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LIBRA is a fluorescent biosensor of inositol 1,4,5-trisphosphate (IP 3 ) and is composed of the ligand-binding domain of the rat type 3 IP 3 receptor and cyan and yellow fluorescent proteins. We examined the responses of LIBRA and its IP 3 -insensitive mutant LIBRA-N to compounds known to inhibit IP 3 -induced Ca 2+ release. Heparin, a competitive antagonist of IP 3 receptors, increased the emission ratio of LIBRA but not that of LIBRA-N. In contrast, 2-aminoethoxydiphenyl borate, a known non-competitive inhibitor of IP 3 receptor, decreased the emission ratios of both LIBRA and LIBRA-N. Thus, the concurrent use of LIBRA-N with LIBRA identifies nonspecific responses. These results indicate that LIBRA and its mutant control can be used to detect specific agonists and antagonists of IP 3 receptors. We also demonstrate the utility of LIBRA and LIBRA-N in discriminating between specific and nonspecific responses in intact cells.

The adsorptive properties of tris(2,2′-bipyridine)ruthenium(II)-clay intercalation compounds, which were synthesized through cation exchange reactions from synthetic saponite (Sumecton SA), synthetic fluoro-tetrasilicic mica and montmorillonite (Kunipia F) for phenols were investigated. The adsorption isotherms of phenols for the tris(2,2′-bipyridine)ruthenium(II)-clays from aqueous solutions followed Langmuir type, indicating strong adsorbate–adsorbent interactions. The basal spacings of the tris(2,2′-bipyridine)ruthenium(II)-clays did not change through the adsorption of phenols. This means that the adsorbed phenols existed in the interlayer nanopore created by the tris(2,2′-bipyridine)ruthenium(II) in the interlayer space of the tris(2,2′-bipyridine)ruthenium(II)-clays. The adsorbed amounts of phenols varied depending upon the nature of clays. One of the factors responsible for the variation in the adsorbed amounts is the layer charge density of smectites. Relatively low-layer charge density of saponite led to relatively large pore volume in the interlayer space. The adsorbed amounts of 2,4-dichlorophenol for the tris(2,2′-bipyridine)ruthenium(II)-saponite and the tris(2,2′-bipyridine)ruthenium(II)-montmorillonite were the largest among three phenols. It is thought that the interactions between tris(2,2′-bipyridine)ruthenium(II) cation and phenols played an important role in the adsorption of these phenols.

The second messenger inositol 1,4,5-trisphosphate (IP(3)) plays a central role in the generation of a variety of spatiotemporally complex intracellular Ca(2+) signals involved in the regulation of many essential physiological processes. Here we describe the development of "LIBRA", a novel ratiometric fluorescent IP(3) biosensor that allows for the quantitative monitoring of intracellular IP(3) concentrations in single living cells in real time. LIBRA consists of the IP(3)-binding domain of the rat type 3 IP(3) receptor fused between the fluorescence resonance energy transfer pair cyan fluorescent protein and yellow fluorescent protein and preceded by a membrane-targeting signal. We show that the LIBRA fluorescent signal is highly selective for IP(3) and unaffected by concentrations of Ca(2+) and ATP in the physiological range. In addition, LIBRA can be calibrated in situ. We demonstrate the utility of LIBRA by monitoring the temporal relationship between the responses intracellular IP(3) and Ca(2+) concentrations in SH-SY5Y cells following acetylcholine stimulation.

This article analyzes the use of elevated urban railways as a contemporary urban development, evaluating such with respect to the problem of permanent barrier effects inside the city created by dep...

In recent years, the damage to Libraries and Museums caused by floods have increased. This is specially true in the underground where great damage can happen easily. And so, it is important to add to the facility management good architectural planning considerations to minimize damages. It is important to take note if there was a previous occurrence of flood damage in the surrounding environment, and the location of the basement specially if the topography is sloping or a hollow low ground. A place where water can flow and accumulate easily are used as dry area, parking, ramps for goods and ramps for disabled people can be given. To prevent flood damages the different patterns of water flow should be predicted and to allow sufficient planning for the drainage of water. Measures should be implemented so that books, works of art, computers would not be damaged. A doubly considered architecture planning provision is then necessary., 京都工芸繊維大学 工芸学部研究報告 第５０巻 理工・欧文(2001) pp.55-64, 図書館と博物館における浸水被害の実態とその対策について論じた研究である。一連の図書館と美術館における浸水被害に関する研究について英文にてまとめたものである。図書館と美術館における浸水被害の実態と浸水被害を受けないようにするための指針を示している。

