scholarly journals Production, characterization, and cross-reactivity of a polyclonal antibody against Arabidopsis TARGET OF RAPAMYCIN

2019 ◽  
Vol 62 (1) ◽  
Author(s):  
Gyeong-Im Shin ◽  
Sun Young Moon ◽  
Song Yi Jeong ◽  
Myung Geun Ji ◽  
Joon-Yung Cha ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Cross Reactivity ◽  
Target Of Rapamycin ◽  
Loss Of Function ◽  
Anticancer Activities ◽  
Purification Method ◽  
E Coli ◽  
Related Family ◽  
Large Gene ◽  
Truncated Variants

AbstractTARGET OF RAPAMYCIN (TOR), a member of the phosphatidylinositol 3-kinase-related family of protein kinases, is encoded by a single, large gene and is evolutionarily conserved in all eukaryotes. TOR plays a role as a master regulator that integrates nutrient, energy, and stress signaling to orchestrate development. TOR was first identified in yeast mutant screens, as its mutants conferred resistance to rapamycin, an antibiotic with immunosuppressive and anticancer activities. In Arabidopsis thaliana, the loss-of-function tor mutant displays embryo lethality, but the precise mechanisms of TOR function are still unknown. Moreover, a lack of reliable molecular and biochemical assay tools limits our ability to explore TOR functions in plants. Here, we produced a polyclonal α-TOR antibody using two truncated variants of TOR (1–200 and 1113–1304 amino acids) as antigens because recombinant full-length TOR is challenging to express in Escherichia coli. Recombinant His-TOR1−200 and His-TOR1113−1304 proteins were individually expressed in E. coli, and a mixture of proteins (at a 1:1 ratio) was used for immunizing rabbits. Antiserum was purified by an antigen-specific purification method, and the purified polyclonal α-TOR antibody successfully detected endogenous TOR proteins in wild-type Arabidopsis and TOR orthologous in major crop plants, including tomato, maize, and alfalfa. Moreover, our α-TOR antibody is useful for coimmunoprecipitation assays. In summary, we generated a polyclonal α-TOR antibody that detects endogenous TOR in various plant species. Our antibody could be used in future studies to determine the precise molecular mechanisms of TOR, which has largely unknown multifunctional roles in plants.

scholarly journals Circular RNA mmu_circ_0005019 inhibits fibrosis of cardiac fibroblasts and reverses electrical remodeling of cardiomyocytes

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Na Wu ◽  
Chengying Li ◽  
Bin Xu ◽  
Ying Xiang ◽  
Xiaoyue Jia ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Cardiac Fibroblasts ◽  
Circular Rna ◽  
Protective Role ◽  
Luciferase Reporter ◽  
Loss Of Function ◽  
Candidate Target ◽  
Paired Control ◽  
Reporter Assays ◽  
And Migration

Abstract Background Circular RNA (circRNA) have been reported to play important roles in cardiovascular diseases including myocardial infarction and heart failure. However, the role of circRNA in atrial fibrillation (AF) has rarely been investigated. We recently found a circRNA hsa_circ_0099734 was significantly differentially expressed in the AF patients atrial tissues compared to paired control. We aim to investigate the functional role and molecular mechanisms of mmu_circ_0005019 which is the homologous circRNA in mice of hsa_circ_0099734 in AF. Methods In order to investigate the effect of mmu_circ_0005019 on the proliferation, migration, differentiation into myofibroblasts and expression of collagen of cardiac fibroblasts, and the effect of mmu_circ_0005019 on the apoptosis and expression of Ito, INA and SK3 of cardiomyocytes, gain- and loss-of-function of cell models were established in mice cardiac fibroblasts and HL-1 atrial myocytes. Dual-luciferase reporter assays and RIP were performed to verify the binding effects between mmu_circ_0005019 and its target microRNA (miRNA). Results In cardiac fibroblasts, mmu_circ_0005019 showed inhibitory effects on cell proliferation and migration. In cardiomyocytes, overexpression of mmu_circ_0005019 promoted Kcnd1, Scn5a and Kcnn3 expression. Knockdown of mmu_circ_0005019 inhibited the expression of Kcnd1, Kcnd3, Scn5a and Kcnn3. Mechanistically, mmu_circ_0005019 exerted biological functions by acting as a miR-499-5p sponge to regulate the expression of its target gene Kcnn3. Conclusions Our findings highlight mmu_circ_0005019 played a protective role in AF development and might serve as an attractive candidate target for AF treatment.

