Recombinant technology allows engineering and production of proteins with desirable properties. Human serum albumin has been developed with recombinant technology, and thus plays an increasing role as a drug carrier in the clinical setting. Genetic variations usually occur on the surface of the protein, and do not impose significant effects on the conformation of albumin. However, binding of fatty acids by genetic variants is affected according to the location of the mutation. Albumin undergoes three major posttranslational modifications, namely, oxidation, glycation, and S-nitrosylation. This review gives an account of the different posttranslational modifications that should be taken into consideration when designing albumin mutant analogues with desirable pharmaceutical properties.
In vivo redox reactions of nitroxyl contrast agents in bile and blood under an oxidative atmosphere were investigated using normal healthy Wistar rats. Differences in intracellular and extracellular volumes in redox environments are discussed. Pharmacokinetic profiles of two nitroxyl contrast agents, 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl (carbamoyl-PROXYL), 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL), in bile and blood were monitored by an electron paramagnetic resonance spectrometer when the rat was breathing 100% O2 or was subcutaneously administrated 0.2 mmol/kg body weight of ferric citrate. Re-oxidation of hydroxylamines to nitroxyl radicals was caused in bile under 100% O2 breathing, but not in blood. The administration of ferric citrate caused marked re-oxidation in bile, but a slight reduction in blood. Tissue H2O2 level may partly play a role in the intracellular re-oxidation process. Tissue Fe3+ concentration can work more effectively for the intracellular re-oxidation of hydroxylamines. The intracellular environment is susceptible to oxidation compared with the extracellular environment under conditions such as 100% O2 breathing or iron overload.
Quantitative detection of free radical reactions induced by low linear-energy-transfer (LET) irradiation in an aqueous solution was attempted using nitroxyl radicals. The stability and reactivity of reaction mixtures containing a nitroxyl radical and a hydrogen donor, i.e., glutathione (GSH), nicotinamide adenine dinucleotide (NADH), or nicotinamide adenine dinucleotide phosphate (NADPH), were tested. X-band electron paramagnetic resonance (EPR) measurements of several reaction mixtures were performed to find a suitable preparation to quantitatively detect free radical reactions produced by low LET irradiation. The EPR signal intensity of nitroxyl radicals was decreased by low LET irradiation when a hydrogen donor coexisted in the reaction mixture. The combination of 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (4-hydroxy-TEMPO, known as TEMPOL) and GSH was most preferable among other preparations tested in this paper, because of the sensitivity and irreversible reaction. The extent of the free radical reaction, i.e., formation of reactive oxygen species, in the reaction mixture depended on the radiation energy when an identical dose was given.
While studying the mechanism of ginsenoside Rg3 (G-Rg3) on tumor inhibition, we produced monoclonal antibody to G-Rg3 for more specific investigation. We immunized Balb/c mice to G-Rg3 conjugated bovine serum albumin (BSA) by intraperitoneal injection and hybridized splenocytes from those immunized mice and myeloma cells. From those fusion cell lines, we selected productive monoclonal clones and obtained culture media containing monoclonal antibody to G-Rg3. After purification, we performed enzyme-linked immunosorbent assay (ELISA) to verify the sensitivity and specificity of the antibody. When compared with G-Rh2 having a very similar structure as a metabolite of G-Rg3, the antibody worked only with G-Rg3 in a concentration-dependent manner. We confirmed that the monoclonal antibody to G-Rg3 can be applied to immunocytochemistry for detection of the treated G-Rg3 inside A549 human lung adenocarcinomas. Thus, the monoclonal antibody to G-Rg3 would be a useful tool for measuring the bioactivity of G-Rg3 in various fields.
The availability and applicability of the combination of paramagnetic nitroxyl contrast agent and T1-weighted gradient echo (GE)-based dynamic magnetic resonance imaging (MRI) measurement for redox imaging are described. The time courses of T1-weighted GE MRI signal intensities according to first-order paramagnetic loss of a nitroxyl contrast agent were simulated for several experimental conditions. The apparent decay rate calculated based on decreasing T1-weighted MRI contrast (kMRI) can show an approximate value of the original decay rate (ktrue) discretionarily given for simulation with suitable experimental parameters. The difference between kMRI and ktrue can be sufficiently small under T1-weighted spoiled gradient echo (SPGR) scan conditions (repetition time=75 ms, echo time=3 ms, and flip angle=45°), with a conventional redox-sensitive nitroxyl contrast agent, such as 4-hydroxy-2,2,6,6,-tetramethylpiperidine-N-oxyl (TEMPOL) and/or 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl (carbamoyl-PROXYL), and with i.v. doses of below 1.5 μmol/g b.w. for mice. The results of this simulation suggest that the kMRI of nitroxyl contrast agents can be the primary index of redox status under biological conditions.
A simplified, rapid, selective HPLC method for determining five cytochrome P450 (CYP) probe drugs in single run is described. The five specific probe substrates of caffeine, chlorzoxazone, tolbutamide, metoprolol and midazolam, together with the internal standard diazepam, were extracted using liquid–liquid extraction in rat plasma, followed by high-performance liquid chromatography (HPLC) using a C18 column (5 μm particle size, 250×4.6 mm i.d.). The mobile phase consisted of a methanol and 50 mM phosphate buffer (pH 3.4, 65 : 35). All analytes were separated simultaneously in a single run that lasted less than 22 min. The detection limits range from 0.2—50 μg/ml for caffeine, 0.5—50 μg/ml for tolbutamide, metoprolol and midazolam, 0.2—100 μg/ml for chlorzoxazone, respectively. The intra- and inter-day precisions for five probe substrates were 1.38—11.10% and 3.39—11.33%, respectively, and the accuracy of five probe substrates ranged from 94.92—113.06% and 92.18—112.62%. The limit of quantification (LOQ) was 0.5 μg/ml for tolbutamide, midazolam and metoprolol, 0.2 μg/ml for caffeine and chlorzoxazone. The present method provides a robust, fast analytical tool for the five-probe drug cocktail. Finally, the method was suitable for determining the plasma concentration of these compounds and evaluating the CYP1A2, 2C9, 2D6, 2E1 and 3A4 activities in rats.
