Microglia, residential macrophages in the central nervous system, can release a variety of factors including cytokines, chemokines, etc. to regulate the communication among neuronal and other types of glial cells. Microglia play immunological roles in mechanisms underlying the phagocytosis of invading microorganisms and removal of dead or damaged cells. When microglia are hyperactivated due to a certain pathological imbalance, they may cause neuronal degeneration. Pathological activation of microglia has been reported in a wide range of conditions such as cerebral ischemia, Alzheimer's disease, prion diseases, multiple sclerosis, AIDS dementia, and others. Nearly 5000 papers on microglia can be retrieved on the Web site PubMed at present (November 2001) and half of them were published within the past 5 years. Although it is not possible to read each paper in detail, as many factors as possible affecting microglial functions in in vitro culture systems are presented in this review. The factors are separated into “activators” and “inhibitors,” although it is difficult to classify many of them. An overview on these factors may help in the development of a new strategy for the treatment of various neurodegenerative diseases.
A sensitive and specific enzyme-linked immunosorbent assay for an antiarrhythmic drug, amiodarone (AMI), was developed, which is capable of measuring levels as low as 16 ng/ml. Anti-AMI antibody was obtained by immunizing rabbits with an antigen conjugated with bovine serum albumin using diazotized 4-amino-1-(2-diethylaminoethoxy)-2,6-diiodobenzene. Enzyme labeling of AMI with β-D-galactosidase was similarly performed using a diazotized 4-amino-1-(2-diethylaminoethoxy)-2,6-diiodobenzene. This enzyme-linked immunosorbent assay was specific for AMI and showed a very slight cross-reactivity (1.25%) with its major metabolite, mono-N-desethylamiodarone. The values of the AMI concentrations measured by this assay were in good correlation to those by HPLC. Its analytical applicability was demonstrated by a kinetic study with human liver microsomes. The enzyme-linked immunosorbent assay should be a valuable tool in therapeutic drug monitoring and pharmacokinetic studies of AMI.
The effects of HM-1 killer toxin (HM-1) on yeast spheroplasts of Saccharomyces cerevisiae were examined under osmotically stabilized conditions. Prolonged incubation of spheroplasts in nutrient-rich media resulted in an increase in volume, accompanied by aberrant morphological changes. By contrast, spheroplasts were enlarged, maintaining a round shape, when incubated in HM-1 media. The required 50% effective dose of HM-1 was as low as 2.2×10−8 M, and this effect by HM-1 was specific to yeast sensitive to HM-1. Some parts of the enlarged spheroplasts were stable, but the round shape was deformed as HM-1 was removed from the medium. In both the control and HM-1-treated spheroplasts, the total protein and DNA content were increased by approximately three and four times in response to their incubations, respectively. Cytochemical analysis by 4′6-diamidino-2-phenylindol (DAPI) staining showed multiple nuclei. Consistently, actin patches of cells were evenly distributed in both the control and HM-1-treated spheroplasts. A similar enlargement of spheroplasts was observed with lipophilic antifungal compounds, aculeacin A and papulacandin B, but the effects were distinct from those of HM-1 because the spheroplasts resulted in lysis after a long incubation. The molecular mechanism(s) behind this unique observation remains to be studied, but it is clear that HM-1 is an excellent tool for studying yeast cell biology.
The effects of gentamicin sulfate on carbonic anhydrase (CA) enzyme activity in in vitro human and in in vivo rat erythrocytes were investigated. For in vitro study, human carbonic anhydrase-I and -II (HCA-I and HCA-II) were purified by affinity-column chromatography, and rats were used for in vivo study. In vivo and in vitro CA enzyme activity was determined colorimetrically using the CO2–hydration method of Wilbur and Anderson as modified by Rickli et al. Gentamicin sulfate (1.98—9.90 mM) showed in vitro inhibitory effects on HCA-I and HCA-II hydratase activity up to a 2 mM concentration, when determined using the CO2–hydratase method. Rat erythrocyte CA activity was significantly inhibited for up to 3 h (p<0.001) following intramuscular administration of gentamicin sulfate to Sprague-Dawley rats (3.2 mg/kg body weight). In conclusion, gentamicin sulfate inhibits CA enzyme activity in vivo and at low concentrations in vitro, but activated it at high concentrations (≥4 mM) in vitro.
Glutathione transferase P (GST-P) gene expression is repressed in normal rats but markedly promoted during the early stage of chemical hepatocarcinogenesis. We have previously identified a silencer region in this gene promoter. The silencer is composed of several cis-elements to which at least three proteins (Silencer factor-A, -B, and -C: SF-A, SF-B, and SF-C) are known to bind. We cloned and characterized the nuclear factor 1 family and the CCAAT/enhancer-binding protein family as SF-A and SF-B, respectively. Recently, zinc finger proteins as candidates for SF-C, which binds to GST-P silencer 2 (GPS2), were isolated. These proteins include four Krüppel-like proteins (BTEB2, EZF, LKLF, and TIEG1) and other factors containing multiple zinc finger motifs (TFIIIA and MZFP). In the present study, we found that the zinc finger proteins showed the same DNA-binding affinities to GPS2. Moreover, transfection analyses revealed that BTEB2, EZF, and TIEG1 repressed the GST-P promoter activity. Therefore, these three factors might contribute to the repression of the GST-P gene expression in normal rat liver.