scholarly journals In Escherichia coli Ammonia Inhibits Cytochrome bo3 But Activates Cytochrome bd-I

Antioxidants ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 13
Author(s):  
Elena Forte ◽  
Sergey A. Siletsky ◽  
Vitaliy B. Borisov
Keyword(s):  
Escherichia Coli ◽  
Molecular Mechanisms ◽  
Dissociation Constants ◽  
Reductase Activity ◽  
Ammonia Concentration ◽  
High Concentration ◽  
E Coli ◽  
Cytochrome Bd ◽  
Cytochrome Bo3 ◽  
Oxygen Reductase

Interaction of two redox enzymes of Escherichia coli, cytochrome bo3 and cytochrome bd-I, with ammonium sulfate/ammonia at pH 7.0 and 8.3 was studied using high-resolution respirometry and absorption spectroscopy. At pH 7.0, the oxygen reductase activity of none of the enzymes is affected by the ligand. At pH 8.3, cytochrome bo3 is inhibited by the ligand, with 40% maximum inhibition at 100 mM (NH4)2SO4. In contrast, the activity of cytochrome bd-I at pH 8.3 increases with increasing the ligand concentration, the largest increase (140%) is observed at 100 mM (NH4)2SO4. In both cases, the effector molecule is apparently not NH4+ but NH3. The ligand induces changes in absorption spectra of both oxidized cytochromes at pH 8.3. The magnitude of these changes increases as ammonia concentration is increased, yielding apparent dissociation constants Kdapp of 24.3 ± 2.7 mM (NH4)2SO4 (4.9 ± 0.5 mM NH3) for the Soret region in cytochrome bo3, and 35.9 ± 7.1 and 24.6 ± 12.4 mM (NH4)2SO4 (7.2 ± 1.4 and 4.9 ± 2.5 mM NH3) for the Soret and visible regions, respectively, in cytochrome bd-I. Consistently, addition of (NH4)2SO4 to cells of the E. coli mutant containing cytochrome bd-I as the only terminal oxidase at pH 8.3 accelerates the O2 consumption rate, the highest one (140%) being at 27 mM (NH4)2SO4. We discuss possible molecular mechanisms and physiological significance of modulation of the enzymatic activities by ammonia present at high concentration in the intestines, a niche occupied by E. coli.

scholarly journals Structure and dynamics of the E. coli chemotaxis core signaling complex by cryo-electron tomography and molecular simulations

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
C. Keith Cassidy ◽  
Benjamin A. Himes ◽  
Dapeng Sun ◽  
Jun Ma ◽  
Gongpu Zhao ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Electron Tomography ◽  
Conformational Dynamics ◽  
Molecular Simulations ◽  
Adaptor Protein ◽  
Signaling Mechanism ◽  
E Coli ◽  
Signaling Complex ◽  
Chemical Gradients ◽  
Cryo Electron Tomography

AbstractTo enable the processing of chemical gradients, chemotactic bacteria possess large arrays of transmembrane chemoreceptors, the histidine kinase CheA, and the adaptor protein CheW, organized as coupled core-signaling units (CSU). Despite decades of study, important questions surrounding the molecular mechanisms of sensory signal transduction remain unresolved, owing especially to the lack of a high-resolution CSU structure. Here, we use cryo-electron tomography and sub-tomogram averaging to determine a structure of the Escherichia coli CSU at sub-nanometer resolution. Based on our experimental data, we use molecular simulations to construct an atomistic model of the CSU, enabling a detailed characterization of CheA conformational dynamics in its native structural context. We identify multiple, distinct conformations of the critical P4 domain as well as asymmetries in the localization of the P3 bundle, offering several novel insights into the CheA signaling mechanism.

