Neuroblastoma (NB) is the most common malignant solid tumor in childhood, and among all childhood malignancies is second in prevalence only to leukemia. In NB we need to both make an accurate diagnosis and rapidly analyze the expression of genetic prognostic factors such as MYCN, H-ras, and trkA. Moreover, it has recently become important to analyze the expression of survivin mRNA, a member of the inhibitor of apoptosis protein family. Expression of the survivin gene is related to tumorigenesis and inhibition of apoptosis in some malignant tumors. We investigated its expression by reverse transcription-polymerase chain reaction (RT-PCR) in NB cell lines (SK-N-SH, NB-39, and IMR-32), two normal blood cell samples, and 13 clinical NB tumor samples. All three NB cell lines had high levels of mRNA expression for this gene, but normal blood cells had no expression. We detected expression of survivin mRNA in 7 of the 13 NB tumor samples (54%). Two NB patients were in stage I disease, 6 in stage II, and 5 in stage IV_A. Quantitative analysis by RT-PCR revealed that the ratio between survivin mRNA and human glyceraldehyde-3-phosphate dehydrogenase (h-GAPDH) mRNA was very low in stages I and II (0—0.017). In contrast, in advanced NBs (stage IV_A) the ratio was much higher (0—0.050). The prognoses of the three patients in the advanced stage who had high ratios of expression were poor. A high level of expression of survivin mRNA indicates a high grade of malignancy, high likelihood of recurrence, and poor prognosis.

View full abstract

In rat tissues, a decrease in spermidine, accompanied by an increase in spermine was induced by the oral administration (once daily for either 1 week or 1 month) of trans-4-methylcyclohexylamine (4MCHA), a spermidine synthase inhibitor. This is similar to the changes observed in polyamine content when cell growth is arrested. The body-weight gain of the rats tended to decrease with increasing doses of 4MCHA. A decrease in spermidine, combined with a moderate increase in spermine, was observed dose-dependently in all of the tissues tested, with a relatively fast clearance of 4MCHA. Manipulating the polyamine content of tissues, by daily administration of 100 μmol 4MCHA for 1 week, made it possible to estimate the effects of simultaneously added spermidine or spermine on endogenous polyamine contents. The altered polyamine levels, obtained after daily administration for 1 week, were maintained during the extended 1-month period, with growth-dependent alteration. The results show it is possible to produce experimental rats with a higher spermine:spermidine ratio than control rats to investigate the physiological significance of spermidine downregulation and spermine upregulation in vivo.

View full abstract

(−)-Epigallocatechin gallate (EGCG), a major component of green tea catechins, is known to inhibit cell growth and to induce apoptosis in a variety of cultured cells. We examined effects of green tea catechins in cultured cells derived from human gastric carcinoma. The proliferation of four cell lines (MKN-1, MKN-45, MKN-74 and KATO-III) was inhibited with EGCG in a dose-dependent manner. The growth of MKN-45 cells was most efficiently inhibited by the treatment (IC₅₀: 40 μM EGCG) among the four cell lines, while KATO-III cells were most insensitive (IC₅₀: 80—150 μM) to the EGCG treatment. In addition, (−)-epicatechin (EC) had a major synergistic effect on the induction of apoptosis in MKN-45 cells treated with EGCG; however it had little effect on the inhibition of cell growth induced by EGCG. To study the molecular mechanisms behind the induction of apoptosis by EGCG, the activity of caspases in MKN-45 cells treated with EGCG was examined. Activity levels of caspases-3, -8 and -9 were elevated in EGCG-treated cells, suggesting that these caspases are involved in the apoptosis induced by EGCG. Furthermore, the synergistic effect of EC with EGCG on the induction of apoptosis was specifically canceled by catalase treatment, suggesting that the synergism involves the extracellular production of reactive oxygen species.

View full abstract

In the present study, by using IPCC-MS3 (GL Sciences Inc. Tokyo, Japan) as the counter-ion in the mobile phase, we established a simple, quick method of analysis that separated and quantified paraquat and diquat on an ODS column by introducing the deproteinized serum sample directly into HPLC. The calibration curve of paraquat and diquat detected at UV 290 nm showed good linearity when the concentration of the injected sample was in the range 0.1—10.0 μg/ml. The detection limit was 0.05 μg/ml, and the mean recoveries (n=5) added 1.0 μg/ml each of paraquat and diquat to standard serum were 87.5% and 89.1%, respectively, while the RSD were 4.52% and 3.85%. All of these were good results, and the time taken for one analysis was less than 30 min. As a result of employing this analytical method for the analyses in four cases of acute poisoning, it was possible to decide promptly on treatment approaches for all of the present cases.

View full abstract

Platelet activating factor (PAF)-acetylhydrolase (PAF-AH) is an enzyme that hydrolyzes the acetyl ester at the sn-2 position of PAF, and converts it to the inactive metabolite, lyso PAF. This enzyme is distributed widely in the intracellular as well as the extracellular matrix and is believed to be a defense mechanism that protects the host against the toxic effects of PAF and other biologically active oxidized phospholipids. Purification and expression of cDNA cloning of the intracellular and extracellular types of PAF-AH from several sources from different species have been reported. In this study, the cDNA for PAF-AH was cloned by reverse transcription (RT)-PCR from total RNA of bovine mammary gland. The complete amino acid sequences from the cDNA contains 444 amino acids and was identical to that of the PAF-AH isolated from the bovine spleen cDNA library except for two mismatches of amino acid residues (Thr-247 to Met and Ile-431 to Thr). Recombinant PAF-AH was expressed in HEK 293 cells, which exhibited enzyme activity in the in vitro assay system. Furthermore, recombinant bovine PAF-AH was identified by western blot using human plasma PAF-AH antibody as a monomeric polypeptide with a molecular weight of approximately 43 kDa. This protein can be applied to in vivo models to test its protective role against the deleterious PAF actions.

