Biology subjects

Hamada, T.

Publications and source records attributed to Hamada, T..

10 recordsLinked to original sources

bioRxiv2025

Metabolism of L-threonate, an ascorbate degradation product, requires a protein with L-threonate metabolizing domains in Arabidopsis

L-Threonate is one of the major degradation products of ascorbate in plants. While bacteria can utilize L-threonate as a sole carbon source by converting it to dihydroxyacetone phosphate, a glycolysis intermediate, through a three- or four-step metabolic pathway, the corresponding processes in plants remain uncharacterized. Remarkably, an Arabidopsis gene encodes a unique protein containing domains homologous to all three enzymes involved in the bacterial three-step pathway. We designated this protein as L-threonate metabolizing domains (LTD) and investigated its functional role in plant L-threonate metabolism. Despite extensive efforts, recombinant expression of LTD was unsuccessful, likely due to its large protein size. Therefore, a reverse genetic approach was employed, using ltd knockout Arabidopsis lines to explore LTD function. Under continuous dark conditions, where ascorbate degradation is facilitated, LTD transcription was significantly upregulated, leading to increased L-threonate dehydrogenase (LtnD) activity. Knockout lines of LTD exhibited no detectable LtnD activity under both light and dark conditions, alongside elevated levels of L-threonate compared to wild-type plants. These results indicate that LTD is essential for L-threonate metabolism in Arabidopsis. The LTD gene is highly conserved among land plants but is absent in green algae, providing a hypothesis that the rise in ascorbate concentrations during plant evolution necessitated a more active metabolism of ascorbate degradation products.

plant biology↗

bioRxiv2025

Foliar ascorbate degradation in the dark occurs independently of ascorbate peroxidase/oxidase, recycling enzymes, and senescence signaling

Ascorbate is a key antioxidant that protects plant cells from oxidative damage. While plants actively synthesize ascorbate during the day, its degradation becomes prominent under prolonged dark conditions. Since ascorbate degradation begins with its oxidized form, dehydroascorbate (DHA), this process inherently requires ascorbate oxidation. However, the molecular mechanisms underlying dark-induced ascorbate oxidation and subsequent degradation remain unclear. In this study, we investigated the role of intracellular and extracellular ascorbate redox regulation in controlling this process. Using Arabidopsis knockout mutants for key enzymes involved in ascorbate oxidation and recycling, including ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), dehydroascorbate reductase (DHAR), and ascorbate oxidase (AO), as well as NADPH oxidases (rbohD and rbohF), we found that none of these enzymes significantly influenced the dark-induced decrease in ascorbate levels. Notably, ascorbate levels decreased similarly in newly generated multiple mutants, including a quintuple mutant ({Delta}dhar pad2 mdar5), which has severely impaired ascorbate recycling capacity, and the ao2 rbohD double mutant, which is strongly expected to exhibit a highly altered apoplastic redox state. Furthermore, we examined the potential involvement of senescence signaling, including ORESARA1 and ethylene signaling components, but found no evidence for their contribution. These findings indicate that the dark-induced decrease in ascorbate levels is not governed by conventional pathways for ascorbate oxidation and recycling or senescence signaling processes, suggesting an unidentified regulatory mechanism.

plant biology↗

bioRxiv2024

Proteomic analysis to classify extracellular vesicles and plasmodesmata in Arabidopsis cell culture supernatants

The plant apoplast contains diverse proteins. In this study, we analyzed the extracellular vesicle proteome using cell culture supernatants. Unexpectedly, many plasmodesmata proteins were identified in the extracellular vesicle fraction. The comparison between the cell culture EV proteome and the plasmodesmata proteome revealed that EV and plasmodesmata proteins were indistinguishable. Highly sensitive imaging analyses showed that PDLP3-GFP, a representative plasmodesmata marker, was localized to plasmodesmata, but it was also present as small granules in apoplast regions. Next, we attempted to distinguish EV proteins from plasmodesmata proteins. The step-wise density gradient ultracentrifugation and clustering analysis separated EV protein clusters (represented by tetraspanins and membrane trafficking proteins) from plasmodesmata clusters (represented by PDLPs and PDCBs). These results provide the basis for future explorations of plant plasmodesmata and EVs.

plant biology↗

bioRxiv2024

Time-course analysis of the transcriptome of Arabidopsis thaliana leaves under high-concentration ammonium sulfate treatment

