Changes in pituitary responses to synthetic ovine corticotrophin releasing factor in fetal sheep

The rise in cortisol in fetal sheep during late pregnancy has been related to increased responsiveness of the adrenal to ACTH. Most reports have suggested that plasma ACTH concentrations rise coincident with or after the prepartum increase in cortisol. To reexamine the relationship of cortisol with basal immunoreactive ACTH (IR-ACTH) throughout the last 40 days of pregnancy and to determine changes in fetal pituitary responsiveness during this time, we measured basal and synthetic ovine corticotrophin-releasing factor (oCRF) (10 ng-10 micrograms) induced rises in ACTH and cortisol in fetal sheep at days 110-115, 125-130, and 135-140 of pregnancy. The fetuses were catheterized on day 105-120 and entered spontaneous labour at greater than 140 days. Basal IR-ACTH (picograms per millilitre +/- SEM) rose from 16.7 +/- 2.9 pg/mL at day 110-115 to 34.8 +/- 8.7 pg/mL at day 141-145. There was a significant effect of time on basal ACTH concentrations with a mean increase of approximately 5 pg ACTH per millilitre of plasma per 5-day sampling interval. Plasma cortisol changed gradually between day 110 and 125 of gestation and then more rapidly to term. At day 110-115 of gestation there was no significant change in plasma ACTH after 10 or 100 ng oCRF, but there was a significant increase in ACTH after 1 microgram of oCRF. Plasma cortisol did not change after any CRF injection. The change in IR-ACTH after oCRF at day 125-130 of gestation was significantly greater than that at day 110-115. Plasma cortisol concentrations were elevated following 1- and 10-micrograms injections of oCRF.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heat-induced inhibition of DNA synthesis in L5178Y-S cells is reversed by caffeine

Heating L5178Y cells for 15 min at 43 degrees C caused a decrease in [3H]thymidine incorporation, which could be reversed by post-treatment with 0.75 mM caffeine in an L5178Y-S (radiation-sensitive, heat-resistant) but not in an L5178Y-R (radiation-resistant, heat-sensitive) strain. The reversal was accompanied by a sparing effect of the treatment: survival of L5178Y-S cells increased by a factor of 1.5. The effect of combined (heat + caffeine) treatment of L5178Y-R cells was cumulative.     

Prediction of titration properties of structures of a protein derived from molecular dynamics trajectories.

This paper explores the dependence of the molecular dynamics (MD) trajectory of a protein molecule on the titration state assigned to the molecule. Four 100-ps MD trajectories of bovine pancreatic trypsin inhibitor (BPTI) were generated, starting from two different structures, each of which was held in two different charge states. The two starting structures were the X-ray crystal structure and one of the solution structures determined by NMR, and the charge states differed only in the ionization state of N terminus. Although it is evident that the MD simulations were too short to sample fully the equilibrium distribution of structures in each case, standard Poisson-Boltzmann titration state analysis of the resulting configurations shows general agreement between the overall titration behavior of the protein and the charge state assumed during MD simulation: at pH 7, the total net charge of the protein resulting from the titration analysis is consistently lower for the protein with the N terminus assumed to be neutral than for the protein with the N terminus assumed to be charged. For most of the ionizable residues, the differences in the calculated pKaS among the four trajectories are statistically negligible and remain in good agreement with the data obtained by crystal structure titration and by experiment. The exceptions include the N terminus, which responds directly to the change of its imposed charge; the C terminus, which in the NMR structure interacts strongly with the former; and a few other residues (Arg 1, Glu 7, Tyr 35, and Arg 42) whose pKaS reflect the initial structure and the limited trajectory lengths. This study illustrates the importance of the careful assignment of protonation states at the start of MD simulations and points to the need for simulation methods that allow for the variation of the protonation state in the calculation of equilibrium properties.     

Impact of Low Dose Prenatal Ethanol Exposure on Glucose Homeostasis in Sprague-Dawley Rats Aged up to Eight Months

