results obtained suggested that a reduction in the amount of MPD was involved in the decrease observed in cholesterol content, and was also important as a risk factor for stroke in SHRSP because the reductions in cholesterol content and MPD protein levels were associated with the sites of strokes. The mechanism responsible for reducing MPD protein levels in the brains of SHRSP differed with each region.

A decrease in renal function leads to the accumulation of various uremic toxins (UTs) which exert unfavorable physiological effects on the body. Indoxyl sulfate (IS), a tryptophan-derived UT, is known to closely associate with the progression of cognitive disorders (CD) including dementia, in addition to chronic kidney disease (CKD) and cardiovascular events. It is, therefore, important to assess blood IS levels in CKD patients with CD. In this study, we assayed serum IS levels in 37 residents who had been admitted to a geriatric health services facility with stage G3b to G5 CKD and evaluated the correlation between serum IS levels and estimated glomerular filtration rate (eGFR). Eighteen out of 37 residents were considered to suffer dementia. When plotting all serum IS levels against eGFR, a weak but significant correlation was observed with a regression coefficient (r) of -0.420. In the non-dementia group, the correlation between serum IS levels and eGFR became stronger (r = -0.720). However, no correlation was observed in the dementia group. At CKD stage G3b, mean serum IS level was higher in the dementia group than in the non-dementia group. These results suggest that eGFR becomes a good marker to predict serum IS level in the case of CKD patients without dementia, but not in those with dementia. Therefore, direct monitoring of serum IS level is essential to assess the onset and/or progression of dementia in the elderly, irrespective of CKD stages.

Non-alcoholic fatty liver disease (NAFLD) is the most common hepatic disorder, characterized by the fat accumulation in hepatocytes without significant alcohol assumption. In lipogenesis in hepatocytes, the tricarboxylic acid cycle citrate plays a crucial role as a carbon source. Citrate is transported into hepatocytes via Na⁺-coupled citrate transporter, NaCT. It has been demonstrated that knockdown of NaCT expression ameliorates diet-induced NAFLD in mice. In addition, NaCT expression in the liver has been reported to be induced in type 2 diabetic mice. Based on these findings, NaCT is considered to be involved in the high prevalence of NAFLD in patients with type 2 diabetes. On the other hand, it is still unclear for the expression level of NaCT under type 1 diabetic condition and its relationship to hepatic lipid accumulation. In this study, we investigated the gene and functional expression level of NaCT in streptozotocin (STZ)-induced type 1 diabetic mice. The mRNA and protein expression levels of NaCT in STZ-treated mice were gradually decreased after STZ treatment. On the other hand, the Na⁺-dependent citrate uptake activity in hepatocytes isolated from STZ-treated mice was not different from that isolated from non-treated mice. Nevertheless, the plasma triglyceride, cholesterol, and nonesterified fatty acid levels were much higher in STZ-treated mice. These results suggest that NaCT expression level is not closely related to the citrate uptake in hepatocytes under type 1 diabetic condition. In conclusion, unlike type 2 diabetes, NaCT may not be responsible for the pathogenesis of NAFLD in type 1 diabetes.

Regenerative medicine aims to replenish damaged tissue. Boosting the capacity of intrinsic stem cells to proliferate is one key for successful regeneration. Adult zebrafish possess tissue resident stem and progenitor cells, which contribute to homeostatic growth and tissue regeneration. In the intact retina, Müller glia sporadically divide to generate fate restricted, proliferative precursors. Cell death reprograms Müller glia into stem cells that divide and produce multi-potent retinal progenitors. Using zebrafish, we evaluated the effect of taurine-conjugated bile acid, Tauroursodeoxycholic acid (TUDCA) on retinal regeneration. In the intact retina, treatment with TUDCA significantly promotes proliferation of the fate restricted precursors, but has no effect on Müller glia. Following constant light exposure, TUDCA attenuates photoreceptor death, indicating that TUDCA is neuroprotective. Following a stab wound, which initiates death of retinal neurons and reprogramming of Müller glia, treatment with TUDCA significantly increases the number of proliferating cells. In the intact retina, TUDCA-induced proliferation was accompanied by decreased expression of cell cycle inhibitors. These results suggest that TUDCA promotes proliferation of actively-cycling stem and progenitors, identifying TUDCA as a potential reagent to promote regeneration of retinal neurons.

