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 AsO-induced apoptosis in BEAS-2B cells. Specifically, AsO 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 AsO-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 AsO-induced apoptosis. Furthermore, AsO increased the level of BCL-2 phosphorylation. These results suggest that in BEAS-2B cells, AsO-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.


Arsenite-induced downregulation of occludin in mouse lungs and BEAS-2B cells via the ROS/ERK/ELK1/MLCK and ROS/p38 MAPK signaling pathways.

inhibits occludin expression in vivo and in vitro at least partially via the ROS/ERK/ELK1/MLCK and ROS/p38 MAPK signaling pathways.


Dysfunction of pulmonary epithelial tight junction induced by silicon dioxide nanoparticles via the ROS/ERK pathway and protein degradation.

Silica nanoparticles (SiNPs) are one of the most widely used types of nanoparticles across many industrial sectors, and are known to be present in the air year-round. In this study, we aimed to evaluate the potential adverse effects of SiNP exposure on pulmonary epithelial tight junctions, which serve as a critical barrier between the respiratory system and the circulatory system. In vivo studies confirmed that SiNPs decreased the protein expression levels of zonula occludens 1 (ZO-1), zonula occludens 2 (ZO-2), and occludin in the lungs of C57BL/6 mice. In vitro studies showed that SiNPs not only decreased the mRNA and protein expression of ZO-1 and ZO-2, but also decreased the protein expression of occludin in human bronchial epithelial (BEAS-2B) cells. In addition, SiNP exposure increased reactive oxygen species (ROS) production and activated extracellular regulated protein kinases (ERKs) and c-Jun N-terminal kinase (JNK). The inhibition of ROS and ERKs effectively protected the SiNP-induced downregulation of ZO-1 mRNA and protein expression, but had no effect on ZO-2 or occludin expression. SiNP-induced matrix metalloproteinase 9 (MMP9) protein expression appeared to be involved in occludin proteolytic degradation, in addition to SiNP-induced direct occludin protein degradation. The present study suggests that SiNPs disturb pulmonary epithelial tight junction structure and function via the ROS/ERK pathway and protein degradation.


Expression and LPS-Induced Elevation of Nod2 and Calprotectin in the Submandibular Gland of Wild-Type and TLR4-Knockout Male Mice

Journal of Research and Practice in Dentistry


Effects of isoproterenol on aquaporin 5 levels in the parotid gland of mice in vivo.

In the membrane fraction of mouse parotid gland (PG), the protein level of aquaporin 5 (AQP5), a member of the water channel family, was increased by injection (ip) of isoproterenol (IPR), a β-adrenergic agonist, at 1 h, and stayed at high levels until 6 h; this change occurred simultaneously as amylase secretion. The AQP5 level then decreased and returned toward the original level at 12-48 h. After IPR injection, the AQP5 mRNA gradually increased and reached a maximum at 24 h. The facts suggest a rapid appearance of AQP5 at plasma membrane by IPR and subsequent degradation/metabolism by activation of proteolytic systems. Pretreatment of animals with two calpain inhibitors, N-Ac-Leu-Leu-methininal (ALLM) and calpeptin, as well as a protein synthesis inhibitor, cycloheximide (CHX), significantly suppressed the IPR-induced AQP5 degradation in the PG membrane fraction; such suppression was not observed by two proteasome inhibitors, MG132 and lactacystin, or the lysosome denaturant chloroquine, although most of these inhibitors increased AQP5 protein levels in unstimulated mice. The AQP5 protein was also degraded by μ-calpain in vitro. Furthermore, we demonstrated that μ-calpain was colocalized with AQP5 in the acinar cells by immunohistochemistry, and its activity in the PG was increased at 6 h after IPR injection. These results suggest that the calpain system was responsible for IPR-induced AQP5 degradation in the parotid gland and that such a system was coupled to the secretory-restoration cycle of amylase in the PG.

American Journal of Physiology, Endocrinology and Metabolism


Roles of AQP5/AQP5-G103D in carbamylcholine-induced volume decrease and in reduction of the activation energy for water transport by rat parotid acinar cells

In order to assess the contribution of the water channel aquaporin-5 (AQP5) to water transport by salivary gland acinar cells, we measured the cell volume and activation energy (E (a)) of diffusive water permeability in isolated parotid acinar cells obtained from AQP5-G103D mutant and their wild-type rats. Immunohistochemistry showed that there was no change induced by carbamylcholine (CCh; 1 μM) in the AQP5 detected in the acinar cells in the wild-type rat. Acinar cells from mutant rats, producing low levels of AQP5 in the apical membrane, showed a minimal increase in the AQP5 due to the CCh. In the wild-type rat, CCh caused a transient swelling of the acinus, followed by a rapid agonist-induced cell shrinkage, reaching a plateau at 30 s. In the mutant rat, the acinus did not swell by CCh challenge, and the agonist-induced cell shrinkage was delayed by 8 s, reaching a transient minimum at around 1 min, and recovered spontaneously even though CCh was persistently present. In the unstimulated wild-type acinar cells, E (a) was 3.4 ± 0.6 kcal mol(-1) and showed no detectable change after CCh stimulation. In the unstimulated mutant acinar cells, high E (a) value (5.9 ± 0.1 kcal mol(-1)) was detected and showed a minimal decrease after CCh stimulation (5.0 ± 0.3 kcal mol(-1)). These results suggested that AQP5 was the main pathway for water transport in the acinar cells and that it was responsible for the rapid agonist-induced acinar cell shrinkage and also necessary to keep the acinar cell volume reduced during the steady secretion in the wild-type rat.

Pflügers Archiv : European Journal of Physiology


Induction of Sca-1 via activation of STAT3 system in the duct cells of the mouse submandibular gland by ligation of the main excretory duct

To examine the very initial step that takes place immediately after tissue injury and is linked to tissue regeneration, we employed the submandibular gland (SMG), which was injured by ligation of its main excretory duct (MED). Ligation of the MED of the SMG in mice induced the expression of Sca-1, a protein marker of hematopoietic stem cells. In the normal gland, a low level of Sca-1 was expressed, which was localized predominantly in the excretory duct cells. At 1 day after ligation, Sca-1 expression increased prominently in almost all of cells in the duct system, but not in the acinar cells. The level of Sca-1 mRNA had begun to increase at 6 h after ligation and continuously rose thereafter until it reached a plateau, which occurred ∼12 h after ligation. STAT3 phosphorylated at its tyrosine-705 (p-STAT3) in the ligated gland increased immediately after ligation, and it was localized in the nuclei of all duct cells. The results of an EMSA revealed the specific binding of a nuclear extract to the sequence of the γ-interferon activation site (GAS) present in the Sca-1 promoter and confirmed that such binding increased after ligation. Thus the present study suggests that STAT3, having been phosphorylated following MED ligation, was transferred to the nucleus, where it bound to the GAS element in the promoter of Sca-1 gene, resulting in promotion of Sca-1 gene expression. Actual prevention of STAT3 phosphorylation reduced the ligation-induced Sca-1 elevation.

American Journal of Physiology, Gastrointestinal and Liver Physiology


Lipopolysaccharide-mediated induction of calprotectin in the submandibular and parotid glands of mice

S100A8 and S100A9 constitute a heterodimeric protein, calprotectin. The mRNAs of S100A8 and S100A9, being expressed at minimal levels in the submandibular and parotid glands (SMG and PG, respectively) of C3H/HeN mice, were induced strongly and transiently by lipopolysaccharide (LPS). Among the mRNAs of members of the S100 protein family examined, those of S100A8 and S100A9 were specifically induced by LPS in the salivary glands. The induction was assumed to be mediated via toll-like receptor 4 (TLR4), since their elevation was limited in C3H/HeJ mice, a TLR4-mutant strain. These proteins became expressed in the granular convoluted tubular cells and striated duct cells in the SMG, and in both acinar and duct cells in the PG (all in the cytoplasm). The salivary calprotectin level was not increased by LPS treatment, implying that elevated calprotectin was not secreted into the saliva and that they may function in microcellular environment of the salivary gland.


Novel phosphorylation of aquaporin-5 at its threonine 259 through cAMP signaling

Aquaporin-5 (AQP5), a water channel, plays key roles in salivary secretion. The novel phosphorylation of AQP5 was investigated by using human salivary gland (HSG) cells and mouse salivary glands. In the HSG cells stably transfected with a wild-type mouse AQP5 construct, a protein band immunoreactive with antibody against phosphorylated PKA substrate was detected in the AQP5 immunoprecipitated sample, and its intensity was enhanced by short-term treatment of the cells with 8-bromo-cAMP, forskolin, or phorbol 12-myristate 13-acetate, but not by that with A23187 calcium ionophore. Such enhancement was inhibited in the presence of H-89, a PKA inhibitor. An AQP5 mutant (AQP5-T259A) expressed by transfection of HSG cells was not recognized by anti-phosphorylated PKA substrate antibody, even when the cells were stimulated with the protein kinase activators. Immunoblotting and immunofluorescence studies using a specific antibody detecting AQP5 phosphorylated at its Thr259 demonstrated that AQP5 was rapidly and transiently phosphorylated at the apical membrane of acinar cells in the submandibular and parotid glands after administration of isoproterenol, but not pilocarpine. Furthermore, both AQP5 and AQP5-T259A were constitutively localized at the plasma membrane in HSG cells under the resting and forskolin-stimulated conditions. These results suggest that AQP5 is phosphorylated at its Thr259 by PKA through cAMP, but not Ca(2+), signaling pathways, and that this phosphorylation does not contribute to AQP5 trafficking in the salivary gland cells.

