Fluctuations in energy-related metabolites during the peri-parturition period in Lori-Bakhtiari ewes

This study describes the fluctuations in serum energy-related metabolites during a period of 2 weeks before, to 2 weeks after parturition in Lori-Bakhtiari ewes. The effect of parity was also studied. Blood profiles were determined in 60 healthy pregnant ewes with single (n = 30) and twin (n = 30) lambings. Blood was collected from each ewe on days 14 and 7 prepartum, and days 7 and 14 postpartum to determine the serum non-esterified fatty acid (NEFA), β-hydroxybutyrate (BHBA), glucose, cholesterol, blood urea nitrogen (BUN) and calcium (Ca) levels. The age of the ewes had no significant effect on the energy metabolism indicators. Serum NEFA, BHBA, glucose, BUN and calcium concentrations recorded peak levels 7 days before parturition. However, NEFA and BHBA recorded significant changes (P < 0.05) during the peri-parturition period. All metabolites changed significantly in ewes carrying twin-bearing ewes, compared to single-bearing ewes. Serum BHBA concentrations recorded positive correlations with the serum NEFA (P < 0.01) and cholesterol (P < 0.05), while blood glucose had negative correlations (P < 0.01) with NEFA, BHBA and Ca. Blood NEFA and BHBA recorded positive correlations (P < 0.05) with the BUN levels and negative correlations (P < 0.05) with Ca. The results showed that blood NEFA and BHBA levels are sensitive indicators of the energy balance during the peri-parturition period in ewes.     

Acceleration Data Reveal Highly Individually Structured Energetic Landscapes in Free-Ranging Fishers (Pekania pennanti)

Investigating animal energy expenditure across space and time may provide more detailed insight into how animals interact with their environment. This insight should improve our understanding of how changes in the environment affect animal energy budgets and is particularly relevant for animals living near or within human altered environments where habitat change can occur rapidly. We modeled fisher (Pekania pennanti) energy expenditure within their home ranges and investigated the potential environmental and spatial drivers of the predicted spatial patterns. As a proxy for energy expenditure we used overall dynamic body acceleration (ODBA) that we quantified from tri-axial accelerometer data during the active phases of 12 individuals. We used a generalized additive model (GAM) to investigate the spatial distribution of ODBA by associating the acceleration data to the animals'' GPS-recorded locations. We related the spatial patterns of ODBA to the utilization distributions and habitat suitability estimates across individuals. The ODBA of fishers appears highly structured in space and was related to individual utilization distribution and habitat suitability estimates. However, we were not able to predict ODBA using the environmental data we selected. Our results suggest an unexpected complexity in the space use of animals that was only captured partially by re-location data-based concepts of home range and habitat suitability. We suggest future studies recognize the limits of ODBA that arise from the fact that acceleration is often collected at much finer spatio-temporal scales than the environmental data and that ODBA lacks a behavioral correspondence. Overcoming these limits would improve the interpretation of energy expenditure in relation to the environment.     

Direct identification of human oxytocin receptor-binding domains using a photoactivatable cyclic peptide antagonist: comparison with the human V1a vasopressin receptor

Understanding of the molecular determinants responsible for antagonist binding to the oxytocin receptor should provide important insights that facilitate rational design of potential therapeutic agents for the treatment of preterm labor. To study ligand/receptor interactions, we used a novel photosensitive radioiodinated antagonist of the human oxytocin receptor, d(CH(2))(5) [Tyr(Me)(2),Thr(4),Orn(8),Phe(3(125)I,4N(3))-NH(2)9]vasotocin. This ligand had an equivalent high affinity for human oxytocin and V(1a) vasopressin receptors expressed in Chinese hamster ovary cells. Taking advantage of this dual specificity, we conducted photoaffinity labeling experiments on both receptors. Photolabeled oxytocin and V(1a) receptors appeared as a unique protein band at 70-75 kDa and two labeled protein bands at 85-90 and 46 kDa, respectively. To identify contact sites between the antagonist and the receptors, the labeled 70-75- and the 46-kDa proteins were cleaved with CNBr and digested with Lys-C and Arg-C endoproteinases. The fragmentation patterns allowed the identification of a covalently labeled region in the oxytocin receptor transmembrane domain III consisting of the residues Leu(114)-Val(115)-Lys(116). Analysis of contact sites in the V(1a) receptor led to the identification of the homologous region consisting of the residues Val(126)-Val(127)-Lys(128). Binding domains were confirmed by mutation of several CNBr cleavage sites in the oxytocin receptor and of one Lys-C cleavage site in the V(1a) receptor. The results are in agreement with previous experimental data and three-dimensional models of agonist and antagonist binding to members of the oxytocin/vasopressin receptor family.     

