Research on ticks and tick-borne pathogens in Missouri--an interim research report.
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Animals↗
Biomedical subjects
R D Hall
Publications and source records attributed to R D Hall.
Asymmetric somatic cell hybridization in plants. I. The early effects of (sub)lethal doses of UV and gamma radiation on the cell physiology and DNA integrity of cultured sugarbeet (Beta vulgaris L.) protoplasts.
An investigation into the possible application of UV radiation as a pretreatment for the donor cells in asymmetric plant cell hybridization protocols has been carried out. A comparison was made between the effects of UV doses in the range 700-4200 J/m2 and those of 60Co gamma radiation over the range 0.15-1 kGy on Beta vulgaris suspension cell protoplasts. The investigation had two aspects. Firstly, alterations to cell physiology (cell wall resynthesis, viability, division and colony formation) in irradiated protoplasts were examined during a 4-week culture period. Results have indicated that a dose of 700 J/m2 UV is necessary to prevent further cell division and colony formation in these cells. A dose of 0.15 kGy gamma radiation generally prevented colony formation, although some early cell division did occur (as was also observed even after 0.45 kGy had been applied). Membrane integrity, as measured after 6 days, using fluorescein diacetate staining, was not affected by either treatment within the dose ranges applied. Secondly, denaturing (alkaline) gel electrophoresis, in association with a pulsed field gel DNA preparation technique, was used to determine the degree of in vivo DNA damage following the radiation treatments. After UV radiation, considerable fragmentation of the DNA was observed, the extent of which was dose-dependent. Gamma radiation, however, appeared to result in fewer DNA lesions, with only the 1 kGy treatment revealing a pattern significantly altered from that of the control. These results augur well for the potential use of UV radiation in asymmetric fusion experiments.
Cell Fusion↗
Asymmetric somatic cell hybridization in plants. II. Electrophoretic analysis of radiation-induced DNA damage and repair following the exposure of sugarbeet (Beta vulgaris L.) protoplasts to UV and gamma rays.
As part of an investigation into whether it would be possible to use UV radiation as a suitable pretreatment of the donor cells in asymmetric hybridization experiments, the effects of this treatment on sugarbeet (Beta vulgaris L.) protoplast DNA have been determined and compared with those of gamma radiation. Both nuclear and mitochondrial DNAs have been examined. The dose ranges chosen had previously been determined to be potentially applicable for fusion experiments. Pulsed field gel electrophoresis and standard agarose gel electrophoresis have been used in combination with laser scanning densitometry to gain an insight into the precise nature and degree of DNA damage resulting from irradiation. It was observed that UV radiation introduced substantial modifications to sugarbeet DNA. Double-strand breaks were detected, the number of which was found to be directly proportional to the dose applied. Such breaks indicate that UV radiation results in substantial chromosome/chromatid fragmentation in these cells. Chemical modifications to the DNA structure could be revealed by a significant reduction in DNA hybridization to specific mitochondrial and nuclear DNA probes. Following gamma irradiation at equivalent biological doses (i.e. those just sufficient to prevent colony formation) much less damage was detected. Fewer DNA fragments were produced indicating the presence of fewer double-strand breaks in the DNA structure. In comparison to UV treatments, DNA hybridization to specific probes following gamma radiation was inhibited less. For both treatments, mitochondrial DNA appeared more sensitive to damage than nuclear DNA. The possibility that DNA repair processes might account for these differences has also been investigated. Results indicate either that repair processes are not involved in the effects observed or that DNA repair occurs so fast that it was not possible to demonstrate such involvement with the experimental system used. The general relevance of such processes to asymmetric cell hybridization is discussed.
Cell Fusion↗
Research on ticks and tickborne pathogens in Missouri--an interim research report.
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Animals↗
The biphotonic photoionization of chlorpromazine during conventional flash photolysis: spin trapping results with 5,5-dimethyl-1-pyrroline-N-oxide.
