CYTOSOL AND NUCLEAR [3H]OESTRADIOL BINDING IN THE FOETAL TISSUES OF GUINEA PIG

1976 ◽  
Vol 83 (4) ◽  
pp. 811-828 ◽  
Author(s):  
J. R. Pasqualini ◽  
C. Sumida ◽  
C. Gelly
Keyword(s):  
Guinea Pig ◽  
Specific Binding ◽  
Nuclear Extract ◽  
Radioactive Material ◽  
Cytosol Fraction ◽  
Nuclear Extracts ◽  
Foetal Kidney ◽  
Foetal Lung

ABSTRACT The formation of [3H]oestradiol macromolecule complexes was studied in vivo and in vitro in the kidney, lung and liver of intact foetal guinea pig. Specific binding of [3H]oestradiol was demonstrated in the cytosol and nuclear fractions of foetal kidney. Intensive binding was found in the cytosol of foetal lung but most of the bound radioactive material (71 %) was [3H]oestrone. Some binding was found in the cytosol of foetal liver but not in the nucleus of this tissue. In the in vivo experiments, the binding of radioactive material to plasma proteins was studied: 22 % of the plasma radioactivity was bound of which 67 % was identified as oestrone sulphate. Oestradiol sulphate represented 7 % and unconjugated oestradiol only 0.6 % (0.1 % of the total plasma radioactivity). On the other hand, 2–3 % of the foetal plasma radioactivity was found as unbound [3H]oestradiol. In the kidney, the formation of [3H]oestradiol complexes in the cytosol fraction does not depend on temperature while nuclear [3H]oestradiol complexes increase with increasing temperature. Maximal formation of [3H]oestradiol complexes in the cytosol fraction and the 0.1 m TRIS and 0.3 m NaCl nuclear extracts was reached after 15 min but binding in the 1 m NaCl nuclear extract continued to increase up to 30 min. After incubation of purified nuclei of foetal kidney with 1.1 × 10−7 m [3H]oestradiol, specific binding was found in the different nuclear fractions. Specific binding was also detected in isolated nuclei previously extracted by 0.1 m TRIS and 0.3 m NaCl before being incubated with 1.1 × 10−7 m [3H]oestradiol. The Kd of binding of [3H]oestradiol in the renal cytosol fraction is 2.5 × 10−10 m with n = 4.5 × 10−14 moles/mg protein. Incubation of isolated 1 m NaCl nuclear extract from foetal kidney with [3H]oestradiol gives a Kd of 3.3 × 10−10 m with n = 2.5 × 10−14 moles/mg protein. It is concluded that the nuclear complexes in the foetal kidney could be formed either through an intermediate cytosol complex or that the "two step" mechanism could take place in the nucleus. Furthermore, direct binding of [3H]oestradiol with high affinity was observed in the 1 m NaCl nuclear extract.

Cytoplasmic and nuclear progesterone receptors in mouse mammary tumours during pregnancy determined by an improved hydroxylapatite assay

1982 ◽  
Vol 94 (1) ◽  
pp. 111-123 ◽  
Author(s):  
Y. Koseki ◽  
M. E. Costlow ◽  
D. Cole ◽  
A. Matsuzawa
Keyword(s):  
Progesterone Receptors ◽  
Nuclear Extract ◽  
Scatchard Analysis ◽  
Normal Mammary Gland ◽  
Single Class ◽  
Heat Stable ◽  
Nuclear Extracts ◽  
Complete Exchange

The binding of [3H] 17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione (R5020) to progesterone receptors in cytosol and nuclear extracts (0·6 m-KCl) of the pregnancy-dependent, TPDMT-4 mouse mammary tumour was measured at various stages of pregnancy. Compared with conventional dextran-coated charcoal (DCC) assays, a hydroxylapatite assay with DCC pretreatment and precharging of the cytosol with unlabelled R5020 (4 × 10−8 mol/l, for 3–4 h at 4 °C) showed the highest level of binding. The DCC treatment markedly increased the level of R5020 binding in both cytosol and nuclear extracts by allowing the receptor to bind to hydroxylapatite. The DCC pretreatment apparently removed a heat-stable and non-dialysable factor which prevented the receptor from binding to the hydroxylapatite. Using this assay R5020 binding reached a steady state in 24 h at 4 °C, with complete exchange of radioactive for non-radioactive ligand by 20 h. Nuclear extracts did not require precharging and complete exchange was more rapid. Scatchard analysis (without precharging) disclosed a single class of binding sites with a dissociation constant for cytosol of 3·2 ± 0·8 (s.e.m.) × 10−9 mol/l (n = 3) and 4·7 ± 0·6 × 10−9 mol/l (n = 5) for the nuclear extract. Binding was hormone-specific and progesterone translocated binding from the cytoplasm to the nucleus both in vivo and in vitro. Translocation, however, led to a substantial loss of total (nuclear + cytoplasmic receptors. During pregnancy, cytoplasmic progesterone receptor levels were unchanged and low compared to nuclear progesterone receptors which increased by sevenfold from days 1 to 11 and then decreased at day 16. Compared with recent data on cytoplasmic progesterone receptors in normal mammary gland, our results suggested that this tumour may have a reduced sensitivity to the down-regulatory activity of progesterone. This lesion may, in part, account for the failure of the tumour to differentiate during pregnancy.