Formation, translocation and resolution of Holliday junctions during homologous genetic recombination

1995 ◽  
Vol 347 (1319) ◽  
pp. 21-25 ◽  
Keyword(s):  
Molecular Mechanisms ◽  
Genetic Recombination ◽  
Holliday Junctions ◽  
The Novel ◽  
E Coli ◽  
The Past ◽  
Endonucleolytic Cleavage ◽  
Insight Into ◽  
Made In

Over the past three or four years, great strides have been made in our understanding of the proteins involved in recombination and the mechanisms by which recombinant molecules are formed. This review summarizes our current understanding of the process by focusing on recent studies of proteins involved in the later steps of recombination in bacteria. In particular, biochemical investigation of the in vitro properties of the E. coli RuvA, RuvB and RuvC proteins have provided our first insight into the novel molecular mechanisms by which Holliday junctions are moved along DNA and then resolved by endonucleolytic cleavage.

Structure of the O-chain polysaccharide of the phenol-phase soluble lipopolysaccharide of Escherichia coli 0:157:H7

1986 ◽  
Vol 64 (1) ◽  
pp. 21-28 ◽  
Author(s):  
Malcolm B. Perry ◽  
Leann MacLean ◽  
Douglas W. Griffith
Keyword(s):  
Escherichia Coli ◽  
Brucella Abortus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Extraction Procedure ◽  
Periodate Oxidation ◽  
Cross Reactivity ◽  
E Coli ◽  
Structure Formula ◽  
Phenol Water

The phenol-phase soluble lipopolysaccharide isolated from Escherichia coli 0:157 by the hot phenol–water extraction procedure was shown by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, periodate oxidation, methylation, and 13C and 1H nuclear magnetic resonance studies to be an unbranched linear polysaccharide with a tetrasaccharide repeating unit having the structure:[Formula: see text]The serological cross-reactivity of E. coli 0:157 with Brucella abortus, Yersinia enterocolitica (serotype 0:9), group N Salmonella, and some other E. coli species can be related immunochemically to the presence of 1,2-glycosylated N-acylated 4-amino-4,6-dideoxy-α-D-mannopyranosyl residues in the O-chains of their respective lipopolysaccharides.

scholarly journals Assessment of phagocytic activity in live macrophages-tumor cells co-cultures by Confocal and Nomarski Microscopy

2017 ◽  
Vol 2 (1) ◽  
Author(s):  
Dalia Martinez-Marin ◽  
Courtney Jarvis ◽  
Thomas Nelius ◽  
Stéphanie Filleur
Keyword(s):  
Tumor Microenvironment ◽  
Tumor Cells ◽  
Phagocytic Activity ◽  
Molecular Mechanisms ◽  
Cell Types ◽  
Functional Assay ◽  
Loss Of Function ◽  
Multiple Cancer

Abstract Macrophages have been recognized as the main inflammatory component of the tumor microenvironment. Although often considered as beneficial for tumor growth and disease progression, tumor-associated macrophages have also been shown to be detrimental to the tumor depending on the tumor microenvironment. Therefore, understanding the molecular interactions between macrophages and tumor cells in relation to macrophages functional activities such as phagocytosis is critical for a better comprehension of their tumor-modulating action. Still, the characterization of these molecular mechanisms in vivo remains complicated due to the extraordinary complexity of the tumor microenvironment and the broad range of tumor-associated macrophage functions. Thus, there is an increasing demand for in vitro methodologies to study the role of cell–cell interactions in the tumor microenvironment. In the present study, we have developed live co-cultures of macrophages and human prostate tumor cells to assess the phagocytic activity of macrophages using a combination of Confocal and Nomarski Microscopy. Using this model, we have emphasized that this is a sensitive, measurable, and highly reproducible functional assay. We have also highlighted that this assay can be applied to multiple cancer cell types and used as a selection tool for a variety of different types of phagocytosis agonists. Finally, combining with other studies such as gain/loss of function or signaling studies remains possible. A better understanding of the interactions between tumor cells and macrophages may lead to the identification of new therapeutic targets against cancer.

scholarly journals Human Sex Matters: Y-linked lysine demethylase 5D drives accelerated male osteogenic differentiation

