Bloodstain age could be estimated from the ratio of oxyhemoglobin/total hemoglobin (fractional oxyhemoglobin) in the bloodstain by this present method, if the temperature at which the bloodstain had been kept was known. The oxyhemoglobin was determined with an oxygen electrode immersed in water in which the oxygen had been depleted, and the total hemoglobin was determined by conventional colorimetry (cyanomethemoglobin method). Ages of prepared bloodstain samples (within 24h after bleeding) were estimated by this present method, which requires only 20μl of bloodstain and only 5 min for the whole analysis.

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Nine murine monoclonal antibodies (MAbs) raised against recombinant soluble intercellular adhesion molecule-1 (sICAM-1) were evaluated by means of ELISAs, and their neutralizing activity was investigated in two bioassays employing Raji cells activated with phorbol myristate acetate. Three of the MAbs inhibited autoaggregation of the activated cells and adhesion to sICAM-1 fixed on a plastic plate. Non-neutralizing antibody SM1-255 is directed to an epitope that was not recognized by the other eight MAbs. This property enabled us to develop two ELISAs employing SM1-255 as a liquid-phase antibody for the quantitation of sICAM-1 circulating (cICAM-1) in human plasma. One assay, employing non-neutralizing antibody WIS2-11 as a solid-phase, has a sensitivity of two pg/well with coefficients of varlation of 3.6-5.8% (within assay) and 5.5-9.5% (between assay). The other assay, employing neutralizing antibody WIS5-85 as a solid-phase, has a sensitivity of four pg/well with coefficients of variation of 2.7-7.2% (within assay) and 9.2-11.2% (between assay). There was a discrepancy between cICAM-1 levels of human plasma determined by these two assays. All samples showed 2-to 5-times higher levels in the assay using WIS2-11 than in that using WIS5-85. The result from the gel electrophoresis employing Western blotting suggests that ICAM-1 circulates in at least two molecular forms with molecular masses of about 85 and 130kDa and with different reactivities to WIS2-11 and WIS5-85.

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From a stereochemical point of view, the dehydrogenation mechanism of the biotransformation of 3α, 7α, 12α-trihydroxy-5β-cholestanoic acid (THCA) into (24E)-3α, 7α, 12α-trihydroxy-5β-cholest-24-enoic acid (Δ²⁴-THCA) has been studied with capillary gas chromatography (GC)/negative ion chemical ionization (NICI)-mass spectrometry. After incubation of (24R, 25R)-or (24S, 25S)-[24, 25-²H₂] THCA, synthesized from (24E)-Δ²⁴-THCA by a deuterated diimide reduction, with a rat liver light mitochondrial fraction, 5β-cholestanoic acids were extracted and derivatized into a pentafluorobenzyl (PFB) ester-dimethylethylsilyl (DMES) ether. Subsequent resolution into THCA and Δ²⁴-THCA was attained by GC on a cross-linked 5% phenylmethyl silicone fused-silica capillary column monitored with a corresponding characteristic carboxylate anion [M-PFB]^- in the NICI mode. The stereospecific elimination of a pro-R hydrogen at C-24 in both (25R)-and (25S)-THCA indicated syn-elimination for the former, whereas anti-elimination for the latter was observed.

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The reconstitution of the iron-sulfur cluster of spinach ferredoxin was examined in vitro using low and high molecular bound sulfur components as sulfur donors. Bound sulfur was rapidly converted to acid-labile sulfur to form an iron-sulfur center in the presence of dihydrolipoate and iron. Reconstitution yields of above 95% were obtained with cystine trisulfide (CT, 0.25mM) and sulfur-bound albumin (SBA, 1.0mM) at 37°C, pH 7.3, following 60min incubation. Spectroscopic features and biological activity of the reconstituted ferredoxin were identical to those of the native holo-protein. The acid-labile sulfur content found in the isolated reconstituted ferredoxin was 2atoms/mol protein, similar to the theoretical value. A possible role for bound sulfur in mammalian cells is indicated and discussed.