In this study, we evaluated the effect of 5-hydroxytryptophan on anti-inflammatory and analgesic activity in RAW 264.7 cells. Cells were treated with different concentrations of 5-hydroxytryptophan for either 1 h or for 24 h. The anti-inflammatory effect was then analyzed by enzyme-linked immunosorbent assay (ELISA), Western blotting and reverse transcription polymerase chain reaction (RT-PCR). In addition, the analgesic activity was evaluated by measuring the acetic acid-induced writhing response. We found that 5-hydroxytryptophan significantly reduced the acetic acid-induced writhing response. Moreover we evaluated the effects of 5-hydroxytryptophan on the release of several inflammatory mediators including nitric oxide (NO) and interleukin-6 (IL-6). Our results demonstrated that 5-hydroxytryptophan inhibited the lipopolysaccharide (LPS)-induced expression of NO and IL-6. Furthermore, we found that 5-hydroxytryptophan played a role in LPS induced inducible nitric oxide synthase (iNOS), cyclo oxygenase-2 (COX-2) and extracellular-signal regulated protein kinase (ERK) activation. Taken together, these results indicate that 5-hydroxytryptophan has the potential for use in the treatment of inflammatory disease and as an analgesic.
Two forms of a self-sufficient lanosterol 14-demethylase fused enzyme consisting of Saccharomyces cerevisiae CYP51 and S. cerevisiae reduced nicotinamide-adenine dinucleotide phospahte (NADPH)-P450 reductase were constructed and characterized. The two forms of fused enzymes, F1 and F2, which had slight differences in the linker regions between their P450 and reductase domains, were expressed in Escherichia coli cells. Both F1 and F2 were purified to homogeneity. The purified preparations of F1 and F2 showed spectral properties of not only P450 but also flavoprotein. F1 and F2 showed lanosterol 14-demethylase activity with kinetic parameters comparable to those obtained with a reconstituted system consisting of S. cerevisiae CYP51 and S. cerevisiae NADPH-P450 reductase. These facts indicate that F1 and F2 are self-sufficient lanosterol 14-demethylases that can catalyze three successive monooxygenations with comparable activity to naturally occurring CYP51. The enzymatic reduction of the CYP51 in F1 and F2 was faster than that of the CYP51 in the reconstituted system. The results of dilution experiments suggested that the electron transfer from the reductase domain to the CYP51 domain in F1 and F2 occurred both intra- and intermolecularly. Two fused self-sufficient lanosterol 14-demethylases were successfully constructed. This is the first example of the purified preparation of an artificial self-sufficient P450 monooxygenase that catalyzes the oxidative cleavage of C–C bond via three successive monooxygenations.
The mechanism of macrophage recognition of oxidatively damaged cells was investigated. Jurkat T cells exposed to various concentrations of H2O2 were bound and phagocytosed by macrophages. The cells exposed to 0.1 mM H2O2 were best bound. The cell-surface ligands recognized by macrophages were suggested to be sialylpolylactosaminyl sugar chains of a major sialoglycoprotein CD43 because 1) the cell binding was inhibited by oligosaccharides containing sialylpolylactosaminyl chains, and their inhibitory activity was destroyed by a polylactosamine-cleaving enzyme endo-β-galactosidase, and by neuraminidase; 2) the oxidized Jurkat cells pretreated with either glycosidase or with anti-CD43 antibody were not bound. The macrophage receptor involved in the binding was suggested to be cell-surface nucleolin because 1) anti-nucleolin antibody inhibited the binding; 2) nucleolin-transfected HEK293 cells bound the oxidized cells; and 3) this binding was inhibited by anti-nucleolin antibody and by anti-CD43 antibody. CD43 on oxidized Jurkat cells tended to form clusters in good accordance with their susceptibility to the macrophage binding. CD43 clustering and the oxidized-cell binding to macrophages were prevented by a caspase inhibitor Z-VAD-fmk, suggesting that the oxidized and bound cells were undergoing apoptosis. Indeed, caspase-3 activity of Jurkat cells increased by the oxidation. These results suggest that moderately oxidized cells undergo apoptosis and are recognized by macrophages as early apoptotic cells.
Glioblastoma is the most malignant and common brain tumor. To promote their growth, these glioma cells secrete a variety of soluble factors including plasminogen activator inhibitor-1 (PAI-1), which functions as an inhibitor of plasminogen activators. We report here with the basis of microarray gene expression analysis that CXCR4 expressing glioma cells are capable of expressing PAI-1 mRNA and protein upon CXCL12 stimulation. Pretreatment with U0126, an inhibitor of mitogen activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) 1/2, abrogated CXCL12-induced PAI-1 expression. Pertussis toxin (PTX), an inhibitor of Gαi proteins, also had inhibitory effects, indicating that the activation of Gαi and ERK MAPK are required for this response. Interestingly, CXCL12 showed additive effects with another PAI-1 inducers, tumor necrosis factor (TNF)-α and/or tumor growth factor (TGF)-β1, in increasing PAI-1 expression. These results indicate that CXCL12/CXCR4 signaling in glioma cells may be another mechanism for these cells to express PAI-1, which may be involved in angiogenesis and tumor invasion in brain tumors.
This study was designed to investigate whether hydroxytyrosol (HT) may ameliorate oxidative stress and nuclear factor kappaB (NF-κB) activation in the lipopolysaccharide (LPS)-stimulated THP-1 cell line. We measured the intracellular reactive oxygen species (ROS) formation using 2,7-dichlorofluorescein diacetate (DCFH-DA) as a fluorescent probe. Intracellular glutathione (GSH) level was estimated by fluorometric methods. Nitric oxide (NO) production was measured as nitrite (a stable metabolite of NO) concentrations using the Griess reagent system following Jiancheng Institute of Biotechnology protocols. To study the effect of HT on LPS-induced NF-κB activation in THP-1 cells, Western blot analysis of the nuclear fraction of cell lysates was performed. The results showed that treatment of THP-1 cells with HT significantly reduced LPS-stimulated NO production and ROS formation in a concentration-dependent manner. HT at 50 and 100 μM concentrations increased the GSH level. The specific DNA-binding activities of NF-κB on nuclear extracts from 50 and 100 μM HT treatments were significantly suppressed. The antioxidant N-acetylcysteine (NAC) also showed the same effects as HT on LPS-induced ROS and NO generation, change of GSH level, and NF-κB activation. These findings suggest that HT has antioxidant activity to suppress intracellular oxidative stress and NF-κB activation in THP-1 cells.
