A simple and rapid assay method for analysis of the metabolic activity of viable but non-culturable (VBNC) Salmonella was established. An environmental isolate of Salmonella Enteritidis (SE), grown to the logarithmic phase, rapidly lost its culturability during incubation with 1–10 mM H2O2 in Luria–Bertani (LB) medium. To assess the viability of the bacteria, we measured 3 different metabolic activities: Respiratory activity by 5-cyano-2,3-ditolyl-tetrazolium chloride (CTC) reduction, glucose uptake assessed with 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG), and DNA synthesis activity evaluated by 5-ethynyl-2′-deoxyuridine (EdU) incorporation. These activities were analyzed by both confocal laser-scanning microscopy and flow cytometry, together with colony-formation assays on LB-agar plates. The results showed that some of the H2O2-treated SE cells were in the VBNC state and that the extent of H2O2-induced decrease in each metabolic activity varied according to the activity. That is, glucose-uptake activity was not markedly changed, being kept at the highest level; whereas the respiratory activity was less than that of the glucose-uptake, and DNA synthesis activity was the lowest among them. These results suggest that the VBNC state might be characterized by different metabolic activities that vary and correspond to the kind and strength of the stress, threatening bacterial survival in an adverse environment.
This study describes the population pharmacokinetics and dose personalization of cyclosporine in 36 patients with connective tissue diseases. A one-compartment open model with absorption was adopted as a pharmacokinetic model, and a nonlinear mixed effects model was used to analyze the population pharmacokinetic models. In the final model, age (AGE) and total body weight (TBW) were influential covariates on clearance (CL/F), which was expressed as CL/F (L/h)=17.8×(AGE/60)−0.269×(TBW/46.9)0.408, in addition to the volume of distribution (Vd/F), (L)=98.0 and absorption rate constant (Ka) (h−1)=0.67 (fixed). The results of the present study provide novel insights into factors involved in determining the most suitable dose and dosing strategy for individual patients with connective tissue disease.
The effects of decitabine (DAC), a DNA methyltransferase (DNMT) inhibitor, on metastasis and exosomal expression of microRNAs were examined in SW620/OxR cells, a human colorectal cancer (CRC) cell line (SW620) with acquired resistance to oxaliplatin. This cell line shows an invasive phenotype by epithelial–mesenchymal transition. Two CRC cell lines, SW480, derived from primary CRC, and SW620, derived from lymph node metastasis, which were obtained from the same patient, as well as SW620/OxR, were also used in the present study. Cytarabine (Ara-C), a non-DNMT-inhibiting cytidine analog, was used as negative control of DAC. No significant difference was observed in the invasion abilities of SW480 cells treated with DAC or Ara-C. On the other hand, invasion ability was suppressed by treatment with DAC in SW620 and SW620/OxR cells. Up-regulated expression of E-cadherin, microRNA-200c (miR-200c), and miR-141 following DAC treatment indicated the acquisition of epithelial cell-like characteristics in SW620 and SW620/OxR cells. Exosomal expression levels of miR-200c and miR-141 were also up-regulated by DAC treatment in SW620 and SW620/OxR but not in SW480 cells. This increase in exosomal miRNA expression negatively correlated with invasion ability. These results suggest that DNA demethylation treatment caused acquisition of epithelial cell-like characteristics in SW620 and SW620/OxR cells. Furthermore, the observed increased exosomal expression of miR-200c and miR-141 may be an indicator or biomarker candidate for mesenchymal–epithelial transition of CRC cells.
Nitric oxide (NO) may act as either a pro-oxidant or an antioxidant in biological systems. Previous work has found inhalation of NO improved survival in a high altitude rat model. NO donor isosorbide mononitrate derivants might have a protective effect against hypoxia. We synthesized a series of isosorbide mononitrate derivant compounds to test their anti-hypoxia activities. Normobaric hypoxia and hypobaric hypoxia models were used to study the protective role of NO donor in mice. The results showed isosorbide mononitrate derivants had protective effects in hypoxia mice. Among those compounds, acetyl ferulic isosorbide mononitrate (AFIM) was the most effective. It prolonged the survival time during the normobaric hypoxia test. It decreased malondialdehyde (MDA) and H2O2 in hypobaric hypoxia mice. The antioxidase activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) remained in normal ranges in the AFIM group. As a sign of mitochondrial dysfunction, the activities of ATPase were down regulated in mice under hypobaric hypoxia conditions. AFIM also protected ATPase activities. The protective effects of AFIM might come from a sustained NO supply and the release of acetyl ferulic acid with anti-oxidant activity.