View full abstract

We have already reported that WBB6F1-W/W^v (W/W^v) mice, which have mutations in the c-kit gene, are highly susceptible to oral sensitization, and that the proportion of TCRγδ-T cells among the intraepithelial lymphocytes (IELs) (γδ-IELs) of W/W^v is much lower than in congenic wild-type (+/+) mice. In this study we examined an inhibitory role of γδ-IELs in oral sensitization using two different methods. First, wild-type (+/+) mice were sensitized by oral administration of 1.0 mg ovalbumin (OVA) by gavage every day for 9 weeks after anti-TCRγδ antibody treatment 4 times. The treatment resulted in an enhanced OVA-specific IgG1 antibody production, active systemic anaphylaxis (ASA), and Th2-dominant cytokine production. Next, W/W^v mice whose bone marrow cells were reconstituted from C57BL/6J mice for 5 months were sensitized by oral administration of OVA. The OVA-specific IgG1 antibody titer in the bone marrow-reconstituted W/W^v mice was neither significantly enhanced, nor ASA was induced. The proportion of γδ-IELs in the reconstituted mice was much higher than that in the untreated W/W^v mice. The above findings suggest that the decrease or increase in number of γδ-IELs enhances or decreases oral sensitization respectively. These results show that γδ-IELs have an important role in the oral tolerance to food antigens.

View full abstract

Inhibitors of the fungal enzyme N-myristoyltransferase (Nmt) reduce fungal growth, as this enzyme is essential for viability. We found that a newly synthesized benzothiazole derivative, (1R,3S)-N-{2-[(cyclopeanthylcarbonyl) amino]-benzothiazol-6-yl}-3-[(2-naphthylmethyl) amino] cyclohexanecarboxamide (FTR1335), preferentially inhibited Candida albicans Nmt (CaNmt) in a dose-dependent manner. The 50% inhibitory concentration (IC₅₀) for CaNmt was 0.49 nM, which was much lower than the 5400 nM IC₅₀ for human Nmt (HsNmt1). The mode of CaNmt inhibition was competitive with the substrate peptide and non-competitive with myristoyl-CoA. Moreover, FTR1335 showed strong antifungal activity in vitro, and the minimum fungicidal concentration for C. albicans was 0.78 μM. These results indicate that FTR1335 might represent a novel family of Nmt inhibitors with fungicidal activity.

View full abstract

Mangiferin, 2-β-D-glucopyranosyl-1,3,6,7-tetrahydroxy-9H-xanthen-9-one, obtained directly from methanolic extracts of Bombax ceiba leaves in substantial amounts demonstrated strong antioxidant activity (EC₅₀ 5.8±0.96 μg/ml or 13.74 μM) using DPPH assay comparable to rutin, commonly used as antioxidant for medical purposes. The acetyl and cinnamoyl derivatives were found to be less active than mangiferin whereas, methyl and 3,6,7-trimethylether tetraacetate derivatives were inactive implying that for antioxidant activity, free hydroxyl groups and catechol moiety are essential. Moreover, mangiferin showed hepatoprotective activity against carbon tetrachloride induced liver injury further supporting the free radical scavenging property in the in vivo system. Additionally, plant extracts and mangiferin failed to exhibit acute anti-inflammatory activity whereas, it displayed significant analgesic effect in acetic acid-induced writhing and hot plate tests in mice. Using naloxone, it was revealed that plant extracts induced analgesia was independent of opioid receptor, whereas, mangiferin demonstrated significant interaction with it at peripheral site with a slight contribution at the neuronal level.

View full abstract

The aim of the present study was to examine the effects of Avena Rhealba^® (AR) oatmeal extract on the metabolism of arachidonic acid (AA) and eicosanoids as well as on the expression of cytosolic phospholipase A₂ (cPLA₂) in the human keratinocyte cell line HaCaT. For this purpose, we examined the effects of AR on basal and A23187-triggered release of [³H]-AA from phospholipids and on the production of [³H]-labeled metabolites of the cyclooxygenase (CO) and 5-lipoxygenase (LO) pathways. AR was found to inhibit A23187-triggered [³H]-AA mobilization from phospholipids (p<0.05) and production of [³H]-labeled metabolites of CO (p<0.05) and LO (p<0.05) pathways. These results suggest AR decreases PLA₂-dependent mobilization of AA from phospholipids. A closer examination of the effects of AR on prostaglandin 6KF1α (6KPGF1α), the stable metabolite of prostacyclin, revealed dose-dependent inhibition of this AA metabolite. AR also decreased A23187- and tumor necrosis factor α-induced cPLA₂ overexpression, as shown by cPLA₂ immunodetection and mRNA expression. These results demonstrate the high potential of AR in the treatment of inflammatory diseases of the skin.

View full abstract

Transcription factor nuclear factor-kappa B (NF-κB) has been demonstrated to be important in regulating various gene expressions such as cytokines, adhesion molecules, and endothelin-1 (ET-1) in vascular endothelial cells. In the present study, we show the effects of xanthoangelol, xanthoangelol D, E, and F, which isolated from the root of Angelica keiskei KOIDZUMI (Umbelliferae), on NF-κB activation and ET-1 gene expression in cultured porcine aortic endothelial cells (PAECs). Treatments of xanthoangelol D but not xanthoangelol, xanthoangelol E and F markedly suppressed both of basal and tumor necrosis factor-α (TNF-α)-induced NF-κB activation in PAECs. To clarify the mechanism of xanthoangelol D-induced suppression on NF-κB activation, we evaluated the effects of xanthoangelol D on phosphorylation and degradation of IκBα, an inhibitory protein bound to NF-κB, and obtained evidence that xanthoangelol D selectively suppresses the phosphorylation of IκBα rather than the degradation of phosphorylated IκBα. In addition, xanthoangelol D significantly attenuated basal and TNF-α-induced prepro ET-1 mRNA expression in PAECs. These results suggest that xanthoangelol D may be useful for the treatment of various vascular diseases involved NF-κB activation.

