scholarly journals Directed evolution of the PcaV allosteric transcription factor to generate a biosensor for aromatic aldehydes

2019 ◽  
Author(s):  
Leopoldo F. M. Machado ◽  
Andrew Currin ◽  
Neil Dixon
Keyword(s):  
Substrate Specificity ◽  
Directed Evolution ◽  
Aromatic Aldehydes ◽  
Phenotypic Analysis ◽  
Naturally Occurring ◽  
Potential Applications ◽  
Substituted Benzoic Acids

AbstractBackgroundGenetically encoded biosensors are useful tools for the detection of metabolites and industrially valuable molecules, and present many potential applications in biotechnology and biomedicine. However, the most common approach to develop biosensors relies on employing a limited set of naturally occurring allosteric transcript factors (aTFs). Therefore, altering the substrate specificity of aTFs towards the detection of new effectors is an important goal.ResultsHere, the PcaV repressor, a member of the MarR aTF family, was used to develop a biosensor for the detection of hydroxyl-substituted benzoic acids, including protocatechuic acid (PCA). The PCA biosensor was further subjected to directed evolution to alter its substrate specificity towards vanillin and other closely related aromatic aldehydes, to generate the Van2 biosensor. Substrate recognition of Van2 was explored in vitro using a range of biochemical and biophysical analyses, and extensive in vivo genetic-phenotypic analysis was performed to determine the role of each amino acid change upon biosensor performance.ConclusionsThis is the first study to report directed evolution of a member of the MarR aTF family, and demonstrates the plasticity of the PCA biosensor by altering its substrate specificity to generate a biosensor for aromatic aldehydes.

scholarly journals Directed evolution of the PcaV allosteric transcription factor to generate a biosensor for aromatic aldehydes

2019 ◽  
Vol 13 (1) ◽  
Author(s):  
Leopoldo F. M. Machado ◽  
Andrew Currin ◽  
Neil Dixon
Keyword(s):  
Transcription Factor ◽  
Directed Evolution ◽  
Aromatic Aldehydes ◽  
Ligand Specificity ◽  
Phenotypic Analysis ◽  
Potential Applications ◽  
Substituted Benzoic Acids

Abstract Background Transcription factor-based biosensors are useful tools for the detection of metabolites and industrially valuable molecules, and present many potential applications in biotechnology and biomedicine. However, the most common approach to develop biosensors relies on employing a limited set of naturally occurring allosteric transcription factors (aTFs). Therefore, altering the ligand specificity of aTFs towards the detection of new effectors is an important goal. Results Here, the PcaV repressor, a member of the MarR aTF family, was used to develop a biosensor for the detection of hydroxyl-substituted benzoic acids, including protocatechuic acid (PCA). The PCA biosensor was further subjected to directed evolution to alter its ligand specificity towards vanillin and other closely related aromatic aldehydes, to generate the Van2 biosensor. Ligand recognition of Van2 was explored in vitro using a range of biochemical and biophysical analyses, and extensive in vivo genetic-phenotypic analysis was performed to determine the role of each amino acid change upon biosensor performance. Conclusions This is the first study to report directed evolution of a member of the MarR aTF family, and demonstrates the plasticity of the PCA biosensor by altering its ligand specificity to generate a biosensor for aromatic aldehydes.

scholarly journals GAGA Factor Isoforms Have Distinct but Overlapping Functions In Vivo

2001 ◽  
Vol 21 (24) ◽  
pp. 8565-8574 ◽  
Author(s):  
Anthony J. Greenberg ◽  
Paul Schedl
Keyword(s):  
Fusion Protein ◽  
Transcriptional Activation ◽  
Functional Difference ◽  
Wild Type ◽  
Gaga Factor ◽  
Phenotypic Analysis ◽  
Alternatively Spliced

ABSTRACT The Drosophila melanogaster GAGA factor (encoded by the Trithorax-like [Trl] gene) is required for correct chromatin architecture at diverse chromosomal sites. The Trl gene encodes two alternatively spliced isoforms of the GAGA factor (GAGA-519 and GAGA-581) that are identical except for the length and sequence of the C-terminal glutamine-rich (Q) domain. In vitro and tissue culture experiments failed to find any functional difference between the two isoforms. We made a set of transgenes that constitutively express cDNAs coding for either of the isoforms with the goal of elucidating their roles in vivo. Phenotypic analysis of the transgenes in Trl mutant background led us to the conclusion that GAGA-519 and GAGA-581 perform different, albeit largely overlapping, functions. We also expressed a fusion protein with LacZ disrupting the Q domain of GAGA-519. This LacZ fusion protein compensated for the loss of wild-type GAGA factor to a surprisingly large extent. This suggests that the Q domain either is not required for the essential functions performed by the GAGA protein or is exclusively used for tetramer formation. These results are inconsistent with a major role of the Q domain in chromatin remodeling or transcriptional activation. We also found that GAGA-LacZ was able to associate with sites not normally occupied by the GAGA factor, pointing to a role of the Q domain in binding site choice in vivo.

