S-100 Protein in Clonal Astroglioma Cells Is Released by Adrenocorticotropic Hormone and Corticotropin-Like Intermediate-Lobe Peptide

S-100 protein in clonal GA-1 and C6 rat glioma cell lines was released in serum-free medium supplemented with adrenocorticotropic hormone (ACTH). The induction of S-100 protein release by ACTH was dose-dependent, showing a half-maximal release at about 5 microM, and the S-100 protein concentration in the medium increased sharply within 3 min, but slightly during further incubation. The S-100 protein release was apparently accompanied by a decrease in the membrane-bound form of S-100 protein in the cell. The S-100 protein release was induced not by the ACTH1-24 fragment, which exhibits the known effects of ACTH, but by the ACTH18-39 fragment, which is designated as corticotropin-like intermediate-lobe peptide (CLIP). These results indicate that the C-terminal half of ACTH is responsible for the S-100 protein release. The enhancement of S-100 protein release by ACTH was also observed in normal rat glioblasts. The release induced by ACTH was apparently specific to S-100 protein, because little release of the cytoplasmic enzymes, creatine kinase, and enolase was observed under the same conditions. High concentrations (5 mM) of dibutyryl cyclic AMP or dibutyryl cyclic GMP were also found to induce S-100 protein release; however, catecholamines (epinephrine, norepinephrine, isoproterenol, and dopamine), acetylcholine, and glutamic acid did not enhance the release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lectin histochemical study on the infraorbital gland of the Japanese serow (Capricornis crispus)

Histology and lectin histochemistry were performed in the infraorbital gland of the Japanese serow. The gland is composed of glandular tissues and a pouch filled with the secretion. The tissues consist of an inner layer of sebaceous glands and an outer layer of apocrine glands. The male sebaceous layer is made up of the ordinary type, whereas the female's layer consists of the ordinary and modified types. In the apocrine gland stained with Arachis hypogaea (PNA), nine different patterns of glandular tubules were distinguished on the basis of staining of the cytoplasm, the Golgi area of secretory cells and secretion. Secretory modes of apocrine secretion and exocytosis were included in these stainings. Myoepithelial cells stained constantly with Glycine max (SBA) except when only the Golgi area of secretory cells was positive. The modified sebaceous gland was stained with PNA, SBA, Ricinus communis I (RCA), Triticum vulgaris (WGA), Canavalia ensiformis (Con A) and Ulex europaeus I (UEA), while the ordinary type was positive in PNA, RCA, SBA, WGA and Con A. The secretion in the pouch was stained with PNA, RCA, SBA, Dolichos biflorus (DBA), WGA and Con A. These findings suggest that the modified sebaceous gland contains large amounts of glycoconjugates and the apocrine gland shows a cyclic secretory process of apocrine secretion and exocytosis.     

The role of phospholipase in beta-agonist-induced down regulation in guinea pig lungs

It has been observed that repeated and prolonged beta-agonist treatment causes the impairment of beta-adrenergic function, so-called "desensitization" or "down regulation". To clarify the mechanism of down regulation, the following experiment was performed using guinea pig lungs. Animals were divided into four groups: In the metaproterenol groups, guinea pigs were treated with metaproterenol (10 mg/kg/day) by intraperitoneal injection once a day for 1 day or for 7 successive days In the control groups, guinea pigs were treated with saline by the same procedure as in the metaproterenol groups. In the group treated with metaproterenol for 7 days, there was a 45% reduction in the number of beta-adrenoceptors and a 62% reduction in adenylate cyclase activity, compared with those of the control group. However, there were no significant changes in the dissociation constant (Kd) of the receptors. On the other hand, no reduction in the number of beta-adrenoceptors and adenylate cyclase activity was observed in the group treated with metaproterenol once a day for 1 day, compared with those of the control group. Phospholipase (PLase) activity in the lung microsomes of guinea pigs injected with metaproterenol for 1 day and for 7 days was elevated by 14.4 and 33.1%, respectively, compared with that of the control groups. Phospholipid contents of lung membranes prepared from the animals treated with metaproterenol for 7 days were significantly decreased compared with those of the control group, though in the group treated with metaproterenol once a day for 1 day, phospholipid contents did not differ from those of the control. Lung membranes treated with PLase A2 revealed decreases both in the number of beta-adrenoceptors and adenylate cyclase activity, dose dependently. These results and the fact that membrane phospholipids are involved in the beta-adrenoceptor system suggest that down regulation observed during beta-agonist administration is, at least in part, attributed to degradation of phospholipids of lung membranes by the persistent activation of PLase in the tissue.     

Purification of bleomycin hydrolase with a monoclonal antibody and its characterization

We established a hybridoma clone that produced anti-bleomycin hydrolase antibody. The subclass of the monoclonal antibody was immunoglobulin M. The antibody significantly reacted with bleomycin hydrolase from rabbit tissues, mouse livers, sarcoma 180, and adenocarcinoma 755 but not significantly with that from MH 134 and Ehrlich carcinoma. The enzyme from L5178Y cells showed an intermediate reactivity. Bleomycin hydrolase was purified from rabbit liver by immunoaffinity with the monoclonal antibody and DEAE gel chromatography. Approximately 1300-fold-purified bleomycin hydrolase was obtained. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing on a polyacrylamide slab gel of purified bleomycin hydrolase showed a single band with an apparent Mr of 48K and an isoelectric pH of 5.2. The molecular weight of bleomycin hydrolase determined on gel filtration high-performance liquid chromatography was ca. 300K, suggesting a hexameric enzyme. The enzyme showed an optimum pH of 6.8-7.8 and gave a Vmax value of 6.72 mg min-1 mg-1 for peplomycin and 9.24 mg min-1 mg-1 for bleomycin B2 and a Km value of 0.79 mM for both substrates. The enzyme was inhibited by E-64, leupeptin, p-tosyl-L-lysine chloromethyl ketone, N-ethylmaleimide, Fe2+, Cu2+, and Zn2+ but was enhanced by dithiothreitol. The results suggest that bleomycin hydrolase is a thiol enzyme.     

