The guanidine group present in the amino acid arginine was found to react with the lipid hydroper... more The guanidine group present in the amino acid arginine was found to react with the lipid hydroperoxide-derived bifunctional electrophile, 4-oxo-2-nonenal. The reaction between N Rtert-butoxycarbony-L-arginine and 4-oxo-2-nonenal resulted in the formation of an adduct (adduct A) that subsequently dehydrated on heating to adduct B. Liquid chromatography/ mass spectrometry and nuclear magnetic resonance spectroscopy were used to assign the structure of adduct B as (N δ ,N ω′ -etheno-2′-heptanon-2′′-one)-N R -t-Boc-arginine. The reaction proceeded from initial reaction of the primary N ω -amino group at the C-1 aldehyde of 4-oxo-2-nonenal. Subsequently, an intramolecular Michael addition of a secondary N δ -amino group occurring at C-3 resulted in formation of the cyclic carbinolamine adduct A. Dehydration and rearrangement of the exocyclic imine resulted in the formation of adduct B, which contained a stable imidazole ring. The tetra peptide LRDE reacted with 4-oxo-2-nonenal primarily at arginine rather than at the amino terminus. This suggests that arginine-containing proteins can react with lipid hydroperoxide-derived 4-oxo-2-nonenal to form a novel imidazole modification.
Benzo[a]pyrene (B[a]P), a representative polycyclic aromatic hydrocarbon (PAH), is metabolically ... more Benzo[a]pyrene (B[a]P), a representative polycyclic aromatic hydrocarbon (PAH), is metabolically activated by three enzymatic pathways; by peroxidases (e.g. cytochrome P450-peroxidase) to yield radical cations; by P4501A1/1B1 monoxygenation plus epoxide hydrolase to yield diol-epoxides; and by P4501A1/1B1 monoxygenation, epoxide hydrolase plus aldo-keto reductases (AKRs) to yield o-quinones. In humans, a major exposure site for environmental and tobacco smoke PAH is the lung, however, the profile of B[a]P metabolites formed at this site has not been well characterized. In this study, human bronchoalveolar H358 cells were exposed to B[a]P, and metabolites generated by peroxidase (B[a]P-1,6-and B[a]P-3,6-diones), from cytochrome P4501A1/1B1 monooxygenation (3-hydroxyl-B[a]P, B[a]P-7,8-and 9,10-trans-dihydrodiols, and B[a]P -r-7,t-8,t-9,c-10tetrahydrotetrol (B[a]P -tetrol-1)), and from AKRs (B[a]P-7,8-dione) were detected and quantified by RP-HPLC-with in line photo-diode array and radiometric detection, and identified by LC-MS. Progress curves showed a lag-phase in the formation of 3-hydroxy-B[a]P, B[a]P-7,8-transdihydrodiol, B[a]P-tetraol-1 and B[a]P-7,8-dione over 24 h. Northern blot analysis showed that B
Amyotrophic lateral sclerosis is a rapidly progressing neurodegenerative disease associated with ... more Amyotrophic lateral sclerosis is a rapidly progressing neurodegenerative disease associated with protein misfolding and aggregation. Most cases are characterized by TDP-43 positive inclusions, while a minority of familial ALS cases are instead FUS and SOD1 positive respectively. Cells can generate inclusions of variable type including previously characterized aggresomes, IPOD or JUNQ structures depending on the misfolded protein. SOD1 invariably forms JUNQ inclusions but it remains unclear whether other ALS protein aggregates arise as one of these previously described inclusion types or form unique structures. Here we show that FUS variably partitioned to IPOD, JUNQ or alternate structures, contain a mobile fraction, were not microtubule dependent and initially did not contain ubiquitin. TDP-43 inclusions formed in a microtubule independent manner, did not contain a mobile fraction but variably colocalized to JUNQ inclusions and another alternate structure. We conclude that the RNA binding proteins TDP-43 and FUS do not consistently fit the currently characterised inclusion models suggesting that cells have a larger repertoire for generating inclusions than currently thought, and imply that toxicity in ALS does not stem from a particular aggregation process or aggregate structure.
Absolute quantification of protein biomarkers such as serum apolipoprotein A1 by both immunoassay... more Absolute quantification of protein biomarkers such as serum apolipoprotein A1 by both immunoassays and LC-MS can provide misleading results. Recombinant ApoA-1 internal standard was prepared using stable isotope labeling by amino acids in cell culture with [(13)C6(15)N2]-lysine and [(13)C9(15)N1]-tyrosine in human cells. A stable isotope dilution LC-MS method for serum ApoA-1 was validated and levels analyzed for 50 nonsmokers and 50 smokers. The concentration of ApoA-1 in nonsmokers was 169.4 mg/dl with an 18.4% reduction to 138.2 mg/dl in smokers. The validated assay will have clinical utility for assessing effects of smoking cessation and therapeutic or dietary interventions in high-risk populations.
