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Biomedical subjects

S Lynn

Publications and source records attributed to S Lynn.

At least 19 recordsLinked to original sources
PubMed2003

Heteroplasmic ratio of the A3243G mitochondrial DNA mutation in single pancreatic beta cells.

AIMS/HYPOTHESIS: To examine whether there is a high content of mutated mitochondrial DNA in individual pancreatic beta cells from a patient with the A3243G mitochondrial DNA mutation. METHODS: Tissues were available from a patient with diabetes and the A3243G mutation including pancreatic tissue. We quantified the amount of mutated mitochondrial DNA in tissue homogenates and single pancreatic beta cells using hot last cycle PCR. RESULTS: The percentage ratio of mutated to wild-type mtDNA was high in tissues such as muscle and brain (>60%), but surprisingly low in both pancreatic islets and in individual beta cells from these islets. The islets were smaller in the patient than in control subjects in keeping with a decreased beta-cell mass. CONCLUSIONS/INTERPRETATION: These observations suggest that either the beta cells show increased sensitivity to the effects mtDNA mutations on respiratory chain function, and/or cells with a high mutant load are preferentially removed leading to a progressive decrease in the islet beta-cell mass.

Brain↗
PubMed2001

Avoiding the 'costs' of testosterone: ecological bases of hormone-behavior interactions.

A combination of laboratory and field investigations of birds has shown that expression of behavior such as territorial aggression can occur throughout the year in many species and in different life history stages. Although it is well known that testosterone regulates territorial aggression in males during the breeding season, the correlation of plasma testosterone and aggression appears to be limited to periods of social instability when a male is challenged for his territory by another male, or when mate-guarding a sexually receptive female. How essentially identical aggression is modulated in non-breeding life history stages is not fully resolved, but despite low circulating levels of testosterone outside the breeding season, expression of territorial aggression does appear to be dependent upon aromatization of testosterone and an estrogen receptor-mediated mechanism. There is accumulating evidence that prolonged high levels of circulating testosterone may incur costs that may potentially reduce lifetime fitness. These include interference with paternal care, exposure to predators, increased risk of injury, loss of fat stores and possibly impaired immune system function and oncogenic effects. We propose six hypotheses to explain how these costs of high testosterone levels in blood may be avoided. These hypotheses are testable and may reveal many mechanisms resulting from selection to avoid the costs of testosterone. It should also be noted that the hypotheses are applicable to vertebrates in general, and may also be relevant for other hormones that have a highly specialized suite of actions in one life history stage (such as breeding), but also have a limited action in other life history stages when the full spectrum of effects would be inappropriate.

Aggression↗
PubMed2000

Modified high-performance liquid chromatography assay for the measurement of 2'-deoxyuridine in human plasma and its application to pharmacodynamic studies of antimetabolite drugs.

A new method is presented for the HPLC determination of plasma 2'-deoxyuridine (dUrd). Briefly, 1 ml of human plasma is deproteinised with perchloric acid followed by purification by solid-phase extraction using a non-polar high-capacity polymeric sorbent. The dUrd is separated on a C18 reversed-phase column using a mobile-phase of 0.05% v/v trifluoroacetic acid in water, with a retention time of 8.5 min at a flow-rate of 1.25 ml min(-1). Quantitation is by UV detection at 261 nm using a photodiode array detector. The limit of quantitation is 6 nM with a linear response over the measured range 6-400 nM. Both intra- and inter-day RSD and bias are typically less than 13%. Chromatograms and pharmacodynamic data from a Phase 1 Clinical Trial of a new antifolate drug, ZD9331 are included to illustrate the utility of the method. They show the increase in circulating dUrd as a result of drug inhibition of the target enzyme thymidylate synthase. The method has the significant advantages of ease and simplicity over earlier methods and may be applied to the analysis of other nucleoside species.

Antimetabolites, Antineoplastic↗
PubMed2000

NADH oxidase activation is involved in arsenite-induced oxidative DNA damage in human vascular smooth muscle cells.

