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.     

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.     

A single point mutation confers tetrodotoxin and saxitoxin insensitivity on the sodium channel II

A single point mutation of the rat sodium channel II reduces its sensitivity to tetrodotoxin and saxitoxin by more than three orders of magnitude. The mutation replaces glutamic acid 387 with a glutamine and has only slight effects on the macroscopic current properties, as measured under voltage-clamp in Xenopus oocytes injected with the corresponding cDNA-derived mRNA.     

Synthesis of the vasoconstrictor peptide endothelin in kidney cells

The expression plasmid containing human prepro-endothelin cDNA was constructed and introduced into COS-7 cells. Mature endthelin, consisting of 21 amino acid residues, was secreted into the culture medium of the transfected cells and was also synthesized by non-transfected COS-7 cells. Normal kidney cells derived from other species also synthesized and secreted endothelin. Partial characterization of endothelins produced by kidney cells suggested that existence of new types of endothelin. This is the first report of the vasoconstrictor peptide endothelin being synthesized in kidney cells.     

Proteinic prorenin-releasing-stimulator (PRS) in the rat submandibular gland

The release of prorenin as well as renin from rat renal slices was confirmed by a rat prorenin-prosegment ELISA system and an assay system for determining the renin activity. A significant increase of the prorenin release was found by adding rat submandibular gland extract to the slice medium, indicating the existence of a prorenin-releasing stimulator (PRS) in the extract. The pI and molecular mass of PRS were 8.5-8.7 and 28-30 kDa, respectively. The PRS was completely inactivated by boiling or a proteinase treatment.     

Synthesis of human endothelin-1 precursors in Escherichia coli

Endothelin, the most potent vasoconstrictor found in nature, is thought to be important in the regulation of blood pressure and/or local blood distribution. Human placenta cDNA fragment encoding preproendothelin-1 (preproET-1) and its carboxyl terminal mature precursor (C-matured precursor) was expressed in E. coli. These products were characterized by both enzyme immunoassay and Western blot analysis.     

Dual loading of the fluorescent indicator fura-2 and 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) in isolated myocytes

Isolated rat heart myocytes were loaded with both the Ca2+ sensitive fluorescent probe fura-2/AM and the fluorescent pH indicator 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF/AM). Changes in [Ca2+]i and pHi were measured simultaneously using digitized video fluorescence microscopy. In measurement of [Ca2+]i and pHi, the ratios of dual-loaded cells were not different from single-loaded cells. Using this method, [Ca2+]i and pHi in myocytes were 48 +/- 7 nM and 7.17 +/- 0.05. It is concluded that [Ca2+]i and pHi could be measured simultaneously in isolated myocyte using dual-loading of fura-2 and BCECF.