Molecular mechanisms of cyclic phosphatidic acid-induced lymphangiogenic actions in vitro.

The lymphatic system plays important roles in various physiological and pathological phenomena. As a bioactive phospholipid, lysophosphatidic acid (LPA) has been reported to function as a lymphangiogenic factor as well as some growth factors, yet the involvement of phospholipids including LPA and its derivatives in lymphangiogenesis is not fully understood. In the present study, we have developed an in-vitro lymphangiogenesis model (termed a collagen sandwich model) by utilizing type-I collagen, which exists around the lymphatic endothelial cells of lymphatic capillaries in vivo. The collagen sandwich model has revealed that cyclic phosphatidic acid (cPA), and not LPA, augmented the tube formation of human dermal lymphatic endothelial cells (HDLECs). Both cPA and LPA increased the migration of HDLECs cultured on the collagen. As the gene expression of LPA receptor 6 (LPA6) was predominantly expressed in HDLECs, a siRNA experiment against LPA6 attenuated the cPA-mediated tube formation. A synthetic LPA1/3 inhibitor, Ki16425, suppressed the cPA-augmented tube formation and migration of the HDLECs, and the LPA-induced migration. The activity of Rho-associated protein kinase (ROCK) located at the downstream of the LPA receptors was augmented in both the cPA- and LPA-treated cells. A potent ROCK inhibitor, Y-27632, suppressed the cPA-dependent tube formation but not the migration of the HDLECs. Furthermore, cPA, but not LPA, augmented the gene expression of VE-cadherin and ß-catenin in the HDLECs. These results provide novel evidence that cPA facilitates the capillary-like morphogenesis and the migration of HDLECs through LPA6/ROCK and LPA1/3 signaling pathways in concomitance with the augmentation of VE-cadherin and ß-catenin expression. Thus, cPA is likely to be a potent lymphangiogenic factor for the initial lymphatics adjacent to type I collagen under physiological conditions.

Shock-absorbing effect of flooring-adopted mechanical metamaterial technology and its influence on the gait and balance of older adults.

OBJECTIVE: To elucidate the performance of a shock-absorbing floor material with a mechanical metamaterial (MM-flooring) structure and its effect on the gait and balance of older adults. METHODS: The drop-weight impact was applied to evaluate the shock-absorbing performance. The falling weight was adjusted equivalent to the energy exerted on the femur of an older woman when she falls, which was evaluated on the MM-flooring and six other flooring materials.Nineteen healthy people over the age of 65 years participated in the gait and balance evaluations. The timed up and go and two-step tests were adopted as gait performance tests, and the sway-during-quiet-balance test with force plates and the functional reach test (FRT) were adopted as balance tests. All the participants underwent these tests on the MM-flooring, shock-absorbing mat and rigid flooring. RESULTS: The shock-absorbing performance test revealed that MM-flooring has sufficient shock-absorbing performance, and suggesting that it may reduce the probability of fractures in the older people when they fall. The results of the gait performance test showed that the participants demonstrated the same gait performance on the MM-flooring and the rigid floor. In the quiet standing test, MM-flooring did not affect the balance function of the participants to the same extent as the rigid floor, compared with the shock-absorbing mat. In the FRT, no significant differences were found for any of the flooring conditions. CONCLUSIONS: MM-flooring has the potential to prevent fractures attributed to falls and does not affect the gait or balance of older adults.

Lysophosphatidic Acid Augments the Gene Expression and Production of Matrix Metalloproteinases-1 and -3 in Human Synovial Fibroblasts in Vitro.

Rheumatoid arthritis (RA) is an inflammatory disease with joint dysfunction following cartilage degradation. The level of lysophosphatidic acid (LPA) has been reported to be augmented in human synovial fluid from patients with RA. However, it remains to be elucidated whether LPA participates in cartilage destruction. In the present study, we have demonstrated that the production of promatrix metalloproteinases (proMMPs)-1 and -3 was augmented along with an increase of extracellular signal-regulated kinase (ERK)1/2 phosphorylation through LPA receptor 1 (LPAR1) in human synovial fibroblasts. These results suggest that LPA transcriptionally increases MMP production by the activation of an LPAR1/ERK1/2 signal pathway in human synovial fibroblasts. Thus, LPA is likely to be a pathological candidate for cartilage degradation in RA.

