JoVE Visualize What is visualize?
Stop Reading. Start Watching.
Advanced Search
Stop Reading. Start Watching.
Regular Search
Find video protocols related to scientific articles indexed in Pubmed.
[Investigation of the action mechanisms of poly-ADP-ribosylation in hexavalent chromium induced cell damage].
Zhonghua Yu Fang Yi Xue Za Zhi
PUBLISHED: 11-13-2014
Show Abstract
Hide Abstract
To investigate the effect of poly-ADP-ribosylation in hexavalent chromium Cr(VI) induced cell damage.
Related JoVE Video
Gene coexpression measures in large heterogeneous samples using count statistics.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 10-06-2014
Show Abstract
Hide Abstract
With the advent of high-throughput technologies making large-scale gene expression data readily available, developing appropriate computational tools to process these data and distill insights into systems biology has been an important part of the "big data" challenge. Gene coexpression is one of the earliest techniques developed that is still widely in use for functional annotation, pathway analysis, and, most importantly, the reconstruction of gene regulatory networks, based on gene expression data. However, most coexpression measures do not specifically account for local features in expression profiles. For example, it is very likely that the patterns of gene association may change or only exist in a subset of the samples, especially when the samples are pooled from a range of experiments. We propose two new gene coexpression statistics based on counting local patterns of gene expression ranks to take into account the potentially diverse nature of gene interactions. In particular, one of our statistics is designed for time-course data with local dependence structures, such as time series coupled over a subregion of the time domain. We provide asymptotic analysis of their distributions and power, and evaluate their performance against a wide range of existing coexpression measures on simulated and real data. Our new statistics are fast to compute, robust against outliers, and show comparable and often better general performance.
Related JoVE Video
High red blood cell distribution width is closely associated with nonalcoholic fatty liver disease.
Eur J Gastroenterol Hepatol
PUBLISHED: 08-29-2014
Show Abstract
Hide Abstract
The red blood cell distribution width (RDW) has been reported to be a risk marker of morbidity and mortality for cardiovascular diseases in various study populations. Nonalcoholic fatty liver disease (NAFLD) is also a risk factor for cardiovascular diseases. However, the relationship between RDW and NAFLD is less certain.
Related JoVE Video
Chronic Copper Exposure Causes Spatial Memory Impairment, Selective Loss of Hippocampal Synaptic Proteins, and Activation of PKR/eIF2? Pathway in Mice.
J. Alzheimers Dis.
PUBLISHED: 08-26-2014
Show Abstract
Hide Abstract
Copper is an essential element for human growth and development; however, excessive intake of copper could contribute to neurotoxicity. Here we show that chronic exposure to copper in drinking water impaired spatial memory with simultaneous selective loss of hippocampal pre-synaptic protein synapsin 1, and post-synaptic density protein (PSD)-93/95 in mice. Copper exposure was shown to elevate the levels of nitrotyrosine and 8-hydroxydeoxyguanosine (8-OHdG) in hippocampus, two markers of oxidative stress. Concurrently, we also found that copper exposure activated double stranded RNA-dependent protein kinase (PKR) as evidenced by increased ratio of phosphorylated PKR at Thr451 and total PKR and increased the phosphorylation of its downstream signaling molecule eukaryotic initiation factor 2? (eIF2?) at Ser51 in hippocampus. Consistent with activation of PKR/eIF2? signaling pathway which was shown to mediate synaptic deficit and cognitive impairment, the levels of activating transcription factor 4 (ATF-4), a downstream signaling molecule of eIF2? and a repressor of CREB-mediated gene expression, were significantly increased, while the activity of cAMP response elements binding protein (CREB) was inactivated as suggested by decreased phosphorylation of CREB at Ser133 by copper exposure. In addition, the expression of the pro-apoptotic target molecule C/EBP homology protein (CHOP) of ATF-4 was upregulated and hippocampal neuronal apoptosis was induced by copper exposure. Taken together, we propose that chronic copper exposure might cause spatial memory impairment, selective loss of synaptic proteins, and neuronal apoptosis through the mechanisms involving activation of PKR/eIF2? signaling pathway.
Related JoVE Video
Role of poly(ADP-ribose) glycohydrolase silencing in DNA hypomethylation induced by benzo(a)pyrene.
Biochem. Biophys. Res. Commun.
PUBLISHED: 08-20-2014
Show Abstract
Hide Abstract
Benzo(a)pyrene (BaP) is a known carcinogen cytotoxic which can trigger extensive cellular responses. Many evidences suggest that inhibitors of poly(ADP-ribose) glycohydrolase (PARG) are potent anticancer drug candidates. However, the role of PARG in BaP carcinogenesis is less understood. Here we used PARG-deficient human bronchial epithelial cell line (shPARG cell) as an in vitro model, and investigated the role of PARG silencing in DNA methylation pattern changed by BaP. Our study shows, BaP treatment decreased global DNA methylation levels in 16HBE cells in a dose-dependent manner, but no dramatic changes were observed in shPARG cells. Further investigation revealed PARG silencing protected DNA methyltransferases (DNMTs) activity from change by BaP exposure. Interestingly, Dnmt1 is PARylated in PARG-null cells after BaP exposure. The results show a role for PARG silencing in DNA hypomethylation induced by BaP that may provide new clue for cancer therapy.
Related JoVE Video
Effect and mechanism of tacrolimus on melanogenesis on A375 human melanoma cells.
Chin. Med. J.
PUBLISHED: 08-19-2014
Show Abstract
Hide Abstract
Topical tacrolimus has been used for vitiligo as a common treatment option for more than ten years while the underlying mechanism is still uncertain. The aim of this study was to investigate the direct effects of tacrolimus on the melanogenesis and migration on human A375 melanoma cells. The expression of c-KIT mRNA and protein of human A375 cells were also investigated.
Related JoVE Video
Neuroprotective effects of Buyang Huanwu decoction on cerebral ischemia-induced neuronal damage.
Neural Regen Res
PUBLISHED: 08-04-2014
Show Abstract
Hide Abstract
Among the various treatment methods for stroke, increasing attention has been paid to traditional Chinese medicines. Buyang Huanwu decoction is a commonly used traditional Chinese medicine for the treatment of stroke. This paper summarizes the active components of the Chinese herb, which is composed of Huangqi (Radix Astragali seu Hedysari), Danggui (Radix Angelica sinensis), Chishao (Radix Paeoniae Rubra), Chuanxiong (Rhizoma Ligustici Chuanxiong), Honghua (Flos Carthami), Taoren (Semen Persicae) and Dilong (Pheretima), and identifies the therapeutic targets and underlying mechanisms that contribute to the neuroprotective properties of Buyang Huanwu decoction.
Related JoVE Video
Comparative analysis of regulatory information and circuits across distant species.
Nature
PUBLISHED: 07-10-2014
Show Abstract
Hide Abstract
Despite the large evolutionary distances between metazoan species, they can show remarkable commonalities in their biology, and this has helped to establish fly and worm as model organisms for human biology. Although studies of individual elements and factors have explored similarities in gene regulation, a large-scale comparative analysis of basic principles of transcriptional regulatory features is lacking. Here we map the genome-wide binding locations of 165 human, 93 worm and 52 fly transcription regulatory factors, generating a total of 1,019 data sets from diverse cell types, developmental stages, or conditions in the three species, of which 498 (48.9%) are presented here for the first time. We find that structural properties of regulatory networks are remarkably conserved and that orthologous regulatory factor families recognize similar binding motifs in vivo and show some similar co-associations. Our results suggest that gene-regulatory properties previously observed for individual factors are general principles of metazoan regulation that are remarkably well-preserved despite extensive functional divergence of individual network connections. The comparative maps of regulatory circuitry provided here will drive an improved understanding of the regulatory underpinnings of model organism biology and how these relate to human biology, development and disease.
Related JoVE Video
Evidence for a hexaheteromeric methylenetetrahydrofolate reductase in Moorella thermoacetica.
J. Bacteriol.
PUBLISHED: 07-07-2014
Show Abstract
Hide Abstract
Moorella thermoacetica can grow with H? and CO?, forming acetic acid from 2 CO? via the Wood-Ljungdahl pathway. All enzymes involved in this pathway have been characterized to date, except for methylenetetrahydrofolate reductase (MetF). We report here that the M. thermoacetica gene that putatively encodes this enzyme, metF, is part of a transcription unit also containing the genes hdrCBA, mvhD, and metV. MetF copurified with the other five proteins encoded in the unit in a hexaheteromeric complex with an apparent molecular mass in the 320-kDa range. The 40-fold-enriched preparation contained per mg protein 3.1 nmol flavin adenine dinucleotide (FAD), 3.4 nmol flavin mononucleotide (FMN), and 110 nmol iron, almost as predicted from the primary structure of the six subunits. It catalyzed the reduction of methylenetetrahydrofolate with reduced benzyl viologen but not with NAD(P)H in either the absence or presence of oxidized ferredoxin. It also catalyzed the reversible reduction of benzyl viologen with NADH (diaphorase activity). Heterologous expression of the metF gene in Escherichia coli revealed that the subunit MetF contains one FMN rather than FAD. MetF exhibited 70-fold-higher methylenetetrahydrofolate reductase activity with benzyl viologen when produced together with MetV, which in part shows sequence similarity to MetF. Heterologously produced HdrA contained 2 FADs and had NAD-specific diaphorase activity. Our results suggested that the physiological electron donor for methylenetetrahydrofolate reduction in M. thermoacetica is NADH and that the exergonic reduction of methylenetetrahydrofolate with NADH is coupled via flavin-based electron bifurcation with the endergonic reduction of an electron acceptor, whose identity remains unknown.
Related JoVE Video
Comparison of D. melanogaster and C. elegans developmental stages, tissues, and cells by modENCODE RNA-seq data.
Genome Res.
