Migratory marker expression in fibroblast foci of idiopathic pulmonary fibrosis

BACKGROUND: Fibroblast foci (FF) are considered a relevant morphologic marker of idiopathic pulmonary fibrosis/usual interstitial pneumonia (IPF/UIP), and are recognised as sites where fibrotic responses are initiated and/or perpetuated in this severe disease. Despite their relevance, the cellular and molecular mechanisms responsible for the formation of FF and their role in tissue remodelling are poorly defined. In previous studies we have provided evidence of abnormal activation of the wnt-signaling-pathway in IPF/UIP that is centred on FF and the overlying epithelium. This important morphogenetic pathway is able to trigger epithelial-mesenchymal-transition (EMT), a mechanism involved in developmental and metastatic processes, which is also potentially involved in pulmonary fibrosis. METHODS: Since EMT is characterised by enhancement of migratory potential of cells, we investigated the molecular profile of FF in 30 biopsies of IPF/UIP and a variety of control samples, focussing on the immunohistochemical expression of three molecules involved in cell motility and invasiveness, namely laminin-5-γ2-chain, fascin, and heat-shock-protein-27. RESULTS: We provide evidence that in UIP these three molecules are abnormally expressed in discrete clusters of bronchiolar basal cells precisely localised in FF. These cellular clusters expressed laminin-5-γ2-chain and heat-shock-protein-27 at very high levels, forming characteristic three-layered lesions defined as "sandwich-foci" (SW-FF). Upon quantitative analysis SW-FF were present in 28/30 UIP samples, representing more than 50% of recognisable FF in 21/30, but were exceedingly rare in a wide variety of lung pathologies examined as controls. In UIP, SW-FF were often observed in areas of microscopic honeycombing, and were also found at the interface between normal lung tissue and areas of dense scarring. CONCLUSION: These molecular abnormalities strongly suggest that SW-FF represent the leading edge of pulmonary remodelling, where abnormal migration and re-epithelialisation take place, and that abnormal proliferation and migration of bronchiolar basal cells have a major role in the remodelling process characterising IPF/UIP. Further investigations will assess their possible use as reliable markers for better defining the UIP-pattern in difficult cases

Fasting plasma mannose levels are associated with insulin sensitivity independent of BMI in Japanese individuals with diabetes

Abstract Background Recently, an integrated network analysis has revealed dysregulation in the metabolism of mannose, a glucose epimer, in severely obese individuals without diabetes. In addition, fasting plasma mannose levels (M0) are associated with insulin resistance independent of BMI. Since the association between mannose and insulin sensitivity (IS) in those with impaired glucose tolerance remains unknown, we aimed to investigate this association in individuals without severe obesity but with varying degrees of glucose tolerance. Methods Based on 75 g OGTT data in Japanese individuals without diabetic medication, individuals were classified as having normal glucose tolerance (NGT), impaired glucose metabolism (IGM), or diabetes (DM). In each group, 25 individuals were consecutively recruited [total 75 individuals, age: 65 ± 11 (mean ± SD); BMI: 24.9 ± 3.8 kg/m2]. QUICKI and Matsuda index (MI) were calculated as IS indices. M0 was assayed using HPLC. Normally-distributed loge-transformed (ln-) values were used for MI and leptin. Results In the simple regression analysis, ln-MI was negatively correlated with BMI (NGT: r = − 0.639, IGM: r = − 0.466, DM: r = − 0.613) and ln-leptin (NGT: r = − 0.480, IGT: r = − 0.447, DM: r = − 0.593) in all 3 groups. Ln-MI was not significantly correlated with M0 in NGT (r = 0.241, P = 0.245) and IGT (r = − 0.296, P = 0.152) groups, it was moderately and negatively correlated in the DM group (r = − 0.626, P < 0.001). Similar results were obtained, when QUICKI was used instead of MI as an index of IS. In multiple regression analysis in the DM group, QUICKI (Q) and ln-MI (M) were independently predicted by BMI (Q: β = − 0.413; M: β = − 0.400) and M0 (Q: β = − 0.413, M: β = − 0.426), accounting for 51.2% (P = 0.0004) and 51.2% (P = 0.0004) of the variability, respectively, which was larger than the prediction for BMI alone (Q: 38.4%, M: 37.6%). Conclusion Fasting plasma mannose was associated with IS independent of BMI in Japanese individuals with DM

MUTYH prevents OGG1 or APEX1 from inappropriately processing its substrate or reaction product with its C-terminal domain

