Biology subjects

Kinoshita, T.

Publications and source records attributed to Kinoshita, T..

At least 37 records · Page 2Linked to original sources

bioRxiv2022

Requirement of sequential hydrolysis by CD73 and ALP for uptake of vitamin B2 into cells

Extracellular hydrolysis of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) to riboflavin is thought to be important for cellular uptake of vitamin B2 because FAD and FMN are hydrophilic and do not pass the plasma membrane. However, it is not clear whether FAD and FMN are hydrolyzed by cell surface enzymes for vitamin B2 uptake. Here, we show that in human cells, FAD, a major form of vitamin B2 in plasma, is hydrolyzed by CD73 (also called ecto-5' nucleotidase) to FMN, then FMN is hydrolyzed by alkaline phosphatase to riboflavin, which is efficiently imported into cells. This process is impaired on the surface of glycosylphosphatidylinositol (GPI)-deficient cells due to lack of these GPI-anchored enzymes. During culture of GPI-deficient cells with FAD or FMN, hydrolysis of these forms of vitamin B2, intracellular levels of vitamin B2, vitamin B2-dependent pyridoxal 5'-phosphate formation, and mitochondrial functions were significantly decreased compared with those in GPI-restored cells. These results suggest that inefficient uptake of vitamin B2 might account for mitochondrial dysfunction seen in some cases of inherited GPI deficiency.

biochemistry↗

bioRxiv2022

Photosynthetic-product-dependent Activation of Plasma Membrane H+-ATPase and Nitrate Uptake in Arabidopsis Leaves.

Plasma membrane (PM) H+-ATPase is a pivotal enzyme for plant growth and development that acts as a primary transporter and is activated by phosphorylation of the penultimate residue, threonine, at the C-terminus. Photosynthetically active radiation activates PM H+-ATPase via phosphorylation in mesophyll cells of Arabidopsis thaliana, and phosphorylation of PM H+-ATPase depends on photosynthesis and photosynthesis-related sugar supplementation, such as sucrose, fructose and glucose. However, the molecular mechanism and the physiological role of photosynthesis-dependent PM H+-ATPase activation are still unknown. Analysis using sugar analogues, such as palatinose, turanose, and 2-deoxy glucose, revealed that sucrose metabolites and products of glycolysis such as pyruvate induce phosphorylation of PM H+-ATPase. Transcriptome analysis showed that novel isoform of the Small Auxin Up RNA genes, SAUR30, is upregulated in a light- and sucrose-dependent manner. Time course analyzes of sucrose supplementation showed that phosphorylation level of PM H+-ATPase increased within 10 min, but expression level of SAUR30 increased later than 10 min. The results suggest two temporal regulations may participate in the regulation of PM H+-ATPase. Interestingly, a 15NO3- uptake assay in leaves showed that light increases 15NO3- uptake, and that increment of 15NO3- uptake depends on PM H+-ATPase activity. The results opened the possibility of physiological role of photosynthesis-dependent PM H+-ATPase activation in the uptake of NO3-. We speculate that PM H+-ATPase may connect photosynthesis and nitrogen metabolism in leaves.

plant biology↗

bioRxiv2022

F-actin regulates polarized secretion of pollen tube attractants in Arabidopsis synergid cell.

Pollen tube attraction is a key event of sexual reproduction in flowering plants. In the ovule, two synergid cells neighboring the egg cell control pollen tube arrival via the active secretion of attractant peptides such as AtLURE1 and XIUQIU from the filiform apparatus facing toward the micropyle. Distinctive cell polarity together with longitudinal F-actin and microtubules are hallmarks of the synergid cell in various species, though functions of these cellular structures are still unclear. In this study we used genetic and pharmacological approaches to elucidate the roles of cytoskeletal components in filiform apparatus formation and pollen tube guidance in Arabidopsis thaliana. Inhibition of microtubule formation reduced invaginations of the plasma membrane but did not abolish micropylar AtLURE1.2 accumulation. In contrast, the expression of a dominant-negative form of ACTIN8 induced disorganization of the filiform apparatus and loss of polar AtLURE1.2 distribution toward the filiform apparatus. Interestingly, after pollen tube reception, F-actin became unclear for a few hours in the persistent synergid cell, which may be involved in pausing and resuming pollen tube attraction during early polytubey block. Our data propose the central role of F-actin in the maintenance of cell polarity and function of male-female communication in the synergid cell.