View full abstract

Ascidians (sea squirts) contain a wealth of alkaloids, but their influence over neuronal nicotinic acetylcholine receptors (nAChRs) has not been evaluated. In this study, we examined the effects of two synthetic compounds, (−)-pictamine, a quinolizidine alkaloid from Clavelina picta, and (−)-lepadin B, a decahydroquinoline alkaloid from Clavelina lepadiformis, on major types of neuronal nicotinic receptors (α4β2 and α7) expressed in Xenopus oocytes. We found that these alkaloids are potent blockers at these receptors: acetylcholine-elicited currents through α4β2 and α7 receptors were blocked by (−)-pictamine with IC₅₀ values of 1.5 μM and 1.3 μM, respectively, and by (−)-lepadin B with IC₅₀ values of 0.9 μM and 0.7 μM, respectively. Interestingly, no recovery was observed after the removal of (−)-pictamine in oocytes expressing α4β2 receptors, whereas the inhibited α7 currents quickly recovered after the removal of (−)-pictamine. Since there are few compounds that elicit irreversible blocks of α4β2 receptors, (−)-pictamine will be a novel, valuable tool to remove the α4β2-nAChR action from neuronal activities mediated by these two major types of nAChRs.

View full abstract

The senescence-accelerated mouse (SAM) is known as a murine model for accelerated aging. The SAMP8 shows age-related deficits of learning and memory at an earlier age than control mice (SAMR1). We investigated the changes in oligodendrocytes in the brain of SAMP8, using immunohistochemistry for myelin basic protein (MBP) and 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP) as an oligodendrocyte marker. SAMP8 at 10 months old showed a decrease in MBP-immunoreactivity (IR) and CNP-IR in the hippocampal CA1 subfield, compared with SAMR1. There were no significant differences in MBP and CNP old in the cerebral cortex and the optic tract between SAMR1 and SAMP8 at 10 months. Furthermore, we measured the area of MBP-IR in the CA1 subfield of both strains and found that the area of MBP-IR in SAMP8 had decreased progressively with age, compared with SAMR1. These results suggest that age-related degeneration of oligodendrocytes had occurred in the hippocampus of SAMP8.

View full abstract

Hepatocyte growth factor (HGF) stimulates the proliferation of hepatocytes and biliary epithelial cells and protects hepatocytes from apoptosis induced by various stimuli. In view of HGF induction by interferons, substances used for the treatment of chronic hepatitis C, this study was conducted to determine whether ursodeoxycholic acid (UDCA), which is widely used for the treatment of cholestatic liver diseases, modulates HGF production. UDCA did not induce HGF production in human dermal fibroblasts, but it potently inhibited phorbol-12-myristate-13-acetate (PMA)- and cholera-toxin-induced HGF production without affecting cell viability. The inhibitory effects of UDCA were as potent as those of transforming growth factor-β1 and dexamethasone. Up-regulations of HGF gene expression induced by PMA and cholera toxin were also inhibited by UDCA. Moreover, UDCA dose-dependently inhibited high constitutive HGF production by MRC-5 cells without decreasing cell viability. Deoxycholate, chenodeoxycholate, taurochenodeoxycholate and glycochenodeoxycholate also inhibited cholera-toxin-induced HGF production at non-cytotoxic doses. UDCA, however, had no apparent effect on PMA-induced phosphorylation of mitogen-activated protein kinase, which is crucial for HGF induction by PMA. These results indicate that non-cytotoxic doses of UDCA inhibited constitutive and induced HGF production and suggest that UDCA supplemented with HGF or HGF inducers could have a more potential therapeutic effect.

View full abstract

It is known that RhoA is translocated from cytoplasm to cell membrane in bronchial smooth muscle when activated by acetylcholine (ACh) stimulation. In the present study, the effects of selective muscarinic receptor antagonist methoctramine, AF-DX116 (for M₂) and 4-diphenylacetoxy N-methylpiperidine (4-DAMP; for M₃) on the ACh-induced rat bronchial smooth muscle contraction and increase in membrane-associated RhoA were investigated to elucidate the muscarinic receptor subtype participating in these responses. To evaluate ACh-induced contraction of bronchial smooth muscle, bronchial ring of rat was prepared, suspended in an organ bath and the tension was measured isometrically. To quantify the ACh-induced increase in membrane-associated RhoA protein, western blot analysis was performed by using homogenates of membrane and cytosolic fractions of the rat bronchi. The muscarinic M₂ and M₃ receptors were detected by using RT-PCR in rat bronchial smooth muscle. Both the ACh-induced smooth muscle contraction and increase in membrane-associated RhoA were markedly inhibited by 4-DAMP, but not by methoctramine or AF-DX116. In conclusion, these results indicated contraction for the first time that the activation of RhoA occurs via M₃ receptor in rat bronchial smooth muscle.

View full abstract

DY-9760e (3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate), a calmodulin antagonist, provides protection against Ca²⁺ overload-associated cytotoxicity and brain injury after cerebral ischemia in rats. In this study, we assessed the effect of DY-9760e on ischemic infarct volume in cats subjected to permanent focal cerebral ischemia. DY-9760e was infused for 6 h, beginning 5 min after occlusion of the middle cerebral artery. The infarct volume was measured at the end of drug infusion. DY-9760e, at the dose of 0.25 but not 0.1 mg/kg/h, significantly reduced cerebral infarct volume without affecting any physiological parameters, and its protective effect was mainly evident in the cerebral cortex, where the penumbra, a salvageable zone, exists. The present study demonstrates that DY-9760e protects against brain injury after focal ischemia in a gyrencephalic animal as well as in the rodents reported previously and suggests its therapeutic value for the treatment of acute stroke.

View full abstract

Inhibitory effects of desipramine (DMI) on rat and human CYP2D enzymes were studied using bufuralol (BF) 1″-hydroxylation as an index. Inhibition was examined under the following two conditions: 1) DMI was co-incubated with BF and NADPH in the reaction mixture containing rat or human liver microsomes or yeast cell microsomes expressing rat CYP2D1, CYP2D2 or human CYP2D6 (co-incubation); 2) DMI was preincubated with NADPH and the same enzyme sources prior to adding the substrate (preincubation). When either rat liver microsomes or recombinant CYP2D2 was employed, the preincubation with DMI (0.3 μM) caused a greater inhibition of BF 1″-hydroxylation than the co-incubation did, whereas BF 1″-hydroxylation by rat CYP2D1 was not markedly affected under the same conditions. The inhibitory effect of DMI on BF 1″-hydroxylation by human liver microsomal fractions or recombinant CYP2D6 was much lower than that on the hydroxylation by rat liver microsomes or CYP2D2. Kinetic studies demonstrated that the inhibition-type changed from competitive for the co-incubation to noncompetitive for the preincubation in the case of CYP2D2, whereas the inhibition-type was competitive for both the co-incubation and the preincubation in the case of CYP2D6. Furthermore, the loss of activity of rat CYP2D2 under the preincubation conditions followed pseudo-first-order kinetics. Binding experiments employing the recombinant enzymes and [³H]-DMI revealed that CYP2D2 and CYP2D6 were the only prominent proteins to which considerable radioactive DMI metabolite(s) bound. These results indicate that rat CYP2D2 biotransforms DMI into reactive metabolite(s), which covalently bind to CYP2D2, resulting in inactivation of the enzyme. In contrast, human CYP2D6 may also biotransform DMI into some metabolite(s) that covalently bind to CYP2D6, but that do not inactivate the enzyme.

