When phenylalanine was incubated with myeloperoxidase (MPO) and NADH in citrate buffer (pH 4.5), o-, m-, and p-tyrosines were identified as hydroxylated products. Tyrosine formation was dependent on the reaction time and MPO concentration. No significant quantities of tyrosines were formed on if MPO was omitted and inactivated MPO was added instead of active MPO. The tyrosine formation by the MPO-NADH system was greatly reduced under anaerobic conditions, and significantly inhibited by hydroxyl radical scavengers. Superoxide dismutase was a potent inhibitor, but catalase was less effective. Even though the superoxide radical (O^-₂)-producing ability of the MPO-NADH system was about 29% of that of the hypoxanthine-xanthine oxidase system, under the experimental conditions employed, the rate of tyrosine formation from phenylalanine by two systems was found to be a similar. The above results suggest that the formation of a hydroxyl radical (OH·) may occur in the MPO-NADH system under aerobic conditions and a superoxide radical may be involved in the OH· formation, with MPO promoting the OH· formation from O^-₂.

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A self-complementary decadeoxyribonucleotide, 5'd(GAAGFGCTTC)3', containing 5-fluorocytosine (F) in substitution for cytosine at the methylation site of DNA-cytosine methylase Hha I (MHha I) has been synthesized. MHha I was inhibited by the pre-incubation of the enzyme with d(GAAGFGCTTC).

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The macular mutant mouse is a model of Menkes' kinky hair disease, which is characterized by a deficiency of ceruloplasmin in the serum. In hemizygotic mice (ml/y), the antibody production against sheep red blood cells (SRBC) was decreased. Treatment with normal (+/y) mouse serum increased in a dose-dependent manner the antibody response of ml/y mouse spleen cells in vitro. However, +/y mouse serum had no effect on +/y mouse spleen cells, even at high doses up to 5%. In contrast, ml/y mouse serum decreased antibody production in +/y mouse spleen cells in a dose-dependent manner. Antibody production in +/y mouse spleen cells decreased with time, after pretreatment with ml/y mouse serum. However, serum from ml/y mice injected with ceruloplasmin did not decrease antibody production in +/y mouse spleen cells but rather increased it in ml/y mouse spleen cells. Ceruloplasmin had no effect on the production of antibodies against SRBC in ml/y and +/y mouse spleen cells in vitro. These findings suggest that one or more enhancers of antibody production exist in normal mouse serum and that one or more suppressors of normal antibody production exist in ml/y mouse serum. It is proposed that the activities of these factors in serum may be regulated by ceruloplasmin.

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A simple analytical method, using capillary gas chromatography-mass spectrometry (GC-MS) was used to investigate the oxidative modification of cholesterol in copper-oxidized low-density lipoprotein (oxidized LDL). The incubation of LDL and 5 μM CuSO₄ in 0.15M NaCl/0.02M Tris-HCl buffer at pH 7.4 led to the peroxidation of LDL as shown by the detection of a thiobarbituric acid-reactive substance (TBA-RS). Oxidized LDL was shown to cause a significant fall in the unesterified and esterified cholesterol content, while oxysterols such as 7-hydroxycholesterol (7-OH) and 7-ketocholesterol (7-Keto) were formed from cholesterol, these conversions occurred in a dose- and time-dependent manner. Both oxysterols were identified by GC-MS on a fused-silica capillary column. In addition, copper-oxidized high-density lipoprotein (oxidized HDL) also resulted in the generation of TBA-RS and oxysterols. In addition, changes in the composition and amount of oxysterols during the incubation of macrophages with oxidized LDL were investigated. The incubation of macrophages with oxidized LDL resulted in the accumulation of cholesterol, 7-OH and 7-Keto derivatives in macrophages and this accummulation also occurred in a dose-dependent manner.These results suggest that the measurement of oxysterols may afford an additional index of oxidized LDL, and that foam cell formation and early atherosclerotic lesions may be responsible for the accumulation of oxysterols in macrophages. In addition, this study shows that HDL is also modified when submitted to an oxidative process.

