A rhamnose-binding lectin isolated from Silurus asotus (catfish) roe by DEAE-cellulose ion exchange and galactose-Sepharose affinity chromatographies predominantly agglutinated human type B and rabbit erythrocytes. S. asotus lectin (SAL) also agglutinated sarcoma 180 ascites carcinoma cells, but not AH109A cells. The most effective saccharide in hemagglutination inhibition assay was L-rhamnose. The monosaccharides possessing steric similarity to the hydroxyl group orientation at C2 and C4 of the pyranose ring structure of L-rhamnose, such as L-mannose and L-lyxose, were also effective. The molecular weight of SAL was determined to be 38000 by size exclusion chromatography on TSK gel G3000SW and 33000 by SDS-polyacrylamide gel electrophoresis under reducing conditions. SAL did not require a Ca²⁺ ion or free thiol group for its agglutination activity. The N-terminal 29 amino acid sequence was determined by a gas-phase sequencer as follows, ANMITCYGDVQKLHXETGLIIVKSXLYGR (X : not determined). It has no homology to the sequences of well known vertebrate lectins.

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Sodium selenate (selenate), as well as insulin, increased the lipoprotein lipase (LPL) activity in isolated rat fat pads in a time- and dose-dependent manner. The increase effect of selenate was not additive to that of insulin. The action of selenate and insulin was decreased by amiloride and disappeared when Ca²⁺ was omitted from the incubation medium. Loading of a chelator of intracellular Ca²⁺ to the fat pads also greatly inhibited the action of selenate. The maximal increase in inositol 1, 4, 5-trisphosphate (IP₃) content was observed with a 30-s incubation of the fat pads with selenate. Dibutyryl cyclic AMP, 3-isobutyl-1-methylxanthine, carbonyl cyanide m-chlorophenylhydrazone, tunicamycin, and monensin all inhibited the increase effect of selenate on the LPL activity to various extents. These results suggest that selenate increases the LPL activity via amiloride- and monensin-sensitive processes, involving the Ca²⁺ mobilization linked to a rapid increase in the IP₃ content in fat pads.

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The mechanism of inactivation of papain (EC 3.4.22.2) by ascorbic acid (AsA) in the presence of cupric ions (Cu²⁺) was investigated. The aerobic combination of Cu²⁺ and AsA resulted in an irreversible loss of enzyme activity. The inactivation was found to be an apparent first order reaction. The prior mixing of Cu²⁺ and AsA caused the complete disappearance of the inactivation. The addition of iron ions led to significant suppression against the inactivation. Cu²⁺ was bound to the enzyme in a molar ratio of 1 : 1. At lower concentrations of Cu²⁺ (molar ratio of enzyme to Cu²⁺ of 1 : <1), the extent of inactivation showed the same dependence against the extent of oxidation of AsA. The rate of inactivation increased as the concentration of AsA was increased. Saturation kinetics were observed with respect to the concentration of AsA. Changes in the concentration of Cu²⁺ had no effect on the dissociation constant of the enzyme-AsA complex (K_I), though the rate constant of inactivation (k₂) showed a linear relationship with the concentration of Cu²⁺. At various pH values tested, no change of k₂ was found, whereas the value of K_I increased when the pH bacame lower. At higher concentrations of Cu²⁺, the rate of inactivation fell beyond a certain concentration of AsA.The present results suggest that both Cu²⁺ and AsA bind to the enzyme to form a ternary complex and that free radicals are site-specifically formed and react preferentially with the enzyme, at the site of their formation, impairing its activity.

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The effects of lipid peroxidation on the surface charge density of the porcine intestinal brush-border membranes were studied using an oxygen-radical-generating system consisting of ascorbic acid, ferrous ion and tert-butyl hydroperoxide (tert-BuOOH). Changes in the membrane surface charge density were monitored using a fluorescent dye, 8-anilino-1-naphthalenesulfonate (ANS). The incubation of the membranes with ascorbic acid/Fe²⁺/tert-BuOOH resulted in a decrease of the fluorescence intensity of the ANS-membrane complex with a red-shift in the emission maximum, depending on the hydroperoxide concentration and the incubation time. The kinetic studies on ANS-binding showed that the apparent dissociation constant of ANS-membrane complex decreased by treatment with ascorbic acid/Fe²⁺/tert-BuOOH. Similar results were also obtained by treatment of the membranes with other oxidizing systems, hematin/tert-BuOOH and dipyridyl/Fe²⁺/tert-BuOOH. These results proposed the possibility that lipid peroxidation of the membranes causes an increase of the positive charge on the membrane surface. The results of the dependence of the ionic strength with increasing KCl concentrations in the medium upon the ANS-binding affinity for the membranes further supported this interpretation.

