Dilution Of Cytosol Research Articles

Minimizing Cytosol Dilution in Whole-Cell Patch-Clamp Experiments

During a conventional whole-cell patch clamp experiment, diffusible cytosolic ions or molecules absent in the pipette solution can become diluted by a factor of one million or more, leading to diminished current or fluorescent signals. Existing methods to prevent or limit cytosol diffusion include reducing the diameter of the pipette's orifice, adding cytosolic extract or physiological entities to the pipette solution, and using the perforated patch clamp configuration. The first method introduces measurement error in recorded signals from increased series resistance and the latter two are cumbersome to perform. In addition, most perforated patch configurations, prevent investigators from using test compounds in the pipette solution. We present a method to overcome these limitations by minimizing cytosol dilution using a novel pipette holder. Cell-attached configuration is obtained with the pipette filled with pipette solution. Most of the pipette solution is then replaced with mineral oil so that cytosol dilution can be minimized in whole-cell configuration. To accomplish this requires a suction line and two Ag/AgCl electrodes inside the pipette. Testing our novel pipette holder with Chinese Hamster Ovarian cells, we demonstrate cytosol dilution factors between 76 and 234. For large cells with somas greater than 40μm, cytosol dilution factors of 10 or less are achievable.

Redox regulation of glutenin subunit assembly in the plant endoplasmic reticulum

The glutenin fraction of wheat storage proteins consists of large polymers in which high- and low-molecular-weight subunits are connected by inter-chain disulfide bonds. We found that assembly of a low-molecular-weight glutenin subunit in the endoplasmic reticulum is a rapid process that leads to accumulation of various oligomeric forms, and that this assembly is sensitive to perturbation of the cellular redox environment. In endoplasmic reticulum-derived microsomes, low-molecular-weight glutenin subunits are subjected to hyper-polymerization, indicating that cytosolic factors play an important role in limiting polymer size. Addition of physiological concentrations of reduced glutathione is sufficient to maintain the original polymerization pattern of the glutenin subunits upon cytosol dilution. Furthermore, we show that a low-molecular-weight glutenin subunit can be glutathionylated in endoplasmic reticulum-derived microsomes, and that it can be directly reduced by glutathione in vitro. These results indicate that glutenin polymerization is sensitive to changes in the redox state of the cell, and suggest that the presence of a reducing cytosolic environment plays an important role in regulating disulfide bond formation in the endoplasmic reticulum of plant cells.

Multielement analysis in the fish hepatic cytosol as a screening tool in the monitoring of natural waters

The possibility of direct measurement of trace elements in hepatic cytosol of European chub (Squalius cephalus) by high-resolution inductively coupled plasma mass spectrometry (HR ICP-MS) after cytosol dilution with Milli-Q water and subsequent acidification was investigated. Due to low detection limits of this procedure, determination of 13 elements (As, Cd, Co, Cu, Fe, Mn, Mo, Pb, Sb, Sn, Sr, V and Zn) was possible in the chub hepatic cytosol, exhibiting excellent measurement repeatability in duplicates. Some of these elements were also measured by HR ICP-MS in acid digested cytosols (Cd, Co, Cu, Fe, Mn, Mo, Sr, V and Zn). Good agreement of the results obtained after sample dilution and sample digestion indicated that complex organic matrix of hepatic cytosol did not affect measurement reliability. Cytosolic concentrations of 13 trace elements in the chub liver were quantified in the following order: Fe, Zn>Cu>Mn>Mo>Sr, V, Cd>Co>As, Pb>Sn>Sb. Unlike Cd, Cu, Fe, Mn and Zn for which the cytosolic concentrations were previously reported after measurement by AAS, cytosolic concentrations of eight additional trace elements characteristic for the liver of chubs inhabiting the low contaminated river water were reported here for the first time (in nanogrammes per gramme)-Mo, 136.8-183.6; Sr, 32.7-63.0; V, 17.5-69.0; Co, 24.3-30.7; As, 9.9-29.5; Pb, 5.8-35.6; Sn, 5.5-12.4; and Sb, 0.9-2.6. The simultaneous measurement of large number of trace elements in the cytosolic fractions of fish tissues, which comprise potentially metal-sensitive sub-cellular pools, could be beneficial as a screening tool in the monitoring of natural waters, because it would enable timely recognition of increased fish exposure to metals.

