The hemoglobin binding activity of Bacillus cereus cells was measured with fluoresceinisothiocyanate (FITC)-conjugated hemoglobin using flow cytometry. Growth of B. cereus was markedly inhibited by the addition of apo-transferrin. B. cereus could not use transferrin-bound iron as an iron source in serum. The growth inhibition was reversed by the addition of a FeCl₃ solution, erythrocytes or hemoglobin. B. cereus released hemolysin; these findings suggested that the hemoglobin released from erythrocytes by B. cereus hemolysin binds to B. cereus and is thus used as an iron soruce.

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The effect of regucalcin, a calcium-binding protein, on Ca²⁺ -and phospholipid-dependent protein kinase (protein kinase C) activity in the cytosol of rat kidney cortex was investigated. With increasing concentratoins of Ca²⁺, phosphatidylserine or dioctanoylglycerol in the reaction mixture, regucalcin (10^-8M) caused a remarakble inhibition of protein kinase C activity. Regucalcin did not have a significant effect on protein kinase C activity in the presence of phosphatidylserine or dioctanoylglycerol without Ca²⁺ addition. Moreover, regucalcin significantly inhibited phorbol 12-myristate 13-acetate (PMA)-increased protein kinase C activity. Meanwhile, staurosporine (10^-9M) caused a signidficant inhibition of protein kinase C activity. This inhibition was further enhanced by regucalcin addition. Regucalcin itself did not have protein kinase activity in either the presence or the absence ot both Ca²⁺ and phospholipids. These results clearly indicate that regucalcin has an inhibitory effect on protein kinase C activity in the cytosol of rat kidney cortex. This inhibitory effect may be partly due to the regucalcin-induced Ca²⁺ binding.

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Generation of blue fluorescence together with phospholipid hydroperoxides and aldehyde species in rat liver microsomes during oxidation with FeCl₂-ADP-ascorbic acid was monitored, and the kind of lipid oxidation products participating in the formation of blue fluorescence was investigated. Contents of phospholipid hydroperoxides were incresed in an early stage of oxidation, and were decreased in an advanced stage of oxidation. Contents of components that liberated malonaldehyde, 4-hydroxyalkenals and other unsaturated aldehydes under the acidic assay conditions were increased in the advanced stage of oxidation. Water-soluble blue fluorescence with a maximum at 440-450nm determined after separation through gel filtration accumulated in the advanced stage of oxidation, and was characterized as resistant to borohydride treatment and to be little dependent on pH values of the solvent. Wavelength of the maximum fluorescence and characteristics of the fluorescence were similar to those of fluorescence with maxima at 440-450nm formed by reaction of unoxidized microsomes, bovine serum albumin or methylamine with alkenals, and different from those of fluorescence with maxima at above 460nm obtained by the reaction with a mixture containing malonaldehyde. Hence, blue fluorescence accumulated in oxidized microsomes cannot be derived from free malonaldehyde but can be from other aldehyde species including alkenals.

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Escherichia coli BM2506 is highly resistant to macrolide antibiotics; it produces macrolide 2'-phosphotransferase II [MPH(2')II] which inactivates such drugs. We investigated the localization and the transfer of the macrolide-resistance determinant that encoded the mphB gene for MPH(2')II in strain BM2506. Although we detected no clear band of plasmid DNA after agarose gel electrophoresis, transformation analysis using satellite DNA that corresponded to plasmid DNA after CsCl-ethidium bromide gradient centrifugation and restriction analysis of plasmid DNA in transformants showed that strain BM2506 harbored two plasmids, pTZ3721 (84kb) and pTZ3723 (24kb), that specified resistance to macrolides, ampicillin, streptomycin, tetracycline and sulfonamide and to macrolides and ampicillin, respectively. Southern hybridization showed that the mphB gene hybridized to both plasmids. Furthermore, pTZ3721 was transferred by conjugation to another strain of E. coli and pTZ3723 was mobilized with a self-transferable plasmid RP1 to other strains of E. coli. Therefore, it appears that the mphB gene is located on two plasmids in BM2506 and can be transferred to other strains of E. coli by conjugation or mobilization.