Friedreich's ataxia (FRDA) is an autosomal recessive disease with metabolic abnormalities tha... more Friedreich's ataxia (FRDA) is an autosomal recessive disease with metabolic abnormalities that have been proposed to play an important role in the resulting neurodegeneration and cardiomyopathy. The inability to access the highly affected neuronal and cardiac tissues has hampered metabolic evaluation and biomarker development. Employment of a LC-MS-based method to determine whether platelets isolated from patients with FRDA exhibit differentiable metabolism compared with healthy controls. Isotopologue analysis showed a marked decrease in glucose incorporation with a concomitant increase in palmitate-derived acyl-CoA thioesters in FRDA platelets compared with controls. Our findings demonstrate that platelets can be used as a surrogate tissue for in vivo biomarker studies to monitor new therapeutic approaches for the treatment of FRDA.
A group ofwomen were fed two separate diets in a crossover study and urinary eicosanoids were qua... more A group ofwomen were fed two separate diets in a crossover study and urinary eicosanoids were quantified. One diet contained 3. 1% oftotal energy (en%) as polyunsaturated fatty acids (3.0 en% linoleic acid) and the other contained 8.4 en% polyunsaturated fatty acids (8.3 en% linoleic acid). Car- bohydrate replaced fat in the low-polyunsaturated-fat diet. No changes were observed in the
Replication stress induced by nucleotide deficiency plays an important role in cancer initiation.... more Replication stress induced by nucleotide deficiency plays an important role in cancer initiation. Replication stress in primary cells typically activates the cellular senescence tumor-suppression mechanism. Senescence bypass correlates with development of cancer, a disease characterized by metabolic reprogramming. However, the role of metabolic reprogramming in the cellular response to replication stress has been little explored. Here, we report that ataxia telangiectasia mutated (ATM) plays a central role in regulating the cellular response to replication stress by shifting cellular metabolism. ATM inactivation bypasses senescence induced by replication stress triggered by nucleotide deficiency. This was due to restoration of deoxyribonucleotide triphosphate (dNTP) levels through both upregulation of the pentose phosphate pathway via increased glucose-6-phosphate dehydrogenase (G6PD) activity and enhanced glucose and glutamine consumption. These phenotypes were mediated by a coordi...
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder primarily affecting mot... more Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder primarily affecting motor neurons. Mutations in optineurin cause a proportion of familial ALS cases, and wildtype optineurin is misfolded and forms inclusions in sporadic ALS patient motor neurons. However it is unknown how optineurin mutation or misfolding leads to ALS. Optineurin acts an adaptor protein connecting the molecular motor myosin VI to secretory vesicles and autophagosomes. Here we demonstrate that ALS-linked mutations p.Q398X and p.E478G disrupt the association of optineurin with myosin VI, leading to an abnormal diffuse cytoplasmic distribution, inhibition of secretory protein trafficking, endoplasmic reticulum (ER) stress and Golgi fragmentation in motor neuron-like NSC-34 cells. We also provide further insight into the role of optineurin as an autophagy receptor. Wildtype optineurin associated with lysosomes and promoted autophagosome fusion to lysosomes in neuronal cells, implying it mediates...
Metabolism of propionate involves the activated acyl-thioester propionyl-CoA intermediate. We emp... more Metabolism of propionate involves the activated acyl-thioester propionyl-CoA intermediate. We employed LC-MS/MS, LC-selected reaction monitoring/MS, and LC-high-resolution MS to investigate metabolism of propionate to acyl-CoA intermediates. We discovered that propionyl-CoA can serve as a precursor to the direct formation of a new six-carbon mono-unsaturated acyl-CoA. Time course and dose-response studies in human hepatocellular carcinoma HepG2 cells demonstrated that the six-carbon mono-unsaturated acyl-CoA was propionate-dependent and underwent further metabolism over time. Studies utilizing [(13)C1]propionate and [(13)C3]propionate suggested a mechanism of fatty acid synthesis, which maintained all six-carbon atoms from two propionate molecules. Metabolism of 2,2-[(2)H2]propionate to the new six-carbon mono-unsaturated acyl-CoA resulted in the complete loss of two deuterium atoms, indicating modification at C2 of the propionyl moiety. Coelution experiments and isotopic tracer stu...