Nitrogen is essential for plant growth and is sourced primarily from nitrate and ammonium in the soil. Even though plants can take ammonium up for nutrition, it often results in toxic effects such as growth suppression and chlorosis. To elucidate the mechanism of ammonium toxicity, a time-course analysis of the transcriptome was performed on A. thaliana leaves treated with high concentrations of ammonium sulfate in the presence of sufficient nitrate. The expression of nitrate-inducible genes tended to be downregulated by the treatment. The expression of genes relating to abscisic acid, jasmonic acid (JA), salicylic acid (SA), and membrane trafficking was upregulated, whereas that of photosynthesis-, auxin-, and cytokinin-related genes involved in growth and development was downregulated. The induction of many osmotic stress-responsive genes suggests the involvement of osmotic stress in ammonium toxicity. Furthermore, the upregulation of nitric oxide (NO)-inducible genes and the simultaneous upregulation of genes involved in JA biosynthesis, glutathione metabolism, and SA response suggested the involvement of endogenous NO and protein S-nitrosylation in response to high concentrations of ammonium sulfate. This study provides a novel and comprehensive overview of transcriptional changes occurring in response to high ammonium sulfate concentrations and proposes possible mechanisms of ammonium toxicity that can be explored in future research.

plant biology↗

bioRxiv2023

The role of hnRNP A3 on age-related increased expression of FIX in the Liver

HnRNP A3 is a protein that binds the age-related increase element (AIE) of blood coagulation factor IX (FIX) and that plays critical roles in age-related gene expression, likely through an epigenetic mechanism as yet unidentified. In a previous study, we found that Ser359 phosphorylated hnRNP A3 proteins do not bind to the AIE of FIX although both unphosphorylated and Ser359 phosphorylated hnRNP proteins exist in the liver. In the present study, to explore the relationship between hnRNP A3 and FIX, we examined the age-related expression pattern of 14 single spots of hnRNP A3 detected by 2DE and subsequent MALDI-TOF/TOF/MS analyses in mouse liver. We found that the level of all four Ser359 phosphorylated hnRNP A3 proteins increased with age (from 1-21 months), while the 10 unphosphorylated hnRNP A3 proteins showed various expression patterns with age. We then examined the functional role of hnRNP A3 in FIX expression using siRNA knockdown technology targeting the hnRNP A3 gene in aged mice (12-17 months old). Inhibition of hnRNP A3 expression induced an increase in the circulating FIX level in aged mice. These results suggested that hnRNP A3 inhibits age-related FIX protein expression and that age-dependent modification of hnRNP A3, including its phosphorylation at Ser359, might be involved in the age-dependent increase in FIX expression in vivo.

physiology↗

bioRxiv2022

Fission yeast Dis1 is an unconventional TOG/XMAP215 that induces microtubule catastrophe to drive chromosome pulling

The shortening of microtubules attached to kinetochores is the driving force of chromosome movement during cell division. Specific kinesins are believed to shorten microtubules but are dispensable for viability in yeast, implying the existence of additional factors responsible for microtubule shortening. Here, we demonstrate that Dis1, a TOG/XMAP215 ortholog in fission yeast, promotes microtubule shortening to carry attached chromosomes. Although TOG/XMAP215 orthologs are generally accepted as microtubule polymerases, Dis1 promoted microtubule catastrophe in vitro and in vivo. Notably, microtubule catastrophe was promoted when the tip was attached to kinetochores, as they steadily anchored Dis1 at the kinetochore-microtubule interface. Engineered Dis1 oligomers artificially tethered at a chromosome arm region induced the shortening of microtubules in contact, frequently pulling the chromosome arm towards the vicinity of spindle poles in meiocytes. Thus, unlike Alp14 and other TOG/XMAP215 orthologs, Dis1 plays an unconventional role in promoting microtubule catastrophe, thereby driving chromosome movement.

cell biology↗

bioRxiv2020

Tumor-Immune Partitioning and Clustering (TIPC) algorithm reveals distinct signatures of tumor-immune cell interactions within the tumor microenvironment

Growing evidence supports the importance of understanding tumor-immune spatial relationship in the tumor microenvironment in order to achieve precision cancer therapy. However, existing methods, based on oversimplistic cell-to-cell proximity, are largely confounded by immune cell density and are ineffective in capturing tumor-immune spatial patterns. Here we developed a novel computational algorithm, termed Tumor-Immune Partitioning and Clustering (TIPC), to offer an effective solution for spatially informed tumor subtyping. Our method could measure the extent of immune cell partitioning between tumor epithelial and stromal areas as well as the degree of immune cell clustering. Using a U.S. nation-wide colorectal cancer database, we showed that TIPC could determine tumor subtypes with unique tumor-immune spatial patterns that were significantly associated with patient survival and key tumor molecular features. We also demonstrated that TIPC was robust to parameter settings and readily applicable to different immune cell types. The capability of TIPC in delineating clinically relevant patient subtypes that encapsulate tumor-immune spatial relationship, immune density, and tumor morphology is expected to shed light on underlying immune mechanisms. Hence, TIPC can be a useful bioinformatics tool for effective characterization of the spatial composition of the tumor-immune microenvironment to inform precision immunotherapy.

cancer biology↗

bioRxiv2019

Autologous adipose-derived stem cell transplantation enhances healing of wound with exposed bone in a rat model

