The formation of cyclic 1,N2-propano guanine (CPr-Gua) adduct is significantly accelerated by the addition of arginine or histone in the reaction of calf thymus DNA with acetaldehyde (AA) or crotonaldehyde (CA). Histone proteins, containing a large amount of basic amino acids such as arginine, are essential as nucleosome cores to package DNA. In the presence of 0.60% (w/v) histone in the reaction mixture, 8-times and 25-times larger amounts of the CPr-Gua adduct were formed in the reaction of the DNA with AA and CA, respectively, compared with those in the absence of histone. Furthermore, for the DNA incubated at 95 °C for 10 min and cooled on ice to make the single-stranded moieties, 72-times and 178-times larger amounts of the CPr-Gua adduct were formed by AA and CA, respectively, in the presence of 0.60% (w/v) histone. These results strongly suggest that DNA in vivo should be exposed to a much more dangerous situation, compared with DNA alone, from the viewpoint of reactivity with the aldehydes. During DNA replication and transcriptional events of cells, the danger will be further increased markedly because of opening of double-strands. Semi-micro HPLC-ESI-MS measurements following deprination of DNA samples were performed for quantification of the adduct as the corresponding base form, CPr-Gua, in evaluation of the reactivity of DNA with AA and CA.
Catechin (epicatechin (EC), epicatechin gallate (ECg), epigallocatechin (EGC) and epigallocatechin gallate (EGCg)), which occur in green tea and black tea, possess strong bactericidal action. We observed a reactive oxygen species that was generated from the catechins as the active mechanism: and this reactive oxygen was identified. EGCg reacted with the dissolved oxygen in aqueous solution, resulting in the generation of hydrogen peroxide. Hydrogen peroxide production derived from EGCg rose with increasing pH. EGCg (0.22 mmol/l) in neutral solution (0.1 mol/l phosphate buffer pH 7.0: PBS) quantitatively generated 0.2 mmol/l hydrogen peroxide after 60 min incubation. The bactericidal effect of EGCg is dependent on hydrogen peroxide levels produced by EGCg; moreover, EGCg action was inhibited by treatment with catalase. Both bactericidal effects correlated closely when the effects of EGCg and hydrogen peroxide for the bacterium (9 of 10 kinds of bacterial strains) were examined. Therefore, hydrogen peroxide, which is generated by EGCg, appears to be involved in the bactericidal action of EGCg.
Rheumatoid arthritis (RA) is characterized by proliferation of synoviocytes that produce proinflammatory cytokines, which is implicated in the pathogenesis of the disease. Among the cytokines, IL-1 is the critical mediator of the disease. When human fibroblast-like synoviocytes line, MH7A, was treated with 3-methylcholanthrene (3-MC), a polycyclic aromatic hydrocarbon (PAH), mRNA of IL-1β was up-regulated. MH7A cells express functional aryl hydrocarbon receptor (AhR) as shown by 3-MC-inducible CYP1A1 mRNA expression. The effect of 3-MC was inhibited by α-napthoflavone, an AhR antagonist, indicating that the effect of 3-MC is mediated via AhR. Benzo[a]pyrene (B[a]P) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) also up-regulated mRNA level of IL-1β in the cells via AhR. As PAHs are much contained in cigarette smoke, these findings provide the possible basis for epidemiological studies indicating a strong association between heavy cigarette smoking and outcome of RA.
The phosphorylation of human C3a (hC3a, anaphylatoxin) by two distinct protein kinases (PKA and CK-I) and the effect of cholesterol-3-sulfate (CH-3S) on this phosphorylation were biochemically investigated in vitro. It was found that (i) hC3a functions as a phosphate acceptor for PKA and CK-I, but not for CK-II; (ii) the CK-I-mediated phosphorylation of hC3a requires the presence of 3 μM CH-3S in a manner similar to the phosphorylation of HMG1 (CH-3S-binding protein) by CK-I; and (iii) CH-3S inhibits the PKA-mediated phosphorylation of hC3a in a dose-dependent manner (ID50=approx. 2 μM). As expected, hC3a containing high levels of Arg- and Lys-residues stimulated approx. 3-fold CK-II activity (phosphorylation of α-casein) in vitro. However, no significant effect of hC3a on CK-II activity was observed when hC3a was preincubated with CH-3S or fully phosphorylated by PKA in vitro. Furthermore, preincubation of hC3a with CH-3S diminished the ability of hC3a to induce vascular permeability in rats. The results provided here suggest that (i) hC3a is a CH-3S-binding protein; and (ii) CH-3S functions as a potent inhibitor for its physiological activities, including phosphorylation by PKA and CK-I, in vitro.
