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T J Lyons

Publications and source records attributed to T J Lyons.

At least 19 recordsLinked to original sources
PubMed2000

Effect of collagen turnover on the accumulation of advanced glycation end products.

Collagen molecules in articular cartilage have an exceptionally long lifetime, which makes them susceptible to the accumulation of advanced glycation end products (AGEs). In fact, in comparison to other collagen-rich tissues, articular cartilage contains relatively high amounts of the AGE pentosidine. To test the hypothesis that this higher AGE accumulation is primarily the result of the slow turnover of cartilage collagen, AGE levels in cartilage and skin collagen were compared with the degree of racemization of aspartic acid (% d-Asp, a measure of the residence time of a protein). AGE (N(epsilon)-(carboxymethyl)lysine, N(epsilon)-(carboxyethyl)lysine, and pentosidine) and % d-Asp concentrations increased linearly with age in both cartilage and skin collagen (p < 0.0001). The rate of increase in AGEs was greater in cartilage collagen than in skin collagen (p < 0.0001). % d-Asp was also higher in cartilage collagen than in skin collagen (p < 0.0001), indicating that cartilage collagen has a longer residence time in the tissue, and thus a slower turnover, than skin collagen. In both types of collagen, AGE concentrations increased linearly with % d-Asp (p < 0.0005). Interestingly, the slopes of the curves of AGEs versus % d-Asp, i.e. the rates of accumulation of AGEs corrected for turnover, were identical for cartilage and skin collagen. The present study thus provides the first experimental evidence that protein turnover is a major determinant in AGE accumulation in different collagen types. From the age-related increases in % d-Asp the half-life of cartilage collagen was calculated to be 117 years and that of skin collagen 15 years, thereby providing the first reasonable estimates of the half-lives of these collagens.

Adolescent↗
PubMed2000

Genome-wide characterization of the Zap1p zinc-responsive regulon in yeast.

The Zap1p transcription factor senses cellular zinc status and increases expression of its target genes in response to zinc deficiency. Previously known Zap1p-regulated genes encode the Zrt1p, Zrt2p, and Zrt3p zinc transporter genes and Zap1p itself. To allow the characterization of additional genes in yeast important for zinc homeostasis, a systematic study of gene expression on the genome-wide scale was used to identify other Zap1p target genes. Using a combination of DNA microarrays and a computer-assisted analysis of shared motifs in the promoters of similarly regulated genes, we identified 46 genes that are potentially regulated by Zap1p. Zap1p-regulated expression of seven of these newly identified target genes was confirmed independently by using lacZ reporter fusions, suggesting that many of the remaining candidate genes are also Zap1p targets. Our studies demonstrate the efficacy of this combined approach to define the regulon of a specific eukaryotic transcription factor.

Gene Expression Profiling↗
PubMed2000

X-ray crystallographic and analytical ultracentrifugation analyses of truncated and full-length yeast copper chaperones for SOD (LYS7): a dimer-dimer model of LYS7-SOD association and copper delivery.

Copper-zinc superoxide dismutase (CuZnSOD) acquires its catalytic copper ion through interaction with another polypeptide termed the copper chaperone for SOD. Here, we combine X-ray crystallographic and analytical ultracentrifugation methods to characterize rigorously both truncated and full-length forms of apo-LYS7, the yeast copper chaperone for SOD. The 1.55 A crystal structure of LYS7 domain 2 alone (L7D2) was determined by multiple-isomorphous replacement (MIR) methods. The monomeric structure reveals an eight-stranded Greek key beta-barrel similar to that found in yeast CuZnSOD, but it is substantially elongated at one end where the loop regions of the beta-barrel come together to bind a calcium ion. In agreement with the crystal structure, sedimentation velocity experiments indicate that L7D2 is monomeric in solution under all conditions and concentrations that were tested. In contrast, sedimentation velocity and sedimentation equilibrium experiments show that full-length apo-LYS7 exists in a monomer-dimer equilibrium under nonreducing conditions. This equilibrium is shifted toward the dimer by approximately 1 order of magnitude in the presence of phosphate anion. Although the basis for the specificity of the LYS7-SOD interaction as well as the exact mechanism of copper insertion into SOD is unknown, it has been suggested that a monomer of LYS7 and a monomer of SOD may associate to form a heterodimer via L7D2. The data presented here, however, taken together with previously published crystallographic and analytical gel filtration data on full-length LYS7, suggest an alternative model wherein a dimer of LYS7 interacts with a dimer of yeast CuZnSOD. The advantages of the dimer-dimer model over the heterodimer model are enumerated.

