Characterization of soluble CD39 (SolCD39/NTPDase1) from PiggyBac nonviral system as a tool to control the nucleotides level

2019 ◽  
Vol 476 (11) ◽  
pp. 1637-1651
Author(s):  
Liziane Raquel Beckenkamp ◽  
Isabele Cristiana Iser ◽  
Giovana Ravizzoni Onzi ◽  
Dieine Maira Soares da Fontoura ◽  
Ana Paula Santin Bertoni ◽  
...  
Keyword(s):  
Purinergic Signaling ◽  
Hydrolytic Activity ◽  
Soluble Form ◽  
Purinergic System ◽  
In Vivo Experiments ◽  
High Doses ◽  
And Migration

Abstract Extracellular ATP (eATP) and its metabolites have emerged as key modulators of different diseases and comprise a complex pathway called purinergic signaling. An increased number of tools have been developed to study the role of nucleotides and nucleosides in cell proliferation and migration, influence on the immune system and tumor progression. These tools include receptor agonists/antagonists, engineered ectonucleotidases, interference RNAs and ectonucleotidase inhibitors that allow the control and quantification of nucleotide levels. NTPDase1 (also called apyrase, ecto-ATPase and CD39) is one of the main enzymes responsible for the hydrolysis of eATP, and purified enzymes, such as apyrase purified from potato, or engineered as soluble CD39 (SolCD39), have been widely used in in vitro and in vivo experiments. However, the commercial apyrase had its effects recently questioned and SolCD39 exhibits limitations, such as short half-life and need of high doses to reach the expected enzymatic activity. Therefore, this study investigated a non-viral method to improve the overexpression of SolCD39 and evaluated its impact on other enzymes of the purinergic system. Our data demonstrated that PiggyBac transposon system proved to be a fast and efficient method to generate cells stably expressing SolCD39, producing high amounts of the enzyme from a limited number of cells and with high hydrolytic activity. In addition, the soluble form of NTPDase1/CD39 did not alter the expression or catalytic activity of other enzymes from the purinergic system. Altogether, these findings set the groundwork for prospective studies on the function and therapeutic role of eATP and its metabolites in physiological and pathological conditions.

scholarly journals Isoflurane Preconditioning Promotes the Survival and Migration of Bone Marrow Stromal Cells

2015 ◽  
Vol 36 (4) ◽  
pp. 1331-1345 ◽  
Author(s):  
Yu Sun ◽  
Qi-fang Li ◽  
Jia Yan ◽  
Rong Hu ◽  
Hong Jiang
Keyword(s):  
Bone Marrow ◽  
Hypoxia Inducible Factor ◽  
Volatile Anesthetic ◽  
Protective Effects ◽  
In Vivo Experiments ◽  
High Doses ◽  
And Migration ◽  
And Function

Background: Preconditioning with the volatile anesthetic isoflurane exerts protective effects in animal models of ischemia. The cytoprotective effects of isoflurane are dependent on the expression of hypoxia inducible factor-1 (HIF-1), a dimeric transcription factor that mediates cellular responses to hypoxia. Methods: We investigated the effect of isoflurane preconditioning on bone marrow stromal cell (BMSC) survival and function. Results: Short exposures to low isoflurane concentrations promoted in vitro survival and migration of BMSCs, whereas long exposures and high doses had the opposite effect. At specific doses and times, isoflurane upregulated the expression of HIF-1α and the stromal-derived factor-1 receptor CXCR4, and induced the activation of Akt, similar to hypoxia, and the effect of isoflurane was abrogated by silencing of HIF-1α or inhibition of PI3K/Akt signaling. In vivo experiments showed that isoflurane preconditioning increased the engraftment of BMSCs into the ischemic brain and improved functional recovery in a mouse model of stroke. Conclusion: Isoflurane preconditioning at specific doses and times improves the survival and function of BMSCs through the upregulation of CXCR4 via a mechanism involving HIF-1α expression and the PI3K/Akt pathway, suggesting that anesthetic preconditioning could be developed as a strategy to improve the efficiency of cell therapy.

