A powerful GABA(B) receptor-mediated inhibition of GABAergic neurons in the arcuate nucleus

We combined histofluorescence with in situ hybridization to identify GABAergic neurons in the arcuate nucleus (ARC) following electrophysiological recordings, using GAD65 as a marker. Intracellular recordings were made in hypothalamic slices prepared from ovariectomized guinea pigs. Over 90% of ARC neurons tested with the GABA(B) receptor agonist baclofen responded with a membrane hyperpolarization or an outward current. The hyperpolarization was dose-dependent, and the GABA(B) receptor antagonist CGP 35,348 produced a rightward shift in the agonist dose-response curve. Agonist potency was lower, and the efficacy greater, in GAD-positive neurons. The use of this novel technique for identifying GABAergic neurons thus reveals differences in the pharmacodynamics of GABA(B) receptor activation GABAergic and non-GABAergic ARC neurons.     

Classifying meconium-stained liquor: is it feasible?

Four samples each of clear and lightly (thin), moderately, and heavily (thick) meconium-stained amniotic fluid were divided in two portions and submitted twice for assessment to 20 midwives (a total of 320 case assessments). None of the midwives completely agreed with the standard assessment for more than 85 percent of the cases. When disregarding clear samples, for which there was good agreement, each of the midwives classified on average only 35.8 percent of the meconium-stained samples in the same category on each of the four occasions that they were presented to them. Calculation of kappa statistics, which express proportional agreement corrected for chance, indicated that none of the midwives showed very good agreement (kappa > 0.81) with the standard and that fewer than 10 percent showed very good agreement with themselves. The data indicate that grading the severity of meconium staining by visual assessment has such poor accuracy and precision that it cannot provide a valid basis for assigning different care policies to different degrees of meconium staining.     

Cholinergic blockade and the mesenteric artery response to head-up tilt-induced central hypovolaemia

The methylation of phosphatidylethanolamine is an auxiliary pathway for phosphatidylcholine biosynthesis in liver. Two forms of the enzyme, phosphatidylethanolamine N-methyltransferase, which catalyses this reaction, are located on the endoplasmic reticulum and mitochondria-associated membranes. Both forms are encoded by a single murine gene, Pempt, located on chromosome 11. The expression of the gene begins at birth. An inverse relationship exists between the rate of liver growth and the expression of phosphatidylethanolamine N-methyltransferase. However, disruption of the Pempt gene does not alter liver growth in mice or cause any other obvious phenotype.     

A Golgi localization signal identified in the Menkes recombinant protein

Menkes disease arises from a genetic impairment in copper transport. The gene responsible for the phenotype has been identified as a copper transporting ATPase ( ATP7A ). Recently, the protein encoded by the ATP7A gene has been localized to the Golgi complex. In order to investigate the role of the Menkes disease protein in copper transport, recombinant constructs containing both the full-length open reading frame and an alternatively spliced form have been successfully expressed and localized in mammalian cells. Other studies of a patient with occipital horn syndrome, an allelic variant of Menkes disease, have demonstrated that only this alternatively spliced isoform and not the full-length form is expressed in this patient. The milder form of this patient's phenotype suggests that the alternatively spliced isoform has some functional role in copper transport. In the present study the full-length recombinant Menkes protein was shown by immunofluorescence to localize to the Golgi apparatus and the alternatively spliced form, lacking sequences for transmembrane domains 3 and 4 encoded by exon 10, was shown to localize to the endoplasmic reticulum. Using sequences from exon 10 fused to a non-Golgi reporter molecule, a 38 amino acid sequence containing transmembrane domain 3 of the Menkes protein was found to be sufficient for localization to the Golgi complex. Therefore, the protein sequence encoded by exon 10 may be responsible for this differential localization and both isoforms may be required for comprehensive transport of copper within the cell.     