plant biology↗

bioRxiv2021

Microplastic pollution of commercial fishes from coastal and offshore waters, Japan

Microplastics (MPs) pollution is a worldwide issue in the marine environment. There is growing concern of consuming MPs through fish, yet the current contamination status of fish collected from the deep sea surrounding Japan remains limited. We present baseline data on MPs in commercially important fishes from the coastal and offshore waters near Kyushu, Japan (East China Sea). We examined the MPs in the digestive tracts of two pelagic (n = 150 in total) and five demersal species (n = 235 in total). The fish were caught by pole and line, and bottom trawl at different geographical positions. The MPs in pelagic fish (39.1 %) were higher than demersal fish (10.3 %) and of larger sizes. The MPs correlated with habitat depth and type. There was species variation in the shape and polymer composition of MPs. These results increase our understanding of the heterogeneous uptake of MPs by fishes.

ecology↗

bioRxiv2021

Temporal Changes in Transcripts of MITE Transposable Elements during Rice Endosperm Development

The repression of transcription from transposable elements (TEs) by DNA methylation is necessary to maintain genome integrity and prevent harmful mutations. However, under certain circumstances, TEs are thought to escape from the host defense system and reactivate their transcription. In Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), DNA demethylases target the sequences derived from TEs in the central cell, the progenitor cell for the endosperm in the female gametophyte. This genome-wide DNA demethylation is also observed in the endosperm after fertilization. In this study, we used a custom microarray to survey the transcripts generated from TEs during the rice endosperm development and at selected timepoints in the embryo as a control. The expression patterns of TE transcripts are dynamically up- and downregulated during endosperm development, especially for miniature inverted-repeat transposable elements (MITEs). Surprisingly, some TE transcripts were directionally controlled, while the other DNA transposons and retrotransposons were not. We also discovered the NF-Y binding motif, CCAAT, in the region near the 5' terminal inverted repeat of Youren, one of the transcribed MITEs in the endosperm. Our results uncover dynamic changes in TE activity during endosperm development in rice.

plant biology↗

bioRxiv2021

CLPTM1L is a lipid scramblase involved in glycosylphosphatidylinositol biosynthesis

Glycosylphosphatidylinositols (GPIs) are membrane anchors of many eukaryotic cell surface proteins. Biosynthesis of GPIs is initiated at the cytosolic face of the endoplasmic reticulum (ER) and the second intermediate, glucosaminyl-phosphatidylinositol (GlcN-PI), is translocated across the membrane to the lumenal face for later biosynthetic steps and attachment to proteins. The mechanism of the lumenal translocation of GlcN-PI is unclear. We report that Cleft lip and palate transmembrane protein 1-like protein (CLPTM1L), an ER membrane protein of unknown function, is a lipid scramblase involved in GPI biosynthesis. Purified CLPTM1L scrambles GlcN-PI, PI, and several other phospholipids in vitro. Knockout of CLPTM1L gene in mammalian cultured cells partially decreased GPI-anchored proteins due to impaired usage of GlcN-PI, suggesting a major role of CLPTM1L in lumenal translocation of GlcN-PI. One-Sentence SummaryCLPTM1L translocates glucosaminyl-phosphatidylinositol across the membrane during glycosylphosphatidylinositol biosynthesis.

biochemistry↗

bioRxiv2021

Mechanosensory trichome cells evoke a mechanical stimuli-induced immune response in plants

