The epididymis has three major regions ; the caput, corpus and cauda. Sperm were obtained from each region, and their binding ability to egg zona pellucida was assayed. In the case of intact sperm : cauda sperm bound in high number, corpus sperm bound at an intermediate level (an average of 30% of cauda sperm binding level), and caput sperm bound rarely (1% of cauda sperm binding level). In the case of sperm immobilized by pretreatment with LaCl₃, when eggs and sperm were shaken in order to allow them to come into collision with each other, the caput and corpus sperm were able to bind to zona pellucida as well as the caudal sperm could. These results suggest that the difference in binding efficiency of the native caput and corpus sperm to bind to the zona pellucida was not due to a defect in a ligand molecule on the plasma membrane but rather to their lack of motility. When a sperm-egg binding assay was done in the presence of a binding inhibitor of caudal sperm, the binding of caudal sperm was significantly inhibited by fetuin or N-acetylgalactosamine, but the binding of caput and corpus sperm was only slightly inhibited by them. We prepared a plasma membrane fraction from each epididymal sperm, and solubilized it with sodium dodecyl sulfate (SDS). Each SDS-solubilizable fraction inhibited the binding of corresponding epididymal sperm to egg zona pellucida but didn't inhibit the binding of sperm from different regions of epididymis. These results suggested that a ligand molecule of caudal sperm was different from that of caput and corpus sperm.

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One of the ligand molecules was purified from mouse cauda epididymal sperm for an indication of its inhibitory activity in sperm-egg binding. It was isolated in an electrophoretically homogeneous form by ionophore A23187 treatment of sperm, sedimentation by ultracentrifugation, solubilization by 2% Nonidet P-40 (NP-40), repeated ion exchange chromatography on DEAE-Sepharose CL-6B and gel filtration on Sephacryl S-200. The purified ligand exhibited a molecular mass of 60000 dalton by gel filtration and 67000 dalton by sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE). This molecule was radioiodinated by the lactoperoxidase method and assayed the binding ability to egg zona pellucida. The labeled ligand specifically bound to both cumulus-free egg zona pellucida and isolated-zona pellucida of unfertilized egg in a similar extent, but didn't bind to 2-cell egg zona pellucida. The maximal binding site of ligand per egg zona pellucida was estimated about 660000 molecules by Scatchard plot analysis.

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Mouse sperm bound to egg zona pellucida and attached to unfixed rabbit erythrocytes specifically, but not to fixed-erythrocytes. When mouse sperm were treated with ionophore A23187, the sperm lost both the binding ability to the zona pellucida and the attachment ability to rabbit erythrocytes. The membrane vesicles released from A23187-treated sperm agglutinated rabbit unfixed erythrocytes. Sperm binding to egg zona pellucida was also inhibited by the membrane vesicles. The inhibition spectrum of a hemagglutination and of an inhibitory activity of sperm-egg binding by various carbohydrates was studied. The hemagglutination was inhibited by N-acetyl-glucosamine, N-acetylgalactosamine and glyco-proteins, such as fetuin and orosomucoid, but not by N-acetylneuraminic acid (NeuNAc) in saccharides and asialo-transferrin in glycoproteins. On the other hand, the sperm-egg binding was inhibited most effectively by NeuNAc and also by asialo-transferrin. The inhibition spectrum of hemagglutination and sperm-egg binding didn't agree with each other. These results suggest that a lectin-like molecule is expressed on mouse sperm surface but is not directly concerned in sperm-egg binding.

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Using a radioligand binding assay, we assessed the affinity and selectivity of the antagonistic effects of bopindolol (4-(benzoyloxy-3-tert-butylaminopropyl)-2-methylindole hydrogenmalonate) and its two metabolites, (18-502, (4-(3-tert-butylamino-2-hydroxypropoxy)-2-methyl indole) and 20-785, (4-(3-tert-butylaminopropoxy)-2-carboxyl indole)), for β₁-and β₂-adrenoceptors and on 5HT_1B-receptors in rat brain and heart. In addition, we also determined the pA₂ values of these agents for their antagonistic effects toward positive chronotropic and inotropic actions (β₁-adrenoceptors) and for their antagonistic effects toward isolated tracheal relaxation (β₂-adrenoceptors), using isoproterenol as an agonist. The data showed that bopindolol was more selective for β₂-adrenoceptors than for β₁-adrenoceptors and 5HT_1B-receptors, but its two metabolites (18-502 and 20-785) did not have significant selectivity for β₁-and β₂-adrenoceptors, using both [³H]CGP12177 and [¹²⁵I] ICYP ([¹²⁵I] iodocyanopindolol) bindings. In contrast, the results from pharmacological assessment for antagonistic potencies, using atria and trachea, showed that bopindolol and its two metabolites did not have significant selectivity on β₁-and β₂-adrenoceptors. A major metabolite of bopindolol, 18-502, had higher pK₁ and pA₂ values for β-adrenoceptors and 5HT_1B-receptors than those of bopindolol and 20-785. These results indicate, first, that a radioligand binding assay for the assessment of the selectivity of bopindolol and its two metabolites for β₁-and β₂-adrenoceptors is more effective than pharmacological experiments, and that there is a possibility that its two metabolites also contribute to the strong β-blocking action of bopindolol.