RS-7897 is a novel antianginal nitrate containing an L-2-oxothiazolidine-4-carboxylic acid (OTCA) and unlike other organic nitrates does not induce nitrate tolerance. OTCA is known to be converted to L-cysteine (L-Cys) by rat 5-oxo-prolinase (5-OPase) and was detected in the plasma of RS-7897-treated dogs. Nitrate tolerance is considered to develop mainly through sulfhydryl depletion. We hypothesized that RS-7897-derived OTCA was converted into L-Cys by 5-OPase and supplied sulfhydryl groups in vascular smooth muscle cells, the targets of the nitrate. As the initial step for clarifying the presumed role of 5-OPase in RS-7897 metabolism, we established a non-radiochemical assay method highly specific for 5-OPase. Using this assay method, we purified 5-OPase from bovine kidney cytosol until homogeneous results were obtained in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The catalytic activity of bovine 5-OPase to convert OTCA to L-Cys was confirmed, as was the case for the rat enzyme. Then the cDNA encoding bovine 5-OPase was cloned. The deduced amino acid sequence revealed that bovine 5-OPase was highly homologous to rat 5-OPase. Based on the bovine cDNA sequence, oligonucleotide primers were synthesized and used for RT-PCR. 5-OPase mRNA was significantly detected in the RT-PCR product of the bovine coronary artery, a major target organ of RS-7897. These results suggest that OTCA may be converted to L-Cys by 5-OPase in the mammalian coronary artery when treated with RS-7897, and thus generated L-Cys may reduce sulfhydryl depletion and contribute to the prevention of the development of nitrate tolerance.
A phenolic antioxidant 3-tert-butyl-4-hydroxyanisole (BHA) is a widely used food additive. BHA had cytotoxicity in human monocytic leukemia U937 cells. BHA at 0.75 mM caused nuclear condensation and fragmentation, structural damage in mitochondria, decrease in mitochondrial transmembrane potential, and internucleosomal DNA cleavage. It induced the activities of caspase-3 and/or -7, -6, -8 and -9, especially high when DEVD-MCA was the substrate (caspase-3 and/or -7). DEVDase activity increased in time- and dose-dependent manner and high activity was observed in lysates of cells treated for 3 h at 0.75 mM. Addition of GSH (reduced glutathione) during the treatment of cells with BHA inhibited the induction of DEVDase activity, and the intracellular GSH level decreased as the concentration of BHA was raised. Intracellular ATP levels decreased in time- and dose-dependent manner when the cells were treated with BHA in the presence or absence of glucose. Enzyme activities involved in the respiratory chain were assayed with the mitochondrial fraction prepared from U937 cells. BHA distinctly inhibited NADH–ubiquinone oxidoreductase (complex I) and cytochrome c oxidase (complex IV) at low concentrations. Succinate–ubiquinone oxidoreductase (complex II) was also inhibited, but to somewhat less extent. Without mitochondrial enzymes, BHA stimulated the ubiquinol-dependent reduction of cytochrome c (complex III), but it might have some detrimental effects on the mitochondrial enzyme reaction of complex III. The inhibition of mitochondrial oxidative phosphorylation might corroborate the mechanistic evidence for apoptosis of leukemia cells by BHA. Cell death induced by BHA is primarily ascribable to apoptosis.
Infection with human papillomavirus, particularly type 16 (HPV16), is highly associated with the development of cervical intraepithelial neoplasia and cervical cancer. The two early viral oncogenes, E6 and E7, are selectively retained and constitutively expressed in tumor cells and are therefore attractive immunotherapeutic targets. Thus a vaccine strategy based on recombinant HPV16 E6/E7 fusion protein represents an efficient approach against HPV16-associated tumors. Although the expression level of HPV16 E6/E7 fusion protein was presumed to be low, direct experimental proof in vivo was lacking. To enhance the expression level and investigate its antitumor efficacy in vivo, we constructed a modified HPV16 E6/E7 fusion gene with three point-mutations and expressed it in Escherichia coli. The encoded protein, denoted mE61—120/mE71—60, comprises 120 N-terminus amino acids of E6 and 60 N-terminus amino acids of E7 plus a histine tag, was purified on an affinity column, and subsequently characterized by Western blotting. Immunization of mice with mE61—120/mE71—60 completely protected them against subsequent challenge and rechallenge with TC-1 tumor cells expressing HPV16 E6 and E7 proteins. In the therapeutic experiments, most mice eliminated the preexisting tumors and had a long-term protection. Consistent with the results of in vivo experiments, the splenocytes from immunized mice elicited cytotoxic T lymphocytes and specifically lysed TC-1 cells in vitro. More importantly, the expression level of mE61—120/mE71—60 was significantly improved, meeting the necessary quantity required for a vaccine clinical trial. In conclusion, these data provide a scientific basis for the use of modified mE61—120/mE71—60 in future human trials.
C-Terminal truncated α-crystallins have been found in lenses of hereditary cataractous rat ICR/f, including two truncated αB-crystallins and several truncated αA-crystallins. These truncated crystallins probably resulted from degradation by m-calpain and Lp82. The αB-crystallin with five amino acid residues deleted showed decreased chaperone activity. Compared with α-crystallins from the normal rat lenses, overall chaperone activity of α-crystallins from the mutant lenses, including the above truncated αB-crystallin, was remarkably reduced. The decreased chaperone activity accompanying the increase in C-terminal truncated α-crystallins may cause the insolubilization of many proteins in the mutant lenses, which it is likely to lead to the progression of cataract formation.
