Biology subjects

Kobayashi, K.

Publications and source records attributed to Kobayashi, K..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Acute oral toxicity and angiotensin-converting enzyme (ACE) inhibitory activity of the aqueous fractions of the methanol leaf extract of Psychotria luzoniensis

This study investigated the acute oral toxicity, in vitro angiotensin-converting enzyme (ACE) inhibitory activity, and metabolite profile of the aqueous fraction of the methanol leaf extract of Psychotria luzoniensis (PLAF), a plant endemic to the Philippines. ACE inhibition was evaluated in the crude extract, fractions, and sub-fractions through IC50 determination. The most bioactive fraction was further subjected to acute oral toxicity testing in mice at the limit dose of 2000 mg/kg body weight, while metabolite profiling was performed using UHPLC-QTOF-MS/MS. PLAF exhibited the strongest ACE inhibition (IC50 34.38 {+/-} 1.86 {micro}g/ml), prompting subsequent fractionation. Among the sub-fractions (PLAF-1A - 1E), PLAF-1D demonstrated the most potent activity (IC50 9.70 {+/-} 0.10 {micro}g/ml). Acute toxicity evaluation of PLAF revealed no mortality, behavioral abnormalities, or significant alterations in body weight, organ weights, serum biochemical parameters (SGPT, SGOT, BUN, creatinine, LDH), or histopathological changes in the liver, kidney, and heart. These findings indicate an LD50 greater than 2000 mg/kg. Metabolite profiling showed candidate flavonoids previously associated with ACE inhibition, including quercetin, kaempferol, rutin, and hyperin. Other putative compounds detected include 4-(3,4-dihydroxyphenyl)-7-hydroxy-2-oxo-2H-chromen-5-yl-{beta}-D-glucopyranoside, juncein, and butin which are rarely reported in other Psychotria species. In this study, the ACE inhibitory activity and favorable toxicity profile of P. luzoniensis highlight its pharmacological potential to support further isolation of bioactive compounds and in vivo blood pressure studies.

pharmacology and toxicology↗

bioRxiv2025

Anatomical and Neurochemical Profiles of GABAergic Projection Neurons in the Mouse Inferior Colliculus

The inferior colliculus (IC) is a critical hub for the integration of auditory signals in the midbrain. Although both glutamatergic and GABAergic neurons in the IC project to the medial geniculate body (MGB), the detailed neurocircuitry and neurochemical properties of GABAergic projection neurons remain poorly understood. In this study, glutamate decarboxylase 67 (GAD67)-Cre mice and the viral vector were utilized to selectively visualize the axonal projections of GABAergic neurons in the IC. These neurons project predominantly to the medial division of MGB (MGv) and contralateral IC and minorly to the ventral nucleus of the trapezoid body. Importantly, GABAergic projections from both lemniscal and non-lemniscal regions of the IC primarily targeted the lemniscal division of the MGB, whereas those projections from the external cortex of the IC--part of the non-lemniscal pathway--additionally extended into non- lemniscal MGB subregions. Using a retrograde tracer, a substantial proportion of GABAergic projection neurons targeting the MGB and contralateral IC were positive for several neurochemical markers, implying that some GABAergic neurons send axon collaterals to both targets. Notably, GABAergic neurons constituted 10% of the total neuronal population in the IC, whereas GABAergic neurons accounted for approximately 20% of IC neurons projecting to the MGB or contralateral IC. Our results suggest that GABAergic projection neurons are more involved in the IC-MGB pathway than would be expected based on their proportion in the IC and may exert widespread influence on auditory processing via direct inhibition of the MGv and indirect modulation through suppression of the contralateral IC.

