Biotin-binding IgG in human sera was quantitated using F(ab')2anti-human IgG-coated multiwell microplates (Muratsugu, M. et al. 2003, Biol. Pharm. Bull., 26, 1605—1608). The biotin–protein ratio of biotinylated IgG, which was used as standard in the assay, was very important to quantitate the level of biotin-binding IgG. We investigated a synthesis method of biotinylated human immunoglobulins, how to determine the biotin–protein ratio of the biotinylated proteins, and their stability to prepare standards for measuring biotin-binding IgG, IgA, and IgM.
A simple and sensitive method for the determination of triptolide, wilforlide A and triptonide in human plasma was developed and validated, using high-performance liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS). Plasma samples were purified using solid-phase extraction (SPE) columns. The HPLC separation of the analytes was performed on a MACHEREY-NAGEL C18 column (2.0 mm×125 mm, 3 μm), using 2.7 mM formic acid containing 10 mM ammonium acetate–acetonitrile (55 : 45, v/v) as mobile phase, with a flow-rate of 0.25 ml/min. The compounds were ionized in the electrospray ionization (ESI) ion source of the mass spectrometer and were detected in the selected ion recording (SIR) mode. The calibration curves were linear in the 0.80—300 ng/ml range for all the three analytes, and the limits of detection were 0.25, 0.40, and 0.35 ng/ml for triptolide, wilforlide A, and triptonide, respectively. The average absolute recoveries for all the three analytes were above 81%. The methodology recoveries were greater than 91% and the relative standard deviations (RSD) of intra-day and inter-day were less than 15%. The developed method was successfully applied to the determination of triptolide, wilforlide A and triptonide concentration in patients' plasma after taking the medicament containing Tripterygium wilfordii HOOK. F.
For the amplification and ultrafast separation of the genetic markers and DNA sequences that are related to human male infertility, a multiplex PCR for amplifying three DNA sequence-tagged sites (STS) located on the human Y chromosome with possible roles in the spermatogenesis process has been designed and applied followed by separation on a microchip. First, the optimum Tm degree for the three DNA markers was optimized and determined experimentally, and the three DNA STS were amplified. These three DNA markers were then separated on a 12-lane microchip electrophoresis system, which can analyze the DNA markers on 12 channels simultaneously. The combination of these two technologies, multiplex PCR and microchip electrophoresis, allows the analysis of 36 DNA markers (12×3) within only 180 s.
Ovarian hormone deficiency increases the generation of reactive oxygen species. Their excess induces oxidative stress, which results in the cell damage or death. It causes the aging diseases—atherosclerosis, rheumatoid arthritis, osteoporosis, etc. Ovariectomized rats are used as oxidative stress models. We verified the effects of ovariectomy-induced oxidative stress on free radical production as evaluated by DPPH elimination, lipoperoxidation evaluated by malondialdehyde levels, and antioxidant activation of superoxide dismutase, catalase, glutathione peroxidase, and estradiol in the liver and sera. Ovariectomized rats were given Salicornia herbacea (SH) intraperitoneally at the dose of 100 mg/kg daily for 2 months. Free radical-scavenging activity of SH was measured in comparison with that of L-ascorbic acid. The histopathology of liver tissue was also investigated. Antioxidative values in the ovariectomized group decreased, but those in the SH-treated group increased due to the free radical-scavenging activity of SH. Moreover, inflammation and cirrhosis in the liver tissue of SH-treated rats decreased significantly. These results suggest that SH may be a potential candidate for an antioxidative reagent.
An alcoholic extract of Phyllanthus amarus (P. amarus) was found to inhibit cytochrome P450 (P450) enzymes both in vivo as well as in vitro. This was studied using specific resorufin derivatives, as substrate for isoenzymes in the P450 super family. Concentration needed for 50% inhibition of 7-ethoxyresorufin-O-deethylase (EROD), CYP1A1 was 4.6 μg/ml while concentration needed for 7-methoxyresorufin-O-demethylase (MROD) CYP1A2 was 7.725 μg/ml and 7-pentoxyresorufin-O-depentylase (PROD), CYP2B1/2 was found to be 4.18 μg/ml indicating that the extract inhibited the P450 enzymes at very low concentration. Extract also inhibited the activity of aniline hydroxylase (an indicator of CYP 2E1 activity, IC50 50 μg/ml) and aminopyrine demethylase (an indicator of CYP 1A, 2A 2B, 2D and 3A activity, IC50 >1000 μg/ml). Oral administration of the extract was also found to reduce the elevated P450 enzyme activities produced by phenobarbitone by 50% at 250 mg/kg body weight. The implication of these results on the inhibition of carcinogenesis produced by the extract is discussed.
Cloning of polyketide synthase (PKS) gene for amphidinolide biosynthesis was attempted from a dinoflagellate Amphidinium sp. (strain Y-42). Fourteen β-ketoacyl synthase gene fragments were obtained by Polymerase Chain Reaction (PCR) amplification from degenerated primer sets designed on the basis of the conserved amino acid sequences of β-ketoacyl synthase domains in known type I PKSs. The PCR analysis using primer sets designed from these fourteen β-ketoacyl synthase gene fragments revealed that these DNA sequences exist only in the dinoflagellates producing amphidinolides. The DNA sequence of the positive clone, which was isolated from genomic DNA library of Amphidinium sp. (strain Y-42) by PCR detection using the specific primer set, was analyzed by shotgun sequencing. The deduced gene products in the positive clone showed similarity with β-ketoacyl synthase (KS), acyl transferase (AT), dehydratase (DH), ketoreductase (KR), and acyl carrier protein (ACP) in known type I PKSs and thioesterase (TE).
We have shown that anorexic response is induced by intraperitoneal injection of zymosan in mice, although the role of prostaglandins in this response is relatively unknown as compared with lipopolysaccharide (LPS)-induced anorexic response. Indomethacin (0.5 and 2.0 mg/kg), a non-selective cyclooxygenase (COX) inhibitor, as well as meloxicam (0.5 mg/kg), a selective COX-2 inhibitor, but not FR122047 (2.0 mg/kg), a selective COX-1 inhibitor, attenuated zymosan-induced anorexia. Zymosan injection elevated COX-2 expression in brain and liver but not in small intestine and colon. Meloxicam (0.5 mg/kg) and FR122047 treatment (2.0 mg/kg) similarly suppressed the generation of brain prostaglandin E2 (PGE2) and peritoneal prostacyclin (PGI2) upon zymosan injection. PGE2 generation in liver upon zymosan injection was suppressed by meloxicam (0.5 mg/kg) but not by FR122047 treatment (2.0 mg/kg). Our observations suggest that COX-2 plays an important role in zymosan-induced anorexia, which is a similar feature in LPS-induced anorexic response. However, non-selective inhibition by selective COX-1 and COX-2 inhibitors of brain PGE2 generation upon zymosan injection does not support the role of COX-2 expressed in brain in zymosan-induced anorexic response. PGE2 generation in liver may account for peripheral role of COX-2 in zymosan-induced anorexic response.
