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A possible cure for diabetes is explored by using non-pancreatic cells such as fetal hepatocytes. The expression of insulin and transcription factors for insulin is investigated in mouse fetal liver. We detected mRNAs for insulin I ( Ins1) and insulin II ( Ins2) and proinsulin- and mature insulin-positive cells in mouse fetal liver by reverse transcription plus the polymerase chain reaction and immunohistochemistry. Glucagon, somatostatin and pancreatic polypeptide were not expressed throughout development. Mouse Ins2 and Ins1 promoters were transiently activated in mouse fetal hepatocytes of embryonic days 13.5 and 16.5, respectively. Pancreatic and duodenal homeobox 1 ( Pdx1) mRNA was not expressed during development of the liver. In contrast, mRNAs and proteins of neurogenic differentiation (NeuroD)/β cell E-box transactivator 2 (Beta2) and v-maf musculoaponeurotic fibrosarcoma oncogene homolog (MafA) were almost simultaneously expressed with insulin genes in the liver. Ins2 and Ins1 promoters were activated in hepatoma cells by the transfection of the expression vector for NeuroD/Beta2 alone and by the combination of NeuroD/Beta2 and MafA, respectively. These results indicate that the expression of NeuroD/Beta2 and MafA is linked temporally with the transcription of Ins2 and Ins1 genes in mouse fetal liver and suggest the potential usage of fetal hepatocytes to make insulin-producing β cells by introducing transcription factors. [ABSTRACT FROM AUTHOR]

Background: CD133 was recently reported to be a cancer stem cell marker and a prognostic marker for several tumors. However, few studies have investigated CD133 expression in esophageal squamous cell carcinoma (ESCC). Therefore, we examined whether CD133 could serve as a prognostic marker of ESCC and investigated the correlation between CD133 expression and the clinicopathological findings of ESCC patients and several markers. Methods: We studied 86 ESCC patients who underwent curative surgery without neoadjuvant treatment at Tohoku University Hospital (Sendai, Japan) between January 2000 and December 2005. We analyzed tissue specimens by immunohistochemical staining for CD133, p53, p16, p27, murine double minute 2 (MDM2), Ki-67, and epidermal growth factor receptor (EGFR). Results: Pathological tumor depth and tumor stage were significantly more advanced among CD133-negative patients than among CD133-positive patients. A log-rank test showed that CD133 immunoreactivity was significantly correlated with the overall survival of the patients (P = 0.049). However, multivariate analysis showed that it was not significantly correlated (P = 0.078). Moreover, CD133 was significantly positively correlated with p27 immunoreactivity (P = 0.0013) and tended to be positively correlated with p16 immunoreactivity (P = 0.057). In addition, p16 immunoreactivity was correlated with smoking history (P = 0.018), pathological lymph node status (P = 0.033), and lymphatic invasion (P = 0.018). Conclusions: This study indicated that CD133 immunoreactivity is a good predictor of prognosis in ESCC patients. In addition, CD133 may play a role in the regulation of tumor cell cycle through p27 and p16 in ESCC. At present, it thus remains controversial whether CD133 expression is a valid prognostic marker for ESCC. To elucidate this relationship, further investigations are required. [ABSTRACT FROM AUTHOR]

Interstitial collagenases, including matrix metalloproteinase-1 (MMP-1) and -8 (MMP-8), serve as initiators of extracellular matrix destruction in periodontal disease. Collagenase activities are mainly regulated by tissue inhibitors of metalloproteinases (TIMPs). We tested the effects of inflammation on MMP-1 and MMP-8 gene expression in periodontal disease. To determine the relative abundance of these mRNAs in gingiva, we used a reverse transcription-polymerase chain reaction (RT-PCR) assay Gingival biopsies were divided into 2 groups; a control group and an inflamed group with severe gingivitis or periodontitis. The MMP-1 mRNA levels were significantly elevated in inflamed gingiva, while the levels of the MMP-8 transcript were not different in the 2 groups and barely detectable by RT-PCR assay. The expression of the TIMP-1 gene was not altered, and remained higher than any of these other genes in both control and diseased gingivae. These results suggest that MMP-1 rather than MMP-8 may play an important rôle in the initiation of collagen degradation in periodontal disease. However, the possibility remains that MMP-8 plays an important role in periodontal tissue destruction, since the mRNA abundance and not the enzyme activity was assessed. [ABSTRACT FROM AUTHOR]

Recent studies have reported that growth factors, such as PDGF or TGFβ stimulate the biochemical and pathological changes of diabetic nephropathy. However, the relationship between the expression of growth factors and the pathological changes has not been clearly demonstrated in in vivo diabetic model. In this study, we produced transgenic mice expressing constitutively active form of Calmodulin-dependent protein kinase IIα (CaMK II α) specifically in pancreatic β cells (TG mice). We found that these mice had the dramatically reduced number of pancreatic β cells and insulin level in pancreas was negligible at 4 weeks of age. Blood glucose level (>600mg/dl) and HbA1c (>HbA1c9.9%) were markedly elevated from 4 and 8 weeks, respectively. The urinary excretion of albumin was increased from 8 weeks (5 times of control mice), but serum BUN and creatinine were not significantly altered compared to that of the control mice. Histological analysis of glomeruli showed diabetic changes, such as glomerular hypertrophy (2 times of control) or mesangial expansion (3 times of control) from 8 weeks. Using this model of diabetic nephropathy, we then examined the expression of PDGF and PDGF receptors. PDGF-B chain in glomeruli was detected by immunostaining from 8 weeks. The expression of PDGF-β receptor was observed in mesangial area of both TG and control mice. The PDFD-β receptor-positive area was increased in TG mice in accordance with the enlarged mesangial area. In conclusion, CaMKIIα-transgenic mice could be considered a good model for diabetic nephropathy observed in type 1 diabetes. Because the expression of PDGFB chain and PDGF-β receptor in glomeruli was enhanced with its biochemical and pathological changes as diabetic nephropathy, it was suggested that the activation of PDGF signaling may play important roles in the occurrence of diabetic nephropathy in these model mice. [ABSTRACT FROM AUTHOR]