Effects of growth hormone, insulin-like growth factor-I, and cortisol on periparturient antibody response profiles of dairy cattle

The objectives of this study were to determine hormone and antibody response profiles from the prepartum period to peak lactation, and evaluate potential immunomodulatory effects of the classic endocrine hormones, growth hormone (GH), insulin-like growth factor-I (IGF-I) and cortisol. Specifically, 33 Holstein cows were immunized with ovalbumin (OVA) and Escherichia coli J5 at weeks -8 and -3 prior to parturition. At parturition (week 0), cows received an additional immunization of OVA. Blood was collected at weeks -8, -3, 0, 3 and 6 relative to parturition and various samples were used to determine plasma hormone concentration, serum immunoglobulin (Ig), and specific antibody response to OVA and E. coli. Colostrum and milk samples were also collected post-parturition to monitor local immunoglobulin and antibody responses. Results indicated that not all periparturient cows exhibited depressed immune response, and that antibody response to OVA could be used to partition cows into 3 groups recognizing animals with sustained measurable antibody response before and after parturition (Group 1), animals which responded poorly to immunization at parturition (Group 2), and animals which did not respond to immunizations at week -3 or parturition (Group 3). Cows with the highest antibody response to OVA (Group 1) also tended (P < or = 0.10) to have the highest response to E. coli J5 at parturition and had the lowest incidence of disease, particularly mastitis. Antibody response to OVA measured in milk tended to be higher in Group 1 cows, particularly at week 0 (P < or = 0.06) compared to cows of Group 3. IGF-I was higher (P < or = 0.05) in cows of Group 1 than Group 3 at peak lactation (week 6).     

Crystal structure of TNF-alpha mutant R31D with greater affinity for receptor R1 compared with R2

Crystal structures have been determined of recombinant human tumor necrosis factor-alpha (TNF-alpha) and its R31D mutant that preferentially binds to TNF receptor R1 with more than seven times the relative affinity of binding to receptor R2. Crystals of the wild-type TNF were of space group P4(1)2(1)2 and had unit cell dimensions of a = b = 94.7 and c = 117.4 A. Refinement of the structure gave an R-factor of 22.3% at 2.5 A resolution. The crystals of TNF R31D mutant diffracted to 2.3 A resolution, and were of identical space group to the wild type with unit cell dimensions of a = b = 95.4 and c = 116.2 A, and the structure was refined to an R-factor of 21.8%. The trimer structures of the wild-type and mutant TNF were similar with a root mean square (r.m.s.) deviation of 0.56 A for Calpha atoms; however, the subunits within each trimer were more variable with an average r.m.s. deviation of 1.00 A on Calpha atoms for pairwise comparison of subunits. Model complexes of TNF with receptors R1 and R2 have been used to predict TNF-receptor interactions. Arg31 in all three subunits of wild-type TNF is predicted to form an ionic interaction with the equivalent glutamic acid in both receptors R1 and R2. Asp31 of the TNF R31D mutant is predicted to interact differently with the two receptors. The side chain of Asp31 in two subunits of the TNF mutant is predicted to form hydrogen bond interactions with Ser59 or Cys70 of R1, while it has no predicted interactions with R2. The loss of three strong ionic interactions of Arg31 and the electrostatic repulsion of Asp31 with Glu in the receptors is consistent with the reduced binding of the R31D mutant to both receptors relative to wild-type TNF. The replacement of these ionic interactions by two weaker hydrogen bond interactions between Asp31 of the R31D mutant and R1, compared with no interactions with R2, is in agreement with the observed preferential binding of the R31D mutant to R1 over R2. Analysis of the structure and function of receptor-discriminating mutants of TNF will help understand the biological role of TNF and facilitate its use as an antitumor agent.      

Uncertainty quantification via bayesian inference using sequential monte carlo methods for CO2 adsorption process

This work presents the uncertainty quantification, which includes parametric inference along with uncertainty propagation, for CO₂ adsorption in a hollow fiber sorbent, a complex dynamic chemical process. Parametric inference via Bayesian approach is performed using Sequential Monte Carlo, a completely parallel algorithm, and the predictions are obtained by propagating the posterior distribution through the model. The presence of residual variability in the observed data and model inadequacy often present a significant challenge in performing the parametric inference. In this work, residual variability in the observed data is handled by three different approaches: (a) by performing inference with isolated data sets, (b) by increasing the uncertainty in model parameters, and finally, (c) by using a model discrepancy term to account for the uncertainty. The pros and cons of each of the three approaches are illustrated along with the predicted distributions of CO₂ breakthrough capacity for a scaled‐up process. © 2016 American Institute of Chemical Engineers AIChE J, 62: 3352–3368, 2016