The non-immune phage antibody library system is one of the most attractive technologies available to current therapeutic, diagnostic and basic scientific research. This system allows the rapid isolation of antibodies of interest that could subsequently be applied directly to drug delivery systems and antibody therapy. Previously, we reported the primer sets to encompass the antibody repertoire and thus improve library quality. However, a wide number of varying primer sets cause to decrease the amplification efficiency of antibody genes. In the present study, we re-generated the library primer sets newly and constructed an improved library from non-immune mice that was far superior in terms of variety and quality. This new library contained 2.4 billion independent clones. In addition, we optimized the selection step from this library to isolate high-affinity antibodies. The optimization of an affinity panning protocol by the incorporation of an automated Microfluidics instrument led to the successful isolation of three different monoclonal antibodies for human vascular endothelial growth factor receptor 2 (KDR). These antibodies were demonstrated to exhibit high specificity and were able to detect a mere 0.6 fmol of KDR by dot blot analysis. Previously reported antibodies for luciferase were also isolated successfully from this library. Our results clearly demonstrate the importance of the improved protocol for the library preparation of antibodies and the resulting isolation of antibodies for clinical and research applications.
Although Akt is known to be associated with drug resistance, its role in cyclic AMP (cAMP)-related inhibition of cell proliferation is not clear. Here, we report that Akt modulates the sensitivity of hepatocellular carcinoma cells to cAMP. Treatment of hepatocellular carcinoma cell lines (HepG2 and Bel-7402) with cAMP inhibited proliferation, with HepG2 cells showing lower sensitivity to cAMP. Biochemical studies showed that cAMP increased FBS-stimulated Akt phosphorylation in HepG2 cells, but completely inhibited FBS-stimulated Akt phosphorylation in Bel-7402 cells, suggesting that the differential response of Akt to cAMP in these two cell lines might contribute to their differential sensitivity. LY294002, a phosphatidylinositol 3-kinase inhibitor that inhibits FBS-stimulated Akt phosphorylation, restored the sensitivity of HepG2 cells to cAMP and API-2 (Akt/protein kinase B signaling inhibitor-2) also showed similar effect. These results collectively indicate that the response level of Akt to cAMP may play a critical role in determining drug sensitivity.
Several chemically synthesized compounds were examined for protective effects against the cell damage in tunicamycin-treated human neuroblastoma IMR-32 cells. Among the compounds tested, an antioxidant, Norbergenin-11-caproate (10 μM), exhibited complete protection against the cell growth inhibitory effect of tunicamycin but did not inhibit the induction of Bip/GRP78 mRNA by tunicamycin. Both norbergenin-11-caproate and α-tocopherol completely inhibited the production of reactive oxygen species induced by tunicamycin, however, α-tocopherol inhibited tunicamycin-induced cell damage only partially, even at 100 μM. These findings suggest the potential of Norbergenin-11-caproate for therapeutic application in endoplasmic reticulum (ER) stress-dependent diseases implicating a specific mechanism other than anti-oxidative one.
We have recently reported that annexin (Anx) A3 expression is necessary for hepatocyte growth in cultured rat hepatocytes seeded at half the subconfluent density on collagen. In the present study, we investigated the effects of various regulatory factors of hepatocyte growth on AnxA3 expression. AnxA3 expression was significantly reduced in hepatocytes cultured under various growth inhibitory conditions such as presence of dexamethasone, culture at subconfluent cell density, and on EHS-Matrigel and lactose-carrying styrene polymer. On the other hand, hepatocyte growth factor and epidermal growth factor, stimulators of hepatocyte growth, significantly increased AnxA3 expression in hepatocytes cultured on EHS-Matrigel. These results show close correlation between known stimulatory or inhibitory actions of various factors to hepatocyte growth and increase or decrease in AnxA3 expression, and suggest the involvement of AnxA3 in their regulation of hepatocyte growth.
We identified antimycin A1 as an inhibitor of the hypoxia-response element (HRE) from screening using a reporter under the control of HRE under hypoxic conditions. Antimycin A1 was effective at 20 pg/ml in inhibiting the reporter activity. The expression of vascular endothelial growth factor (VEGF) mRNA during hypoxia was also inhibited by antimycin A1. Angiogenesis induced by implantation of mouse sarcoma-180 cells was significantly inhibited by non-toxic doses of antimycin A1. Hypoxia inducible factor (HIF)-1α protein levels were significantly decreased by antimycin A1, but its mRNA level was not affected. Antimycin A1 is known to be an inhibitor of mitochondrial electron transport system, and depletion of mitochondria abolished antimycin A1-effect, at least in part. Inhibitors of proteasome or protein synthesis did not affect the decrease in HIF-1α level induced by antimycin A1. These results indicate that antimycin A1 inhibited angiogenesis through decrease in VEGF production caused by inhibition of HIF-1α activation.
Fifty-six stilbenoids isolated from the families of Welwitschiaceae and Gnetaceae were screened for growth inhibitory activity against HL60 cells, and two compounds (gnemonol G and gnetin I) among them exhibited a strong activity with IC50 of 10.0 μM and 12.2 μM at 48 h incubation, respectively. The growth suppression by gnemonol G and gnetin I was found to be in part due to apoptosis which was assessed by morphological findings such as nuclear condensation and fragmentation, and DNA ladder formation in human leukemia HL60 cells.
The different characteristics of Liu-Jun-Zi-Tang, a spray-dried powdered extract from 8 Chinese herbs, and domperidone, a D2 antagonist, both of which are used clinically for the treatment of gastritis, were validated in isolated guinea pig ileal longitudinal muscle. Liu-Jun-Zi-Tang at 0.05 mg/ml did not affect the twitch response by electrical field stimulation, but it concentration-dependently inhibited the twitch response at concentrations of 0.1 and 1.0 mg/ml, and maximum inhibition occurred 5 min after application of Liu-Jun-Zi-Tang. In the presence of Liu-Jun-Zi-Tang, the concentration–response curve for ACh-contraction showed inhibition in a non-competitive manner, which is the same as domperidone. In the presence of Liu-Jun-Zi-Tang (1.0 mg/ml), ACh (10−7 M)-, histamine (5×10−7 M)- and barium chloride (10−4 M)-induced basal contractions were inhibited by 24, 19 and 54 %, respectively. On the other hand, in the presence of domperidone (2×10−5 M), ACh- and barium chloride-induced basal contractions were inhibited by 28 and 63%, respectively, similar to that in the presence of Liu-Jun-Zi-Tang. However, the inhibition of histamine-induced contraction in the presence of domperidone was significantly increased (81%) compared with that in the presence of Liu-Jun-Zi-Tang. These findings suggest that Liu-Jun-Zi-Tang has milder effects on histamine-induced disorders than domperidone. In other words, domperidone has a more potent effect on histamine-induced disorders.