Knockout of Ajuba Attenuates the Growth and Migration of Hepatocellular Carcinoma Cells

2020 ◽  
Vol 160 (11-12) ◽  
pp. 650-658
Author(s):  
Yichen Le ◽  
Yi He ◽  
Meirong Bai ◽  
Ying Wang ◽  
Jiaxue Wu ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Growth ◽  
Cancer Progression ◽  
Normal Liver ◽  
Cell Growth And Migration ◽  
And Migration ◽  
Hcc Cells

Ajuba has been found to be mutated or aberrantly regulated in several human cancers and plays important roles in cancer progression via different signaling pathways. However, little is known about the role of Ajuba in hepatocellular carcinoma (HCC). Here, we found an upregulation of Ajuba expression in HCC tissues compared with normal liver tissues, while a poor prognosis was observed in HCC patients with high Ajuba expression. Knockout of Ajuba in HCC cells inhibited cell growth in vitro and in vivo, suppressed cell migration, and enhanced the cell apoptosis under stress. Moreover, re-expression of Ajuba in Ajuba-deficient cells could restore the phenotype of Ajuba-deficient cells. In conclusion, these results indicate that Ajuba is upregulated in HCC and promotes cell growth and migration of HCC cells, suggesting that Ajuba could possibly be a new target for HCC diagnosis and treatment.

scholarly journals Regorafenib-Attenuated, Bleomycin-Induced Pulmonary Fibrosis by Inhibiting the TGF-β1 Signaling Pathway

2021 ◽  
Vol 22 (4) ◽  
pp. 1985
Author(s):  
Xiaohe Li ◽  
Ling Ma ◽  
Kai Huang ◽  
Yuli Wei ◽  
Shida Long ◽  
...  
Keyword(s):  
Pulmonary Fibrosis ◽  
Signaling Pathway ◽  
Transforming Growth Factor ◽  
Kinase Inhibitor ◽  
In Vivo Experiments ◽  
Age Related ◽  
And Migration ◽  
Tgf Β1

Idiopathic pulmonary fibrosis (IPF) is a fatal and age-related pulmonary disease. Nintedanib is a receptor tyrosine kinase inhibitor, and one of the only two listed drugs against IPF. Regorafenib is a novel, orally active, multi-kinase inhibitor that has similar targets to nintedanib and is applied to treat colorectal cancer and gastrointestinal stromal tumors in patients. In this study, we first identified that regorafenib could alleviate bleomycin-induced pulmonary fibrosis in mice. The in vivo experiments indicated that regorafenib suppresses collagen accumulation and myofibroblast activation. Further in vitro mechanism studies showed that regorafenib inhibits the activation and migration of myofibroblasts and extracellular matrix production, mainly through suppressing the transforming growth factor (TGF)-β1/Smad and non-Smad signaling pathways. In vitro studies have also indicated that regorafenib could augment autophagy in myofibroblasts by suppressing TGF-β1/mTOR (mechanistic target of rapamycin) signaling, and could promote apoptosis in myofibroblasts. In conclusion, regorafenib attenuates bleomycin-induced pulmonary fibrosis by suppressing the TGF-β1 signaling pathway.

scholarly journals Progression and Differentiation of Alveolar Rhabdomyosarcoma Is Regulated by PAX7 Transcription Factor—Significance of Tumor Subclones

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1870
Author(s):  
Klaudia Skrzypek ◽  
Grażyna Adamek ◽  
Marta Kot ◽  
Bogna Badyra ◽  
Marcin Majka
Keyword(s):  
Transcription Factor ◽  
Cell Lines ◽  
Migration Activity ◽  
Id Proteins ◽  
Rh30 Cell ◽  
Str Profile ◽  
And Migration

Rhabdomyosarcoma (RMS), is the most frequent soft tissue tumor in children that originates from disturbances in differentiation process. Mechanisms leading to the development of RMS are still poorly understood. Therefore, by analysis of two RMS RH30 cell line subclones, one subclone PAX7 negative, while the second one PAX7 positive, and comparison with other RMS cell lines we aimed at identifying new mechanisms crucial for RMS progression. RH30 subclones were characterized by the same STR profile, but different morphology, rate of proliferation, migration activity and chemotactic abilities in vitro, as well as differences in tumor morphology and growth in vivo. Our analysis indicated a different level of expression of adhesion molecules (e.g., from VLA and ICAM families), myogenic microRNAs, such as miR-206 and transcription factors, such as MYOD, MYOG, SIX1, and ID. Silencing of PAX7 transcription factor with siRNA confirmed the crucial role of PAX7 transcription factor in proliferation, differentiation and migration of RMS cells. To conclude, our results suggest that tumor cell lines with the same STR profile can produce subclones that differ in many features and indicate crucial roles of PAX7 and ID proteins in the development of RMS.