Computer Simulation↗
PubMed2000

The metal binding properties of the zinc site of yeast copper-zinc superoxide dismutase: implications for amyotrophic lateral sclerosis.

We have investigated factors that influence the properties of the zinc binding site in yeast copper-zinc superoxide dismutase (CuZnSOD). The properties of yeast CuZnSOD are essentially invariant from pH 5 to pH 9. However, below this pH range there is a change in the nature of the zinc binding site which can be interpreted as either (1) a change in metal binding affinity from strong to weak, (2) the expulsion of the metal bound at this site, or (3) a transition from a normal distorted tetrahedral ligand orientation to a more symmetric arrangement of ligands. This change is strongly reminiscent of a similar pH-induced transition seen for the bovine protein and, based on the data presented herein, is proposed to be a property that is conserved among CuZnSODs. The transition demonstrated for the yeast protein is not only sensitive to the pH of the buffering solution but also to the occupancy and redox status of the adjacent copper binding site. Furthermore, we have investigated the effect of single site mutations on the pH- and redox-sensitivity of Co2+ binding at the zinc site. Each of the mutants H46R, H48Q, H63A, H63E, H80C, G85R, and D83H is capable of binding Co2+ to a zinc site with a distorted tetrahedral geometry similar to that of wild-type. However, they do so only if Cu+ is bound at the copper site or if the pH in raised to near physiological levels, indicating that the change at the zinc binding site seen in the wild-type is conserved in the mutants, albeit with an altered pKa. The mutants H71C and D83A did not bind Co2+ in a wild-type-like fashion under any of the conditions tested. This study reveals that the zinc binding site is exquisitely sensitive to changes in the protein environment. Since three of the mutant yeast proteins investigated here contain mutations analogous to those that cause ALS (amyotrophic lateral sclerosis) in humans, this finding implicates improper metal binding as a mechanism by which CuZnSOD mutants exert their toxic gain of function.

Amyotrophic Lateral Sclerosis↗
PubMed2000

Native and modified LDL activate extracellular signal-regulated kinases in mesangial cells.

Glycation and/or oxidation of LDL may promote diabetic nephropathy. The mitogen-activated protein kinase (MAPK) cascade, which includes extracellular signal-regulated protein kinases (ERKs), modulates cell function. Therefore, we examined the effects of LDL on ERK phosphorylation in cultured rat mesangial cells. In cells exposed to 100 microg/ml native LDL or LDL modified by glycation, and/or mild or marked (copper-mediated) oxidation, ERK activation peaked at 5 min. Five minutes of exposure to 10-100 microg/ml native or modified LDL produced a concentration-dependent (up to sevenfold) increase in ERK activity. Also, 10 microg/ml native LDL and mildly modified LDL (glycated and/or mildly oxidized) produced significantly greater ERK activation than that induced by copper-oxidized LDL +/- glycation (P < 0.05). Pretreatment of cells with Src kinase and MAPK kinase inhibitors blocked ERK activation by 50-80% (P < 0.05). Native and mildly modified LDL, which are recognized by the native LDL receptor, induced a transient spike of intracellular calcium. Copper-oxidized (+/- glycation) LDL, recognized by the scavenger receptor, induced a sustained rise in intracellular calcium. The intracellular calcium chelator (EGTA/AM) further increased ERK activation by native and mildly modified LDL (P < 0.05). These findings demonstrate that native and modified LDL activate ERKs 1 and 2, an early mitogenic signal, in mesangial cells and provide evidence for a potential link between modified LDL and the development of glomerular injury in diabetes.

