Oxidized low-density lipoprotein (OxLDL) has previously been thought to promote atherogenesis through foam cell formation. However, the actual nature of OxLDL present in vivo remained obscure until recently. We have produced a monoclonal antibody, DLH3, which specifically binds to OxLDL but not to native LDL. The presence of OxLDL in the LDL fraction of human plasma was demonstrated by introducing a sandwich ELISA procedure using DLH3 together with an anti-apoB antibody. Furthermore, OxLDL levels appeared to increase in certain pathological conditions including acute myocardial infarction and carotid artery atherosclerosis. Accumulation of OxLDL in atherosclerotic lesions has also been demonstrated by immunohistochemical and biochemical studies using the DLH3 antibody. This antibody recognizes oxidized phosphatidylcholines (OxPC) generated during oxidative modification of LDL, and OxPC-apoB adducts formed in OxLDL are the presumed antigens. Measuring OxLDL in plasma would be a useful diagnostic tool for cardiovascular diseases. However, there still remain some major questions related to OxLDL, the answers to which are crucial for understanding the mechanisms of atherogenesis.
A crude extract was prepared with ethanol from dried ripened Vitex agnus-castus fruits growing in Israel (Vitex extract). Cytotoxicity of the extract against human uterine cervical canal fibroblast (HCF), human embryo fibroblast (HE-21), ovarian cancer (MCF-7), cervical carcinoma (SKG-3a), breast carcinoma (SKOV-3), gastric signet ring carcinoma (KATO-III), colon carcinoma (COLO 201), and small cell lung carcinoma (Lu-134-A-H) cells was examined. After culture for 24 h (logarithmic growth phase) or 72 h (stationary growth phase), the cells were treated with various concentrations of Vitex extract. In both growth phases, higher growth activity of cells and more cytotoxic activity of Vitex extract were seen. The cytotoxic activity against stationary growth-phase cells was less than that against logarithmic growth-phase cells. DNA fragmentation of Vitex extract-treated cells was seen in SKOV-3, KATO-III, COLO 201, and Lu-134-A-H cells. The DNA fragmentation in Vitex extract-treated KATO-III cells was inhibited by the presence of the antioxidative reagent pyrrolidine dithiocarbamate or N-acetyl-L-cysteine (NAC). Western blotting analysis showed that in Vitex extract-treated KATO-III cells, the presence of NAC also inhibited the expression of heme oxygenase-1 and the active forms of caspases-3, -8 and -9. It is concluded that the cytotoxic activity of Vitex extract may be attributed to the effect on cell growth, that cell death occurs through apoptosis, and that this apoptotic cell death may be attributed to increased intracellular oxidation by Vitex extract treatment.
The mechanism of the elevation of intracellular glutathione induced by low-dose γ-rays was examined in RAW 264.7 cells. The expression of mRNA for γ-glutamylcysteine synthetase (γ-GCS) increased soon after γ-ray (0.5 Gy) irradiation, and peaked between 3 h and 6 h post-irradiation. A dose of 0.25 to 0.5 Gy was optimum for induction of γ-GCS mRNA expression at 3 h post-irradiation. The effect of inhibitors of activator protein-1 (AP-1) and nuclear factor κB (NF-κB) on the radiation-induced γ-GCS gene expression was then examined. The induction of γ-GCS mRNA expression was significantly suppressed when AP-1 DNA binding, but not NF-κB DNA binding, was inhibited. Finally, electrophoretic mobility shift assay showed that the low-dose radiation markedly increased the DNA binding of AP-1, but not NF-κB, soon after irradiation. These results suggest that the increase of glutathione levels in RAW 264.7 cells by low-dose γ-ray irradiation is mediated by transcriptional regulation of the γ-GCS gene, predominantly through the AP-1 binding site in its promoter.
We previously reported that several gastric irritants, including ethanol, hydrogen peroxide, and hydrochloric acid, induced both necrosis and apoptosis in cultured gastric mucosal cells. In the present study, we examined the effects of sucralfate, a unique gastroprotective drug, on gastric irritant-induced necrosis and apoptosis produced in vitro. Sucralfate strongly inhibited ethanol-induced necrosis in primary cultures of guinea pig gastric mucosal cells. The preincubation of cells with sucralfate was not necessary for its cytoprotective effect to be observed, thus making its mechanism of action different from that of other gastroprotective drugs. Necrosis of gastric mucosal cells induced by hydrogen peroxide or indomethacin was also suppressed by sucralfate. On the other hand, sucralfate only weakly inhibited ethanol-induced apoptosis. These results suggest that the cytoprotective effect of sucralfate on gastric mucosa in vivo can be explained, at least in part, by its inhibitory effect on gastric irritant-induced necrosis.
