Identification of Muscle Strength-Related Gut Microbes through Human Fecal Microbiome Transplantation.

The gut microbiome is well known for its influence on human physiology and aging. Therefore, we speculate that the gut microbiome may affect muscle strength in the same way as the host's own genes. To demonstrate candidates for gut microbes affecting muscle strength, we remodeled the original gut microbiome of mice into human intestinal microbiome through fecal microbiome transplantation (FMT), using human feces and compared the changes in muscle strength in the same mice before and three months after FMT. After comparing before and after FMT, the mice were divided into three groups based on the observed changes in muscle strength: positive, none, and negative changes in muscle strength. As a result of analyzing the α-diversity, ß-diversity, and co-occurrence network of the intestinal microbial community before and after FMT, it was observed that a more diverse intestinal microbial community was established after FMT in all groups. In particular, the group with increased muscle strength had more gut microbiome species and communities than the other groups. Fold-change comparison showed that Eisenbergiella massiliensis and Anaeroplasma abactoclasticum from the gut microbiome had positive contributions to muscle strength, while Ileibacterium valens and Ethanoligenens harbinense had negative effects. This study identifies candidates for the gut microbiome that contribute positively and those that contribute negatively to muscle strength.

Protective effects of sciadopitysin against methylglyoxal-induced degeneration in neuronal SK-N-MC cells.

The accumulation of advanced glycation end products (AGEs) causes metabolic dysfunction and neuronal cell damage. Methylglyoxal (MG) is a major glycating agent that reacts with basic residues present in proteins and promotes the formation of AGEs. Sciadopitysin, a type of biflavonoid, exerts protective effects against neuronal cell damage; however, the underlying mechanisms have not been studied. This study aimed to investigate the mechanisms underlying the protective effects of sciadopitysin against MG-mediated cytotoxicity in SK-N-MC neuroblastoma cells. Our results demonstrated that pretreatment of SK-N-MC cells with sciadopitysin improved the cell viability that was inhibited by MG and inhibited the apoptosis induced by MG. Sciadopitysin attenuated intracellular Ca2+ , NOX4 levels, oxidative stress, and MG-protein adduct levels, and increased nuclear Nrf2 and glyoxalase 1 levels in the presence of MG. These results suggest that sciadopitysin exerts neuroprotective effects against MG-induced death of human SK-N-MC cells via its antioxidative action. This study highlights sciadopitysin as a promising candidate for antioxidant therapy and designing natural drugs against AGE-induced neurodegenerative disorders.

Spironolactone Attenuates Methylglyoxal-induced Cellular Dysfunction in MC3T3-E1 Osteoblastic Cells.

BACKGROUND: Methylglyoxal (MG) is associated with the pathogenesis of age- and diabetes-related complications. Spironolactone is a competitive antagonist of aldosterone that is widely employed in the treatment of hypertension and heart failure. This study examined the effects of spironolactone on MG-induced cellular dysfunction in MC3T3-E1 osteoblastic cells. METHODS: MC3T3-E1 cells were treated with spironolactone in the presence of MG. The mitochondrial function, bone formation activity, oxidative damage, inflammatory cytokines, glyoxalase I activity, and glutathione (GSH) were measured. RESULTS: Pretreatment of MC3T3-E1 osteoblastic cells with spironolactone prevented MG-induced cell death, and improved bone formation activity. Spironolactone reduced MG-induced endoplasmic reticulum stress, production of intracellular reactive oxygen species, mitochondrial superoxides, cardiolipin peroxidation, and inflammatory cytokines. Pretreatment with spironolactone also increased the level of reduced GSH and the activity of glyoxalase I. MG induced mitochondrial dysfunction, but markers of mitochondrial biogenesis such as mitochondrial membrane potential, adenosine triphosphate, proliferator-activated receptor gamma coactivator 1α, and nitric oxide were significantly improved by treatment of spironolactone. CONCLUSION: Spironolactone could prevent MG-induced cytotoxicity in MC3T3-E1 osteoblastic cells by reduction of oxidative stress. The oxidative stress reduction was explained by spironolactone's inhibition of advanced glycation end-product formation, restoring mitochondrial dysfunction, and anti-inflammatory effect.

