scholarly journals Detection of surface-bound ligands by freeze-fracture autoradiography.

1985 ◽  
Vol 101 (3) ◽  
pp. 887-890 ◽  
Author(s):  
J L Carpentier ◽  
D Brown ◽  
B Iacopetta ◽  
L Orci
Keyword(s):  
Cholera Toxin ◽  
Accurate Determination ◽  
Freeze Fracture ◽  
Standard Technique ◽  
Coated Pits ◽  
Experimental Conditions ◽  
Surface Distribution ◽  
Surface Localization ◽  
Autoradiographic Technique

This article describes a new freeze-fracture autoradiographic technique for the detection of radioactive ligands associated with the surface of cells in monolayer or suspension culture. Since freeze-fracture replicas are produced in the conventional way, all membrane features normally seen in freeze-fracture are retained, and autoradiographic grains produced by the labeled ligands are seen superimposed on unaltered exoplasmic membrane fracture faces. To assess the feasibility and resolution of this technique, we compared the surface distribution of alpha 2-macroglobulin and cholera toxin, labeled either with 125I or with colloidal gold, on 3T3-L1 fibroblasts. Both by autoradiography and cytochemical gold labeling, alpha 2-macroglobulin was associated specifically with coated pits, whereas cholera toxin was preferentially found over smaller, apparently non-coated membrane invaginations. Together with data on the surface localization of 125I-transferrin on HL-60 myelomonocytic cells, these results demonstrate the application of this technique for the accurate determination of ligand distribution over large areas of plasma membrane. The simplicity and reproducibility of the method should now allow freeze-fracture autoradiography to become a standard technique for investigating the distribution of both endogenous and exogenous cell surface-associated molecules, as well as the redistribution of such molecules under different experimental conditions.

Factors affecting constancy of acetate concentration and correct determination of methanogenic activity in pH-stat experiments

2003 ◽  
Vol 48 (6) ◽  
pp. 111-118
Author(s):  
M. Mösche ◽  
U. Meyer
Keyword(s):  
Accurate Determination ◽  
Original Method ◽  
Experimental Conditions ◽  
Methanogenic Activity ◽  
Factors Affecting ◽  
Acetate Concentration ◽  
Correct Determination ◽  
Ph Stat ◽  
The Stability

The determination of methanogenic activity with a pH-stat titration bioassay is evaluated utilising a mathematical model of this system. For given kinetic parameters and experimental conditions the model calculates the development of titrant flow and acetate concentration during experiments. Simulations of experiments under various conditions are compared. They show that the original method inherently causes a strong drift of acetate concentration during the experiments and a misestimation of methanogenic activity. As a solution to these disadvantages the addition of sodium hydroxide to the titrant and a careful control of pH during flushing the reactor with gas prior to the experiment are recommended. In this way a better constancy of acetate concentration and a more accurate determination of methanogenic activity should be achievable. The accuracy of this method is limited by the stability of pH-electrode calibration parameters.

The Influence of the Cell Geometry on the Signal Shape in Electron Drift Velocity Measurements

1986 ◽  
Vol 41 (7) ◽  
pp. 912-920 ◽  
Author(s):  
A. F. Borghesani ◽  
L. Bruschi ◽  
M. Santini ◽  
G. Torzo
Keyword(s):  
Edge Effects ◽  
Accurate Determination ◽  
Finite Size ◽  
High Mobility ◽  
Drift Chamber ◽  
Cell Geometry ◽  
Experimental Conditions ◽  
Drift Cell ◽  
Cell Dimensions

An accurate determination of the “time-of-flight” in swarm experiments with a parallel plate drift chamber requires that the time evolution of the charge induced on the collector is linear. This is obtained in very large chambers where the edge effects can be neglected. However, the experimental conditions of high-mobility carriers and highly pressurized gases impose some constraints on the acceptable drift cell dimensions. We have numerically calculated the effects of the finite size of the collector by exploiting the methods of the images. The numerical results have been experimentally checked using a suitable drift cell of variable geometry. As a result, a quantitative limit on the ratio between the collector radius and the drift distance has been established in order to design drift cells for which the edge effects can be neglected. P.A.C.S. numbers: 2940. 4110 D, 3480 B

Relation between outer and luminal diameter in cannulated arteries

1999 ◽  
Vol 277 (5) ◽  
pp. H1745-H1753 ◽  
Author(s):  
Gilles Faury ◽  
Gail M. Maher ◽  
Dean Y. Li ◽  
Mark T. Keating ◽  
Robert P. Mecham ◽  
...  
Keyword(s):  
Accurate Determination ◽  
Experimental Conditions ◽  
Cross Sectional ◽  
Luminal Diameter ◽  
Fluorescent Marker ◽  
Wide Range ◽  
Before And After ◽  
Single Determination ◽  
Knockout Animals