American Journal of Physiology, Cell Physiology


Induction of Sca-1 in the duct cells of the mouse submandibular gland by obstruction of the main excretory duct

The effect of ligation of the main excretory duct (MED) of the mouse submandibular gland (SMG) on the expression of Sca-1, a stem cell antigen, was examined by Western blotting and immunohistochemistry. By Western blotting, the expression of Sca-1 with a molecular weight of 18 kDa was identified in the normal gland. At 1 day post-ligation, the expression level of Sca-1 was strongly increased in the experimental gland and weakly in the contralateral gland, and such expression in both glands decreased at 6 days. By immunohistochemistry, Sca-1 was detected weakly in the apical membrane of excretory duct (ED) cells of the SMG under the normal condition. By duct ligation, Sca-1 became expressed strongly in most cells of the two major duct systems, i.e., the striated duct (SD) and granular convoluted tubules (GCT), but was not detected in the acinar (Ac) cells. By fluorescence-activated cell sorter (FACS) analysis, the number of side population (SP) cells in this gland was found to be increased by ligation. These results imply that Sca-1-positive cells may have a role in the duct cell proliferation in the regeneration step elicited by MED ligation-induced injury.


Regulation of antimicrobial peptide expression in human gingival keratinocytes by interleukin-1α

In the oral cavity, mucosal keratinocytes resist bacterial infection, in part, by producing broad-spectrum antimicrobial peptides (AMPs) including defensin, adrenomedullin and calprotectin. Epidermal keratinocyte expression of many AMPs increases in response to interleukin-1α (IL-1α). IL-1α is produced by epidermal keratinocytes and regulates cell differentiation. To better understand innate immunity in the oral cavity, we sought to determine how IL-1α might regulate expression of AMPs by human gingival keratinocytes (HGKs) using DNA microarray and Western blot analyses. HGKs from three subjects expressed eleven AMPs, including S100A7, S100A8, S100A9, S100A12, secretory leucocyte protease inhibitor, lipocalin 2 (LCN2), cystatin C and β-defensin 2. Of the expressed AMPs, S100A7, S100A12 and LCN2 were up-regulated by IL-1α (inducible AMPs); the other AMPs were considered to be constitutive. Human gingival keratinocytes, therefore, express constitutive and IL-1α-inducible AMPs to provide a rapid and robust innate response to microbial infection.


Effects of naturally occurring G103D point mutation of AQP5 on its water permeability, trafficking, and cellular localization in the submandibular gland of rats

AQPs (aquaporins) are water channel proteins that are expressed in almost all living things. In mammalians, 13 members of AQPs (AQP0-12) have been identified so far. AQP5 is known to be expressed mostly in the exocrine cells, including the salivary gland acinar cells. A naturally occurring point mutation (G308A, Gly103 > Asp103) was earlier found in the rat AQP5 gene [Murdiastuti, Purwanti, Karabasil, Li, Yao, Akamatsu, Kanamori and Hosoi (2006) Am. J. Physiol. 291, G1081-G1088]; in this mutant, the rate of initial saliva secretion under stimulated and unstimulated conditions is less than that for the wt (wild-type) animals. Here the mutant molecule was characterized in detail. Using the Xenopus oocyte system, we demonstrated the mutant AQP5 to have water permeability almost the same as that of the wt molecule. Mutant and wt AQP5s, tagged with GFP (green fluorescent protein; GFP-AQP5s) and expressed in polarized MDCK-II (Madin-Darby canine kidney II) cells, first appeared in the vesicular structure(s) in the cytoplasm, and were translocated to the upper plasma membrane or apical membrane during cultivation, with the mutant GFP-AQP5 being translocated less efficiently. Thapsigargin and H-89 both induced translocation in vitro of either molecule, whereas colchicine inhibited this activity; the fraction of cells showing apical localization of mutant GFP-AQP5 was less than that showing that of the wt molecule under any of the experimental conditions used. In the mutant SMG (submandibular gland) tissue, localization of AQP5 in the apical membrane of acinar cells was extremely reduced. Vesicular structures positive for AQP5 and present in the cytoplasm of the acinar cells were co-localized with LAMP2 (lysosome-associated membrane protein 2) or cathepsin D in the mutant gland, whereas such co-localizations were very rare in the wt gland, suggesting that the mutant molecules largely entered lysosomes for degradation. Replacement of highly conserved hydrophobic Gly103 with strongly hydrophilic Asp103 in rat AQP5, though it did not affect water permeability, may possibly have resulted in less efficient membrane trafficking and increased lysosomal degradation, leading to its lower expression in the apical membrane of the acinar cells in the SMG.


Roles of lysosomal proteolytic systems in AQP5 degradation in the submandibular gland of rats following chorda tympani parasympathetic denervation

Chorda tympani denervation (CTD) of rats was earlier shown to result in loss of submandibular gland (SMG) weight (at only 1 wk) and in continued reduction in aquaporin 5 (AQP5) protein expression (until 4 wk), without affecting its mRNA synthesis (Li X, Azlina A, Karabasil MR, Purwanti N, Hasegawa T, Yao C, Akamatsu T, Hosoi K. Am J Physiol Gastrointest Liver Physiol 295: G112-G123, 2008). The present study indicated that despite elevation of bax, a proapoptosis protein, by CTD, the operation also increased the level of bcl-2, an antiapoptosis protein, in the SMG. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL assay) showed no increase in the number of apoptotic cells in the SMG. CTD, however, induced strongly and transiently (at 1-3 days) the protein expression of LC3B-II, a marker protein of autophagosomes, suggesting that the reduction in the gland weight was due to onset of autophagy by CTD. Upon CTD, Lamp2, a lysosomal marker, gradually increased in amount, reaching a peak at the 14th day. Immunohistochemical analysis revealed an increase in the number of lysosome-like structures positive for both AQP5 and Lamp2 in the acinar cells of the SMG after CTD; similar changes were observed also for AQP5 and LC3Bs. These data suggest that AQP5 in the SMG entered autophagosomes and/or lysosomes for degradation upon CTD. In vitro AQP5-degrading activity was found in the SMG extracts, and such activity was shown to be increased by CTD. Inhibitor experiments implied cathepsins B and L to be candidate enzymes for this degradation under normal and CTD conditions, respectively.


Potential down-regulation of salivary gland AQP5 by LPS via cross-coupling of NF-κB and p-c-Jun/c-Fos

The mRNA and protein levels of aquaporin (AQP)5 in the parotid gland were found to be potentially decreased by lipopolysaccharide (LPS) in vivo in C3H/HeN mice, but only weakly in C3H/HeJ, a TLR4 mutant mouse strain. In the LPS-injected mice, pilocarpine-stimulated saliva production was reduced by more than 50%. In a tissue culture system, the LPS-induced decrease in the AQP5 mRNA level was blocked completely by pyrrolidine dithiocarbamate, MG132, tyrphostin AG126, SP600125, and partially by SB203580, which are inhibitors for IkappaB kinase, 26S proteasome, ERK1/2, JNK, and p38 MAPK, respectively. In contrast, the expression of AQP1 mRNA was down-regulated by LPS and such down-regulation was blocked only by SP600125. The transcription factors NF-kappaB (p65 subunit), p-c-Jun, and c-Fos were increased by LPS given in vivo, whereas the protein-binding activities of the parotid gland extract toward the sequences for NF-kappaB but not AP-1-responsive elements present at the promoter region of the AQP5 gene were increased by LPS injection. Co-immunoprecipitation by using antibody columns suggested the physical association of the three transcription factors. These results suggest that LPS-induced potential down-regulation of expression of AQP5 mRNA in the parotid gland is mediated via a complex(es) of these two classes of transcription factors, NF-kappaB and p-c-Jun/c-Fos.


Trafficking of GFP-AQP5 chimeric proteins conferred with unphosphorylated amino acids at their PKA-target motif (152SRRTS) in MDCK-II cells

Three constructs having mutated PKA-target motif at (152)SRRTS of AQP5, an exocrine type water channel, were prepared and fused to C-terminus of green fluorescence protein cDNA to examine the effects of blocking of phosphorylation at (152)SRRTS (a consensus PKA-target motif of AQP5) on translocation or trafficking of the chimeric proteins expressed in the Madin-Darby canine kidney-II (MDCK-II) cells. H-89 treatment increased translocation of wild-type GFP-AQP5 to the apical membrane. All 3 mutant molecules translocated 1.5 to 2 times more than the control wild-type GFP-AQP5. Colchicine but not cytochalasin B inhibited the translocation of wild-type GFP-AQP5. Present results suggest dephosphorylation of this consensus sequence increase GFP-AQP5 translocation, and that microtubules but not microfilaments are involved in this event.

The Journal of Medical Investigation : JMI


Immunocytochemical study of granular duct cells with a hormonally enhanced granular cell phenotype in the mouse parotid gland

In the parotid glands (PGs) of intact male mice (12 weeks of age, ICR strain), immunofluorescence labels for a true tissue kallikrein, mK1, and for nerve growth factor (NGF) were recognized through the subluminal edges of the striated duct (SD) segments and interlobular duct segments. Because of their small size, secretory granules were not detectable by light microscopy in any of the duct cells. Full-fledged granular cells, containing large secretory granules that were visible by light microscopy, were induced in the SD segments of male mice after the injection of 5alpha-dehydrotestosterone (DHT) and triiodothyronine (T(3)), given either alone or in combination every other day for 2 weeks. A stronger effect was detected in the mice that were concomitantly injected with DHT and T(3), and more abundant, fully developed granular cells appeared in the SD segments of these mice. These full-fledged granular cells were immunoreactive for mK1, NGF, and epidermal growth factor, but not for renin. The present results indicate that some of the SD cells with small granules in the mouse PG can develop a granular cell phenotype, producing more kinds of growth factors, as a result of the actions of androgen and thyroid hormone.