Neuroblastoma (NB) is the most common malignant solid tumor in childhood and, among all childhood malignancies, is second only to leukemia. NB originates before birth in the neural crest, which develops into the adrenal medullae and sympathetic ganglia. In the adrenal medulla, tyrosine hydroxylase (TH) is the first enzyme in the pathway of catecholamine synthesis. We used reverse transcription polymerase chain reaction (RT-PCR) to examine the expression of TH mRNA in NB and Ewing's sarcoma cell lines, small round cell tumors (SRCTs) containing NB, and other clinical tumor samples (osteosarcoma, osteochondroma, and Wilms' tumor). In total, we analyzed 33 clinical tumor samples. TH mRNA was expressed in all three NB cell lines examined, but not in two ES cell lines or in a breast cancer cell line. We detected TH mRNA in 23 of 25 NB tumor samples (92%), but in none of the SRCTs or other clinical tumor samples. This RT-PCR technique showed a sensitivity for TH mRNA of one NB cell per 105 negative cells. Based on these results, the detection of TH mRNA is very useful both as a tumor marker for NB and for detecting minimal residual disease. Therefore, we can use this method to detect tumor cell contamination before hematopoietic stem cell transplantation.
Mice were fed a diet supplemented with palm oil (control diet), n-3 polyunsaturated fatty acids (PUFA)-, or n-9 PUFA-rich oil for 3 weeks. The n-3 PUFA-rich diet suppressed the generation of both leukotrienes (LT) and prostaglandins (PG), but the n-9 PUFA-rich diet did LT but not PG generation during acute inflammation. Leukocyte accumulation during acute inflammation was not different in the n-3 or n-9 PUFA-rich diet group as compared with the control group. The n-3 PUFA-rich diet but not the n-9 PUFA-rich diet suppressed Freund's adjuvant-induced granuloma formation. The n-9 PUFA-rich diet significantly attenuated galactosamine/lipopolysaccharide-induced liver injury more effectively than the n-3 PUFA-rich diet as compared with the control diet. The present study revealed the differential modification of experimentally induced inflammation in mice by dietary n-3 PUFA and n-9 PUFA, which may be due to their different effects on 5-lipoxygenease and cyclooxygenase metabolism of arachidonic acid during inflammatory processes.
We have constructed a recombinant cDNA encoding the chimeric protein between human IFN-β (HuIFN-β) and enhanced green fluorescent protein (EGFP) to elucidate the intracellular localization of IFN-β. Transient expression of the chimeric molecule, HuIFN-β-EGFP, in L cells demonstrated that the chimeric molecule secreted from the cells had an intact biological activity as far as antiviral effect was concerned. Immunostaining of the transfected cells using anti-HuIFN-β antibody demonstrated that green-fluorescence was co-localized with the IFN signal and its profile was similar to IFN signals in the cells transfected with HuIFN-β expressing plasmid DNA. These results indicate that the HuIFN-β-EGFP chimeric gene was expressed as a chimeric protein and the chimera was transported via the regular secretory pathway in the cells. In other cell types, the fluorescence derived from the chimeric protein was also seen on cytoplasmic vesicular structures. These results suggest that HuIFN-β-EGFP will be a useful tool to investigate the intracellular trafficking processes of HuIFN-β in a variety of cell types.
The effects of nilvadipine, a dihydropyridine calcium antagonist, on cytochrome P450 (CYP) activities in human hepatic microsomes were investigated. Nilvadipine competitively inhibited CYP1A2-mediated 7-ethoxyresorufin O-deethylase, CYP2A6-mediated coumarin 7-hydroxylase, CYP2C8/9-mediated tolbutamide methylhydroxylase, CYP2C19-mediated S-mephenytoin 4′-hydroxylase, and CYP3A4-mediated nifedipine oxidase activities, and the inhibition constant (Ki) values were 13.0, 35.8, 5.02, 24.5 and 44.3 μM, respectively. On the other hand, no inhibition of CYP2B6-mediated 7-benzyloxyresorufin O-debenzylation, CYP2D6-mediated bufuralol 1′-hydroxylation, or CYP2E1-mediated chlorzoxazone 6-hydroxylation by nilvadipine at 40 μM concentration was observed. The free fractions of nilvadipine in the incubation mixture estimated by ultracentrifugation were 18.9—27.4%. These results suggest that nilvadipine would not cause clinically significant interactions with other drugs, which are metabolized by CYPs, via the inhibition of metabolism.
Atractylodes japonica has traditionally been used for the treatment of pain and arthritis. The effect of Atractylodes japonica against lipopolysaccharide-induced inflammation was investigated using reverse transcription-polymerase chain reaction (RT-PCR), nitric oxide detection, and prostaglandin E2 (PGE2) immunoassay in mouse RAW 264.7 macrophages. The aqueous extract of Atractylodes japonica suppressed nitric oxide production and PGE2 synthesis by inhibition of the lipopolysaccharide-stimulated enhancement of inducible nitric oxide synthase and cyclooxygenase-2 mRNAs expressions in RAW 264.7 macrophages. These results suggest that Atractylodes japonica exerts anti-inflammatory and analgesic effects probably by suppression of the inducible nitric oxide synthase and cyclooxygenase-2 expressions.
