Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is the principal source of reducing power in numerous processes of physiological importance. We examined the influence of oxidative stress on the relative amounts of NADPH in human red blood cells (RBCs). To determine the homeostasis of the NADPH existing in the reduced form following oxidation, we developed an improved method for measurement of NADPH in human RBCs using high-performance liquid chromatography (HPLC). The present method with fluorescent detection is reproducible and selective than enzymatic cycling method and HPLC methods with spectrometric detection. The calibration curve is linear over the range of 0.1—5.0 μM with a correlation coefficient of 0.999. The within-run precision of the assays for total NADPH (NADPH+NADP+) and NADPH concentrations in human RBC were 2.4% and 8.6%, respectively (n=5). After the RBC suspension was exposed to tert-butyl hydroperoxide (t-BHP), which is scavenged by glutathione peroxidase (GPX) along with NADPH consumption, a significant decrease in the NADPH ratio [(NADPH/(NADPH+NADP+)] was observed after a transient decrease and rapid recovery of the reduced form of glutathione. The marked decrease in the NADPH ratio induced by t-BHP exposure was observed in the absence of glucose. However, the NADPH ratio was not decreased by t-BHP exposure after pre-treatment with a glutathione reductase inhibitor. This method may be useful for the measurement of small amounts of NADPH from various biological sources.
To screen for unauthorized genetically modified organisms (GMO) in the various crops, we developed a multiplex real-time polymerase chain reaction high-resolution melting-curve analysis method for the simultaneous qualitative detection of 35S promoter sequence of cauliflower mosaic virus (35SP) and the nopaline synthase terminator (NOST) in several crops. We selected suitable primer sets for the simultaneous detection of 35SP and NOST and designed the primer set for the detection of spiked ColE1 plasmid to evaluate the validity of the polymerase chain reaction (PCR) analyses. In addition, we optimized the multiplex PCR conditions using the designed primer sets and EvaGreen® as an intercalating dye. The contamination of unauthorized GMO with single copy similar to NK603 maize can be detected as low as 0.1% in a maize sample. Furthermore, we showed that the present method would be applicable in identifying GMO in various crops and foods like authorized GM soybean, authorized GM potato, the biscuit which is contaminated with GM soybeans and the rice which is contaminated with unauthorized GM rice. We consider this method to be a simple and reliable assay for screening for unauthorized GMO in crops and the processing food products.
The rare ginsenosides compound K, compound Y, and compound Mc were produced from the major ginsenosides Rb1, Rb2, Rc, and Rd by a thermostable β-glycosidase from Sulfolobus acidocaldarius via three pathways: Rb1→Rd→compound K, Rb2→compound Y→compound K, and Rc→compound Mc. Each of the ginsenosides was identified by high-performance liquid chromatography using standards and liquid chromatography-mass spectrometry based on their molecular weights. The catalytic efficiency of the enzyme for ginsenosides followed the order Rb1 (4.8)>Rc (4.5)>Rd (1.0)>Rb2 (0.77 mM−1 min−1). The enzyme converted 1 mg/ml reagent-grade Rb1, Rb2, and Rc to 0.53 mg/ml compound K, 0.56 mg/ml compound Y, and 0.70 mg/ml compound Mc, respectively, at pH 5.5 and 85 °C after 180 min, corresponding to mole conversion yields of 94, 80, and 100% (mol/mol), respectively. The enzyme converted the major ginsenosides Rb1, Rb2, Rc, and Rd in 10% (w/v) ginseng root extract to the rare ginsenosides with a mole yield of 99% after 24 h. These results suggest that β-glycosidase from S. acidocaldarius can be used to produce compound K, compound Y, and compound Mc.