Dipyrone is a non-narcotic analgesic and antipyretic drug used in both pediatric and adult patients. Dipyrone solution can be used intranasally as an antipyretic agent for infants. However, dipyrone is not stable in liquid state. Therefore, a stable dipyrone formulation was developed and the antipyretic effect of the formulation was studied after intranasal administration in rabbits and rats, respectively. To guarantee dose accuracy in animal studies, effect of dose volume on the distribution of dipyrone solution in rabbit nasal cavities were studied, using gentian violet as an indicator. Animal fever model and intranasal administration methods were established. In addition, the potential toxicity of the dipyrone formulation was studied. It was shown that the nasal volume of rabbits is large enough to hold 100 μl solution. After intranasal administration, improved pharmacodynamics was obtained with the new developed dipyrone formulation compared to the normal dipyrone solution, and significantly decreased body temperature was observed 10 min after dosing. The toxicity was negligible. In conclusion, the dipyrone formulation is effective and safe for clinical medication.
Mast cell-mediated anaphylactic reaction is involved in many allergic diseases such as asthma and allergic rhinitis. Phellinus linteus has been used as a traditional herb medicine in oriental countries and is known to have anti-tumor, immunomodulatory, anti-inflammatory, and anti-allergic activities. However, roles of Phellinus linteus in the mast cell-mediated anaphylactic reactions have not fully been examined. In the present study, we have investigated the effects of water extract from the fruiting body of Phellinus linteus (WEPL) on mast cell-mediated anaphylaxis-like reactions. Oral administration of WEPL inhibited the compound 48/80-induced systemic anaphylaxis-like reaction and ear swelling response. WEPL also inhibited the anti-dinitrophenyl (DNP) IgE-mediated passive systemic and cutaneous anaphylaxis. WEPL had no cytotoxicity on rat peritoneal mast cells (RPMC). WEPL dose-dependently reduced histamine release from RPMC activated by compound 48/80 or anti-DNP IgE. Moreover, WEPL decreased the compound 48/80-induced calcium uptake into RPMC. Furthermore, WEPL increased the level of intracellular cAMP and significantly inhibited the compound 48/80-induced cAMP reduction in RPMC. These results suggest that WEPL may serve as an effective therapeutic agent for allergic diseases.
Agaricus blazei is a medicinal mushroom native to Brazil. It used to be a source of anti-tumor and immunmoactive compounds and considered a health food in many countries. However, its specific effect against mast cell-mediated anaphylactic reactions is still unknown. In the present study, we investigated the effect of Agaricus blazei water extract (ABWE) on mast cell-mediated anaphylaxis-like reactions. We examined whether ABWE could inhibit systemic anaphylaxis-like reaction, ear swelling response, passive cutaneous anaphylaxis, and mast cell activation. ABWE inhibited compound 48/80-induced systemic anaphylaxis-like reaction, ear swelling response, and passive cutaneous anaphylaxis-like reaction in mice. ABWE also inhibited anti-dinitrophenyl (DNP) IgE-mediated passive cutaneous anaphylaxis. ABWE dose-dependently inhibited compound 48/80-induced or anti-DNP IgE-mediated histamine release from rat peritoneal mast cells (RPMC). Moreover, pretreatment with ABWE reduced compound 48/80-induced calcium uptake into RPMC. When ABWE was added, the level of intracellular cAMP in RPMC showed a significant increase compared with that of control cells. In addtion, ABWE significantly inhibited compound 48/80-induced cAMP reduction in RPMC. These results propose that ABWE may be beneficial in the treatment of mast cell-mediated anaphylactic reactions.
Increasing evidence has suggested an important role for rotenone in the pathogenesis of Parkinson's disease (PD). In this report, sequential linking of two culture systems, monocytic THP-1 cell line and SH-SY5Y neuroblastoma, was utilized. The supernatant from rotenone-stimulated THP-1 cells was used as the incubating medium for the second culture which adopted cells of the SH-SY5Y neuroblastoma. At 6.25—50 nM, concentrations that were nontoxic to SH-SY5Y directly, rotenone induced dose-dependent cell death on SH-SY5Y through stimulating monocyte THP-1 within a period of 48 h. Cytotoxicity was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Hoechst 33258 staining revealed that the treatment of SH-SY5Y with rotenone-stimulated THP-1 supernatant resulted in condensed nuclei and a decrease in cell size. Apoptotic rate measured by flow cytometric analysis indicated that at 25 and 50 nM, the percentage of apoptotic SH-SY5Y cells accumulated to 31.5% and 37.0% respectively. We further investigated whether rotenone (50 nM) activated mitogen-activated protein kinase (MAPK) cascades, and found it had effect on p38 MAPK and ERK in THP-1 cells, but not JNK. Pretreatment of THP-1 cells with the MAPK kinase inhibitor, PD98059, inhibited THP-1 cell-mediated rotenone neurotoxicity towards SH-SY5Y, whereas the p38 MEK inhibitor, SB203580, had no effect. These results suggested that activation of microglia intracellular signaling pathway may also involve in microglia-enhanced rotenone neurotoxicity.
Effects of four Si-Wu-Tang (SWT)'s constituents, fructose (Fru), paeoniflorin (Pae), ferulic acid (FA), tetramethyl pyrazine (TP), and their combination on irradiated mice as model of anaemia were investigated, with the purpose of further understanding the relationship between SWT's constituents and activities. Similarly to SWT, oral administration of Fru, Pae, FA, TP and their combination, to some extent, all showed effects of increasing the number of peripheral leukocyte and increasing four types of progenitor cells in bone marrow, including colony-forming unit-granulocyte-macrophage (CFU-GM), colony-forming unit-mature erythroid (CFU-E), colony-forming unit-immature erythroid (BFU-E) and colony-forming unit-multipotential (CFU-mix). Pae and FA showed significant body weight reducing effect, which were largely abolished when they were combined with Fru and TP. The SWT, Fru and combination significantly increased the thymus index while Pae significantly decreased it. Both SWT and TP significantly increased the spleen index but the combination did not. The results suggested that multiple constituents contribute to the promoting effect of SWT on hematopoiesis. Although being a very common compound in plants, the Fru has a special contribution to SWT's effect, which cannot be neglected. It may be an important active constituent that is responsible for SWT's promoting effect on hematopoiesis and immunity. Another suggestion is that when being combined, some effect of one constituent, sometimes is unexpected side effect, may be abolished by other. This may reflect the advantage of multiple constituent characteristics possessed by most TCMs.
