A sensitive enzyme immunoassay for the determination of serum or urinary cyclazocine was developed. Horseradish peroxidase was used as the labelling enzyme ; it was conjugated with cyclazocine derivatives by the mixed anhydride method. After the immune reaction, bound and free fractions were separated by a double-antibody solid-phase method, using Sepharose 4B gel coupled with purified IgG from goat anti-rabbit IgG serum. The enzyme activity was measured fluorophotometrically, with p-hydroxyphenyl propionic acid and hydrogen peroxide as substrates. The correlation coefficients verified that there was excellent agreement between the results obtained by the new enzyme immunoassay and those of radioimmunoassay (r=0.92) and gas chromatography-mas spectrometry (r=0.94).

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The anti-inflammatory activities of oleanane triterpenoid glycosides, papyrioside L-IIa, L-IIb, L-IIc and L-IId, extracted from Tetrapanax papyriferum (Araliaceae), and their aglycones, papyriogenin A and C and propapyriogenin A₁ and A₂, were investigated by using the carrageenin-induced edema and the cotton pellet granuloma tests in mice. Of these compounds, papyriogenin A and C (30 mg/kg p.o.) manifested in these tests almost the same potency as prednisolone (25 mg/kg p.o.). The structure-activity relationships of these and related triterpenes have been investigated. This takes into account particularly variation in the oxygen function and the molecular conformation. We have found that the anti-inflammatory activity of these triterpenes was favoured when the molecules tended to take a planar conformation ; this was induced by the heteroanulardiene group between C and D ring.

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The effect of stannous chloride on bone metabolism was examined in weanling male rats given oral dose of 1.0 mg Sn/kg at 12-h intervals for 28 d. Hydroxyproline content in the femoral diaphysis but not epiphysis was significantly decreased by tin administration for 14 and 28 d, while free proline contents in the femoral diaphysis and epiphysis were not changed significantly. ³H-Hydroxyproline contents in the femoral diaphysis and epiphysis labeled with ³H-proline injection were not significantly altered by tin administration. In vitro collagen synthesis in the femoral epiphysis tissue but not diaphysis was markedly reduced by tin administration between 3 and 14 d, although ³H-proline incorporations into the femoral diaphysis and epiphysis tissues were not changed significantly. On the other hand, in vitro incorporation of ³H-thymidine into the femoral epiphysis but not diaphysis was significantly decreased by tin administration between 7 and 28 d. A significant decrease of DNA content in the femoral epiphysis but not diaphysis was also observed by tin administration for 14 and 28 d. The present study suggests that tin administration causes the inhibition of collagen synthesis prior to the suppression of DNA synthesis in the femoral epiphysis of rats.

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A highly sensitive enzyme immunoassay system for the determination of cuprozinc-superoxide dismutase (Cu, Zn-SOD) in serum and urine by using β-galactosidase as a labeling enzyme is reported. This assay had a greater sensitivity than that of previously reported radioimmunoassay methods and could measure from 0.05 to 10.0 ng of Cu, Zn-SOD with good reproducibility. Coefficients of variation for this enzyme immunoassay were 2.8-8.3% within a day and 5.6-16.1% between days. The average recoveries were 96-103% from sera and 96-105% from urines, respectively. Using this enzyme immunoassay, serum Cu, Zn-SOD concentrations increased significantly in patients with renal diseases and were slightly elevated in patients with liver diseases. Urinary Cu, Zn-SOD appeared to be the highest in all renal diseases. By immunofluorescent staining, Cu, Zn-SOD was located in the thickened portions of the glomerular basement membrane and proliferating mesangial cells of the kidney tissue from a patient with membranous glomerulonephritis.

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Immunosuppressive effects of trichothecenes of Fusarium solani and Fusarium nivale, T-2 toxin and fusarenon-X, and also of a mycotoxin of Penicillium islandicum, cyclochlorotine, were studied by measuring the anti-2, 4-dinitrophenyl (DNP) antibody responses in guinea pigs immunized with DNP-bovine serum albumin. Among these mycotoxins, T-2 toxin alone suppressed strongly the anti-DNP antibody responses at a certain sublethal dose. With other mycotoxins, no effect was observed at any sublethal doses tested. All of the mycotoxins, on the other hand, inhibited the in vitro blast transformation of guinea pig splenic cells stimulated with lipopolysaccharide of Escherichia coli (B cell mitogen) or concanavalin A (T cell mitogen), when measured by incorporation of ³H-thymidine into DNA. Their inhibitory activities were independent of the sort of mitogen used. As in the case of the antibody responses, T-2 toxin was most potent in reducing the DNA synthesis, and exhibited about 10 and 1000 times as potent inhibitory activity as fusarenon-X and cyclochlorotine, respectively.

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It was shown that the intestinal absorption clearance of D₂O (CL_D2O) could be a more appropriate index to study the solvent drag effect than water volume flow which was the difference between water influx and outflux in the intestinal lumen. Then, the correlation between the intestinal abosrption clearances of drugs (CL_drug) and CL_D2O were studied using the in situ recirculating method in the rat small intestine. The drugs used were low molecular drugs, that is, benzoic acid, salicylic acid, p-hydroxybenzoic acid and antipyrine, and comparably high molecular drugs, that is, cephalexin (CEX), cefroxadine (CXD) and cephalothin (CET). CL_drug and CL_D2O were obtained in hypertonic, isotonic and hypotonic perfused solution adjusted with sodium chloride. Consequently, the correlations for all drugs except CET were significant and high solvent drag effects were observed. CL_drug of benzoic acid, salicylic acid and antipyrine were approximately equal to CL_D2O, suggesting that the intestinal mucosa could not distinguish these lower molecular drugs from water. For the high molecular drugs such as cephalosporins, however, some extent of reflection from the membrane was certainly found in CEX and CXD, and the extent in CET was assumed much larger than CEX and CXD, resulting that the contribution of solvent drag in CET could not be found. Consequently, it was suggested that the solvent drag had some important role in the intestinal absorption of cephalosporins.

