Use of Arctium lappa Extract Against Acetaminophen-Induced Hepatotoxicity in Rats

AbstractBackgroundSevere destructive hepatic injuries can be induced by acetaminophen overdose and may lead to acute hepatic failure.ObjectiveTo investigate the ameliorative effects of Arctium lappa root extract on acetaminophen-induced hepatotoxicity.MethodsRats were divided into 4 groups: normal control group, Arctium lappa extract group, acetaminophen-injected group, and acetaminophen treated with Arctium lappa extract group.ResultsThe treatment with Arctium lappa extract reduced serum alanine transaminase, aspartate aminotransferase, and alkaline phosphatase in the acetaminophen group when compared with the control group. DNA fragments in the acetaminophen-injected group were also significantly increased (P < 0.05). The comet assay revealed increased detaching tail length and DNA concentration during the hepatic toxicity in the acetaminophen group. The malondialdehyde content was inhibited by Arctium lappa treatment (12.97±0.89 nmol/mg) when compared with the acetaminophen-treated-only group (12.97±0.89 nmol/mg). Histopathologic examination revealed that acetaminophen administration produced hepatic cell necrosis, infiltrate of lymphocytes, and vacuolation that were associated with the acetaminophen-treated animal group, but the degree of acetaminophen-induced hepatotoxicity was mediated by treatment with Arctium lappa extract.ConclusionsArctium lappa can prevent most of the hepatic tissue damage caused by acetaminophen overdose in rats

Genome-wide studies of mRNA synthesis and degradation in eukaryotes

In recent years, the use of genome-wide technologies has revolutionized the study of eukaryotic transcription producing results for thousands of genes at every step of mRNA life. The statistical analyses of the results for a single condition, different conditions, different transcription stages, or even between different techniques, is outlining a totally new landscape of the eukaryotic transcription process. Although most studies have been conducted in the yeast Saccharomyces cerevisiae as a model cell, others have also focused on higher eukaryotes, which can also be comparatively analyzed. The picture which emerges is that transcription is a more variable process than initially suspected, with large differences between genes at each stage of the process, from initiation to mRNA degradation, but with striking similarities for functionally related genes, indicating that all steps are coordinately regulated. This article is part of a Special Issue entitled: Nuclear Transport and RNA Processing

Transcriptional run-on: Measuring nascent transcription at specific genomic sites in yeast

DNA transcription by RNA polymerases has always interested the scientific community as it is one of the most important processes involved in genome expression. This has led scientists to come up with different protocols allowing analysis of this process in specific locations across the genome by quantitating the amount of RNA polymerases transcribing that genomic site in a cell population. This can be achieved by either detecting the total number of polymerases in contact with that region (i.e., by chromatin immunoprecipitation (ChIP) with anti-RNA polymerase antibodies) or by measuring the number of polymerases that are effectively engaged in transcription in that position. This latter strategy is followed using transcription run-on (TRO), also known as nuclear run-on (NRO), which was first developed in mammalian cells over 40 years ago and has since been adapted to many other different organisms and high-throughput methods. Here, we detail the procedure for performing TRO in Saccharomyces cerevisiae for single genomic regions to study active transcription on a single gene scale. To do so, we wash the cells in the detergent sarkosyl, which prevents new initiations at the promoter level, and then perform an in situ reaction, leading to the radiolabeling of transcripts by RNA polymerases that were already engaged in transcription at the moment of harvesting. By subsequently quantitating the signal of these transcripts, we can determine the level of active transcription in a single gene. This presents a major advantage over other forms of transcription quantitation such as RNA polymerase ChIP, since in the latter, both active and inactive polymerases are measured. By combining both ChIP and TRO, the amount of inactive or paused polymerases on a particular gene can be estimated

The Prefolding Complex Regulates Chromatin Dynamics during Transcriptional Elongation

Trabajo presentado en la 26th International Conference on Yeast Genetics and Molecular Biology, celebrada en Frankfurt (Alemania) del 29 de agosto al 3 de septiembre de 2013Peer Reviewe

The absence of Pfd1 causes alterations in the intragenic distribution of RNA polymerase II.

