Hepatic Stellate Cells Express Thymosin Beta 4 in Chronically Damaged Liver

Although the various biological roles of thymosin β4 (Tβ4) have been studied widely, the effect of Tβ4 and Tβ4-expressing cells in the liver remains unclear. Therefore, we investigated the expression and function of Tβ4 in chronically damaged livers. CCl4 was injected into male mice to induce a model of chronic liver disease. Mice were sacrificed at 6 and 10 weeks after CCl4 treatment, and the livers were collected for biochemical analysis. The activated LX-2, human hepatic stellate cell (HSC) line, were transfected with Tβ4-specific siRNA and activation markers of HSCs were examined. Compared to HepG2, higher expression of Tβ4 in RNA and protein levels was detected in the activated LX-2. In addition, Tβ4 was up-regulated in human liver with advanced liver fibrosis. The expression of Tβ4 increased during mouse HSC activation. Tβ4 was also up-regulated and Tβ4-positive cells were co-localized with α-smooth muscle actin (α-SMA) in the livers of CCl4-treated mice, whereas such cells were rarely detected in the livers of corn-oil treated mice. The suppression of Tβ4 in LX-2 cells by siRNA induced the down-regulation of HSC activation-related genes, tgf-β, α-sma, collagen, and vimentin, and up-regulation of HSC inactivation markers, ppar-γ and gfap. Immunofluorescent staining detected rare co-expressing cells with Tβ4 and α-SMA in Tβ4 siRNA-transfected cells. In addition, cytoplasmic lipid droplets were observed in Tβ4 siRNA-treated cells. These results demonstrate that activated HSCs expressed Tβ4 in chronically damaged livers, and this endogenous expression of Tβ4 influenced HSC activation, indicating that Tβ4 might contribute to liver fibrosis by regulating HSC activation.     

MqsR, a Crucial Regulator for Quorum Sensing and Biofilm Formation, Is a GCU-specific mRNA Interferase in Escherichia coli

The mqsR gene has been shown to be positively regulated by the quorum-sensing autoinducer AI-2, which in turn activates a two-component system, the qseB-qseC operon. This operon plays an important role in biofilm formation in Escherichia coli. However, its cellular function has remained unknown. Here, we found that 1 base downstream of mqsR there is a gene, ygiT, that is co-transcribed with mqsR. Induction of mqsR caused cell growth arrest, whereas ygiT co-induction recovered cell growth. We demonstrate that MqsR (98 amino acid residues), which has no homology to the well characterized mRNA interferase MazF, is a potent inhibitor of protein synthesis that functions by degrading cellular mRNAs. In vivo and in vitro primer extension experiments showed that MqsR is an mRNA interferase specifically cleaving mRNAs at GCU. The mRNA interferase activity of purified MqsR was inhibited by purified YgiT (131 residues). MqsR forms a stable 2:1 complex with YgiT, and the complex likely functions as a repressor for the mqsR-ygiT operon by specifically binding to two different palindromic sequences present in the 5′-untranslated region of this operon.It has been reported that quorum sensing is involved in biofilm formation (1–4). mqsR expression was found to be induced by 8-fold in biofilms (5) and also by the quorum-sensing signal autoinducer AI-2, which is a species-nonspecific signaling molecule produced by both Gram-negative and Gram-positive bacteria, including Escherichia coli (6). It has been reported that induction of mqsR activates a two-component system, the qseB-qseC operon, which is known to play an important role in biofilm formation (6). Thus, it has been proposed that MqsR (98 amino acid residues) is a regulator of biofilm formation because it activates qseB, which controls the flhDC expression required for motility and biofilm formation in E. coli (6). However, the cellular function of MqsR has remained unknown.Interestingly, all free-living bacteria examined to date contain a number of suicide or toxin genes in their genomes (7, 8). Many of these toxins are co-transcribed with their cognate antitoxins in an operon (termed toxin-antitoxin (TA)² operon) and form a stable complex in the cell, so their toxicity is subdued under normal growth conditions (9–11). However, the stability of antitoxins is substantially lower than that of their cognate toxins, so any stress causing cellular damage or growth inhibition that induces proteases alters the balance between toxin and antitoxin, leading to toxin release in the cell.To date, 16 (12) TA systems have been reported on the E. coli genome, including relB-relE (13, 14), chpBI-chpBK (15), mazEF (16–18), yefM-yoeB (19, 20), dinJ-yafQ (21, 22), hipBA and hicAB (23, 24), prlF-yhaV (25), and ybaJ-hha (26). Interestingly, all of these TA operons appear to use similar modes of regulation: the formation of complexes between antitoxins and their cognate toxins to neutralize toxin activity and the ability of TA complexes to autoregulate their expression. The cellular targets of some toxins have been identified. CcdB directly interacts with gyrase A and blocks DNA replication (27, 28). RelE, which by itself has no endoribonuclease activity, appears to act as a ribosome-associating factor that promotes mRNA cleavage at the ribosome A-site (13, 29, 30). PemK (31), ChpBK (15), and MazF (32) are unique among toxins because they target cellular mRNAs for degradation by functioning as sequence-specific endoribonucleases to effectively inhibit protein synthesis and thereby cell growth.MazF, ChpBK, and PemK have been characterized as sequence-specific endoribonucleases that cleave mRNA at the ACA, ACY (Y is U, A, or G), and UAH (H is C, A, or U) sequences, respectively. They are completely different from other known endoribonucleases such as RNases E, A, and T1, as these toxins function as protein synthesis inhibitors by interfering with the function of cellular mRNAs. It is well known that small RNAs, such as mRNA-interfering cRNA (33), microRNA (34), and small interfering RNA (35), interfere with the function of specific RNAs. These small RNAs bind to specific mRNAs to inhibit their expression. Ribozymes also act on their target RNAs specifically and interfere with their function (36). Therefore, MazF, ChpBK, and PemK homologs form a novel endoribonuclease family that exhibits a new mRNA-interfering mechanism by cleaving mRNAs at specific sequences. Thus, they have been termed “mRNA interferases” (2).During our search for TA systems on the E. coli genome, we found that the mqsR gene is co-transcribed with a downstream gene, ygiT. These two genes appear to function as a TA system, as their size is small (98 residues for MqsR and 131 residues for YgiT) and their respective open reading frames are separated by 1 bp. In this study, we demonstrate that MqsR-YgiT is a new E. coli TA system consisting of a toxin, MqsR, and an antitoxin, YgiT. Moreover, we identify MqsR as a novel mRNA interferase that does not exhibit homology to MazF. This toxin cleaves RNA at GCU sequences in vivo and in vitro. The implication of this finding as to how this mRNA interferase is involved in cell physiology and biofilm formation will be discussed.     

