A high-density linkage map with 2560 markers and its application for the localization of the male-sterile genes <Emphasis Type="Italic">ms3</Emphasis> and <Emphasis Type="Italic">ms4</Emphasis> in <Emphasis Type="Italic">Cryptomeria japonica</Emphasis> D. Don

Cryptomeria japonica pollinosis is one of the most serious allergic diseases in Japan; this is a social problem because C. japonica is the most important Japanese forestry species. In order to reduce the amount of pollen dispersed, breeding programs using trees with male-sterile genes have been implemented. High-density linkage maps with stable ordering of markers facilitate the localization of male-sterile genes and the construction of partial linkage maps around them in order to develop markers for use in marker-assisted selection. In this study, a high-density linkage map for C. japonica with 2560 markers was constructed. The observed map length was 1266.2 cM and the mean distance between adjacent markers was 0.49 cM. Using information from this high-density map, we newly located two male-sterile genes (ms3 and ms4) on the first and fourth linkage groups, respectively, and constructed partial linkage maps around these loci. We also constructed new partial linkage maps around the ms1 and ms2 loci using additional SNP markers. The closest markers to the ms1, ms2, ms3, and ms4 male-sterile loci were estSNP04188 (1.8 cM), estSNP00695 (7.0 cM), gSNP05415 (3.1 cM), and estSNP01408 (7.0 cM) respectively. These results allowed us to develop SNP markers tightly linked to the male sterile genes for use in MAS; this will accelerate the future isolation of these genes by map-based cloning approaches.     

Normal Lung Quantification in Usual Interstitial Pneumonia Pattern: The Impact of Threshold-based Volumetric CT Analysis for the Staging of Idiopathic Pulmonary Fibrosis

BackgroundAlthough several computer-aided computed tomography (CT) analysis methods have been reported to objectively assess the disease severity and progression of idiopathic pulmonary fibrosis (IPF), it is unclear which method is most practical. A universal severity classification system has not yet been adopted for IPF.ObjectiveThe purpose of this study was to test the correlation between quantitative-CT indices and lung physiology variables and to determine the ability of such indices to predict disease severity in IPF.MethodsA total of 27 IPF patients showing radiological UIP pattern on high-resolution (HR) CT were retrospectively enrolled. Staging of IPF was performed according to two classification systems: the Japanese and GAP (gender, age, and physiology) staging systems. CT images were assessed using a commercially available CT imaging analysis workstation, and the whole-lung mean CT value (MCT), the normally attenuated lung volume as defined from −950 HU to −701 Hounsfield unit (NL), the volume of the whole lung (WL), and the percentage of NL to WL (NL%), were calculated.ResultsCT indices (MCT, WL, and NL) closely correlated with lung physiology variables. Among them, NL strongly correlated with forced vital capacity (FVC) (r = 0.92, P <0.0001). NL% showed a large area under the receiver operating characteristic curve for detecting patients in the moderate or advanced stages of IPF. Multivariable logistic regression analyses showed that NL% is significantly more useful than the percentages of predicted FVC and predicted diffusing capacity of the lungs for carbon monoxide (Japanese stage II/III/IV [odds ratio, 0.73; 95% confidence intervals (CI), 0.48 to 0.92; P < 0.01]; III/IV [odds ratio. 0.80; 95% CI 0.59 to 0.96; P < 0.01]; GAP stage II/III [odds ratio, 0.79; 95% CI, 0.56 to 0.97; P < 0.05]).ConclusionThe measurement of NL% by threshold-based volumetric CT analysis may help improve IPF staging.     