Arsenic is atherogenic, carcinogenic, and genotoxic. Because atherosclerotic plaque has been considered a benign smooth muscle cell tumor, we have studied the effects of arsenite on DNA integrity of human vascular smooth muscle cells. By using single-cell alkaline electrophoresis, apparent DNA strand breaks were detected in a 4-hour treatment with arsenite at a concentration above 1 micromol/L. DNA strand breaks of arsenite-treated cells were increased by Escherichia coli formamidopyrimidine-DNA glycosylase and decreased by diphenylene iodinium, superoxide dismutase, catalase, pyruvate, DMSO, or D-mannitol. Extract from arsenite-treated cells showed increased capacity for producing superoxide when NADH was included in the reaction mixture; however, addition of arsenite to extract from untreated cells did not increase superoxide production. The superoxide-producing ability of arsenite-treated cells was also suppressed by diphenylene iodinium, 4,5-dihydroxy-1, 2-benzenedisulfonic acid disodium salt (Tiron), or superoxide dismutase. Superoxide production and DNA strand breaks in arsenite-treated cells were also suppressed by transfecting antisense oligonucleotides of p22phox, an essential component of NADH oxidase. Treatment with arsenite also increased the mRNA level of p22phox. These results suggest that arsenite activates NADH oxidase to produce superoxide, which then causes oxidative DNA damage. The result that arsenite at low concentrations increases oxidant levels and causes oxidative DNA damage in vascular smooth muscle cells may be important in arsenic-induced atherosclerosis.

Aorta↗
PubMed2000

Decreased insulin responsiveness of glucose uptake in cultured human skeletal muscle cells from insulin-resistant nondiabetic relatives of type 2 diabetic families.

To investigate the contribution of inherited biochemical defects to the peripheral insulin resistance of type 2 diabetes, we studied cultured skeletal muscle from 10 insulin-resistant nondiabetic first-degree relatives of type 2 diabetic families and 6 control subjects. Insulin stimulation of glucose uptake and glycogen synthesis was maximal in myoblasts. Insulin-stimulated glucose uptake (fold-stimulation over basal uptake) was decreased in relative compared with control myoblasts at 0.001 micromol/l (0.93 +/- 0.05 [mean +/- SE] vs. 1.15 +/- 0.06, P < 0.05) and 0.1 micromol/l (1.38 +/- 0.10 vs. 1.69 +/- 0.08, P = 0.025) insulin. Insulin responsiveness was markedly impaired in 5 of the relative myoblast cultures, and in 4 of these, there was an associated increase in basal glucose uptake (76.7 +/- 7.0 vs. 47.4 +/- 5.5 pmol x min(-1) x mg(-1) protein, relative vs. control; P < 0.02). Expression of insulin receptor substrate 1, phosphatidylinositol 3-kinase, protein kinase B, and glycogen synthase was normal in the relative cultures with impaired insulin responsiveness. Glycogen synthesis was also normal in the relative cultures. We conclude that the persistence of impaired insulin responsiveness in some of the relative cultures supports the role of inherited factors in the insulin resistance of type 2 diabetes and that the association with increased basal glucose uptake suggests that the 2 abnormalities may be linked.

Adult↗
PubMed1999

Vicinal-thiol-containing molecules enhance but mono-thiol-containing molecules reduce nickel-induced DNA strand breaks.

Several thiol-containing molecules (TCM) are currently used as antidotes for nickel, and vicinal TCM seem to be more effective in mobilizing tissue nickel than are mono TCM. Using single cell alkaline electrophoresis, we have shown that the vicinal TCM, meso-2, 3-dimercaptosuccinic acid (DMSA), 2,3-dimercaptopropane-1-sulfonate, and 2,3-dimercaptopropanol markedly enhanced, whereas the mono TCM, D-penicillamide, glutathione, beta-mercaptoethanol, and diethyl dithiocarbomate, reduced nickel chloride (Ni)-induced DNA breaks in a human leukemia cell line, NB4 cells. Ni or TCM alone did not induce plasmid DNA breaks in test tubes and neither did Ni plus mono TCM; however, Ni plus vicinal TCM did. Vicinal TCM did, but mono TCM did not generate H(2)O(2) in solution. H(2)O(2) alone did not, but H(2)O(2) plus Ni induced plasmid DNA breaks. Although Ni plus glutathione did not break DNA, Ni plus glutathione plus H(2)O(2) did. The Ni-DMSA-induced DNA breaks in NB4 cells, as well as in plasmids, were completely prevented by d-mannitol or partially prevented by several antioxidants. Therefore, the DNA breaks induced by Ni plus vicinal TCM seem to be due to the complex of Ni with TCM in concert with the H(2)O(2) produced by the vicinal TCM. The results that DMSA at a concentration as low as 5 microM enhanced the Ni-induced DNA breaks suggest a further evaluation of the TCM as nickel chelators is needed.