<p><b>A.</b> Comparative distribution of total RNA polymerase II (as measured by Rpb3 ChIP) and active transcription (as measured by run-on) across genes, expressed as 3′/5′ ratios. A large set of highly expressed genes were investigated in <i>pfd1Δ</i> and compared to the wild type, as described in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003776#s4" target="_blank">Materials and methods</a> section. Average results for the specific group of genes are shown according to their length (longer or shorter than 4 kbp) or promoter type (exhibiting a canonical TATA box or a TATA-like box). Any P-values of the Student's t-test lower than 0.05 are shown. Error bars indicate standard errors. <b>B.</b> Time courses reflecting the induction of the <i>GAL1</i> gene. Cells of the indicated isogenic strains were grown in glycerol-lactate medium for two generations and then 2% galactose was added to the cultures. RNA samples were then taken at the indicated times. A significant Northern experiment is shown. The quantification of three biological replicates is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003776#pgen.1003776.s006" target="_blank">Figure S6A</a>. <b>C.</b> The absence of Pfd1 alters the kinetics of RNA polymerase II occupancy during <i>GAL1</i> induction. RNA polymerase II occupancy was measured by anti-Rpb3 ChIP, after the addition of galactose to wild-type or <i>pfd1Δ</i> cells grown in glycerol-lactate medium, at the indicated times. Values correspond to the mean and the standard deviation of three biological replicates. <b>D.</b> The absence of prefoldin does not affect Ser5-phosphorylation of RNA polymerase II. The levels of Ser5-phosphorylated Rpb1 across <i>GAL1</i> were analyzed by ChIP in wild-type or <i>pfd1Δ</i> cells exponentially growing in galactose. Values were normalized to total RNA polymerase levels, as measured by anti-Rpb3 ChIP performed with the same extracts. Values correspond to the mean and the standard deviation of three biological replicates. <b>E.</b> The absence of prefoldin decreases Ser2-phosphorylation of RNA polymerase II. The levels of Ser2-phosphorylated Rpb1 across <i>GAL1</i> were analyzed and normalized as in D. Values correspond to the mean and the standard deviation of three biological replicates.</p

The absence of Pfd1 alters RNA polymerase II elongation across <i>GAL1</i>::YLR454w.

<p><b>A.</b> mRNA levels of GAL1p-YLR454w are more intensively affected than those of <i>GAL1</i> in <i>pfd1Δ</i>. GAL1p-YLR454w and <i>GAL1</i> mRNA levels were measured by Northern in cells growing exponentially in galactose, as described in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003776#s4" target="_blank">Materials and methods</a> section. A significant blot and the quantification of three biological replicas are shown. <b>B.</b> Distribution of the amplicons and probes utilized to measure RNA polymerase II occupancy across <i>GAL1p</i>-YLR454w. <b>C.</b> The profile of total RNA polymerase II along GAL1p-YLR454w is biased towards the 3′ end in <i>pfd1Δ</i>. Occupancy of RNA polymerase II was measured by anti-Rpb3 ChIP in the indicated isogenic strains. Each bar represents one of the amplicons, from 5′ (left) to 3′ (right), described in B. Values were normalized to the 5′ amplicon and to the wild type. The mean and the standard deviation of three biological replicates are shown. Non-normalized data are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003776#pgen.1003776.s006" target="_blank">Figure S6B</a>. <b>D.</b> The profile of active RNA polymerase II along GAL1p-YLR454w is biased towards the 3′ end in <i>pfd1Δ</i>. Distribution of transcriptionally active RNA polymerase II along the GAL1p-YLR454w transcription unit was measured by transcriptional run-on. Cells of the indicated isogenic strains, exponentially growing in YPGAL, were tested for the presence of transcriptionally active RNA polymerases by transcriptional run-on, as it is described in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003776#s4" target="_blank">Materials and methods</a> section. Each bar represents one of the amplicons, from 5′ (left) to 3′ (right), described in B. Values were normalized to the first probe and to the wild type. The mean and the standard deviation of three biological replicates are shown. Non-normalized data are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003776#pgen.1003776.s006" target="_blank">Figure S6C</a>. <b>E.</b> The elongation rate of RNA polymerase II in GAL1p-YLR454w is affected by the absence of Pfd1. RNA polymerase II distribution is shown, 2 min after adding 2% glucose to cells of the indicated isogenic strains that were exponentially growing in galactose medium. Each bar represents one of the amplicons, from 5′ (left) to 3′ (right), described in B. RNA polymerase II levels were measured by anti-Rpb3 ChIP. Values were normalized to time 0 and correspond to the mean and the standard deviation of three biological replicates. <b>F.</b> Relative levels of RNA polymerase II occupancy of the 4 kb region of GAL1p-YLR454w during the last wave of transcription, in the indicated isogenic strains. The plot represents the time course of the normalized values of RNA polymerase II occupancy, measured by anti-Rpb3 ChIP, upon the addition of glucose to cells growing in galactose medium.</p