Construction of DNAzyme-Encapsulated Fibermats Using the Precursor Network Polymer of Poly(γ-glutamate) and 4-Glycidyloxypropyltrimethoxysilane.

Here, we developed functional nucleic acid (FNA)-encapsulated electrospun fibermats. To facilitate stable FNA encapsulation in the γ-PGA/GPTMS fibermats, we used the FNA as an FNA/streptavidin complex, and as a representative FNA, we selected a DNAzyme, the DNA/hemin complex, which is composed of G-quadraplex-forming single-stranded DNA and hemin and exhibits oxidation activity with the aid of a cocatalyst, H2O2. Scanning electron microscopy and Fourier-transform infrared spectroscopy measurements revealed that encapsulation of the DNA/hemin complex (∼1 wt % against the γ-PGA/GPTMS hybrid) in the nanofibers of the γ-PGA/GPTMS fibermats did not affect the structure of the original nanofibers. However, because a unique MW-dependent molecular permeability originated from the 3D network structure of the γ-PGA/GPTMS hybrid, low-MW substrates such as 4-aminoantipyrine, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline, and luminol were able to reach the encapsulated DNA/hemin complex by permeating to the inside of the nanofibers from an immersion buffer and then underwent catalytic oxidation. Conversely, nucleases, which are proteins featuring high MWs (>5 kDa), could not penetrate the γ-PGA/GPTMS nanofibers, and the encapsulated DNA/hemin complex was therefore effectively protected against nuclease digestion. Thus, encapsulating FNAs on the inside of the nanofibers of fibermats offers clear advantages for the practical application of FNAs in sensors and drugs, particularly for use in the in vivo circumstances.

Construction and Characterization of Protein-Encapsulated Electrospun Fibermats Prepared from a Silica/Poly(γ-glutamate) Hybrid.

Protein-encapsulated fibermats are an attractive platform for protein-based bioactive materials. However, the choice of methods is still limited and not applicable to a wide range of proteins. In this study, we studied new polymeric materials for constructing protein-encapsulated fibermats, in which protein molecules are encapsulated within the nanofibers of fibermats without causing deleterious changes to protein structure or function. We constructed a protein-encapsulated fibermat using the poly(γ-glutamate) (PGA)/(3-glycidyloxypropyl)-trimethoxysilane (GPTMS) hybrid as a precursor for electrospinning. Because the PGA/GPTMS hybrid is water-soluble, protein molecules can be added to the precursor in an aqueous solution, significantly enhancing protein stability. Polycondensation during electrospinning (in-flight polycondensation) makes the obtained fibermats water-insoluble, which stabilizes the fibermat structure such that it is resistant to degradation in aqueous buffer. The molecular structure of the PGA/GPTMS hybrid gives rise to unique molecular permeability, which alters the selectivity and specificity of biochemical reactions involving the encapsulated enzymes; lower molecular-weight (MW) substrates can permeate the nanofibers, promoting enzyme activity, but higher MW substrates such as inhibitor peptides cannot permeate the nanofibers, suppressing enzyme activity. We present an effective method of encapsulating bioactive molecules while maintaining their structure and function, increasing the versatility of electrospun fibermats for constructing various bioactive materials.

Nicotinic acetylcholine receptors regulate type 1 inositol 1,4,5-trisphosphate receptor expression via calmodulin kinase IV activation.