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The interaction of a protein-bound polysaccharide (PSK) with caldesmon was studied in detail using sedimentation, low-shear viscometry, electron microscopy and affinity chromatography. The binding of caldesmon to F-actin was concentration-dependently inhibited by PSK. Bundle formation of actin filaments owing to caldesmon was also inhibited by PSK. Caldesmon bound to a PSK-Sepharose 4B column at low ionic strength was released at about 400 mM NaCl, whereas G-actin was not retained by the column. Treatment of caldesmon with chymotrypsin produced major fragments near 100, 80, 60, 38 and 25 kDa. In contrast, 60 and 25 kDa fragments were rarely formed by this treatment in the presence of PSK. Fragments of 80 and 38 kDa, major products produced by chymotrypsin, bound individually to a PSK-Sepharose column, indicating that caldesmon has at least two binding sites for PSK. Addition of calcium and calmodulin partially released caldesmon from actin filaments. PSK-dependent release of caldesmon was also observed in the presence of calcium and calmodulin.

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Two clones of the K562 human leukemic cell line were isolated according to hemoglobin (Hb) expression. One clone was expressed less than 5% (K562-L) and the other more than 90% (K562-H). The two clones did not exhibit any difference in cell growth or cell cycle. However, the Hb expression of K562-H cells was reduced by succinylacetone (S.A.). The above results suggested that the difference in the Hb production of K562-L and K562-H cells depended on the heme synthetic activity. On the other hand, glycophorin A was expressed to a greater extent on K562-L cells than on K562-H cells. These findings suggested that heme synthesis and the expression of glycophorin A on K562 cells were not always related. The CD11b and the CD61 were also expressed to a greater extent on K562-L cells than on K562-H cells, but the CD34 was not expressed on these cells.

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trans-4-Guanidinomethylcyclohexanecarboxylic acid 4-tert-butylphenyl ester (GMCHA-OPh^tBu), a trypsin inhibitor, dose-dependently inhibited the growth of Escherichia coli K-12 IAM1264. Growth inhibition was preceded by dose- and time-dependent inhibition of DNA synthesis. These results strongly suggested participation of a trypsin-like proteinase in DNA synthesis. To clarify this suggestion, the effects of GMCHA-OPh^tBu on the doubling time and on the uptake of [methyl-³H]thymidine into DNA were examined with E. coli K-12 IAM1264 synchronized by a modified version of phosphate starvation. The synchrony lasted for two or three cycles with a doubling time of 55 min and a cell division period of 15 min. The cell cycle of E. coli was divided into three periods, cell division period (P), the period between cell division and initiation of chromosome replication (Q) and the period between initiation of chromosome replication and cell division (R). The R period was subdivided into two periods, R1 in which the rate of thymidine uptake into DNA was increasing, and R2 in which it was constant. The addition of GMCHA-OPh^tBu at the R1 period did not affect the already-initiated round of cell division, however, it retarded the next round. The addition at P, Q or R2 retarded the cell division in the same round, causing prolongation of the R1 period. A sharp and momentary appearance of trypsin-like proteinase activity peaked at the Q/R1 boundary in one cell cycle, and inhibition of the activity prolonged the R1 period. These results suggest that the proteinase probably participates in the onset of the chromosome replication in the synchronized E. coli cells. The proteinase was prepared from exponentially growing E. coli cells and characterized.

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Release of HeLa cells arrested at the G1/S boundary by double-thymidine block caused abrupt uptake of [methyl-³H]thymidine into DNA after 5 min, and two sharp high activity peaks, peak I and peak II, were observed 8 and 23 min after removal of the thymidine block and this was follwed by a gradual uptake of [³H]thymidine. The duration of the cell cycle was 23 h, and definite changes in cell density were observed between 12 and 13 h and also between 35 and 36 h after removal of the thymidine. Addition of amidinopiperidine-4-carboxylic acid 4-tert-butylphenyl ester (APCA-OPh^tBu), a trypsin inhibitor, immediately after removal of the arrest strongly suppressed DNA synthesis and mitosis. In contrast, addition of APCA-OPh^tBu 10 min after removal of the arrest, and hence also after the appearance of peak I, had no effect on peak II nor on the uptake of thymidine occurring during the remainder of the first cell cycle, nor on mitosis. However, it strongly suppressed the second DNA synthesis and mitosis. These results suggest participation of a trypsin-like proteinase at the onset of DNA synthesis. Removal of thymidine from the arrested cells at a cell density of 2% (4×10³ cells/cm²) induced an immediate and rapid rise in trypsin-like proteinase activity. However, the activity decreased with increasing cell density. No clear increase in the activity was seen at a cell density of 20% (4×10⁴ cells/cm²). However, both trypsin-like proteinases obtained at cell densities of 2% and 20% were strongly inhibited by APCA-OPh^tBu and these inhibitory effects were similar. The proteinase was named proteinase In.