View full abstract

The effects of tempol, a superoxide dismutase mimetic, on ischemia/reperfusion-induced acute renal failure (ARF), noradrenaline (NA) overflow and endothelin-1 (ET-1) overproduction in rats were examined. Ischemic ARF was induced by occlusion of the left renal artery and vein for 45 min followed by reperfusion, 2 weeks after contralateral nephrectomy. Renal functional parameters such as blood urea nitrogen, plasma creatinine concentration, and fractional excretion of sodium, NA concentrations in renal venous plasma, and renal ET-1 contents were determined. Renal function in ARF rats markedly decreased at 1 d after reperfusion. Pre-ischemic treatment with tempol (10, 100 mg/kg, i.v.) dose-dependently attenuated the ischemia/reperfusion-induced renal dysfunction. Histopathological examination of the kidney of ARF rats revealed severe renal damages, such as tubular necrosis, proteinaceous casts in tubuli and medullary congestion, which were also significantly suppressed by the tempol treatment. There was a significant increase in NA concentrations in renal venous plasma after the ischemia/reperfusion, and this increase was markedly suppressed by the treatment with tempol. In addition, tempol treatment significantly attenuated the increment of ET-1 content in the kidney exposed to the ischemia/reperfusion. These findings suggest that tempol improves the post-ischemic renal injury by inhibiting the neural activity of renal sympathetic nerve and ET-1 overproduction.

View full abstract

The hypoglycemic and antihyperglycemic effect of alcoholic extract of root of Gmelina asiatica LINN. (G. asiatica) was investigated in normal and in alloxan induced diabetic rats. The blood glucose levels were measured at 0 h and 1, 2, 4, 6, 8 and 16 h after the treatment. The alcoholic extract of G. asiatica showed significant (p<0.05) dose dependent percentage blood glucose reduction in normal (25.8% at 100 mg/kg, 28.9% at 250 mg/kg and 32.4% at 500 mg/kg body weight) and in diabetic rats (26.6% at 100 mg/kg, 32.1% at 250 mg/kg and 48.2% at 500 mg/kg body weight) respectively at 6 h. The antihyperglycemic effect of G. asiatica was compared with the reference standard drug tolbutamide (40 mg/kg).

View full abstract

Effects of serotonergic anxiolytic buspirone on immobilization-induced hyperglycemia were studied in mice. Stress elicited hyperglycemia in mice. Pretreatment with buspirone significantly reduced immobilization-induced hyperglycemia. Buspirone increased serum insulin levels in both non- and stressed mice. The major metabolite of buspirone, 1-(2-pyrimidinyl)piperazine (1-PP) also increased and this further inhibited immobilization-induced hyperglycemia, since 1-PP increased serum insulin levels in both non-stressed and stressed mice, similar to the increases induced by buspirone. These results suggest that buspirone can reduce stress-induced hyperglycemia by facilitating insulin release. Moreover, 1-PP, a metabolite of buspirone may participate in the effects of buspirone. Since 1-PP is an antagonist of α₂ receptors, α₂ receptors may be related to effects of 1-PP.

View full abstract

Prophylactic oral administration of a novel immunomodulator (immunosuppressant), FTY720 (1 mg/kg, three times a week), completely prevented the development of experimental autoimmune myasthenia gravis (EAMG) in C57BL/6 mice. EAMG has been used as an animal model for human myasthenia gravis, and was established by immunizing the mice with acetylcholine receptor (AChR) from Torpedo californica. FTY720 also suppressed the production of both anti-Torpedo californica AChR antibody and anti-mouse AChR autoantibody by the mice, which were observed in mice in which EAMG had become established. These results strongly suggest that FTY720 is a promising candidate for treatment of human myasthenia gravis.

View full abstract

Prostate cancer is the most predominant cancer in men and prostate cancer related death increases every year. Till date, there is no effective therapy other than androgen ablation therapy. At this stage, induction of apoptosis is considered as a better strategy to control cancer. Previous studies reported that aged garlic extract suppresses cancer growth and enhances immune system against cancer. In the present study, diallyl disulfide, oil soluble organosulfur compound of garlic, was studied for its antiproliferative effect on prostate cancer cells in vitro. The suppression of cell growth was demonstrated by [³H]thymidine incorporation assay. Induction of DNA damage was assessed by agarose gel electrophoresis. The results showed that diallyl disulfide inhibited the growth of prostate cancer cells in a dose dependent manner, compared to the control. At 50 μM and 100 μM concentrations, diallyl disulfide induced DNA damage in PC-3 cells. It is concluded that diallyl disulfide, component of aged garlic extract, inhibits proliferation of prostate cancer cells through the induction of apoptosis.

View full abstract

Choto-san is a Kampo medicines that has been used clinically for the treatment of dementia. We measured the mRNA expressions of some factors related to Alzheimer's disease in a dementia model rat brain. The expressions of β-amyloid precursor protein, γ-secretase, α7 nicotinic acetylcholine receptor, neprilysin, and insulin degrading enzyme (IDE) were significantly increased on day 4 after permanent occlusion of the bilateral common carotid arteries (2VO). Choto-san inhibited the enhancement of IDE expression caused by 2VO, although it failed to show any effects on the expressions of the other molecules. These results suggest that Choto-san may produce a state in which it is not necessary to induce IDE expression to demonstrate the anti-dementia effects.

