GATA binding protein 2 mediated ankyrin repeat domain containing 26 high expression in myeloid-derived cell lines.

BACKGROUND: Thrombocytopenia 2, an autosomal dominant inherited disease characterized by moderate thrombocytopenia, predisposition to myeloid malignancies and normal platelet size and function, can be caused by 5'-untranslated region (UTR) point mutations in ankyrin repeat domain containing 26 (ANKRD26). Runt related transcription factor 1 (RUNX1) and friend leukemia integration 1 (FLI1) have been identified as negative regulators of ANKRD26. However, the positive regulators of ANKRD26 are still unknown. AIM: To prove the positive regulatory effect of GATA binding protein 2 (GATA2) on ANKRD26 transcription. METHODS: Human induced pluripotent stem cells derived from bone marrow (hiPSC-BM) and urothelium (hiPSC-U) were used to examine the ANKRD26 expression pattern in the early stage of differentiation. Then, transcriptome sequencing of these iPSCs and three public transcription factor (TF) databases (Cistrome DB, animal TFDB and ENCODE) were used to identify potential TF candidates for ANKRD26. Furthermore, overexpression and dual-luciferase reporter experiments were used to verify the regulatory effect of the candidate TFs on ANKRD26. Moreover, using the GENT2 platform, we analyzed the relationship between ANKRD26 expression and overall survival in cancer patients. RESULTS: In hiPSC-BMs and hiPSC-Us, we found that the transcription levels of ANKRD26 varied in the absence of RUNX1 and FLI1. We sequenced hiPSC-BM and hiPSC-U and identified 68 candidate TFs for ANKRD26. Together with three public TF databases, we found that GATA2 was the only candidate gene that could positively regulate ANKRD26. Using dual-luciferase reporter experiments, we showed that GATA2 directly binds to the 5'-UTR of ANKRD26 and promotes its transcription. There are two identified binding sites of GATA2 that are located 2 kb upstream of the TSS of ANKRD26. In addition, we discovered that high ANKRD26 expression is always related to a more favorable prognosis in breast and lung cancer patients. CONCLUSION: We first discovered that the transcription factor GATA2 plays a positive role in ANKRD26 transcription and identified its precise binding sites at the promoter region, and we revealed the importance of ANKRD26 in many tissue-derived cancers.

Potential target-related proteins in rabbit platelets treated with active monomers dehydrocorydaline and canadine from Rhizoma corydalis.

BACKGROUND: Dehydrocorydaline (DHC) and canadine (THB) are two active alkaloid compounds in Corydalis yanhusuo (Y.H. Chou & Chun C. Hsu) W.T. Wang ex Z.Y. Su & C.Y. Wu (Papaveraceae) (Rhizoma Corydalis). DHC and THC were previously shown to exert anti-platelet aggregation effect dose-dependently, but their exact mechanisms had not yet been addressed. Therefore, it is essential to study the mechanisms of DHC and THB affecting on platelet's function. PURPOSE: To investigate the anti-platelet effects and corresponding signal cascades of DHC and THB on platelet aggregation. METHODS: Firstly, in vitro anti-platelet aggregation of DHC and THB induced by different agonists including thrombin (THR), adenosine diphosphate (ADP) and arachidonic acid (AA) were determined through turbidimetry method. Then the possible target-related platelet proteins after treated with DHC/THB were separated and identified by two dimensional gel electrophoresis (2-DE) and MALDI-TOF-MS/MS analysis, respectively. Finally, the signal cascades network induced by DHC/THB were predicted through functional analysis of these proteins along with the determination of platelet DAG concentration. RESULTS: The platelet aggregation stimulated by THR, ADP and AA were inhibited by DHC and THB dose-dependently to a certain degree. Meanwhile, DHC and THB had the strongest effect on ADP- and THR-induced platelet aggregation respectively. In addition, treatment of these two compounds caused regulations of about sixty proteins in platelet, including cytoskeleton proteins, cell signaling proteins, proteins related to material energy metabolism, etc. CONCLUSIONS: Using proteomic analysis combined with platelet aggregation test and ELISA, this study was successful in exploring the possible mechanisms of DHC/THB on platelet aggregation. DHC might inhibit platelet aggregation by a mechanism involving the ADP receptors P2Y1 and P2Y12, and the effect of THB on platelet function may be related to its binding to THR receptor PAR1 for mediated Gi signaling pathway. These results provide fundamental information for the anti-thrombotic effect of RC.

Comparative Analysis of Soluble Proteins in Four Medicinal Aloe Species by Two-Dimensional Electrophoresis and MALDI-TOF-MS.

Background: Aloe barbadensis Miller 1768, A. vera L. var. chinensis (Haw.) Berger 1908, A. ferox Miller 1768, and A. arborescens Miller 1768 are the most widely cultivated species of Aloe and are used in Asia along with 400 other Aloe species worldwide because of their potent and potential bioactivity. Objective: The objective was to analyze and compare the soluble proteins of four commonly used medicinal Aloe species. Methods: Aloe protein samples were obtained by TCA/acetone-saturated phenol-methanol/ammonium acetate combined extraction (phenol extraction), and then were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. Finally, the differentially expressed proteins of four Aloe species were identified by matrix-assisted laser desorption ionization-time-of-flight-MS analysis. Results: The phenol extraction method was the most suitable method for the protein extraction of Aloe. Fifty differentially expressed proteins in four Aloe species were successfully identified and divided into eight functional categories. Furthermore, Malate dehydrogenase and ran-binding protein in A. barbadensis, cytoskeletal-related protein tubulin in A. vera var. chinensis and auxin-induced protein PCNT-115 in A. arborescens are closely related to their morphological characteristics. Conclusions: There are differences in the soluble proteins of the four Aloe species. Those proteins, related to the difference of their morphology of Aloe, might be used to identify different species. Highlights: Fifty differentially expressed proteins in four medicinal Aloe species were identified, and these proteins were classified into eight categories according to their biological functions. Four special proteins closely related to the morphological characteristics of Aloe were found and might be used to identify these four Aloe species.

Adsorbed hollow fiber-based biological fingerprinting for the discovery of platelet aggregation inhibitors from Danshen-Honghua decoction.

For lead compound discovery from natural products, hollow fiber cell fishing with chromatographic analysis is a newly developed method. In this study, an adsorbed hollow fiber-based biological fingerprinting method was firstly developed to discover potential platelet aggregation inhibitors from Danshen-Honghua decoction. Platelets were seeded on the fiber and their survival rate was tested. Results indicated that more than 92% platelets survived during the whole operation process. Ranitidine and tirofiban were used as positive and negative control respectively to verify the reliability of the presented approach. The main variables such as amount of extract and stirring time that affect the adsorbed hollow fiber-based biological fingerprinting process were optimized, and the repeatability of this method was also investigated. Finally, 12 potential active compounds in Danshen-Honghua decoction were successfully detected using the established approach and structures for nine of them were tentatively identified by liquid chromatography with mass spectrometry analysis. In addition, the in vitro platelet aggregation inhibition test was carried out for five of the nine hit compounds, and three active components, namely, lithospermic acid, salvianolic acid A, and salvianolic acid B were confirmed. These results proved that the proposed method could be an effective approach for screening platelet inhibitors from plant extracts.