FACT, a Factor that Facilitates Transcript Elongation through Nucleosomes

AbstractThe requirements for transcriptional activation by RNA polymerase II were examined using chromatin templates assembled in vitro and a transcription system composed of the human general transcription factors and RNA polymerase II. Activator-induced, energy-dependent chromatin remodeling promoted efficient preinitiation complex formation and transcription initiation, but was not sufficient for productive transcription. Polymerases that initiated transcription on remodeled chromatin templates encountered a block to transcription proximal to the promoter. Entry into productive transcription required an accessory factor present in HeLa cell nuclear extract, FACT (fa cilitates c hromatin t ranscription), which we have purified. FACT acts subsequent to transcription initiation to release RNA polymerase II from a nucleosome-induced block to productive transcription. The biochemical properties and polypeptide composition of FACT suggest that it is a novel protein factor that facilitates transcript elongation through nucleosomes

The Need to Decide If All Estrogens Are Intrinsically Similar

We used gene expression profiling to investigate whether the molecular effects induced by estrogens of different provenance are intrinsically similar. In this article we show that the physiologic estrogen 17β-estradiol, the phytoestrogen genistein, and the synthetic estrogen diethylstilbestrol alter the expression of the same 179 genes in the intact immature mouse uterus under conditions where each chemical has produced an equivalent gravimetric and histologic uterotrophic effect, using the standard 3-day assay protocol. Data are also presented indicating the limitations associated with comparison of gene expression profiles for different chemicals at times before the uterotrophic effects are fully realized. We conclude that the case has yet to be made for regarding synthetic estrogens as presenting a unique human hazard compared with phytoestrogens and physiologic estrogens

Antioksidacijski i antimikrobni učinak ekstrakata tršlje (Pistacia lentiscus L.) u kobasicama od svinjskog mesa

Pistacia lentiscus fruits are ingredients of traditional Cypriot sausages. The objective of this study is to evaluate P. lentiscus extracts as natural additives to the sausages. First, the phenolic content and antioxidant activity of fruit and leaf extracts were determined. Results revealed that leaves are richer source of polyphenolic antioxidants than fruits, with methanol being the better extraction solvent. In the next step, the antioxidant and antimicrobial effects of methanolic extracts (300 mg/kg) in the pork sausage formulation were investigated. Peroxide, acid and thiobarbituric acid-reactive substance values demonstrated that both fruit and leaf extracts reduced the rate of lipid oxidation of sausages at 4 °C. Total viable count revealed significant differences on the fifth day of storage, with better microbial inhibition by leaf extract. No significant differences between the extracts were observed after the tenth day of storage. Overall, the extracts can be used to prevent lipid oxidation and reduce microbial spoilage during the first days of storage of fresh traditional pork sausages.Plod tršlje (Pistacia lentiscus) koristi se kao dodatak tradicionalno proizvedenim ciparskim kobasicama. Svrha je ovoga rada bila ispitati djelovanje ekstrakata tršlje kao prirodnih dodataka kobasicama. Prvo je određen udjel fenola u ekstraktima ploda i lista te je izmjerena njihova antioksidacijska aktivnost. Rezultati pokazuju da je lišće tršlje bogatije polifenolnim antioksidansima od plodova, a za njihovu je ekstrakciju najbolje upotrijebiti metanol. Zatim je ispitan antioksidacijski i antimikrobni učinak metanolnih ekstrakata (300 mg/kg) u kobasicama od svinjskog mesa. Peroksidni i kiselinski brojevi te koncentracija reaktivnih spojeva tiobarbiturne kiseline potvrđuju da se dodatkom oba ekstrakta smanjila oksidacija lipida u kobasicama na temperaturi skladištenja od 4 °C. Nakon pet dana skladištenja je u uzorcima kobasica s ekstraktima tršlje u usporedbi s kontrolnim uzorkom znatno smanjen ukupni broj živih stanica mikroorganizama, pri čemu je ekstrakt lišća imao bolji inhibicijski učinak. Nakon deset dana skladištenja nisu uočene bitne razlike u inhibicijskom učinku između ekstrakta ploda i lišća. Iz dobivenih rezultata moguće je zaključiti da se ekstrakti ploda i lišća tršlje mogu primijeniti za sprečavanje oksidacije lipida i produljenje trajnosti tradicionalnih kobasica od svježeg svinjskog mesa

Phenotypic Anchoring of Gene Expression Changes during Estrogen-Induced Uterine Growth

A major challenge in the emerging field of toxicogenomics is to define the relationships between chemically induced changes in gene expression and alterations in conventional toxicologic parameters such as clinical chemistry and histopathology. We have explored these relationships in detail using the rodent uterotrophic assay as a model system. Gene expression levels, uterine weights, and histologic parameters were analyzed 1, 2, 4, 8, 24, 48, and 72 hr after exposure to the reference physiologic estrogen 17β-estradiol (E(2)). A multistep analysis method, involving unsupervised hierarchical clustering followed by supervised gene ontology–driven clustering, was used to define the transcriptional program associated with E(2)-induced uterine growth and to identify groups of genes that may drive specific histologic changes in the uterus. This revealed that uterine growth and maturation are preceded and accompanied by a complex, multistage molecular program. The program begins with the induction of genes involved in transcriptional regulation and signal transduction and is followed, sequentially, by the regulation of genes involved in protein biosynthesis, cell proliferation, and epithelial cell differentiation. Furthermore, we have identified genes with common molecular functions that may drive fluid uptake, coordinated cell division, and remodeling of luminal epithelial cells. These data define the mechanism by which an estrogen induces organ growth and tissue maturation, and demonstrate that comparison of temporal changes in gene expression and conventional toxicology end points can facilitate the phenotypic anchoring of toxicogenomic data