Determination of oestrogen receptors by enzyme immunoassay. Technical differences between laboratories and their consequences

When multicentre breast cancer trials are performed, receptor analyses must be comparable both over time and in the participating laboratories. However, we show for the first time a high variability for the distribution of oestradiol receptor (ER) values measured by enzyme immunoassay (EIA) from 1987 to 1991. This variability could be explained by calibration changes in the immunoassay kits. We have also analysed the influence on ER-EIA levels of technical differences between laboratories apart from the assay itself. Many steps emerged as being critical, i.e. homogenisation buffer, homogenisation procedure and cytosol dilution. Finally, we show that addition of 4-monohydroxytamoxifen increases the apparent ER content measured by EIA in 92% of cytosols. Thus, many factors must be controlled to ensure high precision with ER-EIA assays. We have to be particularly cautious with the conformational changes that could occur during cytosol preparation and that could also preexist in the tumour samples. Quality controls of cytosol preparation are essential.

The Glucocorticoid Receptor of the Neural Retina of the Chick Embryo: Gradual Changes in Charge of the Glucocorticoid Receptor Complex During Transformation, Detected by Electrofocusing

Abstract Two main difficulties impede the monitoring of the changes in the surface charge of the triamcinolone acetonide-glucocorticoid-receptor (TA-GR) complex concomitant with GR-transformation by electrofocusing (EF). One difficulty relates to the high sensitivity of the nonactivated [3H]TA-GR complex to temperature. The second difficulty relates to the transforming effects of basic carrier ampholytes, pI′ range 8-10 (basic SCAMs) on GR. Performing EF at strictly controlled cold temperature, and omitting the use of basic SCAMS revealed that the nontransformed molybdate-stabilized TA-GR complexes in the cytosol of the neural retina consist of a homogeneously charged GR population exhibiting an apparent pI′ (pI) value of 5.0 ± 0.2 (contrary to the previously reported heterogeneity of GR in ‘nonactivated’ state1). Exposure of the [3H]TA-GR to activation by physiological temperatures, or to activation by 0.4M KC1 in the cold, (in the presence of PMSF, which inhibits proteolytic degradation of GR in the neural retina2) transformed the acidic nonactivated GR complex (designated TA-GR I) to more basic GR species: TA-GR II with a pI′ of 5.6 ± 0.2 and TA-GR III with a pI′ of 6.2 ± 0.1. TA-GR II appeared as a minor component, and TA-GR III appeared as the major form in the activated state. Molybdate inhibited the formation of TA-GR III induced by 0.4M KC1, while it augmented TA-GR II. The formation of TA-GR II from molybdate-stabilized TA-GR I was enhanced by cytosol dilution. The increase in the amount of TA-GR II in molybdate-stabilized cytosol correlated with the decrease in the amount of TA-GR I and the absence of III, suggesting that TA-GR II represents an intermediate state of GR transformation. The suppression of TA-GR III by molybdate is reversible at 30°C. Inhibitors of proteases, leupeptin, antipain and PMSF, caused a slight decrease in the pI′ value of TA-GR II found in the activated cytosol, (5.4 instead of 5.7), but did not change the heterogeneity of GR species found in the activated state. The omission of basic SCAMs in this study reduced the previously reported pI′ values1 of TA-GR species which had been artifactually increased by basic SCAMs. The pI′ values reported in this study were confirmed by replacing SCAMs with simple buffers as carrier constituents, and appear to reflect the authentic surface net charge of TA-GR. EF analysis of [3H]TA-GR complexes that were formed in the intact tissue at 0°C (cell-bound [3H]TA-GR) confirmed the charge homogeneity of the nonactivated GR complexes in the nonactivated cytosol but showed that the cell-bound nonactivated [3H]TA-GR is more acidic than the TA-GR I formed under cell-free conditions, exhibiting a pI′ value of 4.2 ± 0.1. These results are consistent with the hypothesis that GR transformation is a multistage process characterized by a progressive decrease in the negative net charge of the GR complex. The nature of the changes in negative net charge is discussed elsewhere2 in relation to the changes in size of the GR complex occurring in the process of GR transformation.

