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Objective: In living tissue, it has been difficult to make microscopic-level observations without damaging the tissue., Summary Background Data: We have invented a novel intravital fluorescent observation method (IFOM) for real-time tissue observation, combining multi-photon laser scanning microscopy (MPLSM) with curcumin vital staining (CVS-IFOM). The aim of this study was to use CVS-IFOM to analyze the enteric nervous system (ENS) in mice and human patients with hypoganglionosis and Hirschsprung disease., Methods: In an initial viability study, we compared live ENS images from non-fluorescent C57BL6 mice stained with curcumin (n=5) and GFP mice (n=5) using MPLSM. We then explored CVS-IFOM for the live examination of resected colon tissues from one hypoganglionosis and three Hirschsprung disease patients., Results: In the viability study, detailed ENS histological features were only observed in the curcumin-stained mice. In the hypoganglionosis patient, CVS-IFOM provided ENS details that were not visualized under H&E staining or calretinin immunohistochemistry, allowing the analysis of ENS size, neural bundle number, and neural cell number per plexus. In Hirschsprung disease patients, CVS-IFOM showed a gradual hypoplastic change in the ENS from the oral wedge to the anal wedge, detecting disproportionate changes in the ENS within the same intestinal level, supporting a circumferentially uneven distribution of the intestinal ENS., Conclusion: CVS-IFOM may be supportive for intraoperative pathological diagnosis during surgeries in Hirschsprung disease., Competing Interests: Conflict of interest statement: All authors have declared that no conflict of interest exists., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.) more...

Objectives. The incidence of occupational traumatic injuries caused by human error has been reported to occur at 11:00 and 8-9 h after commencing work. Impaired attention is closely related to the incidence of these accidents. Therefore, this study aimed to clarify the changes in blood glucose, fatigue and stress response hormone levels over time among workers in a secondary industry. Methods. The blood glucose and subjective fatigue levels of 26 male secondary-industry workers were measured on workdays. In addition, the cortisol and dehydroepiandrosterone levels in saliva were measured on one workday and one holiday. Results. Blood glucose levels at 11:00 and 17:30 on the workday were significantly lower than those at 09:30. Moreover, hypoglycemia was observed in some participants. A significant increase in subjective fatigue levels was observed during the workday. However, no significant differences in salivary cortisol levels were observed between the workday and the holiday at any time point. Conclusions. Blood glucose levels decreased and subjective fatigue levels increased at the time points that occupational accidents were reported to occur most frequently during work. These factors may contribute to human errors due to impaired attentional function. more...

This study aimed to identify predictors of gait independence in three successive patient cohorts that received inpatient rehabilitation for at least 30 days, 60 days, or 90 days post-stroke. A total of 26 independent variables were collected within 3 days of stroke onset, including basic information (age, sex, stroke type), sensorimotor function (Stroke Impairment Assessment Set), gait function, balance function, and cognitive function. The dependent variable was walking independence (without assistance from another person) at 30, 60, or 90 days post-stroke. A decision tree was developed for predicting gait independence at each assessment time point. The predictors of walking independence differed among the cohorts that received inpatient rehabilitation for at least 30, 60, and 90 days post-stroke. Specifically, the Short Form Berg Balance Scale score was in the higher layer and was a strong predictor of gait at all time points. The cognitive Functional Independence Measure progressed to the higher layer at later time points. The lower extremity motor function was an additional predictor in the 30-day cohort. For later cohorts, the predictive value of balance and cognitive function declined whereas the contribution of the paralyzed grip strength and trunk function increased. These results suggest that sensorimotor and cognitive function within 3 days of stroke can predict walking independence between 1 and 3 months post-stroke; however, the prognostic value of predictors varies among the patients who receive inpatient rehabilitation for shorter versus longer time., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.) more...

Introduction: Many women wearing high-heeled footwear are at high risk of falls. Past studies have examined the balance on level ground or balance during walking. We measured the standing balance on the ground and side slopes for 18 healthy women., Method: Body sway was evaluated based on the center of pressure (COP) while participants stood on level ground on a side slope. The total locus length as well as rectangular and outer peripheral areas were then measured using a Zebris system. Measurements were compared under bare feet, normal shoe, and high-heeled shoe conditions., Results: On level ground, there were no significant differences among the three conditions. On the side slope, the total locus length (TLL), rectangular area (RA) and outer peripheral areas (OPA) were significantly greater for the high-heeled shoes than for the bare feet and normal shoes. Standing on the side slope caused larger body sway than on the level ground, along with a higher risk of falling., Discussion: In TLL, OPA, and RA, the COP moved outside substantially when participants stood on a slope in high heels than in shoes. High heels were highly unstable for standing on a slope since the ankle joint of one leg is in plantar flexion, the foot is pronated, and the other side is plantarflexed at the ankle with pronation of the foot., Conclusion: High-heeled shoes significantly alter a person's balance when standing on a side slope, suggesting a high risk of falling., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.) more...

Objective: Preoperative factors related to perceived leg length discrepancy (PLLD) after total hip arthroplasty (THA) are not well studied. This study aimed to examine the preoperative factors, including hip abductor modulus, related to PLLD one month after THA., Methods: The study included 73 patients diagnosed with osteoarthritis secondary to developmental dysplasia of the hip and a posterior approach to surgery. Multiple logistic regression analysis was performed using the presence or absence of PLLD as the dependent variable and preoperative hip abductor's modulus of elasticity, pain, hip abduction range of motion, hip abductor muscle strength and pelvic obliquity as the independent variable. Additionally, receiver operating characteristic curves were used for the extracted variables for calculating the cutoffs, sensitivity, specificity and area under the curve (AUC) to determine the presence or absence of PLLD. The significance level was set at p<0.05., Results: The hip abductor modulus (odds ratio=1.13; 95% confidence interval=1.06-1.21; p<0.001) was selected as a preoperative factor. The cutoff value to determine the presence or absence of a PLLD was 16.32 kPa. The sensitivity and specificity were 81.8% and 72.5%, respectively, and the AUC was 0.8137., Conclusion: The hip abductor muscle elastic modulus affected PLLD one month after THA. If the preoperative hip abductor elastic modulus is higher than the cutoff value, it may affect the appearance of PLLD at one month postoperatively., Competing Interests: None of the authors has any conflict of interest to disclose., (2021, JAPANESE PHYSICAL THERAPY ASSOCIATION.) more...

