Kent D. Miller

Production and properties of staphylococcus aureus (strain newman D2C) with uniform clumping factor activity

Abstract The most active CF+ Staphylococcus aureus (strain Newman D 2 C) cells were recovered during the exponential through the early resting phases of the growth cycle. Fermentation conditions are described for preparation of large quantities of those cells on BHI. Extractions with 70 per cent ethanol produced sterile particles with intact CF activity. Subsequent ethanol and acetone drying steps completed procedures for production of uniformly active, stable CF+ particles. Congo red stained the killed staphylococci without affecting CF activity, and the stained particles enhanced visualization of the agglutination end-points. Tris buffers (50 mM, pH 7.3) were excellent diluents for CF reagents, but only if freshly prepared or sterilized before storage.

Activation of guanylate cyclase by α-toxins from krait and cobra venom

Abstract The effects of purified neurotoxins from krait and cobra venom were tested on guanylate cyclase activity in rat tissues. Both α-bungarotoxin and α-cobrotoxin stimulated the activity of the soluble form of this enzyme in lung, spleen and kidney, but were without effect in liver, muscle and brain. Half maximal stimulation of guanylate cyclase occurred at about 1 μM with α-bungarotoxin and at about 0·25 μM with α-cobrotoxin. Reduction and alkylation, or oxidative detoxification of the toxins abolished their ability to stimulate the guanylate cyclase enzyme. Treatment with dithiothreitol also greatly reduced the effect of the toxins. These data suggest that the toxins may act in vitro by altering the redox state of guanylate cyclase. Neither curare nor atropine blocked the stimulatory effect of these toxins on the enzyme.

Inhibition of virus-induced plaque formation by atoxic derivatives of purified cobra neutrotoxins

Venom from Naja naja siamensis was resolved into 16 toxic and nontoxic fractions by chromatography on SP-Sephadex, C-25. The principal neurotoxin preparations were chromatographically and electrophoretically homogeneous. Of the purified constituents, only the principal neurotoxin and minor neurotoxins were precursors of inhibitors of plaque formation among baby hamster kidney fibroblasts infected with Semliki Forest Virus.

NONENZYMIC CONTROL OF PROTHROMBIN ACTIVATION

Although considerable attention has been given to the enzymic mechanisms of prothrombin activation, Waugh et a1-I suggested that nonenzymic mechanisms should not be ruled out. Landaburu and Seegers had described activation of purified prothrombin in protamine sulfate solutions,* a process that, like an extrinsic thromboplastic system,3 was resistant to (and progressed in) diisopropylfluorophosphate (DFP) .4 Evidence for a nonenzymic control mechanism stemmed from subsequent studies of thrombin generation from purified horse prothrombin mixed with synthetic polylysine (polyLYS) preparation^.^ Activation occurred only within a relatively high, optimum weight ratio of polyLYS to prothrombin. Other characteristics of the polyLYS reaction were a low temperature optimum, high sensitivity to salt. and insensitivity to DFP. Also, the maximum rate of thrombin formation at optimum ratios of polyLYS to prothrombin varied with the average molecular weight of each polyLYS preparation and with the concentrations of the two reactants. However, the smallest amount of each polyLYS preparation just sufficient to initiate activation was constant despite wide variations in the average molecular weights of the polymers. A number of high-molecular-weight basic compounds substituted for the POIYLYS.~ The present studies are based on the hypothesis that some basic substances of relatively high molecular weight represent models for a physiologic, nonenzymic substance, the latter substance required for initiation of prothrombin activation regardless of the rate of that activation. The studies delineate distinctive parameters of the nonenzymic reaction, unusual solvent effects, and the significance of the purity and species of the prothrombin activated. Activators of known molecular weight are identified that will permit detailed kinetic studies at known molar concentrations of the reactants.

Antiseea to Carcinoembryonic Antigen: Removal of Persistant Impurities (1)

Antisera to carcinoembryonic antigen preparations frequently contained antibodies to a distinct normal serum protein(s). The latter antibodies persisted following absorptions with cross-linked human plasma proteins, normal colon, and Group 0 red cells. A simple method for removal of the contaminating antibodies involved absorptions with the perchloric acid-soluble fraction of normal serum. Excesses of the absorbant and its soluble immune complexes were then removed on anion exchange columns.