scholarly journals Regulation of peripheral lipogenesis by glucagon. Inability of the hormone to inhibit lipogenesis in rat mammary acini in vitro in the presence or absence of agents which alter its effects on adipocytes

1984 ◽  
Vol 217 (3) ◽  
pp. 743-749 ◽  
Author(s):  
N A Robson ◽  
R A Clegg ◽  
V A Zammit
Keyword(s):  
Concentration Ratio ◽  
Response Curve ◽  
Potent Inhibitor ◽  
Specific Binding ◽  
Insulin Dose ◽  
Mammary Glands ◽  
Key Steps ◽  
Glucagon Concentration

The rate of lipogenesis in acini isolated from mammary glands of mid-lactating rats was studied by measuring the rate of incorporation of 3H from 3H2O into total lipid and fatty acids, with glucose as substrate. Glucagon did not affect the rate of lipogenesis in acini. Glucagon did not antagonize the maximal stimulatory effect of insulin, nor did it alter the insulin dose-response curve. Theophylline, at concentrations up to 20 mM, was a potent inhibitor of lipogenesis in acini. Glucagon did not augment the degree of inhibition of lipogenesis induced by 5 mM-theophylline. The results suggest that mammary-gland acini do not respond to glucagon in vitro under conditions in which the hormone induces inhibition of lipogenesis (the present paper) and of individual key steps in the lipogenic pathway in adipocytes [Zammit & Corstorphine (1982) Biochem. J. 208, 783-788; Green (1983) Biochem. J. 212, 189-195]. In agreement with these observations, we could detect only a minimal degree of specific binding of 125I-labelled glucagon to acini which bound insulin normally. This difference in responsiveness of mammary and adipose cell preparations in vitro to glucagon suggests that the two tissues may be differentially responsive to changes in the circulating insulin/glucagon concentration ratio in vivo. The significance of these findings for the regulation of substrate utilization for lipogenesis in the two tissues during lactation is 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.

scholarly journals Predicting the in vivo developmental toxicity of benzo[a]pyrene (BaP) in rats by an in vitro–in silico approach

2021 ◽  
Author(s):  
Danlei Wang ◽  
Maartje H. Rietdijk ◽  
Lenny Kamelia ◽  
Peter J. Boogaard ◽  
Ivonne M. C. M. Rietjens
Keyword(s):  
Dose Response ◽  
In Silico ◽  
Response Curve ◽  
Developmental Toxicity ◽  
Toxicity Testing ◽  
Maximal Effect ◽  
Blood Concentrations ◽  
Reverse Dosimetry

AbstractDevelopmental toxicity testing is an animal-intensive endpoints in toxicity testing and calls for animal-free alternatives. Previous studies showed the applicability of an in vitro–in silico approach for predicting developmental toxicity of a range of compounds, based on data from the mouse embryonic stem cell test (EST) combined with physiologically based kinetic (PBK) modelling facilitated reverse dosimetry. In the current study, the use of this approach for predicting developmental toxicity of polycyclic aromatic hydrocarbons (PAHs) was evaluated, using benzo[a]pyrene (BaP) as a model compound. A rat PBK model of BaP was developed to simulate the kinetics of its main metabolite 3-hydroxybenzo[a]pyrene (3-OHBaP), shown previously to be responsible for the developmental toxicity of BaP. Comparison to in vivo kinetic data showed that the model adequately predicted BaP and 3-OHBaP blood concentrations in the rat. Using this PBK model and reverse dosimetry, a concentration–response curve for 3-OHBaP obtained in the EST was translated into an in vivo dose–response curve for developmental toxicity of BaP in rats upon single or repeated dose exposure. The predicted half maximal effect doses (ED50) amounted to 67 and 45 mg/kg bw being comparable to the ED50 derived from the in vivo dose–response data reported for BaP in the literature, of 29 mg/kg bw. The present study provides a proof of principle of applying this in vitro–in silico approach for evaluating developmental toxicity of BaP and may provide a promising strategy for predicting the developmental toxicity of related PAHs, without the need for extensive animal testing.

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 CHIR-124, a Novel Potent Inhibitor of Chk1, Potentiates the Cytotoxicity of Topoisomerase I Poisons In vitro and In vivo

2007 ◽  
Vol 13 (2) ◽  
pp. 591-602 ◽  
Author(s):  
Archie N. Tse ◽  
Katherine G. Rendahl ◽  
Tahir Sheikh ◽  
Haider Cheema ◽  
Kim Aardalen ◽  
...  
Keyword(s):  
Topoisomerase I ◽  
Potent Inhibitor

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.

Postreceptor defect in insulin action in streptozotocin-induced diabetic rats

1989 ◽  
Vol 256 (5) ◽  
pp. E624-E630 ◽  
Author(s):  
H. Nishimura ◽  
H. Kuzuya ◽  
M. Okamoto ◽  
K. Yamada ◽  
A. Kosaki ◽  
...  
Keyword(s):  
Glucose Transporter ◽  
Insulin Dose ◽  
Insulin Binding ◽  
Diabetic Rats ◽  
Glucose Disposal ◽  
Maximal Response ◽  
Metabolic Clearance ◽  
Glucose Clamp Technique

To clarify the mechanism(s) responsible for the insulin resistance in streptozotocin (STZ)-treated diabetic rats, we studied insulin-induced glucose disposal by using the glucose clamp technique and measured insulin receptor and glucose transporter of muscles. The insulin dose-response curve of the metabolic clearance rate (MCR) of glucose revealed a decrease of the maximal response without a rightward shift in STZ rats. Maximal MCR was even lower when clamped at 300 rather than 150 mg/dl of blood glucose levels. Insulin binding to the crude plasma membrane of muscles from STZ rats was increased compared with controls. The number of glucose transporter of the plasma and microsomal membranes were significantly decreased in STZ rats. These in vivo and in vitro studies using skeletal muscles suggest that in STZ-treated diabetic rats 1) a defect or defects exist in the signal transduction mechanism of insulin in postbinding steps, 2) the decreased maximal MCR is related at least partly to the decrease of glucose transporter numbers, and 3) a defect in glucose metabolism (postglucose transport defect) is also present.

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