Okadaic acid (OA) decreased the leptin content in isolated mouse fat pads in a time and dose-dependent manner. MG-132, a membrane-permeable proteasome inhibitor, prevented the decrease by OA, suggesting the involvement of proteasome in the OA action. No significant decrease in the incorporation of [3H]leucine into leptin was observed with a 4-h incubation, although the amino acid incorporation was stimulated by insulin and decreased by cycloheximide. These results suggest that the OA action is independent of the decrease in protein synthesis. The proteasome fraction, which had been separated from the fat pads pretreated with OA, enhanced the proteolytic degradation of exogenous [125I]leptin in the presence of an ATP-regenerating system together with an ubiquitination system. No enhancement of hydrolytic activity against Suc–Leu–Leu–Val–Tyr–AMC was detected in the OA-treated proteasome fraction, suggesting that the activation of proteasome is not involved in the OA action. The OA-treated proteasome fraction had decreased phosphatase activity against p-nitrophenyl phosphate, suggesting that OA entering the cells may exert its action by preventing dephosphorylation of key molecules. OA may reduce the intracellular leptin content through the increased ubiquitination and proteolytic turnover of leptin by the proteasome, based on the decreased phosphatase activity.
Paecilomyces tenuipes is believed to contain potential oncostatic and tumor-reducing components. Molecular mechanism, however, is poorly understood concerning the potential antitumor components and their biological function. We purified acetoxyscirpendiol (ASD) from methanolic extracts (MPT) of the fungus and tested the two compounds for the molecular profile of their antitumor potential. Using a differential display protocol, cyclin C and Mad-1 were identified as candidate genes responding to MPT. When a quantitative PCR was performed on the total RNA from MCF-7 treated by MPT or ASD, gene expressions of cyclin C and Mad-1 were greatly augmented. In terms of protein expression, cyclin C level increased up to 12 folds in response to ASD as well as MPT. Similar as MPT treatments, ASD-treated cells synthesize cyclin C as 2—4 fold compared to the control treatments. In terms of Mad-1 expression in cells treated with ASD, the level of Mad-1 expression increased up to 2.5 folds by MPT treatment. Cyclin C expression was compared with non-treated cells in various cell lines. MCF-7 cell was shown highly responsive to the MPT or ASD treatment. Taken together, these results strongly indicate that MPT contains potential antitumor components which might exert their action by modulating cell cycle-related genes such as cyclin C and Mad-1 in MCF-7. The major antioncogenic component in MPT may be ASD which modulates cyclin C and Mad-1 expression.
The effects of exogenously applied gibberellic acid (GA3) or brassinolide (BL) on the H+-pumps and aquaporin in the vacuolar membrane of rice seedling leaf sheath were investigated. Antibodies against mung bean H+-PPase, the B subunit of V-ATPase, and radish tonoplast intrinsic protein (TIP) cross-reacted with the vacuolar membrane proteins of rice seedling leaf sheath. The results of immunoblot analysis showed that the amounts of H+-PPase and V-ATPase were retained at a high level for two days in the presence of GA3, although the level gradually decreased without phytohormones, indicating that GA3 has a promotive effect on the activities of vacuolar H+-pumps. However, the levels of V-ATPase and H+-PPase were increased on day 2 and then decreased when treated with BL. There were no visible differences in the level of TIP upon treatment with GA3 and BL. In comparison with the water control, the activity of H+-PPase treated with BL was also retained at a relatively high level, suggesting that BL has a stimulative effect on the activities of H+-PPase. These results indicate that GA3 and BL might be involved in the regulation of the quantity and activities of plant vacuolar H+-pumps.
To determine whether or not the expression of mevalonate pyrophosphate decarboxylase (MPD) depends on the proliferation of peroxisomes, we examined change in the protein level of MPD in the crude extract, the cytosol and the peroxisome-enriched fraction of the livers of rats administered peroxisome proliferative drugs. No increase of MPD was observed in any of these fractions. These data suggest that the expression of MPD is independent of the proliferation of peroxisomes and may be maintained via a specific regulatory mechanism different from that of the expression of peroxisome proliferator-activated receptor alpha.