SA1 is a mixture of 9 Oriental herbs (Korean red ginseng, fermented soybean, Tribulus terrestris, Fructus Rubi, Fructus Lycii, Semen Cuscutae, Dioscorea Rhizome, Fructus Corni and Fructus Crataegi) that are widely used as energizers and vitalizers in the indigenous system of medicine and have been alleged to improve the sexual functions in men. This study evaluated SA1 using both in vitro and in vivo experiments on laboratory animals in order to determine its effect on the sexual behavior and penile erection. The male rats used to examine the copulatory behavior were administered either the vehicle or SA1 (30, 100, 300, 600 mg/kg) orally for 2 weeks. The intracavernous pressure and systemic blood pressure were recorded in anesthetized rats. The responses to acetylcholine and SA1 of rabbit corpus cavernosum strips were also examined. There was an overall increase in the copulatory behavior parameters in the SA1-treated rats, which was reflected by a decrease in the mount and intromission latencies and an increase in the ejaculation latency and mount frequency. SA1 significantly increased the ratio of the intracavernous pressure to mean arterial pressure. In vitro, SA1 significantly enhanced the relaxation responses to acetylcholine. These results suggest that SA1 improves the sexual activity and erectile function.
We have reported that transferrin (Tf)-unbound Gallium-67 (67Ga) was taken up into hepatocytes 1 d after carbon tetrachloride (CCl4)-treatment in rats. It had been reported that the binding affinity of Iron-59 (59Fe) to Tf was greater than that of 67Ga. In the present study, we investigated whether or not Tf was involved in 59Fe uptake by hepatocytes of CCl4-damaged rats. The results showed that the uptake of 59Fe by hepatocytes and the number of Tf-receptor decreased 1 d after CCl4-treatment and increased 2 d after the treatment. Our data demonstrated that the uptake of 59Fe by hepatocytes differed from that of 67Ga and Tf was involved in that of 59Fe. We expect that these finding could serve to analyze the inflammatory stages, disorder stage at 1 d and regeneration stage at 2 d after CCl4-treatment.
The Opuntia ficus-indica var. saboten MAKINO (OFI) has been traditionally used as health food and herbal agent in folk medicine in Korea. In this study, we investigated whether the OFI glycoprotein has antioxidative activity and hypolipidemic effect on Triton WR-1339-induced A/J mice. The OFI glycoprotein inhibits the production of reactive oxygen species (ROS) generated by glucose/glucose oxidase (G/GO) in BNL CL.2 cells. With its antioxidative property, the mice were orally administered in the OFI glycoprotein [50 mg/kg body weight (BW)] for two weeks. Our finding resulted in a significant decrease of plasma lipid levels in Triton WR-1339-treated mice such as total cholesterol (TC), triglyceride (TG), and low-density lipoprotein (LDL). Indeed, mice which induced by Triton WR-1339 were significantly increased the levels of TC, TG and LDL, whereas the high-density lipoprotein (HDL) level obviously decreased. However, the values were reversed at pretreatment with OFI glycoprotein in Triton WR-1339-treated mice. The data also showed that pretreatment with OFI glycoprotein resulted in decrease of thiobarbituric acid-reactive substances (TBARS) level and in increase of nitric oxide (NO) amount in presence of Triton WR-1339-treated mice, while the activities of antioxidant enzyme [superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)] were augmented. Therefore, we speculate that the OFI glycoprotein would be effective in lowering of plasma lipid levels.
Anticholinergic agents such as oxybutynin are clinically useful in the treatment of overactive bladder. However, oral administration of oxybutynin is frequently accompanied by side effects such as dry mouth, and novel bladder-selective anticholinergic agents such as solifenacin and tolterodine are now under development. The aim of the present study was to characterize the suppression of cholinergic salivation and exocrine muscarinic receptor binding of solifenacin on oral administration to mice in comparison with those of oxybutynin. Results showed that both drugs produced a significant increase in Kd values for specific [N-Methyl-3H]scopolamine methyl chloride ([3H]NMS) binding in the mouse submaxillary gland, compared with control values. However, this enhancement in Kd values was significantly smaller with solifenacin than with oxybutynin. Moreover, the inhibitory effect of solifenacin on pilocarpine-induced salivary secretion was significantly weaker than that of oxybutynin. Solifenacin dissociated more readily from muscarinic receptors in the mouse submaxillary gland than oxybutynin. In conclusion, the present study indicates that the weak suppression of cholinergic salivation by solifenacin compared with oxybutynin may be partially attributed to its relatively fast dissociation kinetics from exocrine muscarinic receptors.
1-Methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ), an endogenous parkinsonism-preventing substance, and its 5-, 6-, and 7-hydroxylated derivatives are reported to show in vitro neuroprotective activity against toxicity due to salsolinol in SH-SY5Y human neuroblastoma cells. In the present study, we tested the parkinsonism-preventing potential of these derivatives by means of the pole test in C57BL mice in vivo, and measured brain dopamine contents by liquid chromatography-tandem mass spectrometry. Parkinsonism was induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydroisoquinoline(MPTP), and pretreatment with any of the 1MeTIQ derivatives prevented its induction. 6-Hydroxy-1MeTIQ showed the greatest preventive activity. The amount of dopamine in the brain was reduced by MPTP treatment, and this reduction was suppressed by pretreatment with 1MeTIQ derivatives. These hydroxy-1MeTIQ derivatives may have potential for the treatment of Parkinson's disease as well as 1MeTIQ itself.
We evaluated the cytotoxicity and underlying mechanisms of cardiac glycosides, including digoxin, ouabain and proscillaridin A, on the proliferation of breast cancer MCF-7 cells. In terms of inhibition of cell proliferation of MCF-7 cells, the compounds rank in the order proscillaridin A>digoxin>ouabain. While both digoxin and ouabain inhibited topoisomerase II catalytic activity at nanomolar concentrations (100 nM), neither agent inhibited topoisomerase I catalytic activity even at concentrations as high as 100 μM. On the other hand, proscillaridin A was a potent poison of topoisomerase I and II activity at nanomolar drug concentrations (30 nM, 100 nM, respectively), suggesting that this agent may produce its cytotoxic activity by targeting both enzymes simultaneously. These studies suggest that the stabilization of DNA-topoisomerase II complexes is closely linked to the mechanism of digoxin, ouabain and proscillaridin A cytotoxicity. The potential DNA-binding properties of the cardiac glycosides have been assessed by measuring the displacement of ethidium bromide from calf thymus DNA. These results indicate that digoxin, ouabain and proscillaridin A neither intercalate nor interact with the minor groove of DNA.
