PubMed Health⌕ Search

Biomedical subjects

T G Newcomb

Publications and source records attributed to T G Newcomb.

9 recordsLinked to original sources
PubMed2000

Levels of DNA damage are unaltered in mice overexpressing human catalase in nuclei.

Two types of transgenic mice were generated to evaluate the role of hydrogen peroxide in the formation of nuclear DNA damage. One set of lines overexpresses wild-type human catalase cDNA, which is localized to peroxisomes. The other set overexpresses a human catalase construct that is targeted to the nucleus. Expression of the wild-type human catalase transgene was found in liver, kidney, skeletal muscle, heart, spleen, and brain with muscle and heart exhibiting the highest levels. Animals containing the nuclear-targeted construct had a similar pattern of expression with the highest levels in muscle and heart, but with lower levels in liver and spleen. In these animals, immunofluorescence detected catalase present in the nuclei of kidney, muscle, heart, and brain. Both types of transgenic animals had significant increases of catalase activities compared to littermate controls in most tissues examined. Despite enhanced activities of catalase, and its presence in the nucleus, there were no changes in levels of 8OHdG, a marker of oxidative damage to DNA. Nor were there differences in mutant frequencies at a Lac Z reporter transgene. This result suggests that in vivo levels of H(2)O(2) may not generate 8OHdG or other types of DNA damage. Alternatively, antioxidant defenses may be optimized such that additional catalase is unable to further protect nuclear DNA against oxidative damage.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1999

Detection of tandem CC-->TT mutations induced by oxygen radicals using mutation-specific PCR.

DNA lesions caused by reactive oxygen species (ROS) are considered to be one of the major contributors to DNA damage and mutagenesis. In this study, we developed a modification of allele-specific PCR to detect CC-->TT mutations caused by oxidative damage. These tandem mutations have been previously demonstrated to be indicative of oxygen damage in the absence of UV-irradiation. Using a CC target site in the rat DNA polymerase beta (pol beta) gene and a thermostable restriction enzyme that cuts the wild type sequence but not the TT mutation, we demonstrate that the TT mutation can be preferentially amplified from plasmid DNA damaged by oxygen radicals but not other DNA-damaging agents. We evaluated the potential utility of this assay in screening for mutations in cells and in analyzing those that arise during clonal proliferation in carcinogenesis.

DNA Damage↗
PubMed1999

Metallothionein overexpression suppresses hepatic hyperplasia induced by hepatitis B surface antigen.

Transgenic mice that express the viral coat proteins of hepatitis B virus (HBV) in the liver display hepatocellular damage, inflammation, regeneration, hyperplasia, and, eventually, neoplasia that is similar to that of people with chronic, active hepatitis caused by HBV infection. Hepatocellular regeneration, in the context of chronic injury and inflammation, is thought to expose dividing cells to excessive oxygen radicals, which are believed to lead to DNA damage and, ultimately, neoplasia. Because metallothioneins scavenge free radicals in vitro, we generated mice that express excess (>10-fold) metallothionein I (MT-I* mice) and the HBV surface antigens (HBsAg) to ascertain whether MT-I* would ameliorate aspects of the pathology induced by HBsAg. Markers of hepatocyte injury and tumorigenesis in HBsAg mice were compared to those in double transgenic (HBsAg and MT-I*) mice. Hepatic hyperplasia, histology, aneuploidy, and accumulation of an oxidative DNA adduct, 8-oxo-2'-deoxyguanosine, were examined. Although hepatitis and neoplasia were not prevented by MT-I* expression in the HBsAg mice, there was less hyperplasia and less aneuploidy. We conclude that MT-I produces a beneficial effect in this in vivo model of HBV-induced hepatitis.

Animals↗
PubMed1996

Creation of drug-specific herpes simplex virus type 1 thymidine kinase mutants for gene therapy.

Herpes simplex virus type 1 (HSV-1) thymidine kinase is currently used as a suicide agent in the gene therapy of cancer. This therapy is based on the preferential phosphorylation of nucleoside analogs by tumor cells expressing HSV-1 thymidine kinase. However, the use of HSV-1 thymidine kinase is limited in part by the toxicity of the nucleoside analogs. We have used random sequence mutagenesis to create new HSV-1 thymidine kinases that, compared with wild-type thymidine kinase, render cells much more sensitive to specific nucleoside analogs. A segment of the HSV-1 thymidine kinase gene at the putative nucleoside binding site was substituted with random nucleotide sequences. Mutant enzymes that demonstrate preferential phosphorylation of the nucleoside analogs, ganciclovir or acyclovir, were selected from more than one million Escherichia coli transformants. Among the 426 active mutants we have isolated, 26 demonstrated enhanced sensitivity to ganciclovir, and 54 were more sensitive to acyclovir. Only 6 mutant enzymes displayed sensitivity to both ganciclovir and acyclovir when expressed in E. coli. Analysis of 3 drug-sensitive enzymes demonstrated that 1 produced stable mammalian cell transfectants that are 43-fold more sensitive to ganciclovir and 20-fold more sensitive to acyclovir.