Cardiotoxin III (CTX III), a 60-amino acid basic polypeptide isolated from Naja venom, showed potential therapeutic activity toward cancer cells. Here we report that CTX III inhibited proliferation of human leukemia K562 cells by G2/M phase arresting and apoptosis which was associated with the activation of caspase-8 and cytochrome c release as well as the p38 and c-Jun N-terminal protein kinase (JNK) phosphorylation signaling pathway. We further demonstrated that daily administration of CTX III for 2 d to chicken chorioallantoic membrane (CAM) bearing tumours derived from the CAM at E10 administration of K562 cells resulted in inhibition of the tumours in vivo. Importantly, this in vivo inhibition was also associated with caspase-8 activation and cytochrome c release. Our results suggest that CTX III-induced apoptosis is mediated via the p38 and JNK pathway as well as the caspase-8-dependent Bid-Bax pathway in human K562 cells.
μ-Opioid receptor agonists, such as morphine, are widely applied in pain therapy clinical practice. However, the effects exerted by morphine via receptor are influenced by individual specificity. Single nucleotide polymorphisms (SNPs) in μ-opioid receptor gene (OPRM1) have been reported to influence receptor expression and function. Subsequently, we analyzed SNPs frequency and linkage disequilibrium associated with OPRM1 transcriptional region and 4 exons among healthy Japanese individuals. Consequently, we detected 10 SNPs (−1748G/A, −1565T/C, −1045A/G, −172G/T, −38C/A, 118A/G, ivs2+31 G/A, ivs2+691 C/G, ivs4+274 A/G, and ivs4+435 G/A). Moreover, linkage analysis revealed novel linkage between −1748G/A and −172G/T, which was not observed in studies performed in other nations. In contrast, SNPs frequency detected in this study was similar to previously reported results on Asians; however, linkage disequilibrium reports from different nations differed. These results possibly provide useful information for OPRM1 genotyping in the Japanese population.
DNA topoisomerase I (Top1) is a ubiquitous nuclear enzyme that plays essential roles in various cellular processes, such as transcription or replication. Agents that target Top1, involving camptothecin and its derivatives, are among the most effective anticancer drugs used in the clinic. Previous work has suggested that the level of Top1 expression correlates with the cytotoxicity of camptothecin, but no direct evidence has been provided thus far in the context of human cells with a strictly isogenic genetic background. In this study, we perform heterozygous disruption of the Top1 gene (TOP1) by gene targeting in a human pre-B cell line, Nalm-6, which is karyotypically stable and normal for p53 status. We show that the heterozygous loss of the TOP1 gene does confer cellular resistance to camptothecin, to an extent comparable to that observed in the absence of functional p53 protein. Such a tolerance was not observed with other agents that target DNA topoisomerase II. Our results provide direct evidence that human cells with decreased Top1 levels are significantly more resistant to killing by camptothecin than are otherwise isogenic cells.
We previously reported that eupatilin (5,7-dihydroxy-3,4,6-trimethoxyflavone) extracted from Artemisia asiaitica, augmented the cellular antioxidant defense capacity through induction of the antioxidant protein heme oxygenase-1 (HO-1), thereby protecting ileal smooth muscle cells from nonsteroidal anti-inflammatory drug (NSAID)-induced intestinal toxicity. In the present study, we used cultured feline esophageal epithelial cells (EEC) to investigate the ability of eupatilin to induce expression of HO-1 and to analyze its cytoprotective effect against indomethacin-induced damage, since NSAID users have a higher risk of esophageal ulcers or esophagitis than non-NSAID users. A culture of EEC from cat was prepared. The identity of the cultures was confirmed by immunocytochemistry using cytokeratin antibodies. Western blot analysis showed a concentration- and time- dependent expression of HO-1 in response to eupatilin. Phosphorylation of extracellular regulating protein kinase (ERKs) and Akt, and nuclear translocation of nuclear related factor 2 (Nrf2) were induced by 150 μM eupatilin in a time-dependent manner. Eupatilin-induced HO-1 expression and Nrf2 were partly attenuated by MEK inhibitor PD98059 and almost completely by phosphatidyl-inactiol 3 kinase (PI3K) inhibitor LY294002, but not by c-Jun N-terminal kinase (JNK) inhibitor SP600125 or p38 mitogen activated protein kinase (MAPK) inhibitor SB202190. MTT assay showed that treatment with 2 mM indomethacin for 2 h decreased cell viability to about 41%. Pre-treatment of cells with eupatilin resulted in the dose-dependent inhibition of indomethacin-induced cell damage. We confirmed that ZnPP, an HO-1 inhibitor, repressed eupatilin-induced HO-1 activity and showed the protective effect of eupatilin against indomethacin-induced cell injury. The data suggested that HO-1 was partly responsible for the eupatilin-mediated protective action of esophageal epithelial cells against indomethacin via both ERKs and PI3K/Akt pathways as well as Nrf2 translocation.
Auditory neuropathy (AN) is a hearing disorder characterized by an abnormal auditory brainstem response (ABR). This study examined experimental AN model induced in mice following increased dosages of pyridoxine. Induced AN was examined for ≤10 weeks following the last pyridoxine treatment. To assess AN, we evaluated the ABR, auditory middle latency response (AMLR), otoacoustic emission (OAE), and histochemical morphology of the auditory nerve. Pyridoxine-treated mice exhibited an increase in the hearing threshold shift and delayed latency of both ABR and AMLR in proportion to pyridoxine dosage. Additionally, the extent of auditory nerve fiber loss increased in a dose-dependent manner following pyridoxine intoxication. Coffee or trigonelline treatment ameliorated the hearing threshold shift, delayed latency of the auditory evoked potential, and improved sensory fiber loss induced by pyridoxine intoxication. The present findings demonstrate that high-dose pyridoxine administration can be used to produce a new mouse model for AN, and coffee or trigonelline as a main active compound of coffee extract can potentially facilitate recovery from pyridoxine-induced auditory neuropathy.
Inhibition of cytokine production is the main immunosuppressive effect of cyclosporine (CsA), which is widely used in organ transplantation. Pharmacodynamic (PD) assay for evaluating the inhibition of interleukin-2 (IL-2) production for each patient could provide a more appropriate dosing regimen. We measured the suppression of IL-2 mRNA expression in whole blood following the addition of a range of CsA concentrations by a real-time reverse transcription-polymerase chain reaction (RT-PCR) method. Individual CsA sensitivity on the IL-2 mRNA expression was assessed with healthy subjects both in vitro and ex vivo. We also evaluated it in pre-transplant patients before taking immunosuppressive drugs. Sigmoid Emax model was used to analyze the relationship between CsA concentration and IL-2 mRNA expression. The assay was completed within 8 h. The concentration that resulted in IC50 showed high reproducibility and specificity among the healthy subjects (p<0.005, n=5). Ex vivo study indicated similar inhibition profiles to those of in vitro studies (n=3). The values of IC50 obtained from patients (n=22) also showed large variations and were significantly lower than those from healthy subjects (p<0.05). Semi-quantitative RT-PCR was considered to be a rapid and reliable assay. Our data imply that measurement of IL-2 mRNA levels in whole blood could be valuable in monitoring CsA PD in transplant patients.
