A new fuzzy immunoassay method generally applied to ten Glycyrrhizae Radix (GR) preparations of four different botanical origins was studied. Four kinds of antisera were elicited in rabbits immunized with GRs of different botanical origins. The presence of the characteristic GR protein (GPR) was shown using Western blot analyses and selected antibody enzyme immunoassay (SAEIA) methods. A GRP was isolated from one of the GR specimens which was selected using SAEIA methods. The isolated GRP was heated to reduce its binding activity to an anti-GR serum. A new fuzzy SAEIA method generally applicable for assay of the extract of the ten GR specimens was developed using heat-treated GRP as the solid-phase antigen. The fuzzy SAEIA method was successfully applied for the detection and quantitative analysis of the GR component contained in traditional Chinese medicines.

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In vitro glucuronidation of 25-hydroxyvitamin D₃ and its pro-form has been investigated by means of HPLC with UV detection. Although both substrates gave 3- and 25-glucuronides in the presence of the ray liver microsomal fraction and uridine-5'-diphosphoglucuronic acid, 25-hydroxyvitamin D₃ and its pro-form yielded 3- and 25-glucuronide as the main product, respectively. The latter glucuronide is the one in which the tert-hydroxy group is conjugated. Each glucuronide was identified by its chromatographic behavior in comparison with an authentic sample and data obtained from liquid chromatography/mass spectrometry (LC/MS) using atmospheric pressure chemical ionization.

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The induction of in vivo antioxidant potential following small doses of γ-ray irradiation was investigated in C57BL/6 mice. The antioxidant capacity of various organs was assessed in terms of the scavenging activity of cytosol fractions against 1, 1-diphenyl-2-picrylhydrazyl (DPPH), a chemically stable radical. Significant elevations in the scavenging activity were recognized in several organs, including the liver, pancreas and brain, soon after post-irradiation with 50cGy of γ-ray. These increases persisted for 24 h. γ-Radiation of the liver at 25-50 cGy elevated its cytosolic antioxidant capacity, but this was lowered at 200 cGy. In order to assess which antioxidants underlie this phenomenon, the content of a reduced form of glutathione (GSH) in liver was assayed as a function of time after γ-irradiation at a dose of 50 cGy. The GSH content was significantly increased from 6 h after irradiation, and this change was consistent with that of the total radical scavenging potency of the liver against DPPH. Further, the elevation of GSH content was accompanied by elevated GSSG reductase activity induced by γ-irradiation.

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The maintenance of intracellular osmotic pressure is of fundamental importance for cell survival. Since osmoregulatory processes are important to all living organisms, large fluctuations in environmental osmolarity may elicit or modulate stress responses. We examined whether hyperosmotic stress induced or modulated stress responses in human HeLa cells. When HeLa cells were incubated in medium supplemented with 50-150 mM NaCl or 0.2-1.0 M glycerol for 3-27 h, the stress response, analyzed at the levels of HSP70 synthesis, HSP70 mRNA accumulation, and heat shock transcription factor (HSF) activation, was not induced by either hyperosmotic stress. In hypersomotic 150 mM NaCl or 1.0 M glycerol medium, the stress response to heat shock was inhibited at the levels of HSF activation, HSP70 mRNA accumulation, and HSP70 synthesis. In vitro activation of HSF showed that inhibition of this activation by hyperosmotic NaCl or glycerol stress was not irreversible. Furthermore, addition of the physiological osmolyte betaine to medium reversed the inhibition of heat-induced HSP70 synthesis under hyperosmotic NaCl stress but not under hyperosmotic glycerol stress. The effect of betaine against hyperosmotic NaCl stress was observed mainly at the translation level, and betaine seemed to enable cells to translate HSP70 mRNA specifically under heat shock conditions by restoring protein synthetic ability.