scholarly journals The Extracellular Small Leucine-Rich Proteoglycan Biglycan Is a Key Player in Gastric Cancer Aggressiveness

Cancers ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1330
Author(s):  
Filipe Pinto ◽  
Liliana Santos-Ferreira ◽  
Marta T. Pinto ◽  
Catarina Gomes ◽  
Celso A. Reis
Keyword(s):  
Clinical Value ◽  
Knock Out ◽  
Angiogenic Potential ◽  
In Vivo Experiments ◽  
Cancer Aggressiveness ◽  
Advanced Stages ◽  
Oncogenic Gene

Biglycan (BGN gene), an extracellular proteoglycan, has been described to be associated with cancer aggressiveness. The purpose of this study was to clarify the clinical value of biglycan as a biomarker in multiple independent GC cohorts and determine the in vitro and in vivo role of biglycan in GC malignant features. We found that BGN is commonly over-expressed in all analyzed cohorts, being associated with disease relapse and poor prognosis in patients with advanced stages of disease. In vitro and in vivo experiments demonstrated that biglycan knock-out GC cells display major phenotypic changes with a lower cell survival, migration, and angiogenic potential when compared with biglycan expressing cells. Biglycan KO GC cells present increased levels of PARP1 and caspase-3 cleavage and a decreased expression of mesenchymal markers. Importantly, biglycan deficient GC cells that were supplemented with exogenous biglycan were able to restore biological features, such as survival, clonogenic and migratory capacities. Our in vitro and in vivo findings were validated in human GC samples, where BGN expression was associated with several oncogenic gene signatures that were associated with apoptosis, cell migration, invasion, and angiogenesis. This study provided new insights on biglycan role in GC that should be taken in consideration as a key cellular regulator with major impact in tumor progression and patients’ clinical outcome.

Paradoxical effect of salbutamol in a model of acute organophosphates intoxication in guinea pigs: role of substance P release

2007 ◽  
Vol 292 (4) ◽  
pp. L915-L923 ◽  
Author(s):  
Jaime Chávez ◽  
Patricia Segura ◽  
Mario H. Vargas ◽  
José Luis Arreola ◽  
Edgar Flores-Soto ◽  
...  
Keyword(s):  
Substance P ◽  
Guinea Pigs ◽  
Smooth Muscle Contraction ◽  
In Vivo Experiments ◽  
Intracellular Ca ◽  
Neurokinin 1 ◽  
Substance P Release

Organophosphates induce bronchoobstruction in guinea pigs, and salbutamol only transiently reverses this effect, suggesting that it triggers additional obstructive mechanisms. To further explore this phenomenon, in vivo (barometric plethysmography) and in vitro (organ baths, including ACh and substance P concentration measurement by HPLC and immunoassay, respectively; intracellular Ca2+ measurement in single myocytes) experiments were performed. In in vivo experiments, parathion caused a progressive bronchoobstruction until a plateau was reached. Administration of salbutamol during this plateau decreased bronchoobstruction up to 22% in the first 5 min, but thereafter airway obstruction rose again as to reach the same intensity as before salbutamol. Aminophylline caused a sustained decrement (71%) of the parathion-induced bronchoobstruction. In in vitro studies, paraoxon produced a sustained contraction of tracheal rings, which was fully blocked by atropine but not by TTX, ω-conotoxin (CTX), or epithelium removal. During the paraoxon-induced contraction, salbutamol caused a temporary relaxation of ∼50%, followed by a partial recontraction. This paradoxical recontraction was avoided by the M2- or neurokinin-1 (NK1)-receptor antagonists (methoctramine or AF-DX 116, and L-732138, respectively), accompanied by a long-lasting relaxation. Forskolin caused full relaxation of the paraoxon response. Substance P and, to a lesser extent, ACh released from tracheal rings during 60-min incubation with paraoxon or physostigmine, respectively, were significantly increased when salbutamol was administered in the second half of this period. In myocytes, paraoxon did not produce any change in the intracellular Ca2+ basal levels. Our results suggested that: 1) organophosphates caused smooth muscle contraction by accumulation of ACh released through a TTX- and CTX-resistant mechanism; 2) during such contraction, salbutamol relaxation is functionally antagonized by the stimulation of M2 receptors; and 3) after this transient salbutamol-induced relaxation, a paradoxical contraction ensues due to the subsequent release of substance P.

