Anti-Ro52/TRIM21 is independently associated with pulmonary arterial hypertension and mortality in a cohort of systemic sclerosis patients.

Objective: We undertook a comprehensive cross-sectional analysis of a multicentred Australian cohort of systemic sclerosis (SSc) patients to evaluate the associations of anti-Ro52/TRIM21 with SSc pulmonary involvement.Method: The study included 596 patients from the Australian Scleroderma Cohort Study database whose anti-Ro52/TRIM21 status was known. Anti-Ro52/TRIM21 was measured via line immunoassay. Data on demographic variables, autoantibody profiles, presence of interstitial lung disease (ILD), presence of pulmonary arterial hypertension (PAH), oxygen saturation, Six-Minute Walk Test distance, Borg dyspnoea score, and lung function tests were extracted. SPSS software was used to examine associations using univariate and multivariate analyses.Results: Anti-Ro52/TRIM21 was present in 34.4% of SSc patients. In the cross-sectional analysis, anti-Ro52/TRIM21 was independently associated with PAH [odds ratio 1.75, 95% confidence interval (CI) 1.05-2.90], but not ILD or other surrogate measures of pulmonary involvement such as average patient oxygen saturation. The antibody, however, was also associated with a higher forced vital capacity/diffusing capacity of the lung for carbon monoxide ratio. Prospectively, anti-Ro52/TRIM21 was also associated with an increased risk of death in patients with SSc (hazard ratio 1.62, 95% CI 1.11-2.35), independent of confounding factors. The primary cause of death appeared to be related to PAH and/or ILD, and anti-Ro52/TRIM21 was associated with PAH-related complications.Conclusion: Anti-Ro52/TRIM21 was independently associated with PAH and mortality in SSc patients. Future longitudinal studies are recommended to investigate the timing and pathogenic mechanisms of this autoantibody in PAH.

Clustering of PCOS-like traits in naturally hyperandrogenic female rhesus monkeys.

Study question: Do naturally occurring, hyperandrogenic (≥1 SD of population mean testosterone, T) female rhesus monkeys exhibit traits typical of women with polycystic ovary syndrome (PCOS)? Summary answer: Hyperandrogenic female monkeys exhibited significantly increased serum levels of androstenedione (A4), 17-hydroxyprogesterone (17-OHP), estradiol (E2), LH, antimullerian hormone (AMH), cortisol, 11-deoxycortisol and corticosterone, as well as increased uterine endometrial thickness and evidence of reduced fertility, all traits associated with PCOS. What is known already: Progress in treating women with PCOS is limited by incomplete knowledge of its pathogenesis and the absence of naturally occurring PCOS in animal models. A female macaque monkey, however, with naturally occurring hyperandrogenism, anovulation and polyfollicular ovaries, accompanied by insulin resistance, increased adiposity and endometrial hyperplasia, suggests naturally occurring origins for PCOS in nonhuman primates. Study design, size, duration: As part of a larger study, circulating serum concentrations of selected pituitary, ovarian and adrenal hormones, together with fasted insulin and glucose levels, were determined in a single, morning blood sample obtained from 120 apparently healthy, ovary-intact, adult female rhesus monkeys (Macaca mulatta) while not pregnant or nursing. The monkeys were then sedated for somatometric and ultrasonographic measurements. Participants/materials, setting, methods: Female monkeys were of prime reproductive age (7.2 ± 0.1 years, mean ± SEM) and represented a typical spectrum of adult body weight (7.4 ± 0.2 kg; maximum 12.5, minimum 4.6 kg). Females were defined as having normal (n = 99) or high T levels (n = 21; ≥1 SD above the overall mean, 0.31 ng/ml). Electronic health records provided menstrual and fecundity histories. Steroid hormones were determined by tandem LC-MS-MS; AMH was measured by enzymeimmunoassay; LH, FSH and insulin were determined by radioimmunoassay; and glucose was read by glucose meter. Most analyses were limited to 80 females (60 normal T, 20 high T) in the follicular phase of a menstrual cycle or anovulatory period (serum progesterone <1 ng/ml). Main results and the role of chance: Of 80 monkeys, 15% (n = 12) exhibited classifiable PCOS-like phenotypes. High T females demonstrated elevations in serum levels of LH (P < 0.036), AMH (P < 0.021), A4 (P < 0.0001), 17-OHP (P < 0.008), E2 (P < 0.023), glucocorticoids (P < 0.02-0.0001), the serum T/E2 ratio (P < 0.03) and uterine endometrial thickness (P < 0.014) compared to normal T females. Within the high T group alone, anogenital distance, a biomarker for fetal T exposure, positively correlated (P < 0.015) with serum A4 levels, while clitoral volume, a biomarker for prior T exposure, positively correlated (P < 0.002) with postnatal age. Only high T females demonstrated positive correlations between serum LH, and both T and A4. Five of six (83%) high T females with serum T ≥2 SD above T mean (0.41 ng/ml) did not produce live offspring. Large scale data: N/A. Limitations, reasons for caution: This is an initial study of a single laboratory population in a single nonhuman primate species. While two biomarkers suggest lifelong hyperandrogenism, phenotypic expression during gestation, prepuberty, adolescence, mid-to-late reproductive years and postmenopause has yet to be determined. Wider implications of the findings: Characterizing adult female monkeys with naturally occurring hyperandrogenism has identified individuals with high LH and AMH combined with infertility, suggesting developmental linkage among traits with endemic origins beyond humans. PCOS may thus be an ancient phenotype, as previously proposed, with a definable pathogenic mechanism(s). Study funding/competing interest(s): Funded by competitive supplement to P51 OD011106 (PI: Mallick), by P50 HD028934 (PI: Marshall) and by P50 HD044405 (PI: Dunaif). The authors have no potential conflicts of interest.

