Hepatocarcinogens induce gene mutations in rats in fibroblast-like cells from a subcutaneous granulation tissue

Gene-mutations at the 6-thioguanine locus, in fibroblast locus, in fibroblast like-cells rapidly proliferating on the inside of rat subcutaneous air pouches were analysed (Granuloma Pouch Assay). Target cells were exposed directly by injection into the air pouch, or systemically by oral or intraperitoneal administration to three hepatocarcinogens aflatoxin B1, 2-acetylaminofluorene (2-AAF) and vinylchloride. In addition ethylnitrosourea (ENU) was used as a positive control, and 2-aminofluorene to characterize the pharmacokinetic behaviour of 2-AAF. When administered directly, all chemicals except 2-AAF induced a dose-dependent increase in gene-mutation frequencies. However, 2-AAF was mutagenic after oral administration. The highest inducible mutation frequencies found with the hepatocarcinogens were 8 to 10 times the spontaneous mutation frequency, but only ∼10% of that found with the control mutagen ENU. The known enzymic activation capacity of the granulation tissue, and the mutagenic activity of other chemicals in this system already reported, suggest that after direct exposure, the prostaglandin H synthetase pathway or lipoxygenases are involved in the activation of the hepatotoxins to gene mutation inducing species in vivo. 2-AAF seems to be converted via stable intermediates to the ultimate mutagenic species in the extra-hepatic target tissue

Alterations in the cellular DNA and protein content determined by flow cytometry as indicators for chemically induced structural and numerical chromosome aberrations

Cellular DNA and protein content were determined simultaneously in freshly isolated fibroblast-like rat cells by flow cytometry. After exposure to doxorubicin, nitrofurantoin, propranolol and practolol at a low, tissue like oxygen concentration (5% O2), drug-induced alterations in cell cycle kinetics, in the distribution of DNA and in the protein content of G1-phase cells (nucleus/cytoplasm ratio) were analysed. Optimal exposure time (5 or 24 h) and recovery interval (24 or 48 h) were determined. Variation in the exposure time and recovery period can affect cell cycle kinetics both qualitatively and quantitatively, whereas the distribution of DNA and protein content are affected quantitatively only. A 24-h exposure combined with a 24-h recovery period proved to be the most efficient approach. Each of the tested chemicals induced a specific, dose-dependent pattern of altered cellular DNA and protein content. Comparison with results obtained in other genotoxicity tests, and with data reported earlier, showed that this two-parameter protocol can be used to recognize and to characterize chemicals as clastogens, or as compounds with a combined cytostatic/clastogenic activity, or as spindle-poison-like compound

Ellbogendysplasie beim Hund: Finite-Elemente-Analyse

Ellbogengelenkserkrankungen gewinnen bei jungen Hunden großer, schnellwüchsiger und bewegungsfreudiger Rassen seit Jahren zunehmend an Bedeutung. Als mögliche Ursachen der Ellbogendysplasie wurden - neben genetischer Veranlagung - Übergewicht und Überlastung der gelenkbildenden Knochen benannt. In der vorliegenden Untersuchung wurde der Einfluss verschiedener biomechanischer Parameter auf die Lastübertragung in gesunden und pathologischen Hundeellbogen mit einem zweidimensionalen Finite-Elemente-Modell analysiert. Pathologische Veränderungen der Ellbogenstruktur, wie veränderte Materialeigenschaften oder asynchrones Knochenwachstum, veränderten deutlich die Kontaktdrücke in den Artikulationen, die Knochendeformation und die Spannungen in den Knochen. Die gewonnenen Erkenntnisse unterstützen die langjährigen empirischen Beobachtungen und bieten eine Erklärung für die bis anhin kaum verstandenen klinischen Erscheinungsbilde

Telomere and ribosomal DNA repeats are chromosomal targets of the bloom syndrome DNA helicase

BACKGROUND: Bloom syndrome is one of the most cancer-predisposing disorders and is characterized by genomic instability and a high frequency of sister chromatid exchange. The disorder is caused by loss of function of a 3' to 5' RecQ DNA helicase, BLM. The exact role of BLM in maintaining genomic integrity is not known but the helicase has been found to associate with several DNA repair complexes and some DNA replication foci. RESULTS: Chromatin immunoprecipitation of BLM complexes recovered telomere and ribosomal DNA repeats. The N-terminus of BLM, required for NB localization, is the same as the telomere association domain of BLM. The C-terminus is required for ribosomal DNA localization. BLM localizes primarily to the non-transcribed spacer region of the ribosomal DNA repeat where replication forks initiate. Bloom syndrome cells expressing the deletion alleles lacking the ribosomal DNA and telomere association domains have altered cell cycle populations with increased S or G2/M cells relative to normal. CONCLUSION: These results identify telomere and ribosomal DNA repeated sequence elements as chromosomal targets for the BLM DNA helicase during the S/G2 phase of the cell cycle. BLM is localized in nuclear bodies when it associates with telomeric repeats in both telomerase positive and negative cells. The BLM DNA helicase participates in genomic stability at ribosomal DNA repeats and telomeres

Distribution of lactate dehydrogenase in healthy and degenerative canine stifle joint cartilage