Excessive exposure to alcohol prenatally has a myriad of detrimental effects on the health and well-being of the offspring. It is unknown whether chronic low-moderate exposure of alcohol prenatally has similar and lasting effects on the adult offspring’s health. Using our recently developed Sprague-Dawley rat model of 6% chronic prenatal ethanol exposure, this study aimed to determine if this modest level of exposure adversely affects glucose homeostasis in male and female offspring aged up to eight months. Plasma glucose concentrations were measured in late fetal and postnatal life. The pancreas of 30 day old offspring was analysed for β-cell mass. Glucose handling and insulin action was measured at four months using an intraperitoneal glucose tolerance test and insulin challenge, respectively. Body composition and metabolic gene expression were measured at eight months. Despite normoglycaemia in ethanol consuming dams, ethanol-exposed fetuses were hypoglycaemic at embryonic day 20. Ethanol-exposed offspring were normoglycaemic and normoinsulinaemic under basal fasting conditions and had normal pancreatic β-cell mass at postnatal day 30. However, during a glucose tolerance test, male ethanol-exposed offspring were hyperinsulinaemic with increased first phase insulin secretion. Female ethanol-exposed offspring displayed enhanced glucose clearance during an insulin challenge. Body composition and hepatic, muscle and adipose tissue metabolic gene expression levels at eight months were not altered by prenatal ethanol exposure. Low-moderate chronic prenatal ethanol exposure has subtle, sex specific effects on glucose homeostasis in the young adult rat. As aging is associated with glucose dysregulation, further studies will clarify the long lasting effects of prenatal ethanol exposure.     

Does the Establishment of Sustainable Use Reserves Affect Fire Management in the Humid Tropics?

Tropical forests are experiencing a growing fire problem driven by climatic change, agricultural expansion and forest degradation. Protected areas are an important feature of forest protection strategies, and sustainable use reserves (SURs) may be reducing fire prevalence since they promote sustainable livelihoods and resource management. However, the use of fire in swidden agriculture, and other forms of land management, may be undermining the effectiveness of SURs in meeting their conservation and sustainable development goals. We analyse MODIS derived hot pixels, TRMM rainfall data, Terra-Class land cover data, socio-ecological data from the Brazilian agro-census and the spatial extent of rivers and roads to evaluate whether the designation of SURs reduces fire occurrence in the Brazilian Amazon. Specifically, we ask (1) a. Is SUR location (i.e., de facto) or (1) b. designation (i.e. de jure) the driving factor affecting performance in terms of the spatial density of fires?, and (2), Does SUR creation affect fire management (i.e., the timing of fires in relation to previous rainfall)? We demonstrate that pre-protection baselines are crucial for understanding reserve performance. We show that reserve creation had no discernible impact on fire density, and that fires were less prevalent in SURs due to their characteristics of sparser human settlement and remoteness, rather than their status de jure. In addition, the timing of fires in relation to rainfall, indicative of local fire management and adherence to environmental law, did not improve following SUR creation. These results challenge the notion that SURs promote environmentally sensitive fire-management, and suggest that SURs in Amazonia will require special attention if they are to curtail future accidental wildfires, particularly as plans to expand the road infrastructure throughout the region are realised. Greater investment to support improved fire management by farmers living in reserves, in addition to other fire users, will be necessary to help ameliorate these threats.     

Reduced fetal, placental, and amniotic fluid PTHrP in the growth-restricted spontaneously hypertensive rat

Evidence implicates pivotal roles for parathyroid hormone-related protein (PTHrP) in stimulating cell growth and differentiation, placental calcium transport, and placental vasodilatation. As spontaneously hypertensive rat (SHR) fetuses are growth restricted compared with those of its normotensive control, the Wistar Kyoto (WKY) rat, we examined intrauterine PTHrP and total and ionic calcium concentrations in these rats. Fetal plasma PTHrP concentrations, but not total calcium concentrations, were lower in the SHR compared with WKY (P < 0.05). SHR placental concentrations of PTHrP were lower than in WKY (P < 0.03) and failed to show the increase observed in WKY near term (P < 0.05). PTHrP concentrations in amniotic fluid from SHR were not raised near term and were lower compared with WKY (P < 0.0005). The increased ionic calcium concentrations in amniotic fluid in the WKY near term (P < 0.05) were not detected in the SHR. Thus SHR fetal plasma, placental, and amniotic fluid PTHrP concentrations were reduced and associated with fetal growth restriction. We suggest that PTHrP may play a role in the etiology of both growth restriction during pregnancy and hypertension later in life.     

