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.

scholarly journals Biochemical properties of a highly purified v-rasH p21 protein overproduced in Escherichia coli and inhibition of its activities by a monoclonal antibody.

1985 ◽  
Vol 5 (6) ◽  
pp. 1449-1455 ◽  
Author(s):  
S Hattori ◽  
L S Ulsh ◽  
K Halliday ◽  
T Y Shih
Keyword(s):  
Escherichia Coli ◽  
Monoclonal Antibody ◽  
Active Center ◽  
Biochemical Properties ◽  
Scatchard Analysis ◽  
Single Class ◽  
P21 Protein ◽  
High Level

The v-rasH oncogene of Harvey murine sarcoma virus encodes a 21,000-dalton p21 protein which has been expressed at a high level as a fusion protein in Escherichia coli. We have purified the p21 to over 90% in purity without the use of any detergent or protein denaturant. The purified p21 possesses full biochemical activities of GTP/GDP binding, autokinase, and GTPase. Scatchard analysis indicates a single class of binding sites with Kd values of 0.83 X 10(-8)M for GTP and 1.0 X 10(-8)M for GDP. The binding site can be specifically labeled with a [3H]GTP photoaffinity analog, P3-(4-azidoanilido)-5' GTP. To probe for the active center of p21, we used a battery of six monoclonal antibodies to p21 to examine their effects on p21 activities. We found that only one monoclonal antibody, Y13-259, was capable of inhibiting both GTP/GDP binding and autokinase enzymatic activities, suggesting that these p21 activities are related activities conferred by a single active center within the p21 molecule. These observations together with the recent finding that microinjection of the same monoclonal antibody into NIH 3T3 cells specifically blocks p21 in vivo function (Mulcahy et al., Nature [London] 313:241, 1985) strongly suggest that p21 in vitro activities are responsible for its cellular function.

scholarly journals Reproductive tissue selective actions of progesterone receptors

Reproduction ◽  
2004 ◽  
Vol 128 (2) ◽  
pp. 139-146 ◽  
Author(s):  
Biserka Mulac-Jericevic ◽  
Orla M Conneely
Keyword(s):  
Mammary Gland ◽  
Target Genes ◽  
Progesterone Receptors ◽  
Normal Mammary Gland ◽  
Female Reproduction ◽  
Biological Responses ◽  
Gland Morphogenesis ◽  
The Individual

The steroid hormone, progesterone, plays a central coordinate role in diverse events associated with female reproduction. In humans and other vertebrates, the biological activity of progesterone is mediated by modulation of the transcriptional activity of two progesterone receptors, PR-A and PR-B. These receptors arise from the same gene and exhibit both overlapping and distinct transcriptional activitiesin vitro. To delineate the individual roles of PR-A and PR-Bin vivo, we have generated mouse models in which expression of a single PR isoform has been ablated. Analysis of the reproductive phenotypes of these mice has indicated that PR-A and PR-B mediate mostly distinct but partially overlapping reproductive responses to progesterone. While selective ablation of the PR-A protein (PR-A knockout mice, PRAKO mice) shows normal mammary gland response to progesterone but severe uterine hyperplasia and ovarian abnormalities, ablation of PR-B protein (PRBKO mice) does not affect biological responses of the ovary or uterus to progesterone but results in reduced pregnancy-associated mammary gland morphogenesis. The distinct tissue-specific reproductive responses to progesterone exhibited by these isoforms are due to regulation of distinct subsets of progesterone-dependent target genes by the individual PR isoforms. This review will summarize our current understanding of the selective contribution of PR isoforms to the cellular and molecular actions of progesterone in reproductive tissues.

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.

Biochemical properties of a highly purified v-rasH p21 protein overproduced in Escherichia coli and inhibition of its activities by a monoclonal antibody

1985 ◽  
Vol 5 (6) ◽  
pp. 1449-1455
Author(s):  
S Hattori ◽  
L S Ulsh ◽  
K Halliday ◽  
T Y Shih
Keyword(s):  
Escherichia Coli ◽  
Monoclonal Antibody ◽  
Active Center ◽  
Biochemical Properties ◽  
Scatchard Analysis ◽  
Single Class ◽  
P21 Protein ◽  
High Level