Lipid bodies accumulation in Leishmania infantum-infected C57BL/6 macrophages.

Lipid bodies (LBs) are intracellular accumulations of neutral lipids surrounded by a single membrane. These organelles are involved in the production of eicosanoids, which modulate immunity by either promoting or dampening inflammatory responses. Leishmania infantum, the etiological agent of visceral leishmaniasis in Brazil, is an intracellular parasite that causes disease by suppressing macrophage microbicidal responses. C57BL/6 mouse bone marrow-derived macrophages infected with L. infantum strain LcJ had higher numbers of LB+ cells (P<.0001) and total LBs than noninfected cultures. Large (>3 µm) LBs were present inside parasitophorous vacuoles (PVs). These results contrast with those of L. infantum-infected BALB/c macrophages, in which the only LBs are derived from parasite, not macrophage origin. Increased LBs in C57BL/6 macrophages in close association with parasites would position host LBs where they could modulate L. infantum infection. These results imply a potential influence of the host genetics on the role of LBs in host-pathogen interactions. Overall, our data support a model in which the expression, and the role of LBs upon infection, ultimately depends on the specific combination of host-pathogen interactions.

Distance examination of livestock with drones - an effective method for assessing health and welfare.

Distance examination is an important part of veterinary investigation into ruminant herd health and welfare. The Department of Primary Industries and Regional Development (DPIRD) explored the use of drones to conduct assessments of the health and welfare status of sheep and cattle. Three methods of distance examination were compared comprising observations; from a vehicle, a "micro" category drone and a "very small" category drone. The disturbance and behavioural reactions caused by the methods were compared. Assessments of adverse health and welfare conditions by each method were compared to observations made at yarding. The preferred method was the use of the very small drone which had the best sensitivity for detection of conditions potentially associated with adverse health or welfare and the best optics at a distance that did not disturb the animals. The optics of the very small drone enabled distance examination without disturbance in both cattle and sheep. Cattle were more sensitive to the presence of the drones than sheep. The micro drone was unable to approach cattle close enough to allow undisturbed distance examination. All methods had similar specificity, however, sensitivity varied markedly. The very small drone had the best sensitivity 86% which was statistically greater than the micro drone (44%, P = 0.05) and better than the vehicle observations, which had sensitivity of 77% (not statistically significant). The selection of an appropriate drone model is essential for accurate distance examination. Distance examination of livestock with drones of suitable optic quality and resolution represents an effective method for assessing animal health and welfare.

Effect of dietary lysine on hepatic lysine catabolism in broilers.

