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田中治教授 古稀記念論集, Dedicated to Professor Osamu Tanaka on his 70th birthday, Article

Genetically-encoded calcium indicators such as G-GECO are useful for studying Ca2+ responses during long-term processes. In this study, we employed a lentiviral vector and established a rat dental epithelial cell line that stably expressed G-GECO (SF2-G-GECO). Ca2+ imaging analysis under cell culture conditions revealed that SF2-G-GECO cells exhibited spontaneous Ca2+ responses, which could be classified into the following three major patterns depending on the cell density: localized Ca2+ responses at cell protrusions at a low density, a cell-wide spread of Ca2+ responses at a medium density, and Ca2+ responses in clusters of 3-20 cells at a high density. The P2Y receptor inhibitor suramin (10 μM), the ATP-degrading enzyme apyrase (5 units/mL), and the fibroblast growth factor (FGF) receptor inhibitor FIIN-2 (1 μM) decreased the frequency of spontaneous Ca2+ responses. These results indicate that ATP and FGF are involved in the spontaneous Ca2+ responses. SF2 cells differentiate into ameloblasts via interactions with mesenchymal cells. Therefore, SF2-G-GECO cells are expected to be a useful tool for studying the functions of Ca2+ responses in regulating gene expression during tooth development.

An 84-year-old man developed motor aphasia and right hemiparesis on postoperative day 1 after orchiectomy for suspected malignant lymphoma. He had a history of thoracic endovascular aortic repair for aortic aneurysm using a bypass graft from the right subclavian artery to the left common carotid artery (CCA); however, the graft had become occluded six months later. Brain magnetic resonance imaging revealed acute cerebral infarctions in the left frontal lobe. Carotid ultrasonography revealed a stump at the left CCA, just below the bifurcation, formed by the occluded graft with an oscillating thrombus. This case was rare in that a CCA stump was identified as the embolic source of ischemic stroke.

57-year-old woman with sequelae of cerebral infarction was admitted to our hospital because her left-sided hemiparesis was worsened. The right internal carotid artery (ICA) was not visualized by carotid duplex sonography and brain MRA. Arterial spin labeling (ASL) perfusion MR images showed reduced signals in the bilateral ICA territories at post labeling delay 1,525 ms. Her neurological symptoms improved on the day after hospitalization. On day 3, the bilateral ICAs were well visualized on MRA, while cerebral perfusion in the ICA territories appeared to be normalized on ASL. We diagnosed cervical ICA vasospasm, based on the findings of cervical MRA and cerebral angiography. Three months later, the recurrence of ICA vasospasm occurred. ASL was useful for the serial non-invasive evaluation of cerebral hemodynamics from the onset to improvement in a patient with ICA vasospasm.

Near-infrared photoimmunotherapy (NIR-PIT) is a promising cancer therapy based on a monoclonal antibody conjugated to a photosensitizer (IR700Dye) that is activated by near-infrared light irradiation. We previously reported on the use of NIR-PIT with a small protein mimetic, the Affibody molecule (6–7 kDa), instead of a monoclonal antibody. In this study, we investigated a combination of NIR-PIT for HER2-positive breast cancer cells (SK-BR3, MDA-MB361, and JIMT1) with HER2 Affibody-IR700Dye conjugate and trastuzumab-IR700Dye conjugate. HER2 Affibody and trastuzumab target different epitopes of the HER2 protein and do not compete. In vitro, the combination of NIR-PIT using both HER2 Affibody-IR700Dye conjugate and trastuzumab-IR700Dye conjugate induced necrotic cell death of HER2-positive breast cancer cells without damage to HER2-negative breast cancer cells (MCF7). It was more efficient than NIR-PIT using either the HER2 Affibody-IR700Dye conjugate alone or the trastuzumab-IR700Dye conjugate alone. Additionally, this combination of NIR-PIT was significantly effective against HER2 low-expressing cancer cells, trastuzumab-resistant cells (JIMT1), and brain metastatic cells of breast cancer (MDA-MB361). Furthermore, in vivo imaging exhibited the strong fluorescence intensity of both HER2 Affibody-IR700Dye conjugates and trastuzumab-Alexa488 conjugates in HER2-positive tumor, indicating that both HER2 Affibody and trastuzumab specifically bind to HER2-positive tumors without competing with each other. In conclusion, the combination of NIR-PIT using both HER2 Affibody and trastuzumab expands the targeting scope of NIR-PIT for HER2-positive breast cancer.

