Diffusion of DNA at very low concentrations

The diffusion behavior of DNA samples of molecular weights between 1 × 10⁶ and 25 × 10⁶ Daltons was investigated under standard conditions at mean concentrations c? between 0.0009 and 0.017 g/dl. Special techniques described previously were used and supplemented. The sensitivity required was accomplished by multiple passage through the sample cells (effective path length of 10–45 cm) and application of the Gouy interference method. The maximum DNA refraction index difference has been determined more precisely from Gouy interference fringes by applying a systematic variation procedure and a linear-plot criterion. Convection was prevented by a temperature constancy better than 0.002°C/day, vibrationless operation, and by application of a slight density gradient of heavy water, which also improved the boundary-forming procedure. The corresponding optical HDO gradient was compensated. The concentration dependence of the DNA diffusion coefficient average D_A was found to be positive and very small at extremely low concentrations, that is, below c? = 0.008 g/dl, for the sample of highest molecular weight investigated. With beginning penetration of different DNA molecules, D_A increases markedly. The diffusion constant averages of our polydisperse samples will be corrected for monodisperse subfractions in a following paper. The resulting molecular weights M from diffusion and sedimentation constants (D⁰, s⁰) together with data from literature are the basis of new s⁰–M, D⁰ ? M, and [η]–M relations for monodisperse DNA samples.     

Aquatic nitrogen transformations at low oxygen concentrations

Nitrite and nitrous oxide made up 40% of the hypolimnetic dissolved inorganic nitrogen in mesotrophic Lake Rotoiti, New Zealand, prior to hypolimnetic anoxia. Up to 120 mg of N m-3 as nitrite and 20 mg of N m-3 as nitrous oxide accumulated, whereas dissolved-oxygen concentrations remained between 1.0 and 0.2 g m-3 and were totally consumed when the hypolimnion became completely anoxic. Assays of water column nitrification potentials, together with measurements of the relative rates of nitrate and nitrite reduction, suggested that at low dissolved-oxygen concentrations both nitrite and nitrous oxide were produced mainly by ammonium-oxidizing bacteria, with nitrous oxide being a product of nitrifier denitrification.     

Aerobic microbial growth at low oxygen concentrations

Sterilizable membrane probes were used to study the relation between oxygen concentration and respiration rate in Candida utilis growing on acetate. When the organism was grown in a continuous fermentor at various dissolved oxygen concentrations (0.23 x 10(-6) to 32 x 10(-6)m), with time allowed for full adaptation to each oxygen concentration, the relationship between oxygen concentration and growth rate simulated Michaelis-Menten behavior, giving an apparent K(m) for oxygen of 1.3 x 10(-6)m. When respiration rate was measured at various oxygen concentrations without allowing time for adaptation, it was found that the respiration rate was directly proportional to O(2) concentration at low O(2) concentrations, and independent of O(2) concentration at high O(2) concentrations. Transition from one type of behavior to the other was fairly abrupt. The respiration rate in the presence of excess oxygen depended on the O(2) concentration at which the cells were grown, but the rate at low O(2) concentrations did not. There was evidence that, at low oxygen concentrations, oxygen diffusion through the cell substance limits respiration rate, at least in part.     

Aggregation of deoxyhemoglobin S at low concentrations.

The self-association of deoxyhemoglobin S was measured in dilute solutions (0 to 5 g/dl) by Rayleigh light scattering at 630 nm and osmometry in 0.05 M potassium phosphate buffer (pH 7.35). Weight and number average molecular weights (Mw and Mn, respectively) and the second or higher virial coefficients, B' were determined. No experimentally significant differences were observed between oxy- and deoxy-Hb S up to the concentration of 2 g/dl; their apparent average molecular weights were within experimental error. Above that concentration, both Mn and Mw of deoxy-Hb S were significantly different from that of oxy-Hb S. The negative second viral coefficent of deoxy-Hb S, observed by both techniques, is consistent with the self-association of this protein. The lack of effect of 0.4 M propylurea on the state of aggregation and the significant influence of 0.1 M NaCl suggests that polar interactions are involved in formation of these aggregates.     