Store-operated Ca2+ entry (SOCE) is a ubiquitous Ca2+ entry pathway in non-excitable cells. It is activated by the depletion of Ca2+ from intracellular Ca2+ stores, notably the endoplasmic reticulum (ER). In the past 9 years, it has been established that two key proteins, stromal interacting molecule 1 (STIM1) and Orai1, play critical roles in SOCE. STIM1 is a single-pass transmembrane protein located predominantly in the ER that serves as a Ca2+ sensor within the ER, while Orai1 is a tetraspanning plasma membrane (PM) protein that functions as the pore-forming subunit of store-operated Ca2+ channels. A decrease in the ER Ca2+ concentration induces translocation of STIM1 into puncta close to the PM. STIM1 oligomers directly interact with Orai1 channels and activates them. This review summarizes the molecular basis of the interaction between STIM1 and Orai1 in SOCE. Further, we describe current findings on additional regulatory proteins, such as Ca2+ release–activated Ca2+ regulator 2A and septin, novel roles of STIM1, and modulation of SOCE by protein phosphorylation. Keywords:: store-operated Ca2+ entry, STIM1, Orai1, calcium channel

The ATP-induced oscillatory changes in cytosolic Ca(2+) concentration ([Ca(2+)](i)) were analysed in HSY cells, a salivary ductal cell line from human parotid, using a fluorescence ratio imaging system. At concentrations higher than 1 microM, ATP caused sinusoidal [Ca(2+)](i) oscillations due to the periodic release and reuptake of Ca(2+) by intracellular Ca(2+) stores. The phorbol ester 4beta-phorbol 12,13-dibutyrate (PDBu) changed the [Ca(2+)](i) oscillations to a single spike. The inhibitory effect of PDBu on the [Ca(2+)](i) signals was reversed by protein kinase C (PKC) inhibitors such as staurosporine and chelerythrine chloride. However, preincubation of the cells with the PKC inhibitors did not affect the pattern of the ATP-induced [Ca(2+)](i) oscillations. The desensitization of the [Ca(2+)](i) response observed during prolonged stimulation with ATP was also not prevented by the PKC inhibitors. Incubation of HSY cells with the sulphydryl reagent thimerosal, which enhances the sensitivity of inositol 1,4,5-trisphosphate (IP(3)) receptors, caused repetitive Ca(2+) release from intracellular Ca(2+) stores resulting in baseline spikes of [Ca(2+)](i). The thimerosal-induced [Ca(2+)](i) oscillations did not change in the presence of PDBu and the phospholipase C inhibitor U73122. Thus, we could not provide evidence that negative feedback by PKC plays a central role in the regulation of ATP-induced [Ca(2+)](i) oscillations. These results suggest that the [Ca(2+)](i) oscillations, at least the baseline spikes, in HSY cells can be generated without stimulating the formation of IP(3).

It is well known that mechanical stimulation elicits Ca responses in various cell types. Touch stimulation on a single cell induces an increase in the cytosolic Ca concentration ([Ca]i), which subsequently propagates to neighboring cells as an intercellular Ca wave. In addition to Ca entry through mechanosensitive channels, this Ca response is thought to be induced, at least in part, by inositol 1,4,5-trisphosphate (IP3)-dependent Ca release from intracellular Ca stores (1-3). However, this hypothesis has not been tested directly due to a lack of the quantitative method to measure the cytosolic concentration of IP3 ([IP3]i) in a single living cell. Recently, we developed a FRET-based IP3 biosensor, LIBRAvIII, and a method to measure [IP3]i quantitatively in the single living cell (4). In order to examine the involvement of IP3 in mechanical stimulation-induced Ca responses, we monitored IP3 and Ca responses simultaneously in HSY-EA1 cells, a human parotid cell line, using LIBRAvIII and the Ca indicator fura-2.

Continuous milling tests with diamond-coated and uncoated cemented carbide tools and polycrystalline diamond tools were carried out on air-dried and wet melapi (Shorea sp.) and western redcedar (Thuja plicata D. Don). These tools were examined for corrosive-wear characteristics of the tool-edge appearance, cutting-edge profile, edge recession, and cutting-power consumption. The tool surfaces were observed with a scanning electron microscope and were analyzed by energy-dispersive X-ray spectroscopy. Based on these examinations, the occurrence of corrosive wear while cutting wet woods was confirmed for the uncoated and polycrystalline diamond tools. In contrast, the coated tools did not exhibit corrosive wear, nor did delamitation or wear of the diamond film occur with any of the work materials. The diamond-coated tools showed high resistance not only to mechanical wear but also to corrosive wear.

