Sodium dodecyl sulfate (SDS)-linear polyacrylamide solution-filled capillary electrophoresis using a stable coated fused silica capillary has been developed for the separation and molecular weight determination of proteins. A fused silica capillary was coated with linear polyacrylamide through Si-C bond which suppressed the electroosmotic flow and reduced the adsorption of proteins. Acrylamide solution containing SDS was filled into the coated capillary and polymerized. Compared with a conventional fused silica capillary coated with 3-methacryloxypropyltrimethoxysilane through siloxane linkage (Si-O-Si-C), the developed capillary was much more stable, even in the alkaline condition. Consequently, the durability of the polymer matrices and the reproducibility of separation were markedly improved. An excellent linear relationship was obtained between the mobility and the logarithm of the protein molecular weight. The relative standard deviation of migration times was below 1%.
The molecular-weight distribution of commercially available low-molecular-weight heparins (LMWHs) was estimated by high-performance gel permeation chromatography (HPGPC) using a LMWH molecular-weight calibrant, supplied by the National Institute for Biological Standards and Control for the calibration of columns. This calibrant is a mixture of many polysaccharides with a disaccharide unit prepared by a random depolymerization of unfractionated heparin, and the ends of the fragments have a UV absorption. The ratio of the refractive index to the UV absorption is a relative measure of molecular weight. Various molecular-weight parameters such as weight-, number- or z-average molecular-weights, polydispersity and relative distributions of high, middle and low molecular-weight components were estimated, indicating that LMWHs have a wide variation in molecuar-weight distribution. This variation was ascribed to the difference in preparation methods from unfractionated heparins.
Rats and mice were fed a diet, prepared with soybean oil (SO), perilla oil (PO) or fish oil (FO), for 4 weeks. Compared with the groups of SO-feeding, FO-feeding increased content of eicosapentaenoic acid (20 : 5 n-3), docosapentaenoic acid (22 : 5 n-3) and docosahexaenoic acid (22 : 6 n-3) and PO-feeding elevated the content of α-linolenic acid (18 : 3 n-3), 20 : 5 n-3 and 22 : 5 n-3 of hepatic lipids of both rats and mice. FO-feeding increased the activity of peroxisomal β-oxidation in the livers of both rats and mice. The activities of peroxisomal β-oxidation in rats and mice that were fed FO diet at a high fat (40% of energy) concentration corresponded to 20% and 30%, respectively, of the maximum activities induced by peroxisome proliferators (4-chlorophenoxyisobutyric acid or perfluorooctanoic acid). To a lesser extent, PO-feeding elevated this activity too. There were significant correlations between peroxisomal β-oxidation activity and the content of either 20 : 5 n-3, 22 : 5 n-3 or 22 : 6 n-3 in hepatic lipids. FO-feeding decreased effectively serum level of cholesterol of both rats and mice. The reduction in serum cholesterol by feeding PO was less pronounced than that observed with FO-feeding. A high correlation was found between 22 : 6 n-3 content in hepatic lipids and serum concentration of cholesterol. Although FO-feeding lowered the level of circulating triacylglycerol, PO-feeding produced no change. No substantial correlation was observed between the hepatic content of n-3 fatty acid and the concentration of serum triacylglycerol. These results suggest that manipulation of the acyl composition of hepatic lipids by polyunsaturated fatty acids of the n-3 series lead to alterations in peroxisomal β-oxidation activity and circulating a cholesterol level. The physiological significance of n-3 fatty acid-mediated enhancement of the activity of peroxisomal β-oxidation was discussed.