Human serum albumin (HSA) dimer, where two molecules of HSA are genetically fused with a linker of 10 amino acid, has superior blood retention property, compared with HSA monomer. Moreover, HSA dimer derivative, s-nitrosated HSA dimer, functions as an enhanced permeability and retention effects enhancer. HSA dimer has gained considerable attention as drug delivery system carrier based on its prominent function. However, for the HSA dimer to be used clinically, the safety profile of the HSA dimer is required in order to exclude any potential toxicity or unwanted effects. Thus, the present study was undertaken to investigate the occurance of tissue damage and serologic changes due to repeated administration of HSA dimer (66.5 mg/kg) to mice every 3 d for 56 d, as part of a basic consideration on safety evaluation. The evaluation on survival, behavior and body weight indicate that HSA dimer has no effect on physical growth and physiological functions. Hematological tests suggest that HSA dimer has no direct influence on hemocytes, such as hemolysis and platelet aggregation. Moreover, plasma clinical chemistry and histological examinations indicate that the HSA dimer has no deleterious effect on liver and renal functions. The results obtained here indicate HSA dimer is safe and should be useful for medical and pharmaceutical applications.

Calcined Ni–Al complex hydroxide (NA12) was produced through calcination at 400°C, and its capability on phosphate ion adsorption was examined. Initially, the physicochemical characteristics including specific surface area, the number of hydroxyl groups, pore volume, scanning electron microscope images, and X-ray diffraction patterns of calcined Ni–Al complex hydroxides were evaluated. The level of phosphate ion adsorbed onto NA12 in the value of 128.5 mg/g was higher than that of other compared adsorbents. This study indicated that the level of phosphate ion adsorbed using calcined Ni–Al complex hydroxide was correlated to the properties of an adsorbent surface. Moreover, the binding energy of the NA12 surface before and after the phosphate ion adsorption was also determined, and phosphorus energy (2p and 2s) could be detected after adsorption. The results demonstrated that the NA12 surface properties were important for phosphate ion removal from the aqueous solution. Additionally, the effects of pH, temperature, and contact time on the phosphate ion adsorption were also investigated. The results confirmed a potent recovery of the phosphate ion (over 90%) when using a NaOH solution at 1000 mmol/L in this experiment. Thus, NA12 is a promising adsorbent for the phosphate ion.

Curcumin is a polyphenol compound derived from the roots of Curcuma longa. Although the biological activities of curcumin, such as its anti-inflammatory, anti-oxidant, anti-microbial and anti-cancer effects, have been well applied, its poor chemical stability is a major problem. Pentagamavunon-0 (PGV-0) and Pentagamavunon- 1 (PGV-1) were developed as curcumin analogues with higher bioavailability; however, their anti-cancer activity has not yet been assessed. In this study, we evaluated the anti-metastatic activity of PGV-0 and PGV-1 in 4T1 breast cancer cells. Although both curcumin analogues demonstrated similar anti-proliferative effects to curcumin in 4T1 breast cancer cells, they did not inhibit nuclear factor kappa B (NF-ĸB) activity which is a well-defined molecular target of curcumin for its anti-cancer effects. As PGV-0 and PGV-1 exhibited stronger inhibition of the metastatic capacity in a 4T1 breast cancer model than curcumin, PGV-0 and PGV-1 may be promising curcumin analogues to target cancer metastasis having a distinct molecular mechanism from that of curcumin.

Myeloid-derived suppressor cells (MDSCs), which are derived from immature bone marrow cell (BMC) populations that proliferate in the tumor microenvironment, suppress T cell immune responses. Transient receptor potential vanilloid (TRPV) 4, which is a Ca²⁺ channel, is involved in tumor growth, but the role of TRP channels in MDSC differentiation and function remains unclear. Here, we first investigated the involvement of TRP channels in the differentiation of MDSCs. The selective TRPV4 channel antagonist RN-1734 increased the population of MDSCs (CD11b⁺Gr-1⁺) at Day 3, while the TRPV4 agonist GSK1016790A decreased it, suggesting that stimulation of TRPV4 suppresses the differentiation of BMCs to MDSCs. GSK1016790A also increased the production of nitric oxide and reactive oxygen species, but suppressed the expression of Arg-1 mRNA, which encodes arginase-1, in MDSCs. Furthermore, GSK1016790A decreased the phosphorylation of signal transducer and activator of transcription 3 (STAT3) in MDSCs, thereby attenuating STAT3 signaling. Our results suggest that TRPV4 plays a role in regulating both the differentiation and function of MDSCs, and therefore could be a promising target for cancer immunotherapy.