Oral dryness as a side effect of certain drugs is increasing. The aim of this study was to examine the change of the protein ingredient in saliva of oral dryness patients caused by calcium blocker. Six patients taking calcium blocker and six healthy elderly were enrolled. Unstimulated salivary flow rate, protein concentration, and flow rate of protein were measured and compared between the patients taking calcium blocker and healthy elderly. iTRAQ (Isobaric Tag for Relative and Absolute Quantitation) proteomic analysis was performed to extract the salivary protein changed in patient taking calcium blocker, and the intensities of Western blotting products were quantified (unpaired t-test). Unstimulated salivary flow rate was significantly lower on patients taking calcium blocker (p &lt, 0.01). Protein concentration tended to be higher and the flow rate of protein tended to be lower on patients. As the result of iTRAQ proteomic analysis, calmodulin-like protein 3, glutathione S-transferase P, and keratin type I cytoskeletal 13 increased characteristically in patient taking calcium blocker, and the expression in calmodulin-like protein 3 was significantly larger (p &lt, 0.01). The results of this study indicated that calmodulin-like protein 3 increased in patients taking calcium blocker and could be a salivary biomarker for oral dryness caused by calcium blocker.

Early detection of oral disease is important to reduce its severity and increase the likelihood of successful treatment. This study aimed to perform a quantitative assessment of the saliva components as a first stage of the research to screen oral homeostasis. Here, saliva secretions collected from children were evaluated, and their constituents were analyzed to investigate the potential correlations between the buffering capacity and a range of salivary factors. Subjects aged 3-16 years in the primary, mixed, or permanent dentition stage, were selected for this study. The following salivary factors were analyzed: flow rate, total protein, total sugar quantifications, and constituent analyses using RT-PCR and western blotting. The associations between each factor and the buffering capacity were then analyzed using multiple regression analysis. Flow rate, BPIFA2 RNA level, histatin 1 and BPIFB1 protein levels as well as female sex were positively associated with buffering capacity. In contrast, total sugar concentration and MUC7 RNA levels showed a negative relationship with the buffering capacity. Some of these constituents may indicate oral homeostasis and are therefore potential biomarkers of oral health status. These results suggest that the analyses of the correlations between oral homeostasis and salivary factors are an effective strategy for identifying the susceptibility to oral diseases.

New findings What is the central question of this study? The effects of Ca2+ responses on salivary fluid secretion have been studied indirectly by monitoring ion channel activities and other indices. Therefore, Ca2+ responses during salivary secretion remain poorly understood. What is the main finding and its importance? Herein, we developed a simultaneous monitoring system for Ca2+ responses and salivary secretion in live animals using a YC-Nano50-expressing submandibular gland and a fibre-optic pressure sensor. This new approach revealed a clear time lag between the onset of Ca2+ responses and salivary secretion. We also estimated the [Ca2+ ]i and provided direct evidence for the regulation of salivary secretion by small increases in [Ca2+ ]i in submandibular gland acinar cells. Abstract We monitored changes in [Ca2+ ]i during salivary secretion in the rat submandibular gland in live animals using a combination of intravital Ca2+ imaging with the ultrasensitive Ca2+ indicator YC-Nano50 and a fibre-optic pressure sensor. Intravenous infusion of ACh (10-720 nmol min-1 ) increased [Ca2+ ]i and salivary flow rate in a dose-dependent manner. Repetitive stimulation with ACh induced equivalent Ca2+ responses and salivary secretion in the same individual animals. The accurate ACh stimulation experiments revealed a clear time lag between the onset of the increase in [Ca2+ ]i and salivary secretion. The time lag with the lowest dose of ACh (30 nmol min-1 ) was 106 s, which shortened to 19 s with the dose used for maximal salivary secretion (360 nmol min-1 ). This time lag might reflect the time required for [Ca2+ ]i to reach the level required to activate molecules for fluid secretion. The resting [Ca2+ ]i in submandibular gland was 37 nm, and [Ca2+ ]i at the onset of salivary secretion was 45-57 nm, irrespective of ACh dose. These results indicate that low [Ca2+ ]i is sufficient to trigger fluid secretion in the rat submandibular gland in vivo.

