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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.

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.

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.

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.

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 .

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.

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.

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.

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.

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.

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.

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...

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.

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

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.

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.

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.

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.

The complete amino-acid sequences of two zebrafish emx homeoproteins containing a homeodomain homologous to that of the Drosophila empty spiracles head gap gene product have been determined by cloning and sequencing of cDNAs. The zebrafish emx1 and emx2 homeoproteins consist of 233 and 247 amino acids, respectively. The zebrafish emx1 and emx2 mRNAs are present at 12 h after fertilization, when the presumptive brain is in a simple tubular structure, and before first primary neurons appear. During brain development, the emx1 mRNA is localized in the dorsal telencephalon, whereas the emx2 mRNA is distributed in the dorsal telencephalon, parts of the diencephalon and the otocyst. The differential expression patterns of the two emx homeoprotein mRNAs may define the subdivisions of the zebrafish telencephalon.

Various kinds of 5'-O-unsaturated acyl 5-fluorouridines were synthesized to obtain 5-fluorouridine derivatives with low toxicity and high antitumor activity. Antitumor activity of the compounds against L-1210 leukemia in mice was examined, and the 5'-O-4-opentenoyl derivative showed the highest antitumor activity.

Although operative methods and management have been improved, but an appreciable number of cases are still subject to reoperation due to postoperative complication. We examined 92 cases of reoperative gastrointestinal surgery experienced in our department during a 14-years period.The reuslts were as follows: 1) Incidence of reoperation was 3% and there was difference between the previous and latter terms. 2) Although sutural insufficiency was decreased, postoperative bleeding and ileus were increased in the latter term. 3) Reoperation was done more frequently between 1 and 8 days in the previous term and between 14 and 21 days in the latter term. 4) Patients aged between 30 and 80 years accounted for as much as 77% of reoperative cases. 5) The prognosis for reoperation was unfavorable in malignant than in benign primary cases. 6) Of the 92 examined cases, 30 resulted in death, most of which involved reoperation in the early postoperative period. Old persons accounted for 50% of the cases. The prognosis in old patients depends upon the presence of asymptomatic functional disorders of organs.

A polyacrylamide gel system for human serum lipoprotein electrophoresis was applied successfully to the separation and isolation of canine serum lipoproteins employing a preparative gel slab method combined with slicing technique.With our gel system in which serum lipoproteins are stained prior to electrophoretic separation, the separation of lipoproteins was visible on polyacrylamide gel and the procedure was simple and accurate. Prestaining of serum lipoproteins did not disturb lipoprotein or lipoprotein lipid analyses. Canine serum lipoproteins were separated always into 4 fractions, i. e., 3 subfractions of α lipoprotein (albumin, α-1 and α-2 lipoproteins) and β lipoprotein.Alpha-1 lipoprotein is the principal fraction of serum lipoproteins of normal dogs. Lipoproteins and lipoprotein lipids were extracted from the gel blocks with sufficient recoveries to conduct further studies. Lipid extracts from each lipoprotein showed unique lipid composition differentiable from the extracts of other lipoproteins. Cross contamination of lipoprotein extracts seemed negligible if any as far as examined by lipoprotein lipid analysis or immunoelectrophoresis of the isolated lipoproteins. However, immunoelectrophoresis of whole sera and lipoprotein extracts against rabbit anti-canine immune sera showed partially discordant results suggesting complicated distribution of antigenic components.It was concluded that with the method explored in this work further studies on canine serum lipoproteins seem to yield valuable results.

A patient, female, 67 years old, with adenocarcinoma of Vater's papilla, in addition to arteriographic evidence of occlusion of the celiac axis underwent pancreaticoduodenectomy. In oder to assure sufficient blood flow to upper abdominal viscera, dorsal pancreatic artery was preserved. She has experienced no severe postoperative complications and lives a healthy life now, over 5 years after operation. Occlusion of the celiac axic itself causes no clinical problems, because upper abdominal viscera can be maintained by collateral blood flow through the pancreatic arcades from the superior mesenteric artery. But in case of pancreaticoduodenectomy sacrificing the pancreatic arcades may cause ischemia of upper abdominal viscera and attendant severe postoperative complications. The potential contribution of ischemia of upper abdominal viscera to postoperative complications remains unclear now. But the incidence of severe stenosis of the celiac axis due to arteriosclerotic diseases is high, so that we should consider the ischemic state of upper abdominal viscera after operation such as pancreaticoduodenectomy and total pancreatectomy to be a cause of postoperative complications. We also emphasize the need for preoperative celiac and superior mesenteric arteriography, especially that of on lateral view.

Intramural hematoma of duodenum is seldom reported in the Japanese literature. This disease occurs in children or following abdominal trauma in the majority of cases. A 18-year-old man with traumatic duodenal hematoma underwent successful conservative treatment for 59 days. In this report, we review the diagnosis and therapy of the 47 cases previously reported in Japan. In addition to upper gastrointestinal contrast roentgenogram, CT and ultrasonography were useful in the diagnosis and follow-up of this disease. As a result, conservative treatment is recently on the increase.

Plasma renin activity in acute respiratory acidosis and the effect of hexamethonium bromide was studied. Fourteen mongrel dogs were anesthetized with sodium pentobarbiturate and given 5% and 15% carbon dioxide in room air, successively. Hexamethonium bromide was given to 8 dogs prior to carbon dioxide inhalation. Arterial carbon dioxide partial pressure, oxygen partial pressure and pH were measured in addition to the determination of plasma renin activity. Plasma renin activity was elevated in moderate respiratory acidosis induced by 5% carbon dioxide inhalation from 37.5 +/- 8.8 ng/ml to 52.8 +/- 7.0 ng/ml. In severe respiratory acidosis produced by 15% carbon dioxide inhalation, plasma renin activity elevated further to 85.8 +/- 8.6 ng/ml. Plasma renin activity of the hexamethonium bromide treated dogs was 19.0 +/- 3.5 ng/ml during room air breathing. The activity was elevated to 26.0 +/- 6.4 ng/ml by 5% carbon dioxide inhalation and further to 57.3 +/- 5.9 ng/ml by 15% carbon dioxide inhalation. These findings may suggest that the elevation of plasma renin activity in acute respiratory acidosis induced by carbon dioxide inhalation is independent from sympathetic stimulation.