It is believed that neuronal death caused by abnormal deposition of amyloid-beta peptide is the major cause of the cognitive decline in Alzheimer’s disease. Adult neurogenesis plays a key role in the rescue of impaired neurons and amelioration of cognitive impairment. In the present study, we demonstrated that osthole, a natural coumarin derivative, was capable of promoting neuronal stem cell (NSC) survival and inducing NSC proliferation in vitro. In osthole-treated APP/PS1 transgenic mice, a significant improvement in learning and memory function was seen, which was associated with a significant increase in the number of new neurons (Ki67+/NF-M+) and a decrease in apoptotic cells in the hippocampal region of the brain. These observations suggested that osthole promoted NSC proliferation, supported neurogenesis, and thus efficiently rescued impaired neurons in the hippocampus and ameliorated cognitive impairment. We also found that osthole treatment activated the Notch pathway and upregulated the expression of self-renewal genes Notch 1 and Hes 1 mRNA in NSCs. However, when Notch activity was blocked by the γ-secretase inhibitor DAPT, the augmentation of Notch 1 and Hes 1 protein was ameliorated, and the proliferation-inducing effect of osthole was abolished, suggesting that the effects of osthole are at least in part mediated by activation of the Notch pathway.
Lung cancer is the leading cause of cancer-related death worldwide, and non-small cell lung cancer (NSCLC) is the most common pathological type with a reported frequency of about 85% of all cases. Despite recent advances in therapeutic agents and targeted therapies, the prognosis for NSCLC remains poor, and therefore it is important to identify the biological targets of this complex disease since a blockade of such targets would affect multiple downstream signaling cascades. β-Lapachone (β-Lap) is an antiproliferative agent that selectively induces apoptosis-related cell death in a variety of human cancer cells. However, the mechanisms of its action require further investigation. In this study, we show that treatment with β-lap triggers apoptosis and cell-cycle arrest in two NSCLC cell lines: H1299 and NCI-H358. The transcription factor specificity protein 1 (Sp1) was markedly inhibited by β-lap in a dose- and time-dependent manner. Furthermore, β-lap modulated the protein expression levels of the Sp1 regulatory genes, including cell-cycle regulatory proteins and antiapoptotic proteins, resulting in apoptosis. Taken together, our results indicate that β-lap may be a potential antiproliferative agent candidate by inducing apoptotic cell death in NSCLC tissue through downregulation of Sp1.
Doxorubicin (DOX) is widely used for the treatment of a wide range of cancers such as breast and lung cancers, and malignant lymphomas, but is generally less efficacious in gastrointestinal cancers. The most accepted explanation for the DOX refractoriness is its resistance development. Here, we established DOX-resistant phenotypes of human gastric MKN45 and colon LoVo cells by continuous exposure to incremental concentrations of the drug. While the parental MKN45 and LoVo cells expressed carbonyl reductase 1 (CBR1) highly and moderately, respectively, the gain of DOX resistance further elevated the CBR1 expression. Additionally, the DOX-elicited cytotoxicity was lowered by overexpression of CBR1 and inversely strengthened by knockdown of the enzyme using small interfering RNA or pretreating with the specific inhibitor quercetin, which also reduced the DOX refractoriness of the two resistant cells. These suggest that CBR1 is a key enzyme responsible for the DOX resistance of gastrointestinal cancer cells and that its inhibitor is useful in the adjuvant therapy. Although CBR1 is known to metabolize DOX to a less toxic anticancer metabolite doxorubicinol, its overexpression in the parental cells hardly show significant reductase activity toward low concentration of DOX. In contrast, the overexpression of CBR1 increased the reductase activity toward an oxidative stress-derived cytotoxic aldehyde 4-oxo-2-nonenal. The sensitivity of the DOX-resistant cells to 4-oxo-2-nonenal was lower than that of the parental cells, and the resistance-elicited hyposensitivity was almost completely ameliorated by addition of the CBR1 inhibitor. Thus, CBR1 may promote development of DOX resistance through detoxification of cytotoxic aldehydes, rather than the drug’s metabolism.