We examined the effect of carnosine (β-alanyl-histidine) on nitric oxide (NO) production and endothelial NO synthase (eNOS) activation in endothelial F-2 cells. Carnosine enhanced NO production in a dose-dependent manner, and the stimulatory effect of carnosine was observed at concentrations exceeding 5 mM. The carnosine-stimulated NO production was inhibited by NG-nitro-L-arginine methyl ester, but not by NG-nitro-D-arginine methyl ester. In contrast, β-alanine, histidine (carnosine components) and anserine (N-methyl carnosine) failed to increase NO production. Carnosine had no effect on NO production for the initial 5 min, but thereafter resulted in a gradual increase in NO production up to 15 min. Carnosine did not induce phosphorylation of eNOS at Ser1177. The carnosine-induced increase in NO production was observed even when extracellular Ca2+ was depleted by ethylene glycol bis(2-aminoethyl ether)-N,N,N′-N′-tetraacetic acid however, the effect was abolished upon depletion of intracellular Ca2+ by BAPTA. After F-2 cells were incubated with carnosine for 4 min, intracellular Ca2+ concentration gradually increased. The carnosine-induced increase in intracellular Ca2+ concentration occurred even in the absence of extracellular Ca2+. These results indicate that carnosine facilitates NO production in endothelial F-2 cells. It is also suggested that eNOS is activated by Ca2+, which might be released from intracellular Ca2+ stores in response to carnosine.
We examined the rapid effect of resveratrol on nitric oxide (NO) production in endothelial F-2 cells. During an incubation period of 10 min, resveratrol at low concentrations (<20 μM) had no effect on NO production, whereas it significantly increased NO production at high concentrations (>50 μM). In contrast, pretreatment with resveratrol at low concentrations caused a significant decrease in vascular endothelial growth factor (VEGF)-stimulated NO production. Resveratrol failed to induce phosphorylation of endothelial NO synthase (eNOS) and VEGF receptor and did not affect autophosphorylation of the VEGF receptor. However, resveratrol markedly suppressed VEGF-induced eNOS phosphorylation. Resveratrol at high concentrations reduced the viability of F-2 cells as determined by 3-(4,5-dimethyl-2-thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and dye exclusion methods. Resveratrol-stimulated NO production was completely abolished by the depletion of extracellular Ca2+. These results indicate that resveratrol stimulates NO production by a Ca2+-dependent mechanism and reduces VEGF-stimulated NO production by impairing a Ca2+-independent mechanism in endothelial F-2 cells. However, the stimulatory effect of resveratrol may be partly attributed to its cytotoxicity.
Prostaglandin E2 (PGE2) stimulates osteoclast formation by increasing receptor activator of nuclear factor (NF)-κB ligand (RANKL) mRNA expression via cAMP-protein kinase A (PKA) pathways in osteoblasts. Since phosphodiesterases (PDEs) balance the concentration of intracellular cAMP stimulated by PGE2, we investigated the role of PDEs in PGE2-mediated osteoclast formation using various cAMP-specific PDEs inhibitors. In the presence of PGE2, PDE3 and 4 inhibitors were shown to dose-dependently increase the osteoclast formation in cocultures of mouse bone marrow cells and calvarial osteoblasts. In agreement with this finding, they stimulated PGE2-induced cAMP production followed by increased RANKL mRNA expression in osteoblasts, suggesting that PDE3 and 4 negatively regulate PGE2-mediated RANKL expression in osteoblasts. RT-PCR analysis revealed that PDE3A, 3B, 4A, 4B and 4D are expressed in osteoblasts. The PDE8 inhibitor did not increase osteoclast formation, although it stimulated PGE2-induced RANKL mRNA expression in osteoblasts. The four subtypes of PGE receptors are designated EP1, EP2, EP3, and EP4. PDE3 and 4 inhibitors were found to increase EP1/3, EP4 and/or EP2 agonist-stimulated RANKL expression, indicating that PDE3 and PDE4 negatively regulate PGE2-induced RANKL mRNA expression through four EPs. Taken together, these data suggest that PDE3 and PDE4 could have important pharmacological and clinical implications in bone-related diseases.