A novel combination of conventional flash photolysis and electron spin resonance (ESR) spin-trapping has been used to demonstrate that photoionization of chlorpromazine (CPZ), and the concomitant production of hydrated electron, occurs through a stepwise biphotonic mechanism during conventional flash photolysis at wavelengths above 290 nm. The production of hydrated electron in the flash photolysis experiment has been monitored and quantified through the use of the spin trapping agent, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The effects of nitrous oxide, varying concentrations of CPZ and DMPO, and a range of flash intensities on the ESR spectra of the observed spin adducts of DMPO are discussed. The use of ESR spin trapping to monitor hydrated electron yields in flash photolysis experiments has the potential to permit the use of a much wider range of flash intensities than is typically possible with conventional optical experiments. Thus, there is a greater possibility of distinguishing between monophotonic and biphotonic processes.
Chlorpromazine↗
Detection of singlet molecular oxygen during chloride peroxidase-catalyzed decomposition of ethyl hydroperoxide.
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Chloride Peroxidase↗
Estimation of surviving spiral ganglion cells in the deaf rat using the electrically evoked auditory brainstem response.
A procedure was developed to record the electrically evoked auditory brainstem response (EABR) in the rat with sufficiently little stimulus artifact to permit systematic measurements of the first positive wave (P1), the compound action potential (CAP) of the auditory nerve. Our principal aim was to verify the theoretical prediction that maximum P1 amplitude is directly proportional to the number of excitable auditory nerve fibers. This was carried out in animals with graded lesions of the spiral ganglion induced by perfusion of the cochlea with different concentrations of neomycin. Two series of observations confirmed the theoretical prediction. Several measures of P1, including maximum amplitude, and slopes of the P1 and P1-N1 growth functions, were highly correlated with the number of surviving spiral ganglion cells. Correlation coefficients (r) ranged from 0.75 to 0.92. Amplitudes of the later waves exhibited much lower correlations with spiral ganglion cell counts. These findings suggest that measurement of the CAP in deaf humans, possibly as wave I of the EABR, should provide quantitative information about the status of the nerve, which could be useful in screening candidates for cochlear implants, prescribing the optimum device for individual patients, and determining how benefits derived from such devices relate to the condition of the auditory nerve.
Animals↗
Estimation of surviving spiral ganglion cells in the deaf rat using the electrically evoked auditory brainstem response.
A procedure was developed to record the electrically evoked auditory brainstem response (EABR) in the rat with sufficiently little stimulus artifact to permit systematic measurements of the first positive wave (P1), the compound action potential (CAP) of the auditory nerve. Our principal aim was to verify the theoretical prediction that maximum P1 amplitude is directly proportional to the number of excitable auditory nerve fibers. This was carried out in animals with graded lesions of the spiral ganglion induced by perfusion of the cochlea with different concentrations of neomycin. Two series of observations confirmed the theoretical prediction. Several measures of P1, including maximum amplitude, and slopes of the P1 and P1-N1 growth functions, were highly correlated with the number of surviving spiral ganglion cells. Correlation coefficients (r) ranged from 0.75 to 0.92. Amplitudes of the later waves exhibited much lower correlations with spiral ganglion cell counts. These findings suggest that measurement of the CAP in deaf humans, possibly as wave I of the EABR, should provide quantitative information about the status of the nerve, which could be useful in screening candidates for cochlear implants, prescribing the optimum device for individual patients, and determining how benefits derived from such devices relate to the condition of the auditory nerve.
Animals↗
Spectroscopic studies of cutaneous photosensitizing agents--XV. Anthralin and its oxidation product 1,8-dihydroxyanthraquinone.
The photochemistry (Type I and II) of anthralin and its photo-oxidation product 1,8-dihydroxyanthraquinone (1,8-DHAQ) has been studied in ethanol, acetonitrile and dimethylsulfoxide using spin-trapping and direct detection of singlet oxygen (1O2) luminescence techniques. In ethanol, where it exists in its neutral form (AN), anthralin does not undergo either Type I or II reactions upon UV-irradiation. In contrast, irradiation of anthralin in acetonitrile, a solvent in which anthralin is partially converted to its corresponding mono-anion (AN-), generates both superoxide and singlet oxygen. Irradiation of anthralin in dimethylsulfoxide, where the AN- form is present in substantial quantity, generates superoxide and solvent derived radicals but no detectable singlet oxygen. UV-irradiation of 1,8-DHAQ in ethanol and acetonitrile produces both superoxide and singlet oxygen in significant yields. In dimethylsulfoxide, on the other hand, only superoxide and solvent derived radicals are observed. The 1O2 quantum yield for AN- and 1,8-DHAQ in acetonitrile were determined to be 0.14 and 0.88 relative to rose bengal in the same solvent. These findings suggest that the AN photosensitization occurs via Type I and II pathways, is solvent dependent and involves AN- as well as its oxidation product 1,8-DHAQ, which is a more potent generator of both singlet oxygen and superoxide.