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Poricine gelatin (heat-denatured collagen) was digested with a bioreactor using an enzyme-coupled matrix (ECM) with purified collagenase. The diegsted gelatin, FreAlagin type R (M.W. range 200-10000 Da), was further purified by an HPLC system depending upon molecular size. The molecular weight range of the purified fractions, FreAlagin type P and type AD, were 200-500 and 2000-10000 Da, respectively, and glycine was the N-terminal amino acid of both types (≥93%). ECM has the capability of digesting gelatin at a specific point in the sequence before glycine, and it was determined that FreAlagin type P consists of a tri-peptide fraction with the amino acid sequence Gly-X-Y. No types of FreAlagin exhibited any reactivity with gelatin-specific IgC antibody raised in guinea pigs, and they also possessed an extremely low reactivity with gelatin-specific IgG antibody from the sera of patients who had experienced an anaphylactic reaction against gelatin after vaccination or after eating gelatin-containing foods. From these results, it was determined that FreAlagin types R and AD were non-antigenic, low-allergic gelatins. FreAlagin type R, and especially type AD, had strong adsorption-blocking activity comparable to the level of bovine serum albumin, whereas type P and glycine had virtually no adsorption-blocking activity. Therefore, the new types of gelatin, FreAlagin types R and AD, are suitable for pharmaceutical use to avoid gelatin allergy.

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Previously, we reported that dehydroleucodine (DhL), a sesquiterpene lactone, protected the gastric mucosa of rats from absolute ethanol-induced lesions in a dose-dependent fashion. The mechanism is not mediated by an antiacid secretory action and DhL stimulated mucous production. In the present study, we report the effect of DhL on the mucosal production of prostaglandin E (PGE) and the mucosal release of PGE₂ in rats stomach. DhL in acute treatment does not modify these values decreased by previous treatment with indomethacin or absolute ethanol. However, DhL in subchronic treatment significantly enhanced the mucosal production of PGE and the mucosal release of PGE₂. Also, indomethacin pretreatment resulted in a significant reduction of the cytoprotective action of DhL. These results indicate the participation of endogenous prostaglandins in DhL protection against ethanol damage. Moreover, we suggest that the gastric protective activity of DhL against ethanol induced gastric mucosal damage is mediated, at least in part, through PGE and PGE₂ in subchronic treatment.

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We investigated the antitumor effect of vitamin A(VA) using the double grafted tumor technique to examine whether VA administered into a primary tumor (intralesionally or i.l.) accelerates antitumor immune reactions so that growth of the secondary tumor may be more effectively inhibited than by other systemic administration routes. In the double grafted tumor system, where BALB/c mice were inoculated with MethA fibrosarcoma cells into the right inguinal region (1×10⁶ cells) on day 0 and later into the left (3×10⁶ cells) on day 10, the injection of VA at a dose of 1000 IU/mouse i.l., s.c., i.p., and i.v. on days 3 through 7 inhibited the growth of the secondary tumor to the same extent, while VA at the i.l. dose of 100 IU/mouse into the primary tomor inhibited more effectively than by any other administration route. VA did not inhibit the secondary MethA growth in BALB/c (nu/nu) mice. The spleen cells taken from VA-treated tumor-bearing mice prevented the growth of MethA tumors in naive BALB/c mice when given as a mixture with the MethA inoculum (the Winn assay). The delayed-type hypersensitivity (DTH) response to methylated bovine serum albumin (MBSA) antigen was augmented when VA (1000 IU) was injected at the site of the antigen injection. These results suggest that the direct interaction of VA with the tumor cells may be necessary for the tumor immunity-potentiating effect of VA, and that T-lymphocyte-mediated tumor immunity is involved in the anti-tumor effect of VA. The antitumor mechanism of VA seems to involve retinoid receptors, because the benzoic acid derivative Am80, which has been reported to exert retinoidal activity by binding to specific retinoid receptors, also showed activity.