Lysine is frequently a first- or second-limiting amino acid in poultry diets. Improving the efficiency of lysine use for protein synthesis would effectively lower the lysine requirement and decrease feed costs. Understanding how lysine is degraded and how the degradation is regulated would identify potential molecular targets for interventions to decrease lysine degradation. To better understand lysine degradation in poultry, 3 experiments were conducted. In experiment 1, one-day-old chicks were fed 1.07, 1.25, 1.73, or 3.28% dietary lysine for 2 wk. In experiments 2 and 3, fourteen-day-old chicks were fed 1.07 or 1.25% dietary lysine for 2 wk. Measures of liver lysine catabolism including lysine α-ketoglutarate reductase (LKR) and lysine oxidation (LOX) were assessed. The α-aminoadipate δ-semialdehyde synthase (AASS) is a bifunctional enzyme composed of both LKR and saccharopine dehydrogenase activities, and the relative abundance of this protein and mRNA were likewise assessed. Moreover, potential alternative pathways of lysine catabolism that depend on l-amino acid oxidase (AAOX) and on lysyl oxidase (LYLOX) were considered. In experiment 1, chicks fed lysine-deficient diets had decreased (P < 0.05) LKR activities compared with chicks fed at or above the requirement. However, the lowered LKR activities were not associated with a decreased (P > 0.05) LOX as measured in vitro. In experiments 2 and 3, chicks 28 d of age did not decrease LKR activity (P > 0.05) in response to a lysine-deficient diet. No changes in AASS protein abundance or mRNA were detected. Likewise, no differences in the mRNA abundances of AAOX or LYLOX were detected. The activity of AAOX did increase (P < 0.05) in birds fed a lysine-adequate diets compared with those fed a lysine-deficient diet. Based on kinetic parameters and assumed concentrations, AAOX could account for about 20% of liver lysine oxidation in avians.

Hormonal programming across the lifespan.

Hormones influence countless biological processes across an animal's lifespan. Many hormone-mediated events occur within developmental sensitive periods, during which hormones have the potential to cause permanent tissue-specific alterations in anatomy and physiology. There are numerous selective critical periods in development with different targets being affected during different periods. This review outlines the proceedings of the Hormonal Programming in Development session at the US-South American Workshop in Neuroendocrinology in August 2011. Here we discuss how gonadal steroid hormones impact various biological processes within the brain and gonads during early development and describe the changes that take place in the aging female ovary. At the cellular level, hormonal targets in the brain include neurons, glia, or vasculature. On a genomic/epigenomic level, transcription factor signaling and epigenetic changes alter the expression of critical hormone receptor genes across development and following ischemic brain insult. In addition, organizational hormone exposure alters epigenetic processes in specific brain nuclei and may be an important mediator of sexual differentiation of the neonatal brain. Brain targets of hormonal programming, such as the paraventricular nucleus of the hypothalamus, may be critical in influencing the development of peripheral targets, such as the ovary. Exposure to excess hormones can cause abnormalities in the ovary during development leading to polycystic ovarian syndrome (PCOS). Exposure to excess androgens during fetal development also has a profound effect on the development of the male reproductive system. In addition, increased activity of the sympathetic nerve and stress during early life have been linked to PCOS symptomology in adulthood. Finally, we describe how age-related decreases in fertility are linked to high levels of nerve growth factor (NGF), which enhances sympathetic nerve activity and alters ovarian function.

Impairment of ovarian function and associated health-related abnormalities are attributable to low social status in premenopausal monkeys and not mitigated by a high-isoflavone soy diet.

BACKGROUND: Psychological stress may impair premenopausal ovarian function and contribute to risk for chronic disease. Soy isoflavones may also influence ovarian function and affect health. Here, we report the effects of a psychological stressor (subordinate social status) and dietary soy on reproductive function and related health indices in female monkeys. We hypothesized that reproductive compromise and adverse health outcomes would be induced in subordinate when compared with dominant monkeys and be mitigated by exposure to soy. METHODS: Subjects were 95 adult cynomolgus monkeys (Macaca fascicularis) housed in social groups of five or six. Animals consumed a soy-free, animal protein-based diet during an 8-month Baseline phase and then, during a 32-month Treatment phase, consumed either the baseline diet or an identical diet that substituted high-isoflavone soy protein for animal protein. RESULTS: Across more than 1200 menstrual cycles, subordinate monkeys consistently exhibited ovarian impairment [increased cycle length (P < 0.02) and variability (P < 0.02) and reduced levels of progesterone (P < 0.04) and estradiol (P < 0.04)]. Subordinate status was confirmed behaviorally and was associated with elevated cortisol (P < 0.04) and relative osteopenia (P < 0.05). Consumption of the soy diet had no significant effects. CONCLUSIONS: (i) Psychological stress adversely affects ovarian function and related health indices in a well-accepted animal model of women's health; (ii) Similar effects may extend to women experiencing reproductive impairment of psychogenic origin; (iii) soy protein and isoflavones neither exacerbate nor mitigate the effects of an adverse psychosocial environment; and (iv) this study was limited by an inability to investigate the genetic and developmental determinants of social status.