Catalpol protects against 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced cytotoxicity in osteoblastic MC3T3-E1 cells.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a well-known environmental contaminant that produces a wide variety of adverse effects in humans. Catalpol, a major bioactive compound enriched in the dried root of Rehmannia glutinosa, is a major iridoid glycoside that alleviates bone loss. However, the detailed mechanisms underlying the effects of catalpol remain unclear. The present study evaluated the effects of catalpol on TCDD-induced cytotoxicity in osteoblastic MC3T3-E1 cells. Catalpol inhibited TCDD-induced reduction in cell viability and increases in apoptosis and autophagic activity in osteoblastic MC3T3-E1 cells. Additionally, pretreatment with catalpol significantly decreased the nitric oxide and nitrite levels compared with a control in TCDD-treated cells and significantly inhibited TCDD-induced increases in the levels of cytochrome P450 1A1 and extracellular signal-regulated kinase. Pretreatment with catalpol also effectively restored the expression of superoxide dismutase and extracellular signal-regulated kinase 1 and significantly enhanced the expression of glutathione peroxidase 4 and osteoblast differentiation markers, including alkaline phosphatase and osterix. Taken together, these findings demonstrate that catalpol has preventive effects against TCDD-induced damage in MC3T3-E1 osteoblastic cells.

Glabridin attenuates antiadipogenic activity induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in murine 3T3-L1 adipocytes.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has various toxicological effects in adipose tissue. Evidence is accumulating that glabridin, a flavonoid extracted from licorice, has beneficial effects on the regulation of glucose homeostasis. In this study, we investigated whether glabridin suppresses TCDD-induced loss of adipogenic action using 3T3-L1 adipocytes as a cell culture model of wasting syndrome. Glabridin effectively suppressed TCDD-induced loss of lipid accumulation in this model. Pretreating cells with glabridin increased the gene expression of not only the adipogenesis-associated key transcription factors peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer binding protein alpha, but also lipoprotein lipase in the presence of TCDD. TCDD decreased insulin-stimulated glucose uptake, which was effectively restored by pretreatment with glabridin. Glabridin also inhibited the TCDD-driven decreased production of insulin receptor substrate 1 and glucose transporter 4. TCDD increased the production of mitochondrial superoxides, prostaglandin E2 , phospholipase A2 , cyclooxygenase-1 and intracellular calcium concentrations, while reducing the production of PPARγ coactivator 1 alpha and glycolysis. However, glabridin treatment reduced these TCDD-induced effects. We conclude that glabridin suppresses the TCDD-induced loss of lipid accumulation in 3T3-L1 adipocytes by regulating the levels of PPARγ, CCAAT/enhancer binding protein alpha, lipoprotein lipase, glucose uptake, prostaglandin E2 and energy metabolism. These results also provide in vitro evidence of the effects of glabridin on adipocyte metabolism, which suggests a protective effect against dioxin exposure in the development of insulin resistance and diabetes.

27-Deoxyactein prevents 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced cellular damage in MC3T3-E1 osteoblastic cells.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a well-known environmental contaminant that exerts its toxicity through a variety of signaling mechanisms. The present study evaluated the effects of 27-deoxyactein, one of the major constituents isolated from Cimicifuga racemosa, on TCDD-induced toxicity in osteoblastic MC3T3-E1 cells. TCDD reduced cell survival, markedly increased apoptosis, and enhanced autophagy activity. However, pre-treatment with 27-deoxyactein attenuated all TCDD-induced effects and significantly decreased intracellular calcium (Ca2+) concentrations, the collapse of the mitochondrial membrane potential (MMP), the level of reactive oxygen species (ROS), and cardiolipin peroxidation compared to the TCDD-treated controls. Additionally, TCDD-induced increases in the levels of aryl hydrocarbon receptor (AhR), cytochrome P450 1A1 (CYP1A1), and extracellular signal-regulated kinase (ERK) were significantly inhibited by 27-deoxyactein. The mRNA levels of superoxide dismutase (SOD), ERK1, and nuclear factor kappa B (NF-κB) were also effectively restored by pre-treatment with 27-deoxyactein. Furthermore, 27-deoxyactein significantly increased the expressions of genes associated with osteoblast differentiation, including alkaline phosphatase (ALP), osteocalcin, bone sialoprotein (BSP), and osterix. Taken together, the present findings demonstrate the preventive effects of 27-deoxyactein on TCDD-induced damage in osteoblasts.