scholarly journals Identification of a Nuclear Stat1 Protein Tyrosine Phosphatase

2002 ◽  
Vol 22 (16) ◽  
pp. 5662-5668 ◽  
Author(s):  
Johanna ten Hoeve ◽  
Maria de Jesus Ibarra-Sanchez ◽  
Yubin Fu ◽  
Wei Zhu ◽  
Michel Tremblay ◽  
...  
Keyword(s):  
Protein Tyrosine Phosphatase ◽  
Tyrosine Phosphatase ◽  
Nuclear Extract ◽  
Null Mouse ◽  
Protein Tyrosine ◽  
Nuclear Extracts ◽  
Unknown Protein ◽  
Stat1 Protein

ABSTRACT Upon interferon (IFN) stimulation, Stat1 becomes tyrosine phosphorylated and translocates into the nucleus, where it binds to DNA to activate transcription. The activity of Stat1 is dependent on tyrosine phosphorylation, and its inactivation in the nucleus is accomplished by a previously unknown protein tyrosine phosphatase (PTP). We have now purified a Stat1 PTP activity from HeLa cell nuclear extract and identified it as TC45, the nuclear isoform of the T-cell PTP (TC-PTP). TC45 can dephosphorylate Stat1 both in vitro and in vivo. Nuclear extracts lacking TC45 fail to dephosphorylate Stat1. Furthermore, the dephosphorylation of IFN-induced tyrosine-phosphorylated Stat1 is defective in TC-PTP-null mouse embryonic fibroblasts (MEFs) and primary thymocytes. Reconstitution of TC-PTP-null MEFs with TC45, but not the endoplasmic reticulum (ER)-associated isoform TC48, rescues the defect in Stat1 dephosphorylation. The dephosphorylation of Stat3, but not Stat5 or Stat6, is also affected in TC-PTP-null cells. Our results identify TC45 as a PTP responsible for the dephosphorylation of Stat1 in the nucleus.

BINDING AND METABOLISM OF 5α-ANDROSTANE-3α,17β-DIOL AND OF 5α-ANDROSTANE-3β,17β-DIOL IN THE PROSTATE, SEMINAL VESICLES AND PLASMA OF MALE RATS: STUDIES IN VIVO AND IN VITRO

1975 ◽  
Vol 64 (3) ◽  
pp. 529-538 ◽  
Author(s):  
M. KRIEG ◽  
H.-J. HORST ◽  
M.-L. STERBA
Keyword(s):  
Skeletal Muscle ◽  
Specific Binding ◽  
Biological Effects ◽  
Sucrose Density Gradient ◽  
Total Radioactivity ◽  
Seminal Vesicles ◽  
Male Rats ◽  
Cytosol Fraction

SUMMARY Binding of 5α-androstane-3α,17β-diol (3α-diol) and 5α-androstane-3β,17β-diol (3β-diol) in vivo and in vitro to the 100000 g cytosol fraction of the rat prostate and seminal vesicles as well as to plasma was studied by agargel electrophoresis and sucrose density gradient ultracentrifugation and the results compared with the corresponding findings for 5α-dihydrotestosterone (5α-DHT). The metabolism of 3α-diol and 3β-diol was also investigated by thin-layer chromatography. The following results were obtained: (1) A specific binding of 3α-diol and 3β-diol by the cytosols could not be demonstrated in vitro, while 5α-DHT was specifically bound. (2) In plasma, 3α-diol was extensively bound, 3β-diol less extensively bound, while 5α-DHT remained unbound. (3) After intravenous injection of 3α-diol, specifically bound radioactivity, increasing within 30 min, was found in the prostate cytosol, while after 3β-diol injection no binding occurred. (4) Parallel to the increased binding, the total radioactivity in the prostate accumulated within 30 min after 3α-diol injection, the uptake being 5·3 times higher than in skeletal muscle. However after 3β-diol injection, total radioactivity decreased in the prostate within 30 min, the uptake being only 1·5 times higher than in skeletal muscle. (5) One minute after injection of 3α-diol, 53% of the extracted radioactivity in the prostate had been converted to 5α-DHT, this increased within 30 min to 81%. Thirty minutes after the injection of 3β-diol, about 32% of the extracted radioactivity in the prostate had been converted to 5α-DHT. (6) From the in-vivo and in-vitro experiments it was concluded that 3α-diol exerts its biological effects mainly by its conversion into 5α-DHT.