Type 1 inositol 1,4,5-trisphosphate receptors (IP3 R-1) are among the important calcium channels regulating intracellular Ca(2+) concentration in the central nervous system. In a previous study, we showed that drugs of abuse, such as cocaine, methamphetamine, and ethanol, induced IP3 R-1 upregulation via the calcium signal transduction pathway in psychological dependence. Although nicotine, a major component in tobacco smoke, participates in psychological and/or physical dependence, it has not yet been clarified how nicotine alters IP3 R-1 expression. The present study, therefore, seeks to clarify the mechanism bgy which nicotine modifies IP3 R-1 expression by using mouse cerebral cortical neurons in primary culture. Nicotine induced dose- and time-dependent upregulation of IP3 R-1 protein following its mRNA increase, and the latter was significantly suppressed by a nonselective nicotinic acetylcholine receptors (nAChR) antagonist, mecamylamine. Both cFos and phosphorylated-cJun (p-cJun) were immediately increased in the nucleus, together with an increase of calmodulin kinase (CaMK) IV but not CaMKII expression after nicotine exposure. A nonselective inhibitor of CaMKs, KN-93, and a calcium chelating regent, BAPTA-AM, completely suppressed the expression of cFos and p-cJun in the nucleus as well as the nicotine-induced IP3 R-1 upregulation. These results indicate that nAChR activation by nicotine upregulates IP3 R-1 via increase of activator protein-1, which is a cFos and cJun dimmer, in the nucleus, with activation of Ca(2+) signaling transduction processes.

[Role of intracellular Ca2+ dynamics in the development of drug dependence--Participation of Inositol 1,4,5-trisphosphate receptors].

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are classified to a multigene family of channel proteins that mediate Ca2+ release from endoplasmic reticulum, and are one of regulators to modify intracellular Ca2+ concentration. Little is known about functional relationship between rewarding effects due to drugs of abuse and IP3Rs. This report reviews the roles and regulatory mechanisms of intracellular Ca2+ channels, especially type 1 IP3Rs (IP3Rs-1), in brain of animals with rewarding effects produced by drugs of abuse. Our recent studies have reported that the blockade of IP3Rs suppresses the development of rewarding effects on methamphetamine or cocaine, suggesting that functional up-regulation of IP3R-1 occurs during the development of rewarding effects. Moreover, the critical expression of IP3R-1 in the development of methamphetamine- and cocaine-induced rewarding effects are regulated by Ca2+ participating in signal transduction pathways via both dopamine D1 and D2 receptors. Taken together these results it is suggested that the changes in IP3R-1 play an essential role in the development of drug dependence.

Sensitization of ethanol-induced place preference as a result of up-regulation of type 1 inositol 1,4,5-trisphosphate receptors in mouse nucleus accumbens.

This study involved mice that received 4 days of ethanol (EtOH) vapor inhalation and then were assessed for type 1 inositol 1,4,5-trisphosphate receptor (IP3 Rs-1) expression and the development of EtOH-induced place preference at various time points in withdrawal. IP3 R-1 protein was found to be significantly increased in the nucleus accumbens (NAcc) of mice immediately after 4-day EtOH vapor inhalation, while it significantly reduced to the control level during the next 3 days of withdrawal from EtOH inhalation. EtOH (2 g/kg, i.p.)-induced place preference after 3 days of withdrawal from EtOH vapor inhalation increased dose dependently for 4 days, which was significantly inhibited by 2-aminophenoxyethane-borate, an antagonist for IP3 Rs. EtOH conditioning significantly increased, compared to alcohol-naïve control mice, both IP3 R-1 protein and the release of dopamine in the NAcc of mice after 3 days of withdrawal from EtOH vapor inhaled for 4 days, and this increase of IP3 R-1 protein was completely abolished by intracerebroventricular injection of FK506, an inhibitor for calcineurin. These results indicate that the sensitization of EtOH-induced place preference is due to up-regulated IP3 R-1 via calcineurin-mediated pathway after enhanced release of dopamine in the NAcc on EtOH administration during EtOH conditioning. We revealed signal transduction pathways that may promote sensitization of ethanol (EtOH)-induced place preference. EtOH facilitated the release of dopamine (DA) in the Nucleus accumbens (NAcc), enhancing calcineurin function via dopamine D1-like and D2-like receptor activation, which in turn resulted in increased NFATc4 expression. Increase in NFATc4 may further facilitate transcription factor binding to IP3 R-1 promoter domain to stimulate IP3 R-1 synthesis. Such increased IP3 R-1 elevates intracellular Ca(2+) concentration via facilitated mobilization of Ca(2+) from the intracellular Ca(2+) stores to the cytosol.

Increase of type 2 ryanodine receptors in mouse nucleus accumbens in the development and expression of morphine-induced place preference.