Although the foot is involved in load-bearing and shock absorption, foot pressure (FP), ground contact area (CA), and gait cycle (GC) in flatfeet (FF) have not been examined in detail. We aimed to analyze the influence of FF on FP, CA, and GC. We included 20 and 21 women with FF and normal feet (NF), respectively. A Footscan plantar pressure plate (RsScan International, Belgium) was used to analyze FP, CA, and GC. FP was applied to the unit area of 10 compartments. GC analysis was performed using phase-time measurements by dividing the GC into four phases. In the analysis, FP and CA were compared between the FF and NF groups. A comparison of GC was similarly performed between the two groups. The data provided in this article will be useful when designing studies on the effect of foot shape on FP, CA, and GC during gait., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships, which have or could be perceived to have influenced the work reported in this article., (© 2021 The Authors.) more...

A hippocampal mossy fiber synapse, which is implicated in learning and memory, has a complex structure in which mossy fiber boutons attach to the dendritic shaft by puncta adherentia junctions (PAJs) and wrap around a multiply-branched spine, forming synaptic junctions. Here, we electron microscopically analyzed the ultrastructure of this synapse in afadin-deficient mice. Transmission electron microscopy analysis revealed that typical PAJs with prominent symmetrical plasma membrane darkening undercoated with the thick filamentous cytoskeleton were observed in the control synapse, whereas in the afadin-deficient synapse, atypical PAJs with the symmetrical plasma membrane darkening, which was much less in thickness and darkness than those of the control typical PAJs, were observed. Immunoelectron microscopy analysis revealed that nectin-1, nectin-3, and N-cadherin were localized at the control typical PAJs, whereas nectin-1 and nectin-3 were localized at the afadin-deficient atypical PAJs to extents lower than those in the control synapse and N-cadherin was localized at their nonjunctional flanking regions. These results indicate that the atypical PAJs are formed by nectin-1 and nectin-3 independently of afadin and N-cadherin and that the typical PAJs are formed by afadin and N-cadherin cooperatively with nectin-1 and nectin-3. Serial block face-scanning electron microscopy analysis revealed that the complexity of postsynaptic spines and mossy fiber boutons, the number of spine heads, the area of postsynaptic densities, and the density of synaptic vesicles docked to active zones were decreased in the afadin-deficient synapse. These results indicate that afadin plays multiple roles in the complex ultrastructural morphogenesis of hippocampal mossy fiber synapses., (© 2017 Wiley Periodicals, Inc.) more...

The objective of this study was to investigate the protection level offered by a Powered Air-Purifying Respirator (PAPR) equipped with an improperly sized or stretched-out loose-fitting facepiece using constant and cyclic flow conditions. Improperly sized PAPR facepieces of two models as well as a stretched-out facepiece were tested. These facepieces were examined in two versions: with and without exhaust holes. Loose-fitting facepieces (size "large") were donned on a small manikin headform and challenged with sodium chloride (NaCl) aerosol particles in an exposure chamber. Four cyclic flows with mean inspiratory flows (MIFs) of 30, 55, 85, and 135 L/min were applied using an electromechanical Breathing Recording and Simulation System (BRSS). The manikin Fit Factor (mFF) was determined as the ratio of aerosol concentrations outside (Cout) to inside (Cin) of the facepiece, measured with a P-Trak condensation particle counter (CPC). Results showed that the mFF decreased exponentially with increasing MIF. The mFF values of the stretched-out facepiece were significantly lower than those obtained for the undamaged ones. Facepiece type and MIF were found to significantly affect the performance of the loose-fitting PAPR. The effect of the exhaust holes was less pronounced and depended on the facepiece type. It was concluded that an improperly sized facepiece might potentially offer relatively low protection (mFF < 250) at high to strenuous workloads. The testing was also performed at a constant inhalation flow to explore the mechanism of the particle-facepiece interaction. Results obtained with cyclic flow pattern were consistent with the data generated when testing the loose-fitting PAPR under constant flow conditions. The time-weighted average values of mFF calculated from the measurements conducted under the constant flow regime were capable of predicting the protection under cyclic flow regime. The findings suggest that program administrators need to equip employees with properly sized facepieces and remove stretched-out ones from workplace. Manufacturers should emphasize the importance of proper sizing with their user instructions. more...

We have developed a real-time imaging technique for diagnosis of kidney diseases which is composed of two steps, staining renal cells safely with food dyes and optical sectioning of living renal tissue to obtain histological images by multiphoton microscopy (MPM). Here, we demonstrated that the MPM imaging with food dyes, including erythrosine and indigo carmine, could be used as fluorescent agents to visualize renal functions and structures such as glomerular bloodstreams, glomerular filtration, and morphology of glomeruli and renal tubules. We also showed that the kidneys of IgA nephropathy model-mice stained with the food dyes presented histopathological characteristics different from those observed in normal kidneys. The use of the food dyes enhances the quality of tissue images obtained by MPM and offers the potential to contribute to a clinical real-time diagnosis of kidney diseases., (© 2015 Wiley Periodicals, Inc.) more...