Fresh bovine faeces were inoculated with a non-toxigenic, antibiotic resistant strain of Escheric... more Fresh bovine faeces were inoculated with a non-toxigenic, antibiotic resistant strain of Escherichia coli O157:H7, spread on the rump areas of 30 heifers and allowed to dry for 24 h. Ten of the cattle then entered the normal slaughter process without further treatment. The remaining cattle were washed with a powerhose for 1 min (10 animals) and 3 min (10 animals) before entering the normal slaughter process. Both washing treatments removed all visible faecal materials on the live animals although a significant reduction (P < 0.05) in E. coli O157:H7 levels on the hides was only observed on those animals which were powerhosed for 3 min. After slaughter, E. coli O157:H7 was detected on carcasses and on the knives and hands of operatives. Preslaughter washing for 3 min did not statistically reduce the numbers of E. coli O157:H7 transferred from the hide to the carcass during slaughter. However, the organism was not detected on three of the four areas of the carcass sampled, indicati...
Our previous work demonstrated that the 12-lipoxygenase metabolite of arachidonic acid, 12(S)-hyd... more Our previous work demonstrated that the 12-lipoxygenase metabolite of arachidonic acid, 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] induced a nondestructive and reversible retraction of cultured endothelial cells. In the current study we tested the hypothesis that tumor cells produce 12(S)-HETE during their interactions with endothelial cells which in turn induces endothelial cell retraction. Coincubation of Lewis lung carcinoma cells or elutriated B16 amelanotic melanoma (B16a) cells but not 3T3 fibroblasts with microvascular endothelial cells (CD3) resulted in a time- and concentration-dependent retraction of the CD3 monolayers as revealed by quantitative binding assays and phase contrast microscopy. Lewis lung carcinoma cell-induced endothelial cell retraction was blocked by specific lipoxygenase inhibitors but not by cyclooxygenase inhibitors, suggesting the involvement of a lipoxygenase metabolite(s). Radioimmunoassay and high-performance liquid chromatography analysis of t...
Charcot-Marie-Tooth disease type 1A (CMT1A) is a hereditary peripheral neuropathy with a genetic ... more Charcot-Marie-Tooth disease type 1A (CMT1A) is a hereditary peripheral neuropathy with a genetic locus on chromosome 17p11.2. The majority of patients carry a duplicated DNA segment that encompasses the gene PMP22, which encodes a peripheral myelin protein. PMP22 is the crucial gene involved in the pathogenesis of CMT1A. Molecular diagnosis of CMT1A requires detection of this duplicated segment. Existing methods for detection of the duplication are laborious and time consuming. We have developed a set of polymorphic (AC)n repeat markers (contained within the duplication) for use in the polymerase chain reaction, which give a high probability of detecting three unique alleles in affected individuals. This test detected 85% of a panel of 52 CMT1A patients in which the duplication had previously been demonstrated.
A highly specific stable isotope dilution assay for plasma 6-oxo-prostaglandin F1 alpha has been ... more A highly specific stable isotope dilution assay for plasma 6-oxo-prostaglandin F1 alpha has been developed. The method employs capillary column gas chromatography coupled with negative ion chemical ionisation mass spectrometry. The limit of sensitivity of the assay is 0.5 pg.m1(-1). Concentrations of 6-oxo-prostaglandin F1 alpha in the plasma of 20 healthy volunteers determined by this assay were all below 3 pg.m1(-1). The levels were much lower than any previously reported and confirms that prostacyclin is not a circulating hormone in man under normal physiological conditions.
Considerable consumer concerns about chilled food production, distribution and storage systems ha... more Considerable consumer concerns about chilled food production, distribution and storage systems have, during the last decade, led to increased surveillance and legislative control of most elements of the chilled food chain. However, whilst the rest of the temperature control chain is ever ...
American journal of obstetrics and gynecology, 2014
We sought to identify serum biomarkers of early spontaneous preterm birth (SPTB) using semiquanti... more We sought to identify serum biomarkers of early spontaneous preterm birth (SPTB) using semiquantitative proteomic analyses. This was a nested case-control study of pregnant women with previous SPTB. Maternal serum was collected at 19-24 and 28-32 weeks' gestation, and analyzed by liquid chromatography-multiple reaction monitoring/mass spectrometry. Targeted and shotgun proteomics identified 31 candidate proteins that were differentially expressed in pooled serum samples from spontaneous preterm (cases [<34 weeks]) and term (controls) deliveries. Candidate protein expression was compared in individual serum samples between cases and controls matched by age and race groups, and clinical site. Protein expression was verified by Western blot in the placenta and fetal membranes from cases and controls. Serum samples were available for 35 cases and 35 controls at 19-24 weeks, and 16 cases and 16 controls at 28-32 weeks. One protein, serpin B7, yielded serum concentrations that diff...