Continuous expression of corticotropin-releasing factor in the central nucleus of the amygdala emulates the dysregulation of the stress and reproductive axes.

An increase in corticotropin-releasing factor (CRF) is a putative factor in the pathophysiology of stress-related disorders. As CRF expression in the central nucleus of the amygdala (CeA) is important in adaptation to chronic stress, we hypothesized that unrestrained synthesis of CRF in CeA would mimic the consequences of chronic stress exposure and cause dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis, increase emotionality and disrupt reproduction. To test this hypothesis, we used a lentiviral vector to increase CRF-expression site specifically in CeA of female rats. Increased synthesis of CRF in CeA amplified CRF and arginine vasopressin peptide concentration in the paraventricular nucleus of the hypothalamus, and decreased glucocorticoid negative feedback, both markers associated with the pathophysiology of depression. In addition, continuous expression of CRF in CeA also increased the acoustic startle response and depressive-like behavior in the forced swim test. Protein levels of gonadotropin-releasing hormone in the medial preoptic area were significantly reduced by continuous expression of CRF in CeA and this was associated with a lengthening of estrous cycles. Finally, sexual motivation but not sexual receptivity was significantly attenuated by continuous CRF synthesis in ovariectomized estradiol-progesterone-primed females. These data indicate that unrestrained CRF synthesis in CeA produces a dysregulation of the HPA axis, as well as many of the behavioral, physiological and reproductive consequences associated with stress-related disorders.Molecular Psychiatry (2009) 14, 37-50; doi:10.1038/mp.2008.91; published online 12 August 2008.

Effect of cytochrome P450 and aldo-keto reductase inhibitors on progesterone inactivation in primary bovine hepatic cell cultures.

Progesterone is required for maintenance of pregnancy, and peripheral concentrations of progesterone are affected by both production and inactivation. Hepatic cytochrome P450 (EC 1.14.14.1) and aldo-keto reductase (EC 1.1.1.145-151) enzymes play a pivotal role in the first step of steroid inactivation, which involves the addition of hydroxyl groups to various sites of the cyclopentanoperhydrophenanthrene nucleus. The current objective was to discern the proportional involvement of hepatic progesterone inactivating enzymes on progesterone decay using specific enzyme inhibitors. Ticlopidine, diltiazem, curcumin, dicumarol, and naproxen were used because of their selective inhibition of cytochrome P450s, aldo-keto reductases, and glucuronosyltransferases. Liver biopsies were collected from 6 lactating Holstein dairy cows, and cells were dissociated using a nonperfusion technique. Confluent wells were preincubated for 4 h with enzyme inhibitor and then challenged with progesterone for 1 h. Cell viability was unaffected by inhibitor treatment and averaged 84±1%. In control wells, 50% of the progesterone had been inactivated after a 1-h challenge with 5 ng/mL of progesterone. Preincubation with curcumin, ticlopidine, or naproxen caused the greatest reduction in progesterone inactivation compared with controls and averaged 77, 39, or 37%, respectively. Hydroxylation of 4-nitrophenol to 4-nitrocatechol in intact cells was inhibited by approximately 65% after treatment with curcumin or ticlopidine. Glucuronidation of phenol red or 4-nitrocatechol in intact cells was inhibited by treatment with curcumin, dicumarol, or naproxen. In cytoplasmic preparations, aldo-keto reductase 1C activity was inhibited by curcumin, dicumarol, or naproxen treatment. Microsomal cytochrome P450 2C activity was inhibited by treatment with curcumin or ticlopidine, whereas cytochrome P450 3A activity was inhibited by treatment with curcumin or diltiazem. The contribution of cytochrome P450 2C and cytochrome P450 3A enzymes to progesterone inactivation in bovine hepatic cell cultures was 40 and 15%, respectively. Depending on the inhibitor used, it would appear that the aldo-keto reductase enzymes contribute approximately 40% to the observed progesterone inactivation, although a portion of this inactivation may be attributed to the loss of glucuronosyltransferase activity. Future work focusing on decreasing the activity of these enzymes in vivo could lead to an increase in the bioavailability of progesterone.