Delta-opioid receptor activation before ischemia reduces gap junction permeability in ischemic myocardium by PKC-epsilon-mediated phosphorylation of connexin 43

The aim of this study was to examine the hypothesis that delta-opioid receptor activation before ischemia suppresses gap junction (GJ) permeability by PKC-mediated connexin 43 (Cx43) modulation, which contributes to infarct size limitation afforded by the delta-opioid receptor activation. A delta-opioid receptor agonist, [D-Ala(2),D-Leu(5)]-enkephalin acetate (DADLE, 300 nM), was used in place of preconditioning (PC) ischemia to trigger PC mechanisms in rat hearts. GJ permeability during ischemia, which was assessed by Lucifer yellow, was reduced by DADLE to 47% of the control level, and this effect of DADLE was almost abolished by a PKC-epsilon inhibitor [PKC-epsilon translocation inhibitory peptide (PKC-epsilon-TIP)] but was not affected by a PKC-delta inhibitor (rottlerin). After DADLE infusion, PKC-epsilon, but not PKC-delta, was coimmunoprecipitated with Cx43, and the level of phosphorylation of Cx43 at a PKC-dependent site (Ser(368)) was significantly elevated during ischemia. DADLE reduced infarct size after 35 min of ischemia followed by 2 h of reperfusion by 69%, and PKC-epsilon-TIP and rottlerin eliminated 48% and 63%, respectively, of the infarct size-limiting effect of DADLE. Infusion of a GJ blocker, heptanol, before reperfusion reduced infarct size by 36%, and this protection was not enhanced by preischemic infusion of rottlerin + DADLE, which allows PKC-epsilon activation by DADLE. These results suggest that phosphorylation of Cx43 by PKC-epsilon plays a crucial role in delta-opioid-induced suppression of GJ permeability in ischemic myocardium and that this modulation of the GJ is possibly an adjunct mechanism of infarct size limitation afforded by preischemic delta-opioid receptor activation.     

Altered localization of amyloid precursor protein under endoplasmic reticulum stress

Recent reports have shown that the endoplasmic reticulum (ER) stress is relevant to the pathogenesis of Alzheimer disease. Following the amyloid cascade hypothesis, we therefore attempted to investigate the effects of ER stress on amyloid-beta peptide (Abeta) generation. In this study, we found that ER stress altered the localization of amyloid precursor protein (APP) from late compartments to early compartments of the secretory pathway, and decreased the level of Abeta 40 and Abeta 42 release by beta- and gamma-cutting. Transient transfection with BiP/GRP78 also caused a shift of APP and a reduction in Abeta secretion. It was revealed that the ER stress response facilitated binding of BiP/GRP78 to APP, thereby causing it to be retained in the early compartments apart from a location suitable for the cleavages of Abeta. These findings suggest that induction of BiP/GRP78 during ER stress may be one of the regulatory mechanisms of Abeta generation.     

Multipotent cells from the human third molar: feasibility of cell-based therapy for liver disease

Abstract Adult stem cells have been reported to exist in various tissues. The isolation of high-quality human stem cells that can be used for regeneration of fatal deseases from accessible resources is an important advance in stem cell research. In the present study, we identified a novel stem cell, which we named tooth germ progenitor cells (TGPCs), from discarded third molar, commonly called as wisdom teeth. We demonstrated the characterization and distinctiveness of the TGPCs, and found that TGPCs showed high proliferation activity and capability to differentiate in vitro into cells of three germ layers including osteoblasts, neural cells, and hepatocytes. TGPCs were examined by the transplantation into a carbon tetrachloride (CCl₄)-treated liver injured rat to determine whether this novel cell source might be useful for cell-based therapy to treat liver diseases. The successful engraftment of the TGPCs was demonstrated by PKH26 fluorescence in the recipient's rat as to liver at 4 weeks after transplantation. The TGPCs prevented the progression of liver fibrosis in the liver of CCl₄-treated rats and contributed to the restoration of liver function, as assessed by the measurement of hepatic serum markers aspartate aminotransferase and alanine aminotransferase. Furthermore, the liver functions, observed by the levels of serum bilirubin and albumin, appeared to be improved following transplantation of TGPCs. These findings suggest that multipotent TGPCs are one of the candidates for cell-based therapy to treat liver diseases and offer unprecedented opportunities for developing therapies in treating tissue repair and regeneration.     