neuroscience↗

bioRxiv2025

Thalamo-insular pathway regulates tic generation via motor-limbic crosstalk

Tic disorders accompanied by premonitory urges are hallmark symptoms of Tourette syndrome (TS), yet the underlying neuronal mechanisms remain elusive. Here, we establish a mouse model of motor tics by unilateral striatal injection of a GABAA receptor antagonist. This model induces c-Fos activation in both motor and limbic structures, including the insular cortex (IC). Fiber photometry reveals tic-associated activity in IC as well as the primary motor cortex (M1). Viral tracing demonstrates that basal ganglia outputs from the substantia nigra pars reticulata are transmitted to IC via the intralaminar thalamic nuclei (ITN). Chemogenetic inhibition of IC or the thalamo-insular pathway suppresses tic-related cortical activity and alleviates tic-like behaviors. These findings identify IC as a key node in tic generation and highlight ITN as critical relay stations linking motor and limbic circuits. Aberrant thalamo-insular signaling thus contributes to the pathophysiology of tic disorders and represents a potential therapeutic target in TS. HighlightsO_LIc-Fos activation occurs in both motor and limbic structures in a mouse tic model C_LIO_LIBasal ganglia outputs are transmitted to insula via intralaminar thalamic nuclei C_LIO_LIChemogenetic inhibition of thalamo-insular pathway suppresses motor tics C_LIO_LIThalamo-insular inhibition diminishes tic-associated activity in primary motor cortex C_LI In briefAbnormal basal ganglia activity originating from the striatum drives dysfunction of the insular cortex via the intralaminar thalamic nuclei, leading to motor tics. Chemogenetic inhibition of this pathway suppresses tic-like behaviors, highlighting motor-limbic circuit pathology in tic disorders.

neuroscience↗

bioRxiv2025

Open-source modular FPGA system for two-photon mesoscope enabling multi-layer, multi-depth neural activity recording and lifetime imaging

Large field-of-view (FOV) two-photon microscopy makes it possible to record a large number of neural activities from multiple brain regions simultaneously. However, the larger the field of view, the longer it takes to scan the entire FOV. To increase imaging speed, we have developed open-source software to digitize analogue signals from a photomultiplier tube using a field-programmable gate array (FPGA) at a rate of 3.2 GS/s. By combining this with a newly developed a circular delay-path module for a custom two-photon mesoscope (Diesel2p), we succeeded in simultaneous recording of >10,000 neurons from the entire bilateral dorsal cortex at up to four depths. We also demonstrated large FOV lifetime imaging using the same system. Our modular, open-source FPGA system can be readily integrated into any type of two-photon microscope and will accelerate the biomedical application of multi-scale two-photon imaging in a wide range of pathophysiological investigations.

neuroscience↗

bioRxiv2025

Neural Circuit Remodeling Underlying Enhanced Feeding During Pregnancy in Mice

Pregnancy represents a unique physiological state marked by substantial adaptations influencing both behavior and metabolism1. Alterations have been observed across multiple levels--from changes in cellular morphology, synaptic density, and neuroglial interactions to a brain-wide reduction in gray matter volume2-7. While these adaptations are thought to support offspring survival and promote maternal physiological and psychological well-being8, their functional significance remains largely unresolved. In particular, linking localized synaptic plasticity to a broader brain-wide circuit dynamics has posed a major challenge. Using virus-mediated screening in mice, we investigated the pregnancy-induced remodeling of the presynaptic landscape of paraventricular hypothalamic (PVH) oxytocin neurons, a central hub of maternal physiology9,10. Here we show a selective and reversible elimination of excitatory synaptic inputs from the medial preoptic nucleus to PVH oxytocin neurons, displaying region-, cell-type-, and pathway-specificity. This reduction in excitatory drive attenuated the anorexigenic activity of oxytocin neurons during feeding, thereby promoting the increased food intake characteristic of pregnancy. These data demonstrate that the selective suppression of anorexigenic PVH oxytocin neuron activity plays a critical role in appetite regulation during pregnancy. More broadly, our data identify a pregnancy-associated remodeling of hypothalamic neural circuits with direct physiological relevance.

neuroscience↗

bioRxiv2025

Stem cell-derived cardiomyocyte migration through TGF-β signalling in the context of myocardial infarction