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Two acidic polysaccharides, named ginsenan PA and ginsenan PB, were isolated from the root of Panax ginseng C. A. MEYER. They were homogeneous on electrophoresis and gel chromatography, and their molecular masses were estimated to be 1.6×10⁵ and 5.5×10⁴, respectively. They are composed of L-arabinose : D-galactose : L-rhamnose : D-galacturonic acid : D-glucuronic acid in the molar ratios of 11 : 22 : 1 : 6 : 1 (ginsenan PA) and 3 : 7 : 2 : 8 : 1 (ginsenan PB), in addition to small amounts of O-acetyl groups. Almost all (ginsenan PA) and part (ginsenan PB) of the hexuronic acid residues exist as methyl esters. Reduction of carboxyl groups, methylation analysis, nuclear magnetic resonance and periodate oxidation studies indicated that their structural features include mainly both α-arabino-β-3, 6-galactan type and rhamnogalacturonan type structural units. Both polysaccharides showed remarkable reticuloendothelial system-potentiating activity in a carbon clearance test, pronounced anti-complementary activity and alkaline phosphatase-inducing activity in a dose dependent manner.

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A segment of the neocarzinostatin apoprotein gene corresponding to T³⁰ to A⁹¹ of the protein was amplified using a polymerase chain reaction (PCR) with total DNA from Streptomyces carzinostaticus subsp. neocarzinostaticus E-793 (ATCC 15944) as the template and with 5'- and 3'-primers synthesized in consideration of the codon usage of streptomyces. The PCR product was cloned, sequenced and confirmed to direct an amino acid sequence reasonably well matching that reported. Using the PCR product as a probe, we cloned a DNA segment (2580bp) spanning an open reading frame (ORF) for preapoprotein (leader peptide plus apoprotein) and its upstream and downstream flanking regions. The amino acid sequence deduced from the base sequence of the DNA clearly identified those amino acid residues which had remained inconsistent among different research groups. The base sequence homology with other apoprotein genes of related antibiotics was analyzed and was found to be limited within the structural gene.

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The peroxidation of lipids and changes in the activities of related enzymes, such as xanthine-xanthine oxidase (XOD), superoxide dismutase (SOD), and glutathione peroxidase (GSH-px) in the gastric mucosa were studied in rat model of ischemia-reperfusion with pylorus ligation. Myeloperoxidase (MPO), a marker enzyme of leucocytes, was also studied. Thiobarbituric and reactive substances (TBA RS) in gastric mucosa were signifucantly increased by clamping the celiac artery for 30min and reperfusion for 60min after 3h of pylorus ligation. XOD activity in gastric mucosa increased with the development of gastric mucosal injury. Allopurinol significantly suppressed XOD activity but did not inhibit mucosal injury as the increase in TBA RS. MPO activity in the gastric mucosa was significantly increased by gastric mucosal injury. Famotidine significantly inhibited the increase in MPO activity in gastric mucosa, while allopurinol did not. SOD and GSH-px activities in the gastric mucosa were decreased significantly by gastric mucosal injury. SOD activity was normal following treatment with famotidine and allopurinol. Moreover, GSH-px activity recovered to the normal level with famotidine and allopurinol treatment. These findings suggest that oxygen radicals and lipid peroxidation can cause gastric mucosal injury by ischemia-reperfusion in the pylorus-ligated rat. The generation of oxygen free radicals may be derived mainly from activated polymorphonuclear leukocytes (PMN), and the decrease in SOD and GSH-px activity in gastric mucosa seems to aggravate mucosal injury by free radicals and lipid peroxidation.