Animals↗
PubMed2000

Aminoguanidine and the effects of modified LDL on cultured retinal capillary cells.

PURPOSE: Compared with normal low density lipoprotein (N-LDL), LDL minimally modified in vitro by glycation, minimal oxidation, or glycoxidation (G-, MO-, GO-LDL) decreases survival of cultured retinal capillary endothelial cells and pericytes. Similar modifications occurring in vivo in diabetes may contribute to retinopathy. The goal of this study was to determine whether low concentrations of aminoguanidine might prevent cytotoxic modification of LDL and/or protect retinal capillary cells from previously modified LDL. METHODS: Minimal in vitro modification of LDL (3 days, 37 degrees C) was achieved with glucose (0, 50 mM), under antioxidant conditions (for N-LDL, G-LDL), or under mild oxidant conditions (for MO-, GO-LDL) in the presence/absence of aminoguanidine (0, 1, 10, 100 microM). Glucose and aminoguanidine were then removed by dialysis. Confluent bovine retinal capillary endothelial cells (n = 13) and pericytes (n = 14) were exposed to LDL (100 mg/l) for 3 days, with and without aminoguanidine (100 microM) in media. Cell counts were determined by hemocytometer. RESULTS: A decrease in cell counts after exposure to modified compared with N-LDL was confirmed (P < 0.001) but was significantly mitigated if LDL had been modified in the presence of aminoguanidine (P < 0.001). Aminoguanidine was as effective at 1 microM as at the higher concentrations. Aminoguanidine (100 microM) present in culture media conferred no additional protection, and showed slight evidence of toxicity. Aminoguanidine present during LDL modification had no effect on measured glycation or oxidation products, or on LDL oxidizability. CONCLUSIONS: Very low concentrations of aminoguanidine mitigate toxicity of LDL exposed to stresses that simulate the diabetic environment. This action may contribute to the beneficial effects of aminoguanidine observed in experimental diabetic retinopathy.

Animals↗
PubMed1999

Quantification of N-(glucitol)ethanolamine and N-(carboxymethyl)serine: two products of nonenzymatic modification of aminophospholipids formed in vivo.

Chemical, nonenzymatic modification of protein and lipids by reducing sugars, such as glucose, is thought to contribute to age-related deterioration in tissue protein and cellular membranes and to the pathogenesis of diabetic complications. This report describes the synthesis and quantification of N-(glucitol)ethanolamine (GE) and N-(carboxymethyl)serine (CMS), two products of nonenzymatic modification of aminophospholipids. GE is the product of reduction and hydrolysis of glycated phosphatidylethanolamine (PE), while CMS is formed through reaction of phosphatidylserine (PS) with products of oxidation of either carbohydrate (glycoxidation) or lipids (lipoxidation). Gas chromatography/mass spectrometry procedures for quantification of the N,O-acetyl methyl ester derivatives of the modified head groups were developed. GE and CMS were quantified in samples of PE and PS, respectively, following incubation with glucose in vitro; CMS formation was dependent on the presence of oxygen during the incubation. Both GE and CMS were detected and quantified in lipid extracts of human red blood cell membranes. The content of GE, but not CMS, was increased in the lipids from diabetic compared to nondiabetic subjects. Measurement of these modified lipids should prove useful for assessing the role of carbonyl-amine reactions of aminophospholipids in aging and age-related diseases.

Aging↗
PubMed1999

The role of computed tomography in the diagnosis of arterial gas embolism in fatal diving accidents in Tasmania.