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An antifungal protein (AFP), holotricin 3, was purified from the hemolymph of the coleopteran insect Holotrichia diomphalia larvae. Analysis of its cDNA showed that holotricin 3 is a novel Gly-and His-rich protein consisting of 84 amino acid residues. This protein was similar to AFP, an antifungal protein of Sarcophaga peregrina that was reported previously, in terms of molecular size and high content of Gly and His residues. However, no appreciable sequence similarity was detected between the two proteins.

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We investigated the inhibitory effects of four gastric proton pump inhibitors (PPIs) : rabeprazole, a novel benzimidazole PPI, omeprazole, lansoprazole and AG-2000, on the urease activity of Helicobacter pylori (H. pylori). Their 50% inhibitory concentrations (I₅₀s) were found to be 0.29, 5.4, 9.3 and 0.3 μM respectively. Rabeprazole and omeprazole were also potent inhibitors of Jack bean and Proteus mirabilis cellular ureases. The thioether derivative of rabeprazole, one of its metabolites, had no inhibitory effect on H.pylori urease, despite being reported as a more potent inhibitor of H. pylori growth than rabeprazole. The inhibitory effect of rabeprazole was prevented completely and reversed considerably by the addition of sulfhydryl compounds, such as β-mercaptoethanol, glutathione and dithiothreitol. Moreover, the addition of β-mercaptoethanol recovered the urease activity inhibited by rabeprazole. From these results, we expected that rabeprazole inhibited H. pyloriurease activity by forming disulfide bonds between it and the active site of the enzyme.

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The primary structure of HYI killer toxin produced by Hansenula saturnus was determined in its reduced and pyridylethylated from, as well as the peptides resulting from protease digestion. It was found that the HYI killer toxin consisted of 87 amino acid residues and the molecular weight was calculated to be 9543 Da. It showed 87% homology with HM-1 killer toxin produced by H. mrakii, while there were multiple mutations including one amino acid deletion which, nevertheless, did not alter the strong cytocidal effect on sensitive yeasts.

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Alprenolol 4-hydroxylation and N-desisopropylation in liver microsomes from male Wistar rats were kinetically analyzed to be biphasic. In the 4-hydroxylation at a low substrate concentration (5μM), significant strain [Wistar>Dark Agouti (DA)] and sex (male>female) differences were observed, and the differences decreased at a high substrate concentration (1mM). In the N-desisopropylation, only a strain difference (Wistar>DA) was observed at the low substrate concentration. Cytochrome P450BTL (P450BTL, corresponding to CYP2D2) in a reconstituted system with 5μM alprenolol had high 4-hydroxylase activity, which was about 10 times that of P450ml corresponding to CYP2C11, and N-desisopropylase activity at a similar extent to P450ml. The two microsomal activities at 5μM alprenolol were efficiently decreased by antibodies against P450BTL and by sparteine, a typical substrate of the CYP2D subfamily. Polyclonal antibodies against P450ml and P450PB-1 (corresponding to CYP3A2) partially suppressed only N-desalkylation at 5μM, whereas they reduced the two activities at 1mM. P450ml showed a high N-desisopropylase activity at a substrate concentration of 1mM, where the sex difference was not observed. Furthermore, P450PB-2 corresponding to CYP2C6, which is one of the major P450 isozymes in female rats, also had 4-hydroxylase and N-desalkylase activities. These results suggest that a CYP2D isozyme(s) is the primary enzyme in alprenolol 4-hydroxylation and N-desisopropylation in a lower substrate concentration range, and that the involvement of some male-specific P450 isozyme(s) other than CYP2C11 or CYP3A2 may cause the sex difference in the 4-hydroxylation. In a higher substrate concentration range, CYP2C11 is thought to play a major role particularly in N-desisopropylation in male rats. In female rats, some major constitutive P450 isozyme(s) with a relatively high K_m value (e.g., CYP2C6) may be involved in the metabolism of alprenolol, resulting in the disappearance of the sex difference.