Evaluation of toxicological biomarkers in secreted proteins of HepG2 cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin and their expressions in the plasma of rats and incineration workers.

Toxicological biomarkers of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) were investigated in proteins secreted by HepG2 cells and their expression levels were determined in the plasma of rats exposed to 2,3,7,8-TCDD and in the plasma of incineration workers exposed to dioxins. HepG2 cells were treated with various concentrations of 2,3,7,8-TCDD (0, 0.25, 0.5, 1, 2.5, 5, 10, 25 nM) for 24 or 48 h. MTT and Comet assays were performed to determine cytotoxicities and genotoxicities to select exposure concentrations for the proteomic analysis of proteins secreted by 2,3,7,8-TCDD-treated cells. In the proteomic analysis, dose- and time-dependent toxicological biomarkers were evaluated using two pI ranges (4-7 and 6-9) using a large gel 2-DE system. Fifteen secreted proteins were identified by a nano-LC-ESI-MS/MS and nano-ESI on a Q-TOF2 MS and the identities of eight secreted proteins including glyoxalase 1 (GLO 1), homogentisate dioxygenase (HGD), peroxiredoxin 1 (PRX 1), proteasome subunit beta type (PSMB) 5 and 6, UDP-glucose 6-dehydrogenase (UDP-GlcDH), hydroxyacyl-coenzyme A dehydrogenase (HADH) and serotransferrin (STF) were confirmed by western blotting. Of these, PSMB 5 and PRX 1 were also found in the plasma of rats exposed to 2,3,7,8-TCDD, whereas GLO 1, HGD, PSMB 6 and PRX 1 were found in the plasma of incineration workers exposed to dioxins.

Actein alleviates 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated cellular dysfunction in osteoblastic MC3T3-E1 cells.

The environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is known to affect bone metabolism. We evaluated the protective effects of the triterpene glycoside actein from the herb black cohosh against TCDD-induced toxicity in MC3T3-E1 osteoblastic cells. We found that TCDD significantly reduced cell viability and increased apoptosis and autophagy in MC3T3-E1 osteoblastic cells (P < .05). In addition, TCDD treatment resulted in a significant increase in intracellular calcium concentration, mitochondrial membrane potential collapse, reactive oxygen species (ROS) production, and cardiolipin peroxidation, whereas pretreatment with actein significantly mitigated these effects (P < .05). The effects of TCDD on extracellular signal-related kinase (ERK), aryl hydrocarbon receptor, aryl hydrocarbon receptor repressor, and cytochrome P450 1A1 levels in MC3T3-E1 cells were significantly inhibited by actein. The levels of superoxide dismutase, ERK1, and nuclear factor kappa B mRNA were also effectively restored by pretreatment with actein. Furthermore, actein treatment resulted in a significant increase in alkaline phosphatase (ALP) activity and collagen content, as well as in the expression of genes associated with osteoblastic differentiation (ALP, type I collagen, osteoprotegerin, bone sialoprotein, and osterix). This study demonstrates the underlying molecular mechanisms of cytoprotection exerted by actein against TCDD-induced oxidative stress and osteoblast damage.

The ORF3 protein of porcine circovirus type 2 promotes secretion of IL-6 and IL-8 in porcine epithelial cells by facilitating proteasomal degradation of regulator of G protein signalling 16 through physical interaction.