Inhibition and transcriptional silencing of a subtilisin-like proprotein convertase, PACE4/SPC4, reduces the branching morphogenesis of and AQP5 expression in rat embryonic submandibular gland

The submandibular gland (SMG) develops through the epithelial-mesenchymal interaction mediated by many growth/differentiation factors including activin and BMPs, which are synthesized as inactive precursors and activated by subtilisin-like proprotein convertases (SPC) following cleavage at their R-X-K/R-R site. Here, we found that Dec-RVKR-CMK, a potent inhibitor of SPC, inhibited the branching morphogenesis of the rat embryonic SMG, and caused low expression of a water channel AQP5, in an organ culture system. Dec-RVKR-CMK also decreased the expression of PACE4, a SPC member, but not furin, another SPC member, suggesting the involvement of PACE4 in the SMG development. Heparin, which is known to translocate PACE4 in the extracellular matrix into the medium, and an antibody specific for the catalytic domain of PACE4, both reduced the branching morphogenesis and AQP5 expression in the SMG. The inhibitory effects of Dec-RVKR-CMK were partially rescued by the addition of recombinant BMP2, whose precursor is one of the candidate substrates for PACE4 in vivo. Further, the suppression of PACE4 expression by siRNAs resulted in decreased expression of AQP5 and inhibition of the branching morphogenesis in the present organ culture system. These observations suggest that PACE4 regulates the SMG development via the activation of some growth/differentiation factors.


Hypophysectomy and hormonal therapy modulate mK1-immunoreactive duct cells in the mice sublingual glands

The immunocytochemical localization of a true tissue kallikrein, mK1, in mouse sublingual glands (SLGs) was examined following hypophysectomy and hormonal replacement therapy. In the glands of intact mice (14 weeks of age), mK1 was detected in the striated ducts (SDs). Full-fledged granular cells were scattered in the SDs of male mice (but not in those of female mice), showing a cellular mosaic distribution of mK1 with some being positive and others being negative. mK1 was also detected in transitional-type granular cells, though the secretory granules were too small and scarce to be visible by a light microscopy. Hypophysectomy in male mice resulted in the atrophy and loss of secretory granules in many SD cells. Granulation recovered after the repeated injection of 5alpha-dihydrotestosterone (DHT), 3,5,3'-triiodo-L: thyronine (T3), and dexamethasone (Dex), given either alone or in combination to the hypophysectomized mice. The concomitant injection of DHT and T3, with or without Dex, resulted in the reappearance of the full-fledged granular cells, only some of which were mK1-positive. Electron microscopy revealed mK1 to be present exclusively in the secretory granules of these mK1-positive cells, and no ultrastructural differences were observed between mK1-positive and mK1-negative full-fledged granular cells. These results show that the differentiation of the granular cell phenotype in the mouse SLG duct system requires the concomitant action of androgen and thyroid hormone and retards mK1 synthesis.


Degradation of submandibular gland AQP5 by parasympathetic denervation of chorda tympani and its recovery by cevimeline, an M3 muscarinic agonist

By chorda tympani denervation (CTD, parasympathectomy), the aquaporin 5 (AQP5), but not AQP1, protein level in the rat submandibular gland (SMG) was significantly decreased, dropping to 37% of that of the contralateral gland at 4 wk. The protein levels of AQP5 and AQP1 were not significantly affected by denervation of the cervical sympathetic trunk (sympathectomy). Administration of cevimeline hydrochloride, an M3 muscarinic receptor agonist (10 mg/kg for 7 days po), but not pilocarpine (0.3 mg/kg for 7 days po), recovered the AQP5 protein level reduced by CTD and increased the AQP1 protein level above the control one. The mRNA level of AQP5 was scarcely affected by CTD and cevimeline hydrochloride administration. Administration of chloroquine (50 mg/kg for 7 days po), a denaturant of lysosomes, increased the AQP5 protein level reduced by CTD. An extract obtained from the submandibular lysosomal fraction degraded the AQP5 protein in the total membrane fraction in vitro. These results suggest the possible regulation of the AQP5 protein level in the SMG by the parasympathetic nerves/M3 muscarinic receptor agonist and imply the involvement of lysosomal enzymes, but not a transcriptional mechanism, in this regulation.

American Journal of Physiology, Gastrointestinal and Liver Physiology


Temporospatially regulated expression of subtilisin-like proprotein convertase PACE4 (SPC4) during development of the rat submandibular gland

The temporospatial expression of PACE4, a member of the mammalian subtilisin-like proprotein convertase family involved in the activation of growth/differentiation factors, was investigated by in situ hybridization during the development of the rat submandibular gland (SMG). At the initiation stage (day 15.5 of gestation; E15), PACE4 was intensely expressed in the submandibular epithelium, but weakly expressed in the mesenchymal cells. At E16 when the branching morphogenesis becomes obvious, the expression of PACE4 in the mesenchyme was further decreased, although its level in the submandibular epithelium had not changed remarkably from that at E15. During the next stage of embryonic development (E17-E20), PACE4 was expressed in the cells derived from the submandibular epithelium, which include the proacinar, terminal tubular, and presumptive ductal cells. In the perinatal SMG, PACE4 was still expressed intensely in the terminal portion of the SMG containing the proacinar and terminal tubular cells, whereas its expression in the ductal cells was obviously decreased at the second postnatal day (P2) and at P6. Acinar cells expressing no PACE4 appeared, and their numbers increased following their development (P9-P20). At P30 when the PACE4 expression in the acinar cells was completely suppressed, its expression in the ductal cells became intense again. This temporospatially regulated expression of PACE4 suggests its apparent association with the proliferation, differentiation, and establishment of functional acinar and ductal cells of the SMG.


A naturally occurring point mutation in the rat aquaporin 5 gene, influencing its protein production by and secretion of water from salivary glands

A greater than twofold diversity in the expression level of aquaporin 5 (AQP5) has been observed in the membrane fraction of the submandibular gland (SMG) in Sprague-Dawley rats (Murdiastuti K, Miki O, Yao C, Parvin MN, Kosugi-Tanaka C, Akamatsu T, Kanamori N, and Hosoi K. Pflügers Arch 445: 405-412, 2002). In the present study, breeding between brother and sister rats was repeated within high AQP5 producers and low ones to obtain inbred offspring. High- and low-producer rats from 3rd to 18th generations were used for experiments. By Western blotting, levels of AQP5 proteins in the parotid and lacrimal glands, and lungs were all low in low producers, whereas they were all high in high producers, implying genetic variations of the gene for this water channel. Despite this implication, AQP5 mRNA levels were almost the same between the two groups by Northern blotting, suggesting the irrelevance of transcriptional regulation for this diversity. AQP5 cDNAs from the SMGs of the two groups were sequenced. The nucleotide sequence of AQP5 cDNA from low producers indicated the existence of a point mutation at nt 308 (G308A), leading to a replacement of (103)Gly with (103)Asp in the third transmembrane domain, but no alteration was detected in the Kozak area. The existence of such a mutation was confirmed by the assessment of genomic DNA also. This mutation may have resulted in an abnormal membrane insertion or ineffective trafficking of AQP5, since the rats having this mutation showed extremely low membrane expression of AQP5 in the SMG acinar cells and decreased water secretion from their salivary glands.


Protein kinase A-regulated membrane trafficking of a green fluorescent protein-aquaporin 5 chimera in MDCK cells

The green fluorescent protein (GFP) of the jellyfish, Aeqorea victoria, was used as an autofluorescent tag to track the trafficking of aquaporin 5 (AQP5), an exocrine gland-type water channel. Two groups of chimeric proteins were constructed; one in which GFP was fused to the amino-terminus of AQP5 (GFP-AQP5) and the other, in which it was fused to the carboxyl terminus of it (AQP5-GFP). In each group, 2 chimeras were produced, a wild-type AQP5 with its normal sequence and a mutant AQP5 having a mutated amino acid at 259, i.e., GFP-AQP5-T259A and AQP5-GFP-T259A. They were used to transfect Madin-Darby canine kidney (MDCK) cells. The GFP-AQP5 chimera was localized in the intracellular vesicles, which trafficked to the plasma membrane in response to N(6), 2'-O-dibutyryladenosine 3', 5'-cyclic monophosphate (dbcAMP). Membrane trafficking was inhibited by N-[2-(p-bromocinnamylamino)ethyl]-5-isoquimolinesulfonamide (H-89) but not by palmitoyl-dl-carnitine chloride (PCC). In contrast, the AQP5-GFP chimera expressed in MDCK cells was localized constitutively on the plasma membrane. The cellular localization of the latter chimera was not affected by stimulation with dbcAMP in the presence or absence of H-89 or PCC. Replacement of Thr-259 with Ala-259 did not affect the dbcAMP-induced translocation of the chimeric protein, suggesting that phosphorylation of Thr-259 was not necessary for AQP5 trafficking under the present experimental conditions. Thus, the GFP-AQP5 chimera will be a useful tool to study AQP5 trafficking in vitro, whereas the constitutive membrane localization of the AQP5-GFP chimera suggests the importance of the carboxyl terminus of the AQP5 protein for its sorting, whether it is translocated to intracellular vesicles or to the plasma membrane.

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research


AQP and the control of fluid transport in a salivary gland

Experiments were performed with the perfused rat submandibular gland in vitro to investigate the nature of the coupling between transported salt and water by varying the osmolarity of the source bath and observing the changes in secretory volume flow. Glands were submitted to hypertonic step changes by changing the saline perfusate to one containing different levels of sucrose. The flow rate responded by falling to a lower value, establishing a new steady-state flow. The rate changes did not correspond to those expected from a system in which fluid production is due to simple osmotic equilibration, but were much larger. The changes were fitted to a model in which fluid production is largely paracellular, the rate of which is controlled by an osmosensor system in the basal membrane. The same experiments were done with glands from rats that had been bred to have very low levels of AQP5 (the principal aquaporin of the salivary acinar cell) in which little AQP5 is expressed at the basal membrane. In these rats, salivary secretion rates after hypertonic challenges were small and best modelled by simple osmotic equilibration. In rats which had intermediate AQP5 levels the changes in flow rate were similar to those of normal rats although their AQP5 levels were reduced.Finally, perfused normal glands were subject to retrograde ductal injection of salines containing different levels of Hg(2+) ions (0, 10 and 100 microM: ) which would act as inhibitors of AQP5 at the apical acinar membrane. The overall flow rates were progressively diminished with rising Hg(2+) concentration, but after hypertonic challenge the changes in flow rates were unchanged and similar to those of normal rats. All these results are difficult to explain by a cellular osmotic model but can be explained by a model in which paracellular flow is controlled by an osmosensor (presumably AQP5) present on the basal membrane.