Cell Line↗
PubMed1999

Dithiothreitol enhances arsenic trioxide-induced apoptosis in NB4 cells.

Recently, arsenic trioxide (As2O3) was reported to induce clinical remission in patients with acute promyelocytic leukemia. Modulation of protein phosphorylation by binding to the vicinal thiols has been suggested as a possible mechanism. We found that phenylarsine oxide, a strong vicinal thiol-binding agent, neither induced nuclear fragmentation or DNA laddering nor increased caspase activity in NB4 cells; however, As2O3 and a weak thiol-binding agent, dimethylarsinic acid, did increase activity. Dithiothreitol (DTT) effectively suppressed the phenylarsine oxide-inhibited cellular reductive capacity, but unexpectedly, enhanced As2O3-induced apoptosis in NB4 cells. As2O3-induced and As2O3-plus-DTT-induced apoptosis in NB4 cells was modulated by oxidant modifiers, but not by nitric oxide synthase inhibitors. These results demonstrate that DTT, a dithiol agent and known antidote for trivalent inorganic arsenic, enhances the toxicity of As2O3, thereby opening a new research direction for the mechanisms of arsenic toxicity and perhaps also helping in the development of new therapeutic strategies for treating leukemias.

Apoptosis↗
PubMed1999

Parent-offspring trios: a resource to facilitate the identification of type 2 diabetes genes.

The transmission disequilibrium test with use of trios (an affected proband with both parents) is a robust method for assessing the role of gene variants in disease that avoids the problem of population stratification that may confound conventional case/control studies and allows the detection of parent-of-origin effects. Trios have played a major role in defining genes in a number of polygenic conditions, including type 1 diabetes. We assessed the prevalence, clinical characteristics, and suitability for defining type 2 susceptibility genes of European type 2 diabetes trios. In a Caucasian population in the U.K., only 2.5% of type 2 patients had both parents alive. Using a nationwide strategy, we collected 182 trios defined by strict clinical criteria. Immunological and genetic testing resulted in the exclusion of 25 trios as a result of latent autoimmune diabetes (n = 13), inconsistent family relationships (n = 7), and maternally inherited diabetes and deafness (n = 5). The 157 remaining probands had similar treatment requirements to familial type 2 diabetic subjects but presented at a younger age, were more obese, and more frequently had affected parents. Using this resource, we have not found any evidence for linkage disequilibrium between type 2 diabetes and the glucokinase gene markers GCK1 and GCK2 and the chromosome 20 marker D20S197. We conclude that European type 2 diabetes trios are difficult to collect but provide an important additional approach to dissecting the genetics of type 2 diabetes.

Adult↗
PubMed1998

Calcium-dependent nitric oxide production is involved in arsenite-induced micronuclei.

Arsenic, a human carcinogen is known to induce sister-chromatid exchanges, chromosome aberrations and micronuclei (MN), but its mechanisms remain unknown. Recently, independent studies have suggested that intracellular calcium and reactive oxygen species are involved in arsenite-induced MN, and nitric oxide (NO) is involved in arsenite-induced poly(ADP-ribosylation). The aim of this research is to investigate the involvement of these molecules in arsenite-induced MN. The intracellular oxidant level and calcium level were monitored with a flow cytometer by using dichlorofluorescein diacetate and fluo3-AM, respectively. The NO production was estimated from the nitrite in cell culture medium with a spectrophotometer by using diaminonaphthalene. The results show that a 4-h treatment with arsenite above 5 microM, caused a dose-dependent increase of oxidant, NO, as well as intracellular calcium level. The arsenite-increased intracellular oxidant level was inhibited by NO synthase inhibitors, S-methyl-l-thiocitrulline and Nomega-nitro-l-arginine methyl ester and calcium chelators, ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and 2-[(2-bis-[carboxymethyl]-amino-5-methylphenoxy)-methyl]-6-methoxy-8- bis[carboxy-methyl]aminoquinoline, but not by catalase inhibitor, 3-aminotriazole. The arsenite-increased NO could also be suppressed by NO synthase inhibitors and calcium chelator. However, the arsenite-increased intracellular calcium level was inhibited by calcium chelators, but not by NO synthase inhibitors. A 4-h treatment with arsenite above 10 microM, also induced MN dose-dependently. The arsenite-increased MN could be reduced by NO synthase inhibitors, calcium chelators, as well as superoxide dismutase and uric acid. These results suggest the involvement of peroxynitrite in arsenite-induced MN. We surmise that the disturbance of NO production may cause cardio/peripheral vascular disorders, and the peroxynitrite-mediated DNA damages may cause genetic instability and, hence, cancers in arsenic-exposed humans.