PUBLISHED: 07-03-2014
Show Abstract
Hide Abstract
We report a statistical study to discover transcriptome similarity of developmental stages from D. melanogaster and C. elegans using modENCODE RNA-seq data. We focus on "stage-associated genes" that capture specific transcriptional activities in each stage and use them to map pairwise stages within and between the two species by a hypergeometric test. Within each species, temporally adjacent stages exhibit high transcriptome similarity, as expected. Additionally, fly female adults and worm adults are mapped with fly and worm embryos, respectively, due to maternal gene expression. Between fly and worm, an unexpected strong collinearity is observed in the time course from early embryos to late larvae. Moreover, a second parallel pattern is found between fly prepupae through adults and worm late embryos through adults, consistent with the second large wave of cell proliferation and differentiation in the fly life cycle. The results indicate a partially duplicated developmental program in fly. Our results constitute the first comprehensive comparison between D. melanogaster and C. elegans developmental time courses and provide new insights into similarities in their development . We use an analogous approach to compare tissues and cells from fly and worm. Findings include strong transcriptome similarity of fly cell lines, clustering of fly adult tissues by origin regardless of sex and age, and clustering of worm tissues and dissected cells by developmental stage. Gene ontology analysis supports our results and gives a detailed functional annotation of different stages, tissues and cells. Finally, we show that standard correlation analyses could not effectively detect the mappings found by our method.
Related JoVE Video
Effect on metabolic enzymes and thyroid receptors induced by BDE-47 by activation the pregnane X receptor in HepG2, a human hepatoma cell line.
Toxicol In Vitro
PUBLISHED: 06-06-2014
Show Abstract
Hide Abstract
2,2',4,4'-Tetra-bromodiphenyl ether (BDE-47), an important congener among polybrominated diphenyl ether (PBDE) compounds, has been predominantly in environmental samples and human tissue. Thyroid disruption is the most sensitive endpoint effect among a number of health effects of exposure to BDE-47 in animals and humans. However, the detailed underlying mechanisms in humans are not well understood. In the present study, human pregnane X receptor (hPXR)-overexpressing HepG2 cell model and a dual-luciferase reporter assay system were constructed to investigate the role of hPXR in BDE-47-induced alterations of expression of metabolic enzymes and TR in vitro. The results showed that hPXR was significantly activated by BDE-47, and expression levels of both mRNA and protein of the thyroid receptor (TR) isoforms TR?1 and TR?1 were decreased in hPXR-overexpressing HepG2 cells after BDE-47 treatment. However, the increased expression of hepatic microsomal phase I enzyme CYP3A4 and phase II enzymes, UGT1A3 and SULT2A1 were also found. Taken together, the results indicated that BDE-47 was a strong hPXR activator, activation of hPXR played an important role in BDE-47-induced down-regulation of TR, and up-regulations of CYP3A4, UGT1A3, and SULT2A1 participated in the process, which may provide more toxicological evidence on mechanisms of disruption of thyroid hormone induced by BDE-47.
Related JoVE Video
[Effect of poly-ADP-ribosylation on the alteration of DNA methylation level of human bronchial epithelial cells induced by Cr (VI)].
Zhonghua Yu Fang Yi Xue Za Zhi
PUBLISHED: 05-22-2014
Show Abstract
Hide Abstract
To reveal the role of poly-ADP-ribosylation and DNA methylation in carcinogenic process induced induced by Cr (VI), and to discuss the relations between them.
Related JoVE Video
Prediction of LDL cholesterol response to statin using transcriptomic and genetic variation.
Genome Biol.
PUBLISHED: 05-16-2014
Show Abstract
Hide Abstract
Statins are widely prescribed for lowering LDL-cholesterol (LDLC) levels and risk of cardiovascular disease. There is, however, substantial inter-individual variation in the magnitude of statin-induced LDLC reduction. To date, analysis of individual DNA sequence variants has explained only a small proportion of this variability. The present study was aimed at assessing whether transcriptomic analyses could be used to identify additional genetic contributions to inter-individual differences in statin efficacy.
Related JoVE Video
Comparative analysis of the transcriptome across distant species.
Nature
PUBLISHED: 04-30-2014
Show Abstract
Hide Abstract
The transcriptome is the readout of the genome. Identifying common features in it across distant species can reveal fundamental principles. To this end, the ENCODE and modENCODE consortia have generated large amounts of matched RNA-sequencing data for human, worm and fly. Uniform processing and comprehensive annotation of these data allow comparison across metazoan phyla, extending beyond earlier within-phylum transcriptome comparisons and revealing ancient, conserved features. Specifically, we discover co-expression modules shared across animals, many of which are enriched in developmental genes. Moreover, we use expression patterns to align the stages in worm and fly development and find a novel pairing between worm embryo and fly pupae, in addition to the embryo-to-embryo and larvae-to-larvae pairings. Furthermore, we find that the extent of non-canonical, non-coding transcription is similar in each organism, per base pair. Finally, we find in all three organisms that the gene-expression levels, both coding and non-coding, can be quantitatively predicted from chromatin features at the promoter using a 'universal model' based on a single set of organism-independent parameters.
Related JoVE Video
Characterization of the zebrafish Ugt repertoire reveals a new class of drug-metabolizing UDP glucuronosyltransferases.
Mol. Pharmacol.
PUBLISHED: 04-11-2014
Show Abstract
Hide Abstract
The zebrafish genome contains a gene superfamily of 40 Ugt genes that can be divided into Ugt1, Ugt2, and Ugt5 families. Because the encoded zebrafish UDP glucuronosyltransferase (UGT) proteins do not display orthologous relationships to any of the mammalian and avian UGT enzymes based on molecular phylogeny, it is difficult to predict their substrate specificity. Here, we mapped their tissue-specific expression patterns. We showed that the zebrafish UGT enzymes can be glycosylated. We determined their substrate specificity and catalytic activity toward diverse aglycone substrates. Specifically, we measured mRNA levels of each of the 40 zebrafish Ugt genes in 11 adult tissues and found that they are expressed in a tissue-specific manner. Moreover, functional analyses with the donor of UDP glucuronic acid (UDPGA) for each of the 40 zebrafish UGT proteins revealed their substrate specificity toward 10 important aglycones. In particular, UGT1A1, UGT1A7, and UGT1B1 displayed good glucuronidation activities toward most phenolic aglycones (4-methylumbelliferone, 4-nitrophenol, 1-naphthol, bisphenol A, and mycophenolic acid) and the two carboxylic acids (bilirubin and diclofenac). Importantly, some members of the UGT5, a novel UGT family identified recently, are capable of glucuronidating multiple aglycones with the donor cofactor of UDPGA. In particular, UGT5A5, UGT5B2, and UGT5E1 glucuronidate phenols and steroids with high specificity toward steroid hormones of estradiol and testosterone and estrogenic alkylphenols 4-tert-octylphenol. These results shed new insights into the mechanisms by which fish species defend themselves against vast numbers of xenobiotics via glucuronidation conjugations and may facilitate the establishment of zebrafish as a model vertebrate in toxicological, developmental, and pathologic studies.
Related JoVE Video
Serum proteomic analysis reveals potential serum biomarkers for occupational medicamentosa-like dermatitis caused by trichloroethylene.
Toxicol. Lett.
PUBLISHED: 04-04-2014
Show Abstract
Hide Abstract
Trichloroethylene (TCE) is an industrial solvent with widespread occupational exposure and also a major environmental contaminant. Occupational medicamentosa-like dermatitis induced by trichloroethylene (OMLDT) is an autoimmune disease and it has become one major hazard in China. In this study, sera from 3 healthy controls and 3 OMLDT patients at different disease stages were used for a screening study by 2D-DIGE and MALDI-TOF-MS/MS. Eight proteins including transthyretin (TTR), retinol binding protein 4 (RBP4), haptoglobin, clusterin, serum amyloid A protein (SAA), apolipoprotein A-I, apolipoprotein C-III and apolipoprotein C-II were found to be significantly altered among the healthy, acute-stage, healing-stage and healed-stage groups. Specifically, the altered expression of TTR, RBP4 and haptoglobin were further validated by Western blot analysis and ELISA. Our data not only suggested that TTR, RBP4 and haptoglobin could serve as potential serum biomarkers of OMLDT, but also indicated that measurement of TTR, RBP4 and haptoglobin or their combination could help aid in the diagnosis, monitoring the progression and therapy of the disease.
Related JoVE Video
Extracellular signal-regulated kinase activation during cardiac hypertrophy reduces sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) transcription.
J. Mol. Cell. Cardiol.
PUBLISHED: 03-02-2014
Show Abstract
Hide Abstract
Pathologic cardiac hypertrophy can lead to heart failure, but the mechanisms involved are poorly understood. SERCA2 is critical for normal cardiac calcium handling and function and SERCA2 mRNA and protein levels are reduced by cardiac hypertrophy. We hypothesized that extracellular signal-regulated kinase (ERK) 1/2 activation during hypertrophy reduced SERCA2 transcription. Using a neonatal rat ventricular myocyte model of hypertrophy, we found that pharmacologic inhibitors of ERK activation preserve SERCA2 mRNA levels during hypertrophy. ERK activation is sufficient to reduce SERCA2 mRNA. We determined that ERK represses SERCA2 transcription via nuclear factor-kappaB (NFkB), and activation of NFkB is sufficient to reduce SERCA2 mRNA in cardiomyocytes. This work establishes novel connections between ERK, NFkB, and SERCA2 repression during cardiac hypertrophy. This mechanism may have implications for the progression of hypertrophy to heart failure.
Related JoVE Video
Analysis of trichloroethylene-induced global DNA hypomethylation in hepatic L-02 cells by liquid chromatography-electrospray ionization tandem mass spectrometry.
Biochem. Biophys. Res. Commun.
PUBLISHED: 02-27-2014
Show Abstract
Hide Abstract
Trichloroethylene (TCE), a major occupational and environmental pollutant, has been recently associated with aberrant epigenetic changes in experimental animals and cultured cells. TCE is known to cause severe hepatotoxicity; however, the association between epigenetic alterations and TCE-induced hepatotoxicity are not yet well explored. DNA methylation, catalyzed by enzymes known as DNA methyltransferases (DNMT), is a major epigenetic modification that plays a critical role in regulating many cellular processes. In this study, we analyzed the TCE-induced effect on global DNA methylation and DNMT enzymatic activity in human hepatic L-02 cells. A sensitive and quantitative method combined with liquid chromatography and electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) was validated and utilized for assessing the altered DNA methylation in TCE-induced L-02 cells. Quantification was accomplished in multiple reaction monitoring (MRM) mode by monitoring a transition pair of m/z 242.1 (molecular ion)/126.3 (fragment ion) for 5-mdC and m/z 268.1/152.3 for dG. The correlation coefficient of calibration curves between 5-mdC and dG was higher than 0.9990. The intra-day and inter-day relative standard derivation values (RSD) were on the range of 0.53-7.09% and 0.40-2.83%, respectively. We found that TCE exposure was able to significantly decrease the DNA methylation and inhibit DNMT activity in L-02 cells. Our results not only reveal the association between TCE exposure and epigenetic alterations, but also provide an alternative mass spectrometry-based method for rapid and accurate assessment of chemical-induced altered DNA methylation in mammal cells.
