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Biomedical subjects

W D Wright

Publications and source records attributed to W D Wright.

At least 19 recordsLinked to original sources
PubMed1992

Resolution of DNA topoisomerase II by two-dimensional polyacrylamide gel electrophoresis and western blotting.

Eukaryotic DNA Topoisomerase II (Topo II) has been studied using high-resolution two-dimensional polyacrylamide electrophoresis (2D-PAGE) and immunodetection of resolved proteins using specific antisera (Western blotting). Traditional methods of 2D-PAGE failed to resolve Topo II and neither nonequilibrium nor equilibrium pH gradients allowed Topo II to enter the first dimension gel. Exhaustive nuclease digestion and alternate protein solubilization strategies also produced negative results. We have developed altered first dimension pH gradient profiles and employed a more aggressive protein solubilization procedure which resulted in the resolution of Topo II. The 170-kDa polypeptide focuses with an apparent isoelectric point of approximately 6.5.

Blotting, Western↗
PubMed1992

Effects of radiation on TNF alpha-mediated cytolysis of cell lines derived from cervical carcinomas.

The effect of radiation, a primary mode of treatment for cervical malignancies, on the tumor necrosis alpha (TNF alpha)-mediated cytolysis of five cell lines derived from human cervical carcinoma cell lines (C-33 A, ME-180, HT-3, MS751, and SiHa) was analyzed. Results of this analysis showed that all of the cell lines were resistant to the cytolytic effects of TNF alpha. Although resistant when protein synthesis proceeds normally, ME-180, HT-3, MS751, and SiHa cells were sensitive to TNF alpha-mediated cytolysis in the presence of protein synthesis inhibitors. The cytolytic response of these cells to radiation was heterogeneous, with C-33 A cells being the most radiosensitive and SiHa cells being the least radiosensitive. The cell lines ME-180, MS751, and HT-3 were intermediate in their sensitivities to radiation. Because radiation is known to inhibit protein synthesis, the ability of radiation to enhance TNF alpha cytolytic activity was examined. The cell lines with intermediate sensitivities to radiation (ME-180, HT-3, and MS751) demonstrated statistically significant synergistic increases in cytolysis when exposed to TNF alpha in combination with radiation. Neither the radioresistant SiHa cell line nor the radiosensitive C-33 A cell line displayed increased cytolysis with increasing concentrations of TNF alpha at any dose of radiation. Possible mechanisms which may explain the synergy in ME-180, HT-3, and MS751 cells and lack of synergy in C-33 A and SiHa cells by TNF alpha and radiation are discussed.

Carcinoma, Squamous Cell↗
PubMed1992

The suppression of the synthesis of a nuclear protein in cells blocked in G2 phase: identification of NP-170 as topoisomerase II.

Previous studies of a nuclear protein of molecular weight 170 kDa (NP-170) have shown it to have two interesting properties. First, NP-170 synthesis began in mid- to late S phase and became maximal in G2 phase. Second, the synthesis of NP-170 was suppressed in cells blocked in G2 phase following irradiation with 6.8 Gy (J. M. Holland et al., Radiat. Res. 122, 197-208, 1990). The molecular weight of NP-170 is the same as that of Topoisomerase II (Topo II), an enzyme involved in the alteration of DNA supercoiling status with a double-strand passing function. This study was undertaken to determine whether NP-170 could be Topo II. The results from the present study show that both the proteins have identical cell cycle synthesis patterns. The synthesis of both these proteins is suppressed following irradiation. NP-170 was found to be recognized by a Topo II antibody in both Western blots and immunoprecipitation. This study characterizes NP-170 as Topo II.

Autoradiography↗
PubMed1991

Differences in the DNA supercoiling response of irradiated cell lines from ataxia-telangiectasia versus unaffected individuals.

In this study the manifestation of DNA damage at the nucleoid level was examined in several AT cell lines using an image analysis system to directly visualize and measure the changes in DNA loop size which occur when increasing concentrations of propidium iodide (PI) are used to titrate the DNA supercoiling response (the 'fluorescent halo assay'). This response consists of a relaxation (0.5-7.5 micrograms/ml PI) and rewinding phase (10-50 micrograms/ml PI), the latter of which is impaired by the presence of DNA strand breaks in irradiated cells. In addition to the inhibition of DNA rewinding seen immediately after irradiation at 0 degrees C, the supercoiling response of AT diploid fibroblasts indicated an increased amount of DNA unwinding compared to fibroblasts from unaffected individuals. This difference appeared to saturate, since the excess in DNA loop size over that seen in irradiated fibroblasts from unaffected individuals remained constant after 5 Gy. These results may reflect a greater instability of the DNA-nuclear matrix attachment points in irradiated AT fibroblasts. The DNA supercoiling response in irradiated transformed AT fibroblasts and AT lymphoblasts did not differ from that observed in unaffected cells of the same type. However, all of the immortalized cell lines (AT and unaffected) had inherently larger DNA loop sizes than diploid fibroblasts and exhibited excess unwinding after irradiation.

