RESUMEN
Kynurenine 3-monooxygenase (KMO) is an enzyme central to the kynurenine pathway of tryptophan metabolism. KMO has been implicated as a therapeutic target in several disease states, including Huntington's disease. Recombinant human KMO protein production is challenging due to the presence of transmembrane domains, which localise KMO to the outer mitochondrial membrane and render KMO insoluble in many in vitro expression systems. Efficient bacterial expression of human KMO would accelerate drug development of KMO inhibitors but until now this has not been achieved. Here we report the first successful bacterial (Escherichia coli) expression of active FLAG™-tagged human KMO enzyme expressed in the soluble fraction and progress towards its purification.
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Quinurenina 3-Monooxigenasa/aislamiento & purificación , Quinurenina 3-Monooxigenasa/metabolismo , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Escherichia coli/genética , Histidina , Humanos , Cinética , Quinurenina 3-Monooxigenasa/química , Quinurenina 3-Monooxigenasa/genética , Redes y Vías Metabólicas , Oligopéptidos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , SolubilidadRESUMEN
INTRODUCTION: Measuring cortisol during military training offers insights into physiological responses to stress. We attempted precisely timed, cortisol awakening response (CAR) and pre-sleep cortisol (PSC), and diurnal slope (peak morning minus evening cortisol), during a British Army exercise. We aimed to understand cortisol dynamics and evaluate the feasibility of CAR and PSC in this environment. METHOD: Setting: high-intensity, 10-day infantry exercise. Participants: regular infantry soldiers exercising (EX, n=25) or headquarters-based (HQ, n=6). Participants undertook PSC and WAKE and WAKE+30 min samples after 1-2 days, 5-6 days and 9-10 days. Wrist-worn GENEActiv accelerometers were used to assess sleep duration in EX only. Samples taken ±15 min from prespecified time points were deemed adherent. Validated questionnaires were used to measure resilience and perceived stress. Cortisol and cortisone were measured simultaneously by liquid chromatography tandem mass spectrometry. RESULTS: From adherent participants' samples, CAR was positive and tended to decrease as the exercise progressed. From all available data, HQ demonstrated greater diurnal slope than EX (F=7.68, p=0.02), reflecting higher morning cortisol (F=4.72, p=0.038) and lower PSC (p=0.04). No differences were seen in cortisol:cortisone ratio. 26.1% of CAR samples were adherent, with moderately strong associations between adherence and stress (r=0.41, p=0.009) but no association between adherence and day of exercise (χ2=0.27, p=0.8), sleep duration (r=-0.112, p=0.43) or resilience (r=-0.79, p=0.75). Test-retest reliability ratings for CAR were Cronbach's α of 0.48, -11.7 and 0.34 for the beginning, middle and end of the exercise, respectively. CONCLUSIONS: We observed a reduction in morning cortisol and decreased diurnal slope during a high-intensity military exercise, compared with the HQ comparator cohort in whom diurnal slope was preserved. A carefully timed CAR was not feasible in this setting.