The gene structure of bovine adrenodoxin reductase, a component of the mitochondrial steroid hydroxylating system in the adrenal cortex, was determined. When we screened a bovine genomic DNA library, using bovine adrenodoxin reductase cDNA as a probe, we isolated 10 genomic clones covering a continuous 30 kilobase (kb) region of bovine chromosomal DNA in which the adrenodoxin reductase gene spanned approximately 12 kb. The adrenodoxin reductase gene consisted of 12 exons and all the donor and acceptor sites of the exon/intron junction followed the GT/AG rule. The transcription initiation site was found to be 79 bases upstream of the translation initiation site by primer extension analysis and putative CAAT and GC boxes, but no typical TATA box, were present in the 5'-flanking region. The 5'-flanking region of this gene showed several features characteristic of promoters of house-keeping genes. The CCAGGG sequence present in the 5'-flanking region of genes for adrenodoxin and the steroid hydroxylating cytochromes P-450 in the adrenal cortex was present 13 base upstream from the transcription initiation site. We analyzed the promoter activity of the 5'-upstream region of this gene, using as a reporter the chloramphenicol acetyltransferase gene. The region between position -757 to -376 was proved necessary for expression of this gene in mouse Y-1 tumor cells. Adrenocorticotropin did not enhance the expression of this bovine gene in the cells.
A neutral and an acidic polysaccharide, called peonan SA and peonan SB, were isolated from the root of Paeonia lactiflora PALLAS. They were homogeneous on electrophoresis and gel chromatography, and their molecular masses were estimated to be 11.2×104 and 25.0×104, respectively. Peonan SA is composed of L-arabinose : D-galactose : D-glucose in the molar ratio of 1 : 1 : 28, and peonan SB is composed of L-arabinose : D-galactose : D-galacturonic acid in the molar ratio of 4 : 3 : 2, in addition to small amounts of peptide moieties. About forty percent of the hexuronic acid residues in peonan SB exist as methyl esters. Reduction of carboxyl groups, methylation analysis, nuclear magnetic resonance and periodate oxidation studies indicated that their structural features include mainly 3, 4- and 4, 6-branched α-D-glucan type units (peonan SA) and principally α-1, 5-linked L-arabino-β-3, 6-branched D-galactan type structural units (peonan SB). Both polysaccharides, especially in peonan SB, showed remarkable reticuloendothelial system-potentiating activity in a carbon clearance test and considerable anti-complementary activity.
Various aromatic esters of trans-4-guanidinomethylcyclohexanecarboxylic acid (GMCHA), trypsin inhibitors, strongly inhibited the growth of Bacillus subtilis 558 and their effects were markedly affected by the species of substitution on the phenyl mucleus of the GMCHA phenyl esters. 4-tert-Butylphenyl ester of GMCHA (GMCHA-OPh'Bu), a representative of various GMCHA esters, dose-dependently inhibited the growth of B. subtilis and DNA, RNA and protein syntheses in the cells. The growth inhibition was preceded by suppressive effect of GMCHA-OPh'Bu on DNA synthesis. These results suggested the possible involvement of a trypsin-like proteinase in DNA synthesis. A trypsin-like proteinase was partially purified from B. subtilis 558 by DEAE-cellulose column chromatography, ammonium sulfate fractionation and successive chromatographics on Sephadex G-200, phenyl-Sepharose CL-4B, L-arginine-Sepharose 4B and Sephadex G-200 columns. The properties were compared with those of proteinase In, which momentarily appears just before the onset of DNA synthesis and seems to participate in the initiation of DNA replication, and which was purified from E. coli K-12 IAM 1264. The properties of the proteinase from B. subtilis 558 were similar to proteinase In, however, the molecular mass (110000) was different from that of the latter (66000). Various GMCHA esters strongly inhibited the proteinase activity and the order of the effects was closely correlated with that on the cell growth. The proteinase was tentatively called subtilis proteinase In.
The enzymatic formation of D-cysteinolic acid conjugated chenodeoxycholic acid in liver preparation from a marine teleost, wild and cultured red seabream, Pagrosomus major, was investigated. [24-14C]Chenodeoxycholic acid was incubated with taurine, glycine, or D-cysteinolic acid in the liver preparation in the presence of CoA, ATP, NAT+ and FAD. D-Cysteinolic acid could be conjugated efficiently with chenodeoxycholic acid to give chenodeoxycholyl-D-cysteinolic acid in both wild and cultured red seabream liver preparations, though the production rate was slower than that of the formation of chenodeoxycholytaurine. Under the conditions empolyed, glycine was not utilized as the substrate for the conjugation at all. The formation of chenodeoxycholyl-D-cystenolic acid was decreased by the addition of various concentrations of taurine to the incubation mixture. These results suggest that bile acid-CoA : amino acid N-acyltransferase in red seabream is not able to distinguish taurine and D-cysteinolic acid as the substrate, probably because of their structural similarity. Consequently, D-cystenolic acid conjugated bile acids found in the bile of wild red seabream were thought to be synthesized in the liver of the fish utilizing the unusual amino acid, which originated from foods, prior to secretion into the bile.