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold significant promise for cardiac regeneration therapies. However, the efficacy of such treatments depends on the ability of transplanted cells to migrate and integrate into the damaged myocardium, a process that remains poorly understood. In this study, we investigated the migratory behaviour of hiPSC-CMs using homogenised rat MI tissue to simulate myocardial infarction (MI) in vitro. Transwell migration assays demonstrated a concentration-dependent chemotactic response, with hiPSC-CM migration increasing up to threefold toward MI tissue homogenate. Wound healing assays further confirmed enhanced migration under MI-mimetic conditions. Bulk RNA sequencing revealed activation of the TGF-{beta} signalling pathway as a key regulator of this response. Inhibition of TGF-{beta} signalling, both pharmacologically and through antibody neutralisation, significantly reduced hiPSC-CM migration. These findings uncover a previously underappreciated chemotactic capability of hiPSC-CMs and identify TGF-{beta} signalling as a central mediator, offering new mechanistic insights and potential therapeutic targets to improve the integration and efficacy of hiPSC-CM-based cardiac regeneration strategies.

cell biology↗

bioRxiv2025

Sequence grammar and dynamics of subcellular translation revealed by APEX-Ribo-Seq

Local translation at specific subcellular regions is proposed to define appropriate protein destinations and functions. However, our understanding of local translation still remains far from complete due to a lack of versatile analytical tools. Here, we developed a new method, termed APEX-Ribo-Seq, that integrates ribosome profiling (Ribo-Seq) with APEX2-based proximity labeling. We mapped local translation across 14 distinct organelles and identified over 3,000 genes undergoing localized translation, encoding components of complexes that function at these specific sites. RNA language-model predictors captured compartment-associated information beyond protein sequence, and model-derived attribution nominated sequence-sensitive transcript regions for mechanistic follow-up. Gene-set-level RBP enrichment and knockdown experiments separately identified RBPs associated with compartment-specific translation. The utility of APEX-Ribo-Seq was demonstrated in primary neurons, revealing compartment-specific translatomes in dendrites, axons, and presynapses. Extending this approach in vivo, we applied APEX-Ribo-Seq to germ granules in Drosophila embryos, profiling the temporal transition from translationally silent mRNA storage to localized translation during early embryogenesis. Our results provide a comprehensive view of the spatiotemporal translatome and its regulation.

molecular biology↗

bioRxiv2025

Data-efficient protein mutational effect prediction with weak supervision by molecular simulation and protein language models

Machine learning-based protein mutational effect prediction is widely used in protein engineering and pathogenicity prediction, but training data scarcity remains a major challenge due to high costs of experimental measurements. A previous study proposed data augmentation using computational estimates by molecular simulation. However, this approach has been limited to predicting mutational effects on thermostability. Here, we present a new data augmentation method that combines molecular simulation with zero-shot prediction computed by protein language models. These computational estimates serve as "weak" training data to supplement experimental training data. Our method dynamically adjusts the weight and inclusion of weak training data based on available experimental training data. This reduces potential negative impacts of weak training data while extending applicability to diverse protein properties such as binding affinity and enzymatic activity. Benchmark tests demonstrate that our method improves prediction accuracy particularly when experimental training data are scarce. These results indicate the capability of our approach to advance protein engineering and pathogenicity prediction in small data regimes.

bioinformatics↗

bioRxiv2025

GABA mediates experience-dependent regulation of myelination in the mouse visual pathway.

Myelination in the visual pathway is critical for transmitting visual information from retina to the brain. Reducing visual experience shortens myelin sheath length and slows the conduction velocity of the optic nerve. However, the mechanism underlying such experience-dependent myelination is unclear. Here, we found that closing both eyes, binocular deprivation (BD), during the juvenile period less affects the optic nerve myelination than monocular deprivation (MD) via GABA signaling. RNA-seq analysis of optic nerves from MD and BD mice revealed that GABAergic signaling is downregulated on the deprived side of MD compared to the intact side and BD. Inhibition of GABAergic signaling during the juvenile period resulted in myelin sheath shortening and excessive oligodendrocyte generation in normal mice, similar to the changes observed in MD mice. Enhancing GABAergic signaling rescued the myelin sheath shortening and excessive oligodendrocyte generation in the optic nerve of MD mice. Furthermore, we identified novel GABAergic neurons located within the optic nerve, whose neurites form belt-like presynaptic structures with the oligodendrocyte lineage cells, suggesting a potential source of the GABAergic inputs into oligodendrocytes. Our results indicate that the myelination of visual pathway is maintained by binocular visual inputs via intra-nerve GABA signaling.