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This study used radioligand binding assay methods and pharmacological experiments to examine whether bopindolol, possessing a long-lasting action in addition to potent β-adrenoceptor antagonistic effects, is a slowly dissociating antagonist. In addition, the slow dissociation of two of its metabolites, 18-502 (4-(3-tert-butylamino-2-hydroxypropoxy)-2-methyl indole) and 20-785 (4-(3-tert-butylaminopropoxy)-2-carboxyl indole), which have potent β-blocking activities, was also assessed. The blockade of ³H-CGP12177 binding sites in rat heart and brain induced by pindolol was readily reversed by washing, whereas this inhibition by bopindolol and 18-502 was not easily reversed by washing. In addition, specific bindings of the hearts of the treatment animals with 20-785, atenolol, (±) propranolol, nadolol and celiprolol and of washout were 86.7, 78.8, 77.5, 82.3 and 79.9% of the control, respectively. These blockades by the treatment of each drug and washout in the brain were, however, lower than those in the hearts. On the other hand, when the left and right atria were pretreated with propranolol, bopindolol and 18-502, the inotropic and chronotropic actions of isoprenaline were inhibited by these drugs even though they were not present in the extracellular medium. Pretreatment with 20-785, atenolol and nadolol was readily reversed for both inotropic action and chronotropic rate, and inhibition by celiprolol and pindolol remained at 25% of the control at 240 min after treatment with these drugs. A good correlation between inhibitory binding percentage in the hearts and inhibitory inotropic or chronotropic actions were observed, although it was not observed in the brain. Thus, these results suggest that bopindolol and 18-502 are slowly dissociating β-adrenoceptor antagonists and this property may explain its long-lasting anti-hypertensive effects when compared to other β-blockers.

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Characterization of the serotonin-induced increase in guanosine 3', 5'-cyclic monophosphate (cyclic GMP) was investigated and compared with that induced by atrial natriuretic peptide (ANP) in NG108-15 cells. The cyclic GMP formed by serotonin or ANP was transported in a similar manner to the extracellular medium, although the cyclic GMP formed by bradykinin was not. Serotonin and ANP raised cyclic GMP additively. Serotonin-induced cyclic GMP formation was completely inhibited by pretreatment with 100nM 12-o-tetradecanoylphorbol 13-acetate (TPA), although that induced by ANP was only partialy inhibited and the effects were blocked by pretreatment with staurosporin. In membrane preparations, ANP stimulated cyclic GMP formation in the presence of ATP, but serotonin did not. Serotonin-stimulated cyclic GMP formation was found to occur in neuroblastoma N18TG-2, but not in glioma C6Bu-1. These results suggest that a novel subtype of serotonin receptors (5-HT_GC) which stimulates membrane-bound guanylyl cyclase, different from that stimulated by natriuretic peptide, may exist especially in neurons.

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Sodium 5, 6-benzylidene ascorbate (SBA) is a conjugate of ascorbic acid (Asc) with benzyaldehyde. It has been found that the antioxidant activity of SBA is more stable and has a longer lifetime in living cells and organs than Asc. In this study, we investigated the effect of SBA on the induction of melanin in cultured melanoma (B-16) cells irradiated by UV-A. Melanin content of B-16 cells was significantly increased by UV-A irradiation. The induction was abolished by mannitol and particularly by superoxide dismutase, suggesting the involvement of O^-₂ in the biosynthesis of melanin in cultured melanoma cells. This was theorized by the fact that the induction was also observed in B-16 cells treated with superoxide anion radicals chemically generated in the hypoxanthine/xanthine oxidase-reaction system, instead of UV-A irradiation. The induction of melanin caused by UV-A irradiation was suppressed by SBA in a dose-dependent manner. To elucidate the mechanism of this suppressive effect, the scavenging activity against O^-₂, and the inhibitory effect of SBA on tyrosinase activity were examined. ESR spectrometric analysis showed that SBA strongly scavenged O^-₂, and the presence of SBA in the medium remarkably inhibited the tyrosinase activity in cultured B-16 melanoma cells. It can be concluded that SBA effectively inhibits the melanin biosynthesis in B-16 melanoma cells induced by reactive oxygen species (ROS) generated by UV-A irradiation via tyrosinase.