Ataxia Telangiectasia↗
PubMed1990

Flow cytometric methods for studying isolated nuclei: DNA accessibility to DNase I and protein-DNA content.

Two FCM methods utilizing isolated nuclei were described. A DNase I sensitivity assay, employing changes in binding and digestion kinetics of the enzyme as well as the binding of intercalating fluorochrome was used to observe structural changes of chromatin rendered by physical and chemical agents. A nuclear DNA-protein staining method was used to study changes in nuclear protein content, redistribution of populations in the cell cycle, and unbalanced growth, manifested as an extraordinary accumulation of nuclear protein brought about by physical and chemical perturbation.

Animals↗
PubMed1990

Fluorescent methods for studying subnuclear particles.

Fluorescence assays can be used to reveal molecular interactions through rapidly demonstrable particle-associated events. The additional fact that in many cases fluorescent particles may be analyzed on a per-event basis lends credence to such techniques as probes for biologically significant perturbations and their resolution. Perhaps more importantly, the sorting capability of the flow cytometer enables detailed study of these events in cells in relation to their positions in the cell cycle. Further studies on the effect of drugs and other modalities on the organization of the genome and the nuclear matrix should prove of interest because the interactions of chromatin and this subnuclear particle could be predictive of the state of DNA metabolism under such conditions. With the additional ability of following such organizational changes through the cell cycle, the mechanisms of reversal of perturbing events might be elucidated.

Cell Cycle↗
PubMed1990

Repair of radiation-induced DNA damage in thermotolerant and nonthermotolerant HeLa cells.

The effect of heat exposure on the repair of radiation-induced DNA damage which inhibits the ability of nuclear DNA to undergo supercoiling changes was studied using the fluorescent halo assay in thermotolerant and nonthermotolerant (normal) cells. The assay utilizes an intercalating, fluorescent dye to unwind and rewind endogenous DNA supercoils. When HeLa cells are exposed to 17.3 Gy radiation the ability of DNA to be rewound into supercoils is completely inhibited. However, the ability of DNA to rewind is 70% restored by 30 min after irradiation. Both thermotolerant and normal cells exposed to 45 degrees C for 30 min prior to irradiation had a rewinding ability intermediate between control and unheated cells, but there was no restoration of rewinding ability up to 3 h postirradiation. Thus, when irradiation immediately followed heating, there was no difference between thermotolerant and normal cells. However, when various time intervals were imposed between heating and irradiation, a difference in the ability of the cells to recover from heat-induced alterations became apparent. In normal cells after 6 h of postheat incubation the cells' ability to restore DNA supercoiling was approximately the same as that of control cells, while in thermotolerant cells only 2 h was required to repair the ability to restore supercoiling at the same rate. The rate of repair of DNA remained correlated with relative nuclear protein content as measured by fluorescein isothiocyanate staining in both thermotolerant and normal cells, indicating a possible relationship between the two.

Acclimatization↗
PubMed1990

Effects of irradiation on nuclear protein synthesis in G2 phase of the cell cycle.

A method was developed to determine the synthesis of nuclear proteins throughout the cell cycle which was resolved into six compartments on the basis of DNA and nuclear protein content (i.e., early and late G1, early and late S, etc). Using this technique cell-cycle-specific synthesis of certain nuclear proteins was observed. Of particular interest was a 170-kDa protein(s) whose synthesis was initiated in early S phase and reached a maximum rate in late G2. Following irradiation with 6.8 Gy of 137Cs gamma rays the synthesis of the 170-kDa protein(s) declined in the G2 population with near total inhibition seen by 24 h. Synthesis of the 170-kDa protein(s) appeared to be slightly enhanced, and the postirradiation inhibition of its synthesis was reversed, in the presence of 3 mM caffeine. Also, the synthesis of 55-kDa nuclear protein(s) was stimulated throughout the cell cycle in the presence of 3 mM caffeine. These observations suggest new possibilities regarding the mechanism of the X-ray-induced G2 block and its reversal by caffeine. However, the exact role of these nuclear proteins in cellular events remains to be ascertained.

Caffeine↗
PubMed1989

Flow cytometric studies of the nuclear matrix.