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The inhibitory effects of tetrandrine (an alkaloid isolated from the Chinese medicine Stephania tetrandrae S. Moore) were investigated in terms of the angiogenesis in an adjuvant-induced chronic inflammation model of mouse and tube formation of rat vascular endothelial cells (EC). Tetrandrine (7.5-30 mg/kg) reduced the carmine content, granuloma weight, inflammatory cell count and pouch fluid weight in the inflammation model in a dose-dependent manner. The inhibitory pattern of tetrandrine on these parameters was similar to that of hydrocortisone. The inhibitory effect of tetrandrine on carmine content was 0.56-fold smaller than that of hydrocortisone. Tetrandrine (0.1-10 μM) also inhibited 2% fetal bovine serum (FBS)-stimulated tube formation of vascular EC. The inhibitory effect of tetrandrine on tube formation was more than 100-fold greater than that of hydrocortisone. Tetrandrine (10-30 nM) inhibited the tube formation stimulated by interleukin (IL)-1α and platelet-derived growth factor (PDGF)-BB to a greater extent than FBS-stimulated tube formation. The inhibitory effects of tetrandrine on the action of IL-1α and PDGF-BB were non-competitive. These results demonstrate that tetrandrine may reduce the tube formation of EC in the angiogenic process through inhibition on the post-receptor pathway of IL-1α and PDGF-BB in chronic inflammation.

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The pharmacological profile of a newly synthesized histamine H₁ receptor antagonist, KAA-276 (1-[1-(4-fluorophenylmethyl)-1H-benzimidazol-2-yl]-5-[2-[4-(2-carboxyethyl)-phenyl]ethyl]-1, 5-diazacyclooctane sulfate), was characterized. In a H₁ receptor binding assay in vitro, KAA-276 inhibited [³H]mepyramine binding to guinea pig cerebellar membrane preparations with an IC₅₀ of 0.66 nM. The inhibitory potency of KAA-276 was greater than that of terfenadine, but similar to that of astemizole and letotifen. KAA-276 antagonized the histamine-induced constriction of ileum and trachea isolated from guinea pigs in a dose-dependent manner with a concomitant reduction in the maximum response. Furthermore, the inhibitory effect of KAA-276 on histamine induced contraction was potentiated depending on the duration of preincubation time and revealed an irreversible property. KAA-276 given orally, intraduodenally, and by inhalation significantly inhibited histamine-induced bronchoconstriction dose-dependently in guinea pigs. Inhalation of KAA-276 exhibited inhibitory activity with a rapid onset and long duration, while intraduodenal administration resulted in action with a slow onset. Therefore, KAA-276, an irreversible and selective histamine, H₁ receptor antagonist, was shown to be a useful drug for therapeutic strategies against bronchial asthma when administered by the aerosol route.

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The structural and electronic properties of seventeen alkylxanthine derivatives were calculated using the MO program PM3 to elucidate the key features related to their inhibitory activity on phosphodiesterase (PDE) IV isoenzyme. Except for 7-alkylxanthine derivatives, a good correlation could be established between the distance between the tops of the two alkyl groups at the N¹ and N³ positions of the xanthine skeleton (molecular length) and the PDE IV inhibitory activity (r=0.973, n=13). The same inhibitory activity could also be significantly correlated with the following electronic parameters of alkylxanthines : HOMO energy (r=0.850), absolute hardness (r=-0.806), and absolute electronegativity (r=-0.825). These results suggest that the electronic properties are partly responsible for PDE IV inhibition as far as the effects of structural properties associated with molecular length are concerned. Alkylxanthines may also act as electron donors in the charge-transfer interaction with the active sites on PDE IV isoenzyme.

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To investigate the relationship between the intestinal bacterial metabolism of kalopanaxsaponin B and H from Kalopanix pictus (Araliaceae), and their antidiabetic effect, kalopanaxsaponin B and H were metabolized by human interstinal microfiora and the antidiabetic activity of their metabolites was measured. Human intestinal microflora metabolized kalopanaxsaponin B to kalopanaxaponin A, hederagenin 3-O-α-L-arabinopyranoside and hedaragenin. The main metabolites of kalopanaxsaponin B were kalopanaxsaponin A and hederagenin. Kalopanaxsaponin H was metabolized to kalopanaxsaponin A and I, hederagenin 3-O-α-L-arabinopyranoside and hederagenin. The main metabolites of kalopanaxsaponin H were kalopanaxsaponin I and hederagenin. Among kalopanaxsaponin B, H and their metabolites, kalopanaxsaponin A showed the most potent antidiabetic activity, followed by hederagenin. However, the main components, kalopanaxsaponin B and H, in K. pictus were inactive.