Recombinant human granulocyte colony-stimulating factor (rhG-CSF) has been used for treatment of neutropenia. Filgrastim, Nartograstim, and Lenograstim are clinically available in Japan. However, the differences in potential benefit for radiation-induced disorder between these types of rhG-CSFs remain unknown. Therefore, the effects of three different types of rhG-CSFs on granulocyte progenitor cells and expansion of neutrophils from nonirradiated or 2 Gy X-irradiated human CD34+ hematopoietic progenitor cells were examined. For analysis of granulocyte colony-forming units (CFU-G) and a surviving fraction of CFU-G, nonirradiated or X-irradiated CD34+ cells were cultured in methylcellulose containing rhG-CSF. These cells were cultured in serum-free medium supplemented with rhG-CSF, and the expansion and characteristics of neutrophils were analyzed. All three types of rhG-CSFs increased the number of CFU-G in a dose-dependent manner; however, Lenograstim is superior to others because of CFU-G-derived colony formation at relatively low doses. The surviving fraction of CFU-G was independent of the types of rhG-CSFs. Expansion of neutrophils by rhG-CSF was largely attenuated by X-irradiation, though no significant difference in neutrophil number was observed between the three types of rhG-CSFs under both nonirradiation and X-irradiation conditions. In terms of functional characteristics of neutrophils, Lenograstim-induced neutrophils produced high levels of reactive oxygen species compared to Filgrastim, when rhG-CSF was applied to nonirradiated CD34+ cells. In conclusion, different types of rhG-CSFs lead to different effects when rhG-CSF is applied to nonirradiated CD34+ cells, though Filgrastim, Nartograstim, and Lenograstim show equal effects on X-irradiated CD34+ cells.
Control of cell cycle progression in somatic cells or terminally differentiated cells is a key technology for cell-based therapies such as regenerative therapy. We have prepared an artificial cell cycle progression peptide composed of a human immunodeficiency virus-derived Tat protein transduction domain (PTD) and a p53 genetic suppressor element (GSE123). The peptide significantly promoted hepatocyte growth factor-induced DNA synthesis and the proliferation of primary mouse hepatocytes, which are highly differentiated somatic cells. The addition of a nuclear localization signal (NLS) sequence to the peptide increased the internalization of the peptide to the nuclear fraction. Distribution analysis using a fluoresein isothiocyanate-labeled peptide indicated that the NLS enabled the peptide to escape from the lysosomes to the cytosol. As a result, the NLS-Tat-GSE123 peptide induced potent cell proliferation of primary mouse hepatocytes in vitro. The use of this peptide as an artificial cell growth enhancer, bypassing a specific receptor, is a useful tool for the study of regenerative therapy and cell signaling.
We detected the β-1,2-mannosyltransferases (β-1,2-MTs), which participate in the biosynthesis of oligomannosyl side chains in the mannan acid-labile fraction, in a particulate insoluble fractions prepared from Candida albicans NIH B-792 strain cells grown at 27 °C and at 37 °C in a yeast extract-added Sabouraud liquid medium (YSLM). The β-1,2-MT VI-6 prepared from the cells grown at 27 °C exhibited the maximum activity at pH 7.0 and at 30 °C. The β-1,2-MT VI-6 activity was only slightly affected by Mn2+, Mg2+, Ca2+, and ethylenediaminetetraacetic acid, but completely inhibited by Zn2+ and Ni2+. The β-1,2-MT activities from the cells grown at 37 °C were lower than that from the cells grown at 27 °C, especially on the longer β-1,2-mannooligosaccharides than tetraose.