Syngonanthus arthrotrichus SILVEIRA, popularly known as “sempre-vivas mini-saia,” is found in mountains of the Espinhaço range in the Brazilian states of Bahia and Minas Gerais. Extracts of this species contain several constituents, including flavonoids which may have antiulcerogenic activity. An ethanolic extract (EEOH), and flavonoid-rich (FRF) and flavonoid-deficient (FDF) fractions obtained from the scapes of S. arthrotrichus were investigated for their ability to prevent ulceration of the gastric mucosa in mice and rats. In the ethanol/HCl-induced ulcer model, lansoprazole (30 mg/kg), EEOH (50, 100, 250 mg/kg) given orally protected the gastric mucosal against injury in mice by 79%, 78%, 73%, and 64% respectively. In the ethanol-induced gastric ulcer model in rats, the lansoprazole (30 mg/kg), FRF and FDF (100 mg/kg) significantly protected the gastric mucosal of rats by 65%, 38% and 25% respectively when compared with the negative control group. In indomethacin/bethanechol-induced gastric ulcers, cimetidine (100 mg/kg) and the EEOH (100, 250 mg/kg) inhibited gastric ulcer formation by 73%, 55% and 32% respectively. In this exactly model other treatments as cimetidine, FRF and FDF (100 mg/kg) each caused 54%, 36% and 45% inhibition, respectively. In the stress-induced gastric ulcer model, cimetidine (100 mg/kg) and the EEOH (50, 100, 250 mg/kg), inhibited gastric ulcer formation by 63%, 73%, 68% and 69% respectively. In the same model, cimetidine, FRF and FDF (100 mg/kg) significantly protected the gastric mucosal of the mice by 60%, 51% and 47% when compared to the control group. In pylorus-ligated mice, cimetidine (positive control) and FRF significantly decreased gastric acid secretion, increased gastric pH and reduced the acid output when compared to the negative control. FDF had no significant effect on these parameters. The protection provided by FRF probably involved an antisecretory mechanism mediated by flavonoids which were absent in FDF. The amount of adherent mucous in the stomach contents was also evaluated with the treatments carbenoxolone (200 mg/kg), FRF and FDF (100 mg/kg) treatment. Each treatments significantly increased the amount of adherent mucous in the gastric juice (8.67±1.73, 3.35±1.59, 2.1±0.41 mg/g of wet tissue, respectively) compared to the control group, indicating a cytoprotective action on the gastric mucosa. Treatment with FRF plus indomethacin and FDF plus indomethacin reduced the prostaglandin biosyntesis (13.6±6.5, 27±5.5 pg/well) by the mucosa, indicating that the cytoprotective action on the gastric mucosa was not related to the level of prostaglandins. Only FDF (38±17 pg/well) maintained the level of prostaglandins and guaranteed the integrity of the mucosa. The results indicate that the EEOH, FRF and FDF have antisecretory and cytoprotective actions, that may be related to the presence of luteoline in the extract and active fractions.
We determined the effect of baicalein on prostatic hyperplasia in experimental animal models. Prostatic hyperplasia was induced by testosterone propionate in mice and castrated rats and by transplantation of homologous strain fetal mice urogenital sinus in mice. With the histopathological examination, the efficacy of baicalein on prostate hyperplasia in experimental animals was evaluated by the activity of serum acid phosphatase (ACP) and the following norm of the prostate gland: the volume, wet weight, wet weight index, dry weight index, DNA contents and prostatic epithelial height and cavity diameter. Results showed that baicalein at doses of 260 and 130 mg/kg administrated intragastrically (i.g.) significantly inhibited prostatic hyperplasia in castrated rats induced by testosterone propionate compared with the negative control group (p<0.01). Baicalein at doses of 520 and 260 mg/kg (i.g.) also significantly inhibited prostatic hyperplasia in mice induced by transplantation of homologous strain fetal mouse urogenital sinus and by testosterone propionate (p<0.01). These results suggested that baicalein has an inhibitory effect on prostatic hyperplasia in experimental animals.
In the present study we investigated the effects of a long-term treatment with vitamin E, an antioxidant vitamin, insulin or their combination on cataracts of streptozotocin (STZ)-induced diabetic rats fed a high cholesterol diet. Each rat was checked for cataracts at 0, 1, 2, 4, 8, 12 and 15 weeks after STZ injection. Cataracts were observed from 8 weeks in the control diabetic rats and their incidence of catarats increased to 100% by 12 weeks. The incidence of cataracts in rats treated with vitamin E, insulin and their combination was first seen at 12 weeks and 56%, 20% and 10%, respectively, at 12 weeks and 78%, 50% and 20%, respectively, at 15 weeks. The preventive effects of either agent alone and their combination on the cataracts were in agreement with those obtained by histopathological evaluation of eyeballs. The combined treatment with both agents markedly improved hyperglycemia, hyperlipidemia and increased serum lipid peroxide levels. These results indicate that the combined treatment with vitamin E and insulin is useful in preventing the development and progression of diabetic cataracts.