Although the administration of clonidine, an alpha-2 adrenoceptor agonist, significantly attenuates nociception and hyperalgesia in several pain models, clinical trials of clonidine are limited by its side effects such as drowsiness, hypotension and sedation. Recently, we determined that the sigma-1 receptor antagonist BD1047 dose-dependently reduced nociceptive responses in a mouse orofacial formalin model. Here we examined whether intraperitoneal injection of clonidine suppressed the nociceptive responses in the orofacial formalin test, and whether co-administration with BD1047 enhances lower-dose clonidine-induced anti-nociceptive effects without the disruption of motor coordination and blood pressure. Formalin (5%, 10 µL) was subcutaneously injected into the right upper lip, and the rubbing responses with the ipsilateral fore- or hind-paw were counted for 45 min. Clonidine (10, 30 or 100 µg/kg) was intraperitoneally administered 30 min before formalin injection. Clonidine alone dose-dependently reduced nociceptive responses in both the first and second phases. Co-localization for alpha-2A adrenoceptors and sigma-1 receptors was determined in trigeminal ganglion cells. Interestingly, the sub-effective dose of BD1047 (3 mg/kg) significantly potentiated the anti-nociceptive effect of lower-dose clonidine (10 or 30 µg/kg) in the second phase. In particular, the middle dose of clonidine (30 µg/kg) in combination with BD1047 produced an anti-nociceptive effect similar to that of the high-dose clonidine, but without a significant motor dysfunction or hypotension. In contrast, mice treated with the high dose of clonidine developed severe impairment in motor coordination and blood pressure. These data suggest that a combination of low-dose clonidine with BD1047 may be a novel and safe therapeutic strategy for orofacial pain management.
The plant Millettia pulchra was commonly used in folk medicine for the management of inflammation. However, there was no scientific rationale for these effects and the mechanism of action remained incompletely understood. The present study was designed to investigate the antiinflammatory and analgesic activities of an ethanol extract of the stem of M. pulchra (EMP) in vivo, and to explore the antiinflammatory activity of compounds isolated from EMP in vitro. We found that EMP reduced xylene-induced ear edema and relieved both acetic acid-induced pain and pain in the hot plate test. Additionally, a significant decrease in nitric oxide (NO) production was observed in cells treated with the isolated compounds. Lanceolatin B, which showed the greatest inhibition of NO synthesis among the compounds tested, also reduced levels of interleukin-1 beta (IL-1β), IL-6, tumor necrosis factor-alpha (TNF-α), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), nuclear factor-kappa B (NF-κB), and phosphorylation inhibitory kappa B alpha (p-IκBα) in a dose-dependent manner. These findings provide convincing evidence that EMP and the individual isolated compounds possess significant antiinflammatory and analgesic activities.
Poria cocos is a well-known medicinal plant widely used in China and other East Asian countries owing to its various therapeutic effects. Pachymic acid (PA) is a bioactive lanostrane-type triterpenoid from Poria cocos. In this paper, a method of high-performance liquid chromatographic (LC) coupled with triple quadrupole tandem mass spectrometry (QQQ-MS/MS) was developed and validated to investigate the concentration of PA in rat plasma. Samples were prepared by a liquid–liquid extraction, and chromatographic separation was achieved with a Phenomenex Gemini C18 column (50 mm×2.0 mm i.d.) using a mobile phase consisting of acetonitrile and 0.05% formic acid (85 : 15, v/v) at a flow rate of 0.3 mL/min. The detection was performed on an Applied Bio-Systems API 4000 MS/MS with an electrospray ionization (ESI) inlet in negative multiple reaction monitoring (MRM) mode. Standard curves of samples in plasma were linear (R2=0.9948) over the concentration range of 5–500 ng/mL, and acceptable accuracy and precision were achieved. The lower limit of quantification and detection were 5 and 0.5 ng/mL, respectively. The method was used successfully to study the pharmacokinetics of PA in rats for oral administration. The main pharmacokinetic parameters of elimination half-life (t1/2), area under the plasma concentration–time curve from time zero to infinity (AUC0→∞), plasma clearance (CL), and apparent volume of distribution (Vd) for the PA group were 4.96±1.33 h, 1466.9±361.7 ng·h/mL, 6.82±1.73 L/h, and 48.85±9.47 L, respectively. This LC-MS/MS method can be developed further for clinical investigation of PA-containing products.