Antibiotics influence the anticoagulation effect of warfarin and increase the bleeding risk in patients who are receiving warfarin. Piperacillin/tazobactam (PIPC/TAZ) is commonly used to treat infections such as healthcare-associated infection. However, there have been few reports about the interaction between warfarin and PIPC/TAZ. In this study, we investigated the influence of PIPC/TAZ on the anticoagulation effect of warfarin in hospitalized patients. The primary outcome was elevation of prothrombin time international normalized ratio (PT-INR) after PIPC/TAZ administration. Secondary outcomes were the proportion of patients with supratherapeutic levels of PT-INR, discontinuation of administration or reduction in the dose of warfarin, bleeding, transfusion, and vitamin-K rescue. Fifteen patients were enrolled in this study. PT-INR elevation occurred in 11 (73.3%) of the 15 patients. The median value of PT-INR after administration of PIPC/TAZ was significantly higher than the value before administration: 2.22 (interquartile range (IQR), 2.05-2.76) and 1.90 (IQR, 1.36-2.45), respectively (p = 0.024). Three (20%) of the 15 patients had PT-INR over 4, and discontinuation of administration or reduction in the dose of warfarin was needed in 6 (40%) of the 15 patients. Bleeding occurred in one patient, transfusion was performed in one patient and vitamin-K rescue was performed in one patient. This study showed that PIPC/TAZ induced elevation of PT-INR in patients receiving warfarin and that discontinuation or reduction in the dose of warfarin was needed in 40% of the patients. Therefore, we recommend to close monitoring of PT-INR in patients treated with warfarin during PIPC/TAZ administration.

Dysregulation of the biological clock disrupts the homeostasis of physiological functions, which may lead to the development of various disorders. To investigate the relationship between biological rhythms and disorders, an efficient monitoring method of the biological clock is necessary. In this study, we analyzed the circadian rhythmicity profile of bile acids in urine and investigated whether urinary bile acid (UBA) could reflect the circadian rhythm in liver physiology. Male Wistar-Hannover rats were maintained in metabolic cages under ad lib feeding and later subjected to restricted feeding (in which rats were fed only during light periods). Excreted urine was collected in each session, and bile acid contents were analyzed via an enzyme-based total bile acid assay. UBA content showed diurnal fluctuations under both ad lib and restricted feeding conditions and reached a peak during the dark period of ad lib feeding. In contrast, with restricted feeding, the peak was observed during light periods. Restricted feeding induced an 8-12 h phase shift. Diurnal fluctuation and phase shift by restricted feeding are distinctive features controlled by biological clocks in peripheral tissues. Since bile acids are synthesized in the liver, we propose that UBA might reflect the circadian fluctuation in liver physiology.

Recent findings established (–)-isostemonamine as an anti-proliferator for estrogen receptor α-negative human breast cancer cells, MDA-MB-231, known to grow/divide at an aggressive rate. However, ST-4, a thioamide derivative of (–)-isostemonamine, is believed to not affect the viability of these cells. Epigenetic changes, such as DNA methylation and histone modification, are involved in the progression of many cancers, including breast cancer. In the present study, we investigated whether ST-4 and its related compounds (ST-3 and ST-5) can potentiate the anti-proliferative activity of the established epigenetic modifiers, 5-aza-2’-deoxycytidine (5-aza-dC; a DNA methyltransferase 1 inhibitor), trichostatin A (TSA; a class I/II histone deacetylase inhibitor), and etoposide (a DNA topoisomerase IIα inhibitor). Data obtained from this study demonstrate that, among the studied compounds, ST-4 displays the strongest enhancement of the anti-proliferative activity of TSA, against MDA-MB-231 cells (IC₂₀ of TSA = 14 ± 3.4 nM versus that of TSA/ST-4 combination = 7.8 ± 1.1 nM). However, this effect was not observed at higher concentrations of above 25 nM of TSA, at which the IC₅₀ values of TSA with or without ST-4 were not significantly different (30 ± 4.4 nM versus 28 ± 1.4 nM, respectively). Results from the study suggest that combining ST-4 with established anti-cancer agents could potentiate the latter’s anti-proliferative activity, thereby potentially minimizing the concentration of these agents needed for optimal clinical efficacy and safety.