Inositol 1,4,5-trisphosphate (IP3) is an important intracellular messenger produced by phospholipase C via the activation of G-protein-coupled receptor- or receptor-tyrosine-kinase-mediated pathways, and is involved in numerous responses to hormones, neurotransmitters, and growth factors through the releases of Ca2+ from intracellular stores via IP3 receptors. IP3-mediated Ca2+ signals often exhibit complex spatial and temporal organizations, such as Ca2+ oscillations. Recently, new methods have become available to measure IP3 concentration ([IP3]) using AlphaScreen technology, fluorescence polarization, and competitive ligand binding assay (CFLA). These methods are useful for the high throughput screening in drug discovery. Calcium ions generate versatile intracellular signals such as Ca2+ oscillations and waves. Fluorescent sensors molecules to monitor changes in [IP3] in single living cells are crucial to study the mechanism for the spatially and temporally regulated Ca2+ signals. In particular, FRET-based IP3 sensors are useful for the quantitative monitoring intracellular [IP3], and allowed to uncovered the oscillatory IP3 dynamics in association with Ca2+ oscillations. A mathematical model of coupled Ca2+ and IP3 oscillations predicts that Ca2+ oscillations are the result of modulation of the IP3 receptor by intracellular Ca2+, and that the period is modulated by the accompanying IP3 oscillations. These model predictions have also been confirmed experimentally. At present, however, usefulness of FRET-based IP3 sensors are limited by their relatively small change in fluorescence. Development of novel IP3 sensors with improve dynamic range would be important for understanding the regulatory mechanism of Ca2+ signaling and for in vivo IP3 imaging.

A 45-year old man with untreated diabetes mellitus (HbA1c 11.0%) was admitted with headache and left limb weakness. Findings of diffusion-weighted and FLAIR MR images of the brain were unremarkable. However, cortical vein dilation and occlusion of the upper sagittal sinus were visualized on T2* and magnetic resonance venography images, respectively. Perfusion CT revealed increased mean transit-time in the right frontal lobe. Cerebral venous thrombosis was diagnosed and treated with intravenous heparin. The neurological symptoms disappeared on post onset day (POD) 8. Contrast CT on POD 13 revealed sagittal sinus recanalization and he was discharged 10 days later. Perfusion CT helped to identify cerebral venous thrombosis that might have been associated with untreated diabetes mellitus.

Based on the results of our previous adenophostin A structure-activity relationship studies, two fluorescent inositol 1,4,5-trisphosphate (IP3 ) receptor ligands, fluorescent adenophostin A (FADA) and fluorescent low-affinity ligand (FLL), were designed. Binding of the fluorescent ligands to the fluorescent IP3 sensor in protein-expressing cells caused FRET. This principle was extended to a cell-free assay system by using the fluorescent IP3 sensor bound to agarose beads. The effect of FLL on the FRET signal was reduced by subsequent addition of IP3 . The IC50 values of IP3 on the FRET signals were 139.7 and 352.1 nm for 30 and 100 nm FLL, respectively. This method allowed quantitative measurement of IP3 concentrations below 10 nm and was applied to measure cytosolic IP3 concentrations in COS-7 cells and to examine the potency of synthesized adenophostin A analogues.

Genetically encoded calcium indicators (GECIs) are suitable for long-term imaging studies. In this study, we employed a highly sensitive GECI, G-GECO, and achieved efficient gene delivery with an adenoviral vector. The adenoviral vector allowed us to express G-GECO in more than 80% of cells. More than 80% of G-GECO-expressing cells showed an ATP-induced increase in fluorescence intensity due to Ca2+ release from intracellular stores and subsequent Ca2+ entry. The fluorescence intensity of these cells was increased more than 2-fold by stimulation with 10 μM ATP. We applied long-term imaging (for ~10 h) to monitor Ca2+ responses in SF2, a rat dental epithelial cell line, in culture conditions. SF2 cells showed intermittent rises in the intracellular Ca2+ concentration in the presence of 100 nM 1,25-dihydroxyvitamin D3. Many of these Ca2+ responses began at a specific location in the cytoplasm and spread throughout the entire cytoplasm. The combination of efficient gene delivery with an adenoviral vector and long-term imaging with a highly sensitive GECI enabled detection of intermittent Ca2+ responses that occur only 3-10 times/h/100 cells. This method could be useful to study the effects of Ca2+ responses for regulating longterm processes, such as gene expression, cell migration, and cell division, in many cell types.

