Biology subjects

Kimura, T.

Publications and source records attributed to Kimura, T..

49 records · Page 3Linked to original sources

bioRxiv2021

Drug metabolism activity is a Critical Intrinsic Selector for Hepatocytes to elongate cell lifespan by using a cell-killing antibiotic

Hepatocytes can be used to study the pathogenesis of liver diseases and drug discovery research. Human hepatocytes are, however, hardly expandable in vitro making it difficult to secure large numbers of cells from one donor. In this study, we aimed to establish an in vitro long-term culture system that enables stable proliferation and maintenance of human hepatocytes to ensure a constant supply. We purified human hepatocytes by selection with cytocidal puromycin, and cultured them for more than 60 population doublings over a span of more than 350 days. These results show that this simple culture system with usage of the cytocidal antibiotics enables efficient hepatocyte proliferation and is an effective method for generating a stable supply of hepatocytes for drug discovery research at a significant cost reduction.

cell biology↗

bioRxiv2021

Effect of luminal surface structure of decellularized aorta on thrombus formation and cell behavior

As the number of cardiovascular diseases increases, artificial heart valves and blood vessels have been developed. Although cardiovascular application using decellularized tissue have been studied, the mechanism of their functionality is still unknown. To find the important factor for preparing decellularized cardiovascular prothesis which shows good in vivo performance, the effect of luminal surface structure of decellularized aorta on thrombus formation and cell behavior was investigated. Various luminal surface structures of decellularized aorta were prepared by heating, drying and peeling. The luminal surface structure and collagen denaturation was evaluated by immunohistological staining, collagen hybridizing peptide (CHP) staining and scanning electron microscopy (SEM) analysis. To evaluate the effect of luminal surface structure of decellularized aorta on thrombus formation and cell behavior, blood clotting test and recellularization of endothelial cells and smooth muscular cells were performed. The results of blood clotting test showed that the closer the luminal surface structure is to native aorta, the higher the anti-coagulant property. From the result of cell seeding-test, it was suggested that vascular cells recognize the luminal surface structure and regulate adhesion, proliferation and functional expression. These results will provide important factor for preparing decellularized cardiovascular prothesis and lead to future development on decellularized cardiovascular applications.

bioengineering↗

bioRxiv2020

The Phosphorylation Status of NPH3 Affects Photosensory Adaptation During the Phototropic Response

Photosensory adaptation, which can be classified as sensor or effector adaptation, optimizes the light sensing of living organisms by tuning their sensitivity to changing light conditions. During the phototropic response in Arabidopsis (Arabidopsis thaliana), the light-dependent expression controls of blue-light photoreceptor phototropin1 (phot1) and its modulator ROOT PHOTOTROPISM2 (RPT2) are known as the molecular mechanisms underlying sensor adaptation. However, little is known about effector adaption in plant phototropism. Here we show that control of the phosphorylation status of NONPHOTOTROPIC HYPOCOTYL3 (NPH3) leads to effector adaptation in hypocotyl phototropism. We identified seven phosphorylation sites of NPH3 proteins in the etiolated seedlings of Arabidopsis and generated unphosphorable and phosphomimetic NPH3 proteins on those sites. Unphosphorable NPH3 showed a shortening of its subcellular localization in the cytosol and caused an inability to adapt to very low fluence rates of blue light ([~]10-5 {micro}mol m-2 s-1) during the phototropic response. In contrast, the phosphomimetic NPH3 proteins had a lengthened subcellular localization in the cytosol and could not lead to the adaptation for blue light at fluence rates of 10-3 {micro}mol m-2 s-1 or more. Our results suggest that the activation levels of phot1 and the corresponding phosphorylation levels of NPH3 determine the rate of plasma membrane-cytosol shuttling of NPH3, which moderately maintains the active state of phot1 signaling across a broad range of blue-light intensities and contributes to the photosensory adaptation of phot1 signaling during the phototropic response in hypocotyls. One sentence summaryThe phosphorylation status of NON-PHOTOTROPIC HYPOCOTYL3 proteins affects their subcellular localization and the photosensory adaptation of phot1 signaling.

plant biology↗

bioRxiv2020

KDM2B promotes cell viability by enhancing DNA damage response in canine hemangiosarcoma

