Invasive mycosis has significantly increased in frequency among immunocompromised hosts leading to excessive morbidity and mortality. The combination of sulfamethoxazole (SMX) and trimethoprim (TMP) has been used extensively for the treatment and prophylaxis of infections by various microbes. The purpose of this study is to estimate the anti-fungal activity of SMX-TMP and examine the mechanism of activity. To investigate the antimicrobial activity of SMX-TMP in vitro, a mixture of SMX and TMP at 5 : 1 was serially diluted and added to potato dextrose agar medium or C-limiting agar medium. Aspergillus species were inoculated on the medium plate with SMX-TMP. The growth of A. fumigatus and A. oryzae was inhibited by addition of SMX-TMP. The anti-Aspergillus effect depended on not TMP but SMX and that was inhibited by p-aminobenzoic acid (PABA). A. niger was not sensitive against SMX-TMP in PDA medium, but sensitive in C-limiting medium. Those results showed that the activity depends on culture medium. Furthermore, addition of human serum did not influence the activity of SMX. The finding in this study suggested that SMX might be effective against Aspergillus species in clinical practice and prophylaxis treatment.
Cells respond to oxidative stress including nitric oxide (NO) by increasing cellular glutathione concentration, as a part of adaptive response against oxidative injury. To elucidate the mechanism by which NO induces glutathione we investigated the reactive oxygen species (ROS) generated in the cell. Treatment of RAW264.7 cells with NO donor, sodium nitroprusside (SNP), resulted in a temporary increase in glutathione in a dose-dependent manner, which peaked between 6 h and 12 h after treatment, whereas expression of γ-glutamylcysteine synthetase (γ-GCS) mRNA peaked around 3 h after treatment. The increase was inhibited by NO scavengers, oxyhemoglobin and carboxyl-2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO). N-Acetyl-L-cysteine (NAC) also reduced the increase in glutathione to some extent, whereas both peroxynitrite scavenger ebselen and hydroxyl radical scavenger DMSO inhibited the increase in glutathione in a dose-dependent manner and complete inhibition was observed. Hydrogen peroxide exogenously added to the cell did not increase either glutathione or γ-GCS expression at any concentration, indicating that involvement of hydrogen peroxide is not likely. Flow cytometric analysis showed that SNP induced a marked dose-dependent increase in Rhodamine123 fluorescence, which was completely inhibited by ebselen in a dose-dependent manner, whereas, little increase in 2′,7′-dichlorofluorescin (DCF) fluorescence was observed. Generation of peroxynitrite in mitochondria by SNP was confirmed by elevated level of nitrotyrosine in a mitochondria fraction isolated from SNP-treated cells, and the elevation was completely inhibited by ebselen as well. These results suggest that induction of glutathione (GSH) synthesis by SNP treatment is mediated by peroxynitrite generated in mitochondria.
Inhibitory effects of six fungal bis(naphtho-γ-pyrone) derivatives on nitric oxide (NO) production by a murine macrophage-like cell line, RAW 264.7, which was activated by lipopolysaccharide and interferon-γ were examined. Among these derivatives, chaetochromin (4) (IC50: 0.8 μM), cephalochromin (1) (IC50 1.5 μM), and dihydroisoustilaginoidin A (6) (IC50 2.8 μM) exhibited strong inhibitory activity. The bis(naphtho-γ-pyrone) derivatives did not affect the enzyme activity of inducible nitric oxide synthase (iNOS). However, these derivatives significantly reduced both the induction of iNOS protein and iNOS mRNA expression. These results suggest that the bis(naphtho-γ-pyrone) derivatives have the pharmacologic ability to suppress NO production by activated macrophages.
Two C-type lectins (OLLafs and OLLafl) were isolated from Osmerus (Spirinchus) lanceolatus eggs using asialofetuin-Sepharose column. OLLafs and OLLafl were eluted with 0.2 M sucrose and 0.2 M lactose from the same column, respectively. OLLafl has been estimated to be a heterodimeric protein composed of H- and L-subunit and involved C-type lectin like domain (CTLD). In this study we revealed that OLLafs was a homodimeric protein composed of L-subunit of OLLafl. Although adding EDTA diminished the hemagglutinating activity of OLLafs, the activity of OLLafl was not influenced. Recombinant lectins (rOLLafl-H and -L) and mutant lectins replaced Cys123, 131 and 136 with Ala (mOLLafl-L123, 131 and 136) were established. The activity of mOLLafl-L136 was comparable to rOLLafl-L, and rOLLafl-H was 15 times lower than rOLLafl-L. On the other hand, the activity of mOLLafl-L123 and mOLLafl-L131 were lower than that of rOLLafl-H. Therefore, Cys136 may not participate in hemagglutinating activity of rOLLafl-L. In contrast, Cys123 and Cys131 may partially contribute this activity. Although hemagglutination inhibition profiles of rOLLafl-L, rOLLafl-H and mOLLafl-L136 were similar, m-OLLafl-L131-induced hemagglutination was not inhibited by any sugars tested even at a concentration of 150 mM. Then, Cys131 may directly contribute to the sugar-binding capacity of OLLafl. Affinities of mOLLafl-L123 for these sugars were lower than the others. These results suggest that Cys136 might contribute to the intermolecular disulfide bond in the rOLLafl-L dimer, and that the intramolecular disulfide bond concerning Cys131 might important for lectin activity.
