ガクカセン ニオケル エンショウセイ サイトカイン IL-β ノ プロセッシング

We have shown earlier that the expression of interleukin-1β (IL-1β) mRNA is increased in the submandibular gland (SMG) after injection of lipopolysaccharide (LPS). In this present study we suggest that tissue kallikrein mK13 is a candidate of the processing enzyme for the precursor of IL-1β (pro-IL-1β) in the SMG. High levels of 17.5- and 20-kDa IL-1β proteins were detected by Western blotting in the SMG, and saliva from LPS-injected mice. Despite this fact, pro-IL-1β with a molecular size of 35-kDa was not detected in this tissue. The protein for IL-1β-converting enzyme (ICE) was expressed only at a low level in the SMG as compared with its level in various epithelial tissues or LPS-stimulated macrophages. We detected the strong expression of members of kallikrein family (mK1, mK9, mK13, and mK22) in the SMG but not other tissues. By incubation with mK13, but not with mK1, mK9, nor mK22, the 35-kDa pro-IL-1β was cleaved to give two major products with molecular masses of 17.5- and 22- kDa, which production was inhibited by PMSF, a serine protease inhibitor, but not by ICE inhibitors. The pro-IL-β peptide segment, which includes the putative processing site, was hydrolyzed at Leu^ and Leu^ by incubation with mK13. The immunohistochemistry and an autonomic therapy experiment showed that IL-1β and kallikrein mK13 were co-localized in the secretory granules of granular convoluted tubular cells. Our present results thus suggest that kallikrein mK13 is a plausible candidate for the processing enzyme for pro-IL-1β in the SMG of mice

Phenotype Conversion of Salivary Gland Cells

Four major kallikreins (mK1, mK22, mK9, and mK13) were identified in the mouse submandibular gland (SMG). mK1, a true tissue kallikrein, was used as a protein marker to identify different types of SMG granular convoluted tubule (GCT) cells along with epidermal growth factor (EGF), nerve growth factor (NGF), and renin. Kallikrein mK1 was localized in a very small number (~5%) of GCT cells, which were scattered throughout the GCT, indicating that the majority of GCT cells are mK1-negative. Among mK1-positive cells, particularly strong signals were observed in a small number of narrow cells, recognized as slender granular cells (SG cells, Type IV), in the GCT. After postnatal development of the SMG, GCT cells are no longer uniform based on the bioactive substances (mK1, EGF, NGF, and renin) that they produce and secrete. GCT cells were classified into four subtypes, Types I–IV, and it became clear that these subtypes are complicatedly and reversibly converted by the endocrine hormones 5α-dihydrotestosterone (DHT) and triiodothyronine (T3). Duct segments with similar morphology or hormone dependency were recognized in the sublingual and parotid glands. The presence of duct cells with such characteristics is therefore a common feature of the three major salivary glands of rodents

Aquaporin-1 Deficiency Protects Against Myocardial Infarction by Reducing Both Edema and Apoptosis in Mice

Many studies have determined that AQP1 plays an important role in edema formation and resolution in various tissues via water transport across the cell membrane. The aim of this research was to determine both if and how AQP1 is associated with cardiac ischemic injury, particularly the development of edema following myocardial infarction (MI). AQP1+/+ and AQP1−/− mice were used to create the MI model. Under physiological conditions, AQP1−/− mice develop normally; however, in the setting of MI, they exhibit cardioprotective properties, as shown by reduced cardiac infarct size determined via NBT staining, improved cardiac function determined via left ventricular catheter measurements, decreased AQP1-dependent myocardial edema determined via water content assays and decreased apoptosis determined via TUNEL analysis. Cardiac ischemia caused by hypoxia secondary to AQP1 deficiency stabilized the expression of HIF-1α in endothelial cells and subsequently decreased microvascular permeability, resulting in the development of edema. The AQP1-dependent myocardial edema and apoptosis contributed to the development of MI. AQP1 deficiency protected cardiac function from ischemic injury following MI. Furthermore, AQP1 deficiency reduced microvascular permeability via the stabilization of HIF-1α levels in endothelial cells and decreased cellular apoptosis following MI