A discrepancy between published values of PGI2 production by human umbilical artery in vitro meas... more A discrepancy between published values of PGI2 production by human umbilical artery in vitro measured by platelet bioassay, compared with values of 6-oxo-PGF1 alpha by radioimmunoassay, raised the possibility that another anti-aggregatory prostanoid was produced by this tissue. To test this hypothesis, umbilical artery rings were incubated in buffer and PGI2 determined by platelet bioassay and by a more specific radioimmunoassay based on comparison of 6-oxo-PGF1 alpha in hydrolysed and non-hydrolysed samples. 6-oxo-PGF1 alpha, PGF2 alpha and TXB2 were also measured by gas chromatography negative ion chemical ionisation mass spectrometry. PGI2 concentrations by radioimmunoassay and bioassay were significantly correlated (r = 0.92, p less than 0.01). There was no difference between them, disproving the presence of an additional antiaggregatory substance. PGI2 production determined by bioassay (mean 1.21 ng/mg wet weight/h, range 0.59-1.53 ng/mg/h) differed from previously reported values (range 70-325 ng/mg/h). 6-oxo-PGF1 alpha concentrations were confirmed by gas chromatography negative ion chemical ionisation mass spectrometry. Previous determinations of PGI2 production by this tissue overestimated it by approximately 100 times.
Human alveolar macrophages, obtained during diagnostic bronchoscopy, were maintained in monolayer... more Human alveolar macrophages, obtained during diagnostic bronchoscopy, were maintained in monolayer culture. Challenge of these cells (greater than 95% purity) with 1.2 mg/ml zymosan A particles (opsonized with human serum) was followed by a rapid release of leukotriene B4 into the medium, 7.28 +/- 5.99 ng/mg cell protein at 2 h (mean +/- S.D.4, n = 4). Leukotriene B4 was identified and measured by a novel technique employing capillary column gas chromatography coupled to negative ion chemical ionization mass spectrometry. The release of thromboxane B2, prostaglandins D2, E2, F2 alpha and the lysosomal enzyme N-acetyl-beta-D-glucosaminidase was also measured. Thromboxane B2 was the most abundant metabolite of arachidonic acid released into the culture medium (65.2 +/- 14.8 ng/mg cell protein 2 h after the addition of zymosan A, n = 4), and the synthesis of thromboxane B2 was inhibited by greater than 90% in 1 microM Na flurbiprofen. Inhibition of cyclooxygenase activity was accompanied by a 2-fold increase in leukotriene B4 synthesis.
Journal of pulmonary & respiratory medicine, Jan 30, 2013
The standard of care in Locally-Advanced Non-Small Cell Lung Cancer (LA-NSCLC) is chemotherapy an... more The standard of care in Locally-Advanced Non-Small Cell Lung Cancer (LA-NSCLC) is chemotherapy and radiation; however, Radiation-Induced Lung Injury (RILI), which may be prevented by the anti-inflammatory and anti-oxidant properties of Flaxseed (FS), impedes its maximum benefit. Patients with LA-NSCLC requiring definitive RT were randomized to one FS or control muffin daily from start to 2 weeks after RT. Blood and urine were collected to quantify plasma FS metabolites, Enterodione (ED) and Enterolactone (EL), and urinary oxidative stress biomarkers, 8, 12-iso-iPF2a-VI (isoprostane) and 8-oxo-7,8-dihydro-2'deoxyguanosine (8-oxo-dGuo). Tolerability was defined as consuming ≥ 75% of the intended muffins and no ≥ grade 3 gastrointestinal toxicities. Fourteen patients (control,7; FS,7) were enrolled. The tolerability rates were 42.9 versus 71.4% (p=0.59) for FS and control, respectively. Mean percentages of intended number of muffins consumed were 37% versus 73% (p=0.12). ED and EL ...
Proceedings of the National Academy of Sciences, 2014
The cardiovascular safety of nonsteroidal antiinflammatory drugs (NSAIDs) may be influenced by in... more The cardiovascular safety of nonsteroidal antiinflammatory drugs (NSAIDs) may be influenced by interactions with antiplatelet doses of aspirin. We sought to quantitate precisely the propensity of commonly consumed NSAIDs—ibuprofen, naproxen, and celecoxib—to cause a drug-drug interaction with aspirin in vivo by measuring the target engagement of aspirin directly by MS. We developed a novel assay of cyclooxygenase-1 (COX-1) acetylation in platelets isolated from volunteers who were administered aspirin and used conventional and microfluidic assays to evaluate platelet function. Although ibuprofen, naproxen, and celecoxib all had the potential to compete with the access of aspirin to the substrate binding channel of COX-1 in vitro, exposure of volunteers to a single therapeutic dose of each NSAID followed by 325 mg aspirin revealed a potent drug-drug interaction between ibuprofen and aspirin and between naproxen and aspirin but not between celecoxib and aspirin. The imprecision of estimates of aspirin consumption and the differential impact on the ability of aspirin to inactivate platelet COX-1 will confound head-to-head comparisons of distinct NSAIDs in ongoing clinical studies designed to measure their cardiovascular risk.