Infection of human cell with human immunodeficiency virus type-1 (HIV-1) was suppressed by cellular genetic factor(s) at reverse transcription step. Although same amount of virus adsorbed on both cells, small amount of HIV-1 (IIIB strain) infected HeLa (MAGI/CCR5) cell, while large amount of HIV-1 infected HOS (GHOST/CXCR4) cell. Regulation of virus replication at postentry level by cellular factor(s) had an important role for low efficiency of HIV-1 infection to MAGI/CCR5 cell. Provirus DNA formation in MAGI/CCR5 cell was less efficient than in GHOST/CXCR4 cell. Once GHOST/CXCR4 cell was fused with MAGI/CCR5 cell, susceptibility against HIV-1 decreased. Further, HIV-1 reverse transcriptase (RT) activity was strongly inhibited by cytosolic protein, derived from MAGI/CCR5 cell, in vitro. This research cleared a certain human cell genetically carries some factor(s) which inhibits the activity of HIV-1 RT.
A novel protein, AAT-1, was identified as a AMY-1-binding protein and three splicing variants of AAT-1, AAT-1α, -β and -γ were identified. The function of AAT-1 is thought to be related to spermatogenesis. In this study, we further identified other splicing isoforms of AAT-1, AAT-1L, AAT-1M and AAT-1S, consisting of 767, 603 and 252 amino acids, respectively. These isoforms were found to use a promoter different from that used by AAT-1α, -β and -γ in the aat-1 gene, which contains 20 exons. Only 60 amino acids in the C-terminal portion of AAT-1 derived from exons 15—17 are common among AAT-1L, AAT-1M, AAT-1S and AAT-1α. While AAT-1α is specifically expressed in the testis, AAT-1L, AAT-1M, AAT-1S were found to be differentially expressed in human tissues. All of the isoforms of AAT-1 were found to bind to and colocalized with AMY-1 in human cells. While AAT-1L and AAT-1M were found to be localized diffusely in the cytoplasm, AAT-1S, like AAT-1α, was found to be localized in the mitochondria-like structure, suggesting different roles of AAT-1 isoforms in cells.
The aim of this study was to investigate the changes in mRNA level of embryonic form of myosin heavy chain (SMemb), endothelin-1 (ET-1) and plasminogen activator inhibitor-1 (PAI-1), which are considered to be involved in the angiogenesis and atherosclerosis in diabetic blood vessels, in human umbilical vein endothelial cells (HUVECs) caused by high ambient glucose, and the effects of 2-aminophenoxazine-3-one (Phx-3), which was produced by the reaction of bovine hemoglobin with o-aminophenol, on them. The mRNA level of SMemb, ET-1 and PAI-1 and the level of SMemb protein were extensively upregulated in HUVECs treated with high concentration of glucose (15 mM), compared with those in the cells with normal concentration of glucose (5 mM). The migration activity of HUVECs evaluated by the cell migration assay was accelerated by 15 mM glucose. When 10 μM Phx-3, at the concentration of which the proliferation of HUVECs was not affected, was administered to HUVECs with 15 mM glucose, the mRNA level of SMemb, ET-1 and PAI-1 and the level of SMemb protein were significantly downregulated to the normal levels in the cells. However, when 10 μM Phx-3 was administered to HUVECs with 5 mM of glucose, the mRNA level of SMemb, ET-1 and PAI-1 and the level of SMemb protein were not affected. The migration activity of HUVECs, which was accelerated by high glucose, was reversed by 10 μM Phx-3. The present results suggest that Phx-3 may be a drug to prevent the high glucose-associated endothelial damage, vascular angiogenesis in diabetic patients, by inhibiting the expression of angiogenic factors, such as SMemb, ET-1 and PAI-1, in the endothelial cells.
The volatile extract from dried pericarp of Zanthoxylum schinifolium that was obtained by simultaneous distillation with dichloromethane and water was composed of 29.9% geranyl acetate, 15.8% citronella, 15.4% sabinene and the minor volatile components included β-myrcene, linalool, (−)-isopulegol, citronellyl acetate, 1,4-dimethyl pyrazole, α-terpinene, 3-methyl-6-(1-methylethyl)-2-cyclo-hexene-1-o1 and trans-geraniol. The volatile extract decreased the cell viability and induced apoptotic death in HepG2 human hepatoma cells in a concentration- and time-related manner. In addition, the volatile extract increased the production of reactive oxygen species in a dose-dependent manner. Pretreatment of the cells with Trolox, a well-known antioxidant, significantly suppressed the generation of reactive oxygen species and cell death induced by the extract. However, caspase-3 activity was not changed in the extract-treated cells, suggesting that the extract-induced apoptosis of HepG2 cells is caspase-3 independent. Furthermore, in nude mice inoculated with Huh-7 human hepatoma cells, the extract significantly inhibited tumor development. These results suggest that the volatile extract from Zanthoxylum schinifolium pericarpium is a good candidate for hepatocellular carcinoma (HCC) therapy and that reactive oxygen species are the key signaling molecules in the volatile extract-induced cell death in HepG2 cells.