Olfactory mitral cells extend lateral secondary dendrites that contact the lateral secondary and apical primary dendrites of other mitral cells in the external plexiform layer (EPL) of the olfactory bulb. The lateral dendrites further contact granule cell dendrites, forming dendrodendritic reciprocal synapses in the EPL. These dendritic structures are critical for odor information processing, but it remains unknown how they are formed. We recently showed that the immunoglobulin-like cell adhesion molecule nectin-1 constitutes a novel adhesion apparatus at the contacts between mitral cell lateral dendrites, between mitral cell lateral and apical dendrites, and between mitral cell lateral dendrites and granule cell dendritic spine necks in the deep sub-lamina of the EPL of the developing mouse olfactory bulb and named them nectin-1 spots. We investigated here the role of the nectin-1 spots in the formation of dendritic structures in the EPL of the mouse olfactory bulb. We showed that in cultured nectin-1-knockout mitral cells, the number of branching points of mitral cell dendrites was reduced compared to that in the control cells. In the deep sub-lamina of the EPL in the nectin-1-knockout olfactory bulb, the number of branching points of mitral cell lateral dendrites and the number of dendrodendritic reciprocal synapses were reduced compared to those in the control olfactory bulb. These results indicate that the nectin-1 spots regulate the branching of mitral cell dendrites in the deep sub-lamina of the EPL and suggest that the nectin-1 spots are required for odor information processing in the olfactory bulb., (Copyright © 2015 Elsevier Inc. All rights reserved.) more...

Mitral cells project lateral dendrites that contact the lateral and primary dendrites of other mitral cells and granule cell dendrites in the external plexiform layer (EPL) of the olfactory bulb. These dendritic structures are critical for odor information processing, but it remains unknown how they are formed. In immunofluorescence microscopy, the immunofluorescence signal for the cell adhesion molecule nectin-1 was concentrated on mitral cell lateral dendrites in the EPL of the developing mouse olfactory bulb. In electron microscopy, the immunogold particles for nectin-1 were symmetrically localized on the plasma membranes at the contacts between mitral cell lateral dendrites, which showed bilateral darkening without dense cytoskeletal undercoats characteristic of puncta adherentia junctions. We named the contacts where the immunogold particles for nectin-1 were symmetrically accumulated "nectin-1 spots." The nectin-1 spots were 0.21 μm in length on average and the distance between the plasma membranes was 20.8 nm on average. In 3D reconstruction of serial sections, clusters of the nectin-1 spots formed a disc-like structure. In the mitral cell lateral dendrites of nectin-1-knockout mice, the immunogold particles for nectin-1 were undetectable and the plasma membrane darkening was electron-microscopically normalized, but the plasma membranes were partly separated from each other. The nectin-1 spots were further identified between mitral cell lateral and primary dendrites and between mitral cell lateral dendrites and granule cell dendritic spine necks. These results indicate that the nectin-1 spots constitute a novel adhesion apparatus that tethers mitral cell dendrites in a dendritic meshwork structure of the developing mouse olfactory bulb., (© 2015 Wiley Periodicals, Inc.) more...

Breathing machines are widely used to evaluate respirator performance but they are capable of generating only limited air flow patterns, such as, sine, triangular and square waves. In order to evaluate the respirator performance in practical use, it is desirable to test the respirator using the actual breathing patterns of wearers. However, it has been a difficult task for a breathing machine to generate such complicated flow patterns, since the human respiratory volume changes depending on the human activities and workload. In this study, we have developed an electromechanical breathing simulator and a respiration sampling device to record and reproduce worker's respiration. It is capable of generating various flow patterns by inputting breathing pattern signals recorded by a computer, as well as the fixed air flow patterns. The device is equipped with a self-control program to compensate the difference in inhalation and exhalation volume and the measurement errors on the breathing flow rate. The system was successfully applied to record the breathing patterns of workers engaging in welding and reproduced the breathing patterns. more...

Purpose: Sevoflurane is the most widely used volatile anesthetic of general anesthesia. In children and neonates, it is commonly used alone or in combination with thiopental or propofol. A few recent studies reported that sevoflurane induced neuronal death in the developing rodent brain. We measured the neurotoxicity of these anesthetics at clinical doses, alone and in combination, in the developing mouse brain., Methods: Seven-day-old C57BL/6 mice were randomly assigned to 6 treatment groups. Three groups were exposed to 3% sevoflurane for 6 h after injection of saline, thiopental (5 mg/kg), or propofol (10 mg/kg), whereas three groups were exposed to room air for 6 h after injection of equal doses of saline, thiopental, or propofol. Apoptosis in the hippocampal CA1 region (CA1) and retrosplenial cortex (RC) was assessed using caspase-3 immunostaining., Results: Sevoflurane alone caused significantly higher apoptosis in the CA1 compared with saline plus air (P = 0.04). Sevoflurane in combination with propofol resulted in significantly greater numbers of apoptotic neurons than sevoflurane alone in both the CA1 and the RC (P = 0.04). However, there was no significant difference in apoptotic neuron density in both the regions between the groups treated with sevoflurane alone and in combination with thiopental (P = 0.683)., Conclusion: Sevoflurane alone can induce neuronal apoptosis, and this effect is enhanced by propofol. Thiopental did not exacerbate the neurotoxicity of sevoflurane. There is the possibility that the combination of sevoflurane and propofol is a more harmful anesthetic technique than sevoflurane alone in pediatric patients. more...

Incretins, hormones released by the gut after meal ingestion, are essential for maintaining systemic glucose homeostasis by stimulating insulin secretion. The effect of incretins on insulin secretion occurs only at elevated glucose concentrations and is mediated by cAMP signaling, but the mechanism linking glucose metabolism and cAMP action in insulin secretion is unknown. We show here, using a metabolomics-based approach, that cytosolic glutamate derived from the malate-aspartate shuttle upon glucose stimulation underlies the stimulatory effect of incretins and that glutamate uptake into insulin granules mediated by cAMP/PKA signaling amplifies insulin release. Glutamate production is diminished in an incretin-unresponsive, insulin-secreting β cell line and pancreatic islets of animal models of human diabetes and obesity. Conversely, a membrane-permeable glutamate precursor restores amplification of insulin secretion in these models. Thus, cytosolic glutamate represents the elusive link between glucose metabolism and cAMP action in incretin-induced insulin secretion. more...