Meeting Report: Validation of Toxicogenomics-Based Test Systems: ECVAM–ICCVAM/NICEATM Considerations for Regulatory Use

This is the report of the first workshop “Validation of Toxicogenomics-Based Test Systems” held 11–12 December 2003 in Ispra, Italy. The workshop was hosted by the European Centre for the Validation of Alternative Methods (ECVAM) and organized jointly by ECVAM, the U.S. Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), and the National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM). The primary aim of the workshop was for participants to discuss and define principles applicable to the validation of toxicogenomics platforms as well as validation of specific toxicologic test methods that incorporate toxicogenomics technologies. The workshop was viewed as an opportunity for initiating a dialogue between technologic experts, regulators, and the principal validation bodies and for identifying those factors to which the validation process would be applicable. It was felt that to do so now, as the technology is evolving and associated challenges are identified, would be a basis for the future validation of the technology when it reaches the appropriate stage. Because of the complexity of the issue, different aspects of the validation of toxicogenomics-based test methods were covered. The three focus areas include a) biologic validation of toxicogenomics-based test methods for regulatory decision making, b) technical and bioinformatics aspects related to validation, and c) validation issues as they relate to regulatory acceptance and use of toxicogenomics-based test methods. In this report we summarize the discussions and describe in detail the recommendations for future direction and priorities

Probing the structure and mechanism of DNA gyrase.

DNA gyrase is the enzyme from bacteria which is unique among type II topoisomerases in its ability to introduce negative supercoils into DNA. The enzyme acts as an A2B2 tetramer of molecular weight 374 kDa. The supercoiling reaction of gyrase involves wrapping of DNA around the enzyme and the coupling of ATP binding and hydrolysis to the passage of a DNA segment through a transient double strand break stabilised by gyrase, although its details are undefined. This reaction of gyrase is inhibited by two classes of anti-bacterial compounds, the quinolones and the coumarins. Hydroxyl radical footprinting was used to probe the complex between gyrase and a 198 bp DNA fragment containing the preferred gyrase cleavage site from plasmid pBR322. Gyrase protects 128 bp from the hydroxyl radical with the central 13 bp (adjacent to the gyrase cleavage site) being most strongly protected. Flanking the central region are arms showing periodic protection from the reagent suggesting a helical repeat of 10.6 bp, consistent with the DNA being wrapped upon the enzyme surface. The presence of 5'-adenylyl,Y-imidodiphosphate (ADPNP) or a quinolone drag causes alteration of the protection pattern consistent with a conformational change in the complex involving one arm of the wrapped DNA. This is thought to represent an intermediate in the supercoiling cycle of gyrase. Protein-DNA crosslinking using the photoactivatable thymine analogue 4-thiothymidine was used in an attempt to identify regions of gyrase involved in DNA binding. Complexes containing gyrase and 4-thiothymidine-substituted DNA were irradiated with long-wave UV light to activate the photoreactive reagent. However, no protein-DNA crosslinks were detected. An attempt to radiolabel DNA-binding lysine residues of gyrase was also unsuccessful due to the dissociation of the gyrase-DNA complex upon modification of its lysines. Irradiation of gyrase DNA complexes with short-wave laser-UV light results in the formation of covalent protein-DNA complexes at an efficiency of ~1%. Primer extension analysis was used to tentatively assign the crosslinks to two positions along the 147 bp DNA fragment used. The 43 kDa N-terminal domain of the gyrase B protein is responsible for ATP hydrolysis and also interacts with the coumarin class of gyrase inhibitors. To gain insight into the nature of the ATP-induced conformational change in the gyrase A2B2 tetramer, the effect of the non-hydrolysable analogue ADPNP on the conformation and oligomeric state of the 43 kDa domain was examined. Protein crosslinking studies suggest that the protein exists as a monomer but dimerises in the presence of ADPNP. Limited trypsin proteolysis in the presence of ADPNP results in the protection of a 33 kDa N-terminal fragment of the protein (residues 2-307), consistent with an altered conformation of the 43 kDa domain in the presence of the nucleotide. The effects of the coumarin drugs novobiocin and coumermycin A1 on the 43 kDa domain were also investigated. Novobiocin does not cause oligomerisation of the 43 kDa protein but coumermycin A1 induces dimerisation when present at molar concentrations approaching half that of the 43 kDa protein. Limited trypsin digestion in the presence of either drug results in the protection of a 16 kDa proteolytic fragment from digestion (residues 111-247). Moreover, the products of trypsin digestion in the presence of novobiocin can stably bind novobiocin when denatured and renatured. The 16 kDa fragment was cloned and overexpressed as a direct gene product but was found to be incapable of stable novobiocin binding

FCAWarehouse, a prototype online data repository for FCA

This paper presents FCAWarehouse, a prototype online data repository for FCA. The paper explains the motivation behind the development of FCAWarehouse and the features available, such as the ability to donate datasets and their respective formal contexts, the ability to generate artificial formal contexts on-the-fly, and how these features are also available through a set of web services. The paper concludes by suggesting future work in order to enhance it's usability

Toxicogenetics: Applications and opportunities

The response to drugs and environmental chemicals varies with genotype. Some patients react well to drugs, while others may not benefit, or may even respond adversely. Individuals also experi-ence different reactions to environmental agents, such as allergens. The sequencing of the human genome and the large-scale identi-fication of genome polymorphisms have provided opportunities for understanding the genetic basis for individual differences in response to potential toxicants: an area of study that has come to be known as toxicogenetics. In this article, we discuss the potential applications and implications of this evolving branch of toxicol-ogy. Variability in the Human Genome Only approximately 0.1 % of the three billion base pairs of DNA that comprise the human genome varies in sequence between individuals. However, the information encoded in thi