In this study, we gave the soybean powder-added food pellets (soybean pellets) to investigate anti-anxious effects of soybean in male mice. Twenty eight days after feeding control pellets or soybean pellets, we observed the behavioral changes in the elevated plus maze. There was no significant difference on the time spent in the open arms (%) between mice fed the control and soybean pellets. When we administered m-chlorophenylpiperazine (m-CPP, 2.5 mg/kg, i.p.) to mice, the mice fed control pellets showed the decrease in the time spent in the open arms, suggesting that anxiety-like behavior was induced by m-CPP. On the other hand, we could not observe the m-CPP-induced anxiety-like behavior in mice fed soybean pellets in this test. These results suggest that soybean pellets may attenuate anxiety-like behavior in mice.
We investigated the superoxide anion scavenging effects of 2-amino-1,3- selenazoles and bis-(2-amino-5-selenazoyl) ketones using a highly sensitive quantitative chemiluminescence method. At 166 μM, the 2-amino-1,3-selenazoles and bis-(2-amino-5-selenazoyl) ketones scavenged in the range of 10.0 to 80.0% of O2−. Bis[2-dimethylamino-5-(1,3-selenazolyl)] ketone exhibited the strongest superoxide anion-scavenging activity among the six kinds of 2-amino-1,3-selenazoles and three kinds of bis-(2-amino-5-selenazoyl) ketones. The 50% inhibitory concentration (IC50) of bis[2-dimethylamino-5-(1,3-selenazolyl)] ketone was determined to be 37.1 μM. Thus, bis[2-dimethylamino-5-(1,3-selenazolyl)] ketone acted in vitro as effective and potentially useful O2− scavenger.
Articular cartilage is a potential target for drugs designed to inhibit the activity of matrix metalloproteinases (MMPs) to stop or slow the destruction of proteoglycan and collagen in the cartilage extracellular matrix. The purpose of this study was to investigate the effects of Betula platyphylla var. japonica on inhibiting the release of glycosaminoglycan (GAG), the degradation of collagen, and MMP expression and activity in rabbit articular cartilage explants. Interleukin-1α (IL-1α) rapidly induced GAG, but collagen was much less readily released from cartilage explants. Betula platyphylla var. japonica significantly inhibited GAG and collagen release in a concentration-dependent manner. Betula platyphylla var. japonica dose-dependently inhibited MMP-3 and MMP-13 expression and activities from IL-1α-treated cartilage explant culture when tested at concentrations ranging from 0.02 to 0.2 mg/ml. Betula platyphylla var. japonica had no harmful effect on chondrocyte viability or cartilage morphology in cartilage explants. Histological analysis indicated that Betula platyphylla var. japonica reduced the degradation of the cartilage matrix compared with that of IL-1α-treated cartilage explants. These results indicate that Betula platyphylla var. japonica inhibits the degradation of proteoglycan and collagen through the down regulation of MMP-3 and MMP-13 expression and activities without affecting the viability or morphology of IL-1α-stimulated rabbit articular cartilage explants.
Fossilia Mastodi OSSIS, which is a skeletal fossil of a Mastodon, an ancient mammal, has been found to have anxiolytic, sedative and anticonvulsant activities in Oriental medicine. In this study, in vivo characterization of the sedative activities of Fossilia Mastodi OSSIS was performed in order to obtain basic information for the development of a putative natural sedative. The 80% methanol extract of Fossilia Mastodi OSSIS given per os at a dose of 3 g/kg in mice showed anxiolysis, potentiation of pentobarbital sleeping time, reduced locomotor activity, and anticonvulsive activity. Fossilia elicited GABAA receptor-mediated anxiolysis. The data obtained suggest that the 80% methanol extract of Fossilia Mastodi OSSIS contains some biologically active principles with sedative activity.
The genus Aloe in the family Liliaceae is a group of plants including Aloe vera (Aloe barbadensis MILLER) and Aloe arborescens (Aloe arborescens MILLER var. natalensis BERGER) that are empirically known to have various medical efficacies. In the present study, we evaluated the anti-hyperglycemic effect of Aloe vera gel and isolated a number of compounds from the gel. On the basis of spectroscopic data, these compounds were identified as lophenol, 24-methyl-lophenol, 24-ethyl-lophenol, cycloartanol, and 24-methylene-cycloartanol. These five phytosterols were evaluated for their anti-hyperglycemic effects in type 2 diabetic BKS.Cg-m+/+Leprdb/J (db/db) mice. In comparison with the hemoglobin A1c (HbA1c) levels of vehicle-treated mice, statistically significant decreases of 15 to 18% in HbA1c levels were observed in mice treated with 1 μg of the five phytosterols. Considering the ability to reduce blood glucose in vivo, there were no differences between the five phytosterols. Administration of β-sitosterol did not reduce the blood glucose levels in db/db mice. After administration of the five phytosterols for 28 d, fasting blood glucose levels decreased to approximately 64%, 28%, 47%, 51%, and 55% of control levels, respectively. Severe diabetic mice treated with phytosterols derived from Aloe vera gel did not suffer weight reduction due to glucose loss in the urine. These findings suggest that Aloe vera gel and phytosterols derived from Aloe vera gel have a long-term blood glucose level control effect and would be useful for the treatment of type 2 diabetes mellitus.
Cartilage loss in osteoarthritis is characterized by cartilage degradation and chondrocyte death. Cartilage degradation is induced by activation of matrix-metalloproteinases (MMPs) activity and degradation of glycosaminoglycan (GAG) and collagen. Also, chondrocyte death is induced by the apoptosis through the activation of MAP kinase and caspases activities. On the basis of this background, our study was designed to examine the cartilage protective and anti-apoptotic effect of Aralia cordata. Cartilage explants and Chondrocytes were cultured from rabbit knee joint cartilage and treated by 5 ng/ml IL-1α. Cartilage and chondroprotective effects of Aralia cordata were determined by measuring (1) GAG and collagen expression, (2) GAG and collagen degradation, (3) TIMP and MMPs expression, and (4) TIMP and MMPs activity. Anti-apoptotic effects of Aralia cordata were determined by measuring (1) JNK and p38 MAP kinase expression, (2) apoptotic cells by flow cytometry, and (3) caspase-3 activity. In cartilage explants and chondroctyes treated by IL-1α, Aralia cordata showed the decrease of GAG and collagen degradation, decrease of MMPs (MMP-1, -3, -13) activity, and increase of TIMP-1 activity in a dose-dependent manner. Aralia cordata also showed anti-apoptotic effect by inhibition of early and late apoptotic cells, sub-G1 phase cells, and caspase-3 activity through the downregulation of JNK and p38 MAP kinase signaling pathway. Aralia cordata inhibited the cartilage and chondrocyte destruction through the downregulation of MMPs activities and the inhibition of proteoglycan and collagen degradation. Also, Aralia cordata inhibited the chondrocyte apoptosis through the downregulation of JNK and p38 MAP kinase signal, and the inhibition of caspase-3 activity.