Porcine circovirus type 2 (PCV2) is the main aetiological agent of postweaning multisystemic wasting syndrome. The mechanism of pathogenicity associated with PCV2 infection is still not fully understood. Nevertheless, the fact that large amounts of proinflammatory cytokines within lymphoid tissues are released during the early stage of PCV2 infection may induce chronic inflammatory responses followed by the destruction of lymphoid tissues. However, how PCV2 infection causes an excessive inflammatory response in the host immune system during the early stage of PCV2 infection has still not been elucidated. In this study, we show that direct interaction between the PCV2 ORF3 and regulator of G protein signalling 16 (RGS16) within the cytoplasm of host cells leads to ubiquitin-mediated proteasomal degradation of RGS16. Facilitated degradation of the RGS16 by PCV2 ORF3 further enhances NFκB translocation into the nucleus through the ERK1/2 signalling pathway and increased IL-6 and IL-8 mRNA transcripts. Consequently, more severe inflammatory responses and leukocyte infiltration occur around host cells. This evidence may be the first clue explaining the molecular basis of how excessive amounts of proinflammatory cytokines within lymphoid tissues are released during the early stage of PCV2 infection.

Plasma proteomic analysis of patients infected with H1N1 influenza virus.

This study profiled the plasma proteins of patients infected by the 2011 H1N1 influenza virus. Differential protein expression was identified in plasma obtained from noninfected control subjects (n = 15) and H1N1-infected subjects (n = 15). Plasma proteins were separated by a 2DE large gel system and identified by nano-ultra performance LC-MS. Western blot assays were performed to validate proteins. Eight plasma proteins were upregulated and six proteins were downregulated among 3316 plasma proteins in the H1N1-infected group as compared with the control group. Of 14 up- and downregulated proteins, nine plasma proteins were validated by Western blot analysis. Putative protein FAM 157A, leucine-rich alpha 2 glycoprotein, serum amyloid A protein, and dual oxidase 1 showed significant differential expression. The identified plasma proteins could be potential candidates for biomarkers of H1N1 influenza viral infection. Further studies are needed to develop these proteins as diagnostic biomarkers.

Toxicological biomarkers of 2,3,4,7,8-pentachlorodibenzofuran in proteins secreted by HepG2 cells.

Using a proteomic approach, a study was conducted for determination of the effects of 2,3,4,7,8-pentachlorodibenzofuran (2,3,4,7,8-PCDF) on proteins secreted by HepG2 cells. Briefly, HepG2 cells were exposed to various concentrations of 2,3,4,7,8-PCDF for 24 or 48h. MTT and comet assays were then conducted for determination of cytotoxicity and genotoxicity, respectively. Results of an MTT assay showed that 1nM of 2,3,4,7,8-PCDF was the maximum concentration that did not cause cell death. In addition, a dose- and time dependent increase of DNA damage was observed in HepG2 cells exposed to 2,3,4,7,8-PCDF. Therefore, two different concentrations of 2,3,4,7,8-PCDF, 1 and 5nM, were selected for further analysis of proteomic biomarkers using two different pI ranges (4-7 and 6-9) and large two dimensional gel electrophoresis. Results showed identification of 32 proteins ( 29 up- and 3 down-regulated) by nano-LC-ESI-MS/MS and nano-ESI on a Q-TOF2 MS. Among these, the identities of pyridoxine-5'-phosphate oxidase, UDP-glucose 6-dehydrogenase, plasminogen activator inhibitor I precursor, plasminogen activator inhibitor-3, proteasome activator complex subunit 1, isoform 1 of 14-3-3 protein sigma, peptidyl-prolyl cis-trans isomerase A, 14-3-3 protein gamma, protein DJ-1, and nucleoside diphosphate kinase A were confirmed by western blot analysis. The differential expression of protein DJ-1, proteasome activator complex subunit 1 and plasminogen activator inhibitor-3 was further validated in plasma proteins from rats exposed to 2,3,4,7,8-PCDF. These proteins could be used as potential toxicological biomarkers of 2,3,4,7,8-PCDF.