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Twelve species of pyridobenzazoles and pyrimidobenzimidazole were examined as inhibitors of the replication of respiratory syncytial virus (RSV) in HeLa cells. From the pyridobenzazoles studied, 2-benzamido-4-cyano-1-oxo-1H, 5H-pyrido [1, 2-α] benzimidazol emerged as a potent inhibitor of the RSV. Based on its inhibitory effect on the cytopathogenicity of the RSV in HeLa cells, the 50% effective dose was found to be 0.95μg/ml. The cytotoxicity of this compound for HeLa cells was examined by monitoring the incorporation of radiolabeled uridine into cellular RNA. The selectivity index was ca. 30, the same as Virazole.

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We investigated the effects of intranasal (i.n.) administration of recombinant human granulocyte colonystimulating factors (rhG-CSF) on the total count of leukocytes in peripheral blood (total blood leukocyte count) of rabbits with leukopenia who received cyclophosphamide (CPA). When CPA (30mg/kg per d) was administered intravenously, the total blood leukocyte count decreased to levels below 5000/μl approximately 4d after the initiation of CPA multiple dosing. The decreased level of the total blood leukocyte count was maintained throughout the period of CPA dosing. RhG-CSF was given once a day for 3 d in CPA-treated rabbits via i.n. administration of aqueous preparations containing rhG-CSF with or without α-cyclodextrin (α-CyD). The total blood leukocyte count increased from levels below 5000/μl to the normal physiological level following i.n. administration of rhG-CSF preparation and reduced the period of leukopenia induced by CPA. The coadministration of rhG-CSF and α-CyD was more effective in increasing the total blood leukocyte count. It is suggested that i.n. administration of rhG-CSF is promising for reducing the risk of cytotoxic chemotherapy (CPA)-induced leukopenia as an adverse side effect.

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Time-dependent changes in the pharmacokinetics and renal handling of enprofylline induced by bacterial endotoxin (Klebsiella pneumoniae LPS) were investigated in rats. To evaluate the early effect of LPS on kidney functions and the renal excretion of enprofylline, which is an organic anion drug excreted primarily by an active tubular secretion, LPS (250μg/kg) was infused for 5 min under constant infusion at rates of 2.3 and 23 μg/min/kg for inulin and enprofylline, respectively. LPS caused a drop in the glomerular filtration rate (GFR), estimated as the renal clearance of inulin, to 65-75% of that observed in the control rats within 30 min after the LPS treatment. The renal clearance (CL_r) of enprofylline decreased in conjunction with GFR, while the percentage of decrease in the CL_r was slightly greater than that in GFR. LPS-induced decreases in the CL_r for enprofylline and GFR continued over the testing period of 120min. The time-dependent effect of LPS on the pharmacokinetics of enprofylline was examined by a single injection of enprofylline (2.5 mg/kg) to rats pretreated 2, 10 or 24h earlier with or without LPS. The pharmacokinetic parameters of enprofylline were determined by a model-independent method. Significant changes in the systemic clearance for enprofylline were observed in rats pretreated 2 and 10h earlier with LPS. but no such changes were observed in rats pretreated 24h earlier with LPS. These findings indicate the existence of a time-dependent effect of LPS on the pharmacokinetics of enprofylline, and suggest that LPS at a dose of 250 μg/kg, at least, does not induce cytotoxicity to kidney cells.

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Electroencephalogram (EEG) alterations in rat after the i.v. administration of pentobarbital (PTB) and chlorpromazine (CPZ) were measured by power spectral analysis. The time courses of PTB concentrations in plasma, cerebrospinal fluid (CSF) and brain were determined after the i.v. administration of PTB (20, 40mg/kg) by GC-MS. The PTB concentrations in plasma, CSF and brain could be described by a biexponential equation, a CSF model and a blood flow limited model, respectively. The relationship between the alteration of EEG and the PTB concentrations in the CSF or brain or the effect compartment were analyzed using the sigmoid E_max model. The alteration of EEG after PTB administration could be described by the PTB concentration in these compartments using the sigmoid E_max model. These results indicated that the site of action for the alteration of EEG after PTB administration is in instantaneous equilibrium with the CSF, the brain and the effect compartment. Thus, alterations in EEG after PTB administration can be predicted by monitoring the total PTB concentration in plasma. The alteration of EEG after i.v. administration of CPZ (4mg/kg) showed a two-phase variation. Although the relationship between the alteration of EEG and the CPZ concentrations in CSF or the striatum or the effect compartment (total and free drug) were analyzed using the linear model, the E_max model or the sigmoid E_max model, the two-phase alteration of EEG after CPZ administration could not be described by any of these models. These results indicated that the pharmacokinetic and pharmacodynamic modeling of CPZ during the alteration of EEG may be complicated due to several pharmacokinetic and pharmacodynamic factors, such as an alteration of the free fraction of CPZ in the striatum, the formation of active metabolites, and two different intrinsic effects of CPZ on the EEG (one in an increase and the other in a decrease of the brain's electrical activity).

