A novel calcium-chelating agent, N"-ursodeoxycholyldiethylenetriamine-N, N, N'-triacetic acid (UDCA-DTTA), was synthesized to study its ability to dissolve calcified gallstones. The chelating activity of the compound was demonstrated by dissolving calcium carbonate in vitro at a high dissolution rate. In the presence of the agent, sliced human gallstone with a composition of more than 50% calcium bilirubinate was thoroughly dissolved, indicating that calcium bilirubinate was dissolved from the gallstone. The ability to dissolve calcium was comparable to that of EDTA. However, the laminar structure of the sliced gallstone did not disappear in the presence of EDTA, whereas the structure disappeared in the presence of UDCA-DTTA. All these results indicate that UDCA-DTTA is an interesting compound as a parent substance for developing a prodrug for an oral or intravenous agent to dissolve calcium-containing gallstones.
The fatty acid composition of rat polymorphonuclear leukocytes (PMN) was modified by diets supplemented with a high linoleate (LA) safflower oil (76% LA), mixtures of eicosapentaenoate (EPA) and safflower oil (EPA(20) containing 20% EPA and 61% LA, EPA(40) containing 40% EPA and 46% LA), mixtures of docosahexaenoate (DHA) and safflower oil (DHA(20) containing 20% DHA and 61% LA, DHA(40) containing 40% DHA and 46% LA) or a high α-linolenate (α-LNA)perilla oil (57% α-LNA and 13% LA), and then lipid mediator production in casein-induced peritoneal PMN were compared. EPA and DHA were relatively ineffective in reducing platelet-activating factor (PAF) production; a statistically significant reduction was observed only in the DHA(40) group. In contrast, perilla oil reduced PAF production by 50% as compared with safflower oil. Arachidonate (AA) in the PAF precursor, 1-alkyl-2-acyl-glycerophosphocholine, was roughly correlated with PAF production, but EPA and DHA in the precursor lipid were relatively unrelated. On the other hand, both PGE2 and LTB4 production correlated positively with AA and negatively with EPA and DHA in PMN phospholipids; EPA tended to be somewhat more effective than DHA in reducing PGE2 and LTB4 formation; the activity of perilla oil was no less than EPA(20). Thus, replacing safflower oil with perilla oil was no less effective than supplementing safflower oil with EPA or DHA (at 40% of total fatty acids) in reducing lipid mediator production in rat PMN.
In order to investigate the mechanisms of body temperature change from a thermodynamic aspect, we investigated the specific gravity, specific heat and surface area of rabbit in situ. We obtained the following results.1. The specific gravity of normal, shorn and ecdysed rabbits is 0.94, 0.95 and 0.97, respectively. 2. The specific heat of normal, shorn and ecdysed rabbits is 0.95, 0.89 and 0.69, respectively. 3. The surface area of the rabbit was also measured by using the weight of aluminum foil which covered the surface.
A 5-fluorouracil (5-FU)-resistant subline, DLD-1/5-FU, was established by repeated 5-d exposures of human colon cancer DLD-1 cells to 5-FU. DLD-1/5-FU cells were 41- and more than 75-fold resistant to 96-h and 1-h exposures to 5-FU, respectively. When exposed to 5-FU, DLD-1/5-FU cells exhibited marked resistance to in situ thymidylate synthase (TS) inhibition by 5-FU as compared to DLD-1 cells, and incorporation of 5-FU into cellular RNA in DLD-1/5-FU cells decreased to 25% of that in DLD-1 cells. As causes of resistance to DNA and RNA-directed actions of 5-FU, remarkable reduction of intracellular levels of both 5-fluorouridine 5'-triphosphate (FUTP) and 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) in DLD-1/5-FU cells was confirmed. It was found that activities of uridine kinase, orotate phosphoribosyltransferase and thymidine kinase of DLD-1/5-FU cells were significantly lower than those of the parent cells. Intracellular levels of TS were similar between the two cell lines. These results indicated that the mechanism of resistance to 5-FU in DLD-1/5-FU cells involves reduced enzymatic activation of 5-FU.
