CHARACTERIZATION AND ASSAY OF THE PROGESTERONE RECEPTOR IN RAT UTERINE NUCLEI

1978 ◽  
Vol 76 (1) ◽  
pp. 33-41 ◽  
Author(s):  
M. T. VU HAI ◽  
E. MILGROM
Keyword(s):  
Progesterone Receptor ◽  
Nuclear Receptors ◽  
Specific Binding ◽  
Biological Response ◽  
Fold Increase ◽  
Ovariectomized Rats ◽  
Experimental Conditions ◽  
Receptor Complexes ◽  
Synthetic Progestogen ◽  
Nuclear Progesterone Receptor

SUMMARY Rat uterine nuclei containing unlabelled progesterone–receptor complexes were incubated at 0 °C with the synthetic progestogen [3H]R5020 (17,21-dimethyl-19-norpregna-4,9-diene-3,20-dione). Complete exchange of steroid bound to the receptor was observed after 5–6 h. For longer times (up to 26 h) there was no further change in the concentration of steroid–receptor complexes, but the non-specific binding was increased. Saturation of nuclear receptors was obtained with a concentration of 5–10 nm-[3H]R5020. Competition with unlabelled steroids showed that only progestogens inhibited the binding of [3H]R5020 to nuclei. Optimum experimental conditions to reduce the non-specific binding to nuclei were established, and a method for the assay of nuclear progesterone–receptor complexes was devised, based on these characteristics. The concentration of nuclear receptors was low in oestradiol-primed ovariectomized rats; adrenalectomy gave rise to slightly lower values. Injection of the rats with progesterone resulted in a 5·5-fold increase in the number of nuclear receptors and a parallel decrease in the number of cytosol receptors. Similar injections of corticosterone and testosterone were without effect. Nuclear receptors were also shown to be stable in uteri kept in liquid nitrogen for up to 3 weeks. This assay may be used to study the correlation of a biological response to progesterone with the extent of receptor occupation in the nuclei.

CHARACTERIZATION AND ASSAY OF THE PROGESTERONE RECEPTOR IN RAT UTERINE CYTOSOL

1978 ◽  
Vol 76 (1) ◽  
pp. 21-31 ◽  
Author(s):  
M. T. VU HAI ◽  
E. MILGROM
Keyword(s):  
Progesterone Receptor ◽  
Receptor Complexes ◽  
High Affinity ◽  
Receptor Sites ◽  
Natural Hormone ◽  
Corticosteroid Binding Globulin ◽  
Steroid Binding Proteins ◽  
Steroid Binding ◽  
High Concentrations ◽  
Synthetic Progestogen

SUMMARY The synthetic progestogen R5020 (17,21-dimethyl-19-norpregna-4,9-diene-3,20-dione) binds with high affinity (Ka = 8·8 × 108 1/mol at 0 °C) to the progesterone receptor from rat uterine cytosol. At nanomolar concentrations, equilibrium is attained in less than 90 min. R5020 has a very low affinity for other specific steroid-binding proteins (corticosteroid-binding globulin and oestrogen receptors) present in relatively high concentrations in the uterine cytosol. The affinity of the receptor for the natural hormone progesterone is remarkably low (Ka= 1 × 108−1·7 × 1081/mol at 0 °C) which explains the instability of progesterone–receptor complexes. Advantage may be taken of this property to remove endogenous progesterone easily by charcoal treatment at 0 °C, a treatment which does not modify the concentration of receptors. A method based on these characteristics is described for the assay of the total number (progesterone-bound and unbound) of receptor sites in uterine cytosol. This assay may be used in various physiological situations where endogenous progesterone is present at unknown concentrations.

scholarly journals The effects of oestrogens and progesterone on oestrogen receptors in female rat liver

1980 ◽  
Vol 190 (3) ◽  
pp. 563-570 ◽  
Author(s):  
W Marr ◽  
M G Elder ◽  
L Lim
Keyword(s):  
Nuclear Receptors ◽  
Oestrogen Receptor ◽  
Specific Binding ◽  
Female Rat ◽  
Receptor System ◽  
Tissue Responses ◽  
Differential Retention ◽  
Norethisterone Acetate ◽  
Synthetic Progestogen