Tissue kallikrein mK13 is a candidate processing enzyme for the precursor of interleukin-1b in the submandibular gland of mice

By using Western blot analysis, high levels of 17.5- and 20-kDa interleukin-1beta (IL-1beta) proteins were detected in the submandibular gland (SMG) of mice. Despite this fact, the amount of pro-IL-1beta protein, a precursor of IL-1beta, with a molecular size of 35 kDa in this tissue was below the detectable level, although strong expression of pro-IL-1beta mRNA was observed. A large amount of 17.5-kDa IL-1beta also appeared in the saliva of mice injected with lipopolysaccharide, suggesting that this IL-1beta is a secretory form produced by the SMG. The protein for IL-1beta-converting enzyme, a processing enzyme for pro-IL-1beta, was expressed only at a low level in the SMG as compared with its level in various epithelial tissues or lipopolysaccharide-stimulated macrophages. On the other hand, mK1, mK9, mK13, and mK22, members of the kallikrein family, were detected strongly in the SMG but not in other tissues. By incubation with mK13, but not with mK1, mK9, or mK22, the 35-kDa pro-IL-1beta was cleaved into two major products with molecular masses of 17.5 and 22 kDa, and production was inhibited by phenylmethylsulfonyl fluoride, a serine protease inhibitor, but not by IL-1beta-converting enzyme inhibitors. A peptide segment corresponding to amino acid residues 107-121 of mouse pro-IL-1beta (107WDDDDNLLVCDVPIR) was cleaved by incubation with mK13, generating two peptides, 107WDDDDNL and 114LVCDVPIR. Therefore, kallikrein mK13 would appear to hydrolyze pro-IL-1beta between its Leu113 and Leu114 residues. The results of immunohistochemistry and an autonomic therapy experiment showed that IL-1beta and kallikrein mK13 were co-localized in the secretory granules of granular convoluted tubular cells. Our present results thus suggest kallikrein mK13 is a plausible candidate for the processing enzyme for pro-IL-1beta in the SMG of mice.


Altered allergic cytokine and antibody response in mice treated with Bisphenol A

The objective of this study was to elucidate if Bisphenol A (BPA) administration modulates T helper (Th) cell component of immune responses in a mouse challenged with ovalbumin (OVA), a major food antigen. BALB/c mice, (6 weeks old, female) were orally given either OVA (OVA-fed) or water (Water-fed), immunized intraperitoneally with OVA and injected with either BPA in corn oil or the vehicle alone. After subsequent 2nd immunization, serum titers of total IgE, OVA-specific IgE, IgG, IgG1 IgG2a and ability of their splenocytes for production of interferon (IFN) -gamma, interleukin (IL) -4 and IL-12 were examined by ELISA. Lymphocyte proliferation assay against concanavalin A (Con A) or OVA was also performed for 3H-Thymidine incorporation. In Water-fed groups, treatment with BPA resulted in lower titers of total IgE (P<0.01) and higher levels IgG2a (P<0.05) followed by a higher IFN-gamma (P<0.05) and IL-12 (P<0.05) with an intact IL-4. When OVA-fed groups were examined, the compound did not change production of total and OVA-specific IgE and -IgG2a but resulted in lower production of IFN-gamma (P<0.05). Also, BPA resulted in impaired lymphocyte proliferation to Con A in Water-fed groups (P<0.05) but not in tolerated animals. The findings indicate that BPA results in augmentation of Th1 immune responses but no significant effect on an established tolerance to OVA.

The Journal of Medical Investigation : JMI


Downregulation of AQP2 expression in the kidney of polydipsic STR/N mice

Aquaporin-2 (AQP2) is responsible for the concentration of urine in the kidney collecting tubule under the regulation of vasopressin. The mRNA level of this water channel in polydipsic STR/N mice was extremely reduced compared with that in normal ICR mice. In male mice, reduction of the AQP2 mRNA level was not evident at 3 wk of age, at which time water intake was not increased. At 10 wk of age, however, the AQP2 mRNA level was reduced to 10% of that in control mice, whereas water intake was increased by 36%. At 44 wk, the water intake became five times that of the control ICR mice, and the AQP2 mRNA level in these polydipsic mice was only approximately 5% of control. Similar changes were observed in the AQP2 protein level, suggesting that the mRNA level of AQP2 reflects the protein level of AQP2. These inverse changes in the AQP2 mRNA level and water intake were also evident in female mice. The data imply that polydipsia in STR/N mice may have affected AQP2 mRNA transcription in the kidney, resulting in reduced AQP2 expression, which would contribute to a reduction in overretention of water.

American Journal of Physiology, Renal Physiology


Lipopolysaccharide-induced elevation and secretion of Interleukin-1b in the submandibular gland of male mice

The intraperitoneal injection of lipopolysaccharide (LPS) (400 microg/kg body weight) induced the expression of mRNAs of inflammatory cytokines such as interleukin (IL)-1beta, IL-6 and tumour necrosis factor (TNF)-alpha in the submandibular gland (SMG) of C3H/HeN mice but not that of C3H/HeJ mice, a mutant strain for Toll-like receptor-4 (TLR-4(-) mutant). The mRNA levels of these cytokines in the SMG of the wild-type mice increased as early as 3 hr after injection, peaked at 3-6 hr, and had decreased again by 24 hr. In this study, we particularly focused on IL-1beta, and induction by this endotoxin was investigated in detail. Denervation of the superior cervical trunk and chorda tympani nerve did not diminish the LPS-induced elevation of IL-1beta mRNA in the SMG, indicating the irrelevance of the central nervous system in this induction. TLR-4 mRNA and protein were shown to be strongly expressed in the SMG, suggesting the direct action of LPS on this gland. IL-1beta proteins were localized in the secretory granules of granular convoluted tubular (GCT) cells, and their molecular weights in the gland were 17.5 and 20 kDa. IL-1beta of the same size appeared in the saliva 6 hr after LPS injection in C3H/HeN but not in C3H/HeJ mice. The present study thus suggests that IL-1beta, an inflammation cytokine, is induced and secreted into the saliva in response to endotoxin injected intraperitoneally.


Acute phase protein induction by experimental inflammation in the salivary gland

The submandibular gland (SMG) is a major salivary gland, which plays an important role in maintenance the oral health. In this study, we intended to explore the role of the SMG's defense system of the animals in which experimental inflammation is induced. The levels of mRNAs for inflammation cytokines and acute phase proteins were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). The mRNAs for acute phase proteins were found to be increased in the SMG and extraorbital and intraorbital lacrimal gland (ELG and ILG) of rats at 24 h after subcutaneous injection of turpentine oil. The induction of mRNA for these inflammatory proteins by turpentine oil was preceded by a transient increase in the level of mRNAs for IL-1beta, IL-6 and TNF-alpha at 6 h after subcutaneous injection of the oil. Such cytokine induction was similarly seen by lipopolysaccharide (LPS) injection, and involvement of Toll-like receptor 4 (TLR4) was strongly suggested from experiment using C3H/HeJ mice, a TLR4-deficient mutant strain. The up-regulation of acute phase proteins and inflammation cytokines in the SMG, ELG and ILG by experimental inflammation suggests the existence of a strict defense system via the innate immune system in the SMG and other exocrine gland.


Subcellular redistribution of AQP5 by vasoactive intestinal polypeptide (VIP) in the Brunner's gland of the rat duodenum

Aquaporin (AQP)5, an exocrine-type water channel, was detected in the rat duodenum by Western blot analysis, and was localized by immunohistochemistry in the secretory granule membranes as well as in the apical and lateral aspects of the plasma membrane of Brunner's gland cells. Incubation of duodenal slices with vasoactive intestinal polypeptide (VIP) in vitro significantly increased the amount of AQP5 in the apical membrane fraction in a dose- and time-dependent manner with the amount reaching a plateau at 100 nM VIP and becoming near maximal after a 30-s incubation. Protein kinase inhibitors, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7, 50 muM), and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89; PKA-specific, 1 muM) blocked this increase, but PKC-specific inhibitor calphostin C did not, implying the involvement of PKA but not PKC in this cellular event. Intravenous injection with VIP (40 mug/kg body wt) provoked dilation of the lumen of the Brunner's gland at 2 and 7 min and increased the staining intensity of AQP5 in the apical and lateral membranes. AQP1 (both nonglycosylated and glycosylated forms) was also found to localize in the apical and basolateral membranes of cells of Brunner's gland. VIP, however, did not provoke any significant change in the AQP1 level in the apical membrane, as judged from the results of the above in vitro and in vivo experiments. These results suggest that VIP induced the exocytosis of granule contents and simultaneously caused translocation of AQP5 but not of AQP1 to the apical membrane in Brunner's gland cells.