The present study investigates the effects of Yukmijihwang-tang Derivatives (YMJd) on learning and memory through the Morris water maze task and the central cholinergic system of rats with excitotoxic medial septum (MS) lesion. In the water maze test, the animals were trained to find a platform in a fixed position for 6 d and then received a 60-s probe trial in which the platform was removed from the pool on the 7th day. Ibotenic lesion of the MS showed the impaired performance in the Morris water maze test and severe cell losses in the MS, as indicated by decreased choline acetyltransferase-immunoreactivity in the medial septum. Daily administrations of YMJd (100 mg/kg, i.p.) for 21 consecutive days produced significant reversals of ibotenic acid-induced deficit in learning and memory. These treatments also reduced the loss of choline acetyltransferase (ChAT) immunoreactivity in the MS induced by ibotenic acid. These results suggest that impairments of spatial learning and memory might be attributable to the degeneration of septohippocampal cholinergic (SHC) neurons and that YMJd treatment ameliorated learning and memory deficits partly due through neuroprotective effects on the central acetylcholine system. Our studies suggest that YMJd might be useful in the treatment of Alzheimer's disease.
Brickellia paniculata has been used as spasmolytic in Mexican traditional medicine. Xanthomicrol and 3α-angeloyloxy-2α-hydroxy-13,14Z-dehydrocativic acid (AAHDD) are two of the main leaf components with antispasmodic activity. However, their mechanism of action remains unknown. An in vitro comparative study between xanthomicrol and AAHDD on rat uterus precontracted by either KCl (60 mM) or oxytocin (10 mIU/ml) was carried out to investigate the mechanism of action of these compounds on smooth muscle. Relaxant effect was measured as median inhibitory concentration (IC50) and maximal effect as maximal relaxant response (Rmax). Xanthomicrol was significantly more potent than AAHDD in inhibiting contractions induced by KCl 60 mM, whereas AAHDD was more potent than xanthomicrol in inhibiting contractions induced by oxytocin 10 mIU/ml. These results suggest that xanthomicrol induces a greater blocking effect on voltage-operated calcium channels than on receptor-operated gates.
The inhibitory activity of 40 stilbene oligomers isolated from six plant species against topoisomerase II was evaluated, of which nine compounds showed a potent inhibitory effect, stronger than daunorubicin, a topoisomerase II inhibitor, used as an anti-cancer drug. The specificity of active stilbene oligomers on topoisomerase II was assessed by their effect on DNA restriction enzyme. In particular, specific inhibitory activity was observed in α-viniferin 13-O-β-glucopryranoside (2) and hemsleyanol C (13).
We investigated the pharmacokinetic behavior of palmitoyl prednisolone (Pal-PLS) and its liposomes with L-α-distearoylphosphatidylcholine (DSPC) and cholesterol (Chol) with or without L-α-distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG 2000) after their intravenous administration in rats. Pal-PLS rapidly disappeared from the systemic circulation and prednisolone (PLS) was regenerated after the administration of DSPC/Chol liposomes. PEGylated liposomes including DSPE-PEG 2000, however, successfully maintained high blood concentrations of Pal-PLS and PLS. The blood profiles of drugs after the administration of liposomal Pal-PLS were analyzed according to a two-compartment model. The larger content of DSPE-PEG 2000 in DSPC/Chol liposomes showed a lower first order elimination rate constant from the central compartment (Kel) and clearance (CL). The area under the concentration–time curve (AUC) of Pal-PLS and PLS in PEGylated liposomes was larger than DSPC/Chol liposomes. The mean resident time (MRT) of Pal-PLS and PLS was also prolonged by PEGylated liposomes. Although DSPC/Chol liposomes showed a high distribution of Pal-PLS in the liver and spleen, PEGylated liposomes significantly decreased the liver distribution of Pal-PLS. The biliary and urinary excretions of drugs for 240 min after drug administration were less than 1% of the administrated dose in any formulations. In conclusion, PEGylated liposomes, including Pal-PLS, are useful for maintain the PLS concentration in the blood after intravenous administration.
For hormone refractory prostate carcinoma, a combination therapy of paclitaxel and carboplatin is used to expect life extention. We investigated the pharmacokinetics of carboplatin in Japanese prostate cancer patients (n=10, 55—72 years), and evaluated the usefulness of Calvert's formula in the individualized dosing adjustment. They were intravenously administered carboplatin (area under the free plasma concentration versus time curve (AUC)=5 mg·min/ml), following the intravenous administration of paclitaxel (175 mg/m2). The dosage of carboplatin for each patient was determined with Calvert's formula using individual creatinine clearance values. Plasma concentration of total platinum was measured sequentially and the pharmacokinetic parameters of carboplatin were determined in each patient. Plasma concentration of total carboplatin after intravenous infusion well fitted the two-compartment model. Carboplatin clearance was 62.0±12.7 ml/min (mean±S.D.), and linearly related to the individual creatinine clearance (r2=0.64, p<0.01). The actual AUC for total carboplatin was 8.20±1.11 mg·min/ml, and its inter-individual variability was decreased to 65% of that in carboplatin clearance, indicating the effectiveness of Calvert's formula for dosage adjustment of carboplatin. Leucopenia of grade 4 according to the National Cancer Institute's Common Toxicity Criteria was found in one patient, but no patient demonstrated thrombocytopenia. In conclusion, determining carboplatin dosage based on Calvert's formula decreased the inter-individual variability in the actual AUC compared with that in the carboplatin clearance, and a target AUC of 5 mg·min/ml of carboplatin was comparatively safe for Japanese patients with prostate cancer.
The effect of lecithin coating on the pulmonary absorption of furosemide after application by metered dose inhalers (MDI) containing HFA 227 was evaluated in rats. The plasma concentration of furosemide after application of lecithin-coated furosemide was higher than that after application of the un-coated form. Since the disposition in the lung 2 min after application of un-coated furosemide was significantly lower than that after application of the coated form and the adsorption to a polyethylene tube used for the application of the un-coated form was significantly higher than that of the coated form, the higher plasma concentration after application of lecithin-coated furosemide could be partly related to the efficient delivery of the furosemide particles to the lung. The permeation-enhancing effect of the lecithin coating was investigated using Calu-3 cell monolayers. The cumulative amount of furosemide permeated over 2 h from a suspension containing lecithin-coated furosemide through the monolayers was significantly higher than that from a conventional furosemide suspension. This enhancing effect could also contribute to the high plasma concentration of furosemide in rats. The lecithin-coated furosemide will be useful for the formulation of MDI offering high bioavailability.