The ineffectiveness of anticancer drugs is frequently observed in cancer chemotherapy. The resistance of tumor cells to various cytotoxic drugs is defined as multidrug resistance (MDR). The purpose of our present study was to investigate the inhibitory effects of L1EPO synthesized by our group on P-glycoprotein (P-gp)-mediated MDR in K562/A02 and KBv200 cell lines, which expressed high levels of P-gp. Both the cytotoxicity of the compound and its ability to inhibit K562/A02 and KBv200 cells were determined by sulforhodamine B sodium salt (SRB) assay. Morphologic apoptosis was detected by Hoechst33342 staining assay. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect mdr-1 gene transcription, and Western blot assay was used to assess P-gp expression. Interestingly, we found that the K562/A02 cell line was rendered resistant toward Adriamycin but not towards L1EPO when compared with the parental cells. Furthermore, L1EPO could down-regulate the mdr-1 gene, and it reduced the expression of P-gp and displayed a perfect dose dependence. Moreover, it had less cytotoxicity in normal human cell lines (fibroblast, VEC), GI50>10 μmol/l. Consequently, L1EPO has the potential to overcome P-glycoprotein-mediated MDR in the K562/A02 cell line.
Kv1.4 channel belongs to the family of voltage-gated potassium channels that mediate transient and rapidly inactivating A-type currents and N-type inactivation. This N-type inactivation can be removed by the deletion of N-terminal domains, which exhibit non-inactivating currents and C-type inactivation. In our previous report, we demonstrated that 20(S)-ginsenoside Rg3 (Rg3), one of the active ingredients of ginseng saponins, inhibits human Kv1.4 (hKv1.4) channel currents through the interaction with amino acids, including Lys (K) residue, which is known as K+ activation and the extracellular tetraethylammonium (TEA) binding site. In the present study, we examined the effects of Rg3 on hKv1.4 channel currents without the N-terminal rapid inactivation domain. We constructed hKv1.4Δ2-61 channels by N-terminal deletion of 2-61 amino acid residues. We investigated the effect of Rg3 on hKv1.4Δ2-61 channel currents. We found that Rg3 preferentially inhibited non-inactivating outward currents rather than peak outward currents of hKv1.4Δ2-61 channels. The mutation of K531 hKv1.4Δ2-61 to K531Y hKv1.4Δ2-61 and raising of extracellular [K+]o abolished Rg3 inhibitions on non-inactivating outward currents. Rg3 treatment increased the C-type inactivation rate, but raising the extracellular [K+]o reversed Rg3 action. These results provide additional evidence that K531 residue also plays an important role in the Rg3-mediated non-inactivating current blockages and in Rg3-mediated increase of the C-type inactivation rate in hKv1.4Δ2-61 channels.
We investigated the effects of embryoid body (EB) forming conditions on the expression of hepatocyte marker genes such as alpha-fetoprotein, albumin and CYP7A1 in cells cultured on Matrigel-coated plates for 15 d. The expression levels of hepatocyte marker genes in the cells cultured for 2 d for EB formation from cynomolgus monkey embryonic stem (cmES) cells was higher than those in cells cultured for 5 d. However, the fragment-size of cmES colonies did not markedly affect the expression levels. The expression levels of hepatocyte marker genes, and CYP1A1 and CYP2C43 in cells cultured on Matrigel were considerably higher than those on Matrigel reduced and collagen I. CYP1A1 and CYP3A8 mRNAs were significantly induced by 3-methylcholanthrene and rifampicin, respectively. However, CYP2C43 and CYP2D17 were not induced by these compounds. These results suggested that the differentiation into hepatocytes is affected by the incubation period for EB formation, and that Matrigel successfully promoted in vitro differentiation of cmES cells to hepatocytes.
The subventricular zone (SVZ) generates an immense number of neurons, which migrate to the main olfactory bulb (MOB) and differentiate into granule cells and periglomerular cells in the MOB, even during adulthood. Pheromonal signals, which are mainly received by the vomeronasal organ, provide specific information concerning the reproductive state in a variety of mammal. Vomeronasal sensory neurons project to the accessory olfactory bulb (AOB) located on the dorso-caudal surface of the MOB. In the present study, bromodeoxyuridine-immunoreactive (BrdU-ir) structures in the sagittal section of the MOB including the AOB of young and old male rats were studied to explore the roles of newly generated cells at the region near the rostral end of AOB in the MOB. The density of BrdU-ir cells in the granule cell layer of the MOB of young rats was higher than that of old rats. In young rats, the density of BrdU-ir cells at the region near the rostral end of the AOB was higher than that at the region distant from the AOB. In old rats, the density of BrdU-ir cells at the near region was lower than that at the distant region. The density of BrdU-ir cells at the region near the AOB in the MOB in old rats may be concerned with age-dependent changes in ability of discrimination and memory of general odors and odors related to the reproduction.
In the quest for potent anti-Helicobacter pylori agents, we found 70% EtOH extract of Mallotus philippinensis (LAM.) MUELL. (MPM) with strong bactericidal activity at the concentration of 15.6—31.2 mg/l against eight H. pylori strains. Further fractionation and purification of 70% EtOH extract of MPM led to the isolation of 5 compounds, namely 5,7-dihydroxy-8-methyl-6-prenylflavanone (1), 3′-prenylrubranine (2), red compound (3), isorottlerin (4), and rottlerin (5) which were elucidated on the basis of nuclear magnetic resonance and mass spectroscopy. Among the isolated compounds, rottlerin exhibited most potent bactericidal activity with minimum bactericidal concentration (MBC) value of 3.12—6.25 mg/l against several clinical H. pylori isolates including Japanese and Pakistani strains, nine clarithromycin resistant (CR), and seven metronidazole resistant (MR) strains. Minimum inhibitory concentration (MIC) values of CR (8—>256 mg/l) and MR (>256 mg/l) strains were analyzed by E test. Moreover, the clarithromycin resistant strains were evaluated for A2143G and A2144G point mutations of 23s rRNA gene to correlate the MBC values with mutation type. Our results revealed the potent in vitro anti-H. pylori activity of MPM and rottlerin, specially against CR and MR strains, which could be gainfully utilized for the development of novel antimicrobials to prevent H. pylori related disorders.
