Studies on the production of indole-3-acetic acid with auxin-heterotrophic mutants derived from cultured crown gall cells

Mutants in the indole-3-acetic acid metabolism derived fromcultured crown gall cells were tested to see whether they couldutilize any one of eight indolic compounds in place of indole-3-aceticacid. Two auxin-heterotrophic mutant cell lines could not utilizeindolepyruvic acid, but growth recovered when there was a supplementof indole-3-acetic acid. Indoleacetonitril and indoleacetaldoximeinhibited the growth of mutant cell lines and their parentalcrown gall cells. Cultured crown gall cells may have synthesizedindole-3-acetic acid from tryptophan via indolepyruvic acidand indole-acetaldehyde, and also may be able to produce indole-3-aceticacid from tryptophan via tryptamine (Received May 6, 1980; )     

Reaginic antibody formation in the mouse. IX. Enhancement of suppressor and helper cell activities of primed spleen cells.

Attempts were made to increase the activity of suppressor cells in vitro. Antigen-specific suppressor cells were induced by i.v. injections of urea-denatured ovalbumin (UD-OA) into OA-primed mice. Nonadherent splenic lymphocytes from the UD-OA-treated mice were incubated at 37 degrees C for 24 hr with either OA or OA-bearing macrophages and lymphocytes harvested from the culture were examined for the ability to suppress primary anti-hapten antibody response on nonirradiated mice to DNP-OA. The results showed that the suppressive activity of the lymphocytes increased after culture of the cells with OA or OA-bearing macrophages. Similar results were obtained when nylon column-purified T cell-rich fraction of the lymphocytes were similarly cultured. The suppressive activity was associated with theta-bearing lymphocytes and was specific for OA. Suppressor cells were not induced by the culture of OA-primed lymphocytes with OA. The helper function of splenic lymphocytes from both UD-OA-treated mice and OA-primed mice was enhanced by the culture of the cells with OA-bearing macrophages but not by culture with OA in the absence of macrophages.     

Segregation of modified bacteriorhodopsin aggregations in reconstituted vesicle membrane induced by the change of thermodynamical parameters

It was clearly shown that the change in thermodynamical parameters could cause the segregation of membrane protein aggregations in the phospholipid membrane. At first, reconstituted vesicles were prepared with a membrane protein, bacteriorhodopsin and a constituent phospholipid of biomembranes, L-alpha-dimyristoyl phosphatidylcholine. When the temperature of the suspension was decreased or the osmotic pressure was increased by adding poly(ethylene glycol) to this vesicle suspension at 23 degrees, the circular dichroism spectra showed a typical band indicating bacteriorhodopsin trimer formation implying their aggregation. This suggests that the aggregation of trimers proceeded by adding poly(ethylene glycol) into vesicle suspension, just as it proceeded by decreasing the temperature. Next, vesicles were prepared with fluorescein isothiocyanate-labeled bacteriorhodopsin, photoemissive bacteriorhodopsin and L-alpha-dimyristoyl phosphatidylcholine. The excitation energy transfer between the two modified proteins was measured by fluorescence spectroscopy. In this case, however, when poly(ethylene glycol) was added into the suspension, the yield of the excitation energy transfer decreased. This result indicates that modified proteins aggregate separately in a segregated form in the vesicle membrane.     

The relationship between auxin concentration, auxin protection and auxin destruction in crown gall cells cultured in vitro

Auxin content and IAA-protector activities in cultured crowngall cells were measured at various points of growth in subculture.A coincidence was observed between the change in IAA contentand IAA-protector activities in the cells: high auxin contentswith high IAA-protector activities were obtained at the latelog stage, and low auxin contents with low IAA-protector activitiesat the middle log and at the stationary stages. On the otherhand, IAA-destruction activities in the cells increased slightlythroughout the growth period of the cells. Addition of 2,4-Dto the culture depressed the auxin content and accelerated theIAA-destruction activity in the cells. These results suggestthe existence of a mechanism of negative-feed-back control ofauxin in crown gall cells. (Received May 1, 1978; )     

Demonstration of Fcgamma receptors on human basophil granulocytes.

Fcgamma receptors were detected on human basophil granulocytes. The mononuclear cell fraction of human peripheral blood was incubated with heat-aggregated human IgG (HGG) followed by 125I-anti-HGG. Autoradiography of the cells showed that the majority of basophil granulocytes gave a significant number of grains. Basophils were not labeled by preincubation of the same cells with monomeric HGG followed by 125I-anti-HGG. However, the binding of aggregated HGG to basophils was inhibited by the presence of a high concentration of monomeric HGG or its Fc fragment but not by the Fab fragment. Evidence was obtained that Fcgamma receptors are distinct from IgE receptors on the same cells: i) Saturation of basophils with IgE did not affect the binding of aggregated HGG to the cells. ii) Preincubation with and the presence of aggregated HGG failed to affect the binding of 125I-IgE to basophils, or to block passive sensitization of the cells with IgE antibodies. iii) The Fcgamma receptors did not co-cap with IgE receptors. Aggregated HGG failed to induce histamine release from basophils even in the presence of D2O. It was also found that the presence of aggregated HGG on basophils did not modulate IgE-mediated histamine release from the cells.     

Effects of carrageenan on immune responses. Studies on the macrophage dependency of various antigens after treatment with carrageenan.

Carrageenan, a sulfated polygalactose having macrophage toxic properties, elicited a marked suppression of IgM response to T cell-dependent antigens such as sheep red blood cells (SRBC), dinitrophenylated bovine serum gamma-globulin (DNP-BGG), and trinitrophenylated concanavalin A (TNP-Con A). In contrast, carrageenan did not inhibit antibody responses to such T cell-independent antigens as trinitrophenylated DEAE-dextran (TNP-DEAE-dextran), trinitrophenylated polyvinyl pyrrolidone(TNP-PVP), and trinitrophenylated Ficoll (TNP-Ficoll). Compared to total spleen cells, spleen cells from which macrophages had been removed by adhesion to plastic Petri dishes had less effect on the production of antibody against T cell-dependent antigens, but no change or a rather stimulated effect was observed in in vitro antibiody synthesis against T cell-independent antigens. These results strongly suggest that macrophages are involved in antibody responses to T cell-dependent antigens but not in those to T cell-independent antigens. However, the antibody response to trinitrophenylated lipopolysaccharide (TNP-LPS), a T cell-independent antigen, was inhibited by carrageenan treatment, suggesting that the response is macrophage dependent. Moreover, antibody response to higher doses of dinitrophenylated phytohemagglutinin (DNP-PHA), a T cell-dependent antigen, was shown to be macrophage independent by carrageenan treatment, although the antibody response to low doses of the antigen was macrophage dependent. Considering all these results, carrageenan treatment seems to be a very useful method to determine whether immune response to various antigens are macrophage dependent or not.