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At least 397 records · Page 22Linked to original sources
PubMed1990

An in vitro model of cell migration: evaluation of vascular endothelial cell migration.

In vivo vascular endothelial cell (VEC) migration is thought to play a central role in the development of new capillaries as well as the resurfacing of large vessels. Recently, we have developed an in vitro VEC migration assay system based on the ability of VEC to migrate off of tissue culture microcarrier beads. For these studies, bovine pulmonary artery VEC were grown to confluence on Cytodex 3 microcarrier beads (MCB). Next, the confluent VEC covered microcarrier beads were pipetted into 4-cm2 wells of a tissue culture plate and incubated at 37 degrees C/5% CO2. At various time intervals, the movement of the VEC off of the MCB onto the tissue culture surface was evaluated microscopically. Using this assay, we have studied the effect of endothelial cell growth supplement and various matrices (i.e., fibronectin, gelatin, and Matrigel) on VEC migration. These studies demonstrated that: (i) gelatin had no effect on normal or mitomycin C-pretreated VEC migration; (ii) fibronectin had no effect on normal VEC migration, but stimulated the relative migration of mitomycin pretreated VEC; and (iii) Matrigel significantly suppressed both normal and mitomycin C-pretreated VEC migration. Endothelial cell growth supplement (ECGS) stimulated both normal and mitomycin C-pretreated VEC migration on fibronectin at concentrations of 10 micrograms/ml ECGS. Pretreatment with ECGS had no effect of normal or mitomycin C VEC migration on gelatin. Finally, ECGS stimulated a statistically significant increase in the migration of normal and mitomycin C-pretreated VEC migration on Matrigel.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Microscale, filtration-type binding assay for studying myosin-erythrocyte protein 4.1 interactions.

In vitro binding of skeletal muscle myosin and the erythrocyte cytoskeleton linker protein, band 4.1, was evaluated in a novel small-volume, filtration-based binding assay. The assay equipment consisted of a plastic grid containing several buffer-filled wells into which were placed small nylon screens. Myosin was covalently tethered to an agarose (Sepharose) support and aliquots of this resin were pipetted onto the surface of the submerged nylon screen. Following addition of radiolabeled protein 4.1, and an appropriate incubation period, the myosin-Sepharose beads and bound protein 4.1 were separated by wicking the buffer from beneath the nylon screen with a piece of filter paper. Nylon screens, with adherent resin beads, and the filter paper wicks were then counted to give the amounts of bound and free protein 4.1, respectively. This system proved to be a rapid, simple, and quantitative method for evaluating the behavior of a myosin binding protein under conditions in which free myosin would be prone to assemble into filaments. Moreover, since the assay separates bound and free components within a few seconds, it is well suited for the analysis of low-affinity interactions.

Cytoskeletal Proteins↗
PubMed1991

An assay procedure for solubilized thyroid hormone receptor: use of Lipidex.

A method using Lipidex-1000 is reported for the assay of thyroid hormone receptor activity. The receptor was extracted from rat liver nuclei, incubated with [125I]T3, and applied to 1-ml disposable pipet tip columns containing a small volume of Lipidex. The resin absorbed the free hormone, and the receptor-bound hormone was recovered in the eluate. The method allowed accurate assay of the receptor activity, satisfying a linear relationship between the activity and the receptor protein concentration. The usefulness of this method was demonstrated by determining the pH optimum and the Kd and Bmax of T3 binding by the receptor. The Lipidex column can be used for the preparation of an unoccupied receptor after dissociation of the endogenously bound hormone.

Absorption↗
PubMed1991

General proteinase assay by formation of SDS-protein gels of proteolyzed substrate proteins.

