On the rotational brownian motion of a bacterial idle motor. II. Theory of fluorescence correlation spectroscopy

The photon flux autocorrelation function of a fluorescent label attached to a bacterial motor shaft is calculated for the case in which the bacterial motor is considered to be actively but idly rotating. It is shown that even when the fluorescent label has a very short lifetime, fluorescence correlation spectroscopy should provide a useful tool for determining the rate of revolution of the bacterial motor under various solution conditions.     

Methylation strongly enhances DNA bending in the replication origin region of the Escherichia coli chromosome

Summary Two-dimensional gel electrophoresis, at high and low temperatures, and gel mobilities of circularly permuted DNA segments showed a large bending locus about 50 bp downstream from the right border of the 245 by oriC box, a minimal essential region of autonomous replication on the Escherichia coli chromosome. Bending was strongly enhanced by Dam methylation. In DNA from a Dam^– strain, the mobility anomaly arising from altered conformation was much reduced, but was raised to the original level by methylation in vivo or in vitro. Enhancement of the mobility anomaly was also observed by hybrid formation of the Dam^– strand with the Dam⁺ strand. Near the bending center, GATC, the target of Dam methylase, occurs seven times arranged essentially on the same face of the helix with 10.5 by per turn. We concluded that small bends at each Dam site added up to the large bending detectable by gel electrophoresis.     

Immunocytochemical observation of paraquat-induced alveolitis with special reference to class II MHC antigens.

The expression of class II major histocompatibility complex (MHC) antigens on alveolar epithelial cells and macrophages was investigated immunocytochemically in paraquat-induced alveolitis in the rat lung. Until 2 days after paraquat injection, class II MHC antigens were expressed on the type II alveolar epithelium without any inflammatory cellular infiltration. From the 4th to the 7th day after paraquat injection, class II MHC antigen-positive macrophages increased in the alveolar spaces, whereas the expression on the type II alveolar epithelium became obscure. Over 10 days after the injection, interstitial fibrosis progressed and the intra-alveolar inflammatory infiltrates decreased. Epithelial cells lining the thickened fibrous septa no longer expressed class II MHC antigens. These results suggest that chemical stimuli can induce class II MHC antigen expression on the type II alveolar epithelium in the early stage of cellular injury, followed by inflammatory infiltration and interstitial fibrosis.     

Heterogeneity among microtubules of the cytoplasmic microtubule complex detected by a monoclonal antibody to alpha tubulin

Three monoclonal antibodies specific for tubulin were tested by indirect immunofluorescence for their ability to stain cytoplasmic microtubules of mouse and human fibroblastic cells. We used double label immunofluorescence to compare the staining patterns of these antibodies with the total microtubule complex in the same cells that were stained with a polyclonal rabbit antitubulin reagent. Two of the monoclonal antitubulin antibodies bound to all of the cytoplasmic microtubules but Ab 1-6. 1 bound only a subset of cytoplasmic microtubules within individual fixed cells. Differential staining patterns were observed under various fixation conditions and staining protocols, in detergent-extracted cytoskeletons as well as in whole fixed cells. At least one physiologically defined subset of cytoplasmic microtubules, those remaining in cells pretreated for 1 h with 5 microM colcemid, appeared to consist entirely of Ab 1-6. 1 positive microtubules. The same was not true of the microtubules that remained in either cold-treated cells or in cells that had been exposed to hypotonic medium. The demonstration of antigenic differences among microtubules within single fixed cells and the apparent correlation of this antigenic difference with at least one "physiologically" defined subset suggests that mechanisms exist for the differential assembly or postassembly modification of individual microtubules in vivo, which may endow them with different physical or functional properties.     

Do protozoa conceal a high potency of nucleoside triphosphate hydrolysis present in Toxoplasma gondii?

ATP hydrolytic activity in whole cell homogenates of some protozoa was assayed in the presence or absence of dithiothreitol. The activities in all protozoan cell homogenates, except Toxoplasma gondii, ranged from 0.6 to 32 mumol/mg protein/hr, irrespective of the presence or absence of dithiothreitol. A remarkably higher activity, 11,690 mumol/mg protein/hr, was observed for T. gondii in the presence of dithiothreitol. These results indicate that the higher ATP hydrolytic potency observed for T. gondii is not universal to protozoa, rather it is unique to T. gondii.     

