Wet and dry bacterial spore densities determined by buoyant sedimentation.

The wet densities of various types of dormant bacterial spores and reference particles were determined by centrifugal buoyant sedimentation in density gradient solutions of three commercial media of high chemical density. With Metrizamide or Renografin, the wet density values for the spores and permeable Sephadex beads were higher than those obtained by a reference direct mass method, and some spore populations were separated into several density bands. With Percoll, all of the wet density values were about the same as those obtained by the direct mass method, and only single density bands resulted. The differences were due to the partial permeation of Metrizamide and Renografin, but not Percoll, into the spores and the permeable Sephadex beads. Consequently, the wet density of the entire spore was accurately represented only by the values obtained with the Percoll gradient and the direct mass method. The dry densities of the spores and particles were determined by gravity buoyant sedimentation in a gradient of two organic solvents, one of high and the other of low chemical density. All of the dry density values obtained by this method were about the same as those obtained by the direct mass method.     

Adduct formation between the carcinogen N-acetoxy-2-acetylaminofluorene and synthetic polydeoxyribonucleotides.

The chemical carcinogen N-acetoxy-2-acetylaminofluorene (NA-AAF) was reacted with poly(dG-dC) - poly(dG-dC); poly dG - poly dC; poly(dA-dT) - poly (dA-dT); and poly dA - poly dT under a variety of conditions. Poly (dG-homo GC polymer and 10--20 more reactive the A + T polymers. Lowering the ionic strength increased the extent of reaction, while pH change (8.9 vs. 5.5) had only a small effect. If ionic strength was adjusted so that the two guanine-containing polymers showed equal thermal stability (as judged by Tm) then the alternating copolymer was 7 times as reactive as the homopolymer. In aggreement with previous investigators, the major product was found to be 8-(N-2-fluorenylacetamido) deoxyguanosine.     

Effect of silver ions on transport and retention of phosphate by Escherichia coli.

Silver ions inhibited phosphate uptake and exchange in Escherichia coli and caused efflux of accumulated phosphate as well as of mannitol, succinate, glutamine, and proline. The effects of Ag+ were reversed by thiols and, to a lesser extent, by bromide. In the presence of N-ethylmaleimide and several uncouplers, Ag+ failed to cause phosphate efflux, but still inhibited exchange of intracellular and extracellular phosphate, indicating an interaction at more than one site. It is unlikely that Ag+ caused metabolite efflux by acting solely as an uncoupler, as an inhibitor of the respiratory chain, or as a thiol reagent.     

Properties and origins of infectious rhinovirus type 14 particles of different buoyant densities.

Isopycnic centrifugation of rhinovirus type 14 (RV14), purified from infected HeLa or KB cell cultures, into CsCl gradients resolved two bands of infectious virus particles with buoyant density values of 1.409 +/- 0.007 (H virus) and 1.386 +/- 0.004 (L virus) g/ml. Only H virus was detected by incorporation of radiolabeled uridine into viral RNA, and H virus accounted for the majority of infectivity in gradients. H and L virus could not be differentiated by plaque morphology, extent of neutralization by RV14-specific antiserum, or particle size. Electron microscope studies showed that most L-virus particles were associated with an amorphous material. Treatment of L virus with proteolytic enzymes or rebanding L virus in CsCl gradients resulted in recovery of the majority of infectivity as H virus. Virus purified from cell-free fluids from infected HeLa or KB cell cultures banded only as H virus. HeLa cell cultures challenged with purified H virus and harvested at 3 h postinoculation for virus purification yielded only infectious H virus. Both H and L viruses were detected in cell cultures that had been challenged with purified H virus and harvested at 12 h postinoculation. The data suggest that H virus represents progeny virus, whereas L virus represents sequestered infectious virus particles which become associated with an amorphous material and do not enter into viral replicative processes.     

Proline transport activity in Escherichia coli membrane vesicles of different buoyant densities.