Alcohol consumption is known to cause substantial neuronal loss in several regions of the brain. In Oriental medicine, medications based on Puerariae radix have been known to be of efficacy in the treatment of alcohol-related problems. In the present study, the effect of the aqueous extract of Puerariae radix on the expression of c-Fos, an immediate early gene whose expression is sometimes used as a marker for stimulus-induced changes in the metabolic activity of neurons, in the hippocampus of acutely alcohol-intoxicated juvenile rats was investigated via immunohistochemistry. In the first part of the experiment, Sprague-Dawley rats were divided into six groups: the control group, the alcohol-treated group, the alcohol- and 0.3 mg/kg Puerariae radix-treated group, the alcohol- and 3 mg/kg Puerariae radix-treated group, the alcohol- and 30 mg/kg Puerariae radix-treated group, and the alcohol- and 300 mg/kg Puerariae radix-treated group. In the second part of the experiment, animals were divided into four groups: the control group, the 30 mg/kg Puerariae radix-treated group, the alcohol-treated group, and the alcohol- and 30 mg/kg Puerariae radix-treated group. From the results, it was demonstrated that alcohol administration significantly decreases the number of Fos-positive cells in the various regions of the hippocampus, and Puerariae radix treatment inhibits the alcohol-induced suppression of the expression of Fos in the hippocampus in a dose-dependent manner. Puerariae radix exerted no significant effect on Fos expression in the hippocampus of normal rats. The results presented in this study suggest that Puerariae radix may alleviate alcohol-induced disruption of hippocampal functions.
A novel constituent, shamimicin, 1′′′, 1′′′′′′′-bis-2-(3,4-dihydroxyphenyl)-3,4-dihydro-3,7-dihydroxy-5-O-xylopyranosyloxy-2H-1-benzopyran alongwith lupeol, which possesses potent hypotensive activity has been isolated from Bombax ceiba stem bark. BCBMM—one of the most active hypotensive fractions has revealed its adverse effects on heart, liver and kidneys of mice at the dose of 1000 mg/kg/d.
In recent years, it has been reported that bisphosphonates inhibited the cell cycle of myeloma cells to inhibit cell proliferation directly, and it was also reported that bisphosphonates induced apoptosis of myeloma cells in vitro. Recently, YM529 was developed as a new third-generation bisphosphonate. In our experiment, we investigated whether YM529 showed an antitumor effect on hematopoietic tumor cell lines other than myeloma, and we compared YM529 with YM175, which had a relatively more potent antitumor effect than that of existing bisphosphonates. We found that YM529 inhibited cell proliferation in various hematopoietic tumor cell lines (acute promyelocytic leukemia cell line HL-60, chronic myeloid leukemia cell line K562, histiocytic lymphoma cell line U937, lymphoblastic leukemia T cell line Jurkat, acute lymphoblastic leukemia T cell line MOLT-4, lymphoblastic leukemia B cell line CCRF-SB) including myeloma (myeloma cell line HS-Sultan) dose-dependently and time-dependently to a degree equivalent or superior to that in myeloma, and induced apoptosis at a lower concentration as compared with YM175. We confirmed many dead cells as well as apoptosis based on the detection of the nuclei with separate globular structure, the activation of caspase-3, and the decrease in mitochondrial transmembrane potential. Therefore, it is concluded that further utilization of YM529 can be expected against hematopoietic tumor cells in the future.
Rikkunshi-to, a gastrointestinal function regulatory traditional Chinese herbal (Kampo) medicine, has recently been evaluated for its clinical usefulness in stress and depression. This medicine has modulatory effects on the hypothalamo-pituitary-adrenal axis and autonomic nervous function. We examined the effect of Rikkunshi-to and the other gastrointestinal function regulatory Kampo medicines, Hange-shashin-to, Hange-koboku-to, and Ninjin-to, on the plasma levels of adrenocorticotropic hormone (ACTH) and cortisol under stress conditions by repetitive blood sampling. Rikkunshi-to, Hange-shashin-to, and Hange-koboku-to significantly suppressed increases in plasma ACTH-immunoreactive substance (IS) levels compared with the response to a placebo. Rikkunshi-to and Hange-shashin-to significantly suppressed increases in plasma cortisol levels compared with the response to placebo. Ninjin-to had no significant effect on plasma ACTH-IS and cortisol levels. In this study, Rikkunshi-to, Hange-shashin-to, and Hange-koboku-to (partially) regulated plasma ACTH and cortisol levels under stress. These modulatory effects might be beneficial in stress-related disease and suggest that these medicines have clinical pharmacologic activity.