2021 ◽  
Author(s):  
Madlen Merten ◽  
Johannes F.W. Greiner ◽  
Tarek Niemann ◽  
Meike Grosse Venhaus ◽  
Daniel Kronenberg ◽  
...  
Keyword(s):  
Osteogenic Differentiation ◽  
Molecular Mechanisms ◽  
Female Rats ◽  
Sexually Dimorphic ◽  
Loss Of Function ◽  
Calvarial Bone ◽  
Sexual Dimorphisms ◽  
Increased Risk ◽  
Elevated Expression ◽  
Lysine Demethylase

Female sex is increasingly associated to a loss of bone mass during aging and an increased risk for fractures developing nonunion. Hormonal factors and cell-intrinsic mechanisms are suggested to drive these sexual dimorphisms, although underlying molecular mechanisms are still a matter of debate. Here, we observed a decreased capacity of calvarial bone recovery in female rats and a profound sexually dimorphic osteogenic differentiation human adult neural crest-derived stem cells (NCSCs). Next to an elevated expression of pro-osteogenic regulators, global trancriptomics revealed Lysine Demethylase 5D (KDM5D) to be highly upregulated in differentiating male NCSCs. Loss of function by siRNA or pharmacological inhibition of KDM5D significantly reduced the osteogenic differentiation capacity of male NCSCs. In summary, we demonstrate craniofacial osteogenic differentiation to be sexually dimorphic with the expression of KDM5D as a prerequisite for accelerated male osteogenic differentiation, emphasizing the analysis of sex-specific differences as a crucial parameter for treating bone defects.

scholarly journals Role of PIF4 and FLC in controlling flowering time under daily variable temperature profile

2020 ◽  
Author(s):  
Jutapak Jenkitkonchai ◽  
Poppy Marriott ◽  
Weibing Yang ◽  
Napaporn Sriden ◽  
Jae-Hoon Jung ◽  
...  
Keyword(s):  
Flowering Time ◽  
Molecular Mechanisms ◽  
Transcriptional Regulatory Network ◽  
Variable Temperature ◽  
Loss Of Function ◽  
Environmental Signals ◽  
Regulatory Interactions ◽  
Flowering Genes ◽  
Gene Regulatory ◽  
Flowering Locus

ABSTRACTInitiation of flowering is a crucial developmental event that requires both internal and environmental signals to determine when floral transition should occur to maximize reproductive success. Ambient temperature is one of the key environmental signals that highly influence flowering time, not only seasonally but also in the context of drastic temperature fluctuation due to global warming. Molecular mechanisms of how high or low constant temperatures affect the flowering time have been largely characterized in the model plant Arabidopsis thaliana; however, the effect of natural daily variable temperature outside laboratories is only partly explored. Several groups of flowering genes have been shown to play important roles in temperature responses, including two temperature-responsive transcription factors (TFs), namely PHYTOCHROME INTERACTING FACTOR 4 (PIF4) and FLOWERING LOCUS C (FLC), that act antagonistically to regulate flowering time by activating or repressing floral integrator FLOWERING LOCUS T (FT). In this study, we have demonstrated that the daily variable temperature (VAR) causes early flowering in both natural accessions Col-0, C24 and their late flowering hybrid C24xCol, which carries both functional floral repressor FLC and its activator FRIGIDA (FRI), as compared to a constant temperature (CON). The loss-of-function mutation of PIF4 exhibits later flowering in VAR, suggesting that PIF4 at least in part, contributes to acceleration of flowering in response to the daily variable temperature. We find that VAR increases PIF4 transcription at the end of the day when temperature peaks at 32 °C. The FT transcription is also elevated in VAR, as compared to CON, in agreement with earlier flowering observed in VAR. In addition, VAR causes a decrease in FLC transcription in 4-week-old plants, and we further show that overexpression of PIF4 can reduce FLC transcription, suggesting that PIF4 might also regulate FT indirectly through the repression of FLC. To further conceptualize an overall model of gene regulatory mechanisms involving PIF4 and FLC in controlling flowering in response to temperature changes, we construct a co-expression – transcriptional regulatory network by combining publicly available transcriptomic data and gene regulatory interactions of our flowering genes of interest and their partners. The network model reveals the conserved and tissue-specific regulatory functions of 62 flowering-time-relating genes, namely PIF4, PIF5, FLC, ELF3 and their immediate neighboring genes, which can be useful for confirming and predicting the functions and regulatory interactions between the key flowering genes.