Perception of pathogen-derived ligands by corresponding host receptors is a pivotal strategy in eukaryotic innate immunity. In plants, this is complemented by circadian anticipation of infection timing, promoting basal resistance even in the absence of pathogen threat. Here, we report that trichomes, hair-like structures on the epidermis, directly sense external mechanical forces caused by raindrops to anticipate waterborne infections in Arabidopsis thaliana. Exposure of leaf surfaces to mechanical stimuli initiates the concentric propagation of intercellular calcium waves away from trichomes to induce defence-related genes. Propagating calcium waves enable effective immunity against pathogenic microbes through the calmodulin-binding transcription activator 3 (CAMTA3) and mitogen-activated protein kinases. We propose a novel layer of plant immunity in which trichomes function as mechanosensory cells to detect potential risks.

plant biology↗

bioRxiv2021

Linear causal filtering: definition and theory

1.This work provides a framework based on multivariate autoregressive modeling for linear causal filtering in the sense of Granger. In its bivariate form, the linear causal filter defined here takes as input signals A and B, and it filters out the causal effect of B on A, thus yielding two new signals only containing the Granger-causal effect of A on B. In its general multivariate form for more than two signals, the effect of all indirect causal connections between A and B, mediated by all other signals, are accounted for, partialled out, and filtered out also. The importance of this filter is that it enables the estimation of directional measures of causal information flow from any non-causal, non-directional measure of association. For instance, based on the classic coherence, a directional measure of strength of information flow from A to B is obtained when applied to the linear causal filtered pair containing only A to B connectivity information. This particular case is equivalent to the isolated effective coherence (doi.org/10.3389/fnhum.2014.00448). Of more recent interest are the large family of phase-phase, phase-amplitude, and amplitude-amplitude cross-frequency coupling measures which are non-directional. The linear causal filter makes it now possible to estimate the directional causal versions these measures of association. One important field of application is in brain connectivity analysis based on cortical signals of electric neuronal activity (e.g. estimated sources of EEG and MEG, and invasive intracranial ECoG recordings). The linear causal filter introduced here provides a novel solution to the problem of estimating the direction of information flow from any non-directional measure of association. This work provides definitions, non-ambiguous equations, and clear prescriptions for implementing the linear causal filter in diverse settings.

neuroscience↗

bioRxiv2021

Cubam receptor-mediated endocytosis in hindgut-derived pseudoplacenta of a viviparous teleost Xenotoca eiseni

Nutrient transfer from mother to the embryo is essential for reproduction in viviparous animals. In the viviparous teleost Xenotoca eiseni belonging to the family Goodeidae, the intraovarian embryo intakes the maternal component secreted into the ovarian fluid via the trophotaenia. Our previous study reported that the epithelial layer cells of the trophotaenia incorporate a maternal protein via vesicle trafficking. However, the molecules responsible for the absorption were still elusive. Here, we focused on Cubam (Cubilin-Amnionless) as a receptor involved in the absorption, and cathepsin L as a functional protease in the vesicles. Our results indicated that the Cubam receptor is distributed in the apical surface of the trophotaenia epithelium and then is taken into the intracellular vesicles. The trophotaenia possesses acidic organelles in epithelial layer cells and cathepsin L-dependent proteolysis activity. This evidence does not conflict with our hypothesis that receptor-mediated endocytosis and proteolysis play roles in maternal macromolecule absorption via the trohotaenia in viviparous teleosts. Such nutrient absorption involving endocytosis is not a specific trait in viviparous fish. Similar processes have been reported in the larval stage of oviparous fish or the suckling stage of viviparous mammals. Our findings suggest that the viviparous teleost acquired trophotaenia-based viviparity from a modification of the intestinal absorption system common in vertebrates. This is a fundamental study to understand the strategic variation of the reproductive system in vertebrates. Summary statementHere, we report that an endocytic pathway is a candidate for nutrient absorption in pseudoplacenta of a viviparous teleost. The trait may have developed from common intestinal mechanism among vertebrates.