Whether idarucizumab, an antidote of dabigatran, can be used effectively and safely before thrombolytic therapy with recombinant tissue plasminogen activator (rt-PA) in patients with stroke undergoing treatment with dabigatran remains unknown. We herein describe a 57-year-old man who developed severe cardioembolic stroke with a National Institutes of Health Stroke Scale score of 22 in the left middle cerebral artery territory while undergoing treatment with dabigatran for nonvalvular atrial fibrillation and who was treated with rt-PA after the reversal of dabigatran with idarucizumab. The thrombolytic therapy following the use of idarucizumab significantly improved the patient's neurological symptoms without hemorrhagic complications, although acute arterial occlusion of the right lower limb was found during the clinical course.

What is the central question of this study? The effects of CaWe monitored changes in [Ca

New Findings What is the central question of this study? Pilocarpine stimulates salivary secretion via muscarinic ACh receptors (mAChRs), although the Ca2+-mobilizing effect of pilocarpine in salivary gland cells is extremely small. Therefore, we examined the effect of pilocarpine on Ca2+ responses in submandibular gland cells and on secretion in vitro and in vivo. What is the main finding and its importance? Pilocarpine induces small Ca2+ responses and reduces the effects of other mAChR agonists on Ca2+ responses via its partial agonistic effects. These effects of pilocarpine on Ca2+ responses in the submandibular gland were further established in vivo with a novel Ca2+ imaging system and a genetically encoded Ca2+ indicator. Pilocarpine stimulates salivary secretion via muscarinic ACh receptors (mAChRs), although the effect of pilocarpine on Ca2+ responses in dispersed salivary gland cells is extremely small. Here, we demonstrate the effect of pilocarpine on Ca2+ responses and salivary secretion in the rat submandibular gland (SMG). In fura-2-loaded SMG cells, the maximal effect of pilocarpine on [Ca2+]i elevation was 16% of that of carbachol, and pilocarpine attenuated carbachol- and bethanechol (Bet)-induced [Ca2+]i increases, indicating that pilocarpine acts as a partial agonist for mAChR-mediated Ca2+ responses. The partial agonistic effect of pilocarpine on Ca2+ dynamics in the SMG was also confirmed in live animals using the genetically encoded Ca2+ indicator, YC-Nano50. Administration of pilocarpine (3 mg kg−1, i.p.) elicited a small increase in [Ca2+]i in the SMG. Quantitative analyses demonstrated that resting [Ca2+]i was ∼37 nm, which was increased by pilocarpine (3 mg kg−1) and Bet (10 mg kg−1) to 44 and 69 nm, respectively. The inhibitory effects of pilocarpine on Bet-induced Ca2+ responses were also elucidated in vivo. We further examined real-time changes in pilocarpine-induced SMG salivary secretion and showed that pilocarpine induced an extremely weak secretory response and reduced Bet-induced secretion. Unlike Ca2+ responses, pilocarpine failed to reduce the effect of Bet on SMG blood flow. Our results demonstrate that pilocarpine acts as a partial agonist of mAChRs to induce weak salivary secretion that is correlated with small increases in [Ca2+]i. Furthermore, pilocarpine exhibits an antagonistic effect on mAChR-induced Ca2+ responses and salivary secretion.

Background Salivary glands are comprised predominantly of two epithelial cell types, acinar and ductal cells. Activation of muscarinic acetylcholine receptors on acinar cells induces inositol 1,4,5-trisphosphate-mediated Ca 2+ release from intracellular Ca 2+ stores, followed by Ca 2+ influx across the plasma membrane. The increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ) activates ion channels and transporters involved in fluid secretion; thus [Ca 2+ ] i acts as the primary fluid secretion signal in acinar cells. Highlight The development of Ca 2+ imaging techniques has enabled extensive study of spatiotemporal Ca 2+ dynamics in isolated acinar cells; however, the relationship between Ca 2+ responses and fluid secretion in vivo is not yet fully understood. In vivo Ca 2+ imaging in salivary gland cells is necessary for determining Ca 2+ responses during salivary secretion. Recently, we developed an intravital imaging system for monitoring Ca 2+ responses in rat submandibular gland (SMG) acinar cells in live animals using retrograde ductal injection of adenoviral vectors expressing the genetically encoded Ca 2+ indicator, YC-Nano50. Expression of YC-Nano50 was observed throughout the SMG, and we confirmed that YC-Nano50 was functional for monitoring Ca 2+ responses in SMG cells. Administration of acetylcholine significantly increased [Ca 2+ ] i in the SMG, and this increase gradually decreased to resting levels. Conclusion We successfully visualized agonist-induced Ca 2+ dynamics in SMG acinar cells in live animals. This novel experimental system represents a powerful tool for understanding the relationships between Ca 2+ responses and salivary secretion in vivo .