Acyclovir↗
PubMed1995

Potential sources of multiple mutations in human cancers.

Increasing evidence indicates that most human cancers contain multiple chromosomal alterations. These aberrations are the result of mutations produced not only during the initiation of cancer but also during tumor progression. Since the rates of spontaneous mutations exhibited by normal human cells cannot account for the large numbers of mutations routinely reported in human cancers, we argued that cancer cells are genetically unstable; i.e., they express a mutator phenotype. In this review, we consider potential endogenous sources of these mutations and the recent evidence demonstrating that multiple mutations are present in human cancers. These studies, which connect mismatch repair, genomic instability, and cancer, support the mutator phenotype hypothesis. We conclude that, if multiple mutations are necessary for the progression of cancer, then agents designed to delay their accumulation could significantly reduce cancer deaths.

Chromosome Aberrations↗
PubMed1994

Arachidonic acid and eicosapentaenoic acid stimulate type II pneumocyte surfactant secretion.

Arachidonic acid and its leukotriene metabolites have been shown to stimulate surfactant secretion by alveolar type II cells. The present study was undertaken to determine the effects of various unsaturated fatty acids, including eicosapentaenoic acid, on surfactant secretion. Surfactant secretion was expressed as the percent of [3H]choline-derived phospholipids released into culture medium by type II pneumocytes of adult rats. Consistent with the earlier findings, arachidonic acid stimulated secretion in a concentration-dependent fashion (3.5-21 microM), doubling baseline secretion at 21 microM. Eicosapentaenoic acid was found to be equally effective as arachidonic acid in stimulating secretion. A comparison with palmitic, oleic and linoleic acids revealed that highly unsaturated fatty acids stimulated secretion to the greatest extent. This was supported by a positive correlation between degree of unsaturation (i.e., 0, 1, 2, 4 and 5 double bonds) and stimulation of surfactant secretion. In the present study, exogenous leukotriene E4 (10(-13)-10(-6) did not stimulate surfactant secretion. Neither nordihydroguairetic acid (0.1 microM) nor indomethacin (0.1 microM) affected arachidonic acid-stimulated secretion. The stimulatory effects of arachidonic acid and eicosapentaenoic acid on surfactant secretion were related to the highly unsaturated nature of the fatty acids and were not mediated by increased levels of cyclic adenosine monophosphate or calcium.

Animals↗
PubMed1993

Altered calcium regulation in SV40-transformed Swiss 3T3 fibroblasts.

Calcium homeostasis has long been thought to be altered in transformed cells but mechanisms have not been established. In this study, the photoprotein, aequorin, was used to examine calcium regulation in 3T3 and SV40-transformed 3T3 cells. It was found that calcium transients induced by bradykinin or serum in serum-starved cells are lower and delayed in the transformed cells and decay kinetics are altered. These changes are not related to differences in cell cycle distribution. Though the serum transient is insensitive to nifedipine, verapamil, or lanthanum, removal of extracellular calcium accelerates transient decay in both cell types. Treatment of unstimulated cells with the ER Ca(2+)-ATPase inhibitor, thapsigargin, causes a 4-5-fold greater increase in [Ca2+]i in the transformed than in the nontransformed cells. Following serum stimulation, transformed cells still exhibit a large thapsigargin-induced increase in [Ca2+]i whereas the response in nontransformed cells is nearly abolished. When the 3T3 or SV3T3 cells are exposed to serum or thapsigargin in the absence of extracellular calcium and subsequently exposed to 11.8 mM Ca2+, a much greater influx of calcium again occurs in the SV3T3 cells. The observed changes in SV3T3 cells are most likely due to an alteration in a capacitative mechanism which regulates influx of calcium through the plasma membrane.

3T3 Cells↗
PubMedDate not supplied

Serum- and bradykinin-induced calcium transients in familial Alzheimer's fibroblasts.

The calcium-sensitive photoprotein, aequorin, was used to examine serum- and bradykinin-induced transient increases in free cytosolic calcium ions in skin fibroblasts from 10 individuals with early onset familial AD (FAD), including four who were biopsied before their clinical symptoms would allow a diagnosis of AD, 2 individuals with late onset FAD, 8 at-risk but nonsymptomatic individuals, and 13 controls. The data show that (a) among controls, the peaks of the calcium transients increase in height as a function of donor age; (b) transients induced by 10% serum, 10 nM bradykinin (BK) or 100 nM BK were generally lower in FAD fibroblasts, including those from donors in the early stages of the disease, than in age-matched control cells; (c) such transients are reduced in cells from a proportion of the nonsymptomatic, at-risk individuals. Thus, serum- and BK-induced calcium transients are reduced in fibroblasts from both early and more advanced stage FAD donors and perhaps even from donors who are presymptomatic carriers of the defective gene. The data also suggest that changes in calcium transients in FAD fibroblasts neither mimic nor exaggerate the effects of normal aging.

Adult↗