Resistance in blood vessels is directly related to the inner (luminal) diameter (ID). However, ID can be difficult to measure during physiological experiments because of poor transillumination of thick-walled or tightly constricted vessels. We investigated whether the wall cross-sectional area (WCSA) in cannulated arteries is nearly constant, allowing IDs to be calculated from outer diameters (OD) using a single determination of WCSA. With the use of image analysis, OD and ID were directly measured using either transillumination or a fluorescent marker in the lumen. IDs from a variety of vessel types were calculated from WCSA at several reference pressures. Calculated IDs at all of the reference WCSA were within 5% (mean <1%) of the corresponding measured IDs in all vessel types studied, including vessels from heterozygote elastin knockout animals. This was true over a wide range of transmural pressures, during treatment with agonists, and before and after treatment with KCN. In conclusion, WCSA remains virtually constant in cannulated vessels, allowing accurate determination of ID from OD measurement under a variety of experimental conditions.

scholarly journals Binding of NAD+ by cholera toxin

1987 ◽  
Vol 244 (1) ◽  
pp. 225-230 ◽  
Author(s):  
T S Galloway ◽  
S van Heyningen
Keyword(s):  
Binding Site ◽  
Dissociation Constant ◽  
Cholera Toxin ◽  
Equilibrium Dialysis ◽  
Accurate Determination ◽  
Polyacrylamide Gel ◽  
Nad Glycohydrolase ◽  
Competitive Inhibitors ◽  
Dissociation Constant Kd

1. The Km for NAD+ of cholera toxin working as an NAD+ glycohydrolase is 4 mM, and this is increased to about 50 mM in the presence of low-Mr ADP-ribose acceptors. Only molecules having both the adenine and nicotinamide moieties of NAD+ with minor alterations in the nicotinamide ring can be competitive inhibitors of this reaction. 2. This high Km for NAD+ is also reflected in the dissociation constant, Kd, which was determined by a variety of methods. 3. Results from equilibrium dialysis were subject to high error, but showed one binding site and a Kd of about 3 mM. 4. The A1 peptide of the toxin is digested by trypsin, and this digestion is completely prevented by concentrations of NAD+ above 50 mM. Measurement (by densitometric scanning of polyacrylamide-gel electrophoretograms) of the rate of tryptic digestion at different concentrations of NAD+ allowed a more accurate determination of Kd = 4.0 +/- 0.4 mM. Some analogues of NAD+ that are competitive inhibitors of the glycohydrolase reaction also prevented digestion.

Trace Organic Silicon Analysis Using Atomic Absorption Spectroscopy

1968 ◽  
Vol 22 (5) ◽  
pp. 520-526 ◽  
Author(s):  
Concetta M. Paralusz
Keyword(s):  
Atomic Absorption ◽  
Accurate Determination ◽  
Organic Matrix ◽  
Absorption Spectrometry ◽  
Polydimethyl Siloxane ◽  
Experimental Conditions ◽  
Silicon Compounds ◽  
Organic Silicon ◽  
Organic Matrices

Trace silicon analysis in organic matrix materials is complicated by time-consuming concentration schemes and/or a lack of suitable organic standards. This paper describes an improved technique for quantitative determination of trace amounts of silicon in organic matrices. The method utilizes recent improvements in atomic absorption spectrometry and can be used to determine silicon in levels as low as 0.1 μg/ml in organic solution. Matrix interferences have been overcome by controlling solids of polymers and preparing standards using Si-free matrix materials. Suitable instrumentation and experimental conditions are described. Model organic silicon compounds and polydimethyl siloxane oils have been examined for use as standards. Experimental data, including a comparison of this method with wet-chemical and x-ray results, is presented. This method can be used for accurate determination of trace silicone contamination in the 1–10 ppm range in (1) paper products, (2) resinous and elastomeric polymers, and (3) oils.

Multiple Internal Standard Technique for the Gas-Liquid Chromatographic Determination of Indole in Shrimp

1978 ◽  
Vol 61 (1) ◽  
pp. 136-138
Author(s):  
Walter F Staruszkiewicz ◽  
John F Bond
Keyword(s):  
Silica Gel ◽  
Room Temperature ◽  
Internal Standard ◽  
Standard Technique ◽  
Chromatographic Determination ◽  
Experimental Conditions ◽  
Liquid Chromatographic Determination ◽  
Standard Solution ◽  
Liquid Chromatographic

Abstract A multiple internal standard technique has been developed for the official first action gas-liquid chromatographic (GLC) method for determining indole in shrimp. The modification was developed because interfering GLC peaks are occasionally observed when 2-methylindole is used as the internal standard. An internal standard solution containing 1-methylindole, 2- methylindole, and diphenylamine was added to extracts of shrimp before silica gel cleanup and separation by GLC. All of the compounds were quantitatively recovered and were separated on the GLC column under identical experimental conditions. Extracts of acceptable shrimp to which indole was added at levels of 3–10 μg/ 100 g and extracts of decomposed shrimp were stored at room temperature for 2 weeks. Average and maximum changes (μg indole/100 g) during storage were, respectively, for each internal standard: average 0.6, 0.4, and 1.1; maximum 1.7, 0.9, and 2.9.

