Pleckstrin homology (PH) domains, comprised of rather weakly conserved sequences of about 100 amino acid residues, are a protein motif found in many signaling and cytoskeletal proteins. PH domains have been shown to bind to the βγ subunits of heterotrimeric GTP-binding proteins (Gβγ), but the affinity of PH domains for Gβγ has not been quantitatively estimated in detail. To characterize the nature of the interaction between PH domains and Gβγ, its kinetic parameters were analyzed using a BIAcoreTM instrument. All PH domains tested (PH domains of ras-specific guanine nucleotide exchange factor (ras-GRF), phospholipase (PLC) γ1, and Son of sevenless protein (Sos)) appeared to bind to Gβ1γ2 with affinity constants KD of 0.108, 0.318, and 0.208 μM, respectively. The binding of PH domains to Gβγ was inhibited by preincubation of Gβγ with the GDP-bound but not the GTP-bound form of Giα. This study showed a high affinity interaction between PH domains and Gβγ and suggests a potential role of PH domains in Gβγ-mediated signal transduction in intact cells.
A sulfotransferase (ST) cDNA was isolated from a mouse intestinal cDNA library using a probe which was generated by reverse transcription (RT)-PCR based on the conserved amino acid sequences of the ST molecules. The isolated cDNA (1.1 kb) contained an 858 bp open reading frame encoding a 286 amino acid polypeptide with molecular weight of 33439. The deduced amino acid sequence shares 55.1% and 40.2% identiry with mouse liver aryl/phenol (mSTp1) and alcohol (mSTa1 or mSTa2) STs, respectively, and it is highly similar to those of rat and human liver phenol ST (P-ST) isozymes, ST1B1 (87.8%) and ST1B2 (71.0%), respectively. RT-PCR analyses showed abundant expression of the P-ST mRNA in the intestine as well as in the liver in the mouse tissues examined (brain, heart, intestine, kidney, liver and lung) of both sexes. E. coli-expressed enzyme is capable of catalyzing the sulfation of 2-naphthol at Km=3.3 μM and Vmax=3.33 nmol/min/mg protein and also showed sulfation activity for L-3, 4-dihydroxyphenylalanine (L-DOPA) and dopamine. Among food constituents tested, tannic acid and epigallocatechin gallate strongly inhibited the P-ST activity in vitro.
NADH-quinone oxidoreductase is classified into two groups, NADH dehydrogenase-1 (NDH-1) and NADH dehydrogenase-2 (NDH-2). Animal mitochondrial complex I is an NDH-1 type enzyme. Previously, we isolated potent inhibitors from plants to both NDH-1 and NDH-2. We have now examined detailed inhibitory effects of three tannins (pentagalloylglucose, sanguiin H-11, and oolonghomobisflavan A) on NDH-1 using bovine heart mitochondrial complex I and a subcomplex flavoprotein (containing 3 subunits) derived from complex I. Although many specific inhibitors of NDH-1 (e.g. rotenone and piericidin A) have been reported, the reactive sites are at or near to, the ubiquinone-binding site. NADH-ubiquinone-1 oxidoreductase activity of complex I was inhibited by the three tannins, among which sanguiin H-11 was the most potent inhibitor. NADH-menadione oxidoreductase activity of complex I was susceptible to the three tannins, but completely resistant to rotenone. The inhibitory effects of tannins were all noncompetitive with respect to NADH, ubiquinone-1, and menadione. The NADH-menadione oxidoreductase of flavoprotein was also inhibited by the three tannins, but not by rotenone, which is consistent with the fact that flavoprotein does not contain a native ubiquinone-binding site. The study of the NADH reduced-minus-oxidized difference spectrum of flavoprotein under steady-state conditions indicated that the inhibitory sites of sangiin H-11 and oolonghomobisflavan A exist between the NADH binding site and the FMN site, and that for pentagalloylglucose exists between FMN and an artificial electron acceptor-binding site. These results suggest that the tannins are potent inhibitors of NADH dehydrogenases, and that the inhibitory mechanisms are novel.
