Measurement of phagocyte chemiluminescence using a microtitre plate luminometer

The use of chemiluminescence techniques to study the interaction between bacteria and phagocytes has been useful for examining the extent to which serum factors, such as opsonins, are important in internalization of the organisms and the response of the cell to phagocytosed bacteria. However, such methods have been limited by the number of experiments which can be performed at one time using most commercial luminometers. However, the recent introduction of the Amerlite microtitre plate luminometer allows the measurement of chemiluminescence responses in 96-well microtitre plates. Using this instrument, lucigenin-enhanced chemiluminescence can be detected from as few as 5000 cells (polymorphonuclear leukocytes or monocytes) per well with a 1:10 ratio of cells to zymosan particles opsonized with 10% serum. The opsonic capacity of up to 100 sera can be measured in triplicate wells in a single experiment using four microtitre plates and polymorphonuclear leukocytes prepared from less than 40 ml freshly obtained venous blood. We are currently using this technique to investigate the effect of serum opsonins on the interaction between normal human polymorphonuclear leukocytes and monocytes with mycobacteria of three species (Mycobacterium leprae, M. tuberculosis, and M. aviumintracellulare). Other possible applications of this method are discussed.     

Effect of beta-adrenergic stimulation on uterine contraction in response to arginine vasotocin and prostaglandin F2 alpha in the gecko Hoplodactylus maculatus.

The effects of beta-adrenergic stimulation on uterine contractions occurring in response to arginine vasotocin (AVT) and prostaglandin F2 alpha (PGF2 alpha) were compared during late pregnancy in the viviparous gecko Hoplodactylus maculatus. High doses of AVT (150 or 1,500 ng/g body weight) induced birth in vivo, but PGF2 alpha at doses of up to 2,000 ng/g did not induce birth. The effect of AVT (150 ng/g) on birth rate in vivo was not enhanced by pretreatment 20 min beforehand with the beta-adrenoreceptor antagonist dichloroisoproterenol (2 micrograms/g), whereas the effect of PGF2 alpha (200 ng/g) was markedly enhanced: geckos treated with dichloroisoproterenol and then with PGF2 alpha showed rapid birth-related behavior and gave birth. Isolated uteri showed a tonic contraction in response to AVT (100 ng/ml) and to PGF2 alpha (1,000 ng/ml). Pre-exposure of isolated uteri to the beta-adrenoreceptor agonist isoproterenol (1 microgram/ml) caused relaxation; this pre-exposure did not block the tonic contraction occurring in response to AVT, whereas it completely blocked the tonic contraction induced by PGF2 alpha. We conclude that in H. maculatus, beta-adrenergic stimulation inhibits uterine contractions induced by PGF2 alpha but not those induced by AVT. These data are the first to show that beta-adrenergic stimulation inhibits uterotonic responses to PGF2 alpha in a reptile, and they suggest that the cellular mechanisms by which AVT and PGF2 alpha induce contraction may differ in this species. They also provide further evidence for similarities between mammals and reptiles in the effects of beta-adrenergic stimulation on uterine relaxation.     

Stimulation of net muscle protein synthesis by whey protein ingestion before and after exercise

Timing of nutrient ingestion has been demonstrated to influence the anabolic response of muscle following exercise. Previously, we demonstrated that net amino acid uptake was greater when free essential amino acids plus carbohydrates were ingested before resistance exercise rather than following exercise. However, it is unclear if ingestion of whole proteins before exercise would stimulate a superior response compared with following exercise. This study was designed to examine the response of muscle protein balance to ingestion of whey proteins both before and following resistance exercise. Healthy volunteers were randomly assigned to one of two groups. A solution of whey proteins was consumed either immediately before exercise (PRE; n = 8) or immediately following exercise (POST; n = 9). Each subject performed 10 sets of 8 repetitions of leg extension exercise. Phenylalanine concentrations were measured in femoral arteriovenous samples to determine balance across the leg. Arterial amino acid concentrations were elevated by approximately 50%, and net amino acid balance switched from negative to positive following ingestion of proteins at either time. Amino acid uptake was not significantly different between PRE and POST when calculated from the beginning of exercise (67 +/- 22 and 27 +/- 10 for PRE and POST, respectively) or from the ingestion of each drink (60 +/- 17 and 63 +/- 15 for PRE and POST, respectively). Thus the response of net muscle protein balance to timing of intact protein ingestion does not respond as does that of the combination of free amino acids and carbohydrate.     