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Transcriptional regulatory sequences often influence the efficiency of DNA replication, directly or indirectly, in bacteria, yeast, and animal virus systems. We have tested several transcriptional regulatory sequences for affecting DNA replication, based on pUC vector, in transient systems. Autonomous replication of transfected plasmids was assayed by PCR amplification of the fragments derived from the plasmids, which had replicated in mammalian cells. By this highly efficient method of detecting replicated molecules, pUC19, but not pUC18, showed a weak replication activity in transfected cells. Nucleotide sequences around the HindIII site in pUC19 were required for replication. Monomers or dimers of the octamer transcription motif of the mouse immunoglobulin heavy chain gene, inserted in multicloning sites of pUC19, could stimulate replication, while the 4- or 6-mers did not, in contrast to the results on its transcription activity. Other transcriptional elements including AP1, HSE, and E2F also stimulated replication, but neither CRE nor Sp1 binding motif did. These results suggest that at least some of the transcriptional regulatory sequences function as modulators of DNA replication as well as of transcription.

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Everted membrane vesicles were prepared from cells of Pseudomonas aeruginosa, and cation/H⁺ antiport was measured. We observed activities of Na⁺/H⁺ antiport, Li⁺/H⁺ antiport and K⁺/H⁺ antiport. Judging from the competition pattern, it seems that there are at least two types of antiporter, a Na⁺(Li⁺)/H⁺ antiporter and a K⁺/H⁺ antiporter. Na⁺ was a good substrate for the Na⁺(Li⁺)/H⁺ system, whereas Li⁺ was a poor substrate. Although the K_m value for Na⁺ (or Li⁺) was similar to those in Escherichia coli Na⁺/H⁺ antiporters, the V_max value for Na⁺ (or Li⁺) was much smaller in the P. aeruginosa antiporter than in the E. coli antiporters. Growth of P. aeruginosa was strongly inhibited by 0.4 M, LiCl, but not by NaCl or KCl.

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The inhibitory effects of vapiprost hydrochloride (vapiprost), a novel thromboxane A₂ receptor antagonist, on platelet aggregation and ATP release were studied using platelet rich plasma (PRP) of humans, guinea pigs, rabbits and rats. In in vitro experiments with human platelet, vapiprost inhibited the aggregation and ATP release stimulated with U-46619, collagen or arachidonic acid (AA) at an IC₅₀ of less than 2.1×10^-8M. Vapiprost did not inhibit the primary aggregation or ATP release of human platelets stimulated with adenosine 5'-diphosphate (ADP), epinephrine (Epi) or platelet activating factor (PAF), but inhibited the secondary aggregation stimulated with those agonists at an IC₅₀ of less than 1.3×10^-7M. The sensitivity of platelets in various species of animals to vapiprost was in the following order : human>guinea pigs>rats>rabbits. In ex vivo experiments with guinea pigs which received a single oral dose of vapiprost, the agent demonstrated strong inhibition of ATP release from platelets stimulated with U-46619, collagen or AA at an ID₅₀ of less than 25.8 μg/kg. These inhibitory effects were observed within 30 min and sustained for 24 h at a single dosage of 5 mg/kg of vapiprost. In AA-induced pulmonary infarction models of mice, the sudden death rates decreased significantly with the oral administration of 10 mg/kg or more of vapiprost.These results indicate that vapiprost effectively inhibits the secondary aggregation and ATP release of human platelets stimulated with various agonists, and that guinea pig and human platelets are similar in response to vapiprost. Furthermore, it was demonstrated in ex vivo experiments with guinea pigs that the inhibitory action of vapiprost appears rapidly and lasts for long periods.

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We previously reported that the topical application of ascorbic acid 2-O-α-glucoside (AA-2G) suppressed the cutaneous inflammation by ultraviolet irradiation in human and guinea pigs (Miyai et al., Nishinihon J. Dermatol., 58, 439-443 (1996)). In this paper, the effect of AA-2G on the lethal damage induced by ultraviolet B (UVB) was studied using a human keratinocyte cell line, SCC, established from squamous cell carcinoma. The photoprotective effect of AA-2G on cytotoxicity of UVB in SCC cells was dose dependent (0.125-1 mM) and more effective than that of ascorbic acid (AsA) at 1mM. This protection was completely abolished in the presence of an α-glucosidase inhibitor, castanospermine, indicating that release of AsA from this derivative was essential for reduction of the actinic injury. AA-2G significantly suppressed cytotoxicities of hydrogen peroxide and superoxide anion produced by xanthine and xanthine oxidase. AA-2G exhibited a preventive effect against the cytotoxicity produced by tert-butylhydroperoxide, an inducer of lipid peroxidation, in the presence of α-tocopherol, but not in the absence of α-tocopherol. Cytotoxicity of UVB was also effectively by the combination of AA-2G and α-tocopherol. In addition, AA-2G reduced UVB-promoted formation of lipid peroxide and accumulation of lipofuscin, which is known to be a complex of cellular proteins and metabolites of lipid peroxide.These data suggest that AA-2G prevents the acute inflammation induced by UVB irradiation partly through scavenging reactive oxygen species and protentiating the antioxidative activity of α-tocopherol.

