Ligand binding and thermostability of different allosteric states of the insulin zinc-hexamer

The influence of ligand binding and conformation state on the thermostability of hexameric zinc-insulin was studied by differential scanning calorimetry (DSC). The insulin hexamer exists in equilibrium between the forms T6, T3R3, and R6. Phenolic ligands induce and stabilize the T3R3- and R6-states which are further stabilized by binding of certain anions that do not stabilize the T6-state. It was shown that the thermostability of the resorcinol-stabilized R6-state was significantly higher than that of the T6-state. Further analysis showed that phenol- and m-cresol-stabilized R6-hexamer loses three ligands before reaching the unfolding temperature and hence unfolds from the T3R3-state. The relative affinity of the four tested anionic ligands was found, by DSC, to be thiocyanate > or = 4-hydroxy-3-nitrobenzoate > p-aminobenzoate > chloride. The results correlate with other methods and demonstrate that DSC provides a general and useful method of evaluation of both phenolic and anionic ligand binding to insulin without the use of probes or other alterations of the system of interest. However, it is a prerequisite that the binding is strong enough to saturate the binding sites at temperatures around the unfolding transition.     

Freezing at -800 degrees C distorts the DNA composition of bacterial communities in intestinal samples

Terminal-restriction fragment length polymorphism (T-RFLP) was used to evaluate how to store intestinal specimens for bacterial community analysis. Bacterial communities are increasingly often described by means of DNA-based methods and it is common practice to store intestinal or faecal specimens either at -20 degrees C or -80 degrees C. In this study, samples of intestines from five different pigs were stored at -80 degrees C and -20 degrees C, respectively and a thawing and freezing procedure was carried out three times for each intestinal per pig per temperature. The cumulative sum of the T-RFLP peak heights (T-RF intensities) decreased as the temperature decreased. The composition of the bacterial community changed when stored at -80 degrees C compared to the samples stored at -20 degrees C. Thus it is recommended from this study that samples of intestinal content are stored at -20 degrees C before use for bacterial community analysis, instead of the current practice at -80 degrees C.     

Niche overlap of mountain hare subspecies and the vulnerability of their ranges to invasion by the European hare; the (bad) luck of the Irish

Niche conservatism is the tendency of related species to retain ancestral tolerances after geographic separation. We used Ecological Niche Modelling and Principal Components Analysis of bioclimatic and habitat variables to describe the extent of the species niche, and degrees of bioclimatic–habitat niche conservatism within the mountain hare (L. timidus) clade. Mountain hare niche space was contrasted with that of the European hare (L. europaeus), to shed light on species interactions in contact zones throughout Europe. All five subspecies of mountain hare had quantifiably distinct niches. Fennoscandian (L.t. sylvaticus, L.t. timidus) and highland (L.t. scoticus, L.t. varronis) subspecies, however, were most similar, exhibiting greatest apparent niche conservatism. They inhabit tundra, boreal forest and uplands, and, hence are presumed most similar to the ancestral form. The Irish hare was distinct, being consistently distinguished from other mountain hares in both 2D and nth dimensional (4D) niche space. The ecological distinctiveness of the Irish hare provides further evidence that it is an Evolutionarily Significant Unit, particularly vulnerable to displacement by introduced European hares with which it competes and hybridises. Projections under global climate change suggest that, by 2070, bioclimatic space for invasive European hares in Ireland will expand (by 79%) but contract for endemic Irish hares (by 75%), further facilitating their replacement. The near complete species replacement of the heath hare (L.t. sylvaticus) in southern Sweden, where the European hare has also been introduced, may suggest a similar fate may be in store for the Irish hare.     

Glucose,Lactate and Glutamine but not Glutamate Support Depolarization-Induced Increased Respiration in Isolated Nerve Terminals

