scholarly journals Functional studies on human Pex7p: subcellular localization and interaction with proteins containing a peroxisome-targeting signal type 2 and other peroxins

2002 ◽  
Vol 365 (1) ◽  
pp. 41-50 ◽  
Author(s):  
Karen GHYS ◽  
Marc FRANSEN ◽  
Guy P. MANNAERTS ◽  
Paul P. Van VELDHOVEN
Keyword(s):  
Fluorescent Protein ◽  
Specific Binding ◽  
Chinese Hamster ◽  
Translation System ◽  
Chondrodysplasia Punctata ◽  
Rhizomelic Chondrodysplasia Punctata ◽  
Functional Studies ◽  
Targeting Signal

Pex7p is a WD40-containing protein involved in peroxisomal import of proteins containing an N-terminal peroxisome-targeting signal (PTS2). The interaction of human recombinant Pex7p expressed in different hosts/systems with its PTS2 ligand and other peroxins was analysed using various experimental approaches. Specific binding of human Pex7p to PTS2 could be demonstrated only when Pex7p was formed in vitro by a coupled transcription/translation system or synthesized in vivo in Chinese hamster ovary K1 cells transfected with a construct coding for a Pex7p-green fluorescent protein (GFP) fusion protein. Apparently, no cofactors are required and only monomeric Pex7p binds to PTS2. The interaction is reduced upon cysteine alkylation and is impaired upon truncation of the N-terminus of Pex7p. Interaction of Pex7p with other peroxins could not be demonstrated in bacterial or yeast two-hybrid screens, or in pull-down binding assays. The GFP fusion proteins, tagged at either the N- or C-terminus, were able to restore PTS2 import in rhizomelic chondrodysplasia punctata fibroblasts, and Pex7p-GFP was located both in the lumen of peroxisomes and in the cytosol.

scholarly journals The m6A demethylase FTO promotes esophageal cancer progression through YTHDF1-dependent posttranscriptional silencing of AKT3

2021 ◽  
Author(s):  
Chunbao Zang ◽  
Fangfang Zhao ◽  
Dabing Huang ◽  
Lingsuo Kong ◽  
Minghua Xie ◽  
...  
Keyword(s):  
Esophageal Cancer ◽  
Cancer Progression ◽  
Target Genes ◽  
Specific Binding ◽  
Messenger Rnas ◽  
Rna Seq ◽  
Functional Studies ◽  
Rna Immunoprecipitation

Abstract Background : N 6 -methyladenosine (m 6 A) is the most abundant modification in eukaryotic messenger RNAs (mRNAs), and plays important roles in many bioprocesses. However, its functions in esophageal cancer remain elusive. Methods : Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and transcriptomic RNA sequencing (RNA-seq) were used to screen the target genes of FTO. Western blot, quantitative real-time PCR (RT-qPCR) and immunohistochemical (IHC) were used to detect FTO expression in cell lines and patient tissues. The biological functions of FTO were investigated in vitro and in vivo . RNA pull-down and RNA immunoprecipitation assays were conducted to explore the specific binding of target genes. Results : We discovered that the RNA demethylase FTO was significantly up-regulated in esophageal cancer patients. Knockdown of FTO drastically reduced esophageal cancer cells (ESCCs) proliferation, migration, invasion, and apoptosis. On the other hand, overexpression of FTO significantly promoted ESCCs growth and invasion. Moreover, we found that the m 6 A methyltransferase METTL14 negatively correlates with FTO function on esophageal cancer progression. By using transcriptome-wide m 6 A-Seq and RNA-Seq assays, we identified AKT3 is the target of FTO, which acts in concert in esophageal cancer tumorigenesis and metastasis. Moreover, loss and gain functional studies confirm that YTHDF1 mediates m 6 A-increased translation of AKT3 mRNA. Conclusion : Our results uncovered an METTL14/FTO/YTHDF1/AKT3 signaling network that regulates the esophageal cancer progression.

Activation of EGFP expression by Cre-mediated excision in a new ROSA26 reporter mouse strain

Blood ◽  
2001 ◽  
Vol 97 (1) ◽  
pp. 324-326 ◽  
Author(s):  
Xiaohong Mao ◽  
Yuko Fujiwara ◽  
Aimée Chapdelaine ◽  
Haidi Yang ◽  
Stuart H. Orkin
Keyword(s):  
Fluorescent Protein ◽  
Threshold Level ◽  
Egfp Expression ◽  
Mouse Strains ◽  
Reporter Strain ◽  
Egfp Gene ◽  
Reporter Mouse ◽  
Functional Studies