In the present study, we investigated hypnotic activities of chamomile and passiflora extracts using sleep-disturbed model rats. A significant decrease in sleep latency was observed with chamomile extract at a dose of 300 mg/kg, while passiflora extract showed no effects on sleep latency even at a dose of 3000 mg/kg. No significant effects were observed with both herbal extracts on total times of wakefulness, non-rapid eye movement (non-REM) sleep and REM sleep. Flumazenil, a benzodiazepine receptor antagonist, at a dose of 3 mg/kg showed a significant antagonistic effect on the shortening in sleep latency induced by chamomile extract. No significant effects were observed with chamomile and passiflora extracts on delta activity during non-REM sleep. In conclusion, chamomile extract is a herb having benzodiazepine-like hypnotic activity.
The antianginal effects of lercanidipine, a newly synthesized 1,4-dihydropyridine derivative calcium channel antagonist, were evaluated in experimental angina model rats and the effects were compared with those of nifedipine, benidipine and amlodipine. In the vasopressin-induced angina model, intravenous administration of lercanidipine dose-dependently suppressed vasopressin-induced ST-depression. Amlodipine barely suppressed it, while benidipine, at the same dose, completely suppressed it. Nifedipine had a potency between that of amlodipine and benidipine. Oral administration of lercanidipine showed similar effects to the intravenous administration test on ST change. High doses of amlodipine, benidipine and nifedipine suppressed ST-depression by almost 100%. In the methacholine-induced angina model, lercanidipine suppressed the ST elevation dose dependently. Amlodipine barely suppressed it, while benidipine at 30 μg/kg effected almost total suppression. Nifedipine had a potency between that of amlodipine and benidipine. Intraduodenal administration of lercanidipine also suppressed the ST-elevation dose dependently. Nifedipine, benidipine and amlodipine at 10 mg/kg all markedly suppressed the elevation. Lercanidipine was more potent than the other calcium channel antagonists tested. In conclusion, it was explicitly demonstrated that lercanidipine exerts potent protective effects on the ischemic electrocardiography (ECG) changes in a variety of putative angina pectoris models in rats. An antispasmolytic coronary vasodilating action may be involved in the mechanism. It is expected that lercanidipine will be useful as an antianginal agent.
Chuling, sclerotia of Polyporus umbellatus FRIES, has long been used for urological disorders in traditional medicine. In this study, we demonstrated that Chuling 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 50 to 1000 μg/ml. Moreover, tests were carried out to identify the main ingredient of Chuling with scavenging effect on free radicals. Triterpene carboxylic acids isolated from the methanol extract of Chuling, namely, polyporusterone A and polyporusterone B, were found to have inhibitory activities against AAPH-induced lysis of red blood cells. The anti-hemolytic effect was significantly stronger in polyporusterone B compared with polyporusterone A. Furthermore, the ingestion of 150 mg of Chuling was associated with a significant increase in free-radical scavenging effect of plasma in rats.
Multidrug resistance (MDR) is a major obstacle to successful clinical cancer chemotherapy. A novel doxorubicin anti-resistant Stealth liposomes (DARSLs), prepared by co-encapsulating doxorubicin (DOX) and verapamil (VER) into stealth liposomes, has been developed. The average particle size of DARSLs was 118.1±22.3 nm. Encapsulation efficiencies of DOX and VER in DARSLs were greater than 95% and 70%, respectively. The IC50 of DARSLs as measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT) assay in multidrug resistant rat prostate cancer Mat-LyLu-B2 (MLLB2) cells was 0.079±0.017 μM, 13 fold less than that for liposomal DOX with free VER (LDFV 0.96±0.46 μM) but only about 2 times less than FDFV. The IC50 cytotoxicity on MLLB2 cells of the various formulations was as follows: DARSLs∼LDLV<FDFV<FDLV<LDFV<LD<FD, (LD: liposomal DOX; LV: liposomal VER; FD: free DOX; FV: free VER). Similar cytotoxicities were shown between DARSLs and FDFV in DOX-resistant human uterus sarcoma MES-SA/DX5 cells, reversing DOX-resistance to that shown by FD on DOX-sensitive MES-SA cells. For MLLB2 cells, DARSLs was the most cytotoxic, but its intracellular concentration of DOX, measured as mean cellular fluorescence with flow cytometry was lower (p<0.01) than that observed with the FDFV formulation. In conclusion, DARSLs was an effective DOX formulation which could overcome drug resistance in DOX-resistant tumor cells, but its mechanisms of action may be complex.