Effect of high oxygen on placental function in short-term explant cultures

Ex situ culture of human gestational tissues has been routinely used as a model to investigate tissue function. The objective of this study was to determine the effect of varying oxygen concentrations on human term placental explants over a 24-h time period. Specifically, the effect of incubating placental explants in oxygen concentrations of 8%, 21% or 95% on tissue viability, metabolism and cell death was measured by assessing glucose consumption, lactate production, release of lactate dehydrogenase, parathyroid hormone-related protein (PTHrP), tumour necrosis factor-alpha (TNF-α) and 8-isoprostane, immunoreactivity for cleaved-caspase-9 and immunohistochemistry for the caspase-3-cleaved cytokeratin-18 neoepitope, M30. Exposure to higher oxygen concentrations significantly increased the rates of glucose consumption and lactate production. Apoptosis was significantly increased under conditions of higher oxygen as evidenced by increased M30 in placental explant sections. Similarly, hyperoxia significantly increased the releases of PTHrP, TNF-α and 8-isoprostane. Thus, incubation of placental explants with oxygen concentrations of 95% and, to a lesser extent, 21% oxygen was associated with the modulation of multiple cellular response pathways including those associated with tissue viability and cell death. These data are consistent with the hypothesis that hyperoxia activates pathways and mechanisms involved in cellular metabolism, necrosis and apoptosis, thereby shifting the balance from a steady state towards cell death.     

Effect of Pregnancy for Females Born Small on Later Life Metabolic Disease Risk

There is a strong inverse relationship between a females own birth weight and her subsequent risk for gestational diabetes with increased risk of developing diabetes later in life. We have shown that growth restricted females develop loss of glucose tolerance during late pregnancy with normal pancreatic function. The aim of this study was to determine whether growth restricted females develop long-term impairment of metabolic control after an adverse pregnancy adaptation. Uteroplacental insufficiency was induced by bilateral uterine vessel ligation (Restricted) or sham surgery (Control) in late pregnancy (E18) in F0 female rats. F1 Control and Restricted female offspring were mated with normal males and allowed to deliver (termed Ex-Pregnant). Age-matched Control and Restricted Virgins were also studied and glucose tolerance and insulin secretion were determined. Pancreatic morphology and hepatic glycogen and triacylglycerol content were quantified respectively. Restricted females were born lighter than Control and remained lighter at all time points studied (p<0.05). Glucose tolerance, first phase insulin secretion and liver glycogen and triacylglycerol content were not different across groups, with no changes in β-cell mass. Second phase insulin secretion was reduced in Restricted Virgins (−34%, p<0.05) compared to Control Virgins, suggestive of enhanced peripheral insulin sensitivity but this was lost after pregnancy. Growth restriction was associated with enhanced basal hepatic insulin sensitivity, which may provide compensatory benefits to prevent adverse metabolic outcomes often associated with being born small. A prior pregnancy was associated with reduced hepatic insulin sensitivity with effects more pronounced in Controls than Restricted. Our data suggests that pregnancy ameliorates the enhanced peripheral insulin sensitivity in growth restricted females and has deleterious effects for hepatic insulin sensitivity, regardless of maternal birth weight.     

Acetylcholinesterase: Role of the enzyme's charge distribution in steering charged ligands toward the active site

The electrostatic steering of charged ligands toward the active site of Torpedo californica acetylcholinesterase is investigated by Brownian dynamics simulations of wild type enzyme and several mutated forms, in which some normally charged residues are neutralized. The simulations reveal that the total ligand influx through a surface of 42 Å radius centered in the enzyme monomer and separated from the protein surface by I-14 Å is not significantly influenced by electrostatic interactions. Electrostatic effects are visible for encounters with a surface of 32 Å radius, which is partially hidden inside the protein, but mostly within the solvent. A clear accumulation of encounter events for that sphere is observed in the area directly above the entrance to the active site gorge. In this area, the encounter events are increased by 40% compared to the case of a neutral ligand. However, the differences among the encounter rates for the various mutants considered here are not pronounced, all rate constants being within ±10% of the average value. The enzyme charge distribution becomes more important as the charged ligand moves toward the bottom of the gorge, where the active site is located. We show that neither the enzyme's total charge, nor its dipole moment, fully account for the electrostatic steering of ligand to the active site. Higher moments of the enzyme's charge distribution are also important. However, for a series of mutations for which the direction of the enzyme dipole moment is constant within a few degrees, one observes a gradual decrease in the diffusional encounter rate constant with the number of neutralized residues. On the other hand, for other mutants that change the direction of the dipole moment from that of the wild type, the calculated encounter rate constants can be very close to that of the wild type. The present work yields two new insights to the kinetics of acetylcholinesterase. First, evolution appears to have built a redundant electrostatic steering capability into this important enzyme through the overall distribution of its thousands of partially charged atoms. And second, roughly half of the rate enhancement due to electrostatics arises from steering of the substrate outside the enzyme; the other half of the rate enhancement arises from improved trapping of the substrate after it has entered the gorge. The computational results reproduce qualitatively, and help to rationalize, many surprising experimental results obtained recently for human acetylcholinesterase. © 1996 John Wiley & Sons, Inc.