Simultaneous Fluorescent Identification of Odontoblasts and Ameloblasts.

The tooth is mainly composed of dentin and enamel. Identification of dentin-producing odontoblasts and enamel-producing ameloblasts using reporter techniques is useful to study tooth development and regeneration with tissue engineering. Ameloblasts express Amelogenin, Ameloblastin, Enamelin, and Amelotin, whereas odontoblasts express Dentin sialophosphoprotein (Dspp) and Dentin matrix protein1 (Dmp1). Although there are several transgenic lines using promoter elements or bacterial artificial chromosomes (BACs) to label odontoblasts and ameloblasts, there is a possibility that the expression patterns vary from the endogenous genes. Here, we established 2 lines of mice where tdTomato was knocked into the second exon of X-chromosomal Amelogenin (Amelx), and green fluorescent protein (GFP) was knocked into the second exon of Dspp. tdTomato and GFP were highly expressed on secretory ameloblasts and secretory and fully differentiated odontoblasts, respectively. In addition, DSPP and AMELX were not produced in the dentin matrix and enamel matrix of DsppGFP/GFP and AmelxtdTomato male mice (as representative of AmelxtdTomato/Y hemizygous male mice), respectively. Moreover, micro-computed tomography analysis of AmelxtdTomato male mice revealed a notable reduction in enamel volume but increased dentin mineral density. DsppGFP/GFP mice had reduced dentin mineral density. To identify odontoblasts and ameloblasts from developing tooth, we examined the expression of mesenchymal cell surface molecules CD90, CD166 and epithelial cell surface molecules CD49f, Epcam1 with fluorescence on odontoblasts and ameloblasts in these mice. We found that GFP+ odontoblasts and tdTomato+ ameloblasts in tooth germ from 0.5-d-old DsppGFP/+ mice and AmelxtdTomato male mice were enriched in CD45-/Ter119-/Epcam1-/CD90+/Integrin α4+cell fractions and CD45-/Ter119-/Epcam1+/CD49f+/CD147+ cell fractions, respectively. By using antibodies against mesenchymal and epithelial cell surface molecules and fluorescence, we can easily distinguish odontoblasts from ameloblasts and isolate each cell for further studies. These mice would serve as useful models for tooth development and regeneration as well as provide concurrent observation for the differentiation processes of odontoblasts and ameloblasts in vivo and in vitro.

Crosstalk between NRF2 and HIPK2 shapes cytoprotective responses.

Homeodomain interacting protein kinase-2 (HIPK2) is a member of the HIPK family of stress-responsive kinases that modulates cell growth, apoptosis, proliferation and development. HIPK2 has several well-characterised tumour suppressor roles, but recent studies suggest it can also contribute to tumour progression, although the underlying mechanisms are unknown. Herein, we have identified novel crosstalk between HIPK2 and the cytoprotective transcription factor NRF2. We show that HIPK2 is a direct transcriptional target of NRF2, identifying a functional NRF2 binding site in the HIPK2 gene locus and demonstrating for the first time a transcriptional mode of regulation for this kinase. In addition, HIPK2 is required for robust NRF2 responsiveness in cells and in vivo. By using both gain-of-function and loss-of-function approaches, we demonstrate that HIPK2 can elicit a cytoprotective response in cancer cells via NRF2. Our results have uncovered a new downstream effector of HIPK2, NRF2, which is frequently activated in human tumours correlating with chemoresistance and poor prognosis. Furthermore, our results suggest that modulation of either HIPK2 levels or activity could be exploited to impair NRF2-mediated signalling in cancer cells, and thus sensitise them to chemotherapeutic drugs.

Living Donor Liver Transplantation for Progressive Familial Intrahepatic Cholestasis Type 1: Two Reported Cases.