Effects of ecabet sodium (TA-2711), a locally acting antiulcer agent, on prostanoid production and the morphology of the rat gastrointestinal mucosa were studied in comparison with sucralfate. Ecabet, at therapeutic doses (25 and 100 mg/kg, p.o.), dose-dependently increased the gastric mucosal level of prostaglandin E2 (PGE2) : sucralfate (100 mg/kg, p.o.) showed a tendency to increase the PGE2 level. In an ex vivo study, ecabet (25 and 100 mg/kg, p.o.) dose-dependently increased the capacity of the gastric mucosa to synthesize PGE2 and PGI2 without modifying tromboxane A2 (TXA2) synthesis, and the 100 mg/kg dose persisted for up to 3 h. Ecabet (400 mg/kg, p.o.) also significantly increased PGE2 synthesis and there was a tendency to increase PGI2 synthesis by the duodenal mucosa, without affecting TXA2 synthesis. PGE2 synthesis by the colonic mucosa was not affected, even at a high dose of ecabet (1000 mg/kg, p.o.). When the rat gastric mucosa was examined by light microscopy and scanning electron microscopy, ecabet (100 and 400 mg/kg, p.o.) had caused no morphological change to the gastric mucosa, while sucralfate (100 and 400 mg/kg, p.o.) produced apical rupture of the epithelial cells and subepithelial edema. The present study indicates that ecabet locally stimulates PGE2 and PGI2 production in the gastroduodenal mucosa and this effect is not attributable to a local irritant action accompanied by superficial epithelium damage.
We examined the effects of intravenous anesthetics (thiopental, fentanyl and morphine) on the ventricular activation in a canine myocardial infarction model. Thiopental at 5 and 10 mg/kg delayed or abolished the delayed activation in the infarcted zones with slight delay of activation of the normal zones. Fentanyl at 30 μg/kg slightly but significantly prolonged the activation time in both normal and infarcted zones. Morphine at 1 mg/kg did not produce any significant effect. Thiopental, but neither fentanyl nor morphine, inhibited ventricular stimulation-induced arrhythmias. Thus, thiopental, but not fentanyl nor morphine, markedly depressed the delayed activation in myocardial infarction, which may affect and probably inhibits the ventricular arrhythmias in myocardial infarction. It also should be kept in mind that thiopental may have arrhythmogic effects in myocardial infarction.
The diuretic effects of KW-3902 (8-(noradamantan-3-yl)-1, 3-dipropylxanthine), a novel adenosine A1 receptor antagonist, were determined and compared with those of trichlormethiazide (TCM) and furosemide in saline-loaded conscious dogs. KW-3902, at doses higher than 0.1 mg/kg (p.o.), produced dose-dependent increases of urine volume and sodium excretion and these effects were statistically significant at doses of 1-100 mg/kg. The increase in potassium excretion was lower than that of sodium, and the ratio of sodium to potassium excretion (Na/K) tended to be elevated. TCM (0.3 mg/kg) and furosemide (3 mg/kg) also induced increases in urine volume and sodium excretion. The diuretic effects of KW-3902 lasted for 4 h after administration, whereas TCM and furosemide caused significant natriuresis for 2 h after administration. Thus, KW-3902 exhibited a longer lasting natriuresis than TCM and furosemide. These results indicate that adenosine A1 receptor blockade by KW-3902 causes consistent diuresis and natriuresis in dogs and suggest that adenosine A1 receptor blockade is a promising approach to diuretic therapy.
A correlation was found between the carbonic anhydrase II activating power and the pKa values for a series of azoles, bisazolylmethanes and bisazolylethanes. Strong activations were found for compounds with pKa's in the interval 6.5-8.0. The mechanism of action for such activators is discussed.