TRANSFORMATION OF [1,2-3H] ALDOSTERONE IN THE FOETAL AND PLACENTAL COMPARTMENTS OF THE GUINEA PIG

1976 ◽  
Vol 82 (4) ◽  
pp. 831-841 ◽  
Author(s):  
J. R. Pasqualini ◽  
M. Bedin ◽  
A. M. Cogneville
Keyword(s):  
Enzymatic Hydrolysis ◽  
Guinea Pig ◽  
Radioactive Material ◽  
Foetal Liver ◽  
Acidic Compound

ABSTRACT In vivo and in vitro transformation of labelled d-aldosterone (11β,21-dihydroxy-3,20-dioxo-4-pregnen-18-al) was studied in the foetal tissues and placenta of guinea pig (35–45 days of gestation). The principal metabolite found in all of these tissues was tetrahydroaldosterone (3α,18,21-trihydroxy-20-oxo-5β-pregnan-18-al). A large transformation into this metabolite was found in foetal liver (67–68 %) and in the placenta (69–82%). Incubation of [3H]- or [14C] aldosterone with the isolated placenta also showed a large conversion to the tetrahydroderivative suggesting that the reductase(s) of ring A of aldosterone is present in this tissue. Most of the unmetabolized aldosterone and tetrahydroaldosterone was found in the unconjugated fraction. Very little of the radioactive material was present in the polar steroids extracted with n-butanol. Aldosterone and tetrahydroaldosterone were liberated from this fraction after enzymatic hydrolysis (β-glucuronidase and sulphatase) or by solvolysis, suggesting that part of these conjugates is present in the foetal compartment as sulphate esters and glucuronides. Moreover, in this fraction a polar, unconjugated aldosterone metabolite with the characteristics of an acidic compound was detected.

STUDIES ON THE MECHANISM OF FORMATION OF [3H]ALDOSTERONE MACROMOLECULE COMPLEXES IN THE FOETAL GUINEA PIG KIDNEY

1974 ◽  
Vol 77 (2) ◽  
pp. 356-367 ◽  
Author(s):  
Jorge R. Pasqualini ◽  
Charlotte Sumida ◽  
Chantal Gelly
Keyword(s):  
Guinea Pig ◽  
Total Cell ◽  
Nuclear Fraction ◽  
Mechanism Of Formation ◽  
High Concentration ◽  
Cytosol Fraction ◽  
Pig Kidney ◽  
Nuclear Extracts ◽  
Foetal Kidney ◽  
Period Of Gestation

ABSTRACT The mechanism of the formation of [3H] aldosterone macromolecule complexes in the cytosol and in the different nuclear extracts of the foetal kidney of the guinea pig was studied after incubation of the total cell, the purified nuclei and the partially extracted nuclei. The nuclear aldosterone macromolecule complexes of the 0.1 m Tris and 0.3 m NaCl extracts (soluble nuclear fractions) and those of the 1 m NaCl extract (chromatin) can be formed after the incubation of the purified nuclei. The addition of the cytosol fraction to the nucleus has no significant effect on the formation of the nuclear [3H] aldosterone macromolecule complexes of both the soluble fractions and the chromatin. Furthermore, the [3H] aldosterone complexes of the chromatin can be formed after extraction of the soluble nuclear fraction. The present data suggest that at this period of gestation (30–50 days) one of the pathways in the formation of [3H] aldosterone macromolecule complexes in the nuclear fractions (0.1 m Tris, 0.3 m NaCl and 1 m NaCl) in the foetal guinea pig kidney could be through unbound aldosterone, independent of the cytosol macromolecule complexes. Another possible explanation is that due to the high concentration of endogenous aldosterone circulating in the foetal compartment, the cytosol receptor is already present inside the nucleus.