ObjectivesSoft tissue wounds with exposed bone often require extended healing times and can be associated with severe complications. We describe the ability of artificial dermis with autogenic adipose-derived stem cells (ADSCs) to promote the healing of wounds with exposed bone in a rat model.\n\nMethodsAdipose tissues harvested from the bilateral inguinal regions of Wistar rats were used as ADSCs. Rats were randomly divided into control and ADSC groups to investigate the efficacy of ADSC transplantation for wound healing (n=20 per group). Soft tissue defects were created on the heads of the rats and were covered with artificial dermis with or without the seeded ADSCs. Specimens from these rats were evaluated using digital image analysis, histology, immunohistochemistry, cell labeling, and real-time reverse-transcription polymerase chain reaction (Real-time RT-PCR).\n\nResultsThe average global wound area was significantly smaller in the ADSC group than in the control group on days 3, 7, and 14 after surgery (p<0.05). After 14 days, the blood vessel density in the wound increased by 1.6-fold in the ADSC group compared with that in the control group (p<0.01). Real-time RT-PCR results showed higher Fgfb and Vegf expression levels at all time points, and higher Tgfb1 and Tgfb3 expression levels until 14 days after surgery, in the ADSC group than in the control group (p<0.05).\n\nConclusionsIn wounds with exposed bone, autogenic ADSCs can promote vascularization and wound healing. Use of this cell source has multiple benefits, including convenient clinical application and lack of ethical concerns.

animal behavior and cognition↗

bioRxiv2018

Rac-dependent signaling from keratinocytes promotes differentiation of intradermal white adipocytes

Rac signaling affects numerous downstream targets; however, few studies have established in vivo levels. We generated mice with a single knockout (KO) of Rac1 (Keratin5 (K5)-Cre;Rac1flox/flox, Rac1-KO) and double KO of Rac1 and Rac3 (K5-Cre;Rac1flox/flox;Rac3-/-, Rac1/Rac3-DKO) in keratinocytes. Strikingly, Rac1-KO mice exhibited thinner dermal white adipose tissue, which was considerably further reduced in Rac1/Rac3-DKO mice. DNA microarray using primary keratinocytes from Rac1/Rac3-DKO mice exhibited decreased mRNA levels of Bmp2, Bmp5, Fgf20, Fgf21, Fgfbp1, and Pdgf. Combinational treatment with BMP2 and FGF21 or BMP2 and FGF20 in culture medium, but not individual purified recombinant proteins, could differentiate 3T3-L1 fibroblasts into adipocytes, as could culture media obtained from primary keratinocytes. Conversely, addition of anti-BMP2 or anti-FGF21 antibodies into the culture medium inhibited fibroblast differentiation. Furthermore, combinational treatment with BMP2 and FGF21 promoted adipocyte differentiation only of rat primary white, but not brown, adipocyte precursors. Notably, brown adipogenesis by FGF21 was inhibited by BMP2. Thus, we proposed novel paracrine pathways from keratinocytes to intradermal pre-adipocytes, which function as Rac-dependent modulators of white adipogenesis, but also brown adipogenesis.

cell biology↗

bioRxiv2018

Inherited DNA Repair Defects in Colorectal Cancer

Colorectal cancer (CRC) heritability has been estimated to be around 30%. However, mutations in the known CRC susceptibility genes explain CRC risk in under 10% of the cases. Germline mutations in DNA-repair genes (DRGs) have recently been reported in CRC but their contribution to CRC risk is largely unknown. We evaluated the gene-level germline mutation enrichment of 40 DRGs in 680 unselected CRC individuals compared to 27728 ancestry-matched cancer-free adults. Significant findings were then examined in independent cohorts of 1661 unselected CRC cases and 1456 early-onset CRC cases. Of 680 individuals in the discovery set, 31 (4.56%) individuals harbored germline pathogenic mutations in known CRC susceptibility genes while another 33 (4.85%) individuals had DRG mutations that have not been previously associated with CRC risk. Germline pathogenic mutations in ATM and PALB2 were enriched in both the discovery (OR= 2.81; P= 0.035 and OR= 4.91; P= 0.024, respectively) and validation sets (OR= 2.97; Adjusted P= 0.0013 and OR= 3.42; Adjusted P= 0.034, for ATM and PALB2 respectively). Biallelic loss of ATM was evident in all cases with matched tumor profiling. CRC cases also had higher rates of actionable mutations in the HR pathway that can substantially increase the risk of developing cancers other than CRC. Our analysis provides evidence for ATM and PALB2 as CRC risk genes, underscoring the importance of the homologous recombination pathway in CRC. In addition, we identified frequent complete homologous recombination deficiency in CRC tumors, representing a unique opportunity to explore targeted therapeutic interventions such as PARPi.

genetics↗