Based on phylogenetic analysis of rDNA internal transcribed spacer (ITS) sequences, a pair of specific primers were designed for differentiating the Chinese traditional medicine Hericium species from other mushrooms by PCR. PCR was performed, with total DNAs as a template at an annealing temperature of 52—57 °C. Positive amplification was obtained from H. erinaceus with all DNA templates from different resources, but not from other related species. The result indicated that H. erinaceus could be clearly distinguished from other fungi by detection PCR, and no incorrect discrimination was found under the same reaction conditions. The primers were also successfully employed to identify H. erinaceus with different tissue types.
4-Acetyltropolone, a minor component of Thujopsis dolabrata SIEB. et ZUCC. hondai MAKINO, showed antimicrobial activity against various microorganisms including wood-rotting fungi, a phytogrowth-inhibitory effect with chlorophyll biosynthesis inhibition, cytotoxic effect and inhibitory activity on metalloproteases. This compound had strong antifungal activity on Daedalea dickinsii IFO-4979 [minimum inhibitory concentration (MIC): 0.2 μg/ml] and Coriolus versicolor IFO-4940 (MIC: 0.39 μg/ml). Its cytotoxic effect at 20.0 μg/ml on human stomach cancer KATO-III and Ehrich's ascites carcinoma was stronger than those of podophyllotoxin, vincristine and vinblastine, the anticancer agents isolated from higher plants and used clinically. This compound also had potent antibacterial activity against Staphylococcus epidermidis IFO-12993, its MIC being 1.56 μg/ml. However, other biological activities of 4-acetyltropolone were lower than those of hinokitiol which is the main component of this plant, suggesting that the contribution of the acetyl group at C-4 to biological activity is smaller than that of the isopropyl group at that position. The acute toxicity of 4-acetyltropolone (LD50: 335.2 mg/kg) to mice was much lower than that of hinokitiol (LD50: 191 mg/kg).
An easy and rapid ELISA system, Filtration ELISA, to detect antibodies against bacterial cell surface antigens was developed using a 96-well filtration plate fitted with a 0.22 μm membrane (MultiScreen®-GV, Millipore). Bacterial whole cells were used as antigens without fixing the cells with formalin etc. The whole cell antigens were washed by vacuum filtration through a filter and resuspended in washing buffer. Assay reactions could be done in the wells without losing the solution. The technique was established using antisera of mice immunized with Escherichia coli, and then evaluated by assaying antibodies to Shiga-toxin producing E. coli O157:H7 (STEC), Staphylococcus aureus and Lactobacillus acidophilus in fecal extracts of 157 children who had eaten school lunches contaminated with STEC in comparison with 25 age-matched control children. The lunch group showed significantly higher IgA antibody titers against STEC than the control group (p<0.0005), but not against L. acidphilus. The results indicate that Filtration ELISA is a quantitative and specific technique for measuring antibodies against antigens on the surface of bacteria without extracting antigens from the bacteria. This technique is widely applicable to the assay of antibodies in various samples including serum and fecal extract against various kinds of bacteria.
Ewing's sarcoma (ES), most commonly an undifferentiated tumor of bone, belongs to the enigmatic diagnostic category of small round cell tumors (SRCT) of childhood. The consistent presence of the translocation t (11; 22) in the vast majority of tumors provides evidence for a common histogenesis in ES and its family of tumors (ESFT), and also provides a unique diagnostic characteristic to discriminate this tumor family from SRCT. Molecular analysis of this translocation has revealed that it forms a chimeric gene between EWS on chromosome 22 and FLI-1 on chromosome 11. Similarly, the variant t (21; 22), t (7; 22), t (17; 22), and t (2; 22) rearrangements also form chimeric genes between regions of EWS and the ETS gene family (ERG, ETV1, E1AF, and FEV). Detection of these specific chimeric genes would provide a method for diagnosis of ESFT. We have developed a procedure for simultaneous detection of the chimeric genes by reverse transcription polymerase chain reaction (RT-PCR) with a mixture of primers. We conclude that the detecting those chimeric genes by this method can be easy and useful for diagnosis of ESFT. Moreover, by defining the specific chimeric gene it is possible to detect the tumor cell contamination in autologous blood stem cell transplantation.