Histone dynamics during transcription elongation is impaired in <i>pfd1Δ</i>.

<p><b>A.</b> The absence of Pfd1 impairs the characteristic difference in histone occupancy along the transcribed region that occurs between active and inactive genes. Binding of H3 and H4 to transcribed (YPGAL) and untranscribed (YPD) GAL1p-YLR454w was measured by ChIP in wild-type (left) and <i>pfd1Δ</i> (right) exponentially growing cells. Values correspond to the mean and the standard deviation of three biological replicates. <b>B.</b> Sensitivity of GAL1p-YLR454w chromatin to micrococcal nuclease, under activating (YPGAL) and non activating (YPD) conditions is not affected by the absence of Pfd1. Note that the characteristic up and down pattern caused by the positioned nucleosomes present in the 5′ and 3′ ends of the gene under repressed conditions is equally absent in the wild type and in <i>pfd1Δ</i>. Cells exponentially growing in the indicated media were permeabilized and treated with MNase, as described in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003776#s4" target="_blank">Materials and methods</a> section. Mononucleosomal DNA was then extracted and analyzed by quantitative PCR. Values were normalized to naked DNA digested with MNase and processed in parallel to the chromatin samples. Values correspond to the mean and the standard deviation of two biological replicates. <b>C.</b> The kinetics of the increase in histone occupancy that occurs after transcriptional repression is not influence by the absence of Pfd1. Histone occupancy in the 4 kb region of GAL1p-YLR454w was analyzed during the last wave of transcription after switching the <i>GAL1</i> promoter off. Glucose was added at time 0 to wild-type and <i>pfd1Δ</i> cells growing exponentially in galactose. At the indicated times samples were taken to measure H3 binding to the 4 kb region by ChIP. Values were normalized to the levels of H3 occupancy measured 60 min after the addition of glucose to the cultures. The mean and the standard deviation of three biological replicates are shown. <b>D and E.</b> The difference in H3 occupancy produced by <i>pfd1Δ</i> and <i>dst1Δ</i> across GAL1p-YLR454w does not correlate with their effect on RNA polymerase II occupancy. Ratios of H3 occupancy between YPGAL and YPD in the indicated mutants across GAL1p-YLR454w (D), and levels of RNA polymerase II bound to GAL1p-YLR454w (E). H3 and Rpb3 occupancy was measured by ChIP, as described in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003776#s4" target="_blank">Materials and methods</a> section. Values correspond to the mean and the standard deviation of three biological replicates.</p

Systematic genetic analysis of budding yeast <i>Saccharomyces cerevisiae</i> based on the GLAM assay.