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Local cerebral glucose utilization (LCGU) was investigated in rats with decerebrate rigidity using the [¹⁴C]2-deoxyglucose (2DG) method in order to identify the site responsible for the rigidity. LCGU was increased in the medial vestibular nucleus, the fastigial nucleus, the interpositus nucleus and the dentate nucleus during decerebrate rigidity. Diazepam and tizanidine, centrally acting muscle relaxants, reduced the increase of LCGU in the vestibular and cerebellar nuclei. These results suggest that the vestibular and cerebellar nuclei are the possible sites responsible for the development of decerebrate rigidity and the action of some centrally acting muscle relaxants.

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Of the 21 compounds evaluated for antiimplantation and abortifacient activities, compounds (A₁, A₂, A₄ and B₁) and compounds (C₁, C₂, D₁ and D₃) were found to exhibit 40% and 30% antimplantation activity, respectively, in female rats when given orally on days 1-5 postcoitum. The remaining 13 compounds were found to be inactive. All of the 21 compounds were also tested for the abortifacient activity, but all were found to be inactive.

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To ascertain if there is stereoselectivity in peroxisomal proliferation induced by chiral peroxisome proliferators, induction by stereoisomers of 2-methyl-4'-chlorophenoxyacetic acid and 2-methyl-2-(2', 4'-dichlorophenoxy)acetic acid were studied in rat in vivo and in vitro with isolated rat hepatocytes. No significant differences in the inducing potencies of the stereoisomers of the above two phenoxyacetic acid derivatives were found for cyanide-insensitive fatty acyl-CoA oxidizing system, fatty acyl-CoA oxidase, carnitine acetyltransferase or carnitine palmitoyltransferase. There was also no significant difference in the degree of hepatomegaly or lipid-lowering effect between the isomers. The findings with cultured rat hepatocytes agreed with those of the studies in vivo.

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A high-performance liquid chromatographic method to determine (Me)Arg-Lys-Pro-Trp-tert-Leu-Leu (NT-2) with neurotensin (NT) activity in rat plasma was developed and a pharmacokinetic study was performed in rats. Quantitative analysis with high reproducibility was achieved for NT-2 over the concentration range of 1.14-500ng/ml.(Me)Arg-Lys-Pro-Trp-tert-Leu-Leu-OEt (NT-1) was rapidly hydrolyzed to NT-2 in rat plasma at 37°C. This degradation of NT-1 was observed as a pseudo first-order reaction, and the pseudo first-order rate constant was calculated to be 7.26min^-1 (t_1/2=0.095min).The pharmacokinetic profiles of NT-2 after intravenous administration of NT-1 at doses of 0.1 and 0.3mg/kg were compatible with that of NT-2 after intravenous administration of NT-2 at 0.5mg/kg. Range of the half-life of the terminal phase (t_1/2β) of NT-2 was 0.36-0.53h.The absolute bioaveilabilities after oral administrations of NT-1 and NT-2 at a dose of 20mg/kg were 0.61±0.17 (mean ±S.E.) and 0.19±0.08%, respectively. It was found that NT-1 was more suitable for oral administration than NT-2.

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The conjugate between N-succinyl-chitosan (Suc-chitosan) and mitomycin C (MMC), named Suc-chitosan-MMC, and that between carboxymethyl-chitin (CM-chitin) and MMC, named CM-chitin-MMC, were investigated in vivo. As for the intraperitoneal drug administration using rats, the order of the maximum blood concentration of MMC was unconjugated MMC>CM-chitin-MMC>Suc-chitosan-MMC. The plasma concentration of MMC for Suc-chitosan-MMC was maintained at an almost constant level over 24h. Pharmacokinetic analysis of each plasma concentration indicated that for each conjugate, the in vitro drug release reported previously was useful for the approximate estimation of the in vivo regeneration of MMC. The chemotherapeutic effect of MMC and the conjugates was investigated using mice bearing L1210 leukemia of B16 melanoma. Concerning antitumor activity against L1210 leukemia, the conjugates exhibited a marked effect at higher doses, and their effect increased even in the dose range where the effect of MMC decreased. MMC and the conjugates exhibited a good growth-inhibitory effect against B16 melanoma. Complete inhibition of growth of B16 melanoma was observed at the dose of 10mg eq MMC/kg for CM-chitin-MMC.

