Nectin-1 spots regulate the branching of olfactory mitral cell dendrites.

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.

Performance of an improperly sized and stretched-out loose-fitting powered air-purifying respirator: Manikin-based study.

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.

Sevoflurane in combination with propofol, not thiopental, induces a more robust neuroapoptosis than sevoflurane alone in the neonatal mouse brain.

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.

Low blood glucose and fatigue accumulation at peak hours of occupational trauma in secondary industry workers.

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.

Behavioral alterations associated with targeted disruption of exons 2 and 3 of the Disc1 gene in the mouse.

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.

Laboratory evaluation of the particle size effect on the performance of an elastomeric half-mask respirator against ultrafine combustion particles.

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.

Stroke impairment, balance, and cognitive status on admission predict walking independence up to 90 days post-stroke but their contributions change over time.

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.

Wearing high-heeled shoes on a side slope makes standing balance unstable.

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.

Distinct distribution and localization of Rho-kinase in mouse epithelial, muscle and neural tissues.

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.

Comparison of flatfeet and normal feet using data of the gait cycle, contact area, and foot pressure.

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.

Relationship between Perceived Leg Length Discrepancy at One Month and Preoperative Hip Abductor Muscle Elasticity in Patients after Total Hip Arthroplasty.

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.

Triple N-glycosylation in the long S5-P loop regulates the activation and trafficking of the Kv12.2 potassium channel.

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.

Complementation of placental defects and embryonic lethality by trophoblast-specific lentiviral gene transfer.

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.

Prostaglandin E receptor EP1 enhances GABA-mediated inhibition of dopaminergic neurons in the substantia nigra pars compacta and regulates dopamine level in the dorsal striatum.

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.

Nectin: an adhesion molecule involved in formation of synapses.

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.

Impaired cerebellar development and function in mice lacking CAPS2, a protein involved in neurotrophin release.

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.

PASK (proline-alanine-rich Ste20-related kinase) binds to tubulin and microtubules and is involved in microtubule stabilization.

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.

Implications of the aquaporin-4 structure on array formation and cell adhesion.

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.

TTYH2, a human homologue of the Drosophila melanogaster gene tweety, is up-regulated in colon carcinoma and involved in cell proliferation and cell aggregation.

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.

Multiple roles of afadin in the ultrastructural morphogenesis of mouse hippocampal mossy fiber synapses.

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.