Peter Arthur

Detecting Changes in the Thiol Redox State of Proteins Following a Decrease in Oxygen Concentration Using a Dual Labeling

Publisher: American Chemical Society (ACS)

Date: 25-11-2010

DOI: 10.1021/PR900702Z

Abstract: Cells are routinely exposed to hyperoxic conditions when cultured in the presence of 95% air and 5% carbon dioxide. Hyperoxic conditions can increase the generation of reactive oxygen species and cause oxidative stress. Oxidative stress has been proposed to cause cells in culture to behave differently from cells in vivo. One route by which oxidative stress could affect cellular function is through alterations in protein function caused by the oxidation of thiol groups (-SH) of redox-sensitive cysteine residues. To test whether changes in oxygen concentration were sufficient to cause changes in the thiol redox state of proteins, we developed a sensitive method involving the labeling of reduced and oxidized cysteine residues with fluorescent tags. Using this dual labeling method, we found 62 of 411 protein spots that were significantly more reduced following a 30 min decrease in oxygen concentration. We conclude that the elevated oxygen concentration characteristic of typical cell culture conditions has the potential to affect cellular behavior through changes in the thiol redox state of proteins.

Erythropoietin preconditioning in neuronal cultures: Signaling, protection from in vitro ischemia, and proteomic analysis

Publisher: Wiley

Date: 2006

DOI: 10.1002/JNR.20755

Abstract: In this study we confirmed the presence of the erythropoietin (EPO) receptor on both cultured cortical neurons and PC12 cells and showed that EPO can induce changes in p38, ERK, and JNK signaling molecules in these cells. We induced EPO preconditioning in cortical neuronal cultures that protected neurons from a subsequent in vitro ischemic insult (transient oxygen-glucose deprivation). To investigate downstream changes in protein expression in EPO-preconditioned cortical neuronal cultures, we used two-dimensional gel electrophoresis. Overall, EPO preconditioning resulted in protein up-regulation, and, from 84 of the most differentially expressed proteins selected for identification, the proteins or tentative proteins were identified in 57 cases, representing 40 different proteins. Different protein spots representing the same or closely related protein(s) occurred for 13 of the identified proteins and are likely to represent posttranslational modifications or proteolytic fragments of the protein. Two proteins (78-kD glucose-regulated protein and tropomyosin, fibroblast isoform 1) were detected in control neuronal cultures, but not following EPO preconditioning treatment, whereas one protein (40S ribosomal protein SA) was detected only following EPO preconditioning. Most of the other proteins identified had not previously been associated with EPO preconditioning and will aid in the understanding of EPO's neuroprotective response and possibly the development of new therapeutic interventions to inhibit neuronal death in acute and chronic neurodegenerative diseases.

Hypoxia causes downregulation of protein and RNA synthesis in noncontracting mammalian cardiomyocytes

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 19-04-2002

DOI: 10.1161/01.RES.0000015592.95986.03

Abstract: The aim was to identify energy-consuming processes, other than contraction, downregulated during moderate hypoxia (≈5 μmol/L, 0.5% O 2 ) and severe hypoxia ( .5 μmol/L, .05% O 2 ) in isolated neonatal cardiomyocytes. The metabolic response of cardiomyocytes to moderate and severe hypoxia was assessed by measuring rates of energy consumption and energetic status of cells maintained under these conditions. We found that the rates of energy production were decreased during both forms of hypoxia. Decreased rates of energy production under moderate hypoxia were associated with reduced energy wastage through a downregulation of proton leak in the mitochondria. Cellular protein synthesis and RNA synthesis, major energy-consuming pathways, were downregulated only during severe hypoxia, when oxygen concentrations were low enough to induce energetic stress (quantitatively defined as being any situation in which phosphocreatine concentrations had fallen by ≥40%). Our results suggest that energetic stress is the signal responsible for this downregulation.

The location of protein oxidation in dystrophic skeletal muscle from the mdx mouse model of Duchenne muscular dystrophy

Publisher: Elsevier BV

Date: 12-2022

DOI: 10.1016/J.ACTHIS.2022.151959

Abstract: Duchenne muscular dystrophy (DMD) is a severe childhood disease characterised by progressive muscle wasting caused by widespread myofibre necrosis. Implicated in the pathology of DMD is oxidative stress, caused by excessive generation of reactive oxygen and nitrogen species (RONS). One consequence of RONS exposure is post-translational oxidative modifications to proteins, which can cause loss of protein function. This study used the dystrophic mdx mouse model for DMD to visualise the precise location of different oxidative modifications to proteins in dystrophic muscles, including both reversible (protein thiol oxidation and s-nitrosylation) and irreversible (carbonylation and dityrosine formation) oxidation at various stages of dystrophic muscle necrosis and regeneration. High levels of protein oxidation were observed in mdx myofibres undergoing degeneration and immune cell infiltration (myonecrosis). Since irreversible protein oxidation, especially dityrosine formation, was only colocalised to areas of myonecrosis, we suggest that this specific measurement could be a useful biomarker of myonecrosis. To test this we quantified dityrosines in muscle homogenates this analysis showed significantly higher levels of dityrosines in mdx (compared with control normal) mice aged 23 days, an age when acute onset of extensive myonecrosis occurs in mdx muscles. These results indicate a major localised role of immune cells in RONS generation in dystrophic muscle, and strongly support a role for protein oxidation in myonecrosis and associated dystropathology. Consequently, the measurement of protein oxidation (specifically dityrosines) in dystrophic muscles may be a useful biomarker for indirectly quantifying myonecrosis in research studies using mdx mice and other animal models for DMD.

Exercise intensity and metabolic response in singles tennis

Publisher: Informa UK Limited

Date: 1998

DOI: 10.1080/026404198366371

Abstract: The aim of this study was to determine exercise intensity and metabolic response during singles tennis play. Techniques for assessment of exercise intensity were studied on-court and in the laboratory. The on-court study required eight State-level tennis players to complete a competitive singles tennis match. During the laboratory study, a separate group of seven male subjects performed an intermittent and a continuous treadmill run. During tennis play, heart rate (HR) and relative exercise intensity (72 +/- 1.9% VO2max estimated from measurement of heart rate) remained constant (83.4 +/- 0.9% HRmax mean +/- s(x)) after the second change of end. The peak value for estimated play intensity (1.25 +/- 0.11 steps x s(-1) from video analysis) occurred after the fourth change of end (P< 0.005). Plasma lactate concentration, measured at rest and at the change of ends, increased 175% from 2.13 +/- 0.32 mmol x l(-1) at rest to a peak 5.86 +/- 1.33 mmol x l(-1) after the sixth change of end (P < 0.001). A linear regression model, which included significant terms for %HRmax (P< 0.001), estimated play intensity (P < 0.001) and subject (P < 0.00), as well as a %HRmax subject interaction (P < 0.05), accounted for 82% of the variation in plasma lactate concentration. During intermittent laboratory treadmill running, % VO2peak estimated from heart rate was 17% higher than the value derived from the measured VO2 (79.7 +/- 2.2% and 69.0 +/- 2.5% VO2peak respectively P< 0.001). The %VO2peak was estimated with reasonable accuracy during continuous treadmill running (5% error). We conclude that changes in exercise intensity based on measurements of heart rate and a time-motion analysis of court movement patterns explain the variation in lactate concentration observed during singles tennis, and that measuring heart rate during play, in association with preliminary fitness tests to estimate VO2, will overestimate the aerobic response.

MEASUREMENT OF THE MILK INTAKE OF BREAST-FED INFANTS

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 09-1987

DOI: 10.1097/00005176-198709000-00017

Abstract: Infant test weighing and maternal test weighing are two independent methods for determining milk intake by the breast-fed infant. The sources of error in both these test weighing methods were examined with particular emphasis on the importance of evaluating and correcting for evaporative water loss (EWL). EWL ranged from 3 to 94% of the mother's change in weight after a single breast feed and from 3 to 55% of the infant's change in weight after a single breast feed. Correcting for EWL during a breast feed involved determining the time between the pre- and postfed weighings and measuring the rate of EWL after breast feeding. Significant correlations (p less than 0.001) were found between milk volume intake measured by test weighing the mother and correcting for maternal EWL, and milk volume intake measured by test weighing the infant and correcting for infant EWL. An improved method for measuring 24-h milk intakes by maternal test weighing using a sensitive electronic balance and correcting for EWL is described. The milk intakes, corrected for EWL, ranged from 690-1,041 g/24 h. If no correction for EWL was made then the average overestimate of milk intake by maternal test weighing was 14 +/- 6%.

Prepare, do, review: A skills-based approach for laboratory practical classes in biochemistry and molecular biology

Publisher: Wiley

Date: 06-05-2016

DOI: 10.1002/BMB.20951

Abstract: A new laboratory practical system is described which is comprised of a number of laboratory practical modules, each based around a particular technique or set of techniques, related to the theory part of the course but not designed to be dependent on it. Each module comprises an online recorded pre-lab lecture, the laboratory practical itself and a post-lab session in which students make oral presentations on different aspects of the practical. Each part of the module is assessed with the aim of providing rapid feedback to staff and students. Each laboratory practical is the responsibility of a single staff member and through this "ownership," continual review and updating is promoted. Ex les of changes made by staff to modules as a result of student feedback are detailed. A survey of students who had experienced both the old-style laboratory course and the new one provided evidence of increased satisfaction with the new program. The assessment of acquired shills in the new program showed that it was much more effective than the old course. © 2016 by The International Union of Biochemistry and Molecular Biology, 44:276-287, 2016.

A single 30min treadmill exercise session is suitable for ‘proof-of concept studies’ in adult mdx mice: A comparison of the early consequences of two different treadmill protocols

Publisher: Elsevier BV

Date: 02-2012

DOI: 10.1016/J.NMD.2011.07.008

Abstract: The extent of muscle pathology in sedentary adult mdx mice is very low and treadmill exercise is often used to increase myofibre necrosis however, the early events in dystrophic muscle and blood in response to treadmill exercise (leading to myofibre necrosis) are unknown. This study describes in detail two standardised protocols for the treadmill exercise of mdx mice and profiles changes in molecular and cellular events after a single 30 min treadmill session (Protocol A) or after 4 weeks of (twice weekly) treadmill exercise (Protocol B). Both treadmill protocols increased multiple markers of muscle damage. We conclude that a single 30 min treadmill exercise session is a sufficient and conveniently fast screening test and could be used in 'proof-of-concept' studies to evaluate the benefits of pre-clinical drugs in vivo. Myofibre necrosis, blood serum CK and oxidative stress (specifically the ratio of oxidised to reduced protein thiols) are reliable markers of muscle damage after exercise many parameters demonstrated high biological variation including changes in mRNA levels for key inflammatory cytokines in muscle. The s ling (sacrifice and tissue collection) time after exercise for these parameters is critical. A more precise understanding of the changes in dystrophic muscle after exercise aims to identify biomarkers and new potential therapeutic drug targets for Duchenne Muscular Dystrophy.

Taurine deficiency, synthesis and transport in the mdx mouse model for Duchenne Muscular Dystrophy

Publisher: Elsevier BV

Date: 09-2015

DOI: 10.1016/J.BIOCEL.2015.07.016

Abstract: The amino acid taurine is essential for the function of skeletal muscle and administration is proposed as a treatment for Duchenne Muscular Dystrophy (DMD). Taurine homeostasis is dependent on multiple processes including absorption of taurine from food, endogenous synthesis from cysteine and reabsorption in the kidney. This study investigates the cause of reported taurine deficiency in the dystrophic mdx mouse model of DMD. Levels of metabolites (taurine, cysteine, cysteine sulfinate and hypotaurine) and proteins (taurine transporter [TauT], cysteine deoxygenase and cysteine sulfinate dehydrogenase) were quantified in juvenile control C57 and dystrophic mdx mice aged 18 days, 4 and 6 weeks. In C57 mice, taurine content was much higher in both liver and plasma at 18 days, and both cysteine and cysteine deoxygenase were increased. As taurine levels decreased in maturing C57 mice, there was increased transport (reabsorption) of taurine in the kidney and muscle. In mdx mice, taurine and cysteine levels were much lower in liver and plasma at 18 days, and in muscle cysteine was low at 18 days, whereas taurine was lower at 4: these changes were associated with perturbations in taurine transport in liver, kidney and muscle and altered metabolism in liver and kidney. These data suggest that the maintenance of adequate body taurine relies on sufficient dietary intake of taurine and cysteine availability and metabolism, as well as retention of taurine by the kidney. This research indicates dystrophin deficiency not only perturbs taurine metabolism in the muscle but also affects taurine metabolism in the liver and kidney, and supports targeting cysteine and taurine deficiency as a potential therapy for DMD.

