Plasma Membrane Processes in Smooth Muscle: Characterization of Ca\u3csup\u3e2+\u3c/sup\u3e Transport and Muscarinic Cholinergic Receptors: A Thesis

The thesis research was designed to study the characteristics of two important physiological processes in smooth muscle: Ca2+ transport mediated by the plasmalemmal Ca2+-ATPase and muscarinic receptor-G protein interactions. In resting smooth muscle, several Ca2+ extrusion or sequestration processes offset the passive inward leak of Ca2+. Although biochemical evidence suggests that the plasmalemmal Ca2+ pump plays a key role in this process, the precise role of this enzyme could not be proven until a reliable estimate of the inward Ca2+ leak was measured. Recent studies using dispersed smooth muscle cells from the toad stomach provided an estimate of the basal transmembrane Ca2+ flux rate; thus, we examined the transport capacity of the plasmalemmal Ca2+pump in this tissue. Gastric smooth muscle tissue was disrupted by homogenization and nitrogen cavitation. Membranes enriched 20 fold for plasma membrane markers were obtained using differential centrifugation and purification by flotation on discontinuous sucrose gradients. The membrane vesicles exhibited an ATP-dependent 45Ca uptake that was insensitive to azide or oxalate but sensitive to stimulation by calmodulin or inhibition by orthovanadate and the calmodulin antagonists trifluoperazine (TFP) or calmidazolium (CMZ). 45Ca accumulated in the presence of ATP was rapidly released by Ca2+ ionophore but not by agents that stimulate Ca2+ release from the sarcoplasmic rettculum (caffeine, inositol trisphosphate, GTP). However, both CMZ and TFP evoked a Ca2+ release that was comparable to that observed in the presence of Ca2+ ionophore, suggesting that these compounds have profound effects on membrane Ca2+permeability. 45Ca transport exhibited a high affinity for Ca2+ (KD 0.2 μM) and a high transport capacity, producing a \u3e 12,000-fold gradient for Ca2+and a transmembrane flux rate at least 3-fold greater than that observed in resting smooth muscle cells. As a first step toward understanding the biochemical basis for the diversity of muscarinic cholinergic actions on smooth muscle, we examined the distribution of muscarinic receptor subtypes and coupling to guantne nucleotide-binding (G) proteins in airway and gastric smooth muscle. Receptor subtypes were classified in membranes prepared from bovine trachea and toad stomach based on the relative abilities of the selective antagonists pirenzepine (M1), AF-DX 116 (M2) and 4-DAMP (M3) to displace the binding of nonselective antagonist [3H]QNB (quinuclidinyl benzilate). Based on the binding profiles for these antagonists, it was concluded that both smooth muscle types contain a mixture of M2 and M3 subtypes. In trachea the majority of receptors (86%) were M2, whereas in stomach the majority of receptors (88%) were M3. The displacement of [3H]QNB binding by the agonist oxotremorine indicated a mixed population of high affinity (KD = 4 nM) and low affinity (KD = 2-4 μM) binding sites. The addition of GTPγS abolished all high affinity agonist binding, suggesting that coupling of the receptors to G proteins may confer high affinity. Reaction of membranes with pertussis toxin in the presence of [32P]NAD caused the [32P]-labelling of a ~ 41 kD protein in both gastric and tracheal smooth musc1e. Pretreatment of the membranes with pertussis toxin and NAD completely abolished high affinity agonist binding in gastric smooth muscle, but produced little if any decrease in high affinity agonist binding in trachea. We conclude that, although muscarinic receptor activation leads to the elevation of intracellular Ca2+ and to contraction of both airway and gastric smooth muscle, the dissimilar distributions of receptor subtypes and distinct patterns of coupling to G proteins may indicate that each smooth muscle type uses different receptor-G protein interactions to regulate intracellular signalling pathways

Min-Max Theorems for Packing and Covering Odd (u,v)(u,v)-trails

We investigate the problem of packing and covering odd $(u,v)$-trails in a graph. A $(u,v)$-trail is a $(u,v)$-walk that is allowed to have repeated vertices but no repeated edges. We call a trail odd if the number of edges in the trail is odd. Let $\nu(u,v)$ denote the maximum number of edge-disjoint odd $(u,v)$-trails, and $\tau(u,v)$ denote the minimum size of an edge-set that intersects every odd $(u,v)$-trail. We prove that $\tau(u,v)\leq 2\nu(u,v)+1$. Our result is tight---there are examples showing that $\tau(u,v)=2\nu(u,v)+1$---and substantially improves upon the bound of $8$ obtained in [Churchley et al 2016] for $\tau(u,v)/\nu(u,v)$. Our proof also yields a polynomial-time algorithm for finding a cover and a collection of trails satisfying the above bounds. Our proof is simple and has two main ingredients. We show that (loosely speaking) the problem can be reduced to the problem of packing and covering odd $(uv,uv)$-trails losing a factor of 2 (either in the number of trails found, or the size of the cover). Complementing this, we show that the odd-$(uv,uv)$-trail packing and covering problems can be tackled by exploiting a powerful min-max result of [Chudnovsky et al 2006] for packing vertex-disjoint nonzero $A$-paths in group-labeled graphs

Will the recently approved LARES mission be able to measure the Lense-Thirring effect at 1%?

