Biomedical subjects

D L Busbee

Publications and source records attributed to D L Busbee.

At least 19 recordsLinked to original sources

PubMed1992

Decreased fidelity of DNA polymerases and decreased DNA excision repair in aging mice: effects of caloric restriction.

Hepatic DNA polymerases from calorie restricted and ad libitum 26 month old C57BL/6 mice showed a decline in fidelity of nucleotide incorporation compared with weanling animals. Both alpha and beta polymerases from calorie restricted aged mice exhibited a higher level of fidelity than polymerases from ad libitum aged mice. UV-initiated unscheduled DNA synthesis was significantly higher in hepatocytes from weanling and 18 month old calorie restricted animals compared with cells from 18 month old ad libitum animals, while MMS-initiated unscheduled DNA synthesis did not differ significantly between cells from young and old or ad libitum and calorie restricted animals. These data suggest that calorie restriction could play a significant role in decreasing the age-related decline of cellular mechanisms expected to reduce the rate at which mutations accumulate during aging, and could potentially prolong the onset age of mutation-associated diseases of the elderly.

Aging↗

PubMed1992

Inhibition of DNA synthesis and decreased reproductive capacity in cattle maintained on semi-arid rangeland with Acacia species as a major component of available browse.

The ability of bovine lymphocytes to initiate in vitro blastogenesis in response to mitogen stimulation or to initiate DNA excision repair after treatment with a mutagen was evaluated as a function of the differing environmental conditions under which donor animals were maintained. Crossbred Brahman-Hereford F1 females were held on either a humid, coastal bermudagrass, improved pasture at Overton, TX, or in low or high grazing pressure herds on a semi-arid rangeland (Acacia-dominated shrubland) at Uvalde, Tx. Peripheral blood lymphocytes (PBL) from these animals were examined to determine their in vitro ability to engage in phytohemagglutinin (PHA)-stimulated blastogenesis and to initiate excision repair of DNA damage after exposure of the cells to the model polynuclear hydrocarbon carcinogen 7,8-dihydrodiol-9, 10-epoxy-benzo(a) pyrene (BPDE). PBL from cattle at both locations were compared, with significantly decreased blastogenesis and DNA excision repair observed in PBL from Uvalde high grazing pressure cattle. Cattle in the Uvalde high grazing pressure herd also exhibited significantly decreased reproductive efficiency. The data indicate that ingestion of sufficient quantities of palatable, but toxic, forage species available at the Uvalde test site is sufficient to decrease DNA synthesis associated with either mitogen-stimulated blastogenesis or DNA excision repair in bovine PBL, and suggest that the reduction in PBL DNA synthesis may be correlated with the changed reproductive efficiency in animals ingesting an increased ratio of forage from Acacia species.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed1991

Effect of dietary restriction on the fidelity of DNA polymerases in aging mice.

DNA polymerases purified from hepatic tissues of C57BL/6 mice showed an age-related decrease in both specific activity and fidelity of the various enzyme forms. Polymerases from dietary restricted mice exhibited less of a decline in specific activity and copied synthetic DNA templates with relatively higher fidelity than did enzymes from animals fed ad libitum. Polymerases treated with inositol-1,4-bisphosphate [I(1,4)P2] showed varying levels of increased activity, with fidelity increases up to 3-fold. These data indicate that aging is associated with decreases in both specific activity and fidelity of DNA polymerases isolated from a nondividing tissue, and that dietary restriction impedes the age-related decline in both specific activity and fidelity of these polymerases. The data further indicate that DNA polymerases may interact with phosphoinositide hydrolysis products resulting in increased specific activity and fidelity of the enzymes. Phosphoinositide interactions with polymerases could constitute an important mechanism moderating the age-related decrease in function and accuracy of DNA polymerases.

Aging↗

PubMed1990

Comparative uptake, vascular transport, and cellular internalization of aflatoxin-B1 and benzo(a)pyrene.

