Organellar Glue: A Molecular Tool to Artificially Control Chloroplast–Chloroplast Interactions

細胞小器官を接着する新技術「オルガネラグルー」を開発 --オルガネラ間コミュニケーションの操作に期待--. 京都大学プレスリリース. 2022-09-30.Organelles can physically interact to facilitate various cellular processes such as metabolite exchange. Artificially regulating these interactions represents a promising approach for synthetic biology. Here, we artificially controlled chloroplast–chloroplast interactions in living plant cells with our organelle glue (ORGL) technique, which is based on reconstitution of a split fluorescent protein. We simultaneously targeted N-terminal and C-terminal fragments of a fluorescent protein to the chloroplast outer envelope membrane or cytosol, respectively, which induced chloroplast–chloroplast interactions. The cytosolic C-terminal fragment likely functions as a bridge between two N-terminal fragments, thereby bringing the chloroplasts in close proximity to interact. We modulated the frequency of chloroplast–chloroplast interactions by altering the ratio of N- and C-terminal fragments. We conclude that the ORGL technique can successfully control chloroplast–chloroplast interactions in plants, providing a proof of concept for the artificial regulation of organelle interactions in living cells

N-Cadherin Expressed on Malignant T Cell Lymphoma Cells is Functional, and Promotes Heterotypic Adhesion Between the Lymphoma Cells and Mesenchymal Cells Expressing N-Cadherin

Cadherins are Ca2+-dependent cell–cell adhesion molecules, and are involved in the formation and maintenance of the organocellular architecture. Using a combination of molecular biologic and biochemical methods, we analyzed cadherins expressed on cultured human malignant lymphoma cell lines (adult T cell lymphomas, human T cell leukemia virus type 1-negative T cell lines, and thymus-derived lymphoma cell lines), and obtained evidence that N-cadherin is the major cadherin expressed on these cells. These cells were found to form cell aggregates in a Ca2+-dependent manner, and more importantly to coaggregate and adhere with cells expressing N-cadherin, suggesting that N-cadherin on lymphoma cells is functionally active. Therefore, N-cadherin expressed on lymphoma cells could underlie the frequent invasion of these cells into the mesenchymal tissue in the skin and the central nervous system

Highly stomach-selective gene transfer following gastric serosal surface instillation of naked plasmid DNA in rats.

BACKGROUND: The purpose of this study was to achieve stomach-selective gene transfer in rats by our simple and novel administration method, which is gastric serosal surface instillation of naked plasmid DNA (pDNA). METHODS: Naked pDNA encoding firefly luciferase as a reporter gene was instilled onto the gastric serosal surface in male Wistar rats. As controls, we performed intraperitoneal, intragastric and intravenous administration of naked pDNA. At appropriate time intervals, we measured luciferase activities in the stomach and other tissues. RESULTS: Gene expression in the stomach 6 h after gastric serosal surface instillation of naked pDNA (5 microg) was significantly higher than that after using other administration methods. The present study is the first report on stomach-selective gene transfer following instillation of naked pDNA onto the gastric serosal surface in rats. Also, the gene expression level in the stomach 6 h after gastric serosal surface instillation of naked pDNA was markedly higher than that in other tissues. In a dose-dependent study, the gene expression level was saturated over 5 microg. Gene expression in the stomach was detected 3 h after gastric serosal surface instillation of naked pDNA. The gene expression level peaked 12-24 h after instillation of naked pDNA, then decreased to a level similar to 3 h at 48 h. CONCLUSIONS: Gastric serosal surface in stillation of naked pDNA can be a highly stomach-selective gene transfer method in rats

Potential of a Novel Chemical Compound Targeting Matrix Metalloprotease-13 for Early Osteoarthritis: An In Vitro Study