Mitochondrial hydrogen peroxide production alters oxygen consumption in an oxygen-concentration-dependent manner

Publisher: Elsevier BV

Date: 06-2005

DOI: 10.1016/J.FREERADBIOMED.2005.02.028

Abstract: Metabolic responses of mammalian cells toward declining oxygen concentration are generally thought to occur when oxygen limits mitochondrial ATP production. However, at oxygen concentrations markedly above those limiting to mitochondria, several mammalian cell types display reduced rates of oxygen consumption without energy stress or compensatory increases in glycolytic ATP production. We used mammalian Jurkat T cells as a model system to identify mechanisms responsible for these changes in metabolic rate. Oxygen consumption was 31% greater at high oxygen (150-200 microM) compared to low oxygen (5-10 microM). Hydrogen peroxide was implicated in the response as catalase prevented the increase in oxygen consumption normally associated with high oxygen. Cell-derived hydrogen peroxide, predominately from the mitochondria, was elevated with high oxygen. Oxygen consumption related to intracellular calcium turnover was shown, through EDTA chelation and dantrolene antagonism of the ryanodine receptor, to account for 70% of the response. Oligomycin inhibition of oxygen consumption indicated that mitochondrial proton leak was also sensitive to changes in oxygen concentration. Our results point toward a mechanism in which changes in oxygen concentration influence the rate of hydrogen peroxide production by mitochondria, which, in turn, alters cellular ATP use associated with intracellular calcium turnover and energy wastage through mitochondrial proton leak.

Muscle Pathology in Dystrophic Rats and Zebrafish Is Unresponsive to Taurine Treatment, Compared to the mdx Mouse Model for Duchenne Muscular Dystrophy

Publisher: MDPI AG

Date: 04-02-2023

DOI: 10.3390/METABO13020232

Abstract: Inflammation and oxidative stress are strongly implicated in the pathology of Duchenne muscular dystrophy (DMD), and the sulphur-containing amino acid taurine ameliorates both and decreases dystropathology in the mdx mouse model for DMD. We therefore further tested taurine as a therapy using dystrophic DMDmdx rats and dmd zebrafish models for DMD that have a more severe dystropathology. However, taurine treatment had little effect on the indices of dystropathology in both these models. While we and others have previously observed a deficiency in taurine in mdx mice, in the current study we show that the rat and zebrafish models had increased taurine content compared with wild-type, and taurine treatment did not increase muscle taurine levels. We therefore hypothesised that endogenous levels of taurine are a key determinate in potential taurine treatment efficacy. Because of this, we felt it important to measure taurine levels in DMD patient plasma s les and showed that in non-ambulant patients (but not in younger patients) there was a deficiency of taurine. These data suggest that taurine homeostasis varies greatly between species and may be influenced by age and disease progression. The potential for taurine to be an effective therapy may depend on such variables.

THE APPLICATION OF RATE DIALYSIS TO THE DETERMINATION OF FREE STEROIDS IN PLASMA

Publisher: Elsevier BV

Date: 12-1983

DOI: 10.1016/0003-2697(83)90687-5

Abstract: Rate dialysis is used to obtain the free steroid fraction in undiluted plasma at 37 degrees C. The free steroid fraction is determined from the rate at which a small amount of tritiated steroid diffuses from plasma on one side of a semipermeable membrane into an identical plasma s le on the other side which lacks radioactive steroid. The method may be generally applicable to steroids since the cell permeability constant, which is a function of the volume of the dialysis cell and the area and diffusion properties of the membrane, was similar for seven steroids tested. The method requires only 0.3 ml of plasma, is simple and economical to perform, and enables up to 120 determinations to be made in one day. The free fractions of cortisol, progesterone, and estradiol-17 beta were measured in plasma pooled from pregnant and non-pregnant women and pregnant and lactating sows. The results were compared with those obtained for the same plasma pools by centrifugal ultrafiltration.

Pre‐clinical evaluation of N‐acetylcysteine reveals side effects in the mdx mouse model of Duchenne muscular dystrophy

Publisher: Wiley

Date: 30-09-2017

DOI: 10.1113/JP274229

Oxidative stress causes a decline in lysosomal integrity during hypothermic incubation of rat hepatocytes

Publisher: American Geophysical Union (AGU)

Date: 10-2008

DOI: 10.1029/2007WR005959

Increased taurine in pre-weaned juvenile mdx mice greatly reduces the acute onset of myofibre necrosis and dystropathology and prevents inflammation

Publisher: Public Library of Science (PLoS)

Date: 2016

DOI: 10.1371/CURRENTS.MD.77BE6EC30E8CAF19529A00417614A072

Abstract: The mdx mouse model for the fatal muscle wasting disease Duchenne Muscular Dystrophy (DMD) shows a very mild pathology once growth has ceased, with low levels of myofibre necrosis in adults. However, from about 3 weeks of post-natal age, muscles of juvenile mdx mice undergo an acute bout of severe necrosis and inflammation: this subsequently decreases and stabilises to lower adult levels by about 6 weeks of age. Prior to the onset of this severe dystropathology, we have shown that mdx mice are deficient in the amino acid taurine (potentially due to weaning), and we propose that this exacerbates myofibre necrosis and inflammation in juvenile mdx mice. The purpose of this study was to increase taurine availability to pre-weaned juvenile mdx mice (from 14 days of age), to evaluate the impact on levels of myofibre necrosis and inflammation (at 22 days) during the acute period of severe dystropathology. Untreated 22 day old mdx muscle was not deficient in taurine, with similar levels to normal C57 control muscle. However taurine treatment, which increased the taurine content of young dystrophic muscle (by 40%), greatly reduced myofibre necrosis (by 75%) and prevented significant increases in 3 markers of inflammation. Taurine was very effective at preventing the acute phase of muscle damage that normally results in myofibre necrosis and inflammation in juvenile mdx mice, supporting continued research into the use of taurine as a therapeutic intervention for protecting growing muscles of young DMD boys.

Novel Regulatory Mechanism of STAT1 through Redox Post-translational Modification of Cysteines

Publisher: Elsevier BV

Date: 11-2017

DOI: 10.1016/J.FREERADBIOMED.2017.10.295

The protective effect of hypoxic preconditioning on cortical neuronal cultures is associated with increases in the activity of several antioxidant enzymes

Publisher: Elsevier BV

Date: 08-2004

DOI: 10.1016/J.BRAINRES.2004.05.031

Proteome Mapping of Human Skim Milk Proteins in Term and Preterm Milk

Publisher: American Chemical Society (ACS)

Date: 22-02-2012

DOI: 10.1021/PR2008797

Abstract: The abundant proteins in human milk have been well characterized and are known to provide nutritional, protective, and developmental advantages to both term and preterm infants. However, relatively little is known about the expression of the low abundance proteins that are present in human milk because of the technical difficulties associated with their detection. We used a combination of electrophoretic techniques, ProteoMiner treatment, and two-dimensional liquid chromatography to examine the proteome of human skim milk expressed between 7 and 28 days postpartum by healthy term mothers and identified 415 in a pooled milk s le. Of these, 261 were found in human skim milk for the first time, greatly expanding our understanding of the human skim milk proteome. The majority of the proteins identified were involved in either the immune response (24%) or in cellular (28%) or protein (16%) metabolism. We also used iTRAQ analysis to examine the effects of premature delivery on milk protein composition. Differences in protein expression between pooled milk from mothers delivering at term (38-41 weeks gestation) and preterm (28-32 weeks gestation) were investigated, with 55 proteins found to be differentially expressed with at least 90% confidence. Twenty-eight proteins were present at higher levels in preterm milk, and 27 were present at higher levels in term milk.

Calcium, phosphate and citrate in human milk at initiation of lactation

Publisher: Cambridge University Press (CUP)

Date: 05-1992

DOI: 10.1017/S0022029900030405

Abstract: The onset of copious milk secretion (lactogenesis II) in women occurs between 1 and 3 d after birth, and during this period the composition of breast milk changes. During the first 5 d of lactation we measured the concentrations of total, diffusible and ionized Ca (Ca tot , Ca d , Ca 2+ ), diffusible phosphate (Pi d ), diffusible citrate (Cit d ) and lactose in the breast milk. On day 1 after birth the concentrations (mean ± SEM) were Ca tot , 5·71±0·30 mM Ca d , 2·66±0·19 mM Ca 2+ , 2·90 ±0·18 mM Pi d , 0·26±0·16 mM Cit d , 0·25±0·03 mai and lactose, 76±11 mM. Between day 1 and day 4 the concentration of Ca tot increased 1·7-fold to 9·56 ±0·39 mai, Ca d increased 1·8-fold to 4·75±0·26 mM, Ca 2+ decreased by 20% to 2·33 ± 0·13 mM, Pi d increased 6·6-fold to 1·69±0·11 mM, Cit d increased 20-fold to 5·06±0·21 mM, and lactose increased 2·3-fold to 173±4 mM. A high correlation has been found between [Ca d ] and [Cit d ] in the milk of both ruminant and non-ruminant species, which show a wide range in concentrations of [Ca d ] and [Cit d ], and the data fit a simple physicochemical model of ion equilibria in the aqueous phase of milk. The results of the present study confirm the relationship between [Ca d ] and [Cit d ] in human milk, even during lactogenesis II when the composition of the milk is changing very rapidly.

The effect of hydrogen peroxide concentration and water temperature on yellowtail kingfish Seriola lalandi in a repeated bathing treatment

Publisher: Elsevier BV

Date: 11-2022

DOI: 10.1016/J.AQUACULTURE.2022.738545

Reply from Gavin J. Pinniger, Jessica R. Terrill, Miranda D. Grounds and Peter G. Arthur

Publisher: Wiley

Date: 19-01-2018

DOI: 10.1113/JP275662

N-Acetyl Cysteine Restores Sirtuin-6 and Decreases HMGB1 Release Following Lipopolysaccharide-Sensitized Hypoxic-Ischemic Brain Injury in Neonatal Mice

Publisher: Frontiers Media SA

Date: 15-11-2021

DOI: 10.3389/FNCEL.2021.743093

Abstract: Inflammation and neonatal hypoxia-ischemia (HI) are important etiological factors of perinatal brain injury. However, underlying mechanisms remain unclear. Sirtuins are a family of nicotinamide adenine dinucleotide (NAD)+-dependent histone deacetylases. Sirtuin-6 is thought to regulate inflammatory and oxidative pathways, such as the extracellular release of the alarmin high mobility group box-1 (HMGB1). The expression and role of sirtuin-6 in neonatal brain injury are unknown. In a well-established model of neonatal brain injury, which encompasses inflammation (lipopolysaccharide, LPS) and hypoxia-ischemia (LPS+HI), we investigated the protein expression of sirtuin-6 and HMGB1, as well as thiol oxidation. Furthermore, we assessed the effect of the antioxidant N-acetyl cysteine (NAC) on sirtuin-6 expression, nuclear to cytoplasmic translocation, and release of HMGB1 in the brain and blood thiol oxidation after LPS+HI. We demonstrate reduced expression of sirtuin-6 and increased release of HMGB1 in injured hippoc us after LPS+HI. NAC treatment restored sirtuin-6 protein levels, which was associated with reduced extracellular HMGB1 release and reduced thiol oxidation in the blood. The study suggests that early reduction in sirtuin-6 is associated with HMGB1 release, which may contribute to neonatal brain injury, and that antioxidant treatment is beneficial for the alleviation of these injurious mechanisms.

Longitudinal analysis of protein glycosylation and β-casein phosphorylation in term and preterm human milk during the first 2 months of lactation

Publisher: Cambridge University Press (CUP)

Date: 27-11-2013

DOI: 10.1017/S0007114512004588

Abstract: Human milk proteins provide term and preterm infants with both nutrition and protection. The objective of the present study was to examine longitudinal changes in the protein composition of term and preterm milk during the first 2 months of lactation, focusing on protein phosphorylation and glycosylation. Using gel electrophoresis, the relative concentration and glycosylation status of lactoferrin, secretory Ig A, β-casein, α-lactalbumin, serum albumin, bile salt-stimulated lipase, xanthine oxidoreductase, tenascin and macrophage mannose receptor 1 were measured in milk collected on days 7, 10, 14, 18, 21, 28 and 60 postpartum from preterm mothers (28–32 weeks gestation, n 17). The phosphorylation status of β-casein was also investigated. To determine if these variables differ in term and preterm milk, s les from term mothers (38–41 weeks gestation, n 8) collected on days 7, 14 and 30 of lactation were also analysed. The concentration of the abundant milk proteins decreased during lactation in term and preterm milk ( P 0·05). No difference in protein glycosylation was observed, except for the glycoproteins serum albumin and tenascin. The phosphorylation of β-casein varied significantly between term and preterm milk. Further investigation is required to determine whether these modifications affect protein function and are clinically important to preterm infants.