BACKGROUND: Progressive familial intrahepatic cholestasis type 1 (PFIC1) is an inherited disease characterized by cholestatic features. We report two patients with PFIC1 who underwent liver retransplantation. CASE REPORT: One patient was a 3-year-old female who underwent liver transplantation for PFIC1. She presented with severe diarrhea and fatty liver, and went into liver failure. She therefore underwent liver retransplantation and external biliary diversion 8 years after the initial liver transplantation. The explanted liver was histologically diagnosed with chronic rejection. Her intractable diarrhea stopped after the retransplantation. She was diagnosed with a fatty liver 8 months after the retransplantation and died 4 years after retransplantation due to bleeding from an ileostomy. The other patient was a 3-year-old male. This patient underwent liver retransplantation due to liver cirrhosis caused by steatohepatitis 9 years after the initial liver transplantation. The biliary tract was not diverted. He also experienced severe diarrhea after the retransplantation and requires home parenteral nutrition due to an eating disorder. CONCLUSIONS: Liver transplantation is the only treatment to resolve life-threatening issues due to PFIC1, but requires further improvement as a therapeutic modality.

Haploinsufficiency of Sf3b1 leads to compromised stem cell function but not to myelodysplasia.

SF3B1 is a core component of the mRNA splicing machinery and frequently mutated in myeloid neoplasms with myelodysplasia, particularly in those characterized by the presence of increased ring sideroblasts. Deregulated RNA splicing is implicated in the pathogenesis of SF3B1-mutated neoplasms, but the exact mechanism by which the SF3B1 mutation is associated with myelodysplasia and the increased ring sideroblasts formation is still unknown. We investigated the functional role of SF3B1 in normal hematopoiesis utilizing Sf3b1 heterozygous-deficient mice. Sf3b1(+/-) mice had a significantly reduced number of hematopoietic stem cells (CD34(-)cKit(+)ScaI(+)Lin(-) cells or CD34(-)KSL cells) compared with Sf3b1(+/+) mice, but hematopoiesis was grossly normal in Sf3b1(+/-) mice. When transplanted competitively with Sf3b1(+/+) bone marrow cells, Sf3b1(+/-) stem cells showed compromised reconstitution capacity in lethally irradiated mice. There was no increase in the number of ring sideroblasts or evidence of myeloid dysplasia in Sf3b1(+/-) mice. These data suggest that SF3B1 plays an important role in the regulation of hematopoietic stem cells, whereas SF3B1 haploinsufficiency itself is not associated with the myelodysplastic syndrome phenotype with ring sideroblasts.

Role of epidermal growth factor receptor in maintaining airway goblet cell hyperplasia in rats sensitized to allergen.

BACKGROUND: Stimulation of epidermal growth factor receptor (EGFR) induces airway goblet cell hyperplasia, but the role of this molecule in the maintenance of this pathologic change remains uncertain. OBJECTIVE: To determine the mechanisms by which goblet cell hyperplasia is maintained in airway epithelium, we investigated EGFR-induced signalling pathways that lead to both mucin production and antiapoptosis in vitro. We also tested whether the inhibition of EGFR tyrosine kinase speeds reversal of established goblet cell hyperplasia to normal epithelial phenotype in vivo. METHODS: MUC5AC production was measured by immunoassay, and antiapoptotic responses were determined by Bcl-2 expression and terminal deoxynucleotidyl transferase-mediated dUTP-biotin Nick End Labelling staining using NCI-H292 cells. The effect of an inhibitor of EGFR tyrosine kinase (AG1478) on goblet cell hyperplasia was also determined in rats sensitized with ovalbumin (OVA). RESULTS: MUC5AC was constitutively expressed and few apoptotic cells were observed in NCI-H292 cells under non-stimulated condition. TGF-alpha increased MUC5AC and Bcl-2 expression, an effect that was prevented by inhibitors of EGFR tyrosine kinase (AG1478), MEK (PD98059), and NF-kappaB (CAPE). After the addition of TGF-alpha, AG1478 and an inhibitor of phosphatidylinositol 3 kinase/Akt (LY294002), but not PD98059, induced a marked apoptotic response, which was prevented by the caspase inhibitor Z-VAD fmk. Goblet cell hyperplasia and EGFR expression in airway epithelium were noted in the OVA-sensitized rats. Intratracheal instillation of AG1478 induced apoptosis of goblet cells, reverting the airway epithelium to normal epithelial phenotype. CONCLUSION: These findings indicate that EGFR plays an important role in the maintenance of goblet cell hyperplasia. We speculate that inhibitors of the EGFR cascade might be an effective therapy of airway remodelling.

