Complexes formed between the restriction endonuclease EcoK and heteroduplex DNA

The complexes between the Escherichia coli K restriction endonuclease and heteroduplex DNA (one strand methylated and one unmethylated) have been characterized and shown to have different properties from those formed with unmodified DNA. The nature of the heteroduplex complex appears to commit the enzyme to its methylase mode.     

ERp57 is up‐regulated in free fatty acids‐induced steatotic L‐02 cells and human nonalcoholic fatty livers

Pathogenesis of nonalcoholic fatty liver disease (NAFLD) is not clear. In this study we aimed to identify proteins involved in NAFLD development in free fatty acids (FFA)‐induced hepatosteatotic cells and in human liver biopsies. Steatosis was induced by incubating a normal human hepatocyte‐derived cell line L‐02 with FFA. Differentially expressed proteins in the steatotic cells were analyzed by two‐dimensional gel electrophoresis‐based proteomics. Involvement of one of the up‐regulated proteins in steatosis was characterized using the RNA interference approach with the steatotic cells. Protein expression levels in liver biopsies of patients with NAFLD were assessed by immunohistochemistry. Proteomic analysis of L‐02 steatotic cells revealed the up‐regulation of ERp57, a condition not previously implicated in NAFLD. Knockdown of ERp57 expression with siRNA significantly reduced fat accumulation in the steatotic cells. ERp57 expression was detected in 16 out of 17 patient biopsies and correlated with inflammation grades or fibrosis stages, while in 5 normal biopsies ERp57 expression was not detectable in hepatocytes. In conclusion, ERp57 was up‐regulated in FFA‐induced steatotic hepatic cells and in NAFLD patient livers and demonstrated steatotic properties in cultured cells. Further investigations are warranted to verify the involvement of ERp57 in NAFLD development. J. Cell. Biochem. 110: 1447–1456, 2010. © 2010 Wiley‐Liss, Inc.     

Serum pleiotrophin as a diagnostic and prognostic marker for small cell lung cancer

Pleiotrophin (PTN) is involved in tumour progression, angiogenesis and metastasis. The purpose of this study was to investigate the expression level of PTN in the serum of patients with small cell lung cancer (SCLC) and to explore the clinical significance of PTN. Serum samples from 128 patients with SCLC, 120 healthy volunteers (HV) and 60 patients with benign lung disease (BLD) were collected. The levels of serum PTN were determined with ELISA and its correlation with the clinical data was examined. The serum PTN levels in SCLC patients were significantly higher than that in BLD patients (P < 0.05) or HV (P < 0.05). With a cutoff value of 258.18 ng/mL, the sensitivity and specificity of PTN to SCLC patients and BLD patients, SCLC patients and HV were 79.2% and 91.7%, 86.7% and 95.8% respectively. An area under the curve for all stages of SCLC resulting from PTN, which was significantly better than the other tumour markers tested including progastrin‐releasing peptide and neuron‐specific enolase. High serum PTN levels appear to correlate with poor survival in patients with SCLC. These results suggest that PTN levels in the serum could be a new effective biomarker for the diagnosis and prognosis of SCLC.     

Isorhamnetin attenuates osteoarthritis by inhibiting osteoclastogenesis and protecting chondrocytes through modulating reactive oxygen species homeostasis

Increasing evidence indicates that osteoarthritis (OA) is a musculoskeletal disease affecting the whole joint, including both cartilage and subchondral bone. Reactive oxygen species (ROS) have been demonstrated to be one of the important destructive factors during early‐stage OA development. The objective of this study was to investigate isorhamnetin (Iso) treatment on osteoclast formation and chondrocyte protection to attenuate OA by modulating ROS. Receptor activator of nuclear factor‐kappa B ligand (RANKL) was used to establish the osteoclast differentiation model in bone marrow macrophages (BMMs) in vivo. H₂O₂ was used to induce ROS, which could further cause chondrocyte apoptosis. We demonstrated that Iso suppressed RANKL‐induced ROS generation, which could mediate osteoclastogenesis. Moreover, we found that Iso inhibited osteoclast formation and function by suppressing the expression of osteoclastogenesis‐related genes and proteins. We proved that Iso inhibited RANKL‐induced activation of mitogen‐activated protein kinase activation of mitogen‐activated protein kinase (MAPK), nuclear factor‐kappa B (NF‐κB) and AKT signalling pathways in BMMs. In addition, Iso inhibited ROS‐induced chondrocyte apoptosis by regulating apoptosis‐related proteins. Moreover, Iso was administered to an anterior cruciate ligament transection (ACLT)‐induced OA mouse model. The results indicated that Iso exerted beneficial effects on inhibiting excessive osteoclast activity and chondrocyte apoptosis, which further remedied cartilage damage. Overall, our data showed that Iso is an effective candidate for treating OA.     

