Four types of experimental chronic ulcer models, named acetic acid ulcer models, have been developed to examine the healing process of peptic ulcers, screen anti-ulcer drugs, and better evaluate the adverse effects of various anti-inflammatory drugs on the gastrointestinal mucosa. The model easily and reliably produces round, deep ulcers in the stomach and duodenum, allowing acetic acid ulcer production in mice, rats, Mongolian gerbils, guinea pigs, cats, dogs, miniature pigs, and monkeys. These ulcer models highly resemble human ulcers in terms of both pathological features and healing process. The models have been established over the past 35 years and are now used throughout the world by basic and clinical scientists. One of the characteristic features of acetic acid ulcers in rats is the spontaneous relapse of healed ulcers >100 d after ulceration, an endoscopically confirmed phenomenon. Indomethacin significantly delays the healing of acetic acid ulcers, probably by reducing endogenous prostaglandins and inhibiting angiogenesis in ulcerated tissue. Helicobacter pylori significantly delays healing of acetic acid ulcers and causes relapse of healed ulcers at a high incidence in Mongolian gerbils. Anti-secretory drugs (e.g. omeprazole), prostaglandin analogs, mucosal defense agents (e.g. sucralfate), and various growth factors all significantly enhance healing of acetic acid ulcers. Gene therapy with epidermal growth factor and vascular endothelial growth factor applied to the base of acetic acid ulcers in rats is effective in enhancing ulcer healing. Since an inhibitor of nitric oxide syntase prevents ulcer healing, nitric oxide might be involved in the mechanism underlying ulcer healing. We conclude that acetic acid ulcer models are quite useful for various studies related to peptic ulcers.
Much has been learned about the activity-dependent synaptic modifications that are thought to underlie memory storage, but the mechanism by which these modifications are stored remains unclear. A good candidate for the storage mechanism is Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). CaM kinase II is one of the most prominent protein kinases, present in essentially every tissue but most concentrated in brain. Although it has been about a quarter of a century since the finding, CaM kinase II has been of the major interest in the region of brain science. It plays a multifunctional role in many intracellular events, and the expression of the enzyme is carefully regulated in brain regions and during brain development. Neuronal CaM kinase II regulates important neuronal functions, including neurotransmitter synthesis, neurotransmitter release, modulation of ion channel activity, cellular transport, cell morphology and neurite extension, synaptic plasticity, learning and memory, and gene expression. Studies concerning this kinase have provided insight into the molecular basis of nerve functions, especially learning and memory, and indicate one direction for studies in the field of neuroscience. This review presents the molecular structure, properties and functions of CaM kinase II, as a major component of neurons, based mainly developed on findings made in our laboratory.
Parkinson's disease involves the progressive degeneration of dopaminergic neurons in the substantia nigra. However, the etiology of the disease remains to be elucidated. Endogenous amines, such as 1,2,3,4-tetrahydroisoquinoline (TIQ) derivatives present in the mammalian brain, are known to participate in the pathogenesis of Parkinson's disease. These endogenous neurotoxins have been extensively studied because of their structural resemblance to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), an agent widely used for generating animal models of Parkinson's disease-like symptoms. Investigations of the synthesis and pharmacological properties of TIQ derivatives are expected to contribute to the development of new therapeutic agents for treating Parkinson's disease. In the present study, we describe more efficient synthesis methods for TIQ derivatives via Pummerer-type cyclization of the substrate N-acyl sulfoxide. Furthermore, the modified Pummerer reaction provided a convenient and efficient method for synthesizing various TIQs. TIQ and its derivative, 1-benzyl-TIQ, can induce parkinsonism in primates and rodents. On the other hand, one TIQ derivative, 1-methyl-TIQ, has been shown to prevent MPTP, TIQ, and 1-benzyl-TIQ induced behavioral abnormalities. Therefore, TIQ derivatives are considered to play an important role in both the onset and prevention of Parkinson's disease. In this article, we focus on the synthesis and pharmacological aspects of 1,2,3,4-tetrahydroisoquinoline derivatives in Parkinson's disease.
A liquid chromatographic-mass spectrometric (LC-MS) method was developed and validated for determination of guanfu base I (GFI), and the pharmacokinetics of GFI in Sprague-Dawley rats was examined. The method was linear in the 0.05—20 μg/ml concentration range (r=0.9994). The recovery of guanfu base I was more than 80%. The intraday and interday precision, expressed as the relative standard deviation (RSD), was generally good (<15%). After i.v. dosing, plasma GFI concentration declined in a bi-phasic manner with a terminal elimination half-life of 2.49 h. The total plasma clearance values was 1.46 l/h/kg. After oral dosing, the plasma GFI concentration reached a maximum within 0.5 h. The absolute bioavailability of GFI was 71.31%.
Flavonoids possess anti-inflammatory activity in vitro and in vivo. In order to find the anti-inflammatory flavone derivatives having optimum chemical structures, various flavones were previously synthesized and evaluated for their inhibitory activity of prostaglandin E2 (PGE2) production from lipopolysaccharide (LPS)-treated mouse macrophage cell line, RAW 264.7 cells. Through this screening procedure, 2′,4′,7-trimethoxyflavone (TMF) was selected for further pharmacological study. From the present investigation, it was found that TMF potently inhibited PGE2 production from LPS-treated RAW cells with an IC50 of 0.48 μM, compared to the IC50 values of 0.07 and 1.09 μM for NS-398 and wogonin. TMF, however, did not inhibit cyclooxygenase-2 (COX-2) activity or COX-2 expression level. Instead, TMF was proved to be a phospholipase A2 (PLA2) inhibitor. The IC50 values of TMF against secretory PLA2-IIA (sPLA2-IIA) and cytosolic PLA2 (cPLA2) were 70.5 and 70.4 μM, respectively. At doses of 10—250 μg/ear, TMF also showed in vivo anti-inflammatory activity by topical application against mouse croton oil-induced ear edema assay, suggesting a potential for new anti-inflammatory agent.