The authors described experimental data in hemostatic vascular transection using a STATLase-SDL with a 780-865 nm GaA/As diode wavelength and its handpiece, Dual Hook (DH).The STATLase-SDL and DH are newly developed devices for endoscopic surgery. It seems that the device has improved hemostatic cutting ability.The DH was applied to the abdominal aorta (2 mm) of Japanese white rabbits (n = 15) under general anesthesia. Tissue temperature during laser transection and interluminal bursting pressures were measured. Histopathological examinations at the cut end were carried out by hematoxylin-eosin (HE) and elastica Van Gleson's (EVG) stainings. To investigate short-term effects 11 days postoperatively, the cut ends of the vessels were examined histologically in the heminephrectomy group.The DH hemostatistically cut through arteries up to 2 mm in diameter. Temperatures at the inner hook and in adjunct tissue were higher than 160 degrees C, while that at the outer hook was lower than 80 degrees C. The bursting pressures immediately after transection was 307.2 +/- 35.2 mm Hg at a laser power of 8 W (n = 5), and 295.6 +/- 28.7 mm Hg at 10 W (n = 5). Histological examination of the transected sites on the vessels revealed well-opposed tissue welding of the vascular wall. Absence of fragmentation in the welding of the internal elastic lamina is crucial for closure of the vessel stump. Up to 11 days postoperatively, there were no direct complications related to the use of the DH.The STATLase-SDL and DH have good hemostatic cutting ability and might be suitable for endoscopic surgery.

Background In autosomal recessive early-onset Parkinsonism (PARK2), the pathogenetic process from the loss of function of a ubiquitin ligase parkin to the death of dopamine neurons remains unclear. A dominant hypothesis attributes the neurotoxicity to accumulated substrates that are exempt from parkin-mediated degradation. Parkin substrates include two septins; SEPT4/CDCrel-2 which coaggregates with α-synuclein as Lewy bodies in Parkinson’s disease, and its closest homolog SEPT5/CDCrel-1/PNUTL1 whose overload with viral vector can rapidly eliminate dopamine neurons in rats. However, chronic effects of pan-neural overload of septins have never been examined in mammals. To address this, we established a line of transgenic mice that express the largest gene product SEPT454kDa via the prion promoter in the entire brain. Results Histological examination and biochemical quantification of SEPT4-associated proteins including α-synuclein and the dopamine transporter in the nigrostriatal dopamine neurons found no significant difference between Sept4 Tg/+ and wild-type littermates. Thus, the hypothetical pathogenicity by the chronic overload of SEPT4 alone, if any, is insufficient to trigger neurodegenerative process in the mouse brain. Intriguingly, however, a systematic battery of behavioral tests revealed unexpected abnormalities in Sept4 Tg/+ mice that include consistent attenuation of voluntary activities in distinct behavioral paradigms and altered social behaviors. Conclusions Together, these data indicate that septin dysregulations commonly found in postmortem human brains with Parkinson’s disease, schizophrenia and bipolar disorders may be responsible for a subset of behavioral abnormalities in the patients.

The mechanism of progression of appendicitis has not been clarified. We examined tissue superoxide dismutase (SOD) activity, thiobarbituric acid reactive substance (TBARS), and the localization of Cu, Zn-SOD in 56 inflamed appendices in relation to histopathological classification. There was a significant difference in SOD activity between catarrhal appendix and phlegmonous and gangrenous appendix (2.3 +/- 0.1 vs 5.0 +/- 0.2 and 4.6 +/- 0.6 units/mg protein, respectively P < 0.05). TBARS value was highest in gangrenous appendix, being significantly different from the levels in the other two types (0.47 +/- 0.04 vs 0.19 +/- 0.01 n mol/mg protein, in catarrhal and 0.20 +/- 0.02, in phlegmonous appendix P < 0.05). Positive staining for Cu, Zn-SOD was demonstrated in 64% of catarrhal appendices, 96% of phlegmonous appendices, and 75% of gangrenous appendices, and intense positive staining was recognized in 9%, 28%, and 40% of these appendices, respectively. These results indicated that active oxygen influences the degree of inflammation in phlegmonous and gangrenous appendicitis. Gangrenous appendicitis and the other two types of appendicitis seemed to be different entities.

Store-operated Ca²⁺ entry (SOCE) from the extracellular space plays a critical role in agonist-mediated Ca²⁺ 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 Ca²⁺ release from intracellular Ca²⁺ stores and Ca²⁺ entry from the extracellular space. Ca²⁺ release was not affected by treatment with ST, but Ca²⁺ entry continued in the ST-treated cells even after the removal of ATP. ST did not inhibit Ca²⁺ sequestration into Ca²⁺ stores. The Ca²⁺ entry induced by cyclopiazonic acid (CPA), a reversible ER Ca²⁺ pump inhibitor, was maintained in ST-treated cells even after the removal of CPA, but was not maintained in the control cells. The sustained Ca²⁺ entry in ST-treated cells was completely attenuated by the SOCE inhibitors, La³⁺ and 2-APB. The large increase in Ca²⁺ 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 Ca²⁺ stores. These results show that SOCE remains activated even after the refilling of Ca²⁺ stores in ST-treated cells and that the effect of ST on SOCE may result from a stabilization of the Orai1-STIM1 interaction.