scholarly journals MiR-125b-5p Inhibited The Progression of Hepatoblastoma As A Shared miRNA of The lncRNA NEAT1 and YES1

2020 ◽  
Author(s):  
Zhu Jin ◽  
Yutong Chen ◽  
Yuchen Mao ◽  
Mingjuan Gao ◽  
Zebing Zheng ◽  
...  
Keyword(s):  
Clinicopathological Characteristics ◽  
Luciferase Reporter ◽  
Tumor Growth Inhibition ◽  
Invasion And Migration ◽  
In Vivo Experiments ◽  
Reporter Assays ◽  
And Migration ◽  
Relationship Of

Abstract Background: microRNAs have been studied widely in hepatoblastoma. However, the role of miR-125b-5p and its relationship with the lncRNA sNEAT1 and YES1 in hepatoblastoma have not been reported previously. We aimed to reveal the role of NEAT1/miR-125b-5p/YES1 in the progression of hepatoblastoma.Methods: We collected tumor tissues and their adjacent tissues from 12 hepatoblastoma patients. qRT-PCR was applied to detect the expression of miR-125b-5p, and the relationship of miR-125b-5p with clinicopathological characteristics was analyzed. Dual luciferase reporter assays and RNA pull down assays were used to identify the relationships among NEAT1, miR-125b-5p and YES1. CCK8, Transwell assays and wound healing assays were used to examine cell viability, invasion and migration. In vivo experiments were also applied to detect the effect of miR-125b-5p on hepatoblastoma.Results: miR-125b-5p was significantly downregulated in hepatoblastoma tissue and cells. The higher the PRETEXT grade, the lower the miR-125b-5p level. NEAT1 could bind to miR-125b-5p and inhibit its expression. miR-125b-5p could target YES1 and inhibit its expression. Overexpression of miR-125b-5p decreased the proliferation, invasion, and migratory ability of hepatoblastoma cells. YES1 could rescue the above effects. At the same time, overexpression of miR-125b-5p resulted in decreased YES1 and tumor growth inhibition in vivo.Conclusion: miR-125b-5p acted as a shared miRNA of NEAT1 and YES1 in hepatoblastoma. Overexpression of miR-125b-5p could target YES1 and inhibit its expression, therefore inhibiting the progression of hepatoblastoma.

A role for the EphA family in the topographic targeting of vomeronasal axons

Development ◽  
2001 ◽  
Vol 128 (6) ◽  
pp. 895-906
Author(s):  
B. Knoll ◽  
K. Zarbalis ◽  
W. Wurst ◽  
U. Drescher
Keyword(s):  
Tyrosine Kinases ◽  
Expression Patterns ◽  
Eph Receptors ◽  
Accessory Olfactory Bulb ◽  
In Vivo Experiments ◽  
Receptor Concentration ◽  
High Ligand

We have investigated the role of the Eph family of receptor tyrosine kinases and their ligands in the establishment of the vomeronasal projection in the mouse. Our data show intriguing differential expression patterns of ephrin-A5 on vomeronasal axons and of EphA6 in the accessory olfactory bulb (AOB), such that axons with high ligand concentration project onto regions of the AOB with high receptor concentration and vice versa. These data suggest a mechanism for development of this projection that is the opposite of the repellent interaction between Eph receptors and ligands observed in other systems. In support of this idea, when given the choice of whether to grow on lanes containing EphA-F(c)/laminin or F(c)/laminin protein (in the stripe assay), vomeronasal axons prefer to grow on EphA-F(c)/laminin. Analysis of ephrin-A5 mutant mice revealed a disturbance of the topographic targeting of vomeronasal axons to the AOB. In summary, these data, which are derived from in vitro and in vivo experiments, indicate an important role of the EphA family in setting up the vomeronasal projection.

scholarly journals LINC00319 acts as a microRNA-335–5p sponge to accelerate tumor growth and metastasis in gastric cancer by upregulating ADCY3

2020 ◽  
Vol 318 (1) ◽  
pp. G10-G22
Author(s):  
Jun Zou ◽  
Kun Wu ◽  
Chao Lin ◽  
Zhi-Gang Jie
Keyword(s):  
Gastric Cancer ◽  
Adenylate Cyclase ◽  
Tumor Growth ◽  
Micro Rna ◽  
In Vivo Experiments ◽  
Tumorigenesis And Progression ◽  
Tumor Growth And Metastasis ◽  
And Migration