The aim of this study was to evaluate the relationship between the neurologic side effects associated with serum aprindine concentrations and the safety range of aprindine for the prevention of neurologic side effects in 142 Japanese inpatients. Serum aprindine concentrations were determined by high-performance liquid chromatography. A poor positive correlation was observed between dose and serum aprindine concentration (r2=0.0419, p=0.0114), and between age and ratio of serum aprindine concentration to the dose per body weight of aprindine (r2=0.0159, p=0.121). When aprindine concentration was <1 μg/ml, almost no patients showed neurologic side effects associated with aprindine. On the other hand, about 50% of the patients showed neurologic side effects when aprindine concentrations were >1 μg/ml. Here, the side effects associated with aprindine such as dizziness or intention tremors were observed in 15 patients, which later disappeared after discontinuance of aprindine therapy or a decrease in the dose. In conclusion, serum aprindine concentration should be maintained under approximately 1 μg/ml in Japanese patients to prevent neurologic side effects.
The structural similarity between papaverine and berberine, a known inhibitor of human protein tyrosine phosphatase 1B (h-PTP 1B), prompted us to investigate the potential of papaverine as h-PTP 1B inhibitor. The investigation included simulated docking experiments to fit papaverine into the binding pocket of h-PTP 1B. Papaverine was found to readily dock within the binding pocket of h-PTP 1B in a low energy orientation via an optimal set of attractive interactions. Experimentally, papaverine illustrated potent in vitro inhibitory effect against recombinant h-PTP 1B (IC50=1.20 μM). In vivo, papaverine significantly decreased fasting blood glucose level of Balb/c mice. Our findings should encourage screening of other natural alkaloids for possible anti-h-PTP 1B activities.
Saw palmetto extract (SPE), used widely for the treatment of benign prostatic hyperplasia (BPH) has been shown to bind α1-adrenergic, muscarinic and 1,4-dihydropyridine (1,4-DHP) calcium channel antagonist receptors. Major constituents of SPE are lauric acid, oleic acid, myristic acid, palmitic acid and linoleic acid. The aim of this study was to investigate binding affinities of these fatty acids for pharmacologically relevant (α1-adrenergic, muscarinic and 1,4-DHP) receptors. The fatty acids inhibited specific [3H]prazosin binding in rat brain in a concentration-dependent manner with IC50 values of 23.8 to 136 μg/ml, and specific (+)-[3H]PN 200-110 binding with IC50 values of 24.5 to 79.5 μg/ml. Also, lauric acid, oleic acid, myristic acid and linoleic acid inhibited specific [3H]N-methylscopolamine ([3H]NMS) binding in rat brain with IC50 values of 56.4 to 169 μg/ml. Palmitic acid had no effect on specific [3H]NMS binding. The affinity of oleic acid, myristic acid and linoleic acid for each receptor was greater than the affinity of SPE. Scatchard analysis revealed that oleic acid and lauric acid caused a significant decrease in the maximal number of binding sites (Bmax) for [3H]prazosin, [3H]NMS and (+)-[3H]PN 200-110. The results suggest that lauric acid and oleic acid bind noncompetitively to α1-adrenergic, muscarinic and 1,4-DHP calcium channel antagonist receptors. We developed a novel and convenient method of determining 5α-reductase activity using LC/MS. With this method, SPE was shown to inhibit 5α-reductase activity in rat liver with an IC50 of 101 μg/ml. Similarly, all the fatty acids except palmitic acid inhibited 5α-reductase activity, with IC50 values of 42.1 to 67.6 μg/ml. In conclusion, lauric acid, oleic acid, myristic acid, and linoleic acid, major constituents of SPE, exerted binding activities of α1-adrenergic, muscarinic and 1,4-DHP receptors and inhibited 5α-reductase activity.
Methylsulfonylmethane (MSM), also known as dimethyl sulfone and methyl sulfone, is an organic sulfur-containing compound that occurs naturally in a variety of fruits, vegetables, grains, and animals, including humans. In the present study, we demonstrated the anti-inflammatory effects of MSM in lipopolysaccharide (LPS)-stimulated murine macrophages, RAW264.7 cells. MSM significantly inhibited the release of nitric oxide and prostaglandin E2 by alleviating the expression of inducible nitric oxide synthase and cyclooxygenase-2 in LPS-stimulated RAW264.7 cells. Furthermore, the levels of interleukin-6 and tumor necrosis factor-α were decreased by MSM treatment in cell culture supernatants. Further study indicated that the translocation of the p65 subunit of nuclear factor (NF)-κB to the nucleus was inhibited by MSM treatment in LPS-stimulated RAW264.7 cells, in which it helped block degradation of inhibitor of NF-κB. In addition, in vivo studies demonstrated that topical administration of MSM at 500—1250 μg/ear resulted in similar inhibitory activities in 12-O-tetradecanoylphorbol 13-acetate-induced mouse ear edema. Collectively, theses results indicate that MSM inhibits LPS-induced release of pro-inflammatory mediators in murine macrophages through downregulation of NF-κB signaling.
Psychostimulants induce hyperlocomotion in normal subjects, although, they are effective in producing a calming effect in hyperactive subjects. This paradoxical effect has been related to changes in serotonin (5-HT) neurotransmission in hyperactive dopamine transporter-knockout mice. In addition, we observed that hyperlocomotion in mice lacking pituitary adenylate cyclase-activating polypeptide was attenuated by amphetamine dependent on 5-HT1A receptor signaling and that amphetamine, when co-administered with a 5-HT1A agonist, produced a calming effect in wild-type mice. Here, in an attempt to address how 5-HT1A receptor signaling exerts the calming action of psychostimulants, we examined c-Fos expression in several brain regions after administration of methamphetamine and osemozotan, a selective 5-HT1A receptor agonist. The number of c-Fos-positive cells was increased in the medial prefrontal cortex, striatum and nucleus accumbens in methamphetamine (3 mg/kg body weight)-injected mice. Osemozotan (1 mg/kg) significantly reduced the methamphetamine-induced c-Fos expression in the medial prefrontal cortex and striatum, but not in the nucleus accumbens. This osemozotan action was completely blocked by the 5-HT1A receptor antagonist WAY-100635 (1 mg/kg). As the prefrontal cortex is considered to be involved in the beneficial actions of psychostimulant medications for attention-deficit/hyperactivity disorder, the present result showing 5-HT1A-mediated inhibition of corticostriatal activity may partly be related to this psychostimulant action.