判例研究(Case Study)

研究ノート(Note)

Genetically encoded calcium indicators (GECIs) are suitable for long-term imaging studies. In this study, we employed a highly sensitive GECI, G-GECO, and achieved efficient gene delivery with an adenoviral vector. The adenoviral vector allowed us to express G-GECO in more than 80% of cells. More than 80% of G-GECO-expressing cells showed an ATP-induced increase in fluorescence intensity due to Ca

Background and objective Amelogenins are major components of extracellular matrix proteins in developing teeth, and regulate the growth of enamel crystals. They also function as signaling molecules in cell differentiation. This study aimed to determine the biological effects of amelogenins on the differentiation of HAT-7 dental epithelial cells and MC3T3-E1 pre-osteoblastic cells using full-length recombinant human amelogenin (rh-AMEL). Design rh-AMEL was expressed in a mammalian cell line (Expi293F™) and was purified by DDK agarose beads. Effects of rh-AMEL on differentiation were evaluated by Mineralization and Alkaline phosphatase (ALP) activity using Alizarin Red S staining and colorimetric substrate p-nitrophenol, respectively. Results Western blotting and silver staining confirmed the successful purification of rh-AMEL. Mineralization and ALP activity in HAT-7 cells were significantly higher after treatment with 4 μg/mL rh-AMEL, but not after treatment with Emdogain ® (EMD). In MC3T3-E1 cells, on the other hand, rh-AMEL showed biphasic effects on differentiation. Treatment with low concentrations of rh-AMEL (0.001–0.1 μg/mL) and EMD (0.01–1 μg/mL) increased mineralization and ALP activity in MC3T3-E1 cells, whereas treatment with high concentrations of rh-AMEL (4 μg/mL) and EMD (100 μg/mL) had the opposite effect. Conclusion High concentrations of rh-AMEL and EMD decreased the differentiation of MC3T3-E1 cells. By contrast, a high concentration of rh-AMEL, but not that of EMD, promoted the differentiation of HAT-7 cells. This study demonstrates that the effects of rh-AMEL on cell differentiation differ between HAT-7 and MC3T3-E1 cells, and suggests that different regions on AMEL may induce the differentiation of these cell types.

Adenoviruses have been used for gene transfer to salivary gland cells in vivo . Their use to study the function of salivary acinar cells was limited by a severe inflammatory response and by the destruction of fluid-secreting acinar cells. In the present study, low doses of adenovirus were administered to express Stim1-mKO1 by retrograde ductal injection to submandibular glands. The approach succeeded in increasing muscarinic stimulation-induced Ca 2+ responses in acinar cells without inflammation or decreased salivary secretions. This increased Ca 2+ response was notable upon weak muscarinic stimulation and was attributed to increased Ca 2+ release from internal stores and increased Ca 2+ entry. The basal Ca 2+ level was higher in Stim1-mKO1-expressing cells than in mKO1-expressing and non-expressing cells. Exposure of permeabilized submandibular acinar cells, where Ca 2+ concentration was fixed at 50 nM, to inositol 1,4,5-trisphosphate (IP 3 ) produced similar effects on the release of Ca 2+ from stores in Stim1-mKO1-expressing and non-expressing cells. The low toxicity and relative specificity to acinar cells of the mild gene transfer method described herein are particularly useful for studying the molecular functions of salivary acinar cells in vivo , and may be applied to increase salivary secretions in experimental animals and human in future.