Platonin, a cyanine photosensitizing dye, is a potent macrophage-activating agent and an immunomodulator. In this study, we compare the inhibitory effects of platonin with those of the three clinical drugs minocycline, clindamycin, and cyclosporin, on hypotension, tachycardia, and nitric oxide (NO) formation in a rat model of circulatory shock induced by Escherichia coli lipopolysaccharide (LPS). We also evaluate the effect of drugs on the 6 h survival rate in LPS-treated rats. Administration of LPS (15 mg/kg) caused a rapid drop in mean arterial blood pressure (MAP). Minocycline (10 mg/kg, i.v.) significantly prevented the fall of MAP at 3 h, and platonin (100 μg/kg, i.v.) markedly prevented the fall of MAP within the 0—3 h period after LPS administration. However, neither clindamycin (10 mg/kg, i.v.) nor cyclosporin (15 mg/kg, i.v.) had any effects in this study. On the other hand, an inducible NO synthase inhibitor, NG-nitro-L-arginine ester (L-NAME), caused a significantly increase in MAP and a moderate bradycardia after LPS administration. In addition, an increase in plasma nitrate formation elicited by endotoxemia was significantly reduced by pretreatment with either minocycline (10 mg/kg) or platonin (100 μg/kg). However, only platonin (100 μg/kg) markedly reduced the mortality and prolonged the mean survival time in LPS-treated rats. Minocycline, clindamycin, and cyclosporin had no effects under the same conditions. Further studies using an electron spin resonance (ESR) method were conducted on the scavenging activity of platonin on the free radicals formed. Platonin (10 μM) greatly reduced the ESR signal intensity of superoxide anion, hydroxyl radical, and methyl radical formation. In conclusion, platonin has beneficial effects on ameliorating endotoxaemia. This protective effect of platonin may be mediated, at least partly, by the reduced drop in MAP and the inhibition of NO and free radical formation in rat models of endotoxemia.
We investigated the effects of the aqueous extract of Epimedii Herba (AEEH) on the induction of oral tolerance. Oral tolerance was induced in mice by giving an oral administration of 20 mg ovalbumin (OVA) 7 d before immunization with the antigen. AEEH at 40 mg/kg was given orally daily for 6 d from 24 h after the feeding of OVA. The results showed that oral administration of OVA greatly suppressed total serum and antigen-specific immunoglobulin (Ig) levels, phagocytic activity and delayed-type hypersensitivity (DTH) reaction to the antigen. The suppression of these immune responses to OVA by the oral antigen was associated with a marked reduction of the production of interferon-γ (IFN-γ) and interleukin-4 (IL-4) from spleen cells. However, AEEH treatment significantly blocked the suppression of total serum and antigen-specific IgG2a antibodies, phagocytic activity and DTH response by the oral OVA. The suppression of IFN-γ production by the oral antigen was also greatly decreased by AEEH treatment. Therefore, AEEH appears to be effective in preventing the induction of oral tolerance to OVA.
The effects of YM337, the Fab fragment of a humanized anti-glycoprotein IIb/IIIa (GPIIb/IIIa) monoclonal antibody C4G1, on in vitro platelet function and binding properties were compared with those of abciximab, the Fab fragment of the human/murine chimeric anti-GPIIb/IIIa monoclonal antibody 7E3. Both agents completely inhibited platelet aggregation caused by all agonists tested except ristocetin. Further, both inhibited human platelet adhesion to von Willebrand factor, fibrinogen, fibronectin and subendothelial matrix with similar potency. Fibrinogen binding to washed platelets was dose-dependently inhibited by both agents. In binding assay using 125I-YM337 and 125I-abciximab, Kd values determined with platelet-rich plasma were 6.74±0.56 nM for YM337 and 6.65±1.45 nM for abciximab, and the number of binding sites were 42700±3000 for YM337 and 76000±5400 for abciximab. GPIIb/IIIa was precipitated from the solubilized fraction of platelets by both agents. In contrast, integrin αvβ3 was precipitated from the solubilized fraction of human umbilical vein endothelial cells by abciximab but not by YM337. Fibrinogen binding to purified GPIIb/IIIa was dose-dependently inhibited by both agents. In contrast, vitronectin binding to purified integrin αvβ3 was dose-dependently inhibited by abciximab but not by YM337, supporting the idea that abciximab reacts to integrin αvβ3. Therefore, YM337 was suggested to bind to a different epitope of GPIIb/IIIa from abciximab. These results suggest that YM337 specifically acts on platelet GPIIb/IIIa receptors and has similar inhibitory properties on platelet aggregation and platelet adhesion to abciximab.
This paper deals with the effects of elenoside, (3-hidroxymethyl-1-methoxy-5,6-methylene-dioxy-4-(3,4-methylenedioxyphenyl)-2-naftoic acid lactone-β-D-glucoside) an arylnaphthalene lignan with broad spectrum cytotoxicity in a human tumor cell line panel, isolated from Justicia hyssopifolia (Acanthaceae) grown in the Canary Islands (Spain), on isolated cardiac auricle of rabbits, urinary excretion of rats, and on isolated rat ileum. These effects, using a vehicle (propylene glycol–ethanol–plant oil–Tween 80 (40 : 10 : 50 : 2) as a standard, are presented. Elenoside at concentrations of 3.2×10−4, 6.4×10−4, and 1.2×10−3 M produced an increase in the contraction force of auricles in a concentration-dependent way. At doses of 25 and 50 mg/kg, an antidiuretic effect and a decrease in sodium excretion were observed. Elenoside at concentrations of 3.2×10−4, 6.4×10−4 and 1.2×10−3 M produced an increase in the contraction force of ileum in a concentration-dependent manner. Elenoside produced the concentration dependent inhibition of 86Rb uptake. These results indicate that elenoside has digitalis-like activity similar to mammalian lignans. Moreover, this lignan has an irritant effect on the gastrointestinal tract.