MutY homolog (MUTYH) excises adenine opposite 8-oxoguanine (8-oxoG) in DNA, thus preventing occurrence of G:C to T:A transversion. In cell-free extract prepared from the thymocytes of wild type but not MUTYH-null mice, adenine opposite 8-oxoG in DNA was excised by MUTYH, however, the generated apurinic (AP) site opposite 8-oxoG mostly remained unincised. Recombinant mouse MUTYH (mMUTYH) efficiently excised adenine opposite 8-oxoG and prevented mouse AP endonuclease (mAPEX1) from incising the generated AP site. In contrast, an AP site opposite 8-oxoG created by uracil DNA glycosylase or tetrahydrofuran opposite 8-oxoG was efficiently incised by mAPEX1 in the presence of an excess amount of mMUTYH. Mutant mMUTYH with R361A or G365D substitution, excised adenine opposite 8-oxoG as efficiently as did wild-type mMUTYH, but failed to prevent mAPEX1 from incising the generated AP site. Wild-type mMUTYH bound duplex oligonucleotides containing A:8-oxoG pair with a lower apparent K(d) than that of the mutants, and prevented OGG1 from excising 8-oxoG opposite adenine or the generated AP site. The G365D mutant failed to prevent OGG1 from excising 8-oxoG opposite the generated AP site, thus indicating that the protection of its own product by mMUTYH is an intrinsic function which depends on the C-terminal domain of mMUTYH

Identification and characterization of two forms of mouse MUTYH proteins encoded by alternatively spliced transcripts

There are three types of mouse Mutyh mRNAs (type a, b and c) generated by alternative splicing, and type b mRNA is a major form among the three in most of the tissues examined. The level of type c mRNA is relatively high in brain. Type a and b mRNAs were expected to encode 57.7 kDa protein (MUTYHα), while type c mRNA had a partly different open reading frame encoding a 50.2 kDa protein (MUTYHβ). An in vitro translation of type b and c mRNAs produced a 50 kDa MUTYHα and 47 kDa MUTYHβ, respectively. MUTYHα and MUTYHβ were detected in wild-type embryonic stem (ES) cells or thymocytes prepared from wild-type mice, but neither MUTYH-null ES cells nor thymocytes prepared from MUTYH-null mice. Both MUTYHα and MUTYHβ were mainly localized in the nuclei and some in mitochondria in wild-type ES cells. Recombinant MUTYHα and β were expressed as fusion proteins with thioredoxin in Escherichia coli, but only MUTYHα was partly soluble and thus could be purified. Recombinant MUTYHα possessed DNA glycosylase activities to excise adenine opposite 8-oxoguanine and guanine but not AP lyase activity

Analysis of factors influencing glucose tolerance in Japanese patients with non-alcoholic fatty liver disease

Abstract Background While the association of the prevalence of non-alcoholic fatty liver disease (NAFLD) with impaired glucose metabolism has been reported, the factors influencing glucose tolerance in NAFLD remain to be clarified. Methods Glucose tolerance of 131 Japanese patients diagnosed as NAFLD by histological findings of liver biopsy specimen was examined using 75 g-OGTT. According to Matteoni’s classification, patients were divided to 4 groups [M1 ~ 4, M1, 2: non-alcoholic fatty liver (NAFL); and M3, 4: non-alcoholic steatohepatitis (NASH)]. Based on the OGTT data, insulinogenic index (IGI) and QUICKI were calculated as indices of insulin secretion and insulin sensitivity, respectively. Plasma glucose 120 min after glucose loading (G120) was used as the index for glucose intolerance. Results Stepwise multiple regression analysis using G120 as a dependent variable and loge-IGI, QUICKI, sex, BMI, age, NAFL/NASH as independent variables revealed that loge-IGI (β = −0.595) and QUICKI (β = −0.323) are significant factors predicting glucose intolerance (R2 = 0.403), indicating an important role of insulin secretion in glucose tolerance. These findings accord with glucose intolerance as high as 89.7% in patients with impaired insulin secretion defined by ≤43.2 pmol/mmol (40 μU/mg) IGI. Stepwise multiple regression analysis using QUICKI as a dependent variable and NAFL/NAFLD, sex, BMI, and age as independent variables revealed that BMI (β = −0.469) and NAFL/NAFLD (β = −0.204) are significant factors predicting insulin sensitivity (R2 = 0.248). Conclusion Impairment of insulin secretion is the most important factor to predict glucose intolerance in NAFLD; severity of histological findings is associated with insulin sensitivity independent of adiposity in NAFLD

Fanconi Anemia Protein FANCD2 Promotes Immunoglobulin Gene Conversion and DNA Repair through a Mechanism Related to Homologous Recombination

Recent studies show overlap between Fanconi anemia (FA) proteins and those involved in DNA repair mediated by homologous recombination (HR). However, the mechanism by which FA proteins affect HR is unclear. FA proteins (FancA/C/E/F/G/L) form a multiprotein complex, which is responsible for DNA damage-induced FancD2 monoubiquitination, a key event for cellular resistance to DNA damage. Here, we show that FANCD2-disrupted DT40 chicken B-cell line is defective in HR-mediated DNA double-strand break (DSB) repair, as well as gene conversion at the immunoglobulin light-chain locus, an event also mediated by HR. Gene conversions occurring in mutant cells were associated with decreased nontemplated mutations. In contrast to these defects, we also found increased spontaneous sister chromatid exchange (SCE) and intact Rad51 foci formation after DNA damage. Thus, we propose that FancD2 promotes a subpathway of HR that normally mediates gene conversion by a mechanism that avoids crossing over and hence SCEs