Inositol 1,4,5-trisphosphate (IP₃) receptors consist of three subtypes: IP₃R1, IP₃R2, and IP₃R3. Although numerous IP₃ receptor ligands have been synthesized, none of the subtype-selective ligands are known. We have developed a simple fluorescence method to examine the subtype selectivity of IP₃ receptor ligands using FRET-based IP₃ biosensors LIBRAvI, LIBRAvII, and LIBRAvIII. The addition of IP₃ or adenophostin A (ADA) to permeabilized biosensor-expressing cells increased the fluorescence ratios of these biosensors in a concentration-dependent manner, and the potency of ADA relative to that of IP₃ in terms of the changes in the fluorescence ratios of LIBRAvI, LIBRAvII, and LIBRAvIII was 43-, 22-, and 28-fold, respectively. This fluorescence-based method further showed that several ADA analogs had significant differences with respect to subtype selectivity and potency. These results highlight the important role played by the O-glycosidic structure of ADA in the selectivity of the ligands for IP₃R1, as evidenced by the modified selectivity following replacement of the 5'-hydroxyl with a phenyl or phenethyl group. We also found that one ADA analog 5'-deoxy-5'-phenyladenophostin A possessed a partial agonistic effect on IP₃R1. Together, the novel fluorescent methods described herein are useful for the evaluation of properties of IP₃R ligands, including potency, efficacy, and subtype selectivity.

Store-operated Ca 2+ entry (SOCE) from the extracellular space plays a critical role in agonist-mediated Ca 2+ signaling in non-excitable cells. Here we show that SOCE is enhanced in COS-7 cells treated with staurosporine (ST), a protein kinase inhibitor. In COS-7 cells, stimulation with ATP induced Ca 2+ release from intracellular Ca 2+ stores and Ca 2+ entry from the extracellular space. Ca 2+ release was not affected by treatment with ST, but Ca 2+ entry continued in the ST-treated cells even after the removal of ATP. ST did not inhibit Ca 2+ sequestration into Ca 2+ stores. The Ca 2+ entry induced by cyclopiazonic acid (CPA), a reversible ER Ca 2+ pump inhibitor, was maintained in ST-treated cells even after the removal of CPA, but was not maintained in the control cells. The sustained Ca 2+ entry in ST-treated cells was completely attenuated by the SOCE inhibitors, La 3+ and 2-APB. The large increase in Ca 2+ entry produced in the cells co-expressing Venus-Orai1 and STIM1-mKO1 was stabilized with ST treatment, and confocal imaging of these cells suggested that the complex between Orai1 and STIM1 did not completely dissociate following the refilling of Ca 2+ stores. These results show that SOCE remains activated even after the refilling of Ca 2+ stores in ST-treated cells and that the effect of ST on SOCE may result from a stabilization of the Orai1–STIM1 interaction.

研究ノート(Note)

In the present study, the contribution of inositol (1,4,5)-trisphosphate [Ins(1,4,5)P(3)] generation on the mechanical-stimulation-induced Ca(2+) response was investigated in HSY-EA1 cells. Mechanical stimulation induced a local increase in the cytosolic concentration of Ins(1,4,5)P(3) ([IP(3)](i)), as indicated by the Ins(1,4,5)P(3) biosensor LIBRAvIII. The area of this increase expanded like an intracellular Ins(1,4,5)P(3) wave as [IP(3)](i) increased in the stimulated region. A small transient [IP(3)](i) increase was subsequently seen in neighboring cells. The phospholipase C inhibitor U-73122 abolished these Ins(1,4,5)P(3) responses and resultant Ca(2+) releases. The purinergic receptor blocker suramin completely blocked increases in [IP(3)](1) and the Ca(2+) release in neighboring cells, but failed to attenuate the responses in mechanically stimulated cells. These results indicate that generation of Ins(1,4,5)P(3) in response to mechanical stimulation is primarily independent of extracellular ATP. The speed of the mechanical-stimulation-induced [IP(3)](i) increase was much more rapid than that induced by a supramaximal concentration of ATP (1 mM). The contribution of the Ins(1,4,5)P(3)-induced Ca(2+) release was larger than that of Ca(2+) entry in the Ca(2+) response to mechanical stimulation in HSY-EA1 cells.

We made a hearing investigation to residents besides alleys in Higashiyama area, Kyoto city, about the experiences of criminal damages and how they think about security of alleys. As results, residents besides alleys feel uneasy about earthquakes and fires, but they feel easy about criminal acts. Characteristics of the alleys with which residents can feel ease highly are followings: 1. possible to flee for safety in two directions, 2. possible to recognize entrances, 3. residents associate on the terms of neighbors. We found a tendency that the residents of new houses besides an alley which have a certain width or without barriers, and less association with neighbors, are easily damaged by criminal acts.