Lumin was orally administered to mice daily for 3 d, and on the day following the final administration, mice were sacrificed and splenocytes were stimulated with lipopolysaccharide (LPS). Splenocytes obtained from lumin-treated mice showed enhanced production of interferon γ (IFN-γ) and increased percentages of CD3+ cells. Although T cells are considered to be the source of IFN-γ, it is unlikely that LPS directly stimulates T cells. Next we performed neutralization experiments using a monoclonal antibody (mAb) against interleukin (IL-)12 because this cytokine, which is produced by macrophages, has the direct ability to induce IFN-γ production and the proliferation of activated T cells. This antibody inhibited IFN-γ production by splenocytes. We thus show that orally administered lumin enhances IFN-γ production by splenocytes when the latter are stimulated with LPS, a phenomenon that was observed in correlation with activation of T cells by IL-12, that is produced by macrophages.
Effect of docosahexaenoic acid (DHA) [22 : 6(n-3)]-fortified Chlorella oil fraction on radial maze performance was studied in aged mice. Male ICR mice aged 9 months were fed a diet containing 2 g DHA-fortified Chlorella oil fraction/100 g diet or normal diet (Control group) for 2 months. Two months after the start of feeding, the mice were tested for learning ability related to 2 types of memory, reference memory and working memory, with the partially (4 of 8) baited eight-arm radial maze. Reference memory is a kind of information that should be retained until the next trial. Working memory is a kind of information that disappears in a short time. Entry into the unbaited arms and repeated entry into the visited arms were defined as reference memory errors and working memory errors, respectively. DHA-fortified Chlorella oil fraction administration to mice for 2 months resulted in a significant decrease in the number of working memory errors without affecting the number of reference memory errors. A significant increase in the DHA content in the brain was also observed. These results suggest that the intake of DHA-fortified Chlorella oil fraction effectively enhances working memory in maze performance.
The suppressive effect of flavonoids on the cytotoxicity of linoleic acid hydroperoxide (LOOH) toward rat phenochromocytoma PC12 cells was examined. The extent of cytotoxicity was shown on the basis of % survival determined by the trypan blue exclusion test. On preincubation of cells with either 3-hydroxyflavone, quercetin, or luteolin prior to LOOH exposure, the cytotoxicity was considerably suppressed. In contrast, on coincubation of cells with either eriodictyol, quercetin, kaempherol, luteolin, or 3-hydroxyflavone and LOOH, it was markedly suppressed. Regardless of incubation conditions, quercetin, 3-hydroxyflavone, and luteolin were thus more effective as protective agents against the cytotoxicity than the other flavonoids. These flavonoids further showed a suppressive effect on coincubation rather than on preincubation. These results suggest that such flavonoids are beneficial for cells under oxidative stress.
The aqueous leaf extract of rinbacin was tested for toxic effects on prepubertal rat kidneys following chronic administration. Two doses of rinbacin extract (26.25 g/l and 52.50 g/l) were administered in the rats' drinking water for 13 weeks, and various toxicologic indices tested. Histological study of the kidneys was also carried out at the expiration of the test period. Rinbacin at both dose sizes significantly (p<0.05) increased the absolute and relative kidney weights. Also the serum HCO3− level was significantly (p<0.05) increased, while the serum K+ level was decreased significantly at both dose levels. Only the high dose significantly (p<0.05) increased the serum urea level of the rats. Histological study showed that rinbacin at both dose sizes caused renal pathologic changes, which included necrosis and cellular infiltration of glomeruli and epithelia of the tubules. The effects were less marked in the low dose than the high dose group. Chronic administration of rinbacin extract induces nephrotoxicity in young rats.
Chikusaku-eki is an acidic dark brown liquid obtained as a by-product from bamboo charcoal burners. The solution diluted with water is gaining widespread popularity in Japan as a folk medicine for skin diseases such as scabies, eczema, and atopic dermatitis. In this study, the carcinogenic and tumor-promoting potential of chikusaku-eki was determined using the BALB/c 3T3 A31-1-1 cell transformation system. Carcinogenic activity was tested by treating A31-1-1 cells for 24 h with 0.06% solution, a dose resulting in 35% clonogenic cell survival. In both 2-O-tetradecanoylphorbol-13-acetate (TPA)-treated and non-treated groups, chikusaku-eki did not initiate carsinogenesis. Following initiation with 3-methylcholanthrene (3-MCA), A31-1-1 cells were chronically treated with a non-toxic concentration range of chikusaku-eki (≤0.01%), but chikusaku-eki did not act as a tumor promoter. Thus, chikusaku-eki was not carcinogenic/co-carcinogenic in the in vitro cell transformation assay examined in this study after being diluted more than 104-fold with water.