Effect of a high cornstarch diet on hepatic cytochrome P450 2C and 3A activity and progesterone half-life in dairy cows.

In the cow, inadequate concentrations of progesterone during gestation may lead to an abrupt termination of pregnancy. The primary organ involved in progesterone catabolism is the liver, which contains an abundance of cytochrome P450 isozymes (EC 1.14.14.1; mixed-function monooxygenases). The objectives of the current experiment were to determine the effect of feeding 2 isoenergetic and isonitrogenous diets, formulated to cause divergent insulin secretion, on hepatic cytochrome P450 2C (CYP2C) and 3A (CYP3A) activity as well as the resulting biological half-life of progesterone. Twenty-two Holstein cows averaging 80+/-7 d in milk were randomly assigned to either a high cornstarch diet or a high fiber diet in a crossover experimental design consisting of two 14-d periods. Dry matter intake, milk yield, milk lactose yield, and milk lactose percentage were similar between the 2 diets. Milk fat yield and milk fat percentage were higher in cows fed the high fiber diet, whereas milk protein yield tended to be higher and milk protein percentage was higher in cows fed the high cornstarch diet. Energy balance tended to be improved by 57% in cows consuming the high cornstarch diet. Insulin concentrations at the time of liver biopsy (3.16+/-0.04h post-feeding) were increased by 44% in cows consuming the high cornstarch diet compared with cows consuming the high fiber diet. Cytochrome P450 2C activity was decreased by 45%, whereas CYP3A activity tended to be lowered by 34% in cows consuming the high cornstarch diet. Cytochrome P450 2C mRNA expression tended to be decreased by 21% in cows fed the high cornstarch diet, whereas CYP3A mRNA expression was not different between the dietary treatments. The fractional rate constant of progesterone decay was not different between the 2 diets; however, the half-life of progesterone tended to be longer in cows fed the high cornstarch diet compared with cows fed the high fiber diet (85 vs. 64min, respectively). In summary, cows consuming the high cornstarch diet had increased insulin concentrations and lower hepatic CYP2C and CYP3A activity and tended to have a longer progesterone half-life compared with cows consuming the high fiber diet. Feeding diets that stimulate insulin secretion could alter progesterone clearance during lactation, when dairy cows have increased rates of progesterone inactivation because of high energy demands and increased DMI.

Alveolar macrophage phenotypes in severe equine asthma.

Because the alveolar macrophage (AM) phenotype of horses with severe equine asthma (SEA) is unknown, the cytokines expressed by M1- and M2-polarized AM were determined and the hypothesis that natural hay/straw challenge (NC) induces divergent AM phenotypes in control horses and horses with SEA was tested. Macrophages from control horses were activated either with eIFNγ + lipolysaccharide (LPS) or eIL-4 to characterize M1- or M2-polarized AM gene expression, respectively and determine the response of polarized cells to pathogen-associated molecular patterns (PAMPS): LPS, zymosan, peptidoglycan and hay dust. Subsequently, gene expression was explored in AM of control horses and horses with SEA at pasture and after NC. M1 polarization increased expression of pro-inflammatory cytokines (TNFα, IL-8, IL-12p40), IL-10, and CD80. M2 polarization increased CD206 and down-regulated arginase-II and IL-10. Expression of pro-inflammatory cytokines and CD80 in response to PAMPS was further increased by M1 pre-polarization whereas M2 pre-polarization down-regulated expression of pro-inflammatory cytokines and IL-10 but increased CD206. In horses with SEA, AMs had elevated expression of IL-10 both at pasture and after NC, but only after NC in control horses. CD206 expression increased in both groups during NC. At pasture, stimulation by PAMPS augmented expression of IL-8 and IL-10 in horses with SEA compared to control horses. NC eliminated this difference by selectively increasing expression of IL-10 in control horses. A fundamental shift in the macrophage phenotype in SEA is supported by consistently elevated production of IL-10. A similar non-canonical phenotype develops temporarily in control horses upon NC suggesting that AMs in horses with SEA have lost the ability to respond dynamically to environmental cues.

Tectonic tilt rates derived from lake-level measurements, salton sea, california.