zoology↗

bioRxiv2021

Pervasive false brain connectivity from electrophysiological signals

1.Signals of brain electric neuronal activity, either invasively measured or non-invasively estimated, are commonly used for connectivity inference. One popular methodology assumes that the neural dynamics follow a multivariate autoregression, where the autoregressive coefficients represent the couplings among regions. If observation noise is present and ignored, as is common in practice, the estimated couplings are biased, affecting all forms of Granger-causality inference, both in time and in frequency domains. Significant nonsense coupling, i.e., nonsense connectivity, can appear when in reality there is none, since there is always observation noise in two possible forms: measurement noise, and activity from other brain regions due to volume conduction and low spatial resolution. This problem is critical, and is currently not being addressed, calling into question the validity of many Granger-causality reports in the literature. An estimation method that accounts for noise is based on an overdetermined system of high-order multivariate Yule-Walker equations, which give reduced variance estimators for the coupling coefficients of the unobserved signals. Simulation-based comparisons to other published methods are presented, demonstrating its adequate performance. In addition, simulation results are presented for a zero connectivity case with noisy observations, where the new method correctly reports no connectivity while classical analyses (as found in most software packages) report nonsense connectivity. For the sake of reproducible research, the supplementary material includes, in human readable format, all the time series data used here.

neuroscience↗

bioRxiv2020

Ethanolamine phosphate on the second mannose as bridge in GPI anchored proteins: Towards understanding inherited PIGG deficiency

Glycosylphosphatidylinositols (GPIs) are glycolipids that anchor many proteins (GPI-APs) on the cell surface. The core glycan of GPI precursor has three mannoses, which in mammals, are all modified by ethanolamine-phosphate (EthN-P). It is postulated that EthN-P on the third mannose (EthN-P-Man3) is the bridge between GPI to the protein and the second (EthN-P-Man2) is removed after GPI-protein attachment. However, EthN-P-Man2 may not be always transient, as mutations of PIGG, the enzyme that transfers EthN-P to Man2, result in inherited GPI deficiencies (IGDs), characterized by neuronal dysfunctions. Here, we show EthN-P on Man2 is the preferential bridge in some GPI-APs, among them, the ect-5-nucleotidase and netrin G2. We found that CD59, a GPI-AP, is attached via EthN-P-Man2 both in PIGB-knockout cells, in which GPI lacks Man3 and with a small fraction, in wild type cells. Our findings modify the current view of GPI anchoring and provide mechanistic bases of IGDs caused by PIGG mutations.

molecular biology↗

bioRxiv2020

A unique small molecule pair controls the plant circadian clock

Circadian clocks are the biological time keeping systems that coordinate genetic, metabolic, and physiological behaviors with the external day-night cycle. Previous studies have suggested possible molecular mechanisms for the circadian clock in Arabidopsis thaliana (Arabidopsis), but there might be additional mechanisms that have been hidden due to genetic redundancy. A clock reporter line of Arabidopsis was screened against the 10,000 chemicals in the Maybridge Hitfinder10K chemical library, and a structure-activity relationship study of hit compounds was conducted. Clock mutants were treated with two of the small molecules to gain insight into their mode of action. The screening identified 5-(3,4-dichlorophenyl)-1-phenyl-1,7-dihydro-4H-pyrazolo[3,4-d]pyrimidine-4,6(5H)-dione (TU-892) as a period lengthening molecule. From a structure-activity relationship study, we found that a molecule possessing 2,4-dichlorophenyl instead of a 3,4-dichlorophenyl group (TU-923) had period shortening activity. The period shortening activity of TU-923 was reversed to a lengthening activity in double mutants lacking PSEUDO-RESPONSE REGULATOR 9 (PRR9) and PRR7 (prr9-10 prr7-11). Our study provides a unique small molecule pair that regulates the pace of the clock in opposite ways, likely by targeting unknown factors. Small differences at the atomic level can reverse the period tuning activities. PRR9 and PRR7 are essential for the activity of TU-923 in period shortening.