Epigenetic regulators have been implicated in tumorigenesis of many types of cancer; however, their roles in endothelial cell cancers such as canine hemangiosarcoma (HSA) have not been studied. In this study, we found that lysine-specific demethylase 2B (Kdm2b) was highly expressed in HSA cell lines compared to normal canine endothelial cells. Silencing of Kdm2b in HSA cells resulted to increased cell death in vitro compared to the scramble control by inducing apoptosis through the inactivation of the DNA repair pathways and accumulation of DNA damage. Similarly, doxycycline-induced Kdm2b silencing in tumor xenografts resulted to decreased tumor sizes compared to the scramble control. Furthermore, Kdm2b was also highly expressed in clinical cases of HSA, and its expression levels was higher than in hemangioma, a benign counterpart of HSA. Based on these results, we hypothesized that pharmacological Kdm2b inhibition can also induce HSA cell death and can be used as an alternative treatment for HSA. We treated HSA cells with GSK-J4, a histone demethylase inhibitor, and found that GSK-J4 treatment also induced apoptosis and cell death. On top of that, GSK-J4 treatment in HSA tumor-bearing mice decreased tumor sizes without any obvious side-effects. In this study, we demonstrated that Kdm2b acts as an oncogene in HSA by enhancing DNA damage response and can be used as a biomarker to differentiate HSA from hemangioma. Moreover, we indicated that histone demethylase inhibitor GSK-J4 can be used as a therapeutic alternative to doxorubicin for HSA treatment.

cancer biology↗

bioRxiv2020

D-Serine, an emerging biomarker of kidney diseases, is a hidden substrate of sodium-coupled monocarboxylate transporters

Transporter research primarily relies on the canonical substrates of well-established transporters. This approach has limitations when studying transporters for the low-abundant micromolecules, such as micronutrients, and may not reveal physiological functions of the transporters. While D-serine, a trace enantiomer of serine in the circulation, was discovered as an emerging biomarker of kidney function, its transport mechanisms in the periphery remain unknown. Here, using a multi-hierarchical approach from body fluids to molecules, combining multi-omics, cell-free synthetic biochemistry, and ex vivo transport analyses, we have identified two types of renal D-serine transport systems. We revealed that the small amino acid transporter ASCT2 serves as a D-serine transporter previously uncharacterized in the kidney and discovered D-serine as a noncanonical substrate of the sodium-coupled monocarboxylate transporters (SMCTs). These two systems are physiologically complementary, but ASCT2 dominates the role in the pathological condition. Our findings not only shed light on renal D-serine transport, but also clarify the importance of non-canonical substrate transport. This study provides a framework for investigating multiple transport systems of various trace micromolecules under physiological conditions and in multifactorial diseases.

biochemistry↗

bioRxiv2020

CAMSAP3 is required for mTORC1-dependent ependymal cell growth and lateral ventricle shaping in mouse brains

Microtubules (MTs) regulate numerous cellular processes, but their roles in brain morphogenesis are not well known. Here we show that CAMSAP3, a non-centrosomal microtubule regulator, is important for shaping the lateral ventricles. In differentiating ependymal cells, CAMSAP3 became concentrated at the apical domains, serving to generate MT networks at these sites. Camsap3-mutated mice showed abnormally narrow lateral ventricles, in which excessive stenosis or fusion was induced, leading to a decrease of neural stem cells at the ventricular and subventricular zones. This defect was ascribed at least in part to a failure of neocortical ependymal cells to broaden their apical domain, a process necessary for expanding the ventricular cavities. mTORC1 was required for ependymal cell growth but its activity was downregulated in mutant cells. Lysosomes, which mediate mTORC1 activation, tended to be reduced at the apical regions of the mutant cells, along with disorganized apical MT networks at the corresponding sites. These findings suggest that CAMSAP3 supports mTORC1 signaling required for ependymal cell growth via MT network regulation, and, in turn, shaping of the lateral ventricles. Summary statementCAMSAP3, which mediates non-centrosomal microtubule assembly, is required for mTORC1-dependent maturation of ependymal cells at the neocortex of developing mouse brains. Loss of CAMSAP3 causes deformation of the lateral ventricles.

developmental biology↗

bioRxiv2020

Puromycin selection of cells with a high expression of the cytochrome P450 CYP3A4 gene activity from a patient with drug-induced liver injury (DILI) and their lifespan prolongation using a combination of CDK4R24C, cyclin D1 and TERT

Many drugs have the potential to induce the expression of drug-metabolizing enzymes, particularly cytochrome P450 3A4 (CYP3A4), in hepatocytes. Hepatocytes can accurately evaluate drug-mediated CYP3A4 induction as the gold standard for in vitro hepatic toxicology test, but their lot variation is an issue to be solved. Only a limited number of immortalized hepatocyte cells have been reported. In this study, we generated an immortalized cell expressing CYP3A4 from a patient with drug-induced liver injury (DILI). To generate DILI-derived cells with a high expression of CYP3A4, we employed a three-step approach: 1. Differentiation of DILI-induced pluripotent stem cells (DILI-iPSCs); 2. Immortalization of the differentiated cells; 3. Selection of the cells with puromycin. We hypothesize that cells with a high expression of cytochrome P450 genes can survive even after exposure to cytotoxic antibiotics because of high drug-metabolism activity. Puromycin, one of the cytotoxic antibiotics, was used in this study because of its rapid cytocidal effect at a low concentration. Phenotypic studies in vitro revealed that the puromycin-selected cells (HepaSM or SI cells) constitutively expressed the CYP3A4 gene at an extremely high level, and continued to proliferate at least up to 34 population doublings for more than 250 days. The expression profiles were independent of population doublings. Drug-mediated induction test revealed that the cells significantly increased CYP3A4 after exposure to rifampicin, suggesting that the immortalized cells would serve as another useful source for in vitro examination of drug metabolism and CYP3A4 induction.