A proteolytic method for distinguishing between lipid-free and lipid-bound apolipoprotein A-I

Recent studies indicate that certain lipid-poor forms of apolipoprotein (apo)A-I may be particularly important in promoting cholesterol release from overburdened cells in the periphery. However, a detailed understanding of the physiological relevance of these species has been hampered by the difficulty in measuring them. As part of a search for a rapid assay for these forms of apoA-I, we have observed that the protease enteropeptidase can specifically cleave human lipid-free apoA-I but not its lipid-bound form. Enteropeptidase cleaved lipid-free apoA-I at a single site at amino acid 188, resulting in an N-terminal fragment of 22 kDa. However, apoA-I was not susceptible to enteropeptidase when present in reconstituted high-density lipoprotein (rHDL) particles as small as 6 nm in diameter or in human HDL(3) particles, even at extremely high enzyme-to-protein ratios and extended reaction times. We capitalized on this observation to develop an assay for the measurement of lipid-poor apoA-I in in vitro systems. Densitometry was used to generate a standard curve from sodium dodecyl sulfate polyacrylamide gels to determine the amounts of the N-terminal proteolytic fragment in unknown samples treated with enteropeptidase. This system could accurately quantify apoA-I that had been displaced from rHDL particles and human HDL(3) with purified apoA-II. On the basis of the results, a system of nomenclature is proposed for "lipid-free," "lipid-poor," and "lipid bound" apoA-I.The reported method distinguishes forms of apoA-I by a conformational parameter without previous separation of the species. This simple and inexpensive method will be useful for understanding the characteristics of plasma HDL that are favorable for the dissociation of apoA-I.     

Escherichia coli heat shock protein DnaK: production and consequences in terms of monitoring cooking

Through use of commercially available DnaK proteins and anti-DnaK monoclonal antibodies, a competitive enzyme-linked immunosorbent assay was developed to quantify this heat shock protein in Escherichia coli ATCC 25922 subjected to various heating regimens. For a given process lethality (F(70)(10) of 1, 3, and 5 min), the intracellular concentration of DnaK in E. coli varied with the heating temperature (50 or 55 degrees C). In fact, the highest DnaK concentrations were found after treatments at the lower temperature (50 degrees C) applied for a longer time. Residual DnaK after heating was found to be necessary for cell recovery, and additional DnaK was produced during the recovery process. Overall, higher intracellular concentrations of DnaK tended to enhance cell resistance to a subsequent lethal stress. Indeed, E. coli cells that had undergone a sublethal heat shock (105 min at 55 degrees C, F(70)(10) = 3 min) accompanied by a 12-h recovery (containing 76,786 +/- 25,230 molecules/cell) resisted better than exponentially growing cells (38,500 +/- 6,056 molecules/cell) when later heated to 60 degrees C for 50 min (F(70)(10) = 5 min). Results reported here suggest that using stress protein to determine cell adaptation and survival, rather than cell counts alone, may lead to more efficient heat treatment.     

Interplay between influenza A virus and the innate immune signaling

Pathogens such as influenza A viruses (IAV) have to overcome a number of barriers defined and maintained by the host, to successfully establish an infection. One of the initial barriers is collectively characterized as the innate immune system. This is a broad anti-pathogen defense program that ranges from the action of natural killer cells to the induction of an antiviral cytokine response. In this article we will focus on new developments and discoveries concerning the interaction of IAV with the cellular innate immune signaling. We discuss new mechanisms of interference of IAV with the pathogen recognition receptor RIG-I and the type I IFN antagonist NS1 in the background of already known and established concepts. Further we summarize progress related to recently identified IFN induced proteins and the role of RNA interference in the context of IAV infection.     

Anti-malarial drug targets: Screening for inhibitors of 2C-methyl-d-erythritol 4-phosphate synthase (IspC protein) in Mediterranean plants

The recently discovered non-mevalonate pathway of isoprenoid biosynthesis serves as the unique source of terpenoids in numerous pathogenic eubacteria and in apicoplast-type protozoa, most notably Plasmodium, but is absent in mammalian cells. It is therefore an attractive target for anti-infective chemotherapy. The first committed step of the non-mevalonate pathway is catalyzed by 2C-methyl-D-erythritol 4-phosphate synthase (IspC). Using photometric and NMR spectroscopic assays, we screened extracts of Mediterranean plants for inhibitors of the enzyme. Strongest inhibitory activity was found in leaf extracts of Cercis siliquastrum.