Saponins from soy bean and mung bean inhibit the antigen specific activation of helper T cells by blocking cell cycle progression.

Treatment of helper T (Th) cells with saponins from soy bean and mung bean prevented their activation by inhibiting cell proliferation and cytokine secretion. However, the saponins did not affect the expression of major histocompatibility complex class II (A(b)) and co-stimulatory molecule (CD86) on professional antigen-presenting cells. Instead, the saponins directly inhibited Th cell proliferation by blocking the G(1) to S phase cell cycle transition. Moreover, blocking of the cell cycle by the saponins was achieved by decreased expression of cyclin D1 and cyclin E, and constitutive expression of p27(KIP1). Saponins also increased stability of p27(KIP1) in Th cells after antigenic stimulation.

Performance Comparison Between Fourier-Transform Infrared Spectroscopy-based IR Biotyper and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry for Strain Diversity.

Background: Development of an accessible method to routinely evaluate the clonality of strains is needed in microbiology laboratories. We compared the discriminatory power of the Fourier-transform infrared (FTIR) spectroscopy-based IR Biotyper (Bruker Daltonics GmbH, Bremen, Germany) to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), using whole-genome sequencing (WGS) as the reference method. Methods: Eighty-three extended-spectrum ß-lactamase-producing Escherichia coli isolates were tested using WGS, MALDI-TOF MS, and IR Biotyper. Simpson's diversity index (SDI), a statistical analysis for testing the homogeneity of a dendrogram, and the adjusted Rand index (aRI) were used to compare the discriminatory ability between typing tests. Results: The SDI (95% confidence interval) was 0.969 (0.952-0.985) for WGS, 0.865 (0.807-0.924) for MALDI-TOF MS, and 0.974 (0.965-0.983) for IR Biotyper. Compared with WGS, IR Biotyper showed compatible diversity, whereas MALDI-TOF MS did not. The concordance and aRI improved from 66.3% to 84.3% and from 0.173 to 0.538, respectively, for IR Biotyper versus MALDI-TOF MS with WGS as the reference method. IR Biotyper showed substantially improved performance in strain typing compared with MALDI-TOF MS. Conclusions: IR Biotyper is useful for diversity analysis with improved discriminatory power over MALDI-TOF MS in comparison with WGS as a reference method. IR Biotyper is an accessible method to evaluate the clonality of strains and could be applied in epidemiological analysis during an outbreak of a health care facility, as well as for research on the transmission of resistant bacteria in community settings.

Induction of Heat Shock Proteins and Antioxidant Enzymes in 2,3,7,8-TCDD-Induced Hepatotoxicity in Rats.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) is an environmental toxicant with a polyhalogenated aromatic hydrocarbon structure and is one of the most toxic man-made chemicals. Exposure to 2,3,7,8-TCDD induces reproductive toxicity, immunotoxicity, and hepatotoxicity. In this study, we evaluated how 2,3,7,8-TCDD-induced hepatotoxicity affect the expression of heat shock proteins and antioxidant enzymes using the real-time polymerase chain reaction (PCR) in rat. 2,3,7,8-TCDD increased heat shock protein (Hsp27, α-B-crystallin, Mortalin, Hsp105, and Hsp90s) and antioxidant enzymes (SOD-3, GST and catalase) expression after a 1 day exposure in livers of rats, whereas heat shock protein (α-B-crystallin, Hsp90, and GRP78) and antioxidant enzymes (SOD-1, SOD-3, catalase, GST, and GPXs) expression decreased on day 2 and then slowly recovered back to control levels on day 8. These results suggest that heat shock proteins and antioxidant enzymes were induced as protective mechanisms against 2,3,7,8-TCDD induced hepatotoxicity, and that prolonged exposure depressed their levels, which recovered to control levels due to reduced 2,3,7,8-TCDD induced hepatotoxicity.