Asthma, a common, chronic lung disease in industrialized countries, is characterized by the production of large quantities of IgE antibody by B cells and a decrease of the IFN-γ/IL-4 (Th1/Th2) ratio. Gyokuheifusan (GHS) is a classical formulation of traditional Chinese medicine (TCM) that is usually prescribed to prevent or treat respiratory tract diseases, such as respiratory infection and bronchial asthma. In order to evaluate the possible effectiveness of GHS on bronchial asthma, its immunomodulatory activity was examined in ovalbumin (OVA)-induced asthma model mice. All mice, except those in the normal group, were sensitized by intraperitoneal (IP) administration of OVA emulsified with Al(OH)3, and a second immunization was given 6 d later. After a further 13, 17 and 21d, mice were challenged with inhalation of aerosolized OVA solution, except for the normal group, which received mock sensitization using saline-Al(OH)3 emulsion and were challenged with an aerosol of saline without OVA. Allergen-specific IgE and total IgE in plasma were both significantly increased in the disease-control group. These increases were markedly blocked by GHS treatment. IFN-γ released by splenocytes was significantly increased after co-culture with OVA for 24 h, 48 h, and 72 h. GHS treatment further elevated the IFN-γ content compared with the disease-control group. The production of IL-4 was significantly increased when splenocytes were simulated with OVA for 72 h, but this increase was blocked by GHS treatment, so that GHS returned the decreased IFN-γ/IL-4 (Th1/Th2) ratio of the disease-control group to the normal range. These results indicate that GHS may inhibit the development and severity of asthma.
This study describes the antispasmodic activity of some fractions and cynaropicrin, a sesquiterpene lactone from Cynara scolymus, cultivated in Brazil, against guinea-pig ileum contracted by acetylcholine. The dichloromethane fraction showed the most promising biological effects, with an IC50 of 0.93 (0.49—1.77) mg/ml. Its main active component, the sesquiterpene lactone cynaropicrin, exhibited potent activity, with IC50 of 0.065 (0.049—0.086) mg/ml, being about 14-fold more active than dichloromethane fraction and having similar potency to that of papaverine, a well-known antispasmodic agent. The results confirm the popular use of artichoke for the treatment of gastrointestinal disturbances, and encourage new studies on this compound, in order to obtain new antispasmodic agents.
The present study aimed at investigating the antiviral effects of 2-amino-4,4α-dihydro-4α-7-dimethyl-3H-phenoxazine-3-one (Phx-1) and 3-amino-1,4α-dihydro-4α-8-dimethyl-2H-phenoxazine-2-one (Phx-2) on 6 representative viruses: poliovirus, porcine parvovirus, simian virus 40 (SV-40), herpes simplex virus-1 (HSV-1), Sindbis virus, and vesicular stomatitis virus (VSV). Phx-1 and Phx-2 suppressed the proliferation of poliovirus in Vero cells and that of porcine parvovirus in ESK cells at concentrations between 0.25 μg/ml and 2 μg/ml, when the cells were treated with Phx-1 and Phx-2 for 1 h and then inoculated with these viruses. The proliferation of the other viruses, SV-40, HSV-1, Sindbis virus, and VSV, in the host cells was not influenced by Phx-1 or Phx-2 at concentrations less than 20 μg/ml. The results suggest that Phx-1 and Phx-2 may be useful to prevent the proliferation of poliovirus and porcine parvovirus infection and may contribute to developing new antiviral drugs in future.
Toll-like receptor 4 (TLR4) is known to play an important role in innate immune responses. In the present study, chemically synthetic compound of N1-benzyl-4-methylbenzene-1,2-diamine (BMD) was discovered to inhibit nitric oxide (NO) production in macrophages RAW 264.7 stimulated with lipopolysaccharide (LPS) or fibronectin as TLR4 activators. The BMD compound attenuated LPS-induced synthesis of both mRNA and protein of NO synthase (iNOS), and inhibited LPS or fibronectin-mediated iNOS promoter activity, indicating that the compound down-regulated iNOS expression at transcription level. As a mode of the anti-inflammatory action shown by BMD compound, inhibitory effect on nuclear factor (NF)-κB activation was also investigated in macrophages RAW 264.7 stimulated with the TLR4 activators.
Three flavonoids were isolated from the whole plants of Waltheria indica and biological properties investigated. On the basis of their spectroscopic data, these compounds were identified as (−)-epicatechin, quercetin, and tiliroside. These flavonoids significantly and dose-dependently inhibited the production of the inflammatory mediator nitric oxide (NO), and the cytokines (tumor necrosis factor (TNF)-α and interleukin (IL)-12), in lipopolysaccharide (LPS) and interferon (IFN)-γ activated murine peritoneal macrophages, without displaying cytotoxicity. The order of inhibitory activity was quercetin>tiliroside>(−)-epicatechin. Furthermore, peritoneal macrophages were pre-activated with LPS/IFN-γ for 24 h, and the inhibitory effects of the above mentioned isolates on the production of NO were determined after a further 24 h, to address the possible mechanisms of their action. The present study supports the use of W. indica for the treatment of inflammatory diseases in traditional medicine.