The present study investigated the effect of ryanodine receptors (RyRs) in the development and expression of morphine-induced conditioned place preference (CPP). Type 2 RyRs (RyRs-2) in the nucleus accumbens (NAcc) significantly increased in morphine-conditioned mice, whereas type 1, 2, and 3 RyRs in the frontal cortex and ventral tegmental area showed no changes. Intracerebroventricular pretreatment with dantrolene, a RyRs antagonist, during the conditioning phase of CPP, dose-dependently inhibited morphine-induced CPP. The expression of morphine-induced CPP was abolished by dantrolene administration before the post-conditioning test. These findings suggest that RyRs-2 in the NAcc participate in the development and expression of morphine-induced CPP.

[Role of alteration in intracellular Ca2+ dynamics in the development of drug dependence].

Ca2+ influx into neuron through L-type voltage-gated Ca2+ channels (VDCCs) plays an important role in psychostimulant-induced behaviroal and neuronal plasticity. On the other hand, Ca2+ release from ryanodine receptors in the endoplasmic reticulum is one mechanism altering the intracellular Ca2+ concentration. Little is known about functional relationship between psychological dependence due to drugs of abuse and L-type VDCCs or ryanodine receptors. In this paper, we review the roles and regulatory mechanisms of intracellular Ca2+ channels, especially L-type VDCCs and ryanodine receptors in brain of animals with drug dependence. Our recent study have reported that blockade of L-type VDCCs inhibits the development of rewarding effects on drugs of abuse (methamphetamine, cocaine and morphine), suggesting that up-regulation of L-type VDCCs (alpha 1C and alpha 1D subunits) occurs during the development of psychological dependence. Moreover, the critical expression of type-1 and -2 ryanodine receptors in the development of methamphetamine- and cocaine-induced rewarding effects are regulated by dopamine D1 receptors. These results suggest that changes in intracellular Ca2+ dynamics play an essential role in the development of drug dependence.

Finite element analysis of hip fracture risk in elderly female: The effects of soft tissue shape, fall direction, and interventions.

This study investigates the effects of fall configurations on hip fracture risk with a focus on pelvic soft tissue shape. This was done by employing a whole-body finite element (FE) model. Soft tissue thickness around the pelvis was measured using a standing CT system, revealing a trend of increased trochanteric soft tissue thickness with higher BMI and younger age. In the lateroposterior region from the greater trochanter, the soft tissues of elderly females were thin with a concave shape. Based on the THUMS 5F model, an elderly female FE model with a low BMI was developed by morphing the soft tissue shape around the pelvis based on the CT data. FE simulation results indicated that the lateroposterior fall led to a higher femoral neck force for the elderly female model compared to the lateral fall. One reason may be related to the thin soft tissue of the pelvis in the lateroposterior region. Additionally, the effectiveness of interventions that can help mitigating hip fractures in lateroposterior falls on the thigh-hip and hip region was assessed using the elderly female model. The attenuation rate of the femoral neck force by the hip protector was close to zero in the thigh-hip fall and high in the hip fall, whereas the attenuation rate of the compliant floor was high in both falls. This study highlights age-related changes in the soft tissue shape of the pelvis in females, particularly in the lateroposterior regions, which may influence force mitigation for the hip joint during lateroposterior falls.

The influence of seated postures and anthropometry on lap belt fit in vehicle occupants: A 3D computed tomography study.