Various derivatives of human calcitonin have been synthesized and their biological characteristics compared with those of existing calcitonins. The acute effects of these analogues in reducing serum calcium levels and suppressing appetite were assessed in rats. A calcitonin analogue, PO-1 (CGNLSTCMLGKLSQELHKLQTYPQTAIGVGAP-NH2), having both the N- and C-terminal ten amino acid sequences those of human calcitonin, and the 12 amino acid central region that of salmon calcitonin, was found to have equal effectiveness with salmon calcitonin and elcatonin for reducing serum calcium lavels. Strong hypocalcemic activity was also exhibited by PO23 ([cyclo-Asp1, Lys7]-[des-Gly2]-[Leu8]-PO-1) and PO-29 ([Asp15, Asn17, Phe19, His20]-PO-23). PO-23 was prepared by replacing the N-terminal Cys-Cys S-S bond of PO-1 with a ring structure composed of an Asp-Lys peptide bond to enhance physicochemical stability. PO-29 was prepared by modifying the central area of the PO-23 molecule to more closely mimic human calcitonin. When tested in vitro, human calcitonin analogues with a [cyclo-Asp1, Lys7] structure showed biological activities on osteoclast-like cells comparable to those of existing calcitonins (salmon calcitonin and elcatonin) in keeping with their relative potencies for in vivo hypocalcemic action. Acute anorectic activity in rats was strong with salmon calcitonin and elecatonin but relatively reduced with human calcitonin analogues having a [cyclo-Asp1, Lys7] structure. The activities of these analogues on kidney cells were also weaker than that of salmon calcitonin or elecatonin. These results suggest that stable human calcitonin analogues with a [cyclo-Asp1, Lys7] structure suppress bone resorption to a degree similar to that of salmon calcitonin or elcatonin with weaker activities on non-osseous tissues which might be related to adverse reaction.
Imipramine (IMI) N-oxidase activity in brain microsomes from rats of both sexes was determined by high performance liquid chromatography, and compared with the results in rat liver microsomes. Brain and liver microsomal IMI N-oxidation was sensitive to thermal inactivation and had an optimal pH at around 9.0. IMI N-oxidase activity (15.54 pmol/mim/mg protein) in brain microsomes was about one-hundredth that of liver microsomes (2.08 nmol/min/mg protein) at a substrate concentration of 5 mM. IMI N-oxidase activities in both brain and liver microsomes displayed biphasic kinetics that associated a low Km-low Vmax element with a high Km-high Vmax component. Furthermore, a significant sex difference was observed in Vmax values (male>female) in both phases, but Km values were similar between male and female rats, resulting in a sigmificant sex difference (male>female) in intrinsic clearance values (Vmax/Km) of the low-Km and the high-Km phases.
The purpose of this study was to investigate the characteristics of arachidonic acid-induced peripheral vascular disease in rats. Injecting arachidonic acid (2 mg/leg) into the femoral artery caused hind limb gangrene. Histopathological examination revealed occlusive thrombi and marked vascular injury, including denudation of the endothelium and degeneration of the media in the paw arteries. Arachidonic acid injection markedly enhanced the platelet response to both U-46619 and collagen. Although the number of circulating platelets did not differ between sham-operation rats and arachidonic acid-injected rats, the numbers of circulating white blood cells and red blood cells were raised 10 d after arachidonic acid injection. Thrombocytopenia, induced before arachidonic acid injection, markedly suppressed arachidonic acid-induced hind limb gangrene in rats. In addition, the combined administration of aspirin (100 mg/kg/d, p.o.) and ticlopidine (300 mg/kg/d, p.o.) prevented the progression of arachidonic acid-induced hind limb gangrene. These results suggest that platelets are involved in the progression of arachidonic acid-induced hind limb gangrene. This experimental rat model may be suitable for developing novel drugs for the treatment of peripheral vascular disease.
