Characterization by saturation studies of the in vivo binding of [3H]flunitrazepam in mouse brain

1991 ◽  
Vol 69 (2) ◽  
pp. 176-180 ◽  
Author(s):  
Peter T.-H. Wong
Keyword(s):  
Mouse Brain ◽  
Incubation Temperature ◽  
Specific Binding ◽  
Protein A ◽  
Receptor Occupancy ◽  
Acute Tolerance ◽  
Alternative Methods ◽  
In Vivo Binding

The in vivo binding of [3H]flunitrazepam ([3H]Fln) was characterized in seven regions of the mouse brain. The binding showed saturability and linear Scatchard plots. Hill coefficients were close to unity. Data fitting to a hyperbola by least squares yielded consistent Kd values for all regions studied (0.36–0.6 pmol/mg protein). Bmax values ranged from 0.14 to 0.89 pmol/mg protein, a sixfold regional variation. The order of binding is as follows: cortex > hippocampus > midbrain = thalamus/hypothalamus > striatum ≥ cerebellum > brainstem, consistent with that obtained by in vitro binding. The in vivo receptor density and affinity are apparently lower in comparison with in vitro parameters. This is consistent with the observation that the Kd increases and Bmax decreases in vitro when the incubation temperature is increased from 0 °C. Non-specific binding has been estimated by displacement of in vivo binding by unlabelled ligand in vitro as well as by pretreatment with unlabelled ligand. The two alternative methods were compared and evaluated. It is concluded that the displacement method provides more reliable estimates of the nonspecific binding. Diazepam-sensitive mice did not differ from the control mice in the in vivo [3H]Fln binding. However, mice pretreated with diazepam 1 or 2 days before have binding reduced by 70 or 30%, respectively. The reduced binding may be explained by receptor occupancy by residual oxazepam. However, the low concentration of the residual oxazepam is an unlikely cause of the phenomenon of "acute tolerance" observed in these mice.Key words: benzodiazepines, in vivo binding, characterization, diazepam-sensitive mice, acute tolerance.

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.

A Study on UV Filter Chemicals from Annex VII of European Union Directive 76/768/EEC, in the In Vitro 3T3 NRU Phototoxicity Test

1998 ◽  
Vol 26 (5) ◽  
pp. 679-708 ◽  
Author(s):  
Horst Spielmann ◽  
Michael Balls ◽  
Jack Dupuis ◽  
Wolfgang J. W. Pape ◽  
Odile de Silva ◽  
...  
Keyword(s):  
Neutral Red ◽  
Optimum Concentration ◽  
Alternative Methods ◽  
Uv Filter ◽  
Eu Directive ◽  
Optimum Concentration Range ◽  
The Impact ◽  
Positive Results

In 1996, the Scientific Committee on Cosmetology of DGXXIV of the European Commission asked the European Centre for the Validation of Alternative Methods to test eight UV filter chemicals from the 1995 edition of Annex VII of Directive 76/768/EEC in a blind trial in the in vitro 3T3 cell neutral red uptake phototoxicity (3T3 NRU PT) test, which had been scientifically validated between 1992 and 1996. Since all the UV filter chemicals on the positive list of EU Directive 76/768/EEC have been shown not to be phototoxic in vivo in humans under use conditions, only negative effects would be expected in the 3T3 NRU PT test. To balance the number of positive and negative chemicals, ten phototoxic and ten non-phototoxic chemicals were tested under blind conditions in four laboratories. Moreover, to assess the optimum concentration range for testing, information was provided on appropriate solvents and on the solubility of the coded chemicals. In this study, the phototoxic potential of test chemicals was evaluated in a prediction model in which either the Photoirritation Factor (PIF) or the Mean Photo Effect (MPE) were determined. The results obtained with both PIF and MPE were highly reproducible in the four laboratories, and the correlation between in vitro and in vivo data was almost perfect. All the phototoxic test chemicals provided a positive result at concentrations of 1μg/ml, while nine of the ten non-phototoxic chemicals gave clear negative results, even at the highest test concentrations. One of the UV filter chemicals gave positive results in three of the four laboratories only at concentrations greater than 100μg/ml; the other laboratory correctly identified all 20 of the test chemicals. An analysis of the impact that exposure concentrations had on the performance of the test revealed that the optimum concentration range in the 3T3 NRU PT test for determining the phototoxic potential of chemicals is between 0.1μg/ml and 10μg/ml, and that false positive results can be obtained at concentrations greater than 100μg/ml. Therefore, the positive results obtained with some of the UV filter chemicals only at concentrations greater than 100μg/ml do not indicate a phototoxic potential in vivo. When this information was taken into account during calculation of the overall predictivity of the 3T3 NRU PT test in the present study, an almost perfect correlation of in vitro versus in vivo results was obtained (between 95% and 100%), when either PIF or MPE were used to predict the phototoxic potential. The management team and participants therefore conclude that the 3T3 NRU PT test is a valid test for correctly assessing the phototoxic potential of UV filter chemicals, if the defined concentration limits are taken into account.

