TM2D3, a mammalian homologue of Drosophila neurogenic gene product Almondex, regulates surface presentation of Notch receptors.

Notch signaling is an evolutionarily conserved mechanism required for numerous types of cell fate decisions in metazoans. It mediates short-range communication between cells with receptors and ligands, both of which are expressed on the cell surfaces. In response to the ligand-receptor interaction, the ligand and the extracellular domain of the Notch receptor (NECD) in the complex are internalized into ligand-expressing cells by endocytosis, a prerequisite process for the conformational change of the membrane proximal region of Notch to induce critical proteolytic cleavages for its activation. Here we report that overexpression of transmembrane 2 (TM2) domain containing 3 (TM2D3), a mammalian homologue of Drosophila melanogaster Almondex (Amx), activates Notch1. This activation requires the ligand-binding domain in Notch1 and the C-terminal region containing TM2 domain in TM2D3. TM2D3 physically associates with Notch1 at the region distinct from the ligand-binding domain and enhances expression of Notch1 on the cell surface. Furthermore, cell surface expression of Notch1 and Notch2 is reduced in Tm2d3-deficient cells. Finally, amx-deficient Drosophila early embryos exhibit impaired endocytosis of NECD and Delta ligand, for which surface presentation of Notch is required. These results indicate that TM2D3 is an element involved in Notch signaling through the surface presentation.

Primitive Myxoid Mesenchymal Tumor of Infancy With Fatal Hemorrhage In Utero: A Case Report and Literature Review.

Primitive myxoid mesenchymal tumor of infancy (PMMTI) is a rare soft tissue sarcoma in childhood. We present the case of a newborn male who experienced a severe hemorrhage in utero from the tumor on the scalp. He died at the age of 24 hours owing to hemorrhagic shock. The tumor was posthumously diagnosed as PMMTI. A literature search indicated that cases of severe hemorrhage from soft tissue sarcomas in utero or at birth are limited to infantile fibrosarcoma. This is the first case of PMMTI with massive hemorrhage. Clinicians must be aware of hemorrhagic complications of PMMTI.

Refractory Acute Antibody Mediated Rejection in Liver Transplant After Desensitization of Preformed Donor Specific Antibody-Validity of Bortezomib and Everolimus: A Case Report.

Here, we report a case of living donor liver transplantation (LDLT) complicated with severe acute antibody-mediated rejection (aAMR), although desensitization was performed for preformed donor-specific anti-human leukocyte antigen antibody (DSA). LDLT was performed in a 59-year-old woman with alcoholic cirrhosis with a graft from her 60-year-old husband as a living donor. She had reproductive history of 4 gravidity and parity with her husband. Preoperative serologic studies showed positive complement-dependent cytotoxic crossmatch and anti-human leukocyte antigen-A26 antibody was identified as DSA. Desensitization for preformed DSA with rituximab and plasma exchange was performed before LDLT. We decided to perform LDLT using her husband right liver as living donor graft since the DSA mean fluoro-intensity was down to negative range. The immunosuppressive regimen was comprised with steroid and tacrolimus. However, the recipient developed acute cellular rejection on day 5 after LDLT, followed by severe aAMR. Re-administration of rituximab followed by 4 courses of plasma exchange failed to treat aAMR. The DSA mean fluoro-intensity was successfully suppressed after bortezomib was administered however impaired serologic liver function test and cholestasis were remained. The liver function test and cholestasis in the graft were improved after Everolimus was administered. The recipient was discharged on postoperative day 196. In conclusion, we report a case of LDLT who developed aAMR after desensitization of preformed DSA and was successfully treated with intensive therapy with bortezomib and everolimus.

[A Case of Malignant Peripheral Nerve Sheath Tumor of the Anus Resected by Robot Assisted-APR].