Direct fluorimetric measurement on the hydrolysis of β-naphthyl triphosphate, a fluorescent ATP analog, by heavy meromyosin

A fluorescent ATP analog, β-naphthyl triphosphate, was hydrolyzed to β-naphthyl diphosphate and orthophosphate by heavy meromyosin ATPase. In the process of hydrolysis the fluorescence intensity of β-naphthyl triphosphate changed remarkably. Thus, the rate of β-naphthyl triphosphate hydrolysis is evaluated directly and continuously by measuring the time course of fluorescence intensity.In the presence of Ca²⁺, the Michaelis constant (K_m) of β-naphthyl triphosphate hydrolysis by heavy meromyosin was similar to that of ATP hydrolysis. While, in the presence of Mg²⁺ the K_m of β-napthyl triphosphate hydrolysis was 9.0·10⁻⁶ M, much larger than the value of ATP hydrolysis, indicating that the apparent affinity of the enzyme for β-naphthyl triphosphate is less than that for ATP.The pH dependence of β-naphthyl triphosphatase activity resembled that of ATPase activity, suggesting a similarity in the mechanism of hydrolysis of the two substrates.     

Bacterial dissolution of pyrite by Thiobacillus ferrooxidans

The kinetics of the dissolution of pure pyrite (FeS₂) particles by Thiobacillus ferrooxidans were studied both theoretically and experimentally. Adsorption and dissolution experiments were carried out at 30 °C and pH=2, by using a batch reactor. The adsorption process of T. ferrooxidans to pyrite surface was rapid in comparison with the bacterial dissolution process. The experimental results for the adsorption equilibrium were well correlated by the Langmuir type isotherm. The growth rate of adsorbed bacteria was found to be proportional to the product of the number of adsorbed cells and the fraction of solid surface unoccupied by cells. A new kinetic model for the bacterial dissolution was presented, and shown to correlate well with the experimental data for the rate of bacterial dissolution and for the time variation in the number of cells in the liquid phase. The specific growth rate of adsorbed bacteria was also evaluated.List of Symbols f weight fraction of iron in pyrite - K _A m³/cells equilibrium constant for cell adsorption - R _A cells/d m³-mixture growth rate of bacteria adsorbed on solid surface - R _L cells/d m³-mixture growth rate of free bacteria in the liquid phase - t d time - V m³ volume of solid-liquid mixture - W kg weight of pyrite - W ₀ kg initial weight of pyrite - X _A cells/kg-solid number of adsorbed cells on solid surface - X _Am cells/kg-solid maximum adsorption capacity - X _L cells/m³-liquid number of free cells existing in the liquid phase - X _T cells/m³-mixture total number of cells - X _TO cells/m³ initial total number of cells - Y _A cells/kg-FeS₂ growth yield of adsorbed bacteria - Y _L cells/kg-Fe²⁺ growth yield of free bacteria - [Fe] _T kg/m³-liquid concentration of total iron in the liquid phase - fraction of pyrite dissolved - _V fraction of adsorption sites unoccupied by cells - _A d^–1 specific growth rate of adsorbed bacteria - _L d^–1 specific growth rate of free bacteria - volume fraction of solid particles in solid-liquid mixture     

Retrograde but not anterograde bead movement in intact axons requires dynein

Dynein and kinesin have been implicated as the molecular motors that are responsible for the fast transport of axonal membranous organelles and vesicles. Experiments performed in vitro with partially reconstituted preparations have led to the hypothesis that kinesin moves organelles in the anterograde direction and dynein moves them in the retrograde direction. However, the molecular basis of transport directionality remains unclear. In the experiments described here, carboxylated fluorescent beads were injected into living Mauthner axons of lamprey and the beads were observed to move in both the anterograde and retrograde directions. The bead movement in both directions required intact microtubules, occurred at velocities approaching organelle fast transport in vivo, and was inhibited by vanadate at concentrations that inhibit organelle fast transport. When living axons were injected with micromolar concentrations of vanadate and irradiated at 365 nm prior to bead injections, a treatment that results in the V1 photolysis of dynein, the retrograde movement of the beads was specifically abolished. Neither the ultraviolet irradiation alone nor the vanadate alone produced the retrograde-specific inhibition. These results support the hypothesis that dynein is required for retrograde, but not anterograde, transport in vivo. © 1995 John Wiley & Sons, Inc.