Gastric cancer (GC) is one of the most common cancers in the world and remains a heavy burden of health worldwide. Adenylate cyclase 3 ( ADCY3) is a widely expressed membrane-associated protein in human tissues and has been identified to be a new molecular target of GC. Long noncoding RNAs have a substantial influence on tumorigenesis and progression of tumors by binding to microRNAs. Therefore, this study is to clarify the mechanism by which LINC00319 sponges micro RNA-335–5p ( miR-335–5p) to influence the development of GC. Initially, microarray analysis identified GC-related differentially expressed LINC00319 and ADCY3 for this study. The interaction was confirmed that LINC00319 interacted with miR-335–5p to regulate ADCY3. Next, SGC-7901 cells presenting with the lowest LINC00319 expression and the highest miR-335–5p expression were transfected with LINC00319, miR-335–5p inhibitor, or ADCY3 vector to examine their roles in growth and metastasis of GC cells, which was further ascertained by in vivo experiments. LINC00319 was upregulated and miR-335–5p was downregulated in GC cells. LINC00319 overexpression, miR-335–5p inhibitor, or ADCY3 overexpression was shown to significantly elevate the expression of cyclin-dependent kinase 4 and metastasis associated 1, decrease that of growth arrest-specific 1, and promote tumor growth and metastasis by increasing proliferation and migration and reducing cell apoptosis. Importantly, it was found that overexpressed miR-335–5p exerted its tumor suppressive role in GC through downregulating ADCY3. Collectively, LINC00319 expedited growth and metastasis of GC by upregulating miR-335–5p-mediated ADCY3. NEW & NOTEWORTHY This study is carried out based on in vivo and in vitro studies in mice and gastric cancer (GC) cells with the aim of clarifying the role of LINC00319 on GC growth and metastasis, which associated with micro RNA-335–5p-mediated adenylate cyclase 3. Altogether, we identified LINC00319 to be a potential therapy to treat GC.

Binding of 63Ni(II) to Ultrafiltrable Constituents of Rabbit Serum In Vivo and In Vitro

1975 ◽  
Vol 21 (4) ◽  
pp. 521-527 ◽  
Author(s):  
Noritake Asato ◽  
Maria van Soestbergen ◽  
F William Sunderman
Keyword(s):  
Rabbit Serum ◽  
Total Serum ◽  
In Vivo Experiments ◽  
The Mean ◽  
Layer Chromatography ◽  
In Vivo Administration ◽  
Mean Concentration

Abstract Binding of 63Ni(Il) to ultrafiltrable constituents of rabbit serum was studied (a) after in vitro incubation (2 h, 37 °C) of rabbit serum with 63NiCl2 (10-100 µmol/liter), and (b) at intervals (0.25-2 h) after in vivo administration of 63NiCl2 (40-160 µmol/kg body wt, i.v.). Serum ultrafiltrates were fractionated by thin-layer chromatography, and the separated compounds made visible by autoradiography and by ninhydrin staining. Several (≃5) ultrafiltrable 63Ni-complexes were demonstrable as distinct radiodense 63Ni-bands with chromatographic mobilities corresponding to those of ninhydrin-positive bands. Unbound 63Ni(II) was not detected in serum ultrafiltrates in either the in vitro or in vivo experiments. In sera (n = 10) incubated in vitro with 63Ni(II) (10 µmol/ liter), the mean percentage of ultrafiltrable 63Ni was 36% (range = 33-38) of total serum 63Ni. In contrast, in sera (n = 10) obtained 2 h after i.v. injection of 63Ni(II) (40 µmol/kg), the mean concentration of total serum 63Ni was 10.8 µmol/liter (range = 6-14), and the mean percentage of ultrafiltrable 63Ni was 15% (range = 9-21) of total serum 63Ni. The disparity between the percentages of ultrafiltrable 63Ni obtained in vitro and in vivo was obviated when the in vivo experiments were performed in rabbits bilaterally nephrectomized, with ligated common bile ducts. This investigation confirms the existence of several nickel receptors in serum ultrafiltrates and substantiates the role of ultrafiltrable complexes in the excretion of nickel.

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