Cholesterol organization in membranes at low concentrations: effects of curvature stress and membrane thickness

Cholesterol is often found distributed nonrandomly in domains in biological and model membranes and has been reported to be distributed heterogeneously among various intracellular membranes. Although a large body of literature exists on the organization of cholesterol in plasma membranes or membranes with high cholesterol content, very little is known about organization of cholesterol in membranes containing low amounts of cholesterol. Using a fluorescent cholesterol analog (25-[N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-methyl]amino]-27-norcholesterol, or NBD-cholesterol), we have previously shown that cholesterol may exhibit local organization even at very low concentrations in membranes, which could possibly be attributable to transbilayer tail-to-tail dimers. This is supported by similar observations reported by other groups using cholesterol or dehydroergosterol, a naturally occurring fluorescent cholesterol analog which closely mimics cholesterol. In this paper, we have tested the basic features of cholesterol organization in membranes at low concentrations using spectral features of dehydroergosterol. More importantly, we have investigated the role of membrane surface curvature and thickness on transbilayer dimer arrangement of cholesterol using NBD-cholesterol. We find that dimerization is not favored in membranes with high curvature. However, cholesterol dimers are observed again if the curvature stress is relieved. Further, we have monitored the effect of membrane thickness on the dimerization process. Our results show that the dimerization process is stringently controlled by a narrow window of membrane thickness. Interestingly, this type of local organization of NBD-cholesterol at low concentrations is also observed in sphingomyelin-containing membranes. These results could be significant in membranes that have very low cholesterol content, such as the endoplasmic reticulum and the inner mitochondrial membrane, and in trafficking and sorting of cellular cholesterol.     

Aquatic nitrogen transformations at low oxygen concentrations.

Nitrite and nitrous oxide made up 40% of the hypolimnetic dissolved inorganic nitrogen in mesotrophic Lake Rotoiti, New Zealand, prior to hypolimnetic anoxia. Up to 120 mg of N m-3 as nitrite and 20 mg of N m-3 as nitrous oxide accumulated, whereas dissolved-oxygen concentrations remained between 1.0 and 0.2 g m-3 and were totally consumed when the hypolimnion became completely anoxic. Assays of water column nitrification potentials, together with measurements of the relative rates of nitrate and nitrite reduction, suggested that at low dissolved-oxygen concentrations both nitrite and nitrous oxide were produced mainly by ammonium-oxidizing bacteria, with nitrous oxide being a product of nitrifier denitrification.     

Considerations in the determination of boron at low concentrations

Experimental evidence now supports the nutritional essentiality of boron (B) in some biological systems, and accordingly, the need for reliable analytical B data is increasing. However, the accurate determination of B in biological materials is a formidable challenge at low concentrations (<1 mg B/kg). Recent studies still show significant analytical discrepancies in the analysis of animal tissues and fluids, despite the development of instrumental techniques such as TIMS, ICP-MS, ICP-ES, ICAP, SIMS, NA-MS, PGAA, NRA, and so forth, which have demonstrated detection limits approaching or exceeding (μgB/kg concentrations. Since boric acid is both volatile and ubiquitous in nature, the chemical and physical pathways for B contamination and its loss are manifold, especially during sample preparation. An added obstacle is the inadequacy of biological reference materials certified for B below mg B/kg. With an emphasis toward sample preparation and ICP-MS analysis, examples are provided in this article to help the analyst avoid common problems associated with the analysis of B from biological sources. Topics that are discussed include contamination from Teflon vessels during microwave digestion, losses owing to freeze-drying, B isotopic variations, standards preparation, reagent backgrounds, and instrumental interferences.     

Selection of resistant bacteria at very low antibiotic concentrations

The widespread use of antibiotics is selecting for a variety of resistance mechanisms that seriously challenge our ability to treat bacterial infections. Resistant bacteria can be selected at the high concentrations of antibiotics used therapeutically, but what role the much lower antibiotic concentrations present in many environments plays in selection remains largely unclear. Here we show using highly sensitive competition experiments that selection of resistant bacteria occurs at extremely low antibiotic concentrations. Thus, for three clinically important antibiotics, drug concentrations up to several hundred-fold below the minimal inhibitory concentration of susceptible bacteria could enrich for resistant bacteria, even when present at a very low initial fraction. We also show that de novo mutants can be selected at sub-MIC concentrations of antibiotics, and we provide a mathematical model predicting how rapidly such mutants would take over in a susceptible population. These results add another dimension to the evolution of resistance and suggest that the low antibiotic concentrations found in many natural environments are important for enrichment and maintenance of resistance in bacterial populations.     

The effects of gas sparging on growth and glucose utilization of Zymomonas mobilis at low glucose concentrations

Summary In a batch culture of Zymomonas mobilis the specific growth and specific glucose uptake rates and the molar growth yields were similar under CO₂ and N₂ sparging. The use of N₂ or N₂ with nucleation agents to strip dissolved CO₂ from the medium did not improve growth rates or yields or enhance glucose consumption rates. Carbon dioxide produced no direct feedback inhibition on metabolic rates. The catabolic enzymes of Z. mobilis are not allosterically controlled by CO₂.     