The administration of oestradiol-17 beta or ethynyloestradiol as well as the synthetic progestogen norethisterone acetate resulted in translocation of the oestrogen receptor. Progesterone and the synthetic progestogen (+)-norgestrel were ineffective. The increases in nuclear oestrogen receptor content 1 h after injection of each steroid were similar but different subsequently. The increase with oestradiol-17 beta extended for 3—6 h and for at least 9 h with ethynyloestradiol. With norethisterone acetate, nuclear content was still increased after 24 h. Oestrogen injection resulted in cytosol receptor depletion and a ‘deficit’ in receptor content extending for 6 h, whereas norethisterone acetate-induced translocation was quantitative. With injections of norethisterone acetate + ethynyloestradiol the increase at 1 h and retention of the nuclear receptors were similar to that with norethisterone acetate alone. In contrast, the depletion of cytosol receptor and its restoration were similar to that seen with ethynyloestradiol alone, suggesting that norethisterone acetate did not interfere with the oestrogen receptor replenishment. Specific binding in vitro of [3H]oestradiol-17 beta in liver cytosols was inhibited by (+)-norgestrel and norethisterone acetate, but not progesterone, at concentrations of 10—100 microM. Nuclear receptors present after norethisterone acetate injection bound oestrogen with high affinity (Kd = 1.52 nM), similar to receptors of oestrogen-injected animals. In the uterus, differential retention of nuclear receptors in response to oestrogens is associated with different cellular responses. The differences in the response of the receptor system in liver to the various steroids suggests that the corresponding tissue responses may also be dissimilar. These results are discussed in relation to the problems of liver dysfunction in oral-contraceptive users.

Effects of seven different oestrogen sulphates on uterine growth and on progesterone receptor in the foetal uterus of guinea pig after administration to the mother

1982 ◽  
Vol 101 (4) ◽  
pp. 630-635 ◽  
Author(s):  
J. R. Pasqualini ◽  
A. Lanzone ◽  
A. Tahri-Joutei ◽  
B. L. Nguyen
Keyword(s):  
Progesterone Receptor ◽  
Guinea Pig ◽  
Specific Binding ◽  
Biological Effects ◽  
Biological Response ◽  
The Other ◽  
Specific Binding Sites ◽  
Other Hand ◽  
Uterine Growth ◽  
Two Parameters

Abstract. The biological effect of seven different oestrogen sulphates: oestrone-3-sulphate (E1-3-S), oestradiol-3-sulphate (E2-3-S), oestradiol-17-sulphate (E2-17-S), oestradiol-3,17-disulphate (E23,17-DS), oestriol-3-sulphate (E3-3-S), oestriol-17-sulphate (E3-17-S), oestriol-3,16,17-trisulphate (E3-3,16,17-TS), was studied in the foetal uterus of guinea pig (55–65 days of gestation) after sc administration for 3 consecutive days of each sulphate to the mother. On day 4, two response parameters were investigated, the uterotrophic effect and the action on the progesterone receptor. The monosulphates at the C3 position of the oestrogens (E1-3-S, E2-3-S and E3-3-S) provoked an increase in uterine weight of 1.9–2.4 times in relation to the non-treated animals. These oestrogen sulphates also very significantly stimulated the number of progesterone specific binding sites, 7–10 times in relation to the non-treated animals. On the other hand, when the sulphate was at C17 of the oestrogens (E2-17-S, E3-17-S, E2-3, 17-DS, E3-3,16,17-TS), very little or no effect on the foetal uterus was observed on the two parameters studied. It is concluded that oestrogen (oestrone, oestradiol, oestriol) sulphates in position C3 can be involved in the biological response to the hormone and it is suggested that the effect is carried out after the hydrolysis of the sulphate. On the other hand, sulphates in C17 are not hydrolysed and no significant biological effects were observed in the uterine growth or in the progesterone receptor.

Concanavalin A-induced Aggregation of Human Blood Platelets

1975 ◽  
Vol 33 (02) ◽  
pp. 354-360 ◽  
Author(s):  
Heinrich Patscheke ◽  
Reinhard Brossmer
Keyword(s):  
Concanavalin A ◽  
Binding Capacity ◽  
Specific Binding ◽  
Blood Platelets ◽  
Residual Effect ◽  
Independent Effect ◽  
Con A ◽  
Experimental Conditions ◽  
Main Effect ◽  
Induced Aggregation