neuroscience↗

bioRxiv2025

Nectriatides with no antifungal activity bind to ergosterol and potentiate the antifungal activity of amphotericin B against Candida albicans

Fungal cyclotetrapeptide nectriatide and its synthetic linear derivatives (nectriatide-based compounds, NCTs), having no antifungal activity, potentiated antifungal activity of amphotericin B (AmB) against Candida albicans. The mechanism of action was investigated using fluorescein-conjugated and biotin-tagged probes. Microscopy revealed fluorescent-probe localization at the C. albicans cell membrane. The biotinyl probe binding assay towards membrane lipids showed the highest affinity for ergosterol but no affinity for cholesterol. Ergosterol binding was confirmed by a liposome disruption assay. LC-MS quantification of AmB in C. albicans revealed that NCTs increased AmB binding to C. albicans. These results indicate that NCTs bind to ergosterol on the fungal plasma membrane, subsequently localizing AmB, explaining the observed potentiation of AmB fungicidal activity.

microbiology↗

bioRxiv2025

The Existence and Uniqueness of Solutions in the Leaf Photosynthesis-Transpiration-Stomatal Conductance Model

The An-E-gs model, which consistently describes leaf photosynthesis (An), transpiration (E), and stomatal conductance (gs), is widely recognized and utilized as a "standard model" for quantifying these processes in terrestrial plants. However, since its proposal over 30 years ago, the model has faced a longstanding challenge: the "solution selection" problem, arising from the existence of multiple solutions with no guarantee that the obtained solution is correct. In this study, we mathematically proved that the An-E-gs model always has a unique solution satisfying the criteria gs > 0 and Ci > 0, where Ci represents the CO2 concentration inside the leaf. This result establishes a rigorous mathematical theorem on the existence and uniqueness of solutions in the model. The theorem resolves the longstanding "solution selection" problem by enabling the unambiguous identification of the correct solution through selecting the unique solution that satisfies these criteria. Furthermore, the theorem ensures the validity of past estimations that satisfy these criteria and guarantees that future studies applying these criteria will yield correct estimations. These findings provide a robust mathematical foundation for the An-E-gs model, reinforcing its role as the standard model for estimating leaf photosynthesis, transpiration, and stomatal conductance across diverse disciplines, from plant biology to climate science and beyond.

plant biology↗

bioRxiv2024

Protonation/deprotonation-driven switch for the redox stability of low-potential ferredoxin

Ferredoxin is a small iron-sulfur protein and acts as an electron carrier. Low-potential ferredoxins harbor [4Fe-4S] cluster(s), which play(s) a crucial role as the redox center. Low-potential ferredoxins are able to cover a wide range of redox potentials (-700 to -200 mV); however, the mechanisms underlying the factors which control the redox potential are still enigmatic. Here, we determined the neutron structure of ferredoxin from Bacillus thermoproteolyticus, and experimentally revealed the exact hydrogen-bonding network involving the [4Fe-4S] cluster. The density functional theory calculations based on the hydrogen-bonding network revealed that protonation states of the sidechain of Asp64 close to the [4Fe-4S] cluster critically affected the stability of the reduced state in the cluster. These findings provide the first identification of the intrinsic control factor of redox potential for the [4Fe-4S] cluster in low-potential ferredoxins.

biochemistry↗

bioRxiv2024

Distinct roles of two thalamostriatal systems in learning processes of visual discrimination in common marmosets

The thalamostriatal projections arising from the intralaminar thalamic nuclei (ILN) constitute the principal source of input information to specified subregions of the striatum, a key structure of the cortico-basal ganglia circuitry. However, the roles of primate ILN in cortico-basal ganglia circuit functions remain unclear. Here, we performed immunotoxin-induced selective targeting of two representative structures of the ILN, the parafascicular nucleus (Pf) and centre median nucleus (CM) projecting to the caudate nucleus (Cd) and putamen (Pu), respectively, in common marmosets. Elimination of Pf-Cd neurons resulted in impaired reversal learning of a two-choice visual discrimination task, whereas removal of CM-Pu neurons disturbed the task acquisition. No marked impact of such manipulations was observed on either motor skill learning or spontaneous locomotor activity. Our findings reveal that the two thalamostriatal systems play distinct roles in the learning processes of external cue-dependent decision-making in nonhuman primates.