OBJECTIVE: In vehicle frontal collisions, it is crucial that the lap belt is designed to engage with the anterior superior iliac spine (ASIS) of occupants for a reliable restraint. This study aims to understand the influence of different seated postures on the geometrical relationship of the seat belt and the pelvis for various occupants using 3D upright and supine computed tomography (CT) systems. METHODS: The 3D shapes of bones and soft tissues around the pelvis were acquired through a CT scan for 30 participants. They were seated in a rigid seat equipped with a lap belt simulating the front seat of a small car, and wore a lap belt in three seated postures: upright, slouched and reclined. Parameters related to the likelihood of submarining occurrences, such as belt-ASIS overlap (an index for assessing the potential engagement of the lap belt with the ASIS) and the belt-pelvis angle (the difference between the belt angle and the normal direction of the anterior edge of the ilium) were compared. RESULTS: It was observed that the pelvis angle tilted rearward as the hip point was positioned forward and seatback angle increased. This can be seen in the slouched and reclined posture. The belt-pelvis angle was comparable between the slouched and the reclined postures, and was closer to zero (indicating that the lap belt path is closer to perpendicular to the anterior edge of the ilium) compared to the upright posture. In contrast, the belt-ASIS overlap increased with an increasing flesh margin of the ASIS and shallower belt angle. This suggests that the belt-pelvis angle is influenced by the seated posture whereas the belt-ASIS overlap is dependent more on an individual's anthropometry. The plot of belt-pelvis angle and belt-ASIS overlap exhibited significant variability among participants. CONCLUSIONS: The belt-pelvis angle and the belt-ASIS overlap of individuals will provide valuable information for understanding the current belt-fit location and predicting submarining occurrences for individuals in various postures when designing restraint systems.

Predictive modeling of submarining risk in car occupants based on pelvis angle and lap belt positioning.

OBJECTIVE: The engagement of the lap belt with the pelvis is critical for occupant safety during vehicle frontal crashes to prevent occupant submarining. This study aims to develop a predictive model for submarining risk based on anthropometric parameters and lap belt positioning using finite element (FE) analyses. METHODS: FE analyses were conducted using human body models representing various body shapes (a 50th percentile male, low and high BMI males, and a 5th percentile female) in three seated postures (standard, reclined, and slouched). The lap belt-ASIS overlap and the belt-pelvis angle were used as key parameters for predicting submarining risk. A logistic regression analysis was utilized to correlate submarining occurrence with the initial values of these two parameters at the beginning of impact. Subsequently, this submarining prediction model was applied to computer tomography (CT) measurements of human subjects in different seated postures (upright, reclined, and slouched), and submarining risks were calculated based on the developed model. RESULTS: FE simulations indicated that submarining was more likely to occur as the initial belt-pelvis angle approached zero and there was a smaller initial belt-ASIS overlap. The logistic regression analysis demonstrated that the initial belt-pelvis angle and belt-ASIS overlap were statistically significant for predicting submarining risk. The derived model effectively distinguished submarining occurrence based on the initial values of these two parameters. The application of the submarining model to CT measurements of human subjects showed that submarining risk was lower in the order of upright, slouched, and reclined postures. In the reclined posture, the high submarining risk was attributed to a small belt-ASIS overlap and a rearward-tilted pelvis angle; whereas in the slouched posture, the risk was mostly associated with a rearward-tilted pelvis angle. CONCLUSIONS: The submarining prediction model was developed based on the belt-pelvis angle and the belt-ASIS overlap. This predictive model may help to design restraint systems for various body types and seated postures of occupants.

Activation of GABA(A) receptors suppresses ethanol-induced upregulation of type 1 IP(3) receptors.

Although Type 1 inositol 1,4,5-trisphosphate receptors (IP(3) Rs-1) are one of the major calcium channels to regulate intracellular Ca(2+) concentration, there have been few available data how their expression is modified by long-term exposure to ethanol. The present study attempted to clarify mechanisms of modification of IP(3) R-1 expression during long-term ethanol exposure by γ-aminobutyric acid (GABA)A receptors using mouse cerebral cortical neurons. Long-term exposure to ethanol induced IP(3) R-1 protein upregulation following increased expression of its mRNA. Pretreatment with muscimol, a selective GABA(A) receptor agonist, significantly suppressed the ethanol-induced upregulation of IP(3) R-1 protein and its mRNA, which was significantly abolished by bicuculline, a selective GABA(A) receptor antagonist. These results indicate that GABA(A) receptors negatively regulate the ethanol-induced upregulation of IP(3) R-1 protein expression via the suppression of gene transcription.

Dopamine D1 receptor signaling system regulates ryanodine receptor expression in ethanol physical dependence.