Protein kinase CK2 (“casein kinase II”) is a protein serine/threonine kinase that plays critical roles in biological processes such as cell growth, cell cycle progression, and apoptosis. So far, we have identified that one catalytic isozyme of CK2, CK2α, is over-expressed in the kidney during the progression of glomerulonephritis (GN). Moreover, we have shown that in vivo inhibition of CK2 by administration of CK2 inhibitors was effective in the treatment of experimental GN. Hence the development of potent CK2 inhibitors should be considered in therapeutic strategies for GN. In the present study we identified compound 13, a pyrazine derivative, as a potent CK2 inhibitor. By performing enzyme kinetics analysis in vitro, we characterized the inhibition of compound 13 toward each CK2 catalytic isozyme. Furthermore, in vivo, we demonstrated that compound 13 is effective in attenuating proteinuria, decreasing the enhanced level of blood urea nitrogen and serum creatinine, and ameliorating glomerular crescent formation in an experimental GN rat model. On the other hand, cellular apoptosis was detected in the rat testis following administration of compound 13. This study provides clues for new strategies for developing applicable compounds into CK2-targeted GN treatments.
Aerobic glycolysis is an established hallmark of cancer. Neoplastic cells display increased glucose consumption and a corresponding increase in lactate production compared to the normal cells. Aerobic glycolysis is regulated by the phosphatidylinositol-3-kinase (PI3K)/Akt/ mammalian target of rapamycin (mTOR) signaling pathway, as well as by oncogenic transcription factors such as c-Myc and hypoxia inducible factor 1α (HIF-1α). γ-Tocotrienol is a natural isoform within the vitamin E family of compounds that displays potent antiproliferative and apoptotic activity against a wide range of cancer cell types at treatment doses that have little or no effect on normal cell viability. Studies were conducted to determine the effects of γ-tocotrienol on aerobic glycolysis in mouse +SA and human MCF-7 breast cancer cells. Treatment with γ-tocotrienol resulted in a dose-responsive inhibition of both +SA and MCF-7 mammary tumor cell growth, and induced a relatively large reduction in glucose utilization, intracellular ATP production and extracellular lactate excretion. These effects were also associated with a large decrease in enzyme expression levels involved in regulating aerobic glycolysis, including hexokinase-II, phosphofructokinase, pyruvate kinase M2, and lactate dehydrogenase A. γ-Tocotrienol treatment was also associated with a corresponding reduction in the levels of phosphorylated (active) Akt, phosphorylated (active) mTOR, and c-Myc, but not HIF-1α or glucose transporter 1 (GLUT-1). In summary, these findings demonstrate that the antiproliferative effects of γ-tocotrienol are mediated, at least in the part, by the concurrent inhibition of Akt/mTOR signaling, c-Myc expression and aerobic glycolysis.
A major challenge in stem cell therapy for cardiac repair is how to obtain normally functioning stem cell-derived cardiomyocytes. We aim to address the effects of C-reactive protein (CRP) on the cardiac differentiation of embryonic stem (ES) cells. Immunostaining, Western blotting and electrophysiology were employed. A hundred fifty milligran/liters CRP significantly reduced the percentage of cardiomyocytes differentiated from mouse ES cells, while it may also promote sarcomere development compared to 30 mg/L CRP treatment. Further examination of the action potential (AP) in individual ES cell-derived cardiomyocytes showed that there exist three types of cardiomyocytes: artial-like (A-like), ventricular-like (V-like), and pacemaker-like (P-like). A hundred fifty milligran/liters CRP treatment decreased the P-like cardiomyocytes, whereas it increased the A-like. Such inhibitory effect and alteration were not significant at 30 mg/L CRP treatment. Moreover, 150 mg/L CRP significantly increased the APD90 (90% of duration of AP) and decreased the spontaneous firing rate of AP in P-like cells, while had little effect on other electrophysiological characteristics, including APA (AP amplitude) and MDP (maximum diastolic potential). This study revealed the effect of CRP on the cardiac differentiation of ES cells. It provides an in vitro pathological model and may be of importance to the future work of ES cell-based therapy in clinical applications and in vivo pathological studies.