To understand the effects of lipid-lowering agents on the ethanol-induction of hepatic CYP2E1, clofibrate and L-carnitine were administrated to adult male rats. The administration of ethanol in the diet (containing 21% calories as ethanol, given for 3 weeks) increased levels of hepatic CYP2E1 protein (1.9-fold that of untreated controls) and mRNA (2.1-fold). In contrast, the administration of clofibrate (0.1% v/v) in an ethanol-containing diet did not significantly increase either CYP2E1 protein (1.1-fold) or mRNA (0.8-fold), in spite of the significant increases in blood ketone bodies. Administration of L-carnitine alone had no clear effect on CYP2E1 and blood ketone body levels. Co-administration of L-carnitine, however, increased liver microsomal CYP2E1 protein (2.5-fold) in rats given an ethanol-containing diet. No difference was observed in the mRNA levels in rats receiving ethanol with and without L-carnitine. These results indicate that clofibrate and L-carnitine modulate ethanol-mediated induction of hepatic CYP2E1 independent of blood levels of ketone bodies. It is also suggested that these lipid-lowering agents affected hepatic CYP2E1 through particular mechanisms, suppression of the specific mRNA and post-translation stabilization.
Induction of single strand breaks in DNA was assessed by the conversion of supercoiled closed circular plasmid DNA into the open circular form. Euflavine produced single-strand breaks following irradiation but not in the control maintained in the dark. The single strand breaking activity of photoactivated euflavine was found to be dose-dependent. The effective dose convertion 50% (ED50) of the closed circular DNA to the open circular form was 0.53 μM. A comparison of 8 acridine compounds revealed that the ED50 of diaminoacridines such as euflavine, proflavine and acridine yellow or the 3, 6-dimethylamino-derivative (acridine orange) was less than 1 μM while the ED50 values of the other acridines were greater than 80 μM. Euflavine was markedly inhibited by singlet oxygen scavengers such as NaN3, histidine, α-tocopherol or β-carotene and partly inhibited by superoxide dismutase, mannitol or catalase. These results suggest that enflavine induces single strand breaks in DNA mainly by a type II photodynamic mechanism. Photodynamic single strand breaking activities appeared related to their mutagenic activities on yeast.This experimental system described here is useful for the quantitative assessment of the single strand breaking activities of various photosensitizers in vitro and for the determination of active oxygen species involved in those processes.
The effect of carbon tetrachloride (CCl4) on the induction and activity of 3-methylcholanthrene (MC)-inducible P450IA isozymes (P450IA1 and P450IA2) in the liver was examined by treating male Sprague-Dawley rats with simultaneous i.p. injections of MC and CCl4. Following the treatment, the quantities of P450IA mRNAs determined by Northern blotting and P450IA proteins by Western blotting were significantly reduced as compared with those in rats treated with MC alone. As determined by a bacterial mutation test using carcinogenic aromatic amines are P450IA enzyme substrates, CCl4 treatment selectively inhibited the activity of P450IA2 in the MC-induced isozymes. These results indicate that CCl4 exerts on inhibitory effect on the induction of P4501A isozymes and that it selectively inhibits P450IA2 activity.
The usual therapeutic doses for the treatment of both angina pectoris and cardiac arrhythmia vary widely among beta-blocking agents, with a maximum of about a 200-fold difference, despite subjects' same clinical improvement at the varying doses. In order to clarify the mechanism of this difference, we analyzed retrospectively the cardiac pharmacological activities of beta-blocking agents based on the receptor occupancy theory by using both their unbound concentrations in plasma at steady state (Cssf), as well as dissociation constants (KB and KI, which were determined by in vitro binding experiments and by in vitro pharmacological experiments, respectively) for a beta 1 receptor. A significant log-linear relationship between Cssf and the KB values was obtained with a slope of regression line of 0.91 (r=0.83, p<0.01). On the other hand, the correlation coefficient of the relationship between Cssf and the KI values was low, with a slope of about 0.5 (r=0.80, p<0.01). The beta 1 receptor occupancies calculated from KB values at the steady state condition after the oral administration of usual doses were almost constant (80.5±16.8%), regardless of the wide variation of usual doses of the drugs. This result indicated that the receptor occupancy may be an appropriate indicator for the pharmacological activity of the drug. Furtheremore, there were significant relationships between the primary pharmacokinetic parameters : Cb/Cf, CLtot/F, and Vdssf, and the octanol/water partition coefficient (PC), with correlation coefficients of 0.80, 0.50 and 0.85, respectively. Accordingly, it is suggested that the prediction of a ususal dose of a new beta-blocking agent can be carried out by using the KB and PC values based on quantitative structure-Pharmacokinetic/pharmacodynamic relationships. This methodology should be very useful for estimating the rational usual dose of a new beta-blocking agent from the animal experimental and physicochemical data in the preclinical study.