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cis-Diamminedichloroplatinum (II) (cisplatin : CDDP) suppositories containing NaCl at different concentrations were prepared as a local chemotherapeutic agent for the treatment of uterine endometrial carcinoma and were adminstered to rabbits implanted with uterine VX₂ tumor. The intrauterine CDDP histological level, as well as the antitumor effects and side effects of the suppositories to the liver and kidney were studied. The results showed high intrauterine tissue CDDP level in all suppository administrations.In particular, the NaCl-added suppositories enhanced the intrauterine CDDP level. As for antitumor effects, while the tumor growth rate of the NaCl-added suppository group was likely to be suppressed, the suppositories could not suppress tumor growth completely. The plasma platinum (Pt) level was 1.5μg/ml or less and that of the liver and kidney was as low as 0.31 to 0.48μg/g. No difference in levels depending on NaCl concentration was observed, nor was any abnormality found in the biochemical analysis including glutamate oxaloacetate transaminase (GOT) and blood urea nitrogen (BUN). Histopathological study revealed the degeneration of tumor cells in the NaCl-added suppository group. Minimal congestion and hemorrhage were observed in the endometria, possibly resulting from CDDP. By adding NaCl to CDDP suppositories, the uterine CDDP level and antitumor effects increased while no serious renal dysfunction was noted. Therefore, we conclude that NaCl-added CDDP suppositories are a useful local chemotherapy for endometrial carcinoma.

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The population pharmacokinetics of theophylline during constant-rate intravenous infusion has been studied in 66 children (8.7±4.7 year of age; 26.0±12.8kg, mean±S.D.) with an episode of acute asthma. One hundred and twelve theophylline serum concentrations (13.9±4.8μg/mi) collected retrospectively were analyzed using a nonlinear mixed-effect model. The influence of hepatic dysfunction, age, gender, days after admission, blood gas measurements (PaO₂, PaCO₂, blood pH) and clinical analyses data (total serum protein, albumin concentration, haemoglobin concentration, haematocrit) on theophylline clearance was examined by the likelihood ratio test. A final estimate of population mean clearance was 58.6ml/kg/h, which was decreased by 36% in patients with hepatic dysfunction. Other factors tested displayed no statistically significant effect on theophylline clearance. The inter-individual variability in clearance was 26% while the intra-individual variability in theophylline concentrations was 2.6μg/ml as a standard deviation, which was almost double that observed for stable patients. Taking into account that the therapeutic window of this drug is 10-20μg/ml, this value indicates a relatively large intra-individual variability and suggests that frequent (daily)monitoring of serum concentrations is necessary for patients with an episode of acute asthma.

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The effect of the increased plasma protein binding of quinidine on its disposition was investigated in turpentine-treated rats, since turpentine treatment is known to increase the plasma concentration of α₁-acid glycoprotein which preferentially binds basic drugs. The plasma free fraction of quinidine 16 and 48h after turpentine treatment was decreased by 30 and 76%, respectively, compared to the control value. The treatment did not cause liver injury nor alter the hepatic blood flow. The disappearance of quinidine in plasma after an intravenous injection (3.0, 7.0, 12.5mg/kg) was analyzed by a two-compartment open model in both control and turpentine-treated rats. The blood total body clearance (CL_b) of quinidine at 48h after the treatment was decreased by 30 to 65% in a dose-dependent manner, compared to that in control rats. The distribution volume (V_dss) of quinidine (12.5mg/kg) at 16 and 48h after turpentine treatment was decreased by 30 and 79%, respectively.Hepatic extraction ratio (HER) of quinidine, which was determined at steady state blood concentrations from 0.5 to 2.3μg/ml, was decreased from 0.8 to 0.35 with an increase in the quinidine concentration in control rats. The HER value 48h after turpentine treatment was consistently reduced by 15 to 40% in a concentration-dependent manner compared to the corresponding control value. These findings indicate that the increased plasma binding of quinidine caused a reduction of HER of the drug, and the reduced HER resulted in the decrease in CL_b in turpentine-treated rats.Tissue-to-plasma partition coefficient (K_p) for the lung, kidney, spleen, liver and heart, which was determined at a steady state plasma concentration (1μg/ml), was decreased after the turpentine treatment to the same extent as the decrease in V_dss (16h, 28-39%; 48h, 76-81%). The K_p value in each tissue was proportional to the free fraction of quinidine in the plasma. These results indicate that V_dss and K_p were reduced due to the increase in the plasma protein binding of quinidine in turpentine-treated rats.