Enzymatic amplification and characterization of large DNA fragments from genomic DNA

Conditions for DNA amplification in vitro using modified T7 DNA polymerase have been devised to obtain 2000-bp DNA fragments of the HGPRT gene directly from human genomic DNA. The DNA obtained from a 1.2 x 10(5)-fold amplification has been used for direct sequencing.     

Resveratrol alters microRNA expression profiles in A549 human non-small cell lung cancer cells

Resveratrol is a plant phenolic phytoalexin that has been reported to have antitumor properties in several types of cancers. In particular, several studies have suggested that resveratrol exerts antiproliferative effects against A549 human non-small cell lung cancer cells; however, its mechanism of action remains incompletely understood. Deregulation of microRNAs (miRNAs), a class of small, noncoding, regulatory RNA molecules involved in gene expression, is strongly correlated with lung cancer. In this study, we demonstrated that resveratrol treatment altered miRNA expression in A549 cells. Using microarray analysis, we identified 71 miRNAs exhibiting greater than 2-fold expression changes in resveratrol-treated cells relative to their expression levels in untreated cells. Furthermore, we identified target genes related to apoptosis, cell cycle regulation, cell proliferation, and differentiation using a miRNA target-prediction program. In conclusion, our data demonstrate that resveratrol induces considerable changes in the miRNA expression profiles of A549 cells, suggesting a novel approach for studying the anticancer mechanisms of resveratrol.     

Ambient Temperature and Prevalence of Obesity: A Nationwide Population-Based Study in Korea

Background

Recent studies have suggested a possible association between outdoor or indoor temperature and obesity. We aimed to examine whether ambient temperature is associated with the prevalence of obesity or abdominal obesity in the Korean population.

Methods

Data on anthropometric, socio-demographic, laboratory and lifestyle factors were retrieved from National Health Insurance System data obtained in 2009–2010. Thirty years (1981 to 2010) of meteorological parameters for 71 observation areas were acquired from the Korea Meteorological Administration. Included in this analysis were 124,354 individuals. A body mass index (BMI) ≥ 25 kg/m² and a waist circumference (WC) ≥ 90 cm (men) or 85 cm (women) were considered to represent obesity and abdominal obesity, respectively.

Results

The mean annual temperature (MAT) ranged from 6.6°C to 16.6°C, and BMI was positively correlated with MAT (r = 0.0078, P = 0.0065). WC was positively correlated with MAT (r = 0.0165, P < 0.0001) and negatively correlated with the number of days with mean temperature < 0°C (DMT0; r = –0.0129, P = 0.0002). After adjusting for age, sex, smoking status, alcohol consumption, exercise, income, residential area and altitude, the odds ratios (95% CI) for obesity and abdominal obesity in the highest quintile MAT group were 1.045 (1.010, 1.081) and 1.082 (1.042, 1.124), respectively, compared with the lower four quintiles of the MAT group. Similarly, subjects in the area of the lowest quintile of DMT0 had significantly higher odds of abdominal obesity compared with the higher four quintile groups of DMT0.