Related JoVE Video
Identification of the proteins related to SET-mediated hepatic cytotoxicity of trichloroethylene by proteomic analysis.
Toxicol. Lett.
PUBLISHED: 02-27-2014
Show Abstract
Hide Abstract
Trichloroethylene (TCE) is an effective solvent for a variety of organic materials. Since the wide use of TCE as industrial degreasing of metals, adhesive paint and polyvinyl chloride production, TCE has turned into an environmental and occupational toxicant. Exposure to TCE could cause severe hepatotoxicity; however, the toxic mechanisms of TCE remain poorly understood. Recently, we reported that SET protein mediated TCE-induced cytotoxicity in L-02 cells. Here, we further identified the proteins related to SET-mediated hepatic cytotoxicity of TCE using the techniques of DIGE (differential gel electrophoresis) and MALDI-TOF-MS/MS. Among the 20 differential proteins identified, 8 were found to be modulated by SET in TCE-induced cytotoxicity and three of them (cofilin-1, peroxiredoxin-2 and S100-A11) were validated by Western-blot analysis. The functional analysis revealed that most of the identified SET-modulated proteins are apoptosis-associated proteins. These data indicated that these proteins may be involved in SET-mediated hepatic cytotoxicity of TCE in L-02 cells.
Related JoVE Video
Review on statistical methods for gene network reconstruction using expression data.
J. Theor. Biol.
PUBLISHED: 01-26-2014
Show Abstract
Hide Abstract
Network modeling has proven to be a fundamental tool in analyzing the inner workings of a cell. It has revolutionized our understanding of biological processes and made significant contributions to the discovery of disease biomarkers. Much effort has been devoted to reconstruct various types of biochemical networks using functional genomic datasets generated by high-throughput technologies. This paper discusses statistical methods used to reconstruct gene regulatory networks using gene expression data. In particular, we highlight progress made and challenges yet to be met in the problems involved in estimating gene interactions, inferring causality and modeling temporal changes of regulation behaviors. As rapid advances in technologies have made available diverse, large-scale genomic data, we also survey methods of incorporating all these additional data to achieve better, more accurate inference of gene networks.
Related JoVE Video
Serum amyloid A and clusterin as potential predictive biomarkers for severe hand, foot and mouth disease by 2D-DIGE proteomics analysis.
PLoS ONE
PUBLISHED: 01-01-2014
Show Abstract
Hide Abstract
Hand, foot, and mouth disease (HFMD) affects more than one million children, is responsible for several hundred child deaths every year in China and is the cause of widespread concerns in society. Only a small fraction of HFMD cases will develop further into severe HFMD with neurologic complications. A timely and accurate diagnosis of severe HFMD is essential for assessing the risk of progression and planning the appropriate treatment. Human serum can reflect the physiological or pathological states, which is expected to be an excellent source of disease-specific biomarkers. In the present study, a comparative serological proteome analysis between severe HFMD patients and healthy controls was performed via a two-dimensional difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) strategy. Fifteen proteins were identified as differentially expressed in the sera of the severe HFMD patients compared with the controls. The identified proteins were classified into different groups according to their molecular functions, biological processes, protein classes and physiological pathways by bioinformatics analysis. The up-regulations of two identified proteins, serum amyloid A (SAA) and clusterin (CLU), were confirmed in the sera of the HFMD patients by ELISA assay. This study not only increases our background knowledge about and scientific insight into the mechanisms of HFMD, but also reveals novel potential biomarkers for the clinical diagnosis of severe HFMD.
Related JoVE Video
6-hydroxy-3-succinoylpyridine hydroxylase catalyzes a central step of nicotine degradation in Agrobacterium tumefaciens S33.
PLoS ONE
PUBLISHED: 01-01-2014
Show Abstract
Hide Abstract
Nicotine is a main alkaloid in tobacco and is also the primary toxic compound in tobacco wastes. It can be degraded by bacteria via either pyridine pathway or pyrrolidine pathway. Previously, a fused pathway of the pyridine pathway and the pyrrolidine pathway was proposed for nicotine degradation by Agrobacterium tumefaciens S33, in which 6-hydroxy-3-succinoylpyridine (HSP) is a key intermediate connecting the two pathways. We report here the purification and properties of an NADH-dependent HSP hydroxylase from A. tumefaciens S33. The 90-kDa homodimeric flavoprotein catalyzed the oxidative decarboxylation of HSP to 2,5-dihydroxypyridine (2,5-DHP) in the presence of NADH and FAD at pH 8.0 at a specific rate of about 18.8 ± 1.85 µmol min-1 mg protein-1. Its gene was identified by searching the N-terminal amino acid residues of the purified protein against the genome draft of the bacterium. It encodes a protein composed of 391 amino acids with 62% identity to HSP hydroxylase (HspB) from Pseudomonas putida S16, which degrades nicotine via the pyrrolidine pathway. Considering the application potential of 2,5-DHP in agriculture and medicine, we developed a route to transform HSP into 2,5-DHP with recombinant HSP hydroxylase and an NADH-regenerating system (formate, NAD+ and formate dehydrogenase), via which around 0.53 ± 0.03 mM 2,5-DHP was produced from 0.76 ± 0.01 mM HSP with a molar conversion as 69.7%. This study presents the biochemical properties of the key enzyme HSP hydroxylase which is involved in the fused nicotine degradation pathway of the pyridine and pyrrolidine pathways and a new green route to biochemically synthesize functionalized 2,5-DHP.
Related JoVE Video
Interleukin-6 first plays pro- then anti-inflammatory role in early versus late acute renal allograft rejection.
Ann. Clin. Lab. Sci.
PUBLISHED: 11-20-2013
Show Abstract
Hide Abstract
This study aimed to investigate the potential role of IL-6 in acute T-cell-mediated renal rejection (ACR) during different periods post-transplantation. Fifty-three patients with ACR (32 of whom developed ACR within the first month; 12, between 2 and 6 months; and 9, between 7 and 12 months post-transplantation), 31 patients with delayed graft function (DGF), and 38 recipients with stable renal allograft function were recruited. Luminex analysis was used to monitor levels of IL-6, sIL-6R, IL-1?, IL-1?, and IL-1 receptor antagonist (IL-1Ra) in 228 serum samples from 122 patients, including ACR patients before and during rejection, and after rejection reversal, DGF patients, and stable controls. The associations between IL-6 levels and sIL-6R, IL-1?, IL-1?, and IL-1Ra levels were analyzed using Spearman correlation analysis. In patients who developed ACR within the first month post-transplantation, serum IL-6 concentrations increased significantly compared to the stable control group, but decreased in patients who developed ACR between 2 and 12 months post-transplantation. Concomitantly, levels of sIL-6R gradually increased when ACR occurred between 2 and 12 months post-transplantation. IL-6 levels correlated with IL-1? levels in early stage ACR and with levels of IL-1Ra in late stage ACR. Our results suggest that IL-6 acts as a pro-inflammatory cytokine during early-stage ACR, and plays an anti-inflammatory role during later stages post-transplantation.
Related JoVE Video
A rapid and sensitive LC-ESI-MS/MS method for the detection of YF-49-92.MLS in rat plasma.
Bioanalysis
PUBLISHED: 10-22-2013
Show Abstract
Hide Abstract
YF-49-92.MLS is a novel candidate for TB treatment. An accurate, precise and specific LC-MS/MS method for the quantification of YF-49-92.MLS in rat plasma using verapamil as an IS is reported in this paper.
Related JoVE Video
NADP-specific electron-bifurcating [FeFe]-hydrogenase in a functional complex with formate dehydrogenase in Clostridium autoethanogenum grown on CO.
J. Bacteriol.
PUBLISHED: 07-26-2013
Show Abstract
Hide Abstract
Flavin-based electron bifurcation is a recently discovered mechanism of coupling endergonic to exergonic redox reactions in the cytoplasm of anaerobic bacteria and archaea. Among the five electron-bifurcating enzyme complexes characterized to date, one is a heteromeric ferredoxin- and NAD-dependent [FeFe]-hydrogenase. We report here a novel electron-bifurcating [FeFe]-hydrogenase that is NADP rather than NAD specific and forms a complex with a formate dehydrogenase. The complex was found in high concentrations (6% of the cytoplasmic proteins) in the acetogenic Clostridium autoethanogenum autotrophically grown on CO, which was fermented to acetate, ethanol, and 2,3-butanediol. The purified complex was composed of seven different subunits. As predicted from the sequence of the encoding clustered genes (fdhA/hytA-E) and from chemical analyses, the 78.8-kDa subunit (FdhA) is a selenocysteine- and tungsten-containing formate dehydrogenase, the 65.5-kDa subunit (HytB) is an iron-sulfur flavin mononucleotide protein harboring the NADP binding site, the 51.4-kDa subunit (HytA) is the [FeFe]-hydrogenase proper, and the 18.1-kDa (HytC), 28.6-kDa (HytD), 19.9-kDa (HytE1), and 20.1-kDa (HytE2) subunits are iron-sulfur proteins. The complex catalyzed both the reversible coupled reduction of ferredoxin and NADP(+) with H2 or formate and the reversible formation of H2 and CO2 from formate. We propose the complex to have two functions in vivo, namely, to normally catalyze CO2 reduction to formate with NADPH and reduced ferredoxin in the Wood-Ljungdahl pathway and to catalyze H2 formation from NADPH and reduced ferredoxin when these redox mediators get too reduced during unbalanced growth of C. autoethanogenum on CO (E0 = -520 mV).
Related JoVE Video
Clostridium acidurici electron-bifurcating formate dehydrogenase.
Appl. Environ. Microbiol.
PUBLISHED: 07-19-2013
Show Abstract
Hide Abstract
Cell extracts of uric acid-grown Clostridium acidurici catalyzed the coupled reduction of NAD(+) and ferredoxin with formate at a specific activity of 1.3 U/mg. The enzyme complex catalyzing the electron-bifurcating reaction was purified 130-fold and found to be composed of four subunits encoded by the gene cluster hylCBA-fdhF2.