Cry j 1 is one of the major allergens in Japanese cedar pollen. We attempt high throughput analysis and comprehensive identification of the linear IgE epitopes of Cry j 1. A series of overlapping synthetic Cry j 1 peptides chemically spotted on cellulose membrane was probed with sera from patients in Japan and United States, which were reactive to Cry j 1, and the reactivity of one of the detected sequences was confirmed by means of competitive ELISA using peptide as coated antigen. The peptide 331NGNATPQLTKNA342 (peptide 166) was detected by all three pooled sera used, and peptide 103NGGPCVFIKRVS114 (peptide 52) was detected by two of the three pools of sera. In addition, several peptides reacted with one of the pooled sera. IgE binding to peptide 166-coated wells was inhibited by addition of peptide 166 for several individual patient sera, suggesting that peptide 166 is one of the linear epitopes of Cry j 1. Since patients in United States were suggested to be rarely sensitized with Japanese cedar, they were sensitized with the similar tree pollen allergens such as Cup s 1 and Jun a 1, and cross-reacted with Cry j 1. We have comprehensively investigated human IgE epitopes of Cry j 1 and succeeded in identifying a common linear epitope, 331NGNATPQLTKNA342.
It has been reported that QM was highly expressed by cells isolated from epiphyseal cartilage as opposed to proliferative chondrocytes. In vitro investigation of the expression of QM revealed higher QM expression in nonmineralizing osteoblast and pericyte cultures as compared with mineralizing cultures. These evidences suggest that QM may play an essential role in cell differentiation before mineralization. However, our research results showed that QM overexpression in MC3T3-E1 enhanced cell differentiation and mineralization. In this study, alkaline phosphatase (ALP) activity and nodule mineralization were increased in MC3T3-E1 from QM overexpression cultures relative to normal expression QM cultures. RT-PCR revealed upregulation of the marker genes type I collagen, ALP, osteocalcin, osterix and BMP-2 and a slight decrease of a negative regulator osteopontin. These results suggest that the increasing of QM expression could stimulate osteoblast differentiation and mineralization in MC3T3-E1.
The cytotoxic activity of 7β-hydroxycholesterol (7β-OHC) was evaluated on human NCI-H460 lung cancer cells. 7β-OHC decreased clonogenic survival of NCI-H460 in a dose dependent pattern. 7β-OHC induced apoptosis in NCI-H460, with the characteristic features like increase in sub-G1 hypodiploid (apoptotic) cells, and apoptotic body formation, as evidenced by flow cytometry and fluorescence microscope, respectively. Apoptosis was also associated with loss of mitochondrial transmembrane potential, and the activation of caspases 9 and 3. 7β-OHC resulted in generation of reactive oxygen species (ROS) during apoptosis. On the whole, the results indicated that 7β-OHC inhibited the proliferation of NCI-H460 cells through apoptosis via caspase activation.
Matrix metalloproteinase 2 (MMP-2) is a metalloproteinase belonging to a family of structurally related zinc-dependent endopeptidases capable of degrading extracellular matrix components. To elucidate the functional promoter of the mouse MMP-2 gene, systematic transient expression analysis of the 5′-flanking region of the MMP-2 gene was performed using serially nested deletions. The deletion analysis indicated that the proximal 327-bp sequence from nucleotide positions −313 to +14 relative to the transcription start site is essential for minimal promoter activity and that a 10-bp sequence of the promoter at positions −939 to −930 is required for high expression level of the MMP-2 gene. The 10-bp fragment functioned as a potent stimulator of heterologous SV40 promoter activity. This element is identical to the YB-1 binding motif (Y-box) present within the responsive element-1 (RE-1), which has been shown to act as a potent cis-activator of transcription of the rat MMP-2 gene. The binding of a nuclear factor(s) to the 10-bp fragment was also revealed by electrophoretic mobility shift assays (EMSAs). Antibody-supershift EMSAs of nuclear extracts from NIH 3T3 cells demonstrated YB-1 binding to the RE-1 sequence. It was concluded that the RE-1 is the conserved element for potent expression of MMP-2 gene among rodents.
Macrophage-derived foam cells play an important role in atherosclerotic lesions. Oxidized low-density lipoprotein (OxLDL) induces macrophage proliferation via the specific uptake of lysophosphatidylcholine (LysoPC) of OxLDL by class A, type I and type II macrophage scavenger receptors. We have previously shown that Asp-hemolysin from Aspergillus fumigatus binds to LysoPC as a typical lipid moiety of OxLDL. This study investigated the effect of the Asp-hemolysin-related peptide (P-21), a synthetic peptide derived from a region of Asp-hemolysin that is rich in positive charges, on macrophage proliferation induced by OxLDL. Mouse peritoneal macrophages were used for proliferation study. OxLDL induced macrophage proliferation in an oxidation time-dependent manner, and P-21 inhibited OxLDL-induced macrophage proliferation in a dose-dependent manner. Furthermore, the binding analysis of P-21 to OxLDL by dissociation-enhanced lanthanide fluorometric immunoassay indicated that P-21 binds to OxLDL. These results indicate that P-21 inhibits the OxLDL-induced macrophage proliferation through binding of P-21 to OxLDL.