Effects of leptin on food intake and nitric oxide (NO) metabolites (nitrite and nitrate, NOx) levels of brain were investigated in mice. Leptin dose-dependently decreased milk intake in food-deprived mice. Administration of leptin at a dose of 1 mg/kg, which induces an apparent hypophagia, did not affect NOx levels in the hypothalamus and frontal cortex. These results suggest that leptin reduces food intake in food-deprived mice without altering NO production in the hypothalamus, which plays an important role in regulation of feeding.
The potency to accumulate triglyceride (TG) was compared between perfluorinated fatty acids (PFCAs) with different carbon chain lengths in the liver of male and female rats and induction of peroxisomal β-oxidation. In male rats, either perfluoroheptanoic acid (C7) or perfluorooctanoic acid (C8) had no effect, although perfluorononanonic acid (C9) and perfluorodecanoic acid (C10) markedly accumulated TG. In female rats, C7, C8, and C9 did not cause TG accumulation, whereas C10 caused TG accumulation at the same level as in male rats. TG accumulation induced by C9 was regulated by the level of testosterone in male rats. In contrast with TG accumulation, peroxisomal β-oxidation was induced by C8, C9, and C10 in male rats and by C9 and C10 in female rats. Only a slight difference was observed in the induction by C9 between male and female rats. The induction of TG accumulation by these PFCAs occurred in a dose-dependent manner and significantly correlated with hepatic concentrations of PFCA regardless of their carbon chain length, as was observed with induction of peroxisomal β-oxidation. There is, however, a striking difference in the hepatic concentration of PFCA required to cause induction of TG accumulation and that of peroxisomal β-oxidation. The concentration of PFCA required to induce TG accumulation is much higher than that to induce peroxisomal β-oxidation. These results strongly suggest that TG accumulation induced by PFCAs, as well as the induction of peroxisomal β-oxidation, is dependent only on the number of PFCA molecules in hepatocytes, but is not due to the difference in their chemical structures, and that there is a marked difference in the PFCA threshold to cause distinct biological effects.
The effect of hesperetin, naringenin and its glycoside form on the Sindbis neurovirulent strain (NSV) replication in vitro was studied. All flavanones tested were not cytotoxic on Baby Hamster cells 21 clone 15 (BHK-21). Antiviral effect was evaluated by a colorimetric assay using MTT (3-(4,5 dimethylthiazol-2-yl)-2,5-dipheyl-tetrazolium bromide) and by plaque reduction assay. Hesperetin and naringenin had inhibitory activity on NSV infection. The 50% inhibitory doses (ID50%) of both compounds were 20.5 and 14.9 μg/ml respectively, as established by plaque assay. However their glycosides, hesperidin and naringin did not have inhibitory activity. Implying that the presence of rutinose moiety of flavanones blocks the antiviral effect. Oxygenation on the 3′ positions at the B rings on the hesperetin skeleton decrease the anti viral activity at 25 μg/ml.
Coenzyme Q10 (CoQ10), a highly lipophilic compound present in the inner mitochondrial membrane, is essential for production of cellular energy in the form of ATP. CoQ10 is used as a dietary supplement and for treatment of various cardiovascular disorders. Our goal was to compare the CoQ10 levels in Asians following multiple oral doses administered as sustained release or regular tablets. Twenty healthy male volunteers (19—23 years old) were divided into two equal groups. Each subject in Group I received 50 mg oral doses of coenzyme Q10 as sustained release tablets once a day for fifteen days, while subject in Group II received 50 mg doses of coenzyme Q10 regular tablets. The CoQ10 levels were measured by HPLC-UV (reverse phase ODS column, 10 μm, 250×4.6 mm; oven temperature 30 °C). Mobile phase was constituted by methanol–ethanol 9 : 1 v/v. Flow rate was 1.5 ml/min and UV detection was carried out at 275 nm. Coenzyme Q9 was used as an internal standard. CoQ10 baseline in the morning was 0.88±0.48 mg/l. Following 1 week 50 mg/d dosing of CoQ10, plasma CoQ10 concentrations increased to 1.85±1.03 mg/l for sustained release tablets and up to 1.37±0.74mg/l for regular tablets. The net increment proportion in AUC for sustained release and regular tablets were 148.26±176.56%, 102.57±130.00%, respectively. Both preparations significantly increased the systemic exposure when compared to endogenous baseline.
