Interferon production in postviral fatigue syndrome.
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Child↗
Biomedical subjects
M Fry
Publications and source records attributed to M Fry.
Rabbit liver factor D, a poly(thymidine) template stimulatory protein of DNA polymerases: purification and characterization.
Factor D, a DNA binding protein that enhances the activities of diverse DNA polymerases with a common restricted set of templates, was initially characterized in mouse liver but has resisted extensive purification. In this paper, we report that a similar stimulatory activity can be obtained in highly purified form from nuclei of rabbit hepatocytes. The rabbit liver protein increases the rates at which several DNA polymerases copy sparsely primed natural DNA templates and primed synthetic poly(dT), but it has no effect on the rates of copying of activated DNA or of poly(dG), poly(dA), and poly(dC). Direct binding of the purified stimulatory protein to an oligomer that contains a (dT)16 base stretch is visualized by retardation of the nucleoprotein complex on nondenaturing electrophoretograms. In the presence of the enhancing factor, Michaelis constants, Km, of responsive polymerase for singly primed bacteriophage M13 DNA and for poly(dT), but not for poly(dA), are decreased. Product analysis of M13 DNA primer extension indicates that the rabbit factor augments the apparent processivity of DNA polymerase by decreasing the extent of enzyme pausing at a tract of four consecutive thymidine residues in the template. Gel filtration of the native stimulatory protein yields an apparent relative molecular size of 58 +/- 2 kilodaltons. Stimulatory activity is readily inactivated by heat or by trypsin digestion, but it is resistant to micrococcal nuclease, N-ethyl-maleimide, or calcium ions.
Animals↗
Factor D is a selective single-stranded oligodeoxythymidine binding protein.
Factor D, a protein purified from rabbit liver that selectively enhances traversal of template oligodeoxythymidine tracts by diverse DNA polymerases, was examined for the sequence specificity of its binding to DNA. Terminally [32P]-labeled oligomers with the sequence 5'-d[AATTC(N)16G]-3', N being dT, dA, dG, or dC, were interacted with purified factor D and examined for the formation of protein-DNA complexes that exhibit retarded electrophoretic mobility under nondenaturing conditions. Whereas significant binding of factor D to 5'-d[AATTC(T)16G]-3' is detected, there is no discernable association between this protein and oligomers that contain 16 contiguous moieties of dG, dA, or dC. Furthermore, factor D does not form detectable complexes with the duplexes oligo(dA).oligo(dT) or poly(dA).poly(dT). The preferential interaction of factor D with single-stranded poly(dT) is confirmed by experiments in which the polymerase-enhancing activity of this protein is protected by poly(dT) against heat inactivation two- and four-fold more efficiently than by poly(dA) or poly(dA).poly(dT), respectively.
Animals↗
Leiomyoma of the female urethra.
Smooth muscle tumors of the female urethra are uncommon lesions. Only a few cases of leiomyoma of the female urethra have been reported in the literature. We describe 2 additional cases, review the literature on this rare neoplasm and discuss its management.
Adult↗
Basic radiobiology.
Experimental studies of the biological effects of radiation were started soon after the discoveries of x-rays in 1895, but there is still much that is not known. This article includes some research objectives that are essentially pragmatic in nature, intended to support and improve the current practice of radiotherapy, but the central thrust is the understanding of the mechanisms involved in the biological effects of radiation at the cellular and molecular levels. The article was written by a consortium of scientists and suffers inevitably from the drawback that writing styles are inconsistent, and coverage is not uniform. However, it benefits from the enormous advantage that it reflects the accumulated wisdom and judgment of more than a dozen scientists who, in their own areas of expertise, are recognized as being at the cutting edge of radiation research. The niceties of style and syntax are sacrificed in favor of the quality of the science and the maturity of judgment. The study of DNA damage as a mechanism for cell injury in early- and late-responding tissues, as well as a comparison of DNA damage that leads to lethality, as opposed to transformation and mutagenesis, are key items. The study of cell lethality with cells in culture led to the identification of repair, both sublethal and potentially lethal, as well as the dose-rate effect, and has had a considerable impact on radiotherapy. Future studies should focus on understanding the factors that determine radiosensitivity/radioresistance. A variety of approaches are available, including the study of genetically deficient cell lines from cancer-prone individuals. A parallel approach is the application of the techniques of molecular biology to clone the repair genes in mammalian cells, and to understand genetic defects that alter gene regulation, or to regulate biochemical factors in the cell. Substantial progress has been made in developing in vitro assays for mutagenesis, particularly using hybrids of rodent and human cells. Better methods are needed to study the effects of mutation on gene expression, and sensitive systems are needed that can detect low doses of radiation. Assays of oncogenic transformation, the in vitro counterpart of carcinogenesis, have been used to investigate the oncogenic potential of various types of radiation and chemotherapy agents. Key topics in future will include the investigation of supra-additivity between different agents, the identification and characterization of oncogenes that may be activated by radiation, the development of quantitative assays based on human cells, and further studies involving cell-to-cell communication.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
The DNA sequence specificity of stimulation of DNA polymerases by factor D.