Indenopyridine hydrochloride (IH), an antispermatogenic agent, was tested to determine the testicular pathological changes, seminal spermatozoa concentrations and seminal plasma alkaline phosphatase levels in male dogs. A single oral dosage of 30 mg IH/kg BW induced the dissociation and premature release of germ cells into the lumens of seminiferous tubules. Ring-shaped spermatid nuclei, nuclear pykonosis of spermatocytes and multinucleated cell associations were also observed. Thereafter, the spermatogenic index (SI) significantly decreased one day after IH administration. Moreover, seminal spermatozoa concentrations decreased two weeks after drug treatment; and there was a statistically significant difference in spermatozoa production inhibited by IH compared to the control. Reversible spermatogenesis was noted 7 weeks after IH treatment in male dogs. Meanwhile, seminal plasma alkaline phosphatase levels also significantly increased two weeks after IH treatment. These data confirm that IH might induce a two-month inhibition of spermatogenesis in male dogs.
We previously reported that (+)-matrine and (+)-allomatrine have antinociceptive properties mediated mainly through the activation of κ-opioid receptors. 1-Acyl-4-dialkylaminopiperidines were synthesized as the simplest derivatives of matrine, and the structure–activity relations were examined by the acetic acid-induced abdominal contraction test. The antinociceptive potencies of 1-alkyl-4-dialkylaminopiperidines were significantly lower than those of the corresponding 1-acyl-4-dialkylaminopiperidines. These findings suggest that the amide group of (+)-matrine is an essential functional group that influences antinociceptive potency.
We tested the constituents of two Rhodiola plants, Rhodiola sacra S. H. FU and R. sachalinensis A. BOR, and an Oriental crude drug, Tokaku-joki-to, for their neuroprotective effects. Of the 58 compounds tested, six had considerable protective effects against beta-amyloid-induced death of B103 neuronal cells in vitro. These six compounds also showed protective effects against staurosporine-induced cell death, and two of the six compounds protected neurons from H2O2-induced cell death. These results suggest that some of the tested compounds protect neurons from beta-amyloid toxicity based on antiapoptotic and antioxidative activity.
Two pairs of diagnostic primers, IHm01-L/IHm01-H and IHm02-L/IHm02-H, for distinguishing the Chinese crude drug Oviductus Ranae from its substitutes were designed based on sequences of Cyt b gene fragment of the original animals of the drug and substitutes. Total DNAs were extracted from crude drugs purchased from five drugstores in different regions, as well as from original animals of the drug, Rana chensinensis, and seven species of related ranid species. Diagnostic polymerase chain reactions (PCRs) were performed using the two pairs of primers with the total DNAs of the original animals as a template. The result showed that a 240 bp DNA segment was clearly amplified from all templates of Rana chensinensis using primers IHm01-L and IHm01-H, whereas no DNA band appeared from other templates. While using primers IHm02-L and IHm02-H, we got a clear 140 bp DNA band from all the templates of R. huanrenensis and 3 oviducts of the same species, no PCR product was observed from the other samples. A set of PCR reactions was employed to identify crude drugs from the five drugstores using the two pairs of primers together with HsmL1 and HsmH1 reported in our previous study. The results show that only 20% of the Oviductus Ranae currently sold in markets are qualified products and the rest are not.
We screened the differentiation-inducing activities of 39 mushroom extracts from Akita prefecture, Japan, on the mouse osteoblastic cell line, MC3T3-E1. Sixteen phosphate buffered saline (PBS), 8 boiled PBS, 14 ethanol and 12 methanol extracts induced alkaline phosphatase (ALP) activities, an indicator of MC3T3-E1 cell differentiation. The enzyme activities were markedly induced by extracts of Tricholoma auratum, and we isolated the active compound from methanol extracts of this mushroom. Physical data for the isolated active compound were identical to those for (22E,24R)-ergosta-7,22-diene-3β,5α,6β-triol (1). 1 induced ALP activities of MC3T3-E1 cells and promoted cell proliferation. To investigate the relationships between the chemical structure and differentiation-inducing activity of the compound, ALP-inducing activities of MC3T3-E1 cells by 1, ergosterol (2), ergocalciferol (3), cholesta-3β,5α,6β-triol (4), 7-dehydrocholesterol (5) and cholecalciferol (6) were tested. The enzyme activities of MC3T3-E1 cells were increased 3.0-fold by 10 μM 1 and 2.4-fold by 10 μM 4. However, 2, 3, 5 and 6 did not induce MC3T3-E1 cell ALP activity at 0.1—10 μM. These results suggested that the OH groups at C-5 and/or C-6 of 1 and 4 played an important role in their differentiation-inducing activities on MC3T3-E1 cells. Furthermore, 1 suppressed induction of MC3T3-E1 cell apoptosis by serum starvation.
