Intracellular Signaling Pathways Involved in the Regulation of Gene Expression by Pilocarpine.

OBJECTIVES: Pilocarpine is commonly used clinically to treat dry mouth. The long-term administration of pilocarpine reportedly improves salivary secretion more effectively than short-term administration. Therefore, we hypothesized that pilocarpine alters gene expression in salivary glands via muscarinic receptor stimulation. This study aimed to investigate the effects of pilocarpine use on gene expression mediated by mitogen-activated protein kinase (MAPK) activity. METHODS: The effects of pilocarpine on gene expression were investigated in rats and human salivary gland (HSY) cells using several inhibitors of intracellular signaling pathways. Gene expression in the rat submandibular gland and HSY cells was determined using reverse transcription-quantitative polymerase chain reaction analysis of total RNA. RESULTS: In animal experiments, at 7 days after pilocarpine stimulation, Ctgf and Sgk1 expressions were increased in the submandibular gland. In cell culture experiments, pilocarpine increased Ctgf expression in HSY cells. The mitogen-activated protein kinase kinase inhibitor trametinib, the Src inhibitor PP2, and the muscarinic acetylcholine receptor antagonist atropine suppressed the effect of pilocarpine on gene expression. CONCLUSIONS: Pilocarpine enhances Ctgf and Sgk1 expressions by activating Src-mediated MAPK activity. Although further studies are required to fully understand the roles of Ctgf and Sgk1, changes in gene expression may play an important role in improving salivary secretions.

Serum perampanel levels in patients with seizures are not affected by hemodialysis.

Perampanel belongs to a novel class of antiseizure medications (ASMs). Studies examining the effect of hemodialysis on perampanel serum levels in clinical settings are lacking. We aimed to evaluate the changes in serum perampanel levels during hemodialysis. We studied patients with seizures who received oral perampanel between April 2020 and March 2023 and whose serum concentration of perampanel was measured before and after hemodialysis. We analyzed the serum concentrations of levetiracetam and lacosamide for comparison. Fourteen patients, with a mean age of 76.1 ± 7.88 years, were included. The dose of perampanel was 2.14 ± 1.27 mg. The hemodialysis clearance rate of perampanel, levetiracetam, and lacosamide was 0 ± 13%, 69 ± 11%, and 59.6 ± 8.2%, respectively. The post-dialysis CD ratio decreased significantly with levetiracetam but not with perampanel. Adverse but acceptable effects of perampanel were observed in two patients. The serum concentrations of several ASMs have been shown to be reduced during hemodialysis. Our study revealed that the serum perampanel concentration does not decrease during hemodialysis. Owing to the low rate of adverse effects and the stability of perampanel serum concentration during hemodialysis, perampanel could be a favorable choice as an ASM for patients with seizures undergoing hemodialysis. PLAIN LANGUAGE SUMMARY: Our study looked at how hemodialysis affects the serum levels of perampanel, a new type of medication for seizures. In 14 patients who started treatment between April 2020 and March 2023, perampanel serum levels did not decrease during hemodialysis, unlike other seizure medications. This shows that perampanel can be a good option for patients with seizures who need hemodialysis, with fewer side effects compared to other medications.

Local anesthetics inhibit muscarinic acetylcholine receptor-mediated calcium responses and the recruitment of ß-arrestin in HSY human parotid cells.