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Nineteen cytochalasans including 6 natural cytochalasins, 2 natural aspochalasins, 7 natural chaetoglobosins and 4 their acetates were investigated by viscometry to examine their effects on in vitro polymerization and depolymerization of microtubules. Cytochalasin A was proved as an only active cytochalasan, confirming the studies of Himes et al., and others showed very weak or no noticeable effects. This results indicate that a wide range of effects of cytochalasans are not related to interactions with microtubules.

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The present study demonstrated by DEAE-cellulose column chromatography that oxygen-insensitive nitrofuran reductases in Salmonella typhimurium TA100 consisted of at least two reductases, NADPH-and NAD (P) H-linked enzymes. The NADPH-and NADH-linked activities of the latter enzyme seemed to originate from a single enzyme, because both activities were similarly inactivated by heat and urea treatments, and also inhibited by dicumarol. On the other hand, the NADPH-linked enzyme was less sensitive to heat, urea and dicumarol. Furthermore, the study showed that the NAD (P) H-linked enzyme was a flavoenzyme which could be inactivated by dialysis against 1 M potassium bromide and reactivated by FMN.

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An N-xyloside derivative of p-aminobenzoic acid, K-247, was investigated for the ability to induce changes of Phospholipid metabolism and membrane transport in murine splenic lymphocytes and leukemic cells. K-247 induced an increase of [³H] methyl group incorporation into phospholipid in both normal lymphocytes and leukemic cells (L-1210 and M1 cells). However, K-247 accelerated the turnover of phosphatidylinositol (PI) measured by [³²P] incorporation into PI in L-1210 cells and M1 cells but not in normal lymphocytes ⁴⁵Ca²⁺ influx into normal lymphocytes and leukemic cells was also increased by K-247. A methyltransferase inhibitor, 5'-deoxy-5'-S-isobutyl adenosine (SIBA), suppressed both the increase of phospholipid methylation and that of Ca²⁺ influx. It seemed that Ca²⁺ transport might be regulated by membrane phospholipid methylation. On the other hand, K-247 was found to suppress [³H] aminoisobutylic acid (AIB) uptake into L-1210 cells and M1 cells. Protein synthesis in L-1210 cells and M1 cells slightly decreased but RNA and DNA syntheses in both normal and leukemic cells were not affected by K-247. These results suggest that K-247 mainly acts on cell membranes, which are more sensitive to K-247 in leukemic cells than in normal lymphocytes. K-247 also induced differentiation of M1 cells into macrophages and granulocytes with phagocytic activity and morphological characteristics. Moreover, K-247 elevated the Con A response of murine thymocytes, most of which were immature T cells and had low reactivity to Con A, and caused a decrease of Thy 1.2 antigen on thymocytes. It seemed that K-247 also affected maturation of thymocytes.

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Augmentation of antitumor activity by combined in situ freeze-destruction of tumor (cryosurgery) and administration of antitumor active substances isolated from a hot water extract of an edible mushroom, Flammulina velutipes (Curt. ex Fr.) SING., was investigated in sarcoma 180 bearing ICR mice. Antitumor active substances of the mushroom included β-(1, 3)-glucan (EA₃) and protein-bound polysaccharide (EA₆). Autitumor activity was evaluated by the growth rate of the solid tumor rechallenged subcutaneously (s. c.) or by cumulative mortalities of the mice rechallenged intraperitoneally (i. p.) with the ascites tumor, on day 7 after cryosurgery. Weak antitumor effect induced by cryosurgery against challenged solid tumor of sarcoma 180 was markedly augmented by i.p. administration of EA₆ (10 mg/kg). Cryosurgery of the solid sarcoma 180 apparently did not induce any antitumor effect against challenged ascites sarcoma 180. However, when cryosurgery was combined with oral administration of the polysaccharides (50 mg/kg), prolonged survival of the mice challenged with ascites sarcoma 180 i.p. was recognized by EA₆, but not by EA₃. Timing of the administration of EA₆ (i. p.) with cryosurgery was best in pre-and post-cryosurgery. Immunological activity of EA₆ to the host was discussed.

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A drug absorption model was developed by using the two-dimensional laminar flow in a circular tube, considering a small water absorption or secretion in the intestinal perfusion experiment. The concentration in the intestinal tract was determined by the axial component of velocity, the radial component of velocity, the membrane permeability coefficient, the reflection coefficient and so on. According to the calculated values, the concentration decreased not only from the inlet to the outlet but also from the center of the intestinal tract to the intestinal wall. The concentration at the aqueous-intestinal membrane interface was changed by the radial component of velocity, so it was suggested that the effective thickness of the aqueous diffusion layer (unstirred water layer) varies with a small water absorption or secretion.

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In vitro O-methylation of 4-hydroxyestrone monosulfates has been examined by means of high-performance liquid chromatography with electrochemical detection. When 4-hydroxyestrone or its 3-sulfate was incubated with rat liver homogenate in the presence of S-adenosyl-L-methionine, the 4-methyl ether was formed in twelve times larger amount than the 3-methyl ether. 4-Hydroxyestrone 4-sulfate served for O-methylation to much less extent as a substrate namely, only a small amount of the 4-methyl ether was formed. Enzymic sulfation of 4-hydroxyestrone with rat liver 105000 g supernatant fortified with adenosine 3'-phosphate 5'-phosphosulfate provided solely catechol estrogen 4-sulfates. The participation of catechol O-methyltransferase and aryl sulfatase in the formation of guaiacol estrogens has been discussed.

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