Conclusion

This study finds an association between ambient temperature and prevalence of obesity in the Korean population when controlling for several confounding factors. Adaptive thermogenesis might be a possible explanation for this phenomenon.     

Effect of Cheonggukjang supplementation upon hepatic acyl-CoA synthase, carnitine palmitoyltransferase I, acyl-CoA oxidase and uncoupling protein 2 mRNA levels in C57BL/6J mice fed with high fat diet

This study investigated the effect of Cheonggukjang on mRNA levels of hepatic acyl-CoA synthase (ACS), carnitine palmitoyltransferase I (CPT-I), acyl-CoA oxidase (ACO) and uncoupling protein 2 (UCP2), and on serum lipid profiles in C57BL/6J mice. Thirty male C57BL/6J mice were divided into three groups; normal diet (ND), high fat diet (HD) and high fat diet with 40% Cheonggukjang (HDC). Energy intake was significantly higher in the HDC group than in the ND and HD groups. The HDC group normalized in weight gain, epididymal and back fat (g/100 g) accumulation which are increased by high fat diet. Serum concentrations of triglyceride and total cholesterol in the HDC were significantly lower than those in the HD group. These results were confirmed by hepatic mRNA expression of enzymes and protein (ACS, CPT-1, ACO, UCP2) which is related with lipid metabolism by RT-PCR. Hepatic CPT-I, ACO and UCP2 mRNA expression was increased by Cheonggukjang supplementation. We demonstrated that Cheonggukjang supplement leads to increased mRNA expressions of enzymes and protein involved in fatty acid oxidation in liver, reduced accumulation of body fat and improvement of serum lipids in high fat diet fed mice.     

Proangiogenic TIE2+/CD31+ macrophages are the predominant population of tumor-associated macrophages infiltrating metastatic lymph nodes

Tumor-associated macrophages (TAMs) accumulate in various cancers and promote tumor angiogenesis and metastasis, and thus may be ideal targets for the clinical diagnosis of tumor metastasis with high specificity. However, there are few specific markers to distinguish between TAMs and normal or inflammatory macrophages. Here, we show that TAMs localize in green fluorescent protein-labeled tumors of metastatic lymph nodes (MLNs) from B16F1 melanoma cells but not in necrotic tumor regions, suggesting that TAMs may promote the growth of tumor cells and the progression of tumor metastasis. Furthermore, we isolated pure populations of TAMs from MLNs and characterized their gene expression signatures compared to peritoneal macrophages (PMs), and found that TAMs significantly overexpress immunosuppressive cytokines such as IL-4, IL-10, and TGF-β as well as proangiogenic factors such as VEGF, TIE2, and CD31. Notably, immunological analysis revealed that TIE2⁺/CD31⁺ macrophages constitute the predominant population of TAMs that infiltrate MLNs, distinct from tissue or inflammatory macrophages. Importantly, these TIE2⁺/CD31⁺ macrophages also heavily infiltrated MLNs from human breast cancer biopsies but not reactive hyperplastic LNs. Thus, TIE2⁺/CD31⁺ macrophages may be a unique histopathological biomarker for detecting metastasis in clinical diagnosis, and a novel and promising target for TAM-specific cancer therapy.     

Psoralen attenuates bleomycin‐induced pulmonary fibrosis in mice through inhibiting myofibroblast activation and collagen deposition

Idiopathic pulmonary fibrosis (IPF) is a progressive disease characterized by excessive deposition of extracellular matrix (ECM) and chronic inflammation with limited therapeutic options. Psoralen, a major active component extracted from Psoralea corylifolia L. seed, has several biological effects. However, the role of psoralen in IPF is still unclear. Here, we hypothesized that psoralen played an essential role in IPF in the inhibition of fibroblast proliferation and inflammatory response. A murine model of IPF was established by injecting bleomycin (BLM) intratracheally, and psoralen was administered for 14 days from the 7^th to 21^st day after BLM injection. Our results demonstrated that psoralen treatment reduced body weight loss and improved the survival rate of mice with IPF. Histological and immunofluorescent examination showed that psoralen alleviated BLM‐induced lung parenchymal inflammatory and fibrotic alteration. Furthermore, psoralen inhibited proliferation and collagen synthesis of mouse fibroblasts and partially reversed BLM‐induced expression of α‐smooth muscle actin at both the tissue and cell level. Moreover, psoralen decreased the expression of transforming growth factor‐β1, interleukin‐1β, and tumor necrosis factor‐α in the lungs of BLM‐stimulated mice. Our results reveale for the first time that psoralen exerts therapeutic effects against IPF in a BLM‐induced murine model.