plant biology↗

bioRxiv2020

Effect of nanocarbon molecules on the Arabidopsis thaliana transcriptome

Nanocarbons, such as fullerenes, carbon nanotubes, and graphene have attracted a great deal of attention as next-generation materials because of their unprecedented structures and unique physicochemical properties; however, almost all nanocarbons reported previously were used as mixtures. Thus, there are still many unsolved issues about their biological functions at the molecular level. Our synthetic campaign in the last decade has synthesized structurally uniform and atomically precise nanocarbons, leading to the preparation of a library consisting of eighty structurally diverse nanocarbon molecules. This resource motivated us to explore the as yet uncovered biological functions of these nanocarbon molecules in organisms. Recently, nanotubes were used to deliver genes to plants; however, the effects of the molecules on plants are not well known. To monitor the effects of nanocarbon molecules on plants, we analyzed the transcriptome of Arabidopsis thaliana seedlings treated with [9]cycloparaphenylene (CPP), decaborylated warped nanographene (WNG), and dimethoxyhexabenzotetracene (HBT). Clustering analysis indicated few effects of nanocarbon molecules on the transcriptome, perhaps suggesting a low toxicity of nanocarbon molecules on plants. We found that AT1G05880 (ARIADNE 12) gene, categorized into response to hypoxia genes, was up-regulated by nanocarbon molecules, suggesting that this gene is usable as maker for treatment of nanocarbon molecules.

plant biology↗

bioRxiv2019

Learning from Synthetic Dataset for Crop Seed Instance Segmentation

Incorporating deep learning in the image analysis pipeline has opened the possibility of introducing precision phenotyping in the field of agriculture. However, to train the neural network, a sufficient amount of training data must be prepared, which requires a time-consuming manual data annotation process that often becomes the limiting step. Here, we show that an instance segmentation neural network (Mask R-CNN) aimed to phenotype the barley seed morphology of various cultivars, can be sufficiently trained purely by a synthetically generated dataset. Our attempt is based on the concept of domain randomization, where a large amount of image is generated by randomly orienting the seed object to a virtual canvas. After training with such a dataset, performance based on recall and the average Precision of the real-world test dataset achieved 96% and 95%, respectively. Applying our pipeline enables extraction of morphological parameters at a large scale, enabling precise characterization of the natural variation of barley from a multivariate perspective. Importantly, we show that our approach is effective not only for barley seeds but also for various crops including rice, lettuce, oat, and wheat, and thus supporting the fact that the performance benefits of this technique is generic. We propose that constructing and utilizing such synthetic data can be a powerful method to alleviate human labor costs needed to prepare the training dataset for deep learning in the agricultural domain.

bioinformatics↗

bioRxiv2019

Cross-talks of glycosylphosphatidylinositol biosynthesis with glycosphingolipid biosynthesis and ER-associated degradation

Glycosylphosphatidylinositol (GPI)-anchored proteins and glycosphingolipids interact with each other in the mammalian plasma membranes, forming dynamic microdomains. How their interaction starts in the cells has been unclear. Here, based on a genome-wide CRISPR-Cas9 genetic screen for genes required for GPI side-chain modification by galactose in the Golgi apparatus, we report that {beta}1,3-galactosyltransferase 4 (B3GALT4), also called GM1 ganglioside synthase, additionally functions in transferring galactose to the N-acetylgalactosamine side-chain of GPI. Furthermore, B3GALT4 requires lactosylceramide for the efficient GPI side-chain galactosylation. Thus, our work demonstrates previously unexpected evolutionary and functional relationships between GPI-anchored proteins and glycosphingolipids in the Golgi. Through the same screening, we also show that GPI biosynthesis in the endoplasmic reticulum (ER) is severely suppressed by ER-associated degradation to prevent GPI accumulation when the transfer of synthesized GPI to proteins is defective. Our data demonstrates cross-talks of GPI biosynthesis with glycosphingolipid biosynthesis and the ER quality control system.