DJ-927, currently undergoing Phase I clinical trial, is a new orally effective taxane with potent antitumor effects. The absorption, tissue distribution, and excretion of DJ-927 were investigated in mice, dogs, and monkeys after a single oral administration. After oral administration of [14C]DJ-927, radioactivity was rapidly absorbed, with the Cmax occurring within 1—2 h in all species. The blood and plasma radioactivity elimination was biphasic and species-dependent. Elimination half-life of plasma in dogs was much longer than those in monkeys or mice. In mice, radioactivity was rapidly distributed to all tissues except for the central nervous system, especially to adrenal glands, liver, pituitary glands, kidneys, lungs, and spleen. In all species, radioactivity was mainly excreted in feces. Following a single oral administration to mice, more than 80% of the radioactivity was excreted within 48 h; in dogs and monkeys, 80% of the radioactivity was excreted within 168 h. Urinary excretion was less than 7% of radioactive dose in all species. In vitro plasma protein binding of [14C]DJ-927 in the mouse, dog, and monkey plasma ranged from 92—98%. These studies showed that, the novel oral taxane DJ-927 was rapidly absorbed in all three species when administered by the oral route. The long biological half-life and slow elimination of radioactivity were distinctive in particular, compared with commercial taxanes. DJ-927 (as parent compound and its metabolites) is widely distributed to tissues except the brain. These preclinical data are useful for the design of clinical trials of DJ-927 and also for their interpretation.
SART (specific alternation of rhythm in temperature) stress is known to cause anxiety-like behavior in mice/rats in several anxiety-related behavioral tests. In the present study, we investigated possible roles for corticotropin-releasing factor (CRF) and glucocorticoids in SART stress-induced anxiety-like behavior in two different anxiety-related behavioral tests. In the forced swimming test, CRF, administered intracerebroventricular (i.c.v.) at 0.5—2 pmol/mouse, dose-dependently reduced immobility time in unstressed and SART-stressed mice. α-Helical CRF, a specific CRF receptor antagonist, administered i.c.v. at 0.1—1 nmol/mouse, dose-dependently increased immobility time in SART-stressed mice, but not in unstressed mice. In the elevated plus-maze test, CRF at 10—20 pmol/mouse significantly decreased the time spent in open arms in unstressed mice. CRF at a high dose tended to decrease this time in SART-stressed mice, but this decrease was not statistically significant. α-Helical CRF failed to modify the time in unstressed mice. In contrast, α-helical CRF at 0.38 and 0.75 nmol/mouse increased the time in SART-stressed mice. Both immobility time in the forced swimming test and time spent in open arms in the elevated plus-maze test in unstressed and SART-stressed mice were unaffected by adrenalectomy. These results suggest that CRF plays an important role in anxiety-like behavior caused by SART stress.
Intestinal absorption of low molecular weight heparin (LMWH) as well as unfractionated heparin (UFH) is limited due to its large molecular size and extensive negative charge. Development of its oral formulations would allow outpatient treatment with LMWH and UFH, and contribute a reduction in hospital expenses. The present study was aimed at evaluating the absorption enhancers LabrasolTM and Gelucire 44/14TM, which mainly consist of glycerides and fatty acids esters, to improve the intestinal absorption of LMWH. The absorption effects of saturated fatty acids with several carbon chain lengths (C6—C14) were also investigated. LMWH formulated with or without absorption enhancer was administered to the duodenum of fasted rats. The doses of LMWH and absorption enhancer were 20 mg/kg and 30 mg/kg, respectively. Plasma anti-Xa activity was measured as a marker of the LMWH absorption. By administration of the LMWH formulation with Labrasol but not with Gelucire 44/14, the plasma anti-Xa activity was increased to a level above 0.2 IU/ml which is the critical level for elucidation of its anticoagulant activity. Saturated fatty acids also enhanced the intestinal absorption of LMWH, and the order of absorption-enhancing effect was C10=C12>C14>C16>C8≥C6. These results suggest that the intestinal absorption of LMWH varies with carbon chain lengths of the saturated fatty acids.
To investigate the structure–activity relationship of coumarins for the inhibitory activity on mushroom tyrosinase, the 50% inhibitory concentration (IC50 values) of 18 coumarins and four cinnamic acid derivatives were measured. Among these compounds, esculetin had the strongest inhibitory activity (IC50=43 μM) on mushroom tyrosinase. Introduction of a hydroxy group to the C6 and C7 positions of the coumarin ring and no substitution on the lactone ring played an important role in the expression of the strong inhibitory activity of esculetin. We performed further studies to estimate the in vitro inhibitory effects of esculetin on melanogenesis. Esculetin 5 μM significantly suppressed melanin production in murine B16 melanoma cells without affecting cell growth. Furthermore, the number of 3,4-dihydroxyphenylalanine (DOPA)-positive melanocytes in the split-epidermal sheets treated with 0.05% or 0.1% esculetin was significantly lower than that in the control. From these results, it is suggested that esculetin has inhibitory effects on tyrosinase activity in vitro. However, further detailed studies are necessary to understand the inhibitory mechanism of esculetin.