The guanidine group present in the amino acid arginine was found to react with the lipid hydroper... more The guanidine group present in the amino acid arginine was found to react with the lipid hydroperoxide-derived bifunctional electrophile, 4-oxo-2-nonenal. The reaction between N Rtert-butoxycarbony-L-arginine and 4-oxo-2-nonenal resulted in the formation of an adduct (adduct A) that subsequently dehydrated on heating to adduct B. Liquid chromatography/ mass spectrometry and nuclear magnetic resonance spectroscopy were used to assign the structure of adduct B as (N δ ,N ω′ -etheno-2′-heptanon-2′′-one)-N R -t-Boc-arginine. The reaction proceeded from initial reaction of the primary N ω -amino group at the C-1 aldehyde of 4-oxo-2-nonenal. Subsequently, an intramolecular Michael addition of a secondary N δ -amino group occurring at C-3 resulted in formation of the cyclic carbinolamine adduct A. Dehydration and rearrangement of the exocyclic imine resulted in the formation of adduct B, which contained a stable imidazole ring. The tetra peptide LRDE reacted with 4-oxo-2-nonenal primarily at arginine rather than at the amino terminus. This suggests that arginine-containing proteins can react with lipid hydroperoxide-derived 4-oxo-2-nonenal to form a novel imidazole modification.
Benzo[a]pyrene (B[a]P), a representative polycyclic aromatic hydrocarbon (PAH), is metabolically ... more Benzo[a]pyrene (B[a]P), a representative polycyclic aromatic hydrocarbon (PAH), is metabolically activated by three enzymatic pathways; by peroxidases (e.g. cytochrome P450-peroxidase) to yield radical cations; by P4501A1/1B1 monoxygenation plus epoxide hydrolase to yield diol-epoxides; and by P4501A1/1B1 monoxygenation, epoxide hydrolase plus aldo-keto reductases (AKRs) to yield o-quinones. In humans, a major exposure site for environmental and tobacco smoke PAH is the lung, however, the profile of B[a]P metabolites formed at this site has not been well characterized. In this study, human bronchoalveolar H358 cells were exposed to B[a]P, and metabolites generated by peroxidase (B[a]P-1,6-and B[a]P-3,6-diones), from cytochrome P4501A1/1B1 monooxygenation (3-hydroxyl-B[a]P, B[a]P-7,8-and 9,10-trans-dihydrodiols, and B[a]P -r-7,t-8,t-9,c-10tetrahydrotetrol (B[a]P -tetrol-1)), and from AKRs (B[a]P-7,8-dione) were detected and quantified by RP-HPLC-with in line photo-diode array and radiometric detection, and identified by LC-MS. Progress curves showed a lag-phase in the formation of 3-hydroxy-B[a]P, B[a]P-7,8-transdihydrodiol, B[a]P-tetraol-1 and B[a]P-7,8-dione over 24 h. Northern blot analysis showed that B
Amyotrophic lateral sclerosis is a rapidly progressing neurodegenerative disease associated with ... more Amyotrophic lateral sclerosis is a rapidly progressing neurodegenerative disease associated with protein misfolding and aggregation. Most cases are characterized by TDP-43 positive inclusions, while a minority of familial ALS cases are instead FUS and SOD1 positive respectively. Cells can generate inclusions of variable type including previously characterized aggresomes, IPOD or JUNQ structures depending on the misfolded protein. SOD1 invariably forms JUNQ inclusions but it remains unclear whether other ALS protein aggregates arise as one of these previously described inclusion types or form unique structures. Here we show that FUS variably partitioned to IPOD, JUNQ or alternate structures, contain a mobile fraction, were not microtubule dependent and initially did not contain ubiquitin. TDP-43 inclusions formed in a microtubule independent manner, did not contain a mobile fraction but variably colocalized to JUNQ inclusions and another alternate structure. We conclude that the RNA binding proteins TDP-43 and FUS do not consistently fit the currently characterised inclusion models suggesting that cells have a larger repertoire for generating inclusions than currently thought, and imply that toxicity in ALS does not stem from a particular aggregation process or aggregate structure.
Absolute quantification of protein biomarkers such as serum apolipoprotein A1 by both immunoassay... more Absolute quantification of protein biomarkers such as serum apolipoprotein A1 by both immunoassays and LC-MS can provide misleading results. Recombinant ApoA-1 internal standard was prepared using stable isotope labeling by amino acids in cell culture with [(13)C6(15)N2]-lysine and [(13)C9(15)N1]-tyrosine in human cells. A stable isotope dilution LC-MS method for serum ApoA-1 was validated and levels analyzed for 50 nonsmokers and 50 smokers. The concentration of ApoA-1 in nonsmokers was 169.4 mg/dl with an 18.4% reduction to 138.2 mg/dl in smokers. The validated assay will have clinical utility for assessing effects of smoking cessation and therapeutic or dietary interventions in high-risk populations.