scholarly journals Characterization of the SARS-CoV-2 coronavirus X4-like accessory protein

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Olanrewaju Ayodeji Durojaye ◽  
Nkwachukwu Oziamara Okoro ◽  
Arome Solomon Odiba
Keyword(s):  
Rapid Development ◽  
Protein A ◽  
The Novel ◽  
Quality Model ◽  
A Value ◽  
Model Protein ◽  
Novel Coronavirus ◽  
Global Threat

Abstract Background The novel coronavirus SARS-CoV-2 is currently a global threat to health and economies. Therapeutics and vaccines are in rapid development; however, none of these therapeutics are considered as absolute cure, and the potential to mutate makes it necessary to find therapeutics that target a highly conserved regions of the viral structure. Results In this study, we characterized an essential but poorly understood coronavirus accessory X4 protein, a core and stable component of the SARS-CoV family. Sequence analysis shows a conserved ~ 90% identity between the SARS-CoV-2 and previously characterized X4 protein in the database. QMEAN Z score of the model protein shows a value of around 0.5, within the acceptable range 0–1. A MolProbity score of 2.96 was obtained for the model protein and indicates a good quality model. The model has Ramachandran values of φ = − 57o and ψ = − 47o for α-helices and values of φ = − 130o and ψ = + 140o for twisted sheets. Conclusions The protein data obtained from this study provides robust information for further in vitro and in vivo experiment, targeted at devising therapeutics against the virus. Phylogenetic analysis further supports previous evidence that the SARS-CoV-2 is positioned with the SL-CoVZC45, BtRs-BetaCoV/YN2018B and the RS4231 Bat SARS-like corona viruses.

scholarly journals The Yeast Recombinational Repair Protein Rad59 Interacts With Rad52 and Stimulates Single-Strand Annealing

Genetics ◽  
2001 ◽  
Vol 159 (2) ◽  
pp. 515-525 ◽  
Author(s):  
Allison P Davis ◽  
Lorraine S Symington
Keyword(s):  
Protein A ◽  
Single Strand ◽  
Physical Interaction ◽  
Dna Double Strand Breaks ◽  
Single Stranded Dna ◽  
Single Strand Annealing ◽  
Strand Annealing ◽  
Recombination Pathway

Abstract The yeast RAD52 gene is essential for homology-dependent repair of DNA double-strand breaks. In vitro, Rad52 binds to single- and double-stranded DNA and promotes annealing of complementary single-stranded DNA. Genetic studies indicate that the Rad52 and Rad59 proteins act in the same recombination pathway either as a complex or through overlapping functions. Here we demonstrate physical interaction between Rad52 and Rad59 using the yeast two-hybrid system and co-immunoprecipitation from yeast extracts. Purified Rad59 efficiently anneals complementary oligonucleotides and is able to overcome the inhibition to annealing imposed by replication protein A (RPA). Although Rad59 has strand-annealing activity by itself in vitro, this activity is insufficient to promote strand annealing in vivo in the absence of Rad52. The rfa1-D288Y allele partially suppresses the in vivo strand-annealing defect of rad52 mutants, but this is independent of RAD59. These results suggest that in vivo Rad59 is unable to compete with RPA for single-stranded DNA and therefore is unable to promote single-strand annealing. Instead, Rad59 appears to augment the activity of Rad52 in strand annealing.