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Tablets (300mg) having two different surface areas were prepared from spray-dried particles of curdlan (100mg)/theophylline (200mg). Drug release from the tablets was studied in vitro and in vivo. The in vitro drug release from a tablet with a larger surface area (Tab L) was faster than that with a smaller one (Tab S). The water uptake of Tab L was larger than that of Tab S, probably due to the difference in the tablets' surface areas. However, the water uptake was not a rate-determining step for the drug release from curdlan tablets containing a large amount of theophylline. A straight line was obtained when release % was plotted vs. time. The slope of each curve was calculated as 0.59 for Tab L and 0.58 for Tab S. This indicates that the release mechanism is non-Fickian diffusion controlled. In addition, the curdlan tablets or theophylline powder were administered orally to 5 healthy volunteers, and saliva concentrations of theophylline were determined. Each saliva concentration was converted to plasma concentration using the saliva to plasma ratio of the drug in each subject. The AUC of Tab L was nearly the same as that of powder, while the AUC of Tab S was smaller than that of powder. The mean residence times (MRTs) of theophylline powder, Tab S and Tab L were 11.1±1.5, 25.4±6.3 and 17.1±1.5h (N=4-5, mean±S.D.), respectively. The mean dissolution times (MDTs) of Tab L in vivo and Tab S in vivo were 5.0±2.1 (N=5, mean±S.D.) and 13.9±5.4h (N=4, mean±S.D.), respectively. On the other hand, the MDTs of Tab L in vitro and Tab S in vitro were 4.8 and 11.2h, respectively. In vivo drug release was very similar to in vitro drug release in both tablets. The lower bioavailability of Tab S suggested that the drug release had not been completed during the gastrointestinal transit period. Tab L would thus be a better controlled release form than Tab S.

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The effects of various adjuvants on the cytotoxicity of mitomycin C (MMC) were studied in L1210 mouse leukemia cells. Adjuvants examined in this study were sodium glycocholate (Na-GC), sodium deoxycholate (Na-DC), O-n-dodecyl-β-D-maltopyranoside (LM), and sodium salicylate. Among various additives, bile salts such as Na-GC and Na-DC were the most effective for increasing the cytotoxicity of MMC against L1210 cells. A dose-dependent increase in cytotoxic effect of MMC was observed in the presence of these bile salts. To elucidate a possible mechanism for enhancing the cytotoxic effect of MMC by the bile salts, the cellular uptake of MMC with or without Na-GC was examined using L1210 cells. The cellular concentration of MMC was determined by a reversed-phase HPLC. When Na-GC was coadministered with MMC, the uptake of MMC into L1210 cells was significantly enhanced as compared with MMC alone. Furthermore, the membrane fluidity of L1210 cells, as determined by fluorescence polarization, was increased in the presence of Na-GC. These results suggested that the enhancement of cytotoxicity of MMC by the addition of Na-GC could be attributed to the increasing cellular uptake of MMC due to the increasing membrane fluidity of L1210 cells.