Radiation therapy is an important local treatment for malignant tumors, but ionizing radiation may also facilitate tumor invasion and metastasis. The transient receptor potential (TRP) superfamily, which is a diverse group of ion channels activated by various stimuli, has a variety of pathophysiological functions, including a role in malignancy. However, it is not clear whether TRP channels influence radiation-induced biological effects. Here, we show that TRPV1 and TRPV4 channels contribute to the γ-irradiation-induced enhancement of migration of human lung cancer A549 and mouse melanoma B16 cells. We found that γ-irradiation induced both cell migration and actin stress fiber formation of A549 cells, but both effects were suppressed by the TRPV1 inhibitors capsazepine AMG9810, SB366791, and BCTC, and by the TRPV4 inhibitors RN-1734 and GSK2193874. γ-Irradiation induced migration was also suppressed by knockdown of TRPV1 and TRPV4 channels. Furthermore, γ-irradiation of B16 mouse melanoma cells increased the number of lung metastases in C57BL/6 mice, compared to non-irradiated B16 cells, and TRPV1 and TRPV4 channel inhibitors suppressed this increase. These results suggest that TRPV1 and TRPV4 channels are potential targets for intervention to block the acquisition of motility by cancer cells during radiotherapy in order to reduce the risk of metastasis.

Although morphine-induced modulation of immune cells has been well studied, modulation of immune cells by fentanyl and remifentanil, the latter of which has been used in recent anesthesia procedures, has not been well-studied. Our aim was to identify the effects of fentanyl and remifentanil on phagocytosis and respiratory burst in leucocytes in in vivo and in vitro studies. In the in vivo study, twelve patients were assigned to receive fentanyl-based anesthesia (fentanyl group, n=6) or remifentanil-based anesthesia (remifentanil group, n=6). Blood samples were obtained from before anesthesia to 30 min after anesthesia in each group. In the in vitro study, blood samples were obtained from three healthy volunteers and incubated with various blood concentrations of fentanyl or remifentanil (from 0.3 ng/mL to 9 ng/mL). Phagocytic activity (percentage of phagocyting cells) and respiratory burst activity (percentage of cells producing oxygen radicals) were analyzed. In the in vivo study, phagocytosis was suppressed only before incision in the fentanyl group whereas suppression of phagocytosis was continued until emergence from anesthesia in the remifentanil group. In the in vitro study, incubation with fentanyl or remifentanil tended to enhance phagocytic function of monocytes and had no dose-dependent effect over various concentrations of fentanyl or remifentanil, respectively. Remifentanil-based anesthesia suppressed the phagocytic function of monocytes during its administration.

Few studies have addressed the effects and adverse events associated with daily teriparatide use, as well as the adherence and causes for non-adherence, among Japanese patients with osteoporosis, particularly those older than 80 years. In this study, we aimed to clarify various parameters associated with daily teriparatide use in Japanese patients with osteoporosis in a real-world clinical setting. This retrospective study compared the persistence of daily teriparatide use and the associated effects and adverse events in older (≥80 years, n=52) and younger patients (<80 years, n=106) treated with teriparatide between May 2013 and May 2018 at a single orthopedic clinic. We observed a significantly higher treatment completion rate among younger patients compared to their older counterparts (59.6% vs. 40.6%, p=0.036). Of the 74 patients (both patient groups) who completed a 24-month treatment course, only one (1.35%) developed new vertebral fractures. Our findings suggest that older patients would benefit from consistent osteoporosis treatment, particularly with a generally safe and effective agent, such as teriparatide. However, Log-rank test also shows the older patients exhibits a greater tendency to drop out than the younger patients (p=0.0238). The older patients tended to continue to drop out from the beginning. Accordingly, our results emphasize the importance of interventions, especially continuous encouraging from the first self-injection of teriparatide.