MICROANALYSIS OF THE METABOLIC INTERMEDIATES OF LACTOSE SYNTHESIS IN HUMAN-MILK AND PLASMA USING BIOLUMINESCENT METHODS

Publisher: Elsevier BV

Date: 02-1989

DOI: 10.1016/0003-2697(89)90341-2

Abstract: Sensitive bioluminescent methods were developed to measure the metabolites glucose, glucose 6-phosphate (G6P), glucose 1-phosphate (G1P), UDP-glucose, and UDP-galactose in human milk and lactose and galactose in human plasma. The bioluminescent methods measured NADH produced by coupled enzymatic assays derived from equivalent spectrophotometric methods. We found that the long chain fatty acids in human milk (C10-C16) inhibited the bioluminescent reactions. This inhibition was overcome by adding defatted bovine serum albumin to the reaction mixture containing the bioluminescent enzymes. It also was necessary to modify methods of deproteinizing milk and blood plasma to accommodate small s le volumes. In the development of these assays emphasis was given to simplicity of reagent preparation, minimizing cost, and ease of use. The detection limit for the bioluminescent method for NADH was 0.28 nM for a 20-microliters s le. For the assays of the metabolites, recoveries ranged from 91 to 107%. For s le sizes of 2 to 5 microliters of protein free s le, the detection limits for milk were G1P, 0.09 microM G6P, 0.05 microM UDPhexose, 0.07 microM UDP-Glc, 0.03 microM glucose, 9 microM and for plasma, lactose, 0.76 microM, galactose, 0.31 microM. The bioluminescent methods gave equivalent results to spectrophotometric methods for the measurement of blood lactose and milk glucose.

Assessment of CMV, RSV and SYN1 promoters and the woodchuck post-transcriptional regulatory element in adenovirus vectors for transgene expression in cortical neuronal cultures

Publisher: Elsevier BV

Date: 08-2006

DOI: 10.1016/J.BRAINRES.2006.04.089

Abstract: In order to investigate protein function in rat primary cortical neuronal cultures, we modified an adenoviral vector expression system and assessed the strength and specificity of the cytomegalovirus (CMV), rous sarcoma virus (RSV), and rat and human synapsin 1 (SYN1) promoters to drive DsRed-X expression. We also incorporated the woodchuck post-transcriptional regulatory element (WPRE) and a CMV promoter-enhanced green fluorescent protein (EGFP) reporter cassette. We observed that the RSV promoter activity was strong in neurons and moderate in astrocytes, while the CMV promoter activity was weak-to-moderate in neurons and very strong in astrocytes. The rat and human SYN1 promoters exhibited similar but weak activity in neurons, despite inclusion of the WPRE. We confirmed that the WPRE enhanced RSV promoter-mediated DsRed-X expression in a time-dependent fashion. Interestingly, we observed very weak SYN1-mediated DsRed-X expression in astrocytes and HEK293 cells suggesting incomplete neuronal-restrictive behavior for this promoter. Finally, using our adenoviral expression system, we demonstrated that RSV promoter-mediated Bcl-X(L) overexpression attenuated neuronal death caused by in vitro ischemia and oxidative stress.

Carbohydrate metabolism in human platelets in a low glucose medium under aerobic conditions

Publisher: Elsevier BV

Date: 10-1996

DOI: 10.1016/0304-4165(96)00051-7

Abstract: The metabolism of human platelets has been the subject of investigation for at least three decades, at the level of basic metabolism, and because of the increasing requirement for platelet storage. Platelets are relatively active metabolically and are typical cells in terms of fuels and metabolic pathways. They contain glycogen and utilize glucose and demonstrate aerobic glycolysis and carbohydrate oxidation. Both glycolysis and carbohydrate oxidation contribute significantly to total ATP turnover, so platelets are an ideal system in which to study the partitioning of carbohydrate metabolism between the two available fuels and the two available pathways, in the presence of adequate oxygen. We have designed a system whereby we can study carbohydrate metabolism in relatively pure human platelets, under sterile conditions, over long periods. The system enables us to determine total ATP turnover and, with the aid of a mathematical model, the contribution to this turnover of glycolysis and the oxidation of glucose/glycogen and lactate. When glucose and glycogen are present, most of the glucose and glycogen utilised is converted to lactate, but lactate is being oxidised at this time. When glucose/glycogen stores are exhausted lactate oxidation continues and increases with the result that carbohydrate oxidation accounts for 41% of total ATP turnover over 48 h.

Limiting the Hydrolysis and Oxidation of Maleimide-Peptide Adducts Improves Detection of Protein Thiol Oxidation

Publisher: American Chemical Society (ACS)

Date: 07-04-2017

DOI: 10.1021/ACS.JPROTEOME.6B01060

Abstract: Oxidative stress, caused by reactive oxygen and nitrogen species (RONS), is important in the pathophysiology of many diseases. A key target of RONS is the thiol group of protein cysteine residues. Because thiol oxidation can affect protein function, mechanistic information about how oxidative stress affects tissue function can be ascertained by identifying oxidized proteins. The probes used must be specific and sensitive, such as maleimides for the alkylation of reduced cysteine thiols. However, we find that maleimide-alkylated peptides (MAPs) are oxidized and hydrolyzed under s le preparation conditions common for proteomic studies. This can result in up to 90% of the MAP signal being converted to oxidized or hydrolyzed MAPs, decreasing the sensitivity of the analysis. A substantial portion of these modifications were accounted for by Coomassie "blue silver" staining (∼14%) of gels and proteolytic digestion buffers (∼20%). More than 40% of the MAP signal can be retained with the use of thioglycolic acid during gel electrophoresis, trichloroethanol-UV protein visualization in gels, and proteolytic digestion buffer of pH 7.0 TRIS. This work demonstrates that it is possible to decrease modifications to MAPs through changes to the s le preparation workflow, enhancing the potential usefulness of maleimide in identifying oxidized peptides.

Quantification of Ceroid and Lipofuscin in Skeletal Muscle

Publisher: SAGE Publications

Date: 29-07-2011

DOI: 10.1369/0022155411412185

Abstract: Ceroid and lipofuscin are autofluorescent granules thought to be generated as a consequence of chronic oxidative stress. Because ceroid and lipofuscin are persistent in tissue, their measurement can provide a lifetime history of exposure to chronic oxidative stress. Although ceroid and lipofuscin can be measured by quantification of autofluorescent granules, current methods rely on subjective assessment. Furthermore, there has not been any evaluation of variables affecting quantitative measurements. The article describes a simple statistical approach that can be readily applied to quantitate ceroid and lipofuscin. Furthermore, it is shown that several factors, including magnification tissue thickness and tissue level, can affect precision and sensitivity. After optimizing for these factors, the authors show that ceroid and lipofuscin can be measured reproducibly in the skeletal muscle of dystrophic mice (ceroid) and aged mice (lipofuscin).

Levels of inflammation and oxidative stress, and a role for taurine in dystropathology of the Golden Retriever Muscular Dystrophy dog model for Duchenne Muscular Dystrophy

Publisher: Elsevier BV

Date: 10-2016

DOI: 10.1016/J.REDOX.2016.08.016

A proteomic analysis of the acute effects of high-intensity exercise on skeletal muscle proteins in fasted rats

Publisher: Wiley

Date: 26-09-2006

DOI: 10.1111/J.1440-1681.2006.04470.X

Abstract: Quantitative proteomics is a technique that allows for large-scale comparison of the levels of in idual proteins present in a biological s le. This technique has not previously been applied to examine the response of skeletal muscle proteins to an acute bout of exercise. In the present study, quantitative proteomics was applied to investigate whether the levels of in idual skeletal muscle proteins are acutely affected by a short bout of high-intensity exercise. Gastrocnemius muscle was s led from fasted rats either at rest, immediately following 3 min of high-intensity exercise or after 30 min of recovery. Muscle s les were submitted to two-dimensional gel electrophoresis and 61 of the resulting protein spots were selected for quantitative analysis. It was found that skeletal muscle protein levels were generally not acutely affected by a short bout of high-intensity exercise, with only four of the 61 proteins selected for analysis being significantly altered. These altered proteins were identified using liquid chromatography electrospray ionization-tandem mass spectrometry as creatine kinase, troponin T and a combination of heat shock 20 kDa protein and adenylate kinase 1. In conclusion, quantitative proteomics is sensitive enough to detect acute changes in skeletal muscle protein levels in response to exercise. We have found that the levels of most in idual skeletal muscle proteins are not immediately altered in response to a short bout of high-intensity exercise and recovery in fasted rats.

Lack of oxygen sensing by mitochondria in platelets

Publisher: Wiley

Date: 15-11-1999

DOI: 10.1046/J.1432-1327.1999.00846.X

Abstract: The range over which cells are sensitive to changes in oxygen concentration remains uncertain. Wilson and colleagues [Wilson, D.F. (1994) Med. Sci. Sports Exerc. 26, 37-43] have suggested that cytochrome oxidase is sensitive to oxygen concentrations below about 40 microM, but proposed that this sensitivity is obscured in intact cells because an increase in reduction state of cytochrome c acts to maintain oxygen consumption. We have tested this hypothesis in platelets, which are small cells (2-4 micrometer diameter, < 0.5 micrometer thick) that do not decrease their rate of oxygen consumption until oxygen concentrations fall below 2.5 microM. Contrary to the expectations of the hypothesis, the reduction state of cytochrome c, the concentration of NADH and the rate of glycolytic output are not changed as oxygen concentration declines from 40 microM down to 5 microM. Therefore, we conclude that at least some cell types contain mitochondria that are not capable of sensing oxygen above 5 microM by the mechanism proposed by Wilson and colleagues.

A Blood Biomarker for Duchenne Muscular Dystrophy Shows That Oxidation State of Albumin Correlates with Protein Oxidation and Damage in Mdx Muscle

Publisher: MDPI AG

Date: 03-08-2021

DOI: 10.3390/ANTIOX10081241

Abstract: Duchenne muscular dystrophy (DMD) is a severe X-linked muscle wasting disease with no cure. While the precise mechanisms of progressive dystropathology remain unclear, oxidative stress caused by excessive generation of oxidants is strongly implicated. Blood biomarkers that could track oxidant levels in tissues would be valuable to measure the effectiveness of clinical treatments for DMD our research has focused on developing such biomarkers. One target of oxidants that has the potential to be harnessed as a clinical biomarker is the thiol side chain of cysteine 34 (Cys34) of the blood protein albumin. This study using the mdx mouse model of DMD shows that in plasma, albumin Cys34 undergoes thiol oxidation and these changes correlate with levels of protein thiol oxidation and damage of the dystrophic muscles. A comparison with the commonly used biomarker protein carbonylation, confirmed that albumin thiol oxidation is the more sensitive plasma biomarker of oxidative stress occurring in muscle tissue. We show that plasma albumin oxidation reflects muscle dystropathology, as increased after exercise and decreased after taurine treatment of mdx mice. These data support the use of albumin thiol oxidation as a blood biomarker of dystropathology to assist with advancing clinical development of therapies for DMD.

Hypochlorous acid exposure impairs skeletal muscle function and Ca2+ signalling: implications for Duchenne muscular dystrophy pathology

Publisher: Wiley

Date: 21-10-2023

DOI: 10.1113/JP285263

Oxidative damage to urinary proteins from the GRMD dog and mdx mouse as biomarkers of dystropathology in Duchenne muscular dystrophy

Publisher: Public Library of Science (PLoS)

Date: 08-10-2020

DOI: 10.1371/JOURNAL.PONE.0240317

Decreasing cellular hydrogen peroxide with catalase mimics the effects of hypoxia on the sensitivity of the L-type Ca 2+ channel to β-adrenergic receptor stimulation in cardiac myocytes

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 04-10-2002

DOI: 10.1161/01.RES.0000035528.00678.D5

Abstract: In cardiac myocytes, hypoxia inhibits the basal L-type Ca 2+ current ( I Ca-L ) and increases the sensitivity of I Ca-L to β-adrenergic receptor stimulation. We investigated whether hydrogen peroxide (H 2 O 2 ) is involved in the hypoxic response. Guinea pig ventricular myocytes were dialyzed with catalase, which specifically catalyzes the conversion of H 2 O 2 to H 2 O and oxygen, and then I Ca-L was recorded during exposure to isoproterenol (Iso). Catalase decreased the K 0.5 for activation of I Ca-L by Iso from 2.7±0.3 nmol/L (in cells dialyzed with heat-inactivated catalase) to 0.4±0.1 nmol/L. The increase in sensitivity to Iso by catalase may be attenuated when cells are preexposed to H 2 O 2 . A significant increase in sensitivity of I Ca-L to Iso was recorded when mitochondrial function was inhibited with myxothiazol or FCCP, suggesting that the source of H 2 O 2 was from the mitochondria. Prior exposure of cells to H 2 O 2 attenuated the inhibition of basal I Ca-L during hypoxia and the increase in sensitivity of I Ca-L to Iso during hypoxia. Additionally, extracellularly applied catalase mimicked the effect of hypoxia on basal I Ca-L . Measurement of the rate of production of hydrogen peroxide using 5- (and 6-)chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetate acetyl ester indicated that hypoxia was associated with a significant decrease in the production of hydrogen peroxide in the cells. These data suggest that hypoxia mediates changes in channel activity through a lowering in H 2 O 2 levels and that H 2 O 2 is a key intermediate in modifying basal channel activity and the β-adrenergic responsiveness of the channel during hypoxia.