RESUMEN
11ß-Hydroxysteroid dehydrogenase 1 (11ßHSD1) is a drug target to attenuate adverse effects of chronic glucocorticoid excess. It catalyses intracellular regeneration of active glucocorticoids in tissues including brain, liver and adipose tissue (coupled to hexose-6-phosphate dehydrogenase, H6PDH). 11ßHSD1 activity in individual tissues is thought to contribute significantly to glucocorticoid levels at those sites, but its local contribution vs glucocorticoid delivery via the circulation is unknown. Here, we hypothesised that hepatic 11ßHSD1 would contribute significantly to the circulating pool. This was studied in mice with Cre-mediated disruption of Hsd11b1 in liver (Alac-Cre) vs adipose tissue (aP2-Cre) or whole-body disruption of H6pdh. Regeneration of [9,12,12-2H3]-cortisol (d3F) from [9,12,12-2H3]-cortisone (d3E), measuring 11ßHSD1 reductase activity was assessed at steady state following infusion of [9,11,12,12-2H4]-cortisol (d4F) in male mice. Concentrations of steroids in plasma and amounts in liver, adipose tissue and brain were measured using mass spectrometry interfaced with matrix-assisted laser desorption ionisation or liquid chromatography. Amounts of d3F were higher in liver, compared with brain and adipose tissue. Rates of appearance of d3F were ~6-fold slower in H6pdh-/- mice, showing the importance for whole-body 11ßHSD1 reductase activity. Disruption of liver 11ßHSD1 reduced the amounts of d3F in liver (by ~36%), without changes elsewhere. In contrast disruption of 11ßHSD1 in adipose tissue reduced rates of appearance of circulating d3F (by ~67%) and also reduced regenerated of d3F in liver and brain (both by ~30%). Thus, the contribution of hepatic 11ßHSD1 to circulating glucocorticoid levels and amounts in other tissues is less than that of adipose tissue.
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Cortisona , Glucocorticoides , Masculino , Ratones , Animales , Hidrocortisona , Tejido Adiposo , Esteroides , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/genéticaRESUMEN
The enzyme 11-beta-hydroxysteroid dehydrogenase isoenzyme 2 (11BHSD2) is responsible for converting the active glucocorticoid cortisol to inactive cortisone and in the renal medulla protects the mineralocorticoid receptor (MR) from activation by cortisol. Derangements in 11BHSD2 activity can result in reduced conversion of cortisol to cortisone, activation of the MR by cortisol and, consequently, sodium and water retention. The objective of this study was to examine glucocorticoid metabolism in canine congestive heart failure (CHF), specifically to evaluate whether renal 11BHSD2 activity and expression were altered. Dogs were prospectively recruited into one of two phases; the first phase (n=56) utilized gas chromatography-tandem mass spectrometry to examine steroid hormone metabolites normalised to creatinine in home-caught urine samples. Total serum cortisol was also evaluated. The second phase consisted of dogs (n=18) euthanased for refractory CHF or for behavioural reasons. Tissue was collected from the renal medulla for examination by quantitative reverse transcription polymerase chain reaction, immunohistochemistry and protein immune-blotting. Heart failure did not change urinary cortisol:cortisone ratio (P=0.388), or modify renal expression (P=0.303), translation (P=0.427) or distribution of 11BHSD2 (P=0.325). However, CHF did increase excretion of 5α-tetrahydrocortisone (P=0.004), α-cortol (P=0.002) and α-cortolone (P=0.009). Congestive heart failure modifies glucocorticoid metabolism in dogs by increasing 5α-reductase and 20α-hydroxysteroid dehydrogenase activity. Differences between groups in age, sex and underlying disease processes may have influenced these results. However, 11BHSD2 does not appear to be a potential therapeutic target in canine CHF.