Salubrinal is a selective inhibitor of cellular complexes that dephosphorylate eukaryotic translation initiation factor 2α (eIF2α). In previous reports, salubrinal was shown to have the potential to inhibit the activation of nuclear factor-κB (NF-κB) by several stimuli. However, the effects of salubrinal on NF-κB signaling are largely unknown. In this study, we investigated whether and how salubrinal affects NF-κB activation induced by tumor necrosis factor (TNF)-α and interleukin (IL)-1β. We found that salubrinal selectively blocked TNF-α- but not IL-1β-induced activation of NF-κB. This inhibitory effect occurred upstream of transforming growth factor (TGF)-β-activated kinase 1 (TAK1). Further experiments revealed that salubrinal blocked TNF-α-triggered NF-κB activation independent of its action on eIF2α because knockdown of eIF2α by small interfering RNA (siRNA) did not reverse the inhibitory effect of salubrinal on NF-κB. Moreover, guanabenz, a selective inhibitor of the regulatory subunit of protein phosphatase (PP) 1, also preferentially inhibited TNF-α-triggered activation of NF-κB. These findings raise the possibility that salubrinal may selectively block TNF-α-triggered activation of the NF-κB pathway through inhibition of the PP1 complex.
Membrane type-1 matrix metalloproteinase (MT1-MMP) plays pivotal roles in tumor progression and metastasis, and holds great promise as an early biomarker for malignant tumors. Therefore, the ability to evaluate MT1-MMP expression could be valuable for molecular biological and clinical studies. For this purpose, we aimed to develop short peptide-based nuclear probes because of their facile radiosynthesis, chemically uniform structures, and high specific activity, as compared to antibody-based probes, which could allow them to be more effective for in vivo MT1-MMP imaging. To the best of our knowledge, there have been no reports of radiolabeled peptide probes for the detection of MT1-MMP in cancer tissues. In this study, we designed and prepared four probes which consist of a MT1-MMP-specific binding peptide sequence (consisting of L or D amino acid isomers) and an additional cysteine (at the N or C-terminus) for conjugation with N-(m-[123/125I]iodophenyl) maleimide. We investigated probe affinity, probe stability in mice plasma, and probe biodistribution in tumor-bearing mice. Finally, in vivo micro single photon emission computed tomography (SPECT) imaging and ex vivo autoradiography were performed. Consequently, [123I]I-DC, a D-form peptide probe radioiodinated at the C-terminus, demonstrated greater than 1000-fold higher specific activity than previously reported antibody probes, and revealed comparably moderate binding affinity. [125I]I-DC showed higher stability as expected, and [123I]I-DC successfully identified MT1-MMP expressing tumor tissue by SPECT imaging. Furthermore, ex vivo autoradiographic analysis revealed that the radioactivity distribution profiles corresponded to MT1-MMP-positive areas. These findings suggest that [123I]I-DC is a promising peptide probe for the in vivo detection of MT1-MMP in cancers.
The effects of glycation on skin permeation and accumulation of compounds were evaluated using an in vitro glycated skin model. Glycation of the skin of hairless mice was induced using vertical diffusion cells and incubation with phosphate-buffered saline containing 50 mM glyoxal for 24 h. Flux and accumulation in the skin were determined by applying hydrophilic and lipophilic molecules (Sodium fluorescein; FL-Na and Nile red, respectively) to this in vitro glycated skin model. Furthermore, to investigate the effect of glycation on epidermal–dermal barrier properties, we conducted diffusion experiments with FL-Na and fluorescein isothiocyanate-dextran using stratum corneum (SC)-stripped glycated skin. The in vitro glycated skin model demonstrated characteristic glycation alterations like a yellowish change in skin color and surface roughness. For low-molecular weight (MW) hydrophilic molecules, flux across glycated full-thickness skin was higher than that across normal skin, although there was no difference with lipophilic molecules. However, glycated epidermis–dermis showed lower flux, and the difference increased with the MW of the compound. Furthermore, the amount of high-MW hydrophilic molecules accumulated in glycated epidermis–dermis was decreased. These results suggest that glycated SC and epidermis–dermis differentially regulate the permeability of hydrophilic molecules and highlight the importance of controlling drug delivery by modifying the formulation or method of application depending on skin condition.