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Irinotecan (CPT-11) is a camptothecin derivative used for the treatment of cancer. It is a prodrug that is metabolized to its active form, SN-38 [(+)-(4S)-4, 11-diethyl-4, 9-dihydroxy-1H-pyrano [3', 4' : 6, 7]-indolizino [1, 2-b] quinoline-3, 14 (4H, 12H)-dione]. To clarify the pharmacokinetic difference between CPT-11 and SN-38, the plasma levels, tissue distribution and excretion of SN-38 were investigated after dosing rats with ¹⁴C-labeled SN-38. The plasma radioactivity showed bi-exponential decay with a terminal half-life of 9.91h. TLC separation revealed that the plasma radioactivity consisted mainly of SN-38 at 5 min after dosing ; however, it was soon replaced with SN-38 glucuronide (SN-38 Glu) and an unknown metabolite (M-2). The half-life of unchanged SN-38 after dosing with SN-38 was about 7 min, which was much shorter than that after dosing with CPT-11 (2.8h) as reported previously. Its radioactivity was excreted mainly in feces (70.0% within 168h), and biliary excretion (64.1% within 48h) could account for the fecal excretion. The major component of urinary and biliary radioactivity was found by TLC to be SN-38. Whole body autoradiograms revealed that the tissue distribution of the radioactivity was low except in the liver and kidney. The radioactivity decreased rapidly and little was found in the body 24h after dosing. In conclusion, SN-38 was excreted rapidly from bile and showed poor tissue distribution. These characteristics lead to a shorter SN-38 half-life, more so than dosing with CPT-11.

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The antioxidant effect of C₁₇-sulfoconjugated catechol estrogens was examined under ascorbic acid-or NADPH-dependent lipid peroxidation in rat liver microsomes and compared with that of various estrogens and α-tocopherol. Among the estrogens tested, a free catechol estrogen such as 4-hydroxyestradiol showed the strongest effect, followed by 2-hydroxyestradiol, 2-methoxyestradiol and estradiol. Next to these steroids, 2-hydroxyestradiol 17-sulfate, followed by 4-methoxyestradiol, 4-hydroxyestradiol 17-sulfate and estrone also showed a strong inhibitory effect, which was greater than that of α-tocopherol. Among the C₁₇-sulfates, the guaiacols (2-and 4-methoxyestradiol 17-sulfate) showed a slightly lower effect than α-tocopherol, but estradiol 17-sulfate had almost no effect. The antioxidant activity observed in phenolic or guaiacol steroids was considered to be attributed to the catechols produced by their 2-(or 4-) hydroxylation or their O-demethylation, respectively, during the incubation. This was confirmed by identification of the catechols produced from phenolic or guaiacol estrogens and even from the estrogen C₃-sulfates. The mechanism of the inhibition by catechols on lipid peroxidation was speculated to involve their activity as radical scavengers, because of their strong reducing activity for 1, 1-diphenyl-2-picrylhydrazyl. The above results suggest that C₁₇-sulfoconjugated catechol estrogens (2-and 4-hydroxyestradiol 17-sulfate), although with slightly lower activity than their free catechols, are promising endogenous antioxidants. The physiological role of these estrogen conjugates during pregnancy is discussed.

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The reductive retention mechanism of copper (II)-pyruvaldehyde-bis (N⁴-methylthiosemicarbazone) (Cu-PTSM), a generator-produced positron-emitting ⁶²Cu-labeled radiopharmaceutical, was studied with non-radioactive and radioactive copper. Changes in the chemical form of Cu-PTSM were detected by electron spin resonance spectrometry (ESR) with cold copper. The effects of electron transport chain inhibitors on the reduction of Cu-PTSM were also examined. Rotenone and antimycin A activated the reduction of Cu-PTSM in the brain mitochondria by 1.6-and 1.4-fold, respectively, compared with untreated controls, while thenoyltrifluoroacetone (TTFA) had no effect on the reduction. These results were confirmed with radioactive copper. Furthermore, this reduction of Cu-PTSM was dependent on the protein concentration of mouse brain submitochondrial particle (SMP) with 1mM NADH (0 mg-protein/ml : 1.8±2.5%, 8mg-protein/ml : 69.0±5.5%, each value was % of reduced Cu). Similarly, this reduction depended on NADH concentration at a fixed concentration of SMP (8mg-protein/ml). These results indicated that the electron transport chain, especially complex I, participated in the reduction of Cu-PTSM in brain mitochondria, and this suggested that Cu-PTSM has the potential to act as a functional imaging agent for diagnosis of the electron transport chain.