Interleukin-9 and Interleukin-13 augment UTP-induced Cl ion transport via hCLCA1 expression in a human bronchial epithelial cell line.

BACKGROUND: IL-9 and IL-13 induce airway goblet cell metaplasia, which is associated with expression of a Ca(2+)-activated Cl channel, hCLCA1. OBJECTIVE: As UTP stimulates both mucin secretion and Cl ion transport via a Ca(2+)-dependent pathway, the purpose of this study is to determine whether IL-9 and IL-13 affect UTP-induced Cl ion transport in human bronchial epithelial cell line 16HBE cells, and if they do, to elucidate whether such an effect is associated with hCLCA1 expression. METHODS: The increases in short-circuit current (I(sc)) in response to UTP were measured in the presence of amiloride by the Ussing chamber method. The morphology of epithelial cells was assessed by light microscopic findings, and hCLCA1 expression was investigated by immunocytochemistry and immunoblotting. RESULTS: UTP-induced increases in I(sc) in the cells treated with IL-9 or IL-13 for 48 h were greater than those in non-treated cells, and the potency of IL-13 was greater than that of IL-9. Pre-treatment with Ca(2+)-activated Cl channel inhibitors diisothocyanatostilbene-2, 2-disulphonic acid and niflumic acid completely inhibited the augmenting effects of IL-9 and IL-13 on I(sc). The epithelial layer of the cells treated with IL-9 or IL-13 was thicker than that of non-treated cells. The expression of hCLCA1 protein was induced by IL-13 in a concentration-dependent manner. These effects of IL-13 were more potent than those of IL-9. CONCLUSION: IL-9 and IL-13 augmented UTP-induced Cl ion transport, probably via proliferation of the cells with hCLCA1 expression, and IL-13 was more potent than IL-9 in producing such an effect in 16HBE cells.

Adenocarcinoma of the esophagogastric junction: a summary of responses to a questionnaire on adenocarcinoma of the esophagus and the esophagogastric junction in Japan.

Adenocarcinoma of the esophagogastric junction is recognized as a distinct clinical entity; however, the choice of surgical approaches is controversial. To analyze the results of surgery among patients with adenocarcinoma of the esophagus (type I) and the cardia (type II) based on Siewert's classification in Japan, surgical procedures, histopathologic characteristics, and outcome were re-evaluated according to the TNM classification in 1263 patients with adenocarcinoma of the esophagus (type I) and the cardia (type II) through a questionnaire sent to the members of the Japanese Society of Esophageal Diseases. One hundred and thirty-four (10.6%) patients had type I tumors and 1129 (89.4%) patients had type II tumors. There were significant differences in sex distribution and associated intestinal metaplasia in the esophagus between patients with type I and type II tumors. Although different surgical approaches were performed, the overall 5-year survival rate was 53% without any difference between the two groups. The significant prognostic factors in general linear models were R category, pN category, and differentiation, but not pT category. There was no difference in survival between patients with stage IIB and III disease. The survival rate of the patients who underwent a transhiatal approach was similar to that of those undergoing a transthoracic approach. The results suggest that Siewert's classification (type I and type II) is useful in planning treatment strategy for adenocarcinoma of the esophagogastric junction. Lymph node metastasis was the most important prognostic factor, and staging based on the number of lymph node metastases or the extent of lymph node metastasis is necessary.

Beta-adrenergic receptor-mediated growth of human airway epithelial cell lines.

Abnormal growth of airway epithelium and the resultant thickening of airway walls may produce narrowing of airway calibre, thereby contributing to deterioration of bronchoconstriction in chronic obstructive pulmonary disease (COPD). Beta2-adrenergic agonists have been widely used for the treatment of COPD, but their effects on the growth of airway epithelial cells is unknown. Growth of three human airway epithelial cell lines was studied in vitro. Exposure to salbutamol in serum-free medium increased 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide reduction and intracellular deoxyribonucleic acid (DNA) contents in 16-human bronchial epithelium (16-HBE) cells and NCI-H292 cells, but not in A549 cells. The growth-promoting effect of salbutamol in 16-HBE cells was equipotent to 10% foetal bovine serum and was inhibited by propranolol and a cyclic adenosine monophosphate (cAMP) antagonist, Rp-adenosine 3',5'-cyclic monophosphorothioate triethylammonium salt (Rp-cAMPS). Likewise, forskolin and 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) caused cell growth and DNA synthesis. Western blot analysis showed that salbutamol, forskolin, and 8-Br-cAMP each induced expression of the phosphorylated form of mitogen-activated protein (MAP) kinase, and that the salbutamol-induced phosphorylation was inhibited by propranolol, Rp-cAMPS, and the MAP kinase-kinase inhibitor PD98059. These results suggest that in certain airway epithelial cell lines stimulation of beta2-adrenergic receptors and the consequent production of cyclic adenosine monophosphate may upregulate cell growth, probably through activation of the mitogen-activated protein kinase cascade.