For implant of molecular ions including fluorine atoms or for multi-implantations of F+ and other ions, fluorine depth profiles after anneal change drastically depending on implant conditions. This implies that damage formation during ion implant and defect evolution during anneal are intrinsically different between implant with B atoms and implant without B atoms.

Three zebrafish otx homeoproteins containing a homeodomain homologous to that of the Drosophila orthodenticle head gap gene product have been identified by cloning and sequencing of cDNAs. The zebrafish otx2 homeoprotein shares high amino-acid sequence identity with the mouse Otx2 homeoprotein, whereas the zebrafish otx1 and otx3 homeoproteins exhibit moderate homology with the mouse Otx1 and Otx2 homeoproteins. Three otx homeoprotein mRNAs show different spatio-temporal expression patterns during zebrafish embryogenesis as revealed by Northern blot and whole mount in situ hybridization analyses. Large amounts of the otx1 homeoprotein mRNA are found in fertilized uncleaving eggs. The otx3 homeoprotein mRNA appears in the embryonic shield, the site of the organizer. In the developing brain, three zebrafish otx mRNAs are distributed in the diencephalon and the midbrain, but their fine expression patterns are different. These results suggest that three zebrafish otx homeoproteins, alone or in combination, may play roles in very early embryogenesis, gastrulation, and the development and subdivision of the diencephalon and the midbrain.

The optical laser setting for successful hemostatic transection of vessels using laser bipolar dissector (LBD) and the important factor in closure of the vessel stump were examined in abdo...

天然タイマイ甲の代替材を絹タンパクを用いて開発することを試み, タイマイ甲やホットプレス成形したフィプロインパウダーのDSC, 熱機械特性 (TMA), 曲げ強度などを調べた. その結果, 熱圧処理した含水タイマイ甲では30℃付近から軟化が始まり, 80℃付近から急激に軟化が進むことがTMA測定から分かった. このタイマイ甲のDSCの吸熱ピーク位置は45℃付近と, 70℃前後に存在した. また, 溶融板を作るためのフィブロインパウダーのホットプレス成形条件を検討し, 最適な水分含有率, 圧力, 温度, 処理時間はそれぞれ27%, 450kg/cm2, 160℃, 1分間であることが分かった. この条件で作成した溶融成形板はタイマイ甲と比較して, ほぼ同等の硬度 (ビッカース硬度約30) が得られた. しかし溶融成形板は, 風乾および含水加熱状態での曲げ強さが不足し, 透明度も低かった. これは, このフィブロインパウダーがホットプレスで完全には溶融していないことと, 溶融成形板がタイマイ甲のような多層構造をしていないためと推定した.

Objective Adenoviruses are used for gene transfer to salivary glands cells in vivo . We constructed an adenovirus vector that expressed a fusion protein of human Stim1 and the fluorescent protein mKO1 (Ad-Stim1-mKO1), and used it to investigate the molecular dynamics and functions of exogenously expressed proteins in living salivary acinar cells. Design Ad-Stim1-mKO1 was transferred to rat submandibular glands by retrograde ductal injection. Expression and distribution of Stim1-mKO1 were examined by confocal microscopy. In addition, the effects of Stim1-mKO1 on store-operated Ca 2+ entries were examined in fura-2-loaded cells. Results The expression of Stim1-mKO1 was detected in approximately 40% of rat submandibular acini, whereas the expression in HSY-EA1 cells was ∼80%. Confocal microscopy revealed Stim1-mKO1 fluorescence along the endoplasmic reticulum-like network in the cytoplasm of both HSY-EA1 and dispersed acinar cells. The depletion of intracellular Ca 2+ stores with thapsigargin (ThG), a sarcoplasmic/endoplasmic reticulum Ca 2+ ATPase (SERCA) inhibitor, led to the translocation of Stim1-mKO1 to the peripheral region in these cells. In addition, expression of Stim1-mKO1 enhanced store-operated Ca 2+ entry in these cells. Conclusions We succeeded in expressing Stim1-mKO1 fluorescent protein in the salivary glands of live animals by retrograde ductal injection of an adenoviral vector. This method allowed us to investigate the functions and molecular dynamics of these expressed molecules in living salivary acinar cells.