Sulfotransferase (ST) is considered to be generally not induced by xenobiotics. However, it has been reported that steroids such as dexamethasone (DEX) and pregnenolone-16α-carbonitrile (PCN) are effective ST inducers in rats, and sulfation of xenobiotics is quite different in rats and mice. The present study is primarily aimed at determining the effect of sulfate and steroids on the metabolism of acetaminophen (AA) in vitro using monolayer cultured hepatocytes of Sprague-Dawley rats and ICR mice Hepatocytes of rats and mice. were incubated with inorganic sulfate (0.25, 0.5, 1.0, 2.0, 4.0 mM) and AA in SO4-depleted media. AA sulfation rates increased as the sulfate concentration was raised to 1.0 mM in rats, whereas the addition of inorganic sulfate to the media had a lesser effect in mice hepatocytes. After pretreatment with DEX (0.1, 1.0, 10, 100 μM) and PCN (0.1, 1.0, 10 μM) for 3 d, hepatocytes were incubated with AA in media containing 4.0 mM SO-4. Pretreatment of the hepatocytes with DEX caused an increase in the glucuronidation and sulfation of AA by 2-3 folds in rats, but to a lesser extent in mice. PCN significantly enhanced the formation of AA-glucuronide and AA-sulfate in mice, but had a minimal effect on rat hepatocytes. In summary, sulfate and DEX markedly enhanced the formation of AA-sulfate in rats hepatocytes, and DEX and PCN increased the sulfation of AA in mice hepatocyte. These results partially support the claim that DEX and PCN are effective ST and uridine diphosphate-glucuronyltransferase inducers in vivo.
We studied the structure-activity relationships of lignans from Schisandra chinensis and their derivatives as platelet activating factor (PAF) antagonists. Strong activity was shown in lignans without an ester group at C-6, a hydroxyl group at C-7 or a methylene dioxy moiety and with an R-biphenyl configuration. 6(7)-Dehydroschisandrol A, a derivative of schisandrol A, showed the highest activity (IC50, 2.1×10-6 M) in this study.
By screening water and MeOH extracts of 30 Chinese medicinal plants for their anti-herpes simplex virus (HSV)-1 activity, a MeOH extract of the root tubers of Stephania cepharantha HAYATA showed the most potent activity on the plaque reduction assay with an IC50 value of 18.0 μg/ml. Of 49 alkaloids isolated from the MeOH extract, 17 alkaloids were found to be active against HSV-1, including 13 bisbenzylisoquinoline, 1 protoberberine, 2 morphinane and 1 proaporphine alkaloids, while benzylisoquinoline and hasubanane alkaloids were inactive. Although N-methylcrotsparine was active against HSV-1, as well as HSV-1 thymidine kinase deficient (acyclovir resistant type, HSV-1 TK-)and HSV-2 (IC50 values of 8.3, 7.7 and 6.7 μg/ml, respectively), it was cytotoxic. FK-3000 was found to be the most active against HSV-1, HSV-1 TK- and HSV-2 (IC50 values of 7.8, 9.9 and 8.7 μg/ml) with in vitro therapeutic indices of 90, 71 and 81, respectively. FK-3000 was found to be a promising candidate as an anti-HSV agent against HSV-1, acyclovir (ACV) resistant-type HSV-1 and HSV-2.
Retarded capsules containing 1 mg bumetanide (BN) were prepared and their in vivo absoption and diuretic effect after oral administration in human subjects were studied. For comparison, commercially available tablets of BN (rapid effect) were administered orally. The mean value of the area under the plasma concentration time curve (AUC) after administration of retarded capsules was about one half that of the tablets. The mean maximum plasma concentration (Cmax) and the mean maximum urinary excretion rate of BN after administration of retarded capsules were also about one half compared to those of the tablets. Cumulative urinary volumes for 24 h, however, were not significantly different between retarded capsules and tablets. Peak times for the urinary excretion rate of BN, urine flow rate and the Cmax after administration of retarded capsules were significantly delayed compared to those of tablets. Clockwise hysteresis relationships between the urine flow rate and plasm concentration or urinary excretion rate of BN were observed after administration of retarded capsules. From these studies, retarded capsules of BN possessed a mild diuresis and its diuretic effect was maintained for a few hours after administration.