Nephrotoxicity evaluation and proteomic analysis in kidneys of rats exposed to thioacetamide.

Thioacetamide (TAA) was administered orally at 0, 10, and 30 mg/kg body weight (BW) daily to Sprague-Dawley rats aged 6-7 weeks for 28 consecutive days. Nephrotoxicity and proteomics were evaluated in the kidneys of rats exposed to TAA. The BW decreased, however, the relative kidneys weight increased. No significant histopathologic abnormalities were found in the kidneys. The numbers of monocytes and platelets were significantly increased. However, the mean corpuscular volume and hematocrit values were decreased significantly in rats exposed to 30 mg/kg BW TAA. The expression levels of Kim-1 and NGAL were increased 4 to 5-fold in the kidneys, resulting in significant nephrotoxicity. Proteomic analysis was conducted and a total of 5221 proteins spots were resolved. Of these, 3 and 21 protein spots were up- and downregulated, respectively. The validation of seven proteins was performed by Western blot analysis. The expression level of ASAP2 was significantly upregulated, whereas RGS14, MAP7Dl, IL-3Rα, Tmod1, NQO2, and MUP were reduced. Sixteen isoforms of MUP were found by the 2DE immunoblot assay and were significantly downregulated with increasing exposure to TAA. MUP isoforms were compared in the liver, kidneys, and urine of untreated rats and a total of 43 isoforms were found.

E6 and E7 fusion immunoglobulin from human papilloma virus 16 induces dendritic cell maturation and antigen specific activation of T helper 1 response.

Human papilloma virus (HPV) 16 causes cervical cancer. Induction of oncogenesis by HPV 16 is primarily dependent on the function of E6 and E7 proteins, which inactivate the function of p53 and pRB, respectively. Thus, blocking the activity of the E6 and E7 proteins from HPV 16 is critical to inhibiting oncogenesis during infection. We have expressed and purified soluble HPV 16 E6 and E7 fusion immunoglobulin (Ig), which were combined with the constant region of an Ig heavy chain, in a mammalian system. To assess whether soluble E6 and E7 fusion Igs induce effective cellular immune responses, immature dendritic cells (DCs) were treated with these fusion proteins. Soluble E6 and E7 fusion Igs effectively induced maturation of DCs. Furthermore, immunization with soluble E6 and E7 fusion Igs in mice resulted in antigen-specific activation of T helper 1 (Th1) cells. This is the first comprehensive study to show the molecular basis of how soluble HPV 16 E6 or E7 fusion Igs induces Th1 responses through the maturation of DCs. In addition, we show that DC therapy using soluble HPV E6 and E7 fusion Igs may be a valuable tool for controlling the progress of cervical cancer.

Identification of toxicological biomarkers of di(2-ethylhexyl) phthalate in proteins secreted by HepG2 cells using proteomic analysis.

The effects of di(2-ethylhexyl) phthalate (DEHP) on proteins secreted by HepG2 cells were studied using a proteomic approach. HepG2 cells were exposed to various concentrations of DEHP (0, 2.5, 5, 10, 25, 50, 100, and 250 microM) for 24 or 48 h. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and comet assays were then conducted to determine the cytotoxicity and genotoxicity of DEHP, respectively. The MTT assay showed that 10 microM DEHP was the maximum concentration that did not cause cell death. In addition, the DNA damage in HepG2 cells exposed to DEHP was found to increase in a dose- and time-dependent fashion. Proteomic analysis using two different pI ranges (4-7 and 6-9) and large size 2-DE revealed the presence of 2776 protein spots. A total of 35 (19 up- and 16 down-regulated) proteins were identified as biomarkers of DEHP by ESI-MS/MS. Several differentiated protein groups were also found. Proteins involved in apoptosis, transportation, signaling, energy metabolism, and cell structure and motility were found to be up- or down-regulated. Among these, the identities of cystatin C, Rho GDP inhibitor, retinol binding protein 4, gelsolin, DEK protein, Raf kinase inhibitory protein, triose phosphate isomerase, cofilin-1, and haptoglobin-related protein were confirmed by Western blot assay. Therefore, these proteins could be used as potential biomarkers of DEHP and human disease associated with DEHP.