Abstract Reporter mouse strains are important tools for monitoring Cre recombinase-mediated excision in vivo. In practice, excision may be incomplete in a given population due to threshold level or variegated expression of Cre. Hence, it is desirable in many experimental contexts to isolate cells that have undergone excision to assess the consequences of gene ablation. To generate alternative reporter mice, an enhanced green fluorescent protein (EGFP) gene was targeted to the retroviral-trapped ROSA26 locus. Upon Cre-mediated excision of “Stop” sequences, EGFP was expressed ubiquitously during embryogenesis and in adult tissues (including T cells, B cells, and myeloid cells). Using this new reporter strain, separation of excised from nonexcised cells in vitro was achieved in thymocytes in a noninvasive manner based on activated EGFP expression. This new EGFP reporter strain should facilitate a variety of conditional gene-targeting experiments, including the functional studies of hematopoietic cells in lineage-specific knockout mice.

scholarly journals The C-Terminal Tail of the Polycystin-1 Protein Interacts with the Na,K-ATPase α-Subunit

2005 ◽  
Vol 16 (11) ◽  
pp. 5087-5093 ◽  
Author(s):  
Alessandra Zatti ◽  
Veronique Chauvet ◽  
Vanathy Rajendran ◽  
Thoru Kimura ◽  
Phillip Pagel ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Kinetic Properties ◽  
Α Subunit ◽  
Ovary Cells ◽  
Functional Studies ◽  
Autosomal Dominant Polycystic Kidney

Polycystin-1 (PC-1) is the product of the PKD1 gene, which is mutated in autosomal dominant polycystic kidney disease. We show that the Na,K-ATPase α-subunit interacts in vitro and in vivo with the final 200 amino acids of the polycystin-1 protein, which constitute its cytoplasmic C-terminal tail. Functional studies suggest that this association may play a role in the regulation of the Na,K-ATPase activity. Chinese hamster ovary cells stably expressing the entire PC-1 protein exhibit a dramatic increase in Na,K-ATPase activity, although the kinetic properties of the enzyme remain unchanged. These data indicate that polycystin-1 may contribute to the regulation of Na,K-ATPase activity in kidneys in situ, thus modulating renal tubular fluid and electrolyte transport.

scholarly journals The m6A Demethylase FTO Promotes Esophageal Cancer Progression Through YTHDF1-Dependent Posttranscriptional Silencing of AKT3

2021 ◽  
Author(s):  
Chunbao Zang ◽  
Fangfang Zhao ◽  
Dabing Huang ◽  
Lingsuo Kong ◽  
Minghua Xie ◽  
...  
Keyword(s):  
Esophageal Cancer ◽  
Cancer Progression ◽  
Target Genes ◽  
Specific Binding ◽  
Messenger Rnas ◽  
Rna Seq ◽  
Functional Studies ◽  
Rna Immunoprecipitation

Abstract Background N6-methyladenosine (m6A) is the most abundant modification in eukaryotic messenger RNAs (mRNAs), and plays important roles in many bioprocesses. However, its functions in esophageal cancer (ES) remain elusive. Methods Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and transcriptomic RNA sequencing (RNA-seq) were used to screen the target genes of FTO. Western blot, quantitative real-time PCR (RT-qPCR) and immunohistochemical (IHC) were used to detect FTO expression in cell lines and patient tissues. The biological functions of FTO were investigated in vitro and in vivo. RNA pull-down and RNA immunoprecipitation assays were conducted to explore the specific binding of target genes. Results We discovered that the RNA demethylase FTO was significantly up-regulated in human ES. Knockdown of FTO drastically reduced ESCCs cell proliferation, migration, invasion, and apoptosis in the ESCCs. On the other hand, overexpression of FTO significantly promoted ESCCs growth and invasion. Moreover, we found that the m6A methyltransferase METTL14 negatively correlates with FTO function on ES progression. By using transcriptome-wide m6A-Seq and RNA-Seq assays, we identified AKT3 is the target of FTO, which acts in concert in esophageal cancer tumorigenesis and metastasis. Moreover, loss and gain functional studies confirm that YTHDF1 mediates m6A-increased translation of AKT3 mRNA. Conclusion Our results uncovered an METTL14/FTO/YTHDF1/AKT3 signaling network that regulates the ES progression.