BACKGROUND: Ryanodine receptors (RyRs) amplifying activity-dependent calcium influx via calcium-induced calcium release play an important role in central nervous system functions including learning, memory, and drug abuse. In this study, we investigated the role and the regulatory mechanisms of RyR expression under continuous exposure of mice to ethanol (EtOH) vapor for 9 days. METHODS: The model of EtOH physical dependence was prepared as follows: 8-week-old male ddY mice were exposed to EtOH vapor for 9 days. Protein and mRNA of RyR-1, RyR-2, and RyR-3 in the frontal cortex and limbic forebrain were determined by Western blot and real-time RT-PCR analysis, respectively. RESULTS: Exposure of mice to EtOH vapor for 9 days induced significant withdrawal signs when estimated with withdrawal score, which was dose-dependently suppressed by intracerebroventricular administration of dantrolene, an RyR antagonist. Protein levels of RyR-1 and RyR-2 in the frontal cortex and limbic forebrain significantly increased during EtOH vapor exposure for 9 days with increased expression of their mRNA, whereas that of RyR-3 in these 2 brain regions showed no changes. Increased proteins and mRNA of RyR-1 and RyR-2 were completely abolished by SCH23390, a selective antagonist of dopamine D1 receptors (D1DRs), but not by sulpiride, a selective antagonist of D2DRs. CONCLUSIONS: RyRs play a critical role in the development of EtOH physical dependence and that the up-regulation of RyRs in the brain of mouse, showing EtOH physical dependence is regulated by D1DRs.

Regulatory mechanisms and pathophysiological significance of IP3 receptors and ryanodine receptors in drug dependence.

Calcium is a ubiquitous intracellular signaling molecule required for initiating and regulating neuronal functions. Ca(2+) release from intracellular stores in the endoplasmic reticulum into intracellular spaces via intracellular Ca(2+)-releasing channels, inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs), is one mechanism altering the intracellular Ca(2+) concentration. Functional abnormalities in endoplasmic calcium channels can disturb cellular calcium homeostasis and, in turn, produce pathological conditions. Indeed, our recent studies have indicated the involvement of these upregulated calcium channels in development of the rewarding effect of a drug of abuse and the suppression of its rewarding effect by calcium-channel inhibitors, which suggests a possible functional relationship between intracellular dynamics and the development of the rewarding effects induced by an abused drug. Although previous reports showed that the most important regulators of both RyR and IP3R channel functions are changes in the intracellular Ca(2+) concentration and in phosphorylation of these channels by numerous kinases and calcium modulators, little information is available to clarify how the expression of intracellular calcium channels is regulated. In this review, we therefore introduce the roles and regulatory mechanisms of intracellular calcium channels in drug dependence, especially in the rewarding effect induced by the abused drug.

Acamprosate suppresses ethanol-induced place preference in mice with ethanol physical dependence.

The present study investigated the effect of acamprosate on ethanol (EtOH)-induced place preference in mice with EtOH physical dependence. The expression of EtOH (2 g/kg, intraperitoneally)-induced place preference in mice without EtOH treatment before the experiment was dose-dependently suppressed by acamprosate. The levels of protein kinase A (PKA) and phospho-cAMP response element binding protein (p-CREB) in the limbic forebrain after EtOH-conditioning in naïve mice was unchanged. Furthermore, mice on the 4th day of withdrawal from continuous EtOH vapor inhalation for 9 days showed transient and significant enhancement of EtOH (1 g/kg, intraperitoneally)-induced place preference, which was significantly suppressed by acamprosate (300 mg/kg, oral administration; p.o., once a day) administered daily for 3 days after withdrawal from EtOH inhalation and during EtOH-conditioning. PKA and p-CREB proteins in the limbic forebrain of EtOH-conditioned mice on 4th day of withdrawal from continuous EtOH inhalation for 9 days significantly increased, which were completely abolished by acamprosate. These findings suggest that the signal transduction pathway via the PKA-p-CREB pathway in the limbic forebrain may be functionally related to the development of sensitization of EtOH-induced place preference and provide a possible molecular basis for the pharmacological effect of acamprosate to prevent or reduce the relapse of alcohol dependence.

Severity of child pedestrian injuries due to bonnet-type-vehicle collision.