Determination of Concentration Dependent Diffusion Coefficients of Carbon in Expanded Austenite

2008 ◽  
Vol 273-276 ◽  
pp. 306-311 ◽  
Author(s):  
Thomas S. Hummelshøj ◽  
Thomas L. Christiansen ◽  
Marcel A.J. Somers
Keyword(s):  
Diffusion Coefficient ◽  
Weight Change ◽  
Diffusion Coefficients ◽  
Input Data ◽  
Accurate Determination ◽  
Experimental Conditions ◽  
Thin Foils ◽  
Expanded Austenite ◽  
Aisi 316

In the present paper various experimental procedures to experimentally determine the concentration dependent diffusion coefficient of carbon in expanded austenite are evaluated. To this end thermogravimetric carburization was simulated for various experimental conditions and the evaluated composition dependent diffusivity of carbon derived from the simulated experiments was compared with the input data. The most promising procedure for an accurate determination is shown to be stepwise gaseous carburizing of thin foils in a gaseous atmosphere; the finer the stepsize, the more accurate the approximation of the diffusivity. Thermogravimetry was applied to continuously monitor the weight change of thin foils of AISI 316 during carburizing in CO-H2 gas mixtures for one of the simulated experimental procedures.

Analysis of periodate oxidation of carbohydrates by polarography

1970 ◽  
Vol 48 (16) ◽  
pp. 2474-2483 ◽  
Author(s):  
R. D. Corlett ◽  
W. G. Breck ◽  
G. W. Hay
Keyword(s):  
Lower Limit ◽  
Limit Of Detection ◽  
Accurate Determination ◽  
Periodate Oxidation ◽  
Experimental Conditions ◽  
Quantitative Studies

Experimental conditions using a variety of pH buffers have been established to permit the rapid, accurate determination of periodate and formaldehyde by polarography. The application of these analyses to quantitative studies in the oxidation of carbohydrates was illustrated and the lower limit of detection presently available found to be equivalent to 0.04 μmole carbohydrate.

Changes Occur in Plasma Membrane Turnover when K562 Leukemia Cells are Induced to Differentiate

1982 ◽  
Vol 40 ◽  
pp. 286-287
Author(s):  
L. M. Marshall
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Intracellular Transport ◽  
Parent Cell ◽  
Membrane Turnover ◽  
Coated Pits ◽  
Surface Distribution ◽  
Specific Proteins ◽  
Erythroleukemic Cell ◽  
Specific Membrane

A human erythroleukemic cell line, metabolically blocked in a late stage of erythropoiesis, becomes capable of differentiation along the normal pathway when grown in the presence of hemin. This process is characterized by hemoglobin synthesis followed by rearrangement of the plasma membrane proteins and culminates in asymmetrical cytokinesis in the absence of nuclear division. A reticulocyte-like cell buds from the nucleus-containing parent cell after erythrocyte specific membrane proteins have been sequestered into its membrane. In this process the parent cell faces two obstacles. First, to organize its erythrocyte specific proteins at one pole of the cell for inclusion in the reticulocyte; second, to reduce or abolish membrane protein turnover since hemoglobin is virtually the only protein being synthesized at this stage. A means of achieving redistribution and cessation of turnover could involve movement of membrane proteins by a directional lipid flow. Generation of a lipid flow towards one pole and accumulation of erythrocyte-specific membrane proteins could be achieved by clathrin coated pits which are implicated in membrane endocytosis, intracellular transport and turnover. In non-differentiating cells, membrane proteins are turned over and are random in surface distribution. If, however, the erythrocyte specific proteins in differentiating cells were excluded from endocytosing coated pits, not only would their turnover cease, but they would also tend to drift towards and collect at the site of endocytosis. This hypothesis requires that different protein species are endocytosed by the coated vesicles in non-differentiating than by differentiating cells.

A quantitative approach to inner shell losses

1978 ◽  
Vol 36 (1) ◽  
pp. 526-527 ◽  
Author(s):  
R.D. Leapman ◽  
P. Rez ◽  
D.F. Mayers
Keyword(s):  
Energy Transfer ◽  
Cross Section ◽  
Cross Sections ◽  
Accurate Determination ◽  
Background Intensity ◽  
Core Excitation ◽  
Quantitative Basis ◽  
Cross Section Differential ◽  
Bound Electrons

Microanalysis by EELS has been developing rapidly and though the general form of the spectrum is now understood there is a need to put the technique on a more quantitative basis (1,2). Certain aspects important for microanalysis include: (i) accurate determination of the partial cross sections, σx(α,ΔE) for core excitation when scattering lies inside collection angle a and energy range ΔE above the edge, (ii) behavior of the background intensity due to excitation of less strongly bound electrons, necessary for extrapolation beneath the signal of interest, (iii) departures from the simple hydrogenic K-edge seen in L and M losses, effecting σx and complicating microanalysis. Such problems might be approached empirically but here we describe how computation can elucidate the spectrum shape.The inelastic cross section differential with respect to energy transfer E and momentum transfer q for electrons of energy E0 and velocity v can be written as

Sign in / Sign up

Export Citation Format

Share Document