Tectonic tilt at the Salton Sea was calculated by differencing lake-level measurements from two points on the sea. During the past 26 years, tilting was down toward the southeast. By 1970 differential vertical movement amounted to 110 millimeters between two gages situated 38 kilometers apart on the southwest shore. A reversal in tilt direction in late 1972 has diminished the net differential vertical movement to 60 millimeters.

Genetics and visceral leishmaniasis: of mice and man.

Ninety per cent of the 500,000 annual new cases of visceral leishmaniasis (VL) occur in India/Bangladesh/Nepal, Sudan and Brazil. Importantly, 80-90% of human infections are sub-clinical or asymptomatic, usually associated with strong cell-mediated immunity. Understanding the environmental and genetic risk factors that determine why two people with the same exposure to infection differ in susceptibility could provide important leads for improved therapies. Recent research using candidate gene association analysis and genome-wide linkage studies (GWLS) in collections of families from Sudan, Brazil and India have identified a number of genes/regions related both to environmental risk factors (e.g. iron), as well as genes that determine type 1 vs. type 2 cellular immune responses. However, until now all of the allelic association studies carried out have been underpowered to find genes of small effect sizes (odds ratios or OR < 2), and GWLS using multicase pedigrees have only been powered to find single major genes, or at best oligogenic control. The accumulation of large DNA banks from India and Brazil now makes it possible to undertake genome-wide association studies (GWAS), which are ongoing as part of phase 2 of the Wellcome Trust Case Control Consortium. Data from this analysis should seed research into novel genes and mechanisms that influence susceptibility to VL.

Coagulation parameters following equine herpesvirus type 1 infection in horses.

BACKGROUND: Equine herpesvirus type 1 (EHV-1) is the cause of respiratory disease, abortion storms, and outbreaks of herpesvirus myeloencephalopathy (EHM). Infection of the spinal cord is characterised by multifocal regions of virally infected vascular endothelium, associated with vasculitis, thrombosis and haemorrhage that result in ischaemia and organ dysfunction. However, the mechanism of thrombosis in affected horses is unknown. OBJECTIVES: To evaluate tissue factor (TF) procoagulant activity and thrombin-antithrombin complex (TAT) levels in horses following infection with EHV-1. STUDY DESIGN: In vitro and in vivo studies following experimental EHV-1 infection. METHODS: Horses were infected with EHV-1 and levels of peripheral blood mononuclear cell (PBMC)-associated TF activity; plasma and cerebrospinal fluid (CSF)-derived microvesicle (MV)-associated TF activity and TAT complexes in plasma were examined. RESULTS: EHV-1 infection increased PBMC TF procoagulant activity in vitro and in vivo. In infected horses, this increase was observed during the acute infection and was most marked at the onset and end of viraemia. However, no significant differences were observed between the horses that showed signs of EHM and the horses that did not develop EHM. Significant changes in MV-associated TF procoagulant activity and TAT complexes were not observed in infected horses. MAIN LIMITATIONS: A small number of horses typically exhibit clinical EHM following experimental infection. CONCLUSIONS: The results indicate that EHV-1 infection increases PBMC-associated TF procoagulant activity in vivo and in vitro. Additional in vivo studies are needed to better understand the role of TF-dependent coagulation during EHM pathogenesis in horses.

Neuroendocrine influences and repercussions of the menopause.

In summary, the evidence that both the ovary and the brain are key pacemakers in the menopause is compelling. Our appreciation that estrogens are important neurotrophic and neuroprotective factors has grown rapidly. Future studies will allow us to better understand the ensemble of factors that interact to maintain regular reproductive cyclicity and how this precise dynamic balance changes with age. Furthermore, understanding how estrogen exerts trophic and protective actions should lead to its use as an important therapeutic agent in the maintenance of normal neural function during aging and after injury.

Epigenetic regulation of the estrogen receptor alpha promoter in the cerebral cortex following ischemia in male and female rats.