An improved capillary electrophoresis method for measuring tissue metabolites associated with cellular energy state

Publisher: Wiley

Date: 05-1999

DOI: 10.1046/J.1432-1327.1999.00324.X

Abstract: An improved method for the measurement of tissue metabolites associated with cellular energetic state by capillary electrophoresis is described. This method allows 17 compounds present in a mixture of standards to be determined simultaneously within 43 min with good reproducibility. ATP, ADP, AMP, UTP, IMP, inosine, hypoxanthine, creatine, phosphocreatine, UDP-galactose, NAD and NADH were detected in s les of either rat heart tissue or rat neonatal cardiomyocytes. This method can detect compounds at concentrations of 5 microm in s les. Recoveries for ATP and phosphocreatine added to cardiomyocyte s les were 99.4 +/- 2.1% and 103.1 +/- 3.3%, respectively (mean +/- SEM, n = 3). Our method has been comprehensively validated and is capable of measuring a wider range of tissue metabolites important in assessing cellular energy status than existing methods.

Stability of oxygen deprivation in glass culture vessels facilitates fast reproducible cell death to cortical neurons under simulated ischemia

Publisher: Elsevier BV

Date: 07-2002

DOI: 10.1006/ABIO.2002.5674

Treatment with cysteine precursors decreases protein thiol oxidation and improves muscle pathology in the mdx mouse

Publisher: Elsevier BV

Date: 10-2012

DOI: 10.1016/J.NMD.2012.06.149

Grade distribution digests

Publisher: Wiley

Date: 29-12-2018

DOI: 10.1002/BMB.21104

Abstract: Assessment is a central component of course curriculums and is used to certify student learning, but it can also be used as a tool to improve teaching and learning. Many laboratory courses are structured such that there is only a grade for a particular laboratory, which limits the insights that can be gained in student learning. We developed a laboratory program that incorporates assessments designed to probe student understanding of different components of the in idual modules making up the program. The challenge was to analyze and present grades from these assessment tasks in a format that was readily interpretable by academics. We show that a simplified synthesis of grade distributions (grade distribution digests) provides sufficient information to make decisions about changes in course components. The main feature of the digests is its data visualization approach, where student grades for in idual laboratory practicals, in idual assessment tasks or in idual assessment items are graphically presented as an overall average grade, an average top quartile grade and an average bottom quartile grade, and relative averages across all assessments. This ability to visualize student grades in variety of contexts enables academics with many other demands on their time (e.g. research and administration) to more efficiently identify ways to improve teaching delivery and learning outcomes. Ex les are presented of the use of such data to identify and improve deficiencies in both student skills and teaching practice, resulting in improved learning outcomes. © 2017 by The International Union of Biochemistry and Molecular Biology, 46(2):130-140, 2018.

LACTOSE IN BLOOD IN NONPREGNANT, PREGNANT, AND LACTATING WOMEN

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 10-1991

DOI: 10.1097/00005176-199110000-00003

Abstract: Lactose synthesized in the mammary gland can pass into the bloodstream by either a paracellular or transcellular pathway. In nonpregnant, nonlactating women, the concentration of lactose in the blood plasma was 1.5 +/- 0.1 microM (mean +/- SEM) in 9 women and undetectable in another 11 women. During pregnancy, this concentration was 3.7 +/- 0.4 microM at 10-21 weeks of gestation, with an increase to 8.7 +/- 1.8 microM by 38-40 weeks of gestation. At the initiation of lactation, the concentration of lactose peaked 3-5 days after birth, with a mean peak concentration of 75 +/- 18 microM, and then decreased to 30 +/- 8 microM when lactation was well established at 6 weeks after birth. These findings suggest that the mammary glands are synthetically active by the beginning of the second trimester of pregnancy and reach maximum synthetic capacity soon after birth. Measurement of concentrations of lactose in the blood plasma during pregnancy and lactation may allow an assessment of the successful initiation of both lactogenesis I and II.

METABOLITES OF LACTOSE SYNTHESIS IN MILK FROM WOMEN DURING ESTABLISHED LACTATION

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 10-1991

DOI: 10.1097/00005176-199110000-00004

Abstract: Metabolites in the lactose synthesis pathway were measured in human milk during established lactation. The mean concentrations (range) were: lactose, 198 mM (175-233) glucose, 1.5 mM (0.1-2.4) glucose 6-phosphate, 11 microM (4-23) glucose 1-phosphate, 2.7 microM (1.6-6.8) UDP-glucose, 2.7 microM (0.6-6.0) UDP-galactose, 0.7 microM (less than 0.1-2.5) inorganic phosphate, 2.2 mM (0.8-2.9). During storage of milk within the breast for 2 h, glucose 6-phosphate concentration increased by 1.4 +/- 0.5 microM, while UDP-glucose decreased by 1.0 +/- 0.4 microM. None of the other metabolites showed significant changes in concentration during this period. Only glucose showed a circadian rhythm in concentration, with the highest concentrations between 1400 and 1800 h. The volumes of milk produced by the mothers ranged from 178 to 1,739 ml/24 h. Despite this variation, there were no significant relationships between the 24-h average concentrations of glucose, glucose 6-phosphate, glucose 1-phosphate, UDP-glucose, UDP-galactose, or inorganic phosphate and the amount of lactose produced over 24 h.

Changes in oxygen tension affect cardiac mitochondrial respiration rate via changes in the rate of mitochondrial hydrogen peroxide production

Publisher: Elsevier BV

Date: 07-2009

DOI: 10.1016/J.YJMCC.2009.03.010

Abstract: The capacity of mitochondria to respond to changes in oxygen delivery has the potential to affect the ability of the heart to tolerate decreased oxygen delivery. Respiration by mitochondria is typically regarded as independent of oxygen tension (pO(2)) until critically low oxygen concentrations limit the activity of cytochrome oxidase. Paradoxically, there is evidence that cellular and mitochondrial oxygen consumption (respiration) can decline at oxygen tensions well above this critical pO(2). We tested the hypothesis that oxygen sensitive decreases in mitochondrial hydrogen peroxide production can decrease cardiac mitochondrial respiration rate. Consistent with previous work, an acute decline in pO(2) from 146 mm Hg to 10-13 mm Hg in less than 10 min did not affect mitochondrial respiration rate. In contrast, sustained incubation of mitochondria at a pO(2) of 10-13 mm Hg for 30 min caused a 50% decrease in mitochondrial respiration rate. This decrease in mitochondrial respiration rate was mimicked by incubation with the hydrogen peroxide scavenger catalase and the decrease in mitochondrial respiration rate was fully reversible by reintroducing oxygen or by adding hydrogen peroxide. Incubation at low pO(2) was also associated with a decreased rate of mitochondrial reactive oxygen species production. These findings indicate that oxygen-dependent decreases in the rate of mitochondrial hydrogen peroxide production can decrease cardiac mitochondrial respiration.

Effects of HOCl oxidation on excitation-contraction coupling

Publisher: Rockefeller University Press

Date: 12-11-2022

DOI: 10.1085/JGP.2021ECC16

Abstract: Duchenne muscular dystrophy (DMD) is a fatal X-linked genetic disease characterized by progressive loss of skeletal muscle. The mechanisms underlying the DMD pathology likely involve the complex interaction between reactive oxygen species (ROS) impaired Ca2+ handling and chronic inflammation, characterized by the presence of immune cells such as neutrophils. Hypochlorous acid (HOCl) is a highly reactive form of ROS produced endogenously via the actions of myeloperoxidase, an enzyme secreted by neutrophils. Myeloperoxidase activity is significantly elevated in dystrophic muscle. This study aimed to determine the effect of HOCl exposure on excitation–contraction coupling and its potential contribution to the dystrophic pathology. Isolated extensor digitorum longus (EDL) muscles and single fibers from C57 (wild type) and mdx (dystrophic) mice were used to investigate the effects of HOCl on whole muscle function, intracellular Ca2+ handling, and myofilament force production. HOCl exposure significantly decreased maximum specific force in isolated EDL muscles by 26% and 49%, respectively, in C57 and mdx mice (P & 0.0001). In single interosseous fibers, HOCl exposure significantly increased resting intracellular Ca2+ concentration by ∼17–19% (P & 0.05) and decreased the litude of electrically induced Ca2+ transients by ∼45% and 50%, respectively, in C57 and mdx fibers (C57, P & 0.05 mdx, P & 0.01). These effects of HOCl on resting Ca2+ could be blocked via application of tetracaine (ryanodine receptor blocker) or Gd3+ (stretch-activated channel blocker C57, P & 0.01 mdx, P & 0.01 for both). The effect of HOCl on Ca2+ transient litude was significantly reduced by Gd3+ (C57, P & 0.05 mdx, P & 0.01). In chemically skinned EDL fibers, HOCl exposure decreased maximum Ca2+-activated force by ∼40% in both C57 and mdx fibers (P & 0.001). These results indicate that HOCl potently affects excitation–contraction coupling via impaired Ca2+ handling and myofilament force production. Hence, HOCl potentially links the chronic inflammation, oxidative stress, and impaired Ca2+ handling that underlies the dystrophic pathology.

Evidence for regulation of mitochondrial function by the L-type Ca2+ channel in ventricular myocytes

Publisher: Elsevier BV

Date: 06-2009

DOI: 10.1016/J.YJMCC.2008.12.015

Abstract: The L-type Ca(2+) channel is responsible for initiating contraction in the heart. Mitochondria are responsible for meeting the cellular energy demands and calcium is required for the activity of metabolic intermediates. We examined whether activation of the L-type Ca(2+) channel alone is sufficient to alter mitochondrial function. The channel was activated directly with the dihydropyridine agonist BayK(-) or voltage-cl of the plasma membrane and indirectly by depolarization of the membrane with high KCl. Activation of the channel increased superoxide production (assessed as changes in dihydroethidium fluorescence), NADH production and metabolic activity (assessed as formation of formazan from tetrazolium) in a calcium-dependent manner. Activation of the channel also increased mitochondrial membrane potential assessed as changes in JC-1 fluorescence. The response was reversible upon inactivation of the channel during voltage-cl of the plasma membrane and did not appear to require calcium. We examined whether the response may be mediated through movement of cytoskeletal proteins. Depolymerization of actin or exposing cells to a peptide directed against the alpha-interacting domain of the alpha(1C)-subunit of the channel (thereby preventing movement of the beta-subunit) attenuated the increase in mitochondrial membrane potential. We conclude that activation of the L-type Ca(2+) channel can regulate mitochondrial function and the response appears to be modulated by movement through the cytoskeleton.

Differences in fuel utilization between trout and human thrombocytes in physiological media

Publisher: Springer Science and Business Media LLC

Date: 25-10-1999

DOI: 10.1007/S003600050250

Abstract: Cell culture preparations now play a significant and essential role in physiological and biochemical studies of cell biology. However, the fuels offered in cell culture media are only glucose and glutamine, plus whatever might be in the added sera. It is currently difficult to find a rational way forward on this problem, as there are few data on what fuels cells use in vivo or even in an in vitro physiological situation. A recent study on human platelets redressed the situation somewhat by finding that 75% of ATP turnover could be accounted for by aerobic glycolysis, and by the oxidation of glucose, hydroxybutyrate, acetate, glutamine, palmitate and oleate. In the present study we used a similar strategy to investigate fuel choices by trout thymocytes, cells with a similar function but from a different phylogenetic group. When these cells were presented with a physiological medium, we found that aerobic glycolysis accounted for 9% of total ATP turnover, glucose and glutamine oxidation made a combined contribution of 2.3%, oleate and palmitate oxidation accounted for 15%, and 74% was unaccounted for. These patterns of fuel use are very different from that in human platelets. They demonstrate the cell- and animal-specific nature of cellular metabolism and again expose the inadequacy of the fuel component in culture media.

Oxidation of cysteine 34 of plasma albumin as a biomarker of oxidative stress

Publisher: Informa UK Limited

Date: 02-01-2020

DOI: 10.1080/10715762.2019.1708347

Differential thiol oxidation of the signaling proteins Akt, PTEN or PP2A determines whether Akt phosphorylation is enhanced or inhibited by oxidative stress in C2C12 myotubes derived from skeletal muscle

Publisher: Elsevier BV

Date: 05-2015

DOI: 10.1016/J.BIOCEL.2015.02.015

Abstract: Oxidative stress, caused by excess reactive oxygen species (ROS), has been hypothesized to cause or exacerbate skeletal muscle wasting in a number of diseases and chronic conditions. ROS, such as hydrogen peroxide, have the potential to affect signal transduction pathways such as the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3 K)/Akt pathway that regulates protein synthesis. Previous studies have found contradictory outcomes for the effect of ROS on the PI3K/Akt signaling pathway, where oxidative stress can either enhance or inhibit Akt phosphorylation. The apparent contradictions could reflect differences in experimental cell types or types of ROS treatments. We replicate both effects in myotubes of cultured skeletal muscle C2C12 cells, and show that increased oxidative stress can either inhibit or enhance Akt phosphorylation. This differential response could be explained: thiol oxidation of Akt, but not the phosphatases PTEN or PP2A, caused a decline in Akt phosphorylation whereas the thiol oxidation of Akt, PTEN and PP2A increased Akt phosphorylation. These observations indicate that a more complete understanding of the effects of oxidative stress on a signal transduction pathway comes not only from identifying the proteins susceptible to thiol oxidation, but also their relative sensitivity to ROS.