Plain tablets containing a model drug, sulfamethizole (SMZ), were coated with triolein (TO), trilaurin (TL) and ethylcellulose (EC). The biological behavior of the coated tablets (TOTL-Tab), which are pH independent and sensitive to pancreatic lipase, was investigated in humans. Results of the administration of the tablets with or without an antacid, under fasting and non-fasting conditions, and at 0.5 h before and 0.5 h after meals, were examined. A comparison of the in vivo behavior of SMZ after the administration of these tablets was done using the following data : the lag time of urinary excretion (Ulag), the total urinary recovery percentage (X∞u), and the mean residence time after Ulag (MRTaf). A typical pH-sensitive tablet coated by cellulose acetate phthalate (CAP-Tab) was used as a reference. For the administration of a CAP-Tab alone, the Ulag obtained under both the non-fasting and fasting condition was longer than that of the plain tablet. However, Ulag after the administration of a CAP-Tab with an antacid became considerably shorter. This lag time was about the same as that obtained from the plain tablet, regardless of food ingestion. The obtained CAP-Tab MRTaf and X∞u values were not significantly different in comparison to the plain tablets. Under the non-fasting condition, Ulag, MRTaf and X∞u of TOTL-Tab were not affected by the co-administration of an antacid, and these values were virtually the same as those obtained from a CAP-Tab without an antacid. The urinary excretion data obtained after the administration of TOTL-Tab alone under fasting was analogues to the non-fasting case. When TOTL-Tab was co-administered with an antacid under fasting, the MRTaf was the much longer than that of the plain tablet, and the X∞u was almost a half that of the plain tablet. These results suggest that TOTL-Tab is useful as an enteric release preparation sensitive to pancreatic lipase in humans, except when antacids are taken under a fasting condition.
The effects of an ethanol/panasate 800 (tricaprylin) (40/60) system as a lipophilic vehicle, and an ethanol/water (60/40) system as a hydrophilic vehicle, with or without permeation enhancers for in vitro skin permeation and in vivo skin absorption of tegafur, alclofenac and ibuprofen with different lipophilicity, were evaluated.The in vitro and in vivo skin permeability of tegafur, alclofenac and ibuprofen was enhanced by the use of ethanol/panasate 800 (40/60) or ethanol/water (60/40) binary vehicles as a donor composition. However, the two vehicles showed contrastive properties in relation to the extent of permeation enhancement of the three drugs : tegafur > alclofenac > ibuprofen for the ethanol/panasate 800 (40/60) system, and ibuprofen ≥ alclofenac > tegafur for the ethanol/water (60/40) system. When lauric acid, as a permeation enhancer, was added to both of the binary vehicles, the in vitro and in vivo skin permeability of three drugs further increased, and the in vivo absorption rate of the drugs from the ethanol/water (60/40) system was larger than that from the ethanol/panasate 800 (40/60) system. In conclusion, it was suggested that the ethanol/panasate 800 (40/60) lipophilic binary vehicle is useful for hydrophilic drugs, and conversely, the skin absorption of lipophilic drugs can be improved by the use of the ethanol/water (60/40) hydrophilic binary vehicle with or without lauric acid as a permeation enhancer.