Protein O-linked mannose β1,2-N-acetylglucosaminyltransferase 1 (POMGNT1) is a Golgi glycosyltransferase that catalyzes the formation of the N-acetylglucosamine (GlcNAc) β1→2Man linkage of O-mannosyl glycan. POMGNT1 is not modified by N-glycans because there are no potential N-glycosylation sites; however, it is not clear whether POMGNT1 is modified by O-glycans. To determine whether POMGNT1 is O-glycosylated, we prepared recombinant human POMGNT1 from HEK293T cells. The recombinant POMGNT1 was recognized by Sambucus sieboldiana lectin (SSA), and sialidase digestion of POMGNT1 decreased SSA reactivity and enhanced the reactivity of Arachis hypogaea lectin (PNA). These results suggest that POMGNT1 is modified by a sialylated core-1 O-glycan. Next, we analyzed the structures of the O-glycans on POMGNT1 by β-elimination and pyrazolone-labeling methods in combination with mass spectrometry. We identified several mucin-type O-glycans containing (NeuAc)1(Hex)1(HexNAc)1, (NeuAc)2(Hex)1(HexNAc)1, and (NeuAc)2(Hex)2(HexNAc)2. To examine whether the O-glycans affect the functions and properties of POMGNT1, we compared glycosylated and non-glycosylated forms of recombinant sPOMGNT1 for their activity and surface hydrophobicity using the hydrophobic probe 1-anilino-8-naphthalene sulfonate (ANS). POMGNT1 activity and surface hydrophobicity were not affected by the presence or absence of O-glycans.
To clarify the seasonal fluctuations in air pollution and the effect of long-range transport, we collected airborne particles (n=118) at Dazaifu in Fukuoka, Japan, from June 2012 to May 2013 and measured Pb and SO42−, which are indicators of the long-range transport of anthropogenic air pollutants, as well as their mutagenicity, and other factors. The levels of airborne particles, Pb, and SO42− were very high on March 4, 8, 9, and 19, and May 13, 21, and 22, 2013. The backward trajectories indicated that air masses had arrived from the Gobi Desert and northern China on those days. The mutagenicity of airborne particles was examined using the Ames test on Salmonella typhimurium YG1024. Highly mutagenic airborne particles were mostly collected in winter, and most of them showed high activity both with and without S9 mix. High levels of polycyclic aromatic hydrocarbons (PAHs) were found in many samples that showed high mutagenicity. For the samples collected on January 30, February 21, and March 4, the levels of Pb, SO42−, PAHs, and mutagenicity were high, and the backward trajectories indicated that air masses present on those days had passed through northern or central China. The Japan Meteorological Agency registered Asian dust events at Fukuoka on March 8, 9, and 19, 2013. The results of the present study suggest that high levels of anthropogenic air pollutants were transported with Asian dust. Similarly, long-range transport of air pollutants including mutagens occurred on days when Asian dust events were not registered.
Statins, 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors, are potential drugs for chronic heart failure treatment in clinical studies. However, there may be differences in the effects on heart failure between lipophilic and hydrophilic statins. In this study, we investigated whether hydrophilic rosuvastatin (RSV) and lipophilic pitavastatin (PTV) exert different effects on the left ventricular diastolic function. Subjects were hypercholesterolemia patients with left ventricular diastolic dysfunction. This was an open-label, randomized, parallel, comparative, prospective study. The subjects received treatment with RSV or PTV for 24 weeks, and their low density lipoprotein (LDL)-cholesterol levels were controlled by these statins according to the guideline. The primary endpoint was defined as the change in left ventricle (LV) diastolic function (E/E′) estimated by echocardiography, and the secondary endpoint was the plasma B-type natriuretic peptide (BNP) level. No serious adverse effects were observed during the entire study period in any patient, nor were there any significant differences in changes in the body mass index, blood pressure, or heart rate. Statin treatment did not significantly alter the primary endpoint, E/E′. The change ratio of BNP was not significantly different between PTV and RSV groups. However, BNP was significantly increased in the RSV (p=0.030) but not the PTV (p>0.999) group. This study revealed that although neither RSV nor PTV improved LV diastolic dysfunction, BNP, a biomarker of LV wall stress, was increased in the RSV but not the PTV group. Observation for a longer period is necessary to clarify the different effects of these statins on LV diastolic dysfunction. (UMIN-ID: UMIN000003571).