OBJECTIVES: Local anesthetics act on G protein-coupled receptors (GPCRs); thus, their potential as allosteric modulators of GPCRs has attracted attention. Intracellular signaling via GPCRs involves both G-protein- and ß-arrestin-mediated pathways. To determine the effects of local anesthetics on muscarinic acetylcholine receptors (mAChR), a family of GPCRs, we analyzed the effects of local anesthetics on mAChR-mediated Ca2+ responses and formation of receptor-ß-arrestin complexes in the HSY human parotid cell line. METHODS: Ca2+ responses were monitored by fura-2 spectrofluorimetry. Ligand-induced interactions between mAChR and ß-arrestin were examined using a ß-arrestin GPCR assay kit. RESULTS: Lidocaine reduced mAChR-mediated Ca2+ responses but did not change the intracellular Ca2+ concentration in non-stimulated cells. The membrane-impermeant lidocaine analog QX314 and procaine inhibited mAChR-mediated Ca2+ responses, with EC50 values of 48.0 and 20.4 µM, respectively, for 50 µM carbachol-stimulated Ca2+ responses. In the absence of extracellular Ca2+, the pretreatment of cells with QX314 reduced carbachol-induced Ca2+ release, indicating that QX314 reduced Ca2+ release from intracellular stores. Lidocaine and QX314 did not affect store-operated Ca2+ entry as they did not alter the thapsigargin-induced Ca2+ response. QX314 and procaine reduced the carbachol-mediated recruitment of ß-arrestin, and administration of procaine suppressed pilocarpine-induced salivary secretion in mice. CONCLUSION: Local anesthetics, including QX314, act on mAChR to reduce carbachol-induced Ca2+ release from intracellular stores and the recruitment of ß-arrestin. These findings support the notion that local anesthetics and their derivatives are starting points for the development of functional allosteric modulators of mAChR.

Effects of Semiconductor Laser Irradiation on Differentiation of Human Dental Pulp Stem Cells in Co-Culture with Dentin.

This study aimed to determine the effect of photobiomodulation therapy induced by semiconductor laser irradiation on human dental pulp stem cell (hDPSC) proliferation and their differentiation into odontoblast-like cells (OLCs). The effects of various semiconductor laser irradiation conditions on hDPSCs were examined. Three groups were evaluated: a single laser irradiation at 6 h post-seeding, multiple laser irradiations up to four times every 4 days after the first dose, and a control with no laser irradiation. The cells were irradiated at 10, 30, and 150 mW using a semiconductor laser. The effect of laser irradiation on hDPSC differentiation into OLCs was also determined. Four groups were evaluated, including co-culture using basic medium and dentin discs, simple culture using OLC differentiation-inducing medium, co-culture using OLC differentiation-inducing medium and dentin discs, and control culture with basic medium. The expression of the nestin, ALP, DSPP, and DMP-1 genes was measured using real-time PCR. The multiple irradiation group irradiated at 30 mW exhibited significantly more cell proliferation than the control. The expression of nestin associated with differentiation into OLCs during each culture period tended to be lower, whereas DSPP and ALP expression was higher compared with that of the control. Multiple laser irradiations at a low power of 30 mW induced significant hDPSC proliferation and might induce differentiation into OLCs.

Near-Infrared Photoimmunotherapy Using a Protein Mimetic for EGFR-Positive Salivary Gland Cancer.

Near-infrared photoimmunotherapy (NIR-PIT) is a novel cancer therapy based on a monoclonal antibody (mAb) conjugated to a photosensitizer (IR700Dye). The conjugate can be activated by near-infrared light irradiation, causing necrotic cell death with high selectivity. In this study, we investigated NIR-PIT using a small protein mimetic (6-7 kDa, Affibody) which has more rapid clearance and better tissue penetration than mAbs for epidermal growth factor receptor (EGFR)-positive salivary gland cancer (SGC). The level of EGFR expression was examined in vitro using immunocytochemistry and Western blotting. Cell viability was analyzed using the alamarBlue assay. In vivo, the volume of EGFR-positive tumors treated with NIR-PIT using the EGFR Affibody-IR700Dye conjugate was followed for 43 days. It was found that NIR-PIT using the EGFR Affibody-IR700Dye conjugate induced the selective destruction of EGFR-positive SGC cells and restricted the progression of EGFR-positive tumors. We expect that NIR-PIT using the EGFR Affibody-IR700Dye conjugate can efficiently treat EGFR-positive SGC and preserve normal salivary function.

[Evaluation of cerebral hemodynamics using arterial spin labeling perfusion MR imaging in a patient with cervical internal carotid artery vasospasm].