Osteoarthritis is a progressive disease characterized by cartilage destruction in the joints. Matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs) play key roles in osteoarthritis progression. In this study, we screened a chemical compound library to identify new drug candidates that target MMP and ADAMTS using a cytokine-stimulated OUMS-27 chondrosarcoma cells. By screening PCR-based mRNA expression, we selected 2-(8-methoxy-2-methyl-4-oxoquinolin-1(4H)-yl)-N-(3-methoxyphenyl) acetamide as a potential candidate. We found that 2-(8-methoxy-2-methyl-4-oxoquinolin-1(4H)-yl)-N-(3-methoxyphenyl) acetamide attenuated IL-1 beta-induced MMP13 mRNA expression in a dose-dependent manner, without causing serious cytotoxicity. Signaling pathway analysis revealed that 2-(8-methoxy-2-methyl-4-oxoquinolin-1(4H)-yl)-N-(3-methoxyphenyl) acetamide attenuated ERK- and p-38-phosphorylation as well as JNK phosphorylation. We then examined the additive effect of 2-(8-methoxy-2-methyl-4-oxoquinolin-1(4H)-yl)-N-(3-methoxyphenyl) acetamide in combination with low-dose betamethasone on IL-1 beta-stimulated cells. Combined treatment with 2-(8-methoxy-2-methyl-4-oxoquinolin-1(4H)-yl)-N-(3-methoxyphenyl) acetamide and betamethasone significantly attenuated MMP13 and ADAMTS9 mRNA expression. In conclusion, we identified a potential compound of interest that may help attenuate matrix-degrading enzymes in the early osteoarthritis-affected joints

Secure and effective gene delivery system of plasmid DNA coated by polynucleotide

Polynucleotides are anionic macromolecules which are expected to transfer into the targeted cells through specific uptake mechanisms. So, we developed polynucleotides coating complexes of plasmid DNA (pDNA) and polyethylenimine (PEI) for a secure and efficient gene delivery system and evaluated their usefulness. Polyadenylic acid (polyA), polyuridylic acid (polyU), polycytidylic acid (polyC), and polyguanylic acid (polyG) were examined as the coating materials. pDNA/PEI/polyA, pDNA/PEI/polyU, and pDNA/PEI/polyC complexes formed nanoparticles with a negative surface charge although pDNA/PEI/polyG was aggregated. The pDNA/PEI/polyC complex showed high transgene efficiency in B16-F10 cells although there was little efficiency in pDNA/PEI/polyA and pDNA/PEI/polyU complexes. An inhibition study strongly indicated the specific uptake mechanism of pDNA/PEI/polyC complex. Polynucleotide coating complexes had lower cytotoxicity than pDNA/PEI complex. The pDNA/PEI/polyC complex showed high gene expression selectively in the spleen after intravenous injection into mice. The pDNA/PEI/polyC complex showed no agglutination with erythrocytes and no acute toxicity although these were observed in pDNA/PEI complex. Thus, we developed polynucleotide coating complexes as novel vectors for clinical gene therapy, and the pDNA/PEI/polyC complex as a useful candidate for a gene delivery system

Factors in glucocorticoid regimens associated with treatment response and relapses of IgG4-related disease: a multicentre study

Glucocorticoids (GC) are effective for treating IgG4-related disease (IgG4-RD); however, relapse is often observed. We conducted a retrospective multicentre study to investigate risk factors in GC regimens associated with relapses of IgG4-RD. Data on 166 patients with definitive IgG4-RD diagnosis were collected from 12 institutions. Comprehensive surveillance of clinical backgrounds and GC regimens as well as multivariate analysis of factors associated with treatment responses and relapses was performed. To determine the initial maximal GC dose, the patients were stratified into three groups depending on the initial prednisolone (PSL) dosage: 0.7 mg/kg/day. The multivariate analysis extracted the disease duration and reduction speed of initial GC dose. Patients treated with initial GC 0.7 mg/kg/day of PSL showed higher relapse rates than those treated with 0.4–0.69 mg/kg/day. The relapse rates were significantly higher in patients with fast reduction of the initial dose (>0.4 mg/day) than in patients with slow reduction (<0.4 mg/day). To avoid relapse, 0.4–0.69 mg/kg/day of initial PSL with slow reduction speed (<0.4 mg/day) is needed in the early treatment of IgG4-RD

Regional delivery of model compounds and 5-Fluorouracil to the liver by their application to the liver surface in rats: its implication for clinical use