American Journal of Physiology, Gastrointestinal and Liver Physiology


Additive and/or synergistic action (downregulation) of androgens and thyroid hormones on the cellular distribution and Localization of a true tissue kallikrein, mK1, in the mouse submandibular gland

We investigated the effects of 5alpha-dihydrotestosterone (DHT), 3,5,3'-triiodo-l-thyronine (T(3)), and dexamethasone (Dex) on the expression of mK1 in the granular convoluted tubule (GCT) cells of the submandibular gland (SMG) of hypophysectomized (Hypox) male mice by indirect enzyme-labeled antibody and immunogold antibody methods for light and electron microscopy. Hypox resulted in considerable atrophy of the GCT cells, which were always immunoreactive for mK1, and the cells were characterized by apical small dense secretory granules labeled with gold particles suggesting the presence of mK1, small Golgi apparatus, sparse rough endoplasmic reticulum (RER), and developed basal infoldings. Each of the hormones, DHT, T(3), and Dex, enhanced the GCT phenotype to various degrees in Hypox male mice. Both DHT alone and T(3) alone moderately inhibited mK1 synthesis by increasing the number of mK1-immunonegative GCT cells in Hypox males, but Dex alone had no inhibitory effect on mK1 synthesis. A significant trophic effect on GCT cells was induced by combined injection of DHT and T(3) or of all three hormones, and was reflected in the appearance of abundant large secretory granules, well-developed Golgi apparatus and RER, and reduced basal infoldings. Only a few such GCT cells were immunopositive for mK1, and the pattern of immunopositive and immunonegative cells very closely resembled the mosaic pattern seen in normal male GCTs. These findings suggested that the sexual dimorphism of mK1 expression and the morphological appearance of GCT cells can be induced by treatment with two hormones, DHT and T(3), but not by either of them alone. T(3) appears to have a permissive effect on committed GCT cells that results in downregulation of mK1 expression in these cells.

The Journal of Histochemistry and Cytochemistry


Immunocytochemical localization of mK1, a true tissue kallikrein, in the mouse parotid gland: sexual dimorphism and effects of castration and hypophysectomy

In the normal parotid glands of mice at 12 weeks of age, mK1, a true tissue kallikrein, was detected at the apical rim of the striated ducts (SDs). Sexual dimorphism in the immunostaining intensity in parotid glands was seen, i.e., immunostaining was more intense in males than in females. Under electron microscopy, secretory granules, being small in size, and condensed at the subluminal cytoplasm, were labeled with immunogold particles showing the presence of mK1. These secretory granules were rather abundant and large in males. Castration in males reduced the immunoreactivity of mK1 in the SD cells because of a decrease in the number and size of secretory granules as revealed by electron microscopy. Hypophysectomy in male mice resulted in considerable loss of immunoreactivity for mK1, which was characterized under electron microscopy by complete disappearance or significant reduction of secretory granules in many SD cells. These results suggest that mK1 expression in the SD cells of murine parotid glands is regulated by pituitary-dependent hormones, and sexual dimorphism of mK1 expression is regulated by androgens.


Difference in substrate and inhibitor sequence specificity of human, mouse and rat tissue kallikreins

The kininogenase activities of mouse (mK1), rat (rK1) and human (hK1) tissue kallikreins were assayed with the bradykinin-containing synthetic peptides Abz-MTEMARRPPGFSPFRSVTVQNH2 (where Abz stands for o-aminobenzoyl) and Abz-MTSVIRRPPGFSPFRAPRV-NH2, which correspond to fragments Met374-Gln393 and Met375-Val393 of mouse and rat LMWKs (low-molecular-mass kininogens) with the addition of Abz. Bradykinin was released from these peptides by the mK1- and rK1-mediated hydrolysis of Arg-Arg and Arg-Ser (or Arg-Ala) peptide bonds. However, owing to preferential hydrolysis of Phe-Arg compared with the Arg-Ala bond in the peptide derived from rat LMWK, hK1 released bradykinin only from the mouse LMWK fragment and preferentially released des-[Arg9]bradykinin from the rat LMWK fragment (Abz-MTSVIRRPPGFSPFRAPRV-NH2). The formation of these hydrolysis products was examined in more detail by determining the kinetic parameters for the hydrolysis of synthetic, internally quenched fluorescent peptides containing six N- or C-terminal amino acids of bradykinin added to the five downstream or upstream residues of mouse and rat kininogens respectively. One of these peptides, Abz-GFSPFRAPRVQ-EDDnp (where EDDnp stands for ethylenediamine 2,4-dinitrophenyl), was preferentially hydrolysed at the Phe-Arg bond, confirming the potential des-[Arg9]bradykinin-releasing activity of hK1 on rat kininogen. The proline residue that is two residues upstream of bradykinin in rat kininogen is, in part, responsible for this pattern of hydrolysis, since the peptide Abz-GFSPFRASRVQ-EDDnp was preferentially cleaved at the Arg-Ala bond by hK1. Since this peptidase accepts the arginine or phenylalanine residue at its S1 subsite, this preference seems to be determined by the prime site of the substrates. These findings also suggested that the effects observed in rats overexpressing hK1 should consider the activation of B1 receptors by des-[Arg9]bradykinin. For further comparison, two short internally quenched fluorescent peptides that bind to hK1 with affinity in the nM range and some inhibitors described previously for hK1 were also assayed with mK1 and rK1.


Expression and localization of aquaporins, members of the water channel family, during development of the rat submandibular gland.

The expression and localization of aquaporins (AQP1-AQP5), members of the water channel family, in the developing rat submandibular gland were analysed using RT-PCR, Northern blotting and immunohistochemistry to explore their relation to the development of this salivary gland. RT-PCR analysis revealed unique expression patterns of each AQP. AQP1 was expressed constitutively during prenatal development, whereas the expression of AQP5 became more intense in the course of development from embryonic day 16.5 (E16) to E20. These expression patterns concurred with the results of Northern blot analysis. AQP3 and AQP4 mRNAs in the prenatal development were not detected in Northern blots, although they were detected by RT-PCR. During postnatal development, AQP5 and AQP1 mRNAs were expressed continuously, but no message for AQP3 or AQP4 was detected. AQP2 mRNA was not detected during either prenatal or postnatal development in this tissue. Immunohistochemical studies revealed that AQP5 was first localized at the apical membrane of proacinar cells at E18, and then became clearly distributed at the apical membrane of acinar cells in accordance with the differentiation and establishment of the mature acini. In addition, some vasculature also showed immunoreactivity for AQP5. AQP1 was immunolocalized in the blood vessels, including capillaries, of the gland throughout development. These observations suggest the existence of transcriptional regulation of rat AQP5, which is one of the most probable regulators of saliva production and secretion, during the establishment of the functional submandibular salivary gland.

ラット顎下腺発生過程でのAQPファミリーの発現を解析した．AQP3,4は胎生期に非常に弱く発現するが，AQP1,5は胎生期から生後を通して発現し続ける．AQP1は血管にのみ局在するのに対して，AQP5は胎生18日の未分化腺房細胞管腔膜への局在以後，分化成熟につれ腺房細胞の他，一部の血管にも局在する．発生過程を通しAQP5が唾液の産生·分泌に中心的役割を果たすと考えられる．

Pflügers Archiv : European Journal of Physiology


Divergent expression and localization of aquaporin 5, an exocrine-type water channel, in the submandibular gland of Sprague-Dawley rats.

正常SDラット唾液腺(顎下腺)のAQP5蛋白質発現レベルを調べた所，高発現ラットおよび低発現ラットが存在する事を見いだした．2種のラットを近交化する目的で兄妹交配を進め，現在高発現ラットは8代目に，また低発現ラットは4代目に到った．これらのラットでは発現レベルの差は100倍以上に達した．AQP5蛋白質発現レベルの差は耳下腺，肺，涙腺においても認められたが，AQP5 mRNAレベルはいずれの臓器においても両グループ間で差はなかった．したがって，上記2グループ間で認められたAQP5蛋白質発現レベルの差異はAQP5遺伝子あるいはそのプロモーターに発生した遺伝的変異に基づくものと考えられた．


Developmental and androgenic regulation of the immunocytochemical distribution of mK1, a true tissue kallikrein, in the granular convoluted tubule of the mouse submandibular gland

The action of androgens on the immunocytochemical distribution of mK1, a true tissue kallikrein, was examined in the submandibular gland (SMG) of developing and adult mice by indirect enzyme-labeled and immunogold-labeled antibody methods for light and electron microscopy, respectively. In both sexes at 3 weeks of age, essentially all of the immature granular convoluted tubule (GCT) cells were uniformly immunostained. At 4 weeks of age (the onset of puberty), morphological differences between the two sexes appeared in the GCTs, in which some cells became immunonegative. Thereafter, the immunonegative GCT cells became more abundant in the SMG of males than of females and considerable intercellular variation in staining intensity for mK1 was seen, especially in males. A few slender GCT cells with strong immunoreactivity appeared in GCT segments only in males. Castration of males resulted in an increase in the number of immunopositive GCT cells, whereas administration of dihydrotestosterone (DHT) decreased the number of immunopositive GCT cells in the SMGs of both sexes. Slender GCT cells immunoreactive for mK1 were seen in females treated with DHT for 6 days. However, there were no immunostained slender GCT cells in female SMGs after injection of DHT for 2 weeks. Immunoelectron microscopy disclosed this type of cell in male SMGs, which closely resembles immature GCT cells of prepubertal mice, with a few small secretory granules uniformly labeled with gold particles, a sparse Golgi apparatus and RER, and basal infoldings. In mature male SMGs and in SMGs of DHT-treated females and castrated males, typical GCT cells had a well-developed Golgi apparatus and a net-like RER but few to no basal infoldings, whereas in the female gland equivalent cells had moderately developed RER and some basal infoldings. These results suggest that mK1 is one of the enzymes characteristically present in immature GCT cells and that its synthesis is inhibited in part by androgens, resulting in decreased numbers of immunopositive cells.


Expression and localization of AOP5 in the stomach and duodenum of the rat.

消化管におけるアクアポリン5(AQP5)の発現を，RT-PCRおよびウェッスタンブロッティングによってしらべ，下部胃および十二指腸にその強い発現を認めた．検出したAQP5蛋白質は従来の報告のとおり27kDaであった．発現部位は胃では幽門腺，十二指腸ではブルーナー腺であった．これらの結果はAQP5は胃および十二指腸における水分泌に関与する可能性を示唆する．

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research


Methylene blue as a vascular filling dye.