The effect of cutaneous metabolism on the skin penetration of drugs was analyzed based on a two-layer skin difusion/metabolism mode. In vitro permeation studies of propylparaben and butylparaben with or without an esterase inhibitor, diisopropyl fluorophosphate (DFP), were performed. Pretreatment of the skin with DFP prolonged the lag time for the penetration of intact parabens. Additionally, DFP signifidcantly decreased the total flux of butylparaben, but not that of propylparaben. Model analysis of the penetration profiles revealed that DFP inhibits the cutaneous metabolism without affecting any other processes. To comprehensively understand the relationships among lipophilicity, metabolic rate, and skin permeation of drugs, simulation studies were performed with newly derived equations concerning the permeability coefficient and the lag time for the penetration of both intact and metabolite forms. The analysis revealed that the lag time for the penetration of both intact and metabolite forms becomes shorter with increasing metabolic rate. As the metabolic rate of the drug increases, skin penetration of the intact form decreases whereas that of the metabolite increases. The total flux of intact and metabolite forms increases with increasing metabolic rate, being more obvious for highly lipophilic drugs. This indicates that the permeation of lipophilic compounds such as butylparaben is more highly affected by cutaneous metabolism in the viable layer because these compounds easily penetrate the stratum corneum layer. Consequently, the balance between the permeability of drug across the stratum corneum and the dermis has been implicated to impose a significant influence on the percutaneous absorption of drugs subjected to cutaneous metabolism.
We have previously reported that brain sensitivity to thiopental with respect to electroencephalogram (EEG) is enhanced in uranyl acetate pretreated renal dysfunction rats. The results were attributed to pharmacodynamic factors. In this study, in vivo EEG and in vitro binding studies for γ-aminobutyric acid (GABA)-benzodiazepine receptor complex were performed to investigate the mechanism of the enhanced effect of thiopenta. The receptor binding properties in the brain membrane from normal and renal dysfunction rats were examined using [3H]tert-buthylbicycloorthobenzoate (TBOB), [3H]flunitrazepam and [3H]muscimol. The effect of plasma dialysate from normal (ND) and renal dysfunction rats (RDD) on the thiopental induced EEG and receptor binding were also examined to confirm the role of endogenous compounds. The intrinsic receptor binding characteristics of various sites and their allosteric interaction with thiopental was similar in membrane preparations from normal and renal dysfunction rats. However, RDD, when compared to ND, enhanced the EEG induced by thiopental. At the receptor level, RDD significantly enhanced the thiopental induced inhibition of TBOB. No difference was found between the influence of ND and RDD on the interaction between thiopental and flunitrazepam or muscimol binding. These results showed that the thiopental induced allosteric inhibition of TBOB binding was potentiated by some endogenous compounds in RDD and suggests that this action might be the mechanism, at least in part, for the increased sensitivity of thiopental in renal dysfunction rats.
We previously reported that SK2 hybridoma cells that secreted anti-human interleukin-6 (hIL-6) monoclonal antibodies (SK2 mAb) were microencapsulated within alginate-poly(L)lysine-alginate (APA) membranes (APA-SK2 cells) for immunoisolation, and a single intraperioneal injection of these APA-SK2 cells remarkably improved IgG1 plasmacytosis in hIL-6 transgenic mice (hIL-6 Tgm). However, the duration of the effectiveness of APA-SK2 cells as a cytomedicine was unfortunately limited. In this study, we attempted to re-inject APA-SK2 cells into hIL-6 Tgm for the purpose of prolonging the cytomedical therapy. In hIL-6 Tgm re-injected with APA-SK2 cells, the plasma IgG1 level did not show any increase in 37 week old mice, and their survival time was at least three times longer than those of untreated hIL-6 Tgm These results suggest that re-injected APA-SK2 cells survived and secreted SK2 mAn in the allogeneic mice. Thus, the limited duration of the cytomedical effects of APA-SK2 cells was probably caused by the disappearance of the inner space of microcapsules for cell proliferation, not by the rejection of the host's immune system. Therefore, if we can regulate the proliferation of the cells microencapsulated within a semipermeable membrane, we may be able to develop a cytomedicine which will continue its function longer after a single injection.