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D-Ala and D-Ser were detected in the sera of both normal subjects and patients with renal dysfunction, and their concentrations were higher in the patients than in the normal subjects. A positive correlation between the concentration of D-Ala or D-Ser and that of creatinine (r=0.733, p<0.001 or r=0.634, p<0.001) or blood urea nitrogen (BUN) (r=0.449, p<0.05 or r=0.629, p<0.001) was observed in sera from 20 patients with renal dysfunction. The fraction (%D) of D-Ala in the total Ala in serum ([D/(D+L)]×100) correlated well with the concentration of creatinine (r=0.811, p<0.001), suggesting that it is a candidate as a marker for renal proximal tubular dysfunction. The correlations of %D of Ser with creatinine and BUN levels were 0.796 (p<0.001) and 0.919 (p<0.001), respectively, indicating that %D of Ser may reflect protein turnover or catabolism in certain tissues as well as renal proximal tubular dysfunction.

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Two fractions of neutral α-glucosidase were partially purified from the insoluble fraction of bovine lens. This is the first report of such an event to the best of our knowledge. The apparent native molecular weights of these fractions were 121 kDa (fraction-I) and 254 kDa (fraction-II). Both fractions contained three polypeptides with molecular weights of 21, 25 and 30kDa, although the proportion of these peptides was different in both fractions. The optimal pH of fraction-I and fraction-II was pH 6.0 and 6.5, and the optimal temperature for both fractions was approximately 50°C. The K_m values of fractions-I and -II for 4-methylumbelliferyl-α-glucopyranoside were 0.086, and 0.192mM. The activities of these enzymes were inhibited strongly by HgCl₂ and slightly by D-iodoacetic acid, but not by D-turanose. From this, we suggest that the enzyme in the insoluble fraction of bevine lens may be a cytoplasmic neutral α-glucosidase which binds to the cell membrane.

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The hepatic microsomal metabolism of cannabinoids was studied using the liver from an old woman. Δ⁸-Tetrahydrocannabinol, Δ⁹-tetrahydrocannabinol and cannabinol were biotransformed to their respective 11-hydroxy metabolites by a microsomal fraction with specific activities (pmol/min/mg protein) of 29.1, 47.1 and 27.9, respectively. In addition, both 11-oxo-Δ⁸-tetrahydrocannabinol and 11-oxo-Δ⁹-tetrahydrocannabinol were metabolized to the corresponding carboxylic acids with the microsomes. An antibody against mouse CYP2C29 almost completely inhibited 11-hydroxylation of the cannabinoids and microsomal aldehyde oxygenase (MALDO) activity for 11-oxo-Δ⁸-tetrahydrocannabinol and 11-oxo-Δ⁹-tetrahydrocannabinol, used as substrates, whereas an antibody against rat CYP3A2 conversely stimulated the 11-hydroxylation of Δ⁸-tetrahydrocannabinol and MALDO activity for 11-oxo-Δ⁸-tetrahydrocannabinol. The results indicate that a member of CYP2C is primarily responsible for the metabolism of the above cannabinoids in the human hepatic microsomes.

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First, the effect of E5110, a non-steroidal anti-inflammatory agent (NSAID), on cytochrome P450 subfamilies in dog hepatocyte culture was examined. E 5110 has been shown to cause a drug interaction in dogs and humans via induction of cytochrome P450 3A (CYP 3A). When dog hepatocytes were cultured for 72h in the presence of 2-30μM E5110, the activitiy levels of ethoxycoumarin O-deethylase (ECOD) and testosterone 6β-hydroxylase (6β-OH-T) rose dose-dependently. Subsequent Western blot analysis of microsomes from hepatocyte cultures indicated the presence of higher amounts of CYP 2B and 3A proteins than those of the control. Next, the P450 inducing potency of E 5110 was compared with those of phenobarbital, rifampicin, phenytoin and carbamazepine, which induce CYP 3A in human subjects and human hepatocyte cultures. E 5110 was found to be nearly as effective as phenytoin, but less potent than rifampicin, on the basis of 6β-OH-T induction.