By means of glycyrrhizin (GL)-affinity and Mono S column chromatographies (HPLC), at least four GL-binding proteins (p25, p17, p15-1 and p15-2) in the two Superdex fractions (P-II and P-III fractions) from Habu snake venom were selectively purified. By determination of their N-terminal partial amino acid sequences, a metalloprotease (p25) and three GL-binding phospholipases A2 (gbPLA2s) [PA2Y (p17), PA21 (p15-1) and PA2B (p15-2)] were identified. PA2B (lysine-49 PLA2) was found to be the most sensitive to GL because (i) it strongly bound to a GL-affinity column; and (ii) its enzyme activity was selectively inhibited by low dose (ID50=approx. 1.5μM) of GL, but not by GA. Furthermore, these three gbPLA2s were phosphorylated by casein kinase II (CK-II) in vitro and GL inhibited the CK-II-mediated stimulation of their enzyme activities in vitro.
The co-genotoxic effect of di-(2-ethylhexyl)phthalate (DEHP) was studied in an in vivo Drosophila genotoxicity test. DEHP is categorized as a non-genotoxic carcinogen. In this study, DEHP also did not show genotoxicity in Drosophila. When larvae were simultaneously treated with DEHP and N-nitrosodimethylamine (NDMA), the DNA damaging activity of NDMA was increased in the Drosophila DNA repair test. Furthermore, DNA double strand breaks were increased by the same treatment of Drosophila. These results suggest that DNA double strand breaks cause the co-genotoxic effect of DEHP in Drosophila.
We investigated the mechanism of the trypanocidal activity of gallic acid (GA). GA-induced trypanocidal activity was significantly reduced by pretreatment with superoxide dismutase (SOD) and/or catalase. The ESR technique with 5, 5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trapping agent revealed that a DMPO-OH adduct was detected in culture medium containing GA. The intensity of ESR signals of the DMPO-OH adduct was increased in a time dependent manner. SOD also inhibited the formation of GA-induced DMPO-OH adducts. Furthermore, GA enhanced DNA single-strand breaks induced by Fenton reagent. These results suggest the possibility that GA acts as a pro-oxidant for trypanocidal activity.
The metabolic fate of saikosaponin b1 (1) was investigated using conventional, germ-free and Eubacterium sp. A-44-infected gnotobiote rats. After the oral administration of 1 to germ-free rats at a dose of 50mg/kg, no metabolite was detected in the plasma, the cecal contents or the cumulative feces through the experiment. On the other hand, when 1 was orally given to the Eubacterium sp. A-44-infected gnotobiote rats, considerable amounts of its metabolites, prosaikogenin A (2) and saikogenin A (3), were detected in the rat plasma with the respective AUC0-10h values of 17 424 and 22 260pmol·min/ml, similar to the case of its oral administration to conventional rats (AUC0-10h values of 9 936 and 12 414pmol·min/ml for 2 and 3, respectively). Furthermore, significant amounts of both metabolites were detected in the cecal contents and the cumulative feces of the gnotobiote and conventional rats, but not in those of the germ-free rats, within 10h after the administration.Fecal and cecal activities of hydrolyzing 1 and 2 were found in the gnotobiote and conventional rats, though there were no detectable activities in the germ-free rats. Accordingly, both hydrolyzing activities in the intestinal bacteria, such as Eubacterium sp. A-44, are essential for the appearance of 2 and 3 in the rat plasma and cumulative feces, since orally administered 1 was poorly absorbed from the gastrointestinal tract.
