Selective production of glutamate by an immobilized marine blue-green alga,Synechococcus sp.

Summary Among 200 strains of marine bluegreen algae isolated from the coastal areas of Japan, the marine blue-green alga Synechococcus sp. NKBG 040607 excreted glutamate at the highest rate, 82.6% of total amino acids production being glutamate. Synechococcus sp. NKBG 40607 was immobilized in calcium alginate gel. Glutamate production by immobilized cells was double that of native cells. Maximal glutamate production (25 g/cm³ gel per day) of the immobilized cells was observed under a light intensity of 144 Einstein/m² per second at a cell concentration of 7.5 mg dry cells/cm³ gel. Immobilized cells of Synechococcus sp. can use nitrate as a nitrogen source. Immobilized marine Synechococcus sp. produced 0265 mg/cm³ gel of glutamate for 7 days in the presence of chloramphenicol.     

Inhibition of bovine adrenocortical mitochondrial cytochrome P-450(11)beta-mediated reactions by imidazole derivatives and mineralocorticoid analogs

The effects of several imidazole antimycotic agents, an imidazole and several mineralocorticoid analogs on the cytochrome P-450(11)beta-catalyzed 11 beta-hydroxylation of 11-deoxycorticosterone and aldosterone synthesis were examined. Ketoconazole, clotrimazole, miconazole and etomidate were found to be potent inhibitors of the reactions, causing 50% inhibition of the 11 beta-hydroxylase activity at concentrations between 10(-8) and 10(-7) M. The potency of etomidate as to the inhibition of aldosterone- and 18-hydroxycorticosterone-production was found to be almost equal to that in the case of 11 beta-hydroxylation. Spironolactone and other newly synthesized mineralocorticoid analogs were also found to inhibit the cytochrome P-450(11)beta-mediated reactions. The ID50 values of these drugs for inhibition of the 11 beta-hydroxylase activity were almost equal to those in the case of the aldosterone- and 18-hydroxycorticosterone-biosynthetic activities. The results of kinetical studies indicated that one of the mineralocorticoid analogs, Compound 23-0586, acts as a competitive inhibitor for the cytochrome P-450(11)beta-mediated reactions.     

Monoclonal Antibodies Specific for NADH-Ferricyanide Reductase Domain of Spinach Leaf Nitrate Reductase

Two monoclonal antibodies, 17(3)9 and 36(79)4, were preparedagainst nitrate reductase from Spinacia oleracea L. leaves.An enzyme-linked immunosorbent assay showed that 17(3)9, butnot 36(79)4, reacted more strongly to heat-denatured than nativeantigen. These antibodies inhibited NADH-nitrate reductase aswell as its various partial activities including reduced methylvilogen-nitrate reductase, reduced flavin mononucleotide-nitratereductase and NADH-cytochrome c reductase activities, but notNADH-ferricyanide reductase activity. Immunoblotting after electrophoreticseparation of nitrate reductase fragments obtained by Staphyrococcusaureus V8 protease digestion of native enzyme revealed thatthe two monoclonal antibodies bind to different epitopes locatedon the 28 kDa of the NADH-ferricyanide reductase domain. (Received October 2, 1987; Accepted June 9, 1988)     

Secretion of soluble functional insulin receptors by transfected NIH3T3 cells

In order to determine whether the human insulin receptor ectodomain can be expressed as a functional protein, the coding regions for the transmembrane and cytoplasmic domain of a full-length human insulin receptor cDNA were deleted by site-directed mutagenesis, and the resultant construct was inserted into a bovine papilloma virus vector under the control of the mouse metallothionein promoter. After transfection of mouse NIH3T3 cells, a cell line secreting an insulin binding protein was isolated. The insulin binding alpha subunit had an Mr of 138,000 and a beta subunit of Mr 48,000 (compared to 147,000 and 105,000 for the full-length human insulin receptor expressed in NIH3T3 cells). This difference in size of the alpha subunit was due to a difference in glycosylation as N-glycanase digestion reduced the apparent size of the alpha subunits of secreted and normal membrane-bound receptors to identical values. The secreted receptor formed disulfide-linked heterotetrameric structures with an Mr of 280,000. It was synthesized as an Mr 160,000 precursor which was cleaved into mature subunits with a t1/2 of 3 h. Increasing expression of the cDNA by induction with sodium butyrate lead to the appearance of an Mr 180,000 protein in the medium as well as the mature alpha and beta subunits. A Scatchard plot of insulin binding to the secreted receptor was curvilinear with a Kd of 7 X 10(-10) M for the high affinity sites and 10(-7) M for the low affinity site (compared to Kd values of 1.1 X 10(-9) M and 10(-7) M, respectively, for human insulin receptors expressed in these cells.     

Inhibition of RNA synthesis in embryo of Xenopus laevis by protease inhibitor

We have studied the role of proteases during the development of Xenopus laevis embryos with the aid of protease inhibitors. The activity of proteases was found to be only minimal in the unfertilized egg and during the initiation of development, but activity began to increase at the morula stage. When the activity of proteases was inhibited by antipain, an inhibitor of endopeptidase activity, RNA synthesis in the embryo was inhibited. To examine the relationship between the inhibitory effect of antipain on protease activity and its effect on RNA synthesis, antipain was reduced with NaBH4 to inactivate its protease inhibitory activity. The reduced antipain did not inhibit RNA synthesis in the embryo. Antipain effectively inhibited synthesis of both rRNA and poly(A)+RNA but not 4S RNA. We therefore suggest that protease activity plays an important role in the initiation and/or continuation of RNA synthesis.     