<p>GLAM ratios were calculated for the mutants present in the collection of viable deletions, growing in microtiter plates, as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003776#s4" target="_blank">Materials and methods</a>. The distribution of the values obtained is represented in <b>A</b>. The deletions exhibiting GLAM ratios lower than 0.3 were significantly enriched in the indicated gene ontology category. Those low-scored mutants that maintained low GLAM ratios in exponentially growing cells are listed in <b>B</b>.</p

Recruitment of prefoldin to transcribed genes depends on the integrity of the complex and is favored by Ser2-phosphorylation of the Rpb1 CTD.

<p><b>A.</b> Pfd1 binding to <i>GAL1</i> partially depends on Gim5. Pfd1-Myc occupancy of <i>GAL1</i> was analyzed in wild-type or <i>gim5Δ</i> cells exponentially growing in galactose-containing medium. The anti-Myc ChIP procedure is described in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003776#s4" target="_blank">Materials and methods</a> section. Western blot experiments were performed in order to rule out any significant influence of the <i>gim5Δ</i> mutation on the Pfd1-Myc level. <b>B.</b> Gim5-Myc binding to <i>GAL1</i> does not depend on Pfd1. Gim5-Myc occupancy of <i>GAL1</i> was analyzed in wild-type or <i>pfd1Δ</i> cells exponentially growing in galactose-containing medium. Western blot experiments were performed in order to rule out any increase of Gim5-Myc level in the <i>pfd1Δ</i> mutant. <b>C.</b> The profile of Pfd1 binding to the transcribed region parallels Ser2-phosphorylation of the Rpb1 CTD. Occupancy of total (Rpb3), Ser5-phosphorylated (Se5-P) and Ser2-phosphorylated RNA polymerase II (Ser5-P) was analyzed across <i>GAL1</i> under activating conditions. ChIP experiments were performed using the conditions and antibodies described in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003776#s4" target="_blank">Materials and methods</a> section. <b>D.</b> Prefoldin recruitment to the transcribed region is diminished in a <i>ctk1Δ</i> mutant. Pfd1-Myc occupancy of <i>GAL1</i> was analyzed by ChIP in wild-type or <i>ctk1Δ</i> cells exponentially growing in galactose-containing medium. In all panels, the results shown represent the mean and the standard deviation of three biological replicates.</p

Prefoldin mutants exhibit transcriptional phenotypes and synthetic interactions with TFIIS.

<p><b>A. </b><i>pfd1Δ</i> was impaired in the accumulation of 4.5 kb long mRNAs (**), but behaved as the wild type when expressing a 1.5 kb mRNA (*) driven by the same promoter. A single short transcription units (1) was utilized, but two different long transcription units were tested in order to exclude sequence-specific defects: <i>GAL1pr-PHO5::lacZ</i> (2) and <i>GAL1pr-PHO5::LAC4</i> (3). A significant Northern experiment is shown on the left. The average ratios calculated dividing the signals of the long transcript by the signal of the short transcript are shown on the right. Values correspond to the mean and the standard deviation of three biological replicates. <b>B.</b> Four out of six prefoldin mutants, lacking the yeast prefoldin complex subunits, exhibited significantly lower GLAM ratios than the wild type. The GLAM ratios of double mutants were no lower than single ones. GLAM ratios were calculated dividing the acid phosphatase activity of cells expressing <i>GAL1pr-PHO5::lacZ</i> by the activity of cells of the same strain expressing <i>GAL1pr-PHO5</i>. Values correspond to the mean and the standard deviation of three biological replicates. <b>C.</b> The <i>pfd1Δ dst1Δ</i> double mutant exhibited hypersensitivity to 2.5 µg/ml mycophenolyc acid. Ten-fold serial dilutions were used for the drop assay. <b>D.</b> The <i>pfd1Δ dst1Δ</i> double mutant showed synergistically lower GLAM ratios, as compared to the corresponding single mutants. The GLAM ratios in B and D were calculated after measuring the acid phosphatase activity encoded in the GLAM transcription units, and were normalized to the wild-type value to facilitate comparisons. Values correspond to the mean and the standard deviation of three biological replicates.</p