The anti-tumor effects of selective serotonin reuptake inhibitors (SSRIs) and serotonin and norepinephrine reuptake inhibitors (SNRIs) on several types of cancer cells have been reported. However, comparison of the anti-tumor effects of these drugs on human hepatocellular carcinoma (HepG2) cells has not been studied. We compared the anti-tumor effects of four SSRIs and two SNRIs on HepG2 cells. SSRIs and duloxetine dose-dependently decreased cell viability. Milnacipran had no effect on cell viability. The half-maximal inhibitory concentration was lower in the order of: sertraline, paroxetine, duloxetine, fluvoxamine, escitalopram, and milnacipran. Exposure to sertraline (2 µM) significantly increased caspase-3/7 activity. These results suggest that, of the agents tested here, sertraline had the highest sensitivity to HepG2 cells, and activation of the caspase pathway is involved in the anti-tumor effects of sertraline in HepG2 cells.
Ginseng (Panax ginseng C.A. MEYER, Araliaceae), which contains protopanaxadiol-type and protopanaxatriol-type ginsenosides, has been used for inflammation, fatigue, stress, and tumor in Asian countries. Orally administered ginsenosides are metabolized to their aglycones 20(S)-protopanaxadiol (PPD) and 20(S)-protopanaxatriol (PPT) by gut microbiota. However, their anti-fatigue effects have not been studied thoroughly. Therefore, we investigated the anti-fatigue activities of PPD and PPT in mice, using the weight-loaded swimming (WLS) and the rota-rod tests. Ginseng water extract (GW), ginseng saponin fraction (GWS) and ginseng polysaccharide fraction (GWP) at concentrations of 50 and 100 mg/kg and PPD and PPT at 5 and 10 mg/kg were orally administered to mice once daily for 5 d. GW, GWS, and PPT significantly increased the WLS time, however, GWP and PPD did not cause any significant change. PPT induced the most significant increase in WLS time. PPD (10 mg/kg) and PPT (5 and 10 mg/kg) inhibited the WLS-induced increase in corticosterone, lactate, lactate dehydrogenase (LDH), and creatinine levels as well as the reduction in glucose level. PPT increased the riding time in the rota-rod test, and also inhibited corticosterone, lactate, and creatinine levels. These findings suggest that the anti-fatigue effect of ginseng may be attributable to its saponins, particularly PPT, rather than to its polysaccharides.
Although macrophage phagocytoses modified low-density lipoprotein (LDL), excessive accumulation of modified LDL induces macrophage foam cell formation, which is a feature of atherosclerotic plaque. Thus, the identification of scavenger receptor for modified LDL will provide better understanding of an atherosclerotic event. We recently showed that nucleolin expressed on macrophages acts as a scavenger receptor for various endogenous discarded products. Here, we investigated whether or not nucleolin is involved in the uptake of acetylated LDL (AcLDL). In contrast to normal LDL, AcLDL directly bound to immobilized nucleolin. AcLDL exhibited a higher affinity for macrophages than normal LDL. This binding of AcLDL was inhibited by anti-nucleolin antibody and antineoplastic guanine-rich oligonucleotide (AGRO), a nucleolin-specific oligonucleotide aptamer. In addition, AcLDL exhibited a higher affinity for HEK cells transfected with nucleolin than those without. Further, intracellular accumulation of AcLDL was also inhibited by anti-nucleolin antibody. The results of this study suggest that nucleolin expressed on macrophages is a receptor for AcLDL.