Studies of the uptake of benzo(a)pyrene (BaP) and aflatoxin-B1 (AFB1) after gastric instillation showed that BaP was absorbed via the intestinal lymphatic drainage and transported to the vascular circulation sequestered within lipoproteins in thoracic duct lymph, while AFB1 was absorbed with water soluble compounds into the gastrointestinal venous drainage and was not transported in association with lipoproteins. BaP was taken up into plasma lipoproteins over a broad concentration range, while AFB1 was not sequestered within lipoproteins over the same concentration range. Low density lipoproteins (LDL) facilitated BaP uptake into fibroblasts and impeded BaP uptake into hepatocytes. High density lipoproteins (HDL) facilitated BaP uptake into hepatocytes and impeded BaP uptake into fibroblasts. The uptake of AFB1 into either fibroblasts or hepatocytes was not affected by lipoproteins.

Aflatoxin B1↗

PubMed1989

Purification of Norman Murine Sarcoma DNA polymerase alpha forms with different DNA template primer binding affinity and different specific activity.

1. DNA polymerase alpha was isolated from Norman Murine Myxosarcoma cells using ion exchange, immunoaffinity, and DNA affinity chromatography, showing two distinct enzyme forms designated A1 and A2. 2. Chromatographic analysis of polymerase alpha forms A1 and A2 indicate a charge difference and a difference in affinity of binding to DNA between polymerase alpha forms which were equally reactive to anti-DNA polymerase alpha monoclonal IgG. 3. Polymerase A1 specific activity was about 3600 U/mg while A2 specific activity was about 40,000 U/mg.

Animals↗

PubMed1989

Interaction of phosphatidylinositol-4-monophosphate with a low activity form of DNA polymerase alpha: a potential mechanism for enzyme activation.

1. DNA polymerase alpha isolated from Norman murine myxosarcoma exhibited two isozyme forms, one with low specific activity and low DNA binding affinity (A1), and one with high specific activity and high DNA binding affinity (A2). 2. DNA polymerase alpha A1, but not A2, showed a significant increase in specific activity after treatment with phosphatidylinositol, ATP and phosphatidylinositol kinase, or with phosphatidylinositol-4-monophosphate. 3. Treatment of DNA polymerase alpha A1 with the phospholipase C hydrolysis product of phosphatidylinositol-4-monophosphate, inositol-1,4-bisphosphate, was sufficient to effect the transient increase in activity of polymerase A1 to a form not chromatographically distinguishable from isozyme form A2.

Animals↗

PubMed1987

The sesquiterpene lactone hymenoxon acts as a bifunctional alkylating agent.

Hymenoxon, a toxic sesquiterpene lactone found in bitterweed, bound deoxyguanosine in a cell-free system and formed adducts with guanine residues in cellular DNA. The reactive dialdehyde form of hymenoxon formed stable Schiff base products with deoxyguanosine which were separable from unreacted hymenoxon and deoxynucleosides by reverse-phase high pressure liquid chromatography. Hymenoxon adducts which eluted as a single impure peak from the octadecylsilane column separated on amino and diphenyl-bonded phases with 10% methanol. Tritiated nucleoside adducts were isolated and purified from CFW mouse sarcoma cells treated with hymenoxon. Proton nuclear magnetic resonance spectra of purified hymenoxon-deoxyguanosine adducts revealed a loss of signals for hydroxyl groups in the bishemiacetal of hymenoxon. 13C-nuclear magnetic resonance spectra revealed that the major adduct has 35 carbon atoms, indicating an interaction of at least two guanine residues per hymenoxon molecule and suggesting that hymenoxon may cross-link DNA. Sedimentation analysis of treated DNA further showed that DNA cross-linking by hymenoxon (30 micrograms/ml) was equivalent to that of a known cross-linking agent, mitomycin C (7.5 micrograms/ml). Hymenoxon was more cytotoxic to DNA cross-link repair-deficient Chinese hamster ovary cell mutants than to repair-proficient strains. These data combine to indicate that hymenoxon acts as a bifunctional alkylating agent which cross-links DNA in mammalian cells.

Alkylating Agents↗

PubMed1987

Repair of benzo[a]pyrene-initiated DNA damage in human cells requires activation of DNA polymerase alpha.

