scholarly journals In Vivo Regulation of Follicle-Stimulating Hormone Receptor by the Transcription Factors Upstream Stimulatory Factor 1 and Upstream Stimulatory Factor 2 Is Cell Specific

Endocrinology ◽  
2008 ◽  
Vol 149 (10) ◽  
pp. 5297-5306 ◽  
Author(s):  
Brian P. Hermann ◽  
Kaori Hornbaker ◽  
Daren A. Rice ◽  
Michele Sawadogo ◽  
Leslie L. Heckert
Keyword(s):  
Granulosa Cells ◽  
Sertoli Cells ◽  
Chromatin Immunoprecipitation ◽  
Leucine Zipper ◽  
Specific Binding ◽  
Pubertal Timing ◽  
Upstream Stimulatory Factor ◽  
Stimulatory Factor

Pituitary FSH promotes pubertal timing and normal gametogenesis by binding its receptor (FSHR) located on Sertoli and granulosa cells of the testis and ovary, respectively. Studies on Fshr transcription provide substantial evidence that upstream stimulatory factor (USF) 1 and USF2, basic helix-loop-helix leucine zipper proteins, regulate Fshr through an E-box within its promoter. However, despite the strong in vitro support for USF1 and USF2 in Fshr regulation, there is currently no in vivo corroborating evidence. In the present study, chromatin immunoprecipitation demonstrated specific binding of USF1 and USF2 to the Fshr promoter in both Sertoli and granulosa cells, in vivo. Control cells lacking Fshr expression showed no USF-Fshr promoter binding, thus correlating USF-promoter binding to gene activity. Evaluation of Fshr expression in Usf1 and Usf2 null mice further explored USF’s role in Fshr transcription. Loss of either gene significantly reduced ovarian Fshr levels, whereas testis levels were unaltered. Chromatin immunoprecipitation analysis of USF-Fshr promoter binding in Usf-null mice indicated differences in the composition of promoter-bound USF dimers in granulosa and Sertoli cells. Promoter-bound USF dimer levels declined in granulosa cells from both null mice, despite increased USF2 levels in Usf1-null ovaries. However, compensatory increases in promoter-bound USF homodimers were evident in Usf-null Sertoli cells. In summary, this study provides the first in vivo evidence that USF1 and USF2 bind the Fshr promoter and revealed differences between Sertoli and granulosa cells in compensatory responses to USF loss and the USF dimeric composition required for Fshr transcription.

scholarly journals Follicle-Stimulating Hormone (FSH) Transiently Blocks FSH Receptor Transcription by Increasing Inhibitor of Deoxyribonucleic Acid Binding/Differentiation-2 and Decreasing Upstream Stimulatory Factor Expression in Rat Sertoli Cells

Endocrinology ◽  
2009 ◽  
Vol 150 (8) ◽  
pp. 3783-3791 ◽  
Author(s):  
Pushpa Viswanathan ◽  
Michelle A. Wood ◽  
William H. Walker
Keyword(s):  
Protein Binding ◽  
Sertoli Cells ◽  
Fsh Receptor ◽  
Upstream Stimulatory Factor ◽  
Binding Studies ◽  
Nuclear Extracts ◽  
E Box ◽  
Stimulatory Factor ◽  
Stimulation Of