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The present study was carried out to compare the accelerating effect of shikonin and alkannin and to elucidate the expression of CD antigen and histological changes on the proliferation of granulation tissue in rats. Shikonin and alkannin produced a dose-dependent acceleration of the cotton pellet-induced granuloma formation and this accelerating potency of both compounds on the proliferation of granulation tissue was about the same 5 and 10 d after implantation of the cotton pellet. Also, both compounds increased the ratio of CD11b⁺ cells in the granulation tissue 5 and 10 d after implantation of the cotton pellet. Both compounds increased the expression of CD11b⁺ cells with granulocytes such as macrophages and histiocytes, and then accelerated the proliferation of fibroblasts and collagen fiber. On the other hand, neither compound increased the ratio of CD3⁺ cells in the granulation tissue after 5 and 10 d. These results suggest that shikonin and alkannin accelerate the proliferation of granulation tissue induced by the cotton pellet and this accelerating effect may be attributed to an increase in the expression of CD11b⁺ cells, and the acceleration of the proliferation of fibroblasts and collagen fiber in the granulation tissue.

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Thirteen novel diarylheptanoids bearing a chalcone or a flavanone moiety (1-13), a new curcumin derivative, 1, 2-dihydrobis(de-O-methyl)curcumin(14), and two known flavonoids(15 and 16) isolated from the seeds of Alpinia blepharocalyx K. Schum. were tested for their inhibitory effects on nitric oxide (NO) production in lipopolysaccaride (LPS)-activated murine macrophages J774.1 in vitro. All the tested compounds inhibited NO production in a concentration-dependent manner (IC₅₀=36-568 μM). Among the compounds examined, blepharocalyxin B (13) was the most potent inhibitor of NO production (IC₅₀=36-568 μM). Analysis of the structure activity relationship among these novel diarylheptanoids led to the conclusion that the position of attachment of a chalcone or a flavanone to a diarylheptanoid does not affect their inhibitory potency although their presence in association causes a substantial enhancement of the inhibitory activity. Moreover, a conjugated double bond in a chalcone moiety potentiated the inhibitory activity. On the other hand, hexamethoxydeoxycalyxin A (17) and pentamethoxycalyxin B (18), a methylated product of calyxin A (1) and an epimeric mixture of calyxin B, showed greatly reduced activity suggesting that phenolic hydroxyl groups are involved in the inhibitory activity.

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Thrombomodulin (TM) is s thrombin receptor on the endothelial cell surface, effective as an anticoagulant by changing procoagulant thrombin to an anticoagulant one. As rabbit TM with glycosaminoglycan (GAG) has a more potent anticoagulant activity than that without GAG, we expressed recombinant GAG-modified urinary thrombomodulin (GAG-UTM) in C-127 cells. The effect of an additional GAG chain on anticoagulant activity was investigated in comparison with unmodified recombinant UTM (r-UTM). In vitro, the activity of cleavage of fibrinogen by thrombin or prothrombinase activity was more potently depressed by GAG-UTM than by r-UTM, and the generation of activited protein C by TM-thrombin complex was accelerated by GAG modification. The acceleration of antithrombin III-dependent anticoagulant activity was shown only by GAG-UTM. Parameters like thrombin time, prothrombin time and activated partial thromboplastin time in human plasma were prolonged by GAG-UTM more than by r-UTM. In vivo, the effect of GAG-UTM and r-UTM in endotoxin-induced disseminated intravascular coagulation (DIC) rats was investigated using hematological parameters. GAG-UTM and r-UTM significantly reduced the decrease in fibrinogen and platelet number induced by endotoxin at the dosage of 0.1 and 1.0 mg/kg/h, respectively, suggesting that the antithrombotic effect of GAG-UTM in endotoxin-induced DIC rats was 10-fold as potent as that of r-UTM. GAG-UTM reduced the prolongation of the bleeding time induced by endotoxin, while r-UTM accelerated it. These results suggest that the addition of a GAG chain may increase availability as an anticoagulant.