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Mice immunized with formalin-killed Candida albicans were resistant to challenge by a lethal amount of viable C. albicans. The growth-inhibitory activity to C. albicans was detected in sera from the immunized mice, and was inhibited by the addition of anti-transferrin antibody or ferric sulfate. Both the amount of transferrin and the unsaturated iron-binding capacity (UIBC) in the serum were significantly increased, indicating that apo-transferrin increased in the immunized mice. Moreover, the intraperitoneal administration of apo-transferrin enhanced the protection from the Candida infection in vivo.

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The high K⁺ -evoked release of acetylcholine (ACh) from electric organ synaptosomes isolated from the Japanese marine ray, Narke japonica, was strongly inhibited by dihydropyridines at micromolar concentrations. However, this inhibition seems to be a non-specific effect sine the agonist (-)-Bay K 8644 also had inhibitory effects. Calciseptine, a peptide toxin specific for L-type Ca channels, inhibited to a lesser extent the evoked acetylcholine release : the maximum inhibition was about 20%. This finding is in accord with our data (Tokumaru et al., J. Neurochem., 65, 831 (1995)) regarding inhibition by a monoclonal antibody against the α₂δ-subunit of the L-type Ca channel and provides evidence for the involvement of an L-type-like Ca channel in ACh release in addition to ω-conotoxin GVIA-sensitive N-type and ω-agatoxin IVA-sensitive P-type Ca channels.

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Nineteen mono- and di-fluorinated derivatives of quinoline, 1, 7-phenanthroline, 1, 10-phenanthroline, benzo-[h]quinoline, and benzo[f]quinoline were subjected to analysis of their structure-mutagenicity relationships. For this purpose, six new fluorinated derivatives were synthesized. The results support that the enamine epoxide structure of the pyridine moiety, as well as the bay-region epoxide structure, is responsible for mutagenicity. Formation of K-region epoxides might involve a detoxification process rather than mutagenic activation.

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Urinary proteins of patients with diabetes mellitus (DM) were analyzed using cellulose acetate membrane electrophoresis, to determine the clinical usefulness of fraction patterns of the proteins in detecting the group at high risk for diabetic nephropathy.We divided the protein patterns into 5 groups. Four groups (I, II, III, IV) were found in the healthy group and a newly classified group was termed group 0 and was characterized by a prominent albumin peak with a negligible or small globulin peak.The incidence of groups, 0, I, II, III and IV, and 36.6%, 13.3%, 18.7%, 10.7% and 22.7%, respectively. This distribution was clearly different from that of healthy subjects and the most characteristic feature of diabetics was that group 0 accounted for 36.6% of the total cases.Characteristic features of each group were examined from the aspect of laboratory and clinical findings.Urinary protein patterns were concluded to be useful not only to predict the high risk group for diabetic nephropathy in the preclinical stage but also to discriminate nephropathic types of glomerular or tubular origin. It is useful for clinicians to know the risk stage and prognosis for diabetic nephropathy.

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An ultrafiltrate (M.W.<10000) of 0.1 M acetic acid extract from Japanese red pine needles was treated with Sep-Pak Vac C₁₈. and an absorbable fraction (pine aqueous components fraction, PAC) was obtained. Since the intraperitoneal administration of PAC to rats decreased plasma triglyceride in a screening test for bioactivity, we tried to further isolate the active substances with respect to this triglyceride-decreasing action. Active substances were precipitated by acid, and we then divided them into fractions using reversed phase HPLC. The active fractions were hydrolyzed, then the hydrolysate was re-separated by the same HPLC system. Negative ion mode FAB-LC/MS of the bioactive fractions of this hydrolysate revealed an [M-H]^- molecular ion peak at m/z 169 and 321. The ¹³C-NMR spectra were consistent with those of authentic gallic acid and galloyl gallic acid, a dimer of gallic acid.Authentic gallic acid also showed a triglyceride-decreasing action, and galloyl gallic acid even more markedly decreased triglyceride. These findings suggest tha tthe triglyceride-decreasing action of Japanese red pine needles is due to these polyphenol compounds. In addition, amino acids were also detected in the hydrolysate of PAC, indicating that the components in Japanese red pine needles that decrease triglycerides are complexes of a hydrolyzable tannin, which contains gallic acid and galloyl gallic acid as components and peptides.