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.

A Case of Huge Scrotal Hematoma during Mechanical Thrombectomy.

Objective: We report a case of huge scrotal hematoma during emergency mechanical thrombectomy. Case Presentation: An 85-year-old man presented with sudden aphasia and right-sided hemiplegia. He was diagnosed with cerebral infarction due to left M1 occlusion and underwent an emergency mechanical thrombectomy. The treatment was completed with full recanalization, but when replacing the long sheath in the right femoral artery with a short sheath, the patient flexed his leg. The sheath could not be replaced, resulting in a massive scrotal hematoma. Shortly after, the patient went into cardiopulmonary arrest but recovered spontaneous circulation after cardiopulmonary resuscitation. The puncture site was treated hemostatically with manual compression, and the scrotal hematoma was not enlarged. He was transferred to another hospital with a modified Rankin Scale score of 5. Conclusion: Scrotal hematoma is a rare but potentially fatal puncture site complication that should be considered during neuro-endovascular treatment.

Multiple Thrombectomies for Cerebral and Coronary Artery Occlusion in Trousseau Syndrome.

Objective: Trousseau syndrome (TS) is a condition of systemic thrombosis generally associated with an underlying malignancy. An ischemic stroke is a representative thrombotic event. Thrombectomy is a useful procedure for the treatment of cerebral large vessel occlusion, and anticoagulation therapy is the main preventive treatment for TS. This case report describes a woman with terminal pancreatic tumor presenting with repeated occlusions of cerebral and coronary arteries necessitating multiple thrombectomies. Case Presentation: A 67-year-old woman was admitted to our hospital with severe right hemiplegia and global aphasia. MRI revealed left M1 occlusion; therefore, a thrombectomy was performed. Her symptoms recovered completely. Body contrast CT revealed pancreatic cancer with multiple metastases, and she was diagnosed with TS. On day 4 after thrombectomy, the same neurological symptoms occurred and re-occlusion of the left M1 was confirmed. Endothelial injury was suspected, and thrombectomy was repeated. Despite continuing anticoagulation therapy, the coronary artery was occluded and she underwent percutaneous coronary intervention on day 13. To treat the primary pancreatic lesion, she was transferred to the Surgery unit on day 20. Conclusion: Hypercoagulability associated with TS and endothelial damage due to rough procedure resulted in repeated vessel occlusions in this case. Careful thrombectomy and anticoagulation therapy with strict monitoring are needed in TS patients.

Combination of Near-Infrared Photoimmunotherapy Using Trastuzumab and Small Protein Mimetic for HER2-Positive Breast Cancer.

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.

Spontaneous calcium responses of SF2 rat dental epithelial cells stably expressing the calcium sensor G-GECO.

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.

Protein Ingredient in Saliva on Oral Dryness Patients Caused by Calcium Blocker.

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 < 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 < 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.

Ischemic Stroke Caused by Carotid Stump at the Common Carotid Artery.

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.

Analysis of salivary factors related to the oral health status in children.

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.

[Case report: cerebral venous thrombosis detected by perfusion CT in a patient with untreated diabetes mellitus].

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.

Effects of full-length human amelogenin on the differentiation of dental epithelial cells and osteoblastic cells.

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.

Simultaneous monitoring of Ca2+ responses and salivary secretion in live animals reveals a threshold intracellular Ca2+ concentration for salivation.

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.

[Advances in methods for analyzing IP3 signaling and understanding of coupled Ca2+ and IP3 oscillations].

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.

Thrombolytic Therapy in Severe Cardioembolic Stroke After Reversal of Dabigatran with Idarucizumab: Case Report and Literature Review.

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.

Effect of 1,25-dihydroxyvitamin D3 on spontaneous calcium responses in rat dental epithelial SF2 cells revealed by long-term imaging.

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.

Highly Sensitive Measurement of Inositol 1,4,5-Trisphosphate by Using a New Fluorescent Ligand and Ligand Binding Domain Combination.

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.