Ulmus pumila L. is a deciduous tree that is widely distributed in East Asia. The stem and root bark of this species have been used in traditional Chinese medicine (TCM) for edema, mastitis, gastric cancer, and inflammation. 1,2) To search for biologically active substances, we separated the 80% ethanol extract of the root bark of U. pumila, and two sesquiterpene o-naphthoquinones, mansonone E (ME) and F (MF), were isolated from this species for the first time. It has been reported that ME and MF have antioxidant activities, 3) and many recent reports have focused on the apoptosis-inducing effects of antioxidants such as magnolol, ursolic acid, oleanic acid, maslinic acid, and others. [4][5][6] Therefore in this study the cytotoxic effects of ME and MF on the human tumor cell lines A375-S2, HeLa, MCF-7, and U937 were investigated. Both ME and MF showed potent antiproliferative effects, and ME induced apoptosis of HeLa cells by downregulation of Bcl-2 and Bcl-XL and upregulation of Bax, leading to the initiation of mitochondria-apoptosome pathways. The effects of ME on normal human cells, human peripheral blood mononuclear cells (PBMCs) and human embryonic lung (HEL) fibroblast cells were also investigated. MATERIALS AND METHODS General Experimental ProceduresThe NMR spectra were recorded on a Bruker ARX-300 NMR spectrometer ( 1 H, 300 MHz; 13 C, 75 MHz), using TMS as an internal standard. EI-MS were recorded on a GC-MS QP5050 spectrometer (Shimadzu, Kyoto, Japan). Column chromatography was performed on silica gel (100-140 and 200-300 mesh, Qingdao Haiyang Chemical, Shandong, China) and Sephadex LH-20 (bead size 25-100 mm, Sigma-Aldrich Chemical, St. Louis, MO, U.S.A.). Analytical TLC was performed using silica gel 60 F 254 plates (Kieselgel 60 F 254 precoated plates, Merck, Darmstadt, Germany). Hemipreparative HPLC was performed on an ODS C-18 column (9.8 mmϫ250 mm, 5 mm, Phenomenex, Torrance, CA, U.S.A.) using a watermethanol system as a mobile phase, monitored with an RID detector. All solvents were of analytical reagent or chromatographic reagent grade.Plant Material The root barks of U. pumila were collected from Fuxin, Liaoning Province, China, in May 2000 and identified by Prof. Zheng Cui of Shengyang Pharmaceutical University, China. Fresh root barks were dried in a dark, well-ventilated place, and a voucher specimen has been deposited in the Department of Traditional China Materia Medica, Shenyang Pharmaceutical University. Extraction and IsolationThe air-dried root barks of U. pumila (10 kg) were milled and extracted three times with 80% ethanol under reflux. The ethanol extract was filtered and concentrated under reduced pressure. The crude product (1195 g) was successively partitioned with EtOAc and n-butanol. The EtOAc layer was concentrated under reduced pressure, and the residue (111.8 g) was subjected to silica gel column chromatography eluted with c-hexane and a c-hexaneacetone mixture with increasing proportions of acetone. The fractions were collected and combined by monitoring with analytical T...
Adoptive cell therapy using TCR-engineered T cells (TCR-T cells) represents a promising strategy for treating relapsed and metastatic cancers. We previously established methods to identify neoantigenspecific TCRs based on patients' PBMCs. However, in clinical practice isolation of PBMCs from advanced-stage cancer patients proves to be difficult. In this study, we substituted blood-derived T cells for tumor-infiltrating lymphocytes (TILs) and used an HLA-matched cell line of antigenpresenting cells (APCs) to replace autologous dendritic cells. Somatic mutations were determined in head and neck squamous cell carcinoma resected from two patients. HLA-A*02:01-restricted neoantigen libraries were constructed and transferred into HLA-matched APCs for stimulation of patient TILs. TCRs were isolated from reactive TIL cultures and functionality was tested using TCR-T cells in vitro and in vivo. To exemplify the screening approach, we identified the targeted neoantigen leading to recognition of the minigene construct that stimulated the strongest TIL response. Neoantigen peptides were used to load MHC-tetramers for T cell isolation and a TCR was identified targeting the KIAA1429 D1358E mutation. TCR-T cells were activated, exhibited cytotoxicity, and secreted cytokines in a dose-dependent manner, and only when stimulated with the mutant peptide. Furthermore, comparable to a neoantigen-specific TCR that was isolated from the patient's PBMCs, KIAA1429 D1358E -specific TCR T cells destroyed human tumors in mice. The established protocol provides the required flexibility to methods striving to identify neoantigen-specific TCRs. By using an MHC-matched APC cell line and neoantigen-encoding minigene libraries, autologous TILs can be stimulated and screened when patient PBMCs and/or tumor material are not available anymore.Abbreviations: Head and neck squamous cell carcinoma (HNSCC); adoptive T cell therapy (ACT); T cell receptor (TCR); tumor-infiltrating lymphocytes (TIL); cytotoxic T lymphocyte (CTL); peripheral blood mononuclear cell (PBMC); dendritic cell (DC); antigen-presenting cells (APC)
Cysteine-rich secretory protein 2 (CRISP2) is an important protein in spermatozoa that plays roles in modulating sperm flagellar motility, the acrosome reaction, and gamete fusion. Spermatozoa lacking CRISP2 exhibit low sperm motility and abnormal morphology. However, the molecular mechanisms underlying the reduction of CRISP2 in asthenoteratozoospermia (ATZ) remain unknown. In this study, low expression of CRISP2 protein rather than its mRNA was observed in the ejaculated spermatozoa from ATZ patients as compared with normozoospermic males. Subsequently, bioinformatic prediction, luciferase reporter assays, and microRNA-27a (miR-27a) transfection experiments revealed that miR-27a specifically targets CRISP2 by binding to its 3’ untranslated region (3’-UTR), suppressing CRISP2 expression posttranscriptionally. Further evidence was provided by the clinical observation of high miR-27a expression in ejaculated spermatozoa from ATZ patients and a negative correlation between miR-27a expression and CRISP2 protein expression. Finally, a retrospective follow-up study supported that both high miR-27a expression and low CRISP2 protein expression were associated with low progressive sperm motility, abnormal morphology, and infertility. This study demonstrates a novel mechanism responsible for reduced CRISP2 expression in ATZ, which may offer a potential therapeutic target for treating male infertility, or for male contraception.
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