Ser9 phosphorylation of mitochondrial GSK-3beta is a primary mechanism of cardiomyocyte protection by erythropoietin against oxidant-induced apoptosis

The aim of this study was to determine the role of GSK-3beta in cardiomyocyte protection afforded by erythropoietin (EPO) against oxidant stress-induced apoptosis. Treatment with EPO (10 units/ml) induced Ser473 phosphorylation of Akt and Ser9 phosphorylation of GSK-3beta and significantly reduced the proportion of apoptotic H9c2 cardiomyocytes after exposure to H2O2 from 38.3 +/- 2.7% to 26.0 +/- 2.9%. This protection was not detected in cells transfected with constitutively active GSK-3beta (S9A), which lacks Ser9 for inhibitory phosphorylation. The antiapoptotic effect of EPO was mimicked completely by GSK-3beta knockdown using small interfering RNA and partly by the transfection with kinase-deficient GSK-3beta (K85R). The level of colocalization of intracellular GSK-3beta with mitochondria assessed by enhanced green fluorescent protein-tagged GSK-3beta or immunocytochemistry was not altered by EPO treatment. However, EPO increased the level of Ser9-phospho-GSK-3beta colocalized with mitochondria by 50% in a phosphatidylinositol 3-kinase-dependent manner. Mitochondrial translocation of Bcl-2-associated X protein (BAX) after exposure to H2O2 was inhibited by EPO pretreatment and by GSK-3beta knockdown. These results suggest that the suppression of GSK-3beta activity by Akt-mediated Ser9 phosphorylation in the mitochondria affords cardiomyocytes tolerance against oxidant-induced apoptosis, possibly by inhibiting the access of BAX to the mitochondria.     

Cholesterol-lowering effect of rice bran protein containing bile acid-binding proteins

Dietary plant protein is well known to reduce serum cholesterol levels. Rice bran is a by-product of rice milling and is a good source of protein. The present study examined whether feeding rats a high-cholesterol diet containing 10% rice bran protein (RBP) for 10 d affected cholesterol metabolism. Rats fed dietary RBP had lower serum total cholesterol levels and increased excretion of fecal steroids, such as cholesterol and bile acids, than those fed dietary casein. In vitro assays showed that RBP strongly bound to taurocholate, and inhibited the micellar solubility of cholesterol, compared with casein. Moreover, the bile acid-binding proteins of the RBP were eluted by a chromatographic column conjugated with cholic acid, and one of them was identified as hypothetical protein OsJ_13801 (NCBI accession No. EAZ29742) using MALDI-TOF mass spectrometry analysis. These results suggest that the hypocholesterolemic action of the RBP may be caused by the bile acid-binding proteins.     

Gastric inhibitory polypeptide modulates adiposity and fat oxidation under diminished insulin action

Gut hormone gastric inhibitory polypeptide (GIP) stimulates insulin secretion from pancreatic β-cells upon ingestion of nutrients. Inhibition of GIP signaling prevents the onset of obesity and consequent insulin resistance induced by high-fat diet. In this study, we investigated the role of GIP in accumulation of triglycerides into adipocytes and in fat oxidation peripherally using insulin receptor substrate (IRS)-1-deficient mice and revealed that IRS-1^−/−GIPR^−/− mice exhibited both reduced adiposity and ameliorated insulin resistance. Furthermore, increased gene expression of CD36 and UCP2 in liver, and increased expression and enzyme activity of 3-hydroxyacyl-CoA dehydrogenase in skeletal muscle of IRS-1^−/−GIPR^−/− mice might contribute to the lower respiratory quotient and the higher fat oxidation in light phase. These results suggest that GIP plays a crucial role in switching from fat oxidation to fat accumulation under the diminished insulin action as a potential target for secondary prevention of insulin resistance.