Partitioning of calcium in human milk after freezing and thawing

Publisher: Springer Science and Business Media LLC

Date: 11-02-2009

DOI: 10.1051/DST/2008035

Protein synthesis during oxygen conformance and severe hypoxia in the mouse muscle cell line C2C12

Publisher: Elsevier BV

Date: 06-2000

DOI: 10.1016/S0304-4165(00)00046-5

Abstract: Oxygen conformance can be described as the ability to reduce energy demand, and hence oxygen consumption, in response to a decline in oxygen availability without a decrease in the concentration of ATP. It has been proposed that oxygen conformance may enhance cellular survival at low oxygen concentrations. We demonstrate that non-contracting C2C12 cells, a mouse skeletal muscle cell line, are capable of oxygen conformance. Typically, we found oxygen consumption to decline by 30-40% as the concentration of oxygen was reduced from 100 microM to 10 microM. Unexpectedly, the rate of protein synthesis, a major energy consumer in the cell, did not decrease significantly during oxygen conformance. Unlike oxygen conformance, severe hypoxia (<0.5 microM) caused a 36% decline in the concentration of PCr, and under these conditions of energy stress, the rate of protein synthesis declined by 43%. We conclude that there are two distinct metabolic responses to declines in oxygen concentration in non-contracting C2C12 cells.

Dimethyl fumarate modulates the dystrophic disease program following short-term treatment

Publisher: American Society for Clinical Investigation

Date: 26-09-2023

DOI: 10.1172/JCI.INSIGHT.165974

Treatment with the cysteine precursor l-2-oxothiazolidine-4-carboxylate (OTC) implicates taurine deficiency in severity of dystropathology in mdx mice

Publisher: Elsevier BV

Date: 09-2013

DOI: 10.1016/J.BIOCEL.2013.07.009

Abstract: Oxidative stress has been implicated in the pathology of the lethal skeletal muscle disease Duchenne muscular dystrophy (DMD), and various antioxidants have been investigated as a potential therapy. Recently, treatment of the mdx mouse model for DMD with the antioxidant and cysteine and glutathione (GSH) precursor n-acetylcysteine (NAC) was shown to decrease protein thiol oxidation and improve muscle pathology and ex vivo muscle strength. This study further investigates the mechanism for the benefits of NAC on dystrophic muscle by administering l-2-oxothiazolidine-4-carboxylate (OTC) which also upregulates intracellular cysteine and GSH, but does not directly function as an antioxidant. We observed that OTC, like NAC, decreases protein thiol oxidation, decreases pathology and increases strength, suggesting that the both NAC and OTC function via increasing cysteine and GSH content of dystrophic muscle. We demonstrate that mdx muscle is not deficient in either cysteine or GSH and that these are not increased by OTC treatment. However, we show that dystrophic muscle of 12 week old mdx mice is deficient in taurine, a by-product of disposal of excess cysteine, a deficiency that is ameliorated by OTC treatment. These data suggest that in dystrophic muscles, apart from the strong association of increased oxidative stress and protein thiol oxidation with dystropathology, another major issue is an insufficiency in taurine that can be corrected by increasing the availability of cysteine. This study provides new insight into the molecular mechanism underlying the benefits of NAC in muscular dystrophy and supports the use of OTC as an alternative drug for potential clinical applications to DMD.

MILK LACTOSE, CITRATE, AND GLUCOSE AS MARKERS OF LACTOGENESIS IN NORMAL AND DIABETIC WOMEN

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 11-1989

DOI: 10.1097/00005176-198911000-00016

Abstract: A study was undertaken to define an appropriate marker of lactogenesis II (the onset of copious milk secretion) in mothers, and to determine the effect of diabetes on this marker. Changes in the concentrations of three milk components--lactose, citrate, and glucose--were measured in 38 normal mothers and 6 type I diabetic mothers up to 10 days after birth. Milk yield was measured in 12 of the normal mothers, and all mothers were asked to note the time of milk "coming in" (the feeling of overfullness of the breasts). The average concentrations of lactose, citrate, and glucose in milk were low for the first 24 h after birth, then between 24 and 48 h after birth there was a rapid increase in the concentrations of lactose and citrate, and this transitional period was followed by a plateau period that began between 60 and 84 h after birth. For in idual mothers the transitional period for citrate began 32 +/- 9 h (n = 13) and finished 77 +/- 10 h (n = 17) after birth, and for lactose the transitional period finished at 53 +/- 12 h (n = 29) after birth. For diabetic mothers these times were significantly later. The average 24-h milk intake by infants increased from 82 to 556 ml/24 h between 24 and 144 h after birth. Milk intakes were correlated with the concentration of lactose (r = 0.52, n = 51, p less than 0.001), citrate (r = 0.47, n = 47, p less than 0.001), and glucose (r = 0.69, n = 50, p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Screening for increased protein thiol oxidation in oxidatively stressed muscle tissue

Publisher: Informa UK Limited

Date: 22-06-2011

DOI: 10.3109/10715762.2011.590136

Abstract: Elevated oxidative stress can alter the function of proteins through the reversible oxidation of the thiol groups of key cysteine residues. This study evaluated a method to scan for reversible protein thiol oxidation in tissue by measuring reduced and oxidized protein thiols. It assessed the responsiveness of protein thiols to oxidative stress in vivo using a dystrophic (mdx) mouse model and compared the changes to commonly used oxidative biomarkers. In mdx mice, protein thiol oxidation was significantly elevated in the diaphragm, gastrocnemius and quadriceps muscles. Neither malondialdehyde nor degree of glutathione oxidation was elevated in mdx muscles. Protein carbonyl content was elevated, but changes in protein carbonyl did not reflect changes in protein thiol oxidation. Collectively, these data indicate that where there is an interest in protein thiol oxidation as a mechanism to cause or exacerbate pathology, the direct measurement of protein thiols in tissue would be the most appropriate screening tool.

Proteomic analysis reveals different protein changes during endothelin-1- or leukemic inhibitory factor-induced hypertrophy of cardiomyocytes in vitro.

Publisher: Elsevier BV

Date: 05-2005

DOI: 10.1074/MCP.M400155-MCP200

Isotope-Coded Maleimide Affinity Tags for Proteomics Applications

Publisher: American Chemical Society (ACS)

Date: 28-04-2023

DOI: 10.1021/ACS.BIOCONJCHEM.1C00206

Abstract: Isotope-coded affinity tags (ICATs) are valuable tools for mass spectrometry-based quantitative proteomics, in particular, for comparison of protein (cysteine-residue) thiol oxidation state in normal, stressed, and diseased tissue. However, the iodoacetamido electrophile used in most commercial ICATs suffers from poor thiol-selectivity and modest rates of adduct formation, which can lead to spurious results. Hence, we designed and synthesized three ICATs containing thiol-selective

Iron and Oxidative Stress in Cold-Initiated Necrotic Death of Rat Hepatocyte

Publisher: Elsevier BV

Date: 06-2010

DOI: 10.1016/J.TRANSPROCEED.2010.03.143

Abstract: Iron chelators and antioxidants have been shown to prevent hypothermia-induced apoptosis in hepatocytes. This study examined whether iron chelation and antioxidants could also prevent hypothermia-induced necrosis. Isolated rat hepatocytes were incubated at 4 degrees C for 6 hours and then rewarmed at 37 degrees C for 18 hours with or without the iron chelator deferoxamine and a selection of antioxidants. There was no evidence of increased cell death or adenosine triphosphate depletion during hypothermic incubation. After hypothermia and rewarming, the majority of rat hepatocytes died of necrosis as indicated by the absence of DNA fragmentation, caspase 3 activity, and apoptotic bodies. Cell death was significantly reduced if deferoxamine or a selection of antioxidants were present during hypothermia and rewarming. Deferoxamine was more effective in preventing cell death when added prior to hypothermia, indicating cell death processes were likely initiated during hypothermia.

Desferrioxamine in warm reperfusion media decreases liver injury aggravated by cold storage

Publisher: Baishideng Publishing Group Inc.

Date: 2013

DOI: 10.3748/WJG.V19.I5.673

Visualizing and quantifying oxidized protein thiols in tissue sections: A comparison of dystrophic mdx and normal skeletal mouse muscles

Publisher: Elsevier BV

Date: 12-2013

DOI: 10.1016/J.FREERADBIOMED.2013.09.024

Abstract: Reactive oxygen species (ROS) are not only a cause of oxidative stress in a range of disease conditions but are also important regulators of physiological pathways in vivo. One mechanism whereby ROS can regulate cell function is by modification of proteins through the reversible oxidation of their thiol groups. An experimental challenge has been the relative lack of techniques to probe the biological significance of protein thiol oxidation in complex multicellular tissues and organs. We have developed a sensitive and quantitative fluorescence labeling technique to detect and localize protein thiol oxidation in histological tissue sections. In our technique, reduced and oxidized protein thiols are visualized and quantified on two consecutive tissue sections and the extent of protein thiol oxidation is expressed as a percentage of total protein thiols (reduced plus oxidized). We tested the application of this new technique using muscles of dystrophic (mdx) and wild-type C57Bl/10Scsn (C57) mice. In mdx myofibers, protein thiols were consistently more oxidized (19 ± 3%) compared with healthy myofibers (10 ± 1%) in C57 mice. A striking observation was the localization of intensive protein thiol oxidation (70 ± 9%) within myofibers associated with necrotic damage. Oxidative stress is an area of active investigation in many fields of research, and this technique provides a useful tool for locating and further understanding protein thiol oxidation in normal, damaged, and diseased tissues.

Evidence that intracellular cyclophilin A and cyclophilin A/CD147 receptor-mediated ERK1/2 signalling can protect neurons against in vitro oxidative and ischemic injury

Publisher: Elsevier BV

Date: 2007

DOI: 10.1016/J.NBD.2006.08.012

Abstract: We previously reported that cyclophilin A protein is up-regulated in cortical neuronal cultures following several preconditioning treatments. In the present study, we have demonstrated that adenoviral-mediated over-expression of cyclophilin A in rat cortical neuronal cultures can protect neurons from oxidative stress (induced by cumene hydroperoxide) and in vitro ischemia (induced by oxygen glucose deprivation). We subsequently demonstrated that cultured neurons, but not astrocytes, express the recently identified putative cyclophilin A receptor, CD147 (also called neurothelin, basigin and EMMPRIN), and that administration of purified cyclophilin A protein to neuronal cultures induces a rapid but transient phosphorylation of the extracellular signal-regulated kinase (ERK) 1/2. Furthermore, administration of purified cyclophilin A protein to neuronal cultures protects neurons from oxidative stress and in vitro ischemia. Interestingly, we detected up-regulation of cyclophilin A mRNA, but not protein in the hippoc us following a 3-min period of sublethal global cerebral ischemia in the rat. Despite our in vivo findings, our in vitro data show that cyclophilin A has both intracellular- and extracellular-mediated neuroprotective mechanisms. To this end, we propose cyclophilin A's extracellular-mediated neuroprotection occurs via CD147 receptor signalling, possibly by activation of ERK1/2 pro-survival pathways. Further characterization of cyclophilin A's neuroprotective mechanisms may aid the development of a neuroprotective therapy.

Citrate, calcium, phosphate and magnesium in sows' milk at initiation of lactation

Publisher: Cambridge University Press (CUP)

Date: 02-1998

DOI: 10.1017/S0022029997002574

Abstract: Colostrum and milk were collected from ten sows at frequent intervals from before farrowing until 9 d after farrowing. Ionized calcium, pH, and total concentrations of citrate, calcium, phosphate and magnesium were measured in whole milk. The diffusible fraction of the mammary secretion was separated by ultrafiltration and was used for the measurement of diffusible citrate, calcium, phosphate and magnesium. The pH before farrowing was 5·7, and increased to 6·5 on day 4 as total calcium and phosphate also increased. Before farrowing, total and diffusible citrate were 7·8 and 7·3 m M respectively, while diffusible phosphate was 11·9 m M , and these concentrations all decreased during the study period. Total magnesium ranged between 3·3 and 4·1 m M , while diffusible magnesium ranged between 2·0 and 3·1 m M . While these concentrations and patterns of change of diffusible calcium and citrate are quite different from those of women's milk during the first week after birth, theoretical physicochemical relationships between diffusible calcium and citrate, and ionized calcium and HPO 4 2− were corroborated by these results. We conclude that diffusible citrate plays an important role in the determination of the concentration of diffusible calcium. However, while citrate may be the major determinant of the total concentration of calcium in women's milk, this is not the case in sows' milk.

