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D Busbee

Publications and source records attributed to D Busbee.

29 records · Page 2Linked to original sources
PubMed1989

Age-related changes in DNA polymerase alpha expression.

DNA polymerase alpha isozymes differing in specific activity and affinity of binding to DNA were purified from human fibroblasts derived from donors of different ages. Fetal-derived fibroblasts expressed a single, high-activity enzyme (A2), with high affinity of binding to DNA. Adult-derived fibroblasts exhibited two forms of DNA polymerase alpha, one identical to the fetal enzyme, and a second with about tenfold less activity showing low affinity of binding to DNA (A1). The ratio of DNA polymerase A2/A1 decreased dramatically with age, from 100% A2 in fetal-derived fibroblasts to about 94% A1 in fibroblasts derived from a 66-year-old donor. The DNA binding affinity of polymerase alpha A1 from adult-derived fibroblasts increased concomitant with a significant increase in activity when the enzyme was treated with phosphatidylinositol-4-monophosphate (PIP), or with inositol-1, 4-bisphosphate (I(1,4)P2). The enzyme reverted back to a less active form, with loss of the noncovalently bound I(1,4)P2, as a function of time. When permeabilized human fibroblasts with low DNA excision repair capacity were treated with 7,8-dihydrodiol-9,10-epoxybenzo(a)-pyrene (BPDE) in the presence of 32P-ATP, phosphatidylinositol, and cycloheximide, excision repair was initiated and 32P-labeled DNA polymerase alpha was recovered in the absence of de novo protein synthesis. DNA synthesis associated with either scheduled DNA synthesis or BPDE-initiated excision repair declined as a function of increased age in human cells. The data suggest that the decline in both DNA excision repair-associated and mitogen-activated DNA synthesis may be correlated with decreased total intracellular levels of DNA polymerase and with the decline in polymerase alpha activity as a function of age, that DNA repair-associated initiation of DNA synthesis in adult-derived cells may increase with activation of a pool of low activity DNA polymerase alpha, and that DNA polymerase alpha activity increases as a function of enzyme interaction with a component of the PI phosphorylation cascade.

Cell Survival↗
PubMed1988

Activation of a low specific activity form of DNA polymerase alpha by inositol-1,4-bisphosphate.

A low activity form of DNA polymerase alpha immunoaffinity-purified from adult-derived human fibroblasts was activated by interaction with phosphatidylinositol-4-monophosphate, while a high activity form of the enzyme did not interact with phosphatidylinositol-4-monophosphate or its derivatives. Phosphatidylinositol-4-monophosphate was apparently hydrolyzed in the presence of a highly purified low activity form of DNA polymerase alpha, effecting the release of diacylglycerol and the retention of inositol-1,4-bisphosphate by the enzyme complex. The resulting inositol-1,4-bisphosphate/protein complex exhibited increased affinity of binding to DNA template/primer and increased deoxynucleotidyltransferase activity. These data indicate that inositol-1,4-bisphosphate may function as an effector molecule in the activation of a low activity form of human DNA polymerase alpha and suggest that it may function as a second messenger during the initiation of mitosis in stimulated cells.

1-Phosphatidylinositol 4-Kinase↗
PubMed1988

DNA polymerase alpha activity in mitogen-activated lymphocytes.

DNA polymerase alpha isolated from adult-derived lymphocytes was separated into isozyme forms with low (A1) and high (A2) specific activity. In quiescent lymphocytes only A1 was detected, while mitogen-stimulated lymphocytes contained both A1 and A2 enzyme. Polymerase alpha A1, but not A2, interacted with phosphatidylinositol, ATP, and phosphatidylinositol kinase to yield an activated enzyme with increased affinity of binding to DNA. Mitogen-stimulated lymphocytes showed increased enzyme protein and total activity for both A1 and A2, but, when pre-treated with cycloheximide, exhibited an apparent increase in A2 specific activity with no increase in activity for A1 polymerase. These data suggest that mitogen stimulation of lymphocytes increased total DNA polymerase alpha activity by the phosphoinositide-related activation of polymerase alpha A1 to an A2-like form and by initiating de-novo synthesis of polymerase alpha A2.

Adult↗
PubMed1987

Lability of DNA polymerase alpha correlated with decreased DNA synthesis and increased age in human cells.