Inclusion complexes of phenytoin (DPH) with 6-O-α-D-glucosyl (G1)- and 6-O-α-D-maltosyl (G2)-β-cyclodextrins (β-CyDs) were prepared in a molecular mixing ratio of 1 : 1. The advantages of these preparations in terms of dissolution characteristics and the oral absorbency of DPH were evaluated in comparison with the known solid dispersions of polyvinylpyrrolidone K-30 and sodium deoxycholate (DC-Na). The results of a phasesolubility study indicated that G1- and G2-β-CyDs provided higher solubility for DPH than 2-hydroxypropyl (HP)-β-CyD. Irrespective of inclusion ability, the DPH/β-CyD complexes allowed faster dissolution rates than those of the known dispersions in JP 1st and 2nd mediums. The dissolution behavior of the DPH/DC-Na dispersion was considerably different between the 1st and 2nd mediums. The complexation by the sugar-modified derivatives yielded a higher stability of dissolved DPH in the JP 2nd medium than that yielded by K-30 or DC-Na. The safe estimation of carriers themselves indicated that G1- and G2-β-CyDs did not damage the small intestine, while 10 mM DC-Na showed some damage. Compared with the DPH/K-30 dispersion, the preparations with the sugar-modified β-CyDs were more effective in enhancing the absobability of DPH after oral administration.These results clearly suggest that complexation with G1- and G2-β-CyDs are useful forms for the oral delivery of DPH. The advantage of these complexes is that they produce an increased level of DPH available for gastrointestinal ansorption. Additionally, G2-β-CyD is recommended as a safe and potent additive for DPH.
In vitro simultaneous transport and metabolism of three ester prodrugs of nicotinic acid (NA), methyl nicotinate (MN), ethyl nicotinate (EN) and butyl nicotinate (BN) were studied using excised skin from hairless mouse. Hydrolysis studies of these esters with and without skin homogenate were also done at 37°C. Both the ester and NA were detected in all receiver solutions in permeation studies, and no chemical hydrolysis of the esters was found, indicating that the esters were hydrolyzed during the skin permeation process. The total (ester+NA) flux from a saturated solution of ester prodrugs was higher than that of NA and was highest for MN, followed by EN and BN, whereas the total permeability coefficient of ester prodrugs increased from MN to BN. A difference in the NA/total flux ratio was found among these prodrugs; thus, esterase activity was also dependent on the alkyl chain length of the esters. The total flux from each ester solution increased linearly with the donor concentration. NA flux from MN and EN solutions increased with an increase in the donor concentration and reached a plateau at the high concentration range, suggesting that metabolic saturation occurred. NA fluxes at the plateau were similar among ester prodrugs and corresponded to the Vmax estimated from the hydrolysis experiment. The order of donor concentration at which NA reched a plateau also corresponded to the order of Km. It was confirmed that a difference in alkyl chain length of the ester prodrugs affected not only permeability but also metabolism in the skin permeation process.
The relation between degranulation and rapid metabolic responses (acidification rate changes) in RBL-2H3 cells was studied using a cytosensor microphysiometer, a silicon-based biosensor system. The metabolic responses in RBL-2H3 cells by antigen stimulation were compared with those by inhibitors of Ca2+ -ATPase. The former resulted in a rapid transient increase in the acidification rate of RBL-2H3 cells while the latter resulted in gradual decreases. When RBL-2H3 cells were costimulated with the inhibitors of Ca<2+> -ATPase and an activator (PMA, phorbol-12-myristoyl-13-acetate) of protein kinase C, the metabolic responses increased again in RBL-2H3 cells. This seems to indicate that degranulation in RBL-2H3 cells was accelerated by costimulation with the inhibitors and PMA. However, costimulation was not able to completely mimic the way antigen stimulated RBL-2H3 cells in degranulation and in rapid metabolic reaponses.