Cadmium is an environmental pollutant. Metallothioneins are cysteine-rich, low-molecular-weight proteins that are induced by cadmium, which they chelate for detoxification. In humans, the functional metallothionein isoforms are MT1A, 1B, 1E, 1F, 1G, 1H, 1M, 1X, 2A, 3, and 4. It has been reported that overexpression of MT1G inhibits all-trans-retinoic acid (ATRA)–induced hematopoietic differentiation. Here, we found that cadmium inhibited ATRA-induced nitroblue tetrazolium reduction activity, a marker of hematopoietic differentiation, in human acute myelocytic leukemia HL-60 cells. Reverse transcription – quantitative polymerase chain reaction analysis of HL-60 cells revealed the expression of the metallothionein isoforms MT1G, MT1X, and MT2A and showed that MT1G expression increased significantly after cadmium treatment. ATRA treatment significantly attenuated this cadmium-induced increase in MT1G expression; however, MT1G expression remained significantly higher than that in untreated cells. No significant changes in MT1X and MT2A expression were observed. We also found that the cadmium-induced increase in MT1G expression was independent of CpG demethylation. ATRA is a standard chemotherapy treatment for acute promyelocytic leukemia; our findings suggest that cadmium may inhibit the effect of this chemotherapy.

Many bacterial species express tyrosine kinases termed BY-kinases that share no homology with eukaryotic enzymes. We have previously reported that the Staphylococcus aureus BY-kinase CapB2 when fused with the C-terminal activation domain of its modulator CapA1, can translate into an active tyrosine kinase in HEK293T cells. In the present study, full-length CapA1 and CapB2 tagged with different fluorescent proteins were transfected into HEK293T cells. When expressed individually, the modulator CapA1, a membrane protein in bacteria, also appeared to localize to the cell membrane in HEK293T cells. In contrast, the catalytic subunit, CapB2, was found to be cytosolic. Coexpression of the two proteins resulted in apparent translocation of CapB2 to the membrane with concomitant activation of tyrosine kinase activity. This translocation and activation of CapB2 did not occur when the cytoplasmic C-terminal tail of CapA1 was deleted. Conversely, the CapA1 cytoplasmic C-terminal tail alone, when attached to a membrane localization sequence, was sufficient for CapB2 translocation and kinase activation. Our results indicate that the kinase activity of CapB2 is stimulated by direct interaction with the C-terminal cytoplasmic domain of CapA1 and that the process can be reconstituted and visualized in a human cell line. We created various mutants of CapA and CapB, and present data that demonstrate the correlation between CapA-CapB interaction and kinase activation.

Owing to the high metal binding affinity of metallothionein (MT), newly synthesized MT was speculated to attenuate the activity of metal-responsive element (MRE)-binding transcriptional factor 1 (MTF-1) by removing Zn from the activated MTF-1. To investigate the potential role of MT in the inactivation of MTF-1, we examined the transcriptional levels of reporter and endogenous MRE-dependent genes using mouse embryonic fibroblasts (MEFs) established from MT-knockout (KO) and wild-type (WT) mice. The activation of MTF-1 by the Cd exposure of MT-KO MEFs was sustained for 12 h, whereas that of MT-WT MEFs showed the rapid attenuation. Consequently, MT was found to negatively regulate MTF-1 activity, which can control the expression of MT itself.

Both NF-E2-related factor 2 (Nrf2) and heat shock protein 70 (HSP70) contribute to cellular defense to various stresses, and have emerged as candidates of therapeutic targets to improve or prevent tissue damage. Cefotaxime (CTX), a third-generation cephalosporin antibiotic, is conceived as a safe drug largely free from side effects. CTX exhibits broad-spectrum antimicrobial activity, and thereby, is most commonly prescribed for the treatment of infectious diseases induced by Gram-positive or Gram-negative bacteria. In this study, we unexpectedly found the beneficial properties of CTX that upregulate both Nrf2 and HSP70 to the extent that stress-induced damage is ameliorated. Non-toxic levels of reactive oxygen species (ROS) induced by CTX activated the Nrf2 pathway without cytotoxicity, which in turn upregulated HSP70. Interestingly, the cytotoxicity of Fas/CD95 ligand (FasL), a cytotoxic cytokine that strongly induces apoptosis, was significantly ameliorated by pre-treatment with CTX, most likely because of the upregulation of Nrf2 and HSP70. Our results therefore show novel properties of CTX, which raise the possibility that CTX works as a non-toxic therapeutic agent for preventing and repairing tissue damage.