neuroscience↗

bioRxiv2024

Responses of chlorophyll fluorescence to CO2 elimination as an indicator of Crassulacean acid metabolism photosynthesis

Crassulacean acid metabolism (CAM) is found in a wide variety of vascular plant species, mainly those inhabiting water-limited environments. Identifying and characterizing diverse CAM species enhances our understanding of the physiological, ecological, and evolutionary significance of CAM photosynthesis. In this study, we examined the effect of CO2 elimination on chlorophyll fluorescence-based photosynthetic parameters in two constitutive CAM Kalanchoe species and six orchids. In CAM-performing Kalanchoe species, the effective quantum yield of photosystem II showed no change in response to CO2 elimination during the daytime but decreased with CO2 elimination at dusk. We applied this method to reveal the photosynthetic mode of epiphytic orchids and found that Gastrochilus japonicus, Oberonia japonica, and Bulbophyllum inconspicuum, but not Bulbophyllum drymoglossum, are constitutive CAM. Although B. drymoglossum had relatively high malate content in leaves, they did not depend on it to perform photosynthesis even under water deficient or high light conditions. Anatomical comparisons revealed a notable difference in the leaf structure between B. drymoglossum and B. inconspicuum; B. drymoglossum leaves possess the large water storage tissue internally, unlike B. inconspicuum leaves, which develop pseudobulbs. Our data propose a novel approach to identify and characterize CAM plants without labor-intensive experimental procedures. HighlightResponses of chlorophyll fluorescence-based photosynthetic parameters to CO2 elimination differ between Crassulacean acid metabolism (CAM) and C3 metabolism, proposing a novel approach to identify and characterize CAM plants.

plant biology↗

bioRxiv2024

Arabidopsis thaliana ACTIN DEPOLYMERIZING FACTORs play a role in leaf senescence regulation

ACTIN DEPOLYMERIZING FACTORs (ADFs) regulate the organization and dynamics of actin microfilaments (AFs) by cleavage and depolymerization of AFs. The Arabidopsis thaliana genome encodes 11 ADF genes grouped into four subclasses. Subclass I ADFs, ADF1, -2, -3, and -4, are expressed in all vegetative tissues and are reportedly involved in the regulation of plant growth, and biotic and abiotic stress responses. Furthermore, the nuclear localization of ADF4 is seemingly important in disease responses. Here, we present data that indicate a previously unknown regulatory role of subclass I ADFs in leaf senescence. ADF4 knockout mutants (adf4) and transgenic lines in which the expression of all members of subclass I ADFs was downregulated (ADF1-4Ri) showed acceleration of both dark-induced and age-dependent leaf senescence. Among the eight ACTIN genes encoded in A. thaliana, ACT2, -7, and -8 are expressed in vegetative tissues. In contrast to adf4 and ADF1-4Ri, neither ACT2 and ACT8 double mutant (act2/8), nor ACT7 knockout mutant (act7), showed accelerated leaf senescence. Upregulation of the expression of senescence-associated genes occurred earlier in adf4 and ADF1-4Ri lines than in wild type. Examination of the expression of subclass I ADFs genes during senescence revealed a reduced expression of ADF4 but not of other subclass I members. Additionally, we showed that nuclear localization of ADF4 was important for regulating leaf senescence. Altogether, our data indicate that subclass I ADFs, particularly ADF4, play an important role in the regulation of leaf senescence.

plant biology↗

bioRxiv2024

A beta-Galactosidase acting on unique galactosides: the structure and function of a beta-1,2-galactosidase from Bacteroides xylanisolvens, an intestinal bacterium