Normal human fibroblasts treated with r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) yielded DNA polymerase alpha with elevated levels of activity, incorporated [3H]thymidine as a function of unscheduled DNA synthesis, and exhibited restoration of normal DNA-strand length as a function of unscheduled DNA synthesis. Lipoprotein-deficient fibroblasts treated with BPDE did not show elevated levels of DNA polymerase alpha activity, exhibited minimal [3H]thymidine incorporation, and had fragmented DNA after 24 h of repair in the absence of lipoprotein or phosphatidylinositol supplementation. When DNA polymerase beta activity was inhibited, cells with normal lipoprotein uptake exhibited [3H]thymidine incorporation into BPDE-damaged DNA but did not show an increase in DNA-strand length. DNA polymerase alpha activity and [3H]thymidine incorporation in lipoprotein-deficient fibroblasts increased to normal levels when the cells were permeabilized and low-density lipoproteins or phosphatidylinositol were introduced into the cells. DNA polymerase alpha isolated from normal human fibroblasts, but not from lipoprotein-deficient fibroblasts, showed increased specific activity after the cells were treated with BPDE. When BPDE-treated lipoprotein-deficient fibroblasts were permeabilized and 32P-ATP was introduced into the cells along with lipoproteins, 32P-labeled DNA polymerase alpha with significantly increased specific activity was isolated from the cells. These data suggest that treatment of human fibroblasts with BPDE initiates unscheduled DNA synthesis, as a function of DNA excision repair, which is correlated with increased activity of DNA polymerase alpha, and that increased DNA polymerase alpha activity may be correlated with phosphorylation of the enzyme in a reaction that is stimulated by low-density lipoprotein or by the lipoprotein component, phosphatidylinositol.

Apolipoproteins B↗

PubMed1987

Cytogenetic analysis of a gossypol-induced murine myxosarcoma.

Cytogenetic analysis of gossypol acetate-induced murine myxosarcoma demonstrated a stemline of 78 chromosomes and the presence of three marker (M) chromosomes produced by robertsonian translocation. Tumor cells at passage 1 that contain chromosomes M1 and M2 were nontumorigenic, whereas cells at passage 3 were tumorigenic in syngeneic mice and showed M1, M2, and M3. The presence of M3 has been implicated to be responsible for the tumorigenic phenotype.

Animals↗

PubMed1986

Monoclonal antibody that blocks phosphoinositide-dependent activation of mouse tumor DNA polymerase alpha.

A monoclonal antibody (MaB) against mouse sarcoma DNA polymerase alpha was isolated from the culture medium of an IgG-secreting hybridoma. The MaB demonstrated reactivity against two murine DNA polymerase alpha preparations and a calf thymus DNA polymerase alpha. Murine sarcoma polymerase was activated in vitro by phosphatidylinositol-4-monophosphate (PIP) showing increased deoxynucleotidyltransferase activity and enhanced binding affinity to activated DNA template. The MaB did not neutralize polymerase activity, but blocked further activation of the enzyme by PIP. Treatment of polymerase with MaB prior to treatment with PIP inhibited both increased enzyme activation and increased binding of the enzyme to DNA template. Treatment of polymerase with MaB subsequent to treatment with PIP did not block enzyme activation or increased DNA template binding. The data suggest that this anti-DNA polymerase alpha IgG is directed against a regulatory subunit of the polymerase rather than the catalytic subunit. The antibody may serve to distinguish between DNA polymerase alpha preparations with distinctly different regulatory subunits.

Animals↗

PubMed1986

Phosphatidylinositol-dependent activation of DNA polymerase alpha.

DNA polymerase alpha was activated in vitro by cAMP-independent, phospholipid-dependent, protein kinase catalytic subunit. Of the phospholipids examined, phosphatidylinositol showed the greatest potential for interaction with protein kinase and ATP to activate DNA polymerase alpha in vitro. DNA polymerase alpha was directly activated by phosphorylated phosphatidylinositol in the absence of protein kinase and ATP. Activation of DNA polymerase alpha as a function of phosphorylation was demonstrated using 32P-ATP as the phosphate donor. In vitro treatment of the enzyme with phosphatidylinositol produced Linweaver-Burk plots showing noncompetitive kinetics of enzyme activation, suggesting that activation occurs prior to binding of the enzyme to DNA template/primer. These data indicate that DNA polymerase alpha may be activated in vitro in the presence of protein kinase, ATP, and phosphatidylinositol, and suggest that phosphorylation of the enzyme may constitute an intracellular mechanism of enzyme activation.