Klebsiella pneumoniae MGH78578, a clinical isolate, showed high level of resistance to many antimicrobial agents. We cloned genes responsible for drug resistance from chromosomal DNA of K. pneumoniae MGH78578 by shotgun method using Escherichia coli KAM32, a drug hypersensitive strain, as host. We obtained 43 hybrid plasmids that made host cells resistant to several antimicrobial agents. We classified them into 17 groups based on growth properties in the presence of each one of 9 antimicrobial agents and on restriction patterns of each hybrid plasmid. Analysis of the cloned genes must be very useful for investigation of major parts of multidrug resistance systems including multidrug efflux pumps in K. pneumoniae MGH78578 in which genome sequence is available.
Nedaplatin is known to exhibit antitumor activity similar to that of cisplatin. However, concerning side effects, nedaplatin causes renal toxicity less frequently than cisplatin. In this study, we compared the incidence of renal toxicity between cisplatin and nedaplatin by investigating the difference in kidney tissue accumulation. Kidney tissue accumulation of cisplatin administered at 3.75 mg/kg was similar to that of nedaplatin administered at 24 mg/kg. At these doses, the plasma creatinine level and urinary excretion of glucose and N-acetyl-β-D-glucosaminidase (NAG) similarly increased. There was a correlation between kidney accumulation of cisplatin and nedaplatin and the increases in plasma creatinine level and urinary excretion of NAG. Therefore, our results suggest that nedaplatin less frequently causes renal toxicity in comparison to cisplatin due to lower kidney accumulation.
Stressors with a physical factor such as immobilization, electric foot shock, cold swim, etc., have been shown to produce oxidative damage to membrane lipids in the brain. In this study, we investigated the effect of protracted social isolation stress on lipid peroxidation activity in the mouse brain and elucidated the protective effect of majonoside-R2, a major saponin component of Vietnamese ginseng, in mice exposed to social isolation stress. Thiobarbituric acid reactive substance levels, one of the end products of lipid peroxidation reaction, were increased in the brains of mice subjected to 6—8 weeks of social isolation stress. Measurements of nitric oxide (NO) metabolites (NOx−) also revealed a significant increase of NO production in the brains of socially isolated mice. Moreover, the depletion of brain glutathione content, an endogenous antioxidant, in socially isolated animals occurred in association with the rise in lipid peroxidation. The intraperitoneal administration of majonoside-R2 (10—50 mg/kg) had no effect on thiobarbituric acid reactive substances (TBARS), NO, or glutathione levels in the brains of group-housed control mice but it significantly suppressed the increase in TBARS and NO levels and the decrease in glutathione levels caused by social isolation stress. These results suggest that mice subjected to 6—8 weeks of social isolation stress produces oxidative damage in the brain partly via enhancement of NO production, and that majonoside-R2 exerts a protective effect by modulating NO and glutathione systems in the brain.
Statin, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, has an anti-inflammatory effect. The aim of this study was to investigate the effect of Lovastatin (statin) on the cholecystokinin-octapeptide (CCK)-induced acute pancreatitis in rats. In statin treated group, the pancreas weight/body weight (pw/bw) ratio in CCK-induced acute pancreatitis was significantly lower than DMSO-treated group. Statin also increased the pancreatic level of HSP 60. Additionally, the secretions of IL-1β, TNF-α and IL-6 and the lipase levels were decreased in statin treated group. These results suggest that statin may play an important role in mitigating the progression of the inflammatory reactions during acute pancreatitis.
Immune activation is an effective as well as protective approach against emerging infectious diseases. The immunomodulatory activities of Triphala (Terminalia chebula, Terminalia belerica and Emblica officinalis) were assessed by testing the various neutrophil functions like adherence, phagocytosis (phagocytic index (P.I) and avidity index (A.I)) and nitro blue tetrazolium (NBT) reduction in albino rats. In recent years much attention is being focused on the immunological changes occur during stress. Noise (100 dB) stress for 4 h/d for 15 d, was employed to alter the neutrophil functions. The neutrophil function tests and corticosterone levels were carried out in eight different groups of animals, namely control, Triphala, noise-stress, Triphala noise-stress, and corresponding immunized groups were used. Sheep red blood cells (SRBC 5×109 cells per ml) were used for immunizing the animals that belongs to immunized groups. In Triphala administration (1 g/kg/d for 48 d), A.I was found to be significantly enhanced in the Triphala group, while the remaining neutrophil functions and steroid levels were not altered significantly. However the neutrophil functions were significantly enhanced in the Triphala immunized group with a significant decrease in corticosterone level was observed. Upon exposure to the noise-stress, the neutrophil functions were significantly suppressed and followed by a significant increase in the corticosterone levels were observed in both the noise-stress and the noise-stress immunized groups. These noise-stress-induced changes were significantly prevented by Triphala administration in both the Triphala noise-stress and the Triphala noise-stress immunized groups. Hence our study has divulged that oral administration of Triphala appears to stimulate the neutrophil functions in the immunized rats and stress induced suppression in the neutrophil functions were significantly prevented by Triphala.
Xanthoangelol, a major chalcone constituent of the stem exudates of Angelica keiskei, was evaluated for cell toxicity and apoptosis-inducing activity in human neuroblastoma (IMR-32) and leukemia (Jurkat) cells. Xanthoangelol concentration-dependently reduced the survival rates of both cell lines as revealed by the trypan blue exclusion test. Early apoptosis induced by 4 h incubation with xanthoangelol was detected using flow cytometry after double-staining with annexin V and propidium iodide (PI). Western blot analysis showed that xanthoangelol markedly reduced the level of precursor caspase-3 and increased the level of cleaved caspase-3, but Bax and Bcl-2 proteins were not affected. These results suggest that xanthoangelol induces apoptotic cell death by activatation of caspase-3 in neuroblastoma and leukemia cells through a mechanism that does not involve Bax/Bcl-2 signal transduction. Therefore, xanthoangelol may be applicable as an effective drug for treatment of neuroblastoma and leukemia.