We evaluated the cardioprotection against myocardial ischemia-reperfusion injury induced by sevoflurane postconditioning (SpostC) in chronically-infarcted rat hearts, and investigated the roles of phosphoinositide 3-kinase (PI3K)-protein kinase B/Akt (PKB/Akt), mitogen-activated extracellular regulated kinase 1/2 (MEK 1/2)-extracellular regulated kinase 1/2 (ERK 1/2), and mitochondrial permeability transition pore (mPTP). Left anterior descending (LAD) coronary artery was ligated to induce myocardial infarction in rats. Six weeks later, chronically-infarcted hearts were isolated and subjected to 30 min of global ischemia, followed by 1 h of reperfusion with Krebs-Henseleit (K-H) buffer. SpostC was administered by perfusing the hearts with K-H buffer saturated with 3% sevoflurane during the first 15 min of reperfusion. To evaluate the role of PI3K-PKB/Akt and MEK 1/2-ERK 1/2 in SpostC, PI3K inhibitor LY294002 (15 μM) and MEK 1/2 inhibitor PD98059 (20 μM) were administered alone or together with sevoflurane during the first 15 min of reperfusion. We found that exposure of 3% sevoflurane during early reperfusion significantly improved functional recovery (improved left ventricular developed pressure (LVDP), ±dp/dt, CF, HR and reduced left ventricular end-diastolic pressure (LVEDP)), decreased myocardial infarct size and reduced LDH and CK-MB release, when compared with unprotected hearts. However, these protective effects were abolished in the presence of either LY294002 or PD98059, which was accompanied by the prevention of PKB/Akt and ERK 1/2 phosphorylation, and reduction of myocardial nicotinamide adenine dinucleotide (NAD+) content. These findings suggest that sevoflurane postconditioning protects chronically-infarcted rat hearts against ischemia-reperfusion injury by inhibiting mPTP opening via recruitment of PKB/Akt and ERK 1/2.
The present study was undertaken to investigate the characteristics of morphine in rat sleep patterns and also the effects of donepezil and memantine on somnolence caused by morphine. Electrodes were chronically implanted into the cortex and dorsal neck muscle of rats for electroencephalogram (EEG) and electromyogram (EMG) recordings, respectively. EEG and EMG were recorded with an electroencephalograph. SleepSigh ver.2.0 was used to analyse the sleep-wake state. Total times of wakefulness, non-rapid eye movement (NREM) sleep and rapid eye movement (REM) sleep were measured from 10:00 to 16:00. Morphine at a high dose caused a significant decrease in sleep latency and total REM sleep time, although the drug at low doses caused significant increases in sleep latency and total awake time, and a significant decrease in NREM sleep time. Donepezil, memantine and methylphenidate antagonized the decrease in sleep latency caused by morphine. From these findings, it can be concluded that morphine caused somnolence, and donepezil and memantine are useful for somnolence caused by morphine, similar to methylphenidate.
Parkinson's disease (PD) is characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra (SN) with the presence of α-synuclein inclusions termed Lewy bodies. The aggregation of α-synuclein into oligomeric species affects neuronal viability, having a causal role in the development of PD. The neuroprotective effects of protocatechuic acid (PAc) have been reported. However, the effects of PAc on tyrosine hydroxylase (TH) and α-synuclein in rat pheochromocytoma (PC12) cells treated with 1-methyl-4-phenylpyridinium ion (MPP+) remains unclear. In this study, we demonstrated that PAc inhibited the cytotoxicity, apoptotic morphology, reduction of TH expression and abnormal oligomeration of α-synuclein in PC12 cells treated with MPP+. Taken together, our results indicate that the neuroprotective effects of PAc on PC12 cells treated with MPP+ is related to the inhibition of the oligomerization of α-synuclein.
Ibudilast (3-isobutyryl-2-isopropylpyrazolo[1,5-α]pyridine) is clinically used as a cerebral vasodilator in Japan. However, the effects of ibudilast on retinal blood vessels have not been fully examined. The aim of this study, therefore, was to examine the effects of ibudilast on retinal blood vessels in rats in vivo. Male Wistar rats (8 to 10 weeks old) were anesthetized with thiobutabarbital (120 mg/kg, intraperitoneally (i.p.)). Retinal vascular images were captured with a fundus camera system for small animals, and the diameter of retinal blood vessels was measured. Ibudilast (0.1 and 1 mg/kg, intravenously (i.v.)) elicited a sustained increase in the diameter of retinal blood vessels and heart rate without altering systemic blood pressure. The effects of ibudilast were significantly reduced by treatment with the nonselective cyclooxygenase inhibitor indomethacin (5 mg/kg, i.p.). These results suggest that ibudilast dilates retinal blood vessels through cyclooxygenase-dependent mechanisms in rats in vivo.