Friedreich's ataxia (FRDA) is an autosomal recessive disease with metabolic abnormalities tha... more Friedreich's ataxia (FRDA) is an autosomal recessive disease with metabolic abnormalities that have been proposed to play an important role in the resulting neurodegeneration and cardiomyopathy. The inability to access the highly affected neuronal and cardiac tissues has hampered metabolic evaluation and biomarker development. Employment of a LC-MS-based method to determine whether platelets isolated from patients with FRDA exhibit differentiable metabolism compared with healthy controls. Isotopologue analysis showed a marked decrease in glucose incorporation with a concomitant increase in palmitate-derived acyl-CoA thioesters in FRDA platelets compared with controls. Our findings demonstrate that platelets can be used as a surrogate tissue for in vivo biomarker studies to monitor new therapeutic approaches for the treatment of FRDA.
A group ofwomen were fed two separate diets in a crossover study and urinary eicosanoids were qua... more A group ofwomen were fed two separate diets in a crossover study and urinary eicosanoids were quantified. One diet contained 3. 1% oftotal energy (en%) as polyunsaturated fatty acids (3.0 en% linoleic acid) and the other contained 8.4 en% polyunsaturated fatty acids (8.3 en% linoleic acid). Car- bohydrate replaced fat in the low-polyunsaturated-fat diet. No changes were observed in the
Replication stress induced by nucleotide deficiency plays an important role in cancer initiation.... more Replication stress induced by nucleotide deficiency plays an important role in cancer initiation. Replication stress in primary cells typically activates the cellular senescence tumor-suppression mechanism. Senescence bypass correlates with development of cancer, a disease characterized by metabolic reprogramming. However, the role of metabolic reprogramming in the cellular response to replication stress has been little explored. Here, we report that ataxia telangiectasia mutated (ATM) plays a central role in regulating the cellular response to replication stress by shifting cellular metabolism. ATM inactivation bypasses senescence induced by replication stress triggered by nucleotide deficiency. This was due to restoration of deoxyribonucleotide triphosphate (dNTP) levels through both upregulation of the pentose phosphate pathway via increased glucose-6-phosphate dehydrogenase (G6PD) activity and enhanced glucose and glutamine consumption. These phenotypes were mediated by a coordi...
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder primarily affecting mot... more Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder primarily affecting motor neurons. Mutations in optineurin cause a proportion of familial ALS cases, and wildtype optineurin is misfolded and forms inclusions in sporadic ALS patient motor neurons. However it is unknown how optineurin mutation or misfolding leads to ALS. Optineurin acts an adaptor protein connecting the molecular motor myosin VI to secretory vesicles and autophagosomes. Here we demonstrate that ALS-linked mutations p.Q398X and p.E478G disrupt the association of optineurin with myosin VI, leading to an abnormal diffuse cytoplasmic distribution, inhibition of secretory protein trafficking, endoplasmic reticulum (ER) stress and Golgi fragmentation in motor neuron-like NSC-34 cells. We also provide further insight into the role of optineurin as an autophagy receptor. Wildtype optineurin associated with lysosomes and promoted autophagosome fusion to lysosomes in neuronal cells, implying it mediates...
Metabolism of propionate involves the activated acyl-thioester propionyl-CoA intermediate. We emp... more Metabolism of propionate involves the activated acyl-thioester propionyl-CoA intermediate. We employed LC-MS/MS, LC-selected reaction monitoring/MS, and LC-high-resolution MS to investigate metabolism of propionate to acyl-CoA intermediates. We discovered that propionyl-CoA can serve as a precursor to the direct formation of a new six-carbon mono-unsaturated acyl-CoA. Time course and dose-response studies in human hepatocellular carcinoma HepG2 cells demonstrated that the six-carbon mono-unsaturated acyl-CoA was propionate-dependent and underwent further metabolism over time. Studies utilizing [(13)C1]propionate and [(13)C3]propionate suggested a mechanism of fatty acid synthesis, which maintained all six-carbon atoms from two propionate molecules. Metabolism of 2,2-[(2)H2]propionate to the new six-carbon mono-unsaturated acyl-CoA resulted in the complete loss of two deuterium atoms, indicating modification at C2 of the propionyl moiety. Coelution experiments and isotopic tracer stu...