Structural features of a wheat plastome as revealed by complete sequencing of chloroplast DNA.

Structural features of the wheat plastome were clarified by comparison of the complete sequence of wheat chloroplast DNA with those of rice and maize chloroplast genomes. The wheat plastome consists of a 134,545-bp circular molecule with 20,703-bp inverted repeats and the same gene content as the rice and maize plastomes. However, some structural divergence was found even in the coding regions of genes. These alterations are due to illegitimate recombination between two short direct repeats and/or replication slippage. Overall comparison of chloroplast DNAs among the three cereals indicated the presence of some hot-spot regions for length mutations. Whereas the region with clustered tRNA genes and that downstream of rbcL showed divergence in a species-specific manner, the deletion patterns of ORFs in the inverted-repeat regions and the borders between the inverted repeats and the small single-copy region support the notion that wheat and rice are related more closely to each other than to maize.

Cooperative functions of the mannoprotein-encoding genes in the biogenesis and maintenance of the cell wall in Saccharomyces cerevisiae.

To elucidate the roles of genes involved in the cell wall biogenesis and function in Saccharomyces cerevisiae, we isolated and characterized mutants that were lethal in a strain in which the SED1 gene encoding a cell wall mannoprotein was disrupted. Thus, double mutants of SED1 and either MNN9 or MNN10 were unable to grow and YOL155c on a multicopy plasmid could suppress their synthetic lethality. A Yol155cp-GFP fusion protein was found to localize to the cell wall, suggesting that it might also be a cell wall mannoprotein. Subsequently, we analysed the effects of the shut-off of SED1 in a sed1 and mnn9 double mutant: cells after the shut-off showed anomalous cellular morphology and died in the mitotic M phase. From these and other results, we postulate that these genes function cooperatively with each other and in a cell cycle-dependent manner in the biogenesis and maintenance of cell wall in S. cerevisiae.

Phase II evaluation of protracted infusion of cisplatin and 5-fluorouracil in advanced squamous cell carcinoma of the esophagus: a Japan Esophageal Oncology Group (JEOG) Trial (JCOG9407).

BACKGROUND: Surgery for advanced esophageal carcinoma has its limits as regards aggressiveness and therapeutic effect, therefore effective multimodality treatment is required to obtain better survival. The objective of this study was to evaluate whether daily continuous infusion of CDDP could achieve a higher clinical response rate with less toxicity than its drip infusion in the previous phase II study that we had conducted. METHODS: Patients with primary extensive or relapsed esophageal carcinoma after esophagectomy, which had distant organ metastasis and histologically proven SCC, were eligible for this study. A dose of 20 mg/m(2) of cisplatin and 800 mg/m(2) of 5-fluorouracil was given by continuous infusion for 24 h on days 1-5. This treatment was repeated every 4 weeks for up to four cycles. A total of 36 men and six women with a median age of 64 (range 39-75) years were registered and 36 patients were eligible. RESULTS: The overall response rate of the registered patients was 33.3% (12/36) and the median response duration was 175 days. Median survival time was 201.5 days and the 1-year survival rate was 27.8%. Change from bolus to continuous infusion of cisplatin affected neither the type nor the degree of toxicity. CONCLUSION: Daily continuous infusion of cisplatin was not associated with higher response or lower toxicity than those seen with the high-dose bolus or multibolus treatment regimens. We conclude that this regimen in this setting is not worthy of further phase III trials. JEOG is now evaluating other drug combination regimens.

Serum p53 antibody as a useful marker for monitoring of treatment of superficial colorectal adenocarcinoma after endoscopic resection.

