The Sub-Division of Alpha-Adrenoceptors in Vascular Smooth Muscle

1) The aim of this study was to examine the sub-division of alpha-adrenoceptors in vascular smooth muscle. These studies were carried out on the pithed rat and isolated rat aorta preparations. 2) Various parameters were investigated in an attempt to discover the physiological conditions which might be optimal for activation of the alpha-adrenoceptor subtypes. In the pithed rat, the influence of blood gases and the differential effects of calcium-entry blockade on the alpha1 - and alpha2-adrenoceptor-mediated pressor responses were examined. The isolated rat aorta was used to study more closely the calcium dependency of the alpha receptor response, in vascular smooth muscle. 3) The influence of blood gases on the alpha-adrenoceptor subtype-mediated pressor response was studied in the pithed rat by varying the inspired gas mixture or the ventilation volume. Acidosis was found to favour the peak responses to the alpha2-adrenoceptor agonist, xylazine, while alkalosis favoured the peak responses to the alpha1-adrenoceptor agonist, phenylephrine. A combination of hypoxia and hypercapnia greatly depressed the alpha1 response to phenylephrine whereas the alpha2 response to xylazine remained relatively unaffected. When PaO2 was varied in either acidotic or alkalotic conditions, the response to phenylephrine increased as PaO2 increased. 4) To prevent hypoxia in air ventilated rats , large stroke volumes were required. This caused alkalosis and hence decreased responsiveness to xylazine. Consequently, air ventilated pithed rats gave poorer responses to xylazine than did those ventilated on 100% O2. The results show that alpha1- and alpha2-adrenoceptor mediated pressor responses can be differentially affected by blood gases. The relative contribution of alpha1- and alpha2-adrenoceptors to vascular tone may be either under- or over-estimated depending on the arterial blood gases. 5) The pithed rat was also used to study the effect of the calcium-entry blocker, nifedipine, on the alpha1- and alpha2-adrenoceptor pressor responses. To do this, alpha1 and alpha2 agonists were either bolus injected or infused into the pithed rat's blood stream. It was found that nifedipine could inhibit the response initiated by both types of agonist, usually for both the bolus and infusion responses. Calcium-entry blockade was significantly greater on the more prolonged, secondary component of the pressor responses; whether produced by alpha1 or by alpha2 receptor activation. This demonstrates that calcium-entry occurs during the secondary component of the alpha response and can be initiated by either alpha1- or alpha2-adrenoceptor subtypes. 6) The shape of the response produced by infusing the alpha-agonists varied greatly. Most alpha1 agonists, with the exception of SGD 101/75, did not achieve a plateau during the 20 minute infusion time. The alpha2 agonists, on the other hand, readily attained a plateau which was maintained throughout the remainder of the infusion. Phenylephrine and noradrenaline produced pressor responses which reached a maximum and then declined during the remainder of the infusion. These results indicate a difference in the mode of action by which the various alpha-agonists produce their infusion pressor responses. 7) The levels of noradrenaline in the rat arterial and venous plasma were measured during the infusion of noradrenaline, by an HPLC system. Arterial noradrenaline levels rose throughout the infusion whereas venous levels remained relatively unaffected. 8) The isolated rat aortic ring produced a biphasic, contractile response when stimulated by noradrenaline. The initial transient component (ITC) was relatively resistant to the removal of calcium from the external buffering saline and to calcium-entry blockade by nifedipine. It was therefore thought to be the result of mobilization of internal calcium. The slower secondary component (SSC) was reduced by the removal of calcium from the external buffering saline and by calcium-entry blockade, when carried out under reduced calcium conditions. It was considered to be the result of calcium-entry into the cell. The inorganic calcium-entry blocker, cadmium, and the intracellular calcium blocker, dantrolene sodium, were both found to reduce the ITC and the SSC equally well. These results indicate that calcium-entry is involved in both components of the response to noradrenaline. (Abstract shortened by ProQuest.)

Redox Signaling through DNA

Biological electron transfer reactions between metal cofactors are critical to many essential processes within the cell. Duplex DNA is, moreover, capable of mediating the transport of charge through its π-stacked nitrogenous bases. Increasingly, [4Fe4S] clusters, generally redox-active cofactors, have been found to be associated with enzymes involved in DNA processing. DNA-binding enzymes containing [4Fe4S] clusters can thus utilize DNA charge transport (DNA CT) for redox signaling to coordinate reactions over long molecular distances. In particular, DNA CT signaling may represent the first step in the search for DNA lesions by proteins containing [4Fe4S] clusters that are involved in DNA repair. Here we describe research carried out to examine the chemical characteristics and biological consequences of DNA CT. We are finding that DNA CT among metalloproteins represents powerful chemistry for redox signaling at long range within the cell

Harmonization in preclinical epilepsy research: A joint AES/ILAE translational initiative

Among the priority next steps outlined during the first translational epilepsy research workshop in London, United Kingdom (2012), jointly organized by the American Epilepsy Society (AES) and the International League Against Epilepsy (ILAE), are the harmonization of research practices used in preclinical studies and the development of infrastructure that facilitates multicenter preclinical studies. The AES/ILAE Translational Task Force of the ILAE has been pursuing initiatives that advance these goals. In this supplement, we present the first reports of the working groups of the Task Force that aim to improve practices of performing rodent videoâ\u80\u93electroencephalography (vEEG) studies in experimental controls, generate systematic reviews of preclinical research data, and develop preclinical common data elements (CDEs) for epilepsy research in animals

