J. Michael Mullins

ROLE OF MODULATION ON THE EFFECT OF MICROWAVES ON ORNITHINE DECARBOXYLASE ACTIVITY IN L929 CELLS

The effect of 835 MHz microwaves on the activity of ornithine decarboxylase (ODC) in L929 murine cell was investigated at an SAR of approximately 2.5 W/kg. The results depended upon the type of modulation employed. AM frequencies of 16 Hz and 60 Hz produced a transient increase in ODC activity that reached a peak at 8 h of exposure and returned to control levels after 24 h of exposure. In this case, ODC was increased by a maximum of 90% relative to control levels. A 40% increase in ODC activity was also observed after 8 h of exposure with a typical signal from a TDMA digital cellular telephone operating in the middle of its transmission frequency range (approximately 840 MHz). This signal was burst modulated at 50 Hz, with approximately 30% duty cycle. By contrast, 8 h exposure with 835 MHz microwaves amplitude modulated with speech produced no significant change in ODC activity. Further investigations, with 8 h of exposure to AM microwaves, as a function of modulation frequency, revealed that the response is frequency dependent, decreasing sharply at 6 Hz an 600 Hz. Exposure with 835 MHz microwaves, frequency modulated with a 60 Hz sinusoid, yielded no significant enhancement in ODC activity for exposure times ranging between 2 and 24 h. Similarly, exposure with a typical signal from an AMPS analog cellular telephone, which uses a form of frequency modulation, produced no significant enhancement in ODC activity. Exposure with 835 MHz continuous wave microwaves produced no effects for exposure times between 2 and 24 h, except for a small but statistically significant enhancement in ODC activity after 6 h of exposure. Comparison of these results suggests that effects are much more robust when the modulation causes low-frequency periodic changes in the amplitude of the microwave carrier.

Analysis of spindle microtubule organization in untreated and taxol-treated PtK1 cells.

Taxol, a microtubule stabilizing agent, has been used to study changes in spindle microtubule organization during mitosis. PtK1 cells have been treated with 5 μg/ml taxol for brief periods to determine its effect on spindle architecture. During prophase taxol induces microtubules to aggregate, particularly evident in the region between the nucleus and cell periphery. Taxol induces astral microtubule formation in prometaphase and metaphase cells concomitant with a reduction in spindle length. At anaphase taxol induces an increase in length in astral microtubules and reduces microtubule length in the interzone. Taxol‐treated telophase cells show a reduction in the rate of furrowing and astral microtubules lack a discrete focus and are arranged more diffusely on the surface of the nuclear envelope. In summary, taxol treatment of cells prior to anaphase produces an increase in astral microtubules, a reduction in kinetochore microtubules and a decrease in spindle length. Brief taxol treatments during anaphase through early G1 promotes stabilization of microtubules, an increase in the length of astral microtubules and a delayed rate of cytokinesis.

Cytochalasin B‐induced redistribution of cytokeratin filaments in PtK1 cells

Indirect immunofluorescence demonstrated a dramatic reorganization of cytokeratin filaments produced by cytochalasin B (CB) treatment of PtK1 cells. Much of the normal cytokeratin network became arranged into a latticework consisting of bundles of cytokeratin filaments that radiated from, and interconnected, distinct foci. Electron microscopy showed foci to be dense granular regions through which bundles of cytokeratin filaments looped. Composition of the foci included actin, myosin, and alpha-actinin, as shown by labeling with rhodamine phalloidin or specific antisera. Simultaneous treatment with CB and colchicine was not required for lattice formation, but did produce more extensive development than did CB alone. In cells treated only with CB, the microtubule network remained intact, even in regions of extensive lattice formation. These results contrast sharply with those of Knapp et al (J. Cell Biol. 97:1788 [1983b]), who found lattice formation dependent upon simultaneous CB and colchicine treatment. Time-course and dose-response studies of CB treatment showed lattice formation to follow disruption of stress fibers and the concentration of actin into distinct patches that marked the location of lattice foci. Overall results suggest a structural association between microfilaments and cytokeratin filaments that produces the lattice pattern upon CB-induced disruption of stress fibers. Lattice formation was not limited to a specific cell-cycle stage, since G1, G2, and M cells displayed the lattice. Treatment of cells with dihydro-CB and experiments with enucleated cells showed that lattice formation was dependent upon neither the inhibition of sugar transport nor the nuclear extrusion effects of CB.

Anaphase progression and furrow establishment in nocodazole-arrested PtK1 cells.

The relationship between progression through anaphase and furrow establishment was investigated in PtK1 cells using the anti-mitotic agent Nocodazole to arrest cells at different points in anaphase. The capacity of cells to furrow was compared to the kinetochore-kinetochore separation attained at the time of arrest. For the stages of anaphase examined, furrowing capacity increased directly with kinetochore-kinetochore separation until complete furrows were formed after kinetochore-kinetochore separations of 14 μm or more were reached. Furrow establishment thus occurs during a definite interval during anaphase in PtK1 cells. Results from electron microscopy of both Nocodazole-treated and control cells suggest that a population of astral microtubules may be important for furrow establishment.

Overview of conventional fluorescence photomicrography.

In an age of digital imaging, photographic film still provides a viable and effective means for recording fluorescence images by photomicrography. To maximize the quality of results that are obtained, a photographic emulsion with sufficient sensitivity for the low light level characteristic of Immunofluorescence must be selected, exposures adjusted for reciprocity failure, and modern, high numerical aperture objective lenses employed to produce the brightest possible image. Mounting media that reduce the effects of photobleaching on fluorochromes also help to maintain image brightness, and so reduce exposure times. Digital scanning of film-based micrographs provides the convenience of utilizing image processing software to adjust image density and contrast, and to produce quality prints.