We describe the realization of oscillators combined with quartz's innate excellent long-term stability of frequency and frequency temperature characteristics by optimization of SAW oscillator's design conditions using quartz crystal substrates in application on groove structure. As a result, we could realize SAW oscillator with cubic frequency temperature curve, inflection point temperature of 28°C at is close to a room temperature and extremely small frequency variation of 11ppm in wide temperature range between −40°Cand +85°C. Further, we could get excellent long-term stability lower than 3ppm of frequency variation in 2000 hours high temperature environment test with 85°C. SAW resonator that is making up oscillator has gained good resonance characteristics such as Q=14,700 and CI=12Ω with 400MHz. We realized high-performance SAW oscillator on practical use with characteristics such as low jitter and low phase noise, in addition excelling in frequency temperature characteristics and frequency long-term stability by oscillating fundamental wave in high-frequency range with 100MHz to 700MHz.

In most non-excitable cells, 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 (B-SOC). In the wild-type (WT) DT40 cells, BCR stimulation with anti-IgM antibodies induced Ca(2+) release and subsequent Ca(2+) entry in the presence of 0.3 microM La(3+) which blocks CCE completely. In the inositol 1,4,5-trisphosphate receptor-deficient (IP(3)R-KO) cells, BCR stimulation elicited neither Ca(2+) release nor Ca(2+) entry. However, under pretreatment of thapsigargin (ThG), BCR stimulation induced La(3+)-resistant Ca(2+) entry into both WT and IP(3)R-KO cells. These results indicate that BCR stimulation and Ca(2+) store depletion work in concert to activate the La(3+)-resistant Ca(2+) entry pathway. B-SOC was inhibited by tyrosine kinase inhibitor, genistein. In addition, B-SOC was completely abolished in Stim1-deficient cells and was restored by overexpression of yellow fluorescent protein (YFP)-tagged Stim1, but was unaffected by double knockdown of Orai1/Orai2. These results demonstrate a unique non-CCE pathway, in which Ca(2+) entry depends on Stim1 and tyrosine kinase activation. It is likely that similar regulation of Ca(2+) entry occurs in other cell types including salivary gland cells.

Inositol 1,4,5-trisphosphate (IP3) 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 the present study, we have developed a series of IP3 biosensors that exhibit strong pH stability and varying affinities for IP3 ,a s well as a method for the quantitative measurement of cytosolic concentrations of IP3 ((IP3)i )i n single living cells. We applied this method to elucidate IP3 dynamics during agonist-induced Ca 2+ oscillations, and demonstrated cell type-dependent differences in IP3 dynamics ; a non-fluctuating rise in (IP3)i and repetitive IP3 spikes during Ca 2+ oscillations in COS-7 cells and HSY-EA1 cells, respectively. The size of the IP3 spikes in HSY-EA1 cells varied from 10 to 100 nM, and the (IP3)i spike peak was preceded by a Ca 2+ spike peak. These re- sults suggest that repetitive IP3 spikes in HSY-EA1 cells are passive reflections of Ca 2+ oscillations, and are unlikely to be essential for driving 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 tempo- ral regulations of IP3 and Ca 2+ . J. Med. Invest. 56 Suppl. : 357-361, December, 2009

The primary structure of the mouse glutamate receptor beta 2 subunit has been deduced by cloning and sequencing cDNA. The beta 2 subunit has structural characteristics common to the subunits of glutamate-gated ion channels. Expression of the cloned cDNA in Xenopus oocytes yields functional glutamate receptor channels selective for kainate.

We have previously identified two membrane proteins (36 kDa and 28 kDa) associated with ω-conotoxin GVIA-sensitive (N-type) calcium channels in the brain. Partial amino acid sequences and immunoblot analysis after sodium dodecyl sulfate-electrophoresis in urea indicated the presence of several isoforms (synaptocanalins) of the 36 kDa protein in the brain. The amino acid sequence of one form of the 36 kDa protein (named synaptocanalin I) deduced from its cDNA indicates that it is a novel protein with significanthomology to epimorphin, a protein involved in epithelial morphogenesis

A monoclonal antibody recognizing the alpha 2 delta complex of the dihydropyridine (DHP)-sensitive calcium channel of skeletal muscle immunoprecipitated most of the DHP receptor solubilized from bovine and rabbit brains, and bovine cardiac muscle. However, it did not significantly immunoprecipitate the high affinity omega-conotoxin receptor solubilized from these brains. These results indicate that the DHP receptor and the high affinity omega-conotoxin receptor are different molecules in mammalian brain.