We investigated the structure–activity relationship of the (R)- and (S)-isomer of 7-methyl- and 8-alkyl-tetrahydroimidazo[2,1-i]purines for phosphodiesterase 4 (PDE4) inhibitors. (S)-8-Isopropyl-3,4-dipropyl-imizazo[2,1-i]purine (S)-2c exhibited both potent and selective PDE4 inhibitory activity.
Two recent studies have demonstrated that clotrimazole, a well-known potential antifungal agent, inhibits the in vitro growth of chloroquine-resistant strains of the malaria parasite, Plasmodium falciparum. In a previous study, we suggested that clotrimazole acts as an anti-malarial agent by inhibiting heme catabolism in the malaria parasite and by enhancing heme-induced membrane damage. In this paper, we examined the mechanism of action by measuring hemolysis as an indicator of membrane damage. Our results showed that clotrimazole does not promote the binding of heme to membranes, and that the enhancement of heme-induced hemolysis by clotrimazole is not caused by lipid peroxidation or by oxidation of thiol groups in membrane proteins. Instead, clotrimazole inhibits glutathione-dependent heme degradation, resulting in an enhancement of heme-induced hemolysis. We also found that clotrimazole increases the susceptibility of erythrocytes to hypotonic lysis in the presence of heme and that sucrose could inhibit hemolysis induced by heme–clotrimazole complexes. Thus, it appears that the enhancement of heme-induced hemolysis by clotrimazole in our experiments is due to a colloid osmotic hemolysis mechanism. The hydrophobicity and the large molecular size of the heme-clotrimazole complex might be key factors for induction of hemolysis.
The present study examined whether the cisplatin-induced nephropathy could be ameliorated by administration of butein isolated from the stems of Rhus verniciflua STOCKS. The present study showed that polyuric profile was revealed in cisplatin-induced acute renal failure (ARF) rats associated with decreases in urinary sodium, potassium, chloride, and creatinine excretion, and osmolality. Among these renal functional parameters, urinary volume and osmolality were partially restored by administration of butein (10 mg/kg, i.p.), but electrolytes and creatinine excretion were not restored. Both solute-free water reabsorption and creatinine clearance were also significantly decreased in rats subjected to cisplatin. When butein was administered in rats with cisplatin-induced ARF for 4 d, solute-free water reabsorption was improved by 91% compared with that of cisplatin-induced ARF rats, but creatinine clearance was not restored. The expression levels of aquaporin 2 (AQP 2) in the inner, outer medulla, and cortex were significantly decreased in the kidney of ARF, which were partially reverted by administration of butein. In histological examination of the kidney, butein treatment partially prevented the lesions at tubules of renal cortex in cisplatin-induced ARF rats, while the lesions at glomeruli were not ameliorated. Taken together, butein ameliorates renal concentrating ability via up-regulation of renal AQP 2 water channel in rats with cisplatin-induced ARF without ameliorating effect on renal filtration defect.
The effects of herbal medicines that constitute Gam-du-tang and Gung-gui-tang on cytokine-induced cytotoxicity and thyroid major histocompatibility complex (MHC) class II antigen expression in FRTL rat thyrocytes were investigated. No effect on cell growth was found with interferon (IFN)-γ. However, tumor necrosis factor (TNF)-α was cytotoxic, and this was increased by preincubation with IFN-γ. Ethanol extract of Glycyrrhizae Radix, black beans, Angelicae Radix, and Cnidii Rhizoma (0.3—15 mg/ml) in both regimens significantly inhibited IFN-γ and TNF-α-mediated cytotoxicity of rat thyroid cells (p<0.05, p<0.01). In addition, IFN-γ (1—100 U/ml) treatment induced class II antigen expression in up to 60% of FRTL cells, as detected by a murine monoclonal antibody to rat MHC class II antigen (FITC-OX6). Aberrant thyroid cell MHC class II antigen expression induced by IFN-γ is suppressed by the extract of herbal medicines. These results indicate that herbal medicines inhibit cytokine-induced thyroid cell destruction, therefore, may have therapeutic potential in the treatment of autoimmune thyroid disease.
Rhei Rhizoma (Dahuang in Chinese) is widely known as a purgative and antiinflammatory agent. In the Japanese Pharmacopoeia, Rhei Rhizoma is prescribed for four Rheum species, Rheum palmatum, R. tanguticum, R. officinale, and R. coreanum, while the first three species are prescribed for Dahuang in the Chinese Pharmacopoeia. Due to the morphologic similarity of the aerial parts and frequent occurrence of intermediate forms, the taxonomy of this genus and the correct identification of Rheum species and their derivative drugs are very difficult. To resolve taxonomic problems of the genus Rheum and develop an ultimate identification method for plants and drugs, molecular analysis of the chloroplast matK gene and nuclear 18S ribosomal RNA gene were performed on nine species. The sequence comparison of the matK gene revealed that most species had variable sequences not only inter- but also intraspecies. However, the specimens of the same species belonged to the same subclade in the phylogenetic tree constructed based on matK gene sequences, except for R. palmatum, in which specimens belonged to three subclades related to their production areas. The nucleotide differences at positions 587, 707, and 838 distinguished official species from others, while specific nucleotides at positions 367 and 937 became identification markers for R. palmatum, R. tanguticum, and R. officinale (or R. coreanum). Moreover, three groups of R. palmatum, each belonging to three subclades, were characterized by the nucleotides at positions 619, 769, 883, and 1061. By detecting marker nucleotides, the botanical origins of Rhei Rhizoma were determined.