Sulfhydryl modification and activation of phenylalanine hydroxylase by dinitrophenyl alkyl disulfide

A new family of asymmetric thiol-disulfide exchange reagents, the dinitrophenyl alkyl disulfides (DNPSSR), was used to modify rat liver phenylalanine hydroxylase. The results indicate that the enzyme has two different types of reactive sulfhydryl (SH) residues per subunit. One SH residue was modified selectively by a DNPSSR having a neutral and hydrophilic alkyl group, and this modification was accompanied by appreciable activation of enzyme; the other SH residue was modified only by an anionic DNPSSR, and this modification did not result in activation. The catalytic properties of phenylalanine hydroxylase activated by DNPSSR were similar to those of the N-ethylmaleimide- (NEM-) modified enzyme, but the process of activation by DNPSSR was quite different from modification with NEM. An analysis of the reaction kinetics of the modification and of catalysis by the modified enzyme suggests that DNPSSR modification causes a change in the subunit interaction leading to a loss of the negative cooperativity normally seen with phenylalanine hydroxylase.     

Suppression of Inhibitory Effects of Copper on Enzymatic Activities by Copper-Binding Substances from Athyrium yokoscense Assayed in vitro

A metal-tolerant fern, Athyrium yokoscense, is capable of growingin highly copper-contaminated soil, but cupric chloride inhibitedthe activities of some enzymes extracted from the fern. Thefunction in the detoxification of copper of two copper-bindingsubstances was investigated by examination of their effectson various enzymes assayed in vitro, i.e. acid phosphatase (orthophosphoric-monoesterphosphohydrolase [acid optimum], EC 3.1.3.2 [EC] ), glucose-6-phosphatedehydrogenase (D-glucose-6-phosphate: NADP⁺ 1-oxidoreductase,EC 1.1.1.49 [EC] ) and isocitrate dehydrogenase (threo-Ds-isocitrate:NADP⁺ oxidoreductase [decarboxylating], EC 1.1.1.42 [EC] ). The twocopper-binding substances, whose apparent molecular weightsare 9.5 kDa and 2 kDa, were previously obtained from the solublecytoplasmic fraction of the fern root. The 9.5-kDa substance,which is a cysteine-rich peptide induced as a result of exposureof the fern to copper, was found to suppress almost entirelythe inhibitory effects of the metal on the enzymes. The suppressoractivity of the peptide was nearly as effective as that of ethylenediaminetetraaceticacid. The 2-kDa substance, which is also found in fern thathas not been exposed to copper, had a more modest suppressoractivity. These results indicate that the 9.5-kDa substancemay contribute to the copper-tolerance of the fern growing incopper-contaminated soil. (Received August 26, 1988; Accepted March 17, 1989)     

Identification of the cell surface molecule involved in sexual cell fusion of Dictyostelium discoideum

Dictyostelium discoideum was used as a model system for elucidating the molecular mechanism of sexual cell fusion. In heterothallic strains NC4 and HM1 of D. discoideum, complements in mating type, amoeboid cells acquire fusion competence only under certain environmental conditions, such as the presence of excess water and a certain period of darkness, to fuse sexually. The surface of cells which acquired fusion competence was found to possess specific antigens. Monovalent antibodies prepared from rabbit antiserum against fusion-competent NC4 cells inhibit the sexual cell fusion of these cells completely. Two specific antigenic proteins, 39 and 138 k Da in relative molecular mass and specific for fusion-competent cells, were detected. Only one, the 138-k Da protein, was capable of neutralizing the fusion-inhibitory activity of the monovalent antibody. These results show that the 139-k Da protein is the one involved in the sexual cell fusion of NC4 and HM1 strains in D. discoideum.     

SPKK, a new nucleic acid-binding unit of protein found in histone.

A new DNA-binding unit of a protein different from the alpha-helix, the beta-sheet and the Zn-finger is proposed based on the analysis of the structure of the N-terminus of sea urchin spermatogenous histone H1. DNA-binding arms of the sea urchin spermatogenous histones, H1 and H2B, are composed of repeats of Ser-Pro-Lys(Arg)-Lys(Arg) (SPKK) residues. A six-times repeat of SPKK (S6 peptide) was isolated from H1 and the competition of S6 for DNA binding with a DNA-binding dye, Hoechst 33258, was analysed. The S6 peptide is shown to be a competitive inhibitor of Hoechst 33258, and it is concluded that the SPKK repeat binds to DNA in its minor groove with a binding constant, KS6 = 1.67 X 10(10) M-1. The circular dichroism (CD) spectrum of a synthetic peptide, SPRKSPRK (S2 peptide), is quite different from those of both the alpha-helix and the beta-sheet and resembles that of a random coil. From statistical consideration of protein structures it is proposed that SPKK forms a compact beta-turn stabilized by an additional hydrogen bond. Since a repeated chain of such turn of SPKK offers a repeat of amides of Ser residues at a distance similar to that of DNA-binding amides of the drugs, Hoechst 33258 and netropsin, and since the amides of these drugs bind to DNA replacing the spine of hydration in a minor groove, it is proposed that a repeat of SPKK binds to DNA in the minor groove using similar hydrogen bonds.