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[Structural changes in the contractile proteins of muscle fiber studied by polarization ultraviolet fluorescence microscopy. VII. The effect of Ca2+ on the nature of the conformational changes in F-actin induced by the binding of heavy meromyosin].

Changes in anisotropy of tryptophan fluorescence and in birefringence of actin filaments induced by the binding to actin of heavy meromyosin (HMM), both containing DTNB light chains and devoid of them, were found in rabbit muscle fibres free of myosin, troponin, and tropomyosin. Ca2+ was shown to affect the pattern of changes in tryptophan fluorescence anisotropy and birefringence of actin filament at the moment of HMM interaction with actin, providing HMM contains DTNB light chains. Anisotropy of tryptophan fluorescence and birefringence of actin filaments rises in the absence of Ca2+ (pCa greater than or equal to 7), while in its presence (pCa less than or equal to 6) these values drop down. Furthermore, these changes become cooperative when Ca2+ concentration increases from pCa = 7 to pCa = 6. It was shown that the binding of HMM devoid of DTNB light chains to F-actin decreases tryptophan fluorescence anisotropy and birefrigence of actin filaments, regardless of Ca2+ concentration. Ca2+-dependent structural changes of F-actin induced by interaction of heads of myosin molecules with actin are assumed to be of great importance in regulation of muscle contraction of vertebrate skeletal muscles.

Actins↗

[Polarization method of fluorescence microscopy for the analysis of the spatial organization of cylindrically symmetrical structures].

Basic clue points and applied mathematics of the simplified variant of the theory for the quantitative analysis of spatial macromolecular organization of anisotropic structures with cylindrical symmetry based on emission polarization characteristics are given. Moreover, a graphical method is presented for the effective analysis of the spatial emission anisotropy of multiple-component systems.

Fluorescence↗

Multiple labeling of cellular constituents by combining surface reflection interference and fluorescence microscopy.

In this paper the technique for visualizing cytoskeleton in detergent-extracted cultured cells by surface reflection interference (SRI) microscopy after staining with the protein dye Coomassie Brilliant Blue (SRI-CooB technique) is used in conjunction with fluorescently-labeled antibodies or with other fluorescent probes to detect a number of constituents in the same cultured cell. Because SRI-CooB technique preferentially visualizes microfilament bundles along the ventral aspect of cells adhering to a glass substratum, we feel that this simple and rapid technique has great potential in studies of cell-substratum adhesiveness and of adhesion-related cytoskeletal organization.

Animals↗

Visualization of RecA filaments and DNA by fluorescence microscopy.

We have developed two experimental methods for observing Escherichia coli RecA-DNA filament under a fluorescence microscope. First, RecA-DNA filaments were visualized by immunofluorescence staining with anti-RecA monoclonal antibody. Although the detailed filament structures below submicron scale were unable to be measured accurately due to optical resolution limit, this method has an advantage to analyse a large number of RecA-DNA filaments in a single experiment. Thus, it provides a reliable statistical distribution of the filament morphology. Moreover, not only RecA filament, but also naked DNA region was visualized separately in combination with immunofluorescence staining using anti-DNA monoclonal antibody. Second, by using cysteine derivative RecA protein, RecA-DNA filament was directly labelled by fluorescent reagent, and was able to observe directly under a fluorescence microscope with its enzymatic activity maintained. We showed that the RecA-DNA filament disassembled in the direction from 5' to 3' of ssDNA as dATP hydrolysis proceeded.

DNA↗

Measurement of the effects of cadmium stress on protozoan grazing of bacteria (bacterivory) in activated sludge by fluorescence microscopy.

The effect of cadmium stress on protozoan bacterivory in sewage sludge was measured by experimentally exposing sludge communities to 0 to 150 mg of Cd per liter for up to 6 h and then determining the rates of protozoan grazing on bacteria, using a double-staining technique and epifluorescence microscopy. Bacterivory was measured by incubating the sludge with fluorescently labeled bacterium-sized latex beads and directly observing ingestion of the beads and bacterial cells in the sludge by epifluorescence microscopy of preserved samples. Staining with 4',6-diamidino-2-phenylindole and acridine orange permitted the simultaneous determination of protozoan numbers and bacterivory activity as estimated by the number of bacterial cells and bacterium-sized latex beads ingested by the representative ciliate Aspidisca costata. Enumeration with latex beads proved to be an effective way of estimating bacterivory in sludges subjected to heavy-metal stress. This technique should prove useful for determining the effects of other chemical stresses on protozoan numbers and bacterivory in organic-rich environments. Although the number of protozoa declined significantly only after exposure to 100 mg of Cd per liter for 4 h, grazing, as indicated by bead ingestion, was significantly inhibited by Cd concentrations of greater than 25 mg/liter in less than 1 h, and exposure to 100 mg of Cd per liter effectively stopped protozoan grazing within 1 h of exposure. Protozoan ingestion of latex beads and bacteria was inversely correlated to Cd concentration and exposure time. The reduction of protozoan bacterivory by Cd provides a possible explanation for the increase in suspended bacteria in the effluents of metal-stressed treatment facilities.

Analysis of Variance↗

Structure of dental glass-ionomer cements by confocal fluorescence microscopy and stereomicroscopy.

The microstructure of four cements, setting by different mechanisms (acid-base, dual cure, triple cure), was studied. The porosity of unpolymerized materials was detected by stereomicroscopy. After polymerization and storage in water or lactic acid solution, the porosity, filler distribution and gel layer, which was formed at the filler/matrix interface of polymerized materials, were examined by confocal laser microscopy. For this purpose, the specimens were treated with fluorescent dye solution before the test. The results showed that hydrolytic degradation (pH 7) mainly involved the resin matrix, and the acid erosion (pH 3.5) involved the gel layer too. As regards the filler, materials with different setting mechanism released the glass particles in different times. The loss of the filler particles occurred quicker in acid-base setting cements, and slower in triple-cured material.

Fluorescent Dyes↗