Adenosine Triphosphate↗

PubMed1986

In vitro cytotoxicity of chrysotile asbestos to human pulmonary alveolar macrophages is decreased by organosilane coating and surfactant.

Human pulmonary alveolar macrophages were used to quantitate the cytotoxic effect of surface-altered chrysotile asbestos. Little difference was observed in mortality between chrysotile asbestos that was surface-treated to a 42% extent by a hydrophobic organosilane or untreated chrysotile. Little or no effect on mortality was observed when human pulmonary alveolar macrophages were cultured with untreated chrysotile or acid-leached asbestos in the presence of 10 mM dipalmitoyl lecithin. However, when human pulmonary alveolar macrophages were cultured with a hydrophobically-treated (to a 42% or 95% extent) chrysotile asbestos in the presence of 10 mM dipalmitoyl lecithin, a statistically significant decrease in mortality was observed compared to untreated chrysotile. No mutagenic activity was observed when V79 cells were cultured with acid-leached, or 42% hydrophobically-treated chrysotile asbestos, even when human pulmonary alveolar macrophages were included as an activation source. The 95% hydrophobically-treated and acid-leached chrysotile also exhibited decreased binding of benzo[a]pyrene compared to untreated chrysotile asbestos.

Adult↗

PubMed1986

Inhibition of DNA polymerase activity by methyl methanesulfonate.

Methyl methanesulfonate (MMS) inhibits both thymidine incorporation into DNA in mitogen-activated human lymphocytes and deoxythymidine triphosphate incorporation into template DNA by DNA polymerase-alpha in a cell-free system. When MMS-modified DNA was used as the template for DNA synthesis utilizing unmodified DNA polymerase-alpha, nucleotide incorporation into template DNA was not inhibited. When unmodified DNA was used as the template for DNA synthesis utilizing MMS-modified DNA polymerase-alpha, nucleotide incorporation was differentially inhibited dependent on the MMS concentration. An analysis of the kinetics of DNA polymerase-alpha inhibition showed that incorporation of all 4 deoxynucleoside triphosphates into DNA template was noncompetitively inhibited by MMS, which is consistent with nonspecific MMS modification of the enzyme. These data indicate that MMS modification of DNA polymerase-alpha alone is sufficient to inhibit the incorporation of deoxynucleoside triphosphates into template DNA in vitro. The data further indicate that alkylation of both DNA polymerase-alpha and DNA template synergistically increases inhibition of DNA synthesis.

Alkylation↗

PubMed1985

DNA polymerase activity in a repair-deficient human cell line.

A human low-density-lipoprotein (LDL) receptor-deficient diploid fibroblast cell line (GM1915) was determined to be short patch competent (DNA polymerase-beta) and long patch deficient (DNA polymerase-alpha) for DNA excision repair. Analysis of DNA from GM1915 cells or from WI38 control cells, following treatment with a mutagen known to initiate long patch excision repair, showed that GM1915 cells exhibited decreased resynthesis of oligonucleotide segments excised during repair. When cells deficient in DNA polymerase-alpha activity were permeabilized to permit LDL entry, repair synthesis immediately increased. These data suggest that DNA polymerase-alpha is not activated by mutagen treatment in GM1915 cells and that introduction of LDL into the cells results in activation of the enzyme.

Cell Line↗

PubMed1985

Alkylation of deoxyguanosine by the sesquiterpene lactone hymenoxon.

Hymenoxon, a toxic sesquiterpene lactone found in the ruminant forage plant Hymenoxys odorata, binds deoxyguanosine in a cell-free system, and forms adducted guanine residues in sheep lymphocyte DNA. Mitogen-stimulated DNA synthesis in lymphocytes was inhibited by hymenoxon at concentrations greater than 100 microM. Unscheduled DNA synthesis in lymphocytes was initiated by hymenoxon concentrations exceeding 50 microM, and inhibited by concentrations above 100 microM. We describe an HPLC method which separates unmodified hymenoxon and deoxyguanosine from the hymenoxon-deoxyguanosine adduct, and allows the quantitation of adducts in hymenoxon-treated cells.

Alkylation↗

PubMed1985

Polychlorinated biphenyl uptake and transport by lymph and plasma components.