Lithium (Li+) salts are commonly used in treating bipolar diseases. As physicians frequently keep the patients on long-term lithium therapy, awareness of the numerous side effects and pathogenesis of this lightest alkali metal is needed for such treatments. The current study was designed to evaluate the toxic effect of small doses of lithium nitrate in rats. In the present study we showed that the oral gavage feeding of lithium nitrate (20 mg Li/kg body wt) for 7 weeks on every alternate day to male albino wistar rats elicited a significant alterations in gross hematological values owing to hypochromic anemia and leucocytosis. Erythrocyte sedimentation rate (ESR) and clotting time depicted higher values and animals exhibited icteric condition. Serum levels of hexose, cholesterol and blood urea elevated; however, proteins depleted markedly. A significant increase in serum calcium and phosphorus has also been registered in lithium salt treated animals. The enzyme activities of alkaline phosphatase (Alpase) and acid phosphatase (Acpase) diminished depicting the disturbed general physiological status while there was a marked rise in the activities of transaminases (GOT and GPT) reflecting a stimulating transamination reaction in hepatic and renal tissues. The histopathological picture of the kidney tissues revealed many deformative alterations. Necrosis, binucleated cells and Kuffer's cells are visible in renal tissue. The epithelium lining of renal tissue was damaged and there were also some marked changes in glomerular region apart from intracellular alterations in corticomedulary region. The results of present study suggest that small doses of lithium induce toxicity in rats.
This study investigated inhibitory effects of N,N-unsubstituted selenourea derivatives on tyrosinase activity. Three types of N,N-unsubstituted selenoureas derivatives exhibited inhibitory effect on dopa (3,4-dihydroxyphenylalanine) oxidase activity of mushroom tyrosinase. Compound D at a concentration of 200 μM exhibited 55.5% of inhibition on dopa oxidase activity of mushroom tyrosinase. This inhibitory effect was higher than that of kojic acid (39.4%), a well known tyrosinase inhibitor. Moreover, the compound D identified as a noncompetitive inhibitor by Lineweaver–Burk plot analysis. In addition, compound D also inhibited the melanin production in melan-a cells.
The adsorption characteristics of eight adsorbents, cholestyramine, colestimide, aluminum silicate, sucralfate, aluminum hydroxide, calcium polystyrene sulfonate, carbon sphere and medicinal carbon, on the drugs such as methotrexate, antidepressants, mizoribine and ciprofloxacin hydrochloride were investigated in vitro. Medicinal carbon showed an excellent adsorption of all the tested drugs while the carbon spheres showed a high but slow adsorption characteristic. Cholestyramine and colestimide showed a higher adsorption in methotrexate than the other adsorbents. Aluminum silicate and calcium polystyrene sulfonate showed higher adsorption in four antidepressants, clomipramine hydrochloride, imipramine hydrochloride, mianserin hydrochloride and trazodone hydrochloride. In mizoribine, there were no adsorbents that showed higher adsorption except for the medicinal carbon. In ciprofloxacin hydrochloride, aluminum preparations and calcium polystyrene sulfonate showed higher adsorption characteristics. It is suggested that several adsorbents are potentially useful treatments for drug overdoses, but that these adsorbents have the possibility of decreasing the effects of the co-administered medicines.
We previously reported that either (+)-matrine (matridin-15-one) or (+)-allomatrine (the C-6 epimer of matrine)-induced antinociceptive effect was attenuated by s.c. pretreatment with a κ-opioid receptor (KOR) antagonist nor-binaltorphimine (nor-BNI), indicating the critical role of KORs in antinociceptive effects induced by these alkaloids. In the present study, we found that i.c.v. administration of either (+)-matrine- or (+)-allomatrine induced antinociceptive effects in the mouse tail-flick and warm-plate test, whereas these alkaloids when given spinally failed to induce antinociception. In the guanosine-5′-O-(3-[35S]thio)trisphosphate ([35S]GTPγS) binding assay, we demonstrated that neither (+)-matrine nor (+)-allomatrine produced the stimulation of [35S]GTPγS binding in the membranes of the spinal cord, indicating that (+)-matrine- and (+)-allomatrine-induced supraspinal antinociceptive actions was not due to a direct stimulation of KORs by these alkaloids. Therefore, we next investigated the involvement of dynorphin A (1-17) release at the spinal or supraspinal site in (+)-matrine- or (+)-allomatrine-induced antinociception. The i.c.v. pretreatment with an antiserum against dynorphin A (1-17) could not affect the antinociceptive effect induced by s.c. treatment of (+)-matrine. In contrast, the s.c.-administered (+)-matrine- and (+)-allomatrine-induced antinociceptive effect was significantly attenuated by i.t. pretreatment of an antiserum against dynorphin A (1-17). The present data suggest that either (+)-matrine or (+)-allomatrine when given i.c.v. may stimulate the descending dynorphinergic neuron, resulting in the stimulation of KORs in the spinal cord, and this phenomenon in turn produces the antinociception in mice.
Two compounds were synthesized which have a structural component other than those of our new series histone deacetylase (HDAC) inhibitors to determine the structure–activity relationship. It was also examined whether the inhibitory effects on cancer cell proliferation by HDAC inhibitors involve p21/WAF1 induction and G1 or G2/M arrest in p53-mutated MG63 human osteosarcoma cells as do other HDAC inhibitors. It was demonstrated that inhibitors with the 2-naphthylcarbonyl group and hydroxamic acid at both termimal sides as well as the phenylene component at the center of molecule markedly induce the p21/WAF1 protein by stimulating p21/WAF1 gene promoter activity. Furthermore, cell cycle analysis revealed that these compounds arrest MG63 cells in the G2/M phase.