The expression level of MDR1 mRNA was evaluated in colorectal adenocarcinomas and adjacent noncancerous colorectal tissues obtained from 21 Japanese patients. It was lower in the former than in the latter (p=0.012), suggesting its down-regulation as a consequence of malignant transformation of colorectal tissues, possibly with the suppression of differentiation. Relatively lower expression was suggested in moderately-differentiated colorectal adenocarcinomas than well-differentiated ones, but there was no statistical difference (p=0.111). MDR1 mRNA up-regulation was found in a colorectal adenocarcinoma cell line, HCT-15, after treatment with two typical differentiating agents, sodium butyrate and all-trans retinoic acid, suggesting its involvement in the cellular events, resulting in differentiation without malignant transformation. MDR1 T-129C, but not G2677A,T and C3435T, was associated with the lower expression of MDR1 mRNA both in colorectal adenocarcinomas (p=0.040) and adjacent noncancerous colorectal tissues (p=0.023), possibly being an useful invasive marker predicting poorly-differentiated colorectal adenocarcinomas and thereby the poor prognosis of the patients, especially when no extra biopsy samples will be obtained. Further investigations with relatively large number of patients should be undertaken to confirm these preliminary results.
A novel and convenient method to predict the pharmacokinetics of several kinds of antibiotic agents in patients with end-stage renal disease (ESRD) was examined based on the in vitro extraction ratios and pharmacokinetic parameters in healthy volunteers. The dializability of 17 antibiotic agents in 4% human serum albumin solution were determined using a high-performance hemodialytic membrane for clinical use. We assumed that the off-hemodialysis clearance approximated the non-renal clearance, while the on-hemodialysis clearance was considered to be sum of the off-hemodialysis clearance and the hemodialytic clearance. The estimated on- and off-hemodialysis clearances were compared with the ones observed in ESRD patients. In order to confirm the method prospectively, an in vivo pharmacokinetic study was performed in dogs with mercury chloride-induced experimental renal failure. The in vitro extraction ratios of 9 β-lactams were broadly ranged from 10.9 to 75.6% depending on their physicochemical properties. In contrast, those of the other antibiotics were consistent with their chemical classes: 60.5—63.2% for fluoroquinolone, 48.8—51.1% for aminoglycoside and 18.7—25.6% for glycopeptide. Both the estimated on- and off-hemodialysis clearances of the 17 antibiotics coincided well with the observed values in the literature, regardless of their physicochemical and pharmacokinetic properties. The validity and applicability of this method to three cefems, cefmetazole, cefotaxime and cefoperazone, was prospectively confirmed in the animal study. In conclusion, this new method enables the prediction of the on- and off-hemodialysis clearances of several kinds of antibiotics in ESRD patients from minimal information of their pharmacokinetics in healthy subjects and their in vitro dializability.
We investigated Staphylococcus aureus (S. aureus) contamination on the surface of working tables in ward staff centers and the effects of disinfection by wiping with 80% (v/v) ethyl alcohol on this contamination. When working tables were not regularly disinfected or washed, S. aureus [methicillin-sensitive S. aureus (MSSA) and/or methicillin-resistant S. aureus (MRSA)] was detected in 29 (51.8%) of 56 tables in 6 wards investigated. MRSA was detected in 17 (30.4%) of 56 tables and in all investigated wards. The S. aureus contamination density on the entire surface of the S. aureus-contaminated tables was 2081±8915 (mean±S.D.) colony-forming units (cfu) (n=29, range, 10—4.8×104 cfu), and the MRSA contamination density on the entire surface of the MRSA-contaminated tables was 158±200 cfu (n=17, range, 10—7.4×102 cfu). The second investigation was performed immediately after working tables not regularly disinfected or washed were disinfected by wiping once with 80% (v/v) ethyl alcohol, and MRSA was detected in 5 (8.9%) of 56 tables. The contamination density on the entire surface of the MRSA-contaminated tables was 36±30 cfu (n=5, range, 10—80 cfu). The third investigation was performed immediately after working tables not regularly disinfected or washed were disinfected by wiping with 80% (v/v) ethyl alcohol twice with a 1-min interval, and no S. aureus contamination was observed in any of 56 tables. These results suggest that disinfection by wiping once with 80% (v/v) ethyl alcohol could not completely eliminate MRSA in particulates. For the disinfection of the MRSA-contaminated surface of working tables and the removal of particulates, regular disinfection by wiping with 80% (v/v) ethyl alcohol is necessary.
An in vitro cell culture model that mimics in vivo extracellular environment would be useful in developing in vivo gene delivery system. In the present study, a parallel flow model was applied to investigate the impact of convective flow on cellular uptake and transfection activity in endothelial cells. LipofectAMINE PLUS and adenovirus were used as model vectors, which bind cells via electrostatic- and ligand-receptor interactions, respectively. Whereas a convective flow increased the total amount of vector passing through the flow chamber by 3 orders of magnitude, uptake was increased by less than 10-fold, suggesting that the flow severely inhibited cellular uptake by reducing the retention time in the chamber and/or by diminishing the affinity between the cell and vector. Moreover, the uptake of both vectors was increased in a shear stress-dependent manner to a comparable extent, suggesting that the effect of flow on the cellular uptake was not significant. In contrast, transfection efficiency (TE), expressed as the transfection activity normalized by the cellular uptake of vectors was dramatically stimulated by shear stress, only when LipofectAMINE PLUS was used. Since the activities of the CMV promoter were unaffected by a shear stress, it is possible that altered intracellular trafficking may responsible for the improvement in lipoplex-mediated TE, presumably related to the cellular uptake pathway.
Activated macrophages are the key effector cells in rheumatoid arthritis (RA) and secrete multiple mediators of inflammation including proinflammatory cytokines. We investigated delivery of a nuclear factor kappa B (NFκB) decoy by folate-linked lipid-based nanoparticles (NP-F) into murine macrophages. The expression of folate receptor (FR) in RAW264.7 cells activated by lipopolysaccaride was confirmed by strong expression of FR mRNA, and association of FITC-labeled folate-BSA conjugate. When transfected via NP-F, the NFκB decoy was strongly detected in the cytoplasm, and an inhibitory effect on the translocation of NFκB into the nucleus was observed at 0.03 μM of the decoy, suggesting that NP-F effectively delivered the NFκB decoy into the cytoplasm. This information is of value for the design of NFκB decoy carrier systems targeting FR in activated macrophages in gene therapy for autoimmune diseases such as RA.