Oral administration of Saiko-ka-Ryukotsu-Borei-To (SRB), a traditional Chinese formulation, dose dependently inhibited intimal thickening in carotid artery injured by balloon endothelial denudation in cholesterol-fed rats. SRB also inhibited vascular smooth muscle cell (VSMC) proliferation, which is assessed by counting the VSMCs immunoreactive with antiproliferating cell nuclear antigen (PCNA) antibody in the intimal area. VSMC proliferation is considered to play a central role in the development of intimal thickening. SRB slightly, but not significantly, reduced serum total cholesterol and low-density lipoprotein cholesterol. These results indicate that the suppressive effect of SRB on intimal thickening may result from its inhibitory effect against VSMC proliferation, but does not depend on lowering of lipid levels. The balloon injury model used in this study has similar pathological processes to restenosis after percutaneous coronary intervention (PCI). Therefore the present results may provide a new therapeutic strategy using SRB to reduce restenosis after PCI in the treatment of patients with ischemic coronary artery disease. Furthermore, since it is considered that artery restenosis after balloon injury in PCI is “accelerated atherosclerosis, ” SRB may have beneficial effects in atherosclerosis that develops over a long clinical course in hyperlipidemia, diabetes, etc.
Nitric oxide (NO) plays an important role in many inflammatory responses and is also involved in carcinogenesis. In the present study, we investigated the inhibitory effect of extracts from Humulus lupulus L. on both the production of NO and the expression of inducible NO synthase (iNOS) in mouse macrophage RAW 264.7 cells. The production of NO was induced by a combination of lipopolysaccharide (LPS) and IFN-γ, and determined by Griess assay. The expression of iNOS was detected by Western blotting. The LPS/IFN-γ-induced production of NO and expression of iNOS were significantly inhibited by the ethyl acetate soluble fraction of Humulus lupulus L. Through bioactivity guided fractionation, humulene, five chalcones, 2,2-di-(3-methyl-2-butyleyl)-4,5-dihydoxy-cyclopent-4-en-1,3-dione, lupulone and three of its derivatives were isolated from the ethyl acetate soluble fraction. The chalcones, including xanthohumol, significantly inhibited the production of NO by suppressing the expression of iNOS.
The effects of berberine in senescence accelerated mice P6 (SAMP6) were investigated to learn whether the alkaloid affects bone mineral density (BMD). Oral administration of berberine (10 mg/kg/d) to male and female mice for 22 weeks resulted in an increase in BMD in both sexes. A decreased concentration of deoxypyridinoline (Dpd) in urine was only observed in female mice. There was no effect on body or tibia weight or on the concentration of procollagen type I carboxyterminal extension peptide (PICP) in serum.
Vernonia spp. (Asteraceae) are used in herbolaria in Latin America in menstrual and stomach disorders, suggesting smooth muscle relaxing properties of some of their chemical constituents. For pharmacological support for this belief, sesquiterpene lactones glaucolides D and E were assayed on isolated rat smooth muscle. Glaucolide E proved more potent than glaucolide D to relax high KCl- or noradrenaline-induced contractions in aorta and to relax the high KCl-contraction in uterus. Hirsutinolide-type sesquiterpene lactone also was tested but displayed no effect. Relaxation of smooth muscle by structurally related sesquiterpene lactone parthenolide has been attributed mainly to the α-methylene γ-lactone moiety; because glaucolides D and E lack this functional group, their relaxant properties may rely on other alkylating sites such as C10 of the germacra-1(10),4-diene-4-epoxide skeleton.
The epidermal permeability barrier appears to be regulated primarily by the lamellar arrangement of lipid bilayers between coneocytes of the stratum corneum and presents a significant barrier to the transdermal delivery of drugs. The aim of the present study was to investigate the effects of oleic acid on the ultrastructure of stratum corneum lipids in rat skin. Wistar rats were treated topically with 10% oleic acid/propylene glycol for 2 h, the structure of stratum corneum was examined by electron microscopy using osmium tetroxide or ruthenium tetroxide postfixation, and the epidermal barrier function was evaluated in a lanthanum tracer study. Ultrastructural examination revealed that there was a marked alteration in the stratum corneum and the tracer penetrated into the intercellular spaces of the stratum corneum after application of oleic acid. These results suggest that ruthenium tetroxide postfixation is a powerful tool for the study of the stratum corneum lipid structure. Oleic acid might increase the epidermal permeability through a mechanism involving the perturbation of stratum corneum lipid bilayers and lacunae formation to enhance transdermal drug delivery.