SummaryConcanavalin A (CON A) causes platelets to aggregate. A Ca++-independent effect of CON A could be separated from a main effect which depends on Ca++. The main effect probably is a consequence of the CON A-induced platelet release reaction and therefore is platelet-specific. The weak residual effect observed in the presence of Na2EDTA may be due to a similar mechanism as has been demonstrated for CON A-induced aggregations of several other normal and malignant transformed animal cells.Na2EDTA did not inhibit the carbohydrate-specific binding capacity of CON A. Therefore, Na2EDTA appears not to demineralize the CON A molecules under these experimental conditions.α-methyl-D-glucoside inhibits the Ca++-independent as well as the Ca++-dependent effect of CON A.Pretreatment by neuraminidase stimulated the platelet aggregation induced by CON A. It is possible that removal of terminal sialic acid residues makes additional receptors accessible for the binding of CON A.

scholarly journals Ultrafiltration Method for Plasma Protein Binding Studies and Its Limitations

Processes ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 382
Author(s):  
Camelia-Maria Toma ◽  
Silvia Imre ◽  
Camil-Eugen Vari ◽  
Daniela-Lucia Muntean ◽  
Amelia Tero-Vescan
Keyword(s):  
Protein Binding ◽  
Plasma Protein Binding ◽  
Plasma Protein ◽  
Specific Binding ◽  
Critical Role ◽  
Volume Ratio ◽  
Binding Studies ◽  
Experimental Conditions ◽  
Key Aspects

Plasma protein binding plays a critical role in drug therapy, being a key part in the characterization of any compound. Among other methods, this process is largely studied by ultrafiltration based on its advantages. However, the method also has some limitations that could negatively influence the experimental results. The aim of this study was to underline key aspects regarding the limitations of the ultrafiltration method, and the potential ways to overcome them. The main limitations are given by the non-specific binding of the substances, the effect of the volume ratio obtained, and the need of a rigorous control of the experimental conditions, especially pH and temperature. This review presents a variety of methods that can hypothetically reduce the limitations, and concludes that ultrafiltration remains a reliable method for the study of protein binding. However, the methodology of the study should be carefully chosen.

waveRAPID—A Robust Assay for High-Throughput Kinetic Screens with the Creoptix WAVEsystem

2021 ◽  
pp. 247255522110138
Author(s):  
Önder Kartal ◽  
Fabio Andres ◽  
May Poh Lai ◽  
Rony Nehme ◽  
Kaspar Cottier
Keyword(s):  
Drug Discovery ◽  
Fold Increase ◽  
Microtiter Plate ◽  
Experimental Conditions ◽  
Short Pulses ◽  
Cycle Times ◽  
Fragment Screening ◽  
New Instrument ◽  
Single Well ◽  
Compound Screening

Surface-based biophysical methods for measuring binding kinetics of molecular interactions, such as surface plasmon resonance (SPR) or grating-coupled interferometry (GCI), are now well established and widely used in drug discovery. Increasing throughput is an often-cited need in the drug discovery process and this has been achieved with new instrument generations where multiple interactions are measured in parallel, shortening the total measurement times and enabling new application areas within the field. Here, we present the development of a novel technology called waveRAPID for a further—up to 10-fold—increase in throughput, consisting of an injection method using a single sample. Instead of sequentially injecting increasing analyte concentrations for constant durations, the analyte is injected at a single concentration in short pulses of increasing durations. A major advantage of the new method is its ability to determine kinetics from a single well of a microtiter plate, making it uniquely suitable for kinetic screening. We present the fundamentals of this approach using a small-molecule model system for experimental validation and comparing kinetic parameters to traditional methods. By varying experimental conditions, we furthermore assess the robustness of this new technique. Finally, we discuss its potential for improving hit quality and shortening cycle times in the areas of fragment screening, low-molecular-weight compound screening, and hit-to-lead optimization.

scholarly journals LED Illumination Spectrum Manipulation for Increasing the Yield of Sweet Basil (Ocimum basilicum L.)