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In order to determine an advantageous site for intestinal insulin absorption, the hypoglycemic effects of insulin after administration to the duodenum, the jejunum, the ileum and the colon were investigated using an in situ loop method. Insulin solution was administered to the various loops of fasted rats with or without aprotinin (AP) as a protease inhibitor, or absorption enhancers such as sodium caprate, Na₂EDTA or sodium glycocholate. An obvious hypoglycemic effect of insulin alone was seen only in the ileum loop washed with phosphate buffered saline. When coadministered with AP, the most remarkably amplified effect was again observed in the ileum. In the ileum, the area under the serum insulin levels vs. time curve from 0 to 4h was linearly related to the logarithm of the AP dose. Both sodium caprate and Na₂EDTA significantly promoted the hypoglycemic effect of insulin at all sites, and their intensity increased towards the distal regions of the intestine. On the other hand, sodium glycocholate improved only colonic insulin efficacy. These results suggest that the ileum seems to be the most useful region in the small intestine for insulin absorption; however, insulin must be protected from proteolysis to enhance its absorption. In addition, the insulin efficacy could be increased by absorption promoters more effectively in the colon than in the small intestine.

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The systemic effects of epidermal growth factor (EGF) ointment containing nafamostat (NM), gabexate, or gelatin was studied in rats with burns or open wounds. At 1d after burn, plasma epinephrine, cortisol, and glutamic-oxalacetic transaminase (GOT) levels were elevated, but treatment with EGF plus NM (EGF+NM) ointment significantly suppressed the increase in these levels. Further, there was no loss of body weight in the open wound model following treatment with EGF+NM ointment, while loss of body weight occurred in animals in which EGF ointments without NM were applied. Increases in plasma epinephrine 1d after open wound formation were also suppressed by the application of EGF+NM ointment. Treatment with EGF ointment containing gabexate (GX) or gelatin (GL) ameliorated changes in body weight that occurred after open wound formation, while loss of body weight in animals with open wounds occurred following the application of ointment base, EGF ointment, GX ointment, or GL ointment. The present study thus indicates that the topical application of EGF ointment containing a protease inhibitor has ameliorative systemic effects.

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Two efficient extraction methods for phospholipids have been established employing a TLC plate with a concentration zone or a solid-phase extraction cartridge. First, small amounts of five authentic phospholipids, 10 to 100μg each, were separated on a TLC plate with a concentration zone. These phospholipids were not adsorbed to the silica gel of this TLC plate and the substrates were recovered in good yields. Second, a solid-phase extraction cartridge containing NH₂-modified silica gel was able to retain all of the authentic phospholipids among C₁₈-, diol-, and CN-modified silica gel and silica gel beds. Elution of neutral and acidic phospholipids from the NH₂-cartridge was carried out with a chloroform-methanol mixture and a chloroform-methanol-28% ammonium mixture containing 0.05M ammonium acetate, respectively.Recovery tests on the above two methods were carried out by conventional capillary gas chromatography. Thus, not less than 95% of each phospholipid was recovered from the TLC plate or NH₂-cartridge in a 10 to 100μg sample range. Also, the standard deviation of repeatability of recovery for phosphatidylcholine by solid phase extraction at a 10μg sample range was favorable at σ=2.8. This method of separation was applied to determination of the fatty acid compositions of phospholipids in small amounts of plasma.

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Tetracycline resistance protein (TET) of Bacillus subtilis plasmid pNS1 was detected by immunoblot analysis using a specific antibody to TET-chloramphenicol acetyltransferase (CAT) fusion protein. In two-dimensional electrophoresis, one major spot which seemed to be the pNS1-encoded TET (pNS1-TET), was detected by immunostaining. Its molecular weight and isoelectric point were approximately 52kDa and 6.2, respectively. Judging from the nucleotide sequence, the pNS1-TET is a very hydrophobic, 50kDa protein. Therefore, the 52kDa protein is thought to be an intact form of the pNS1-TET produced by B. subtilis cells.