View full abstract

The pharmacokinetics of bulleyaconitine A (BLA) after a single dose of 0.2 mg intramuscular injection was evaluated in healthy volunteers. Physical exam, vital signs, clinical laboratory tests and electrocardiogram measurements were monitored to assess the safety and tolerance of the drug. The plasma levels of BLA in serial samples, collected over 15 h, were measured by a validated high-performance liquid chromatography (HPLC)-electrospray ionization tandem mass spectrometry (MS-MS) method. It was demonstrated that BLA was absorbed rapidly after intramuscular injection. The pharmacokinetic parameters were as follows: the t_max value was 0.90±0.68 h, the C_max value was 1.13±0.76 ng/ml, the AUC_0—t was 5.16±2.05 ng·h/ml, and t_1/2 was found to be 4.88±0.97 h. No subject showed any drug-related clinically significant changes on physical examination, vital signs or laboratory tests. Eight of ten subjects reported a distinct feeling of pain at the site of injection starting approximately at the time of their peak plasma concentration and lasting for 2—6 h. The pain was tolerable, and no subject required additional treatment.

View full abstract

This study was undertaken to elucidate the mechanism of anti-inflammatory action of gentianine, a constituent of Gentiana Macrophylla. The effects of gentianine on lipopolysacharide (LPS)-induced production of pro-inflammatory cytokines were investigated in male Sprague-Dawley rats. For the first time, we found that oral administration of gentianine (10—100 mg/kg) suppressed the increases in tumor necrosis factor-α (TNF-α) (ED₅₀, 37.7 mg/kg) and interleukin (IL)-6 (ED₅₀, 38.5 mg/kg) in the sera from the rats challenged with bacterial LPS (100 μg/kg; i.p.). However, LPS induced production of other interleukins, such as IL-α, was not significantly altered by gentianine. These results suggest that the potential anti-inflammatory action of gentianine might be at least partly based on the suppressed production of TNF-α and IL-6.

View full abstract

A new non-alcoholic beverage made of red wine vinegar and grape juice (Budo-no-megumi^TM) has been recently demonstrated to lower the blood pressure of human as well as rats. In this study, we pharmacologically analyzed the mechanism of its hypotensive action. The thoracic aorta with intact endothelium was isolated from Sprague-Dawley rats, and incubated with a Tyrode's solution. The beverage in concentrations of 0.25 to 2% relaxed the pre-contracted aorta with an α-adrenoceptor agonist phenylephrine in a concentration-dependent manner, 2% of which induced 59% relaxation. In contrast, the vasodilator response disappeared in the aorta without endothelium. L-Nitro-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, significantly reduced the vasodilator effect of the beverage, whereas indomethacin, an inhibitor of cyclooxygenase, hardly affected it. These results suggest that the beverage can activate the nitric oxide synthase activity to exert vasodilation, which may partly explain its previously reported hypotensive action.

View full abstract

The NADH-dependent activity by hepatic microsomes of Japanese monkeys for 7-oxo-Δ⁸-tetrahydrocannabinol (7-oxo-Δ⁸-THC) formation from 7β-hydroxy-Δ⁸-THC exhibited about 70% of the NADPH-dependent activity (100%) at the substrate concentration of 72.7 μM, although NADPH was an obligatory cofactor for maximal activity. Both NADH- and NADPH-dependent activities were significantly inhibited by the typical P450 inhibitors, such as SKF525-A and metyrapone. Both activities were almost completely inhibited by the NADPH-P450 reductase inhibitor diphenyliodonium chloride. The ratio of NADH- and NADPH-dependent activities varied significantly according to the substrate concentration. Interestingly, the NADH-dependent activity was higher than that of NADPH at low substrate concentrations of 13—50 μM. The ratio was also affected by the cofactor concentration. In the reconstituted system of CYP3A8 purified from hepatic microsomes of Japanese monkeys as a major enzyme responsible for the NADPH-dependent oxidation, NADH as well as NADPH could sustain the oxidation of 7β-hydroxy-Δ⁸-THC to the corresponding ketone. The NADH-dependent oxidation of 7β-hydroxy-Δ⁸-THC by monkey livers is mainly catalyzed by CYP3A8 as well as the NADPH-dependent oxidation. These results indicate that NADH as a cofactor may be also useful for the oxidation of 7β-hydroxy-Δ⁸-THC, and that the cofactor requirement for the reaction is varied by the concentrations of substrate and/or cofactor.

View full abstract

The transition metal zinc (Zn) is an endogenous regulator of apoptosis. The ability of Zn to modulate apoptosis is believed to be mediated by the regulation of caspase activity. Previously, we reported that an acute influx of labile Zn induced apoptosis via activation of caspase in human leukemia HL-60 cells treated with a Zn ionophore (Py, pyrithione) and Zn at 1 and 25 μM, respectively. In the present study, we investigated the involvement of caspase-3 in Py (1 μM)/Zn (25 μM)-induced apoptosis in HL-60 cells. Pro-caspase-3 is an inactive form of caspase-3. The processing of pro-caspase-3, a sign of caspase-3 activation, occurred 6 h after treatment with Py/Zn. Proteolysis of poly (ADP-ribose) polymerase (PARP), a substrate of caspase-3, was also observed 6 h after treatment with Py/Zn. We also confirmed the elevation of caspase-3 activity as an index of the cleavage of amino acid sequences recognized by activated caspase-3. An inhibitor of caspase-3 attenuated the appearance of the DNA ladder. Taken together, these results indicate that the activation of caspase-3 is partly responsible for the induction of apoptosis in Py/Zn-treated HL-60 cells.

View full abstract

In this study, the antiinflammatory activities of ginsenoside Rb1, which is a main constituent of the root of Panax ginseng (Araliaceae), and of its metabolite compound K, as produced by human intestinal bacteria, on lipopolysaccharide (LPS)-induced RAW264.7 cells were investigated. Compound K potently inhibited the production of NO and prostaglandin E2 in LPS-induced RAW 264.7 cells, with IC₅₀ values of 0.012 and 0.004 mM, respectively. Compound K also reduced the expression levels of the inducible NO synthase (iNOS) and COX-2 proteins and inhibited the activation of NF-kB, a nuclear transcription factor. Compound K inhibited the NO level produced by iNOS enzyme activity in a cell-free system, but did not inhibit COX-1 and 2 activities. When ginsenoside Rb1 was orally administered to rats, compound K, but not ginsenoside Rb1, were excreted in their urine. These findings suggest that ginsenoside Rb1 can be transformed to compound K by intestinal bacteria, and compound K may be effective against inflammation.