メチレンブルー溶液とピクリン酸溶液とを混合すると，色素の濃度に依存して，ゲル状∼ゴム状の沈澱を生ずることを見出した．この沈殿物を回収してその吸収パターンを計測した結果，メチレンブルーそのものであり，ピクリン酸との化合物や共沈殿物ではないことが判明した．すなわち，ピクリン酸による塩析効果によって色素が沈澱する．ピクリン酸を含むブアン液によって潅流固定した実験動物の血管内にメチレンブルー溶液を注入することにより，生成したゴム状またはゲル状の茶色の色素によって血管内を満たすことができる．通常の色素による染色では，すべての血管が染色され，組織は瀰慢性に濃染してしまう．この新たに開発した方法は，色素濃度を加減することによって生成物の粘性を選ぶことができ，色素進入の血管径を選択できるので，太い血管から順に色素を充填することが可能で，血管網のin situ観察に高い利用価値を有する．

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Androgen regulation of the cellular distribution of the true tissue kallikrein mK1 in the submandibular gland of the mouse

The kallikrein gene family encodes for at least four different proteases in the mouse submandibular gland (SMG): mK1 (true tissue kallikrein), mK9, mK13, and mK22. These enzymes and many other biologically active proteins are synthesized by the granular convoluted tubule (GCT), a specialized segment of the SMG duct system. The GCT is under multihormonal regulation by androgens, thyroid hormones, and adrenocortical hormones. Androgens suppress synthesis of mK1 in the SMG but enhance expression of the other three kallikreins. We prepared an antibody with limited immunoreactivity for mK1 and used it to examine the effects of androgen status on the distribution of this isozyme in the SMGs of developing and mature mice by immunoperoxidase staining for the light microscope and immunogold labeling for the electron microscope. In prepubertal mice, every immature GCT cell contains mK1, confined to an accumulation of small granules in the subluminal cytoplasm. In mature mice, not every GCT cell contains mK1, and in those cells that do there is considerable intergranular variation in the intensity of staining for mK1. GCT cells containing mK1 are much more abundant in the glands of females than of males, resulting in a peculiar sexually dimorphic mosaic distribution of this isozyme in the mature SMG. Castration of adult males increases the number of GCT cells expressing mK1. Administration of androgen to intact or castrated males or to intact females reduces the number of cells staining for mK1. In all cases, immunogold labeling for mK1 is confined to secretory granules. No fine structural differences were noted between cells that were positively or negatively stained for mK1. Therefore, although GCT cells appear to be composed of a uniform population of cells on the basis of morphology alone, they are not homogeneous in their content of secretory proteins. These results indicate that androgen regulation of GCT cells is more complex than has been appreciated to date.

The Journal of Histochemistry and Cytochemistry


Induction of C-reactive protein, serum amyloid P component, and kininogens in the submandibular and lacrimal glands of rats with experimentally induced inflammation.

テルペンチン油を投与した実験的炎症モデルラットにおいて，投与24時間後に，C-反応生蛋白質，血清アミロイド蛋白質，H-およびT-キニノーゲンのmRNAが，顎下腺，眼窩内および眼窩外涙腺に誘導されることを見いだした．投与6時間後に，この誘導に先立って，TNF-αmRNAが誘導された．顎下腺，眼窩内および眼窩外涙腺におけるこれら急性期蛋白質の誘導は，外分泌腺に強い防御系が存在することを示唆するものと考えられる．


Expression and locarization of AQP4 in the crypt epithelial cells of the rat duodenum.

RT-PCRによってアクアポリン4(AQP4)の消化管における発現を調べた．AQP4は下部胃，十二指腸，空腸，回腸，盲腸，大腸，および直腸に発現していたが上部胃ニは発現を認めなかった．ウェッスタンブロッティングで十二指腸に30kDaのAQP4蛋白質の発現を認めた．滅め貴組織化学実験によりAQP4は十二指腸上皮の陰窩細胞の基底側膜に局在したが絨毛細胞には局在しなかった．


Search for the dye that stains the vascular system.

血管系の，特に動脈系に特異的なin situ染色法を開発する目的で，数種の染料を選び，その染色性その他の諸性質を検討した．いくつかの候補の中から，ルクソールファストブルーが最も望ましい性質を有することを明らかにした．すなわち，同色素溶液はピクリン酸溶液と接触すると複塩または錯塩を生成し，原色素とは異なる吸収曲線を呈する沈澱を生じる．ピクリン酸を含む固定液で潅流したのち左心室より同色素液を注入すると，動脈系だけが染色される．このようなin situ血管染色法は，今までになかったものである．


Highly regulated expression of subtilisin-like proprotein convertase PACE4 (SPC4) during dentinogenesis.

Expressions of mRNAs for four subtilisin-like proprotein convertases (SPCs: furin, PACE4, PC6, and PC8) and bone morphogenetic protein 4 (BMP4) in the rat molar tooth during development were analyzed by Northern blotting, reverse transcriptase-polymerase chain reaction (RT-PCR), and in situ hybridization to explore the possible involvement of SPCs in the processing of proBMPs. We found a temporospacial expression of PACE4, but not one of the other SPCs, in this tissue; i.e., RT-PCR analysis revealed that the level of PACE4 mRNA, but not that of the other SPC mRNAs became high around the second postnatal day. This increase was in good accordance with the increase in BMP4 mRNA, indicating an apparent association of these molecules with the differentiation and establishment of functional ameloblasts and odontoblasts. During dentinogenesis, PACE4 mRNA was localized in the ameloblasts and odontoblasts. These observations suggest that PACE4 plays a crucial role in dentinogenesis, especially via the activation of BMPs.

サチライシン様プロセシング酵素PACE4は遺伝子プロモーター領域の構造から，その発現は極めて複雑に制御されると考えられたが，生体内での発現制御については不明であった．本研究により，歯牙形成過程において実際に発現が複雑に制御されていること，また， この様な発現制御がファミリーの中でもPACE4に特有であることが初めて明らかになった．

Biochemical and Biophysical Research Communications


Expression of kininogens in the connective tissue-type mast cells of the rats.

The connective tissue-type mast cells present in the submandibular gland (SMG) and peritoneal cavity of rats were found to express kininogens (KGs), the expression of which was demonstrated by Western blotting, reverse transcription-polymerase chain reaction (RT-PCR), RT-PCR Southern blotting, and light- and electron-microscopic immunocytochemistry. In the SMG, the analysis of cDNA amplified by RT-PCR revealed that the molecular species of mRNAs expressed were high-molecular-weight (HMW)-K KG and T-I KG. Light microscopic immunocytochemistry exclusively localized the KG protein(s) in the mast cells present in the SMG. The signals in the mast cells were very strong, but no positive reaction was observed in the granular convoluted tubular cells, acinar cells or striated duct cells. As determined by using electron microscopy, extremely strong labelling with immunogold was observed in the secretory granules of the mast cells, but no labelling in their nucleus or cytoplasm. Analysis by Western blotting and RT-PCR Southern blotting indicated that both protein and mRNA of KGs were present in the mast cells separated from the peritoneal cavity, indicating de novo synthesis of KG in these cells. Preliminary experiments implied that the connective tissue-type mast cells in other rat tissues also expressed KG.

ラット顎下腺および腹腔に存在する結合組織型肥満細胞にキニノーゲン(KG)が発現していることをRT-PCR，ウェッスタンブロッティング，免疫組織化学的手法を用いて明らかにした．発現しているKGは高分子型およびT-1KGであった．KGは肥満細胞分泌顆粒に局在した．RT-PCRサザンブロッティングにより分離腹腔肥満細胞にKG mRNAを検出したのでKGは肥満細胞でde novoの合成が行われて入ることが示唆された．


Study on oral health care and general health of elderly (in Japanese)

高齢者における口腔ケアと健康に関する基礎的研究


Involvement of vesicle-cytoskeleton interaction in AQP5 trafficking in AQP5-gene-transfected HSG cells.

ヒト唾液腺細胞にアクアポリン5(AQP5)を遺伝子導入したところ，AQP5蛋白質は細胞内の小胞に発現した．タプシガージン等により細胞内カルシウムを上昇させると小胞のAQP5蛋白質は細胞膜へ移動した．さらに微小管阻害剤である，コルヒチンおよびビンブラスチンはこの移動を抑制した．AQP5のトラッフィッキングは微小管が関与して入る可能性が示唆された．


Subtilisin-like proprotein convertase PACE4 (SPC4) is a candidate processing enzyme of bone morphogenetic proteins during tooth formation

サチライシン様プロセシング酵素PACE4が歯牙形成過程において時期·空間特異的に発現し，歯牙形成に必須の骨形成蛋白質群(BMPs)と一致して局在することから， PACE4が歯牙形成時の最も有力なBMPsプロセシング酵素で， BMPsの活性化によりエナメル芽細胞と象牙芽細胞の分化成熟と密接に関わることを見い出した．


An unusual sexually dimorphic mosaic distribution of a subset of kallikreins in the granular convoluted tubule of the mouse submandibular gland detected by an antibody with restricted immunoreactivity


Co-localization of estrogen and androgen receptors immunoreactivities in the trigeminal motor nucleus


Expression of an alloenzyme of prorenin-converting enzyme in the submandibular gland of DBA/2N mice.

DBA/2Nマウス顎下腺抽出液に等電点8.7の新種カリクレイン酵素を見いだした．本酵素のアミノ酸配列はmK13およびmK26(プロレニン変換酵素1及2)と酷似していた．本酵素に対応するcDNAをクローニングし，その配列を解析し，このcDNAは全783塩基中，mKlk13およびmKlk26とそれぞれ6および12塩基異なる事を明らかにした．本酵素はmK13アロ酵素(mK13b)であると考えられた．


Prorenin processing and restricted endoproteolysis by mouse tissue kallikrein family ezymes (mK1, mK2, mK13, and mK22).