Fresh bovine faeces were inoculated with a non-toxigenic, antibiotic resistant strain of Escheric... more Fresh bovine faeces were inoculated with a non-toxigenic, antibiotic resistant strain of Escherichia coli O157:H7, spread on the rump areas of 30 heifers and allowed to dry for 24 h. Ten of the cattle then entered the normal slaughter process without further treatment. The remaining cattle were washed with a powerhose for 1 min (10 animals) and 3 min (10 animals) before entering the normal slaughter process. Both washing treatments removed all visible faecal materials on the live animals although a significant reduction (P < 0.05) in E. coli O157:H7 levels on the hides was only observed on those animals which were powerhosed for 3 min. After slaughter, E. coli O157:H7 was detected on carcasses and on the knives and hands of operatives. Preslaughter washing for 3 min did not statistically reduce the numbers of E. coli O157:H7 transferred from the hide to the carcass during slaughter. However, the organism was not detected on three of the four areas of the carcass sampled, indicati...
Our previous work demonstrated that the 12-lipoxygenase metabolite of arachidonic acid, 12(S)-hyd... more Our previous work demonstrated that the 12-lipoxygenase metabolite of arachidonic acid, 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] induced a nondestructive and reversible retraction of cultured endothelial cells. In the current study we tested the hypothesis that tumor cells produce 12(S)-HETE during their interactions with endothelial cells which in turn induces endothelial cell retraction. Coincubation of Lewis lung carcinoma cells or elutriated B16 amelanotic melanoma (B16a) cells but not 3T3 fibroblasts with microvascular endothelial cells (CD3) resulted in a time- and concentration-dependent retraction of the CD3 monolayers as revealed by quantitative binding assays and phase contrast microscopy. Lewis lung carcinoma cell-induced endothelial cell retraction was blocked by specific lipoxygenase inhibitors but not by cyclooxygenase inhibitors, suggesting the involvement of a lipoxygenase metabolite(s). Radioimmunoassay and high-performance liquid chromatography analysis of t...
Charcot-Marie-Tooth disease type 1A (CMT1A) is a hereditary peripheral neuropathy with a genetic ... more Charcot-Marie-Tooth disease type 1A (CMT1A) is a hereditary peripheral neuropathy with a genetic locus on chromosome 17p11.2. The majority of patients carry a duplicated DNA segment that encompasses the gene PMP22, which encodes a peripheral myelin protein. PMP22 is the crucial gene involved in the pathogenesis of CMT1A. Molecular diagnosis of CMT1A requires detection of this duplicated segment. Existing methods for detection of the duplication are laborious and time consuming. We have developed a set of polymorphic (AC)n repeat markers (contained within the duplication) for use in the polymerase chain reaction, which give a high probability of detecting three unique alleles in affected individuals. This test detected 85% of a panel of 52 CMT1A patients in which the duplication had previously been demonstrated.
A highly specific stable isotope dilution assay for plasma 6-oxo-prostaglandin F1 alpha has been ... more A highly specific stable isotope dilution assay for plasma 6-oxo-prostaglandin F1 alpha has been developed. The method employs capillary column gas chromatography coupled with negative ion chemical ionisation mass spectrometry. The limit of sensitivity of the assay is 0.5 pg.m1(-1). Concentrations of 6-oxo-prostaglandin F1 alpha in the plasma of 20 healthy volunteers determined by this assay were all below 3 pg.m1(-1). The levels were much lower than any previously reported and confirms that prostacyclin is not a circulating hormone in man under normal physiological conditions.
Considerable consumer concerns about chilled food production, distribution and storage systems ha... more Considerable consumer concerns about chilled food production, distribution and storage systems have, during the last decade, led to increased surveillance and legislative control of most elements of the chilled food chain. However, whilst the rest of the temperature control chain is ever ...
American journal of obstetrics and gynecology, 2014
We sought to identify serum biomarkers of early spontaneous preterm birth (SPTB) using semiquanti... more We sought to identify serum biomarkers of early spontaneous preterm birth (SPTB) using semiquantitative proteomic analyses. This was a nested case-control study of pregnant women with previous SPTB. Maternal serum was collected at 19-24 and 28-32 weeks' gestation, and analyzed by liquid chromatography-multiple reaction monitoring/mass spectrometry. Targeted and shotgun proteomics identified 31 candidate proteins that were differentially expressed in pooled serum samples from spontaneous preterm (cases [<34 weeks]) and term (controls) deliveries. Candidate protein expression was compared in individual serum samples between cases and controls matched by age and race groups, and clinical site. Protein expression was verified by Western blot in the placenta and fetal membranes from cases and controls. Serum samples were available for 35 cases and 35 controls at 19-24 weeks, and 16 cases and 16 controls at 28-32 weeks. One protein, serpin B7, yielded serum concentrations that diff...