DNA excision repair and mitogen-initiated blastogenesis in human cells declined in efficiency as an apparent function of decreased DNA polymerase alpha specific activity with increased age of the cell donor. DNA polymerase alpha isolated from fetal cells contained a single, high-specific-activity enzyme form that could not be further activated and that was stable with regard to enzyme activity and affinity for DNA template-primer. DNA polymerase alpha isolated from adult-derived cells contained both low-specific-activity and high-specific-activity forms. The low-activity enzyme form, which showed low affinity of binding to DNA template-primer, was activated by treatment with phosphatidylinositol, 32P-ATP, and phosphatidylinositol kinase, resulting in a 32P-labeled enzyme that exhibited high affinity of binding to DNA template-primer. The activated enzyme was unstable, exhibiting a loss of 32P-label correlated with the loss of both specific activity and high affinity of binding to DNA template-primer. The data suggest that DNA polymerase alpha isolated from adult-derived human cells has low-activity and high-activity forms. Decreased specific activity of DNA polymerase alpha correlated with increased age of the donor appears to be a function of loss of an enzyme activator molecule resulting in diminished ability of the enzyme to bind DNA template-primer.

Adult↗
PubMed1985

Uptake and vascular transport of ingested aflatoxin.

The uptake and vascular transport of abomasally instilled aflatoxin B1 (AFB1) was investigated in sheep. Aflatoxin uptake was compared with that of palmitate, a water-insoluble oil known to be absorbed into the intestinal lymphatic drainage which bypasses the liver to enter the peripheral vascular circulation via the thoracic duct. After instillation into the abomasum, aflatoxin was detected in inferior vena cava blood within 30 min, while palmitate was not detected in vena cava blood at any time. Palmitate was detected in thoracic duct lymph after about 2 h. More than 95% of the palmitate in lymph was associated with the chylomicron fraction, while aflatoxin in either plasma or lymph was not detectably associated with any of the circulating lipoproteins. In addition, aflatoxin did not partition into plasma or lymph lipoproteins in vitro. Toxic lipophilic xenobiotics, such as benzo(a)pyrene and polychlorinated biphenyls (PCBs) do partition into lipoproteins, are absorbed into the intestinal lymphatic drainage, bypassing the liver to enter the peripheral vascular circulation directly, and are not specifically hepatotoxic. These data suggest that the mode of aflatoxin absorption from the gastrointestinal system results in its immediate transport to the liver, which may contribute to aflatoxin hepatotoxicity.

Aflatoxin B1↗
PubMed1978

A human plasma component that binds benzo(a)pyrene.

A component capable of binding benzo(a)pyrene was measured in plasma from cigarette smokers and nonsmokers. This plasma fraction was found to have a high specificity of binding to benzo(a)pyrene, bound benzanthracene competitively with benzo(a)pyrene, and was positively correlated (r = 0.861, p less than 0.001) with the capacity of the individual subject's lymphocytes to be induced for AHH activity in culture. An inverse correlation (r = -0.957, p less than 0.001) between the presence of the plasma component in lung cancer patients and the capacity of lung cancer patients' lymphocytes to be induced in culture is unexplained at this time. A benzo(a)pyrene-binding fraction was not found in induced or uninduced cultured lymphocytes from smokers or nonsmokers, or in homogenates of lung excisional tissue from smokers with or without primary lung cancer.

Aryl Hydrocarbon Hydroxylases↗
PubMedDate not supplied

Effects of aging and dietary restriction on DNA polymerases: gene expression, enzyme fidelity, and DNA excision repair.

Hepatic DNA polymerases isolated from young and old C57BL/6N mice fed ad libitum or calorically restricted differed in chromatographic characteristics, binding affinity for DNA template-primer, specific activity, and fidelity of synthesis. DNA polymerase alpha total and specific activity declined slightly, while the nucleotide misincorporation frequency increased dramatically, with increased age of the donor animals. A positive correlation was observed between polymerase alpha specific activity and the affinity of enzyme binding to activated DNA template-primer. Both the age-associated decline in enzyme activity and the decrease in fidelity of synthesis were modified by dietary restriction, with higher specific activity levels and lower misincorporation frequencies for DNA polymerases from dietarily restricted animals compared with ad libitum animals of all ages. Fidelity of both DNA polymerase alpha and beta increased following treatment with the phosphoinositide hydrolysis product inositol-1,4-bisphosphate. The data suggest that dietary restriction could play an important role in decreasing the age-associated decline in function of physiological systems sensitive to decreased or defective DNA synthesis.

Aging↗