We have devised a method to measure the protein and nucleic acid content of the nuclear matrix using flow cytometry. Nuclear matrices were prepared from nuclei by DNase I digestion followed by 3 M NaCl extraction. The resulting particles were stained with fluorescein isothiocyanate (FITC) for protein and propidium iodide (PI) for double-stranded nucleic acids, and fluorescence as well as forward angle light scatter was detected. The matrices were also subjected to additional chemical or enzymatic perturbations, and changes in the above parameters were measured. Results showed that matrices from heat-shocked cells not only retained the majority of heat-induced excess nuclear protein, but also exhibited higher PI signals than controls after RNase A digestion. This observation did not hold if RNase A digestion preceded high-salt extraction, suggesting that a salt-extractable moiety had been replaced or altered by heat so that double-stranded RNA was protected from the nucleolytic attack. The residual PI fluorescence in matrices from heated cells bore a linear relationship to the increased protein content in those matrices, indicating that the excess protein sequesters matrix-associated RNA. Polyacrylamide gel electrophoresis of matrix polypeptides revealed increased amounts of many proteins as a result of heat as well as the appearance of several new proteins, one of which comigrates with the HSP72/73 heat-shock proteins. The results of these studies show that flow cytometry can be used to study the nuclear matrix and is capable of detecting changes that result from alterations in its protein composition.

Cell Nucleus↗
PubMed1989

Changes in the structure of nucleoids isolated from heat-shocked HeLa cells.

Using a technique to detect changes in DNA supercoiling which allows one to visualize both DNA unwinding and rewinding in presence of the intercalating dye, propidium iodide (PI), we showed that hyperthermic treatment (30 min at 45 degrees C) of HeLa S3 cells alters the response to the intercalating dye. Depending on the treatment conditions, we observed a reduction in the maximum size of the DNA loop that can be measured at the relaxation point (PI concentration 5-7.5 micrograms/ml). Cellular heating also affected all degrees of DNA rewinding (measured as a function of PI concentrations between 10 and 50 micrograms/ml). By 6 h after cellular heating these heat effects had disappeared. This time interval correlated with the time necessary for recovery from a heat-induced increase to normal nuclear and nucleoid protein content. Using gel electrophoresis we showed that the nucleoids (DNA plus nuclear matrix proteins) after heat exposure are enriched in several polypeptides and that there is a specific increase in HSP 72/73. We hypothesize that the altered response to the intercalating dye after cellular heat shock is due to an increase in polypeptides associated with the nuclear matrix thereby altering the DNA-nuclear protein matrix anchor points.

DNA, Superhelical↗
PubMed1989

DNA supercoiling changes in nucleoids from irradiated L5178Y-S and -R cells.

DNA supercoiling ability was assayed following irradiation in two cell lines of differing radiosensitivity, L5178Y-S (LY-S) and L5178Y-R (LY-R). Cells treated with NaCl and Triton X-100 were exposed to increasing concentrations of the fluorescent, DNA-intercalating dye, propidium iodide (PI), and the diameter of the resulting fluorescent halo of DNA was measured. As the PI concentration was increased from 0.5 to 5 micrograms/ml, halo diameter increased from 20-25 to 45-55 microns due to the unwinding of the DNA supercoils. This process was similar for both cell lines under all conditions studied. As the PI concentration was increased to 50 micrograms/ml, the halo rewound to a diameter of 25-30 microns in unirradiated cells from both lines. However, following exposure to 3-12 Gy of 137Cs gamma rays, the ability of the DNA to be rewound was inhibited in a dose-dependent manner. Rewinding inhibition was greater in LY-S cells than in LY-R cells. Since the induction of DNA damage (e.g., single-strand DNA breaks) appears to be the same for both cell lines, this result implies that a similar extent of damage results in a greater loss of topological constraints on the DNA loops in LY-S. Such a change might be related to the protein composition of the nucleoid cores. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that nucleoids from LY-S cells were missing a 55-kDa protein present in LY-R.

Animals↗
PubMed1988

The interaction of heat and radiation affecting the ability of nuclear DNA to undergo supercoiling changes.

DNA damage (putatively strand breaks) from ionizing radiation inhibits the ability of intercalating dyes to induce right-handed supercoils in the DNA loops of HeLa nucleoids [Cook and Brazelle, J. Cell Sci. 22, 287-302 (1976); Roti Roti and Wright, Cytometry 8, 461-467 (1987)] while heat-induced changes in the nuclear matrix enhance this ability [Roti Roti and Painter, Radiat. Res. 89, 166-175 (1982)]. Since heat and radiation interact synergistically or additively on most cellular functions which they affect, the rewinding of DNA supercoils is unusual in that these agents alone affect it in an antagonistic manner. When HeLa cells were exposed to 45 degrees C for 30 min and immediately irradiated with 10 Gy of 137Cs gamma rays, the rewinding response was intermediate between that for cells which had been exposed to 10 Gy only and control. When repair of this damage was assayed in control cells, 97% of the initial damage had been repaired at 30 min postirradiation; at the same time only 10% of the initial damage had been repaired in the heat-shocked cells. This apparent dose reduction effect and the inhibition of repair were interpreted to indicate that heat-induced changes in nuclear structure were masking DNA damage from the assay and the repair system. These effects correlated with the amount of heat-induced excess protein associated with the nucleus and the nucleoid.