The tumor distribution and the disposition of serum proteins, such as albumin, fetuin, transferrin, and IgG, were investigated in mice bearing Sarcoma 180. Serum proteins labeled with fluoresceinisothiocyanate (FITC) were administered to the mice. The FITC-labeled proteins acylated with glutaric anhydride were also administered to the mice in order to investigate the effect of chemical modification. The plasma concentration of each glutarylated serum protein was significantly lower, about 15 to 46-fold, in comparison to that of the non-acylated protein at 24 h after administration. The tissue distributions of the glutarylated serum proteins were also decreased compared to those of the non-acylated proteins. Especially, the hepatic distribution of albumin and IgG was significantly reduced with glutarylation. The urinary excretion of albumin and transferrin, and fecal excretion of IgG, were significantly increased with glutarylation. The serum proteins were accumulated effectively in the tumor tissue in mice bearing Sarcoma 180. It was found that the tumor distributions were not impaired by the glutarylation, except involving fetuin. It was suggested, therefore, that the glutarylated serum proteins were valuable for relative tumor-selectivity and might be utilized in a macromolecular carrier system for antitumor drugs.
To investigate the pharmacokinetics of cyproheptadine (CPH) and its metabolites, the plasma concentration and urinary excretion of CPH and its detectable metabolites were determined after intravenous (i.v.) administration of parent or synthesized metabolites to rats. The plasma CPH concentration-time course was subjected to biexponential calculation following the i.v. administration of CPH, producing the temporal and low plasma concentrations of desmethylcyproheptadine (DMCPH) and the sustained plasma concentrations of desmethylcyproheptadine-epoxide (DMCPHepo). DMCPH was also eliminated, according to the biexponential equation, after i.v. administration of preformed DMCPH, forming DMCPHepo in plasma. On the other hand, no detectable DMCPHepo was found in plasma after the i.v. administration of cyproheptadine epoxide (CPHepo). All compounds administered had large distribution volumes and were almost entirely excreted as DMCPHepo in urine; this excretion continued for a long time. However, the urinary excretion pattern of DMCPHepo after CPHepo was different from those after CPH and DMCPH. The mean residence times of the epoxidized metabolites estimated from the urinary data were much longer than those from the plasma concentration data, suggesting either a gradual reflux of the metabolites from a tissue depot into systemic circulation under those plasma concentrations close of detection limit, or some interaction which delays excretion into the urine. This study suggests that both metabolic pathways of CPH, through DMCPH and CPHepo, to DMCPHepo are possible, but that the demethylation of CPH largely occurs prior to epoxidation; also that the extensive and persistent distribution of DMCPHepo to tissues may relate to the toxicity of CPH reported in rats.
The interaction between EMI (eosin-5-maleimide) and band 3 of human erythrocytes was studied under various conditions. It was found that the effects of the ionic strength on the EMI-band 3 interaction strongly depended on pH. At pH 6.0, the ionic strength had remarkable effects on the EMI-bound band 3, whereas at pH 7.4, the EMI-band 3 interaction was independent of ionic strength. From the change in the circular dichroism spectra of the EMI-bound band 3, it was revealed that the conformation or the structure of the EMI-binding sites in the cytoplasmic domain of band 3 was strongly dependent on ionic strength. The thermodynamic parameters for the covalent-binding between EMI and band 3 were calculated on the basis of the difference spectra of the EMI and ghost system. The values of activation energy and activation entropy change at pH 6.0 were extraordinarily small compared with those values of pH 7.4. These findings represent the characteristics of the EMI-binding sites in the cytoplasmic domain of band 3. The interaction of EMI with an isolated fragment of the cytoplasmic domain, 43k fragment, was also examined. The circular dichroism spectra of the EMI-bound 43k fragments was significantly different from those of the EMI-bound band 3. This may indicate that the quaternary structure of the EMI-binding site in the cytoplasmic domain of band 3 is altered by an allosteric connection with the membrane-spanning domain of band 3. Further, from the pH titration of the 43k fragment, it was suggested that lysine residues are responsible for the ionic interaction between EMI and the 43k fragment.