cell biology↗

bioRxiv2020

Ammonia-based enrichment and long-term propagation of zone I hepatocyte-like cells

Zone I and zone III hepatocytes metabolize ammonia through urea cycle and drug by cytochrome P450, respectively. Ammonia has a cytotoxic effect, and can therefore be used as a selection agent for enrichment of hepatocytes. Besides, isolated hepatocytes from livers can be propagated ex vivo under appropriate condition. However, it has not been investigated so far whether ammonia-treated hepatocyte-like cells are able to proliferate in vitro. In this study, we employed the ammonia selection strategy to purify hepatocyte-like cells that were differentiated from human pluripotent stem cells (PSCs) that are embryonic stem cells (ESCs) and induced pluripotent stem cells. Hepatocyte-like cells after exposure to ammonia highly expressed the CPS1 gene that metabolizes ammonia to carbamoyl phosphate. The resistance to cytotoxicity or cell death by ammonia is probably attributed to the metabolic activity of ammonia in the cells. In addition to the ammonia metabolism-related genes, ammonia-selected PSC-derived hepatocytes increased expression of the CYP3A4 gene, one of the cytochrome P450 genes, that is mainly expressed in zone III hepatocytes. Ammonia-selected hepatocyte-like cells derived from both ESCs and iPSCs can be propagated in vitro up to 30 population doublings for more than 190 days without affecting expression of the liver-associated genes, implying that the ammonia-selected cells have immortality or equivalent life span on the appropriate feeder cells like ESCs and iPSCs. The long-term cultivation of ammonia-selected hepatocyte-like cells resulted in the increased expression of hepatocyte-associated genes such as the CPS1 and CYP3A4 genes. The ammonia selection method to enrich a hepatocyte population was also applicable to immortalized cells from the liver. Ammonia treatment in combination with in vitro propagation will be used to obtain large amounts of hepatocytes or hepatocyte-like cells for pharmacology, toxicology and regenerative medicine.

cell biology↗

bioRxiv2019

Arabidopsis ROOT PHOTOTROPISM2 is a Light-Dependent Dynamic Modulator of Phototropin1

Arabidopsis thaliana phototropin1 (phot1) is a blue-light photoreceptor, i.e. a blue-light-activated Ser/Thr-protein kinase that mediates various light responses including phototropism. Phot1 functions in hypocotyl phototropism dependent on the light induction of ROOT PHOTOTROPISM2 (RPT2) proteins within a broad range of blue light intensities. It is not yet known however how RPT2 contributes to the photosensory adaptation of phot1 to high intensity blue light and the second positive phototropism. We here show that RPT2 suppresses the activity of phot1. Yeast two-hybrid analysis indicated RPT2 binding to the LOV1 (light, oxygen or voltage sensing 1) domain of phot1 required for its high photosensitivity. Our biochemical analyses revealed that RPT2 inhibits the autophosphorylation of phot1, suggesting that it suppresses the photosensitivity and/or kinase activity of phot1 through the inhibition of LOV1 function. We found for the first time that RPT2 proteins are degraded via a ubiquitin-proteasome pathway when phot1 is inactive and stabilized under blue-light conditions in a phot1-dependent manner. We propose that RPT2 is a molecular rheostat that maintains a moderate activation level of phot1 under any light intensity conditions.

plant biology↗

bioRxiv2019

Differences in splicing defects between the grey and white matter in myotonic dystrophy type 1