The v-rasH oncogene of Harvey murine sarcoma virus encodes a 21,000-dalton p21 protein which has been expressed at a high level as a fusion protein in Escherichia coli. We have purified the p21 to over 90% in purity without the use of any detergent or protein denaturant. The purified p21 possesses full biochemical activities of GTP/GDP binding, autokinase, and GTPase. Scatchard analysis indicates a single class of binding sites with Kd values of 0.83 X 10(-8)M for GTP and 1.0 X 10(-8)M for GDP. The binding site can be specifically labeled with a [3H]GTP photoaffinity analog, P3-(4-azidoanilido)-5' GTP. To probe for the active center of p21, we used a battery of six monoclonal antibodies to p21 to examine their effects on p21 activities. We found that only one monoclonal antibody, Y13-259, was capable of inhibiting both GTP/GDP binding and autokinase enzymatic activities, suggesting that these p21 activities are related activities conferred by a single active center within the p21 molecule. These observations together with the recent finding that microinjection of the same monoclonal antibody into NIH 3T3 cells specifically blocks p21 in vivo function (Mulcahy et al., Nature [London] 313:241, 1985) strongly suggest that p21 in vitro activities are responsible for its cellular function.

scholarly journals C-Terminal Deletions Can Suppress Temperature-Sensitive Mutations and Change Dominance in the Phage Mu Repressor

Genetics ◽  
1996 ◽  
Vol 142 (3) ◽  
pp. 661-672 ◽  
Author(s):  
Jodi L Vogel ◽  
Vincent Geuskens ◽  
Lucie Desmet ◽  
N Patrick Higgins ◽  
Ariane Toussaint
Keyword(s):  
Binding Activity ◽  
Temperature Sensitive ◽  
Dna Binding Activity ◽  
Heat Stable ◽  
C Terminus ◽  
Acid Domain ◽  
Mutant Forms ◽  
Amino Acid Domain

Abstract Mutations in an N-terminal 70-amino acid domain of bacteriophage Mu's repressor cause temperature-sensitive DNA-binding activity. Surprisingly, amber mutations can conditionally correct the heat-sensitive defect in three mutant forms of the repressor gene, cts25 (D43-G), cts62 (R47-Q and cts71 (M28-I), and in the appropriate bacterial host produce a heat-stable Sts phenotype (for survival of temperature shifts). Sts repressor mutants are heat sensitive when in supE or supF hosts and heat resistant when in Sup° hosts. Mutants with an Sts phenotype have amber mutations at one of three codons, Q179, Q187, or Q190. The Sts phenotype relates to the repressor size: in Sup° hosts sts repressors are shorter by seven, 10, or 18 amino acids compared to repressors in supE or supF hosts. The truncated form of the sts62-1 repressor, which lacks 18 residues (Q179–V196), binds Mu operator DNA more stably at 42° in vitro compared to its full-length counterpart (cts62 repressor). In addition to influencing temperature sensitivity, the C-terminus appears to control the susceptibility to in vivo Clp proteolysis by influencing the multimeric structure of repressor.

scholarly journals In vivo transcriptome analysis provides insights into host-dependent expression of virulence factors by Yersinia entomophaga MH96, during infection of Galleria mellonella

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Amber R Paulson ◽  
Maureen O’Callaghan ◽  
Xue-Xian Zhang ◽  
Paul B Rainey ◽  
Mark R H Hurst
Keyword(s):  
Galleria Mellonella ◽  
Functional Enrichment ◽  
Natural Environments ◽  
Insecticidal Toxin ◽  
Heat Stable ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Cell Densities

Abstract The function of microbes can be inferred from knowledge of genes specifically expressed in natural environments. Here, we report the in vivo transcriptome of the entomopathogenic bacterium Yersinia entomophaga MH96, captured during initial, septicemic, and pre-cadaveric stages of intrahemocoelic infection in Galleria mellonella. A total of 1285 genes were significantly upregulated by MH96 during infection; 829 genes responded to in vivo conditions during at least one stage of infection, 289 responded during two stages of infection, and 167 transcripts responded throughout all three stages of infection compared to in vitro conditions at equivalent cell densities. Genes upregulated during the earliest infection stage included components of the insecticidal toxin complex Yen-TC (chi1, chi2, and yenC1), genes for rearrangement hotspot element containing protein yenC3, cytolethal distending toxin cdtAB, and vegetative insecticidal toxin vip2. Genes more highly expressed throughout the infection cycle included the putative heat-stable enterotoxin yenT and three adhesins (usher-chaperone fimbria, filamentous hemagglutinin, and an AidA-like secreted adhesin). Clustering and functional enrichment of gene expression data also revealed expression of genes encoding type III and VI secretion system-associated effectors. Together these data provide insight into the pathobiology of MH96 and serve as an important resource supporting efforts to identify novel insecticidal agents.

scholarly journals Selective Targeting of Breast Cancer by Tafuramycin a Using SMA-Nanoassemblies