FSH acts through the FSH receptor (FSHR) to modulate cell processes that are required to support developing spermatozoa. Within the testis, only Sertoli cells possess receptors for FSH and are the major targets for this regulator of spermatogenesis. FSH stimulation of Sertoli cells for 24–48 h is known to induce Fshr mRNA expression through an E-box motif (CACGTG) located 25 bp upstream of the transcription start site. In contrast, FSH stimulation for 8 h inhibits Fshr transcription. DNA-protein binding studies performed using nuclear extracts from Sertoli cells show that protein binding to the Fshr promoter E-box was reduced 68% after 6 h of FSH stimulation but increased 191% over basal levels after 48 h of stimulation. The proteins binding to the Fshr E-box were identified as upstream stimulatory factor (USF)-1 and -2. FSH stimulation transiently decreased USF1 levels and increased the expression of the inhibitor of DNA binding/differentiation (ID)-2 repressor protein with the same kinetics as the decreased USF/E-box interactions. Overexpression of ID2 resulted in a dose-dependent decrease in USF-driven Fshr promoter activity in the MSC-1 Sertoli cell line, and ID2 inhibited USF binding to the Fshr E-box. Together, these studies suggest that stimulation of Sertoli cells with FSH transiently decreases expression of the USF1 activator and induces accumulation of the ID2 repressor, to block USF binding to the Fshr promoter and delay activation of Fshr transcription. This FSH-regulated mechanism may explain the cyclical changes in Fshr expression that occurs in Sertoli cells in vivo.

scholarly journals B23/nucleophosmin is involved in regulation of adenovirus chromatin structure at late infection stages, but not in virus replication and transcription

2012 ◽  
Vol 93 (6) ◽  
pp. 1328-1338 ◽  
Author(s):  
Mohammad Abdus Samad ◽  
Tetsuro Komatsu ◽  
Mitsuru Okuwaki ◽  
Kyosuke Nagata
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Infectious Virus ◽  
Core Protein ◽  
Virus Genome ◽  
Virus Particles ◽  
Core Proteins ◽  
Stimulatory Factor ◽  
Interfering Rna

B23/nucleophosmin has been identified in vitro as a stimulatory factor for replication of adenovirus DNA complexed with viral basic core proteins. In the present study, the in vivo function of B23 in the adenovirus life cycle was studied. It was found that both the expression of a decoy mutant derived from adenovirus core protein V that tightly associates with B23 and small interfering RNA-mediated depletion of B23 impeded the production of progeny virions. However, B23 depletion did not significantly affect the replication and transcription of the virus genome. Chromatin immunoprecipitation analyses revealed that B23 depletion significantly increased the association of viral DNA with viral core proteins and cellular histones. These results suggest that B23 is involved in the regulation of association and/or dissociation of core proteins and cellular histones with the virus genome. In addition, these results suggest that proper viral chromatin assembly, regulated in part by B23, is crucial for the maturation of infectious virus particles.

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.

scholarly journals Base-edited CAR T cells for combinational therapy against T cell malignancies

Leukemia ◽  
2021 ◽  
Author(s):  
Christos Georgiadis ◽  
Jane Rasaiyaah ◽  
Soragia Athina Gkazi ◽  
Roland Preece ◽  
Aniekan Etuk ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Specific Binding ◽  
Base Editing ◽  
Car T Cells ◽  
Dna And Rna ◽  
Car T ◽  
T Cell Malignancies

AbstractTargeting T cell malignancies using chimeric antigen receptor (CAR) T cells is hindered by ‘T v T’ fratricide against shared antigens such as CD3 and CD7. Base editing offers the possibility of seamless disruption of gene expression of problematic antigens through creation of stop codons or elimination of splice sites. We describe the generation of fratricide-resistant T cells by orderly removal of TCR/CD3 and CD7 ahead of lentiviral-mediated expression of CARs specific for CD3 or CD7. Molecular interrogation of base-edited cells confirmed elimination of chromosomal translocations detected in conventional Cas9 treated cells. Interestingly, 3CAR/7CAR co-culture resulted in ‘self-enrichment’ yielding populations 99.6% TCR−/CD3−/CD7−. 3CAR or 7CAR cells were able to exert specific cytotoxicity against leukaemia lines with defined CD3 and/or CD7 expression as well as primary T-ALL cells. Co-cultured 3CAR/7CAR cells exhibited highest cytotoxicity against CD3 + CD7 + T-ALL targets in vitro and an in vivo human:murine chimeric model. While APOBEC editors can reportedly exhibit guide-independent deamination of both DNA and RNA, we found no problematic ‘off-target’ activity or promiscuous base conversion affecting CAR antigen-specific binding regions, which may otherwise redirect T cell specificity. Combinational infusion of fratricide-resistant anti-T CAR T cells may enable enhanced molecular remission ahead of allo-HSCT for T cell malignancies.