scholarly journals Fatty acid analogue N-arachidonoyl taurine restores function of IKs channels with diverse long QT mutations

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Sara I Liin ◽  
Johan E Larsson ◽  
Rene Barro-Soria ◽  
Bo Hjorth Bentzen ◽  
H Peter Larsson
Keyword(s):  
Fatty Acid ◽  
Long Qt Syndrome ◽  
Molecular Mechanisms ◽  
Loss Of Function ◽  
Long Qt ◽  
Acid Analogue ◽  
Fatty Acid Analogue ◽  
Qt Syndrome ◽  
Channel Currents ◽  
Iks Channel

About 300 loss-of-function mutations in the IKs channel have been identified in patients with Long QT syndrome and cardiac arrhythmia. How specific mutations cause arrhythmia is largely unknown and there are no approved IKs channel activators for treatment of these arrhythmias. We find that several Long QT syndrome-associated IKs channel mutations shift channel voltage dependence and accelerate channel closing. Voltage-clamp fluorometry experiments and kinetic modeling suggest that similar mutation-induced alterations in IKs channel currents may be caused by different molecular mechanisms. Finally, we find that the fatty acid analogue N-arachidonoyl taurine restores channel gating of many different mutant channels, even though the mutations are in different domains of the IKs channel and affect the channel by different molecular mechanisms. N-arachidonoyl taurine is therefore an interesting prototype compound that may inspire development of future IKs channel activators to treat Long QT syndrome caused by diverse IKs channel mutations.

scholarly journals Long noncoding RNA SNHG25 promotes the malignancy of endometrial cancer by sponging microRNA-497-5p and increasing FASN expression

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Yuhua He ◽  
Shuifang Xu ◽  
Yi Qi ◽  
Jinfang Tian ◽  
Fengying Xu
Keyword(s):  
Endometrial Cancer ◽  
Long Noncoding Rna ◽  
Noncoding Rna ◽  
Fatty Acid Synthase ◽  
Molecular Mechanisms ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Study Cohort ◽  
Loss Of Function ◽  
Ec Cells

Abstract Background Small nucleolar RNA host gene 25 (SNHG25), a long noncoding RNA, has been well-studied in epithelial ovarian cancer. However, the specific functions of SNHG25 in endometrial cancer (EC) have not been studied yet. In this study, we aimed to elucidate the clinical significance of SNHG25 in EC and determine the regulatory activity of SNHG25 on the tumor-associated EC phenotype. We also thoroughly explored the molecular mechanisms underlying SNHG25 function in EC. Methods Gene expression was measured using quantitative real-time polymerase chain reaction. The detailed functions of SNHG25 in EC were examined by performing loss-of-function experiments. Moreover, the regulatory mechanisms involving SNHG25, microRNA-497-5p, and fatty acid synthase (FASN) were unveiled using the luciferase reporter assay and RNA immunoprecipitation. Results We observed a high level of SNHG25 in EC using the TCGA dataset and our study cohort. Patients with a high SNHG25 level had shorter overall survival than those with a low SNHG25 level. SNHG25 deficiency resulted in tumor-repressing activities in EC cells by decreasing cell proliferation, migration, and invasion and promoting cell apoptosis. Furthermore, the function of SNHG25 depletion in impairing tumor growth in vivo was confirmed. SNHG25 sequestered miR-497-5p as a competing endogenous RNA in EC and consequently positively regulated FASN expression. Thus, the decrease in miR-497-5p or increase in FASN could neutralize the modulatory actions of SNHG25 knockdown in EC cells. Conclusions The depletion of SNHG25 impedes the oncogenicity of EC by targeting the miR-497-5p/FASN axis. The newly elucidated SNHG25/miR-497-5p/FASN pathway may be a promising target for the molecular-targeted management of EC.

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