The current study was carried out to investigate the in vitro effects of an 85% methanol extract of dried Morus alba leaves on melanin biosynthesis, which is closely related to hyperpigmentation. These extracts inhibited the tyrosinase activity that converts dopa to dopachrome in the biosynthetic process of melanin. Mulberroside F (moracin M-6,3′-di-O-β-D-glucopyranoside), which was obtained after the bioactivity-guided fractionation of the extracts, showed inhibitory effects on tyrosinase activity and on the melanin formation of melan-a cells. This compound also exhibited superoxide scavenging activity that is involved in the protection against auto-oxidation. But its activity was low and was weaker than of kojic acid. These results suggest that mulberroside F isolated from mulberry leaves might be used as a skin whitening agent.
Prolyl endopeptidase (PEP, EC 188.8.131.52) has been proposed to play a role in degradation of proline-containing neuropeptides involved in the processes of learning and memory, e.g., vasopressin, substance P, and thyrotropin-releasing hormone (TRH). In the course of our search for bioactive constituents in medicinal plants, we studied the PEP inhibitory constituents of the roots of Lindera strychnifolia F. VILL and isolated two known tannins, epicatechin (1) and aesculitannin B (2), and four known sesquiterpenes, linderene (3), linderene acetate (4), linderalactone (5) and isolinderalactone (6) as inhibitors. On the inhibitory activities of six compounds against PEP from Flavobacterium meningosepticum and that from rat brain supernatant, compounds 1, 2 and 4 inhibited the enzyme from Flavobacterium more strongly than that from rat brain supernatant. However, compounds 3, 5 and 6 inhibited the enzymes from both origins to the same extent and furthermore, compound 6 was the strongest natural inhibitor against PEP from rat brain supernatant. The kinetic study of these inhibitors indicated that compounds 1, 2 are noncompetitive inhibitors and compounds 3—6 are competitive inhibitors. This is the first example of non-phenolic constituents showing significant competitive inhibitory activity being isolated from natural medicines.
Eriobotrya japonica is considered a medicinal plant, and its leaves (Eriobotrya folia) have been used to treat skin diseases, as well as to relieve inflammation, pain, coughing, and sputa. In our evaluation of the pharmacological efficacy of the seed extracts, constituents of the seeds were found to contain the unsaturated fatty acids linolenic and linoleic acids and the sterol β-sitosterol in the 70% EtOH and the MeOH extracts. The seed extracts were orally administered to rats with dimethylnitrosamine-induced hepatopathy, and blood L-asparate aminotransferase (AST) and L-alanine aminotransferase (ALT) levels, liver retinoid level, and hydroxyproline level were measured. Liver fibrosis rates calculated after Azan-Mallory staining and evaluation of the liver function-improving effects of extracts were showed that AST, ALT, and hydroxyproline levels and liver fibrosis rates were significantly lower, and retinoid levels were significantly higher in hepatopathic rats treated with 70% EtOH and MeOH extracts of the seed than in water-treated control rats. This suggests that the positive effect on liver function of the extracts varies depending on the extracting solvent used. 70% EtOH and MeOH extract of the seeds inhibited the development of liver fibrosis in hepatopathic rats, thus exhibiting potent improvement. The unsaturated linolenic and linoleic acids and the sterol β-sitosterol contained in these extracts may also contribute to the improvement of liver function.
We examined the inhibitory effects of oleanane-type triterpenoids from fabaceous plants on the TNF-α-induced expression of cell adhesion molecules on THP-1 human monocytic leukemia cells and compared them with a glucocorticoid dexamethasone. In a cell-based ELISA, abrisapogenol E, soyasapogenols B and C, soyasapogenol B, kuzusapogenol B-methyl ester, and oleanolic acid significantly inhibited intercellular adhesion molecule (ICAM-1) expression. Moreover, these triterpenoids showed the same activity as dexamethasone. On the other hand, the absence of hydroxyl group at C-24 position of sapogenin rather to increase ICAM-1 expression compared with the untreated control. We concluded that the activity of oleanane saponins and sapogenins against ICAM-1 expression are dependent upon the position of the hydroxyl group, and in particular upon the status of the C-21 and C-24 positions and of the glycosyl group at C-3 position.
Chinese medicinal prescriptions were screened for their effects on α-amylase activity in mouse plasma. Daio-kanzo-to, Hange-koboku-to, Saiko-ka-ryukotsu-borei-to, Saiko-keishi-kankyo-to and Mao-to changed the activity in isolated mouse plasma greatly and concentration dependently. However, no prescription influenced the elevation of postprandial blood glucose.