scholarly journals Development of 18F-Labeled Radiotracers for PET Imaging of the Adenosine A2A Receptor: Synthesis, Radiolabeling and Preliminary Biological Evaluation

2021 ◽  
Vol 22 (5) ◽  
pp. 2285
Author(s):  
Thu Hang Lai ◽  
Susann Schröder ◽  
Magali Toussaint ◽  
Sladjana Dukić-Stefanović ◽  
Mathias Kranz ◽  
...  
Keyword(s):  
Specific Binding ◽  
Brain Slices ◽  
Total Activity ◽  
Biological Evaluation ◽  
Adenosine A2a Receptor ◽  
Positron Emission ◽  
A2a Receptor ◽  
Adenosine A2a

The adenosine A2A receptor (A2AR) represents a potential therapeutic target for neurodegenerative diseases. Aiming at the development of a positron emission tomography (PET) radiotracer to monitor changes of receptor density and/or occupancy during the A2AR-tailored therapy, we designed a library of fluorinated analogs based on a recently published lead compound (PPY). Among those, the highly affine 4-fluorobenzyl derivate (PPY1; Ki(hA2AR) = 5.3 nM) and the 2-fluorobenzyl derivate (PPY2; Ki(hA2AR) = 2.1 nM) were chosen for 18F-labeling via an alcohol-enhanced copper-mediated procedure starting from the corresponding boronic acid pinacol ester precursors. Investigations of the metabolic stability of [18F]PPY1 and [18F]PPY2 in CD-1 mice by radio-HPLC analysis revealed parent fractions of more than 76% of total activity in the brain. Specific binding of [18F]PPY2 on mice brain slices was demonstrated by in vitro autoradiography. In vivo PET/magnetic resonance imaging (MRI) studies in CD-1 mice revealed a reasonable high initial brain uptake for both radiotracers, followed by a fast clearance.

scholarly journals Ezrin is an Actin Binding Protein That Regulates Sertoli Cell and Spermatid Adhesion During Spermatogenesis

Endocrinology ◽  
2014 ◽  
Vol 155 (10) ◽  
pp. 3981-3995 ◽  
Author(s):  
N. Ece Gungor-Ordueri ◽  
Elizabeth I. Tang ◽  
Ciler Celik-Ozenci ◽  
C. Yan Cheng
Keyword(s):  
Sertoli Cell ◽  
Sertoli Cells ◽  
Adherens Junction ◽  
Actin Binding ◽  
Permeability Barrier ◽  
Tight Junction Permeability ◽  
Residual Bodies ◽  
Cell Interface

Abstract During spermatogenesis, the transport of spermatids and the release of sperms at spermiation and the remodeling of the blood-testis barrier (BTB) in the seminiferous epithelium of rat testes require rapid reorganization of the actin-based cytoskeleton. However, the mechanism(s) and the regulatory molecule(s) remain unexplored. Herein we report findings that unfold the functional significance of ezrin in the organization of the testis-specific adherens junction at the spermatid-Sertoli cell interface called apical ectoplasmic specialization (ES) in the adluminal compartment and the Sertoli cell-cell interface known as basal ES at the BTB. Ezrin is expressed at the basal ES/BTB in all stages, except from late VIII to IX, of the epithelial cycle. Its knockdown by RNA interference (RNAi) in vitro perturbs the Sertoli cell tight junction-permeability barrier via a disruption of the actin microfilaments in Sertoli cells, which in turn impeded basal ES protein (eg, N-cadherin) distribution, perturbing the BTB function. These findings were confirmed by a knockdown study in vivo. However, the expression of ezrin at the apical ES is restricted to stage VIII of the cycle and limited only between step 19 spermatids and Sertoli cells. A knockdown of ezrin in vivo by RNAi was found to impede spermatid transport, causing defects in spermiation in which spermatids were embedded deep inside the epithelium, and associated with a loss of spermatid polarity. Also, ezrin was associated with residual bodies and phagosomes, and its knockdown by RNAi in the testis also impeded the transport of residual bodies/phagosomes from the apical to the basal compartment. In summary, ezrin is involved in regulating actin microfilament organization at the ES in rat testes.

