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  • 1
    Electronic Resource
    Electronic Resource
    Corticotropin-Releasing Hormone (CRH) Expression and Protein Kinase A Mediated CRH Receptor Signalling in an Immortalized Hypothalamic Cell Line (2003)
    Oxford, UK : Blackwell Science, Ltd
    Journal of neuroendocrinology 15 (2003), S. 0 
    Details
    ISSN: 1365-2826
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Corticotropin-releasing hormone (CRH) is a 41 amino acid neuropeptide which plays an important role in the stress response in the hypothalamus. We describe the development of an immortalized hypothalamic cell line which expresses CRH. We hypothesized that this cell line would possess the relevant characteristics of parvocellular CRH-expressing neurones such as glucocorticoid receptor (GR) expression and vasopressin (VP) coexpression. For production of hypothalamic cells, embryonic day 19 rat pup hypothalami were dissected and dissociated into tissue culture dishes. They were immortalized by retrovirus-mediated transfer of the SV40 large T antigen gene at 3 days of culture and then screened for expression of CRH following dilution cloning. One cell line was chosen (IVB) which exhibited CRH-like immunoreactivity (CRH-LI) and expressed CRH, VP and CRH1 receptor RNA via the reverse transcriptase-polymerase chain reaction. In addition, the cell line expressed the neuronal marker, microtubule-associated protein-2. We verified that the CRH-LI from IVB cell lysates coeluted with CRH standard via reversed-phase high-performance liquid chromatography (HPLC). Furthermore, oxidation of the lysate converted its HPLC profile to that identical with oxidized CRH standard. In addition, IVB cells exhibited high affinity binding to CRH. Incubation of IVB cells with CRH lead to increases in cAMP levels and protein kinase A activity in a concentration-dependent manner. Incubation of IVB cells with CRH also resulted in increases in phospho-cyclic-AMP response element binding protein (CREB) imunostaining as detected by immunocytochemical analysis. Finally, CRH treatment of IVB cell lines has been linked to CREB-mediated gene expression as determined via the PathDetect CREB trans-reporting system. The characteristics of IVB cells, such as CRH and VP coexpression, GR expression and a biologically active CRH-R1-mediated signalling pathway, suggest that this neuronal cell line may serve as model of parvocellular CRH neurones.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2826.2003.01026.x
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  • 2
    Electronic Resource
    Electronic Resource
    Effect of Repeated Lipopolysaccharide Administration on Tissue Cytokine Expression and Hypothalamic-Pituitary-Adrenal Axis Activity in Rats (2001)
    Oxford, UK : Blackwell Science, Ltd
    Journal of neuroendocrinology 13 (2001), S. 0 
    Details
    ISSN: 1365-2826
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The effects of chronic immune challenge on cytokine expression and hypothalamic-pituitary-adrenal axis (HPA) axis responses to stress were studied in Wistar rats after administration of increasing doses of lipopolysaccharide (LPS). Repeated LPS (R-LPS) decreased body weight and increased adrenal weight and pituitary pro-opiomelanocortin mRNA levels. LPS injection increased plasma adrenocorticotropic hormone (ACTH) and corticosterone but the effect was attenuated in R-LPS. Plasma corticosterone but not ACTH responses to restraint were also reduced in R-LPS. Basal and restraint-stimulated corticotropin releasing hormone (CRH) mRNA levels were lower in R-LPS, but responses to a new LPS injection were similar to controls. In contrast, type 1 CRH receptor (CRH-R1) mRNA responses to both LPS and restraint were blunted in R-LPS. Vasopressin mRNA levels in parvocellular neurones were higher in R-LPS, and increased further after restraint but not after a new LPS injection. Glucocorticoid receptor (GR) levels in the paraventricular nucleus (PVN) increased after a single LPS or R-LPS (24 h after the last injection) but declined after a new injection in R-LPS. Interleukin (IL)-1β and IL-6 mRNAs increased in the pituitary, spleen and circumventricular organs after single or R-LPS, suggesting that cytokines may contribute to the activation of the HPA axis though pathways from the circumventricular organs as well as paracrine effects in the pituitary. The data show that (i) adaptation of the HPA axis during repeated LPS injection involves increases in vasopressin : CRH expression ratios in parvocellular neurones; (ii) that hypothalamic CRH and vasopressin responses to acute stimulation are independent of CRH-R1 expression in the PVN; and (iii) there is a dissociation between pituitary and adrenal responses to acute stress suggesting a decrease of adrenal sensitivity to ACTH.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2826.2001.00684.x
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  • 3
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    DREAMing: a simple and ultrasensitive method for assessing intratumor epigenetic heterogeneity directly from liquid biopsies (2015)
    Oxford University Press
    Details
    Publication Date: 2015-12-16
    Description: Many cancers comprise heterogeneous populations of cells at primary and metastatic sites throughout the body. The presence or emergence of distinct subclones with drug-resistant genetic and epigenetic phenotypes within these populations can greatly complicate therapeutic intervention. Liquid biopsies of peripheral blood from cancer patients have been suggested as an ideal means of sampling intratumor genetic and epigenetic heterogeneity for diagnostics, monitoring and therapeutic guidance. However, current molecular diagnostic and sequencing methods are not well suited to the routine assessment of epigenetic heterogeneity in difficult samples such as liquid biopsies that contain intrinsically low fractional concentrations of circulating tumor DNA (ctDNA) and rare epigenetic subclonal populations. Here we report an alternative approach, deemed DREAMing (Discrimination of Rare EpiAlleles by Melt), which uses semi-limiting dilution and precise melt curve analysis to distinguish and enumerate individual copies of epiallelic species at single-CpG-site resolution in fractions as low as 0.005%, providing facile and inexpensive ultrasensitive assessment of locus-specific epigenetic heterogeneity directly from liquid biopsies. The technique is demonstrated here for the evaluation of epigenetic heterogeneity at p14 ARF and BRCA1 gene-promoter loci in liquid biopsies obtained from patients in association with non-small cell lung cancer (NSCLC) and myelodysplastic/myeloproliferative neoplasms (MDS/MPN), respectively.