Dynamics of Salivary Gland AQP5 under Normal and Pathologic Conditions

Aquaporin 5 (AQP5) plays an important role in the salivary gland function. The mRNA and protein for AQP5 are expressed in the acini from embryonic days E13-16 and E17-18, respectively and for entire postnatal days. Ligation-reopening of main excretory duct induces changes in the AQP5 level which would give an insight for mechanism of regeneration/self-duplication of acinar cells. The AQP5 level in the submandibular gland (SMG) decreases by chorda tympani denervation (CTD) via activation autophagosome, suggesting that its level in the SMG under normal condition is maintained by parasympathetic nerve. Isoproterenol (IPR), a β-adrenergic agonist, raised the levels of membrane AQP5 protein and its mRNA in the parotid gland (PG), suggesting coupling of the AQP5 dynamic and amylase secretion-restoration cycle. In the PG, lipopolysaccharide (LPS) is shown to activate mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signalings and potentially downregulate AQP5 expression via cross coupling of activator protein-1 (AP-1) and NF-κB. In most species, Ser-156 and Thr-259 of AQP5 are experimentally phosphorylated, which is enhanced by cAMP analogues and forskolin. cAMP-dependent phosphorylation of AQP5 does not seem to be markedly involved in regulation of its intracellular traffcking but seems to play a role in its constitutive expression and lateral diffusion in the cell membrane. Additionally, Ser-156 phosphorylation may be important for cancer development

Relationships between feeding behaviors and emotions : an electroencephalogram (EEG) frequency analysis study

Feeding behaviors may be easily affected by emotions, both being based on brain activity; however, the relationships between them have not been explicitly defined. In this study, we investigated how emotional environments modulate subjective feelings, brain activity, and feeding behaviors. Electroencephalogram (EEG) recordings were obtained from healthy participants in conditions of virtual comfortable space (CS) and uncomfortable space (UCS) while eating chocolate, and the times required for eating it were measured. We found that the more participants tended to feel comfortable under the CS, the more it took time to eat in the UCS. However, the EEG emergence patterns in the two virtual spaces varied across the individuals. Upon focusing on the theta and low-beta bands, the strength of the mental condition and eating times were found to be guided by these frequency bands. The results determined that the theta and low-beta bands are likely important and relevant waves for feeding behaviors under emotional circumstances, following alterations in mental conditions

Protective effect of dehydroandrographolide on obstructive cholestasis in bile duct-ligated mice

Background: Dehydroandrographolide (DA) is the main contributor to the therapeutic properties of the medicinal plant Andrographis paniculata (AP). However, it is unknown whether DA has a hepatoprotective effect on obstructive cholestasis in mice and humans. Methods: We administered DA to mice for 5 days prior to bile duct ligation (BDL) and for the 7 days. Liver function markers, liver histology and necrosis, compensatory responses of hepatocytes, liver fibrosis and the expression of hepatic fibrogenesis markers were evaluated in BDL mice and/or human LX-2 cells. Results: Mice treated with DA demonstrated lower levels of serum alanine transarninase (ALT), milder liver damage, liver necrosis and fibrosis formation than in vehicle control with carboxymethylcellulose (CMC) mice after BDL. DA treatment also enhanced the Mrp3 expression of hepatocytes but not Mrp4 following BDL. Further, DA treatment in BDL mice significantly reduced liver mRNA and/or protein expression of Tgf-β, Col1a1, α-Sma and Mmp2. This result was also supported by hydroxyproline analysis. The molecular mechanisms of DA treatment were also assessed in human hepatic stellate cell line (LX-2 cell). DA treatment significantly inhibited Tgf-β-induced Col1a1, Mmp2 and α-Sma expression in human LX-2 cells. These data suggested that DA treatment reduced liver damage through development of a hepatic adaptive response and inhibition of the activation of HSCs, which led to a reduction in liver fibrosis formation in BDL mice. Conclusions: DA treatment protected against liver damage and fibrosis following BDL and might be an effective therapy for extrahepatic cholestasis due to bile duct obstruction

Aquaglyceroporins are involved in uptake of arsenite into murine gastrointestinal tissues

Aquaglyceroporins (AQGPs) are members of aquaporin (AQP) family and belong to a subgroup of this water channel family ; they are transmembrane proteins that transport water as well as glycerol and other solutes of small molecules. Recent studies have also identified that AQGPs are important transporters of trivalent metalloid in some mammalian cells. However, the uptake routes of arsenite in mammals are still less defined. In this study, to understand the routes of arsenite intake in mammals, mice were treated with Hg(II), glycerol, and As(III) and uptake of As(III) into the gastrointestinal tissues was measured. The level of inorganic arsenic (iAs) in gastrointestinal tissues after As(III) stimulation was much higher than Hg(II) +As(III) or glycerol+As(III) group. RT-PCR results showed that AQGPs were extensively expressed in gastrointestinal tissues of mice. We also treated Caco-2 cells with Hg(II) and As(III) ; the level of iAs in a group treated with Hg(II)+As(III) decreased compared with As(III)-treated group. Our results suggested that AQGPs could be important transporters in arsenite uptake into gastrointestinal tissues of mice, but more data are need to prove if AQGPs is the only pathway involved in As transport in mammals or just one of them