We compared the inhibitory action of gabapentin, which is used to treat neuropathic pain, on mechanical allodynia induced by chemotherapeutic agents, paclitaxel, oxaliplatin, and vincristine, in mice. Single injections of paclitaxel, oxaliplatin, and vincristine at the doses corresponding to doses clinically used caused mechanical allodynia of similar intensity. Oral administration of gabapentin (30, 100 mg/kg) produced a dose-dependent inhibition of allodynia caused by paclitaxel and oxaliplatin, but not vincristine. Intrathecal injection of gabapentin (30, 100μg/site) significantly inhibited allodynia induced by paclitaxel, but not oxaliplatin and vincristine. Intraplantar injection of gabapentin (30, 100 μg/site) did not significantly inhibit allodynia induced by paclitaxel and oxaliplatin. Paclitaxel increased the expression of mRNA of voltage-dependent calcium channel α2δ-1 subunit, an action site of gabapentin, in the dorsal spinal cord, and oxaliplatin increased it in the dorsal root ganglia. Vincristine was without effects on α2δ-1 subunit mRNA in these regions. These results suggest that the efficacy of gabapentin in the treatment of mechanical allodynia is dependent on chemotherapy agent used. It may be partly due to the distinct effects of chemotherapy agents on the expression of α2δ-1 subunit of voltage-dependent calcium channel.
Diabetes is the leading cause of end-stage renal failure, since glucose-dependent metabolic factors are synergistically activated within the diabetic kidney. Accordingly, in Japan, there is much debate over the health benefits of natural therapies to reduce these risk factors. In our previous study, we reported that Cornus officinalis SIEB. et ZUCC. possessed an antidiabetic effect via ameliorating glucose-mediated metabolic disorders as well as aminoguanidine, an inhibitor of advanced glycation endproduct (AGE) formation, with a renoprotective effect. The aim of the present study was to investigate the effect of 7-O-galloyl-D-sedoheptulose (GS) against diabetic oxidative stress and AGE formation. Streptozotocin-induced diabetic rats were orally administered GS for 20 d, and the changes in serum glucose levels, as well as those of body weight every 10 d were evaluated. In addition, glucose, fluorescent AGE, methylglyoxal, glycolaldehyde (GA), and immunoblotting analyses for heme oxygenase-1, receptor for AGE, Nε-(carboxymethyl)lysine, Nε-(carboxyethyl)lysine, and GA-pyridine were performed in the kidney at the end of the experiment. The results obtained in this study demonstrated that 20 d of treatment with GS had beneficial effects on hypoglycemic and renal metabolic abnormalities, including renal glucose, oxidative stress, and AGE formation. Together, our data help to elucidate its potential therapeutic value against diabetic kidney disease.
The stems of Akebia plants, Akebiae Caulis, have long been used in traditional Chinese and Japanese medicines, and are mainly produced in western Japan. Three Akebia plants, Akebia quinata (AQ), A. trifoliata (AT), and A. pentaphylla (AP) grow wild in Japan. With the aim of carrying out molecular biological identification of Akebia plant species and discriminating Akebiae Caulis from other related crude drugs originating from non-Akebia plants, sequencing analysis of Akebia plants collected from various parts of Japan and the southern Korean Peninsula was performed. Specimens identified morphologically as AQ and AT had their respective common internal transcribed spacer one (ITS1) sequences, which could be distinguished. Cloning experiments of AP specimens showed that their ITS1 contained both common sequences of AQ and AT as well as their chimera. These chimeric sequences were not identical between AP specimens, suggesting that AP is not a species with uniform DNA sequences but a group of individuals with hybrid genomes of AQ and AT. Based on the sequences of Akebia species found here, we propose polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) methods to discriminate Akebiae Caulis from the related crude drugs and to distinguish three Akebia plants. Comparison of triterpene-rich fractions of extracts from Akebia plants by TLC showed that AP had an intermediate profile of AQ and AT.
Through screening for natural ligands against peroxisome proliferator-activated receptor γ (PPARγ) using the PPARγ luciferase reporter assay, 6 hydroxy unsaturated fatty acids were isolated from adlay seed (Coix lacryma-jobi L. var. ma-yuen STAPF.) extracts with acetone and 70% ethanol. The structures of these compounds were determined via spectral analysis as 13-hydroxy-(9E,11E)-octadecadienoic acid (13-E,E-HODE) (1), 9-hydroxy-(10E,12E)-octadecadienoic acid (9-E,E-HODE) (2), 9-hydroxy-(10E)-octadecenoic acid (3), 10-hydroxy-(8E)-octadecenoic acid (4), 8-hydroxy-(9E)-octadecenoic acid (5), 11-hydroxy-(9Z)-octadecenoic acid (6). 9-E,E-HODE (2) exhibited the most potent PPARγ agonist activity of the isolated hydroxy unsaturated fatty acids. 9-E,E-HODE (2) and 13-E,E-HODE (1) are the respective geometrical isomers of 9-hydroxy-(10E,12Z)-octadecadienoic acid and 13-hydroxy-(9Z,11E)-octadecadienoic acid, both of which are likely to be natural PPARγ agonists produced in various mammalian cells, suggesting that 9-E,E-HODE may also act as PPARγ agonist.
Flavonoids are divided into flavones, flavonols, flavanones, and isoflavones etc. according to their basal structure, and are known to include compounds with physiological and pharmacological effects such as anti-oxidant, anti-tumor, and anti-inflammation activities. The ingestion of flavonoids may induce pharmacokinetic interactions through the co-administration of drugs. In this study, we investigated the inhibitory potentials on cytochrome P450 (CYP) 3A activity of 23 flavonoids using human liver microsomes, and tried to identify the molecular features that cause the inhibition of CYP3A. The activity of testosterone 6β-hydroxylate was evaluated to quantify CYP3A activity. We analyzed Quantification Theory I, in which extreme values of the inhibitory effects of CYP3A activity were tested with flavonoids supplied at a level of 10 μM. The inhibitory effects of flavonoids ranged widely from 1.5 μM to more than 100 μM for the half maximal inhibitory concentration. Because the inhibitory effects were only weakly correlated with the pKa value, the inhibitory effects could not be accounted for by the molecular characteristics of the flavonoids. On the other hand, flavones with the basal structure and hydroxylation at positions 7 and 4′ showed significantly increased inhibitory effects on CYP3A activity. In addition, the hydroxylation of position 2′ and 3′, methoxylation of position 4′, and the isoflavone basal structure significantly decreased the inhibitory effects on CYP3A activity. In conclusion, the basal structure and the substituents of flavonoids are important in the inhibitory effects of flavonoids on CYP3A activity.