scholarly journals The effects of morin on bone regeneration to accelerate healing in bone defects in mice

2020 ◽  
Vol 34 ◽  
pp. 205873842096290
Author(s):  
Jun Wan ◽  
Tao Ma ◽  
Yun Jin ◽  
Shaodong Qiu
Keyword(s):  
Molecular Level ◽  
Bone Defects ◽  
In Vitro Models ◽  
Marker Genes ◽  
Protein Markers ◽  
Naturally Occurring ◽  
Stems Cells

Restoring bone defects are the major challenge facing clinical trial therapy, particularly skull related problems. Morin, a naturally occurring compound, has pro-osteogenesis. This research focuses on assessing the role of morin for its pro-osteogenesis activities. We utilized in vivo and in vitro models to investigate the molecular-level mechanisms of morin’s osteoblastic biological activity. The effectiveness of morin on pro-osteogenesis (100 mg/kg/day) was assessed by monitoring modifications in the bone histomorphometry score, the development of immature osteoblasts from mesenchymal stems cells and improvements in the expression of pro-osteogenic cytokines in skull defected (SD) mice. Quantitative—PCR, Western blot analysis, and immunofluorescence were studied to investigate the signaling pathways. Morin has a substantial in vivo pro-osteogenesis effect which can facilitate the development of osteoblasts, the production of osteoblast related marker genes, and in vitro protein markers for osteoblasts. From a molecular biology standpoint, morin contributes to the development of osteoblasts and stimulation of the Wnt pathway with the activation and translocation of β-catenin nuclei. Our findings from the study revealed that morin may be a beneficial substitute for helping regenerate bone defects.

Naturally occurring short splice variant of CYLD positively regulates dendritic cell function

Blood ◽  
2009 ◽  
Vol 113 (23) ◽  
pp. 5891-5895 ◽  
Author(s):  
Cathy Cecilia Srokowski ◽  
Joumana Masri ◽  
Nadine Hövelmeyer ◽  
Anna Katharina Krembel ◽  
Christine Tertilt ◽  
...  
Keyword(s):  
Dendritic Cell ◽  
Splice Variant ◽  
Cell Activation ◽  
Cell Function ◽  
Nuclear Translocation ◽  
Naturally Occurring ◽  
Short Splice Variant

Abstract Deubiquitination of NF-κB members by CYLD is crucial in controlling the magnitude and nature of cell activation. The role of the naturally occurring CYLD splice variant in dendritic cell (DC) function was analyzed using CYLDex7/8 mice, which lack the full-length CYLD (flCYLD) transcript and overexpress the short splice variant (sCYLD). Bone marrow–derived DCs from CYLDex7/8 mice display a hyperactive phenotype in vitro and in vivo and have a defect in establishing tolerance with the use of DEC-205–mediated antigen targeting to resting DCs. The combination of sCYLD overexpression and lack of flCYLD in CYLDex7/8 DCs leads to enhanced NF-κB activity accompanied by an increased nuclear translocation of the IκB molecule Bcl-3, along with nuclear p50 and p65. This suggests that, in contrast to flCYLD, sCYLD is a positive regulator of NF-κB activity, and its overexpression induces a hyperactive phenotype in DCs.

scholarly journals Potential Role of Natural Products to Combat Radiotherapy and Their Future Perspectives

Molecules ◽  
2021 ◽  
Vol 26 (19) ◽  
pp. 5997
Author(s):  
Rokeya Akter ◽  
Agnieszka Najda ◽  
Md. Habibur Rahman ◽  
Muddaser Shah ◽  
Sylwia Wesołowska ◽  
...  
Keyword(s):  
Natural Products ◽  
Cancer Cells ◽  
Cell Damage ◽  
Cancer Treatments ◽  
Antitumor Effects ◽  
Potential Applications

Cancer is the second leading cause of death in the world. Chemotherapy and radiotherapy (RT) are the common cancer treatments. In addition to these limitations, the development of adverse effects from chemotherapy and RT reduces the quality of life for cancer patients. Cellular radiosensitivity, or the ability to resist and overcome cell damage caused by ionizing radiation (IR), is directly related to cancer cells’ response to RT. Therefore, radiobiological research is emphasizing chemical compounds ’radiosensitization of cancer cells so that they are more reactive in the IR spectrum. Recent years researchers have seen an increase in interest in natural products that have antitumor effects with minimal side effects. Natural products, on the other hand, are easy to recover and therefore less expensive. There have been several scientific studies done based on these compounds that have tested their ability in vitro and in vivo to induce tumor radiosensitization. The role of natural products in RT, as well as their usefulness and potential applications, is the goal of this current review.

scholarly journals SETD3 protein is the actin-specific histidine N-methyltransferase