Adenosine 5'-triphosphatase (ATPase) plays a role in the process of energy conversion in the controlled hydrolysis of ATP. The effect of Ca2+ -binding protein on ATPase activity in the brain cytosol of rats of different ages was investigated. ATPase activity in the brain cytosol of 50-week-old rats was significantly decreased as compared with that of 5-week-old rats. The presence of calcium chloride (10-5 and 10-4 M) in the enzyme reaction mixture caused a significant increase in ATPase activity in the brain cytosol of rats of different ages. This incrase was not altered by trifluoperazine (2×10-5 M), an antagonist of calmodulin. Calmodulin (5 μg/ml), calbindin (5 μg/ml) or S-100A protein (10 μg/ml), a Ca2+ -binding protein, had no effect on ATPase activity. Meanwhile, regucalcin (10-9 M), which is present in brain, significantly decreased ATPase activity in young and older rats. However, the effect of regucalcin was weakened by the presence of Ca2+ (10-5 M). The addition of anti-regucalcin monoclonal antibody in the reaction mixture caused a significant elevation of ATPase activity; this increase was completely abolished by adition of regucalcin (10-9 M). The present study suggests that regucalcin plays an inhibitory role in the regulation of ATPase activity in the brain cytosol of rats of different ages.
The proteolytic activity of a fusion protein between protein A and human pepsinogen C (PA-PGC) was measured by a modified milk clotting assay on 96-well microtiter plate. The assay ranges for 30 and 120 min-incubation were approximately 0.08-1.25 ng and 0.02-0.16 ng, respectively. Although the absorbance of milk solution decreased by the precipitation of clotted casein, the white precipitate was, if anything, convenient for an easy detection of reaction-positive wells. The borderline between the wells with or without precipitate was very clear and easily detected without the plate reader. This feature was thought to be suitable for the positive-negative judgment of a dilution test in enzyme immunoassay (EIA). Rabbit IgG adsorbed on a microtiter plate was then measured using PA-PGC and this milk assay (designated as PA-PGC assay). Althouth PA-PGC assay needed silightly longer incubation than the conventional color assay, the sensitivity of both systems was almost identical, and the reaction-positive wells containing white precipitate were easily detected at a very low range by the over night incubation without using the plate reader. PA-PGV assay possesses the unique and useful properties, and it can be used as a convenient and easy EIA technique.
By human intestinal bacteria, glycyrrhizin (18β-glycyrrhetinic acid-3-O-[β-D-glucuronopyranosyl-(1→2)-β-D-glucuronopyranoside], GL) was metabolized to 18β-glycyrrhetinic acid (GA) : the main pathway metabolized GL to GA by glucuronidases of Bacteroides J-37 and Eubacterium sp. GLH and the minor pathway metabolized GL to 18β-glycyrrhetinic acid-3-O-β-D-glucuronide (GAMG) by β-glucuronidase of Streptococcus LJ-22. β-Glucuronidase from Streptococcus LJ-22 hydrolyzed GL to GAMG (not GA). The molecular weight and optimal pH of the enzyme were 240 kDa and 5-6.
More than fifty chalcone derivatives were synthesized to examine structure-activity relationships against human aldose reductase. Certain 2', 4'-dihydroxychalcone derivatives inhibited human aldose reductase activities, and 2', 4', 2, 4-tetrahydroxychalcone and 2', 4', 2-trihydroxychalcone showed potent inhibitory activity with IC50 values of 7.4×10-9 M and 1.6×10-7 M, respectively. On the other hand, cis-form chalcones, which were isomerized from the original trans-forms by irradiation of daylight in methanol solution, promoted the activity of human aldose reductase.
The metabolic behavior of orally administered gallic acid was investigated by HPLC and 4-O-methyl gallic acid was found to be the main metabolite in rat peripheral blood and urine. After oral administration of gallic acid, maximum concentration in portal vein and inferior vena cava occurred at 15 and 30 min, respectively. In portal vein, gallic acid was preferentially detected relative to 4-O-methyl gallic acid, whereas gallic acid and 4-O-mehyl gallic acid were equally detected in inferior vena cave. On the other hand, 4-O-methyl gallic acid but not gallic acid was found in liver. The contents of gallic acid and 4-O-methyl gallic acid in urine were nearly 100 times higher than those in blood. The ratio of 4-O-methyl gallic acid to total gallic acid metabolites in urine was from 0.55 to 0.76, indicating that a considerable amount of gallic acid was excreted without being metabolized. In this study we found that gallic acid administered orally existed in the blood for 6 h at most, and more than half was metabolized to 4-O-methyl gallic acid, followed by excretion into urine.