Objectives: This study quantified the particle size effect on the performance of elastomeric half-mask respirators, which are widely used by firefighters and first responders exposed to combustion aerosols., Methods: One type of elastomeric half-mask respirator equipped with two P-100 filters was donned on a breathing manikin while challenged with three combustion aerosols (originated by burning wood, paper, and plastic). Testing was conducted with respirators that were fully sealed, partially sealed (nose area only), or unsealed to the face of a breathing manikin to simulate different faceseal leakages. Three cyclic flows with mean inspiratory flow (MIF) rates of 30, 85, and 135 L/min were tested for each combination of sealing condition and combustion material. Additional testing was performed with plastic combustion particles at other cyclic and constant flows. Particle penetration was determined by measuring particle number concentrations inside and outside the respirator with size ranges from 20 to 200 nm., Results: Breathing flow rate, particle size, and combustion material all had significant effects on the performance of the respirator. For the partially sealed and unsealed respirators, the penetration through the faceseal leakage reached maximum at particle sizes >100 nm when challenged with plastic aerosol, whereas no clear peaks were observed for wood and paper aerosols. The particles aerosolized by burning plastic penetrated more readily into the unsealed half-mask than those aerosolized by the combustion of wood and paper. The difference may be attributed to the fact that plastic combustion particles differ from wood and paper particles by physical characteristics such as charge, shape, and density. For the partially sealed respirator, the highest penetration values were obtained at MIF = 85 L/min. The unsealed respirator had approximately 10-fold greater penetration than the one partially sealed around the bridge of the nose, which indicates that the nose area was the primary leak site. more...

Migrating cells acquire front-rear polarity with a leading edge and a trailing tail for directional movement. The Rac exchange factor Tiam1 participates in polarized cell migration with the PAR complex of PAR3, PAR6, and atypical protein kinase C. However, it remains largely unknown how Tiam1 is regulated and contributes to the establishment of polarity in migrating cells. We show here that Tiam1 interacts directly with talin, which binds and activates integrins to mediate their signaling. Tiam1 accumulated at adhesions in a manner dependent on talin and the PAR complex. The interactions of talin with Tiam1 and the PAR complex were required for adhesion-induced Rac1 activation, cell spreading, and migration toward integrin substrates. Furthermore, Tiam1 acted with talin to regulate adhesion turnover. Thus, we propose that Tiam1, with the PAR complex, binds to integrins through talin and, together with the PAR complex, thereby regulates Rac1 activity and adhesion turnover for polarized migration. more...

A major challenge of cancer biology is to visualize the dynamics of the metastatic process in secondary organs at high optical resolution in vivo real-time. Here, we presented intravital, dual-colored imaging of liver metastasis formation from a single cancer cell to metastatic colonies in the living liver of living mice using two photon laser scanning microscopy (TPLSM). Red fluorescent protein expressing murine (SL4) or human (HT29) colorectal cancer cell lines were inoculated to the spleen of green fluorescent protein expressing mice. Intravital TPLSM was performed by exteriorizing and fixing the liver lobe of living mice. This was repeated several times for the long-term imaging of the same mouse. Viable cancer cells in the living liver of living mice were visualized intravitally at a magnification of over 600×. Single cancer cells were arrested within hepatic sinusoids 2 h after injection. Platelet aggregation surrounding a cancer cell was observed, indicating a phenomenon of tumor-cell induced platelet aggregation. Cancer cells were extravasated from hepatic sinusoids to the space of Disse. Protrusions of Kupffer cells surrounding a cancer cell were observed, indicating that Kupffer cells appear to phagocytose cancer cells. SL4 cells formed liver metastatic colonies with extensive stromal reaction. Liver metastases by HT29 cells were observed as a cluster of micrometastatic nodules. High-resolution, dual-colored, real-time visualization of cancer metastasis using intravital TLPSM can help to understand spatiotemporal tumor-host interactions during metastatic processes in the living organs of living animals., (Copyright © 2011 Wiley Periodicals, Inc.) more...

The small GTP-binding protein Rho plays a crucial role in a wide variety of cellular functions through various effector proteins. Rho-kinase is a key effector protein of Rho, which is composed of two isoforms, ROCK1 and ROCK2. To clarify the site of action of ROCK1 and ROCK2, we performed immunofluorescence and immunoelectron microscopic analyses using isoform-specific antibodies in mouse tissues. In the large and small intestines, ROCK1 immunoreactivity was predominantly identified in epithelial cells, and ROCK2 immunoreactivity was negligible. In these epithelial cells, ROCK1 immunoreactivity was distributed on plasma membranes, while ROCK1 immunogold signals were localized at cell-cell contacts and cell adhesion sites, especially at the adherens junctions at the ultrastructural level. In the bladder epithelium, however, ROCK1 and ROCK2 signals were identified at intermediate filaments, and ROCK2 signals were also observed in nuclei. In the three types of muscular cells-smooth, cardiac, and skeletal muscle cells-ROCK1 and ROCK2 also showed differential distribution. ROCK1 signals were localized at actin filaments, plasma membranes, and vesicles near plasma membranes in smooth muscle cells; at the lysosomes in skeletal muscle cells; and were undetectable in cardiac muscle cells. ROCK2 signals were localized at actin filaments and centrosomes in smooth muscle cells, at intercalated discs in cardiac muscle cells, and at Z-discs and sarcoplasmic reticulum in skeletal muscle cells. In the brain, ROCK1 immunoreactivity was distributed in glia, whereas ROCK2 immunoreactivity was observed in neurons. These results indicate that the two isoforms of Rho-kinase distribute differentially to accomplish their specific functions. more...