An 81-year-old female visited a local hospital with complaints of anal pain. A tumor was found on the right side of her anus, and the histopathological diagnosis was a non-epithelial malignant tumor. Therefore, the patient was referred to our hospital. Result of imaging inspection revealed that the tumor had invaded the lower rectum, but had not distantly metastasized. Based on the findings of another biopsy, the patient was diagnosed with a malignant peripheral nerve sheath tumor (MPNST). Robot-assisted abdominoperineal resection(D1)was performed, and the lesion was resected without any pathological remnants. During the postoperative period, the patient developed perineal wound infection. Subsequently, the patient was discharged from the hospital on postoperative day 10. At the 6-month postoperative follow-up, no recurrence was noted. Most MPNSTs occur in the limbs, trunk, and neck. MPNST in the primary gastrointestinal tract or in the vicinity of the gastrointestinal tract is relatively rare, and in principle, combined resection of the intestinal tract is required for surgical treatment. Here, we report a case of MPNST that occurred near the anus and infiltrated to the lower rectum and was completely resected by robot-assisted abdominoperineal resection.

Maternal almondex, a neurogenic gene, is required for proper subcellular Notch distribution in early Drosophila embryogenesis.

Notch signaling plays crucial roles in the control of cell fate and physiology through local cell-cell interactions. The core processes of Notch signal transduction are well established, but the mechanisms that fine-tune the pathway in various developmental and post-developmental contexts are less clear. Drosophila almondex, which encodes an evolutionarily conserved double-pass transmembrane protein, was identified in the 1970s as a maternal-effect gene that regulates Notch signaling in certain contexts, but its mechanistic function remains obscure. In this study, we examined the role of almondex in Notch signaling during early Drosophila embryogenesis. We found that in addition to being required for lateral inhibition in the neuroectoderm, almondex is also partially required for Notch signaling-dependent single-minded expression in the mesectoderm. Furthermore, we found that almondex is required for proper subcellular Notch receptor distribution in the neuroectoderm, specifically during mid-stage 5 development. The absence of maternal almondex during this critical window of time caused Notch to accumulate abnormally in cells in a mesh-like pattern. This phenotype did not include any obvious change in subcellular Delta ligand distribution, suggesting that it does not result from a general vesicular-trafficking defect. Considering that dynamic Notch trafficking regulates signal output to fit the specific context, we speculate that almondex may facilitate Notch activation by regulating intracellular Notch receptor distribution during early embryogenesis.

Changes of salivary functions in experimental periodontitis model rats.

OBJECTIVE: This study was designed to investigate the mechanism of salivary dysfunction in an experimental periodontitis rat model and to examine the improvements in salivary secretion following treatment of the experimental periodontitis. METHODS: In the experimental periodontitis rat model, which included a unilateral ligature for 4 weeks around the second upper molar, several salivary functions were investigated. Changes in the salivary function were evaluated 4 weeks after removal of the ligature in some rats. RESULTS: The periodontitis model showed significant reductions in the weight of the bilateral major salivary glands and pilocarpine-induced salivary secretion. The model also showed an increase in the number of apoptotic cells in bilateral salivary glands. According to Ca(2+) imaging and Western blotting, there were no differences in the muscarine-induced intracellular Ca(2+) mobilization in acinar cells or in the M3 receptor and AQP5 expression levels in the salivary glands between the sham and the periodontitis model. Following removal of the ligature, differences in the weights of salivary glands and pilocarpine-induced salivary secretion between the sham and the periodontitis model animals were not found. CONCLUSION: These results suggest that experimental periodontitis leads to hyposalivation and that relief from it improves salivary function. It is likely that lower levels of salivary secretion are caused by the decrease of functional acinar cells in salivary glands in the experimental periodontitis model, and the bilateral gland effects in the unilateral periodontitis model are caused by systemic rather than by local effects.

Disruption of NF-κB1 prevents bone loss caused by mechanical unloading.