Plants ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 344
Author(s):  
Md Momtazur Rahman ◽  
Mikhail Vasiliev ◽  
Kamal Alameh
Keyword(s):  
Growth Rate ◽  
Fold Increase ◽  
Photosynthetic Photon Flux Density ◽  
Ocimum Basilicum ◽  
Photon Flux ◽  
Photon Flux Density ◽  
White Led ◽  
Experimental Conditions ◽  
Sweet Basil ◽  
Optimal Illumination

Manipulation of the LED illumination spectrum can enhance plant growth rate and development in grow tents. We report on the identification of the illumination spectrum required to significantly enhance the growth rate of sweet basil (Ocimum basilicum L.) plants in grow tent environments by controlling the LED wavebands illuminating the plants. Since the optimal illumination spectrum depends on the plant type, this work focuses on identifying the illumination spectrum that achieves significant basil biomass improvement compared to improvements reported in prior studies. To be able to optimize the illumination spectrum, several steps must be achieved, namely, understanding plant biology, conducting several trial-and-error experiments, iteratively refining experimental conditions, and undertaking accurate statistical analyses. In this study, basil plants are grown in three grow tents with three LED illumination treatments, namely, only white LED illumination (denoted W*), the combination of red (R) and blue (B) LED illumination (denoted BR*) (relative red (R) and blue (B) intensities are 84% and 16%, respectively) and a combination of red (R), blue (B) and far-red (F) LED illumination (denoted BRF*) (relative red (R), blue (B) and far-red (F) intensities are 79%, 11%, and 10%, respectively). The photosynthetic photon flux density (PPFD) was set at 155 µmol m−2 s−1 for all illumination treatments, and the photoperiod was 20 h per day. Experimental results show that a combination of blue (B), red (R), and far-red (F) LED illumination leads to a one-fold increase in the yield of a sweet basil plant in comparison with only white LED illumination (W*). On the other hand, the use of blue (B) and red (R) LED illumination results in a half-fold increase in plant yield. Understanding the effects of LED illumination spectrum on the growth of plant sweet basil plants through basic horticulture research enables farmers to significantly improve their production yield, thus food security and profitability.

P1279INSULIN RESISTANCE IN HEMODIALYSIS PATIENS

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Pavel Filinyuk ◽  
Aleksander Rumyantsev
Keyword(s):  
Insulin Resistance ◽  
Systemic Inflammation ◽  
Clinical Manifestations ◽  
Biological Response ◽  
Fold Increase ◽  
Atherogenic Dyslipidemia ◽  
Model Assessment ◽  
Peripheral Tissues ◽  
Reactive Protein ◽  
Homeostatic Model Assessment

Abstract Background and Aims insulin resistance (IR) is a decrease in the biological response of sensitive tissues to insulin. IR is known as an adverse risk factor in cardiovascular disease, which largely determines the prognosis of patients receiving hemodialysis (HD). But this issue is not well understood. For the screening of IR, special indices have been developed that characterize the sensitivity of tissues to insulin. The aim of the study was to compare the methods of screening for IR in patients receiving HD in relation to the markers of systemic inflammation and atherogenic dyslipidemia (AtD). Method 124 patients receiving HD for 75.4 ± 44.5 months were examined including 66 men and 58 women aged 57.6 ± 13.6 years. For IR screening, the Homeostatic Model Assessment-1 and 2 indices (HOMA-1 and HOMA-2), the Quantitative Insulin Sensitivity Check Index (QUICKI) and triglycerides / glucose (Tri/G) were used. Patients were examined in accordance with the recommendations of KDIGO. Data analysis was carried out using “STATISTICA 10.0”. Results fasting insulin levels were elevated in 19% of patients. But, the calculated indices were consistent with the idea that IR is much more common. So, the IR index in the HOMA -1 model was increased in 47%, in the HOMA -2 model - in 33%, in the QUICKI model - in 36%, the TriH indicator - in 91%. The sensitivity of peripheral tissues in the HOMA-1 and HOMA-2 models was equally reduced by 35-40%. The results of the correlation analysis between indicators of IR and plasma concentration of C-reactive protein and lipid profile are presented in table 1. Informativeness of IR indicators depending on the presence of obesity is presented in table 2 We were also interested in whether insulin resistance affects the development of clinical manifestations of atherosclerosis, cardiac arrhythmias, and heart failure. An analysis of this relationship did not reveal. Only the IR index in the HOMA-1 model with a value of more than 2.7 units was associated with a 4.5-fold increase in the risk of developing clinical manifestations of atherosclerotic lesions (χ2 = 4.582 p = 0.032). Statistically significant it was only in men. Given our data, perhaps IR is one of the reasons for the higher morbidity and mortality of men at HD. Conclusion a comparison of IR models allows us to distinguish HOMA-2 as the most accurate index. The highest correlation with systemic inflammation and AtD was in the HOMA-1 and HOMA-2 indices.