Disrupted-In-Schizophrenia 1 (DISC1) is a promising candidate gene for susceptibility to psychiatric disorders, including schizophrenia. DISC1 appears to be involved in neurogenesis, neuronal migration, axon/dendrite formation and synapse formation; during these processes, DISC1 acts as a scaffold protein by interacting with various partners. However, the lack of Disc1 knockout mice and a well-characterized antibody to DISC1 has made it difficult to determine the exact role of DISC1 in vivo. In this study, we generated mice lacking exons 2 and 3 of the Disc1 gene and prepared specific antibodies to the N- and C-termini of DISC1. The Disc1 mutant mice are viable and fertile, and no gross phenotypes, such as disorganization of the brain's cytoarchitecture, were observed. Western blot analysis revealed that the DISC1-specific antibodies recognize a protein with an apparent molecular mass of ~100 kDa in brain extracts from wild-type mice but not in brain extracts from DISC1 mutant mice. Immunochemical studies demonstrated that DISC1 is mainly localized to the vicinity of the Golgi apparatus in hippocampal neurons and astrocytes. A deficiency of full-length Disc1 induced a threshold shift in the induction of long-term potentiation in the dentate gyrus. The Disc1 mutant mice displayed abnormal emotional behavior as assessed by the elevated plus-maze and cliff-avoidance tests, thereby suggesting that a deficiency of full-length DISC1 may result in lower anxiety and/or higher impulsivity. Based on these results, we suggest that full-length Disc1-deficient mice and DISC1-specific antibodies are powerful tools for dissecting the pathophysiological functions of DISC1. more...

Background: Intravital three-dimensional (3D) visualization of treatment efficacy in experimental colitis in living mice using two-photon laser scanning microscopy (TPLSM) has not been described., Methods: Colitis was induced with dextran sulfate sodium (DSS) in green fluorescent protein (GFP) transgenic mice. The 3D tomographic image of DSS-induced colitis with or without prednisolone was obtained intravitally using TPLSM. A serosal-approaching method was developed, by which we could observe all layers of the cecum from serosa to luminal mucosa without opening and everting the cecum. The dynamic pathology and treatment efficacy were assessed in the same mouse on several occasions., Results: The time-lapse 3D tomographic movie of DSS-induced colitis was obtained in living mice at a magnification of greater than ×600, which demonstrated irregularity of crypts, disappearance of crypts, inflammatory cell infiltrates in the lamina propria, and abscess formation at the bottom of crypts. Intravital TPLSM in the same mice demonstrated fewer infiltrating leukocytes and crypt abscesses on day 14 in the steroid group compared with the nonsteroid group., Conclusions: Intravital 3D tomographic visualization of experimental colitis using TPLSM in combination with the serosal-approaching method can provide dynamic pathology at a high magnification, which may be useful in evaluating treatment efficacy in the same living mice. more...

Background: Two-photon laser-scanning microscopy (TPLSM) is a powerful diagnostic tool for real-time, high-resolution structural imaging. However, obtaining high-quality in vivo TPLSM images of intra-abdominal organs remains technically challenging., Materials and Methods: An organ-stabilizing system was applied to high-quality TPLSM imaging. Real-time imaging of visceral organs, such as the liver, spleen, kidney and intestine, of transgenic green fluorescent protein (GFP) mice was performed in vivo using TPLSM. The bacterial translocation model using dextran sodium sulfate (DSS)-induced colitis was also investigated in prepared GFP mice following simple surgery. This allowed the capture of morphological real images using in vivo TPLSM. Immunohistochemical analysis of ZO-1 was performed to support the morphological findings of TPLSM., Results and Conclusions: We established an organ-stabilizing system to evaluate the real-time imaging of visceral organs in actin-GFP transgenic mice using in vivo TPLSM. DSS-induced colitis showed irregularity of crypt architecture, disappearance of crypts, inflammatory cell infiltration and increased rolling of white blood cells along the vasculature. In addition, the intercellular distance of mucosal cells in the crypt and vascular endothelial cells in the intestinal wall was increased in the intestinal mucosa during DSS colitis. In DSS colitis, there was remarkable loss of mucosal and vascular endothelial ZO-1 expression, as could be seen by a decrease in ZO-1 staining. In conclusion, our observations suggested the possibility that our TPLSM imaging system can be used to clarify the pathophysiological changes in various diseases using longitudinal studies of microscopic changes in the same animal over long periods of time. more...

Dopamine (DA) is a neuromodulator that is critical for sensory-motor, cognitive and emotional functions. We previously found that mice lacking prostaglandin E receptor EP1 showed impulsive emotional behaviors accompanied by enhanced DA turnover in the frontal cortex and striatum. Given that these behavioral phenotypes were corrected by DA receptor antagonists, we hypothesized that EP1 deficiency causes a hyperdopaminergic state for its behavioral phenotype. Here we tested this hypothesis by examining the EP1 action in the nigrostriatal dopaminergic system. We first used microdialysis and found an elevated extracellular DA level in the dorsal striatum of EP1-deficient mice compared with wild-type mice. Despite the EP1 expression in the striatum, neither deficiency nor activation of EP1 altered the intrastriatal control for DA release, uptake or degradation. Immunohistochemistry revealed punctate EP1 signals apposed with dopaminergic neurons in the substantia nigra pars compacta (SNc). Many EP1 signals were colocalized with a marker for GABAergic synapses. Further, an EP1 agonist enhanced GABA(A)-mediated inhibitory inputs to SNc dopaminergic neurons in midbrain slices. Therefore, the prostaglandin E(2)-EP1 signaling directly enhances GABAergic inputs to SNc dopaminergic neurons. The lack of this EP1 action may lead to a hyperdopaminergic state of EP1-deficient mice. more...