Binding of a cellular protein to the gibbon ape leukemia virus enhancer

1987 ◽  
Vol 7 (8) ◽  
pp. 2735-2744
Author(s):  
J P Quinn ◽  
N Holbrook ◽  
D Levens
Keyword(s):  
Long Terminal Repeat ◽  
Specific Binding ◽  
Leukemia Virus ◽  
Cellular Protein ◽  
Terminal Repeat ◽  
Enhancer Activity ◽  
Repeated Element ◽  
Gibbon Ape Leukemia Virus

The gibbon ape leukemia virus (GALV) contains enhancer activity within its long terminal repeat. In the GALV Seato strain this activity resides in a 48-base-pair (bp) repeated element. We demonstrate the existence of a cellular protein which binds in this region of the Seato strain. A sensitive method for enriching protein-DNA complexes from crude extracts coupled with exonuclease and DNase footprint analysis revealed the specific binding of this protein to a 21-bp region within each repeated element. A 22-bp oligonucleotide fragment defined solely by the 21-bp footprint binds a protein in vitro and displays enhancer activity in vivo, suggesting that this protein is a major determinant of GALV enhancer activity. The protein is present in three cell lines which are positive for enhancer activity and is not detected in Jurkat cells, which are negative for enhancer activity. Only GALV long-terminal-repeat variants which support high levels of enhancer activity in vivo compete with this protein for specific binding in vitro, suggesting a potential role for the protein in determining enhancer activity. This protein binding is not inhibited by competition with heterologous retroviral enhancers, demonstrating that it is not a ubiquitous retroviral enhancer binding protein.

scholarly journals Evaluation of Radiolabeled Girentuximab In Vitro and In Vivo

2018 ◽  
Vol 11 (4) ◽  
pp. 132 ◽  
Author(s):  
Tais Basaco ◽  
Stefanie Pektor ◽  
Josue Bermudez ◽  
Niurka Meneses ◽  
Manfred Heller ◽  
...  
Keyword(s):  
Specific Binding ◽  
Sodium Ascorbate ◽  
Tumor Uptake ◽  
Tetraacetic Acid ◽  
In Vivo Experiments ◽  
Caix Expression ◽  
Tetraazacyclododecane Tetraacetic Acid

Girentuximab (cG250) targets carbonic anhydrase IX (CAIX), a protein which is expressed on the surface of most renal cancer cells (RCCs). cG250 labeled with 177Lu has been used in clinical trials for radioimmunotherapy (RIT) of RCCs. In this work, an extensive characterization of the immunoconjugates allowed optimization of the labeling conditions with 177Lu while maintaining immunoreactivity of cG250, which was then investigated in in vitro and in vivo experiments. cG250 was conjugated with S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (DOTA(SCN)) by using incubation times between 30 and 90 min and characterized by mass spectrometry. Immunoconjugates with five to ten DOTA(SCN) molecules per cG250 molecule were obtained. Conjugates with ratios less than six DOTA(SCN)/cG250 had higher in vitro antigen affinity, both pre- and postlabeling with 177Lu. Radiochemical stability increased, in the presence of sodium ascorbate, which prevents radiolysis. The immunoreactivity of the radiolabeled cG250 tested by specific binding to SK-RC-52 cells decreased when the DOTA content per conjugate increased. The in vivo tumor uptake was < 10% ID/g and independent of the total amount of protein in the range between 5 and 100 µg cG250 per animal. Low tumor uptake was found to be due to significant necrotic areas and heterogeneous CAIX expression. In addition, low vascularity indicated relatively poor accessibility of the CAIX target.

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