Related JoVE Video
Differences in the metabolic status of healthy adults with and without active brown adipose tissue.
Wien. Klin. Wochenschr.
PUBLISHED: 05-01-2013
Show Abstract
Hide Abstract
Previous studies have proven the existence of active brown adipose tissue (BAT) in adults; however, its effect on systematic metabolism remains unclear.
Related JoVE Video
Identification of serum biomarkers for occupational medicamentosa-like dermatitis induced by trichloroethylene using mass spectrometry.
Toxicol. Appl. Pharmacol.
PUBLISHED: 04-18-2013
Show Abstract
Hide Abstract
Occupational medicamentosa-like dermatitis induced by trichloroethylene (OMLDT) is an autoimmune disease and it has become a serious occupational health hazard. In the present study, we collected fasting blood samples from patients with OMLDT (n=18) and healthy volunteers (n=33) to explore serum peptidome patterns. Peptides in sera were purified using weak cation exchange magnetic beads (MB-WCX), and analyzed by matrix-assisted laser desorption ionization time-of-flight-mass spectrometry (MALDI-TOF-MS) and ClinProTools bioinformatics software. The intensities of thirty protein/peptide peaks were significantly different between the healthy control and OMLDT patients. A pattern of three peaks (m/z 2106.3, 2134.5, and 3263.67) was selected for supervised neural network (SNN) model building to separate the OMLDT patients from the healthy controls with a sensitivity of 95.5% and a specificity of 73.8%. Furthermore, two peptide peaks of m/z 4091.61 and 4281.69 were identified as fragments of ATP-binding cassette transporter family A member 12 (ABCA12), and cationic trypsinogen (PRRS1), respectively. Our findings not only show that specific proteomic fingerprints in the sera of OMLDT patients can be served as a differentiated tool of OMLDT patients with high sensitivity and high specificity, but also reveal the novel correlation between OMLDT with ABC transports and PRRS1, which will be of potential value for clinical and mechanistic studies of OMLDT.
Related JoVE Video
Association between Obesity, Serum Lipids, and Colorectal Polyps in Old Chinese People.
Gastroenterol Res Pract
PUBLISHED: 04-12-2013
Show Abstract
Hide Abstract
Background. Colorectal cancer mostly arises from the polyps of colon. The aim of our study was to examine the association of body mass index (BMI) and serum lipids with the colorectal polyps in old Chinese people. Methods. The risk of developing colorectal polyps was studied in 244 subjects (212 men and 32 women, 74.63 ± 11.63 years old) who underwent colonoscopy for the first time from January 2008 to July 2012 at the Navy General Hospital, Beijing, China. According to the results of colonoscopy, all the subjects were divided into 112 normal control, 38 right colorectal polyps, 53 left colorectal polyps, and 41 both right and left colorectal polyps groups. The total plasma cholesterol, plasma triglyceride, plasma creatinine concentration, blood urinary nitrogen, and fasting glucose were determined using a multichannel analyzer. Results. There were significant differences among normal control, right colorectal polyps, left colorectal polyps, and both right and left polyps groups, which were the BMI, total cholesterol, triglycerides, creatinine, and urinary nitrogen. In binary logistic regression analysis, there were two risk factors associated with the occurrence of colorectal polyps, which included BMI and systolic blood pressure. Conclusions. Colorectal polyps were significantly associated with increased BMI, total cholesterol, and triglycerides levels.
Related JoVE Video
Sorting single satellite cells from individual myofibers reveals heterogeneity in cell-surface markers and myogenic capacity.
Integr Biol (Camb)
PUBLISHED: 02-13-2013
Show Abstract
Hide Abstract
Traditional cell-screening techniques such as FACS and MACS are better suited for large numbers of cells isolated from bulk tissue and cannot easily screen stem or progenitor cells from minute populations found in their physiological niches. Furthermore, these techniques rely upon irreversible antibody binding, potentially altering cell properties, including gene expression and regenerative capacity. To address these challenges, we have developed a novel, label-free stem-cell analysis and sorting platform capable of quantifying cell-surface marker expression of single functional organ stem cells directly isolated from their micro-anatomical niche. Using our unique platform, we have discovered a remarkable heterogeneity in both the regenerative capacity and expression of CXCR4, ?1-integrin, Sca-1, M-cadherin, Syndecan-4, and Notch-1 in freshly isolated muscle stem (satellite) cells residing on different, single myofibers and have identified a small population of Sca-1(+)/Myf5(+) myogenic satellite cells. Our results demonstrate the utility of our single-cell platform for uncovering and functionally characterizing stem-cell heterogeneity in the organ microniche.
Related JoVE Video
Serum levels of CXCR3 ligands predict T cell-mediated acute rejection after kidney transplantation.
Mol Med Rep
PUBLISHED: 02-12-2013
Show Abstract
Hide Abstract
The early diagnosis of acute rejection is crucial for graft survival after kidney transplantation. The interferon-? (IFN?)-CXCR3-chemokine-dependent inflammatory loop plays a pivotal role in the recruitment of T lymphocytes during acute rejection. Previously published studies have typically focused on the CXCR3 receptor rather than on its ligands. In the present study, we used Luminex assays to detect the levels of CXCR3 ligands, monokine induced by IFN? (MIG), IFN-induced protein 10 (IP-10) and IFN-induced T?cell chemoattractant (I-TAC), in the serum of renal allograft recipients. According to a renal biopsy performed one month after kidney transplantation, 32 recipients were diagnosed with T cell-mediated acute rejection and 38 patients were evaluated as stable. Serum was collected after the diagnosis of acute rejection or one month after transplantation. The concentrations of MIG (median, 4,271 pg/ml), IP-10 (median, 686.7 pg/ml) and I-TAC (median, 44.32 pg/ml) in the serum during an acute rejection episode were significantly higher compared with those of the stable patients (MIG, P=0.0002; IP-10, P=0.0001; I-TAC, P=0.0103; vs. the stable function group, P<0.05). Based on the receiver-operating characteristic (ROC) curve, the joint detection of MIG, IP-10 and I-TAC in the serum using Luminex analysis may constitute a non-invasive and efficient method for the early prediction of T cell-mediated acute rejection following kidney transplantation.
Related JoVE Video
A reversible electron-bifurcating ferredoxin- and NAD-dependent [FeFe]-hydrogenase (HydABC) in Moorella thermoacetica.
J. Bacteriol.
PUBLISHED: 01-11-2013
Show Abstract
Hide Abstract
Moorella thermoacetica was long the only model organism used to study the biochemistry of acetogenesis from CO(2). Depending on the growth substrate, this Gram-positive bacterium can either form H(2) or consume it. Despite the importance of H(2) in its metabolism, a hydrogenase from the organism has not yet been characterized. We report here the purification and properties of an electron-bifurcating [FeFe]-hydrogenase from M. thermoacetica and show that the cytoplasmic enzyme efficiently catalyzes both H(2) formation and H(2) uptake. The purified heterotrimeric iron-sulfur flavoprotein (HydABC) catalyzed the coupled reduction of ferredoxin (Fd) and NAD(+) with H(2) at 55 °C at pH 7.5 at a specific rate of about 100 ?mol min(-1) mg protein(-1) and the reverse reaction, the coupled reduction of protons to H(2) with reduced ferredoxin and NADH, at a specific rate of about 10 ?mol min(-1) mg protein(-1) in the stoichiometry Fd(ox) + NAD(+) + 2H(2) Fd(red)(2-) + NADH + 3H(+). When ferredoxin from Clostridium pasteurianum, NAD(+), and the enzyme were incubated at pH 7.0 under 100% H(2) in the gas phase (E(0) = -414 mV), more than 95% of the ferredoxin (E(0) = -400 mV) was reduced, which indicated that ferredoxin reduction with H(2) is driven by the exergonic reduction of NAD(+) (E(0) = -320 mV) with H(2). In the absence of NAD(+), ferredoxin was not reduced. We identified the genes encoding HydABC within the transcriptional unit hydCBAX and mapped the transcription start site.
Related JoVE Video
Effect of low dose bisphenol A on the early differentiation of human embryonic stem cells into mammary epithelial cells.
Toxicol. Lett.
PUBLISHED: 01-09-2013
Show Abstract
Hide Abstract
It has been previously reported that bisphenol A (BPA) can disturb the development of mammary structure and increase the risk of breast cancer in experimental animals. In this study, an in vitro model of human embryonic stem cell (hESC) differentiation into mammary epithelial cells was applied to investigate the effect of low dose BPA on the early stages of mammogenesis. A newly established hESC line was directionally differentiated into mammary epithelial cells by a well-established three-dimensional (3D) culture system. The differentiated mammary epithelial cells were characterized by immunofluorescence and western blotting assay, and were called induced differentiated mammary epithelial cells (iDMECs) based on these data. The hESCs were treated with low doses of BPA range 10(-9)-10(-6)M during the differentiation process, with DMSO as the solvent control and 17-?-estrodiol (E2) as the estrogen-positive control. Our results showed that low dose BPA and E2 could influence the mammosphere area of iDMECs and upregulate the expression level of Oct4 and Nanog proteins, while only BPA could downregulate the expression of E-cadherin protein. Taken together, this study provides some insights into the effects of low dose BPA on the early differentiation stage of mammary epithelial cells and suggests an easier canceration status of iDMECs under the effect of low dose BPA during its early differentiation stage.
Related JoVE Video
Diet-induced obesity causes long QT and reduces transcription of voltage-gated potassium channels.
J. Mol. Cell. Cardiol.
PUBLISHED: 01-04-2013
Show Abstract
Hide Abstract
In humans, obesity is associated with long QT, increased frequency of premature ventricular complexes, and sudden cardiac death. The mechanisms of the pro-arrhythmic electrophysiologic remodeling of obesity are poorly understood. We tested the hypothesis that there is decreased expression of voltage-gated potassium channels in the obese heart, leading to long QT. Using implanted telemeters, we found that diet-induced obese (DIO) wild-type mice have impaired cardiac repolarization, demonstrated by long QT, as well as more frequent ventricular ectopy, similar to obese humans. DIO mice have reduced protein and mRNA levels of the potassium channel Kv1.5 caused by a reduction of the transcription factor cyclic AMP response element binding protein (CREB) in DIO hearts. We found that CREB knock-down by siRNA reduces Kv1.5, CREB binds to the Kv1.5 promoter in the heart, and CREB increases transcription of mouse and human Kv1.5 promoters. The reduction in CREB protein during lipotoxicity can be rescued by inhibiting protein kinase D (PKD). Our results identify a mechanism for obesity-induced electrophysiologic remodeling in the heart, namely PKD-induced reduction of CREB, which in turn decreases expression of the potassium channel Kv1.5.
