Biology subjects

Ojima, K.

Publications and source records attributed to Ojima, K..

4 recordsLinked to original sources

bioRxiv2024

Large MAF Transcription Factors Reawaken Evolutionarily Dormant Fast-Glycolytic Type IIb Myofibers in Human Skeletal Muscle

Small mammals rely on type IIb myofibers, expressing the fastest myosin IIb (encoded by MYH4), for rapid muscle contraction. In contrast, larger mammals, including humans, show reduced or absent MYH4 expression and type IIb myofibers, favoring slower-contracting myofibers. The evolutionary mechanisms underlying this shift remain unclear. Here, we identify large MAF transcription factors (MAFA, MAFB, MAF) as key regulators of MYH4 expression in large mammals, including human and bovine. Overexpression of large MAFs induces MYH4 expression and enhances glycolytic capacity in human myotubes, supported by RNA-seq and metabolic flux analyses. RNA-seq of human muscle biopsies reveals a positive correlation between MAFA, MAF, and MYH4 expression, with these genes elevated in power-trained individuals. These findings reveal a conserved mechanism across mammals, showing that large MAFs can induce type IIb myofibers even in humans, with potential applications for enhancing athletic performance and addressing age-related muscle weakness associated with the loss of fast-twitch myofibers.

cell biology↗

bioRxiv2023

A hybrid combination of in vitro cultured buccal mucosal cells using two different methodologies, complementing each other in successfully repairing a stricture-inflicted human male urethral epithelium

BackgroundAutologous buccal mucosal tissue derived cell transplantation techniques in repairing a stricture inflicted male urethral epithelium have been evolving. There was not much of clarity on the cell type, in vitro culture methods and the mode of transplantation, until we reported our buccal epithelium expanded and encapsulated in scaffold-hybrid approach to urethral stricture (BEES-HAUS) clinical study yielding a successful engraftment and repair with a long-term patency. We herein report with technical clarity on the advantages of mixing two-dimensional (2D) monolayer cultured fibroblast like cells and three dimensional (3D) thermo-reversible gelation polymer (TGP) cultured cells; the former secreting IGF-1, a cytokine known for its healing effects and the latter expressing epithelial surface markers in flow cytometry, both sourced from human buccal tissue, together transplanted using TGP as a carrier. MethodsHuman buccal tissues (n=22) redundant after urethroplasty surgery was used after informed consent and IEC approval. They were enzyme digested, divided into two portions; one was cultured as monolayer method (2D) and the other in 3D in TGP. Flowcytometry and quantification of IGF-1 in cell culture supernatant through the culture period were undertaken. ResultsIn flowcytometry, the cells on day 0, lacked AE1/AE3 - pancytokeratin expression indicative of epithelial phenotype of culture, which progressively increased in the 3D-TGP group, during invitro culture of up to 21 days. The 2D showed expression of only fibroblasts like cells that were negative for AE1/AE3 but positive for CD140b. IGF-1 secretion was significantly higher in 2D cultures than in 3D-TGP (p-value < 0.05). ConclusionThe 3D- TGP cultured cells of epithelial nature and the 2D cultured fibroblast like cells secreting IGF-1, together when transplanted using TGP scaffold as a carrier, adapted to a hostile in vivo milieu after releasing the fibrous strands with urethrotomy, successfully engrafted and repaired a stricture-inflicted male urethral epithelium in the BEES-HAUS procedure. While this hybrid combination of cells are considered to have potential in managing other epithelial damages, further research of such hybrid combination and their behaviour in disease affected environments may help to expand this solution in regenerating and repairing other tissues and organs as well. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/555240v1_ufig1.gif" ALT="Figure 1"> View larger version (70K): org.highwire.dtl.DTLVardef@1e00b7eorg.highwire.dtl.DTLVardef@12886aorg.highwire.dtl.DTLVardef@b9fbb3org.highwire.dtl.DTLVardef@3bd247_HPS_FORMAT_FIGEXP M_FIG C_FIG Illustration of pathogenesis of urethral stricture and the contribution of the hybrid combination of two-dimensional (2D) and three dimensional, 3D-TGP (Thermo-reversible gelation polymer) cultured cells to the successful repair of the stricture-inflicted male urethral epithelium in the buccal epithelium expanded and encapsulated in scaffold-hybrid approach to urethral stricture (BEES-HAUS) technique.

synthetic biology↗

bioRxiv2021

Coordination chemogenetics for activation of GPCR-type glutamate receptors in brain tissue

Direct activation of cell-surface receptors is highly desirable for elucidating the physiological roles of receptors. However, subtype-selective ligands are very limited because of the high homology among receptor subtypes. A potential approach for selective activation of a receptor subtype is chemogenetics, in which both point mutagenesis of the receptors and designed ligands are used. However, ligand-binding properties are affected in most current methods. Here, we developed a chemogenetic method for direct activation of metabotropic glutamate receptor 1 (mGlu1), which plays essential roles in cerebellar functions in the brain. Our screening identified a mGlu1 mutant, mGlu1(N264H), that was directly activated by palladium complexes. Notably, a palladium complex showing low cytotoxicity successfully activated mGlu1 in mGlu1(N264H) knock-in mice, revealing that activation of endogenous mGlu1 is sufficient to evoke the critical cellular mechanism of synaptic plasticity, a basis of motor learning in the cerebellum.

neuroscience↗

bioRxiv2020

Ligand-directed two-step labeling to quantify neuronal glutamate receptor trafficking

The regulation of glutamate receptor localization is critical for development and synaptic plasticity in the central nervous system. Conventional biochemical and molecular biological approaches have been widely used to analyze glutamate receptor trafficking, especially for AMPA-type glutamate receptors (AMPARs). However, conflicting findings have been reported because of a lack of useful tools for analyzing endogenous AMPARs. Here, we develop a new method for the rapid and selective labeling of chemical probes to AMPARs by combining affinity-based protein labeling and bioorthogonal click chemistry under physiological conditions. This method allowed us to quantify AMPAR distribution and trafficking, which revealed some unique features of AMPARs, such as a long lifetime and a rapid recycling in neurons. This method was also successfully expanded to selectively label NMDA-type glutamate receptors. Thus, bioorthogonal two-step labeling may be a versatile tool for investigating the physiological and pathophysiological roles of glutamate receptors in neurons.

biochemistry↗