Biochemical studies of intrauterine components of the tammar wallaby Macropus eugenii during pregnancy

Development ◽  
1981 ◽  
Vol 62 (1) ◽  
pp. 325-338
Author(s):  
Elizabeth J. Thornber ◽  
Marilyn B. Renfree ◽  
Gregory I. Wallace
Keyword(s):  
Protein Concentration ◽  
Specific Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Fold Increase ◽  
Tammar Wallaby ◽  
Macropus Eugenii ◽  
Tenfold Increase ◽  
Specific Proteins ◽  
Wet Weight

The in vitro uptake and incorporation of [3H]ui idine by blastocysts of the tammar wallaby showed a 16- and 30-fold increase from day 0 to day 10 after removal of pouch young, respectively. Two of the six non-expanded blastocysts recovered on day 5 showed a tenfold increase in incorporation. During the first ten days after removal of pouch young the diameter of the blastocyst increased threefold. Endometrial exudate from gravid uteri had a higher protein concentration than exudate from nongravid uteri (39·5 ± 0·9 and 32·0 ± 2·0 mg/ml (mean ± s.e.m.), respectively). Endometrial exudates from uteri where the blastocyst was actively growing were found to contain six uterine-specific proteins. These were separated by gradient polyacrylamide gel electrophoresis. Two of the proteins were pre-albumins and the others were larger molecules (M.W. 153000–670000). Two proteins were only present at particular stages of pregnancy: the other four were present at all stages from diapause to birth, in exudate from gravid and nongravid uteri. The specific binding of progesterone and androstenedione to proteins in endometrial exudates or uterine flushings from pregnant wallabies was less than one per cent of the value obtained from day-5 pregnant rabbits. The ability of mouse blastocysts to take up and incorporate [3H]uridine into acidinsoluble material increased threefold in the presence of day-10 endometrial exudates from wallabies. However, this was less than ten percent of the values obtained in the presence of bovine serum albumin. The concentration of calcium in endometrial exudates increased from 23·6 to 45·2 μg/ml during pregnancy; in endometrium it remained at 88·7 μg/g (wet weight) throughout pregnancy, and in plasma it was 53·3 μg/ml. The concentration of zinc in endometrial exudates was 4·5 μg/ml; in endometrium it decreased from 21·8 to 13·3 μg/g (wet weight) during pregnancy and in plasma it was 0·6 μg/ml.

Characterization of a novel 23-kilodalton protein of unactive progesterone receptor complexes

1994 ◽  
Vol 14 (3) ◽  
pp. 1956-1963
Author(s):  
J L Johnson ◽  
T G Beito ◽  
C J Krco ◽  
D O Toft
Keyword(s):  
Progesterone Receptor ◽  
Cdna Clone ◽  
Peptide Fragments ◽  
Receptor Complexes ◽  
Human Cdna ◽  
Partial Clone ◽  
Chicken Oviduct ◽  
Brain Cdna Library ◽  
Carboxy Terminal ◽  
And Function

Immunoprecipitation of unactivated avian progesterone receptor results in the copurification of hsp90, hsp70, and three additional proteins, p54, p50, and p23. p23 is also present in immunoaffinity-purified hsp90 complexes along with hsp70 and another protein, p60. Antibody and cDNA probes for p23 were prepared in an effort to elucidate the significance and function of this protein. Antibodies to p23 detect similar levels of p23 in all tissues tested and cross-react with a protein of the same size in mice, rabbits, guinea pigs, humans, and Saccharomyces cerevisiae, indicating that p23 is a conserved protein of broad tissue distribution. These antibodies were used to screen a chicken brain cDNA library, resulting in the isolation of a 468-bp partial cDNA clone encoding a sequence containing four sequences corresponding to peptide fragments isolated from chicken p23. This partial clone was subsequently used to isolate a full-length human cDNA clone. The human cDNA encodes a protein of 160 amino acids that does not show homology to previously identified proteins. The chicken and human cDNAs are 88% identical at the DNA level and 96.3% identical at the protein level. p23 is a highly acidic phosphoprotein with an aspartic acid-rich carboxy-terminal domain. Bacterially overexpressed human p23 was used to raise several monoclonal antibodies to p23. These antibodies specifically immunoprecipitate p23 in complex with hsp90 in all tissues tested and can be used to immunoaffinity isolate progesterone receptor complexes from chicken oviduct cytosol.

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