Galactosides are major carbohydrates that are found in plant cell walls and various prebiotic oligosaccharides. Studying the detailed biochemical functions of {beta}-galactosidases in degrading these carbohydrates is important. In particular, identifying {beta}-galactosidases with new substrate specificities could help in the production of potentially beneficial oligosaccharides. In this study, we identified a {beta}-galactosidase with novel substrate specificity from Bacteroides xylanisolvens, an intestinal bacterium. The enzyme did not show hydrolytic activity toward natural {beta}-galactosides during the first screening. However, when -D-galactosyl fluoride (-GalF) as a donor substrate and galactose or D-fucose as an acceptor substrate were incubated with a nucleophile mutant, reaction products were detected. The galactobiose produced from the -GalF and galactose was identified as {beta}-1,2-galactobiose using NMR. Kinetic analysis revealed that this enzyme effectively hydrolyzed {beta}-1,2-galactobiose and {beta}-1,2-galactotriose. In the complex structure with methyl {beta}-galactopyranose as a ligand, the ligand is only located at subsite +1. The 2-hydroxy group and the anomeric methyl group of methyl {beta}-galactopyranose faces in the direction of subsite -1 and the solvent, respectively. This observation is consistent with the substrate specificity of the enzyme regarding linkage position and chain length. Overall, we concluded that the enzyme is a {beta}-galactosidase acting on {beta}-1,2-galactooligosaccharides. SynopsisThe structural and functional analysis of {beta}-galactosidase from an intestinal bacterium led to the discovery of a new {beta}-galactosidase hydrolyzing unique {beta}-1,2-galactooligosaccharides.

biochemistry↗

bioRxiv2024

Beta-Glucanase superfamily identified by sequential, functional, and structural analyses

{beta}-1,2-Glucans are natural glucose polymers that play important physiological roles, including as symbiotic or pathogenic factors and in osmoregulation. Phylogenetically new glycoside hydrolase (GH) families have recently been identified from {beta}-1,2-glucanase (SGL) sequences from bacteria (GH144) and a fungus (GH162). In this study, we identified four phylogenetically new groups (Groups 1-4), and determined that these families, together with GH144, GH162, and GH189, a family of transglycosylase domains in cyclic {beta}-1,2-glucan synthases, form a superfamily. Biochemical analysis of six proteins in these groups revealed that the proteins in Groups 1-3 showed hydrolytic activity specific to {beta}-1,2-glucan. The kinetic parameters of the enzymes of Groups 1-3 were similar to GH144 and GH162 SGLs, indicating that these enzymes were SGLs. Optical rotation analysis revealed that the SGLs followed an anomer-inverting mechanism. Structural analysis and prediction of the proteins in Groups 1-4, GH144, GH162, and GH189 suggested that Groups 1-3 and GH144 had the same reaction mechanism. Nevertheless, Groups 1-3 were dispersed irregularly in the superfamily. Overall, we determined that Groups 1-3 were new GH families, GHxxx, GHyyy, and GHzzz, respectively, and proposed that this superfamily be called an SGL superfamily because of the phylogenetical, functional, and structural relationships within the superfamily. HighlightsWide variety of glycoside hydrolases is far beyond our understanding. Functional and structural analysis identified three new glycoside hydrolase families. Molecular evolution with irregular changes in reaction mechanism was revealed.

biochemistry↗

bioRxiv2023

Age-associated aberrations of cumulus-oocyte interaction and microfilamentous structure in the zona pellucida decline female fertility

One of the major age-related declines in female reproductive function is the reduced quantity and quality of oocytes. Here we demonstrate that structural changes in the zona pellucida (ZP) decrease fertilization rates from 34- to 38-week-old female mice, equivalent to the mid-reproductive of human females. In middle-age ovaries, the decline in the number of transzonal projections was accompanied by a decrease in cumulus cell-oocyte interactions, resulting in a deterioration of the oocyte quality. Scanning electron microscopy showed the ZP surface microfilament structure transitioning from rugged to smooth with age, leading to decreased fertilization rates due to impaired sperm binding to the ZP. Moreover, the fertilization rate of middle-age mice was restored to a comparable level to that of young mice by destabilizing the ZP in the presence of glutathione. These results suggest that the age-related structural changes in the ZP is a key for successful fertilization at reproductive age.

developmental biology↗