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The uptake of methylchlorpromazine (MCP), a quaternary derivative of chlorpromazine, was investigated using brush-border membrane vesicles islated from rat small intestine. MCP was taken up rapidly by the vesicles, a major part of the uptake being due to binding to the membrane. Saturable MCP binding to the brush-border membrane was inhibited strongly by chlorpromazine, moderately by propantheline and imipramine, and slightly but significantly by methylbenactyzine and mepenzolate. However, choline and tetramethylammonium failed to exhibit any such inhibitory effect. The movement of MCP into the intravesicular space was driven by an inside-negative transmembrane electrical potential difference (TEPD) induced by NaSCN or valinomycin. There was no effect of TEPD on MCP binding to the brush-border membrane. The data suggested that both rapid binding to the brush-border membrane and inside-negative TEPD, which is present physiologically across the membrane, play a significant role in the absorptive movements of MCP across intestinal epithelium.

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We investigated microbial contamination of in-use antiseptics at a hospital. No microbial contamination was observed in 70 samples of 0.02% benzalkonium chloride solution (500-ml volume), 70 samples of 1% titratable I₂ povidone-iodine solution (250-ml volume), or 15 samples of 0.1% ethacridine lactate solution (500-ml volume) during use in reduced amounts. Nor was any microbial contamination observed in 70 samples of cotton balls soaked in 1% titratable I₂ povidone-iodine solution in canisters or cotton gauze soaked in 70% (w/v) ethanol solution in canisters.However, among 70 samples of cotton balls soaked in 0.02% benzalkonium chloride solution in canisters, 6 (8.6%) were contaminated with 10⁴ to 10⁶ viable cells/ml. The microbial species detected were glucose non-fermentative bacilli such as Alcaligenes xylosoxidans and Pseudomonas putida. The contaminants obtained from cotton balls soaked in 0.02% benzalkonium chloride solution did not proliferate in that solution or in distilled water but showed rapid growth in the cotton balls soaked in either of these liquids. These findings suggested that benzalkonium chloride solution tends to become contaminated when cotton balls are immersed. Therefore, cotton balls soaked in benzalkonium chloride solution are not recommended as an antiseptic. When no other choice is available, the cotton balls should be soaked in benzalkonium chloride solution at the time of usage.

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Photodynamic therapy (PDT) as a cancer treatment is notable for its quite low side effects in comparison with those of chemotherapy and radiotherapy. However, the accumulation of porphyrin derivatives used in PDT into tumor tissues is rather low. Since long-circulating liposomes are known to accumulate passively into tumor tissues, we liposomalized a porphyrin derivative, benzoporphyrin derivative monoacid ring A (BPD-MA), and used these liposomes to investigate the usefulness of PDT for tumor-bearing mice. BPD-MA was liposomalized into glucuronate-modified liposomes, which are known to be long-circulating. These liposomes were injected i.v. into Balb/c mice bearing Meth A sarcoma, and tumor regression and servival time were monitored after irradiation with laser light. Tumor regression and complete curing of tumor (80% cure rate by the treatment with 6 mg/kg BPD-MA) were observed when long circulating liposomalized BPD-MA was injected and laser-irradiated. In contrast, only a 20% cure rate was obtained when the animals were treated with BPD-MA solution or BPD-MA entrapped in conventional liposomes. These results suggest that a long-circulating liposomal formulation of photo-sensitive agents is useufl for PDT.