We describe the synthesis of four novel metabolically stabilized analogues of Ins(1,4,5)P(3) based on the known cyclopentane pentaol tris(phosphate) 2: tris(phosphorothioate) 3, tris(methylenephosphate) 4, tris(sulfonamide) 5, and tris(sulfate) 6. Of these analogues, only the tris(phosphorothioate) 3 and parent tris(phosphate) 2 bound to the type I InsP(3)R construct. In addition, both the tris(phosphorothioate) 3 and parent tris(phosphate) 2 elicited calcium release in MDA MB-435 breast cancer cells. The Ins(1,4,5)P(3) agonist activities of these two compounds can be rationalized on the basis of computational docking of the ligands to the binding domain of the type I InsP(3)R.

The heterogeneity of salivary ductal cells, with regard to their sensitivity to Ca(2+)-mobilizing agonists, was visualized by multi-photon microscopy. Stimulation of isolated parotid ducts with 0.1 and 1 microM epinephrine (Epi) elevated the intracellular Ca(2+) levels ([Ca(2+)](i)) in approximately 30% and90% of the ductal cells, respectively. Of the 0.1 microM Epi-responsive cells, 80% responded rapidly to subsequent stimulation with 1 microM Epi. Similarly, threshold concentrations (0.5 or 1 microM) of phenylephrine (PhL), carbachol (CCh) or ATP, induced responses in approximately 20% of the ductal cells, and subsequent stimulations with 10 microM of the same agonist activated approximately 80% of ductal cells. These observations indicate that parotid ducts contain a certain subpopulation of cells, which exhibits particularly high sensitivity to these Ca(2+)-mobilizing agonists, compared to the remaining ductal cells. Sequential stimulation with threshold concentrations of PhL, CCh, and ATP induced Ca(2+) responses in approximately 33% of ductal cells. Of these responsive cells, the majority (69%) could only respond to one of the three agonists; while a small minority (9%) were capable of responding to all three agonists. These results indicate that low concentrations of PhL, CCh, and ATP activate different subpopulations of parotid ductal cells.

In the recent years, the importance of relatively low temperatures ranging from 400degC to 600degC is growing for the rapid thermal processes in semiconductor manufacturing. Measurement and evaluation techniques for the temperature distribution on the wafer surface within the manufacturing equipments are essential. Matsushita Electric Industrial Co., SENCorporation and NMIJ/AIST have done research on a novel temperature measurement method for this temperature region that utilizes the reordering phenomena of amorphous silicon, called the REAL method (Really Exposed Temperature Evaluation Using Reordering of Implanted Amorphous Si Layers). In this paper, we present the principle, the advantages, a trial calculation of the measurement uncertainty and the application of the REAL method

Human gingival fibroblasts (HGFs) express protease-activated receptor-1 (PAR-1) at high levels. In cultured HGFs, we studied the signaling pathway of thrombin-induced interleukin-6 (IL-6) production. The PAR-1 agonist peptide SFLLRN mimicked the thrombin-induced IL-6 production in the presence of amastatin, an aminopeptidase inhibitor. Thrombin or a combination of SFLLRN and amastatin also strikingly induced the expression of IL-6 mRNA. Although continuous exposure of HGFs to thrombin rapidly desensitized Ca(2+) signaling, the cells did not lose their ability to produce IL-6 in response to thrombin. Similarly, although treatment of HGFs with BAPTA-AM [1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester], an intracellular Ca(2+) chelator, markedly attenuated the thrombin-induced increase in intracellular Ca(2+) concentration, the same treatment did not suppress the thrombin-induced IL-6 production. However, thrombin-induced IL-6 production was strongly inhibited by the p38 mitogen-activated protein (MAP) kinase and tyrosine kinase inhibitors, and Western blotting analyses showed that thrombin stimulates p38 MAP kinase phosphorylation. Specific inhibitors that inhibit extracellular signal-regulated kinase 1/2 kinase, phosphatidylinositol 3-kinase, and RhoA kinase also partially suppressed the thrombin-induced IL-6 production, but the effects were smaller than those of the p38 MAP and tyrosine kinase inhibitors. Thus, thrombin induces HGFs to produce IL-6 by activating PAR-1, and the tyrosine kinase- and p38 MAP kinase-dependent pathways, rather than the Ca(2+) signaling pathway, may play a crucial role in the IL-6 production.