The mechanism of enhancement of DNA polymerase activity by the murine DNA-binding protein factor D was investigated. Extension by Escherichia coli DNA polymerase I and calf thymus DNA polymerase-alpha of 5'-32P-labeled oligodeoxynucleotide primers that are complementary to poly(dT) or to bacteriophage M13 DNA was measured in the absence or presence of factor D. With 5'-[32P](dA)9.poly(dT), factor D enables E. coli polymerase I to fill approximately 15-nucleotide gaps between adjacent primers; whereas in the absence of the stimulatory protein, poly(dT) is not copied significantly. In order to study the nucleotide specificity of synthesis enhancement, we used M13mp10 DNA containing 4 consecutive thymidine residues downstream from the 3-hydroxyl terminus of an oligonucleotide primer. Upon addition of factor D, both polymerase I and polymerase-alpha can traverse this sequence more efficiently and thus generate longer DNA products. Densitometric analysis of nonextended and elongated 5'-32P-labeled M13 primer indicates that, without changing the frequency of primer utilization, factor D enhances the activity of these DNA polymerases by increasing their apparent processivity. By positioning oligonucleotide primers 4, 8, and 12 bases upstream from the (dT)4 template sequence, we show that the enhancement of synthesis by factor D is independent of the position of the oligothymidine cluster. We hypothesize that factor D interacts with oligo(dT).oligo(dA) domains in DNA to alter their conformation, which may normally obstruct the progression of DNA polymerases.
Animals↗
Template-selective stimulation of diverse DNA polymerases by the murine DNA-binding protein factor D.
Factor D, a template-selective DNA polymerase-alpha stimulatory protein from mouse liver (Fry, M., Lapidot, J., and Weisman-Shomer, P. (1985) Biochemistry 24, 7549-7556) is shown here to enhance the activities of diverse DNA polymerases with a cognate template specificity. DNA synthesis catalyzed by Escherichia coli DNA polymerase I, avian myeloblastosis virus polymerase, and some mammalian alpha- and gamma-polymerases was increased by factor D. With every enhanced polymerase, factor D increased the rate of copying of only poly(dT) among various tested synthetic poly-deoxynucleotides. Of the natural DNA templates examined, rates of copying of sparsely primed denatured DNA and of singly primed circular phi X174 or M13 bacteriophage DNA, but not of activated DNA, were enhanced. Michaelis constants (Km) of affected templates with responsive polymerases were decreased by factor D, without alteration in maximum velocity (Vmax). By contrast, factor D increased Vmax of deoxyribonucleoside 5'-monophosphate incorporation without changing Km of deoxyribonucleoside 5'-triphosphate substrates. Binding of factor D to poly(dT), poly(dA).poly(dT), and DNA, but less to poly(dA), was indicated by specific retention of their complexes on a DEAE-cellulose column. That factor D does not bind to DNA polymerase-alpha or to its complex with the DNA template was demonstrated by the failure of the factor to be coprecipitated with alpha-polymerase by anti-polymerase-alpha monoclonal antibodies in either the absence or presence of various templates. Lack of binding of factor D to the polymerase molecule was also indicated by simultaneous maximum stimulation of two competing polymerases by a limiting amount of factor. These combined results suggest that the enhancement of DNA synthesis is exerted through interaction of factor D with the template. It is proposed that this association leads to a tighter binding of the polymerase to the template and facilitates DNA synthesis.
Animals↗
Hydrogen peroxide production in uncoupled mitochondria of the parasitic nematode worm Nippostrongylus brasiliensis.
1. Mitochondria from the parasitic nematode worm Nippostrongylus brasiliensis produce H2O2 in the energized state; higher rates of H2O2 production were observed in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone. 2. Antimycin A inhibits respiration and H2O2 production by 70 and 65% respectively; the residual activities can be attributed to alternative electron-transport pathway(s). 3. o-Hydroxydiphenyl and 1,3,5-trihydroxybenzene, inhibitors of alternative electron transport, inhibit respiration by 37% and H2O2 production by 26%. 4. Another inhibitor of alternative electron transport, salicylhydroxamic acid, shows a complex mode of action; low concentrations (less than 0.5 mM) stimulate respiration and H2O2 production, whereas 2 mM-salicylhydroxamic acid inhibited respiration by 35% and stopped H2O2 production completely. 5. O2 thresholds were observed for the inhibition of respiration at O2 concentrations greater than 57.7 microM and inhibition of H2O2 production (greater than 20.5 microM-O2); apparent Km values for oxygen were 5.5 microM and 3.0 microM respectively. 6. In the presence of antimycin A the O2-inhibition thresholds and apparent Km values for O2 of respiration and H2O2 production matched closely, suggesting that the alternative oxidase is a likely site of H2O2 production. 7. These results are discussed in relation to O2 toxicity to N. brasiliensis.