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Recombinant glycosaminoglycan-modified urinary thrombomodulin (GAG-UTM) expressed in mouse C-127 cells has potent antithrombotic activity available as an anticoagulant. GAG-UTM, a glycoprotein with sialic acid, was investigated regarding the influence of the terminal sialic acid on its pharmacokinetics upon rapid intravenous injection in rat. Asialo GAG-UTM desialated by neuraminidase was cleared rapidly from plasma. Sialyzed GAG-UTM, a sialyzed asialo GAG-UTM with α-2, 6-sialyltransferase, containing sialic acid similarly to native sialo GAG-UTM, had only a short half-life in plasma, suggesting that the binding site of sialic acid on galactose was not only sialyzed with α-2, 6-sialyltransferase but also with 2, 3-sialyltransferase. Asialo GAG-UTM with oxidized terminal galactose, however, had a long half-life. These results suggest that terminal sialic acid may be important to the pharmacokinetics of GAG-UTM; therefore, an analysis of asialo GAG-UTM became significant for quality control. In order to analyze sialo-and asialo-types in the early stage of purification, we investigated separation and analysis methods for both types and found a suitable sample of each : RCA-120-Agarose column for separation and ELISA using anti-thrombomod-ulin antibody and RCA lectin for analysis.

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The selective cytotoxic activity of extracts from two marine green algae, Cladophoropsis vaucheriaeformis and Halimeda discoidea, was examined via a dose response assay against mouse leukemia L1210 cells and normal NIH-3T3 cells. The MeOH-extract from C. vaucheriaedormis showed selective cytotoxicity to L1210 cells at concentrations ranging from 50 to 100 μg/ml. In particular, the greatest selectivity for cytotoxic activitiy was found at the concentration of 50 μg/ml, at which the growth of L1210 cells was inhibited completely and that of NIH-3T3 was not affected at all.However, MeOH, extracts from the red alga Laurencia okamurae and brown alga Dictyopteris undulata, which displayed non-selective cytotoxicity in our previous screening program, did not show similar selective cytotoxicity at any concentrations tested.These results indicate that the marine green alga C. vaucheriaeformis may contain a unique antitumor substance with selective cytotoxic activity against L1210 cells. Our results also suggest that this active substance might be of low molecular weight and therefore MeOH-soluble.

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Our previous studies have shown that the degree of damage to a liposome corresponds to the variability of the animal species from which the serum comes, and that a complement activating factor (CAF) plays an important role in inducing the activation of the complement system, ultimately leading to the lysis of the liposomes. In this study, our attention focused on the characterization of the bovine serum factor (bCAF) that is involved in complement-mediated immune lysis of the liposome. The active fraction containing CAF partially purified with PEG and ammonium sulfate results in marked activation of the complement system via the alternative pathway when interacted with CAF-depleted serum, whereas the active fraction or CAF-depleted serum alone does not activate the complement. The interaction between lipopolysaccharide (LPS), heparin, zymosan or their mixture in place of CAF and CAF-depleted serum does not result in any significant activation of the complement system. Results from pretreatment with rabbit anti-bovine IgM IgG and rabbit anti-bovine IgG IgG indicate that activation of the complement system is not attributable to the antibody which is generally involved in activation of complement via the classical pathway. The results have further been proven by pretreatment with Concanavalin A (Con A) sepharose and protein G sepharose ruling out the possibility of antibody-mediated activation of complement. Our studies on collagenase and trypsin digestion demonstrate that the relative activity of CAF does not diminish with increase in collagenase concentration, and decreases with increase in trypsin concentration, strongly indicating that CAF does not have a collagen-like domain in its structure. The relative activity of CAF is dramatically inhibited after reduction with 2-mercaptoethanol (2-ME), clearly demonstrating that CAF is sensitive to reduction with 2-ME and confirming a sulhydryl-dependent protein. The optimal activity of CAF is observed in the range of 35-45°C and its half-life at 37°C is about 105 h. Furthermore, the relative activity of DAF increases and gradually approaches a plateau level with the increase of Mg²⁺ concentration. Obviously, complement activation induced by CAF depends on adequate Mg²⁺ concentration, confirming that this dependence is characterisitic of the altenative pathway.