Epigenetic inactivation of HOXA5 and MSH2 gene in clear cell renal cell carcinoma.

The high-throughput method using microarray is an easy and fast way to analyze the methylation status of hundreds of preselected genes and to screen them for signatures in methylation. The aim of our study is to detect hypermethylated genes and to analyze the association between methylation status and clinicopathological parameters of clear cell renal cell carcinoma. The genetic substrate included 62 cancer tissues and 62 matched adjacent normal kidney tissues. We adapted the GoldenGate genotyping assay to determine the methylation state of 1505 specific CpG sites in 807 genes. We identified two genes (HOXA5 and MSH2) with ß-value differences of more than 0.3 between cancer and normal tissues. The high methylation group in HOXA5 had high Fuhrman's nuclear grade (P= 0.041). Other data in HOXA5 and MSH2 were not significant with methylation status (P > 0.05). Survival curve of the high methylation group in HOXA5 was slightly lower than that of the low methylation group. However, the statistical significances of overall survival in HOXA5 and MSH2 were low (P > 0.05). We report the hypermethylation of two genes in clear cell renal cell carcinoma. The data we obtained could provide the basis for a diagnostic test pathological assessment, or prognosis in clear cell renal cell carcinoma.

Proteomic analysis of proteins secreted by HepG2 cells treated with butyl benzyl phthalate.

Proteomic changes in proteins secreted by human hepatocellular carcinomas (HepG2) cells exposed to butyl benzyl phthalate (BBP) were evaluated. HepG2 cells were treated with three different concentrations of BBP (0, 10, or 25 µM) for 24 or 48 h. Following incubation, the cells were subjected to proteomic analysis using two different pI ranges (4-7 and 6-9) and large-size two-dimensional gel electrophoresis. Results showed resolution of a total of 2776 protein spots. Of these, 29, including 19 upregulated and 10 downregulated proteins, were identified by electrospray ionization-mass spectrometry-mass spectrometry (ESI-MS/MS). Among these, the identities of cystatin C, Rho guanine nucleotide dissociation inhibitor, gelsolin, DEK protein, Raf kinase inhibitory protein, triose phosphate isomerase, heptaglobin-related protein, inter-alpha-trypsin inhibitor heavy chain H2, and electron transfer flavoprotein subunit beta were confirmed by Western blot analysis. These proteins were found to be involved in apoptosis, signaling, tumor progression, energy metabolism, and cell structure and motility. Therefore, these proteins have potential to be employed as biomarkers of BBP exposure and may be useful in understanding mechanisms underlying the adverse effects of BBP.

Analysis of low molecular weight plasma proteins using ultrafiltration and large gel two-dimensional electrophoresis.

In this study, various solvent systems were applied to obtain a high and consistent recovery rate of low molecular weight plasma proteins (LMPP) from human plasma. A buffer system containing 7 M urea, 2 M thiourea, 25 mM NH(4)HCO(3) + 20% ACN (pH 8.2) produced the highest recovery rate of LMPP. To validate the recovery of cut off membrane (COM) obtained using the urea buffer system, 27 different 30 kDa COMs were used to prepare the LMPP sample which were then subjected to 1-D SDS-PAGE. Statistical analysis showed that the buffer system with COM produced a consistent the recovery of LMPP. In addition, 2-DE analysis was also conducted to determine the relative intensity of each protein spot. When molecular weight ranges over 30 kDa and under 30 kDa were evaluated, 953 and 587 protein spots were observed in the gels, respectively, resulting in a total of 1540 protein spots being resolved. Identification of the major proteins were then performed using a nano-LC/MS system comprised of an HPLC system and an ESI-quadrupole IT MS equipped with a nano-ESI source.