In a search for inhibitory components from natural products against NFAT transcription factor, this study investigated the ethyl acetate extract of the fruits of Liquidambar formosana. Four oleanane triterpenoids were isolated and identified to be liquidambaric acid, oleanolic acid, 3α-acetoxy-25-hydroxy-olean-12-en-28-oic acid and lantanolic acid. Of these compounds, 3α-acetoxy-25-hydroxy-olean-12-en-28-oic acid (IC50: 4.63 μM) and lantanolic acid (IC50: 12.62 μM) exhibited strong inhibitory activity against the NFAT transcription factor.
The antiallergic properties of the 70% ethanol extract from Plumbago zeylanica stems (EPZ) were investigated in the present study. The extract (500, 1000 mg/kg, p.o.) dose-dependently inhibited systemic anaphylactic shock induced by compound 48/80 in mice, reduced homologous passive cutaneous anaphylaxis and skin reactions induced by histamine or serotonin in rats, significant differences were observed at the dose of 1000 mg/kg. In vitro, EPZ (5, 20, 50 μg/ml) concentration-dependently reduced histamine release from rat peritoneal mast cells caused by compound 48/80 and antigen. EPZ (50 μg/ml) markedly increased intracellular cAMP content of rat mast cells. These findings demonstrate that EPZ inhibits mast cell-dependent immediate allergic reactions, which is probably mediated by reducing the release of mediators such as histamine from mast cells via elevating intracellular cAMP level and weakening the inflammatory action of mediators.
Ginseng was incubated under mildly acidic conditions and its inhibitory effect on a rat ischemia-reperfusion model was investigated. When ginseng was treated with 0.1% hydrochloric acid at 60 °C, its protopanaxadiol saponins were transformed to diasteromeric ginsenoside Rg3 and Δ20-ginsenoside Rg3. When the transformed ginseng extract, of which the main component was ginsenosides Rg3, was treated with human intestinal microflora, the main metabolite was ginsenoside Rh2. Orally administered acid-treated ginseng (AG) extract and ginsenoside Rh2 potently protect ischemia-reperfusion brain injury. The ginsenoside Rh2 also inhibited prostaglandin-E2 synthesis in lipopolysaccharide-stimulated RAW264.7 cells, but showed no in vitro antioxidant activity. These results suggest that AG and ginsenoside Rh2 can improve ischemic brain injury.
Clinical approach using tumor necrosis factor-alpha (TNF-α) as selective destruction against tumor endothelial cells and selective enhancer of tumor vascular permeability for effective accumulation of antitumor chemotherapeutic agents has attracted attention. However, the clinical application of TNF-α as a systemic antitumor agent has been limited because of toxic side-effects. To systemically use TNF-α as an antitumor agent and the selective enhancer of tumor vascular permeability, we assessed the usefulness of PEGylated TNF-α (PEG-TNF-α). PEG-TNF-α at a dose of 1000 JRU showed marked hemorrhagic necrosis in S-180 tumors without side-effects due to selective destruction of tumor vasculature, whereas wild-type TNF-α at a dose of 10000 JRU showed a little hemorrhagic necrosis with severe side-effects. PEG-TNF-α induced the enhancement of tumor vascular permeability. The permeability was increased at 1 h, after an i.v. injection of PEG-TNF-α and returned to the basal level at 2 h. In addition, high molecular weight of PEG (molecular weight; 500K) accumulated in tumor tissue as well as low molecular weight of PEG (molecular weight; 12K). On the other hand, PEG-TNF-α didn't affect the permeability of normal tissue and inflammation site. This data suggested that PEG-TNF-α was useful agent as selective enhancer of tumor vascular permeability with safe.
