Rat gastric mucins recognized by monoclonal antibodies RGM21 and HIK1083: isolation of mucin species characteristic of the surface and glandular mucosa.

Whole mucins and reduced subunits were extracted from the corpus of the rat stomach. After purification by Sepharose CL-4B chromatography followed by cesium trifluoroacetate equilibrium centrifugation, they were analyzed by Sepharose CL-2B chromatography, rate-zonal sedimentation centrifugation, and Q-Sepharose chromatography. Monoclonal antibodies RGM21 and HIK1083, which histochemically stained mucins in the surface and glandular mucosa of the rat stomach, respectively, were used to detect the site-specific mucins. Although RGM21- and HIK1083-reactive mucins both had a multimerized structure, the density and size of both the whole mucins and reduced subunits differed, thus indicating the presence of distinct mucin species in the surface and glandular mucosa. The mucin subunits were separated into four fractions, UB, B1, B2a, and B2b, by Q-Sepharose chromatography. HIK1083 reacted mainly with UB, while RGM21 reacted with B1, B2a, and B2b. These results, combined with dot-blot, amino acid, and carbohydrate composition analyses, showed that the surface mucins may consist of three kinds of subunits. In contrast, the glandular mucins may consist of one kind of subunit which differs from that of surface mucins.     

Nucleobase catalysis in the hairpin ribozyme

RNA catalysis is important in the processing and translation of RNA molecules, yet the mechanisms of catalysis are still unclear in most cases. We have studied the role of nucleobase catalysis in the hairpin ribozyme, where the scissile phosphate is juxtaposed between guanine and adenine bases. We show that a modified ribozyme in which guanine 8 has been substituted by an imidazole base is active in both cleavage and ligation, with ligation rates 10-fold faster than cleavage. The rates of both reactions exhibit bell-shaped dependence on pH, with pK(a) values of 5.7 +/- 0.1 and 7.7 +/- 0.1 for cleavage and 6.1 +/- 0.3 and 6.9 +/- 0.3 for ligation. The data provide good evidence for general acid-base catalysis by the nucleobases.     

The unique action for bone metabolism of 1 alpha,25-(OH)2D3-26,23-lactone

[23 (S), 25 (R)]-1 alpha,25-Dihydroxyvitamin D3-26,23-lactone [( 23 (S),25 (R)]-1 alpha,25-(OH) 2D3-26,23-lactone) increased dose-dependently alkaline phosphatase activity in osteoblastic cells, clone MC3T3-E1, in medium containing 0.1% bovine serum albumin. The maximal stimulated enzyme activity per mg protein was 1.6-fold over that of control cultures at 250 pg/ml. The metabolite also increased collagen synthesis in a dose-related fashion. On the other hand, [23 (S),25 (R)]-1 alpha,25-(OH)2D3-26,23-lactone decreased slightly but significantly 45Ca mobilization, and blocked the resorptive action of 1 alpha,25-dihydroxyvitamin D3 but not that of parathyroid hormone, in mouse calvaria in organ culture. These results indicate that [23 (S),25 (R)]-1 alpha, 25-(OH)2D3-26,23-lactone stimulates the differentiation of osteoblasts and inhibits bone resorption in vitro.     

Effect of forskolin on collagen production in clonal osteoblastic MC3T3-E1 cells

The effect of forskolin on collagen production in osteoblasts was investigated by using clonal osteoblastic MC3T3-E1 cells cultured in a-minimum essential medium containing 0.1% bovine serum albumin. Forskolin increased the adenylate cyclase activity in membranes pelleted from homogenates of the cell line in a dose-dependent manner. The drug caused a 13-fold stimulation at 10(-4) M, indicating that the compound directly acts on adenylate cyclase, leading to an increase in the intracellular cAMP content of the cells. Collagen accumulation in the cultures was elevated by one-day treatment with 5 X 10(-5) M forskolin to about twice that in the controls. The stimulation was mainly due to an elevation in collagen synthesis but not to an inhibition of intracellular collagen degradation because forskolin dose-dependently increased collagen synthesis; it also significantly increased the amount of low-molecular-weight hydroxyproline found in the cultures. Cells treated with forskolin produced mainly type I collagen, as found in bone matrix in situ, with only small amounts of other types of collagen. Furthermore, forskolin time-dependently inhibited DNA synthesis in the cells, indicating that the increase in type I collagen synthesis by forskolin was not due to stimulated cell proliferation. These results suggest that cAMP is closely linked to the differentiation of osteoblasts in vitro.     