Salivary gland development : its mediation by a subtilisinlike proprotein convertase, PACE4

The submandibular gland (SMG) develops under the epithelial-mesenchymal interaction. Its process is regulated by various growth/differentiation factors, which are synthesized as inactive precursors and activated via the limited proteolysis at their multi basic amino acid site(s) such as Arg-X-Lys/Arg-Arg. Although many of these processing steps are elucidated to be catalyzed by subtilisin-like proprotein convertases (SPCs), little is known about the role of SPCs in the SMG development. Here, we focused upon the physiological role of PACE4 (SPC4), a member of SPC family, in the SMG development. In the organ culture system of rat embryonic SMG (E15), Dec-RVKR-CMK, a potent inhibitor for SPCs, inhibited the salivary branching and the expression of an exocrine gland type water channel, AQP5. However, other peptidyl-CMKs and inhibitors for trypsin-like serine proteases including leupeptin did not affect the salivary branching and AQP5 expression. Dec-RVKR-CMK also suppressed the expression of PACE4, but not furin, another member of the family. The specific antibody for the catalytic domain of PACE4 suppressed the salivary branching and AQP5 expression similarly. These inhibitory effects of Dec-RVKR-CMK were partially rescued by the addition of recombinant BMP2 whose precursor is a candidate for the physiological substrates of PACE4. Further, the transcriptional silencing of PACE4 by its specific siRNAs caused the suppression of both the salivary branching and AQP5 expression in the present organ culture system. These observations strongly support the idea that PACE4 mediates the SMG development

Arsenic trioxide induces expression of BCL-2 expression via NF-κB and p38 MAPK signaling pathways in BEAS-2B cells during apoptosis

Inorganic arsenic compounds are environmental toxicants that are widely distributed in air, water, and food. B-cell lymphoma 2 (BCL-2) is an oncogene having anti-apoptotic function. In this study, we clarify that BCL-2, as a pro-apoptotic factor, participates in As2O3-induced apoptosis in BEAS-2B cells. Specifically, As2O3 stimulated the expression of BCL-2 mRNA and protein in a dose-dependent manner which was highly accumulated in the nucleus of BEAS-2B cell together with chromatin condensation and DNA fragmentation during apoptosis. Mechanistically, the process described above is mediated through the NF-κB and p38 MAPK signaling pathways, which can be abated by corresponding inhibitors, such as BAY11–7082 and SB203580, respectively. Additionally, BAY11–7082, actinomycin D, and cycloheximide have inhibitory effects on As2O3-induced expression of BCL-2 mRNA and protein, and restore the cell viability of BEAS-2B cells. Suppression of BCL-2 protein activation by ABT-199 also restored viability of BEAS-2B cell in As2O3-induced apoptosis. Furthermore, As2O3 increased the level of BCL-2 phosphorylation. These results suggest that in BEAS-2B cells, As2O3-induced apoptosis is mainly dominated by BCL-2 upregulation, nuclear localization and phosphorylation. The study presented here provides a novel insight into the molecular mechanism of BCL-2-induced apoptosis

Down-regulation of submandibular gland AQP5 following parasympathetic denervation in rats

Following chorda tympani denervation (CTD, parasympathetomy), the protein levels of aquaporin5 (AQP5) as well as AQP1 and Na+K+ATPase α-subunit in the rat submandibular gland (SMG) were found to be decreased significantly. However, the level of another membrane protein, dipeptidyl peptidase IV was not affected by CTD, suggesting a selective reduction of AQP5, AQP1, and Na+K+ATPase1α-subunit proteins by CTD. However, the AQP5 mRNA level was scarcely affected by CTD, which suggested that transcription process of AQP5 was unaffected by this operation. AQP5 protein was shown to be degraded in vitro by the extract of the SMG obtained from normal rat ; inhibitor experiments in vitro suggested cathepsin B was a responsible enzyme. Co-localization of AQP5 and LAMP-2, a lysosomal marker, implicated AQP5 is degraded in lysosomes. A significant increase in the protein levels of LC3-II, an autophagy marker, at day 1 after CTD, and co-localization of the LC3 protein and AQP5, suggested that CTD activated autophagy of SMG, leading to AQP5 degradation