Animals↗
Effects of inhibitors on the oxygen kinetics of Nippostrongylus brasiliensis.
Endogenous respiration of the parasitic nematode Nippostrongylus brasiliensis and the succinate oxidase activity of isolated mitochondria were partially inhibited by antimycin A; the remaining respiratory activity was sensitive to salicylhydroxamic acid (SHAM). Sub-millimolar concentrations of SHAM markedly stimulated respiration by 60% in whole N. brasiliensis and isolated mitochondria; stimulation by SHAM was not observed in the presence of antimycin A. Little change in the relative fluxes of electrons through the classical, antimycin A-sensitive pathway and the alternative SHAM sensitive pathway was observed between low and high O2 concentrations; this may suggest that the O2 affinities of both pathways are similar. O2 dependence of respiration showed O2 thresholds above which respiration decreases; in the absence of inhibitors whole N. brasiliensis and isolated mitochondria had threshold values around 60 microM O2. Increased O2 threshold values were observed in the presence of SHAM and antimycin A. The apparent Km values for O2 of whole N. brasiliensis and isolated mitochondria were 31 +/- 2 microM O2 and 3.5 +/- 0.2 microM O2 respectively; this difference in apparent Km values may reflect the presence of O2 gradients in the whole worm. The Km and O2 inhibition threshold values observed for whole N. brasiliensis are in good agreement with the proposed range of O2 concentrations thought to exist within the worm's natural environment. H2O2 production was detected in respiring uncoupled mitochondria, but H2O2 could not be detected in the medium surrounding whole N. brasiliensis. SHAM-stimulated respiration was accompanied by increased H2O2 production which was prevented by the addition of antimycin A.
Animals↗
DNA polymerase-alpha: enzymology, function, fidelity, and mutagenesis.
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Animals↗
A DNA template recognition protein: partial purification from mouse liver and stimulation of DNA polymerase alpha.
A protein that specifically enhances up to 13-fold the rate of copying of poly(dT) template by DNA polymerase alpha was partially purified from chromatin of regenerating mouse liver cells. This stimulatory protein, designated herein factor D, also increases 2-3-fold the activity of polymerase alpha with heat-denatured DNA and with primed, circular single-stranded phi X174 DNA. However, factor D has no detectable effect on the copying by polymerase alpha of poly(dG), poly(dA), and poly(dC) templates. Activity of mouse DNA polymerase beta is not affected by factor D with all the tested templates. In contrast to polymerase alpha, factor D is resistant to inactivation by N-ethylmaleimide and calcium ions, but it is readily heat-inactivated at 46 degrees C and is inactivated by trypsin digestion. Partially purified factor D is not associated with detectable activities of DNA polymerase, DNA primase, deoxyribonucleotidyl terminal transferase, and endo- or exodeoxyribonuclease.
Animals↗
Fidelity of DNA polymerases isolated from regenerating liver chromatin of aging Mus musculus.
DNA polymerase-alpha and -beta were fractionated from the chromatin of regenerating liver of young and old mice. The DNA polymerases were resolved from each other and partially purified by DEAE-cellulose, phosphocellulose, and DNA-cellulose column chromatography. No significant age-related difference in the kinetics of heat inactivation was observed for either DNA polymerase. No age-dependent difference was found in the fidelity with which these enzymes copied phi X174 DNA. These results suggest that the functional properties of these DNA polymerases do not change with age as is postulated in some theories of aging.
Aging↗
Novel anti-malarial hydroxynaphthoquinones with potent broad spectrum anti-protozoal activity.
Novel hydroxynaphthoquinones are reported with outstanding efficacy against Plasmodium, Eimeria and Theileria species. Biochemical evidence is presented for the selective toxicity of these compounds being due to inhibition of parasite respiratory systems.
Animals↗
Spontaneous colonic drainage. A rare complication of an amebic liver abscess.
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Adult↗
Potent and selective hydroxynaphthoquinone inhibitors of mitochondrial electron transport in Eimeria tenella (Apicomplexa: Coccidia).