Several rodent models of cortical malformation are available for the study of cellular mechanisms associated with early-onset epilepsy, but few are associated with spontaneous seizures. We examined the effect of pilocarpine on the spontaneous seizure development and excitability of the CA1 pyramidal cells of rats after prenatal treatment with methylazoxymethanol (MAM). Pilocarpine induced status epilepticus (SE) onset latency was greater for normal rats than for MAM-treated rats. After several days of normal behavior following pilocarpine treatment, the duration of spontaneous seizures were greater in MAM-pilocarpine rats than in normal-pilocarpine rats. Compared with the normal rats, electrical stimulation of afferent fibers resulted in more robust population responses in the CA1 region in all groups. At interstimulus intervals of 30 and 70 ms, the MAM-pilocarpine rats displayed a decrease in paired pulse inhibition versus the conventional MAM rats. A loss of somatostatin- and parvalbumin-immunoreactive neurons was apparent in the normal-pilocarpine rats, MAM-pilocarpine rats, and conventional MAM rats. These results indicate that pilocarpine induces spontaneous seizures and hyperexcitability in MAM-pilocarpine rats.
The antifungal activity of ZJ-522, a new triazole antifungal agent restructured from fluconazole and butenafine, was compared to that of fluconazole and butenafine against 43 strains of fungi representing 13 fungal species. MICs were determined by using the National Committee for Clinical Laboratory Standards (NCCLS)-recommended broth microdilution method for yeasts, which was modified for filamentous fungi. ZJ-522 was about 50-fold and 2 to 16-fold more potent than fluconazole against yeasts and filamentous fungi respectively, but it was less active than butenafine against filamentous fungi, although butenafine was inactive against most yeasts. Thus, the fashion of ZJ-522 antifungal activity more similar to that of fluconazole than that of butenafine indicates that ZJ-522 should be an inhibitor of lanosterol 14α-demethylase but not of squalene epoxidase, and should be a candidate for clinical development.
Microsomal triglyceride (TG) transfer protein (MTP) is involved in the secretion of TG-rich very low-density lipoprotein (VLDL), a process which leads to the generation of hypertriglyceridemia and atherosclerosis. We investigated the possible role of Ca2+ on MTP activity in hepatocytes. Exogenous CaCl2 and calmodulin increased MTP activity dose-dependently, and calcium ionophore A23187 (A23187) also increased total Ca2+ level and MTP activity in hepatocytes. Moreover, MTP activity increased by CaCl2 or A23187 was abrogated in the presence of EDTA, a Ca2+ chelator. MTP activity was increased by the simultaneous addition of CaCl2 and calmodulin. However, this increase was inhibited by N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7), a Ca2+ antagonist. A23187 increased the release of TG and cholesterol from hepatocytes, and these were inhibited by EDTA. A23187 also increased the ratio of TG to HDL-cholesterol in hepatocytes culture medium, which indicates the release of TG is higher than that of HDL-cholesterol from hepatocytes. Thus, our findings demonstrate that hepatocellular Ca2+ contributes directly or indirectly to MTP activation. In conclusion, the inhibition of MTP activity via the suppression of hepatocellular Ca2+ may result in the inhibition of hypertriglyceridemia.
We have reported that teprenone (geranylgeranylacetone), an anti-ulcer drug, prevents acute gastric mucosal lesion progression in rats treated once with compound 48/80 (C48/80), a mast cell degranulator, possibly by suppressing mucus depletion, neutrophil infiltration, and oxidative stress in the gastric mucosa. Herein, we examined the preventive effect of gefarnate (geranyl farnesylacetate), an anti-ulcer drug, on acute gastric mucosal lesion progression in rats treated once with C48/80 (0.75 mg/kg, i.p.) in comparison with that of teprenone, because the chemical structure and anti-ulcer action of gefarnate are similar to those of teprenone. Gefarnate (50, 100 or 200 mg/kg) administered orally at 0.5 h after C48/80 treatment, at which time gastric mucosal lesions appeared, reduced progressive gastric mucosal lesions at 3 h dose-dependently. At 3 h after C48/80 treatment, the gastric mucosa had decreased adherent mucus and hexosamine contents and increased myeloperoxdiase (an index of neutrophil infiltration) and xanthine oxidase activities and thiobarbituric acid reactive substances (an index of lipid peroxidation) content. Post-administered gefarnate attenuated all these changes dose-dependently. These preventive effects of gefarnate were similar to those of teprenone at a dose of 200 mg/kg. Post-administered gefarnate did not affect the increases in serum serotonin and histamine concentrations and the decrease in gastric mucosal blood flow at 3 h after C48/80 treatment like teprenone. These results indicate that orally administered gefarnate prevents acute gastric mucosal lesion progression in C48/80-treated rats possibly by suppressing mucus depletion, neutrophil infiltration, and oxidative stress in the gastric mucosa like teprenone.