Four cases of fatal diving accidents in Tasmania are presented, highlighting the role of CT in the investigation of diving fatalities. The CT technique allows rapid diagnosis when arterial gas embolism (AGE) is suspected. The traditional method of investigation, underwater autopsy, is a difficult procedure that requires specialized training in which the subtle diagnosis of AGE may be completely missed. Facilities for performing underwater autopsies are normally available only in tertiary referral centres, and therefore the diagnosis of AGE may be missed due to lack of facilities. The use of CT in the diagnosis of AGE in divers was first utilized in the early 1980s but has still not become widely adopted in forensic practice. This radiological technique has the advantage of being sensitive, quick, reliable, readily available and provides a permanent record. For hospitals that do not have a resident forensic pathologist, a CT scan can be easily performed and interpreted to eliminate the possibility of AGE. There are a number of pitfalls in the diagnosis of AGE with CT, particularly intravascular gas production following postmortem fermentation and off-gassing. Awareness of these pitfalls will help the radiologist in making a correct diagnosis of AGE.

Accidents↗
PubMed1999

Pre-enrichment of modified low density lipoproteins with alpha-tocopherol mitigates adverse effects on cultured retinal capillary cells.

PURPOSE: We determined whether pre-enrichment of low density lipoproteins (LDL) with alpha-tocopherol mitigates their adverse effects, following in vitro glycation, oxidation or glycoxidation, towards cultured bovine retinal capillary endothelial cells (RCEC) and pericytes. METHODS: LDL, while still in plasma obtained and pooled from non-diabetic humans, was supplemented in vitro with alpha-tocopherol. It was then isolated and modified in vitro by glycation, minimal oxidation, and glycoxidation. Bovine RCEC and pericytes were exposed to LDL (100mg protein/ ml) for three days. Cell count was determined by cell counting, supernatant levels of plasminogen activator inhibitor-1 (PAI-1) and endothelin-1 (ET-1) by ELISA, and nitrite levels by spectroscopic colorimetric assay. RESULTS: While pre-enrichment of LDL with alpha-tocopherol did not reduce the measured extent of lipoprotein modification, it abolished the reduction in cell count observed with glycated, oxidized and glycoxidized LDL v. normal LDL. Pre-enrichment of LDL with alpha-tocopherol also reduced RCEC supernatant PAI-1 and ET-1 (corrected for cell counts) and increased RCEC and pericyte-associated supernatant nitrite levels: such effects of alpha-tocopherol may inhibit clot formation and favor vasodilatation. CONCLUSIONS: Enrichment of LDL with alpha-tocopherol abolishes adverse effects of glycated, mildly oxidized, and glycoxidized LDL on cultured retinal cell count, and mitigates adverse effects on modulators of fibrinolysis and vascular tone. Direct evidence is required before Vitamin E supplementation is recommended for people with diabetes.

Animals↗
PubMed1999

The transoceanic air evacuation of unstable angina patients.

BACKGROUND: The United States Air Force (USAF) air evacuation system in the Western Pacific frequently transports unstable angina patients on fixed wing aircraft over long distances to reach definitive cardiologic and cardiothoracic capabilities. This study begins to answer the question: Does the transfer of unstable angina (i.e., crescendo angina or rest angina) patients over long distances adversely affect the overall outcome of such patients? METHODS: A case series study was conducted by obtaining a list of urgent and priority unstable angina patients from the Pacific theater from January 1992 through December 1996. Each of these patients' inpatient records were reviewed for cardiac catheterization (cath) result, ejection fraction (EF), previous cardiac history, angioplasty (PTCA) procedures, coronary artery bypass graft (CABG) surgery, and whether a patient had congestive heart failure (CHF), myocardial infarction (MI), death or other complications on this admission. RESULTS: Of the 59 records reviewed, 61% (36/59) had coronary artery disease (CAD) of at least one vessel greater than 90% occlusion, 31% (18/59) had PTCA, 24% (14/59) had a CABG, 5% (3/59) had CHF, 3% (2/59) had an MI, 24% had some type of a complication, and 1.7% (1) died on this admission. CONCLUSION: Patients with significant CAD seem to tolerate air transport well with relatively few complications considering the overall morbidity and mortality associated with the diagnosis of unstable angina.

Adult↗
PubMed1998

The dark side of dioxygen biochemistry.