A new approach to the assay of proteinases is described. The method relies on water-insoluble protein substrates, such as gluten and fibrin, which form expanded gels in the presence of sodium dodecyl sulfate (SDS) reagent. Powdered substrate is dispersed in buffer and aliquots are pipetted into long, narrow, 400-microliters tubes made of clear polypropylene. After the addition of enzyme and a period of incubation, a SDS reagent is added, the tubes are centrifuged, and the height of the SDS-protein gel is measured. Reduction of gel height gives a direct measure of enzyme activity. Salt concentration, pH, and incubation times must be consistent for both test and control reactions in order to obtain reproducible results. Examples of proteinases measured by this method are trypsin, chymotrypsin, elastase, pronase, papain, pepsin, an insect (Nysius huttoni) salivary proteinase, and wheat proteinase. The assay could detect enzyme in crude extracts or in purified form. In 1-h incubations, 10 ng of pepsin and elastase or 20 ng of purified insect proteinase could be detected. The assay was simple, fast, economical, and sensitive.

Endopeptidases↗
PubMed1991

Assay of 3-hydroxy-3-methylglutaryl CoA reductase activity using anionic-exchange column chromatography.

A rapid, easy, and sensitive method is described in this paper for the assay of 3-hydroxy-3-methylglutaryl CoA (HMG CoA) reductase, a key enzyme in cholesterol biosynthesis. [14C]HMG CoA was used as the substrate and the product formed, i.e., [14C]mevalonate, was allowed to be converted to its lactone form (mevalonolactone) in the presence of HCl. The reaction mixture was applied to a column containing an anionic exchanger. The column was made up of QAE-Sephadex (A25, formate form) packed to a height of 4 cm in Pasteur pipets. Under these conditions, mevalonolactone was not retained by the column and was eluted with ammonium formate solution while HMG CoA, being negatively charged, was retained by the gel and eluted by HCl above 0.05 M. Determination of the amount of radioactivity in mevalonolactone was then used to quantitate the activity of HMG CoA reductase. This assay has been successfully used for determining the activity of this enzyme in a microsomal fraction prepared from the liver of the rat.

Anions↗
PubMed1993

Tension-stabilized pores in giant vesicles: determination of pore size and pore line tension.

We present the first observations of giant, long-existing, stabilized pores in vesicle membranes. Using a new experimental technique for studying the electro-permeabilization of lipid membranes, giant liposomes (from 25 to 56 microns in diameter) were subjected to single, square, electric pulses (duration 150 microseconds and electric field strength from 63 to 126 kV/m). The liposomes were held by a micropipet and small membrane tensions were created by controlling the pipet suction pressure. The liposomes were loaded with media having different refractive index from the outside solution, and, under these conditions, the formation of pores in the pressurized liposome could be visualized by the jet of inside solution that flowed out from the membrane pore. By adjusting the membrane tension, pores were kept open, and pore lifetimes could be varied from tenths of a second to several seconds. The pore size was determined from the volumetric flow in the pore region and the measured pressure differences across the bilayer. It was clear from the experiments that only one pore remained opened after the pulse. The estimated pore radii were on the order of one micrometer. The pores were in a quasi-stationary state and when they closed they did so spontaneously in a quick process (in milliseconds). The isotropic membrane tension was determined for the same measurements and from determinations of both pore size and dynamic membrane tension the pore line tension was found. The line tension of the pore region was determined for two lipid compositions, stearoyl-oleoylphosphatidylcholine and stearoyl-oleoylphosphatidylcholine with 50 mol% cholesterol, and the obtained values for single bilayers were (0.92 +/- 0.07) x 10(-11) N and (3.05 +/- 0.12) x 10(-11) N, respectively.

Cell Membrane Permeability↗
PubMed1977

A simple method for calculating Km and V from a single enzyme reaction progress curve.

A method of calculating Km and V from a single reaction progress curve is presented. The integrated Michaelis-Menten equation Vt = So + Km 1n(So/S), may be rearranged to the form 1/v = 1/V + Km/VS, where v = (Si - Sj)/deltat = deltaS/deltat and S = (Si - Sj)/ln(Si/Sj) or S is approximated by (Si + Sj)/2.Si and Sj represent substrate concentrations at two points along a reaction progress curve separated by the time interval, deltat. The error resulting from the approximation depends on the magnitude of deltaSi/Si; when deltaSi/Si less than 0.3, the error is insignificant; when deltaSi/Si greater than 0.3, the error becomes significant. Procedures are presented to correct this error. Simulated data and application to the direct spectrophotometric assay of AMP aminohydrolase and the lactate dehydrogenase coupled assay of pyruvate kinase are provided. The method is recommended when routine Km and V values are desired. Compared to the initial rate method, it is faster, requires less substrate, and eliminates pipetting errors.

