Resolution and reconstitution of Rhodospirillum rubrum pyridine dinucleotide transhydrogenase. Proteolytic and thermal inactivation of the membrane component.

Pyridine dinucleotide transhydrogenase of the Rhodospirillum rubrum chromatophore membrane was readily resolved by a washing procedure into two inactive components, a soluble transhydrogenase factor protein and an insoluble membrane-bound factor. Transhydrogenation was reconstituted on reassociation of these components. The capacity of the membrane factor to reconstitute enzymatic activity was lost after proteolysis of soluble transhydrogenase factor-depleted membranes with trypsin. NADP+ or NADPH, but neither NAD+ nor NADH, stimulated by several fold the rate of trypsin-dependent inactivation of the membrane factor. Substantial protection of the membrane factor from proteolytic inactivation was observed in the presence of Mg2+ ions, an inhibitor of transhydrogenation, or when the soluble transhydrogenase factor was bound to the membrane. Coincident with the loss of enzymatic reconstitutive capacity of the membrane factor was a loss in the ability of the membranes to bind the soluble transhydrogenase factor in a stable complex. The membrane component was inactivated by preincubating soluble transhydrogenase factor-depleted membranes at temperatures above 45 degrees. NADP+, NADPH, or Mg2+ ions, but neither NAD+ nor NADH, protected against inactivation. These studies indicate that (a) the binding of NADP+ or NADPH to the membrane factor promotes a conformational alteration in the protein such that its themostability and susceptibility to proteolysis are increased, and (b) the inhibitory Mg2+ ion-binding site resides in the membrane component.     

Effects of PCB on interspecific competition in natural and gnotobiotic phytoplankton communities in continuous and batch cultures

The toxicity of polychlorinated biphenyls (PCB) to the diatomThalassiosira pseudonana (formerlyCyclotella nana), grown in pure and mixed cultures, was greatest when in competition with other species. Continuous cultures were superior to batch cultures for studying competitive interactions, and PCB caused greater alteration of species composition in continuous cultures than it did in batch cultures. Natural phytoplankton communities from Vineyard Sound, maintained in continuous culture, responded to PCB stress the same as did gnotobiotic communities, withT. pseudonana showing similar responses in both communities. A PCB concentration of 0.1 μg/liter (0.1 part per billion), a level not uncommon in natural waters, did not affect algal growth in pure cultures but caused substantial disruption of continuous culture communities. The possible impact of PCB pollution on natural phytoplankton communities is discussed. Contribution No. 3181 from the Woods Hole Oceanographic Institution.     

DNA replication in Escherichia coli: physical and kinetic studies of the replication point

DNA replication in Escherichia coli was followed using analytical composite agarose-acrylamide gel electrophoresis of native and denatured DNA samples from cells that had been pulse-labeled in vivo. The results obtained with samples of native DNA, combined with the different sensitivities of the various electrophoretic fractions of native DNA to enzymatic attack, led us to conclude that a significant amount of single-strandedness exists in the region of the replication site. Data obtained from kinetic analyses of the labeling of both the high molecular weight DNA and the fragments formed on denaturation of native DNA samples were consistent with the Okazaki model (Okazaki et al., 1968a,b) of DNA replication. Furthermore, the data indicated that eight to ten precursor fragments are present per fork during bacterial growth at both 20 and 37 °C, under the conditions used in this study.     

Stimulation of Cell Division by Croton Oil in Blue-Green Bacteria

The potent tumor-promoting agent croton oil, which has been shown previously to be strongly mitogenic in mammalian cells, stimulates cell division in snake mutants of the blue-green bacterium Agmenellum quadruplicatum.     

GROWTH REGULATION BY ETHYLENE IN FERN GAMETOPHYTES. IV. INVOLVEMENT OF PHOTOSYNTHESIS IN OVERCOMING ETHYLENE INHIBITION OF SPORE GERMINATION

The inhibitory effects of ethylene on spore germination were investigated. In darkness spore germination was completely inhibited by 10 μ1 · 1⁻¹ ethylene. Light partially overcame this inhibition, and the effect of continuous irradiation with white fluorescent light saturated at about 450 μW · cm⁻². Monochromatic red, blue and far-red light were effective in overcoming ethylene inhibition, whereas green was not. Short periodic exposures to red or far-red light were not sufficient to overcome ethylene inhibition. This suggested that phytochrome was not involved. The photosynthetic inhibitor DCMU blocked the effect of light. Infrared gas analysis showed that photosynthesis saturated at about 450 μW · cm⁻² in white light. Red, blue and far-red light were more efficient photosynthetically than green light; DCMU blocked photosynthesis. Normalized curves of photosynthesis and germination vs. light intensity showed a similar dependence on light energy. It was concluded that light appears to overcome the inhibitory effects of ethylene through some process dependent on photosynthesis.     

Reduced toxicity of amphotericin B methyl ester (AME) vs. amphotericin B and fungizone in tissue culture

Summary The comparative toxicities of amphotericin B methyl ester (AME), the parent antibiotic amphotericin B (AB), and the deoxycholate solubilized complex of AB, Fungizone² (FZ), toward five cell lines has been determined as measured by early membrane damage (⁵¹Cr release), 24 hr survival, 72 hr viability, and growth rate. Cells used were of turtle (TH-1), marsupial (PT K2), human MA 160), rabbit (RK-13) and hamster (BHK-21) origin. AME: (a) caused less membrane damage at 1 hr than AB or FZ; (b) was less toxic than AB or FZ as indicated by 24 hr cell survival and 72 hr cell viability; and (c) was required in higher levels than AB or FZ to reduce the growth rate of all five cell lines. Spectrophotometric analysis of residual polyene levels indicated that AME had good stability in tissue culture medium. Previous studies have indicated that AME has the same in vitro antifungal activity as the parent antibiotic AB (1, 2). These findings suggest that AME may prove to be superior to AB and FZ for use as an antifungal agent in tissue culture systems. Fungizone^R. Trade mark. E. R. Squibb and Sons. This investigation was supported in part by Contract NIH 69-2161, NIH Grant No. AI-02095 and NIH Training Grant No. GM 507 from the National Institute of General Medical Sciences.