To develop a transdermal dosage form of propofol (PF), in vitro skin permeability and in vivo absorbability of PF were investigated in rats, and the effectiveness of enhancers on the transdermal delivery of PF was estimated. Propylene glycol (PG), isopropyl myristate and macrogol were used as co-solvent type enhancers. L(−)-Menthol (MEN), D(+)-limonene, oleic acid, stearic acid, sucrose fatty acid esters and sodium dodecyl sulfate (SDS) were used as membrane-acting type enhancers. Among the co-solvent type enhancers, PG showed the highest enhancing effect in vitro. Furthermore, the synergistic effect of the combined use of PG and membrane-acting type enhancers was confirmed. Higher values of permeation parameters were observed with the combined use of PG and MEN, sucrose fatty acid esters or SDS. For the in vivo experiment, the addition of a smaller amount of PG was preferable to the amount used in the in vitro experiment. The synergistic effect of enhancers was observed with the combined use of PG and MEN. Our findings suggest that the combination of PG and MEN was useful as enhancers for the transdermal absorption of PF. These results provide useful information to develop a transdermal dosage form of PF as a sedative or a hypnotic.
A novel iontophoretic system utilizing ion-exchange membranes is effective for selective transdermal delivery of ionized drugs. In the present study, we examined in vivo availability and safety of ion-exchange iontophoresis in the transdermal delivery of anionic diclofenac, a well known anti-inflammatory medication, to rat dorsal skin. While iontophoresis increased the plasma concentration of diclofenac sodium and skin injury was not observed, no anti-inflammatory effect was exerted. To enhance the efficiency of transdermal delivery of diclofenac sodium, iontophoresis was combined with topical application of one of three terpenes (menthol, nerolidol or geraniol) as chemical enhancers. By combining iontophoresis with geraniol, the plasma concentration of diclofenac sodium increased over 20-fold, and suppression of inflammation was achieved. Skin irritation was not seen even after 1.5 h iontophoresis. The enhancing effect of geraniol is attributed to increased penetration of the drug into the stratum corneum as well as enhanced transport across the anion-exchange membrane. Ion-exchange iontophoresis combined with geraniol is a highly effective transdermal delivery system.
We investigated the metabolism of pranlukast, a selective leukotriene agonist, and the potential for drug–drug interactions. Although cytochrome P450 (CYP) 3A4 appeared to be the major cytochrome P450 isoform involved in the metabolism of pranlukast, the results suggested that pranlukast metabolism was inhibited less than 50% by ketoconazole, a reversible CYP3A4 inhibitor, or by anti-CYP3A4 antibodies. Irreversible macrolide CYP3A4 inhibitors, clarithromycin, erythromycin and roxithromycin, exhibited little effect on pranlukast metabolism. On the other hand, pranlukast reversibly inhibited CYP2C8 and/or 2C9, and CYP3A4, with Ki values of 3.9 and 4.1 μmol/l, respectively. The [I]in,max,u/Ki ratios were 0.004 and 0.003, respectively. The Ki values were about 300-fold greater than the [I]in,max,u, therefore it is suggested that, at clinical doses, pranlukast will not affect the pharmacokinetics of concomitantly administered drugs that are primarily metabolized by CYP2C8 and/or 2C9 or CYP3A4.
The growing concomitant consumption of drugs and herbal preparations such as garlic, and the numerous reports about the influence of herbal preparations on intestinal transport, led us to evaluate the influence of aged garlic extract on the transport function and electrophysiological parameters of the small intestinal mucosa. Aged garlic extract induced increase of the absolute value of the transepithelial potential difference and of the short-circuit current in both permeability models tested (rat jejunum, Caco-2 cell monolayers) without affecting transepithelial electrical resistance. It also caused a significant increase of the P-glycoprotein and multidrug resistance associated protein 2 mediated effluxes through rat jejunum of marker substrates Rhodamine 123 and 2,4-dinitrophenyl-S-glutathione, respectively. Rhodamine 123 efflux through the Caco-2 cell monolayers was not altered by aged garlic extract, whereas the efflux of 2,4-dinitrophenyl-S-glutathione increased significantly. So altered activity of the important transport proteins could significantly change the pharmacokinetic properties of conventional medicines taken concomitantly with aged garlic extract.
Lipid rafts on cell membranes have heterogeneity such as cholesterol-rich microdomains and sphingolipids-rich microdomains. We previously reported that β-cyclodextrin (β-CyD) induced morphological changes of red blood cells (RBC) from discocyte to stomatocyte, possibly due to extraction of cholesterol from cholesterol-rich lipid rafts of RBC membranes. In this study, the effects of methyl-β-cyclodextrin (M-β-CyD) and 2,6-di-O-methyl-β-cyclodextrin (DM-β-CyD) on lipid rafts and morphological changes in rabbit RBC (RRBC) were examined, compared to those of β-CyD. In sharp contrast to β-CyD, M-β-CyD and DM-β-CyD induced morphological changes of RRBC from discocyte to echinocyte. At pre-hemolytic concentrations of β-CyDs, M-β-CyD and DM-β-CyD strongly released cholesterol from cholesterol-rich lipid rafts, compared to β-CyD. Meanwhile, the lowering effects of DM-β-CyD on fluorescent sphingomyelin analogue in sphingolipids-rich lipid rafts were more potent than those of β-CyD and M-β-CyD. The magnitude of the abilities of M-β-CyD and DM-β-CyD to extract membrane constituents was higher than that of β-CyD, consistent with that of hemolytic activity. Furthermore, DM-β-CyD and M-β-CyD, not β-CyD, lowered the amount of proteins in cholesterol-rich lipid rafts of RRBC. These results suggest that higher hemolytic activity and morphological changes from discocyte to echinocyte in RRBC induced by M-β-CyD and DM-β-CyD may be due to the extraction of both cholesterol and proteins from cholesterol-rich lipid rafts of RRBC, although DM-β-CyD may interact with sphingolipids-rich lipid rafts on RRBC membranes only slightly.
