A Novel Artificially Humanized Anti-Cripto-1 Antibody Suppressing Cancer Cell Growth

Cripto-1 is a member of the EGF-CFC/FRL1/Cryptic family and is involved in embryonic development and carcinogenesis. We designed a novel anti-Cripto-1 artificial antibody and assessed the recognition to the antigen and the potential to suppress the growth of cancer stem cells. First, single chain antibody clones were isolated by bio-panning with the affinity to recombinant Cripto-1 protein from our original phage-display library. Then, the variable regions of heavy chain VH and light chain VL in each clone were fused to constant regions of heavy chain CH and light chain CL regions respectively. These fused genes were expressed in ExpiCHO-S cells to produce artificial humanized antibodies against Cripto-1. After evaluation of the expression levels, one clone was selected and the anti-Cripto-1 antibody was produced and purified. The purified antibody showed affinity to recombinant Cripto-1 at 1.1 pmol and immunoreactivity to cancer tissues and cell lines. The antibody was available to detect the immunoreactivity in tissue microarrays of malignant tumors as well as in Cripto-1 overexpressing cells. Simultaneously, the antibody exhibited the potential to suppress the growth of human colon cancer derived GEO cells overexpressing Cripto-1 with IC50 at approximately 110 nM. The artificially humanized antibody is proposed to be a good candidate to target cancer cells overexpressing Cripto-1

Stereocontrol in NIPAAm polymerization

Radical polymerizations of N-isopropylacrylamide (NIPAAm) were carried out in toluene at low temperatures in the presence of phosphoric acid esters such as trimethyl, triethyl (TEP), tri-n-propyl, and tri-n-butyl phosphates (TBP). Syndiotactic-rich poly(NIPAAm)s were obtained at the temperature range from –60°C to 0°C, and particularly TEP provided the highest syndiotacticity (racemo diad = 65%) at –40°C. On the other hand, lowering temperature reversed the stereoselectivity of the propagation reaction so that isotactic-rich poly(NIPAAm)s were obtained at –80°C. In particular, TBP exhibited the most isotactic-specificity (meso diad = 57%). Job's plots for NIPAAm-TBP mixtures revealed that NIPAAm and TBP formed 1:1 complex at 0°C and predominantly 1:2 complex at –80°C through a hydrogen-bonding interaction. Therefore, it is considered that the stereospecificity of NIPAAm polymerization should depend on the stoichiometry of the hydrogen-bond-assisted complex. Thus, the mechanism for the present polymerization system was discussed

HYDROGEN-BOND-ASSISTED STEREOCONTROL

Radical polymerization of N-isopropylacrylamide (NIPAAm) in toluene at low temperatures was investigated in the presence of triisopropyl phosphate (TiPP). The addition of TiPP induced a syndiotactic-specificity that was enhanced by lowering polymerization temperature, whereas atactic polymers were obtained in the absence of TiPP regardless of temperature. Syndiotactic-rich poly(NIPAAm) with racemo diad = 65% was obtained at –60°C with a fourfold amount of TiPP, but almost atactic poly(NIPAAm)s were obtained by lowering temperature to –80°C. This result contrasted with the result in the presence of primary alkyl phosphates, such as tri-n-propyl phosphate, that stereospecificity varied from syndiotactic to isotactic by lowering polymerization temperature. NMR analysis at –80°C revealed that TiPP predominantly formed 1:1 complex with NIPAAm, although primary alkyl phosphates preferentially formed 1:2 complex with NIPAAm. Thus, it was concluded that a slight increase in bulkiness of the added phosphates influenced the stoichiometry of the NIPAAm-phosphate complex at lower temperatures and consequently a drastic change in the effect on stereospecificity of NIPAAm polymerization was observed

Cripto-1 as a Potential Target of Cancer Stem Cells for Immunotherapy

Simple Summary Cancer immunotherapy is gaining attention as a potential fourth treatment following surgery, chemotherapy, and radiation therapy. Cancer stem cells have recently been recognized and validated as a key target for cancer treatment. Cripto-1, which is a GPI-anchored membrane-bound protein that functions as a co-receptor of Nodal, is a marker of cancer stem cells. Since Nodal is a member of the TGF-beta family, which performs an important role in stem cells and cancer stem cells, the inhibition of Cripto-1 could be a strategy by which to block Nodal signaling and thereby suppress cancer stem cells. We propose that Cripto-1 may be a novel target for cancer immunotherapy. The immune system has been found to be suppressed in cancer patients. Cancer cells are extremely resistant to chemotherapeutic drugs, conventional immunotherapy, or cancer antigen vaccine therapy. Cancer immunotherapy, which is mainly based on immune checkpoint inhibitors, such as those for PD-1, PD-L1, and CTLA4, is an effective treatment method. However, no immunotherapeutic target has been found that retains validity in the face of tumor diversity. The transforming growth factor (TGF)-beta cytokine family possesses broad biological activity and is involved in the induction and/or transdifferentiation of helper T cells, which are important in immunotherapy. Nodal is a member of the TGF-beta family playing important roles in tissue stem cells and cancer stem cells (CSCs), interacting with the co-receptor Cripto-1, as well as with Activin type IB (Alk4) and Activin typeIIreceptors, and maintaining stemness and Notch and Wnt/beta-catenin signaling in CSCs. In recent years, it has been reported that Cripto-1 could be a potential therapeutic target in CSCs. Here, we review the accumulated literature on the molecular mechanisms by which Cripto-1 functions in CSCs and discuss the potential of Cripto-1 as an immunotherapeutic target in CSCs