We previously found that a methanolic extract of the stems of Sambucus sieboldiana inhibited bone resorption in organ culture. In this study, we further fractionated the methanol extract guided by the activity towards bone resorption stimulated by parathyroid hormone (PTH) in vitro. The ethyl acetate fraction (EtOAc Fr.) of the methanolic extract inhibited PTH-stimulated bone resorption of neonatal mouse bones, and the inhibitory activity was more potent than those of other fractions. Oral administration of the EtOAc Fr. (50 and 100 mg/kg/d) to ovariectomized (OVX) rat prevented the decrease in bone mineral density (BMD) of the lumbar (L2-4) vertebra, indicating that the EtOAc Fr. is effective in vivo. Furthermore, the EtOAc Fr. (50, 100 and 150 mg/kg/d) decreased the serum calcium level elevated in low calcium dietary rats. The phenolic constituents of the EtOAc fraction were examined for their inhibitory effect on bone resorption stimulated by PTH in neonatal mouse bone. Among them, vanillic acid, vanillin and coniferyl alcohol showed significant inhibitory effects on bone resorption. Of the compounds examined, vanillic acid was found to have a significant inhibitory effect on the decrease of BMD in OVX mice. Therefore, the EtOAc Fr. of S. sieboldiana showed a suppressive effect on bone resorption in vitro and in vivo. In addition, the inhibitory effects of the EtOAc Fr. on bone resorption may be at least partly due to the inhibitory action of vanillic acid.
The apparent octanol/water partition coefficient (APC) of salicylate (SA) increased as the concentration of alkylamine (amyl, hexyl, heptyl, octyl and nonylamine) in aqueous phase increased, presumably through intermolecular ion pair formation between the negatively charged SA moiety and the alkylamine cation. The true partition coefficient (TPC) and the formation constant (Kf) of the ion pair were calculated from the partition data. The skin permeability of SA increased as the APC of SA increased, when 20-fold molar excess of alkylamine was added to the donor compartment. Permeability of ion pairs (PAB) from the aqueous phase to a shed snake skin was estimated from the permeability data assuming 1 : 1 ion pair. The methylene group contribution to the free energy of transfer of ion pairs from water to the shed snake skin was less than the reported value for nonionized drugs. This suggests that the ion pair is more polar by nature than nonionized molecules, even if ionic characteristics are masked to some extent by ion pair formation.
We evaluated the dose-dependent (saturable) gastrointestinal absorption of cefatrizine, an aminocephalosporin transported by peptide carriers, in rats by a physiological mechanism-based approach to clarify its absorption characteristics and to examine the in vitro (in situ)-in vivo correlation in intestinal transport. With an increase in oral dose (μmol/5 ml/kg) from 5 (low) to 50 (high), the intestinal absorption rate constant (ka), which was estimated by analysis of gastrointestinal disposition, decreased markedly, from 0.301 to 0.056min-1. This decrease was ascribable to the saturability of intestinal membrane transport, of which the concentration dependency in the perfused intestine was similar in extent to the dose dependency in ka. However, the apparent absorption rate constant (ka'), which was estimated by analysis of plasma concentrations after oral administration, decreased only modestly from 0.037 to 0.023 min-1. This was associated with the result that, at the low dose, ka' was far smaller than ka and comparable with kg (gastric emptying rate constant), suggesting gastric emptying-limited absorption. At the high dose, where intestinal cefatrizine absorption was less efficient, ka' was closer to ka than kg. It was also observed that the bioavailability was close to unity, independent of dose, suggesting that the intestinal transit time is long enough to achieve complete absorption, even at the high dose, where intestinal cefatrizine absorption is less efficient. Thus, it was found that the effect of saturability in the intestinal transport of cefatrizine is apparently attenuated in its overall gastrointestinal absorption because of the involvement of gastric emptying and intestinal transit time as additional physiological factors to define absorption. It was also found that a scaling factor is required to correlate the intestinal membrane transport between in vitro (in situ) and in vivo, though this remains to be verified to be utilized for developing oral drug delivery strategies and optimizing oral drug therapy.