Flavonoids including tea catechins and gallic acid esters were characterized for their ability to inhibit o-methyltranslation of protocatechuic acid (PCA) to form vanillic acid (VA) in rat liver cytosolic preparations and cultured hepatocytes. Flavonols and flavones exhibited different behaviors in inhibiting the formation of VA between the cell-free enzymatic preparations and the intact cells. The underlying mechanism of the inhibitory effects of flavonols and flavones on PCA o-methylation in cultured hepatocytes may not be due to the inhibition of the enzyme activity of catechol o-methyl transferase (COMT). Catechin gallates inhibited PCA o-methylation in liver cytosolic preparations with markedly higher potency than other flavonoids. As compared with catechin gallates, ungallated catechins had two to three orders of magnitude lower efficiency in inhibiting cytosolic PCA o-methylation. Gallic acid esters inhibited cytosolic PCA o-methylation with strong potency almost equal to that of catechin gallates. These results suggest that the COMT-inhibitory activity of catechin gallates is derived from the presence of the galloyl moiety at the C3 position in the C-ring. Catechin gallates and gallic acid esters inhibited PCA o-methylation in cultured hepatocytes with two orders of magnitude lower efficacy than that in cytosolic preparations. The inhibitory effects of catechin gallates and gallic acid esters on cellular PCA o-methylation appear to be due to the direct inhibition of COMT activity.
We used senescence-accelerated mouse prone 8 (SAMP8), a useful model of accelerated aging, to investigate the responsiveness to leptin with aging. The state of leptin-induced STAT3 phosphorylation in the hypothalamus was found to be higher in SAMP8 than in SAMR1, a control mouse showing normal aging, at 14—18 months of age but not at 2 months of age. Moreover, leptin receptor Ob-Rb expression in the hypothalamus was up-regulated in SAMP8. The results indicate that leptin sensitivity increases with aging in the SAM mouse brain.
Changes in the levels of CYP4A1, PPARα, and RXRα mRNA expression in the liver following overdose of fat-free or fat-containing total parenteral nutrition (TPN) were studied in 3-week-old male Sprague–Dawley rats. The rats were divided into three groups: group 1, oral diet; group 2, fat-free TPN; and group 3, TPN with 20% of calories from soybean oil emulsion. Levels of CYP4A1, PPARα, and RXRα mRNA in the fat-free TPN group were significantly lower than those in the other groups. Levels of CYP4A1 and PPARα mRNA were strongly correlated (r=0.849), and levels of CYP4A1 and RXRα mRNA were weakly correlated (r=0.618). This is the first report of a strong correlation between the levels of CYP4A1 and PPARα mRNA following overdose of fat-free or fat-containing TPN in infant rats. Our results also indicate that it is important to include fat in TPN regimens in order to prevent CYP4A1 mRNA down-regulation, which may be related to changes in PPARα mRNA levels in the liver.
2-Amino-4,4α-dihydro-4α-7-dimethyl-3H-phenoxazine-3-one (Phx-1) reversed concentration dependently the contraction of both rat aorta and guinea pig tenia cecum induced by phenylephrine or high-K+. Since Phx-1 suppresses the responses of human mononuclear cells to phytohemagglutinin, (Akazawa et al.Tohoku J. Exp. Med., 196, 185—192, 2002), Phx-1 may be useful for developing new vasorelaxing agents with immunosuppressive action.
R-(−)-1-(Benzofuran-2-yl)-2-propylaminopentane [R-(−)-BPAP] enhances electric field stimulation-induced release of catecholamine from isolated brain stem and ameliorates motor deficits in rats. We evaluated the effects of R-(−)-BPAP on the expression of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), and their receptors, trkB and p75NTR in rat mesencephalic slice cultures. Levels of mRNA and protein were measured at 48 h after R-(−)-BPAP treatment by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. R-(−)-BPAP significantly increased the mRNA and protein levels of BDNF, without affecting the level of NT-3 mRNA. In addition, R-(−)-BPAP significantly increased the mRNA level of trkB, but not that of p75NTR. These effects of R-(−)-BPAP may result in enhanced BDNF/trkB signaling, and could thus underlie the potential neurotrophic and antidepressant actions of this drug.
Our preliminary study demonstrated that 70% ethanol Cortidis Rhizoma extracts (CR) had a hypoglycemic action in diabetic animal models. We determined whether CR fractions acted as anti-diabetic agent, and a subsequent investigation of the action mechanism of the major compound, berberine ([C20H18NO4]+), was carried out in vitro. The 20, 40 and 60% methanol fractions from the XAD-4 column contained the most insulin sensitizing activities in 3T3-L1 adipocytes. The common major peak in these fractions was berberine. Treatment with 50 μM berberine plus differentiation inducers significantly reduced triglyceride accumulation by decreased differentiation of 3T3-L1 fibroblasts to adipocytes and triglyceride synthesis. Significant insulin sensitizing activity was observed in 3T3-L1 adipocytes which were given 50 μM berberine plus 0.2 nM insulin to reach a glucose uptake level increased by 10 nM of insulin alone. This was associated with increased glucose transporter-4 translocation into the plasma membrane via enhancing insulin signaling pathways and the insulin receptor substrate-1-phosphoinositide 3 Kinase-Akt. Berberine also increased glucose-stimulated insulin secretion and proliferation in Min6 cells via an enhanced insulin/insulin-like growth factor-1 signaling cascade. Data suggested that berberine can act as an effective insulin sensitizing and insulinotropic agent. Therefore, berberine can be used as anti-diabetic agent for obese diabetic patients.
The anti-allergic and anti-oxidative activities of curcumin-related compounds (glycosides, reductants and bis-demethoxy analogs) were investigated to elucidate the underlying active mechanisms and structural features of curcumin in exerting these activities. The anti-allergic activities were assessed by measurement of histamine release from rat basophilic leukemia cells, RBL-2H3. Curcumin and tetrahydrocurcumin (THC) caused a marked decrease in histamine release. Glycosides of curcumin, bis-demethoxycurcumin and THC also inhibited the release of histamine, though less potently than curcumin did. The anti-oxidative activities were assessed by measurement of cell-free or cellular radical scavenging. All compounds but diglycosides or bis-demethoxycurcumin analogs distinctly exerted anti-oxidative effects. The relationship between both of these activities revealed that all compounds with potent radical scavenging activities caused a definite decrease in histamine release, but some compounds with non-potent radical scavenging activities also inhibited the histamine release. These results suggest that the hydroxy groups of curcumin play a significant role in exerting both the anti-oxidative and anti-allergic activities, and that most of the compounds develop the anti-allergic activities through mechanisms related to anti-oxidative activities, but some through mechanisms unrelated to anti-oxidation activity.