cell biology↗

bioRxiv2019

Generation of strong casein kinase 1 inhibitor of Arabidopsis thaliana

Casein kinase 1 (CK1) is an evolutionarily conserved protein kinase among eukaryotes. Studies on yeast, fungi, and animals have revealed that CK1 plays roles in divergent biological processes. By contrast, the collective knowledge regarding the biological roles of plant CK1 lags was behind those of animal CK1. One of reasons for this is that plants have more multiple genes encoding CK1 than animals. To accelerate the research for plant CK1, a strong CK1 inhibitor that efficiently inhibits multiple members of CK1 proteins in vivo (in planta) is required. Here, we report a novel strong CK1 inhibitor of Arabidopsis (AMI-331). Using a circadian period-lengthening activity as estimation of the CK1 inhibitor effect in vivo, we performed a structure-activity relationship (SAR) study of PHA767491 (1,5,6,7-tetrahydro-2-(4-pyridinyl)-4H-pyrrolo[3,2-c]pyridin-4-one hydrochloride), a potent CK1 inhibitor of Arabidopsis, and found that PHA767491 analogues bearing a propargyl group at the pyrrole nitrogen atom (AMI-212) or a bromine atom at the pyrrole C3 position (AMI-23) enhance the period-lengthening activity. The period lengthening activity of a hybrid molecule of AMI-212 and AMI-23 (AMI-331) is about 100-fold stronger than that of PHA767491. An in vitro assay indicated a strong inhibitory activity of CK1 kinase by AMI-331. Also, affinity proteomics using an AMI-331 probe showed that targets of AMI-331 are mostly CK1 proteins. As such, AMI-331 is a strong potent CK1 inhibitor that shows promise in the research of CK1 in plants.

plant biology↗

bioRxiv2019

Complement- and inflammasome-mediated autoinflammation-paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic stem cell disorder characterized by complement-mediated hemolysis and thrombosis, and bone marrow failure. Affected cells harbor somatic mutation in X-linked PIGA gene, essential for the initial step in glycosylphosphatidylinositol (GPI) biosynthesis. Loss of GPI biosynthesis results in defective cell-surface expression of GPI-anchored complement regulators CD59 and DAF. The affected stem cells generate many abnormal blood cells after clonal expansion, which occurs under bone marrow failure. Here, we report the mechanistic basis of a disease entity, autoinflammation-paroxysmal nocturnal hemoglobinuria (AIF-PNH), caused by germline mutation plus somatic loss of PIGT on chromosome 20q. A region containing maternally imprinted genes implicated in clonal expansion in 20q-myeloproliferative syndromes was lost together with normal PIGT from paternal chromosome 20. Taking these findings together with a lack of bone marrow failure, the mechanisms of clonal expansion in AIF-PNH appear to differ from those in PNH. AIF-PNH is characterized by intravascular hemolysis and recurrent autoinflammation, such as urticaria, arthralgia, fever and aseptic meningitis. Consistent with PIGTs essential role in synthesized GPIs attachment to precursor proteins, non-protein-linked free GPIs appeared on the surface of PIGT-defective cells. PIGT-defective THP-1 cells accumulated higher levels of C3 fragments and C5b-9 complexes, and secreted more IL-1{beta} than PIGA-defective cells after activation of the complement alternative pathway. IL-1{beta} secretion was dependent upon C5b-9 complexes, accounting for the effectiveness of the anti-C5 drug eculizumab for both intravascular hemolysis and autoinflammation. These results suggest that free GPIs enhance complement activation and inflammasome-mediated IL-1{beta} secretion.

immunology↗

bioRxiv2019

Pathogen-induced pH changes regulate the growth-defense balance of plants

Environmental adaptation of organisms relies on fast perception and response to external signals, which lead to developmental changes. Plant cell growth is strongly dependent on cell wall remodeling. However, little is known about cell wall-related sensing of biotic stimuli and the downstream mechanisms that coordinate growth and defense responses. We generated genetically encoded pH sensors to determine absolute pH changes across the plasma membrane in response to biotic stress. A rapid apoplastic acidification by phosphorylation-based proton pump activation was followed by an acidification of the cortical side of the plasma membrane in response to the fungus Fusarium oxysporum. The proton chemical gradient modulation immediately reduced cellulose synthesis and cell growth and, furthermore, had a direct influence on the pathogenicity of the fungus. All these effects were dependent on the COMPANION OF CELLULOSE SYNTHASE proteins that are thus at the nexus of plant growth and defense. Hence, our discoveries show a remarkable connection between plant biomass production, immunity, and pH control, and advance our ability to investigate the plant growth-defense balance.

plant biology↗