Four members of the tissue kallikrein family, mK1, mK9, mK13, and mK22, all of which exhibit extensive homology in amino acid sequence among themselves, were obtained from the submandibular gland of ICR mice and examined for their ability to cleave prorenin. Tissue kallikrein mK13 was confirmed to be a prorenin-converting enzyme; and mK9, which was earlier shown to be an EGF-binding protein, was found to cleave mouse Ren 2 prorenin specifically and convert it to mature renin with an activity of approximately 1/10 of that of mK13. With the same substrate, mK22 (beta-NGF endopeptidase) gave two products, renin and arginyl-renin; whereas mK1 (true tissue kallikrein) did not process it at all. The endoproteolytic activity of tissue kallikreins was examined with various peptide-MCA substrates. The substrates contained three key structures; X(Y)-Arg-Arg, X(Y)-Lys-Arg and X-Lys-Lys motifs (where X and Y are hydrophilic and hydrophobic amino acids, respectively). We found that mK1, mK9 and mK13 preferentially cleaved the former two types of substrate, except Y-Arg-Arg-MCA. The substrate X-Lys-Lys-MCA was hardly cleaved by these three tissue kallikreins but was preferentially cleaved by mK22. The four tissue kallikreins seem to have the ability to process precursor proteins containing a pair of basic amino acid residues; the specificities of three of the enzymes (mK1, mK9 and mK13) were similar to each other but were different from that of mK22.

マウス顎下腺に存在する4種類のカリクレイン酵素のキニン遊離活性，プロレニン変換活性を調べた．mK1は最も強いキニン遊離活性を有したがプロレニン変換活性は検出限界以下であった，他方mK13はキニン遊離活性を検出できなかったが，最も強いプロレニン変換活性をしめした．レニンアンギオテンシン系とキニンカリクレイン系がファミリーの異なるメンバーが発現することによって調節される可能性が考えられた．

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology


Regulation of Na+,K+-ATPase in submandibular glands of hypophysectomized male mice by steroid and thyroid hormones

The Journal of Histochemistry and Cytochemistry


In vivo modulation of proliferating cell nuclear antigen in growth plate chondrocytes from normal, hypophysectomized, and growth hormone-treated hypophysectomized rats -A comparative immunohistochemical study with image analysis-


ATP- and EGF-stimulated phosphatidylinositol synthesis by two different pathways, phospholipase D and diacylglycerol kinase, in A-431 epidermoid carcinoma cells


Effects of steroids and thyroid hormones on Na+,K+-ATPase activity and localization of its a subunit protein in submandibular gland of hypophysectomized mice (in Japanese)

脳下垂体摘出マウス顎下腺のNa+, K+-ATPase活性およびその局在性に及ぼす甲状腺ホルモン並びにステロイドホルモンの影響

Journal of the Japan Salivary Gland Society


The molecular diversity of salivary kallikreins (prorenin converting enzymes)(in Japanese)

唾液腺カリクレイン(プロレニン変換酵素)の分子多様性


Effects of pertussis toxin on signal transduction via P2-purinergic receptors in A-431 human epidermoidal carcinoma cells


Extracellular calcium causes the release of calcium from intracellular stores in chick osteocytes

Biochemical and Biophysical Research Communications


Effects of FMLP and LPS on [Ca2+]i of peritoneal exudate polymorphonuclear leukocytes following onset of inflammation


The molecular diversity and physiological function of salivary kallikreins (in Japanese)

唾液腺カリクレインの生理機能と分子多様性


Computer-aided image analysis applied to immunogold-silver staining: evaluation of proliferating cell nuclear antigen (PCNA)-reactive sites in paraffin sections


Involvement of high-affinity binding site for EGF receptor in formation of rounding in A-431 epidermoid carcinoma cells


Identification of mK1, a true tissue (glandular) kallikrein of mouse submandibular gland: Tissue distribution and a comparison of kinin-releasing activity with other submandibular kallikreins


Effects of nerve growth factor and dexamethasone on Na+,K+-ATPase of cultured PC12 cells


Bradykinin-stimulated transient modulation of epidermal growth factor receptors in A-431 human epidermoid carcinoma cells


Effects of female hormones and 3,5,3'-triiodothyronine or dexamethasone on induction of epidermal growth factor and proteinases F, D, A, and P in the submandibular glands of hypophysectomized male mice


Effects of single or combined injection of female, thyroid and adrenocortical hormones on the proteinase isozyme (F, D, A, P) activities and EGF contents in the submandibular gland of hypophysectomized mice (in Japanese)

脳下垂体除去マウス顎下腺の各種proteinase (F, D, A, P)及びEGFに対する女性ホルモンと副腎皮質及び甲状腺ホルモンの併用による動態


Characterization of ecto-nucleoside triphosphatase on A-431 human epidermoidal carcinoma cells


P2-Purinergic receptors and cellular calcium metabolism in A 431 human epidermoid carcinoma cells

American Journal of Physiology, Cell Physiology


Additive and/or synergistic effects of 5a-dihydrotestosterone, dexamethasone, and triiodo-L-thyronine on induction of proteinases and epidermal growth factor in the submandibular gland of hypophysectomized mice


Effects of various hormones on a-amylase and proteinase activity in salivary glands of hypophysectomized mice (in Japanese)

脳下垂体除去マウス唾液腺のamylase及びproteinase活性の変動に及ぼす各種ホルモンの影響

Journal of the Japan Salivary Gland Society


Involvement of cellular calcium incorporated from the medium in production of inositol trisphosphate in A431 cells


Effects of Ca2+ and Mg2+ on ATP-dependent 45Ca2+ influx in A-431 human epidermoidal carcinoma cells


Reversal of relative proteinase F activity and onset of androgen-dependent proteinases in the submandibular gland of postnatal mice


A new electrophoretic variant of a subunit of Na+,K+-ATPase from the submandibular gland of rats

The alpha catalytic subunits of Na+/K(+)-ATPase were isolated from the kidney and brain of rats (alpha 1 and alpha 2, respectively). The antisera raised against these subunits were used as probes to analyze the isoform of catalytic subunits of Na+/K(+)-ATPase in various tissues of rats. Of 27 rat tissues examined, most had a catalytic subunit identical to alpha 1 but some, such as the nervous and muscle tissues, had both alpha 1 and alpha 2 isoforms as judged by their reactivities to antisera and their electrophoretic mobility. We found that the submandibular gland contained a new electrophoretic variant of immunoreactive alpha subunit (designated alpha(S) in this report) in addition to alpha 1 identical to those found in kidney and brain. The new variant, alpha(S), strongly cross-reacted with anti-alpha 1 antiserum, but to a lesser extent with anti-alpha 2 antiserum. The alpha(S) had a molecular mass which was found to be slightly less (approx. 90 kDa) than brain and kidney alpha 1. We examined whether or not the alpha(S) is formed by proteolytic cleavage of alpha subunits during preparation and concluded that this is not the case. The alpha(S) reacted with [gamma-32P]ATP, resulting in the formation of radioactive alpha subunit which was stabilized by 2 mM ouabain but which was labile in the presence of 70 mM potassium chloride. Since N-terminal amino acid sequence of alpha(S) protein [G()DKY()PAAVS] corresponds exactly and uniquely with the sequence of the alpha 1 chain between residues 1 and 11, it is very probable that alpha(S) protein originated from alpha 1 protein following the post-translational processing.

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology


A human-mouse hybrid cell line expressing both HLA and H-2 antigens


Measurement of intracellular calcium in single cells by a fluorescence analysis using laser beam (in Japanese)

レーザーを用いた蛍光分析による単一細胞レベルでの細胞内カルシウムの測定

Journal of Clinical and Experimental Medicine


Exogenous ATP and other nucleoside phosphates modulate epidermal growth factor receptors of A-431 epidermoid carcinoma cells

The Proceedings of The National Academy of Sciences USA


Postnatal changes in an a-subunit isoform, a(S), of Na+,K+-ATPase in the submandibular gland of rats


A simple and rapid purification procedure for the tetramethylrhodamine-labeled antibodies

A conjugate preparation of antibody (Fab) and tetramethylrhodamine (TMR) generally adsorbs free TMR which is very difficult to remove because of its strong hydrophobic binding. On the basis of criteria such as sodium dodecyl sulfate (SDS) gel electrophoresis and staining of the plasma membrane of live cells, we found that simple extraction with n-butyl alcohol or iso-amyl alcohol could remove the contaminating free dye. The procedure is especially useful when one needs to prepare conjugates with low nonspecific binding for the study of lateral diffusion of cell membrane-associated antigens.


Modulation of HLA antigens in response to the binding of epidermal growth factor by A431 cells


Androgen responsiveness of proteinase F in the submandibular salivary glands of inbred mice

Using the radioimmunoassay technique, we studied androgenic regulations of proteinase F in the submandibular salivary glands (SMG) of various mouse strains (ICR, BALB/CA, C3H/HeN, C57BL/10N, and DBA/2N). The proteinase contents in BALB/CA and DBA/2N mice were almost the same between males and females. However, prominent sex differences in enzyme content were seen in all other strains; i.e., male glands of C3H/HeN and C57BL/10N contained more proteinase F than female glands did. On the contrary, the enzyme level in the ICR was exceedingly higher in females than in males. The enzyme contents in ICR SMG increased in males following castration and decreased after androgen injection in both castrated males and normal females. On the contrary, the enzyme level in C3H/HeN was decreased by castration and increased by androgen injection. Therefore, these sex differences in ICR and C3H/HeN mice depend on androgen, though the direction of the response is opposite.