Sulfapyridine (SP), one of the metabolites of sulfasalazine (SASP), is further metabolized into N-acetylsulfapyridine (AcSP) by polymorphic N-acetyltransferase 2 (NAT2). NAT2 activity has been diagnosed by phenotyping, that is, evaluating plasma concentrations or urinary excretions of tentatively administered test drugs for dose individualization and avoidance of serious adverse events. Herein, we investigated the relationship between NAT2 genotypes and the pharmacokinetics of SP in healthy Japanese subjects, as well as the adverse events of SASP in patients with inflammatory bowel disease (IBD). Eight healthy subjects and 13 IBD patients were classified into three groups by NAT2 genotyping; the homozygote for the wild-type allele (Rapid Types), the compound heterozygote for the wild-type and mutant alleles (Intermediate Types), and the homozygote for mutant alleles (Slow Types). A single oral dose of 40 mg/kg SASP was administered to each healthy subject, and plasma and urine samples were taken until 51 and 72 h after administration, respectively. Both the SP and AcSP concentrations in each sample were determined by the HPLC method. The NAT2 genotypes were well-correlated with the plasma concentrations or urinary excretions of SP and AcSP in 8 healthy subjects, except for one Slow Type. In patients with IBD, skin rash was seen in 3 of 6 Rapid Types and 1 of 6 Intermediate Types, consistent with the concept that hypersensitive reactions are independent of serum SP concentrations. In contrast, SASP dosing-related acute pancreatitis was found in the Slow Type patient. In this case, the NAT2 activity was diagnosed by genotyping in advance, and the medical staff could pay scrupulous attention, resulting in no serious subjective symptoms such as abdominal pain, anorexia or fever. Further investigations on the relationship between the NAT2 genotype and adverse events are required, although genotyping appeared to be a promising method to avoid such serious adverse events.
The effects of SK-896, a new human motilin analogue ([Leu13]motilin-Hse), on digestive tract motility in postoperative ileus were evaluated in a dog model of ileus after laparotomy. SK-896 was intravenously administered at 0.17, 0.33 and 0.67 μg/kg starting soon after operation and then at 6-h intervals, for a total of 9 times. SK-896 progressively, dose-dependently and significantly increased the duodenal motility from 1 h after operation. The recovery time of the gastrointestinal-interdigestive migrating complex (GI-IMC) activity, which is an indicator of normal gastrointestinal tract activity after laparotomy, was 56.5±5.0 h in the control group. SK-896 significantly shortened this recovery time. On the other hand, the plasma SK-896 concentrations declined diexponentially after administration, and can be described by a linear pharmacokinetic model within the dose range used. In addition, the pharmacokinetics of SK-896 did not change significantly at any postoperative time. There was no correlation between the plasma SK-896 concentrations and the intensity of duodenal motility, because the activity in the duodenum decreased transiently 13 h after laparotomy and increased with time thereafter. The changes in the activity are considered to reflect the progressive changes in the state of ileus. In conclusion, SK-896 increased the duodenal motility significantly, shortening the recovery time of GI-IMC-like activity in dogs with post-laparotomy ileus. Therefore, it is expected from these results that SK-896 would be useful and effective for the treatment of gastroparalysis after abdominal surgery.
The purpose of this study was to modify the biodistribution and pharmacokinetics of tilisolol, a β-blocker, using the palmitoyl prodrug approach. After intravenous administration of tilisolol and O-palmitoyl tilisolol in rats, drug concentrations were determined in blood, bile, urine, and several tissues. The concentration-time profiles of tilisolol and O-palmitoyl tilisolol were analyzed pharmacokinetically. The blood concentrations of O-palmitoyl tilisolol after intravenous administration of O-palmitoyl tilisolol were about 10-fold higher than those of tilisolol after intravenous administration of tilisolol. The biliary excretion rates of O-palmitoyl tilisolol and tilisolol after intravenous administration of O-palmitoyl tilisolol were about 10- to 100-fold larger than those of tilisolol after intravenous administration of tilisolol. In addition, the hepatic uptake clearance of O-palmitoyl tilisolol after intravenous administration of O-palmitoyl tilisolol was 3.6-fold higher than that of tilisolol after the intravenous administration of tilisolol. In the in vitro experiments, it was demonstrated that the distribution ratios between blood cells and plasma (blood/plasma) of O-palmitoyl tilisolol and tilisolol was 95.7 and 55.5%, respectively. These findings suggest that O-palmitoyl tilisolol exists as a binding form with biological components, especially blood cells, in systemic circulation. In conclusion, the palmitoyl prodrug approach is useful as a drug delivery system to deliver the parent drug to the liver.
The purpose of this investigation was to determine whether the pharmacokinetics of the angiotensin II receptor antagonist losartan is altered in renal failure. Male Wistar rats were pretreated with uranyl nitrate or subjected to bilateral ureteral ligation to produce acute renal failure (ARF). Saline-injected and sham-operated rats, respectively, served as controls. Uranyl nitrate-treated rats showed significantly higher serum concentrations of losartan after oral administration and the area under the serum concentration–time curve (AUC0—24) of losartan increased about 3-fold compared to control rats. The systemic clearance of losartan significantly decreased from 410±254 ml/h/kg in control to 177±112 ml/h/kg in uranyl nitrate-treated rats. In order to investigate the mechanisms of reduced clearance of losartan associated with ARF, a hepatic microsome fraction was prepared from normal and ARF rats. No significant difference was found in the metabolism of losartan by hepatic microsomes prepared from ARF and control rats. In addition, the metabolic activity of microsomes was examined in the presence of uremic rat serum. The unbound clearance of losartan and the unbound clearance associated with the formation of EXP3174 in the presence of uremic serum were significantly lower than those in the presence of control serum. Furthermore, the metabolism of losartan was inhibited by indoxyl sulfate, a uremic toxin, in an uncompetitive manner. These results suggest that ARF is associated with reduced clearance of losartan due to the inhibition of hepatic metabolism by accumulated uremic toxin(s).