Related JoVE Video
Development of recombinant antigen array for simultaneous detection of viral antibodies.
PLoS ONE
PUBLISHED: 01-01-2013
Show Abstract
Hide Abstract
Protein microarrays have been developed to study antibody reactivity against a large number of antigens, demonstrating extensive perspective for clinical application. We developed a viral antigen array by spotting four recombinant antigens and synthetic peptide, including glycoprotein G of herpes simplex virus (HSV) type 1 and 2, phosphoprotein 150 of cytomegalovirus (CMV), Rubella virus (RV) core plus glycoprotein E1 and E2 as well as a E1 peptide with the optimal concentrations on activated glass slides to simultaneously detect IgG and IgM against HSV1, HSV2, CMV and RV in clinical specimens of sera and cerebrospinal fluids (CSFs). The positive reference sera were initially used to measure the sensitivity and specificity of the array with the optimal conditions. Then clinical specimens of 144 sera and 93 CSFs were tested for IgG and IgM antibodies directed against HSV1, HSV2, CMV and RV by the antigen array. Specificity of the antigen array for viral antibodies detection was satisfying compared to commercial ELISA kits but sensitivity of the array varied relying on quality and antigenic epitopes of the spotting antigens. In short, the recombinant antigen array has potential to simultaneous detect multiple viral antibodies using minute amount (3 µl) of samples, which holds the particularly advantage to detect viral antibodies in clinical CSFs being suspicious of neonatal meningitis and encephalitis.
Related JoVE Video
Sparse linear modeling of next-generation mRNA sequencing (RNA-Seq) data for isoform discovery and abundance estimation.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 12-01-2011
Show Abstract
Hide Abstract
Since the inception of next-generation mRNA sequencing (RNA-Seq) technology, various attempts have been made to utilize RNA-Seq data in assembling full-length mRNA isoforms de novo and estimating abundance of isoforms. However, for genes with more than a few exons, the problem tends to be challenging and often involves identifiability issues in statistical modeling. We have developed a statistical method called "sparse linear modeling of RNA-Seq data for isoform discovery and abundance estimation" (SLIDE) that takes exon boundaries and RNA-Seq data as input to discern the set of mRNA isoforms that are most likely to present in an RNA-Seq sample. SLIDE is based on a linear model with a design matrix that models the sampling probability of RNA-Seq reads from different mRNA isoforms. To tackle the model unidentifiability issue, SLIDE uses a modified Lasso procedure for parameter estimation. Compared with deterministic isoform assembly algorithms (e.g., Cufflinks), SLIDE considers the stochastic aspects of RNA-Seq reads in exons from different isoforms and thus has increased power in detecting more novel isoforms. Another advantage of SLIDE is its flexibility of incorporating other transcriptomic data such as RACE, CAGE, and EST into its model to further increase isoform discovery accuracy. SLIDE can also work downstream of other RNA-Seq assembly algorithms to integrate newly discovered genes and exons. Besides isoform discovery, SLIDE sequentially uses the same linear model to estimate the abundance of discovered isoforms. Simulation and real data studies show that SLIDE performs as well as or better than major competitors in both isoform discovery and abundance estimation. The SLIDE software package is available at https://sites.google.com/site/jingyijli/SLIDE.zip.
Related JoVE Video
Mice with cardiac overexpression of peroxisome proliferator-activated receptor ? have impaired repolarization and spontaneous fatal ventricular arrhythmias.
Circulation
PUBLISHED: 11-28-2011
Show Abstract
Hide Abstract
Diabetes mellitus and obesity, which confer an increased risk of sudden cardiac death, are associated with cardiomyocyte lipid accumulation and altered cardiac electric properties, manifested by prolongation of the QRS duration and QT interval. It is difficult to distinguish the contribution of cardiomyocyte lipid accumulation from the contribution of global metabolic defects to the increased incidence of sudden death and electric abnormalities.
Related JoVE Video
Characterization of the disposition of melamine in female Sprague-Dawley rats using ultra-performance liquid chromatography-tandem mass spectrometry.
J Anal Toxicol
PUBLISHED: 10-19-2011
Show Abstract
Hide Abstract
This study characterized the concentration-time profile of melamine in the heart, liver, spleen, lungs, kidneys, bladder, feces, urine, and plasma after melamine (MM) administration. Female Sprague-Dawley rats received a single oral dose of 1.0 g/kg body weight. Samples (n=4 per time point) were collected at 12, 24, 48, 72, 96, 120, 144, and 168 h. Based on calculations of the area under the concentration-time curves after dosing, ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) was used to detect MM concentration in tissues. Peak concentrations of MM are reached in the livers and lungs at 12 h after dosing and in hearts, spleens, kidneys, bladders, feces, urine, and plasma at 24 h after dosing. More than 90% of the ingested MM is excreted in feces and urine within 24 h. These results provided initial understanding of the tissue disposition of MM. Moreover, this study demonstrates that UPLC-MS-MS can be used to detect MM in biological samples.
Related JoVE Video
Ultrasound operators confidence influences diagnosis of ovarian tumors - a study in China.
Asian Pac. J. Cancer Prev.
PUBLISHED: 08-31-2011
Show Abstract
Hide Abstract
To assess the effect of ultrasound operators confidence in diagnosis of ovarian cancer, and the factors influencing diagnostic accuracy.
Related JoVE Video
Temporal dissection of tumorigenesis in primary cancers.
Cancer Discov
PUBLISHED: 06-29-2011
Show Abstract
Hide Abstract
Timely intervention for cancer requires knowledge of its earliest genetic aberrations. Sequencing of tumors and their metastases reveals numerous abnormalities occurring late in progression. A means to temporally order aberrations in a single cancer, rather than inferring them from serially acquired samples, would define changes preceding even clinically evident disease. We integrate DNA sequence and copy number information to reconstruct the order of abnormalities as individual tumors evolve for 2 separate cancer types. We detect vast, unreported expansion of simple mutations sharply demarcated by recombinative loss of the second copy of TP53 in cutaneous squamous cell carcinomas (cSCC) and serous ovarian adenocarcinomas, in the former surpassing 50 mutations per megabase. In cSCCs, we also report diverse secondary mutations in known and novel oncogenic pathways, illustrating how such expanded mutagenesis directly promotes malignant progression. These results reframe paradigms in which TP53 mutation is required later, to bypass senescence induced by driver oncogenes.
Related JoVE Video
Serum hematopoietic growth factors as diagnostic and prognostic markers of acute renal allograft rejection: a potential role for serum stem cell factor.
Cytokine
PUBLISHED: 05-27-2011
Show Abstract
Hide Abstract
Hematopoietic growth factors (HGFs) are proliferative and chemotactic agents for hematopoietic stem cells, although their functions on renal transplantation are rarely reported. This study aimed to investigate the association of HGFs with acute T cell-mediated renal rejection.
Related JoVE Video
Gadd45? is an inducible coactivator of transcription that facilitates rapid liver growth in mice.
J. Clin. Invest.
PUBLISHED: 05-24-2011
Show Abstract
Hide Abstract
The growth arrest and DNA damage-inducible 45 (Gadd45) proteins act in many cellular processes. In the liver, Gadd45b (encoding Gadd45?) is the gene most strongly induced early during both compensatory regeneration and drug-induced hyperplasia. The latter response is associated with the dramatic and rapid hepatocyte growth that follows administration of the xenobiotic TCPOBOP (1,4-bis[2-(3,5)-dichoropyridyloxy] benzene), a ligand of the nuclear receptor constitutive androstane receptor (CAR). Here, we have shown that Gadd45b-/- mice have intact proliferative responses following administration of a single dose of TCPOBOP, but marked growth delays. Moreover, early transcriptional stimulation of CAR target genes was weaker in Gadd45b-/- mice than in wild-type animals, and more genes were downregulated. Gadd45? was then found to have a direct role in transcription by physically binding to CAR, and TCPOBOP treatment caused both proteins to localize to a regulatory element for the CAR target gene cytochrome P450 2b10 (Cyp2b10). Further analysis defined separate Gadd45? domains that mediated binding to CAR and transcriptional activation. Although baseline hepatic expression of Gadd45b was broadly comparable to that of other coactivators, its 140-fold stimulation by TCPOBOP was striking and unique. The induction of Gadd45? is therefore a response that facilitates increased transcription, allowing rapid expansion of liver mass for protection against xenobiotic insults.
Related JoVE Video
Effects of long-term low-dose formaldehyde exposure on global genomic hypomethylation in 16HBE cells.
Toxicol. Lett.
PUBLISHED: 04-26-2011
Show Abstract
Hide Abstract
Formaldehyde (FA), a volatile organic compound, is a ubiquitous air pollutant that is classified as Carcinogenic to humans (Group 1) by IARC (2006). As a well-recognized human carcinogen, its carcinogenic mechanisms are still poorly understood. Previous studies have emphasized on genetic changes. However, little is known about the epigenetic mechanisms of FA exposure. In this study, We not only characterized the epigenomic response to long-term low-dose FA exposure in 16HBE cells, but also examined the expression of DNA methyltransferases (DNMTs) and the methyl-CpG-binding protein DNA-binding domain protein 2 (MBD2). Each week the 16HBE cells were treated with 10 ?M FA for 24 h (h). After 24 weeks (W) of exposure to FA, the level of genomic DNA methylation gradually decreased in a time-related manner. Moreover, our results showed that FA exposure down-regulated the expression of DNMT3a and DNMT3b at both mRNA and protein level, and up-regulated the levels of DNMT1 and MBD2 at both mRNA and protein level. Our study indicated that long-term FA exposure could disrupt genomic DNA methylation, which may be one of the possible underlying carcinogenic mechanisms of FA.
Related JoVE Video
Foramen magnum arachnoid cyst induces compression of the spinal cord and syringomyelia: case report and literature review.