    Keywords: Chromatin and Epigenetics
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 4
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    Linker histone H1.0 interacts with an extensive network of proteins found in the nucleolus (2013)
    Oxford University Press
    Details
    Publication Date: 2013-04-14
    Description: The H1 linker histones are abundant chromatin-associated DNA-binding proteins. Recent evidence suggests that linker histones also may function through protein–protein interactions. To gain a better understanding of the scope of linker histone involvement in protein–protein interactions, we used a proteomics approach to identify H1-binding proteins in human nuclear extracts. Full-length H1.0 and H1.0 lacking its C-terminal domain (CTD) were used for protein pull-downs. A total of 107 candidate H1.0 binding proteins were identified by LC-MS/MS. About one-third of the H1.0-dependent interactions were mediated by the CTD, and two-thirds by the N-terminal domain-globular domain fragment. Many of the proteins pulled down by H1.0 were core splicing factors. Another group of H1-binding proteins functions in rRNA biogenesis. H1.0 also pulled down numerous ribosomal proteins and proteins involved in cellular transport. Strikingly, nearly all of the H1.0-binding proteins are found in the nucleolus. Quantitative biophysical studies with recombinant proteins confirmed that H1.0 directly binds to FACT and the splicing factors SF2/ASF and U2AF65. Our results demonstrate that H1.0 interacts with an extensive network of proteins that function in RNA metabolism in the nucleolus, and suggest that a new paradigm for linker histone action is in order.
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 5
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    R2d2 Drives Selfish Sweeps in the House Mouse (2016)
    Oxford University Press
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    Publication Date: 2016-05-17
    Description: A selective sweep is the result of strong positive selection driving newly occurring or standing genetic variants to fixation, and can dramatically alter the pattern and distribution of allelic diversity in a population. Population-level sequencing data have enabled discoveries of selective sweeps associated with genes involved in recent adaptations in many species. In contrast, much debate but little evidence addresses whether "selfish" genes are capable of fixation—thereby leaving signatures identical to classical selective sweeps—despite being neutral or deleterious to organismal fitness. We previously described R2d2 , a large copy-number variant that causes nonrandom segregation of mouse Chromosome 2 in females due to meiotic drive. Here we show population-genetic data consistent with a selfish sweep driven by alleles of R2d2 with high copy number ( R2d2 HC ) in natural populations. We replicate this finding in multiple closed breeding populations from six outbred backgrounds segregating for R2d2 alleles. We find that R2d2 HC rapidly increases in frequency, and in most cases becomes fixed in significantly fewer generations than can be explained by genetic drift. R2d2 HC is also associated with significantly reduced litter sizes in heterozygous mothers, making it a true selfish allele. Our data provide direct evidence of populations actively undergoing selfish sweeps, and demonstrate that meiotic drive can rapidly alter the genomic landscape in favor of mutations with neutral or even negative effects on overall Darwinian fitness. Further study will reveal the incidence of selfish sweeps, and will elucidate the relative contributions of selfish genes, adaptation and genetic drift to evolution.
    Print ISSN: 0737-4038
    Electronic ISSN: 1537-1719
    Topics: Biology
    Published by Oxford University Press
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  • 6
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    Simultaneous Bayesian Estimation of Alignment and Phylogeny under a Joint Model of Protein Sequence and Structure (2014)
    Oxford University Press
    Details
    Publication Date: 2014-08-20
    Description: For sequences that are highly divergent, there is often insufficient information to infer accurate alignments, and phylogenetic uncertainty may be high. One way to address this issue is to make use of protein structural information, since structures generally diverge more slowly than sequences. In this work, we extend a recently developed stochastic model of pairwise structural evolution to multiple structures on a tree, analytically integrating over ancestral structures to permit efficient likelihood computations under the resulting joint sequence–structure model. We observe that the inclusion of structural information significantly reduces alignment and topology uncertainty, and reduces the number of topology and alignment errors in cases where the true trees and alignments are known. In some cases, the inclusion of structure results in changes to the consensus topology, indicating that structure may contain additional information beyond that which can be obtained from sequences. We use the model to investigate the order of divergence of cytoglobins, myoglobins, and hemoglobins and observe a stabilization of phylogenetic inference: although a sequence-based inference assigns significant posterior probability to several different topologies, the structural model strongly favors one of these over the others and is more robust to the choice of data set.
    Print ISSN: 0737-4038
    Electronic ISSN: 1537-1719
    Topics: Biology
    Published by Oxford University Press
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