Permanent middle cerebral artery occlusion (MCAO) causes neuronal cell death in the striatum and cortex. In rodents, estradiol treatment protects the cortex from cell death in an estrogen receptor alpha (ERalpha) dependent manner. ERalpha is only transiently expressed in the cortex during neonatal development and is very low in uninjured adult cortex. Following MCAO, ERalpha mRNA expression is upregulated in the cortex of female rats, but the mechanism of this increase is still unknown. It is also unknown whether a similar increase in ERalpha expression in seen in males. In the following studies, male and vehicle or estradiol-treated ovariectomized (OVX) female rats underwent MCAO to investigate the regulation of ERalpha expression after ischemia. Twenty-four hours after surgery, mRNA or genomic DNA was collected from 1 mm micropunches taken from 300 mum brain sections for quantitative reverse transcription-polymerase chain reaction (RT-PCR) or methylation-specific (MSP) PCR, respectively. Additionally, adjacent 20 mum sections were processed for ERalpha immunohistochemistry. In OVX females, ERalpha mRNA and protein were increased in the ischemic cortex, but unchanged in males. We hypothesized that this increase in ERalpha in females is due to a reversal of gene silencing by DNA methylation. Using MSP targeting of CpG islands within the 5' untranslated region (UTR) of the rat ERalpha gene, we found that ischemia decreased methylation in the ischemic cortex of both groups of females, but there was no change in methylation in males. Using chromatin immunoprecipitation, we found that MeCP2 associates with ERalpha 5'UTR corresponding with the methylation status of the promoter. These data are the first to demonstrate a difference in the regulation of ERalpha expression in response to MCAO between males and females and that methylation of the ERalpha gene corresponds with mRNA levels in the brain.

Short communication: insulin alters hepatic progesterone catabolic enzymes cytochrome P450 2C and 3A in dairy cows.

High proportions of embryonic and early fetal losses in dairy cattle are associated with low peripheral concentrations of progesterone, which could result from increased catabolism, decreased production, or both. Progesterone catabolism occurs primarily in the liver via the cytochrome P450 2C (CYP2C) and cytochrome P450 3A (CYP3A) subfamilies (EC 1.14.14.1; unspecific monooxygenases). Recent observations from our laboratory have shown that the fractional rate constant of progesterone decay can be dramatically reduced by insulin because of a decrease in hepatic CYP2C and CYP3A activity. Little information exists on the regulation of progesterone catabolic enzymes in dairy cows. We hypothesized that elevated insulin concentrations would down-regulate hepatic CYP2C and CYP3A mRNA; therefore, our objectives were to determine the relative abundance of hepatic CYP2C and CYP3A mRNA in dairy cows in response to elevated concentrations of insulin. In the first experiment, 17 mature Holstein cows were drenched daily with 500 mL of water (n = 10) or propylene glycol (a gluconeogenic substrate; n = 7) from 10 d before their expected calving date until d 25 postpartum. Cows drenched with propylene glycol had a 30% increase in peripheral concentrations of insulin. Liver biopsies were collected on d 25 postpartum to determine the relative abundance of CYP2C and CYP3A mRNA. In the second experiment, 19 mature, lactating Holstein cows were randomly assigned to a hyperinsulinemic-euglycemic clamp (0.3 or 1.0 microg of insulin/kg of BW per h; n = 6 each) or remained as controls (saline infused; n = 7) for 96 h beginning on d 10 postpartum. Insulin infusion resulted in a 2.6- or 8- fold increase in peripheral concentrations of insulin, respectively. On d 14 postpartum, a liver biopsy was collected to determine CYP2C and CYP3A mRNA abundance. In experiment 1, the relative abundance of CYP2C mRNA in cows treated with propylene glycol did not differ from controls; however, the relative abundance of CYP3A mRNA in the propylene glycol group was 63% that of controls. For experiment 2, there was a dose-dependent decrease in the relative abundance of both CYP2C and CYP3A mRNA with increasing dosage of insulin. In conclusion, this study demonstrated that, in the cow, either providing a gluconeogenic feed-stuff or treatment with insulin decreased the abundance of mRNA for enzymes responsible for hepatic progesterone catabolism.

Hydrogen peroxide-mediated toxicity for Leishmania donovani chagasi promastigotes. Role of hydroxyl radical and protection by heat shock.

Leishmania must survive despite exposure to the toxic oxidant hydrogen peroxide (H2O2) during phagocytosis by macrophages. We investigated the mechanism of H2O2 toxicity for L. donovani chagasi promastigotes, and factors responsible for their relative H2O2 resistance. There was a dose-dependent toxic effect of H2O2 for promastigotes isolated during logarithmic phase of growth. In contrast, stationary phase promastigotes were less susceptible to H2O2 toxicity, and more infectious for BALB/c mice. By spin trapping we found that hydroxyl radical (.OH) was generated after exposure of promastigotes to H2O2, and the amount of .OH was greater with log-phase than with stationary-phase promastigotes. .OH was generated after the addition of H2O2 to the cytosol but not the membranes of fractionated promastigotes, and the magnitude of .OH was greater in log than in stationary promastigote cytosol. Deferoxamine inhibition suggested that intracellular promastigote iron catalyzes .OH formation via the Fenton reaction. Furthermore, exposure of log-phase promastigotes to heat shock induced a relative H2O2-resistant state, which was not associated with a decrease in .OH formation but which required ongoing transcription. Thus, growth to stationary phase and heat shock both induce a state of relative H2O2 resistance, but these are probably due to different resistance mechanisms.