The depletion of intracellular Ca(2+) stores activates capacitative Ca(2+) entry (CCE), which is a Ca(2+)-selective and La(3+)-sensitive entry pathway. Here, we report a novel mechanism of La(3+)-resistant Ca(2+) entry that is synergistically regulated by B-cell-receptor (BCR) stimulation and Ca(2+) store depletion. In DT40 cells, stimulation of BCRs with anti-IgM antibodies induced Ca(2+) release and subsequent Ca(2+) entry in the presence of 0.3 microM La(3+), a condition in which CCE is completely blocked. This phenomenon was not observed in inositol 1,4,5-trisphosphate receptor-deficient DT40 (IP3R-KO) cells. However, in response to thapsigargin pretreatment, BCR stimulation induced La(3+)-resistant Ca(2+) entry into both wild-type and IP3R-KO cells. These results indicate that BCR stimulation alone does not activate Ca(2+) entry, whereas BCR stimulation and depleted Ca(2+) stores (either due to IP3R-mediated Ca(2+) release or Ca(2+) uptake inhibition) work in concert to activate La(3+)-resistant Ca(2+) entry. This Ca(2+) entry was inhibited by genistein. In addition, BCR-mediated Ca(2+) entry was completely abolished in Stim1-deficient DT40 cells and was restored by overexpression of YFP-Stim1, but was unaffected by double knockdown of Orai1 and Orai2. These results demonstrate a unique non-CCE pathway, in which Ca(2+) entry depends on Stim1- and BCR-mediated activation of tyrosine kinases.

Inositol 1,4,5-trisphosphate (IP(3)) is an intracellular messenger that elicits a wide range of spatial and temporal Ca(2+) signals, and this signaling versatility is exploited to regulate diverse cellular responses. In this study, we have developed a series of IP(3) biosensors that exhibit strong pH stability and varying affinities for IP(3), as well as a method for the quantitative measurement of cytosolic concentrations of IP(3) ([IP(3)](i)) in single living cells. We applied this method to elucidate IP(3) dynamics during agonist-induced Ca(2+) oscillations, and we demonstrated cell type-dependent differences in IP(3) dynamics, a nonfluctuating rise in [IP(3)](i) and repetitive IP(3) spikes during Ca(2+) oscillations in COS-7 cells and HSY-EA1 cells, respectively. The size of the IP(3) spikes in HSY-EA1 cells varied from 10 to 100 nm, and the [IP(3)](i) spike peak was preceded by a Ca(2+) spike peak. These results suggest that repetitive IP(3) spikes in HSY-EA1 cells are passive reflections of Ca(2+) oscillations, and are unlikely to be essential for driving Ca(2+) oscillations. In addition, the interspike periods of Ca(2+) oscillations that occurred during the slow rise in [IP(3)](i) were not shortened by the rise in [IP(3)](i), indicating that IP(3)-dependent and -independent mechanisms may regulate the frequency of Ca(2+) oscillations. The novel method described herein as well as the quantitative information obtained by using this method should provide a valuable and sound basis for future studies on the spatial and temporal regulations of IP(3) and Ca(2+).

Thus far, research on modern city planning in China has focused on international cities such as Shanghai and colonial cities such as Tsingtao. Such urban areas have been considered as representing ...

The inositol 1,4,5-trisphosphate receptors (IP3Rs) form clusters following agonist stimulation, but its mechanism remains controversial. In this study, we visualized the clustering of green fluorescent protein (GFP)-tagged type 3 IP3R (GFP-IP3R3) in cultured living cells using confocal microscopy. Stimulation with ATP evoked GFP-IP3R3 clustering not only in cells with replete Ca2+-stores but also in cells with depleted Ca2+ stores. Thapsigargin (ThG) and ionomycin failed to mimic the ATP-induced cluster formation despite the continuous elevation of intracellular Ca2+ concentration ([Ca2+]i). Application of IP3 caused GFP-IP3R3 clustering in permeabilized cells, and the response was completely inhibited by heparin, a competitive inhibitor of IP3R. Experiments using LIBRAv, an IP3 biosensor, showed that ATP significantly stimulated IP3 generation even in store-depleted cells. We also found that pretreatment with ThG accelerated or enhanced the ATP-induced clustering in both the presence and absence of extracellular Ca2+. When permeabilized cells were stimulated with the threshold of IP3, the GFP-IP3R3 clustering clearly occurred in Ca2+-free medium but not in Ca2+-containing medium. These results strongly support the hypothesis that the agonist-induced clustering of IP3R is triggered by IP3 binding, rather than [Ca2+]i elevation. Although depletion of the Ca2+ store by itself does not cause the clustering, it may increase the sensitivity of IP3R to cluster formation, leading to facilitation of IP3-triggered clustering. Keywords:: inositol 1,4,5-trisphosphate (IP3), clustering of IP3 receptor, Ca2+-store depletion, thapsigargin, green fluorescent protein (GFP)