Immunocytochemical study of proteinase F in the mouse submandibular gland

The Journal of Histochemistry and Cytochemistry


Inhibitory effect of androgen on the synthesis of proteinase F in the male mouse submandibular gland


A new esteroproteinase (proteinase F) from the submandibular gland of female mice

One of the esteroproteinases present in the submandibular glands of female mice was purified and characterized. The enzyme, designated proteinase F in this report, had a pI value of 4.6 and a molecular weight of 27600, being comprised of two subunits of 10000 and 18000 daltons. The amino acid composition of proteinase F resembled that of the epidermal growth factor-binding protein, but antiserum against proteinase F only reacted weakly against the binding protein. Proteinase F had an optimum pH at around 9.0 and was strongly inhibited by Cu2+ and Hg2+ (42 and 76% inhibition, respectively, at a concentration of 4 x 10(-6) M). It was also inhibited by aprotinin, phenylmethylsulfonylfluoride, iodoacetamide, leupeptin, antipain, and benzamidine but neither by trypsin inhibitors from pancrease, soybean, or ovomucoid, nor by TLCK, TPCK, and epsilon-amino-n-caproic acid. Although its actual physiological function has yet to be determined, these properties indicate that proteinase F is a new enzyme, being distinguished from known proteinases, kallikrein, plasmin, trypsin, chymotrypsin, tonin, angiotensin-converting enzyme, proteinase A (beta-nerve growth factor endopeptidase), proteinase D (epidermal growth factor-binding protein), P-esterase, renin A, and renin C. Proteinase F was present in the submandibular glands of female mice more abundantly than in those of males, but it increased in males following castration. Thus, proteinase F appears to be affected by male hormones in vivo.

Biochimica et Biophysica Acta (BBA) - General Subjects


Characterization of two esteroproteases from the male mouse submandibular gland


Induction of epidermal growth factor by tri-iodo-L-thyronine in the submandibular glands of mice with testicular feminization


Induction of various androgen-dependent esteroproteases (trypsin-like and chymotrypsin-like enzymes) by tri-iodo-L-thyronine in the submandibular glands of female mice and mice with testicular feminization


Purification and properties of b-N-acetylhexosaminidase from mouse submandibular gland


Effect of autonomic agents on the secretion of N-acetyl b-glucuronidase of mouse submaxillary gland


Induction of androgen-dependent protease and serous-like granules by tri-iodothyronine in the submandibular gland of mice with testicular feminization


Regulation of delayed-hypersensitivity response by the submandibular gland of male mice


Androgenic regulation of N-acetyl b-glucuronidase activity in the submandibular glands of mice


Affinity adsorption of L-glutamine D-fructose-6-phosphate aminotransferase to Sepharose coupled with p-chloromercuribenzoate

Biochemical and Biophysical Research Communications


Sex difference in L-glutamine D-fructose-6-phosphate aminotransferase activity of mouse submandibular gland

L-Glutamine D-fructose-6-phosphate aminotransferase (2-amino-2-deoxy-D-glucose-6-phosphate ketol-isomerase (amino-transferring), EC 5.3.1.19) activities in the three main salivary glands of male and female mice were measured. It was found that the activity in the submandibular gland was about 10 times more in females than in males, whereas the activities in the sublingual and parotid glands of males and females were similar. The activity in the submandibular gland of female mice was not affected appreciably by ovariectomy but it decreased to the level in males on injection of testosterone. The activity in males was not affected appreciably by injection of progesterone or 17beta-estradiol, but it increased to the level in females after castration. The increased activity in castrated male mice was decreased again to the normal level by testosterone injection. Thus, this sex difference is caused by androgen, not by female hormones. On the basis of in vivo experiments using actinomycin D, it was suggested that testosterone produced an "enzyme inhibitor", which suppressed the enzyme activity in the submandibular glands of androgen-rich animals.


Influence of androgen on b-glucuronidase activity in liver, kidney and submandibular salivary gland of the mouse


Effect of autonomic agents on the amount of androgen-dependent granules in convoluted tubular cells of the mouse submandibular gland

Canadian Journal of Physiology and Pharmacology


Regulation of the secretory process of granular components from the convoluted tubular cells of the mouse submandibular gland


Androgenic control of the submandibular gland of mice

マウス顎下腺のアンドロゲンによる調節


Effects of sex hormones on synthesis of proteins contained in granules present in convoluted tubular cells of mouse submandibular glands


Effect of testosterone on the amount of serous-like granules in convoluted tubular cells of mouse submandibular glands


Binding protein for 5α-dihydrotestosterone in mouse submandibular gland

The changes in the levels of the binding protein for 5 alpha-dihydrotestosterone in cytoplasmic extract of the submandibular glands during development were compared in male and female mice using a DEAE-cellulose filter assay. The binding protein was first detectable 5 days after birth in both sexes, at a time coincident with androgen-independent cytodifferentiation of the convoluted tubular cells in the submadibular gland. The level of the binding protein in female mice was maintained at 5 pmol/mg protein after birth, whereas in males it began to decrease from 3 weeks after birth with inccrease in serum testosterone, becoming much less than a quarter of the level in females or immature mice by 4 weeks after birth. However, after castration, the level of detectable binding protein in mature male mice increased within 7 days to the same level as that in females or immature mice. This suggests that the low binding capacity for exogenous hormone in mature male mice is due to occupancy of the binding sites by endogenous hormone.

Biochimica et Biophysica Acta (BBA) - General Subjects


Regulation of secretion of the components in androgen-dependent granules in convoluted tubular cells of mouse submandibular glands (in Japanese)

マウス顎下腺細管細胞内に存在する男性ホルモン依存性顆粒の分泌機作

Journal of Physiological Society of Japan


Quantitative measurement of androgen dependent granules in the mouse submandibular gland and effects of various sex hormones (in Japanese)

マウス顎下腺の男性ホルモン依存性顆粒の定量化と性ホルモンの効果

Journal of Clinical and Experimental Medicine


Effects of pH indicators on various activities by chromatophores of Rhodospirillum rubrum


Energization of NTP-NDP kinase by acid base transition leading to ATP formation


Electron and proton transports in Rhodospirillum rubrum chromatophores


Competition between Pi and pH indicators in photosynthetic ATP formation with chromatophores of Rhodospirillum rubrum


Formation and decomposition of pyrophosphate related to bacterial photophosphorylation


Effects of injection of chromatins from Rhodamine sarcoma into rats on pI-isozymes of liver pyruvate kinase

Gann : Japanese Journal of Cancer Research


For learners aiming to study science in Japan

日本で理系分野の研究を目指す学習者の方へ


Report: Chosun University School of Dentistry (in Japanese)

朝鮮大学校歯科大学訪問の報告


Effects of hypophysectomy and hormonal replacement on the amylase activity in parotid gland of mice (2)(in Japanese)

マウス耳下腺に対する各種ホルモンの薬理学的研究 第2報マウス耳下腺内のamylase活性における脳下垂体除去およびそのホルモン投与の影響

Journal of Meikai University School of Dentistry


Relation between cell rounding induced by epidermal growth factor (EGF) and high affinity binding sites for EGF in A-431 human epidermoid carcinoma cells (in Japanese)

A-431ヒト類表皮癌細胞のEGF刺激によるrounding形成とEGF高親和性結合部位との関係

Journal of Meikai University, School of Dentistry


Stability of HLA and H-2 expression on the surface of human-mouse hybrid cells prepared by cell hybridization technique

Journal of Meikai University, School of Dentistry


Effects of L-ascorbic acid and bone metabolism factors on alkaline phosphatase activity of and 45Ca2+ incorporation by ROS 17/2.8 cells (in Japanese)

ROS17/2.8細胞のアルカリホスファターゼ活性および45Ca2+取り込みに対する各種骨代謝因子並びにL-アスコルビン酸の影響

The Journal of Meikai University School of Dentistry


Effects of various factors involved in bone metabolism on 45Ca2+ incorporation and alkaline phosphatase activity of ROS 17/2.8 cells (in Japanese)

ROS17/2.8細胞による45Ca2+ 取り込みとアルカリフォスファターゼ活性に与える各種骨代謝因子の作用


Two esteroproteases from mouse submandibular gland (in Japanese)


Developmental changes of various esteroprotease isozymes in the mouse submandibular gland (in Japanese)

マウス顎下腺における esteroprotease isozymes の日齢による変化


Histochemical localization of Na+,K+-ATPase in rat submandibular gland -The effects of mineralocorticoid and sodium ion- (in Japanese)

ラット顎下腺の Na+, K+-ATPase の局在性 -とくに mineralocorticoid および Na+ の影響について-


Induction of androgen-dependent esteroproteases (trypsin-like and chymotrypsin-like) by tri-iodothyronine in the submandibular glands of female mice and mice with testicular feminization (in Japanese)

雌マウスおよび睾丸性女性化症マウスの顎下腺における androgen-dependent esteroproteases (trypsin-likeおよび chymotrypsin-like) の tri-iodothyronine による誘導


Purification and properties of N-acetyl b-D-glucuronidase from mouse submandibular gland (in Japanese)

マウス顎下腺 N-acetyl b-D-glucosaminidase の精製と性質


The effect of mineralocorticoids and sodium ion on Na+,K+-ATPase activity in rat submandibular gland (in Japanese)

ラット顎下腺の Na+,K+-ATPase 活性に及ぼすmineralocorticoid および Na+ の影響


Separation and purification of glutamine fructose-6-phosphate aminotransferase by affinity chromatography (in Japanese)

Affinity chromatography による glutamine fructose-6-phosphate aminotransferase の分離，精製


Sex difference in the activity of L-glutamine D-fructose-6-phosphate aminotransferase in the submandibular glands of mice (in Japanese)

マウス顎下腺 glutamine fructose-6-phosphate aminotransferase にみられる性差について


Studies on secretory granules in the convoluted tubules of mouse submandibular gland (3) A role of a-adrenergic receptor in the secretory process of granular components (in Japanese)

マウス顎下腺顆粒細管内顆粒の研究(3)顆粒成分分泌におけるa-adrenergic receptor の役割


Binding protein for 5a-dihydrotestosterone in mouse submandibular gland (in Japanese)

マウス顎下腺の男性ホルモン結合蛋白


Studies on secretory granules in the convoluted tubules of mouse submandibular gland (2) Changes in granular components determined by immunochemical methods (in Japanese)

マウス顎下腺顆粒細管内顆粒の研究(2)免疫学的方法によって測定した顆粒成分の動態


Studies on secretory granules in the convoluted tubules of mouse submandibular gland (1) Preparation of granule specific anti-serum (in Japanese)"

マウス顎下腺顆粒細管内顆粒の研究(1)顆粒細管内顆粒特異抗血清の調製