Gastrointestinal decontamination by activated charcoal (AC) is the most important treatment for acetaminophen (APAP) overdose. Because AC adsorbs a wide variety of toxins, it may also adsorb the oral antidote, N-acetylcysteine (NAC). NAC is a specific antidote for APAP overdose and administered as a 72-h oral regimen. We evaluated AC adsorption of NAC after APAP adsorption in vitro. Different concentrations of NAC solution diluted with simulated gastric fluids (SGF) and simulated intestinal fluids (SIF) were added to AC and incubated at 37°C for 1 h. The AC was then removed by filtration, and the NAC concentration was determined. This revealed that NAC was not only adsorbed onto the AC but also converted to N,N’-diacetyl-L-cystine (DAC), which is oxidized NAC. We then calculated the maximum adsorption capacity per gram of AC (Qm). The apparent Qm based on the amount of decreased NAC in the SGF was 400 mg/g, and that in the SIF was 714 mg/g. The actual Qm based on only the amount of adsorption in the SGF was 294 mg/g, and that in the SIF was 59 mg/g. We also determined whether or not AC could adsorb the loading and maintenance doses of NAC after APAP adsorption. The residual rate in the SGF was 2.1%, and that in the SIF was 0.3%. The rate of conversion to DAC was higher in the SIF than that in the SGF. By both the actions of adsorption and oxidation, AC may reduce the effect of loading and maintenance doses of NAC.

Peroxisome proliferator-activated receptor α (PPARα) belongs to the nuclear receptor superfamily and exerts hypolipidemic and anti-inflammatory functions when activated by ligand-binding. To screen its ligands, cell-based reporter assays have been widely used, but it is difficult to investigate the effects of the metabolites of test compounds on PPARα due to very low drug-metabolizing capability of cell lines generally used in those assays. The aim of this study was to construct a convenient PPARα reporter assay system with drug-metabolizing capability by using 9,000 x g supernatant (S9) of rat liver homogenate, which abundantly includes various drug-metabolizing enzymes. We used clofibrate as a model compound since it requires hydrolysis to clofibric acid to activate PPARα. In cell-based reporter assays using a PPARα-responsive luciferase reporter plasmid and a rat PPARα expression plasmid, reporter activity was increased by treatment with bezafibrate and clofibric acid, which directly activate PPARα as ligands, but not with clofibrate. The addition of S9 to culture media increased reporter activity of the cells treated with clofibrate, as expected. When heat-denatured S9 was used or a carboxylesterase inhibitor was included in the system, clofibrate-induced PPARα activation was not observed, suggesting that carboxylesterases are responsible for the hydrolysis of clofibrate to clofibric acid. Taken together, we have established a convenient PPARα reporter assay system with drug-metabolizing capability to assess PPARα-activating potency of both test compounds and their metabolites.

Clostridioides difficile (C. difficile) colitis and pseudomembranous colitis are known as healthcare-associated intestinal infections. In this study, the incidence of C. difficile colitis and pseudomembranous colitis was investigated using the Japanese Adverse Drug Event Report (JADER). Using JADER data between April 2004 and September 2017, the patient who developed C. difficile colitis and pseudomembranous colitis were investigated. During the study period, 375 cases of C. difficile colitis and 903 cases of pseudomembranous colitis were reported. The numbers of reported cases of both C. difficile colitis and pseudomembranous colitis were largest in those in their 70s, accounting for 24.7% and 25.6%, respectively. Patients in their 60s-90s comprised the majority of all patients with both C. difficile colitis and pseudomembranous colitis. Both C. difficile colitis and pseudomembranous colitis were caused by antibiotics in many patients, and signals of all antibiotics were detected. In C. difficile colitis, signals of immunosuppressants, corticosteroids, and alkylating drugs were also detected among drugs other than antibiotics. For pseudomembranous colitis, the use of molecularly targeted drugs, antimetabolic drugs, and corticosteroids was reported other than antibiotics. Using JADER, we revealed risk factors for the development of C. difficile colitis and pseudomembranous colitis, and firstly revealed that molecularly targeted drugs other than antibiotics could also be potential risk factors. Our findings may be useful for the early detection of drug-induced C. difficile colitis and pseudomembranous colitis.