We have studied the crude methanolic extract (CME), some fractions (hexane, dichloromethane and ethyl acetate) and four pure compounds: eupomatenoid-3 (1), eupomatenoid-5 (2), conocarpan (3) and orientin (4), from Piper solmsianum, for possible antifungal activity against 12 pathogenic fungi. The minimal inhibitory concentration (MIC) was determined and the experiments showed that the CME exhibited antifungal action against all the dermatophytes tested, with MIC values of between 20 μg/ml to 60 μg/ml. Similar activity also was verified for the hexane, dichloromethane and ethyl acetate fractions. However, the starting material (CME), and all the fractions, did not exert inhibitory effect against hyaline hyphomycetes and were only discretely active against the zigomycetes and yeasts. Compounds 2, 3 and 4 also exhibited pronounced activity against all the dermatophytes tested (MIC≤1 to 9 μg/ml) with potency as high as the standard antifungal drug (ketoconazole). Compound 3 also exhibited activity against all the yeasts tested. In conclusion, the antifungal activity of P. solmsianum seems to be related mainly to the presence of compounds 2, 3 (neolignans) and 4 (flavonoid), however it was verified that another active compound, as yet unidentified, exists in the plant.
A series of novel 1-substituted-4-(3-methylphenyl)-1,2,4-triazolo[4,3-a]quinazolin-5(4H)-ones were synthesized by the cyclization of 2-hydrazino-3-(3-methylphenyl) quinazolin-4(3H)-one with various one carbon donors. The starting material 2-hydrazino-3-(3-methylphenyl)quinazolin-4(3H)-one, was synthesized from 3-methylaniline by a novel innovative route. When tested for their in vivo H1-antihistaminic activity on conscious guinea pigs, all the test compounds protected the animals from histamine induced bronchospasm significantly, whereas the compound 1-methyl-4-(3-methylphenyl)-1,2,4-triazolo[4,3-a]quinazolin-5(4H)-one II was found to be equipotent (percent protection 70.0%) with the reference standard chlorpheniramine maleate (percent protection 71%). Compound II show negligible sedation (7%) when compared to chlorpheniramine maleate (25%). Hence it could serve as prototype molecule for further development as a new class of H1-antihistamines.
The present study was designed to examine whether the methanol extract of Sorbus commixta cortex (MSC) could prevent the development of atherosclerosis through regulating the vascular nitric oxide (NO) and endothelin-1 (ET-1) systems in atherogenic-diet rats. Our findings show that aortic NO production as well as endothelial nitric oxide synthase (ecNOS) expression was significantly decreased in atherogenic-diet rats compared with those in the control group. Aortic ET-1 expression was augmented in rats fed an atherogenic-diet while NF-κB p65 was upregulated. Treatment of atherogenic-diet rats with either low (100 mg/kg/d) or high (200 mg/kg/d) doses of MSC led not only to significant increases in the aortic NOS/NO system, but also to decreases in aortic ET-1 expression. The aortic expression level of NF-κB p65 was also attenuated in atherogenic-diet rats by chronic treatment with low or high doses of MSC. Atherogenic-diet induced increases in the expression of adhesion molecules including intercellular adhesion molecules-1 (ICAM-1), vascular cell adhesion molecue-1 (VCAM-1), and E-selectin were markedly decreased by treatment with MSC. From the histopathological examination, MSC treatment was shown to lessen the thickening noted in the aortic intima and media of the atherogenic-diet rats. These results suggest that MSC affects the atherogenic process via the suppression of proinflammatory and adhesion molecules in atherogenic-diet rats, which may be, at least in part, causally related with the regulation of vasoactive systems such as the NO and ET-1 systems.
This study was designed to investigate the effects of the aqueous ethanol extract of Astragalus membranaceus BUNGE (Leguminosae) on rat thoracic aorta. Isometric tension was recorded in response to drugs in organ bath. In endothelium-intact aortic rings, A. membranaceus extract induced a significant dose-dependent relaxation of the rings precontracted by phenylephrine, which could be inhibited by preincubation with L-N(ω)-nitro-arginine methyl ester or methylthioninium chloride. In endothelium-denuded ones, the extract could dose-dependently relax the rings contracted by phenylephrine, not by KCl; and it could also attenuate contractile response to phenylephrine, not to caffeine or phorbol-12,13-diacetate in Ca2+-free medium; but it failed to affect the CaCl2-induced enhancement of contractile response to phenylephrine in Ca2+-free medium. These results indicate that nitric oxide signaling and Ca2+-handling pathway are involved in the A. membranaceus extract-induced vasodilatation.
The complete amino acid sequences of [2Fe–2S] ferredoxin from Atropa belladonna and Hyoscyamus niger have been determined by automated Edman degradation of the entire S-carboxymethylcysteinyl proteins and of the peptides obtained by enzymatic digestion. These two ferredoxins exhibited 1—8 differences in their amino acid sequences compared to those of other tropane-alkaloid-containing plants (Scopolia japonica, Datura stramonium, D. metel, and D. arborea), and only 1 or 4 differences compared to S. japonica and D. arborea. In contrast, 9—23 differences were observed among the other solanaceous ferredoxins. This suggests that tropane-alkaloid-containing plants are closely related taxonomically.