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E2020 (1-benzyl-4-[(5, 6-dimethoxy-1-indanon)-2-yl] methylpiperidine hydrochloride) is currently being developed as a treatment for senile dementia of the Alzheimer type. Its mechanism of action is to increase central cholinergic activity by inhibiting acetylcholinesterase (AChE) in the brain. In this study, the kinetics of the inhibitory action of E2020 on AChE was examined in comparison with its derivatives (2A1050 and 2A1034) and the reference compound tetrahydroaminoacridine (THA) all of which have a similar action. Analysis of the data using Lineweaver-Burk plots shows that inhibition by the test compounds fell into the category of mixed type. Inhibitor dissociation constants for the free enzyme (K₁) and acetyl-enzyme (K₁) of E2020 are one or two orders of magnitude lower than those of 2A1050, 2A1034 and THA. These findings indicate the strong inhibitory effect of E2020 on AChE.

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The inhibitory effect of a 50% ethanolic extract obtained from the dried leaves and the bark of Myrica rubra, was investigated in vitro on melanin biosynthesis which is closely related to hyperpigmentation. These extracts inhibited tyrosinase activity which converts dopa to dopachrome in the biosynthetic process. Furthermore, the extracts inhibited the production of melanin from dopachrome by autoxidation and also showed superoxide dismutase (SOD)-like activity. After bioassay-guided fractionation, quercetin, myricetin and myricetin 3-O-rhamnoside were isolated from the leaves. As they showed the inhibitory effect on tyrosinase activity, the activity is partially attributable to them in the extract of M. rubra. These results suggested that the leaves or the bark of M. rubra might be used as a whitening agent for the skin.

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Thermally reversible gels of the block copolymer, Synperonic T908, have been evaluated as vehicles for the rectal administration of indomethacin. Prolonged plasma levels of indomethacin following rectal administration in such gels was observed with the 40% w/w Synperonic T908 gels when compared with commercial suppositories. The release rate decreased with an increase in gel concentration over the range 30% to 40% w/w. Histological observation showed no damage to the rectal mucosal membrane in animals at 6h after the administration of a 40% w/w gel.

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The use of curdlan, a natural β-1, 3-glucan, in the preparation of sustained release suppositories was studied in vitro. To prepare the suppositories, indomethacin, prednisolone or salbutamol sulfate was mixed with curdlan gel. Preparation conditions, including heating time and curdlan concentrations of 5 and 10%, had little effect on the drug release. The tonicity (hypotonic or isotonic) of the media for the suppository preparation and for in vitro drug release study also had little effect on drug release. However, the heating temperature during gel preparation, the drug amount in the suppository and the type of release media did affect drug release. It was found that drug release was sustained and diffusion-controlled in the three drugs. And finally, curdlan can be applicable for use in a sustained release suppository.

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The hypoglycemic effect of senegin-II, the main component of Polygala senega (Polygalaceae), was examined in normal and KK-Ay mice, one of the model animals of non-insulin-dependent diabetes mellitus (NIDDM). Senegin-II (2.5mg/kg) reduced the level of blood glucose in normal mice from 220±8 to 131±5mg/dl 4 hours after intraperitoneal administrarion (P<0.001), and also significantly lowered the blood glucose of KK-Ay mice from 434±9 to 142±6mg/dl under similar conditions (P<0.001).

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The reactivity of malondialdehyde (MDA) with spermidine at the physiological pH was considerably higher than that with putrescine, glycine, lysine, methylamine or dimethylamine, and the major reaction product of MDA-spermidine adduct was identified as N⁴-(2-propenal) spermidine.

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Trichosporin (TS)-B-VIa, a peptide consisting of 19 amino acid residues and an amino alcohol, causes Ca²⁺-dependent secretion of catecholamines from bovine adrenal chromaffin cells. The TS-B-VIa-induced secretion was greater under alkaline conditions and at a temperature of 37°C compared with that at 21° or 30°C. It was not observed when the peptide was eliminated from the incubation medium. These results strongly suggest that the stimulatory effect of TS-B-VIa on the secretion is reversible and dependent on the temperature and pH of the incubation medium.

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Spontaneous transfer of protein from either influenza virus-infected or uninfected cells to dimyristoylphosphatidylcholine (DMPC) liposomes was examined. The amount of transferred protein of liposomes incubated with influenza virus-infected cells (I-lipo) was more than that of liposomes incubated with uninfected cells (U-lipo). The ratio of the amount of spontaneous transferred protein of I-lipo to that of U-lipo increased in proportion to the diameter of liposomes except for small unilamellar vesicles.

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