Involvement of Na+/Ca2+ exchanger (NCX) in pentylenetetrazol (PTZ)-induced convulsion by use of NCX knockout mice and the selective ligand SEA0400 to NCX was examined. In the SEA0400-administered group, the latency to clonic convulsion was extended into 210 s, although the latency to clonic convulsion was observed until 100 s in control group. SEA0400 had little effect on bicuculline-induced clonic seizure nicotine-induced wild running and 4-aminopyridine-induced tonic flexion, respectively. Tonic flexion convulsion was occurred three fifth in the wild type mice group by administration of PTZ, but tonic flexion was not observed in NCX1 knockout mice groups. These results suggest that NCX is involved in inhibitory action in PTZ-induced convulsion.
Inflammatory cytokine interleukin-8 (IL-8) and reactive oxygen species (ROS) overexpressed in the gastric mucosa when exposed to Helicobacter pylori, defined as a class I carcinogen. Moreover, infection with H. pylori leads to morphological changes in co-cultured cells known as hummingbird phenomenon along with increased motility. Resveratrol, a highly abundant polyphenol in red grapes, has shown anti-inflammatory, anti-cancer, cardioprotective and neuroprotective activities. However, the effect of resveratrol in H. pylori-infected cells has not been investigated. The present study was, therefore, aimed to evaluate the effect of resveratrol on the induction of IL-8, ROS and hummingbird morphology in H. pylori-infected gastric epithelial cells. The non-toxic concentration of resveratrol for both H. pylori and epithelial cells was determined by brucella broth dilution method and DNA fragmentation assay. The non-toxic resveratrol (≤100 μM) treatment did not demonstrate any inhibitory effect against H. pylori adhesion to gastric epithelial cells. However, preincubation of the cells with 75 and 100 μM of resveratrol significantly (p<0.05 and p<0.01 respectively) inhibited the secretion of IL-8 from H. pylori-infected cells. In addition, resveratrol pretreatment at 1—100 μM suppressed H. pylori-induced ROS generation in a concentration dependent manner. Moreover, H. pylori-initiated morphological changes were markedly blocked by resveratrol. Hence, resveratrol can be considered as a potential candidate against various H. pylori related gastric pathogenic processes.
In order to efficiently develop improved cancer therapies it is important to predict the efficacy of anti-cancer drugs. In this regard, identification of genes that are related to drug sensitivity is vital. We previously established a panel of 39 human cancer cell lines (JFCR39) and a panel aiming for organ-specific analysis of 45 human cancer cell lines (JFCR45). Here, we focus on 20 human gastric cancer cell lines from JFCR45, a panel of human cancer cell lines to predict genes that determine chemosensitivity to anti-cancer drugs. We measured both chemosensitivity to a range of anti-cancer drugs as well as changes in gene expression profile. We then identified genes in which expression is related to chemosensitivity by using a Pearson correlation. As a result, anti-cancer drugs that have similar mechanisms of action showed similar fingerprints against a gastric subpanel of human cancer cell lines, as was the case with JFCR39 and JFCR45. Furthermore, we identified many candidate genes related to the sensitivity of gastric cancer cells to anti-cancer drugs.