We previously demonstrated that β-D-xylopyranosyl-(1→3)-β-D-glucuronopyranosyl echinocystic acid (codonoposide 1c), a biologically active compound isolated from the roots of Codonopsis lanceolata, is cytotoxic to cancer cells. In the present study, we investigated the effects of codonoposide 1c on the induction of apoptosis, and its putative action pathway in HL-60 human promyelocytic leukemia cells. Codonoposide 1c-treated HL-60 cells displayed several features of apoptosis, including DNA fragmentation, formation of DNA ladders by agarose gel electrophoresis, and externalization of annexin-V targeted phosphatidylserine (PS) residues. We observed that codonoposide 1c caused activation of caspase-8, caspase-9, and caspase-3. A broad caspase inhibitor (z-VAD-fmk), caspase-8 inhibitor (z-IETD-fmk), and caspase-3 inhibitor (z-DEVD-fmk) almost completely suppressed codonoposide 1c-induced DNA fragmentation. We further found that codonoposide 1c induces mitochondrial translocation of Bid from cytosol, reduction of cytosolic Bax, and cytochrome c release from mitochondria. Interestingly, codonoposide 1c also triggered the mitochondrial release of Smac/DIABLO (second mitochondria-derived activator of caspases/direct inhibitor of apoptosis-binding protein with a low isoelectric point) into cytosol, and a reduction in X-linked inhibitor of apoptosis protein (XIAP). Taken together, our data indicate that codonoposide 1c is a potent inducer of apoptosis and facilates its activity via Bid cleavage and translocation to mitochondria, Bax reduction in cytosol, release of cytochrome c and Smac/DIABLO into the cytosol, and subsequently caspase activation, providing a potential mechanism for the cytotoxic activity of codonoposide 1c.
The methanol extract of Sorbus commixta cortex (MSC) induced relaxation of the phenylephrine-precontracted aorta in a dose-dependent manner, which was disappeared by removal of functional endothelium. Pretreatment of the aortic tissues with NG-nitro-L-arginine methyl ester (L-NAME), methylene blue, or 1H-[1,2,4]-oxadiazole-[4,3-α]-quinoxalin-1-one (ODQ) inhibited the vascular relaxation induced by MSC. MSC-induced vascular relaxations were also markedly attenuated by addition of verapamil or diltiazem, while the relaxant effect of MSC was not blocked by pretreatment with indomethacine, glibenclamide, tetraethylammonium (TEA), atropine, or propranolol, respectively. Incubation of endothelium-intact carotid arteries or of human umbilical vein endothelial cells (HUVECs) with MSC increased the production of guanosine 3′,5′-cyclic monophosphate (cGMP). Moreover, MSC-induced cGMP production was effect was blocked by pretreatment with L-NAME or ODQ. These results suggest that MSC dilates vascular smooth muscle via endothelium-dependent nitric oxide-cGMP signaling pathway, possible involvement of L-type Ca2+ channel.
We evaluated the effects of the traditional Chinese medicines, Hachimi-jio-gan, Juzen-taiho-to and Unkei-to, on bone loss in murine model of senile osteoporosis (SAMP6). Two-month-old SAMP6 were divided into control and experimental groups. The control mice had the tap water available as the only drinking fluid. The experimental mice were given 0.05% aqueous solution of Hachimi-jio-gan, Juzen-taiho-to or Unkei-to for three months. The solution intake of a mouse averaged 5 ml per day. The bones were studied morphologically and histomorphometrically, together with bone mineral density (BMD), serum parathyroid hormone (PTH) and estradiol levels. In the control group, BMD and the amount of bone forming surface were low, the serum PTH level was high when compared with the normal mice SAMR1. Many osteocytes and osteoblasts showed degenerative changes and numerous mast cells were observed in the bone marrow. Compared with controls, the serum estradiol level was higher in the Unkei-to group. However, we did not find any significant changes of bones. In the Hachimi-jio-gan and Juzen-taiho-to groups, the bone mass and the amount of bone forming surface increased. Most of the osteocytes and osteoblasts appeared normal. As compared with controls, the number of mast cells in bone marrow decreased in the Hachimi-jio-gan group. The serum PTH level had declined in the Juzen-taiho-to group. The present study provides certain evidence that Hachimi-jio-gan and Juzen-taiho-to are effective in preventing bone loss in SAMP6, while Unkei-to can only improve the ovary function.
The inhibitory effects of compounds from Salicornia herbacea (Chenopodiaceae) on rat lens aldose reductase (RLAR) and sorbitol accumulation in streptozotocin-induced diabetic rat tissues were investigated. The various fractions from the MeOH extract of S. herbacea were tested for their effects on RLAR in vitro. Among them, the EtOAc fraction was found to exhibit a potent RLAR inhibition (IC50=0.75 μg/ml), from which an active principle as a potent AR inhibitor was isolated and its chemical structure was elucidated as isorhamnetin-3-O-β-D-glucoside (1) by spectral analysis. Compound 1 exhibited a potent RLAR inhibition in vitro, its IC50 being 1.4 μM. Compound 1, when administered orally at 25 mg/kg in streptozotocin (STZ)-induced diabetic rats, caused not only a significant inhibition of serum glucose concentration but also sorbitol accumulation in the lenses, red blood cells (RBC), and sciatic nerves. These results indicate that compound 1 from S. herbacea is a leading compound for further study as a new drug for the prevention and/or treatment of diabetes and its complications.