Myotonic dystrophy type 1 (DM1) is a multi-system disorder caused by CTG repeats in the myotonic dystrophy protein kinase (DMPK) gene. This leads to sequestration of the splicing factor, muscleblind-like 2 (MBNL2), and aberrant splicing, mainly in the central nervous system. We investigated the splicing patterns of MBNL1/2 and genes controlled by MBNL2 in several regions of the brain and between the grey matter (GM) and white matter (WM) in DM1 patients using RT-PCR. Compared with the control, the percentage of spliced-in parameter (PSI) for most of the examined exons were significantly altered in most of the brain regions of DM1 patients, except for the cerebellum. The splicing of many genes was differently regulated between the GM and WM in both DM1 and control. The level of change in PSI between DM1 and control was higher in the GM than in the WM. The differences in alternative splicing between the GM and WM may be related to the effect of DM1 on the WM of the brain. We hypothesize that in DM1, aberrantly spliced isoforms in the neuronal cell body of the GM may not be transported to the axon. This might affect the WM as a consequence of Wallerian degeneration secondary to cell body damage. Our findings may have implications for analysis of the pathological mechanisms and exploring potential therapeutic targets.

molecular biology↗

bioRxiv2019

Genetic Mapping in Autohexaploid Sweet Potato with Low-coverage NGS-based Genotyping Data

Next-generation sequencing (NGS)-based genotyping methods can generate numerous genetic markers in a single experiment and have contributed to plant genetic mapping. However, the benefits of NGS-based methods are limited in autopolyploids as their genetic segregation mode is complex. Moreover, autopolyploids have large genomes and require abundant sequencing data to obtain sufficient genetic markers. There are several methods for genetic mapping in autopolyploids. These approaches may be impractical for plant genetic studies as they require large amounts of data and are not cost-effective. In the present study, we propose a simple strategy for genetic mapping of polyploids in a cost-effective manner. The allele dosage probabilities calculated from NGS read counts were used in association analyses to detect loci associated with specific phenotypes. This approach is superior to conventional methods of determining allele dosage, which usually result in the filtering of many genetic markers with low read depth. The validity of the strategy was demonstrated using real phenotype data from autohexaploid sweet potato populations to detect genetic loci for both qualitative and quantitative traits, the latter of which required the use of allele dosage probabilities for the detection of loci. We demonstrate that this proposed method is useful with reasonable NGS read counts.

genetics↗

bioRxiv2019

Immortalization of human zone I hepatocytes from biliary atresia with CDK4R24C, cyclin D1, and TERT for cytochrome P450 induction testing

BackgroundHepatocytes are an important tool for in vitro toxicology testing. In addition to primary cultures, a limited number of immortalized cell lines have been developed. We here describe a new cell line, designated as HepaMN, which has been established from a liver associated with biliary atresia.\n\nMethodsHepatocytes were isolated from a liver of 4-year-old girl with biliary atresia and immortalized by inoculation with CSII-CMV-TERT, CSII-CMV-Tet-Off, CSII-TRE-Tight-cyclin D1 and CSII-TRE-Tight-CDK4R24C (mutant CDK4: an INK4a-resistant form of CDK4) lentiviruses at the multiplicity of infection of 3 to 10.\n\nResultsHepaMN cells exhibited morphological homogeneity, displaying hepatocyte-like phenotypes. Phenotypic studies in vivo and in vitro revealed that HepaMN cells showed polarized and functional hepatocyte features along with a canalicular cell phenotype under defined conditions, and constitutively expressed albumin and carbamoyl phosphate synthetase I in addition to epithelial markers. Since HepaMN cells are immortal and subcloned, kinetics and expression profiles were independent of population doublings.\n\nConclusionsHepaMN cells showed increased CYP3A4 expression after exposure to rifampicin, implying that their close resemblance to normal human hepatocytes makes them suitable for research applications including drug metabolism studies.

cell biology↗

bioRxiv2019

miR-26a regulates extracellular vesicle secretion from prostate cancer cells via targeting SHC4, PFDN4 and CHORDC1

Extracellular vesicles (EVs) are known to be involved in intercellular communication during cancer progression; thus, elucidating the detailed mechanism will contribute to the development of a novel strategy for EV-targeted cancer treatment. However, the biogenesis of EVs in cancer cells is not completely understood. MicroRNAs (miRNAs) regulate a variety of physiological and pathological phenomena; thus, miRNAs could regulate EV secretion. Here, we performed high-throughput miRNA-based screening to identify the regulators of EV secretion using an ExoScreen assay. By using this miRNA-based screening, we identified miR-26a, which was reported as a tumor suppressive miRNA, as a miRNA involved in EV secretion from prostate cancer (PCa) cells. In addition, we found that the SHC4, PFDN4, and CHORDC1 genes regulate EV secretion in PCa cells. Suppression of these genes by siRNAs significantly inhibited the secretion of EVs in PCa cells. Furthermore, the progression of PCa cells was inhibited in an in vivo study. On the other hand, injection of EVs isolated from PCa cells partially rescued this suppressive effect on tumor growth. Taken together, our findings suggest that miR-26a regulates EV secretion via targeting SHC4, PFDN4, and CHORDC1 in PCa cells, resulting in the suppression of PCa progression.

cancer biology↗