A matrix-type transdermal therapeutic system was developed for treating diseases of the eye where it is difficult for drug molecules to reach with conventional topical instillation. Prednisolone was employed as a model drug. An in vivo study using rats showed that the daily application of the patch maintained a constant plasma concentration of the drug, which was equivalent the therapeutic plasma level following three times daily oral administration (30 mg), for approximately 24 h. Transdermal delivery provided equivalent to or higher bioavailability (drug distribution) to the eyeball of topical administration. Moreover, pharmacokinetic analysis indicated that the present transdermal therapeutic system may be clinically effective as a new treatment for ocular diseases.
KV-2920 is a novel acyl-CoA: cholesterol acyltransferase (ACAT) inhibitor. To confirm the efficacy of KV-2920–low density lipoprotein (LDL) complex (KV–LDL complex) as a drug-carrier complex on experimental atherosclerosis, we examined its inhibitory effects in vitro and in vivo. LDL was isolated from human plasma and the KV–LDL complex was prepared by incubation in the presence of Celite 545. The complex contained about 800 mol KV-2920 in 1 mol LDL. The cholesterol levels in the serum and aorta of atherogenic mice after 14 weeks of feeding were significantly higher than those of nonatherogenic mice. With the intravenous injection of KV–LDL complex, although the cholesterol levels in the serum were not influenced, the level of cholesterol ester in the aorta of atherogenic mice was significantly reduced. The concentration of cholesterol ester in the macrophages derived from ICR mice was predominantly increased by incubation with LDL for 48 h, this increase was significantly inhibited by incubation with KV–LDL complex. Moreover, the complex also inhibited the increase of cholesterol ester in macrophages following incubation with oxidized LDL. These findings suggest that KV–LDL complex inhibits the foam cell formation of macrophages though action of KV-2920 as an ACAT inhibitor, and prevents the accumulation of cholesterol ester in the aorta of atherogenic mice. Therefore, KV–LDL complex may be useful as a drug–carrier complex in antiatherosclerotic therapy.
Baicalin, baicalein, wogonoside and wogonin are flavone constituents of Scutellariae Radix with various beneficial biological activities. The purpose of this study was to investigate the urinary pharmacokinetics of these flavones after oral administration of Scutellariae Radix commercial powder. Ten healthy male volunteers received a dose of 5.2 g commercial powder (comparable to 9 g crude drug), respectively. The concentrations of baicalin, baicalein and wogonin in the commercial powder as well as their metabolites in urine were assayed by HPLC method. The glucuronides and sulfates of baicalein and wogonin in urine were hydrolyzed with β-glucuronidase and sulfatase, respectively. Our results showed that the mean cumulated renal excretion of baicalein glucuronides and sulfates were 43.1±4.5 μmol (2.9% of dose) and 64.8±6.3 μmol (4.3% of dose), respectively, whereas wogonin glucuronides and sulfates were 21.6±2.0 μmol (5.9% of dose) and 20.7±1.7 μmol (5.7% of dose), respectively. The result indicated that the renal excretion of conjugated metabolites of wogonin (11.6% of dose) were higher than that of baicalein (7.2% of dose). The baicalein sulfates was predominant than the corresponding glucuronides, whereas wogonin sulfates was comparable to the corresponding glucuronides.
Irinotecan-containing nanoparticles (NP) were prepared by coprecipitation with addition of water to acetone solution of poly(DL-lactic acid), poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) and irinotecan, and subsequent evaporation of organic solvent. NP were purified by gel filtration and used for experiments after condensation by evaporation. The obtained NP showed the drug content of 4.5% (w/w) and the mean particle diameter of 118 nm with the particle diameter distribution between 80—210 nm. When the antitumor effect was examined at a repeated dose of 20 mg irinotecan eq/kg for 3 d (3×20 mg/kg) using mice bearing Sarcoma 180 subcutaneously, only NP suppressed tumor growth significantly. After i.v. injection in rats, NP maintained irinotecan plasma concentration longer than CPT-11 aqueous solution. The present nanoparticle formation is suggested as a possibly useful dosage form of irinotecan against solid tumor.