Mammalian voltage-dependent potassium (Kv) channels regulate the excitability of nerve and muscle cells. Kv12.2 features the longest S5-P loop among all known mammalian Kv channels with the most N-linked glycosylation sites (three sites). Despite its unique structural features, Kv12.2 is not well characterized. Because glycosylation plays important roles in the folding, trafficking, and function of various Kv channels, we focused on the N-glycosylation of Kv12.2. We show that Kv12.2 is N-glycosylated in Chinese hamster ovary (CHO) cells and in cultured neurons as well as in the mouse brain. As an effect of N-glycosylation on the function of Kv12.2, we demonstrate that removal of sugar chains causes a depolarizing shift in the steady-state activation without a significant reduction in current amplitude. Unlike the previously reported shift for Shaker-type Kv channels, this shift does not appear to be due to negatively charged sialic acid residues in the sugar chains. We next examined the trafficking in CHO cells to address whether the unglycosylated Kv12.2 channels are utilized in vivo. Although double mutants, retaining only one glycosylation site, are trafficked to the surface of CHO cells irrespective of the position of the glycosylated site, unglycosylated channels are not trafficked to the cell surface. Furthermore, we could not detect unglycosylated channels in the mouse brain. Our data suggest that only glycosylated Kv12.2 channels show proper voltage dependence and are utilized in vivo. more...

We identified that whirlin is localized to chromosome 9q32-33, and is up-regulated in colorectal cancer tissues by using oligonucleotide array techniques and the Sosui system (http://www.tuat.ac.jp/~mitaku/sosui/). The deduced 920-amino acid protein encoded by the whirlin gene contains three PDZ domains and a proline-rich region that separates PDZ2 from PDZ3, which is located at the C terminus. As previously reported, human whirlin gene is alternatively spliced to form a long and a short transcript in situ hybridization. The sequence of the encoded protein shows that the short C-terminal isoform contains one PDZ domain and the proline-rich domain (whirlin isoform 2), whereas the long isoform is composed of all three PDZ domains and the proline-rich domain (whirlin isoform 1). The gene expression of whirlin was found to be up-regulated in colorectal cancer tissues compared with matched normal colon tissues by semi-quantitative RT-PCR (P<0.05). Western blotting detected whirlin protein with a molecular mass of 49.3 kDa in colorectal cancer samples, suggesting that the whirlin protein overexpressed in colorectal cancer samples is the short C-terminal isoform 2. Its expression was recognized in colorectal cancer cell lines and was increased in accordance with tumor progression in colorectal cancer patients. Immunohistochemistry showed high levels of staining for whirlin isoform 2 only in the mucosal glands in colon cancers, but this protein was barely detected in normal colonic glands. Immunoelectron microscopic findings showed that whirlin isoform 2 is localized on plasma membranes and endoplasmic reticulum membranes, but not in the nuclei. Tissue microarrays showed that whirlin isoform 2 is abundantly expressed in colon cancers with lymph node metastasis compared with those without lymph node metastasis, and overexpression of this protein was associated with tumor progression. In conclusion, we demonstrated that whirlin isoform 2 is highly expressed in colon cancer tissues and that it is related to tumor progression. more...

Proline-alanine-rich Ste20-related kinase (PASK, also referred to as SPAK) has been linked to ion transport regulation. Here, we report two novel activities of PASK: binding to tubulin and microtubules and the promotion of microtubule assembly. Tubulin binding assay showed that full-length PASK and its kinase domain bound to purified tubulin whereas the N-terminal or C-terminal non-catalytic domains of PASK did not. The full-length PASK and its kinase domain were sedimented with paclitaxel-stabilized microtubules by ultracentrifugation. These results indicate that the kinase domain of PASK can interact directly with both microtubules and soluble tubulin in vitro. Truncated PASK lacking the N-terminal non-catalytic domain promoted microtubule assembly at a subcritical concentration of purified tubulin. FLAG-PASK expressed in COS-7 cells translocated to the cytoskeleton when the cells were stimulated with hypertonic sodium chloride, and stabilized microtubules against depolymerization by nocodazole. Our findings suggest that PASK may regulate the cytoskeleton by modulating microtubule stability. more...

Aim: To investigate the expression patterns of TTYH2 in the human colon cancer and colon cancer cell lines and to evaluate the inhibitory effect of small interfering RNA (siRNA) on the expression of TTYH2 in colon cancer cell lines., Methods: We investigated the expression patterns of TTYH2 in colon cancer, adjacent non-tumorous colon mucosa, and cancer cell lines (DLD-1, caco-2, and Lovo) by RT-PCR. Furthermore, a siRNA plasmid expression vector against TTYH2 was constructed and transfected into DLD-1 and Caco-2 with Lipofectamine 2000. The down regulation of TTYH2 expression was detected by RT-PCR and the role of siRNA in inducing cell proliferation and cell aggregation was evaluated by MTT and aggregation assay., Results: TTYH2 gene expression in colon cancer tissue was significantly up-regulated compared with normal colonic mucosa (1.23 +/- 0.404 vs 0.655 +/- 0.373, P = 0.0103). Colon cancer derived cell lines including DLD-1, Caco-2, and Lovo also expressed high levels of TTYH2. In contrast, transfection with siRNA-TTYH2 significantly inhibited both proliferation and scattering of these cancer cell lines., Conclusion: The present work demonstrates, for the first time, that the TTYH2 gene expression is significantly up-regulated in colon cancer. The TTYH2 gene may play an important role in regulating both proliferating and metastatic potentials of colorectal cancer. more...

Ca2+-dependent activator protein for secretion 2 (CAPS2/CADPS2) is a secretory granule-associated protein that is abundant at the parallel fiber terminals of granule cells in the mouse cerebellum and is involved in the release of neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF), both of which are required for cerebellar development. The human homolog gene on chromosome 7 is located within susceptibility locus 1 of autism, a disease characterized by several cerebellar morphological abnormalities. Here we report that CAPS2 knock-out mice are deficient in the release of NT-3 and BDNF, and they consequently exhibit suppressed phosphorylation of Trk receptors in the cerebellum; these mice exhibit pronounced impairments in cerebellar development and functions, including neuronal survival, differentiation and migration of postmitotic granule cells, dendritogenesis of Purkinje cells, lobulation between lobules VI and VII, structure and vesicular distribution of parallel fiber-Purkinje cell synapses, paired-pulse facilitation at parallel fiber-Purkinje cell synapses, rotarod motor coordination, and eye movement plasticity in optokinetic training. Increased granule cell death of the external granular layer was noted in lobules VI-VII and IX, in which high BDNF and NT-3 levels are specifically localized during cerebellar development. Therefore, the deficiency of CAPS2 indicates that CAPS2-mediated neurotrophin release is indispensable for normal cerebellar development and functions, including neuronal differentiation and survival, morphogenesis, synaptic function, and motor learning/control. The possible involvement of the CAPS2 gene in the cerebellar deficits of autistic patients is discussed. more...