The aim of the present study was to characterize membrane transport mechanisms of mizoribine in the intestinal epithelial cells. We evaluated the contribution of Na+-dependent and -independent membrane transporters to mizoribine absorption in the rat intestine using an in situ closed loop method. In addition, we evaluated the effects of structurally related compounds, extracellular Na+ concentrations, and an inhibitor of Na+-independent equilibrative nucleoside transporter, nitrobenzylmercaptopurine ribonucleoside (NBMPR), on the uptake of mizoribine in human intestinal epithelial LS180 cells. In the presence and also absence of Na+ in rat intestinal loops, more than 60% of the administered dose (50 μg at the concentration of 100 μg/ml=386 μM) of mizoribine was absorbed in 40 min. In the LS180 cells, ribavirin and inosine reduced the uptake of 400 μM mizoribine with the increasing concentrations (from 5 to 50 mM) of the inhibitors. The cellular uptake of mizoribine in the absence of extracellular Na+ decreased to 72.7% of the uptake in the presence of extracellular Na+, whereas 100 μM NBMPR decreased the uptake of mizoribine markedly to 34.7% of that without NBMPR. These findings suggest that Na+-independent nucleoside transporters are largely responsible for absorption of mizoribine in the intestine.
Reducing sugar, 2-deoxy-D-ribose (dRib), produces reactive oxygen species through autoxidation and protein glycosylation and causes osteoblast dysfunction. Kaempferol, a natural flavonoid, was investigated to determine whether it could influence dRib-induced cellular dysfunction and oxidative cell damage in the MC3T3-E1 mouse osteoblastic cell line. Osteoblastic cells were treated with 30 mM dRib in the presence or absence of kaempferol (10−9—10−5 M) and markers of osteoblast function and lipid peroxidation were subsequently examined. Kaempferol (10−9—10−5 M) significantly inhibited the dRib-induced decrease in growth of MC3T3-E1 osteoblastic cells. In addition, treatment with kaempferol resulted in a significant elevation of alkaline phosphatase (ALP) activity, collagen content, and mineralization in the cells. Treatment with kaempferol increased osteoprotegerin (OPG) secretion and decreased malondialdehyde (MDA) contents of MC3T3-E1 osteoblastic cells in the presence of 30 mM dRib. Taken together, these results suggest that kaempferol inhibits dRib-induced osteoblastic cell damage and may be useful for the treatment of diabetes-related bone disease.
The objective of this study was to propose a clinically effective and safe micafungin (MCFG) treatment for 20 recipients of living-donor liver transplantations (LDLTs), after considering the influence of liver function on its plasma pharmacokinetics. In all patients, an improvement of clinical symptoms was observed after MCFG treatment. Liver and renal functions were not significantly changed by the administration of MCFG. In the recipients, the trough plasma concentration of MCFG was 5.2±2.6μg/ml (mean±S.D.), which was dependent on the dose (p=0.0033). Additionally, there was a good correlation between the trough and peak MCFG plasma concentrations (p<0.0001). The trough concentration of MCFG was significantly correlated with serum total bilirubin levels (p=0.0166). In addition, the MCFG concentration/dose (C/D) ratio was significantly higher in the patients with total bilirubin levels >5 mg/dl than in those with total bilirubin levels ≤5 mg/dl (p<0.0001). The C/D ratio of MCFG was weakly but not significantly correlated with total bilirubin levels at total bilirubin levels >5 mg/dl (p=0.0508). Therefore, a reduced dose of MCFG should be considered when total bilirubin levels are >5 mg/dl. Furthermore, careful monitoring of total bilirubin levels is recommended during MCFG treatment in LDLT-recipients with severe liver dysfunction. These results provide helpful advice about MCFG administration for the treatment of fungal infections in LDLT patients with fluctuating liver function.
The hydrodynamic method by rapid intravenous injection of a large volume of plasmid DNA is known to be an efficient and liver-specific method of in vivo gene delivery and achieves high levels of foreign gene expression, particularly in hepatocytes. Low transgene activities have also been observed in other organs such as the spleen and lung; however, the expression profiles of mRNA and protein are still unknown. Therefore, we investigated the localization of luciferase mRNA by in situ hybridization and luciferase activity in mice after transfection of pCMV-luc encoding the luciferase gene under the control of cytomegalo virus (CMV) promoter. We found that hydrodynamic injection effectively induced mRNA expression of the transgene only in the liver although transgene activities were observed in other organs. The transgene activity observed in other organs may be due to leakage from hepatocyte gene expression by transient increase in the permeability of the hepatocyte cellular membrane caused by increased pressure by hydrodynamic injection.
For designing a complex vector that has the advantages of both polyethylenimine (PEI) and chitosan for gene delivery, a PEI/chitosan/DNA complex was constructed at various N/P ratios (the ratios of moles of the amine groups of cationic polymers to those of the phosphate ones of DNA) and both the cytotoxicity and the transfection efficiency of the vector were evaluated. The results demonstrated that the chitosan/DNA binding degree was depended on the N/P ratio. The mean size of the complex vector was between 100 nm and 150 nm. Compared with PEI/DNA, the complex vector (PEI/chitosan/DNA with chitosan/DNA N/P=4, PEI/DNA N/P=10) appeared to have low cytotoxicity, which maintained the cell survival rate at greater than 80%, and showed higher transfection efficiency of nearly 1000 fold compared with that using chitosan/DNA alone. Furthermore, the expression efficiency of the complex vector carrying enhanced green fluorescent protein was not inhibited in the presence of serum in both HeLa cells and A549 cells. The PEI/chitosan complex may be a promising gene carrier that has high transfection efficiency as well as low cytotoxicity.
The Bifidobacterium breve M-16V strain has previously been shown to be effective in infants in improving the symptoms of allergic hypersensitivity to cow's milk and atopic dermatitis. In the current study, we investigated the effect of an oral administration of M-16V on immunoglobulin (Ig) E production in BALB/c mice. Live M-16V was orally administered to ovalbumin (OVA)-immunized mice for 3 weeks at a dose level of 5×108 colony-forming unit (cfu)/0.5 ml/d/animal. While M-16V treatment significantly reduced the serum levels of total IgE, OVA-specific IgE and OVA-specific IgG1, as compared to controls, it did not affect the serum level of OVA-specific IgG2a. In M-16V-administered mice, there was a significant decrease in the serum OVA-specific IgG1/IgG2a ratio. In addition, while ex vivo production of interleukin (IL)-4 by the splenocytes from M-16V-administered mice was significantly lower as compared to controls, there was no difference in the production of gamma-interferon (IFN-γ) and IL-10. We also examined the effect of M-16V on cytokine and IgE production from OVA-sensitized splenocytes via restimulation with OVA in vitro. While M-16V suppressed OVA-induced total IgE and IL-4 production and induced secretion of IFN-γ and IL-10 in a dose-dependent manner, it was not able to induce IL-12. We concluded that oral administration of M-16V suppressed the T-helper type (Th) 2 immune response and IgE production and modulated the systemic Th1/Th2 balance, and which was at least partially independent of the Th1 cytokine induction. These results suggest that M-16V may potentially have an antiallergic activity.