Contraction of insulin-resistant muscle normalizes insulin action in association with increased mitochondrial activity and fatty acid catabolism

Acute exercise can reverse muscle insulin resistance, but the mechanism(s) of action are unknown. With the use of a hindlimb perfusion model, we have found that acute contraction restores insulin-stimulated glucose uptake in muscle of obese Zucker rats to levels witnessed in lean controls. Previous reports have suggested that obesity-related insulin resistance stems from lipid oversupply and tissue accumulation of toxic lipid intermediates that impair insulin signaling. We reasoned that contraction might activate hydrolysis and oxidation of intramuscular lipids, thus alleviating "lipotoxicity" and priming the muscle for enhanced insulin action. Indeed, analysis of mitochondrial-derived acyl-carnitine esters suggested that contraction caused robust increases in -oxidative flux and mitochondrial oxidation. As predicted, contraction decreased intramuscular triacylglycerol content; however, diacylglycerol and long chain acyl-CoAs, lipid intermediates presumed to trigger insulin resistance, were either unchanged or increased. In muscles from obese animals, insulin-stimulated tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 remained impaired after contraction, whereas phosphorylation of the downstream signaling protein, AS160, was partially restored. These results suggest that acute exercise enables diabetic muscle to circumvent upstream defects in insulin signal transduction via mechanisms that are more tightly coupled to increased mitochondrial energy metabolism than the lowering of diacylglycerol and long chain acyl-CoA. skeletal muscle; intramuscular lipids; signaling; exercise     

Effects of a training program on resting plasma 2-hydroxycatecholestrogen levels in eumenorrheic women

De Crée, Carl, Peter Ball, BärbelSeidlitz, Gerrit Van Kranenburg, Peter Geurten, and Hans A. Keizer.Effects of a training program on resting plasma2-hydroxycatecholestrogen levels in eumenorrheic women.J. Appl. Physiol. 83(5):1551-1556, 1997.Catecholestrogens (CE) represent a majormetabolic pathway in estrogen metabolism. Previous information on CEand training is limited to two cross-sectional studies that did notinvolve standardized training. Our purpose, by means of a prospective design, was to evaluate the effects of a brief, exhaustive training program on resting plasma concentrations of 2-hydroxy CE. The experimental design spanned two menstrual cycles: a control cycle and atraining cycle. The subjects were nine previously untrained, eumenorrheic women [body fat: 24.8 ± 1.0 (SE) %]. Datawere collected during the follicular (FPh) and the luteal phases (LPh).Posttraining FPh and LPh tests were held the day after the last day ofa 5-day period of training on a cycle ergometer. Total2-hydroxyestrogens (2-OHE) averaged 200 ± 29 pg/ml during the FPhand 420 ± 54 pg/ml during the LPh(P < 0.05). Levels of total2-methoxyestrogens (2-MeOE) were 237 ± 32 pg/ml during the FPh and339 ± 26 pg/ml during the LPh (P < 0.05). After training, although the plasma levels of 2-OHEsignificantly decreased (21%;P < 0.05) during the LPh, the actualCE formation (as estimated from the 2-OHE-to-total estrogens ratio)increased (+29%; P < 0.05). CE activity, as expressed by the 2-MeOE-to-2-OHE ratio, showedsignificantly higher values in both phases (FPh, +14%; LPh, +13%;P < 0.05). At the same time, restinglevels of norepinephrine (NE) were increased by 42%(P < 0.05). CE strongly inhibitbiological decomposition of NE by catechol-O-methyltransferase (COMT).Results of the present study suggest that, in response to training, CEare increasingly competing with the enzyme COMT, thus preventingpremature NE deactivation.

     

Ex-vivo models of the Retinal Pigment Epithelium (RPE) in long-term culture faithfully recapitulate key structural and physiological features of native RPE

The Retinal Pigment Epithelium (RPE) forms the primary site of pathology in several blinding retinopathies. RPE cultures are being continuously refined so that dynamic disease processes in this important monolayer can be faithfully studied outside the eye over longer periods. The RPE substrate, which mimics the supportive Bruch’s membrane (BrM), plays a key role in determining how well in-vitro cultures recapitulate native RPE cells. Here, we evaluate how two different types of BrM substrates; (1) a commercially-available polyester transwell membrane, and (2) a novel electrospun scaffold developed in our laboratory, could support the generation of realistic RPE tissues in culture. Our findings reveal that both substrates were capable of supporting long-lasting RPE monolayers with structural and functional specialisations of in-situ RPE cells. These cultures were used to study autofluorescence and barrier formation, as well as activities such as outer-segment internalisation/trafficking and directional secretion of key proteins; the impairment of which underlies retinal disease. Hence, both substrates fulfilled important criteria for generating authentic in-vitro cultures and act as powerful tools to study RPE pathophysiology. However, RPE grown on electrospun scaffolds may be better suited to studying complex RPE-BrM interactions such as the formation of drusen-like deposits associated with early retinal disease.     