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11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/metabolismo , Enfermedades de los Perros/metabolismo , Glucocorticoides/metabolismo , Insuficiencia Cardíaca/veterinaria , Riñón/metabolismo , Animales , Cortisona/orina , Perros , Femenino , Cromatografía de Gases y Espectrometría de Masas/veterinaria , Insuficiencia Cardíaca/tratamiento farmacológico , Hidrocortisona/orina , Masculino , Estudios ProspectivosRESUMEN
Endogenous glucocorticoid action is important in the structural and functional maturation of the fetal heart. In fetal mice, although glucocorticoid concentrations are extremely low before E14.5, glucocorticoid receptor (GR) is expressed in the heart from E10.5. To investigate whether activation of cardiac GR prior to E14.5 induces precocious fetal heart maturation, we administered dexamethasone in the drinking water of pregnant dams from E12.5 to E15.5. To test the direct effects of glucocorticoids upon the cardiovascular system we used SMGRKO mice, with Sm22-Cre-mediated disruption of GR in cardiomyocytes and vascular smooth muscle. Contrary to expectations, echocardiography showed no advancement of functional maturation of the fetal heart. Moreover, litter size was decreased 2 days following cessation of antenatal glucocorticoid exposure, irrespective of fetal genotype. The myocardial performance index and E/A wave ratio, markers of fetal heart maturation, were not significantly affected by dexamethasone treatment in either genotype. Dexamethasone treatment transiently decreased the myocardial deceleration index (MDI; a marker of diastolic function), in control fetuses at E15.5, with recovery by E17.5, 2 days after cessation of treatment. MDI was lower in SMGRKO than in control fetuses and was unaffected by dexamethasone. The transient decrease in MDI was associated with repression of cardiac GR in control fetuses following dexamethasone treatment. Measurement of glucocorticoid levels in fetal tissue and hypothalamic corticotropin-releasing hormone (Crh) mRNA levels suggest complex and differential effects of dexamethasone treatment upon the hypothalamic-pituitary-adrenal axis between genotypes. These data suggest potentially detrimental and direct effects of antenatal glucocorticoid treatment upon fetal heart function.
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Dexametasona/farmacología , Diástole/efectos de los fármacos , Corazón Fetal/efectos de los fármacos , Exposición Materna , Animales , Peso Corporal , Femenino , Corazón Fetal/diagnóstico por imagen , Genotipo , Glucocorticoides/farmacología , Sistema Hipotálamo-Hipofisario/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Liso Vascular/metabolismo , Miocitos Cardíacos/metabolismo , Tamaño de los Órganos , Sistema Hipófiso-Suprarrenal/metabolismo , ARN Mensajero/metabolismo , Receptores de Glucocorticoides/metabolismoRESUMEN
BACKGROUND: Basic military training (BMT) is a useful model of prolonged exposure to multiple stressors. 8-12 week BMT is associated with perturbations in the hypothalamic-pituitary-adrenal (HPA) axis which could predispose recruits to injury and psychological strain. However, characterisations of HPA axis adaptations during BMT have not been comprehensive and most studies included few if any women. METHODS: We studied women undertaking an arduous, 44-week BMT programme in the UK. Anxiety, depression and resilience questionnaires, average hair cortisol concentration (HCC), morning and evening saliva cortisol and morning plasma cortisol were assessed at regular intervals throughout. A 1-h dynamic cortisol response to 1 µg adrenocorticotrophic hormone-1-24 was performed during weeks 1 and 29. RESULTS: Fifty-three women (aged 24 ± 2.5 years) completed the study. Questionnaires demonstrated increased depression and reduced resilience during training (F 6.93 and F 7.24, respectively, both p < 0.001). HCC increased from 3 months before training to the final 3 months of training (median (IQR) 9.63 (5.38, 16.26) versus 11.56 (6.2, 22.45) pg/mg, p = 0.003). Morning saliva cortisol increased during the first 7 weeks of training (0.44 ± 0.23 versus 0.59 ± 0.24 µg/dl p < 0.001) and decreased thereafter, with no difference between the first and final weeks (0.44 ± 0.23 versus 0.38 ± 0.21 µg/dl, p = 0.2). Evening saliva cortisol did not change. Fasting cortisol decreased during training (beginning, mid and end-training concentrations: 701 ± 134, 671 ± 158 and 561 ± 177 nmol/l, respectively, p < 0.001). Afternoon basal cortisol increased during training while there was a trend towards increased peak stimulated cortisol (177 ± 92 versus 259 ± 13 nmol/l, p = 0.003, and 589 ± 164 versus 656 ± 135, p = 0.058, respectively). DISCUSSION: These results suggest a normal stress response in early training was followed quickly by habituation, despite psychological and physical stress evidenced by questionnaire scores and HCC, respectively. There was no evidence of HPA axis maladaptation. These observations are reassuring for women undertaking arduous employment.