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We examined the inhibitory effect of chlorpromazine, a phenothiazine derivative, on SV40 DNA replication in a HeLa cell extract. The inhibition by chlorpromazine was reversed by cardiolipin, phosphatidylinositol, phosphatidylglycerol, and phosphatidylserine. Studies on a staged reaction showed that the pre-elongation step was sensitive to chlorpromazine : chlorpromazine-resistant DNA synthesis was observed when the drug was added after the pre-elongation step. Analysis of replication products by agarose gel electrophoresis revealed that some steps of DNA chain elongation and maturation of closed circular forms were also sensitive to this drug.

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The effect of oxatomide on reactive oxygen species (ROS) generated both by neutrophils and in a cell-free, xanthine-xanthine oxidase system was examined. The species investigated were superoxide radical anion (O^-₂), hydrogen peroxide (H₂O₂) and hydroxyl radical (OH·). Oxatomide significantly decreased neutrophil-generated O^-₂, H₂O₂ and OH· in a dose-dependent manner. H₂O₂ and OH· generated in a cell-free system were also reduced in the presence of oxatomide.The present study indicates that oxatomide decreases ROS generation presumably by inhibiting the neutrophil oxygen metabolism, and has the ability to scavenge H₂O₂ and OH·.

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The activity of imipramine 2-hydroxylase highly correlated with that of desipramine 2-hydroxylase but not with that of desipramine N-demethylase. The correlation was also found between N-demethylation and 2-hydroxylation when imipramine was used as a substrate, whereas no correlation was observed between them when desipramine was used in place of imipramine. Both activities of desipramine and imipramine 2-hydroxylase were markedly inhibited by quinidine but not by quinine. Although the activity of imipramine N-demethylase was slightly inhibited by both quinidine and quinine, the activity of desipramine N-demethylase was unaffected under the same conditions. The activity of imipramine N-demethylase was roughly correlated with the amounts of P450 3A4 immunochemically determined and the activities of testosterone 6β-hydroxylase in human liver microsomes. The P450 3A4 catalyzed imipramine N-demethylation much more efficiently than 2-hydroxylation in a reconstituted system, whereas neither N-demethylation nor 2-hydroxylation of desipramine was catalyzed by P450 3A4. The activity of imipramine N-demethylase was inhibited, to various extents, by anti-P450 3A4 antibodies in human liver microsomes.Taking together these and other results, it is suggested that P450 3A4, other than P450 2Cmp, also partly contributes to N-demethylation of imipramine, depending on human liver microsomes.

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To confirm the inflammatory activity of polymeric photoproducts (CPZ-polymers) of chlorpromazine (CPZ), which were obtained by gel filtration of a UV-pre-irradiated CPZ aqueous solution, the histamine release from rat peritoneal exudate cells was studied and the paw-inflammation in mice induced by these CPZ-polymers was examined. CPZ-polymers induced a dose-dependent histamine release at concentrations of 1, 3 and 10 mg/ml. This effect was approximately one-tenth of that of compound 48/80. Furthermore, CPZ-polymers markedly induced lasting paw-edema in a dose-dependent manner, the swellings remaining for at least 96 h. When intraperitoneally injected into mice, CPZ-polymers induced a significant elevation of histamine release in the peritoneal cavity 0.5 h after the injection, compared with a control group. The histamine levels in the cavities returned to normal within the next 0.5 h, and remained normal for at least 23 h, indicating that histamine release may be caused only in the early stages of CPZ-polymer-induced inflammation. The inflammatory activity of the CPZ-polymers suggests that they are inflammatory substances formed from CPZ by UV-irradiation.