Cytoplasmic membrane vesicles prepared by lysis of Escherichia coli W 3110 spheroplasts in a French press at 0 degrees C are heterogeneous with respect to density due to membrane protein aggregation as a result of lateral phase separation of membrane phospholipids and to the presence of more or less outer membrane. These different vesicle classes can be separated on isopycnic density gradients. Assays for various membrane-associated functions show that the membranes differ not only with respect to density and structure but also with respect to function. The proline transport system (as detected by uptake experiments with the artificial electron donor ascorbate-phenazine methosulfate) shows maximal activities in membrane fractions that have considerably higher densities than the normal cytoplasmic membrane. This is always the case, whether vesicles are isolated from membranes that exhibit a temperature-induced protein aggregation or not. A correlation between high proline transport activity and the presence of vesicles with double membranes (consisting of outer and inner membrane) has been established. The possibility that the outer membrane protects the transport system in the cytoplasmic membrane during the isolation of vesicles is discussed.     

Cromatin and core particles formed from the inner histones and synthetic polydeoxyribonucleotides of defined sequence.

Chicken erythrocyte inner histones (H2A, H2B, H3 and H4) were associated with the two complementary homopolymeric polydeoxyribonucleotides and the two alternating copolymeric polydeoxyribonucleotides. No evidence for formation of chromatin-like structures was obtained for the complexes with poly(dG) . poly(dC) or poly(dA) . poly(dT). Both poly (dGdC) . poly(dGdC) and poly(dAdT) . poly(dAdT) could be folded by histones to yield material digested by DNAase I to multiples of about 10 and by staphylococcal nuclease to 146 bp core particles. Due to the lack of sequence heterogeniety in the complex of histones with poly(dAdT) . poly(dAdT), core particles with remarkable fine structural detail are obtained. The internal organization of DNA in the AT-containing and GC-containing core particles appears not to be identical.     

Translocatable resistance to mercuric and phenylmercuric ions in soil bacteria.

Of a sample of 42 gram-negative Hg-resistant bacteria, three (a Pseudomonas fluorescens, a Klebsiella sp. and a Citrobacter sp.) contained translocatable elements conferring resistance to Hg2+ (all three) and to Hg2+ and phenylmercuric acetate (P. fluorescens). The discovery of transposable phenylmercuric acetate resistance extends the range of known resistance "transposons" from heavy metals and antibiotics to organometallic compounds.     

Kinetic and equilibrium studies of the interactions of silver ions with poly(A)

The interaction of silver ions with poly(A) was studied by potentiometric titration, uv spectrophotometry, and stopped-flow spectroscopy. For 0 < r_b < 0.5, where r_b is moles of silver ion bound per mole of nucleotide base, there exists only one type of binding for poly(A). Using McGhee's theory, the binding parameters, such as intrinsic binding constant, number of sites per nucleotide, and cooperativity, were determined from the potentiometric titration data. Using the stopped-flow method, one relaxation time was observed in 0 < r₀ < 0.5, where r₀ is the moles of silver ions added per mole of nucleotide base. The concentration dependences of the relaxation time suggest that the binding of silver ions to poly(A) proceeds through the following mechanism: where M is free silver ions, P the free binding sites on poly(A), and C and C′ are two forms of the complex. The nature of the binding of silver ions to poly(A) is also discussed.     

Effect of cesium ions on the electrotropic actions of acetylcholine in the rabbit atrium

Cs+ (15-20 mM) decreases the electrotropic vagal effects on an isolated vagal innervated rabbit atrium. By means of investigating the action potentials and the phase plane trajectories of trabeculae from the rabbit atrium using a modified single sucrose gap technique the anomalous rectification disappears and also the effect of acetylcholine (ACh) on action potential duration. We presume that the anomalous rectification should be a necessary condition of electrotropic vagal (ACh) action on the rabbit atrium.     

Effect of polydeoxyribonucleotides on human fibroblasts in primary culture

The effects of a mixture of oligo‐ and polydeoxyribonucleotides (PDRN) on the growth and protein secretion of cultured human skin fibroblasts were investigated. Both intact and DNAase‐digested PDRN stimulated cell proliferation to a similar extent. When cultured fibroblasts were incubated with radioactive amino acids in the presence of intact or digested PDRN the incorporation of the tracer into secreted proteins increased significantly. This stimulation appears to be specific for certain protein components, including fibronectin. These results are interpreted assuming that PDRN and the nucleotides and nucleosides resulting from its degradation, can act as signal transducers or, alternatively, can be internalized and utilized to provide purine and pyrimidine rings for the salvage pathways. Copyright © 1999 John Wiley & Sons, Ltd.     