Energy turnover in the normoxic and anoxic turtle heart

Publisher: Elsevier BV

Date: 05-1997

DOI: 10.1016/S0300-9629(96)00283-6

Abstract: We examined the possibility that the heart of the tuttle Chrysemys scripta is an exceptional anaerobic performer, by measuring myocardial power output, lactate output, and estimated ATP turnover in perfused heart preparations. Over a range of myocardial power outputs at 5 and 15 degrees C we find that turtle hearts perfused with anoxic saline do not show a particularly outstanding ability to produce ATP anaerobically. Furthermore, at 15 degrees C anoxia reduced the ATP turnover rate to 50% of the normoxic rate. At 5 degrees C the anoxia-induced depression of ATP turnover was even more pronounced, being 4-fold lower than the normoxic rate. In addition, anoxia at 5 degrees C reduced the basal metabolic rate of the tuttle heart. We conclude that long-term cardiac tolerance of hypoxia in this species is more likely related to metabolic depression rather than to an exceptional anaerobic performance.

Biomarkers for Duchenne muscular dystrophy: myonecrosis, inflammation and oxidative stress

Publisher: The Company of Biologists

Date: 02-2020

DOI: 10.1242/DMM.043638

Oxidative stress as a therapeutic target during muscle wasting: considering the complex interactions

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 07-2008

DOI: 10.1097/MCO.0B013E328302F3FE

Role of NAD(P)H oxidase in the regulation of cardiac L-type Ca²⁺channel function during acute hypoxia

Publisher: Oxford University Press (OUP)

Date: 09-2005

DOI: 10.1016/J.CARDIORES.2005.04.025

Abstract: The role of NAD(P)H oxidase in regulating cellular production of reactive oxygen species (ROS) and the L-type Ca2+ channel during acute hypoxia was examined in adult ventricular myocytes from guinea pig. The fluorescent indicator dihydroethidium (DHE) was used to detect superoxide and the response of the L-type Ca2+ channel to beta-adrenergic receptor stimulation was used as a functional reporter since hypoxia increases the sensitivity of the L-type Ca2+ channel (I(Ca-L)) to isoproterenol (Iso). Hypoxia caused a 41.2+/-5.2% decrease in the rate of the DHE signal (n=21 p<0.01). Of the classical NAD(P)H oxidase inhibitors, DPI but not apocynin mimicked the effect of hypoxia on the sensitivity of I(Ca-L) to Iso. However, the potent NAD(P)H oxidase agonist angiotensin II had no effect on cellular superoxide or the sensitivity of I(Ca-L) to Iso. Although DPI inhibits NAD(P)H oxidase, it also decreased superoxide in isolated mitochondria in a concentration-dependent manner. Partial inhibition of mitochondrial function with nanomolar concentrations of FCCP or myxothiazol mimicked the effect of hypoxia on cellular superoxide and the sensitivity of I(Ca-L) to Iso. In addition, hypoxia caused a 69.3+/-0.8% decrease in superoxide in isolated mitochondria (n=4 p<0.01), providing direct evidence for a role for the mitochondria. Our data suggest that mitochondria appear to be involved in oxygen sensing, regulation of cellular ROS, and the function of I(Ca-L) during acute hypoxia in cardiac myocytes and NAD(P)H oxidase does not appear to contribute substantially.

The lactation cycle in the sow: physiological and management contradictions

Publisher: Elsevier BV

Date: 10-1997

DOI: 10.1016/S0301-6226(97)00076-6

Contrasting actions of prolonged mitogen-activated protein kinase activation on cell survival

Publisher: Elsevier BV

Date: 06-2006

DOI: 10.1016/J.BBRC.2006.04.161

Abstract: Activation of the ERK mitogen-activated protein kinase pathway has been implicated in pro-survival and cellular protective mechanisms, so that chronic ERK activation may be a useful therapeutic strategy. Here, we further explored the consequences of prolonged ERK activation following expression of constitutively active form of MEK, MEK-EE, in cardiac myocytes. We confirmed that chronic MEK-EE overexpression halved myocyte death following glucose deprivation, but surprisingly this was not associated with preserved intracellular ATP levels. Whilst activities of a number of antioxidant enzymes were not altered upon MEK-EE expression, paradoxically Cu/Zn superoxide dismutase activity was almost halved upon MEK-EE expression. When we then exposed myocytes to the superoxide generator menadione, we observed significantly higher death of MEK-EE expressing myocytes. Pre-incubation with U0126 inhibited menadione-induced death. Our results are the first to show that MEK-ERK signalling can act to increase or decrease cell survival, the outcome depending on the form of stress stimulus encountered.

Investigation of the effect of taurine supplementation on muscle taurine content in the mdx mouse model of Duchenne muscular dystrophy using chemically specific synchrotron imaging

Publisher: Royal Society of Chemistry (RSC)

Date: 2020

DOI: 10.1039/D0AN00642D

Abstract: Sulfur K-edge XANES was used to quantify changes in the taurine content of mouse muscle tissue in a model of muscular dystrophy. The changes could be associated with markers of disease pathology that were revealed by classical H& E histology.

In vivo decrease of elevated protein thiol oxidation and protection of dystrophic muscle from exercise induced damage, using the thiol containing antioxidant N-acetylcysteine in dystrophic mdx mice

Publisher: Elsevier BV

Date: 10-2011

DOI: 10.1016/J.NMD.2011.06.987

The Pasteur effect in human platelets: implications for storage and metabolic control

Publisher: Wiley

Date: 11-1995

DOI: 10.1111/J.1365-2141.1995.TB05381.X

Abstract: The Pasteur effect and the associated acidosis have long been considered a major cause of platelet death during storage. We have investigated this phenomenon using a defined platelet preparation and a system whereby the oxidative and glycolytic contributions to total ATP production can be measured over a range of oxygen concentrations from saturating (pO2 = 158 mmHg) to anoxic (pO2 = 0 mmHg). Platelets do not show a Pasteur effect until the pO2 decreases to < 2.0 mmHg, whereupon lactate production increases 1.5-fold. The Pasteur effect is therefore not a likely cause of platelet death during storage where pO2 in a storage bag typically drops to no less than 50 mmHg. The data also have implications for the role of oxygen diffusion in oxidative metabolism, and for the compensatory nature of the Pasteur effect. As platelets are relatively small cells, and the onset of the Pasteur effect occurs at a relatively low oxygen concentration, diffusion may limit the rate of oxygen consumption in most other (larger) cells. The Pasteur effect is only fully compensative if the P/O2 ratio used for the calculations is lower than the conventional one. Since recent research strongly suggests that the conventional P/O2 ratio is too high, ex les of fully compensative Pasteur effects may be more common than the literature suggests.

Dimethyl fumarate modulates the Duchenne muscular dystrophy disease program following short-term treatment in mdx mice

Publisher: Cold Spring Harbor Laboratory

Date: 17-09-2022

DOI: 10.1101/2022.09.15.508124

Abstract: New medicines are urgently required to treat the fatal neuromuscular disease, Duchenne muscular dystrophy (DMD). DMD involves progressive muscle damage and weakness, which are preceded by oxidative stress, inflammation, and mitochondrial dysfunction. Dimethyl fumarate (DMF) is a potent small molecule nuclear erythroid 2-related factor 2 (Nrf2) activator with current clinical utility in the treatment of multiple sclerosis and psoriasis. Pharmaceutical targeting of Nrf2 by DMF has strong translational potential for DMD, given it: (1) promotes antioxidant defence systems (2) has a potent immuno-modulatory profile and (3) can be rapidly re-purposed into clinical care strategies for DMD patients. Here, we tested two weeks of daily 100mg/kg DMF versus 5mg/kg standard care prednisone (PRED) treatment during the peak muscle degeneration period in juvenile mdx mice, the gold standard murine DMD model. Both drugs modulated seed genes driving the DMD disease program and improved muscle force production in fast-twitch muscle. However, only DMF showed pro-mitochondrial effects that protected contracting muscles from fatigue, improved histopathology and augmented clinically compatible muscle function tests. In contrast, PRED treatment stunted mouse growth, worsened histopathology and modulated many normally expressed inflammatory and extracellular matrix (ECM) genes consistent with pan immunosuppression. These findings suggest DMF could be a more selective modulator of the DMD disease program with better efficacy and fewer side effects than standard care PRED therapy warranting follow-up studies to progress clinical translation.

Mitochondria-derived reactive oxygen species drive GANT61-induced mesothelioma cell apoptosis

Publisher: Impact Journals, LLC

Date: 16-01-2015

DOI: 10.18632/ONCOTARGET.2729

Abstract: Gli transcription factors of the Hedgehog (Hh) pathway have been reported to be drivers of malignant mesothelioma (MMe) cell survival. The Gli inhibitor GANT61 induces apoptosis in various cancer cell models, and has been associated directly with Gli inhibition. However various chemotherapeutics can induce cell death through generation of reactive oxygen species (ROS) but whether ROS mediates GANT61-induced apoptosis is unknown. In this study human MMe cells were treated with GANT61 and the mechanisms regulating cell death investigated. Exposure of MMe cells to GANT61 led to G1 phase arrest and apoptosis, which involved ROS but not its purported targets, GLI1 or GLI2. GANT61 triggered ROS generation and quenching of ROS protected MMe cells from GANT61-induced apoptosis. Furthermore, we demonstrated that mitochondria are important in mediating GANT61 effects: (1) ROS production and apoptosis were blocked by mitochondrial inhibitor rotenone (2) GANT61 promoted superoxide formation in mitochondria and (3) mitochondrial DNA-deficient LO68 cells failed to induce superoxide, and were more resistant to apoptosis induced by GANT61 than wild-type cells. Our data demonstrate for the first time that GANT61 induces apoptosis by promoting mitochondrial superoxide generation independent of Gli inhibition, and highlights the therapeutic potential of mitochondrial ROS-mediated anticancer drugs in MMe.

Inhibitors of c-Jun N-terminal kinases: JuNK no more?

Publisher: Elsevier BV

Date: 2008

DOI: 10.1016/J.BBAPAP.2007.09.013

Peroxiredoxin 2 overexpression protects cortical neuronal cultures from ischemic and oxidative injury but not glutamate excitotoxicity, whereas Cu/Zn superoxide dismutase 1 overexpression protects only against oxidative injury

Publisher: Wiley

Date: 2007

DOI: 10.1002/JNR.21429

Abstract: We previously reported that peroxiredoxin 2 (PRDX2) and Cu/Zn superoxide dismutase 1 (SOD1) proteins are up-regulated in rat primary neuronal cultures following erythropoietin (EPO) preconditioning. In the present study, we have demonstrated that adenovirally mediated overexpression of PRDX2 in cortical neuronal cultures can protect neurons from in vitro ischemia (oxygen-glucose deprivation) and an oxidative insult (cumene hydroperoxide) but not glutamate excitotoxicity. We have also demonstrated that adenovirally mediated overexpression of SOD1 in cortical neuronal cultures protected neurons only against the oxidative insult. Interestingly, we did not detect up-regulation of PRDX2 or SOD1 protein in the rat hippoc us following exposure to either 3 min or 8 min of global cerebral ischemia. Further characterization of PRDX2's neuroprotective mechanisms may aid in the development of a neuroprotective therapy.

Beneficial effects of high dose taurine treatment in juvenile dystrophic mdx mice are offset by growth restriction

Publisher: Public Library of Science (PLoS)

Date: 02-11-2017

DOI: 10.1371/JOURNAL.PONE.0187317

Primary cortical neuronal cultures reduce cellular energy utilization during anoxic energy deprivation

Publisher: Wiley

Date: 26-09-2003

DOI: 10.1046/J.1471-4159.2003.02049.X

Abstract: It has been widely hypothesized that neurons reduce cellular energy use in response to periods of energy deprivation. To test this hypothesis, we measured rates of energy use under normoxia and anoxia in immature (6 days in vitro) and mature (13 days in vitro) neuronal cultures. During anoxic incubation immature and mature cultures reduced cellular energy use by 80% and 45%, respectively. Reduced cellular energy use dramatically affected ATP depletion in neuronal cultures under anoxia. Intracellular ATP stores were expected to deplete within 3 min of anoxia. However, ATP was maintained at decreased but stabilized concentrations for at least 3 h. The capacity of neuronal cultures to reduce cellular energy use during anoxia correlated with their sensitivity towards simulated ischemia. Immature cultures, with the largest capacity to reduce cellular energy use, survived simulated ischemia 2.5 times longer than mature cultures. The addition of glutamate receptor antagonists to mature cultures further decreased cellular energy use during anoxia and significantly extended their survival time under simulated ischemia. This study verifies that primary cortical neuronal cultures reduce cellular energy use during energy deprivation. Additionally, we show that maturation of glutamate receptor activity increases non-depressible energy demand in neuronal cultures.

Increasing taurine intake and taurine synthesis improves skeletal muscle function in the mdx mouse model for Duchenne muscular dystrophy

Publisher: Wiley

Date: 18-01-2016

DOI: 10.1113/JP271418

Plasma albumin thiol oxidation as a biomarker of muscle damage and inflammation

Publisher: Elsevier BV

Date: 10-2019

DOI: 10.1016/J.NMD.2019.06.422

Necrotic death of neurons following an excitotoxic insult is prevented by a peptide inhibitor of c-jun N-terminal kinase.