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In 3, 4-methylenedioxyamphetamine (MDA)-treated rat brain and urine, 3-methyl-6, 7-methylenedioxy-1, 2, 3, 4-tetrahydroisoquinoline (3Me6, 7MDTIQ) was detected as a new metabolite. The content of 3Me6, 7MDTIQ in (R)-MDA-treated rat was significantly higher than that in (S)-MDA-treated brain and urine. This result suggests that this metabolism of MDA contains stereoselective pathways.The pharmacological effects of 3Me6, 7MDTIQ on behavior differed from those of MDA. The ambulation score of 3Me6, 7MDTIQ was significantly decreased compared to the control, and the rearing score of 3Me6, 7MDTIQ was moderately decreased compared to the control in an open-field test.

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To avoid development of a lesion on the small intestine by acemetacin (ACM) following oral administration of the drug, we developed a new device for its percutaneous application. The device for transdermal application of ACM consisted of a silicon O-ring, a backing of aluminum foil and adhesive tape, and rate-controlling membranes with three different pore sizes (HP-1100, 2100 and 4050). Two percent ACM gel ointment was contained in the device. In the in vitro release experiment, the ACM release from the device was limited by these membranes with release rate constants of 0.630±0.052, 0.289±0.012, 0.098±0.11 and 0.083±0.011h^-1 for no membrane, HP-4050, HP-2100 and HP-1100 membranes, respectively. In the in vitro penetration experiment, the ACM penetrating through the skin appeared in the reservoir cell without the metabolic conversion to indomethacin (IM). After the application of the ACM device with the HP-2100 rate-controlling membrane on the rat abdominal skin, ACM was not detected in the plasma but the therapeutic plasma concentration of IM could be maintained over a 54h period. These results indicate that the device with a rate-controlling membrane may be useful for the percutaneous application of ACM as an anti-inflammatory drug and its clinical application. For the percutaneous absorption of ACM after application of the ACM devices, the values estimated by the proposed model which consisted of 6 compartments well fit to the data obtained from this in vivo experiment. It has therefore been proved that the model described in this paper can significantly predict plasma IM profiles after application of the ACM device.

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Iontophoretic and passive transport of an ionized drug (sulfisoxazole) across excised rat skin was studied using a two-chamber cell with four electrodes under successive experimental conditions : without electrical current (stage-I) and with electrical current (stage-II). Two iontophoretic/diffusion models, i.e. a one-layer membrane model and a two-layer membrane model, in which a difference in the electrical potential gradient was taken into account between the stratum corneum and epidermis/dermis layer, were constructed to describe the non-steady-state drug permeation process during iontophoresis. The observed iontophoretic lag-time was two times greater than the calculated value based on the one-layer membrane model. According to the two-layer membrane model, the calculated iontophoretic lag-time agreed with the observed value. It was revealed by model adaptation to the observed data that the stratum corneum fraction of the electro-chemical potential difference across the whole skin caused by the iontophoresis was around 90%. This result was consistent with the observation that the direct current resistance of whole skin was seven times greater than that of stripped skin.

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We newly synthesized a pivaloyloxymethyl ester of ofloxacin (OFLX-PVM) as prodrug in order to avoid the chelate formation between new quinolone and metal cations such as Al³⁺, Mg²⁺, Ca²⁺, or Fe²⁺ in the gastrointestinal tract. This compound was rapidly hydrolyzed in an incubation experiment by 43% in plasma, by 92% in small intestinal mucosal homogenates, and by 97% in liver homogenates during 0.5 h incubation, but was resistant to hydrolysis by pancreatic enzymes. In everted gut sac experiments, this compound was efficiently absorbed even in the presence of aluminium ion, whereas the absorption of ofloxacin (OFLX) was decreased significantly by the presence of aluminium ion. Minimal inhibitory concentration (MIC) values of OFLX-PVM were far higher than OFLX. Effects of aluminium hydroxide on the oral bioavailability of OFLX and OFLX-PVM were investigated in rabbits. The area under the plasma concentration-versus-time curve from zero to 24 h (AUC_{0-24 h}) following oral administration of OFLX was decreased significantly by 47.6% by combined administration with aluminium hydroxide, but AUC_{0-24 h} values of OFLX-PVM coadministered with and without aluminium hydroxide were similar to that of OFLX alone.These observations indicate that this new compound is likely to offer a prodrug for avoidance of interaction between new quinolone and metal cations.