BACKGROUND: The aim of this study was to clarify the pattern of child pedestrian injury, injury severity, and its relation to collision velocity in bonnet-type-vehicle collision. METHODS: In-depth data were retrospectively collected from the Institute for Traffic Accident Research and Data Analysis on pedestrians younger than 13 years old with any bodily injuries from collisions with bonnet-type vehicles between 1993 and 2004. RESULTS: Forty-seven patients from 43 collisions with a mean age of 6.9 ± 2.5 years were included in the study. Injury severity was not significantly different between patients who were hit by the front of the vehicle and those who were hit by the side of the vehicle. In front collisions, impact with the vehicle was associated with significantly higher Abbreviated Injury Scale (AIS) scores than those for impact with the road, especially for the lower extremities (mean: 1.2 vs 0.2, P < 0.001). Injury severity of the lower extremities and collision velocity were examined. The estimated collision velocity of the vehicle was not significantly different between patients with lower extremity AIS scores of 0 or 1 and those of 2 or 3. CONCLUSIONS: Some pediatric pedestrians suffer from collisions with bonnet-type vehicles without lower extremity fractures owing to the characteristics of child pedestrians. Providing injury prevention programs for children in communities and schools, developing active safety devices in the vehicle, and modifying the vehicle body to a pediatric pedestrian-friendly structure may increase pedestrian protection.

[Mechanisms of ethanol-induced type I IP3 receptor expression].

Ethanol has a variety of action on neuronal functions, though its mechanism of action remains uncertain. Previous investigations have demonstrated functional alteration of neurotransmitter receptors and ion channels by ethanol at its concentration observed in the blood of alcoholics. Our recent studies have shown that chronic ethanol treatment up-regulates high voltage-gated L-type calcium channels and ryanodine receptors, both of which regulate intracellular Ca2+ concentration, and that the up-regulation of these calcium channels participates in behavioral changes including the rewarding effect. Among inositol 1,4,5-trisphosphate receptors (IP3Rs) classified into three different subtypes (type 1 (IP3R-1), type 2, and type 3 IP3Rs) with distinct physiological properties, IP3R-1 is the major neuronal member in the central nervous system, predominantly enriched in cerebellar Purkinje cells and abundant in neurons in the cerebral cortex. Although the most important result of IP3R channel functions is the change in intracellular Ca2+ concentration and phosphorylation of IP3Rs, there are few available data on ethanol effect on IP3Rs. In this report, we demonstrate the functional relationship between ethanol-induced rewarding effect and IP3R-1 expression and regulatory mechanisms of IP3R-1 expression after chronic ethanol exposure, especially focusing on Ca(2+)-related signal transduction pathways via dopamine D1 receptors using mouse cerebral cortical neurons.

Dopamine D1 receptors regulate type 1 inositol 1,4,5-trisphosphate receptor expression via both AP-1- and NFATc4-mediated transcriptional processes.

Although our recent report demonstrates the essential involvement of up-regulation of a regulator of intracellular Ca(2+) concentration, type 1 inositol 1,4,5-trisphosphate receptors (IP(3) Rs-1), mediated via dopamine D1-like receptor (D1DR) stimulation in the cocaine-induced psychological dependence, the exact mechanisms of regulation of IP(3) R-1 expression by D1DRs have not yet been clarified. This study attempted to clarify these mechanisms using mouse cerebral cortical neurons. An agonist for phosphatidylinositide-linked D1DRs, SKF83959, induced dose- and time-dependently IP(3) R-1 protein up-regulation following its mRNA increase without cAMP production. U73122 (a phospholipase C inhibitor), BAPTA-AM (an intracellular calcium chelating reagent), W7 (a calmodulin inhibitor), KN-93 (a calmodulin-dependent protein kinases inhibitor), and FK506 (a calcineurin inhibitor), significantly inhibited the SKF83959-induced IP(3) R-1 up-regulation. Furthermore, immunohistochemical examinations showed that SKF83959 increased expression of both cFos and cJun in nucleus as well as enhanced translocation of both calcineurin and NFATc4 complex to nucleus from cytoplasm. In addition, SKF83959 directly recruited binding of both AP-1 and NFATc4 to IP(3) R-1 promoter region. These results indicate that D1DR activation induces IP(3) R-1 up-regulation via increased translocation of AP-1 as well as NFATc4 in Gαq protein-coupled calcium signaling transduction pathway.