Fluorochromes: Properties and Characteristics

Immunofluorescence microscopy provides a sensitive means by which antigens can be localized within tissues or individual cells. For the most effective use of this technique the researcher can draw upon basic information on factors that affect the brightness of the fluorescence image, and how well that image can be distinguished from background fluorescence or interfering fluorescence signals. A wide variety of fluorochromes are available, with emitting wavelengths that range from the blue-violet end of the visible spectrum to the infrared. Individual fluorochromes are characterized by their extinction coefficients, quantum yields, susceptibility to photobleaching, the wavelengths at which they maximally absorb excitatory and emit fluorescent light, and how far apart those wavelength maxima are separated. Additional choices for fluorescent labeling of antibodies are provided by the availability of fluorescent quantum dots. Informed choices of fluorochromes can obviate many problems, particularly with regard to situations in which two or more antigens are to be localized simultaneously within a specimen.

D2O induced alterations of mitosis in PtK1, cells☆

Deuterium oxide (D2O) was applied to PtK1 cells to assess its effect on mammalian mitosis. Cells exposed to culture medium containing up to 50% D2O were able to enter and complete mitosis, but the duration of mitosis was increased proportionally to the concentration of D2O applied. Cells exposed to 50% D2O showed increases of more than 300% for the interval between nuclear envelope breakdown and anaphase onset, and approximately 65% for the interval between anaphase onset and initial furrowing. At a concentration of 80%, D2O acted as an inhibitor of mitosis; after 8 h exposure to this concentration, cultures showed an increase in the proportion of mulinucleate cells and an absence of mitotic figures. When applied early in anaphase, 80% D2O effectively slowed chromosome separation, prolonging anaphase for more than 60 min. Normal chromosome motion was restored when medium containing D2O was replaced with control medium. Mitotic chromosomes remained condensed throughout prolonged anaphase intervals. Immunofluoresence examination of spindles stained using a monoclonal anti-tubulin revealed no pronounced increase in microtubule polymerization after exposure of cells to 20-80% D2O.Deuterium oxide (D2O) was applied to PtK1 cells to assess its effect on mammalian mitosis. Cells exposed to culture medium containing up to 50% D2O were able to enter and complete mitosis, but the duration of mitosis was increased proportionally to the concentration of D2O applied. Cells exposed to 50% D2O showed increases of more than 300% for the interval between nuclear envelope breakdown and anaphase onset, and approximately 65% for the interval between anaphase onset and initial furrowing. At a concentration of 80%, D2O acted as an inhibitor of mitosis; after 8 h exposure to this concentration, cultures showed an increase in the proportion of mulinucleate cells and an absence of mitotic figures. When applied early in anaphase, 80% D2O effectively slowed chromosome separation, prolonging anaphase for more than 60 min. Normal chromosome motion was restored when medium containing D2O was replaced with control medium. Mitotic chromosomes remained condensed throughout prolonged anaphase intervals. Immunofluoresence examination of spindles stained using a monoclonal anti-tubulin revealed no pronounced increase in microtubule polymerization after exposure of cells to 20-80% D2O.

Pdr5-mediated multidrug resistance requires the CPY-vacuolar sorting protein Vps3: are xenobiotic compounds routed from the vacuole to plasma membrane transporters for efflux?

In Saccharomyces cerevisiae several members of the ATP-binding cassette transporter superfamily efflux a broad range of xenobiotic substrates from cells. The vacuole also plays a critical role in multidrug resistance. Mutations in genes such as VPS3 that are essential for vacuolar acidification and carboxypeptidase Y vacuolar protein-sorting are multidrug sensitive. A similar phenotype is also observed with deletions of VPS15, VPS34, and VPS38, which encode essential members of the carboxypeptidase Y vacuolar protein–sorting pathway. Prior to the work described herein, detoxification by transporters and the vacuole were presumed to function independently. We demonstrate that this is not the case. Significantly, Vps3 has an epistatic relationship with Pdr5, a major yeast multidrug transporter. Thus, a double pdr5, vps3 deletion mutant is no more multidrug sensitive than its isogenic single-mutant counterparts. Subcellular fractionation experiments and analysis of purified plasma membrane vesicles indicate, however, that a vps3 mutation does not affect the membrane-localization or ATPase activity of Pdr5 even though rhodamine 6G efflux is reduced significantly. This suggests that Vps3 and probably other members of the carboxypeptidase Y vacuolar protein–sorting pathway are required for relaying xenobiotic compounds to transporters in the membrane.

Fluorescence microscopy of etched methacrylate sections improves the study of mitosis in plant cells

Etched sections of methacrylate infiltrated plant tissue [Gubler (1989) Cell Biol. Int; Rep., 13:137–145; Baskin et al. (1992) Planta, 187:405–413] offer many advantages over the more traditional squash technique of Wick et al. [(1981) J. Cell Biol. 89:685–690] for immunofluorescence microscopic investigation of the plant cytoskeleton, especially during mitosis. These advantages include: (1) unimpeded access of antibody probes, (2) confocal‐like imaging without the expense of confocal equipment, (3) maintenance of organ architecture as well as intracellular structure, (4) the ability to independently examine separate focal planes with the same or multiple antibody(s) or other labelling compounds, and (5) the ability to archive unetched sections, polymerized or non‐polymerized infiltrated tissue. In this paper examples of staining of various microtubule cytoskeletal and mitotic proteins are shown in a variety of methacrylate embedded plant tissues. Microsc. Res. Tech. 40:369–376, 1998.

Immunofluorescence assay of hybridoma supernatants

Methods are described for performing immunofluorescence assay using cultured mammalian cells as target specimens. This procedure uses slides that allow processing of 12 specimens as a unit. Avidin-biotin is employed to provide increased sensitivity.