View full abstract

A furocoumarin derivative, oxypeucedanin, was purified from Angelica dahurica, and its effects on the human Kv1.5 (hKv1.5) channel and on the cardiac action potential duration (APD), were examined using the patch-clamp technique and the conventional microelectrode technique. Oxypeucedanin inhibited the hKv1.5 current in a concentration-dependent manner, with an IC₅₀ value of 76 nM, while it had no effect on human eag-related gene (HERG) current. Oxypeucedanin induced an initial fast decline of hKv1.5 current during depolarizations. The inhibition of hKv1.5 channel by oxypeucedanin was voltage-dependent, especially at depolarizing pulses between −40 and 0 mV which corresponds to the voltage range of the channel's opening. Oxypeucedanin also slowed the deactivation time course, resulting in a tail crossover phenomenon. Additionally, oxypeucedanin prolonged the APD of rat atrial and ventricular muscles in a dose-dependent manner. These results suggest that oxypeucedanin is a kind of open-channel blocker of the hKv1.5 channel and it prolongs the APD; therefore, it is an excellent candidate as an antiarrhythmic drug for atrial fibrillation.

View full abstract

Moutan Cortex Radicis (MCR) is one of the most widely used Oriental medicines. In this study, we assessed the reducing effect of ethanol extract of MCR on hydrogen peroxide-induced reactive oxygen production, the main cause of cell damage or death in PC12 cells. The viability of cells treated with 1 mg/ml of MCR was significantly restored from that of oxidative-stressed PC12 cells. Measurement of intracellular reactive oxygen species (ROS) generation was determined using the H₂DCFDA assay. MCR at 1—0.01 mg/ml concentration inhibited ROS production in oxidative-stressed cells. To identify candidate genes responsible for the anti-oxidative effects of MCR on PC12 cells, an oligonucleotide microarray analysis was performed. The result of gene expression profiles showed that 10 genes were up-regulated and 7 were down-regulated in MCR plus hydrogen peroxide treated cells compared with hydrogen peroxide treated cells. Among them, heme oxygenase (HO) and cathechol-O-methyltransferase (COMT) are related to regulation of ROS generation and the others are known to regulate cell survival and progression. Subsequently, we performed real-time RT-PCR to quantify the ROS related gene. MCR treatment increased the expression of HO by 370% and COMT by 280% at the concentration of 1 mg/ml. These findings suggest that MCR inhibits the production of ROS and cytotoxicity by oxidative-stressed PC12 cells through over-expression of HO and COMT.

View full abstract

Crude preparations of Stephania tetrandra S. MOORE (ST), a traditional herbal medicine, have been used safely for arthritis and silicosis in China. In this study, we demonstrated that ST in vitro protects red blood cells from 2,2-azo-bis (2-amidinopropane) dihydrochloride (AAPH)-induced hemolysis. The inhibitory effect was dose-dependent at concentrations of 10 to 1000 μg/ml. Moreover, tests were carried out to identify the main ingredient of ST that exerts a scavenging effect on free-radicals. Three representative alkaloids, tetrandrine, fangchinoline, and cyclanoline, isolated from ST, were found to have inhibitory activities against AAPH-induced lysis of red blood cells (RBC). Furthermore, the ingestion of 200 mg of ST extract was associated with a significant increase in free-radical scavenging effect of plasma in rats. These results suggest that ST as antioxidant inhibits AAPH-induced hemolysis of RBC both in vitro and in vivo.

View full abstract

In order to identify the existence of Panax species in herbal medicine preparations, the Ginseng specific marker primer was selected and created based on the sequence of Korean ginseng DNA fragment, 359 bp. The gradient PCR was performed on 40 types of the herbal medicines including the 7 types of Araliaceae that are in the same family with the Panax ginseng using the created Ginseng maker primer. As result, Panax notoginseng (Chinese), Panax japonicus (Japanese) and Panax quinquefolius (American), along with Panax ginseng (Korean) were the only ones amplified. However, in the case of Atractylodes lancea, one of the herbal medicines not categorized as Panax species, the DNA was prominently amplified by the Ginseng marker primer. The sequence of the amplified DNA of Atractylodes lancea was identified, resulting in enabling the differentiation from the Panax species by the Restriction Fragment Length Polymorphisms (RFLP) method. In addition, the results of the gradient PCR performed on the herbal medicine preparations that consists of Panax ginseng showed that 290 bp size of the original DNA fragments of Panax ginseng was amplified on the herbal medicine preparations containing Panax ginseng. Therefore, these results suggest a possibility of creating a new testing method for identifying specific herb medicines using the gradient PCR, a molecular biological method not only on Panax ginseng, but also on other herbal medicines and herbal medicine preparations.

View full abstract

Thiopurine methyltransferase (TPMT) metabolizes thiopurine drugs which are used in the treatment of leukemia and some autoimmune diseases. Previously, 11 mutant alleles of TPMT gene (TPMT*1S, *2, *3A, *3B, *3C, *3D, *4, *5, *6, *7, and *8) have been reported. These mutant alleles may cause life-threatening toxicity in patients exposed to thiopurine drugs, 6-mercaptopurine and azathioprine. We have developed a rapid and accurate protocol for TPMT genotype determination using Pyrosequencing^TM technology in 96 Japanese subjects. Five fragments of the TPMT gene (exon 4, 5, 7, 8, 10) were amplified by PCR, and the 10 single-nucleotide polymorphisms (SNPs) for TPMT*1S, *2, *3A, *3B, *3C, *3D, *4, *5, *6, *7, and *8 were sequenced. The results of this pyrosequencing method corresponded exactly with those of the DNA sequencing method using BigDye terminator chemistry. We have demonstrated that typing of 10 SNPs can be performed within 30 min. Pyrosequencing has a wide application in the large-scale identification of individual TPMT genotypes.