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The contribution of hydrophobicity of different types of detergents to disopyramide uptake by rat small intestinal brush-border membrane vesicles was studied in relation to their membrane lipid fluidity and the physicochemical parameters of the detergents, i.e., hydrophile-lipophile balance (HLB). Span-, Tween-type detergents or glycerol esters at non-solubilizing concentrations (0.01-0.05% (w/v)) decreased the extent of maximum uptake of the drug in the presence of outward H⁺ -gradient, but not in the absence of the gradient. The fluorescence anisotropy of the vesicles using diphenylhexatriene (DPH), as reflected by its incorporation into the membrane inner lipid layer, decreased with the addition of all detergents used. In contrast, that of the vesicles using trimethylammoniumphenyl phenylhexatriene (TMA-DPH), which reflected its incorporation into the membrane outer lipid layer, increased depending on the concentration of Tween-type detergents except for Tween 81 and Tween 85, glycerol esters (MO-500, MO-750, ML-500 and ML-750); it decreased with the addition of Span-type detergents, Tween 81 and glycerol ester (MO-310). Therefore, the membrane lipid fluidity change of the outer leaflet, rather than the inner lipophilic domain, of the membrane vesicles caused by the detergents was found to be dependent on the hydrophobicity, but not on the type of detergent. This seems to correlate with the inhibitory effects on the facilitated uptake of the drug by the membrane vesicles.

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The binding and internalization of fractionated [³H]heparin (FH) was kinetically analyzed in rat liver parenchymal cells to clarify its cellular uptake mechanism. The binding of FH to the cell surface was saturable with the dissociation constant (K_d) of 53.5 nM and a maximum binding capacity (B_max) of 19.9 pmol/mg protein. The binding of FH to the cell surface was competitively inhibited not only by heparan sulfate, a polyanion analogous to heparin, but also by rose bengal, an organic anion, suggesting the binding is based on an electric interaction requiring an anionic charge for substrates and consistent with the earlier suggestion of the involvement of the scavenger-like receptor. According to kinetic model analysis, the rate constants of association (k_on), dissociation (k_off), and internalization (k_{int, app}) were estimated to be 0.0005 nM^-1 min^-1, 0.0112 min^-1, and 0.0056 min^-1, respectively. Although both K_d and B_max were larger than those reported in Kupffer cells, suggesting lower affinity and higher capacity in liver parenchymal cells, the apparent internalization rate constant was similar to that in Kupffer cells. We thus provided additional evidence suggesting that a scavenger-like receptor exists in rat liver parenchymal cells, and then kinetically characterized the surface binding and internalization of fractionated heparin by this receptor.

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Pharmacological effect after intravenous (i.v.) or subcutaneous (s.c.) administration of human recombinant granulocyte colony stimulating factor (rhG-CSF) was evaluated by using a physiologically based pharmacokinetic/pharmacodynamic model. The increase of neutrophil counts in blood after s.c. administration of rhG-CSF (0.5-1.0 μg/kg) was larger than that after i.v. administration of the same dose, while area under the curves of plasma concentration of rhG-CSF after s.c. administration was smaller than that after i.v. administration (Azuma et al., J. Clin. Therap. Med., 5, 1579-1603, 1989). Based on the pharmacokinetic/pharmacodynamic model considering metabolic turnover of neutrophil in vitro, time course of absolute neutrophil counts in blood after either type of rhG-CSF administration to normal healthy volunteers was analyzed. The nonlinear relationship between the concentration of rhG-CSF and in vivo activity for proliferation of neutrophil could be explained by the drug-receptor-effector ternary complex model. These in vivo and in vitro models made it possible to understand the above-mentioned discrepancy of pharmacokinetic/pharmacodynamic behavior between i.v. and s.c. administration of rhG-CSF. Simulation of the neutrophil count-time profiles after repeated i.v. and s.c. dosing of rhG-CSF according to these models showed good aggreement with the observed data. In order to obtain the rational dosage regimen of rhG-CSF from pharmacological and economical points of view, a slow constant infusion method may be more useful than rapid infusion.

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We previously isolated from mouse cells an autonomous replicating sequence (ARS) ARS65 (Ariga, Itani and Iguchi-Ariga, Mol. Cell. Biol. 7, 1-6, 1987). Here we report the nucleotide sequence of ARS65. The sequence from BglII to EcoRI sites cloned as ARS was 2568 bp long. There exist three intersting domains : a TA repeat, a myc like box (essential sequence for c-myc ARS, and a T rich region. Cloned DNAs containing various segments of pARS65 were transfected to rat 3Y1 cells together with the hydromycinB resistance expression vector, and hygromycinB resistant clones were isolated. Established cell lines transfected with plasmids carrying either a myc-like box or a T rich region harbored the replicated plasmids, indicating that these two elements are necessary for the ARS function of pARS65.