Characterization of estrogen binding sites in the liver of the newt pleurodeles waltl (urodela: amphibia): Demonstration of a sex-linked heterogeneity

Saturation analysis over a wide range of [ 3H]estradiol-17β concentrations (l–40 nM) in cytosols prepared from liver of the newt Pleurodeles waltl of both sexes revealed a sex-linked heterogeneity of the estradiol-17β binding sites. In females, one type of binding site has been identified as a classical receptor. It exhibited a high affinity for estradiol-17β ( K d = 9 × 10 −9 M), had a high specificity for estrogenic compounds and was stabilized by monothioglycerol. In males, in addition to the receptor found in females, a second estrogen binding component was detected, not observed in female cytosols. It exhibited a ( K d of 4.8 × 10 −8 M for estradiol 17β, higher capacity and displayed the same highly specific estrogen binding as does the estrogen receptor. It was affected by monothioglycerol and its binding was found to be significantly increased on cytosol dilution, as well as by estrogen-treatment.

Quality control of estrogen receptor assays in the Netherlands

Lyophilized receptor-positive tissue powders and cytosols, prepared from calf uterus and human breast tumor tissue, are used to assess the validity of routine dextran-coated charcoal estrogen receptor assays. Since 1978 lyophilized reference preparations have been analyzed twice yearly by 18 laboratories in the Netherlands. During 8 consecutive trials 20 different lyophilized samples were studied. The inter-laboratory variability of estrogen receptor results decreased with time. Most laboratories found receptor values around the median value of all groups together, though some participants consistently reported estrogen receptor values that were higher or lower than the median. The variability of estrogen receptor results between labs seemed to be associated with cytosol dilution, determination of non-specific binding, concentration and volume of dextran-coated charcoal, and the use of single dose assays or Scatchard analysis. The agreement on the presence or absence of estrogen receptors was more than 98% for lyophilized reference samples with high receptor content. For samples with low receptor content 85% agreement was observed, while 12% of the assays performed on receptor-negative material were reported to be estrogen receptor-positive. The use of the same protein determination (Coomassie Brilliant Blue) and human serum albumin standard has decreased the interlaboratory variation coefficient of the protein results to 7.5%.

Transformation of a rabbit progesterone receptor from an 8S form to 5.5S and 4S forms

The 8S form of the rabbit uterine progesterone receptor transforms slowly at 0°C to a 4S form with an intermediate 5.5S form. The transformation is accelerated by either heat, increased ionic strength and dilution of cytosol. The transformation, which is reversibly inhibited by sodium molybdate, is unrelated to total cytosolic alkaline phosphatase activity. The transformation is accompanied by a positive change in receptor surface charge and a decrease in the rate of progesterone dissociation. The stability of the 8S progesterone-receptor complex is reduced and the sedimentation coefficient increased by acidic conditions; acid does not affect the 4S receptor as drastically.

An in vitro study of the stability of the chicken intestinal cytosol 1,25-dihydroxyvitamin D 3-specific receptor

The stability of the chick intestinal cytosol receptor for 1,25-dihydroxyvitamin D 3 has been examined using radiological binding studies and sucrose density gradient ultracentrifugation. Specific binding of 1,25-dihydroxyvitamin D 3 to the 3.7 S binding protein decreases in crude cytosol in a time- and temperature-dependent manner. Increased receptor instability is also observed outside a pH range of 6 to 10. Ionic strength does not seem to be a critical factor in preventing loss of specific 1,25-dihydroxyvitamin D 3 binding activity. However, when KCl is present at a concentration of 300 m m during cytosol preparation, quantitatively more specific binding per unit protein was obtained. Consistent with the idea that loss of specific binding might be due to enzymatic degradation or inactivation of receptor, dilution of cytosol was found to slow the rate of loss of specific 1,25-dihydroxyvitamin D 3 binding. The importance of maintaining a reducing environment for the 1,25-dihydroxyvitamin D 3 binding protein is demonstrated by the destruction of binding activity by n-ethylmaleimide and by the increased stability in the presence of 5.0 m m dithiothreitol. Likewise, 5.0 m m monothioglycerol was partially effective in preventing the loss of specific 1,25-dihydroxyvitamin D 3 binding during in vitro incubation. Several protease inhibitors were not able to exert a stabilizing influence on receptor integrity during in vitro incubations. Albeit, both tosylamide-phenylethylchloromethyl ketone and p-hydroxymercuribenzoate actually decreased specific 1,25-dihydroxyvitamin D 3 binding. This inhibition appeared to be reversible if samples were subsequently incubated in the presence of dithiothreitol. These results clearly demonstrate that the aporeceptor is extremely unstable and the integrity of sulfhydryl constituents is of primary importance.