The uptake and vascular transport of ingested Aroclor 1242, an isomeric mixture of polychlorinated biphenyls (PCB), was investigated in experimental animals. High concentrations of ingested PCB were found in the chylomicron fraction of thoracic duct lymph. When the lymph flow was exteriorized PCB were not subsequently found in the vascular circulation. When lymph was not exteriorized plasma PCB concentrations reached maximal levels 6 hr after ingestion. Less than 1% of total plasma PCB was detected in cellular fractions of blood over a 10-hr period following ingestion. Chylomicrons contained 31% of total plasma PCB 30 min after ingestion, decreasing to less than 6% at 4 hr. A maximum of 10% of plasma PCB at 1 hr, and less than 5% at 6 hr, after ingestion was associated with very low density lipoproteins (VLDL) or low density lipoproteins (LDL). Although PCB enter the vascular circulation with the chylomicron fractions of lymph, delipoproteinated plasma contained 52% of the total PCB in blood collected 30 min after ingestion. This level increased to 78% after 2 hr, and remained constant at about 80% for an additional 8-hr period. High performance liquid chromatographic (HPLC) examinations of delipoproteinated plasma from blood taken 6 hr after PCB ingestion showed elution of greater than 95% of plasma PCB to coincide with the albumin peak. Electrophoretic examinations of delipoproteinated plasma showed the association of PCB with albumin to be noncovalent. The results suggest that apolar PCB are absorbed into intestinal epithelial cells from which they are secreted into the lymphatic drainage sequestered within the apolar core of chylomicrons, that these PCB transit the thoracic duct and enter the vascular circulation within chylomicrons and are metabolized or otherwise released from chylomicrons during hepatic chylomicron clearance, and that resulting PCB or PCB derivatives circulate in association with plasma albumins.

Animals↗

PubMed1984

Human lymphocytes treated with r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene require low-density lipoproteins for DNA excision repair.

Human lymphocytes which were non-mitogen-stimulated, and which were depleted of lipoproteins, were found to be deficient in DNA excision repair typically initiated in these cells in response to treatment with a direct-acting polynuclear aromatic hydrocarbon carcinogen. Lymphocytes either depleted of lipoproteins or supplemented with human low-density lipoproteins formed DNA-carcinogen adducts which were not chromatographically distinguishable. The state of lipoprotein depletion did not alter lymphocyte uptake of thymidine from the medium. Lymphocytes which were depleted of lipoproteins, treated with carcinogen, and subsequently supplemented with low-density lipoproteins, regained the ability to engage in DNA excision repair. The data suggest that either low-density lipoprotein(s), or a component(s) of low-density lipoprotein(s), is required by human lymphocytes in order to initiate excision repair of carcinogen-damaged DNA.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed1984

Inhibition of DNA synthesis by an electrophilic metabolite of benzo[a]pyrene.

Mitogen-stimulated scheduled DNA synthesis and DNA excision repair in human lymphocytes, as well as DNA polymerase a activity in a cell-free system, were inhibited by an electrophilic metabolite of benzo[a]pyrene. This metabolite, (+/-)-anti-(7r,8t)-dihydroxy-(9,10t)-epoxy-7,8,9,10-tetrahyd robenzo[a]pyrene (BPDE), covalently binds to cellular macromolecules and is mutagenic, carcinogenic, and cytotoxic. Human lymphocytes treated with BPDE at concentrations greater than 500-800 ng/ml showed decreases in both mitogen-stimulated DNA synthesis and excision repair of damaged DNA but did not exhibit overt cytotoxicity (excluded trypan blue and maintained an adenylate charge of greater than 0.7). Formation of, and total concentration of, BPDE-DNA adducts was not correlated with inhibition of DNA synthesis. DNA polymerase alpha studies using a cell-free system showed that enzymatic activity was not diminished when purified polymerase was treated with BPDE prior to the addition of template DNA. When the template DNA concentration was varied, BPDE inhibition of enzyme activity was uncompetitive. BPDE inhibition of enzyme activity was found to be noncompetitive when concentrations of dATP, dCTP, or dTTP were varied and competitive when the concentration of dGTP was varied. The data indicate that BPDE competitively inhibits interaction of dGTP with the template-DNA polymerase alpha complex.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