Int J Med Sci
PUBLISHED: 04-21-2011
Show Abstract
Hide Abstract
It is very rare that a foramen magnum arachnoid cyst induces compression of the spinal cord and syringomyelia, and currently there are few treatment experiences available. Here we reported the case of a 43-year-old male patient who admitted to the hospital due to weakness and numbness of all 4 limbs, with difficulty in urination and bowel movement. MRI revealed a foramen magnum arachnoid cyst with associated syringomyelia. Posterior fossa decompression and arachnoid cyst excision were performed. Decompression was fully undertaken during surgery; however, only the posterior wall of the arachnoid cyst was excised, because it was almost impossible to remove the whole arachnoid cyst due to toughness of the cyst and tight adhesion to the spinal cord. Three months after the surgery, MRI showed a reduction in the size of the arachnoid cyst but syrinx still remained. Despite this, the symptoms of the patient were obviously improved compared to before surgery. Thus, for the treatment of foramen magnum arachnoid cyst with compression of the spinal cord and syringomyelia, if the arachnoid cyst could not be completely excised, excision should be performed as much as possible with complete decompression of the posterior fossa, which could result in a satisfying outcome.
Related JoVE Video
Levels and profiles of PCDD/Fs, PCBs in mothers milk in Shenzhen of China: estimation of breast-fed infants intakes.
Environ Int
PUBLISHED: 03-06-2011
Show Abstract
Hide Abstract
Sixty breast milk samples were collected in Shenzhen, China from July to November in 2007. The samples were analyzed of the concentrations of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and polychlorinated biphenyls (PCBs). The range of upper-bound for ?TEQ-(PCDD/Fs+PCBs) in the samples was 4.10-35.3 pg TEQ g(-1) lipid (median: 10.6 pg TEQ g(-1) lipid; mean: 11.9 pg TEQ g(-1) lipid). The levels of the measured contaminants in the breast milk had significant correlations with the length of inhabitation period in Shenzhen (r=0.487, p<0.05 for PCDD/Fs, r=0.431, p<0.05 for PCBs and r=0.478, p<0.05 for ?TEQ-(PCDD/Fs+PCBs)), and the consumption rate of fish (r=0.366, p<0.05 for PCDD/Fs, r=0.486, p<0.05 for PCBs and r=0.416, p<0.05 for ?TEQ-(PCDD/Fs+PCBs)), respectively. Moreover, significant positive correlations were also detected between the participants age (r=0.305, p<0.05 for ?TEQ-PCBs and r=0.275, p<0.05 for ?TEQ-(PCDD/Fs+PCBs)) and the body burdens of these contaminants respectively. It is estimated that the daily intake (EDI) of the sum of PCDD/Fs and DL-PCBs by the breast-fed infants was 5.60-161 pg TEQ kg(-1) bw per day (mean: 48.2 pg TEQ kg(-1) bw per day; median: 42.2 pg TEQ kg(-1) bw per day). The result showed that both the body burdens of PCDD/Fs and PCBs of the recruit population and the calculated EDI of the breast-fed infants were higher than those in the non-exposed areas in mainland China. This suggests that continuous surveillance on PCDD/Fs and PCBs levels in human milk is critical to more precisely evaluate the human health risk posed by the negative environmental impact in Shenzhen in the future.
Related JoVE Video
Cystic cavernous malformation of the cerebellopontine angle: case report and literature review.
World J Surg Oncol
PUBLISHED: 01-13-2011
Show Abstract
Hide Abstract
Cavernous malformations (CMs) in the cerebellopontine angle (CPA) are rare, and most of such CMs reported to date are solid and extend from the internal auditory canal into the CPA. In contrast, cystic CMs that arise in the CPA and do not involve the internal auditory canal and dura of the skull base are extremely rare.
Related JoVE Video
Study of cytoskeletal changes induced by okadaic acid in HL-7702 liver cells and development of a fluorimetric microplate assay for detecting diarrhetic shellfish poisoning.
Environ. Toxicol.
PUBLISHED: 01-09-2011
Show Abstract
Hide Abstract
Diarrhetic shellfish poisoning (DSP) is a gastrointestinal illness with symptoms such as diarrhea, nausea, vomiting, headache, chills and moderate to severe abdominal pain. DSP has been recognized as a worldwide public health problem, causing great concern to the shellfish industry. Accumulation of DSP in shellfish is an unpredictable phenomenon that necessitates the implementation of a widespread collection and thorough monitoring program for mollusk toxicity. Therefore, development of accurate analytical protocols for the rapid determination of toxicity levels would be necessary. In this study we investigated cytoskeletal changes induced by okadaic acid in HL-7702 Liver Cells and developed a new cytotoxicity assay for detection and quantitation of DSP. This assay is based on fluorometric of F-actin depolymerization induced by okadaic acid (OA) compounds in HL-7702 liver cell line. The measurable range of OA was 2.5 ? 40 nmol/L. The detection limit of the F-actin assay for OA was 2.01 ?g/100 g muscles in shellfish extracts. The performance of this assay has been evaluated by comparative analysis of shellfish samples by the fluorescent assay, mouse bioassay, and ELISA assay. Comparison of the results by all three methods revealed excellent consistency, the results of fluorescent assay were in significant correlation with ELISA assay (R(2) = 0.830). Examination of F-actin assay is very convenient, rapid, and sensitive, which can be used to quantify the amount of OA in shellfish samples.
Related JoVE Video
[Expression changes of glutathione transferase omega 1 induced by low dose trichloroethylene].
Wei Sheng Yan Jiu
PUBLISHED: 11-10-2010
Show Abstract
Hide Abstract
To determine the expression of glutathione transferase omega 1 (GSTO-1) during hormesis induced by low dose trichloroethylene (TCE).
Related JoVE Video
[Effects of 2,2,4,4-tetrabrominated diphenyl ethers on the TRalpha1 and TRbeta1 expression in liver in C57BL/6 mice].
Wei Sheng Yan Jiu
PUBLISHED: 08-24-2010
Show Abstract
Hide Abstract
To study the effect of 2,2,4,4-tetrabrominated diphenyl ethers (BDE-47) on the expression change of TRalpha1, TRbeta1 at both mRNA and protein levels.
Related JoVE Video
NADP+ reduction with reduced ferredoxin and NADP+ reduction with NADH are coupled via an electron-bifurcating enzyme complex in Clostridium kluyveri.
J. Bacteriol.
PUBLISHED: 07-30-2010
Show Abstract
Hide Abstract
It was recently found that the cytoplasmic butyryl-coenzyme A (butyryl-CoA) dehydrogenase-EtfAB complex from Clostridium kluyveri couples the exergonic reduction of crotonyl-CoA to butyryl-CoA with NADH and the endergonic reduction of ferredoxin with NADH via flavin-based electron bifurcation. We report here on a second cytoplasmic enzyme complex in C. kluyveri capable of energetic coupling via this novel mechanism. It was found that the purified iron-sulfur flavoprotein complex NfnAB couples the exergonic reduction of NADP+ with reduced ferredoxin (Fdred) and the endergonic reduction of NADP+ with NADH in a reversible reaction: Fdred2-+NADH+2 NADP++H+=Fdox+NAD++2 NADPH. The role of this energy-converting enzyme complex in the ethanol-acetate fermentation of C. kluyveri is discussed.
Related JoVE Video
Reoperation as a result of raised intracranial pressure associated with cyst formation in tumor cavity after intracranial tumor resection: a report of two cases.
Case Rep Med
PUBLISHED: 05-02-2010
Show Abstract
Hide Abstract
Reoperation as a result of increased intracranial pressure (ICP) associated with cyst formation in an intracranial tumor resection cavity is a rare clinical condition. We report two cases of reoperation as a result of raised ICP associated with cyst formation in the tumor resection cavity, one arising after glioma resection and the other after meningioma resection. In both cases, a "valve"-like structure was noted intraoperatively in the roof region of the tumor resection cavity. Surgical resection of the "valve"-like structure led to slow regression over several months after the reoperation rather than to immediate disappearance of the cyst. Both cases illustrate that the "valve"-like structure formed in the roof region of the tumor resection cavity may be responsible for cyst formation. Surgical resection of it provides good long-term outcomes in such patients though short-term outcomes are unsatisfactory; we speculate that if the resection of the cortical tissue around the "valve"-like structure is enough wide, its return may be avoided.
Related JoVE Video
Elevated body burdens of PBDEs, dioxins, and PCBs on thyroid hormone homeostasis at an electronic waste recycling site in China.
Environ. Sci. Technol.
PUBLISHED: 04-23-2010
Show Abstract
Hide Abstract
A cross-sectional study of 25 sample sets (each set consisted of maternal serum and cord whole blood) from 50 pregnant women in zone A (n = 25 from exposed group) and zone B (n = 25 from reference group) was conducted to examine the association between thyroid hormone (TH) levels and PBDE, PCDD/F, and PCB exposures. Thyroid hormones TT3, TT4, and TSH levels were measured in maternal serum at 16 weeks of gestation. The concentrations of PBDEs, PCDD/Fs, and PCBs were determined by isotope dilution HRGC/HRMS in cord blood samples. Body burdens of the three contaminants in cord blood in zone A (median: summation sigma TEQ-PCDD/Fs 0.041, summation operator TEQ-PCBs 0.022 pg WHO-TEQ/g, summation operator PBDEs 23.4 pg/g whole weight, respectively) were significantly higher than those from the reference area (median: summation sigma TEQ-PCDD/Fs 0.014, summation sigma TEQ-PCBs 0.0041 pg WHO-TEQ/g, summation sigma PBDEs 16.15 pg/g, respectively) (p < 0.05). Levels of TT4 and TSH in serum in zone A were significantly lower than those in zone B (p < 0.05). A negative correlation was found between TT4 levels and body burdens of PCDD/Fs and PCBs. However, there was no significant association of concentration of PBDEs and levels of the three thyroid hormones. Our results suggest that electronic waste (e-waste) recycling contributes to high body burdens of PBDEs, PCDD/Fs, and PCBs and affects thyroid hormone homeostasis in humans. The potential health risk for neonates still needs further investigation.