The direct inhibitory potential of twenty five anti-tuberculosis drugs on eight CYP-specific reactions in human liver microsomes was investigated to predict in vivo drug–drug interactions (DDIs) from in vitro data. Rifampicin, rifabutin, and thioacetazone inhibited one CYP reaction. Isoniazid and clofazimine had inhibitory effects on four CYP reactions, and rifapentine, ethionamide, and prothionamide widely inhibited CYP reactions. Based on the inhibition constant (Ki) and the therapeutic total inhibitor concentrations [I]max of eight drugs in human plasma, [I]max/Ki values were calculated to evaluate clinical DDIs. The [I]max/Ki values were 0.20 or less for rifampicin, rifabutin, and thioacetazone; 0.15–2.0 for isoniazid; 0.14–1.5 for rifapentine; 0.29–1.4 for ethionamide; 0.41–2.2 for prothionamide; and 0.12–6.3 for clofazimine. The highest [I]max/Ki values were 2.0 for isoniazid on CYP3A4 [testosterone (T)]; 1.5 for rifapentine on CYP3A4 [midazolam (M)]; 1.4 for ethionamide on CYP2C8; 2.2, 1.8, and 1.3 for prothionamide on CYP2B6, CYP2C19, and CYP2C8, respectively; and 6.3 and 5.7 for clofazimine on CYP3A4 (M) and CYP3A4 (T), respectively. These drugs with high [I]max/Ki values lead to clinical DDIs. Considering the drug regimens for tuberculosis (TB) and co-infection with TB and human immunodeficiency virus, the inhibitory potential for CYP3A4 and CYP2B6 is particularly important. These results suggest that clofazimine and prothionamide are likely to cause clinically relevant DDIs when co-administered with products metabolized by CYP3A4 and CYP2B6, respectively. Isoniazid and rifapentine may cause DDIs with drugs metabolized by CYP3A4.
A 56-year-old woman with systemic lupus erythematosus had bacteremia due to multidrug-resistant Pseudomonas aeruginosa (MDRP). She was initially treated with imipenem–cilastatin, tobramycin, and aztreonam; however, MDRP was still detected intermittently in her plasma. Multidrug-susceptibility tests demonstrated that MDRP was susceptible only to colistin. Therefore, in addition to these antibiotics, the administration of intravenous colistin methanesulfonate, a prodrug formula of colistin, was started at a daily dose of 2.5 mg/kg (as colistin base activity). The initial dose setting was based on the patient’s renal function (baseline creatinine clearance=32.7 mL/min). After initiating colistin, the patient’s C-reactive protein levels gradually decreased. Blood cultures showed no evidence of MDRP on days 8, 14, and 22 after colistin initiation. However, the patient’s renal function went from bad to worse owing to septic shock induced by methicillin-resistant Staphylococcus aureus (MRSA) infection. A few days later, the trough plasma levels of colistin were 7.88 mg/L, which appeared to be higher than expected. After decreasing the colistin dose, the patient’s renal function gradually improved. On the final day of colistin treatment, the plasma levels decreased to 0.60 mg/L. MDRP could not be detected in blood culture after colistin treatment. Therefore, we successfully treated a case of bloodstream infection due to MDRP by therapeutic drug monitoring (TDM) of colistin. It is suggested that the monitoring of blood colistin levels by liquid chromatography-tandem mass spectrometry can contribute to safer, more effective antimicrobial therapy of MDRP because TDM facilitates quick decisions on dose adjustments.
Nitric oxide (NO) is a gaseous regulatory factor produced by NO synthases (NOS) and it plays several critical roles viaS-nitrosylation of protein cysteine residues. Histone deacetylase (HDAC) functions in the maintenance/balance of chromatin acetylation and contributes to transcriptional supression. It has been reported that S-nitrosylation of HDAC2 is involved in the regulation of deacetylase activity. However, it remains unknown whether other subtypes of the HDAC family are S-nitrosylated. In the present study, we found that HDAC6 is a target of NO. A biotin-switch assay revealed that endogenous HDAC6 is S-nitrosylated by both NO donors and NO derived from the inducible type of NOS in cells treated with cytokines. NO led to suppressed deacetylase activity in vitro and increased acetylated α-tubulin, a major substrate for HDAC6, in A549 cells. These findings suggest that S-nitrosylation of HDAC6 plays a pivotal role in the regulation of protein acetylation.