A discrepancy between published values of PGI2 production by human umbilical artery in vitro meas... more A discrepancy between published values of PGI2 production by human umbilical artery in vitro measured by platelet bioassay, compared with values of 6-oxo-PGF1 alpha by radioimmunoassay, raised the possibility that another anti-aggregatory prostanoid was produced by this tissue. To test this hypothesis, umbilical artery rings were incubated in buffer and PGI2 determined by platelet bioassay and by a more specific radioimmunoassay based on comparison of 6-oxo-PGF1 alpha in hydrolysed and non-hydrolysed samples. 6-oxo-PGF1 alpha, PGF2 alpha and TXB2 were also measured by gas chromatography negative ion chemical ionisation mass spectrometry. PGI2 concentrations by radioimmunoassay and bioassay were significantly correlated (r = 0.92, p less than 0.01). There was no difference between them, disproving the presence of an additional antiaggregatory substance. PGI2 production determined by bioassay (mean 1.21 ng/mg wet weight/h, range 0.59-1.53 ng/mg/h) differed from previously reported values (range 70-325 ng/mg/h). 6-oxo-PGF1 alpha concentrations were confirmed by gas chromatography negative ion chemical ionisation mass spectrometry. Previous determinations of PGI2 production by this tissue overestimated it by approximately 100 times.
Human alveolar macrophages, obtained during diagnostic bronchoscopy, were maintained in monolayer... more Human alveolar macrophages, obtained during diagnostic bronchoscopy, were maintained in monolayer culture. Challenge of these cells (greater than 95% purity) with 1.2 mg/ml zymosan A particles (opsonized with human serum) was followed by a rapid release of leukotriene B4 into the medium, 7.28 +/- 5.99 ng/mg cell protein at 2 h (mean +/- S.D.4, n = 4). Leukotriene B4 was identified and measured by a novel technique employing capillary column gas chromatography coupled to negative ion chemical ionization mass spectrometry. The release of thromboxane B2, prostaglandins D2, E2, F2 alpha and the lysosomal enzyme N-acetyl-beta-D-glucosaminidase was also measured. Thromboxane B2 was the most abundant metabolite of arachidonic acid released into the culture medium (65.2 +/- 14.8 ng/mg cell protein 2 h after the addition of zymosan A, n = 4), and the synthesis of thromboxane B2 was inhibited by greater than 90% in 1 microM Na flurbiprofen. Inhibition of cyclooxygenase activity was accompanied by a 2-fold increase in leukotriene B4 synthesis.
Journal of pulmonary & respiratory medicine, Jan 30, 2013
The standard of care in Locally-Advanced Non-Small Cell Lung Cancer (LA-NSCLC) is chemotherapy an... more The standard of care in Locally-Advanced Non-Small Cell Lung Cancer (LA-NSCLC) is chemotherapy and radiation; however, Radiation-Induced Lung Injury (RILI), which may be prevented by the anti-inflammatory and anti-oxidant properties of Flaxseed (FS), impedes its maximum benefit. Patients with LA-NSCLC requiring definitive RT were randomized to one FS or control muffin daily from start to 2 weeks after RT. Blood and urine were collected to quantify plasma FS metabolites, Enterodione (ED) and Enterolactone (EL), and urinary oxidative stress biomarkers, 8, 12-iso-iPF2a-VI (isoprostane) and 8-oxo-7,8-dihydro-2'deoxyguanosine (8-oxo-dGuo). Tolerability was defined as consuming ≥ 75% of the intended muffins and no ≥ grade 3 gastrointestinal toxicities. Fourteen patients (control,7; FS,7) were enrolled. The tolerability rates were 42.9 versus 71.4% (p=0.59) for FS and control, respectively. Mean percentages of intended number of muffins consumed were 37% versus 73% (p=0.12). ED and EL ...
Proceedings of the National Academy of Sciences, 2014
The cardiovascular safety of nonsteroidal antiinflammatory drugs (NSAIDs) may be influenced by in... more The cardiovascular safety of nonsteroidal antiinflammatory drugs (NSAIDs) may be influenced by interactions with antiplatelet doses of aspirin. We sought to quantitate precisely the propensity of commonly consumed NSAIDs—ibuprofen, naproxen, and celecoxib—to cause a drug-drug interaction with aspirin in vivo by measuring the target engagement of aspirin directly by MS. We developed a novel assay of cyclooxygenase-1 (COX-1) acetylation in platelets isolated from volunteers who were administered aspirin and used conventional and microfluidic assays to evaluate platelet function. Although ibuprofen, naproxen, and celecoxib all had the potential to compete with the access of aspirin to the substrate binding channel of COX-1 in vitro, exposure of volunteers to a single therapeutic dose of each NSAID followed by 325 mg aspirin revealed a potent drug-drug interaction between ibuprofen and aspirin and between naproxen and aspirin but not between celecoxib and aspirin. The imprecision of estimates of aspirin consumption and the differential impact on the ability of aspirin to inactivate platelet COX-1 will confound head-to-head comparisons of distinct NSAIDs in ongoing clinical studies designed to measure their cardiovascular risk.
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