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The influence of concentration of each component in l-menthol-ethanol-water ternary solvent system (MEW system) on the skin permeation of morphine hydrochloride (MPH) was investigated in hairless rats. The cumulative amount of MPH permeated through the excised abdominal skin over 8 h (Q-8) was selected as an index of skin permeability. With changing MPH concentration over a wide range from 0.01 to 10% in a MEW system (5% l-menthol and 40% ethanol), the values of Q-8 were proportional to MPH concentration. The concentration was fixed at 1% for the following experiments. For the effect of the concentration of l-menthol at 40% ethanol, the maximum Q-8 was observed at 5% l-menthol, and no greater enhancement of Q-8 was obtained by increasing l-menthol concentration above that. In the ethanol effect at 5% l-menthol, the maximum Q-8 was observed at 45% ethanol. When 2-propanol and methanol, which are more lipophilic and hydrophilic than ethanol, respectively, were used instead of ethanol, the maximum values of Q-8 were observed at 40 and 55%. The maximum values for Q-8 were obtained in the vicinity of the solubility of l-menthol in the MEW system in all cases, suggesting that the skin permeation enhancing effect of the system is dependent on the thermodynamic activity of l-menthol.

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A method has been developed for rapid analysis of the urinary hydroxylysine glycosides [glucosyl-galactosyl-hydroxylysine (GGH) and galactosyl-hydroxylysine (GH)] as collagen metabolites. The glycosides were fluorometrically derivatized with 5-dimethylaminonaphthalene-1-sulfonyl chloride, and then subjected to reversed-phase high-performance liquid chromatography using a gradient system. Suitable standard hydroxylysine glycosides which were not diastereoisomers were prepared from human urine. Urinary GGH and GH were analyzed within 1 h, including column washing. This method was applied to the urine samples of 68 healthy persons of different ages.

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The long-chain fatty acid composition in rat liver, by which peroxisomal β-oxidation system was induced, was analyzed in vitro and in vivo. In primary culture of hepatocytes, treatment with 0.4 mM clofibrate or long-chain fatty acids caused an increase in the peroxisomal β-oxidation activity, in addition the oleic acid [18 : 1(n-9)] content in the cells was increased two-fold by addition of very long-chain fatty acids to the culture medium, but clofibrate had no effect. A two-fold of increase in 18 : 1(n-9) content was observed in the hepatic subcellular fractions from rats fed a diet containing 0.25% clofibric acid. The ratio of cis-vaccenic acid [18 : 1(n-7)] to total 18 : 1 content decreased by more than half compared with control in clofibric acid treated liver. The level of 18 : 1(n-7) was not changed in all experiments. It is suggested that perturbation of C-18 chain length fatty acids occurs in peroxisomal β-oxidation induced liver.

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Total DNA was extracted from leaves of Glehnia littoralis belonging to various geographical strains with different furanocoumarin composition, and digested with restriction enzymes. Hybridization of digoxigenin-labeled probe containing rice ribosomal DNA to the digested DNA showed no difference in the restriction fragment length polymorphism (RFLP) profiles among the plants of different geographical origin. It is concluded that genetic diversity among the geographical strains of G. littoralis is so narrow as to be incapable of detecting RFLPs using rDNA as a probe.

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To learn the influence of polyethylene glycol (PEG) on bioavailability, this study compared the bioavailability of acetaminophen in the presence of 10% ethanol (acetaminophen-ethanol liquid) to that in the presence of 10% PEG (acetaminophen-PEG liquid) since these two preparations are commonly used in hospital formulary. The results in Thai male volunteers showed there was not significantly different.

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The strain-specific antigenicity between Bacillus cereus decreased but the common antigenicity did not change, when the flagellar antigen was treated with pH 2. Flagellar antigen of B. cereus H.1 was separated into two fractions by gel chromatography, one with a specific antigenicity (P1 fraction), and the other with a common antigenicity (P2 fraction). It was revealed that a common antigenic epitope of the P2 fraction was detected on a 61 kDa protein by further purification using a Mono Q column. This common antigenicity of flagella was increased by treatment with sodium dodecyl sulfate (SDS) and 2-mercaptoethanol, suggesting that a common antigenic epitope exists inside the flagellar component.

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