Enzymes↗
PubMed1976

Completely isolated molluscan neurons. An ultrastructural study.

The neurons of the molluscs Lymnaea and Helix isolated by fermentative digestion followed by mechanical treatment do not differ ultrastructurally from intact ones. These cells have sufficient metabolic reserves and incorporate into RNA 8% of the total radioactive pool, even more than neurons in ganglia under equal conditions. Neuronal damage can occur, mainly during the pipetting, and this is usually expressed in vacuolization of the cytoplasm. It is important to note that alterations in cell ultrastructure develop earlier than changes in the membrane electrical properties. The surface of the isolated neurons is enlarged two-fold due to the infoldings of the cell membrane. So, the specific resistance of soma membrane of these neurons was calculated as 78 +/- 13 komega-sq. cm. On the surface of isolated neurons scraps of glial and neuronal processes not connected with their own cell bodies, and as a consequence not powerful, are sometimes found. Some endings of the neuronal processes on the surface of isolated neurons are ultrastructurally similar to the axo-somatic synapses.

Animals↗
PubMed1985

Monitoring of cell viability in suspensions of embryonic CNS tissue and its use as a criterion for intracerebral graft survival.

Neuronal cell suspensions, prepared by trypsination and mechanical disruption from embryonic CNS tissue, are currently used for intracerebral neuronal grafting to deep brain sites. In the present study the viability of suspended neurons from different brain regions has been monitored with a fluorescent vital stain, and studied as a function of time after dissociation and age of the donor rat embryos. Subsequently, the validity of the in vitro viability rates as a criterion for the in vivo survivability of each individual suspension was tested for suspensions prepared from the developing mesencephalon, rich in dopamine-containing neurons. The results indicate that mechanically dissociated embryonic CNS neurons remain viable for several hours in a simple glucose-saline solution at room temperature. The in vitro viability scores declined faster in suspensions prepared from mesencephalon than in those prepared from telencephalon (striatum and basal forebrain), and they declined faster in suspensions prepared from older embryos. The fetal cells were sensitive to the mechanical trauma caused by excessive pipetting, and tissue from older embryos seemed generally more vulnerable in the trypsination-dissociation procedure. The grafting experiments showed a good correlation between the in vitro cell viability counts and in vivo neuronal survival after grafting, indicating that the vital stain, at least under certain conditions, can be used as a simple and practical routine test to check and standardize cell suspensions to be used in intracerebral grafting experiments.

Animals↗
PubMed1987

Inactivating and non-inactivating outward current channels in cell-attached patches of Helix neurons.

Two species of inactivating outward current channels and a non-inactivating voltage-dependent current were seen in cell-attached patches of Helix neurons. Large, slowly inactivating channels had a slope conductance of 44 pS as measured with patch pipets containing the normal extracellular ion concentrations, including 4 mM potassium. Latency to maximal opening was 50-220 ms, and the inactivation time constant averaged 350 ms. Channel opening was decreased by preceding depolarization. The channels were selective for potassium and inhibited by 50 mM TEA. Small, quickly inactivating channels were 14 pS and had kinetics and voltage dependence similar to IA. Patch depolarization also activated a non-inactivating voltage-dependent outward current having channel conductance and/or kinetics such that individual channel openings and closings could not be distinguished. Such current was also seen in the presence of 50 mM TEA, but not in the presence of Co2+, characteristics which are similar to outward hydrogen ion currents, described by others in Helix neurons.

Calcium↗
PubMed1988

Studies on the ionic mechanism for the neuromodulatory actions of adenosine in the brain.