After the approval by the Italian Space Agency of the LARES satellite, which should be launched at the end of 2009 with a VEGA rocket and whose claimed goal is a about 1% measurement of the general relativistic gravitomagnetic Lense-Thirring effect in the gravitational field of the spinning Earth, it is of the utmost importance to reliably assess the total realistic accuracy that can be reached by such a mission. The observable is a linear combination of the nodes of the existing LAGEOS and LAGEOS II satellites and of LARES able to cancel out the impact of the first two even zonal harmonic coefficients of the multipolar expansion of the classical part of the terrestrial gravitational potential representing a major source of systematic error. While LAGEOS and LAGEOS II fly at altitudes of about 6000 km, LARES will be placed at an altitude of 1450 km. Thus, it will be sensitive to much more even zonals than LAGEOS and LAGEOS II. Their corrupting impact \delta\mu has been evaluated by using the standard Kaula's approach up to degree L=70 along with the sigmas of the covariance matrices of eight different global gravity solutions (EIGEN-GRACE02S, EIGEN-CG03C, GGM02S, GGM03S, JEM01-RL03B, ITG-Grace02s, ITG-Grace03, EGM2008) obtained by five institutions (GFZ, CSR, JPL, IGG, NGA) with different techniques from long data sets of the dedicated GRACE mission. It turns out \delta\mu about 100-1000% of the Lense-Thirring effect. An improvement of 2-3 orders of magnitude in the determination of the high degree even zonals would be required to constrain the bias to about 1-10%.Comment: Latex, 15 pages, 1 table, no figures. Final version matching the published one in General Relativity and Gravitation (GRG

Conservative evaluation of the uncertainty in the LAGEOS-LAGEOS II Lense-Thirring test

We deal with the test of the general relativistic gravitomagnetic Lense-Thirring effect currently ongoing in the Earth's gravitational field with the combined nodes \Omega of the laser-ranged geodetic satellites LAGEOS and LAGEOS II. One of the most important source of systematic uncertainty on the orbits of the LAGEOS satellites, with respect to the Lense-Thirring signature, is the bias due to the even zonal harmonic coefficients J_L of the multipolar expansion of the Earth's geopotential which account for the departures from sphericity of the terrestrial gravitational potential induced by the centrifugal effects of its diurnal rotation. The issue addressed here is: are the so far published evaluations of such a systematic error reliable and realistic? The answer is negative. Indeed, if the difference \Delta J_L among the even zonals estimated in different global solutions (EIGEN-GRACE02S, EIGEN-CG03C, GGM02S, GGM03S, ITG-Grace02, ITG-Grace03s, JEM01-RL03B, EGM2008, AIUB-GRACE01S) is assumed for the uncertainties \delta J_L instead of using their more or less calibrated covariance sigmas \sigma_{J_L}, it turns out that the systematic error \delta\mu in the Lense-Thirring measurement is about 3 to 4 times larger than in the evaluations so far published based on the use of the sigmas of one model at a time separately, amounting up to 37% for the pair EIGEN-GRACE02S/ITG-Grace03s. The comparison among the other recent GRACE-based models yields bias as large as about 25-30%. The major discrepancies still occur for J_4, J_6 and J_8, which are just the zonals the combined LAGEOS/LAGOES II nodes are most sensitive to.Comment: LaTex, 12 pages, 12 tables, no figures, 64 references. To appear in Central European Journal of Physics (CEJP

Perspectives in measuring the PPN parameters beta and gamma in the Earth's gravitational fields with the CHAMP/GRACE models