Focusing on the disposition of cyclosporin A (CsA) in the liver and intestine, effects of gentamicin-induced acute renal failure (ARF) on the decreased oral bioavailability of CsA were evaluated in rats. The area under the CsA concentration–time curve (AUC) in ARF rats after oral administration (5 mg/kg) significantly decreased by 43% as compared to the control, while the apparent oral clearance significantly increased by 76% of the control. The portal AUC of CsA in ARF rats with bile flow decreased by 67% as compared to the control rats. Without bile flow, the portal AUC of CsA in control rats decreased by 50% as compared to those with bile flow, whereas ARF rats without bile flow showed no notable change as compared to those with bile flow. The AUC of CsA mono-oxidative metabolite via CYP3A (M-OH) in ARF rats after oral or intravenous administration increased significantly by 84% or 241%, respectively, while there was no difference in the portal M-OH between control and ARF rats, suggesting that the elimination of M-OH was prolonged because of nephrotoxicity. Although the exsorption clearance of CsA from the blood circulation to the intestine after intravenous administration to ARF rats decreased significantly as compared to the control; and basolateral-to-apical transport of CsA through Caco-2 monolayers was significantly retarded in the presence of uremic toxins, there was no significant change in the total body clearance of CsA between ARF and control rats. Moreover, there were no effects of uremic toxins on the protein binding of CsA in plasma. These observations suggest that hepatic or intestinal CYP3A and P-glycoproteine (P-gp) are not likely to be concerned with lowering the oral bioavailability of CsA, and that bile function under the ARF condition induced by gentamicin is responsible for a marked decrease in the fraction absorbed of CsA in the small intestine.
We evaluated the features of cell death induced by CDF (cyclophosphamide [CPA], doxorubicin [DOX], 5-fluorouracil [5-FU]) multi-drug administration in vitro using the human breast cancer cell line MCF-7. Used individually, DOX and 5-FU induced 60% cell death in MCF-7 cells, at 5 μg/ml and 25 μg/ml, respectively, by the 4th day following drug treatment. CPA was the least cytotoxic of the 3 drugs, causing only 20% cell death, even at the high concentration of 500 μg/ml. Treating cells with a mixture of all three anticancer drugs resulted in 60% cell death, on the second and third day following drug treatment. The nature of the cytotoxicity of CPA, DOX, and 5-FU was investigated, because these drugs are sometimes used to induce apoptosis. Biochemical analysis showed faint DNA fragmentation in the case of DOX or all three drugs, but not for treatment with CPA or 5-FU. In contrast, the morphological apoptotic feature of a condensed nucleus was observed only for CPA and 5-FU. Flow cytometric data agreed with the morphological results in that the FACS cytogram for DOX and for all three drugs was different from that for CPA or 5-FU given alone. These observations suggested that the cell death induced by these anticancer drugs in the human breast cancer cell line MCF-7 is a mixture of apoptotic and non-apoptotic, but it becomes completely non-apoptotic in the case of multi-drug administration.
The expression and functional role of CD8 glycoprotein, a marker of cytotoxic/suppressor T lymphocytes and NK cells, were not studied on freshly isolated connective tissue type rat peritoneal mast cells, a rat mucosal type mast cell line (RBL 2H3), or human mast cell line (HMC-1). We used the reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, immunohistochemistry and enzyme-linked immunosorbent assay. Results: RT-PCR and Western blot analysis identified the presence of CD8 α/β chains on the mast cells, and immunohistochemistry confirmed CD8α expression on rat or human mast cells. Functional studies demonstrated that stimulation of CD8 α/β chains on rat mast cells induced the secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), which are regarded as important mediators during infection. However, co-stimulation with stem cell factor had no effect on CD8-induced mediator secretion. Our findings demonstrate novel biological roles of CD8 molecules in mast cells.
When hot-water extracts of mushroom Agaricus brazei MURRILL fruiting bodies (Agaricus extracts) were administered orally into Meth A-bearing mice, tumor growth was significantly inhibited compared with controls given water orally. Treatment with Agaricus extracts for five successive days significantly increased natural killer activity of spleen cells in naïve BALB/c mice. In Meth A-bearing BALB/c mice, Agaricus extracts enhanced the induction of antigen-specific cytotoxic T lymphocytes and interferon γ production, indicating that Agaricus extracts potentiate cytotoxic activity in innate and adaptive immunity.
A calcium-induced alginate gel bead (Alg-CS) containing chitosan (CS) and 2-(4-chlorophenoxy)-2-methylpropionic acid (CMP) was prepared. We then investigated (a) CMP release from Alg-CS, and (b) uptake of bile acid into the Alg-CS, within the gastrointestinal tract. Dried Alg-CS gradually swelled in taurocholate solution, while releasing CMP and taking up bile acid. The amount of bile acid taken up into the Alg-CS increased incrementally according to the degree of deacetylation of CS. Furthermore, the molecular weight of CS also affected the properties of the Alg-CS. An approximately linear relationship was observed between CMP release and bile acid uptake of Alg-CS.
For the purpose of the avoidance of reticuloendothelial system (RES)-trapping, liposome entrapped benzoporphyrin derivative monoacid ring A (BPD-MA), which is used for cancer photodynamic therapy (PDT), was modified with polyethylene glycol (PEG-LipBPD-MA). Tumor accumulation of BPD-MA at 3 h after injection with PEG-LipBPD-MA in Meth A-sarcoma-bearing mice was significantly higher than that after injection with non-modified liposomal BPD-MA (Cont-LipBPD-MA) as expected. On the contrary, significant tumor growth suppression after PDT was observed only for Cont-LipBPD-MA but not for PEG-LipBPD-MA. Thus, PEGylation enhances the passive targeting of liposomal BPD-MA in tumor, but decreases the susceptibility of the drug in PDT.