The pharmacokinetic–pharmacodynamic (PK–PD) relationship of the newly developed drug, (−)-(S)-4-[1-[4-[1-(4-isobutylphenyl)butoxy]benzoyl]indolizin-3-yl]butyric acid (TF-505), was characterized via a population approach in early human study. Healthy volunteers were divided into six groups. The groups received four single doses (25, 50, 75 or 100 mg) and 2 multiple doses (12.5 or 25 mg) of TF-505, respectively. Dihydrotestosterone (DHT) data were collected to assess TF-505 pharmacodynamics. Population PK/PD modeling of TF-505 was performed via mixed-effects modeling using the NONMEM software package. The final PK–PD model incorporates a two-compartment PK model and an extended indirect PD model. The population PK parameters were 0.197 h−1 for the ka, 0.0678 h−1 for ke, 12.5 l for Vc, 0.0645 h−1 for k12, 0.0723 h−1 for k21. Extension of indirect response model by incorporating a time-dependent periodic function for kin takes into account the chronopharmacologic rhythms (Imax: 0.706±0.297, IC50: 1.01±1.64 (μg/ml), kout: 0.221±0.0486 (h−1), Rm: 20.4±8.08 (% h−1), Ramp: 5.06±3.43 (% h−1), Tz: 5.01±0.407 (h) (Population mean±S.E.)). Rm is the mean DHT synthesis rate, Ramp is the amplitude of the DHT synthesis rate, and Tz is the acrophase time, signifying maximum synthesis rate. The present study represents a successful population PK–PD model using the full data from early human studies. The population parameters thus obtained could provide useful indicators for the determination of dosage regimens in exploratory studies in patient populations.
Recent studies have suggested that oral bacteriotherapy with probiotics might be useful in the management of allergic diseases. We investigated the effect of oral administration of Bifidobacterium bifidum G9-1 (BBG9-1) on immunoglobulin (Ig) E production in BALB/c mice. Live BBG9-1 was orally administered to mice for 2 weeks from 1 week before ovalbumin (OVA)-immunization. The treatment of BBG9-1 significantly reduced serum total IgE level. In addition, BBG9-1 significantly and largely reduced the serum level of OVA-specific IgE without lowering of the specific IgG1 and increasing of the specific IgG2a. We also examined T helper type (Th) 1 and Th2 cytokine production from OVA-immunized splenocytes by restimulation with OVA in vitro. Productions of interferon (IFN)-γ, interleukin (IL)-4 and IL-5 from the splenocytes of mice given BBG9-1 were weaker than those of control mice. We conclude that oral administration of BBG9-1 selectively and powerfully suppresses total and antigen specific IgE production in mice. It is suggested that BBG9-1 is useful for the prophylactic treatment in IgE-dependent allergic diseases.
Time-dependent effects of St. John's wort (SJW) on midazolam 1-hydroxylation were investigated in Wistar rats. Wistar rats treated with SJW (1000 mg/kg/d) for 1, 3, and 7 d were administered midazolam orally at a dose of 10 mg/kg. Oral clearance of midazolam in the SJW treated rats increased time dependently, and was significant after 7 d of treatment with SJW. The midazoram-1-hydroxylation activity in liver microsomes obtained from the SJW treated rats was significantly higher than in the control group. Linear correlation was observed between oral clearance and midazolam-1-hydroxylation activity in the liver microsomes, suggesting that CYP3A induction in liver mainly decreased the midazolam concentration in plasma. Immunoblotting revealed that the protein amount of CYP3A was induced within 3 d of SJW treatment. Since the midazolam-1-hydroxylation activity continuously increased for at least 7 d, the induction of CYP3A by SJW continued to cause interactions with drugs metabolized by CYP3A. It is important for persons receiving SJW for an extended time to consider its interactions with prescription drugs.
We recently reported that a cationic liposomal vector, TFL-3, could be used to achieve significant gene expression in primary cultured rat hepatocytes (Nguyen et al., Biol. Pharm. Bull., 26, 880—885 (2003)). A combination of hepatocyte transplantation and hepatocyte-targeted gene transfer represents a potentially important strategy for expanding treatment options for liver disease. A widely applied approach to support cross-species is necessary before human applications can be realized. Therefore, in this study, we examined the utility of TFL-3 in another species of rodent hepatocytes, namely mouse hepatocytes. Gene expression in mouse hepatocytes by TFL-3 was successful and the level was higher than those in rat hepatocytes that we recently reported on. Interestingly, it appears that both the degree and rate of gene expression were dependent on the incubation time prior to lipofection as well as on the density of cells per dish, but these parameters were independent of the amount of pDNA associated with the cells. These significantly suggest that the culture time prior to and following lipofection, which are related to the biological condition of the cells, may be one of major factors that affect gene expression in hepatocytes and non- or less dividing cells.
This study was performed to investigate the effect of CYP2D6*10 on the pharmacokinetics of R- and S-carvedilol in healthy Japanese volunteers. Five or 10 mg of carvedilol was orally administered to 23 subjects (22—44 years old), and blood samples were taken at 2 and 6 h after dosing. We determined the polymorphic alleles of CYP2D6 in each subject. The whole blood concentration of R- and S-carvedilol was measured by an HPLC method. The pharmacokinetic parameters in individual subjects were estimated by the Bayesian method using the nonlinear mixed effects model (NONMEM) program. The mean values of oral clearance for R- and S-carvedilol were estimated to be 1.01 and 2.15 l/h/kg, respectively. The oral clearance was highly correlated with the apparent volume of distribution among the subjects, suggesting that the interindividual difference in bioavailability was largely responsible for the pharmacokinetic variability of carvedilol. The oral clearance and also volume of distribution of both enantiomers were significantly lower in the subjects with the CYP2D6*10 allele than with the CYP2D6*1/*1 or *1/*2 genotype. These results suggested that the systemic and/or pre-systemic metabolism of R- and S-carvedilol in the liver is significantly decreased in Japanese with the CYP2D6*10 allele.