BACKGROUND: Mutation of the p53 gene is a genetic alteration found in human cancers. Overexpression of p53 has been found to induce antibody production in serum, and, recently, the simple detection of serum antibody has been made possible. The aim of this study was to evaluate the potential role of serum p53 antibody in the early diagnosis of superficial colorectal cancer and in the monitoring of its treatment after endoscopic resection. METHODS: In a prospective study, our subjects were 27 patients with superficial colorectal adenocarcinomas, whose results were compared with those in 38 patients with benign adenomas; all patients were treated by endoscopic resection. The correlation between serum p53 antibody levels before and within 3 weeks after resection was determined, using an immunoassay. Immunohistological staining for p53 was also performed, and its sensitivity was compared with that of two other tumor markers. RESULTS: Preoperatively, serum p53 antibody was detected in 63.0% (17/27) patients with adenocarcinoma and in 2.6% (1/38) patients with adenoma, showing a significant difference (P < 0.001). However, the two other markers carcinoembryonic antigen (CEA) and carbohydrate antigen CA19-9, showed no significant difference between superficial colorectal adenocarcinoma and adenoma. The serum p53 antibody status was strongly correlated with p53 immunostaining in adenocarcinoma (P = 0.0065), but there was no significant correlation in adenoma (P = 0.973). Sixteen (94.1%) of 17 seropositive adenocarcinoma patients, showed negative conversion after complete tumor resection, and all these 16 patients remained seronegative. CONCLUSION: The detection of serum p53 antibody is expected to serve as a new genetic marker, determined by serological analyses, for aiding in the early diagnosis of superficial colorectal cancer and indicating its local curability after endoscopic treatment.

Prognosis of patients with advanced carcinoma of the esophagus with complete response to chemotherapy and/or radiation therapy: a questionnaire survey in Japan.

BACKGROUND: We estimated the survival of patients with advanced carcinoma of the esophagus in Japan who achieved complete response (CR) with chemotherapy and/or radiation therapy. METHODS: A questionnaire was designed for patients with cancer of the esophagus with pretreatment stage II-IV (excluding organ metastasis [M1]), who were treated with chemotherapy and/or radiation therapy and achieved either a clinical CR continuing for more than 1 year, or a pathological CR in surgical specimens. All patients were treated between January 1, 1990, and December 31, 1997, in Japan. RESULTS: Of the 169 eligible patients for whom adequate data were available, 106 patients with continuing clinical CR were defined as group A and 63 with pathological CR as group B. The overall survival rates at 5 years were 62.4% in group A and 64.8% in group B. In each of groups A and B, there was no significant difference in overall survival among subgroups of patients classified by initial pretreatment clinical stage. In group A, the survival rate of patients with concurrent chemotherapy and radiation therapy was significantly better than the rates for patients with chemotherapy alone or radiotherapy alone. In group A, the frequency of first failure at the local site of esophageal carcinoma was 7.7%. Of the 12 patients in group B (19%) who died less than 1 year postoperatively, 6 died of postoperative complications. CONCLUSION: The effect of CR to chemotherapy and/or radiation therapy for carcinoma of the esophagus on survival was marked. In patients with esophageal carcinoma who achieve CR, the prognosis may be independent of the initial pretreatment stage. Local failure in group A patients remains a problem, however.

Mouse homologue of coq7/clk-1, longevity gene in Caenorhabditis elegans, is essential for coenzyme Q synthesis, maintenance of mitochondrial integrity, and neurogenesis.

coq7/clk-1 was isolated from a long-lived mutant of Caenorhabditis elegans, which showed sluggish behavior and an extended life span. Mouse coq7 is homologous to Saccharomyces cerevisiae coq7/cat5 that is required for biosynthesis of coenzyme Q (CoQ), an essential cofactor in mitochondrial respiration. Here we generated COQ7-deficient mice to investigate the biological role of COQ7 in mammals. COQ7-deficient mouse embryos failed to survive beyond embryonic day 10.5, exhibiting small-sized body and delayed embryogenesis. Morphological studies showed that COQ7-deficient neuroepithelial cells failed to show the radial arrangement in the developing cerebral wall, aborting neurogenesis at E10.5. Electron microscopic analysis further showed the enlarged mitochondria with vesicular cristae and enlarged lysosomes filled with disrupted membranes, which is consistent with mitochondriopathy. Biochemical analysis demonstrated that COQ7-deficient embryos failed to synthesize CoQ(9), but instead yielded demethoxyubiquinone 9 (DMQ(9)). Cultured embryonic cells from COQ7-deficient mice were rescued by adding bovine fetal serum in vitro, but exhibited slowed cell proliferation, which resembled to the phenotype of clk-1 with delayed cell divisions. The result implied the essential role of coq7 in CoQ synthesis, maintenance of mitochondrial integrity, and neurogenesis in mice.