PURPOSE: The purpose of this study was to examine drug distribution in the liver after drug application to the rat liver surface. METHODS: Phenolsulfonphthalein (PSP) and fluorescein isothiocyanate dextran (MW 4400, FD-4) as model compounds or 5-fluorouracil (5-FU) was applied to the rat liver surface by employing a cylindrical diffusion cell (i.d. 9 mm, 0.64 cm2). Then, blood and the remaining solution in the diffusion cell were collected at selected times, followed by excision of the liver. The excised liver was divided into three sites: the region under the diffusion cell attachment site (site 1), the applied lobe except for site 1 (site 2), and non-applied lobes (site 3). RESULTS: In the case of i.v. administration, there were no differences in PSP concentrations among the three sites of the rat liver, and the concentrations rapidly decreased. On the other hand, the PSP concentration in site 1 after application to the rat liver surface was considerably higher than in site 2 and site 3. In addition, the area under the curve (AUC) value (AUCsite1), calculated from the PSP concentration profile in site 1, was about 10 times larger than that in site 3. A similar trend of regional delivery advantage by liver surface application was observed in the case of the macromolecule model FD-4, with a marked AUCsite1 of about 5 times larger than the other two sites. Moreover, we clarified that the anticancer drug 5-FU preferentially distributed in site 1 after application to the rat liver surface. CONCLUSION: These results demonstrate the possibility of regional delivery of drugs to the liver by application to the liver surface

Improved stomach selectivity of gene expression following microinstillation of plasmid DNA onto the gastric serosal surface in mice

Stomach-selective gene transfer is a promising approach as a therapeutic strategy for refractory gastric diseases. In this study, we improved the stomach selectivity of gene expression following microinstillation of naked plasmid DNA (pDNA) onto the gastric serosal surface in mice. pDNA encoding firefly luciferase was used as a reporter gene. It was confirmed that the gene expression level in the stomach 6 h after gastric serosal surface microinstillation of pDNA was significantlyhigher than after intragastric, intraperitoneal and intravenous administration. Regarding selectivity ofgene expression, the gene expression level in the stomach after gastric serosal surfacemicroinstillation of 1 μg/1 μL (dose/volume) pDNA was 5.7 times higher than that in the spleen. In our previous study (30 μg/30 μL), the expression level in the stomach was 2.7 times higher than that in the spleen; therefore, the selectivity was 2.1 times higher in this study. When we investigated gene expression at various pDNA solution concentrations, the ratio of the gene expression level in the stomach to that in the spleen was the highest as 1 μg/1 μL of pDNA, which was considered the optimal concentration. Information in this study is useful for further development of target organ-selective gene delivery systems

Development of Effective Cancer Vaccine Using Targeting System of Antigen Protein to APCs.

PURPOSE: To develop a novel cancer vaccine using the targeting system of antigen protein to antigen-presenting cells (APCs) for efficient and safe cancer therapy. METHODS: The novel delivery system was constructed with antigen protein, benzalkonium chloride (BK), and γ-polyglutamic acid (γ-PGA), using ovalbumin (OVA) as a model antigen protein and evaluating its immune induction effects and utilities for cancer vaccine. RESULTS: BK and γ-PGA enabled encapsulation of OVA and formed stable anionic particles at nanoscale, OVA/BK/γ-PGA complex. Complex was taken up by dendritic cell line DC2.4 cells efficiently. We subcutaneously administered the complex to mice and examined induction of IgGs. The complex induced not only Th2-type immunoglobulins but also Th1-type immunoglobulins. OVA/BK/γ-PGA complex inhibited tumor growth of E.G7 cells expressing OVA regularly; administered OVA/BK/γ-PGA complex completely rejected tumor cells. CONCLUSION: The novel vaccine could be platform technology for a cancer vaccine

Transition-Metal-Catalyzed Diarylation of Isocyanides with Triarylbismuthines for the Selective Synthesis of Imine Derivatives

The transition-metal-catalyzed diarylation of isocyanides with triarylbismuthines was investigated in detail, and rhodium catalysts such as [RhCl(nbd)]2 were found to selectively afford N-alkyl diaryl ketimines. On the other hand, palladium-catalyzed diarylation proceeded with the incorporation of two molecules of isocyanide, preferentially yielding N,N’-dialkyl or N,N’-diaryl α-diimines. In addition, a cascade synthesis of 2,3-diarylquinoxalines starting from the palladium-catalyzed diarylation of isocyanides with triarylbismuthines was successfully achieved