BAG3‐positive pancreatic stellate cells promote migration and invasion of pancreatic ductal adenocarcinoma

BAG3 is constitutively expressed in multiple types of cancer cells and its high expression is associated with tumour progression and poor prognosis of PDAC . However, little is known about the role of BAG3 in the regulation of stromal microenvironment of PDAC. The current study demonstrated that beside PDAC tumour cells, BAG3 was also expressed in some activated stroma cells in PDAC tissue, as well as in activated PSCs. In addition, the current study demonstrated that BAG3 expression in PSCs was involved in maintenance of PSCs activation and promotion of PDACs invasion via releasing multiple cytokines. The current study demonstrated that BAG3‐positive PSCs promoted invasion of PDACs via IL‐8, MCP1, TGF‐β2 and IGFBP2 in a paracrine manner. Furthermore, BAG3 sustained PSCs activation through IL‐6, TGF‐β2 and IGFBP2 in an autocrine manner. Thereby, the current study provides a new insight into the involvement of BAG3 in remodelling of stromal microenvironment favourable for malignant progression of PDAC, indicating that BAG3 might serve as a potential target for anti‐fibrosis of PDAC.     

Long non‐coding RNA cardiac hypertrophy‐associated regulator governs cardiac hypertrophy via regulating miR‐20b and the downstream PTEN/AKT pathway

Pathological cardiac hypertrophy (CH) is a key factor leading to heart failure and ultimately sudden death. Long non‐coding RNAs (lncRNAs) are emerging as a new player in gene regulation relevant to a wide spectrum of human disease including cardiac disorders. Here, we characterize the role of a specific lncRNA named cardiac hypertrophy‐associated regulator (CHAR) in CH and delineate the underlying signalling pathway. CHAR was found markedly down‐regulated in both in vivo mouse model of cardiac hypertrophy induced by pressure overload and in vitro cellular model of cardiomyocyte hypertrophy induced by angiotensin II (AngII) insult. CHAR down‐regulation alone was sufficient to induce hypertrophic phenotypes in healthy mice and neonatal rat ventricular cells (NRVCs). Overexpression of CHAR reduced the hypertrophic responses. CHAR was found to act as a competitive endogenous RNA (ceRNA) to down‐regulate miR‐20b that we established as a pro‐hypertrophic miRNA. We experimentally established phosphatase and tensin homolog (PTEN), an anti‐hypertrophic signalling molecule, as a target gene for miR‐20b. We found that miR‐20b induced CH by directly repressing PTEN expression and indirectly increasing AKT activity. Moreover, CHAR overexpression mitigated the repression of PTEN and activation of AKT by miR‐20b, and as such, it abrogated the deleterious effects of miR‐20b on CH. Collectively, this study characterized a new lncRNA CHAR and unravelled a new pro‐hypertrophic signalling pathway: lncRNA‐CHAR/miR‐20b/PTEN/AKT. The findings therefore should improve our understanding of the cellular functionality and pathophysiological role of lncRNAs in the heart.     

miR‐20a‐5p regulates pulmonary surfactant gene expression in alveolar type II cells

MicroRNA (miRNA) critically controls gene expression in many biological processes, including lung growth and pulmonary surfactant biosynthesis. The present study was conducted to investigate whether miR‐20a‐5p had such regulatory functions on alveolar type II (AT‐II) cells. To accomplish this, miR‐20a‐5p–overexpressed and miR‐20a‐5p–inhibited adenoviral vectors were constructed and transfected into cultured AT‐II cells that were isolated from rat foetal lungs of 19 days' gestation. Transfection efficiency was confirmed by observing the fluorescence of green fluorescent protein (GFP) carried by the viral vector, whereas miR‐20a‐5p levels were verified by real‐time PCR. The CCK‐8 assay was used to compare the proliferation ability of AT‐II cells that had over‐ or underexpressed miR‐20a‐5p. The expression of surfactant‐associated proteins (SPs) and phosphatase and tensin homolog (PTEN) was measured by real‐time PCR and Western blotting. In AT‐II cells, transfection resulted in over‐ or under‐regulation of miR‐20a‐5p. While overexpression of miR‐20a‐5p promoted pulmonary surfactant gene expression, its underexpression inhibited it. Consistent with its role in negatively regulating the pulmonary surfactant gene, an opposite pattern was observed for miR‐20a‐5p regulation of PTEN. As a result, when miR‐20a‐5p was rendered overexpressed, PTEN was down‐regulated. By contrast, when miR‐20a‐5p was underexpressed, PTEN was up‐regulated. Neither overexpression nor underexpression of miR‐20a‐5p altered the cell proliferation. miR‐20a‐5p plays no role in proliferation of foetal AT‐II cells but is a critical regulator of surfactant gene expression. The latter appears to be achieved through a regulatory process that implicates expression of PTEN.     

Synthesis and Antitumor Evaluation in Vitro of NO‐Donating Ursolic Acid‐Benzylidene Derivatives

Antitumor activity of triterpenoid and its derivatives has attracted great attention recently. Our previous efforts led to the discovery of a series of NO‐donor betulin derivatives with potent antitumor activity. Herein, we prepared eight compounds derived from ursolic acid (UA). All the compounds were evaluated for their in vitro cytotoxicity against four human cancer cell lines (HepG‐2, MCF‐7, HT‐29 and A549). Among the compounds tested, compound 4a was found to be most active against HT‐29 (IC₅₀=4.28 μm ). Further biological assays demonstrated that compound 4a could induce cell cycle arrest at G1 phase and apoptosis in a dose‐dependent manner. In addition, compound 4a was found to upregulate pro‐apoptotic Bax, p53 and downregulate anti‐apoptotic Bcl‐2. All these results suggested that compound 4a is a potential candidate drug for the therapy of colon cancer.