Biology subjects

Nakazawa, M.

Publications and source records attributed to Nakazawa, M..

3 recordsLinked to original sources

bioRxiv2023

Protective role of protease-activated receptor-2 in anaphylaxis model mice

Anaphylaxis is a severe life-threatening hypersensitivity reaction induced by mast cell degranulation. Among the various mediators of mast cells, little is known about the role of tryptase. Therefore, we aimed to elucidate the role of protease-activating receptor-2 (PAR-2), a receptor activated by tryptase, in murine anaphylactic models using PAR-2-deficient mice and newly generated tryptase-deficient mice. Anaphylaxis was induced by IgE-dependent and IgE-independent mast cell degranulation in mice. PAR-2 deficiency exacerbated the decrease in body temperature and hypotension during anaphylaxis; however, the number of skin mast cells, degree of mast cell degranulation, and systemic and local vascular hyperpermeability were comparable in PAR-2 knockout and wild-type mice. Nitric oxide, which is produced by endothelial nitric oxide synthase (eNOS), is an indispensable vasodilator in anaphylaxis. In the lungs of anaphylactic mice, PAR-2 deficiency promoted eNOS expression and phosphorylation, suggesting a protective effect of PAR-2 against anaphylaxis by downregulating eNOS activation and expression. Based on the hypothesis that the ligand for PAR-2 in anaphylaxis is mast cell tryptase, tryptase-deficient mice were generated using CRISPR-Cas9. In wild-type mice, the PAR-2 antagonist exacerbated the body temperature drop due to anaphylaxis; however, the effect of the PAR-2 antagonist was abolished in tryptase-deficient mice. These results suggest that tryptase is a possible ligand of PAR-2 in anaphylaxis and that the tryptase/PAR-2 pathway attenuates the anaphylactic response in mice.

molecular biology↗

bioRxiv2022

Nitrate Assimilation Underlying Kleptoplasty

While photoautotrophic organisms utilize inorganic nitrogen as the nitrogen source, heterotrophic organisms utilize organic nitrogen and thus do not generally have an inorganic nitrogen assimilation pathway. Here we focused on the nitrogen metabolism of Rapaza viridis, a unicellular eukaryote exhibiting kleptoplasty. Although belonging to the lineage of essentially heterotrophic flagellates, R. viridis exploits the photosynthetic products of the kleptoplasts and was therefore suspected to potentially utilize inorganic nitrogen. From the transcriptome data of R. viridis, we identified the gene RvNaRL, which had sequence similarity to nitrate reductases found in plants. Phylogenetic analysis revealed that RvNaRL was acquired by a horizontal gene transfer event. To verify its function of the protein product RvNaRL, we established a RNAi mediated knockdown and a CRISPR-Cas9-mediated knockout experiments for the first time in R. viridis and applied them to this gene. The RvNaRL knockdown and knockout cells exhibited significant growth only when ammonium was supplied but, in contrast to the wild-type cells, no substantial growth when nitrate was supplied. Such arrested growth in absence of ammonium was attributed to impaired amino acid synthesis due to the deficiency of nitrogen supply from the nitrate assimilation pathway; this in turn resulted in the accumulation of excess photosynthetic products in the form of cytosolic polysaccharide grains as observed. These results indicate that RvNaRL is certainly involved in nitrate assimilation by R. viridis. Thus, we infer that R. viridis achieved its advanced kleptoplastic strategy owing to a posteriori acquisition of the nitrate assimilation pathway the horizontal gene transfer.

biochemistry↗

bioRxiv2020

Recovery of cryo-injured rabbit urethras by biofabricated C-shaped adipose-derived mesenchymal cell structures

Urethral tissue damage can cause stress urinary incontinence or urinary retention, and there are few long-term, effective treatments. For structural and functional recovery of urethral tissues, we designed and constructed C-shaped structures composed of adipose-derived mesenchymal cells (AMCs) by using a three-dimensional (3D)-bioprinter system, which could be transplanted in damaged urethras without obstructing the lumen. We determined if transplantation of the biofabricated structures could reconstruct the urethral tissues. AMCs were harvested from rabbits, cultured and labeled with PKH26 to form spheroids. The spheroids were assembled on a custom-designed C-shaped support by a 3D bioprinter. Urethras of rabbits were cryo-injured by spraying with liquid nitrogen for 20 seconds, incised and biofabricated structure was autologously transplanted. Control rabbits were treated similarly but without transplantation structure. Two and four weeks after surgery, the control urethras were partially constricted; however, the structure-transplanted urethras were patent. The AMCs within the structures differentiated into skeletal muscle, smooth muscle, nerve, or endothelial cells. Some cells contained growth factors and cytokines. Therefore, biofabricated C-shaped AMC structures have potential to be an effective treatment for urethral recovery. Summary StatementThree-dimensional bioprinter was used to biofabricate novel C-shaped structures composed of adipose-derived mesenchymal cells (AMCs) and have the potential to be an effective treatment for urethral recovery.

bioengineering↗