Related JoVE Video
Bayesian approach to transforming public gene expression repositories into disease diagnosis databases.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 04-01-2010
Show Abstract
Hide Abstract
The rapid accumulation of gene expression data has offered unprecedented opportunities to study human diseases. The National Center for Biotechnology Information Gene Expression Omnibus is currently the largest database that systematically documents the genome-wide molecular basis of diseases. However, thus far, this resource has been far from fully utilized. This paper describes the first study to transform public gene expression repositories into an automated disease diagnosis database. Particularly, we have developed a systematic framework, including a two-stage Bayesian learning approach, to achieve the diagnosis of one or multiple diseases for a query expression profile along a hierarchical disease taxonomy. Our approach, including standardizing cross-platform gene expression data and heterogeneous disease annotations, allows analyzing both sources of information in a unified probabilistic system. A high level of overall diagnostic accuracy was shown by cross validation. It was also demonstrated that the power of our method can increase significantly with the continued growth of public gene expression repositories. Finally, we showed how our disease diagnosis system can be used to characterize complex phenotypes and to construct a disease-drug connectivity map.
Related JoVE Video
Cloning and comparative analyses of the zebrafish Ugt repertoire reveal its evolutionary diversity.
PLoS ONE
PUBLISHED: 01-24-2010
Show Abstract
Hide Abstract
UDP-glucuronosyltransferases (Ugts) are a supergene family of phase II drug-metabolizing enzymes that catalyze the conjugation of numerous hydrophobic small molecules with the UDP-glucuronic acid, converting them into hydrophilic molecules. Here, we report the identification and cloning of the complete zebrafish Ugt gene repertoire. We found that the zebrafish genome contains 45 Ugt genes that can be divided into three families: Ugt1, Ugt2, and Ugt5. Both Ugt1 and Ugt2 have two unlinked clusters: a and b. The Ugt1a, Ugt1b, Ugt2a, and Ugt2b clusters each contain variable and constant regions, similar to that of the protocadherin (Pcdh), immunoglobulin (Ig), and T-cell receptor (Tcr) clusters. Cloning the full-length coding sequences confirmed that each of the variable exons is separately spliced to the set of constant exons within each zebrafish Ugt cluster. Comparative analyses showed that both a and b clusters of the zebrafish Ugt1 and Ugt2 genes have orthologs in other teleosts, suggesting that they may be resulted from the "fish-specific" whole-genome duplication event. The Ugt5 genes are a novel family of Ugt genes that exist in teleosts and amphibians. Their entire open reading frames are encoded by single large exons. The zebrafish Ugt1, Ugt2, and Ugt5 genes can generate additional transcript diversity through alternative splicing. Based on phylogenetic analyses, we propose that the ancestral tetrapod and teleost Ugt1 clusters contained multiple Ugt1 paralogs. After speciation, these ancestral Ugt1 clusters underwent lineage-specific gene loss and duplication. The ancestral vertebrate Ugt2 cluster also underwent lineage-specific duplication. The intronless Ugt5 open reading frames may be derived from retrotransposition followed by gene duplication. They have been expanded dramatically in teleosts and have become the most abundant Ugt family in these lineages. These findings have interesting implications regarding the molecular evolution of genes with diversified variable exons in vertebrates.
Related JoVE Video
SiO2 nanoparticles induce cytotoxicity and protein expression alteration in HaCaT cells.
Part Fibre Toxicol
PUBLISHED: 01-19-2010
Show Abstract
Hide Abstract
Nanometer silicon dioxide (nano-SiO2) has a wide variety of applications in material sciences, engineering and medicine; however, the potential cell biological and proteomic effects of nano-SiO2 exposure and the toxic mechanisms remain far from clear.
Related JoVE Video
[Recombinant human gapM1 expressed in Pichia pastoris and its anti-diabetic effect].
Sheng Wu Gong Cheng Xue Bao
PUBLISHED: 11-27-2009
Show Abstract
Hide Abstract
Adiponectin is an adipokine predominantly synthesized and secreted by adipocytes in the white adipose tissue, and it can lower the blood glucose level and increase free fatty acid oxidation. In the current study, we developed the globular domain of adiponectin (gapM1) to treat type II diabetes. In both flask and fermentor, we cultivated Pichia pastoris expressing recombinant gapM1 and established the purification procedure by using gel filtration and anion exchange chromatography. To evaluate the biological activity of recombinant gapM1, we used rat type II diabetes model fed high-fat diet in combination with low-dose STZ (Streptozocin) induction. We purified 200 mg gapM1 with purity of 96% from 10 liters of supernatant. The recombinant gapM1 significantly lowered blood glucose (34.2%), serum triglyceride (79.6%) and total cholesterol (62.1%) in type II diabetes induced rat. Therefore, the recombinant human gapM1 is successfully expressed in Pichia pastoris and effectively treated type II diabetes in rat models.
Related JoVE Video
[Effects and toxicity mechanism of 2,2,4,4-tetrabrominated diphenyl ethers on the homeostasis of thyroid hormone system in C57BL/6 mice].
Wei Sheng Yan Jiu
PUBLISHED: 11-03-2009
Show Abstract
Hide Abstract
To investigate the effects and of toxicity mechanism of 2,2,4,4-TETRABDE (BDE-47) on TT3, TT4 and TSH in C57BL/6 mice.
Related JoVE Video
An overview of recent developments in genomics and associated statistical methods.
Philos Trans A Math Phys Eng Sci
PUBLISHED: 10-07-2009
Show Abstract
Hide Abstract
The landscape of genomics has changed drastically in the last two decades. Increasingly inexpensive sequencing has shifted the primary focus from the acquisition of biological sequences to the study of biological function. Assays have been developed to study many intricacies of biological systems, and publicly available databases have given rise to integrative analyses that combine information from many sources to draw complex conclusions. Such research was the focus of the recent workshop at the Isaac Newton Institute, High dimensional statistics in biology. Many computational methods from modern genomics and related disciplines were presented and discussed. Using, as much as possible, the material from these talks, we give an overview of modern genomics: from the essential assays that make data-generation possible, to the statistical methods that yield meaningful inference. We point to current analytical challenges, where novel methods, or novel applications of extant methods, are presently needed.
Related JoVE Video
The maize root stem cell niche: a partnership between two sister cell populations.
Planta
PUBLISHED: 08-21-2009
Show Abstract
Hide Abstract
Using transcript profile analysis, we explored the nature of the stem cell niche in roots of maize (Zea mays). Toward assessing a role for specific genes in the establishment and maintenance of the niche, we perturbed the niche and simultaneously monitored the spatial expression patterns of genes hypothesized as essential. Our results allow us to quantify and localize gene activities to specific portions of the niche: to the quiescent center (QC) or the proximal meristem (PM), or to both. The data point to molecular, biochemical and physiological processes associated with the specification and maintenance of the niche, and include reduced expression of metabolism-, redox- and certain cell cycle-associated transcripts in the QC, enrichment of auxin-associated transcripts within the entire niche, controls for the state of differentiation of QC cells, a role for cytokinins specifically in the PM portion of the niche, processes (repair machinery) for maintaining DNA integrity and a role for gene silencing in niche stabilization. To provide additional support for the hypothesized roles of the above-mentioned and other transcripts in niche specification, we overexpressed, in Arabidopsis, homologs of representative genes (eight) identified as highly enriched or reduced in the maize root QC. We conclude that the coordinated changes in expression of auxin-, redox-, cell cycle- and metabolism-associated genes suggest the linkage of gene networks at the level of transcription, thereby providing additional insights into events likely associated with root stem cell niche establishment and maintenance.
Related JoVE Video
BMP signaling pathway is required for commitment of C3H10T1/2 pluripotent stem cells to the adipocyte lineage.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 07-20-2009
Show Abstract
Hide Abstract
Obesity is accompanied by an increase in both adipocyte number and size. The increase in adipocyte number is the result of recruitment to the adipocyte lineage of pluripotent stem cells present in the vascular stroma of adipose tissue. These pluripotent cells have the potential to undergo commitment and then differentiate into adipocytes, as well as myocytes, osteocytes, and chondrocytes. In this article, we show that both bone morphogenetic protein (BMP)2 and BMP4 can induce commitment of C3H10T1/2 pluripotent stem cells into adipocytes. After treatment of C3H10T1/2 stem cells with these BMPs during proliferation followed by exposure to differentiation inducers at growth arrest, nearly all cells enter the adipose development pathway, express specific adipocyte markers, and acquire the adipocyte phenotype. Overexpression of constitutively active BMP receptor (CA)-BMPr1A or CA-BMPr1B induces commitment in the absence of BMP2/4, whereas overexpression of a dominant-negative receptor dominant-negative-BMPr1A suppresses commitment induced by BMP. Also, knockdown of the expression of Smad4 (coregulator in the BMP/Smad signaling pathway) with RNAi disrupts commitment by the BMPs. However, knockdown of expression of p38 MAPK (an intermediary in the BMP/MAPK signaling pathway) with RNAi had little effect on BMP-induced commitment. Together, these findings indicate that the BMP/Smad signaling pathway has a dominant role in adipocyte lineage determination. Proteomic analysis identified lysyl oxidase (LOX), a bona fide downstream target gene of the BMP signaling pathway. Expression of LOX is induced by BMP2/4 during adipocyte lineage commitment, and knockdown of its expression disrupts the commitment process.
Related JoVE Video
O-linked N-acetylglucosamine modification on CCAAT enhancer-binding protein beta: role during adipocyte differentiation.
J. Biol. Chem.
PUBLISHED: 05-28-2009
Show Abstract
Hide Abstract
CCAAT enhancer-binding protein (C/EBP)beta is a basic leucine zipper transcription factor family member, and can be phosphorylated, acetylated, and sumoylated. C/EBPbeta undergoes sequential phosphorylation during 3T3-L1 adipocyte differentiation. Phosphorylation on Thr(188) by MAPK or cyclin A/cdk2 primes the phosphorylations on Ser(184)/Thr(179) by GSK3beta, and these phosphorylations are required for the acquisition of DNA binding activity of C/EBPbeta. Here we show that C/EBPbeta is modified by O-GlcNAc, a dynamic single sugar modification found on nucleocytoplasmic proteins. The GlcNAcylation sites are Ser(180) and Ser(181), which are in the regulation domain and are very close to the phosphorylation sites (Thr(188), Ser(184), and Thr(179)) required for the gain of DNA binding activity. Both in vitro and ex vivo experiments demonstrate that GlcNAcylation on Ser(180) and Ser(181) prevents phosphorylation on Thr(188), Ser(184), and Thr(179), as indicated by the decreased relative phosphorylation and DNA binding activity of C/EBPbeta delayed the adipocyte differentiation program. Mutation of both Ser(180) and Ser(181) to Ala significantly increase the transcriptional activity of C/EBPbeta. These data suggest that GlcNAcylation regulates both the phosphorylation and DNA binding activity of C/EBPbeta.