Inhibition of Human and Animal Platelet Adhesiveness to Glass Bead Columns by Adenosine, Dipyridamole, Chlorpromazine and Acetylsalicylic Acid

1976 ◽  
Vol 36 (02) ◽  
pp. 401-410 ◽  
Author(s):  
Buichi Fujttani ◽  
Toshimichi Tsuboi ◽  
Kazuko Takeno ◽  
Kouichi Yoshida ◽  
Masanao Shimizu
Keyword(s):  
Guinea Pig ◽  
Acetylsalicylic Acid ◽  
Guinea Pigs ◽  
Glass Bead ◽  
Animal Species ◽  
Inhibitory Effect ◽  
Rabbit Platelet ◽  
Platelet Adhesiveness

SummaryThe differences among human, rabbit and guinea-pig platelet adhesiveness as for inhibitions by adenosine, dipyridamole, chlorpromazine and acetylsalicylic acid are described, and the influence of measurement conditions on platelet adhesiveness is also reported. Platelet adhesiveness of human and animal species decreased with an increase of heparin concentrations and an increase of flow rate of blood passing through a glass bead column. Human and rabbit platelet adhesiveness was inhibited in vitro by adenosine, dipyridamole and chlorpromazine, but not by acetylsalicylic acid. On the other hand, guinea-pig platelet adhesiveness was inhibited by the four drugs including acetylsalicylic acid. In in vivo study, adenosine, dipyridamole and chlorpromazine inhibited platelet adhesiveness in rabbits and guinea-pigs. Acetylsalicylic acid showed the inhibitory effect in guinea-pigs, but not in rabbits.

Studies on Metabolism of Urokinase and Mechanism of Thrombolysis by Urokinase

1979 ◽  
Vol 42 (03) ◽  
pp. 885-894 ◽  
Author(s):  
Tatsuo Ueno ◽  
Norio Kobayashi ◽  
Tadashi Maekawa
Keyword(s):  
Molecular Weight ◽  
Plasma Clearance ◽  
Radioactive Material ◽  
Optimal Dosage ◽  
Plasma Clot ◽  
Optimal Dosage Regimen ◽  
Arterial Thrombus ◽  
Contact Experiment

SummaryPharmacokinetics of intravenously injected 125I-labeled urokinase (125I-UK) of a molecular weight of 33,000 daltons in normal rabbits and patients with various diseases were investigated. The plasma clearance of 125I-UK in rabbits was described by a biexponential curve within six hours with a half-life of 8 minutes, 2.3 hours, respectively. The radioactivity in the liver and kidneys 15 minutes after iv injection with 125I-UK was 9.6% and 14.0% of the radioactivity injected, respectively. Approximately 80% of the total radioactive material injected was excreted in the urine in 18 hours. No increase in activator activity in the urine was observed after a large amount of UK injection. Activity uptake of 125I-UK by experimentally induced arterial thrombus was little. Lysis of the stasis thrombus was produced by injecting 7.5 × 104 IU of UK in only one out of 8 rabbits. In vitro contact experiment revealed that transfer of 125I-UK to plasma clot is slow (24 hours for 10% of 125I-UK by plasma clot). In 4 patients plasma clearance of 125I-UK was essentially similar to that in rabbits. From the results obtained optimal dosage regimen of UK administration for complete thrombolysis in vivo was discussed.

Correlation between in vitro and in vivo Data of Radiolabeled Peptide for Tumor Targeting

2019 ◽  
Vol 19 (12) ◽  
pp. 950-960
Author(s):  
Soghra Farzipour ◽  
Seyed Jalal Hosseinimehr
Keyword(s):  
Tumor Targeting ◽  
Single Photon ◽  
Specific Binding ◽  
Specific Interaction ◽  
Photon Emission ◽  
Emission Computed Tomography ◽  
Mixed Effect ◽  
Radiolabeled Peptides

Tumor-targeting peptides have been generally developed for the overexpression of tumor specific receptors in cancer cells. The use of specific radiolabeled peptide allows tumor visualization by single photon emission computed tomography (SPECT) and positron emission tomography (PET) tools. The high affinity and specific binding of radiolabeled peptide are focusing on tumoral receptors. The character of the peptide itself, in particular, its complex molecular structure and behaviors influence on its specific interaction with receptors which are overexpressed in tumor. This review summarizes various strategies which are applied for the expansion of radiolabeled peptides for tumor targeting based on in vitro and in vivo specific tumor data and then their data were compared to find any correlation between these experiments. With a careful look at previous studies, it can be found that in vitro unblock-block ratio was unable to correlate the tumor to muscle ratio and the success of radiolabeled peptide for in vivo tumor targeting. The introduction of modifiers’ approaches, nature of peptides, and type of chelators and co-ligands have mixed effect on the in vitro and in vivo specificity of radiolabeled peptides.