Anthralin↗
Detection of singlet (1O2) oxygen phosphorescence during chloroperoxidase-catalyzed decomposition of ethyl hydroperoxide.
Evidence for the production of singlet molecular oxygen (1O2) during the chloroperoxidase-catalyzed decomposition of ethyl hydroperoxide has been obtained through the use of optical spectroscopy, oxygen electrode experiments, and electron spin resonance (ESR). ESR spin-trapping experiments with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) demonstrate the production of the ethyl peroxyl free radical during the chloroperoxidase/ethyl hydroperoxide reaction. Oxygen and acetaldehyde concentrations suggest that the production of ethyl peroxyl radicals constitutes less than 2% of the decomposition of ethyl hydroperoxide at the concentrations of reactants used. The phosphorescence of 1O2 at 1268 nm was observed during the chloroperoxidase-catalyzed decomposition of ethyl hydroperoxide in deuterium oxide buffer. Chloroperoxidase also catalyzes the decomposition of tert-butyl hydroperoxide to its corresponding peroxyl radical. Alkoxyl and alkyl-DMPO spin adducts were also detected. A much lower yield of 1O2 phosphorescence was observed during the chloroperoxidase-catalyzed decomposition of tert-butyl hydroperoxide. This phosphorescence probably arises through secondary production of alkyl peroxyl radicals. These results suggest that the initial enzyme-dependent production of ethyl peroxyl radicals is followed by enzyme-independent reaction of two peroxyl radicals through the tetroxide intermediate, as originally proposed by Russell (Russell, G. A. (1957) J. Am. Chem. Soc. 79, 3871-3877), to form acetaldehyde, ethyl alcohol, and molecular oxygen.
Chloride Peroxidase↗
Walk-through trap for control of horn flies (Diptera: Muscidae) on pastured cattle.
A walk-through fly trap designed in 1938 by W. G. Bruce was tested for two field seasons in Missouri. Screened elements along both sides of the device functioned as cone traps, thereby catching horn flies, Haematobia irritans (L.), as they were swept from cattle by strips of carpet hung from the roof. Horn fly control on pastured cattle averaged 54 and 73% when they were afforded access to the trap. Analyses of Diptera captured in the trap indicated that horn flies comprised the most abundant species; face flies, Musca autumnalis De Geer, stable flies, Stomoxys calcitrans (L.), and others were present in smaller numbers. Cattle were not reluctant to use the trap, and no structural problems were observed during the experiment.
Animals↗
The stepwise biphotonic photoionization of chlorpromazine as seen by laser flash photolysis.
It is generally accepted that both promazine (PZ) and chlorpromazine (CPZ) photionize monophotonically to their respective cation radicals and the corresponding hydrated electrons. It is also supposed that this photoinization has a role in the phototoxic effects of these drugs. However, using laser flash photolysis, we have observed that photoionization of CPZ during S1 excitation (lambda greater than 300 nm) is a stepwise biphotonic process. In the case of PZ our flash photolysis results are less clearcut, but are consistent with stepwise biphotonic photoionization for S1 excitation. We demonstrate, using computer simulation of the intramolecular kinetics, that the estimated triplet state lifetime of CPZ is sufficiently long (23 ns at room temperature) to account for the apparent monophotonic photoionization that has been observed by others at high light intensities and short pulse times. Our laser flash photolysis results also suggest that the photo-ionization mechanism of PZ and CPZ is wavelength-dependent. Both drugs exhibit apparent monophotonic photoionization when they are excited at 266 nm under conditions of laser pulse width and intensity similar to those at 355 nm. We suggest that photoionization is not an important mechanism in the observed phototoxic and photoallergic effects of PZ and CPZ in sunlight.
Chlorpromazine↗
A fluorescent indicator for measuring cytosolic free magnesium.