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We evaluated the effects of Z-100, extracted from human type Mycobacterium tuberculosis strain Aoyama B, on collagen-induced arthritis (CIA) in mice. One hundred thirty-five DBA-1K mice, 8 weeks of age, were assigned to 9 groups and immunized with bovine type II collagen (CII) or CFA. From the next day, Z-100 at does of 0.004, 0.04 or 0.4 mg/kg B.W./d for 48 d was intradermally injected into the tail base. Methotrexate (MTX) at daily doses of 0.1, 0.3, or 1.0 mg/kg B.W. and cyclophosphamide (CY) and at a daily dose of 5 mg/kg. B.W. were used as reference drugs. The effects of these drugs on CIA mice were evaluated in terms of the incidence of CIA, the arthritis index (AI), and hind paw edema, after which the animals were sacrificed at 49 d, and both anti-CII antibody titer and delayed-type hypersensitivity (DTH) reaction were measured. In the arthritic control groups, the AI and hind paw edema were significantly increased after the second immunization on day 28. The anti-CII antibody titer and DTH reaction were significantly increased compared to normal mice on day 49. Z-100 significantly inhibited the AI at a dose of 0.4 mg/kg/d on day 49, and suppressed the incidence of both CIA and hind paw edema. Increases in both anti-CII antibody titer and DTH reaction in CIA mice were prevented by treatment with Z-100 at 0.4 mg/kg/d. MTX, in a dose-dependent manner, and CY, at a dose of 5 mg/kg/d, inhibited the incidence of CIA, AI hind paw edema, anti-CII antibody titer and DTH reaction in CIA mice. Z-100 at a dose of 0.4 mg/kg was as effective as MTX was at a dose of 0.3 mg/kg against the DTH reaction, and it had no side effects. These results suggest the usefulness of Z-100 in patients with chronic rheumatoid arthritis.

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We examined in vitro cytotoxic activity of imidazolyl-1, 3, 5-triazine derivatives using human breast cancer cell lines (MCF-7, R-27, T-47D and ZR-75-1) and murine leukemia cell line (P388). The percentage of viable cells was determined by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazorium bromide (MTT) assay. Hexamethyl-melamine (HMM), a 1, 3, 5-triazine derivative has previously been recognized as an antitumor agent effective against lung, ovarian and breast cancer, but failed to show a significant cytotoxic activity in the present study. In contrast, four imidazolyl-1, 3, 5-triazine derivatives, 2-(1-imidazolyl)-4, 6-bis(morpholino)-1, 3, 5-triazine, 2-(1-imidazolyl)-4-morpholino-6-(3-thiazolidinyl)-1, 3, 5-triazine, 2-(4-cyano-4-phenylpiperidino)-4-(imidazolyl)-6-morpholino-1, 3, 5-triazine and 2-(1-imidazolyl)-4-(1-N-methyl-N-phenylamino)-6-morpholino-1, 3, 5-triazine showed cytotoxic activity for most cell lines, which was significantly greater than the activity of hydroxymethylpentamethylmelamine (HMPMM), a major metabolite of HMM.

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The absorption characteristics of azasetron, a serotonin type 3 (5-HT₃) receptor antagonist which is used for the treatment of chemotherapy-induced emesis and nausea, were investigated in rabbits. The serum concentrations of azasetron following rectal administration as a suppository increased rapidly and showed the mean t_max value of 0.18 h. The concentrations were greater after rectal administration than those after oral administration. The absolute bioavailability was significantly different between the rectal, 52.9% and oral doses, 21.6%. The mean C_max and t_max values after the rectal dose were 904.8 ng/ml and 0.18 h, respectively, whereas those after the oral dose averaged 124.7 ng/ml and 0.85 h, respectively.These results indicate that azasetron in absorbed to a greater extent and more rapidly into the systemic circulation via the rectum than via the intestine in rabbits. Consequently, the suppository form azasetron hydrochloride may be feasible for the treatment of chemotherapy-induced acute emesis and nausea.