View full abstract

Intestinal chiral inversion of ibuprofen is still lacking direct evidence. In a preliminary experiment, ibuprofen was found to undergo inversion in Caco-2 cells. This investigation was thus conducted to determine the characteristics and influence of some biochemical factors on the chiral inversion of ibuprofen in Caco-2 cells. The effects of substrate concentration (2.5—40 μg/ml), cell density (0.5—2×10⁶ cells/well), content of serum (0—20%), coexistence of S ibuprofen (corresponding doses), sodium azide (10 mM), exogenous Coenzyme A (CoA) (0.1—0.4 mM), and palmitic acid (5—25 μM) on inversion were examined. A stereoselective HPLC method based on the Chromasil-CHI-TBB column was developed for quantitative analysis of the drug in cell culture medium. The inversion ratio (F_i) and elimination rate constant were calculated as the indexes of inversion extent. Inversion of ibuprofen in Caco-2 cells was found to be both dose and cell density dependent, indicating saturable characteristics. Addition of serum significantly inhibited the inversion, to an extent of 2.7 fold decrease at 20% content. Preexistence of S enantiomer exerted a significant inhibitory effect (p<0.01 for all tests). Sodium azide decreased the inversion ratio from 0.43 to 0.32 (p<0.01). Exogenous CoA and palmitic acid significantly promoted the inversion at all tested doses (p<0.01 for all tests). This research provided strong evidence to the capacity and capability of intestinal chiral inversion. Although long incubation times up to 120 h were required, Caco-2 cells should be a suitable model for chiral inversion research of 2-APAs considering the human-resourced and well-defined characteristics from the present study.

View full abstract

We investigated the pharmacokinetic characteristics of 11-hydroxyaclacinomycin X (ID-6105), a novel anthracycline, after intravenous (i.v.) bolus administration in rats and beagle dogs. We developed an HPLC-based method to analyze ID-6105 levels in plasma, bile, urine, feces, and tissue homogenates and validated the method in a pharmacokinetic study. The plasma concentration of ID-6105 decreased to below the quantifiable limit (0.02 μg/ml) at 4 and 8 h after i.v. administration in rats at doses of 2 and 10 mg/kg, respectively (t_1/2,α and t_1/2,β of 0.78 and 17.8 min at a dose of 2 mg/kg, 0.91 and 176 min at a dose of 10 mg/kg, respectively). The AUC increased with nonlinear pharmacokinetics following the dosage increase from 2 to 10 mg/kg in rats, while the pharmacokinetics were not significantly altered in beagle dogs following a dosage increase from 0.5 to 2.5 mg/kg. Of the various tissues tested, ID-6105 was mainly distributed in the lung, spleen, kidney, adrenal gland, and liver after i.v. bolus administration. ID-6105 levels in the lung or kidney 2 h after i.v. bolus administration were comparable to the initial plasma concentration. However, the ID-6105 concentrations in various tissues 48 h after i.v. bolus administration became too small to measure. The cumulative amounts of ID-6105 found in the bile 48 h after the administration of 2 and 10 mg/kg were calculated to be 26.7 and 18.5% of the initial dose, respectively. The corresponding values in the urine 72 h after i.v. administration were 4.33 and 3.07% of the initial dose, suggesting that ID-6105 is mostly excreted in the bile. In conclusion, our observations indicate that ID-6105 was rapidly cleared from the blood and transferred to tissues such as the lung, spleen, kidney, and liver 2 h after i.v. bolus administration. Moreover, the majority of ID-6105 appears to be excreted in the bile by 24 h after i.v. bolus administration.

View full abstract

Effect of two preparation (Croatian and Brazilian) of water-soluble derivative of propolis (WSDP), caffeic acid, quercetin, chrysin, naringenin (components present in WSDP) on the development of Ehrlich ascites tumour (EAT) was evaluated. Test components (50 mg/kg) were given perorally or intraperitoneally 2 h prior the intraperitonel injection of EAT (2×10⁶) cells. It was observed that all test compounds effectively inhibited tumour growth and the proliferation of EAT. The volume of ascitic fluid induced by EAT cells and total number of cells present in the peritoneal cavity was markedly reduced in EAT-bearing mice treated with test components. In treated mice the number of polymorphonuclear (PMN) cells in the peritoneal cavity was increased while the number of macrophages was decreased. The macrophage spreading activity revealed that WSDP and all test compounds affected the functional state of macrophages increasing their tumorcidal activity; the effect of WSDP was most pronounced indicating synergistic effect of components present in WSDP. Antitumor activity of WSDP may be the result of different specific mechanism(s) of flavonoids present as compared to individual flavonoid given alone. It is likely that the part of antitumor efficacy of test components against EAT cells was the results of increased activity of macrophages.

View full abstract

We recently showed that size, not surface charge, is a major determinant of the in vitro lipofection efficiency of pDNA/TFL-3 complex (lipoplex), even in the presence of serum. In this study, the effect of lipoplex size as a result of interaction with serum proteins on in vitro lipofection and the relationship of this with in vivo lipofection was examined in a murine lung metastasis model. As previously described, the pDNA to lipid ratio (P/L ratio) affected both the size and zeta potential of the lipoplex. In vitro studies also indicated that transgene expression in B16BL6 cells was largely dependent on the size of the lipoplex, both in the absence or presence (50% (v/v)) of serum. An in vivo lipofection experiment showed that predominant gene expression in lungs occurred only in tumor-bearing mice, not in normal mice. Based on the in vitro study, this tumor-related gene expression was not related to lipoplex size in the presence of serum (50% (v/v)), suggesting that the size alteration, as the result of interactions with serum proteins in the blood stream may not play an important role in the case of systemic injections. In addition, the efficient gene expression in tumor-bearing lung was not related to the progression of lung metastases. The area-specific gene expression in tumor-bearing lungs, which was largely dependent on the P/L ratio of the lipoplexes, was observed by fluorescent microscopy. Although the underlying mechanism for the area-specific transgene expression is not clear, it may be related to the interaction of lipoplexes with tumor cells, vascular endothelial cells under angiogenesis and normal cells in the lungs. The possibility that TFL-3 is a useful utility to the targeted delivery of pDNA to lungs and tumor-related lipofection is demonstrated. This result suggests that area-specific gene expression in lung metastases may be achieved by controlling the physicochemical properties of the lipoplex, i.e. the P/L ratio.