scholarly journals An In Vitro Study of the Effect of Viburnum opulus Extracts on Key Processes in the Development of Staphylococcal Infections

Molecules ◽  
2021 ◽  
Vol 26 (6) ◽  
pp. 1758
Author(s):  
Urszula Wójcik-Bojek ◽  
Joanna Rywaniak ◽  
Przemysław Bernat ◽  
Anna Podsędek ◽  
Dominika Kajszczak ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Protein A ◽  
In Vitro Study ◽  
Alternative Methods ◽  
Staphylococcal Protein A ◽  
Viburnum Opulus ◽  
Staphylococcal Infections ◽  
Diet Supplements ◽  
Drug Resistant Strains

Staphylococcus aureus is still one of the leading causes of both hospital- and community-acquired infections. Due to the very high percentage of drug-resistant strains, the participation of drug-tolerant biofilms in pathological changes, and thus the limited number of effective antibiotics, there is an urgent need to search for alternative methods of prevention or treatment for S. aureus infections. In the present study, biochemically characterized (HPLC/UPLC–QTOF–MS) acetonic, ethanolic, and water extracts from fruits and bark of Viburnum opulus L. were tested in vitro as diet additives that potentially prevent staphylococcal infections. The impacts of V. opulus extracts on sortase A (SrtA) activity (Fluorimetric Assay), staphylococcal protein A (SpA) expression (FITC-labelled specific antibodies), the lipid composition of bacterial cell membranes (LC-MS/MS, GC/MS), and biofilm formation (LIVE/DEAD BacLight) were assessed. The cytotoxicity of V. opulus extracts to the human fibroblast line HFF-1 was also tested (MTT reduction). V. opulus extracts strongly inhibited SrtA activity and SpA expression, caused modifications of S. aureus cell membrane, limited biofilm formation by staphylococci, and were non-cytotoxic. Therefore, they have pro-health potential. Nevertheless, their usefulness as diet supplements that are beneficial for the prevention of staphylococcal infections should be confirmed in animal models in the future.

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 UV-induced Hyperphosphorylation of Replication Protein A Depends on DNA Replication and Expression of ATM Protein

2001 ◽  
Vol 12 (5) ◽  
pp. 1199-1213 ◽  
Author(s):  
Gregory G. Oakley ◽  
Lisa I. Loberg ◽  
Jiaqin Yao ◽  
Mary A. Risinger ◽  
Remy L. Yunker ◽  
...  
Keyword(s):  
Dna Replication ◽  
Uv Radiation ◽  
Excision Repair ◽  
Genetic Disorder ◽  
Protein A ◽  
Dna Recombination ◽  
Delayed Response ◽  
Replication Intermediates

Exposure to DNA-damaging agents triggers signal transduction pathways that are thought to play a role in maintenance of genomic stability. A key protein in the cellular processes of nucleotide excision repair, DNA recombination, and DNA double-strand break repair is the single-stranded DNA binding protein, RPA. We showed previously that the p34 subunit of RPA becomes hyperphosphorylated as a delayed response (4–8 h) to UV radiation (10–30 J/m2). Here we show that UV-induced RPA-p34 hyperphosphorylation depends on expression of ATM, the product of the gene mutated in the human genetic disorder ataxia telangiectasia (A-T). UV-induced RPA-p34 hyperphosphorylation was not observed in A-T cells, but this response was restored by ATM expression. Furthermore, purified ATM kinase phosphorylates the p34 subunit of RPA complex in vitro at many of the same sites that are phosphorylated in vivo after UV radiation. Induction of this DNA damage response was also dependent on DNA replication; inhibition of DNA replication by aphidicolin prevented induction of RPA-p34 hyperphosphorylation by UV radiation. We postulate that this pathway is triggered by the accumulation of aberrant DNA replication intermediates, resulting from DNA replication fork blockage by UV photoproducts. Further, we suggest that RPA-p34 is hyperphosphorylated as a participant in the recombinational postreplication repair of these replication products. Successful resolution of these replication intermediates reduces the accumulation of chromosomal aberrations that would otherwise occur as a consequence of UV radiation.

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