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We synthesized various glycolipid derivatives and examined the in vivo behaviors of liposomes modified with these movel glycolipid derivatives. Gal-t-pas (1, {8-(2-hexadecyloctadecanoylamino)-3, 6-dioxaoctyl}-β-D-galactoside), Lac-t-psa (3, 8-(2-hexadecyloctadecanoylamino)-3, 6-dioxaoctyl β-D-lactoside) and GalNAc-t-psa (4, 8-(2-hexadecyloctadecanoylamino)-3, 6-dioxaoctyl 2-acetamido-β-D-galactopyranoside) modified liposomes were recognized by the liver. Lac-t-psa (3) modified liposome was accumulated to the highest degree, followed by GalNAc-t-psa (4) modified liposome and then Gal-t-psa (1) modified liposome. The intrahepatic distributions of Gal-t-psa (1), GalNAc-t-psa (4), Glc-t-psa (2, 8-(2-hexadecyloctadecanoylamido)-3, 6-dioxaoctylβ-D-glucopyranoside) and Lac-t-psa (3) modified liposomes were investigated. GalNAc-t-psa (4) and Lac-t-psa (3) modified liposome were accumulated to greater extents than Gal-t-psa (1) modified liposome in hepatic parenchymal cells. The intrahepatic distribution of these liposomes showed that Lac-t-psa (3) and GalNAc-t-psa (4) were preferable to Gal-t-psa (1) for the selective delivery of liposomes to hepatic parenchymal cells.

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The effectiveness of chitosan, a biocompatible polymer derived by the deacetylation of chitin, as a scaffold of hepatocyte attachment, was examined. Since chitosan gel was too fragile to use for cell culture, its free amino groups were crosslinked by glutaraldehyde to increase its strength. Rat hepatocytes seeded onto glutaraldehyde-crosslinked chitosan (GA-chitosan) gel could stably sttach to the surface, retaining its spherical form, the same as in vivo, and the release a vary small amount of lactate dehydrogenase during the 5 d culture period. By contrast, hepatocytes on a collagen-coated surface spread flat, and they released much more lactate dehydrogenase than those on the GA-chitosan gel. Hepatocytes on GA-chitosan also retained higher urea synthesis activity, a liver-specific function, than those on the collagen-coated surface. These results indicate that chitosan is a promising biopolymer as a scaffold of hepatocyte attachment, which can be applied to an effective bioartificial liver support system.

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A genomic DNA clone of karasurin was isolated using the polymerase chain reaction from Trichosanthes kirilowii var. japonica (Cucurbitaceae). The amino acid sequence deduced from the nucleotide sequence was consistent with previously reported sequences of karasurin-A and karasurin-C except for a putative signal peptide and extra amino acids at the C-terminus, neither of which is present in the natural protein. Recombinant karasurin was synthesized in Escherichia coli, in which the cloned karasurin gene was expressed under the control of the trc promotor.

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We found that an antigen-specific anaphylaxis was induced by antigen challenge to the abdominal wall, ear auricle, or subcutaneous tissue in mice sensitized 9 days previously with antigen and adjuvant. The anaphylactic reaction was detected by vascular permeability at the injected site 7 minutes after challenge, which was the best time for estimation. A novel method (AW method) for induction and detection of the anaphylactic reaction in mice was established using the abdominal wall as the challenge site. This method could detect the anaphylactic response in mice 1 to 3 weeks after sensitization. The increase in vascular permeability was completely inhibited by administration of diphenhydramine.

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We examined the action mechanism of oleanolic acid 3-O-monodesmoside, momordin Ic (1), and oleanolic acid 3-O-glucuronide (2) for the inhibitory effect on the increase in serum glucose levels in oral glucose-loaded rats. Although 1 and 2 dose-dependently inhibited the increase in serum glucose levels in oral glucose-loaded rats, these compounds showed no significant effects on serum glucose levels in normal rats, intraperitoneal glucose-loaded rats, and alloxane-induced diabetic mice. Furthermore, 1 and 2 were found to suppress gastric emptying in rats, and also to inhibit the glucose uptake in rat small intestine concentration dependently in vitro. These results indicate that 1 and 2 given orally have neither insulin-like activity nor insulin releasing-activity. 1 and 2 apparently inhibited glucose absorption by suppressing the transfer of glucose from the stomach to the small intestine and by inhibiting the glucose transport system at the small intestinal brush border.

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