Fraxinellone and sauchinone, isolated from natural substance, are known to have an anti-inflammatory effect in inflammatory conditions. However, the anti-inflammatory actions of these compounds have been insufficiently demonstrated in viral-induced neuroinflammation. A viral component (double-stranded (ds)RNA) triggers a toll-like receptor 3-dependent inflammatory response that stimulates pro-inflammatory mediators in the brain. In present study, we initially examined the biological effects of fraxinellone and sauchinone on anti-inflammatory actions in dsRNA-stimulated microglia. Both compounds inhibited dsRNA-induced inducible nitric oxide synthase (iNOS) expression, a major pro-inflammatory enzyme. To demonstrate the mechanism of inhibitory effect on iNOS expression, we further examined the signaling pathway induced by dsRNA in microglia. Our data show that dsRNA promotes the expression of signal transducers and activators of transcription (STAT)1/3 identified as major inflammatory transcription factors as well as activates c-Jun N-terminal kinase (JNK) in an early time. Moreover, both compounds suppressed activation of JNK-STAT1/3 signaling pathway. These results suggest that an anti-inflammatory effect by fraxinellone and sauchinone is mediated via blockade of the JNK-STAT1/3-iNOS signaling pathway in viral-infected microglia.
Interleukin (IL)-4 has been suggested as a molecular therapeutic target to prevent and/or treat various allergic diseases and several flavonoids have been suggested as anti-allergic agents suppressing IL-4 production. In an effort to find novel candidates for anti-allergic agents from natural sources, we screened several flavonoids affecting on IL-4 production. In this study, we showed that 7,8,4′-trihydroxyflavone (T-412) significantly decreased IL-4 production both in phorbol 12-myristate 13-acetate (PMA) and ionomycin (PI)-activated EL-4 T cells and concanavalin A (ConA)-activated murine CD4+ T cells in a dose- and time-dependent manner. The PI-induced increase of IL-4 mRNA expression was dramatically suppressed by T-412 at 6 h, indicating the suppression is regulated at transcriptional level. T-412 also significantly inhibited IL-4 gene promoter activity in EL-4 T cells transiently transfected with luciferase reporter plasmid containing IL-4 promoter (pGL4.14-IL-4). Western blot analysis of the transcription factors revealed that T-412 suppressed the nuclear expression of nuclear factor of activated T cells (NF-AT)c1, c-Jun and c-Maf, but not c-Fos and nuclear factor kappa B (NF-κB). Our data suggested that T-412 might have potential as a candidate for anti-allergic agent having suppressive effects on IL-4 production in activated T cells by controlling the transcription of IL-4.
Disks of probucol and solid dispersion systems of probucol–polyvinylpyrrolidone (PVP) in various weight ratios were prepared. Dissolution of probucol was markedly increased in the solid dispersion systems in J.P. XV disintegration media No. 1 (pH 1.2) and No. 2 (pH 6.8). The concentrations of probucol after the dissolution of the disks of solid dispersion systems showed supersaturation. Following the administration of disks of solid dispersion systems in rabbits, a marked increase in the area under the plasma concentration time curve (AUC) was observed. When the weight ratio of PVP to probucol was larger, a larger AUC was observed. When disks of the 1 : 9 solid dispersion system (weight ratio of probucol : PVP=1 : 9) containing 50 and 100 mg probucol were respectively administered, AUC values were approximately proportional to the dose. AUC values following the administration of disks of the 1 : 9 solid dispersion systems containing 15 mg probucol (total weight: 150 mg) and 500 mg probucol were approximately equal. The mean half life (t1/2) was 12 h when disks of the 1 : 9 solid dispersion system were administered, whereas the t1/2 was 35 h when probucol disks were administered. The markedly increased dissolution of probucol in solid dispersion systems resulted in a marked increase in its bioavailability.