Distribution of rhodanese in plants

To study the distribution of rhodanese (E.C. 2.8.1.1) in plants, rhodanese activity was assayed on 13 cyanogenic and 12 non-cyanogenic species. All the species tested had the enzyme activity. This phenomenon leads to a hypothesis that the enzyme is generally distributed in plants.     

Increased atrial natriuretic peptide (6-33) binding sites in the subfornical organ of water deprived and Brattleboro rats

Binding sites for rat atrial natriuretic peptide (6-33) (ANP) were quantitated in the subfornical organ of chronically dehydrated homozygous Brattleboro rats unable to synthesize vasopressin; heterozygous Brattleboro rats, their controls, Long Evans rats and Long Evans rats after 4 days of water deprivation. Brain sections were incubated in the presence of 125I-ANP and the results analyzed by autoradiography coupled to computerized microdensitometry and comparison to 125I-standards. Brattleboro rats and water deprived Long Evans rats presented a higher number of ANP binding sites than their normally hydrated controls. Our results suggest a role of ANP binding sites in the subfornical organ in the central regulation of fluid balance and vasopressin secretion.     

Localization of atrial natriuretic peptide, ANP-(99-126) binding sites in the rat thymus and spleen with quantitative autoradiography

Binding sites for atrial natriuretic peptide, ANP-(99-126) were studied in lymphoid organs of the rat with quantitative autoradiography. Tissue sections were incubated in the presence of 0.13 nM 125I-ANP-(99-126) followed by autoradiography using [3H]-Ultrofilm, and the results were analyzed by computerized densitometry and comparison to 125I-standards. Specific ANP binding sites were localized in the medulla and the cortex of the rat thymus and in the white pulp of the rat spleen, with apparent binding sites concentrations of 93, 65, and 126 fmol/mg protein, respectively. The presence of ANP binding sites in areas related to the maturation and function of lymphocytes, and to the production of thymic hormones, suggests the possibility of a role of circulating ANP in the modulation of the immune response.     

Localization of ghrelin-producing cells in the stomach of the rainbow trout (Oncorhynchus mykiss)

Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor (GHS-R), was isolated from the rat stomach and determined to be n-octanoylated 28-amino-acid peptide. In this study, we studied the distribution of ghrelin-producing cells (ghrelin cells) in the gastrointestinal tract of male and female rainbow trout (Oncorhynchus mykiss) by immunohistochemistry using N-terminal region-recognizing antibody and also by in situ hybridization using a trout ghrelin-specific cRNA probe. Ghrelin cells were found in the mucosal layer of the stomach but not in the myenteric plexus, and no ghrelin cells were observed in other regions of the gastrointestinal tract. Ghrelin cells could be classified into two types: closed- and opened-type cells. The density of ghrelin cells increased gradually in the direction from the cardiac to pyloric portions of the stomach in both sexes. The number of ghrelin cells per unit area seemed to be higher in females than in males. In conclusion, trout ghrelin cells exist in the stomach and are classified into two types of cells, closed- and opened-type cells.     

Overexpression of CNP in chondrocytes rescues achondroplasia through a MAPK-dependent pathway

Achondroplasia is the most common genetic form of human dwarfism, for which there is presently no effective therapy. C-type natriuretic peptide (CNP) is a newly identified molecule that regulates endochondral bone growth through GC-B, a subtype of particulate guanylyl cyclase. Here we show that targeted overexpression of CNP in chondrocytes counteracts dwarfism in a mouse model of achondroplasia with activated fibroblast growth factor receptor 3 (FGFR-3) in the cartilage. CNP prevented the shortening of achondroplastic bones by correcting the decreased extracellular matrix synthesis in the growth plate through inhibition of the MAPK pathway of FGF signaling. CNP had no effect on the STAT-1 pathway of FGF signaling that mediates the decreased proliferation and the delayed differentiation of achondroplastic chondrocytes. These results demonstrate that activation of the CNP-GC-B system in endochondral bone formation constitutes a new therapeutic strategy for human achondroplasia.