Principle of Gas Tungsten Arc Welding is quite simple. Electron, which is emitted from Tungsten electrode, hits into the metal, and the heat from this impingement produce enough heat for fusion welding. However, actual behaviors are quite complicated. Electron emission is much influenced by physical condition of cathode and ambient circumstance. Considerable electron emits from unbelievable location of the cathode, especially at the ignition stage when the cathode is not enough hot. The anode spot, where main electrons hit into the metal, is also much influenced by surface condition and purity of shielding gas. The anode spot is usually appeared at the center of molten pool where the temperature seems to be maximum value. But, it sometimes moves irregularly or hits on the solid surface. This situation is mainly caused by oxidized layer on the metal or molten pool. Some minor elements on the metal act as a kind of catalytic agent. It sometimes drastically changes weld geometry. An amount of erosion on cathode surface is increased with increased oxygen contents. The penetration depth is also increased with oxygen contents. Plasma condition is also changed, because cathode and anode conditions are closely connected with plasma condition. Dynamic behavior of arc-cathode and arc-anode reactions was observed by ultra-high speed video system in a chamber from vacuum to high pressure, where contents of gas are controlled. Effects of oxygen and minor elements on anode and cathode reactions were considered.Copyright © 2004 by ASME

We examined the function of GFP-IP(3)R3 (green fluorescent protein-tagged inositol 1,4,5-trisphosphate receptor type 3) in Ca(2+) release and entry using a mutant DT40 cell line (IP(3)R-KO) in which all three IP(3)R genes had been disrupted. GFP-IP(3)R3 fluorescence largely overlapped with the distribution of endoplasmic reticulum, whereas a portion of GFP-IP(3)R3 apparently co-localized with the plasma membrane. The application of IP(3) to permeabilized WT (wild-type) DT40 cells induced Ca(2+) release from internal stores. Although this did not occur in IP(3)R-KO cells it was restored by expression of GFP-IP(3)R3. In intact cells, application of anti-IgM, an activator of the BCR (B-cell receptor), or trypsin, a protease-activated receptor 2 agonist, did not cause any Ca(2+) response in IP(3)R-KO cells, whereas these treatments induced oscillatory or transient Ca(2+) responses in GFP-IP(3)R3-expressing IP(3)R-KO cells, as well as in WT cells. In addition, BCR activation elicited Ca(2+) entry in WT and GFP-IP(3)R3-expressing IP(3)R-KO cells but not in IP(3)R-KO cells. This BCR-mediated Ca(2+) entry was observed in the presence of La(3+), which blocks capacitative Ca(2+) entry. Thapsigargin depleted Ca(2+) stores and led to Ca(2+) entry in IP(3)R-KO cells irrespective of GFP-IP(3)R3 expression. In contrast with BCR stimulation, thapsigargin-induced Ca(2+) entry was completely blocked by La(3+), suggesting that the BCR-mediated Ca(2+) entry pathway is distinct from the capacitative Ca(2+) entry pathway. The present study demonstrates that GFP-IP(3)R3 could compensate for native IP(3)R in both IP(3)-induced Ca(2+) release and BCR-mediated Ca(2+) entry.

We examined the mechanism by which protease-activated receptor (PAR)-1 is desensitized by comparing the effect of thrombin and the soluble agonist peptide SFLLRN on Ca 2+ responses in HSY-EA1 cells. Thrombin-induced increases in cytosolic Ca 2+ concentrations ([Ca 2+ ] i ) returned to basal levels within 60 s, but SFLLRN generated a sustained [Ca 2+ ] i elevation. Interestingly, thrombin-desensitized cells partially retained their ability to respond to SFLLRN. We desensitized PAR-2 by pretreating cells with SLIGKV to confirm that this response was not due to PAR-2, which can recognize SFLLRN. The highly specific PAR-1 agonist peptide TFLLR also increased [Ca 2+ ] i in PAR-2-desensitized cells pretreated with thrombin. These observations indicate that thrombin disarms PAR-1 from further proteolytic activation, but leaves the receptor responsive for non-tethered ligands.

By reverse transcription (RT)-PCR analyses, human gingival fibroblasts (HGFs) were demonstrated to express mRNAs for protease-activated receptor-1 (PAR-1) and PAR-3, although the expression of PAR-3 was much weaker than that of PAR-1. The mRNAs for PAR-2 and PAR-4 were not found by RT-PCR. Furthermore, PAR activation was studied by monitoring cytosolic Ca(2+) concentration ([Ca(2+)]i) in cultured HGFs loaded with fura-2. At concentrations0.1 nM, alpha-thrombin caused a transient increase in [Ca(2+)]i in a concentration-dependent manner, and the maximum response was obtained with 10 nM alpha-thrombin. After the [Ca(2+)]i response, the HGFs were completely desensitized to the second stimulation with alpha-thrombin. The PAR-1 agonist peptide SFLLRN produced approximately the same transient [Ca(2+)]i response as alpha-thrombin. After stimulation with SFLLRN, the HGFs did not respond to alpha-thrombin, indicating that treatment with SFLLRN results in complete desensitization to alpha-thrombin. The PAR-2 and PAR-4 agonist peptides had no effect on [Ca(2+)]i in HGFs. These results suggest that alpha-thrombin-induced Ca(2+) mobilization in HGFs is solely mediated by PAR-1.