Molecules ◽  
2021 ◽  
Vol 26 (12) ◽  
pp. 3532
Author(s):  
Ibrahim M. El-Deeb ◽  
Valeria Pittala ◽  
Diab Eltayeb ◽  
Khaled Greish
Keyword(s):  
Breast Cancer ◽  
Progesterone Receptors ◽  
In Vivo Studies ◽  
Chemotherapy Agent ◽  
Anticancer Effects ◽  
Tumor Tissues ◽  
Potential Strategy ◽  
Tumor Accumulation

Triple-negative breast cancer (TNBC) is a heterogeneous subtype of tumors that tests negative for estrogen receptors, progesterone receptors, and excess HER2 protein. The mainstay of treatment remains chemotherapy, but the therapeutic outcome remains inadequate. This paper investigates the potential of a duocarmycin derivative, tafuramycin A (TFA), as a new and more effective chemotherapy agent in TNBC treatment. To this extent, we optimized the chemical synthesis of TFA, and we encapsulated TFA in a micellar system to reduce side effects and increase tumor accumulation. In vitro and in vivo studies suggest that both TFA and SMA–TFA possess high anticancer effects in TNBC models. Finally, the encapsulation of TFA offered a preferential avenue to tumor accumulation by increasing its concentration at the tumor tissues by around four times in comparison with the free drug. Overall, the results provide a new potential strategy useful for TNBC treatment.

scholarly journals Effect of intron mutations on processing and function of Saccharomyces cerevisiae SUP53 tRNA in vitro and in vivo.

1986 ◽  
Vol 6 (7) ◽  
pp. 2663-2673 ◽  
Author(s):  
M C Strobel ◽  
J Abelson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Trna Gene ◽  
Nuclear Extract ◽  
Nonsense Suppression ◽  
Trna Genes ◽  
Suppressor Activity ◽  
Specific Sequence ◽  
Made In

The Saccharomyces cerevisiae leucine-inserting amber suppressor tRNA gene SUP53 (a tRNALeu3 allele) was used to investigate the relationship between precursor tRNA structure and mature tRNA function. This gene encodes a pre-tRNA which contains a 32-base intron. The mature tRNASUP53 contains a 5-methylcytosine modification of the anticodon wobble base. Mutations were made in the SUP53 intron. These mutant genes were transcribed in an S. cerevisiae nuclear extract preparation. In this extract, primary tRNA gene transcripts are end-processed and base modified after addition of cofactors. The base modifications made in vitro were examined, and the mutant pre-tRNAs were analyzed for their ability to serve as substrates for partially purified S. cerevisiae tRNA endonuclease and ligase. Finally, the suppressor function of these mutant tRNA genes was assayed after their integration into the S. cerevisiae genome. Mutant analysis showed that the totally intact precursor tRNA, rather than any specific sequence or structure of the intron, was necessary for efficient nonsense suppression by tRNASUP53. Less efficient suppressor activity correlated with the absence of the 5-methylcytosine modification. Most of the intron-altered precursor tRNAs were successfully spliced in vitro, indicating that modifications are not critical for recognition by the tRNA endonuclease and ligase.

scholarly journals In Vitro Probitotic Evaluation of Saccharomyces boulardii with Antimicrobial Spectrum in a Caenorhabditis elegans Model

Foods ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1428
Author(s):  
Ramachandran Chelliah ◽  
Eun-Ji Kim ◽  
Eric Banan-Mwine Daliri ◽  
Usha Antony ◽  
Deog-Hwan Oh
Keyword(s):  
Pancreatic Enzyme ◽  
Saccharomyces Boulardii ◽  
Antimicrobial Spectrum ◽  
Antimicrobial Efficiency ◽  
Heat Stable ◽  
Yeast Strains ◽  
Probiotic Yeast ◽  
Probiotic Strains

In the present study, we screened for potential probiotic yeast that could survive under extreme frozen conditions. The antimicrobial and heat-stable properties of the isolated yeast strains Saccharomyces boulardii (S. boulardii) (KT000032, KT000033, KT000034, KT000035, KT000036, and KT000037) was analyzed and compared with commercial probiotic strains. The results revealed that the tested S. boulardii KT000032 strain showed higher resistance to gastric enzymes (bile salts, pepsin, and pancreatic enzyme) at low pH, with broad antibiotic resistance. In addition, the strain also showed efficient auto-aggregation and co-aggregation abilities and efficient hydrophobicity in the in-vitro and in-vivo C. elegens gut model. Further, the KT000032 strain showed higher antimicrobial efficiency against 13 different enteropathogens and exhibited commensal relationships with five commercial probiotic strains. Besides, the bioactive compounds produced in the cell-free supernatant of probiotic yeast showed thermo-tolerance (95 °C for two hours). Furthermore, the thermo-stable property of the strains will facilitate their incorporation into ready-to-eat food products under extreme food processing conditions.

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