Although much evidence exists to support the modulation of neurotransmitter release by adenosine in both the central and peripheral nervous systems, the ionic mechanisms involved in this process are still not established. In our initial series of studies, the effects of adenosine analogs on depolarization-induced 45Ca influx into synaptosomes were examined, but no reduction in 45Ca uptake was observed in the presence of these agents, regardless of whether synaptosomes were depolarized by KCl or veratridine. The possibility that synaptosomal adenosine receptors are coupled directly to K+ channels and that an increase in K+ conductance is the primary ionic event initiated by adenosine was examined in resealed synaptic plasma membranes. We followed the movement of K+ through the use of the labeled lipophilic anion [35S]thiocyanate [( 35S]SCN-), which distributes across membranes according to the existing membrane potential. Both 2-chloroadenosine and adenosine-5'-cyclopropyl carboxamide produced dose-dependent increases in SCN- uptake, indicating enhanced K+ fluxes across the synaptic membranes. These effects of adenosine receptor agonists on membrane K+ conductance were blocked by receptor antagonists such as isobutylmethylxanthine. In addition, the placement of a K+-selective microelectrode in the caudate nuclei of intact, anesthetized rats revealed a substantial increase in the extracellular K+ concentration when adenosine was released onto the cells from a pipet in the assembly containing the ion-selective microelectrode. The results of these studies at both the membrane level and in the intact brain suggest that the initial event in the neuromodulatory actions of adenosine is an increase in the membrane conductance for K+ rather than inhibition of the voltage sensitive Ca2+ channels.

1-Methyl-3-isobutylxanthine↗
PubMed1990

Nystatin-perforated patch recordings disclose NMDA-induced outward currents in cultured neocortical neurons.

Excitatory amino acid-induced responses were studied in cultured rat neocortical neurons using two types of whole-cell patch-clamp recordings. Conventional recording methods, using either KCl or CsCl in the patch pipet, showed N-methyl-D-aspartate (NMDA) currents to be biphasic, consisting of peak and steady-state currents with similar reversal potentials. Recordings obtained with the nystatin-perforated patch method and KCl as the principal intracellular cation disclosed an NMDA-evoked outward current. Outward currents were not seen with either recording method in response to kainate or quisqualate, nor in response to NMDA when CsCl was the major intracellular cation. The NMDA-evoked outward current is attributed to activation of a potassium current by calcium entering through the NMDA channel. This outward current may serve to limit neuronal depolarization during excessive NMDA-mediated excitation.

Administration, Topical↗
PubMed1975

Fluoride determination in plasma by ion selective electrodes: a simplified method for the clinical laboratory.

A potentiometric method for the determination of fluoride (F-) in serum and plasma is proposed; it is based on a combination of the single-known-addition method and the electrode slope-by-dilution method. This procedure provides reliable results in extremely low measuring ranges down to 2.5 mug/l, where the electrode slope deviates markedly from Nernstian behaviour. In this method no electrode calibration is required and only one standard is necessary. 1 ml of plasma is sufficient for one analysis. Apart from a 5% enrichment of all samples with a concentrated total ionic strength adjustment buffer, no further preparation of the sample is required. The simplicity of the various pipetting and analytical steps, and also of the evaluation of the readings, may render this method highly suitable for the clinical laboratory. Investigations into the accuracy and precision of the method produced satisfactory results. The recovery in plasma amounted to 99.7%, even in the low measuring ranges. The discrimination capacity of the method amounts to 0.1 mug/l. With the apparatus and experimental procedures described, 18 plasma analyses per day can be performed even at low F- concentrations with which longer electrode stabilization periods are required. Storage of the plasma samples frozen at --20 degrees C for up to 8 weeks exerts no effect on the F- concentration. Problems of sample contamination and other disturbances are discussed. Determinations of normal values in 20 test subjects resulted in a mean value of 10.4 plus or minus 4.01 mug/l (Mean plus or minus S.D.). The modal value amounted to 9.5 mug/l, and the range was between 5.9 and 18.8 mug/l. The F- content of the drinking water supplied to this group of persons amounted to 180 mug/l. The importance of the method has been illustrated using a clinico-nephrological study as an example.

Adult↗
PubMed1975

Semi-automated radioimmunoassays for total serum thyroxine and triiodothyronine.