Tacrolimus, an immunosuppressive agent, is metabolized mainly in the liver and has shown large intra- and interindividual pharmacokinetic variability. We investigated the effect of liver dysfunctions on the pharmacokinetics of tacrolimus in rats with experimental liver diseases. Experimental hepatic failure was induced by CCl4-treatment or bile duct ligation. Tacrolimus (1 or 0.3 mg/kg) was administered intravenously or intraportally to the rats (n=5-6 per group), and blood samples were collected over a 240-min period. The tacrolimus concentrations in the blood were then measured by a high-performance liquid chromatography-enzyme immunoassay. In the normal rats, the hepatic extraction ratio of tacrolimus (EH) was dose-independent, ranging from 0.566-0.598 at 0.3 and 1.0 mg/kg doses. The EH were dose-dependent in the CCl4-treated rats and in the bile duct-ligated rats : the EH at 1.0 mg/kg dose were 0.158-0.170 and those at 0.3 mg/kg dose were 0.329-0.394. The intermediate EH of tacrolimus suggested that the clearance of tacrolimus depends not only on hepatic intrinsic clearance but also on hepatic blood flow. The present pharmacokinetic study also suggested that the decrease of EH and the dose-dependence of EH contribute to the elevation of blood tacrolimus concentrations and to the large variability in the pharmacokinetics of tacrolimus after oral administration in hepatic dysfunctions.
We studied the enhancing and toxic effects of five different absorption enhancers on the transport of FITC-dextran with an average molecular weight of 4000 (FD-4) across Caco-2 cell monolayers, and their enhancing effects were also compared with those in rat intestine. The enhancing and cytotoxic properties of these enhancers were characterized using the following tests : measurement of the permeability coefficients of FD-4 and the transepithelial electrical resistance (TEER) in Caco-2, the release of cytosolic lactate dehydrogenase (LDH) and intracellular mitochondrial dehydrogenase (MDH) activity. All the absorption enhancers increased the permeability of FD-4 across Caco-2 cell monolayers and a good relationship was observed between the enhancement and their toxic effects. However, EDTA and Na-Cap were effective for improving the transport of FD-4 across Caco-2 cells without serious cytotoxicity. At concentrations with low cytotoxicity, various absorption enhancers exihibited reversible effects on the TEER values in Caco-2 cell monolayers, except for 50mM sodium salicylate (Na-Sal). Moreover, we obtained a good correlation between the enhancement of these enhancers in Caco-2 cell monolayers and in rat large intestine. This finding indicated that the effectiveness of absorption enhancers in the Caco-2 monolayer system was similar to an in vivo rat system. Therefore, the screening system using Caco-2 cell monolayers is useful for examining the effectiveness and toxicity of absorption enhancers.
Viral inactivation in superoxide dismutase (SOD) derived from human red cells was carried out by ultraviolet light C (UVC) irradiation. With 400J/m2 UVC irradiation, the titer of canine parvovirus (CPV, a nonenveloped virus), M13 bacteriophage (M13, a nonenveloped phage) and vesicular stomatitis virus (VSV, an enveloped virus), which were spiked into SOD solution, were reduced by >4.6log10 (detection limit), 7.0log10 and 6.2log10, respectively. The SOD activity was maintained and the band pattern of SOD on an electrophoresis gel was not changed even by 1000J/m2 UVC irradiation. These results indicate that UVC irradiation is a promising method for the inactivation of both enveloped and nonenveloped viruses in SOD preparations while maintaining its activity.
An unknown adenine-related compound (UKC) in rat liver mitochondria was characterized. Based on the sensitivity to periodate oxidation, nuclease P1 digestion, property of fluorescence derivatization, elution behavior on different separation modes of HPLC columns and the mass spectrum of purified UKC, the UKC was identified as adenosine 3'-monophosphate (3'-AMP), an intracellular P-site inhibitor of adenylate cyclase. 3'-AMP may be enzymatically produced from RNA in rat liver mitochondria in temperature- and time-dependent manners. A partial characterization of 3'-AMP forming enzyme is included.
When puerarin or daidzin were incubated for 24h with human intestinal bacteria, two metabolites, daidzein and calycosin, were produced from them, respectively. The metabolic time course of puerarin was as follows : at an early time, puerarin was converted to daidzin, and then calycosin. The metabolic time course of daidzin by human intestinal bacteria was also similar to that of puerarin. The in vitro cytotoxicities of these metabolites, calycosin and dadizein, were superior to those of puerarin and daidzein.