Trehalose sensitivity in Drosophila correlates with mutations in and expression of the gustatory receptor gene Gr5a.

Drosophila taste gene Tre is located on the distal X chromosome and controls gustatory sensitivity to a subset of sugars [1, 2]. Two adjacent, seven-transmembrane domain genes near the Tre locus are candidate genes for Tre. One (CG3171) encodes a rhodopsin family G protein receptor [3, 4], and the other (Gr5a) is a member of a chemosensory gene family encoding a putative gustatory receptor [5-7]. We carried out molecular analyses of mutations in Tre to elucidate their involvement in the gustatory phenotype. Here, we show that Tre mutations induced by P element-mediated genomic deletions disrupt Gr5a gene organization and the expression of Gr5a mRNA, while disruption of the CG3171 gene or its expression was not always associated with mutations in Tre. In flies with the spontaneous mutation Tre(01), both CG3171 and Gr5a mRNAs are transcribed. Coding sequences of these two candidate genes were compared among various strains. A total of three polymorphic sites leading to amino acid changes in CG3171 were not correlated with the gustatory phenotype. Among four nonsynonymous sites in Gr5a, a single nucleotide polymorphism leading to an Ala218Thr substitution in the predicted second intracellular loop cosegregated with Tre(01). Taken together, the mutation analyses support that Gr5a is allelic to Tre.

Molecular cloning, genetic mapping, and expression of the mouse Sf3b1 (SAP155) gene for the U2 snRNP component of spliceosome.

SAP155, a subunit of the U2 snRNP, is essential for prespliceosome assembly and splicing catalysis of the major spliceosome. Moreover, the protein has been identified in the minor (U12-dependent) spliceosome. These facts strongly suggest that SAP155 is shared by two distinct complexes owing to its importance in the removal of any type of intron. Here we have isolated a cDNA encoding the 146-kDa mouse homolog, designated Sf3b1. The amino acid sequence of Sf3b1 is very highly conserved among homologs from Schizosaccharomyces pombe (52.4% identity) to human (99.6%), and the C-terminal 825 residues of these Sf3b1 homologs show even higher identities. This C-terminal region shows significant similarity to the PR65 subunit of protein phosphatase 2A, which is composed of 15 tandem repeats of a 39 amino acid sequence. Mouse genome analyses showed Sf3bh1 to be a single-copy gene mapping to the central part of Chromosome (Chr) 1. Northern blot analysis and whole mount in situ hybridization revealed Sf3b1 to be ubiquitously expressed in a variety of adult tissues and mid-gestation embryos.

Monitoring of p53 autoantibodies after resection of colorectal cancer: relationship to operative curability.

OBJECTIVE: To investigate the clinical use of p53 autoantibodies as a marker in the postoperative monitoring of colorectal cancer. DESIGN: Retrospective study. SETTING: Teaching hospital, Japan. SUBJECTS: 40 patients with colorectal cancer who had p53 autoantibodies in their serum preoperatively. INTERVENTIONS: Serial assay of p53 autoantibodies by ELISA before and after resection. MAIN OUTCOME MEASURES: Interpretation by a qualitative analysis. RESULTS: A significant correlation was observed between curability by surgical resection and postoperative disappearance of p53 autoantibodies. Twenty-seven (96%) of 28 patients, who had p53 autoantibodies and whose cancer was completely removed, had no such antibodies after resection and no recurrence after 7 to 26 months. CONCLUSIONS: Postoperative assays of p53 autoantibodies are potentially useful for predicting recurrence of colorectal cancer in patients who have p53 autoantibodies preoperatively.