Single stage semi-automated radioimmunoassays for total serum thyroxine (T4) and triiodothyronine (T3) are described which employ an automatic pipetting station, automatic gamma counter, and a programmable calculator with paper tape reader and printing facility. Both assays require only a small volume of unextracted serum, and are specific and sensitive. Their sample capacity, precision, speed, and cost are comparable with the measurement of serum protein-bound iodine. Both assays therefore have significant advantages over previous methods for the assessment of thyroid function in the diagnostic laboratory. A Simple method of automating the calculation of results is described, which is applicable to any radioimmunoassay in which the standard curve is approximately linear on a plot of the free/bound fraction against the antigen concentration. In addition, a general method is reported which reveals the relative contributions of intrinsic, systematic, and random error to radioimmunoassay precision.

Analysis of Variance↗
PubMed1980

Dual isotope separation technique for radioassay. The effect of cross counting between two simultaneously counted isotopes.

A theoretical model, describing the effects of cross counting on the dual-isotope-corrected bound count rate and on the precision with which it is estimated, is presented. Experimental support for the validity of the model is given. The bound count rate is shown to be independent of the amount of marker isotope incorrectly counted as label isotope. The precision with which the bound count rate is estimated appears to be dependent on the size of this cross counting correction, although other factors such as pipetting errors are of more importance. Implications for the choice of assay system are discussed.

Cobalt Radioisotopes↗
PubMed1981

Quantification of albumin in urine by a new method: zone immuno-electrophoresis assay (ZIA).

Albumin in urine was determined by a new procedure called zone immunoelectrophoresis assay. A special apparatus was used with 20 vertical glass tubes (diameter 2 mm) which were filled up to 2 cm below their upper orifices with agarose gel containing antibodies against albumin. The samples were pipetted on top of each gel. Albumin was transported by electrophoresis into the agarose gels, in which zones of immunoprecipitates were formed. The amount of albumin in a sample was directly proportional to the distance from the gel surface to the front of the zone. The coefficient of variation was 8.8% within-day and 9.6% between days. The recovery of albumin added to urines was about 101%. The normal range for 40 adult persons was 2--23 mg/l with a mean and a median of 9.9 and 9.5 mg/l, respectively. The detection limit was well below the lowest value. The method was simple to use and the antiserum needed was only 0.5 microliter per test.

Adult↗
PubMed1981

A solid-phase fluoroimmunoassay of tobramycin.

A solid-phase fluoroimmunoassay has been developed for the determination of tobramycin in serum. The principle of this method is based on direct competition of a fluorescein-labeled and unlabeled hapten for a fixed amount of specific antibody immobilized on a dipstick sampler. The bound and free labeled hapten are separated simply by removing the sampler from the reaction mixture. The fluorescence signal from the bound fluorescein-labeled hapten is measured with a FIAX fluorometer. The fluorescein-labeled tobramycin, which is easily prepared, has excellent shelf life. The assay is fast and simple, involves only one pipetting step, and avoids the use of radiochemicals. Within-assay coefficients of variation are less than 10%. The assay correlates well with radioimmunoassay (r = 0.97) and radioenzymatic assay (r = 0.96).

Anti-Bacterial Agents↗
PubMed1987

Assay of sodium-lithium countertransport rate by direct measurement of erythrocyte lithium concentrations.

A method is described for the in-vitro measurement of erythrocytic sodium-lithium countertransport rate (SLC) by direct assay of intra-erythrocytic lithium (Li) concentrations in Li loaded cells during controlled Li efflux. Bicarbonate stimulated erythrocytic Li loading to an approximate intracellular concentration of 3 mmol/l can be achieved within 4 min. Maximum SLC rates are achieved at intra-erythrocytic concentrations greater than 2.5 mmol/l. Lithium efflux is linear over 0-60 min, and independent of pH change. The within-batch coefficient of variation for positive displacement pipetting of Li loaded erythrocytes is 2.8%. Storage of erythrocytes in isosmotic MgCl2 for 24 h had no effect on the SLC. Intra-individual coefficient of variation for SLC was less than 6%. The reference range for Caucasian subjects (n = 43) was 0.14-0.49 mmol Li +/l erythrocytes/h.

Antiporters↗