Ointments of the skin depigmentation agent hydroquinone (HQ) have been prepared by extemporaneous nonsterile compounding in our hospital. The HQ ointments were highly effective in the treatment of various types of skin pigmentations; however, various problems have emerged including chromatic aberration of the ointments, a relatively large variability of efficacy, and mild side effects. Chromatic aberration is expected to induce non-compliance, and this may be the reason for the relatively large variability in efficacy. In this paper, the effects of various storage conditions on the chromatic aberration and HQ content of HQ ointments were evaluated, and it was suggested that the chromatic aberration was accelerated by exposure to high temperature, air and light, although these had no effect on the HQ content. In addition, various types of HQ ointments were prepared to find a formulation to minimize chromatic aberration, and it was found that the concentrations of antioxidants, Na2SO3 and L(+)-ascorbic acid (AsA), seemed to be too high, and that the protective effect of AsA on chromatic aberration was mainly due to its acidifying effect.
The effects of α1-acid glycoprotein (AGP) on human erythrocyte membrane were examined in vitro. Bovine and dog AGP, in addition to human AGP or asialo human AGP were used, and the collected data were compared with that for human serum albumin (HSA). A new technique developed by Kikuchi was used to investigate erythrocyte deformability. The addition of AGPs including human AGP facilitated the passage of human erythrocytes with an average diameter of 7.2 μm suspended in phosphate buffered saline (PBS) through a 5 μm wide microchannel; hemolysis was suppressed after the passage. The stabilizing effects of AGPs on membrane were evaluated. Human AGP prevented hemolysis induced by hypotonic phosphate buffer solution. The effects of human AGP on the oxidative changes in erythrocytes exposed to oxygen radicals were investigated. Human AGP protected erythrocytes from H2O2 and prevented the oxidation of dihydrorhodamine 123 to rhodamine 123 from H2O2. We propose that the antioxidant activity of human AGP is due to the binding of free radicals. In all studies, the effects of human AGP on erythrocytes might not be a function of the negative charge associated with sialyl residues, because the presence of N-acetylneuraminic acid had no effect. However, human AGP may promote microcirculation and antioxidant activity compared with HSA. No species differences in the physiological function of AGP were found. These results suggest that an increase in the AGP content of serum above the normal value found under pathological conditions facilitates the passage of erythrocytes through capillaries, stabilizes erythrocyte membranes and protects against oxidative stress, all of which are favorable for microcirculation.
This study was conducted to investigate the hypocholesterolemic effect of the hot-water fraction (HW) from cultured mycelia of Cordyceps sinensis in a 5 l fermenter. The composition of HW was mainly carbohydrate (83.9%) and protein (11.8%) on a dry basis, and the carbohydrate of HW consisted of glucose, mannose, galactose, and arabinose in the molecular ratio of 1.0 : 0.8 : 0.5 : 0.1, respectively. In mice fed a cholesterol-free diet and those fed a cholesterol-enriched diet, body and liver weights were not significantly different from those of the controls. The serum total cholesterol (TC) of all mice groups administered HW (150 and 300 mg/kg/d, respectively) with the cholesterol-enriched diet decreased more than in the control group. Among the mice fed the cholesterol-enriched diet, HW also increased the high-density lipoprotein (HDL) cholesterol level, but decreased the very low-density lipoprotein plus low-density lipoprotein (VLDL+LDL) cholesterol level. The changes in HDL- and VLDL+LDL-cholesterol levels consequently decreased the atherogenic value. The results indicate that HW in rats administered a cholesterol-enriched diet decreased the plasma cholesterol level. The 300 mg/kg dose had a significant effect on the serum TC level.
It is well-recognized that DNA methylation causes gene silencing. However, although DNA methylation is closely associated with alterations in chromatin structure, it is not clear whether methylation at a particular locus contributes to the formation of an inactive chromatin structure. In this study, we have investigated the chromatin structure of the CpG island of the human CDH1 gene in cancer cells. The CDH1-CpG island was differentially methylated between silenced cells, however, DNase I hypersensitive site that are present in the expressing cell were commonly lost in silenced cells and transcription factors were excluded from their binding sites. These results demonstrate that formation of an inactive structure was not affected by heterogeneous methylation status of the CpG island.