Effect of silver ions on ethylene biosynthesis by tomato fruit tissue

Mature-green tomato fruit (Lycopersicon esculentum Mill.) were treated asymmetrically with 2 millimolar silver thiosulfate (STS) through a cut portion of the peduncle while still attached to the plant. One-half of the fruit received silver and remained green while the other half ripened normally and was silver-free (less than 0.01 parts per billion). Harvested mature-green fruit were also treated with STS through the cut pedicel. Green tissue from silver-treated fruit had levels of 1-aminocyclopropane-1-carboxylic acid (ACC, the immediate ethylene precursor) slightly less or similar to that of turning or red-ripe tissue from the same fruit, and similar to that of mature-green tissue from control fruit. Ethylene production was higher in green tissue from silver-treated fruit than from either red tissue from the same fruit, or mature-green tissue from control fruit. By inhibiting ACC synthesis with aminoethoxyvinyl glycine, and by applying ACC ± silver to excised disks of pericarp tissue from control or silver-treated tomatoes, we showed that short-term silver treatment did not affect the biological conversion of ACC to ethylene, while long-term treatment stimulated both the conversion of ACC to ethylene and the synthesis of ACC.     

Effect of metal ions on growth and sporulation of Clostridium perfringens in a synthetic medium.

All cells, whatever their origin, function in an essentially inorganic environment. In this environment, metal cations play an important role. Attempts were made in the present study to determine if different amounts of five metal ions (MG++, Fe++, Mn++, Zn++, Ca++) are needed for secondary metabolism of Clostridium perfringens than are needed for primary metabolism. Both the vegetative growth stage (primary metabolic stage) and spore stage (secondary metabolic stage) of Clostridium perfringens were studied. Endeavors were made to detect the effects of metal ions, if any, on growth and sporulation of the organisms. Mg++ was required for growth of vegetative cells and maintenance of normal cellular morphology, Fe++ was needed for sporulation as well as for vegetative growth, but the amount needed for spore formation was higher than that needed for growth. Ca++ was essential to refractility and heat-resistance of spores. Zn++ inhibited both growth and sporulation, if the Mg++ concentration was low. Mn++ was required for neither growth nor sporulation. For maximum heat-resistance of the spores, Ca++ plus unidentified organic substances were required.     

Isolation and characterization of microbodies and symphyomicrobodies with different buoyant densities from the fungus Blastocladiella emersonii.

Microbodies isolated from sporangia of the aquatic fungus $\text{Blastocladiella emersonii}$ have a mean buoyant density of 1.222 g/cm³ after centrifugation through a linear sucrose gradient, and contain catalase, isocitrate lyase and malate synthase. Microbodies fuse to produce one symphyomicrobody per zoospore at the time of sporogenesis. An increase in density accompanies this process. The symphyomicrobody has a mean buoyant density of 1.292 g/cm³ while the spore's single mitochondrion has a buoyant density of 1.219 g/cm³. Statistical data are also provided for both starting levels and purification of symphyomicrobody and mitochondrial enzyme markers.     

Prevention of autolysis in suspensions of Neisseria gonorrhoeae by mercuric ions.

Mercuric ions were the only metal ions which prevented autolysis of Neisseria gonorrhoeae, as measured both by stabilization of optical density and by prevention of release of [3H]diaminopimelic acid.     

Enzymatic reduction of mercurous and mercuric ions in Bacillus cereus

A strain of Bacillus cereus, which can grow in nutrient broth containing 50 microM HgCl2, was isolated from soil. Mercurous or mercuric ion dependent oxidation of reduced NADPH was demonstrated in crude extracts of cells grown in nutrient broth containing 10 microM HgCl2. The properties of this mercuric reductase were similar to those of the enzymes from R factor bearing Escherichia coli in substrate specificity, heat stability, requirement of sulfhydryl compounds, sensitivity to some heavy metal ions, and molecular weight.