The Presence of Two Cd-Binding Components in the Roots of Water Hyacinth Cultivated in a Cd2+-Containing Medium

To find why water hyacinth accumulates the toxic heavy metalion Cd²⁺, the plant was cultivated in a mineral medium supplementedwith Cd²⁺. Cd accumulated in the roots against the concentrationgradient, mostly as a soluble form in the cytoplasm. Chromatographywith Sephadex G-25 and G-50 columns showed that the accumulatedCd was present in two forms with mol wt of 2,300 and 3,000.The components carrying Cd showed a high ratio of absorbanceat 254 nm to that at 280 nm, which suggests that they resemblemammalian Cd-thioneins. These components were not detected inthe roots of water hyacinth cultivated in the absence of Cd²⁺,indicating that they are formed in response to the Cd²⁺ supplement. (Received September 6, 1984; Accepted November 30, 1984)     

Cloning and sequencing of rpoH and identification of ftsE-ftsX in Pseudomonas putida PpG1.

The rpoH gene encoding the heat-shock sigma factor of Pseudomonas putida was cloned by using its ability to complement the temperature-sensitive growth of the Escherichia coli rpoH mutant. The cloned DNA contained an open reading frame for a 284 amino acid sequence exhibiting high homology to the sigmaH proteins of P. aeruginosa and E. coli. Moreover, homologs to the cell division genes ftsX and ftsE were found immediately upstream of the rpoH gene.     

Characterization of a novel Le(x)-active ganglioside from chick intestinal tissues recognized by murine monoclonal antibody 188C1.

A monoclonal antibody, 188C1, raised against skin tissue from the back of bullfrogs (Rana catesbeiana) was found to recognize a common antigen in neural and intestinal tissues of chicken (Fujita, S. (1989) in Biological Transduction Mechanisms (Kasai, M., Yoshioka, T., and Suzuki, H., eds) pp. 159-177, Japan Scientific Societies Press, Tokyo Japan). The 188C1 antigen was isolated from chick intestinal tissues and characterized as a novel ganglioside by means of Q-Sepharose and Iatrobeads column chromatography, and chemical, immunochemical, and immunohistochemical analyses. The chemical structure was as follows: Gal beta 1-4 GlcNAc beta 1-3Gal beta 1-3GalNAc beta 1-4Gal beta 1-4Glc beta 1-1'Cer Fuc alpha 1-3 NeuAc alpha 2-3 This represents a novel hybrid structure of type 2 Le(x) epitope and GM1 ganglioside core structure, designated as Le(x)-GM1. Monoclonal antibody 188C1 reacted strongly with Le(x)-GM1 on thin layer chromatography, but its reactivity was greatly reduced when sialic acid was removed from the antigen. This indicated that the internal sialic acid residue might participate in antigenicity of the Le(x) determinant. In addition to 188C1, a more specific antibody reacting with Le(x)-GM1 but not with asialo-Le(x)-GM1 was raised by immunizing a rabbit with Le(x)-GM1. TLC/enzyme immunostaining using this specific antibody showed the presence of Le(x)-GM1 in chick intestinal tissue, but not in chick brain.     

Purification and characterization of Streptomyces griseus metalloendopeptidases I and II.

Two metalloendopeptidases, designated as Streptomyces griseus metalloendopeptidases I and II (SGMPI and SGMPII), were isolated from a commercial Pronase P by a method including affinity chromatography on carbobenzoxy-L-alaninyl-triethylenetetraminyl-Sepharose (Z-Ala-T-Sepharose). The two enzymes differed from each other in behavior on ion-exchange chromatography but showed the same amino-terminal sequence at least up to the 20th residue. Their molecular weights were both estimated to be 37,000 by SDS-polyacrylamide gel electrophoresis. Elemental and amino acid composition analyses indicated that both of them contained about 1 g atom of zinc and one cystine residue per mol of protein. Cleavage specificities of the two enzymes toward synthetic peptide-substrates were very similar to those observed with thermolysin. EDTA, o-phenanthroline, and phosphoramidon strongly inhibited these enzymes, while typical serine-protease inhibitors and cysteine-protease inhibitors had no effect. The findings clearly indicate that SGMPI and SGMPII can be classified into the family of zinc-endopeptidases. It was unexpectedly found, however, that these metalloendopeptidases were strongly inhibited by protein serine-protease inhibitors produced by Streptomycetes, such as Streptomyces subtilisin inhibitor (SSI), alkaline protease inhibitor-2c' (API-2c'), and plasminostreptin (PS).     

Interleukin-1 enhances the response of osteoblasts to platelet-derived growth factor through the alpha receptor-specific up-regulation.

Both platelet-derived growth factor (PDGF) and interleukin-1 (IL-1) are produced by activated macrophages and are thought to contribute to bone remodeling, but their precise roles remain to be clarified. The interaction between PDGF and IL-1 was, therefore, studied in normal osteoblast-like cells (MC3T3-E1). The expression of alpha- and beta-PDGF receptors on MC3T3-E1 cells was detected by RNA blot analysis and confirmed by immunoblot analysis. PDGF-induced chemotactic as well as mitogenic activities were synergistically enhanced by either IL-1 alpha or IL-1 beta (40 units/ml) pretreatment in serum-free medium, although IL-1 alone did not show any detectable chemotactic activities. This biological enhancement by IL-1 was accompanied by a selective increase of alpha-PDGF receptor expression, following the augmentation of alpha receptor autophosphorylation and inositol phosphate hydrolysis induced by PDGF-AA. These findings suggest that PDGF and IL-1 are jointly involved in the bone-remodeling microenvironment as local coupling factors.     

Light and electron microscopic immunocytochemistry on the localization of 3 beta-hydroxysteroid dehydrogenase/isomerase in the bovine adrenal cortical cells

The localization of 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD) was studied in bovine adrenal glands by light as well as electron microscopic immunocytochemistry, using anti-bovine adrenal 3 beta-HSD antibody. With light microscopy the cytoplasm of the glomerulosa cells was weakly immunostained, while that of the fasciculata-reticularis cells was intensely immunostained though both the capsular connective tissue cells and the medullary cells were entirely negative for this reaction. Electron microscopic immunocytochemistry revealed that the positive reaction products for 3 beta-HSD were present on the membrane of smooth endoplasmic reticulum of the cortical cells, especially that of the fasciculata and reticularis cells. Other cell organelles such as mitochondria and Golgi apparatus were entirely negative. The present results indicate that 3 beta-HSD is present in the membrane of smooth endoplasmic reticulum of bovine adrenal cortical cells.