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Sebastian Kwiatkowski ◽  
Agnieszka K Seliga ◽  
Didier Vertommen ◽  
Marianna Terreri ◽  
Takao Ishikawa ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Substrate Specificity ◽  
Post Translational Modification ◽  
Set Domain ◽  
Health And Disease ◽  
New Research ◽  
Glycolytic Phenotype

Protein histidine methylation is a rare post-translational modification of unknown biochemical importance. In vertebrates, only a few methylhistidine-containing proteins have been reported, including β-actin as an essential example. The evolutionary conserved methylation of β-actin H73 is catalyzed by an as yet unknown histidine N-methyltransferase. We report here that the protein SETD3 is the actin-specific histidine N-methyltransferase. In vitro, recombinant rat and human SETD3 methylated β-actin at H73. Knocking-out SETD3 in both human HAP1 cells and in Drosophila melanogaster resulted in the absence of methylation at β-actin H73 in vivo, whereas β-actin from wildtype cells or flies was > 90% methylated. As a consequence, we show that Setd3-deficient HAP1 cells have less cellular F-actin and an increased glycolytic phenotype. In conclusion, by identifying SETD3 as the actin-specific histidine N-methyltransferase, our work pioneers new research into the possible role of this modification in health and disease and questions the substrate specificity of SET-domain-containing enzymes.

The Role of Polyphenols in the Modulation of Plasma Non-Enzymatic Antioxidant Capacity (NEAC)

2012 ◽  
Vol 82 (3) ◽  
pp. 228-232 ◽  
Author(s):  
Mauro Serafini ◽  
Giuseppa Morabito
Keyword(s):  
Antioxidant Capacity ◽  
Dietary Intervention ◽  
Ex Vivo ◽  
The Body ◽  
Dietary Polyphenols ◽  
Enzymatic Antioxidant ◽  
Endogenous Antioxidant

Dietary polyphenols have been shown to scavenge free radicals, modulating cellular redox transcription factors in different in vitro and ex vivo models. Dietary intervention studies have shown that consumption of plant foods modulates plasma Non-Enzymatic Antioxidant Capacity (NEAC), a biomarker of the endogenous antioxidant network, in human subjects. However, the identification of the molecules responsible for this effect are yet to be obtained and evidences of an antioxidant in vivo action of polyphenols are conflicting. There is a clear discrepancy between polyphenols (PP) concentration in body fluids and the extent of increase of plasma NEAC. The low degree of absorption and the extensive metabolism of PP within the body have raised questions about their contribution to the endogenous antioxidant network. This work will discuss the role of polyphenols from galenic preparation, food extracts, and selected dietary sources as modulators of plasma NEAC in humans.

Anti-obesity role of Aster glehni extract: in vivo and in vitro effects

Planta Medica ◽  
2012 ◽  
Vol 78 (11) ◽  
Author(s):  
HM Lee ◽  
TG Ahn ◽  
CW Kim ◽  
HJ An
Keyword(s):  
In Vitro Effects

A Molecular Approach to Immunoscintigraphy: A Study of the T-Antigen Conformation on the Surface of Tumors

1987 ◽  
Vol 26 (01) ◽  
pp. 1-6 ◽  
Author(s):  
S. Selvaraj ◽  
M. R. Suresh ◽  
G. McLean ◽  
D. Willans ◽  
C. Turner ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Tumor Cell ◽  
T Antigen ◽  
Human Carcinomas ◽  
Animal Tumor ◽  
Synthetic Antigens

The role of glycoconjugates in tumor cell differentiation has been well documented. We have examined the expression of the two anomers of the Thomsen-Friedenreich antigen on the surface of human, canine and murine tumor cell membranes both in vitro and in vivo. This has been accomplished through the synthesis of the disaccharide terminal residues in both a and ß configuration. Both entities were used to generate murine monoclonal antibodies which recognized the carbohydrate determinants. The determination of fine specificities of these antibodies was effected by means of cellular uptake, immunohistopathology and immunoscintigraphy. Examination of pathological specimens of human and canine tumor tissue indicated that the expressed antigen was in the β configuration. More than 89% of all human carcinomas tested expressed the antigen in the above anomeric form. The combination of synthetic antigens and monoclonal antibodies raised specifically against them provide us with invaluable tools for the study of tumor marker expression in humans and their respective animal tumor models.

Role of Platelets, Thrombin, Integrin llb-llla, Fibronectin and Von Willebrand Factor on Tumor Adhesion in Vitro and Metastasis in Vivo

1995 ◽  
Vol 74 (01) ◽  
pp. 282-290 ◽  
Author(s):  
Mary Lynn Nierodzik ◽  
Abraham Klepfish ◽  
Simon Karpatkin
Keyword(s):  
Von Willebrand Factor ◽  
Von Willebrand ◽  
Tumor Adhesion ◽  
Willebrand Factor
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