scholarly journals Improved in vivo PET imaging of the adenosine A2A receptor in the brain using [18F]FLUDA, a deuterated radiotracer with high metabolic stability

2021 ◽  
Author(s):  
Thu Hang Lai ◽  
Magali Toussaint ◽  
Rodrigo Teodoro ◽  
Sladjana Dukić-Stefanović ◽  
Daniel Gündel ◽  
...  
Keyword(s):  
Specific Binding ◽  
Radiochemical Yield ◽  
Toxicity Study ◽  
In Vivo Evaluation ◽  
One Pot ◽  
Specific Binding Ratio ◽  
Mri Studies ◽  
The Brain

Abstract Purpose The adenosine A2A receptor has emerged as a therapeutic target for multiple diseases, and thus the non-invasive imaging of the expression or occupancy of the A2A receptor has potential to contribute to diagnosis and drug development. We aimed at the development of a metabolically stable A2A receptor radiotracer and report herein the preclinical evaluation of [18F]FLUDA, a deuterated isotopologue of [18F]FESCH. Methods [18F]FLUDA was synthesized by a two-step one-pot approach and evaluated in vitro by autoradiographic studies as well as in vivo by metabolism and dynamic PET/MRI studies in mice and piglets under baseline and blocking conditions. A single-dose toxicity study was performed in rats. Results [18F]FLUDA was obtained with a radiochemical yield of 19% and molar activities of 72–180 GBq/μmol. Autoradiography proved A2A receptor–specific accumulation of [18F]FLUDA in the striatum of a mouse and pig brain. In vivo evaluation in mice revealed improved stability of [18F]FLUDA compared to that of [18F]FESCH, resulting in the absence of brain-penetrant radiometabolites. Furthermore, the radiometabolites detected in piglets are expected to have a low tendency for brain penetration. PET/MRI studies confirmed high specific binding of [18F]FLUDA towards striatal A2A receptor with a maximum specific-to-non-specific binding ratio in mice of 8.3. The toxicity study revealed no adverse effects of FLUDA up to 30 μg/kg, ~ 4000-fold the dose applied in human PET studies using [18F]FLUDA. Conclusions The new radiotracer [18F]FLUDA is suitable to detect the availability of the A2A receptor in the brain with high target specificity. It is regarded ready for human application.

scholarly journals In vitro and in vivo inhibition of malaria parasite infection by monoclonal antibodies against Plasmodium falciparum circumsporozoite protein (CSP)

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Merricka C. Livingstone ◽  
Alexis A. Bitzer ◽  
Alish Giri ◽  
Kun Luo ◽  
Rajeshwer S. Sankhala ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Monoclonal Antibodies ◽  
Disease Burden ◽  
Vaccine Candidate ◽  
Specific Binding ◽  
Circumsporozoite Protein ◽  
Target Epitope ◽  
Selection Of

AbstractPlasmodium falciparum malaria contributes to a significant global disease burden. Circumsporozoite protein (CSP), the most abundant sporozoite stage antigen, is a prime vaccine candidate. Inhibitory monoclonal antibodies (mAbs) against CSP map to either a short junctional sequence or the central (NPNA)n repeat region. We compared in vitro and in vivo activities of six CSP-specific mAbs derived from human recipients of a recombinant CSP vaccine RTS,S/AS01 (mAbs 317 and 311); an irradiated whole sporozoite vaccine PfSPZ (mAbs CIS43 and MGG4); or individuals exposed to malaria (mAbs 580 and 663). RTS,S mAb 317 that specifically binds the (NPNA)n epitope, had the highest affinity and it elicited the best sterile protection in mice. The most potent inhibitor of sporozoite invasion in vitro was mAb CIS43 which shows dual-specific binding to the junctional sequence and (NPNA)n. In vivo mouse protection was associated with the mAb reactivity to the NANPx6 peptide, the in vitro inhibition of sporozoite invasion activity, and kinetic parameters measured using intact mAbs or their Fab fragments. Buried surface area between mAb and its target epitope was also associated with in vivo protection. Association and disconnects between in vitro and in vivo readouts has important implications for the design and down-selection of the next generation of CSP based interventions.

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