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The uric acid concentration in blood has been widely accepted as a diagnostic indicator of hyperuricemia and gout, and its assay method is well established. In the present study, we developed a simple and rapid method for the determination of uric acid in hair, which can be obtained non-invasively. The concentration (nmol/mg hair) of uric acid extracted from 10-20 mg hair with 0.1 M potassium hydroxide was determined by an enzymatic method using uricase. The concentration of uric acid (nmol/mg hair, mean±S.D. : 0.49±0.157, n=16) in hair from hyperuricemic patients was significantly higher than that (0.26±0.107, n=8) in healthy volunteers (p<0.01). The within-run and between-day precision (CVs) of the assay was 9.6-10.3% (n=10 each) and 11.6-16.3% (n=7 each), respectively. The concentration (nmol/mg hair, y) of uric acid in hair correlated well with that in serum (mg/l, x) : y=0.09x-0.12 (r=0.75, S_yx=0.122, n=23). Changes in the concentration of uric acid in the hair of antihyperucemic drug-treated patient paralleled that in serum, suggesting that the concentration of uric acid in hair is a reliable indicator of the metabolic control in hyperuricemia.

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Nitric oxide (NO) generation from a spin trap, N-tert-butyl-α-phenylnitrone (PBN) under various oxidative conditions was examined. The absorbance of PBN at 295 nm decreased with time of UV-irradiation, showing that PBN was decomposed by UV irradiation. The hydroxyl radical formed from a Fenton reagent also decomposed PBN, but there was little effect by a peroxyl radical and a superoxide. Nitrite, an oxidative product of NO, in PBN solution was determined using a NOx analyzer based on Griess reaction. UV-irradiation and the hydroxyl radical also formed nitrite. Direct detection of NO from the sample on reaction with hydroxyl radical was successful using a GC/MS/SIM on the UV-irradiated sample. NO generated in PBN solutions activated guanylate cyclase. From these results, PBN is viewed as a new kind of medicine which acts as an actioxidant and as an No donor in vivo.

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The sexual difference of calcium-binding protein regucalcin mRNA expression in the liver of rats was investigated by Northern blot analysis. Liver regucalcin cDNA (0.9 kb of open reading frame) was used as a probe.The analysis of total RNAs extracted from various tissues of rats indicated that regucalcin mRNA was present primarily in the liver with a size of 1.8kb. Hepatic regucalcin mRNA was expressed in male rats more than in females. This sexual difference was also seen in aged rats (50 weeks old), although the expression was decreased with increasing age. Ovariectomy did not cause a significant alteration in hepatic regucalcin mRNA levels. The subcutaneous administration of 17β-estradiol (0.2 mg/100 g body weight) in ovariectomized rats did not cause an appreciable increase in hepatic mRNA levels. The results demonstrate that regucalcin mRNA expression in rat liver is based on sex, and that this difference may not be related to estrogen.

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In vitro binding characteristics of Δ⁸-tetrahydrocannabinol (Δ⁸-THC) and its metabolites, 11-hydroxy-Δ⁸-THC (11-OH-Δ⁸-THC) and 11-oxo-Δ⁸-THC, as well as an inactive metabolite, Δ⁸-THC-11-oic acid, as a cannabinoid receptor site from bovine cortex were examined using the specific agonist [³H]CP-55940. 11-OH-Δ⁸-THC and 11-oxo-Δ⁸-THC strongly inhibited the specific binding of [³H]CP-55940. The K_i values of 11-OH-Δ⁸-THC and 11-oxo-Δ⁸-THC for the specific binding of [³H]CP-55940 were 52 and 143 nM, respectively, whereas that of Δ⁸-THC-11-oic acid was 917 nM. Scatchard plot analyses indicated that 11-OH-Δ⁸-THC and 11-oxo-Δ⁸-THC caused a significant increase in the apparent K_D value without changing the apparent B_max. These results reveal that active metabolites of Δ⁸-THC also competitively bind to the cannabinoid receptor as agonists.