Purification of GP57, and auxin-regulated extracellular glycoprotein of carrots,and its immunocytochemical localization in dermal tissues

A glycoprotein (GP57) was purified by ion-exchange and hydroxylapatite column chromatography from the 70%-ethanol precipitate of culture medium of non-embryogenic carrot cells (Daucus carota L.) grown with 2,4-dichlorophen-oxyacetic acid (2,4-D). Its apparent molecular mass (M _r) was estimated to be 57000 by sodium dodecylsulfate-polyacrylamide gel electrophoresis and 50000 by gel filtration. GP57 contained 14% (w/w) carbohydrate; the M _r of the peptide portion was estimated to be 55000 after deglycosylation by trifluoromethanesulfonic acid. GP57 is composed of two polypeptides with the same M_r and with very similar amino-acid composition but different pI values, 8.8 and 9.5. Both are rich in aspartic acid, serine and threonine, and may possess N-linked oligosaccharide chains, including fucose and xylose. A monoclonal antibody (MAb) against the purified GP57 reacted with both the pI 8.8 and the 9.5 components, as well as the deglycosylated GP57. Immunoblotting with the MAb indicated that GP57 is synthesized in and released from cultured cells which have been supplied with auxin. In immunocytochemical studies, GP57 was found in the space between the embryo and the endosperm of dry seeds, and its content decreased during germination. GP57 was also found in the endodermis and epidermis of young roots, the periderm of mature taproots, and the epidermis of petioles and young leaves.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GP57 M _r-57000 glycoprotein - GP65 M _r-65000 glycoprotein - MAb monoclonal antibody - M _r apparent molecular mass - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis - TFMS trifluoromethanesulfonic acid     

Role of cell-mediated immunity in the resistance to experimental sporotrichosis in mice

The in vitro activity of several new imidazoles, cloconazole, sulconazole, butoconazole, isoconazole and fenticonazole, were compared with those of amphothericin B, flucytosine, and three azoles: econazole, miconazole and ketoconazole against isolates of pathogenic Candida. A total of 186 clinical isolates of 10 species of the genus Candida and two culture collection strains were tested by an agar-dilution technique. Isoconazole was the most active azole, followed by butoconazole and sulconazole. Differences between some of the species in their susceptibility to the antifungal agents were noted. Sulconazole and cloconazole had the highest activity in vitro against 106 isolates of C. albicans. Butoconazole and isoconazole were also very active against isolates of C. albicans, and were the most active azole compounds against 80 isolates of Candida spp.     

Induction of interdigitating reticulum cell-like differentiation in human monocytic leukemia cells by conditioned medium from IL-2-stimulated helper T-cells

Monocytic leukemia (MoL) cells were obtained from the peripheral blood of a patient in whom the leukemic cells infiltrating various lymphoreticular organs exhibited features intermediate between interdigitating reticulum cells (IDC) and ordinary phagocytic macrophages, whereas the leukemic cells in the peripheral blood were essentially monocytic and lacked such features. Peripheral blood CD4+ T-cells were established as an interleukin-2-dependent T-cell line. When the MoL cells were exposed for a few days to conditioned medium from the T-cell line, they extended several dendritic cytoplasmic projections and became intensely positive for HLA-DR antigen, cytoplasmic S-100β protein, and CD1 antigen. Functionally, the conditioned medium significantly down-regulated Fc-mediated and Fc-independent phagocytic activities, and the levels of lysosomal enzymes such as lysozyme and nonspecific esterase in the MoL cells. Moreover, the conditioned medium significantly up-regulated the accessory cell function of the MoL cells as measured by the primary allogenic mixed leukocyte reaction (MLR). Furthermore, the conditioned medium significantly down-regulated the expression of CD14 antigen. Biochemical analysis indicated that the factor responsible for these changes is a protein which is distinct from known human cytokines and whose molecular weight is approximately 31 kDa. These findings suggest that IDC are closely related the monocytic lineage and that helper T-cells play an important role in constructing the microenvironment of T-lymphoid tissues which is necessary for the differentiation and maturation of IDC.     

Correlation between the virulence ofFrancisella tularensis in experimental mice and its acriflavine reaction

Correlation between the virulence of Francisella tularensis in experimental mice and its acriflavine reaction was studied. The cultures derived from all four strains (Ebina, CMB2, Schu, and N9) that had long been subcultured on agar media yielded two types of colonies, i.e., acriflavine reaction-positive (acf+) and acriflavine reaction-negative (acf-) colonies. All acf+ colonies, regardless of their parent strains, were shown to be low virulent in mice. Acf- colonies were shown to be either high (Ebina, CMB2) or low (Schu, N9) virulent. The low-virulent acf- colonies gained virulence during several passages in mice, whereas the acf+ colonies remained low virulent even after the animal passages.