A plasmid, pEP2104 (23.9 kilobase pairs), from Staphylococcus aureus carries a gene that specifies inducible resistance to 14-membered (erythromycin, EM, and oleandomycin, OL) and 16-membered macrolide (mycinamicin I and II), but not to all of the latter, and to streptogramin type B antibiotics (partial macrolide- and streptogramin- B-antibiotic resistance : PMS-resistance) (L. Janosi, E. Ban, Acta Microbiol. Acad. Sci. Hung., 29, 187 (1982) and Y. Nakajima et al., J. Pharmacobio-Dyn., 15, 319 (1992)). The induced cells of strain 8325(pEP2104) did not inactivate EM, OL, josamycin, rokitamycin or mikamycin B (MKM-B), and the cell-free extract of the strain did not inactivate EM or MKM-B, either. Ribosomes from the cells whose PMS-resistance was induced by EM were sensitive not only to EM or spiramycin, but also to MKM-B. A 63000-dalton protein increased to a great extent only in the cell membrane fractions of induced 8325(pEP2104), and may be involved in PMS-resistance.
Crude polysaccharides were obtained from a hot-water extract and alkaline extracts of the cultural mycelium of Cordyceps sinensis. They showed significant activity in normal mice and streptozotocin-induced diabetic mice as a result of intraperitoneal (i.p.) injection. A crude polysaccharide (CS-OHEP) obtained from 5% sodium hydroxide extract slightly lowered the plasma glucose level in normal mice by oral (p.o.) administration. A neutral polysaccharide (CS-F30) exhibited higher hypoglycemic activity than its crude polysaccharide (CS-OHEP), exhibited by i.p. injection, and it significantly lowered the glucose level by p.o. administration (50 mg/kg). However, it hardly affected the plasma insulin level in normal mice. CS-F30 ([α]D +21°in water) is composed of galactose, glucose and mannose (molar percent, 62 : 28 : 10), and its molecular weight is about 45000.
The effect of sodium dodecylsulfate (SDS) and deodecyltrimethylammonium chloride (DTAC) on the conformation of bovine pancreatic deoxyribonuclease I (DNase I, EC 126.96.36.199) has been studied by using fluorescence and circular dichroism methods as a function of surfactant concentration. About 60% of the fluorescence was quenched in the presence of 1 mM SDS, indicating the partial conformational transition of DNase I; the conformational susceptibility of DNase I to SDS was reduced by Ca2+ ions. The free energy changes were evaluated at 4.0±0.1 kcal·mol-1 in the presence of 5 mM Ca2+ ions and 2.5±0.1 kcal·mol-1 in the absence of Ca2+ ions. No fluorescence changes occurred in DTAC solution. The residue ellipticity of DNase I at 222 nm decreased in the presence of 1-1.4 mM SDS indicating degradation of the secondary structure of DNase I, while DTAC had not effect on the ellipticity at 222 nm, even in the presence of 10 mM DTAC. The enzymatic activity of DNase I was abolished by SDS, the deactivation profile being compatible with the fluorescence decay profile induced by SDS. On the other hand, no deactivation was induced by DTAC.
Acyclothymidine (AcyT, 5-methyl-1-(2'-hydroxyethoxymethyl)uracil), a potent inhibitor of pyrimidine nucleoside phosphorylase (PyNPase), was co-administered with 5'-deoxy-5-fluorouridine (5'-DFUR), a PyNPase activating prodrug of 5-fluorouracil (5-FU), to rabbits. The absorption and pharmacokinetic parameters of 5'-DFUR and its active metabolite 5-FU, after administration of 5'-DFUR in combination with AcyT, were evaluated in the animals. Animals were given an oral or intravenous administration of 5'-DFUR (50 mg/kg) in combination with an equimolar dose of AcyT (40 mg/kg). The half-lives (t1/2) of 5'-DFUR and 5-FU in plasma were 16.8 and 11.5 min, respectively. AUC (area under the plasma concentration-time curve) of 5'-DFUR and 5-FU following the oral administration of 5'-DFUR (50 mg/kg) was 1710 and 24.3 μg·min/ml, respectively. After the oral co-administration of 5'-DFUR and AcyT (at a molar ratio of 1 : 1), the AUC values for 5'-DFUR and 5-FU increased to 2680 and to 121.1 μg·min/ml, respectively. However, this combination had little effect on the t1/2 of 5'-DFUR.