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 POLRMT mutations impair mitochondrial transcription causing neurological disease

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Monika Oláhová ◽  
Bradley Peter ◽  
Zsolt Szilagyi ◽  
Hector Diaz-Maldonado ◽  
Meenakshi Singh ◽  
...  
Keyword(s):  
Progressive External Ophthalmoplegia ◽  
Global Developmental Delay ◽  
Disease Mechanism ◽  
Mitochondrial Transcription ◽  
Functional Studies ◽  
Mtdna Deletions ◽  
Molecular Nature ◽  
Important Disease

AbstractWhile >300 disease-causing variants have been identified in the mitochondrial DNA (mtDNA) polymerase γ, no mitochondrial phenotypes have been associated with POLRMT, the RNA polymerase responsible for transcription of the mitochondrial genome. Here, we characterise the clinical and molecular nature of POLRMT variants in eight individuals from seven unrelated families. Patients present with global developmental delay, hypotonia, short stature, and speech/intellectual disability in childhood; one subject displayed an indolent progressive external ophthalmoplegia phenotype. Massive parallel sequencing of all subjects identifies recessive and dominant variants in the POLRMT gene. Patient fibroblasts have a defect in mitochondrial mRNA synthesis, but no mtDNA deletions or copy number abnormalities. The in vitro characterisation of the recombinant POLRMT mutants reveals variable, but deleterious effects on mitochondrial transcription. Together, our in vivo and in vitro functional studies of POLRMT variants establish defective mitochondrial transcription as an important disease mechanism.

scholarly journals Development of a Fluorescent Tool for Studying Legionella bozemanae Intracellular Infection

2021 ◽  
Vol 9 (2) ◽  
pp. 379
Author(s):  
Breanne M. Head ◽  
Christopher I. Graham ◽  
Teassa MacMartin ◽  
Yoav Keynan ◽  
Ann Karen C. Brassinga
Keyword(s):  
Growth Kinetics ◽  
Legionella Pneumophila ◽  
Fluorescent Protein ◽  
Disease Incidence ◽  
Acanthamoeba Castellanii ◽  
Infection Model ◽  
Intracellular Infection ◽  
Green Fluorescent

Legionnaires’ disease incidence is on the rise, with the majority of cases attributed to the intracellular pathogen, Legionella pneumophila. Nominally a parasite of protozoa, L. pneumophila can also infect alveolar macrophages when bacteria-laden aerosols enter the lungs of immunocompromised individuals. L. pneumophila pathogenesis has been well characterized; however, little is known about the >25 different Legionella spp. that can cause disease in humans. Here, we report for the first time a study demonstrating the intracellular infection of an L. bozemanae clinical isolate using approaches previously established for L. pneumophila investigations. Specifically, we report on the modification and use of a green fluorescent protein (GFP)-expressing plasmid as a tool to monitor the L. bozemanae presence in the Acanthamoeba castellanii protozoan infection model. As comparative controls, L. pneumophila strains were also transformed with the GFP-expressing plasmid. In vitro and in vivo growth kinetics of the Legionella parental and GFP-expressing strains were conducted followed by confocal microscopy. Results suggest that the metabolic burden imposed by GFP expression did not impact cell viability, as growth kinetics were similar between the GFP-expressing Legionella spp. and their parental strains. This study demonstrates that the use of a GFP-expressing plasmid can serve as a viable approach for investigating Legionella non-pneumophila spp. in real time.

scholarly journals CRISPR/Cas9-based generation of a recombinant double-reporter pseudorabies virus and its characterization in vitro and in vivo

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
Peng-Fei Fu ◽  
Xuan Cheng ◽  
Bing-Qian Su ◽  
Li-Fang Duan ◽  
Cong-Rong Wang ◽  
...  
Keyword(s):  
Pseudorabies Virus ◽  
Fluorescent Protein ◽  
Antiviral Agents ◽  
Firefly Luciferase ◽  
Inhibitory Effect ◽  
Economic Losses ◽  
Green Fluorescent ◽  
Swine Herds

AbstractPseudorabies, caused by pseudorabies virus (PRV) variants, has broken out among commercial PRV vaccine-immunized swine herds and resulted in major economic losses to the pig industry in China since late 2011. However, the mechanism of virulence enhancement of variant PRV is currently unclear. Here, a recombinant PRV (rPRV HN1201-EGFP-Luc) with stable expression of enhanced green fluorescent protein (EGFP) and firefly luciferase as a double reporter virus was constructed on the basis of the PRV variant HN1201 through CRISPR/Cas9 gene-editing technology coupled with two sgRNAs. The biological characteristics of the recombinant virus and its lethality to mice were similar to those of the parental strain and displayed a stable viral titre and luciferase activity through 20 passages. Moreover, bioluminescence signals were detected in mice at 12 h after rPRV HN1201-EGFP-Luc infection. Using the double reporter PRV, we also found that 25-hydroxycholesterol had a significant inhibitory effect on PRV both in vivo and in vitro. These results suggested that the double reporter PRV based on PRV variant HN1201 should be an excellent tool for basic virology studies and evaluating antiviral agents.

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