The purpose of this study was to examine the effect of the size of galactosylated cationic liposome (Gal-liposome)/plasmid DNA complex (Gal-lipoplex) on hepatocyte-selective gene transfection after intraportal administration. pCMV-Luc was selected as a model plasmid DNA. After intraportal administration of Gal-lipoplex to mice, the hepatic and intrahepatic gene expression was evaluated. To evaluate the effect of size, three different sizes of Gal-liposome were prepared. The mean particle sizes of Gal-lipoplex were about 141, 179, and 235 nm, respectively. The hepatic transfection efficacy was significantly enhanced by increasing the size of Gal-lipoplex. However, the gene expression in liver parenchymal cells (PC) of Gal-lipoplex of about 141 nm in size was significantly higher than that in liver non-parenchymal cells (NPC). In contrast, gene expression in PC of Gal-lipoplex of about 235 nm in size was significantly lower than that in NPC. These results highlight the importance of the Gal-lipoplex size for hepatocyte-selective gene transfer in vivo. The information in this study will be valuable for the future use, design, and development of Gal-lipoplex for in vivo applications.
In this study we aimed to determine the role of piracetam (PIR) in preventing radiation induced cochlear damage after total-cranium irradiation (radiotherapy; RT). Male albino guinea pigs used in the study were randomly divided into three groups. Group 1 (Control group) (n=11) received neither PIR nor irradiation, but received saline solution intraperitoneally (i.p.) and received sham irradiation. Group 2 (RT group) (n=32) was exposed to total cranium irradiation of 33 Gy in 5 fractions of 6.6 Gy/d for five successive days, with a calculated (α/β=3.5) biological effective dose of fractionated irradiation equal to 60 Gy conventional fractionation, then received saline solution for five successive days i.p. Group 3 (PIR+RT group) (n=33) received total cranium irradiation, plus 350 mg/kg per day PIR for five successive days i.p. After the last dose of RT, the guinea pigs were all sacrificed at the 4th, 24th and 96th hours, respectively. Their cochleas were enucleated for histopathologic examination. It was observed that total cranium irradiation (RT group) promoted degeneration in stria vascularis (SV), spiral ganglion cells (SG), outer hair cells (OHC) and inner hair cell (IHC) of cochleas at these times (p<0.05). While in the PIR+RT group, there was no statistically significant difference on radiation-induced cochlear degeneration in SV and OHC at 4th (p>0.05) and IHC at 4th, 24th hours (p>0.05), there was a significant difference on radiation-induced cochlear degeneration in SV and OHC at 24th and 96th hours (p<0.05), IHC at 96th hour (p<0.05) and SG at 4th, 24th and 96th hours (p<0.05). There was no any cochlear degeneration in the control group. Piracetam might reduce radiation-induced cochlear damage in the guinea pig. These results are pioneer to studies that will be performed with PIR for radiation toxicity protection.
We examined region-dependent differences and alterations in the levels of protective thiol compounds, glutathione (GSH) and metallothionein (MT)-I and -II, in cultured rat astrocytes under several culture conditions and in brain tissues of rats at postnatal and weaning periods. Regardless of culture conditions, both protein concentrations and mRNA expressions of MT-I and -II were much higher in the cerebral hemisphere than in cerebellar astrocytes, whereas no difference was observed in GSH concentration. In both astrocytes, the GSH concentrations did not change within 12 h but significantly increased 24 h after being maintained in a serum-free defined medium. At 24 h, protein concentrations and mRNA expressions of MT-I and -II also increased in the respective astrocytes, and were further enhanced when maintained in the presence of 50 μM Zn2+. In the brain tissues, the MT-I/-II protein concentrations were significantly higher in the cerebral cortex (a part of the cerebral hemisphere) than in the cerebellum, whereas the GSH concentration was similar at both postnatal day (P)1 and P35. In addition, the concentrations in the respective regions were significantly higher at P35 than at P1. These results suggest that region-dependent differences in the cellular levels of GSH and MTs in cultured astrocytes might reflect the in vivo differences, and that the levels of the respective thiol compounds in cultured astrocytes increase after serum elimination along with the region-dependent differences.
Intestinal epithelial cells (IECs) have been known to produce galactose-α1,4-galactose-β1,4-glucose ceramide (Gb3) which plays a pivotal role in the mucosal immune response. In particular, Shiga-like toxins (Stx) can induce apoptosis of IECs in the development of hemolytic uremic syndrome (HUS) through binding on Gb3. Therefore, it has been hypothesized that down-regulation of Gb3 (or binding of Stx) prevents Stx from damaging in IECs. This study investigated whether curcumin, having various biological properties such as being anti-bacterial, anti-viral and anti-cancer, could decrease binding of Stx and the related signal pathway. Curcumin significantly inhibited the binding of Stx and the production of Gb3 synthase (GalT6) mRNA in HT29 IECs stimulated with TNF-α and IL-1β. Additionally, curcumin was able to inhibit mitogen-activated protein kinases (MAPKs), such as p38 and JNK, but not ERK1/2, degradation of IκB or translocation of NF-κB p65. Furthermore, curcumin significantly attenuated Stx-1 induced cell death and IL-8 expression. In summary, these data link Gb3 expression in HT29 cells stimulated with TNF-α and IL-1β and suggest that blocking of Stx-binding by curcumin may prevent the Stx-associated HUS.
Turmeric, the powdered dry rhizome of the Curcuma longa plant, and curcumin, the major anti-oxidant constituent of turmeric, have been shown to possess chemopreventive activity. To elucidate the possible interaction of turmeric and curcumin with conjugation reactions, which in many cases are involved in the activation of procarcinogens, we measured their effects in the conjugation of 1-naphthol in Caco-2 cells, a human colon carcinoma cell line, within a 24 h period. Turmeric exhibits inhibitory activity toward both sulfo- and glucuronosyl conjugations of 1-naphthol at approximately the same levels (IC50=0.24 and 0.29 mg/ml, respectively). Curcumin inhibits sulfo-conjugation at lower concentrations (IC50=9.7 μg/ml), but only showed weak inhibition toward glucuronosyl conjugation of 1-naphthol in Caco-2 cells. In addition, turmeric was found to strongly inhibit in vitro phenol sulfotransferase (SULT) activity and demonstrate moderate inhibitory properties against UDP-glucuronosyl transferase (UGT) activity in Caco-2 cells (IC50=0.17 mg/ml and 0.62 mg/ml, respectively). Curcumin also strongly inhibits in vitro phenol sulfotransferase activity with an IC50 of 2.4 μg/ml. Moreover, and in contrast to the moderate inhibition of UGT activity by turmeric and curcumin, both induce the expression of the UGT1A1 and UGT1A6 genes, revealed by real-time PCR analysis. These findings are indicative of a possible interaction of both turmeric and curcumin with conjugation reactions in the human intestinal tract and colon. This in turn may affect the bioavailability of therapeutic drugs and toxicity levels of environmental chemicals, particularly procarcinogens.