Abstract: Cyclic ADP-ribose (cADPR), a potent Ca2+ mobilizing intracellular messenger synthesized by CD38, regulates the opening of ryanodine receptors (RyRs). Increases in intracellular Ca2+ concentrations in pancreatic islets, resulting from Ca2+ mobilization from RyRs as well as Ca2+ influx from extracellular sources, are important in insulin secretion by glucose. In the present study, by screening a rat islet cDNA library, we isolated a novel RyR cDNA (the islet-type RyR), which is generated from the RyR2 gene by alternative splicing of exons 4 and 75. When the expression vectors for the islet-type and the authentic RyRs were transfected into HEK293 cells, the islet-type RyR2 as well as the authentic one showed high affinity [3H]ryanodine binding. Intracellular Ca2+ release in the islet-type RyR2-transfected cells was enhanced in the presence of cADPR but not in the authentic RyR2-transfected cells. The islet-type RyR2 mRNA was expressed in a variety of tissues such as in pancreatic islets, cerebrum, and cerebellum, whereas the authentic RyR2 mRNA was predominantly expressed in heart and aorta. These results suggest that the islet-type RyR2 may be an intracellular target for cADPR signaling. [Copyright &y& Elsevier]

Abstract: Dalcetrapib (RO4607381/JTT-705), an agent that targets cholesteryl ester transfer protein, is in development for prevention of cardiovascular events. In vitro studies were performed to identify receptors that mediate an off-target effect of dalcetrapib observed in preclinical models: increased lipid uptake into the lamina propria of the small intestine and into mesenteric lymph node macrophages. Uptake of oxidized low-density lipoprotein (LDL) cholesterol or dalcetrapib-treated chylomicrons was quantitated by triglyceride assay or fluorescent labeling in primary macrophages and the cell lines CHO, J774A.1 (mouse macrophages) and THP-1 (human macrophages). Quantitative reverse-transcriptase polymerase chain reaction and immunoblotting measured candidate receptor expression. Lectin-like oxidized LDL receptor (LOX-1) and scavenger receptor type AI (SR-AI) were excluded as candidate receptors based on lack of association between their expression and uptake of dalcetrapib-treated lipids. In J774A.1 cells, uptake of dalcetrapib-treated chylomicrons was increased by LPS and associated with expression of MAcrophage Receptor with COllagenous domain (MARCO). MARCO was expressed at very low levels in human macrophages and was not inducible by LPS. The MARCO receptor may account for the variable species susceptibility towards dalcetrapib-mediated chylomicron uptake by macrophages. [Copyright &y& Elsevier]

Abstract: Heparan sulfate (HS) binds with several signaling molecules and regulates ligand–receptor interactions, playing an essential role in embryonic development. Here we showed that HS was intensively expressed in pancreatic islet β-cells after 1week of age in mice. The enzymatic removal of HS in isolated islets resulted in attenuated glucose-induced insulin secretion with a concomitant reduction in gene expression of several key components in the insulin secretion machinery. We further depleted islet HS by inactivating the exostosin tumor-like 3 gene specifically in β-cells. These mice exhibited abnormal islet morphology with reduced β-cell proliferation after 1week of age and glucose intolerance due to defective insulin secretion. These results demonstrate that islet HS is involved in the regulation of postnatal islet maturation and required to ensure normal insulin secretion. [Copyright &y& Elsevier]

Abstract: Regenerating gene (Reg) I has been identified as a regenerative/proliferative factor for pancreatic islet cells. We examined Reg I expression in the regenerating liver of a rat model that had been administered 2-acetylaminofluorene and treated with 70% partial hepatectomy (2-AAF/PH model), where hepatocyte and cholangiocyte proliferation was suppressed and the hepatic stem cells and/or hepatic progenitor cells were activated. In a detailed time course study of activation of hepatic stem cells in the 2-AAF/PH model, utilizing immunofluorescence staining with antibodies of Reg I and other cell-type-specific markers, we found that Reg I-expressing cells are present in the bile ductules and increased during regeneration. Reg I-expressing cells were colocalized with CK19, OV6, and AFP. These results demonstrate that Reg I is significantly upregulated in the liver of the 2-AAF/PH rat model, accompanied by the formation of bile ductules during liver regeneration. [Copyright &y& Elsevier]

Abstract: Detailed studies were performed on diabetic kidneys derived from transgenic mice overexpressing the mutant form (Thr286Asp) of Ca2+/calmodulin-dependent protein kinase IIα (CaM kinase IIα) in pancreatic β-cells. Kidney weight/body weight ratio, urinary albumin/creatinine ratio, serum BUN level, and mesangial/glomerular area ratio were all significantly higher in transgenic mice than in wild-type mice. cDNA microarray analysis revealed 17 up-regulated genes and 12 down-regulated genes in transgenic kidney. Among up-regulated genes, cyclin D2 (6.70-fold) and osteopontin (2.35-fold) were thought to play important roles in the progression of diabetic nephropathy. Transgenic glomeruli and tubular epithelial cells were strongly stained for osteopontin, a molecule which induces immune response. In quantitative real-time RT-PCR analyses, expressions of not only M1 macrophage marker genes but also M2 macrophage marker genes were elevated in renal cortex of transgenic mice. Overall results indicate that CaM kinase IIα (Thr286Asp) transgenic mice serve as an excellent model for diabetic nephropathy. [Copyright &y& Elsevier]