Mechanical unloading, such as in a microgravity environment in space or during bed rest (for patients who require prolonged bed rest), leads to a decrease in bone mass because of the suppression of bone formation and the stimulation of bone resorption. To address the challenges presented by a prolonged stay in space and the forthcoming era of a super-aged society, it will be important to prevent the bone loss caused by prolonged mechanical unloading. Nuclear factor κB (NF-κB) transcription factors are activated by mechanical loading and inflammatory cytokines. Our objective was to elucidate the role of NF-κB pathways in bone loss that are caused by mechanical unloading. Eight-week-old wild-type (WT) and NF-κB1-deficient mice were randomly assigned to a control or mechanically unloaded with tail suspension group. After 2 weeks, a radiographic analysis indicated a decrease in bone mass in the tibias and femurs of the unloaded WT mice but not in the NF-κB1-deficient mice. An NF-κB1 deficiency suppressed the unloading-induced reduction in bone formation by maintaining the proportion and/or potential of osteoprogenitors or immature osteoblasts, and by suppression of bone resorption through the inhibition of intracellular signaling through the receptor activator of NF-κB ligand (RANKL) in osteoclast precursors. Thus, NF-κB1 is involved in two aspects of rapid reduction in bone mass that are induced by disuse osteoporosis in space or bed rest.

CD38/ADP-ribosyl cyclase in the rat sublingual gland: subcellular localization under resting and saliva-secreting conditions.

CD38 is a 42-45 kDa transmembrane glycoprotein that exhibits ADP-ribosyl cyclase enzyme activity. In the rat, we have previously reported strong ADP-ribosyl cyclase activity in the sublingual salivary gland (Masuda W. and Noguchi T. Biochem. Biophys. Res. Commun. (2000) 270, 469-472). Here, we have examined the specific localization of CD38/ADP-ribosyl cyclase activity in this gland and whether that localization changes upon saliva-secretary stimulation. Under resting conditions, CD38/ADP-ribosyl cyclase activity in the post-nuclear fraction of SLG homogenates was separated into two major peaks by sucrose density gradient centrifugation. The first peak included the plasma membrane proteins Na+/K+ ATPase and aquaporin 5, while the second peak included mucous secretory protein mucin and vesicle-associated membrane protein 2. When rats were subjected to the muscarinic agonist pilocarpine, the CD38/ADP-ribosyl cyclase activity disappeared from the second peak, as did mucin and vesicle-associated membrane protein 2. Pre-treatment of rats with the muscarinic antagonist atropine before pilocarpine administration, or adrenergic stimulation with isoproterenol, the sucrose density gradient separation profiles were same as that seen under resting condition. Using an immunofluorescent strategy, we observed the preferential localization of CD38 in the basolateral plasma membrane and intracellular granule-like membrane in sublingual acinar cells under resting conditions.

InsP3R-associated cGMP kinase substrate determines inositol 1,4,5-trisphosphate receptor susceptibility to phosphoregulation by cyclic nucleotide-dependent kinases.

Ca(2+) release through inositol 1,4,5-trisphosphate receptors (InsP(3)R) can be modulated by numerous factors, including input from other signal transduction cascades. These events shape the spatio-temporal characteristics of the Ca(2+) signal and provide fidelity essential for the appropriate activation of effectors. In this study, we investigate the regulation of Ca(2+) release via InsP(3)R following activation of cyclic nucleotide-dependent kinases in the presence and absence of expression of a binding partner InsP(3)R-associated cGMP kinase substrate (IRAG). cGMP-dependent kinase (PKG) phosphorylation of only the S2+ InsP(3)R-1 subtype resulted in enhanced Ca(2+) release in the absence of IRAG expression. In contrast, IRAG bound to each InsP(3)R subtype, and phosphorylation of IRAG by PKG attenuated Ca(2+) release through all InsP(3)R subtypes. Surprisingly, simply the expression of IRAG attenuated phosphorylation and inhibited the enhanced Ca(2+) release through InsP(3)R-1 following cAMP-dependent protein kinase (PKA) activation. In contrast, IRAG expression did not influence the PKA-enhanced activity of the InsP(3)R-2. Phosphorylation of IRAG resulted in reduced Ca(2+) release through all InsP(3)R subtypes during concurrent activation of PKA and PKG, indicating that IRAG modulation is dominant under these conditions. These studies yield mechanistic insight into how cells with various complements of proteins integrate and prioritize signals from ubiquitous signaling pathways.