Publisher: Wiley

Date: 10-04-2007

DOI: 10.1111/J.1471-4159.2007.04618.X

Abstract: Peptide inhibitors of c-Jun N-terminal kinase (JNK) have been shown to potently protect against cerebral ischemia. The protective effect has been ascribed to prevention of apoptosis, but cell death following cerebral ischemia is a consequence of both apoptotic and necrotic cell death. We evaluated whether a peptide inhibitor (TAT-TIJIP) of JNK could prevent necrotic cell death in an in vitro model of excitotoxic neuronal death. We find that TAT-TIJIP effectively prevented cell death by interfering with several processes which have been identified as leading to cell death by necrosis. In particular, reactive oxygen species production was reduced, as indicated by an 88% decrease in the rate of dihydroethidium fluorescence in the presence of TAT-TIJIP. Furthermore, TAT-TIJIP attenuated the increase in cytosolic calcium following the excitotoxic insult. The potent neuroprotective properties of JNK peptide inhibitors likely reflects their abilities to prevent cell death by necrosis as well as apoptosis.

The effect of storage at 25 degrees C on proteins in human milk

Publisher: Elsevier BV

Date: 04-2011

DOI: 10.1016/J.IDAIRYJ.2010.12.001

The concentration of creatine and creatine phosphate in sow colostrum and milk throughout lactation and weaning

Publisher: CSIRO Publishing

Date: 1997

DOI: 10.1071/A96077

Abstract: The concentrations of creatine and creatine phosphate were determined by enzymatic analysis of mammary secretions from a total of 63 sows from pre-farrowing (PF), during farrowing, and on Days 1, 2, 3, 4, 5, 7, 11, 18, and 28 (at weaning) of lactation. Three sows were s led on all days and on Days 1, 2, 3, 4, and 5 post weaning. S les were collected from an additional 5 sows on the s ling days PF to Day 28 of lactation. The concentration of creatine remained constant at 1·4±0·02 mM (mean ±s.e.m.) prior to farrowing and throughout lactation. The concentration of creatine phosphate was 0·41±0·05 mM prior to farrowing until Day 2 of lactation. The concentration of creatine phosphate then increased gradually from Day 3 to reach a peak at 1·1± 0·05 mM by Day 7 and then remained unchanged throughout lactation. The concentrations of creatine and creatine phosphate decreased 30-42 h post weaning, and by 118 h post weaning both creatine and creatine phosphate were undetectable in milk s les. The within-sow variation in the concentration of creatine and creatine phosphate in colostrum before farrowing was small (CVs 2-9% and 6-9%, respectively). However, during farrowing, throughout lactation, and post weaning, the variations in the concentration of creatine and creatine phosphate were much larger (CVs 19-27% and 25-80%, respectively). In contrast, the between-sow variations in the concentration of creatine and creatine phosphate in mammary secretion collected at all of these stages of the lactation cycle were large (CVs 15-41% and 19-57%). It was concluded that concentrations of creatine and creatine phosphate may reflect mammary gland metabolic activity and could be involved in the development of the piglet during the suckling period.

A perifusion system to control oxygen concentration in cell suspensions

Publisher: Elsevier BV

Date: 10-1998

DOI: 10.1006/ABIO.1998.2787

Abstract: A system is described for the perifusion of cells with a perifusate containing oxygen at concentrations defined by the user. The metabolic competency of cells in this system was assessed by measuring total ATP turnover for human platelets. Human platelets, which are small (radius of 1-2 micron) anucleate cells involved in blood clotting, maintained ATP turnover over a 4-h period at 37 degrees C. Oxygen concentration in this system was controlled by adjusting the proportions of air-saturated and nitrogen-saturated perifusates. Ex les of oxygen consumption and lactate output as a function of oxygen concentration are described.

Transient exposure to hydrogen peroxide causes an increase in mitochondria-derived superoxide as a result of sustained alteration in L-type Ca2+ channel function in the absence of apoptosis in ventricular myocytes

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 13-04-2007

DOI: 10.1161/01.RES.0000263010.19273.48

Abstract: We sought to understand the effect of a transient exposure of cardiac myocytes to H 2 O 2 at a concentration that did not induce apoptosis. Myocytes were exposed to 30 μmol/L H 2 O 2 for 5 minutes followed by 10 U/mL catalase for 5 minutes to degrade the H 2 O 2 . Cellular superoxide was measured using dihydroethidium. Transient exposure to H 2 O 2 caused a 66.4% increase in dihydroethidium signal compared with controls exposed to only catalase, without activation of caspase 3 or evidence of necrosis. The increase in dihydroethidium signal was attenuated by the mitochondrial inhibitors myxothiazol or carbonyl cyanide p -(trifluoromethoxy)phenyl-hydrazone and when calcium uptake by the mitochondria was inhibited with Ru360. We investigated the L-type Ca 2+ channel ( I Ca-L ) as a source of calcium influx. Nisoldipine, an inhibitor of I Ca-L , attenuated the increase in superoxide. Basal channel activity increased from 5.4 to 8.9 pA F. Diastolic calcium was significantly increased in quiescent and contracting myocytes after H 2 O 2 . The response of I Ca-L to β-adrenergic receptor stimulation was used as a functional reporter because decreasing intracellular H 2 O 2 alters the sensitivity of I Ca-L to isoproterenol. H 2 O 2 increased the K 0.5 required for activation of I Ca-L by isoproterenol from 5.8 to 27.8 nmol/L. This effect and the increase in basal current density persisted for several hours after H 2 O 2 . We propose that extracellular H 2 O 2 is associated with an increase in superoxide from the mitochondria caused by an increase in Ca 2+ influx from I Ca-L . The effect persists because a positive feedback exists among increased basal channel activity, elevated intracellular calcium, and superoxide production by the mitochondria.

N-Acetylcysteine treatment of dystrophic mdx mice results in protein thiol modifications and inhibition of exercise induced myofibre necrosis

Publisher: Elsevier BV

Date: 05-2012

DOI: 10.1016/J.NMD.2011.11.007

Abstract: Oxidative stress is implicated as a factor that increases necrosis of skeletal muscles in Duchenne Muscular Dystrophy (DMD) and the dystrophic mdx mouse. Consequently, drugs that minimize oxidative stress are potential treatments for muscular dystrophy. This study examined the in vivo benefits to mdx mice of an antioxidant treatment with the cysteine precursor N-acetylcysteine (NAC), administered in drinking water. NAC was completely effective in preventing treadmill exercise-induced myofibre necrosis (assessed histologically) and the increased blood creatine kinase levels (a measure of sarcolemma leakiness) following exercise were significantly lower in the NAC treated mice. While NAC had no effect on malondialdehyde level or protein carbonylation (two indicators of irreversible oxidative damage), treatment with NAC for one week significantly decreased the oxidation of glutathione and protein thiols, and enhanced muscle protein thiol content. These data provide in vivo evidence for protective benefits of NAC treatment on dystropathology, potentially via protein thiol modifications.

Protein thiol oxidation does not change in skeletal muscles of aging female mice

Publisher: Springer Science and Business Media LLC

Date: 06-12-2014

DOI: 10.1007/S10522-013-9483-Y

Abstract: Oxidative stress caused by reactive oxygen species is proposed to cause age related muscle wasting (sarcopenia). Reversible oxidation of protein thiols by reactive oxygen species can affect protein function, so we evaluated whether muscle wasting in normal aging was associated with a pervasive increase in reversible oxidation of protein thiols or with an increase in irreversible oxidative damage to macromolecules. In gastrocnemius muscles of C57BL/6J female mice aged 3, 15, 24, 27, and 29 months there was no age related increase in protein thiol oxidation. In contrast, there was a significant correlation (R (2) = 0.698) between increasing protein carbonylation, a measure of irreversible oxidative damage to proteins, and loss of mass of gastrocnemius muscles in aging female mice. In addition, there was an age-related increase in lipofuscin content, an aggregate of oxidised proteins and lipids, in quadriceps limb muscles in aging female mice. However, there was no evidence of an age-related increase in malondialdehyde or F2-isoprostanes levels, which are measures of oxidative damage to lipids, in gastrocnemius muscles. In summary, this study does not support the hypothesis that a pervasive increase in protein thiol oxidation is a contributing factor to sarcopenia. Instead, the data are consistent with an aging theory which proposes that molecular damage to macromolecules leads to the structural and functional disorders associated with aging.

A semiautomated enzymatic method for determination of nonesterified fatty acid concentration in milk and plasma

Publisher: Wiley

Date: 10-1998

DOI: 10.1007/S11745-998-0304-9

Abstract: An enzymatic assay for the determination of nonesterified fatty acid concentrations in milk and plasma is described. The procedure is semiautomated for use with a plate luminometer or plate spectrophotometer and enables routine batch processing of large numbers of small s les (< or =5 microL). Following the activation of nonesterified fatty acids (NEFA) by acylCoA synthetase, the current assay utilizes UDP-glucose pyrophosphorylase to link inorganic pyrophosphate to the production of NADH through the reactions catalyzed by phosphoglucomutase and glucose-6-phosphate 1-dehydrogenase. With this assay sequence the formation of NADH from NEFA is complete within 50 min at 37 degrees C. Enzymatic spectrophotometric techniques were unsuitable for NEFA determination in human milk due to the opacity of the s le. The use of the NADH-luciferase system has overcome this problem, allowing the enzymatic determination of NEFA in human milk. S le collection and treatment procedures for milk and plasma have been developed to prevent enzymatic lipolysis and to limit interference from enzymes present in milk. The recovery of palmitic acid added to milk and plasma s les was 94.9+/-2.9 and 100+/-4.5%, respectively. There was no difference (P = 0.13) in plasma NEFA concentrations determined by the current method and a commercially available enzymatic spectrophotometric technique (Wako NEFA-C kit). Plasma NEFA concentrations determined by gas chromatography were 28% higher compared to both the Wako NEFA-C kit and the current method.

Taurine

Publisher: Elsevier BV

Date: 10-2017

DOI: 10.1016/J.NMD.2017.06.356

The Effect of Acute Taurine Ingestion on Human Maximal Voluntary Muscle Contraction

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 02-2018

DOI: 10.1249/MSS.0000000000001432

The contractile phenotype of a novel dystrophin-negative mouse strain with enhanced voluntary exercise capability

Publisher: Elsevier BV

Date: 10-2017

DOI: 10.1016/J.NMD.2017.06.352

Metabolites of Lactose Synthesis in Milk from Diabetic and Nondiabetic Women During Lactogenesis II

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 07-1994

DOI: 10.1097/00005176-199407000-00017

Abstract: The concentrations of lactose, glucose, glucose 6-phosphate (G6P), glucose 1-phosphate (G1P), UDPglucose (UDPglc), UDPgalactose (UDPgal), and inorganic phosphate (P(i)) (metabolites in the lactose synthesis pathway) were measured in mammary secretion from nondiabetic (ND) and insulin-dependent diabetic (IDD) mothers during the first 10 days postpartum to determine their relationship with the amount of lactose synthesized and their association with the delay in lactose synthesis in IDD mothers. For all mothers the concentrations of all metabolites were low initially, and in ND mothers the first increases occurred as follows: lactose--day 2 glucose, G6P and P(i)--day 3 G1P and UDPglc--day 4 and UDPgal--day 6. The first increases for IDD mothers occurred 1-4 days later than for ND mothers. The concentrations of glucose, G6P, G1P, UDPglc, and P(i) were related to the amount of lactose synthesized. Since the rate of lactose synthesis and concentrations of other metabolites were less than half-maximal while the concentration of glucose was low, and since there was a delay in the increase in mammary gland concentrations of glucose in IDD mothers, it is concluded that glucose availability has the potential to play a role in the regulation of the rate of lactose synthesis at lactogenesis II.

Oxidative stress causes a decline in lysosomal integrity during hypothermic incubation of rat hepatocytes

Publisher: Elsevier BV

Date: 2008

DOI: 10.1016/J.FREERADBIOMED.2007.09.003

Abstract: Oxidative stress during cold preservation has been identified as a significant cause of cell injury but the process by which injury occurs is poorly understood. We examined loss of lysosomal integrity as a possible cause of cell injury during extended cold storage of isolated rat hepatocytes. After 21 h of hypothermia, there was a marked decline in lysosomal integrity, which was correlated with an increase in lipid peroxidation. When lipid peroxidation was prevented with the antioxidant Trolox (a vitamin E analog) or the iron chelator desferrioxamine, lysosomal integrity was preserved. In contrast, increasing lysosomal iron with ferric chloride caused an increase in lipid peroxidation and decreased lysosomal integrity. Loss of lysosomal integrity during cold preservation in this experimental model was consistent with iron-initiated oxidative stress. The progressive loss of lysosomal integrity during hypothermic incubation has the potential to affect liver function after transplantation.