Plasma corticosterone concentrations in wild and captive juvenile tuatara (Sphenodon punctatus)

Abstract

High mortality and abnormal growth patterns commonly found in captive juvenile tuatara were hypothesised to be due in part to the effects of long‐term chronic stress of captivity. This study compared plasma concentrations of the reptilian adrenal steroid, corticosterone, in wild juvenile tuatara on Stephens Island, Cook Strait, and in captive juveniles of Stephens Island origin, held in New Zealand institutions, in February and August 1992. Seasonal variation in plasma concentration of corticosterone in wild juveniles in four seasons of the year was also examined. This is the first study of seasonal cycles in plasma corticosterone in a wild juvenile reptile. Plasma corticosterone concentrations were significantly higher in captive juvenile females (4.21 ± 0.27 ng/ml; mean ± SE) compared with wild juvenile females (2.44 ± 0.42 ng/ml) in February (P < 0.05), but not in August, and there was no difference in concentration between captive and wild juvenile males in either month. There was significant seasonal variation in plasma corticosterone in wild juvenile females (P < 0.05). However, there was no seasonal variation observed in wild juvenile males, and the magnitude of the variation in plasma corticosterone was low in both sexes (1.28 ± 0 ng/ml ‐4.65 ±3.41 ng/ml). Although mean plasma corticosterone was higher in captive juvenile females compared with wild juvenile females in February 1992, the value in captive females was within the range of mean plasma corticosterone concentrations observed in the seasonal study, and may be therefore due to asynchronicity of seasonal cycles, rather than stress. Further research is required; however, lack of correlation between plasma corticosterone concentrations and either growth rate or density indicate that captive juvenile tuatara in New Zealand are not suffering from pronounced chronic stress.     

Short-term success of a translocation of Otago skinks (Oligosoma otagense) to Orokonui Ecosanctuary

Natural habitat is not always available for translocations due to habitat modification or pressure from introduced predators. We followed the release of 30 captive-bred Otago skinks (Oligosoma otagense) into a 109?m² outdoor enclosure of artificial habitat at Orokonui Ecosanctuary in southeastern New Zealand. We evaluated the short-term success of the translocation by assessing visibility and survival of the skinks over the 3 summer months following release in spring 2013, plus survival the following spring. Skinks were visible most days, especially in warm, dry conditions. Survival was high over the first summer; 80% of skinks were seen at least once during February 2014. The following spring 63% of skinks were resighted during three surveys and breeding has since been reported. Thus, it is feasible to maintain captive-bred adults of this Nationally Endangered skink in artificially constructed, outdoor habitat in a coastal location. However, to avoid predation of juveniles by adult skinks, future translocations should avoid releasing juveniles close to adults.     

Differences in dietary and plasma fatty acids between wild and captive populations of a rare reptile, the tuatara (Sphenodon punctatus)

Tuatara (Sphenodon) are rare reptiles endemic to New Zealand. Wild tuatara on Stephens Island (study population) prey on insects as well as the eggs and chicks of a small nesting seabird, the fairy prion (Pachyptila turtur). Tuatara in captivity (zoos) are fed diets containing different insects and lacking seabirds. We compared the fatty acid composition of major dietary items and plasma of wild and captive tuatara. Fairy prions (eaten by tuatara in the wild) were rich in C20 and C22 polyunsaturated fatty acids (PUFA), especially the n-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). In contrast, items from the diet of captive tuatara contained no C20 and C22 PUFA and were higher in medium-chain and less unsaturated fatty acids. Plasma from wild tuatara was higher in n-3 PUFA [including alpha-linoleic acid (C18:3n-3), EPA and DHA], and generally lower in oleic acid (C18:1) and palmitic acid (C16:0), than plasma from captive tuatara in the various fractions (phospholipid, triacylglycerol, cholesterol ester and free fatty acids). Plasma from wild adult tuatara showed strong seasonal variation in fatty acid composition, reflecting seasonal consumption of fairy prions. Differences in the composition of diets and plasma between wild and captive tuatara may have consequences for growth and reproduction in captivity. Accepted: 3 August 1998     

Are viviparous lizards from cool climates ever exclusively nocturnal? Evidence for extensive basking in a New Zealand gecko

Viviparity has evolved numerous times among squamate reptiles; however, the combination of viviparity and nocturnality is apparently rare among lizards. We used time‐lapse photography to examine evidence for diurnal activity in a viviparous lizard often described as nocturnal, the gecko Woodworthia ‘Otago/Southland’ from southern New Zealand (family Diplodactylidae). Evidence for diurnal emergence was extensive. Females have a higher incidence of basking compared to males, although no difference was detected between females in different reproductive conditions. Temperature loggers inserted into calibrated copper models were used to compare the body temperatures available to geckos in two basking positions and in two retreat types. Models in basking positions reached higher mean temperatures than models in retreats, although there was no significant effect of basking position or retreat type on model temperatures. Collectively, our results indicate that pregnant geckos that bask consistently could reduce gestation length by at least 14 days compared with females that remain in retreats. Extensive basking in this species adds to the growing evidence of diurno‐nocturnality in many New Zealand lepidosaurs, including other viviparous geckos. Our results lead us to question whether viviparity in lizards is ever compatible with ‘pure’ nocturnality in a cool climate. © 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, ●● , ●●–●●.