The purpose of this work is to clarify the relationship of mean particle size to the dissolution rate of polydispersed particles for Biopharmaceutical Classification System (BCS) Class II drugs. An equation for the initial dissolution rate of polydispersed particles relative to mean particle diameter was theoretically derived from the Noyes–Whitney equation assuming spherical particles and sink conditions. To verify the relationship of dissolution to the mean particle diameters, the dissolution rates of 6 types of hypothetically-generated log-normal polydispersed particles and 3 different sized particles of aprepitant, a designated BCS class II drug, were compared with known mean diameters calculated according to surface area-weighted mean diameter (D3,2, commonly referred to as the Sauter mean diameter), length-weighted mean diameter (D3,1) and number-weighted mean diameter (D3,0). The results confirmed that the initial dissolution rates of polydispersed particles reflect the mean diameter and correlated best with the reciprocal of D3,2 at the start of dissolution, in accord with our theoretical conclusions. The particle size required for sufficient dissolution of aprepitant was also investigated by examining the relationship between D3,2 and oral absorption predicted using a physiological-based model.
Plant-derived estrogen-like compounds, or phytoestrogens, are given much attention due to their potential therapeutic use. In this study, xanthorrhizol, a natural sesquiterpenoid isolated from the rhizome of Curcuma xanthorrhiza ROXB. (Zingiberaceae), was evaluated for its estrogenic activity. It has been known that compounds acting as ligands for estrogen receptors (ERs) are considered to possess estrogenic activity. Therefore, the Gal-4/ER transactivation assay in transiently transfected African green monkey kidney (COS-7) cells was used to examine the estrogenic activity of xanthorrhizol. Both subtypes of ERs, ERα and ERβ, were involved in this assay. Further transactivation assays and pS2 mRNA analysis were also conducted in estrogen receptor-positive human breast cancer (MCF-7). Our results showed that xanthorrhizol significantly increased Gal-4/ER luciferase activity in a dose-dependent manner and induced the endogenous ER-estrogen response element (ERE) interaction in MCF-7 cells. Xanthorrhizol also significantly enhanced the expression of the pS2 gene in MCF-7 cells. In contrast, treatment using ICI 182780, an ER antagonist, suppressed all activities induced by xanthorrhizol, indicating ER-dependant activities were involved. These results suggest that xanthorrhizol possesses estrogenic activity and its estrogenic effects are mediated by estrogen-induced gene expression.
The effect of Wellsolve, a new solubilizing agent, on the function of intestinal membrane barrier and transporters including P-glycoprotein (P-gp) and peptide transporter (PEPT1) was examined by an in vitro diffusion chamber and an in situ closed loop method. The model drugs used in this study were 5(6)-carboxyfluorescein (CF), rhodamine123 (a P-glycoprotein substrate), cephalexin (a typical substrate for PEPT1) and griseofulvin (a BCS Class II drug). Intestinal absorption of CF was not affected by the addition of 1—10% (v/v) Wellsolve, while 20% (v/v) Wellsolve significantly enhanced its intestinal absorption by the in situ absorption study. Therefore, this finding suggested that high concentration of Wellsolve might alter the intestinal barrier function. The mucosal to serosal (absorptive) and serosal to mucosal (secretory) transport of rhodamine123 was significantly inhibited in the presence of 5.0—20% (v/v) of Wellsolve, suggesting that Wellsolve might not affect the function of P-gp in the intestine. The intestinal transport of cephalexin was not affected in the presence of Wellsolve, suggesting that this solubilizing agent might not change the function of PEPT1 in the intestine. In the toxicity studies, we found that 1—10% (v/v) Wellsolve did not change the release of lactate hydrogenase (LDH) and protein from the intestinal membranes. Furthermore, intestinal absorption of griseofulvin in the presence of 10% (v/v) Wellsolve significantly increased as compared with the control. In summary, Wellsolve at lower concentrations might be a potent and safe solubilizing agent for improving the solubility and absorption of poorly water-soluble drugs including griseofulvin.