RECOVERY METABOLISM OF SKIPJACK TUNA (KATSUWONUS-PELAMIS) WHITE MUSCLE - RAPID AND PARALLEL CHANGES IN LACTATE AND PHOSPHOCREATINE AFTER EXERCISE

Publisher: Canadian Science Publishing

Date: 06-1992

DOI: 10.1139/Z92-170

Abstract: Lactate, glycogen, and high-energy phosphate levels were measured in serial biopsies from tuna white muscle during recovery from 15 min of enforced swimming. Exercise caused glycogen and phosphocreatine levels to decrease sharply and lactate concentration to increase markedly (up to 150 μmol∙g −1 ). Lactate was cleared from white muscle in less than 90 min, at rates comparable to those seen in mammals (about 1.3 μmol∙g −1 ∙min −1 ), and this was accompanied by nearly stoichiometric increases in white muscle glycogen (2 lactate:1 glucosyl unit). The plasma lactate concentration remained elevated (35–40 mM) until lactate clearance from white muscle was completed, whereas the level of plasma glucose was constant (12–16 mM) for the entire 3-h recovery period. The exercise routine caused minimal changes in white muscle purine nucleotides apart from a slight, but significant, increase in IMP content. Transient changes in ATP appear to have resulted from short-term intense swimming activity noted during anesthetization. Unlike other teleosts, lactate clearance in tuna paralleled creatine rephosphorylation during recovery from exercise. We suggest that the postexercise adjustment of intracellular pH is responsible for this relationship. Lactate was seemingly metabolized within the white muscle mass, as indicated by in situ conservation of lactate carbon apparent from stiochiometric increases in white muscle glycogen levels. This prospect is discussed in view of low estimates of lactate utilization rates by other tissues and contrasted with expected high rates of whole-body lactate turnover during recovery.

Oxidative stress and pathology in muscular dystrophies: focus on protein thiol oxidation and dysferlinopathies

Publisher: Wiley

Date: 15-02-2013

DOI: 10.1111/FEBS.12142

Abstract: The muscular dystrophies comprise more than 30 clinical disorders that are characterized by progressive skeletal muscle wasting and degeneration. Although the genetic basis for many of these disorders has been identified, the exact mechanism for pathogenesis generally remains unknown. It is considered that disturbed levels of reactive oxygen species (ROS) contribute to the pathology of many muscular dystrophies. Reactive oxygen species and oxidative stress may cause cellular damage by directly and irreversibly damaging macromolecules such as proteins, membrane lipids and DNA another major cellular consequence of reactive oxygen species is the reversible modification of protein thiol side chains that may affect many aspects of molecular function. Irreversible oxidative damage of protein and lipids has been widely studied in Duchenne muscular dystrophy, and we have recently identified increased protein thiol oxidation in dystrophic muscles of the mdx mouse model for Duchenne muscular dystrophy. This review evaluates the role of elevated oxidative stress in Duchenne muscular dystrophy and other forms of muscular dystrophies, and presents new data that show significantly increased protein thiol oxidation and high levels of lipofuscin (a measure of cumulative oxidative damage) in dysferlin-deficient muscles of A/J mice at various ages. The significance of this elevated oxidative stress and high levels of reversible thiol oxidation, but minimal myofibre necrosis, is discussed in the context of the disease mechanism for dysferlinopathies, and compared with the situation for dystrophin-deficient mdx mice.

Automated Analysis of Cellular Metabolites at Nanomolar to Micromolar Concentrations Using Bioluminescent Methods

Publisher: Elsevier BV

Date: 05-1995

DOI: 10.1006/ABIO.1995.1281

Abstract: Automated bioluminescent assays for lactate, oxoglutarate, pyruvate, ammonia, NAD, glutamate, ATP, phosphocreatine, creatine, and NADH were developed from existing spectrophotometric and fluorometric assays. A key feature was the development of a luminescent reagent that was not only relatively cheap and stable, but also could be stored frozen. With this reagent it was possible to measure NADH concentrations as low as 0.1 nM in a 25-microliters s le. The sensitivity of the other assays was limited by contamination of the enzymes and cofactors used in the assays and ranged from 0.05 to 25 microM with s le volumes of 0.75 - 10 microliters.

Necrotic death without mitochondrial dysfunction-delayed death of cardiac myocytes following oxidative stress.

Publisher: Elsevier BV

Date: 03-2007

DOI: 10.1016/J.BBAMCR.2006.11.013

Abstract: Oxidative stress has been implicated in cell death in range of disease states including ischemia/reperfusion injury of the heart and heart failure. Here we have investigated the mechanisms of cell death following chronic exposure of cardiac myocytes to oxidative stress initiated by hydrogen peroxide. This exposure induced a delayed form of cell death with ultrastructural changes typical of necrosis, and that was accompanied by the release of lactate dehydrogenase and increased lipid peroxidation. However, this delayed death was not accompanied by the loss of mitochondrial membrane potential or caspase-3 activation. Furthermore, we could demonstrate that this delayed necrosis was at least partially prevented by pre-treatment with the hypertrophic stimuli endothelin-1 or leukemic inhibitory factor. Our results suggest that this delayed form necrosis may also comprise an ordered series of events involving pathways amenable to therapeutic modulation.

The coupling of NAD(P)(+)-producing reactions to a semiautomated bioluminescent reaction

Publisher: Elsevier BV

Date: 04-1999

DOI: 10.1006/ABIO.1999.4009

Abstract: Many cellular metabolites can be measured with high sensitivity using bioluminescent techniques. These metabolites are coupled to an appropriate enzyme to produce NAD(P)H, which can then be coupled to the bioluminescent reactions. The sensitivity of bioluminescence cannot be readily applied to methods in which cellular metabolites consume NAD(P)H because of the difficulty in measuring, with sufficient sensitivity, decreases in the concentration of NAD(P)H against a high background NAD(P)H concentration. We have overcome these technical difficulties by developing a bioluminescent reagent to measure the production of NAD(P)+. Assays for creatine/creatine phosphate, pyruvate, and succinate, as well as the kinetic measurement of lactate, are described for a range of biological material. The assays are highly sensitive, quantitative, and reproducible and show no s le-specific inhibition. The range of assays and the erse biological material tested suggests that NAD(P)+ bioluminescence has a wide potential for application.

Effect of work and recovery duration on skeletal muscle oxygenation and fuel use during sustained intermittent exercise

Publisher: Springer Science and Business Media LLC

Date: 09-1999

DOI: 10.1007/S004210050615

Abstract: The purpose of this study was to compare rates of substrate oxidation in two protocols of intermittent exercise, with identical treadmill speed and total work duration, to reduce the effect of differences in factors such as muscle fibre type activation, hormonal responses, muscle glucose uptake and non-esterified fatty acid (NEFA) availability on the comparison of substrate utilisation. Subjects (n = 7) completed 40 min of intermittent intense running requiring a work:recovery ratio of either 6 s:9 s (short-interval exercise, SE) or 24 s:36 s (long-interval exercise, LE), on separate days. Another experiment compared O(2) availability in the vastus lateralis muscle across SE (10 min) and LE (10 min) exercise using near-infrared spectroscopy (RunMan, NIM. Philadelphia, USA). Overall (i.e. work and recovery) O(2) consumption (VO(2)) and energy expenditure were lower during LE (P < 0.01, P < 0.05, respectively). Overall exercise intensity, represented as a proportion of peak aerobic power (VO2(peak)), was [mean (SEM)] 64.9 (2.7)% VO2(peak) (LE) and 71.4 (2.4)% VO2(peak) (SE). Fat oxidation was three times lower (P < 0.01) and carbohydrate oxidation 1.3 times higher (P < 0. 01) during LE, despite the lower overall exercise intensity. Plasma lactate was constant and was higher throughout exercise in LE [mean (SEM) 5.33 (0.53) mM, LE 3.28 (0.31) mM, SE P < 0.001)]. Plasma pyruvate was higher and glycerol was lower in LE [215 (17) microM, 151 (13) microM, P < 0.05, pyruvate 197 (19) microM, 246 (19) microM, P < 0.05, glycerol]. There was no difference between protocols for plasma NEFA concentration (n = 4) or plasma noradrenaline and adrenaline. Muscle oxygenation declined in both protocols (P < 0.001), but the nadir during LE was lower [52.04 (0. 60)%] compared to SE [61.85 (0.51)% P < 0.001]. The decline in muscle oxygenation during work was correlated with mean lactate concentration (r = 0.68 P < 0.05 n = 12). Lower levels of fat oxidation occurred concurrent with accelerated carbohydrate metabolism, increases in lactate and pyruvate and reduced muscle O(2) availability. These changes were associated with proportionately longer work and recovery periods, despite identical treadmill speed and total work duration. The proposal that a metabolic regulatory factor within the muscle fibre retards fat oxidation under these conditions is supported by the current findings.

A fluorescent dual labeling technique for the quantitative measurement of reduced and oxidized protein thiols in tissue samples

Publisher: Elsevier BV

Date: 02-2011

DOI: 10.1016/J.FREERADBIOMED.2010.11.018

Abstract: Oxidative stress can result in the reversible oxidation of protein thiols. Because the activity of numerous proteins is sensitive to thiol oxidation, this has the potential to affect many cellular functions. We describe a highly sensitive, quantitative labeling technique that measures global and specific protein thiol oxidative state in skeletal muscle tissue. The technique involves labeling the reduced and oxidized protein thiols with different fluorescent dyes. The resulting s le is assayed using a 96-well plate fluorimeter, or in idual protein bands are separated using SDS-PAGE. We show that artifactual oxidation during s le preparation and analysis has the potential to confound results, and techniques to prevent this are described. We tested the technique by analyzing the muscles of mdx and c57 mice and found that the muscles of mdx mice were significantly (p<0.05) more oxidized (13.1±1.5% oxidized thiols) than those of c57 mice (8.9±0.7% oxidized thiols). This technique provides an effective means to measure the extent to which oxidative stress affects the oxidation of protein thiols in biological tissues.

A comparison of skeletal muscle oxygenation and fuel use in sustained continuous and intermittent exercise

Publisher: Springer Science and Business Media LLC

Date: 09-1999

DOI: 10.1007/S004210050614

Abstract: In this study we compared substrate oxidation and muscle oxygen availability during sustained intermittent intense and continuous submaximal exercise with similar overall (i.e. work and recovery) oxygen consumption (VO2). Physically active subjects (n = 7) completed 90 min of an intermittent intense (12 s work:18 s recovery) and a continuous submaximal treadmill running protocol on separate days. In another experiment (n = 5) we compared oxygen availability in the vastus lateralis muscle between these two exercise protocols using near-infrared spectroscopy. Initially, overall VO(2) (i.e. work and recovery) was matched, and from 37.5 min to 67.5 min of exercise was similar, although slightly higher during continuous exercise (8% P < 0.05). Energy expenditure was constant (22.5-90 min of exercise) and was not different in intermittent intense [0.81 (0.01) kJ x min(-1). kg(-1)] and continuous submaximal [0.85 (0.01) kJ x min(-1) x kg(-1)] exercise. Overall exercise intensity, represented as a proportion of peak aerobic power (VO2(peak)), was 68.1 (2.5)% VO2(peak) and 71.8 (1.8)% VO2(peak) for intermittent and continuous exercise protocols, respectively. Fat oxidation was almost 3 times lower (P < 0.05) and carbohydrate oxidation was approximately 1.2 times higher (P < 0.05) during intermittent compared to continuous exercise, despite the same overall energy expenditure. Capillary plasma lactate was constant from 15 to 90 min of exercise, and pyruvate was constant from 15 to 75 min, although both were higher (P < 0.0001, lactate P < 0.001, pyruvate) during intermittent [5.05 (0.28) mM, 200 (7) microM, respectively] compared to continuous exercise [2.41 (0.10) mM, 114 (4) microM, respectively]. There was no difference between protocols for either plasma glycerol or non-esterified fatty acids. The decrease in muscle oxygenation during work periods of intermittent exercise resulted in a lower nadir oxygenation [54.62 (0.41)%] compared to continuous exercise [58.82 (0.21)%, P < 0.001]. The decline in oxygenation was correlated with treadmill speed (r = 0.72 P < 0.05). These results show a difference in substrate utilisation and muscle oxygen availability during sustained intermittent intense and continuous submaximal exercise, despite a similar overall VO(2) and identical energy expenditure.

University Of Western Australia

Location: Australia

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Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0882866

Start Date: 12-2008

End Date: 12-2009

Amount: $470,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0560987

Start Date: 03-2005

End Date: 12-2005

Amount: $156,697.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP0347870

Start Date: 06-2003

End Date: 12-2006

Amount: $84,099.00

Funder: Australian Research Council

Industrial Transformation Training Centres - Grant ID: IC170100016

Start Date: 05-2018

End Date: 12-2024

Amount: $3,123,492.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP100100793

Start Date: 03-2010

End Date: 05-2015

Amount: $80,007.00

Funder: Australian Research Council