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.     

Binding of beta-naphthyl triphosphate to human adult hemoglobin accompanying deoxygenation. Investigated by simultaneous measurements of fluorescence, absorbance and partial pressure of oxygen

Binding of a fluorescent allosteric effector, beta-naphthyl triphosphate (beta-NapP3), to human adult hemoglobin (HbA) at various levels of oxygen saturation were investigated by simultaneous measurements of fluorescence, absorbance and oxygen partial pressure. Amounts of beta-NapP3 bound to HbA were easily estimated from the fluorescence intensities of HbA solutions, because it was previously proved that the fluorescence of beta-NapP3 bound to HbA is completely quenched. Exchange reactions of the above fluorescent allosteric effector with 2,3-bisphosphoglycerate (DPG) were also examined at various levels of oxygen saturation. It was found that beta-NapP3 binds to deoxyHbA tetramer in the molar ratio of 2:1, and that one of the two beta-NapP3 competes with DPG. It was also found that beta-NapP3 binds to completely oxygenated HbA tetramer in the molar ratio of 1:1, and that the bound beta-NapP3 was not released by adding DPG. The binding affinity of beta-NapP3 for the noncompetitive site of completely oxygenated HbA, to which DPG does not bind, was smaller than that for the noncompetitive site of deoxyHbA, to which DPG also does not bind. Furthermore, the correlations between oxygen bindings by HbA and the bindings of beta-NapP3 to HbA in the intermediate stages of deoxygenation were investigated. It was revealed that HbA as a tetramer exists in three conformational states rather than simple two states as Monod, Wyman, and Changeux had proposed.     

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.     

Increased choline acetyltransferase activity by Chinese herbal medicine Sho-saiko-to-go-keishi-ka-shakuyaku-to in aged rat brain

The effect of a Chinese herbal medicine Sho-saiko-to-go-keishi-ka-shakuyaku-to (TJ-960) on the brain choline acetyltransferase (CAT) activity was studied in adult (3.5 months of age) and aged (24 months of age) rats. After oral administration of 5% TJ-960 solution for 3 months, CAT activity in the hippocampus, pons-medulla oblongata and striatum of aged rats was significantly lower than that of adult rats. CAT activity in the cerebellum, however, was significantly higher in the aged rats, as compared to the adult rats. TJ-960 significantly increased CAT activity in the hippocampus and striatum of aged rats, but did not affect the activity of the enzyme in the adult rat brain.     

Application of ozone disinfection to remove Enterococcus seriolicida, Pasteurella piscicida, and Vibrio anguillarum from seawater.

Survival of bacterial fish pathogens, including Enterococcus seriolicida, Vibrio anguillarum, and Pasteurella piscicida, in ozonated seawater was determined in a batch system. Bacterial counts of all fish pathogens decreased at more than 0.040 to 0.060 mg of total residual oxidants (TROs) per liter, whereas no decrease in viable counts was observed at less than 0.018 to 0.028 mg of TROs per liter. The 99% inactivation point was achieved at concentrations of 0.111 mg/liter for E. seriolicida, 0.063 mg/liter for P. piscicida, and 0.064 mg/liter for V. anguillarum within 1 min. Moreover, the mean 99 and 99.9% killing concentration-contact time (C.t) products were 0.123 and 0.186 mg.min/liter for E. seriolicida, 0.056 and 0.084 mg.min/liter for P. piscicida, and 0.081 and 0.123 mg.min/liter for V. anguillarum, respectively. However, the mean 99 and 99.9% C.t products for the mixed population in coastal seawater were 0.200 and 0.621 mg.min/liter. These results strongly suggest that ozone treatment at more than 1.0 mg of TROs per liter for several minutes is able to disinfect seawater for mariculture efficiently.     

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     

The DNA replication priming protein, PriA, is required for homologous recombination and double-strand break repair.

The PriA protein, a component of the phiX174-type primosome, was previously shown to be essential for damage-inducible DNA replication in Escherichia coli, termed inducible stable DNA replication. Here, we show that priA::kan null mutants are defective in transductional and conjugational homologous recombination and are hypersensitive to mitomycin C and gamma rays, which cause double-strand breaks. The introduction of a plasmid carrying the priA300 allele, which encodes a mutant PriA protein capable of catalyzing the assembly of an active primosome but which is missing the n'-pas-dependent ATPase, helicase, and translocase activities associated with PriA, alleviates the defects of priA::kan mutants in homologous recombination, double-strand break repair, and inducible stable DNA replication. Furthermore, spa-47, which was isolated as a suppressor of the broth sensitivity of priA::kan mutants, suppresses the Rec- and mitomycin C sensitivity phenotypes of priA::kan mutants. The spa-47 suppressor mutation maps within or very near dnaC. These results suggest that PriA-dependent primosome assembly is crucial for both homologous recombination and double-strand break repair and support the proposal that these processes in E. coli involve extensive DNA replication.     

Repression by a differentiation-specific factor of the human cytomegalovirus enhancer.

We detected a novel nuclear protein, MRF, that binds to multiple sites on the modulator which is located upstream of the human cytomegalovirus major immediate early gene enhancer. The expression of MRF is differentiation specific; the DNA binding activity is present in nuclear extracts from undifferentiated Tera-2 and THP-1 cells, but significantly reduced after these cells are induced to differentiate. In undifferentiated cells the enhancer activity is repressed by the modulator and upon differentiation the enhancer becomes active. Competitive binding assays demonstrate that MRF requires the presence of multiple A+T stretches for binding to DNA, rather than binding to a specific DNA sequence. Mutations of these stretches in the modulator reduce the binding activity of MRF, as well as the repressing activity on the enhancer. These results suggest that MRF may act as a repressor of enhancer function. We propose that MRF binds over the entire modulator and exerts repressor activity.