DNA Damage↗
PubMed1987

Visualization of DNA loops in nucleoids from HeLa cells: assays for DNA damage and repair.

An assay for visualization of DNA loops undergoing supercoiling changes has been developed. The assay utilizes the fluorescent dye, propidium iodide (PI), which intercalates into the DNA and under the proper conditions causes the supercoiling status of the DNA to change. Thus, the DNA can be seen as a fluorescent halo that changes diameter with PI concentration. At low PI concentrations (0-7.5 micrograms/ml) the supercoils are relaxed with increasing PI, while at higher PI concentrations (7.50-50 micrograms/ml) supercoils in the opposite winding sense are rewound with increasing PI. When HeLa cells were irradiated with 1-20 Gy of 137Cs gamma-rays, the ability to rewind the DNA supercoils was inhibited in a dose-dependent manner, presumably because of the presence of radiation-induced DNA strand breakage, which removed the topological constraints on the DNA loops. These lesions were repaired rapidly during post-irradiation incubation. The ability of the DNA loops to be rewound was restored within 8 min after 10 Gy of gamma-irradiation, such that no difference from control cells could be detected. The half-time for repair of the radiation-induced lesions that inhibit DNA rewinding was similar to that for repair of DNA single strand breaks. The assay has certain advantages over current methods for assaying DNA damage in that it involves measurement of single cells and it does not require the DNA to be labeled with radioactive precursors.

DNA↗
PubMed1986

The colour-matching functions and visual research.

The following points are examined: the problem of how to define the colour of a stimulus used in visual research; the limited relation of the colour-matching functions to the spectral sensitivity curves of the individual colour receptors in the retina; and the case for abandoning luminance as a colorimetric variable and lightness as a colour appearance variable.

Color↗
PubMed1985

DNase I sensitivity of nuclear DNA measured by flow cytometry.

The DNase I digestion kinetics of DNA in isolated nuclei (from HeLa or murine mammary carcinoma, 67 cells) were assayed flow cytometrically by measuring the changes in ethidium bromide (EtBr) fluorescence following various digestion time intervals. The DNase I digestion curve was characterized by an initial 25-30% increase in fluorescence upon addition of the enzyme, a rapid reduction in fluorescence to approximately 50-55% in 30 minutes, and a limit digest of 45-50% beyond 45 minutes. Throughout digestion, the DNA histogram retained its characteristic bimodal shape, showing that histogram rearrangement was not responsible for the changes in EtBr fluorescence. Irradiation with 5 X 10(6) rads (137Cs-gamma-rays) or exposure to 50 mM EDTA caused an increase in EtBr fluorescence similar to that caused by DNase I, suggesting that DNA nicking and/or chromatin loosening were responsible for this increase. Residual DNA assayed by the solubilization of 14C-TdR (thymidine)-labeled DNA indicated a similar kinetic pattern without the initial increase. However, at the limit digest, the fraction of DNA remaining trichloroacetic acid (TCA) insoluble (10%) was smaller than that measured by loss of EtBr fluorescence (50% of initial, 40% of maximum). Part of this difference was due to the presence of TCA soluble DNA trapped within the nuclear matrix (15-20%). This trapped DNA was released when the digested nuclei were exposed to 0.5-1.0 M NaCl just prior to EtBr staining. Exposure of HeLa cells to three agents that are believed to cause changes in chromatin structure resulted in alterations in the DNase I digestion kinetics measured flow cytometrically.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1977

The fundamentals of color perception. Charles F. Prentice Award Lecture--1977.

The author raises the question, "Suppose a newly qualified optometrist announced that he wanted to undertake research on the perception of color; what fundamental elements of the subject should he attempt to master before setting out on his research program?" In answering this question, the author traces his own development in the subject of color vision.

Awards and Prizes↗
PubMed1977

A Goldmann perimeter with high luminance chromatic targets.

We have modified the original Goldmann perimeter by replacing the source and shutter by a Xenon arc source with maximum target luminance of 3183 cdm-2, and a quartz iodine lamp yielding maximum background luminance of 150 cdm-2, both measured at the bowl. Colorimetric and spectrophotometric specifications of the new colour targets are comparable to the original, but give luminance intensities equivalent to 318 cdm-2: levels only realized for the white target in the original system. Chromatic profiles are comparable to previous profiles for achromatic targets and the close correspondence between the retinal threshold gradients produced by the two methods simplifies comparison of data obtained with out instrument with those obtained from routine static perimetry. Under high luminance conditions we do not find the relative foveal scotoma for blue targets observed by other researchers. The use of photometrically equated targets well withi; the photopic levels is an improvement on present methods of colour perimetry.

Color↗