Novel hydroxynaphthoquinones have been shown to be potent and selective inhibitors of mitochondrial electron transport in the protozoan Eimeria tenella, inhibiting at concentrations of 10(-10) to 10(-11)M. The primary site of electron transport inhibition has been localized to the ubiquinol-cytochrome c reductase span of the respiratory chain, whereas a secondary site of inhibition occurs in the NADH- and succinate-ubiquinone reductase complexes. Inhibition at the primary site is selective for the E. tenella enzyme; inhibition at the secondary sites is comparable in both E. tenella and chick (Gallus gallus) liver mitochondria. Hydroxynaphthoquinone inhibition of chick liver succinate-cyto-chrome c reductase was fully reversible by addition of the exogenous ubiquinone-2 analogue, 6-decyl-2,3-dimethoxy-5-methyl-1,4-benzoquinone; inhibition of the corresponding E. tenella enzyme was not reversed by this ubiquinone. E. tenella lines made resistant to the anticoccidial agents decoquinate or clopidol showed no cross-resistance to the hydroxynaphthoquinones, either at the level of electron transport or in vivo.
Coccidiostats↗
Effects of decoquinate and clopidol on electron transport in mitochondria of Eimeria tenella (Apicomplexa: Coccidia).
Resistance of the chicken coccidium Eimeria tenella to the anticoccidial agents decoquinate and clopidol, and the synergistic activity of mixtures of these compounds have been confirmed in vivo. Inhibition of electron transport by decoquinate and clopidol has been studied in mitochondria isolated from unsporulated oocysts of the same lines of E. tenella as those used in the in vivo studies. Electron transport in mitochondria of a drug sensitive line was susceptible to inhibition by both decoquinate and clopidol, and mitochondria isolated from lines made resistant to one or the other of these compounds showed a corresponding resistance at the level of electron transport. Combinations of low concentrations of decoquinate and clopidol caused a greater inhibition of electron transport than expected from summation of their individual actions. Isobolograms showed that decoquinate and clopidol in fact potentiated each other's effect on electron transport. Induced resistance to either decoquinate or clopidol resulted in an increased sensitivity of electron transport to inhibition by the other drug. Cytochrome spectra of E. tenella mitochondria and a biphasic response of NADH-oxidase and terminal oxidase activity to inhibition by cyanide or azide suggest the presence of two functional terminal oxidases. There is a correlation between the resistance of electron transport to inhibition by decoquinate or clopidol and the susceptibility to inhibition by cyanide or azide. Mitochondrial electron transport that is more resistant to inhibition by decoquinate exhibits greater sensitivity to cyanide and azide; electron transport that is more resistant to inhibition by clopidol exhibits a decreased sensitivity to cyanide and azide. Resistance to decoquinate and clopidol is discussed in view of a possibly branched electron transport chain in mitochondria of E. tenella.
Animals↗
Delayed and reduced cell replication and diminishing levels of DNA polymerase-alpha in regenerating liver of aging mice.
Kinetics of cell replication was compared in regenerating livers of Mus musculus at ages ranging between 6 and 32 months. Incorporation of [3H]-thymidine into DNA and autoradiographic analysis showed that the maximal extent of DNA replication following partial hepatectomy became delayed with age. Furthermore, the total fraction of parenchymal and nonparenchymal cells in S phase at different intervals during regeneration diminished as mice aged. The specific activity of DNA polymerase-alpha, the putative replicative enzyme, declined progressively during aging. The specific activity of DNA polymerase-beta, the purported repair enzyme, declined to an appreciably lesser extent during the lifespan of the mouse. No evidence was found for the appearance of a specific inhibitor of polymerase-alpha in senescent mouse liver. Also, the bulk of the activities of both hepatic DNA polymerase-alpha and -beta remained localized in the cell nucleus throughout the lifetime of the animal and were mainly associated with chromatin.
Aging↗
Nematoda: aerobic respiratory pathways of adult parasitic species.
Aerobic respiratory pathways have been compared in adult parasitic nematodes, including Trichostrongylus colubriformis, Nippostrongylus brasiliensis, Ostertagia ostertagi, Cooperia oncophora, Haemonchus contortus, Oesophagostomum venulosum, Chabertia ovina, Dictyocaulus filaria, Dictyocaulus viviparus, and Ascaridia galli. Respiration was measured in both whole worm or tissue homogenates and isolated mitochondrial fractions, and delineated into the mammalian type or alternative respiratory pathways on the basis of their inhibition by antimycin A. The alternative, antimycin A-insensitive respiratory pathway was of comparable activity in all parasitic nematodes studied, irrespective of the body diameter or habitat of the worm. The mammalian-type, antimycin A-sensitive respiratory pathway showed variations; the extent of this pathway correlated with both the body diameter and habitat of the worm, being greater in thinner worms and those worms whose habitat is supposedly more aerobic.
Adenosine Diphosphate↗