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The effect of a novel anti-allergic drug, betotastine besilate (betotasitine) on intertleukin (IL)-5 production by human peripheral blood mononuclear cells (PBMC) was investigated. PBMC of Dermatophagoides farinae extract (Df)-sensitive donors produced IL-5 and showed a proliferative responce upon stimulation with relevant antigen (10μg/ml). Df-induced IL-5 production by PBMC was significantly inhibited by betotastine at 10 and 100 μM. Betotastine also suppressed proliferation of PBMC with less potency. The effect of betotastine on IL-5 production was enhanced and significant even at 0.1 μM when the drug was added 120 min before antigen stimulation. Ketotifen and cetirizine also inhibited IL-5 production, but the effects of these drugs were significant only at 100 μM. These findings indicate that the suppression of IL-5 production may be involved in the anti-allergic effect of betotastine.

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Resting L1210 cells were treated with nimustine (ACNU), a bifunctional alkylating anticancer agent, for 2h in a nutrient-depleted medium. The cells were then transferred to a fresh medium and incubated for a further 48 h. Functions of the cells thus prepared were examined in terms of the dye-exclusion of the membrane, 2, 3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide, inner salt, sodium salt (XTT)-reducing ability of the mitochondria, and heat generation due to vital metabolism as the measure of cell viability. The cells treated with ACNU were functioning normally in all the cell functions examined but were completely devoid of proliferating capacity. These results suggest the possibility that ACNU might impair the proliferative capacity of the resting cell population inside a solid tumor without causing such impairment to the cells of normal organs and tissues composed of intrissically non-proliferative cells.

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Drug absorption studies using dogs have been difficult because of different gastrointestina(GI) conditions between dogs and humans, including dogs' shorter intestinal transit time and strong agitation force in the GI tract. We attempted to modify the agitation force and GI transit time in dogs using codeine. The agitation force was examined based on the in vitro/in vivo correlation for a CR tablet of acetaminophen showing agitation-dependant release. Codeine improved the GI condition better than atropine or loperamide, employed previously.

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By determining the formation amount of isonicotinic acid (INA) from isonicotinic acid hydrazide (isoniazid : INH) in isolated rat hepatocytes, we were able to identify the involvement of the oxidative cleavage of the acid hydrazide. INA formation from INH increased significantly using the isolated hepatocytes prepared from rats pretreated with phenobarbital (PB), 3-methylcholanthrene (3MC), dexamethazone (DEX) and rifampicin (RIF), respecitively, in comparison to the control group. On the other hand, a remarkable decrease in INA formation from INH was observed by the addition of such P450 inhibitor as metyrapone or cimetidine as well as an amidase inhibitor bis(p-nitrophenyl)phosphate (BNPP) to the isolated hepatocytes prepared from PB-pretreated rats. By further experiments using rat hepatic microsomes, the oxidative pathway of INA formation in INH metabolism was determined to be P450-dependent, since NADPH and oxygen were both essential for the oxidative pathway of INH to INA and the amount of INA formation was also significantly increased by P450 inducers. Regarding acetylisoniazid (AcINH) and isonicotinic acid amide (INAA), however, INA formation by P450 was little observed in the microsomal experiments.

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Simplified differential display of mRNA was applied to isolate and identify genes transcriptionally regulated in mouse liver by sho-saiko-to administration. A cDNA fragment up-regulated by sho-saiko-to was isolated and characterized. cDNA sequencing and subsequent database analysis revealed that the fragment showed significant sequence similarity with mouse testosterone 16-alpha-hydroxylase (cytochrome P-450_16α) cDNA. The increased level of mRNA expression of cytochrome P-450_16α in association with sho-saiko-to administration suggests the molecular mechanism of the chemopreventive effect of sho-saiko-to. This result indicates the usefulness of the mRNA differential display technique to investigate the molecular mechanism of Kampo medicine.

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