As a basic approach to identifying the distribution mechanism of quinolone antibiotics into saliva, salivary excretion of five fluoroquinolones, ciprofloxacin (CPFX), norfloxacin (NFLX), lomefloxacin (LFLX), ofloxacin (OFLX) and sparfloxacin (SPFX), was compared in rats. Blood, parotid and mandibular saliva were periodically collected from the anesthetized rats after bolus i.v. administration (10 mg/kg) of the quinolones. Quantification of the fluoroquinolones was performed by HPLC methods. The saliva-to-plasma unbound concentration (S/Pu) ratios of the fluoroquinolones in parotid saliva were larger than those of mandibular saliva. These five quinolones had considerably different S/Pu ratios from 0.014 to 1.497, while the S/Pu ratios theoretically calculated by the pH-partition theory were around 1.0 to 1.3, which showed no relationship to the corresponding measured ratios. Satisfactory linear correlations were observed in the plots of measured S/Pu ratios against 1-octanol–water partition coefficients of the fluoroquinolones in both types of saliva. These results indicate that fluoroquinolones possess different diffusibility in salivary distribution among the drugs and between parotid and mandibular glands. It was also clarified that the lipophilicity of the fluoroquinolones primarily determines the extent of salivary excretion.
The effects of probucol, a cholesterol-lowering agent, on several cytochrome P450 (CYP) isoform-specific reactions in human liver microsomes were investigated to predict drug interactions with probucol in vivo from in vitro data. The following eight CYP catalytic reactions were used in this study: CYP1A1/2-mediated 7-ethoxyresorufin O-deethylation, CYP2A6-mediated coumarin 7-hydroxylation, CYP2B6-mediated 7-benzyloxyresorufin O-debenzylation, CYP2C8/9-mediated tolbutamide methylhydroxylation, CYP2C19-mediated S-mephenytoin 4′-hydroxylation, CYP2D6-mediated bufuralol 1′-hydroxylation, CYP2E1-mediated chlorzoxazone 6-hydroxylation, and CYP3A4-mediated testosterone 6β-hydroxylation. Probucol had neither stimulatory nor inhibitory effects on CYP1A1/2, 2A6, 2B6, 2C8/9, 2C19, 2D6, 2E1, and 3A4 activities at concentrations up to 300 μM, indicating that probucol, at the expected therapeutic concentrations, would not be predicted to cause clinically significant interactions with other CYP-metabolized drugs.
Glucocorticoid regulates various physiological processes via the activation and repression of gene expression. The anti-inflammatory effects and the adverse effects are believed to be dependent on the repression and the activation of genes, respectively. Reporter gene assay is a useful technique to separately evaluate these two functions and has been used for in vitro screening of novel ligands for the glucocorticoid receptor (GR). We report here the application of a reporter gene assay for the in vivo determination of the GR-mediated gene activation. A reporter plasmid containing glucocorticoid response elements was introduced to abdominal mouse skin using a gene gun. Administration of prednisolone induced the expression of the reporter gene, only when the GR expression plasmid was co-transfected with the reporter plasmid. Endogenous levels of corticosterone appeared to be negligible in this protocol. The dose response for this induction was comparable to those for the decreases in thymus weight and serum corticosterone. These results suggest that gene gun-mediated skin transfection enables the in vivo reporter gene assay and that this technique can be used to predict the potency of ligands for the GR-mediated gene activation.
The difference in the blood concentration of tacrolimus between the assay methods, microparticle enzyme immunoassay (MEIA) and enzyme linked immunosorbent assay (ELISA) was observed in a liver transplant recipient with anemia. MEIA provided significantly higher concentration than those of ELISA (7.8±1.9 vs. 5.0±1.8 ng/ml, p<0.05) while the patient had low haematocrit <25%. The difference, however, was not observed during the periods with haematocrit >25%. This observation suggested that unknown tacrolimus levels generated from difference in assay methods gave incorrect blood tacrolimus during anemia. False positive concentration of tacrolimus ranging 0.1—3.3 ng/ml was observed in MEIA applying to the blood samples obtained from the patients without receiving tacrolimus. The false positive tacrolimus increased in the samples with lower hematocrit, suggesting that MEIA gave incorrect blood tacrolimus during anemia. Since MEIA potentially overestimates the tacrolimus levels, ELISA should be used for blood tacrolimus monitoring in the patients with anemia.