D. K. Hammer

Further characterization and regulation of malonyl-coenzyme A: Flavonoid glucoside malonyltransferases from parsley cell suspension cultures

Two malonyltransferases, malonyl-CoA:flavone/flavonol 7-O-glucoside malonyltransferase and malonyl-CoA:flavonol 3-O-glucoside malonyltransferase, were purified to apparent homogeneity from uv-irradiated parsley cell cultures. Both purified enzymes appear to be specific for flavonoid glycosides. Additional malonyltransferases, active toward several phenol glucosides other than flavonoids, were present in partially purified 7-O-glucoside malonyltransferase preparations. Antibodies raised against the purified 3-O-glucoside malonyltransferase did not inhibit the activity of the 7-O-glucoside malonyltransferase over a wide antibody concentration range. Determination of the rate of synthesis in vivo of the 3-O-glucoside malonyltransferase after ultraviolet light-pulse induction of parsley cells revealed two maxima at 6 and 30 h, respectively. These results indicate that the induced changes in 3-O-glucoside malonyltransferase activity were the consequence of either a repeated change in the rate of synthesis of one enzyme species or changes in the synthesis rates of more than one enzyme species.

Effect of platelet-activating factor on human polymorphonuclear leukocyte enhancement of chemiluminescence and antibody-dependent cellular cytotoxicity.

The effect of platelet‐activating factor on human granulocytes was determined by luminol‐dependent chemiluminescence (CL), antibody‐dependent cellular cytotoxicity (ADCC), and rosette assay. An activation of the respiratory burst could be measured by CL and be shown to depend on the presence of extracellular calcium. This direct stimulation of PMN was proved to be inhibited by oxygen radical scavengers as well as by the calcium channel blocker diltiazem. Furthermore, the presence of PAF enhanced the activation of PMN via Fc‐ or C3b‐receptors, as demonstrated by CL and ADCC. On the other hand, no influence on the rosette‐forming capacity of PMN could be detected. The results support the concept of the import role of PAF in inflammatory processes.

Bacteriophage Conjugates: The Effect of Coupling Reaction and Specific Antibodies on the Kinetics of Inactivation and Reactivation

The reaction of bacteriophage T4 with 1-fluoro-2,4-dinitrobenzene resulted in a covalent binding of 2,4-dinitrophenyl (DNP) determinants to the phage. From the kinetics of inactivation reflecting the coupling process it is concluded that attachment of more than one DNP group to the critical site(s) of the phage is required for inactivation (multi-hit reaction). Contrary to this the neutralization of DNP-T4 by anti-DNP antibody turned out to be a first order reaction, until 80 %> neutralization fitting one-hit kinetics. If compared with native T4, the susceptibility of DNP-T4 to neutralization by anti-T4 antibody is considerably higher, indicating that attachment of DNP groups to T4 amplifies the sensitivity to neutralization by anti-T4. Comparing neutralization kinetics of DNP-T4 and native T4 by anti-DNP-T4 antibody it is suggested that native determinants and DNP groups, as well as determinants resulting from alteration due to the coupling process, all together may contribute as targets for neutralization. Three characteristics strengthen the view that the velocity of T4 conjugates in infecting the host strain is markedly decreased if compared with that of native T4: (a) considerable discrepancy between direct plating and decision technique (b) increasing variety of plaque size and (c) decreased velocity of the first step of reproduction. The kinetics of neutralization observed can be reconciled with a model proposed by Krummel and Uhr. The kinetics of reactivation of neutralized DNP-T4 by the presence cf DNP-BSA has been investigated and the problems involved in the reaction are discussed.

MOPC 104E IgM/anti-idiotype solid-phase inhibition assay as a model for screening myeloma proteins for ligand binding specificity

The inhibition of binding of 125I-labeled MOPC 104E IgM anti-idiotype to the controlled pore glass-bound IgM by various oligo- and polysaccharides was investigated as a model for the screening of myeloma proteins with unknown hapten binding specificity. The inhibitory efficiencies of the different haptens in this system were found to correlate well with their known abilities to bind to MOPC 104E IgM. Thus B1355S dextran, nigerosyl-alpha (1-3)-nigerose, and nigerosyl-alpha (1-3)-glucose, all known for specific binding to MOPC 104E IgM, were efficient inhibitors in the assay, in contrast to a series of more or less unrelated carbohydrate compounds, which showed only weak inhibitory capacity. According to these results there seems to be no obstacle to applying the anti-idiotype assay system to screening myeloma proteins whose binding specificity has not yet been determined.