The previously developed chelator O-aminophenol-N,N,O-triacetic acid (APTRA) (L. A. Levy, E. Murphy, B. Raju, and R. E. London. Biochemistry 27: 4041-4048, 1988) has been modified to yield a fluorescent analogue which can be utilized as an intracellular probe for ionized Mg2+. The fluorescent analogue, FURAPTRA, with a magnesium dissociation constant of 1.5 mM, is structurally analogous to the calcium chelator fura-2 and exhibits a similar excitation shift on magnesium complexation. Hence, data on the intracellular Mg2+ concentration can be obtained using an analogous ratio method. The acetoxymethyl form of the chelator is readily loaded into cells and has been used to determine a cytosolic free Mg2+ concentration of 0.59 mM for isolated rat hepatocytes. As a consequence of the relatively high levels of cytosolic Mg2+, the problem of ion buffering is much less severe than for the analogous calcium indicators.
Animals↗
Direct observation of singlet oxygen production by merocyanine 540 associated with phosphatidylcholine liposomes.
The production of singlet molecular oxygen (1O2) by the photosensitizing dye merocyanine 540 (MC540) bound to phosphatidylcholine liposomes has been demonstrated by direct detection of 1O2 luminescence at 1268 nm. 1O2 phosphorescence emission was enhanced in deuterated buffer and upon saturation of the sample with oxygen and could be quenched by the addition of sodium azide to the external medium. No 1O2 luminescence was detected in nitrogen-saturated samples, in the absence of dye, or with MC540 in aqueous solution. Photobleaching of liposome-bound MC540 was also observed to be dependent on oxygen concentration. These studies are consistent with 1O2 intermediacy in the mechanism of MC540-mediated photosensitization.
Dimyristoylphosphatidylcholine↗
Superoxide, hydrogen peroxide and singlet oxygen in hematoporphyrin derivative-cysteine, -NADH and -light systems.
Hematoporphyrin derivative and light in the presence of cysteine or glutathione were found to convert oxygen to superoxide and hydrogen peroxide at pH less than approx. 6.5, while at pH greater than 6.5 no superoxide or hydrogen peroxide production was observed. However, at pH values greater than 6.5 the rate of oxygen consumption increased. This rate paralleled the acid dissociation curve of the cysteine thiol group and is consistent with the chemical quenching of 1O2 by cysteine. The superoxide and hydrogen peroxide formation observed below pH 6.5 appeared not to be related to the singlet oxygen production of hematoporphyrin derivative. In addition, superoxide and hydrogen peroxide production was observed with hematoporphyrin derivative and light in the presence of NADH, both above and below pH 6.5. Direct detection of singlet oxygen luminescence at 1268 nm in the hematoporphyrin derivative-light system (2H2O as solvent) revealed an apparent linear increase in the singlet oxygen emission intensity as the p2H was raised from 7.0 to 10.0. Azide efficiently quenched this observed emission. In addition, at p2H 7.4, 1 mM cysteine resulted in a 40% reduction of the singlet oxygen luminescence, while at p2H 9.4 the signal was quenched by over 95% (under the experimental conditions employed). In total, we interpret these results as consistent with the chemical quenching of 1O2 by the ionized thiol group of cysteine.
Cysteine↗
Singlet oxygen intermediacy in the photodynamic action of membrane-bound hematoporphyrin derivative.
The cell-damaging photochemistry of hematoporphyrin derivative (HPD) has been investigated using isolated erythrocyte membranes as a test system. Irradiation of membranes in the presence of the tumor-localizing fraction of HPD resulted in formation of singlet molecular oxygen (1O2) as measured by the phosphorescence at 1268 nm. The authentic product of 1O2 attack on cholesterol, 3 beta-hydroxy-5 alpha-cholest-6-ene-5-hydroperoxide, was identified in this system. Relatively insignificant amounts of free radical-derived hydroperoxides were detected. These results suggest that 1O2 plays a major role in the HPD-sensitized photokilling of tumor cells in vivo.
Cholesterol↗
ESR detection of endogenous ascorbate free radical in mouse skin: enhancement of radical production during UV irradiation following application of chlorpromazine.
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Animals↗
Near-infrared detection of singlet molecular oxygen produced by photosensitization with promazine and chlorpromazine.
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Chlorpromazine↗