This paper discusses the importance of gaps in urban space and cityscape. It proposes that gaps are particularly important in high-density urban areas, testifying of the options for urban densification and presents the case study of Tokyo's J.R. Yamanote railway and Shibuya district to support this idea. In the case of the J.R. Yamanote line – a railway with a concentric layout serving Tokyo's central districts – we see how gaps initially created under elevated sections of this structure gradually disappeared and became occupied by new urban tissues, allowing urban growth to continue taking place in the city centre. In Shibuya, where no more land was available, gaps were used to support a strategy of space usage intensification. While physically transportation and commercial infrastructures appeared absorbing all gaps between them, some amusement facilities such as a cable-car or a planetarium were the devices created to expand the visual horizon beyond the increasingly dense urban space. These findings reveal that in such processes, gaps have appeared, disappeared and in some cases reappeared to promote continuous urban intensification processes essential to the preservation of urban centrality.

More than half of the patients with glomerulonephritis, nephrotic syndrome and renovascular hypertension showed obviously abnormal profiles of serum lipoproteins (Lps). The abnormal profiles returned to normal or near-normal when the disease was ameliorated or corrected surgically. A unique Lp profile (broad midband pattern, BMP) was observed in approximately 89% of uremic patients on hemodialysis therapy. The results of the current investigations indicated that the BMP could probably be formed by the accumulation of catabolic remnants of VLDL.

What is the central question of this study? Pilocarpine stimulates salivary secretion via muscarinic ACh receptors (mAChRs), although the Ca(2+) -mobilizing effect of pilocarpine in salivary gland cells is extremely small. Therefore, we examined the effect of pilocarpine on Ca(2+) responses in submandibular gland cells and on secretion in vitro and in vivo. What is the main finding and its importance? Pilocarpine induces small Ca(2+) responses and reduces the effects of other mAChR agonists on Ca(2+) responses via its partial agonistic effects. These effects of pilocarpine on Ca(2+) responses in the submandibular gland were further established in vivo with a novel Ca(2+) imaging system and a genetically encoded Ca(2+) indicator. Pilocarpine stimulates salivary secretion via muscarinic ACh receptors (mAChRs), although the effect of pilocarpine on Ca(2+) responses in dispersed salivary gland cells is extremely small. Here, we demonstrate the effect of pilocarpine on Ca(2+) responses and salivary secretion in the rat submandibular gland (SMG). In fura-2-loaded SMG cells, the maximal effect of pilocarpine on [Ca(2+) ]i elevation was 16% of that of carbachol, and pilocarpine attenuated carbachol- and bethanechol (Bet)-induced [Ca(2+) ]i increases, indicating that pilocarpine acts as a partial agonist for mAChR-mediated Ca(2+) responses. The partial agonistic effect of pilocarpine on Ca(2+) dynamics in the SMG was also confirmed in live animals using the genetically encoded Ca(2+) indicator, YC-Nano50. Administration of pilocarpine (3 mg kg(-1) , i.p.) elicited a small increase in [Ca(2+) ]i in the SMG. Quantitative analyses demonstrated that resting [Ca(2+) ]i was ∼37 nm, which was increased by pilocarpine (3 mg kg(-1) ) and Bet (10 mg kg(-1) ) to 44 and 69 nm, respectively. The inhibitory effects of pilocarpine on Bet-induced Ca(2+) responses were also elucidated in vivo. We further examined real-time changes in pilocarpine-induced SMG salivary secretion and showed that pilocarpine induced an extremely weak secretory response and reduced Bet-induced secretion. Unlike Ca(2+) responses, pilocarpine failed to reduce the effect of Bet on SMG blood flow. Our results demonstrate that pilocarpine acts as a partial agonist of mAChRs to induce weak salivary secretion that is correlated with small increases in [Ca(2+) ]i . Furthermore, pilocarpine exhibits an antagonistic effect on mAChR-induced Ca(2+) responses and salivary secretion.