Controlled-release morphine suppositories were prepared by utilizing polyglycerol ester of fatty acid (PGEF). The addition of PGEF to fatty suppository base Witepsol H15 resulted in a decrease of morphine release rate from suppositories. Among PGEFs examined, decaglycerol heptabehenate (HB750) was the most effective additive for the controlled-release of morphine from fatty suppositories. The apparent viscosity of suppository bases increased with the increase in HB750 content, and good correlation was observed between the apparent viscosity of suppository bases at 37 °C and the amount of HB750 added in the mixed base. The in vitro release rate of morphine was decreased by the addition of HB750 and the release rate constant (Higuchi's rate constant) for morphine release was significantly correlated with the HB750 content in the mixed bases as well as the apparent viscosity of mixed bases, indicating that the release of morphine from the mixed bases could be regulated by the HB750 content in the mixed bases. After rectal administration of Witepsol H-15-HB750 mixed suppositories to dogs, plasma concentrations of morphine did not increase rapidly at early time periods, but relatively high levels of morphine in plasma were sustained for longer time periods. Mean residence time of morphine was considerably prolonged without changing relative bioavailability in the case of the mixed base suppositories containing 15—17% HB750, compared with the Witepsol H15 suppository, clearly indicating that the mixed bases containing HB750 are expected to be useful for the design of controlled-release morphine suppositories.
The intake of pre-germinated brown rice (PR) instead of white rice (WR) ameliorates the hyperglycemia. To clarify the mechanism(s) to decrease the post-prandial blood glucose concentration, the effect of water-soluble/oil-soluble fraction-depleted PR bran (termed as “DB”; which is destarched and defatted PR bran) on post-prandial blood glucose was compared with that of full-fat PR bran (PB) or WR. The test diets, WR diet, PB diet and DB diet which are containing identical amount of available carbohydrate (1.5 g) were fed to Wistar strain rats. Post-prandial blood glucose concentration and incremental area under the curve (IAUC) for DB diet were lower than those for WR diet, and there was no difference between the DB diet and PB diet. Changes in plasma insulin concentration and the IAUC obtained also revealed the same tendency as those observed in blood glucose concentration. These results indicate that the blood glucose-lowering effect of PB diet may be derived from the properties of PB involving substantially higher content of dietary fiber than WR, and that the potential benefit of intake of PR instead of WR in the prevention of diabetic vascular complications.
Ascorbic acid (AA) is one of the most important endogenous reducing agents and can participate in a variety of cellular events. In vitro, AA can act as a potent oxidant agent in the presence of free metals, promote modifications in protein structures and form reactive oxygen species during its oxidation. We have observed that AA (above 6 mmol/l) inactivates δ-aminolevulinate dehidratase (δ-ALA-D), a sulfhydryl-containing enzyme and that the inhibitory action was considerably decreased when 3-morpholinepropanesulfonic acid buffer (MOPS – pH: 6.8; 100 mmol/l) was used in the δ-ALA-D activity assay instead of potassium phosphate buffer (PB – pH: 6.8; 100 mmol/l). δ-ALA-D inhibition, probably, is mediated by the oxidation of –SH groups caused by the auto-oxidation of AA promoted by metals or another oxidizing system present in liver supernatants. This hypothesis was confirmed by studying dithiothreitol (DTT – 400 μmol/l) oxidation, as a model of enzyme thiols, where we observed that the mechanism underlying DTT and δ-ALA-D oxidation caused by ascorbate is the same. The difference observed between different buffers may be related to the oxidation of Fe(II) to Fe(III) that was more accentuated in PB than in MOPS buffer. The presence of ethilenediamintetraacetic acid (EDTA – 100 μmol/l) and Fe(III) (5 μmol/l) stimulated DTT oxidation more in PB than in MOPS buffer. Deferroxamine (DF – 100 μmol/l) considerably decreased DTT oxidation. Catalase (0.4 mg/ml) and Superoxide dismutase (SOD – 300 U/ml) had only a modest effect on DTT oxidation. The present results suggest that δ-ALA-D inhibition by AA is mediated primarily by the oxidized form of AA and reactive oxygen species play only a modest role in the process.
We discovered a phenomenon in which the blood flow in vein microcirculation markedly decreases in response to hen-egg white lysozyme (HEL)-sensitization without any change in blood pressure. Using this blood flow decrease as a guide, we developed an in vivo assay method to search for substances, which can prevent allergies. Antagonists of histamine, serotonin and platelet activating factor (PAF) did not affect the blood flow decrease in response to HEL-sensitization. On the other hand, cyclooxygenase (COX)-1, COX-2, thromboxane (TX) A2, endothelin-1 (ET-1), prostacyclin (PGI2) and granulocytic elastase (GE) as well as nitric oxide (NO) from inducible NO synthase (iNOS) were involved in the blood flow decrease. Thus, these substances might injure vascular endothelial cells, and cause a decrease in blood flow in vein microcirculation. Our method can be used to search for preventive agents against allergies involving NO, COX-1, 2 and PGI2. This is the first report to applying to an assay method the specific blood flow decrease to occur in the promotion stage of allergy.