Differences in the Ca2+ response resulting from neurotransmitter stimulations of rat parotid acini and ducts.

There are few data available regarding the differences in intracellular Ca2+ responses of parotid acinar and ductal cells. This study investigated the Ca2+ mobilization that was induced by the chemical stimulation of acinar and ductal cells from rat parotid glands. In fura-2 loaded parotid cells, carbachol increased the intracellular Ca2+ concentration ([Ca2+](i)) to a greater extent in the acinar cells than in the ductal cells, but noradrenaline increased the [Ca2+](i) in the ductal cells more than in the acinar cells. Although there was no difference in the alpha1-adrenergic receptor agonist phenylephrine-induced Ca2+ mobilization between acini and ducts, the beta-adrenergic receptor agonist isoproterenol increased the [Ca2+](i) in only the ductal cells. Additionally, the effects of non-adrenergic, non-cholinergic neurotransmitters were investigated. Substance P and ATP increased the [Ca2+](i) in parotid acini and/or ducts. A substance P-induced Ca2+ response was observed in only acini, while the ATP-induced Ca2+ response was significantly higher in ducts than in acini. These results suggest that parotid acini have a greater sensitivity to cholinergic and substance P stimulation and a lesser sensitivity to beta-adrenergic and ATP stimulation than the ductal cells. In light of these results, substance P and isoproterenol will be useful for identifying parotid acini and ducts, respectively.

Relationship of chewing-stimulated whole saliva flow rate and salivary gland size.

OBJECTIVE: We have recently reported that unstimulated whole saliva flow rates (UWSFR) correlate positively with salivary gland sizes and body profiles of weight and body mass indices. In the present study, the correlations of chewing-stimulated whole saliva flow rates (CWSFR) with salivary gland sizes and the body profiles were investigated, and the results were compared with those of UWSFR. DESIGN: Saliva samples were collected from 24 healthy young males and 26 females by the spitting method while chewing paraffin and the CWSFRs were measured. UWSFR and the estimated sizes of the three major salivary glands in our previous study were used. RESULTS: The CWSFRs in all subjects and in males correlated positively with UWSFR, but not in females. The CWSFRs in all subjects correlated positively with parotid and/or submandibular gland sizes, weights and body mass indices, just as with UWSFR; however, the correlation coefficients with salivary gland sizes were smaller than those of UWSFR. In contrast to the results of UWSFR, the correlation coefficients of the CWSFRs with parotid gland sizes in all subjects were larger than those with the sizes of the submandibular glands. The CWSFRs in males correlated only with parotid gland sizes, and those in females did not correlate with any of the parameters. CONCLUSIONS: The results suggest that the larger the size of the salivary glands, the greater the CWSFR, at least in males.

Relationship of the unstimulated whole saliva flow rate and salivary gland size estimated by magnetic resonance image in healthy young humans.

OBJECTIVE: The aim of this study was to examine the relationships between gland sizes and the flow rate and composition of the unstimulated whole saliva in humans. DESIGN: In 28 healthy young adults, the sizes of the three major salivary glands were estimated by use of a magnetic resonance (MR) imaging technique. Unstimulated whole saliva was collected for 5 min by the spitting method, and the flow rate and the concentrations of total protein, Na(+) and K(+) and pH were measured. RESULTS: The estimated sizes of the parotid and submandibular glands showed a significant positive correlation with the flow rate and the secretion rate of total protein in the unstimulated whole saliva, but that of the sublingual glands did not. Concerning the concentrations of Na(+) and K(+) and pH, there were no correlations with the salivary gland sizes. CONCLUSIONS: The results suggest that the larger the sizes of the parotid and submandibular glands, the faster the fluid flow and protein secretion rates in unstimulated whole saliva.

