Under voltage clamp, Paramecium tetraurelia was used to examine the cold-induced inward current and its inhibition by a calmodulin antagonist, W-7 [N-(6 aminohexyl)-5-chloro-1-naphthalenesulphonamide]. Cooling of the cell caused an inward current. The amplitude of the current was increased as the membrane potential was made more positive than the resting potential, and it was significantly blocked by using CsCl-filled electrodes and tetraethylammonium in the bath solution, suggesting that the current was accompanied mainly by a decrease in K⁺ conductance. The cold-induced inward current was reversibly inhibited by the external application of W-7 in a concentration-dependent manner. EGTA-microinjection into the cell also reduced the current. These results indicate that the decrease in K⁺ conductance induced by cooling is Ca²⁺-dependent and is inhibited by W-7.

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DNA-dependent protein kinase (DNA-PK), consisting of the 470-kDa catalytic component (DNA-PKcs) and the DNA-binding regulatory component Ku protein (p70/p80), catalyzes phosphorylation of a variety of DNA replication/transcription/repair factors in the presence of double-stranded DNA. In the resting states of human peripheral blood mononuclear cells, DNA-PK activity and the protein level of DNA-PKcs were very low in the nuclear extracts, but they were high in the whole cell extracts. Depending upon proliferation of the T lymphocytes, DNA-PK activity and the protein level of DNA-PKcs in the nuclear extracts greatly increased. Immunocytochemical analysis suggested translocation of DNA-PKcs from the cytoplasm to the nucleus upon growth stimulation in the T lymphocytes.

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In order to identify the cDNAs responsible for hemopoietic supportive activity, expression of mRNAs in hemopoietic supportive bone marrow stromal cells (MS-5) and non-supportive stromal cells (MS-K) were compared by the cDNA subtraction method. A subtracted MS-5-cDNA library, which contains cDNA clones corresponding to mRNAs present in MS-5 cells but not in MS-K cells, was constructed. Screening of subtracted MS-5-cDNA library resulted in the isolation of some clones. Two of them, lipid binding protein (LBP) and haptoglobin (Hp), were expressed specifically in MS-5 cells but not in MS-K cells. The genes of LBP and Hp were subcloned into mammalian expression vector and transfected into hemopoietic non-supportive stromal cells line, MS-K. Then LBP-expressing stable transformants (MS-K-LBP) and Hp-expressing transformants (MS-K-Hp) were cloned. A rosette formation assay was carried out to investigate whether or not the LBP and Hp cause MS-K cells to adhere to hemopoietic cells. MS-K-LBP formed rosettes with hemopoietic cells as MS-5 cells, although the MS-K-Hp and normal MS-K cells did not form rosettes. These data indicate that LBP expressed in hemopoietic supportive stromal cells is partly responsible for the adhesion of hemopoietic stem cells to stromal cells.

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To investigate basement membrane formation by cooperation between pneumocytes and pulmonary fibroblasts, we cultured type II alveolar epithelial cells obtained from rats transfected with SV40-large T antigen gene (SV40-T2 cells) on type I collagen matrices. On fibroblasts-embedded gel (T2-Fgel), SV40-T2 cells ultrastructurallly formed a continuous and thin layer of lamina densa, while on collagen gel without fibroblasts (T2-gel) SV40-T2 cells produced only discontinuous and diffuse deposits. Stripping SV40-T2 cells off the tissues by H₂O₂ treatment revealed a continuous and plane surface of lamina densa assembled on the T2-Fgel tissue, whereas only amorphous deposits appeared on the T2-gel tissue. Immunolocalization of major basement membrane components showed that type IV collagen, laminin, perlecan and entactin (nidogen) were continuously integrated on the lamina densa in T2-Fgel. In T2-gel, all these components were discontinuously distributed beneath SV40-T2 cells. The contribution of pulmonary fibroblasts to the assembly of basement membrane through reorganization of collagen matrix and/or soluble factors was examined by the cultured of SV40-T2 cells on the freeze-thawed fibroblast-tissue and/or with the fibroblast-conditioned medium. Both SV40-T2 cells on the freeze-thawed fibroblast-tissue and SV40-T2 cells in T2-gel in the fibroblast-conditioned medium failed to produce a lamina densa. SV40-T2 cells could assemble a lamina densa only on the freeze-thawed fibroblast-tissue in the fibroblast-conditioned medium. These results show that the basement membrane components are assembled to a lamina densa by combination of the reorganization of collagen matrix and the supply of soluble factors by pulmonary fibroblasts.

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The biochemical and immunocytochemical properties of peroxisomal and mitochondrial β-oxidation enzymes in bovine adrenal chromaffln cells were investigated. Peroxisomes were detectable by immunofluorescence staining using antibodies against acyl-CoA oxidase, peroxisomal 3-ketoacyl-CoA thiolase and catalase. The mitochondria were abundantly stained with antibody against mitochondrial 3-ketoacyl-CoA thiolase. The biosynthesis and intracellular processing of acyl-CoA oxidase and the peroxisomal 3-ketoacyl-CoA thiolase was slower than that in fibroblasts. The peroxisomal β-oxidation activities shown by [1-¹⁴C] lignoceric acid oxidation were slightly lower than those in fibroblasts, whereas the mitochondrial β-oxidation activities shown by [1-¹⁴C] palmitic acid oxidation were almost identical to those in fibroblasts. Adrenal chromaffin cells are useful materials for investigating the peroxisomal and mitochondrial metabolism of autonomic neurons and may contribute to the clarification of neuronal dysfunction in peroxisomal and mitochondrial disorders.

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We have demonstrated that testosterone-bovine serum albumin conjugate labeled with 2-nm colloidal gold (testosterone-BSA-gold) injected into the vascular system of rat becomes visible as silver deposits on the sections of tissues embedded in epoxy resin after silver enhancement and enters the androgen-target cell nuclei, e.g. round spermatids, but not the non-target cell nuclei. The diameter of the silver deposits depends on the duration of silver enhancement. In this study, to make clear the transfer route of testosterone-BSA-gold into the round spermatid nucleus, the testis of rat injected testosterone-BSA-gold was observed under electron microscope after silver enhancement for short periods of time. The small silver deposits were present on the cell membrane, vesicles, Golgi region, acrosome, subacrosomal space, both the post-acrosomal and the subacrosomal nuclear envelope, and the nucleoplasm in the cap-phase spermatids. The silver deposits were also found in the perinuclear cisterna of post-acrosomal nuclear envelope, but not in the nuclear pore. When the spermatids were observed at high-power magnification without the silver enhancement, the outer nuclear membrane showed many irregular invaginations toward the inner nuclear membrane in the post-acrosomal nuclear envelope. Furthermore, a double-membrane-like vesicle seemed to be present in the nuclear envelope. In the vesicle, the gold particles were present along the inner membrane. These results suggest that testosterone-BSA-gold can enter the nucleoplasm through some route provided by the nuclear double membranes in both the post-acrosomal and the subacrosomal nuclear enve-lope.

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