Effect of melengestrol acetate as an alternative to induce molting in hens on the expression of yolk proteins and turnover of oviductal epithelium.

Inducing hens to molt increases egg quality, egg production and extends the productive life of hens. It has been previously demonstrated that melengestrol acetate (MGA), an orally active progestin, decreased gonadotropic support for the ovary, which decreased the steroidogenic support for the oviduct and resulted in the cessation of lay. Estradiol produced by the theca cells of small follicles stimulates the production of the yolk proteins vitellogenin II and apolipoprotein II by the liver and supports the oviductal epithelial cells. The objective of the present experiment was to determine gene expression for yolk proteins and oviductal epithelial cell turn-over in response to a MGA-induced molt. Hy-Line W-36 laying hens were fed either 0 or 8mg MGA per day for 28 days in a balanced diet and then returned to a standard layer ration until day 36. Four birds per treatment on days 1, 8, 16, 28 and 36 were euthanized and the liver was removed and snap frozen in liquid nitrogen until RNA was extracted. Expression of vitellogenin II and apolipoprotein II genes was determined using real-time RT-PCR. A portion of the magnum was removed to determine proliferation and programmed cell death for secretory and ciliated luminal epithelium. Vitellogenin II and apolipoprotein II gene expression was reduced in hens fed 8mg MGA compared to those fed 0mg MGA. There was no effect of day on gene expression of either yolk protein. Cell proliferation was increased in the ciliated epithelial cells of the oviduct in hens receiving 8mg MGA compared to those receiving 0mg. However, programmed cell death of the ciliated epithelial cells was not different between controls and MGA treatment. Programmed cell death and proliferation increased in the secretory epithelial cells in hens receiving 8mg MGA compared to controls. Therefore, utilizing MGA as an alternative method to induce molt results in some, but not all, of the physiological changes previously described for hens molted by feed withdrawal. These findings lead us to suggest that some of the observed physiological changes resulting from feed withdrawal are required to increase egg quality and egg production following molt and other changes are not necessary, but are just a result of nutrient deprivation.

Motivation of hens to obtain feed during a molt induced by feed withdrawal, wheat middlings, or melengestrol acetate.

Traditionally, molting was initiated by withdrawing feed. However, public criticism of feed deprivation, based on the perception that it inhumanely increases hunger, has led the poultry industry to ban the practice. Thus far, alternatives have not been demonstrated to ameliorate the increase in hunger that led to the ban on inducing molting by feed deprivation. Incorporating melengestrol acetate (MGA), an orally active progestin, into a balanced layer diet induces molting and increases postmolt egg quality. Hy-Line W-98 hens (n = 60) were randomly assigned to a balanced layer ration (control), a balanced layer ration containing MGA, or a 94% wheat middlings diet (wheat) for 20 d, or were feed deprived for 8 d. Hens were trained to peck a switch to receive a feed reward based on a progressive ratio reinforcement schedule. Motivation of hens to acquire feed was measured as the total number of pecks recorded in 15 min on d 0, 4, 8, 12, 16, and 20. On d 20, abdominal fat pad and digesta-free gizzards were weighed. The number of pecks in the feed-deprived group was greater than controls by d 4 and remained greater at d 8, when these hens were removed from the experiment. Hens in the wheat group that were rewarded with a layer diet pecked more than controls from d 8 to 20. Hens in the MGA group pecked for a reward at the same rate as control hens throughout the experiment. Hens fed the wheat diet had heavier gizzards compared with control and MGA-fed hens. Hens fed MGA had greater abdominal fat pad compared with wheat and control hens. Hens molted using a diet containing MGA have a similar motivation to obtain feed as control hens; therefore, this alternative does not appear to increase hunger. However, hens molted with a wheat middling diet appear to be as motivated to obtain feed as did the feed-deprived hens.