The current bounds on the PPN parameters gamma and beta are of the order of 10^-4-10^-5. Various missions aimed at improving such limits by several orders of magnitude have more or less recently been proposed like LATOR, ASTROD, BepiColombo and GAIA. They involve the use of various spacecraft, to be launched along interplanetary trajectories, for measuring the effects of the solar gravity on the propagation of electromagnetic waves. In this paper we investigate what is needed to measure the combination nu=(2+2gamma-beta)/3 of the post-Newtonian gravitoelectric Einstein perigee precession of a test particle to an accuracy of about 10^-5 with a pair of drag-free spacecraft in the Earth's gravitational field. It turns out that the latest gravity models from the dedicated CHAMP and GRACE missions would allow to reduce the systematic error of gravitational origin just to this demanding level of accuracy. In regard to the non-gravitational errors, the spectral noise density of the drag-free sensors required to reach such level of accuracy would amounts to 10^-8-10^-9 cm s^-2 Hz^-1/2 over very low frequencies. Although not yet obtainable with the present technologies, such level of compensation is much less demanding than those required for, e.g., LISA. As a by-product, an independent measurement of the post-Newtonian gravitomagnetic Lense-Thirring effect with a 0.9% accuracy would be possible as well. The forthcoming Earth gravity models from CHAMP and GRACE will further reduce the systematic gravitational errors in both of such tests.Comment: LaTex2e, 14 pages, 3 tables, no figures, 75 references. To appear in Int. J. Mod. Phys.

The contribution of TEM to solving issues in the oogenesis of lower metazoans: a comparison between Acoela and rhabditophoran Platyhelminthes

The chapter reports data on the oogenesis and eggshell formation in some Acoela obtained by our group over the years by means of transmission electron microscopy (TEM) and cytochemical techniques. In this review we show how TEM and appropriate cytochemical methods have been helpful in clarifying issues related to female germ cell differentiation and more in general to the cellular aspects of the reproductive biology in these lower metazoans. The ultrastructural findings have been compared with those from other Acoelomorpha (Acoela + Nemertodermatida) and rhabditophoran Platyhelminthe

Flax for seed or fibre use? Flax capsules from ancient Egyptian sites (3rd millennium BC to second century AD) compared with modern flax genebank accessions

In order to determine whether ancient Egyptians had already selected and cultivated very specialized flax types according to their purpose for textile or oil production, respectively, we compared archaeobotanical flax finds with nearly 3000 diverse genebank accessions kept at the Plant Gene Resources of Canada (PGRC). The main result was that fibre flax was most probably already cultivated during the 3rd millennium BC, whereas in later periods an intermediate flax and/or oil type was in use. The collaboration between archaeobotanists and genebank curators shows how our knowledge about ancient usage of plants can be improved by interpreting observations made on ancient findings using recent characterization data obtained from diverse genebank material

File Specification for M2AMIP Products

The Modern-Era Retrospective analysis for Research and Applications, Version 2 (MERRA-2) is an atmospheric reanalysis computed with the Goddard Earth Observing (EOS) System, Version 5.12.4 (GEOS) data assimilation system (Gelaro et al., 2017). To supplement the reanalysis, the GEOS General Circulation Model (GCM) used in MERRA-2 has been used to generate a 10-member ensemble of simulations, configured following the convention of the Atmospheric Model Intercomparison Project (AMIP; Gates et al., 1992). Each ensemble member was initialized using meteorological fields from a different date in November 1979. The AMIP simulations used the sea-surface temperature (SST) and sea-ice boundary conditions that were used in MERRA-2 (Bosilovich et al., 2016). This 10-member ensemble of AMIP simulations, denoted M2AMIP, is available for download in a group of self-describing files, which are documented in this office note. All data collections are provided on the same horizontal grid as MERRA-2. This grid has 576 points in the longitudinal direction and 361 points in the latitudinal direction, corresponding to a resolution of 0.625 degrees by 0.5 degrees. Although data collections are available at this grid, all fields are computed on a cubed-sphere grid with an approximate resolution of 50 km by 50 km and are then spatially interpolated to the latitude-longitude grid. There are no changes in the vertical grids used: variables are provided on either the native vertical grid of 72 model layers, or interpolated to 42 standard pressure levels. Unlike MERRA, no data collections are available at the vertical layer edges. More details on the grid are provided in Section 4. MERRA-2 introduced observation-based precipitation forcing for the land surface parameterization and the corresponding variable PRECTOTCORR in the MERRA-2 FLX (surface turbulent fluxes and related quantities) and LFO (land-surface forcing) collections (see Section 6; Reichle et al., 2017). While this variable is still available for M2AMIP, there was no observation-based forcing, making the value identical to the model derived precipitation, PRECTOT. Similarly, without data assimilation, the values for the analysis increments, D*DTANA, in the tendency and vertically integrated file collections are zero. The M2AMIP data are available for download online through the NASA Center for Climate Simulation (NCCS) DataPortal (https://portal.nccs.nasa.gov/datashare/gmao_m2amip/). Data are arranged in subdirectories based on ensemble member, followed by year and month. Control files that are compatible with the Grid Analysis and Display System (GrADS) are available in the ctl_daily and ctl_monthly directories for the hourly, three hourly, and monthly mean data. Control files for the monthly mean diurnal cycle can be found in the ctl_diurnal subdirectory within the directory for each individual ensemble member