Mammalian small heat shock protein (s-hsp) has been suggested to participate not only in stress tolerance but also in the growth regulation and differentiation of cells. To confirm the role of s-hsp in cell growth, we investigated the relationship between the expression of hsp26 and yeast cell growth. Cells lacking constitutive hsp70, ssa1ssa2, have been known to have a poor growth rate and to over-express hsp26 and some other hsps. We obtained several cell clones of ssa1ssa2 whose doubling times were different from one another. The amount of hsp26 was closely linked to the doubling time of ssa1ssa2 cells. This result suggests that the expression of hsp26 modulates the growth rate of yeast cells lacking constitutive hsp70, similarly to mammalian cells.
Recombinant RNase LE from tomato and squid liver RNase Tp, typical plant/animal type RNases belonging to the RNase T2 family, were subjected to limited digestion with several proteases, and the cleavage sites were analyzed by Edman degradation. Recombinant RNase LE was cleaved specifically at the 24th Lys by lysylen-dopeptidase and trypsin, and RNase Tp was cleaved at the 21st Glu by V8 protease. These cleavage sites are located very close to those where the cleavage during preparation of several animal RNase T2 family enzymes was observed. From this finding, it was concluded that the short segment around the 20th amino acid residue in plant/animal RNases in located on the surface of the molecules and forms loops, and is thus very sensitive to proteases.
We investigated the in vivo effects of thalidomide on the production of tumor necrosis factor-α(TNF-α). An in vivo systemic release of TNF-αoccurred after the injection of lipopolysaccharide (LPS) in male ddY mice, and the TNF-α serum levels reached 652.2±75.7 pg/ml 90 min after the injection of LPS (0.3 mg/kg, i. p.). When thalidomide (1, 3, or 6 mg/kg) was administered intraperitoneally 3h before the injection of LPS (0.3 mg/kg, i.p.), thalidomide markedly enhanced LPS-induced TNF-α release in a dose-dependent manner. The TNF-α serum levels at 90 min were 640±58.6, 1985±132.6, and 2795±203.5 pg/ml, respcetively, compared to 628.6±64.4 pg/ml in mice treated with LPS-alone. Pretreatment with a single injection of thalidomide (1, 3, or 6 mg/kg. i. p.) dosedependently increased the subsequent mortality caused by a challenge with LPS (15 mg/kg, i. p.), a dose that caused death in 10% of the control mice. We conclude that thalidomide enhances in vivo TNF-α secretion and the lethality of LPS in mice.
In a series of searches fot DNA topoisomerase II inhibitors from naturally occurring compounds, a wood extract of Neonauclea calycina MERR. (Rubiaceae) showed a moderate effect in vitro. Purification of the extract resulted in the isolation of seven known anthraquinones. The structures were characterized as damnacanthal, rubiadin 1-methyl ether, nordamnacanthal, morindone, damnacanthol, lucidin 3-O-primeveroside and morindone 6-O-primeveroside by spectral analysis, respectively. Damnacanthal and morindone showed an intensive inhibitory effect against topoisomerase II (IC50 : 20μg/ml and 21μg/ml).
During the course of study searching for the antiparasitic agents from natural compounds, we found that curcumin showed cytotoxicity against African trypanosomes in vitro. The LD50 values of curcumin were 4.77±0.91μM for bloodstream forms and 46.52±4.94μM for procyclic forms of Trypanosoma brucei brucei (GUTat 3.1 clone).
The tonic action of water Guarana extract, (Paullinia cupana MART.), was investigated in normal, exercised, and epinephrine-induced glycogenolytic mice. A water extract of Guarana (GW) (500 mg/kg) increased the blood glucose level (p<0.001) and decreased the liver glycogen contents of mice 60 min after oral maltose administration (p<0.05). GW also significantly suppressed exercise-induced hypoglycemia (60 min : p<0.05). However, GW did not affect the blood glucose in epinephrine-induced glycogenolytic and exercise mice. These findings indicate that the suppressive mechanism of hypoglycemia might be due to the promotion of glycogen resolution.