The effectivity of water extract from the style of Zea mays on diabetic nephropathy was investigated in the development of new natural medicinal resources. Streptozotocin (STZ) induced diabetic rats were used to evaluate the therapeutic effect of the style. Urinary albumin excretion and creatinine clearance were examined for diagnosis of diabetic nephropathy. From these results it was learned that the style of Z. mays prevented glomerular hyperfiltration. The present findings indicated that the water extract of the title material suppressed the progression of diabetic glomerular sclerosis in STZ-induced diabetic rat.
The AIP1 fraction, a small water-soluble fraction purified from Artemisia iwayomogi, was shown to increase antibody production and suppress transplanted tumor cell growth in mice. In order to understand its immuno-modulating activity, we have examined the effect of the AIP1 on mouse thymocytes in vitro. Treatment of mouse thymocytes in culture with the fraction resulted in the suppression of the cell death and the extension of the cell survival. A mouse gene array provided a profile of gene expression change showing the pattern of up- and down-regulated genes by the AIP1 treatment, suggesting that the Fas/FasL-dependent apoptosis pathway might be modulated by the fraction.
Long term and repeated exposure of ultraviolet (UV) light, a harmful environmental stress, on the skin often induces chronic skin diseases such as skin cancer as well as photoaging (premature skin aging), and the mechanisms of these skin damages are closely associated with up-regulation of matrix metalloproteinases (MMPs) activities. Here we investigated the effect of 2′,4′,7-trihydroxyisoflavone isolated from the whole plants of Viola hondoensis (Violaceae) on the expression of MMPs in UV-irradiated human skin fibroblasts in vitro. 2′,4′,7-Trihydroxyisoflavone markedly reduced UV-induced MMP-1 expression, but not MMP-2, at the both mRNA and protein levels in a dose-dependent manner. Our report is the first description for the ability of 2′,4′,7-trihydroxyisoflavone to regulate MMP-1 expression specifically.
The extract of Ongael [leaves of Phaleria cumingii (MEISN.) F. VILL.], a Palauan medicinal herb, enhanced an in vitro phagocytic activity of mouse macrophages RAW 264.7 cells (RAW 264.7). Activity-guided fractionation of the Ongael extract by the in vitro phagocytosis assay using RAW 264.7 led to the isolation of a mixture of acylglucosylsterols (1) as an active constituent along with other inactive constituents, tetracosanol and mangiferin. On the basis of chemical modifications and spectral analyses, the compound 1 was deduced to be a mixture of the known 3-O-(6-O-acyl-β-D-glucosyl)-β-sitosterols, the acyl moiety being mainly palmitoyl (57%), oleoyl (12%) and α-linolenoyl (12%) with small amount of stearoyl (7%) and linoleoyl (4%).
In order to reveal one of possible mechanisms for neuronal protective action of extract of Ginkgo biloba leaves (EGBL), the effect of EGBL on kainate- and KCl-induced increases in intracellular Ca2+ concentration ([Ca2+]i) of rat cerebellar neurons was examined using a confocal laser microscope with appropriate fluorescent probes. EGBL at 3 μg/ml started to attenuate kainate-induced increase of [Ca2+]i and further increase in EGBL concentration (up to 30 μg/ml) concentration-dependently and significantly inhibited the kainate response. The complete inhibition by EGBL was observed in some neurons when the concentration was 10—30 μg/ml. The kainate-induced increase in [Ca2+]i was mainly due to Ca2+ influx through voltage-dependent Ca2+ channel opened by membrane depolarization via activation of kainate receptor-channel. However, the increase in [Ca2+]i by KCl was not significantly affected by EGBL at concentrations where the kainate response was greatly inhibited. EGBL consisting of flavone glycosides and terpene lactones is known to be an antioxidant. Furthermore, in this study, it is shown that EGBL exerts an inhibitory action on kainate receptor (a subtype of glutamate receptor). Since some of neurodegenerative diseases are due to cell death induced by glutamate excitotoxicity and oxidative stress, EGBL may be very suitable for preventing and/or treating such diseases.
Propofol (PF), a highly lipophilic anesthetic, has several desirable properties, such as the rapid onset and cessation of its effects upon intravenous infusion. In this study, the transdermal absorption of PF was investigated with the aim of the development of an alternative route of administration. PF solutions containing isopropyl myristate (IPM), ethanol or propylene glycol (PG) at various concentrations were prepared and applied to the abdominal skin of rats. Petrolatum and fatty alcohol propylene glycol (FAPG) ointments containing PF were also prepared and applied to the dorsal skin. Eyelid opening was measured and the ratio of the measured value to the initial value was calculated to evaluate the level of the pharmacological effect of the preparation. The PG solution containing 80% PF achieved higher plasma PF concentrations than the 100% PF solution. The PF-FAPG ointment produced a higher plasma PF concentration than the PF-petrolatum ointment. Furthermore, a drowsy state was confirmed after transdermal administration of 42% PF-FAPG ointment. These results indicate that the combination of PF and PG was appropriate for the transdermal absorption of PF, and PF was absorbed through the rat skin to an extent sufficient to cause a continuous sedative effect.