In dogs, degenerative joint diseases (DJD) have been shown to be associated with increased lactate dehydrogenase (LDH) activity in the synovial fluid. The goal of this study was to examine healthy and degenerative stifle joints in order to clarify the origin of LDH in synovial fluid. In order to assess the distribution of LDH, cartilage samples from healthy and degenerative knee joints were investigated by means of light and transmission electron microscopy in conjunction with immunolabeling and enzyme cytochemistry. Morphological analysis confirmed DJD. All techniques used corroborated the presence of LDH in chondrocytes and in the interterritorial matrix of healthy and degenerative stifle joints. Although enzymatic activity of LDH was clearly demonstrated in the territorial matrix by means of the tetrazolium-formazan reaction, immunolabeling for LDH was missing in this region. With respect to the distribution of LDH in the interterritorial matrix, a striking decrease from superficial to deeper layers was present in healthy dogs but was missing in affected joints. These results support the contention that LDH in synovial fluid of degenerative joints originates from cartilage. Therefore, we suggest that (1) LDH is transferred from chondrocytes to ECM in both healthy dogs and dogs with degenerative joint disease and that (2) in degenerative joints, LDH is released from chondrocytes and the ECM into synovial fluid through abrasion of cartilage as well as through enhanced diffusion as a result of increased water content and degradation of collage

Tolerance of intravenous methylprednisolone for relapse treatment in demyelinating CNS disease

In Switzerland, the first course of intravenous steroids for treatment of episodes of demyelinating CNS disease is usually administered in an inpatient setting. We prospectively evaluated short term tolerance of treatment with special emphasis on sleep quality

Dynamic Remodeling of Individual Nucleosomes Across a Eukaryotic Genome in Response to Transcriptional Perturbation

The eukaryotic genome is packaged as chromatin with nucleosomes comprising its basic structural unit, but the detailed structure of chromatin and its dynamic remodeling in terms of individual nucleosome positions has not been completely defined experimentally for any genome. We used ultra-high–throughput sequencing to map the remodeling of individual nucleosomes throughout the yeast genome before and after a physiological perturbation that causes genome-wide transcriptional changes. Nearly 80% of the genome is covered by positioned nucleosomes occurring in a limited number of stereotypical patterns in relation to transcribed regions and transcription factor binding sites. Chromatin remodeling in response to physiological perturbation was typically associated with the eviction, appearance, or repositioning of one or two nucleosomes in the promoter, rather than broader region-wide changes. Dynamic nucleosome remodeling tends to increase the accessibility of binding sites for transcription factors that mediate transcriptional changes. However, specific nucleosomal rearrangements were also evident at promoters even when there was no apparent transcriptional change, indicating that there is no simple, globally applicable relationship between chromatin remodeling and transcriptional activity. Our study provides a detailed, high-resolution, dynamic map of single-nucleosome remodeling across the yeast genome and its relation to global transcriptional changes

The Bloom's syndrome helicase (BLM) interacts physically and functionally with p12, the smallest subunit of human DNA polymerase δ

Bloom's syndrome (BS) is a cancer predisposition disorder caused by mutation of the BLM gene, encoding a member of the RecQ helicase family. Although the phenotype of BS cells is suggestive of a role for BLM in repair of stalled or damaged replication forks, thus far there has been no direct evidence that BLM associates with any of the three human replicative DNA polymerases. Here, we show that BLM interacts specifically in vitro and in vivo with p12, the smallest subunit of human POL δ (hPOL δ). The hPOL δ enzyme, as well as the isolated p12 subunit, stimulates the DNA helicase activity of BLM. Conversely, BLM stimulates hPOL δ strand displacement activity. Our results provide the first functional link between BLM and the replicative machinery in human cells, and suggest that BLM might be recruited to sites of disrupted replication through an interaction with hPOL δ. Finally, our data also define a novel role for the poorly characterized p12 subunit of hPOL δ

Assessing the functional structure of genomic data

Motivation: The availability of genome-scale data has enabled an abundance of novel analysis techniques for investigating a variety of systems-level biological relationships. As thousands of such datasets become available, they provide an opportunity to study high-level associations between cellular pathways and processes. This also allows the exploration of shared functional enrichments between diverse biological datasets, and it serves to direct experimenters to areas of low data coverage or with high probability of new discoveries

Genome-wide analysis of transcriptional dependence and probable target sites for Abf1 and Rap1 in Saccharomyces cerevisiae

Abf1 and Rap1 are general regulatory factors (GRFs) that contribute to transcriptional activation of a large number of genes, as well as to replication, silencing and telomere structure in yeast. In spite of their widespread roles in transcription, the scope of their functional targets genome-wide has not been previously determined. Here, we use microarrays to examine the contribution of these essential GRFs to transcription genome-wide, by using ts mutants that dissociate from their binding sites at 37°C. We then combine this data with published ChIP-chip studies and motif analysis to identify probable direct targets for Abf1 and Rap1. We also identify a substantial number of genes likely to bind Rap1 or Abf1, but not affected by loss of GRF binding. Interestingly, the results strongly suggest that Rap1 can contribute to gene activation from farther upstream than can Abf1. Also, consistent with previous work, more genes that bind Abf1 are unaffected by loss of binding than those that bind Rap1. Finally, we show for several such genes that the Abf1 C-terminal region, which contains the putative activation domain, is not needed to confer this peculiar ‘memory effect’ that allows continued transcription after loss of Abf1 binding