Animals↗
PubMed1998

Arsenite stimulates poly(ADP-ribosylation) by generation of nitric oxide.

Recent studies indicate that arsenic may generate reactive oxygen species to exert its toxicity. Because reactive oxygen species are known to induce poly(ADP-ribosylation), which is implicated in DNA repair, signal transduction, and apoptosis, we have investigated the effect of arsenite on poly(ADP-ribosylation). The results showed that arsenite treatment induced poly(ADP-ribosylation), NAD depletion, DNA strand breaks, and micronuclei in CHO-K1 cells. Increase of nitrite level, a stable product of nitric oxide, was also detected in medium of arsenite-treated cultures. S-methyl-L-thiocitrulline and N omega-nitro-L-arginine methyl ester, inhibitors of nitric oxide synthase, could suppress the arsenite-induced NAD depletion, DNA strand breaks, and micronuclei, whereas 3-aminobenzamide, an inhibitor of poly (ADP-ribose) polymerase, could enhance micronucleus production and NAD depletion in arsenite-treated cells. These results suggest that arsenite treatment may generate nitric oxide to damage DNA and which then stimulate poly(ADP-ribosylation). Because arsenite also induced DNA strand breaks and NAD depletion in bovine aortic endothelial cells, and these could also be suppressed by S-methyl-L-thiocitrulline, the induction of nitric oxide may be important to the etiology of arsenic-induced vascular disorders in humans.

Animals↗
PubMed1998

Isolation and characterisation of an antifungal antibiotic (GR135402) with protein synthesis inhibition.

A novel antifungal antibiotic GR135402 has been isolated from a fermentation broth of Graphium putredinis which inhibited protein synthesis in Candida albicans but not rabbit reticulocytes. The spectrum of activity included C. albicans and Cryptococcus neoformans but not some other Candida species or Aspergillus species. Therapeutic efficacy in a mouse model of systemic candidosis was attained following parenteral dosing.

Animals↗
PubMed1997

Cadmium inhibits DNA strand break rejoining in methyl methanesulfonate-treated CHO-K1 cells.

The cogenotoxicity of Cd has been recognized. This effect may stem from Cd inhibition of DNA repair. We studied the effects of Cd on DNA repair of methyl methanesulfonate (MMS)-damaged Chinese hamster ovary cells (CHO-K1) by single-cell alkaline electrophoresis. The results indicate that in the presence of Cd, DNA strand breaks accumulated in MMS-treated cells. Using hydroxyurea (Hu) plus cytosine-beta-D-arabinofuranoside (AraC) to block DNA polymerization, DNA strand breaks accumulated and Cd had little inhibitory effects on these accumulations. However, Cd inhibited the rejoining of these DNA strand breaks, which could be rejoined 6 hr after release from Hu plus AraC blockage. These results indicate that the potency of Cd inhibition of DNA repair replication and/or ligation may be greater than the inhibition of DNA adduct excision. To further elucidate this mechanism, we used an in vitro cell-free assay system to analyze the Cd effects on DNA repair synthesis, DNA polymerization, and DNA ligation. We have shown a dose-dependent inhibition of these three activities by Cd in CHO-K1 cell extract. The IC50s of Cd were 55, 26, and 10 microM, respectively. Moreover, Cd inhibition of DNA ligation in cell extract could be recovered partially by thiol compounds such as glutathione, beta-mercaptoethanol, dithiothreitol, and metallothionein. Since both in vivo and in vitro studies demonstrated that Cd was more effectively involved in interfering with the DNA ligation step and that thiol agents could partially remove Cd inhibition of DNA ligation, we speculate that part of the Cd inhibition of DNA repair may be through binding of Cd to the proteins participating in DNA ligation.

Animals↗
PubMed1997

Magnesium reduces nickel inhibition of DNA polymerization.