Free D-aspartic acid in rat salivary glands.

Free D-aspartic acid (D-Asp) has been reported to occur in a wide variety of tissues and cells, exclusively in central nervous system and endocrine tissues. In this manuscript, we demonstrate that large amounts of D-Asp are present in the exocrine tissue, salivary glands. In adult male rats, D-Asp concentrations in parotid and submandibular gland were 212+/-68 and 233+/-34 nmol/g wet weight, respectively, and were low (38+/-20 nmol/g wet weight) in sublingual gland. This result indicates that substantial level of D-Asp exists not only in central nervous system and endocrine tissues but also in exocrine tissues. Furthermore, D-Asp concentration in parotid gland increased transiently at 3 weeks of age and decreased thereafter. In contrast, the D-Asp level in submandibular gland continued to increase gradually from 1 to 7 weeks of age and remained at an adult level after 7 weeks of age. Using anti-D-Asp antibody, immunohistochemical study was done against these glands and it showed that the predominant localization of D-Asp in acinar cells in parotid gland, while D-Asp is specifically located in striated duct cells in submandibular gland. These results suggest that D-Asp may play different roles between the two glands.

Hypoxia up-regulates glyceraldehyde-3-phosphate dehydrogenase in mouse brain capillary endothelial cells: involvement of Na+/Ca2+ exchanger.

The molecular regulatory mechanisms and the characterization of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in hypoxia were studied in a mouse brain capillary endothelial cell line, MBEC4. Activation of GAPDH gene expression by hypoxia was suppressed by an intracellular Ca(2+) chelator and inhibited by a non-selective cation channel blocker or a Na(+)/Ca(2+) exchanger (NCX) blocker. Sequencing of reverse transcription-PCR products demonstrated that MBEC4 expressed an mRNA encoding NCX3, which functions even under cellular ATP-depleted conditions, in addition to mRNAs encoding NCX1 and NCX2. The inhibition of Ca(2+)/calmodulin-dependent protein kinases or c-Jun/AP-1 activation caused a significant decrease in the activation of GAPDH mRNA by hypoxia. These results suggest that hypoxia stimulates Ca(2+) influx through non-selective cation channels and causes the reverse operation of the three NCX isoforms, and consequently, increased intracellular Ca(2+) up-regulates GAPDH gene expression through an AP-1-dependent pathway. Furthermore, subcellular fractionation experiments showed that hypoxia increased GAPDH proteins not only in the cytosolic fraction, but also in the nuclear and particulate fractions, in which GAPDH should play no roles in glycolysis. However, the GAPDH activity did not rise in proportion to the increase of GAPDH protein by hypoxia even in the cytosolic fraction. These results suggest that not all hypoxia-induced GAPDH molecules contribute to glycolysis.

D-Aspartic acid in bovine dentine non-collagenous phosphoprotein.

In tooth dentine, owing to its slow metabolism after its formation, racemized and transformed D-aspartic acid remains in the tissue and accumulates with age. However, no dentinal proteins which contain D-aspartic acid have been identified. In this study, a non-collagenous phosphoprotein was purified from bovine dentine. Its molecular mass was about 130 kDa and its amino acid composition was very similar to that of bovine dentine phosphophoryn. The purified protein contained a large proportion of aspartic acid residues and some of them were stereoinverted from the L-isomer to the D-isomer. The D-/L-aspartic acid ratio of dentine non-collagenous phosphoproteins purified from 8-month-old fetal, postnatal and 1-year-old bovine first incisors showed that the stereoinversion tended to increase with age. These results suggest that the purified non-collagenous phosphoprotein is a candidate for the protein in dentine containing D-aspartic acid.