Green Process of Three-Component Prostaglandin Synthesis and Rapid ¹¹C Labelings for Short-Lived PET Tracers: Highly Polished C-Couplings Revolutionizing Advances in Bio- and Medical Sciences

General synthesis of prostaglandins (PGs) has been accomplished based on a one-pot three-component coupling using a combination of organocopper or organozincate conjugate addition to 4-hydroxy-2-cyclopentenone followed by trapping of resulting enolate with an organic halide. Based on the use of this synthetic methodology, biologically significant PG derivatives including ent-Δ7-PGA1, 15SAPNIC ([3H]APNIC), and 15R–TIC have also been synthesized. Ultimately, organozincate conjugate addition combined with the enolate trapping by an organic triflate results in practical green three-component coupling comprising the use of stoichiometric amounts of three components (enone, α- and ω-side chains in a nearly 1:1:1 ratio) without using HMPA and heavy metals. General methodology for introducing short-lived 11C and 18F radionuclides into carbon frameworks has been established by developing rapid C-[11C]methylation and C-[18F]fluoromethylation using Pd0-mediated rapid cross-coupling between [11C]methyl iodide and an organotributylstannane or organoboronate; or [18F]fluoromethyl bromide and organoboronate, respectively, allowing the synthesis of a wide variety of biologically significant and disease-oriented PET probes such as 15R-[11C]TIC. Moreover, PdII-mediated rapid C-[11C]carbonylation using [11C]CO and organoboronate at ambient temperature under atmospheric pressure using conventional helium carrier gas has been explored. Further, C-[11C]carboxylation has been promoted using [11C]CO2 and organoboronate with RhI catalyst under atmospheric pressure

Eosinophil Cationic Protein Shows Survival Effect on H9c2 Cardiac Myoblast Cells with Enhanced Phosphorylation of ERK and Akt/GSK-3β under Oxidative Stress

Eosinophil cationic protein (ECP) is well known as a cationic protein contained in the basic granules of activated eosinophils. Recent studies have reported that ECP exhibits novel activities on various types of cells, including rat neonatal cardiomyocytes. Here we evaluated the effects of ECP on rat cardiac myoblast H9c2 cells. Our results showed that ECP enhanced the survival of the cells, in part by promoting the ERK and Akt/GSK-3β signaling pathways. ECP attenuated the cytotoxic effects of H2O2 on H9c2 cells as well as the production of reactive oxygen species, the number of apoptotic cells and caspase 3/7 activity in the cells. In conclusion, ECP activated the ERK and Akt/GSK-3β pathways, resulting in anti-oxidative effects on H9c2 cells that attenuated apoptosis

The Outline of the High School Trial Corresponding to the University Entrance Selection Reform : Special Examination Subcommittee of the National Six University Collaborative Consortium Education Collaboration Organization

6 大学連携事業として，評価したい資質について掘り下げ質問を行う構造化面接と，面接に代わる筆記試験であるペーパーインタビューについて，県内の高等学校の協力を得て2019 年度にトライアルを行った。評価した資質は「新たなこと追究しようとする態度」と「協働して取り組む態度」であり，まず，構造化面接とペーパーインタビューの結果を本学の教員が評価し，その結果を高校教員が個々の受験生に抱いている日常的評価と比較した。トライアルの結果，構造化面接の有効性ならびに，受験者全員に面接を課すことが，時間的，人員的に実施困難な入試におけるペーパーインタビューの有効性が確認された。同時に，測ろうとする資質によっては，面接との評価結果が異なる部分があることも明らかとなった

Isolation and characterization of cancer stem cells derived from human glioblastoma

Cancer stem cell (CSC) is considered as a cause of cancer recurrence and metastasis. Simultaneously CSCs are responsible for the heterogeneous population in tumor tissues due to their differentiation potential. However, the characterizations of CSCs are still not enough and cancer stem cell lines widely available is desired to be established for the advancement of cancer research. In this study, we tried to isolate and characterize stem like cells from human glioblastoma cell line U-251MG cells. U-251MG P1 cells, which was previously condensed in the presence of hyaluronic acid as CD44 positive population were subjected to single cell isolation procedure. Although 5 clones were isolated, only one clone exhibited high expression of CD44, Nanog, OCT3/4 and SOX2, and named U-251MGSC1. The sphere forming ability of U-251MGSC1 cell was significantly higher than the parental U-251MG cells. Tumorigenicity of U-251MG-SC1 cells were higher than that of U-251MG cells. U-251MGSC1 cells exhibited higher expression of CD44, SOX2, Nestin and A2B5 than U-251MG cells in vitro and in vivo. The expression of GFAP and NF-M was enhanced when the cells were treated with the conditioned medium of U-251MG cells indicating the potential of differentiation. Sphere forming ability was more efficient than that of U-251MG cells and was enhanced in the presence of hyaluronic acid, which enhanced the cell growth as well. U-251MGSC1 cells exhibited rapid growth tumor in nude mice and efficient metastatic ability in transmembrane assay when compared with U-251MG cells. As the result, we concluded U-251MGSC1 cell was a glioblastoma CSC line derived from the parental U-251MG cells. U-251MGSC1 cells will be a good tool to develop effective therapeutic agents against CSCs and to elucidate the properties of glioma derived CSCs and the mechanism of tumor development in brain