The activities of DNA polymerization and DNA ligation in extract of Chinese hamster ovary cells were both stimulated by MgCl2. DNA polymerization was stimulated by MgCl2 above 0.25 mM, whereas, MgCl2 above 2 mM was required to stimulate DNA ligation. The activity of DNA polymerization maintained a plateau at MgCl2 1-12 mM, whereas DNA ligation reached a maximal activity at MgCl2 6 mM and decreased thereafter. NiCl2 0.1-0.2 mM also had a stimulatory effect on DNA polymerization, but was much less potent than MgCl2. However, nickel ion (Ni2+) had no detectable stimulating effect on the activity of DNA ligation. In the presence of MgCl2, the activities of DNA polymerization and DNA ligation decreased with increasing concentration of NiCl2. Ni2+ inhibition of DNA polymerization was reduced by increasing the concentration of MgCl2, but increasing the concentration of MgCl2 did not reduce Ni2+ inhibition of DNA ligation. Preincubating cell extract with MgCl2 decreased the Ni2+ inhibition of DNA polymerization but not DNA ligation. These results suggest that Ni2+ may compete with magnesium ion (Mg2+) to reduce DNA polymerization, but this mechanism seems not applicable to Ni2+ inhibition of DNA ligation.

Animals↗
PubMed1997

Arsenite retards DNA break rejoining by inhibiting DNA ligation.

Arsenic has been shown to inhibit methyl methane-sulphonate (MMS)-induced DNA repair but the exact mechanism remains controversial. The purpose of this investigation is to examine which step of DNA repair is most sensitive to arsenite (As) and how As inhibits it. The results from single-cell alkaline electrophoresis, showing post-treatment with As increased DNA strand breaks in MMS-treated cells, suggest that that the excision step seems to be less sensitive to As than later steps. To test this hypothesis, hydroxyurea (Hu) plus cytosine-beta-D-arabinofuranoside (AraC) were used to block DNA polymerization, allowing the DNA strand breaks to accumulate. These experiments indicated that As had weak inhibitory effects on DNA strand break accumulation. However, As inhibited the rejoining of those DNA strand breaks which could be rejoined within 4 h after release from blockage by Hu plus AraC. To further elucidate this mechanism, a cell extract was used to compare the relative sensitivity of the various steps in DNA repair to As. The potency of the As inhibitory effect as deduced from concentration-response curves were: ligation of poly(rA).oligo(dT) > ligation of poly(dA).oligo(dT) approximately DNA polymerization > or = DNA repair synthesis > excision. As is known to inhibit the activity of pyruvate dehydrogenase by interacting with vicinal dithiol groups. Dithiothreitol could effectively remove As inhibition of both the ligation of poly(rA).oligo(dT) and the activity of pyruvate dehydrogenase but had no obvious effect on As inhibition of poly(dA).oligo(dT) ligation. Since DNA ligase III contains vicinal dithiol groups, we postulate that As may inhibit DNA break rejoining by interacting with the vicinal dithiols to inactivate DNA ligation in MMS-treated cells.

Animals↗
PubMed1997

Reactive oxygen species are involved in nickel inhibition of DNA repair.

Nickel has been shown to inhibit DNA repair in a way that may play a role in its toxicity. Since nickel treatment increases cellular reactive oxygen species (ROS), we have investigated the involvement of ROS in nickel inhibition of DNA repair. Inhibition of glutathione synthesis or catalase activity increased the enhancing effect of nickel on the cytotoxicity of ultraviolet (UV) light. Inhibition of catalase and glutathione peroxidase activities also enhanced the retardation effect of nickel on the rejoining of DNA strand breaks accumulated by hydroxyurea plus cytosine-beta-D-arabinofuranoside in UV-irradiated cells. Since DNA polymerization and ligation are involved in the DNA-break rejoining, we have investigated the effect of ROS on these two steps in an extract of Chinese hamster ovary cells. Nickel inhibition of the incorporation of (3H)dTTP into the DNase I-activated calf thymus DNA was stronger than the ligation of poly(dA) x oligo(dT), whereas H2O2 was more potent in inhibiting DNA ligation than DNA polymerization. Nickel, in the presence of H2O2, exhibited a synergistic inhibition on both DNA polymerization and ligation and caused protein fragmentation. In addition, glutathione could completely recover the inhibition by nickel or H2O2 alone but only partially recover the inhibition by nickel plus H2O2. Therefore, nickel may bind to DNA-repair enzymes and generate oxygen-free radicals to cause protein degradation in situ. This irreversible damage to the proteins involved in DNA repair, replication, recombination, and transcription could be important for the toxic effects of nickel.

Animals↗