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Effects of lipid peroxide breakdown products, (E)-4-hydroxy-2-nonenal (4-HN) and n-hexanal, on mouse lung lesion were examined. When 4-HN was injected i.v., the plasuma level of 4-HN increased just after the injection and then decreased immediately. The amounts of 4-HN increased in the liver and lung were ca. 0.085 and 0.43% to the dose administered, respectively, 5min after the injection. Reduced glutathione (GSH) content and both GSH peroxidase (GSH-Px) and GSH reductase (GSSGR) activities in the lung were decreased significantly by 4-HN treatment. On the other hand, in the case of i.v. injection of n-hexanal into mice, the amount of n-hexanal detected in the lung was 5.0% to that of 4-HN, and no effect on the activities of GSH-Px and GSSGR and the content of GSH was observed.These results suggest that 4-HN generated from lipid peroxides would be transferred into the lung and cause the lung lesion through the inhibition of GSH-dependent antioxidative defense systems.

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We previously identified a factor, designated E1A-stimulating factor 1 (ESF-1), that stimulates transcription of the adenovirus type 12 E1A gene in vitro by binding to a region next to a TATA box. We report here on the partial characterization of the mode of ESF-1 action. Point mutations within the nucleotide sequence 5'-TCAGCTGA-3' abolished the binding of ESF-1. Stimulation of transcription of the adenovirus type 12 E1A gene in vitro was lost when a 7 base pair insertion was introduced between the ESF-1-binding site and a TATA box. These results suggest that ESF-1 stimulates adenovirus type 12 E1A gene transcription by interacting with a palindromic DNA sequence present in a region immediately upstream from a TATA box.

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2'-Chloroflavone (CF) and 4'-CF were studied as inducers of cytochrome P-450-mediated monooxygenases in rat hepatic microsomes by comparison with phenobarbital (PB) and 3-methylcholanthrene (MC). As a result of interaction with the substrate 7-ethoxycoumarin (7-EC), 2'-CF-induced microsomes showed a difference spectrum different from usual type I, whereas 4'-CF-induced microsomes showed a typical type I spectrum. 2'-CF-induced microsomes increased the V_max of aminopyrine (AM) N-demethylase activity about 1.6-fold while not affecting the apparent K_m value. In contrast, 4'-CF-induced microsomes decreased the K_m of 7-EC O-deethylase activity to about one fourth while significantly increasing the V_max value. All the activities of AM N-demethylase, 7-EC O-deethylase and 6, 7-dimethoxycoumarin O-demethylase in 2'-CF-induced microsomes were inhibited strongly by metyrapone and SKF 525-A, and significantly by diphenhydramine. The three enzyme activities in 4'-CF-induced microsomes were inhibitied markedly by α-naphthoflavone and significantly by 2-bromo-4'-nitroacetophenone. These results further support the previous proposal that 2'-CF is a PB-type inducer of cytochrome P-450 while 4'-CF is a MC-type inducer.

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Absorption of recombinant human granulocyte colony-stimulating factors (rhG-CSF) following intranasal administration of aqueous preparations containing rhG-CSF with or without dimethyl-β-cyclodextrin was investigated. It was found that rhG-CSF was absorbed and the total leukocyte numbers in peripheral blood markedly increased after intranasal instillation of rhG-CSF solution (pH 4) without excipients. Simultaneously administered dimethyl-β-cyclodextrin enhanced nasal absorption of rhG-CSF. Consequently, serum rhG-CSF concentrations and total leukocyte numbers were greatly increased.

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Acetone alkylhydrazones were readily autooxidized to 2-alkylazo-2-propyl hydroperoxides, which were directly mutagenic in Salmonella typhimurium TA1535, TA100, TA102 and Escherichia coli WP2hcr^-. The mechanism of this mutagenicity presumes that the hydroperoxides in aqueous solution decompose to alkyl diazonium ions which were observed in the alkylation of 4-(p-nitrobenzyl)pyridine, and also to hydroxyl radical which was detected by ESR.

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