The cellular biochemistry of dioxygen is Janus-faced. The good side includes numerous enzyme-catalyzed reactions of dioxygen that occur in respiration and normal metabolism, while the dark side encompasses deleterious reactions of species derived from dioxygen that lead to damage of cellular components. These reactive oxygen species have historically been perceived almost exclusively as agents of the dark side, but it has recently become clear that they play beneficial roles as well.

Antioxidants↗
PubMed1998

Frequency of the "push-pull effect" in U.S. Air Force fighter operations.

BACKGROUND: Recent investigation into the push-pull effect (PPE), the reduction of +Gz tolerance when preceded by less than +1 Gz, has focused on centrifuge studies to demonstrate the presence of adverse cardiovascular responses. Maneuvers found to cause the Push-Pull Effect (PPEM) have not been studied previously in U.S. Air Force (USAF) fighter aircraft. The frequency of and extent to which PPEMs are performed in fighter aircraft are unknown. METHODS: Head-up display (HUD) videotapes from F-15 and F-16 air combat training missions were reviewed for the presence of PPEMs. The frequency of engagements containing PPEMs and the magnitude of the Gz profiles were noted. RESULTS: PPEMs were found in 11 to 67%, of engagements reviewed, depending on the nature of the training mission, with an overall average of 32%. The PPEMs that were observed contained segments of less than +1 Gz, ranging on average from 0.0 to 0.5 Gz for an average of 3.5 to 5 s duration. CONCLUSIONS: PPEMs are present in air combat training missions performed by today's USAF fighter aircraft and represent an operationally significant source of risk for accidents. These findings support continued research into the physiologic response to PPE and the development of countermeasures.

Acceleration↗
PubMed1998

The "push-pull effect" and G-induced loss of consciousness accidents in the U.S. Air Force.

BACKGROUND: A recent Canadian Forces CF-18 Hornet aircraft accident has focused interest on the push-pull effect (PPE). PPE has not previously been identified in U.S. Air Force (USAF) G-Induced Loss of Consciousness (G-LOC) Accidents. The presence of maneuvers known to cause the push-pull effect (PPEMs) that lead to G-LOC accidents suggests that PPE is operationally significant and is a potential cause of G-LOC accidents. METHODS: USAF accident reports where G-LOC was found to be causal were reviewed for indications of a PPEM immediately prior to the G-LOC. Terminology in the narratives was used to indicate a PPEM in the accident sequences. RESULTS: In 3 of 24 mishaps, the presence of a PPEM was highly probable, while another 4 mishaps were found to have a probable association with PPEMs. The probable presence of PPEMs represents a significant percentage (12.5-29%) of USAF G-LOC accidents. CONCLUSION: USAF G-LOC accident reports contained descriptions that indicated the presence of PPEMs in accident sequences. This finding suggests that the PPE is an operationally significant source of risk for accidents in USAF high-performance aircraft.

Acceleration↗
PubMed1997

Age-dependent increase in ortho-tyrosine and methionine sulfoxide in human skin collagen is not accelerated in diabetes. Evidence against a generalized increase in oxidative stress in diabetes.

The glycoxidation products Nepsilon-(carboxymethyl)lysine and pentosidine increase in skin collagen with age and at an accelerated rate in diabetes. Their age-adjusted concentrations in skin collagen are correlated with the severity of diabetic complications. To determine the relative roles of increased glycation and/or oxidation in the accelerated formation of glycoxidation products in diabetes, we measured levels of amino acid oxidation products, distinct from glycoxidative modifications of amino acids, as independent indicators of oxidative stress and damage to collagen in aging and diabetes. We show that ortho-tyrosine and methionine sulfoxide are formed in concert with Nepsilon-(carboxymethyl)lysine and pentosidine during glycoxidation of collagen in vitro, and that they also increase with age in human skin collagen. The age-adjusted levels of these oxidized amino acids in collagen was the same in diabetic and nondiabetic subjects, arguing that diabetes per se does not cause an increase in oxidative stress or damage to extracellular matrix proteins. These results provide evidence for an age-dependent increase in oxidative damage to collagen and support previous conclusions that the increase in glycoxidation products in skin collagen in diabetes can be explained by the increase in glycemia alone, without invoking a generalized, diabetes-dependent increase in oxidative stress.