Synaptosomes prepared from various aged and gene modified experimental animals constitute a valuable model system to study pre-synaptic mechanisms. Synaptosomes were isolated from whole brain and the XFe96 extracellular flux analyzer (Seahorse Bioscience) was used to study mitochondrial respiration and glycolytic rate in presence of different substrates. Mitochondrial function was tested by sequentially exposure of the synaptosomes to the ATP synthase inhibitor, oligomycin, the uncoupler FCCP (carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone) and the electron transport chain inhibitors rotenone and antimycin A. The synaptosomes exhibited intense respiratory activity using glucose as substrate. The FCCP-dependent respiration was significantly higher with 10 mM glucose compared to 1 mM glucose. Synaptosomes also readily used pyruvate as substrate, which elevated basal respiration, activity-dependent respiration induced by veratridine and the respiratory response to uncoupling compared to that obtained with glucose as substrate. Also lactate was used as substrate by synaptosomes but in contrast to pyruvate, mitochondrial lactate mediated respiration was comparable to respiration using glucose as substrate. Synaptosomal respiration using glutamate and glutamine as substrates was significantly higher compared to basal respiration, whereas oligomycin-dependent and FCCP-induced respiration was lower compared to the responses obtained in the presence of glucose as substrate. We provide evidence that synaptosomes are able to use besides glucose and pyruvate also the substrates lactate, glutamate and glutamine to support their basal respiration. Veratridine was found to increase respiration supported by glucose, pyruvate, lactate and glutamine and FCCP was found to increase respiration supported by glucose, pyruvate and lactate. This was not the case when glutamate was the only energy substrate.     

Identification of a membrane proteomic signature for human embryonic stem cells independent of culture conditions

Proteomic profiling of human embryonic stem cells (hESC) can identify cell fate determination and self-renewal biomarkers. Employing Fourier transform LC-ESI-MS/MS and MS³ mass spectrometry, we obtained a membrane proteomic signature overlapping between hESC cultured on mouse embryonic fibroblast (MEF) feeders and those grown under MEF-free culture conditions. We identified 444 transmembrane or membrane-associated proteins, of which 157 were common between both culture conditions. Functional annotation revealed CD antigens (10%), adhesion proteins (4%), proliferation-associated proteins (4%), receptors (41%), transport proteins (21%), structural proteins (5%), and proteins with miscellaneous functions (15%). In addition, 15 CD antigens and a number of surface marker molecules not previously observed in hESC at a proteome level, e.g., Nodal modulator 1, CD222, transgelin-2, and CD81, were identified. In conclusion, we describe the first membrane proteome profile of hESC that is independent of culture conditions. These data can be used to define the phenotype of hESC.     

Effects of alternative styles of risk information on EMF risk perception

Risk scenarios characterized by exposures to new technologies with unknown health effects, together with limited appreciation of benefits pose a challenge to risk communication. The present report illustrates this situation through a study of the perceived risk from mobile phones and mobile masts in residential areas. Good information should objectively convey the current state of knowledge. The research question is then how to inform lay people so that they trust and understand the information. We used an Internet‐based survey with 1687 Danish participants randomized to three types of information about radiation from mobile phones and masts. The objective was to study whether different types of information were rated as equally useful, informative, comprehensible, and trustworthy. Moreover, an important issue was whether information would influence risk perception and intended behavior. The conclusion is that lay people rate information about risks associated with a new and largely unknown technology more useful and trustworthy when provided with brief statements about how to handle the risk, rather than more lengthy technical information about why the technology may or may not entail health hazards. Further, the results demonstrate that information may increase concern among a large proportion of the population, and that discrepancies exist between expressed concern and intended behavior. Bioelectromagnetics 31:504–512, 2010. © 2010 Wiley‐Liss, Inc.     

Differential patterns of relaxation by synthetic atrial peptide (APII) in the pulmonary artery, ascending and distal abdominal aorta

The vasodilatory effects of the synthetic rat atriopeptin (APII) have been studied in vitro in agonist-contracted, endothelium-denuded segments of the rat pulmonary artery, the ascending, and the distal abdominal aorta. In the pulmonary artery the contractures to methoxamine were inhibited more potently by APII (pD2 = 9.10 +/- 0.40, n = 6) than by the vasodilatory neuropeptide VIP (pD2 = 7.37 +/- 0.66, n = 6). The intrinsic activity of APII was 0.46 +/- 0.16 (n = 6). In segments previously exposed to either VIP or the beta 2-agonist salbutamol, APII was a near complete agonist (alpha = 0.82 +/- 0.17, n = 7 and 0.84 +/- 0.14, n = 6, respectively) without significant changes in the potencies. APII was a complete agonist also for the inhibition of the alpha-agonist-contracted segments of the aorta, however, with potencies 10-fold lower than in the pulmonary artery. VIP was without functionally significant effects in the aorta. The tachykinins (CGRP, SP, Neurokinins A and B) were without effects in all segments tested. In the ascending aorta, APII induced a long-lasting tachyphylaxis to the alpha-agonists, nearly completely abolishing the subsequent responsiveness to NA and methoxamine for more than 4 h.