View full abstract

In order to understand and overcome multidrug resistance (MDR) of human non-small cell lung cancer (NSCLC), mRNA and protein expression levels of P-glycoprotein (MDR1), multidrug resistance-associated protein 1 (MRP1), and lung resistance-related protein (LRP) were investigated and compared with the chemosensitivity and the intracellular/intranuclear cisplatin accumulation of three NSCLC cell lines (Ma-10, Ma-31, and Ma-46). Ma-31 was more resistant than Ma-10 and Ma-46 to cisplatin, carboplatin, etoposide, and paclitaxel. The mRNA level of MDR1 was extremely low, and MDR1 protein was not detected in all cell lines. MRP1 mRNA expression was highest in Ma-31 and lowest in Ma-10, but there was no notable difference between the MRP1 protein expression in three cell lines. LRP mRNA/protein was equally expressed in Ma-10 and Ma-31, but was nominal in Ma-46. The intracellular/intranuclear cisplatin accumulation of the cells was determined to be Ma-31>Ma-46>Ma-10. Thus, MDR1, MRP1, and LRP mRNA and protein expression levels were not correlated with the chemosensitivity or the intracellular/intranuclear cisplatin accumulation of each cell line. The present results indicate that MDR proteins (MDR1, MRP1, and LRP) may not play an important role in the chemoresistance and drug efflux of NSCLC cells.

View full abstract

The contribution of P-glycoprotein (P-gp) to the intestinal absorption of cyclosporin A (CsA) was investigated by comparing the in vivo pharmacokinetics of CsA in P-gp knockout mice versus wild-type mice following both oral and intravenous administration and by examining the transport of CsA across Caco-2 cell monolayers. The apparent oral bioavailability of CsA in P-gp knockout mice was 1.55-fold larger than in wild-type mice, leading to an apparent absolute bioavailability of 41.8%. A concentration dependent efflux transport of CsA across Caco-2 cell monolayers was found, which exhibited saturation at a CsA concentration of 1 μM. These results suggest that the involvement of P-gp in the intestinal absorption of CsA is not as profound as was previously thought.

View full abstract

The cardiovascular drug oxyfedrine HCl revealed noteworthy in vitro antibacterial action against 501 strains of Gram positive and Gram negative bacteria. It also offered significant protection to mice challenged with a mouse-virulent bacterial strain. Prompted by such results, the present study was carried out to ascertain whether this drug could augment the efficiency of an antibiotic when used in combination with it. For this purpose, ten bacterial strains were selected, which were sensitive to oxyfedrine as well as to six antibiotics, like benzyl penicillin, chloramphenicol, ciprofloxacin, erythromycin, streptomycin and tetracycline. Distinct and statistically significant (p<0.01) synergism was observed between oxyfedrine and tetracycline by disc diffusion tests, compared with their individual effects. The fractional inhibitory concentration (FIC) index of this combination, evaluated by checkerboard analysis, was 0.37, which confirmed synergism between the pair. This synergistic drug duo was further dispensed to infected mice. The results of the mouse-protection tests advocated that the combination was significantly synergistic (p<0.0001), according to Student's ‘t’ test. Hence, the capacity of extended antibiotic therapy in several microbial diseases may be improved with the help of this synergistic drug pair, and the study might throw light on newer directions to contest drug-resistant bacterial infections.

View full abstract

We report on a facile and rapid computerized in-vitro assay for the quantification of leukocyte adhesion to endothelial cells under static conditions using bovine polymorphonuclear neutrophils (PMN) or human leukaemic Mono Mac 6 cells (MM6) and bovine aorta endothelial cells (BAEC). Images of leukocytes adherent to BAEC monolayers grown in microtiter plates were obtained by a digital camera attached to a conventional microscope and transferred to the public domain NIH ImageJ program for analysis. Using individually adapted program routines adherent leukocytes are easily discriminated and reproducibly quantified. The results obtained with our assay correspond to previous findings and demonstrate the suitability of the described procedure, which can easily be adapted to further standards as proven by the use of two different leukocyte species. This assay lends itself to the screening of pharmacological substances with different mechanism of action that might act on either leukocytes or endothelial cells.

View full abstract

Recently, some properties of myo-inositol hexakisphosphate (InsP₆) are related to its dermatological use as discolouring agent, on preventing calcinosis cutis or due to its important role on premature aging. Some studies also seem to demonstrate a capacity of InsP₆ to inhibit skin cancer. In this paper, a first study of the absorption of InsP₆ through the skin is developed. Due to the correlation between InsP₆ absorption and its urinary excretion, these last values were used to evaluate this process. It was found that using a moisturizing cream as vehicle, the InsP₆ sodium salt was absorbed at significantly higher amounts than the InsP₆ calcium–magnesium salt. Maximum InsP₆ urinary concentrations were observed approximately at 14 d of 2% InsP₆ topical cream application, and gave 66.35±5.49 mg/l urinary InsP₆ when the sodium salt was used and 16.02±2.61 mg/l urinary InsP₆ when the calcium–magnesium salt was applied. When the InsP₆ topical cream administration ceased, the InsP₆ urinary excretion fell dramatically approximately during a period of 10 d. From these results, it can be deduced that by topical administration InsP₆ can achieve important concentrations in tissues and biological fluids, this demonstrating that it is possible to propose the topic use as a new InsP₆ administration route.

View full abstract

Rat pheochromocytoma PC12 cells undergo neuronal differentiation in response to nerve growth factor. We show here that exposure of PC12 cells to Nardostachys chinensis glycoside induces the outgrowth of neurites, increases the activity of AChE, triggers cell cycle arrest in G1 and enhances the expression of growth associated protein 43 (GAP-43). Both the outgrowth of neurites and the increase in AChE activity are prevented partly by PD98059, a specific inhibitor of MEK1. These results suggest that N. chinensis glycoside induces the characteristics of neuronal differentiation in PC12 cells via the mitogen-activated protein kinase (MAPK)-related signal cascade.

View full abstract