Kinetic analysis of tubulin exchange at microtubule ends at low vinblastine concentrations

We have investigated the effects of vinblastine at micromolar concentrations and below on the dynamics of tubulin exchange at the ends of microtubule-associated-protein-rich bovine brain microtubules. The predominant behavior of these microtubules at polymer-mass steady state under the conditions examined was tubulin flux, i.e., net addition of tubulin at one end of each microtubule, operationally defined as the assembly or A end, and balanced net loss at the opposite (disassembly or D) end. No dynamic instability behavior could be detected by video-enhanced dark-field microscopy. Addition of vinblastine to the microtubules at polymer-mass steady state resulted in an initial concentration-dependent depolymerization predominantly at the A ends, until a new steady-state plateau at an elevated critical concentration was established. Microtubules ultimately attained the same stable polymer-mass plateau when vinblastine was added prior to initiation of polymerization as when the drug was added to already polymerized microtubules. Vinblastine inhibited tubulin exchange at the ends of the microtubules at polymer-mass steady state, as determined by using microtubules differentially radiolabeled at their opposite ends. Inhibition of tubulin exchange occurred at concentrations of vinblastine that had very little effect on polymer mass. Both the initial burst of incorporation that occurs in control microtubule suspensions following a pulse of labeled GTP and the relatively slower linear incorporation of label that follows the initial burst were inhibited in a concentration-dependent manner by vinblastine. Both processes were inhibited to the same extent at all vinblastine concentrations examined. If the initial burst of label incorporation represents a low degree of dynamic instability (very short excursions of growth and shortening of the microtubules at one or both ends), then vinblastine inhibits both dynamic instability and flux to similar extents. The ability of vinblastine to inhibit tubulin exchange at microtubule ends in the micromolar concentration range appeared to be mediated by the reversible binding of vinblastine to tubulin binding sites exposed at the polymer ends. Determination by dilution analysis of the effects of vinblastine on the apparent dissociation rate constants for tubulin loss at opposite microtubule ends indicated that a principal effect of vinblastine is to decrease the dissociation rate constant at A ends (i.e., it produces a kinetic cap at A ends), whereas it has no effect on the D-end dissociation rate constant.     

Chronic effects of low insecticide concentrations on freshwater caddisfly larvae

The insecticide load in surface waters does not ordinarily reach concentrations acutely toxic to aquatic fauna. The effects of the low insecticide concentrations typical of natural habitats are still not clear, for they often appear only after relatively long exposure times. To test such a situation, the insecticides lindane and parathion were introduced into a static-with-renewal outdoor aquaria system at concentrations about four and five orders of magnitude lower than their respective 96-h LC₅₀s, and their chronic (about 90 days) effects on the survival rate of freshwater caddisfly larvae were observed. The emergence and hence survival rate of Limnephilus lunatus Curtis was significantly reduced by lindane at 0.1 ng l^–1, a value nearly five orders of magnitude lower than the 96-h LC₅₀. Parathion, with acute and subacute toxicity similar to that of lindane, did not significantly alter the emergence rate of this species. In contrast, this substance did produce a significant reduction in emergence rate of the closely related species Limnephilus bipunctatus Curtis at 1 ng l^–1, even though this species was significantly less susceptible than L. lunatus to parathion at high concentrations. We conclude that chronic insecticide exposure can be hazardous to freshwater macroinvertebrates even at unexpectedly low concentrations. The low-concentration effects may depend on both species and substance and therefore cannot be predicted from toxicity data at higher concentrations.     

Gramicidin-mediated currents at very low permeant ion concentrations.

Current-voltage relations have been measured for the fluxes of caesium ions through pores formed by gramicidin in lipid bilayer membranes. The ionic currents have been separated from capacitative currents using a bridge circuit with an integrator as null-detector. The conductances during brief voltage pulses were small enough to avoid the effects of diffusion polarization and the ionic strength was raised using choline chloride or magnesium sulfate to reduce the effects of double-layer polarization. Under these conditions the current-voltage relations have the same shape at 0.1 and 1 mM, but different shapes for higher concentrations. These data demonstrate that the fluxes do not obey independence for concentrations above 10 mM, but they cannot be used in isolation to support a particular value of the binding constant. The shape observed at low concentrations suggests that entry of ions into the pore remains weakly potential dependent even at 300 mV.     

Screening method for compounds acting at super low concentrations

At present, there are no simple and reliable methods for selection of compounds with physiological activity in super-low concentrations (SLC). The screening system with high predictability is proposed. At the beginning, experiment is carried out on infusorians--spirostoma. Their motion activity, behavior, living ability and shape are registered at concentrations of substances in a range of 10(-3)-10(-13) mol/l. If the effect is detected on spirostoma, then experiments are performed on laboratory animals or their isolated tissues. Specific deviations on injection substance in SLC are detected, depending on compounds class. Nine representatives of different classes of biologically active compounds are investigated in the work: carbofos, deltametrin, 3-quinuclidinyl benzylate, atropine, phenosan, fenazepam, dopamine receptor antagonist SCH-23390, ciprine, and hexenal. It is accepted that if compound action in SLC is observed on spirostoma, then effects in super-low doses are also detected in a whole animal.