Normal female rat distribution studies showed high and specific uptake of 6-deoxy-6-[131I]iodo-L-ascorbic acid (6-131IAsA) into the adrenal glands, known to highly express the ascorbate sodium-dependent vitamin C transporter-2 (SVCT-2), and the adrenal gland was clearly visualized by whole-body autoradiography. Preinjection of sulfinpyrazone, a known blocker of ascorbate transport, with 6-131IAsA resulted in decreased uptake of radioactivity in rat adrenal glands compared to the control group, seemingly illustrating the participation of the SVCT transporter (probably the SVCT-2 subtype) in the uptake process in vivo. 4-Aminopyrazolo[3,4-d]pyrimidine-induced hypolipidemic rats showed a 1.7-fold increase in adrenal uptake of radioactivity at 30 min postinjection of 6-131IAsA, compared to the control, with increased adrenal-to-liver and adrenal-to-kidney ratios. To further characterize 6-131IAsA for its tumor uptake properties, biodistribution studies were also performed using male nude mice implanted with either Y-1 adrenocortical tumor cells or adrenal medulla-derived PC12 cells. None of these tumors exhibited relevant uptake of 6-131IAsA while normal adrenal glands showed high uptake of radioactivity, suggesting that these tumors in this model have only a poor transport capacity for this agent. The present study demonstrates that the use of radioiodinated 6-IAsA may help to obtain information about functional alterations in diseased adrenal glands, but it does not exhibit desirable properties as a tumor-seeking agent for ascorbic acid bioactivity.
The present study describes the molecular authentication of 21 Korean Artemisia species using PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) technique based on the trnL–F sequences in chloroplast DNA. Five different banding patterns were generated from 21 Artemisia species using HinfI restriction enzyme. A. apiacea, A. keiskeana and A. sieversiana have specific banding patterns. The remaining 18 species had shared two banding patterns. Phylogenetic analysis based on trnL–F sequence variations showed results similar to PCR-RFLP banding patterns. It suggested that the trnL–F region does not have sufficient variations to identify the 21 Artemisia species. However, the specific banding patterns for A. apiacea, A. keiskeana and A. sieversiana can be utilized as a DNA marker for discriminating them from other Artemisia species. These markers will be also useful for developing A. apiacea, A. keiskeana and A. sieversiana into new medicine and food based on their efficacy.
The effects of intracellular ribavirin on morphology and redistribution of phosphatidylserine (PS) in human erythrocytes were examined. Erythrocytes were incubated with 1 mM ribavirin in the presence/absence of dipyridamole, an inhibitor of es-type nucleoside transporter. Intracellular ribavirin was accumulated in erythrocytes with the concentration of 1361 μM, which corresponds to the blood level in patients receiving ribavirin. Dipyridamole reduced ribavirin accumulation by 40.7% (807 μM) via inhibiting es-type nucleoside transporter on erythrocytes. Morphological transformation into echinocytic form was observed in 86.4% of the erythrocytes treated with ribavirin. Dipyridamole pre-treatment decreased the morphological change to 20.0%. Ribavirin increased the PS-exposing cells compared with control (2.15% vs. 0.87%). PS-exposing cells were also decreased by inhibiting ribavirin accumulation with dipyridamole (0.62%). The results suggest that intracellular ribavirin induces morphological change and PS exposure in erythrocytes and accelerates erythrophagocytosis in the reticuloendothelial system.
Retinoic acid (RA) has a variety of biological effects in mammalian cells and tissues. It is well known that RA is a potent anticancer agent that induces differentiation of leukemia cells as well as inhibiting cell growth. The current study examined HL60 proteins using anti-RA monoclonal antibodies (ARMAs) and found that some RA-binding proteins may be histones. These proteins eluted in the void volume fractions following Mono Q anion exchange chromatography and immunostained with ARMAs. These ARMAs showed specific binding to the proteins in a saturable manner that depended on antibody concentration. Certain of these proteins co-migrated with histones on one-dimensional polyacrylamide gel electrophoresis. It was also found that histones isolated from HL60 cells treated with RA also immunostained with ARMAs. These results indicate that basic proteins, including histones, may be bound to RA covalently in HL60 cells and that RA-binding histones may play significant roles in the transcriptional regulation of genes by RA.