Placental dysfunction underlies many complications during pregnancy, and better understanding of gene function during placentation could have considerable clinical relevance. However, the lack of a facile method for placenta-specific gene manipulation has hampered investigation of placental organogenesis and the treatment of placental dysfunction. We showed previously that transduction of fertilized mouse eggs with lentiviral vectors leads to transgene expression in both the fetus and the placenta. Here we report placenta-specific gene incorporation by lentiviral transduction of mouse blastocysts after removal of the zona pellucida. All of the placentas analyzed, but none of the fetuses, were transgenic. Application of this method substantially rescued mice deficient in Ets2, Mapk14 (also known as p38alpha) and Mapk1 (also known as Erk2) from embryonic lethality caused by placental defects. Ectopic expression of Mapk11 also complemented Mapk14 deficiency during placentation. more...

Aquaporin-4 (AQP4) is the predominant water channel in the mammalian brain and an important drug target for treatment of cerebral edema, bipolar disorder and mesial temporal lobe epilepsy. We determined the AQP4 structure by electron crystallography of double-layered, two-dimensional (2D) crystals. The structure allows us to discuss how the expression ratio between the long and short AQP4 splicing variant can determine the size of in vivo orthogonal arrays. Furthermore, AQP4 contains a short 3(10) helix in an extracellular loop, which mediates weak but specific interactions between AQP4 molecules in adjoining membranes. This finding suggests a previously unexpected role for AQP4 in cell adhesion. This notion was corroborated by expression of AQP4 in L-cells, which resulted in clustering of the cells. Our AQP4 structure thus enables us to propose models for the size regulation of orthogonal arrays and channel-mediated cell adhesion. more...

Aim: A few studies have applied genomic-wide gene expression analysis in inflamed colon tissue sample in ulcerative colitis (UC). We reported the first study of non-inflamed mucosal gene expression in UC and explored its clinical implication., Methods: Non-inflamed mucosa was obtained from 6 UC patients who received total colectomy. The gene expression of UC in noninflamed mucosa was monitored with a microarray. For a selected gene, RT-PCR was performed to verify array results and to further examine expression pattern in inflamed mucosa of UC, colorectal cancer tissue and normal mucosa using additional matched pairs., Results: Two genes showing 2.5-fold decreased expression with significance set at in UC samples were homeo box a4 (HOXa4) and mads box transcription enhancer factor 2, polypeptide B (MEF2b). RT-PCR showed that MEF2b expression in non-inflamed mucosa was significantly downregulated, whereas the expression of MEF2b increased in accordance with the severity of mucosal inflammation. HOXa4 expression both in inflamed and non-inflamed mucosa in UC was consistently downregulated, and the downregulation of HOXa4 was also found in colorectal carcinoma., Conclusion: It is suggested that the MEF2b expression in UC which increase in accordance with inflammation may be induced by the inflammatory mediator. In contrast the downregulation of HOXa4 may be partly involved in the pathogenesis of disease including UC and UC-related carcinogenesis. more...

Myosin phosphatase (MP) is a major phosphatase responsible for the dephosphorylation of the regulatory light chain of myosin II. MYPT1, a target subunit of smooth and nonmuscle MP, is responsible for activation and regulation of MP. To identity the physiological roles of MP, we have generated MYPT1-deficient mice by gene targeting. The heterozygous mice showed no changes in expression levels of MYPT1 and no distinct phenotype compared to wild-type mice was observed. None of the F2 mice were homozygous for the MYPT1 deletion, indicating that the targeted disruption of the MYPT1 gene resulted in embryonic lethality. The point of embryonic lethality is before 7.5 dpc. These findings indicate that MYPT1 is essential for mouse embryogenesis. more...

Neurogenesis has been described in limited regions of the adult mammalian brain. In this study, we showed that the ependymal layer of the 3rd ventricle is a neurogenic region in the adult rat brain. DiI labeling of the 3rd ventricle revealed that neural progenitor cells were derived from cells at the ependymal layer of the adult 3rd ventricle. The mitosis of these progenitor cells at the ependymal layer was promoted by bFGF administration. Combination of BrdU administration, nestin/GFAP immunohistochemistry, and labeling by GFP-recombinant adenoviral infection (vGFP) indicated that at least some tanycytes might be neural progenitor cells in the ependymal layer of the 3rd ventricle. Tracing by vGFP indicated that neural progenitor cells may have migrated from the 3rd ventricle to the hypothalamic parenchyma, where they were integrated into neural networks by forming synapses. In addition, some BrdU(+) neurons had immunoreactivity for orexin A in the hypothalamus. These results indicate that neural progenitor cells exist in the ependymal layer of the adult rat 3rd ventricle and that they may differentiate into neurons functioning in the hypothalamus. more...