Substrate Binding Regulates Redox Signaling in Human DNA Primase

Generation of daughter strands during DNA replication requires the action of DNA primase to synthesize an initial short RNA primer on the single-stranded DNA template. Primase is a heterodimeric enzyme containing two domains whose activity must be coordinated during primer synthesis: an RNA polymerase domain in the small subunit (p48) and a [4Fe4S] cluster-containing C-terminal domain of the large subunit (p58C). Here we examine the redox switching properties of the [4Fe4S] cluster in the full p48/p58 heterodimer using DNA electrochemistry. Unlike with isolated p58C, robust redox signaling in the primase heterodimer requires binding of both DNA and NTPs; NTP binding shifts the p48/p58 cluster redox potential into the physiological range, generating a signal near 160 mV vs NHE. Preloading of primase with NTPs enhances catalytic activity on primed DNA, suggesting that primase configurations promoting activity are more highly populated in the NTP-bound protein. We propose that p48/p58 binding of anionic DNA and NTPs affects the redox properties of the [4Fe4S] cluster; this electrostatic change is likely influenced by the alignment of primase subunits during activity because the configuration affects the [4Fe4S] cluster environment and coupling to DNA bases for redox signaling. Thus, both binding of polyanionic substrates and configurational dynamics appear to influence [4Fe4S] redox signaling properties. These results suggest that these factors should be considered generally in characterizing signaling networks of large, multisubunit DNA-processing [4Fe4S] enzymes

Diffuse reflectance spectroscopy-enhanced drill for bone boundary detection

Intramedullary nailing is a routine orthopedic procedure used for treating fractures of femoral or tibial shafts. A critical part of this procedure involves the drilling of pilot holes in both ends of the bone for the placement of the screws that will secure the IM rod to sections of the fractured bone. This step introduces a risk of soft tissue damage because the drill bit, if not stopped in time, can transverse the bone-tissue boundary into the overlying muscle, causing unnecessary injury and prolonging healing time due to periosteum damage. In this respect, detecting the bone-tissue boundary before break-through can reduce the risks and complications associated with intramedullary nailing. Hence, in the present study, a two-wavelength diffuse reflectance spectroscopy technique was integrated into a surgical drill to optically detect bone-tissue boundary and automatically trigger the drill to stop. Furthermore, Monte-Carlo simulations were used to estimate the maximum distance from within the bone at which the bone-tissue boundary could be detected using DRS. The simulation results estimated that the detection distance, termed the “look-ahead-distance” was ∼1.5 mm for 1.3 mm source-detector fiber separation. Experimental measurements with 1.3 mm source-detector fiber separation showed that the look-ahead-distance was in the order of 250 µm in experiments with set drill rate and in the range of 1 mm in experiments where the holes were drilled by hand. Despite this difference, the automated DRS enhanced drill successfully detected the approaching bone tissue boundary when tested on samples of bovine femur and muscle tissue

Redox Signaling through DNA

Biological electron transfer reactions between metal cofactors are critical to many essential processes within the cell. Duplex DNA is, moreover, capable of mediating the transport of charge through its π-stacked nitrogenous bases. Increasingly, [4Fe4S] clusters, generally redox-active cofactors, have been found to be associated with enzymes involved in DNA processing. DNA-binding enzymes containing [4Fe4S] clusters can thus utilize DNA charge transport (DNA CT) for redox signaling to coordinate reactions over long molecular distances. In particular, DNA CT signaling may represent the first step in the search for DNA lesions by proteins containing [4Fe4S] clusters that are involved in DNA repair. Here we describe research carried out to examine the chemical characteristics and biological consequences of DNA CT. We are finding that DNA CT among metalloproteins represents powerful chemistry for redox signaling at long range within the cell

Scratching the Surface: A Marine Sediment Provenance Record From the Continental Slope of Central Wilkes Land, East Antarctica

The geology of Wilkes Land, East Antarctica, is masked by kilometers of ice and remains largely unexplored. Defining the sediment provenance adjacent to this hidden region is important for distinguishing the proximal subglacial basement terranes and refining the dynamic regional glaciological history. This study presents a detrital sediment provenance record spanning c. 23.5 ka from the continental slope of central Wilkes Land. Sediment provenance was characterized using U-Pb geochronology and trace element geochemistry from detrital zircon, titanite and apatite, and Pb isotopic signatures from detrital feldspar. These data were compared with new feldspar Pb-isotopic signatures and existing U-Pb zircon data sets from rare nearby coastal outcrop. A principally igneous source was revealed with dominant age populations between c. 1,360-1,100 Ma and c. 1,620-1,490 Ma, characteristic of rocks of the proximal Wilkes and Banzare provinces, respectively. Minor detritus was additionally sourced from the proximal Nuyina Province (c. 1,450-1,390 Ma). Temporal variation in the climate and ice sheet configuration are likely responsible for subtle downcore changes observed in detrital sediment provenance. High sedimentation rates during the glacial period suggest reworking of continental shelf sediments and downslope transport in debris flows during ice sheet advance. Glacial meltwater fluxes fed largely by the Totten Glacier were responsible for supplying detritus during deglaciation. During interglacials, detritus was derived from a broad coastal region and delivered to the slope via multiple glacial outlets. These results present the first substantial offshore evidence to support recent interpretations that the subglacial crust of central Wilkes Land has a dominantly Mesoproterozoic history.Sian Tooze was supported by a PhD scholarship from the Australian Research Council's Special Research Initiative for the Antarctic Gateway Partnership (Project ID SR140300001). Taryn L. Noble acknowledges support from the Science and Industry Endow-ment Fund, and Australian Antarctic Science Program Grant 441