Peter G. Arthur

Milk Lactose, Citrate, and Glucose as Markers of Lactogenesis in Normal and Diabetic Women

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 individual 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 < 0.001), citrate (r = 0.47, n = 47, p < 0.001), and glucose (r = 0.69, n = 50, p < 0.001). The time of milk “coming in” was highly variable (59 ± 13) (X ± SD) and occurred well after the onset of copious milk secretion between 24 and 48 h after birth. We conclude that for individual mothers, milk lactose and citrate are useful markers of lactogenesis II. The changes in the concentrations of these markers suggested that lactogenesis II was delayed in type I diabetic mothers.

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

Abstract The c-Jun N-terminal kinases (JNKs) have been the subject of intense interest since their discovery in the early 1990s. Major research programs have been directed to the screening and/or design of JNK-selective inhibitors and testing their potential as drugs. We begin this review by considering the first commercially-available JNK ATP-competitive inhibitor, SP600125. We focus on recent studies that have evaluated the actions of SP600125 in lung, brain, kidney and liver following exposure to a range of stress insults including ischemia/reperfusion. In many but not all cases, SP600125 administration has proved beneficial. JNK activation can also follow infection, and we next consider recent examples that demonstrate the benefits of SP600125 administration in viral infection. Additional ATP-competitive JNK inhibitors have now been described following high throughput screening of small molecule libraries, but information on their use in biological systems remains limited and thus these inhibitors will require further evaluation. Peptide substrate-competitive ATP-non-competitive inhibitors of JNK have also now been described, and we discuss the recent advances in the use of JNK inhibitory peptides in the treatment of neuronal death, diabetes and viral infection. We conclude by raising a number of questions that should be considered in the quest for JNK-specific inhibitors.

Exercise intensity and metabolic response in singles tennis

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.

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

We sought to understand the effect of a transient exposure of cardiac myocytes to H2O2 at a concentration that did not induce apoptosis. Myocytes were exposed to 30 &mgr;mol/L H2O2 for 5 minutes followed by 10 U/mL catalase for 5 minutes to degrade the H2O2. Cellular superoxide was measured using dihydroethidium. Transient exposure to H2O2 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 Ca2+ channel (ICa-L) as a source of calcium influx. Nisoldipine, an inhibitor of ICa-L, attenuated the increase in superoxide. Basal channel activity increased from 5.4 to 8.9 pA/pF. Diastolic calcium was significantly increased in quiescent and contracting myocytes after H2O2. The response of ICa-L to &bgr;-adrenergic receptor stimulation was used as a functional reporter because decreasing intracellular H2O2 alters the sensitivity of ICa-L to isoproterenol. H2O2 increased the K0.5 required for activation of ICa-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 H2O2. We propose that extracellular H2O2 is associated with an increase in superoxide from the mitochondria caused by an increase in Ca2+ influx from ICa-L. The effect persists because a positive feedback exists among increased basal channel activity, elevated intracellular calcium, and superoxide production by the mitochondria.

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

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 hippocampus 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 As extracellular-mediated neuroprotection occurs via CD147 receptor signalling, possibly by activation of ERK1/2 pro-survival pathways. Further characterization of cyclophilin As neuroprotective mechanisms may aid the development of a neuroprotective therapy.

MEASUREMENT OF THE MILK INTAKE OF BREAST-FED INFANTS

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 mothers change in weight after a single breast feed and from 3 to 55% of the infants 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%.

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

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.

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

Preconditioning describes a variety of treatments that induce neurons to become more resistant to a subsequent ischemic insult. How preconditioned neurons adapt to subsequent ischemic stress is not fully understood, but is likely to involve multiple protective mechanisms. We hypothesized hypoxic preconditioning induces protection by a coordinated up-regulation of antioxidant enzyme activity. To test this hypothesis, we developed two in vitro models of ischemia/reperfusion, involving oxygen-glucose deprivation (OGD) where neuronal cell death was predominantly by necrosis (necrotic model) or programmed cell death (PCD model). Hypoxic preconditioning 24 h prior to OGD significantly reduced cell death from 83% to 22% in the necrotic model and 68% to 11% in the PCD model. Consistent with the hypothesis, the activity of the antioxidant enzymes glutathione peroxidase, glutathione reductase, and Mn superoxide dismutase were significantly increased by 54%, 73% and 32%, respectively, in neuronal cultures subjected to hypoxic preconditioning. Furthermore, superoxide and hydrogen peroxide concentrations following OGD were significantly lower in the PCD model that had been subjected to hypoxic preconditioning.

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

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 (V˙O2). 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 V˙O2 (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 · min−1 · kg−1] and continuous submaximal [0.85 (0.01) kJ · min−1 · kg−1] exercise. Overall exercise intensity, represented as a proportion of peak aerobic power (V˙O2peak), was 68.1 (2.5)% V˙O2peak and 71.8 (1.8)% V˙O2peak 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) μM, respectively] compared to continuous exercise [2.41 (0.10) mM, 114 (4) μM, 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 V˙O2 and identical energy expenditure.

Decreasing Cellular Hydrogen Peroxide With Catalase Mimics the Effects of Hypoxia on the Sensitivity of the L-Type Ca2+ Channel to β-Adrenergic Receptor Stimulation in Cardiac Myocytes

Abstract— In cardiac myocytes, hypoxia inhibits the basal L-type Ca2+ current (ICa-L) and increases the sensitivity of ICa-L to &bgr;-adrenergic receptor stimulation. We investigated whether hydrogen peroxide (H2O2) is involved in the hypoxic response. Guinea pig ventricular myocytes were dialyzed with catalase, which specifically catalyzes the conversion of H2O2 to H2O and oxygen, and then ICa-L was recorded during exposure to isoproterenol (Iso). Catalase decreased the K0.5 for activation of ICa-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 H2O2. A significant increase in sensitivity of ICa-L to Iso was recorded when mitochondrial function was inhibited with myxothiazol or FCCP, suggesting that the source of H2O2 was from the mitochondria. Prior exposure of cells to H2O2 attenuated the inhibition of basal ICa-L during hypoxia and the increase in sensitivity of ICa-L to Iso during hypoxia. Additionally, extracellularly applied catalase mimicked the effect of hypoxia on basal ICa-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 H2O2 levels and that H2O2 is a key intermediate in modifying basal channel activity and the &bgr;-adrenergic responsiveness of the channel during hypoxia.