Choroid plexus ependymal cells (CPECs) were known to promote axonal growth when choroid plexus is grafted into the adult rat spinal cord. The present study was carried out to examine whether CPECs promote axonal outgrowth from neurons derived from the CNS in vitro. Hippocampal neurons were cocultured on CPEC monolayers. After 24 h, neurite extension was evaluated using various parameters in comparison with cultures grown on poly-L-lysine (PLL)-coated plates and cocultures grown on astrocyte monolayers. The primary neurite length and total neurite length were longest in the cocultures with CPECs. The number of primary neurites and the number of branches were larger in the cultures with CPECs than in the cultures on PLL-coated plates, but almost the same as in the cocultures with astrocytes. Next, we examined whether the neurite extension-promoting effect occurring within 24 h is due primarily to contact with the CPECs or to factors secreted by CPECs into the culture medium. The CPEC monolayers were killed by ethanol fixation, and neurons cultured on them. The neurons extended long neurites with elaborate branching, as in the case of cocultures grown on living CPECs. On the other hand, CPEC-conditioned medium exhibited less promoting effect on neurite outgrowth from hippocampal neurons. These results indicate that CPECs have a capacity to promote neurite outgrowth from CNS neurons in vitro , and that surface plasma membrane-bound components of CPECs strongly contribute to the enhancement of neurite outgrowth in the present coculture system. more...

Sept2 is a member of the septin family of GTPases. Septins form filaments in a GTP-form dependent manner, and are involved in cytokinesis from yeast to mammals; however, some mammalian septins, including Sept2, are expressed in the brain, a tissue in which almost all the cells are postmitotic. Recently, some functions of mammalian septin other than cytokinesis such as vesicle transport have been reported. However, mammalian septin's physiological functions are still unclear. The present study revealed that Sept2 co-localizes with the astrocyte glutamate transporter GLAST in the Bergmann glial processes facing axons and synapses. Biochemical analyses demonstrated that Sept2 bound directly to the carboxy-terminal region of GLAST in a GDP-form dependent manner. Expression of constitutive GDP-form Sept2 mutant reduced the glutamate uptake activity of GLAST via internalization of GLAST from cell surface. Thus Sept2 may regulate GLAST-mediated glutamate uptake by astrocytes, which is important for appropriate transmitter signalling in the cerebellum. more...

Neural stem cells were isolated from deceased early postnatal and adult rats with varying post-mortem intervals. Animals were killed by deep anesthesia and stored in a refrigerator at 4 degrees C for 1-6 days before use. Neurospheres were obtained from the forebrain tissue, including the lateral ventricle in the early postnatal rats, and from the striatal wall of lateral ventricle, including the subventricular zone (SVZ) in adult rats. The number of neurospheres obtained in the primary culture from early postnatal animals was much larger than that from the adult rats. There was no significant difference in the population of neurospheres between the living and the deceased animals at least within 2 days after death. A few neurospheres were still obtainable at 6 days after death in early postnatal animals, but almost no neurospheres were obtained at 5 days after death in the adult rats. The differentiation capacity of neural stem cells in neurospheres was similar between the deceased and the living animals. The rich vascular bed in the SVZ of the lateral ventricle suggests that the vascular architecture might be in part responsible for the survival of the neural stem cells in the deceased animals. Neurosphere cells derived from deceased adult rats survived and differentiated mainly into glial cells in the host spinal cord tissue after transplantation into the injured spinal cord. Therefore, the neural stem cells from deceased animals express the same phenotypes as those from living animals in terms of neurosphere formation, proliferation, and differentiation at least 2 days after death. The neural stem cells from cadavers have great significance in terms of their clinical use as homografts for CNS regeneration., (Copyright 2003 Wiley-Liss, Inc.) more...

Recent studies have suggested that the soluble N-ethylmaleimide-sensitive factor attached protein (SNAP) receptor (SNARE)-mediated membrane fusion system is involved in vesicle fusion with the surface plasma membrane, which leads to neurite elongation. There have been several reports analyzing the effects of neurite outgrowth by inhibition of SNAREs. We studied this mechanism by overexpressing GFP-fusion SNAREs including VAMP-2, SNAP-25A, and syntaxin1A in PC12 cells to investigate the role of SNAREs in neurite outgrowth. When overexpressed in PC12 cells, VAMP-2 promoted neurite elongation, whereas SNAP-25A stimulated neurite sprouting. On the other hand, overexpression of syntaxin1A neither promoted nor inhibited neurite outgrowth. Thus, VAMP-2 and SNAP-25A play different roles in neurite elongation and sprouting. more...

Nogo-A is known as an oligodendrocyte/myelin-associated molecule having an inhibitory effect on neurite outgrowth in the central nervous system. During development, starting from P21 Nogo-A was detected in the cytoplasm of mature oligodendrocytes with compact myelin sheaths in the rat spinal cord. COS7 cells transfected with recNogo-A displayed strong Nogo-A immunoreactivity in their cytoplasm as well as on the mitotic spindle. Nogo-A was not detected in membrane protein fractions from transfected plus biotinylated COS7 cells. Nogo-A was co-immunoprecipitated with alpha-tubulin and myelin basic protein (MBP) from rat brain tissue. These results show that Nogo-A is expressed in association with tubulin and MBP in the mature oligodendrocytes. more...

The nectin-afadin system is a novel cell-cell adhesion system that organizes adherens junctions cooperatively with the cadherin-catenin system in epithelial cells. Nectin is an immunoglobulin-like adhesion molecule, and afadin is an actin filament-binding protein that connects nectin to the actin cytoskeleton. Nectin has four isoforms (-1, -2, -3, and -4). Each nectin forms a homo-cis-dimer followed by formation of a homo-trans-dimer, but nectin-3 furthermore forms a hetero-trans-dimer with nectin-1 or -2, and the formation of each hetero-trans-dimer is stronger than that of each homo-trans-dimer. We show here that at the synapses between the mossy fiber terminals and dendrites of pyramidal cells in the CA3 area of adult mouse hippocampus, the nectin-afadin system colocalizes with the cadherin-catenin system, and nectin-1 and -3 asymmetrically localize at the pre- and postsynaptic sides of puncta adherentia junctions, respectively. During development, nectin-1 and -3 asymmetrically localize not only at puncta adherentia junctions but also at synaptic junctions. Inhibition of the nectin-based adhesion by an inhibitor of nectin-1 in cultured rat hippocampal neurons results in a decrease in synapse size and a concomitant increase in synapse number. These results indicate an important role of the nectin-afadin system in the formation of synapses. more...