The nonlinear mixed effects model (NONMEM) was used to analyze the pharmacokinetics of routinely administered bisoprolol in middle-aged and elderly Japanese patients. The subjects consisted of 29 males and 11 females with a mean age of 63.5±10.1. Data on the plasma concentration of bisoprolol from 94 blood samples obtained at steady-state following repetitive oral administration were analyzed using the NONMEM program, where a one-compartment model with repetitive bolus dosing was parameterized in terms of oral clearance (CL/F) and apparent volume of distribution (V/F). Individual CL/F values were correlated with body weight (WT) and creatinine clearance (CLcr). The relation between CLcr and the CL/F of bisoprolol was not altered by the CYP2D6 and CYP2C19 genotypes, gender, or age. The mean CL/F value estimated with NONMEM was 0.0612·WT+1.15·CLcr (l/h), and the mean V/F value was 2.61·WT (l). The residual interindividual variability of CL/F and V/F were 22.0% and 12.6%, respectively. The pharmacokinetic variability of bisoprolol is small even in routinely treated Japanese patients, provided that both body weight and renal function are taken into account for the prediction of oral clearance of the drug.
To anticipate drug–drug interactions by nicardipine in vivo, cytochrome P450 (CYP) forms responsible for the metabolism of nicardipine and inhibition of CYP-dependent drug metabolism by nicardipine were investigated. Microsomes of human B-lymphoblastoid cells expressing each human CYP form were used for the metabolism of nicardipine. Inhibitory effects of nicardipine on drug metabolism were studied using human liver microsomes. CYP2C8, CYP2D6 and CYP3A4 were identified as major CYP forms for the metabolism of nicardipine in human liver microsomes. Nicardipine strongly inhibited two-pathways of triazolam hydroxylation both catalyzed by CYP3A4. Comparison of three Ca2+ antagonists, nicardipine, nifedipine, and diltiazem revealed that only nicardipine showed such a strong inhibitory potency on the typical CYP2D6-catalyzed drug metabolism. Furthermore, nicardipine inhibited other reactions catalyzed by CYP1A, CYP2A6, CYP2C8, CYP2C9 and CYP2C19 with Ki values ranging from 1.1 to 29.4 μM. In conclusion, nicardipine was a relatively potent inhibitor of human CYP2D6, CYP3A4 and CYP2C (especially for CYP2C8 and CYP2C19) in vitro, suggesting that drug–drug interactions between nicardipine and other drugs metabolized mainly by these CYP forms appear to occur in vivo.
The mRNA expression levels of 10 toll-like receptors (TLRs) and 21 related genes in total RNA from pooled specimens of fetal human tissues (brain and liver), from single and pooled specimens of various adult human tissues (adrenal gland, brain, heart, kidney, liver, lung, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, testis, thymus, thyroid gland, trachea, and uterus), and from two cell lines (Hep G2 and HeLa) were analyzed by real-time reverse transcription PCR. The mRNA expression of the 10 human TLRs was successfully detected in all of the tissues and in HeLa cells. TLR2, TLR3, TLR6, and TLR9 were consistently expressed in Hep G2 cells, but TLR1, TLR4, TLR5, TLR7, TLR8, and TLR10 showed no or very weak expression in these cells. The mRNA expression of many TLR-related genes (ICAM1, CD14, MyD88, LY96, TRIF, TICAM2, TIRAP, CD83, SOCS1, TNFAIP3, TOLLIP, IRAK1, IRAK2, IRAK4, and TRAF6) was successfully detected in all of the tissues and cell lines. The mRNA expression of CD80, CD86, IRAK3, and CCL2 was successfully detected in all of the tissues and cell lines except for Hep G2 cells. The mRNA expression of CCL5 was successfully detected in all of the tissues and cell lines except for fetal brain. The mRNA expression of CXCL10 was successfully detected in all of the tissues except for fetal and adult brain. These results provide valuable information for studies concerning the regulation of TLR-related genes.
The turmeric (Curcuma longa L. rhizomes) EtOH extract significantly suppressed an increase in blood glucose level in type 2 diabetic KK-Ay mice. In an in vitro evaluation, the extract stimulated human adipocyte differentiation in a dose-dependent manner and showed human peroxisome proliferator-activated receptor (PPAR)-γ ligand-binding activity in a GAL4-PPAR-γ chimera assay. The main constituents of the extract were identified as curcumin, demethoxycurcumin, bisdemethoxycurcumin, and ar-turmerone, which had also PPAR-γ ligand-binding activity. These results indicate that turmeric is a promising ingredient of functional food for the prevention and/or amelioration of type 2 diabetes and that curcumin, demethoxycurcumin, bisdemethoxycurcumin, and ar-turmerone mainly contribute to the effects via PPAR-γ activation.