Adolescent↗
PubMed1997

Carboxymethylethanolamine, a biomarker of phospholipid modification during the maillard reaction in vivo.

Nepsilon-(Carboxymethyl)lysine (CML) is a stable chemical modification of proteins formed from both carbohydrates and lipids during autoxidation reactions. We hypothesized that carboxymethyl lipids such as (carboxymethyl)phosphatidylethanolamine (carboxymethyl-PE) would also be formed in these reactions, and we therefore developed a gas chromatography-mass spectrometry assay for quantification of carboxymethylethanolamine (CME) following hydrolysis of phospholipids. In vitro, CME was formed during glycation of dioleoyl-PE under air and from linoleoylpalmitoyl-PE, but not from dioleoyl-PE, in the absence of glucose. In vivo, CME was detected in lipid extracts of red blood cell membranes, approximately 0.14 mmol of CME/mol of ethanolamine, from control and diabetic subjects, (n = 22, p >> 0.5). Levels of CML in erythrocyte membrane proteins were approximately 0.2 mmol/mol of lysine for both control and diabetic subjects (p >> 0.5). For this group of diabetic subjects there was no indication of increased oxidative modification of either lipid or protein components of red cell membranes. CME was also detected in fasting urine at 2-3 nmol/mg of creatinine in control and diabetic subjects (p = 0.085). CME inhibited detection of advanced glycation end product (AGE)-modified protein in a competitive enzyme-linked immunosorbent assay using an anti-AGE antibody previously shown to recognize CML, suggesting that carboxymethyl-PE may be a component of AGE lipids detected in AGE low density lipoprotein. Measurement of levels of CME in blood, tissues, and urine should be useful for assessing oxidative damage to membrane lipids during aging and in disease.

Adult↗
PubMed1997

Quantification of malondialdehyde and 4-hydroxynonenal adducts to lysine residues in native and oxidized human low-density lipoprotein.

Malondialdehyde (MDA) and 4-hydroxynonenal (HNE) are major end-products of oxidation of polyunsaturated fatty acids, and are frequently measured as indicators of lipid peroxidation and oxidative stress in vivo. MDA forms Schiff-base adducts with lysine residues and cross-links proteins in vitro; HNE also reacts with lysines, primarily via a Michael addition reaction. We have developed methods using NaBH4 reduction to stabilize these adducts to conditions used for acid hydrolysis of protein, and have prepared reduced forms of lysine-MDA [3-(N epsilon-lysino)propan-1-ol (LM)], the lysine-MDA-lysine iminopropene cross-link [1,3-di(N epsilon-lysino)propane (LML)] and lysine-HNE [3-(N epsilon-lysino)-4-hydroxynonan-l-ol (LHNE)]. Gas chromatography/MS assays have been developed for quantification of the reduced compounds in protein. RNase incubated with MDA or HNE was used as a model for quantification of the adducts by gas chromatography/MS. There was excellent agreement between measurement of MDA bound to RNase as LM and LML, and as thiobarbituric acid-MDA adducts measured by HPLC; these adducts accounted for 70-80% of total lysine loss during the reaction with MDA. LM and LML (0.002-0.12 mmol/ mol of lysine) were also found in freshly isolated low-density lipoprotein (LDL) from healthy subjects. LHNE was measured in RNase treated with HNE, but was not detectable in native LDL. LM, LML and LHNE increased in concert with the formation of conjugated dienes during the copper-catalysed oxidation of LDL, but accounted for modification of < 1% of lysine residues in oxidized LDL. These results are the first report of direct chemical measurement of MDA and HNE adducts to lysine residues in LDL. LM, LML and LHNE should be useful as biomarkers of lipid peroxidative modification of protein and of oxidative stress in vitro and in vivo.

Aldehydes↗