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Sulfated derivatives of (1→3)-β-D-glucans with different degree of branching (DB) i.e., curdlan (linear, DB 0), grifolan (6-O-substituted by β-D-glucose at every third residue of the main chain, DB 1/3), and SSG (6-O-substituted by β-D-glucose at every second residue, DB 1/2) having DS value (degree of substitution) lower than 0.6 were prepared. Biological activities [clotting of plasma, alternative pathway of complement (APC), proliferative response of murine spleen cells (mitogenic activity), and activation of murine peritoneal macrophages in vitro] of these derivatives were examined. Clotting of plasma was inhibited by the derivatives having DS above 0.2. APC was inhibited by incubation with derivatives and was strongly dependent on substitution. Inhibition of APC was the most significant in sulfated SSG having DS 0.6 [SSG (3)]. Mitogenic activity was observed by most of the derivatives and the highest activity was shown by SSG (1) (DS 0.2). Macrophage was also activated but SSG (1) would lose the capability to recognize the receptor for (1→3)-β-D-glucans. From these results, it appears that the biological activities of (1→3)-β-D-glucans were significantly modulated by sulfation and the effect was dependent on both DS and DB.

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Using an air pouch type allergic inflammation model in rats, a role of circulating leukocytes in allergic inflammation responses was investigated by comparing normal rats with peripheral leukocytopenia rats induced by cyclophosphamide treatment. In the leukocytopenia rats, vascular permeability, edema formation, leukocyte infiltration into the pouch fluid, neutrophil chemotactic activity in the pouch fluid occurred both at the early phase (4 h) and the late phase (8 h) after the antigen challenge were decreased. However, edema formation induced by intradermal injection of arachidonate metabolites, serotonin, or platelet activating factor was not suppressed at all in the leukocytopenia rats. A possible role of peripheral leukocytes in allergic and non-allergic inflammation is discussed.

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Metabolism of two polychlorinated biphenyls, 3, 4, 3', 4'- and 2, 5, 2', 5'-tetrachlorobiphenyl (TCB), was studied using rat liver microsomes and the four forms of cytochrome P-450 (P-450), P-450b, P-450e, P-450c and P-450d. At first, effects of various inducers of P-450 such as phenobarbital (PB), 3-methylcholanthrene (MC), isosafrole (ISF) and pregnenolone 16α-carbonitrile on the formation of metabolites of these TCBs by liver microsomes were compared. 3, 4, 3', 4'-TCB was significantly metabolized by liver microsomes from MC-treated rats to form two previously reported metabolites, 4-hydroxy-3, 5, 3', 4'-TCB and 5-hydroxy-3, 4, 3', 4'-TCB with a relative ratio of 2.5 : 1. Incubation with microsomes from untreated or PB-treated rats produced none of the metabolites. On the other hand, 2, 5, 2', 5'-TCB was metabolized to 3-hydroxy-2, 5, 2', 5'-TCB most easily by liver microsomes from PB-treated rats and at a moderate rate by liver microsomes from ISF-treated rats. Activities of microsomes from untreated or MC-treated rats to hydroxylate 2, 5, 2', 5'-TCB were low or undetectable. When these TCB hydroxylase activities were examined with a reconstituted system consisting of each P-450, reduced nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P-450 reductase, dilauroylphosphatidylcholine and NADPH-generating system, only P-450c catalyzed both the 4-and 5-hydroxylations of 3, 4, 3', 4'-TCB at a ratio of 2.2 : 1. On the contrary, the hydroxylation of 2, 5, 2', 5'-TCB proceeded efficiently with P-450b and P-450e, being more efficient with the former. P-450d did not show any catalytic activity toward 3, 4, 3', 4'-TCB and 2, 5, 2', 5'-TCB. These results indicated that P-450c and P-450b play a major role in the metabolism of 3, 4, 3', 4'-TCB in MC-treated microsomes and of 2, 5, 2', 5'-TCB in PB-treated microsomes, respectively. The inhibition studies using antibodies against P-450c and P-450b further supported the above conclusions.

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