Related JoVE Video
Neuregulin 1 genetic variation and anterior cingulum integrity in patients with schizophrenia and healthy controls.
J Psychiatry Neurosci
PUBLISHED: 05-19-2009
Show Abstract
Hide Abstract
Neuregulin1 (NRG1) influences the development of white matter connectivity and is implicated in genetic susceptibility to schizophrenia. The cingulum bundle is a white matter structure implicated in schizophrenia. Its anterior component is especially implicated, as it provides reciprocal connections between brain regions with prominent involvement in the disorder. Abnormalities in the structural integrity of the anterior cingulum in patients with schizophrenia have been reported previously. The present study investigated the potential contribution of NRG1 variation to anterior cingulum abnormalities in participants with schizophrenia.
Related JoVE Video
Severe hypersensitivity dermatitis and liver dysfunction induced by occupational exposure to trichloroethylene.
Ind Health
PUBLISHED: 04-16-2009
Show Abstract
Hide Abstract
Trichloroethylene-induced hypersensitivity dermatitis is one of the serious occupational health events in China, however, little is known about the clinical features and possible mechanism of this disorder. The objective of the present study was to report some typical trichloroethylene-induced dermatitis patients and investigate their occupational exposure as well as the clinical features. We sampled and tested some cleaning agents from the companies where TCE-induced skin disorder occurred, the trichloroethylene concentrations were also monitored in the workplace air. Additionally, the symptoms, signs and laboratory test results of patients were collected. TCE concentrations varied from 10.2% to 91.4% in the cleaning agent by gas chromatography-mass chromatography analysis, and TCE levels in the workplace air ranged between 18 mg/m(3) and 683 mg/m(3), at most sampled sites TCE levels were higher than China national health standard for TCE. The trichloroethylene exposure time of the patients was 5-90 days (average 38.2 d), the patients with headache, dizziness, skin itch, fever were 90.5%, 100%, 100%, and 61.9%, respectively. 85.7% patients had skin erythema, 90.5% with rashes, and 38.1% with blisters. In addition, liver enlargement occurred in 3 patients, the abnormal rate of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (T-Bil) were 90.5%, 85.7% and 76.2%, respectively. 6 out of 15 patients were with abnormal electrocardiogram, and trichloroacetic acid (TCA) elevated in 14 patients (66.7%). Taken together, the major detrimental effect of trichloroethylene was to induce hypersensitivity dermatitis and liver dysfunction, the occurrence of this disorder is likely related to the individual hypersensitivity to trichloroethylene exposure.
Related JoVE Video
Identification of antigenic proteins associated with trichloroethylene-induced autoimmune disease by serological proteome analysis.
Toxicol. Appl. Pharmacol.
PUBLISHED: 04-01-2009
Show Abstract
Hide Abstract
Although many studies indicated that trichloroethylene (TCE) could induce autoimmune diseases and some protein adducts were detected, the proteins were not identified and mechanisms remain unknown. To screen and identify autoantigens which might be involved in TCE-induced autoimmune diseases, three groups of sera were collected from healthy donors (I), patients suffering from TCE-induced exfoliative dermatitis (ED) (II), and the healed ones (III). Serological proteome analysis (SERPA) was performed with total proteins of TCE-treated L-02 liver cells as antigen sources and immunoglobins of the above sera as probes. Highly immunogenic spots (2-fold or above increase compared with group I) in group II and III were submitted to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and tandem mass spectrometry sequencing. Western blot analysis was followed using commercial antibodies and individual serum. Six proteins were identified. Among them, Enoyl Coenzyme A hydratase peroxisoma 1 and lactate dehydrogenase B only showed stronger immunogenicity for group II sera, while Purine nucleoside phosphorylase, ribosomal protein P0 and proteasome activator subunit1 isoform1 also showed stronger immunogenicity for group III sera. Noteworthy, NM23 reacted only with group II sera. Western blot analysis of NM23 expression indicated that all of the individual serum of group II showed immune activity, which confirmed the validity of SERPA result. These findings revealed that there exist autoantibodies in group II and III sera. Besides, autoantibodies of the two stages of disease course were different. These autoantigens might serve as biomarkers to elucidate mechanisms underlying TCE toxicity and are helpful for diagnosis, therapy and prognosis of TCE-induced autoimmune diseases.
Related JoVE Video
Effect of anluohuaxian tablet combined with gamma-IFN on schistosomal liver fibrosis.
J. Huazhong Univ. Sci. Technol. Med. Sci.
PUBLISHED: 02-18-2009
Show Abstract
Hide Abstract
The therapeutic effects of anluohuaxian tablet combined with gamma-IFN on schistosomal liver fibrosis and its mechanism were studied in a murine model and clinical cases of schistosomal liver fibrosis. Fifty Kunming mice were randomly divided into 5 groups: normal control group, infection control group, anluohuaxian tablet-treated group, gamma-IFN-treated group and combined treatment (anluohuaian tablet+gamma-IFN) group. Pathologic changes in liver, including hepatic pigmentation and the size of schistosomal egg granuloma, were observed by HE staining after treatment for 8 weeks. The expression of the type I and collagen III, and TIMP-1 was detected by immunohistochemistry. TGF-beta1 mRNA expression was examined by real-time fluorescent quantitative PCR. Sixty patients with schistosomal liver fibrosis were divided into treatment group and control group. The patients in treatment group were treated with anluohuaxian tablet in combination with gamma-IFN for 6 months. Before and after treatment, the changes of symptoms and signs, liver function, serum liver fibrosis indexes and imaging indexes were observed. The results showed that as compared with infection control group, all forms of treatments relieved the hepatic pathological injury with apparently diminished size of schistosomal egg nodules and decreased percentage of pigmentation (P<0.05). Furthermore, the expression of collagen I and III, TIMP-1, and TGF-beta1 mRNA in combined treatment group was significantly decreased as compared with anluohuaxian tablet-treated and gamma-IFN-treated groups (P<0.05). In the clinical observation, the serum liver fibrosis indexes, the portal vein width as well as the spleen thickness was significantly reduced in treatment group as compared with control group (P<0.05). It was concluded that the combined use of anluohuaxian tablet with gamma-IFN in schistosomal liver fibrosis could protect liver function, alleviate liver fibrosis, and could be used as a choice in treating patients with schiatosomal liver fibrosis.
Related JoVE Video
Preparation and characterization of murine monoclonal antibodies against rat spinal cord injury and regeneration related protein no. 69.
Hybridoma (Larchmt)
PUBLISHED: 02-12-2009
Show Abstract
Hide Abstract
Spinal cord injury and regeneration related protein No. 69 (SCIRR69) is a rat transmembrane bZIP transcription factor homologous to mice and human transcription factor CREB3L2. Previous work demonstrated the N-terminal region plays a critical role in its transcriptional activity. In this study, a peptide containing 18 amino acids (5-22aa) at the N-terminus of rat SCIRR69 was synthesized and coupled to the carrier protein as immunogen. One hybridoma cell line was obtained by standard cell fusion technique, followed by enzyme-linked immunosorbent assay (ELISA) and Western blot screening. The newly developed monoclonal antibody (MAb) was designated 4B4, the isotype of which was IgG2a. Immunofluorescence and Western blotting results showed that MAb 4B4 could recognize the SCIRR69 protein in both native and denatured forms. 4B4 will be a useful tool for the functional research of SCIRR69 in future studies.
Related JoVE Video
Integrative disease classification based on cross-platform microarray data.
BMC Bioinformatics
PUBLISHED: 01-30-2009
Show Abstract
Hide Abstract
Disease classification has been an important application of microarray technology. However, most microarray-based classifiers can only handle data generated within the same study, since microarray data generated by different laboratories or with different platforms can not be compared directly due to systematic variations. This issue has severely limited the practical use of microarray-based disease classification.
Related JoVE Video
[A fluorimetric microplate assay for detecting diarrheic shellfish poisoning toxins].
Wei Sheng Yan Jiu
Show Abstract
Hide Abstract
To establish a new assay for detecting the cytotoxicity of diarrheic shellfish poisoning (DSP) toxins. The assay is based on the depolymerization of F-actin induced by okadaic acid (OA), which is one of main components of DSP toxins.
Related JoVE Video
Down-regulation of Pol? expression leads to increased DNA damage, apoptosis and enhanced S phase arrest in L-02 cells exposed to hydroquinone.
Toxicol. Lett.
Show Abstract
Hide Abstract
DNA polymerase eta (Pol?), the product of the xeroderma pigmentosum variant gene, is required for translesion DNA synthesis, and plays a pivotal role in preventing genome instability after DNA damage induced by genotoxic agents. Studies have previously suggested a link between Pol? and susceptibility to hydroquinone (HQ)-induced toxicity. To further address the role of Pol? in the response of L-02 cells to HQ, we employed RNA interference to silence Pol? expression in L-02 cells and examined the susceptibility of these Pol?-deficient cells to the toxic effects of HQ. In this study, cell survival rate was determined using the MTT assay, DNA damage was determined by the Comet assay, apoptosis and cell cycle distribution were determined using flow cytometry, the mRNA expression levels of Pol? were determined by real-time PCR, and the protein expression levels of Pol? and ?-H2AX were determined by Western blot, ?-H2AX foci were visualized by confocal laser scanning fluorescence microscopy after cells were exposed to HQ at various concentrations for 24h in vitro. The results showed that stable Pol?-knockdown cells were successfully constructed and more than 80% inhibition of Pol? expression was confirmed. The results also showed that down-regulation of Pol? led to a decrease in cell proliferation and an enhanced susceptibility to HQ-induced cytotoxicity. Pol?-deficient cells were 2-fold more sensitive to HQ when compared with nonspecific siRNA control cells. Moreover, Pol?-silenced L-02 cells treated with HQ displayed an increased level of DNA double-strand breaks as measured by olive tail moment, and an elevated DNA damage response as indicated by the induction of ?-H2AX. In addition, knockdown of Pol? resulted in more enhanced apoptosis and more pronounced S phase arrest following HQ treatment. Together, these results show that Pol? plays an important role in the response of L-02 cells to HQ-induced DNA damage.
Related JoVE Video

What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.