Principle of Gas Tungsten Arc Welding is quite simple. Electron, which is emitted from Tungsten electrode, hits into the metal, and the heat from this impingement produce enough heat for fusion welding. However, actual behaviors are quite complicated. Electron emission is much influenced by physical condition of cathode and ambient circumstance. Considerable electron emits from unbelievable location of the cathode, especially at the stage of the cathode is not enough hot. The anode spot, where main electrons hit into the metal, is also much influenced by surface condition. The anode spot is usually appeared at the center of molten pool where the temperature seems to be maximum value. But, it sometimes moves irregularly or hits on the solid surface. This situation is mainly caused by oxidized layer on the solid metal or molten metal. Some minor elements in/on the metal act as a kind of catalytic agent. It sometimes drastically changes weld geometry. Fundamental experiment to observe arc instability was carried out from vacuum to high pressure, where contents of gas are controlled. Effects of oxygen and minor elements on anode reactions were measured by high speed digital video cameras. Measured molten metal flow, plasma distribution and anode behavior during GTA welding will be discussed in detail.Copyright © 2003 by ASME

The expression of mRNA for amylase was examined using the reverse transcriptase-polymerase chain reaction (RT-PCR). An amylase product was strongly detected in parotid and pancreas, but less strongly in liver. The degree of identity between the PCR products was assessed by restriction-enzyme mapping using two restriction enzymes, EcoRI and ScaI, and DNA sequencing. The PCR product from pancreas was cut by both EcoRI and ScaI, while the products from parotid and liver were cut by EcoRI but not by ScaI. The sequence of the parotid product was 90.4% homologous to that of the pancreas, and 100% homologous to that of the liver. These results indicate that the same amylase mRNA may be expressed in parotid and liver. In addition, the expression of amylase mRNAs in other rat tissues was investigated using RT-PCR, and the sensitivity of each PCR product to ScaI was tested. A weak single band was detected in submandibular gland, sublingual gland and stomach. ScaI digestion cut the stomach product into two fragments, but had no effect on the submandibular and sublingual products. Thus, it may be possible to classify amylase isoenzymes into pancreatic and parotid types based on the sensitivity of their PCR products to ScaI.

Inositol 1,4,5-trisphosphate receptor (IP3R) is an intracellular Ca2+ channel that releases Ca2+ into the cytosol and its subcellular distribution is believed to have significant effects on Ca2+ signalling. We constructed a plasmid vector containing full-length rat type 3 IP3R linked to GFP (GFP-IP3R) for expression in mammalian cells. Western blot analyses revealed that the expressed fusion protein contained both GFP and full-length type 3 IP3R. Fluorescence confocal microscopy showed that the fluorescence of GFP-IP3R3 was distributed to reticular network structures, even after cell permeabilization with saponin. We further visualized intracellular membranes with DiOC6, a vital fluorescent marker for intracellular membranes, and provide evidence that the distribution of GFP-IP3R3 overlaps with the distribution of the endoplasmic reticulum. Our results indicate that GFP-IP3R3 can be used to visualize IP3R in living cells, and pave the way for subsequent mutational and functional studies.

Rat parotid acinar cells lacking zymogen granules were obtained by inducing granule discharge with the beta-adrenoceptor agonist isoproterenol. To assess whether zymogen granules are involved in the regulation of Ca(2+) signalling as intracellular Ca(2+) stores, changes in cytosolic free Ca(2+) ion concentration ([Ca(2+)](i)) were studied with imaging microscopy in fura-2-loaded parotid acinar cells lacking zymogen granules. The increase in [Ca(2+)](i) induced by muscarinic receptor stimulation was initiated at the apical pole of the acinar cells, and rapidly spread as a Ca(2+) wave towards the basolateral region. The magnitude of the [Ca(2+)](i) response and the speed of the Ca(2+) wave were essentially similar to those in control acinar cells containing zymogen granules. Western blot analysis of the inositol 1,4,5-trisphosphate receptor (IP(3)R) was performed on zymogen granule membranes and microsomes using anti-IP(3)R antibodies. The immunoreactivity of all three IP(3)Rs was clearly observed in the microsomal preparations. Although a weak band of IP(3)R type-2 was detected in the zymogen granule membranes, this band probably resulted from contamination by the endoplasmic reticulum (ER), because calnexin, a marker protein of the ER, was also detected in the same preparation. Furthermore, Western blotting and reverse transcriptase-PCR analysis failed to provide evidence for the expression of ryanodine receptors in rat parotid acinar cells, whereas expression was clearly detectable in rat skeletal muscle, heart and brain. These results suggest that zymogen granules do not have a critical role in Ca(2+) signalling in rat parotid acinar cells.