Browsing by Subject "Cell Line"

Now showing 1 - 7 of 7
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    Open Access
    A Point Mutation in the Juxtamembrane Stalk of Human Angiotensin I-converting Enzyme Invokes the Action of a Distinct Secretase
    (2001) Alfalah, Marwan; Parkin, Edward T; Jacob, Ralf; Sturrock, Edward D; Mentele, Reinhard; Turner, Anthony J; HOOPER, Nigel M; Naim, Hassan Y
    Angiotensin I-converting enzyme (ACE) is one of a number of integral membrane proteins that is proteolytically shed from the cell surface by a zinc metallosecretase. Mutagenesis of Asn(631) to Gln in the juxtamembrane stalk region of ACE resulted in more efficient secretion of the mutant protein (ACE(NQ)) as determined by pulse-chase analysis. In contrast to the wild-type ACE, the cleavage of ACE(NQ) was not blocked by the metallosecretase inhibitor batimastat but by the serine protease inhibitor, 1,3-dichloroisocoumarin. Incubation of the cells at 15 degrees C revealed that ACE(NQ) was cleaved in the endoplasmic reticulum, and mass spectrometric analysis of the secreted form of the protein indicated that it had been cleaved at the Asn(635)-Ser(636) bond, three residues N-terminal to the normal secretase cleavage site at Arg(638)-Ser(639). These data clearly show that a point mutation in the juxtamembrane region of an integral membrane protein can invoke the action of a mechanistically and spatially distinct secretase. In light of this observation, previous data on the effect of mutations in the juxtamembrane stalk of shed proteins being accommodated by a single secretase having a relaxed specificity need to be re-evaluated.
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    Open Access
    Adipokinetic hormone signaling through the gonadotropin-releasing hormone receptor modulates egg-laying in Caenorhabditis elegans
    (2009) Lindemans, M; Liu, F; Janssen, T; Husson, S J; Mertens, I; Gäde, G; Schoofs, L
    In mammals, hypothalamic gonadotropin-releasing hormone (GnRH) is a neuropeptide that stimulates the release of gonadotropins from the anterior pituitary. The existence of a putative functional equivalent of this reproduction axis in protostomian invertebrates has been a matter of debate. In this study, the ligand for the GnRH receptor in the nematode Caenorhabditis elegans (Ce-GnRHR) was found using a bioinformatics approach. The peptide and its precursor are reminiscent of both insect adipokinetic hormones and GnRH-preprohormone precursors from tunicates and higher vertebrates. We cloned the AKH-GnRH-like preprohormone and the Ce-GnRHR and expressed the GPCR in HEK293T cells. The GnRHR was activated by the C. elegans AKH-GnRH-like peptide (EC50 = 150 nM) and by Drosophila AKH and other nematode AKH-GnRHs that we found in EST databases. Analogous to both insect AKH receptor and vertebrate GnRH receptor signaling, Ce-AKH-GnRH activated its receptor through a Gαq protein with Ca2+ as a second messenger. Gene silencing of Ce-GnRHR, Ce-AKH-GnRH, or both resulted in a delay in the egg-laying process, comparable to a delay in puberty in mammals lacking a normal dose of GnRH peptide or with a mutated GnRH precursor or receptor gene. The present data support the view that the AKH-GnRH signaling system probably arose very early in metazoan evolution and that its role in reproduction might have been developed before the divergence of protostomians and deuterostomians.
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    Open Access
    Characterisation of the global transcriptional response to heat shock and the impact of individual genetic variation
    (2016) Humburg, Peter; Maugeri, Narelle; Lee, Wanseon; Mohr, Bert; Knight, Julian C
    Abstract Background The heat shock transcriptional response is essential to effective cellular function under stress. This is a highly heritable trait but the nature and extent of inter-individual variation in heat shock response remains unresolved. Methods We determined global transcription profiles of the heat shock response for a panel of lymphoblastoid cell lines established from 60 founder individuals in the Yoruba HapMap population. We explore the observed differentially expressed gene sets following heat shock, establishing functional annotations, underlying networks and nodal genes involving heat shock factor 1 recruitment. We define a multivariate phenotype for the global transcriptional response to heat shock using partial least squares regression and map this quantitative trait to associated genetic variation in search of the major genomic modulators. Results A comprehensive dataset of differentially expressed genes following heat shock in humans is presented. We identify nodal genes downstream of heat shock factor 1 in this gene set, notably involving ubiquitin C and small ubiquitin-like modifiers together with transcription factors. We dissect a multivariate phenotype for the global heat shock response which reveals distinct clustering of individuals in terms of variance of the heat shock response and involves differential expression of genes involved in DNA replication and cell division in some individuals. We find evidence of genetic associations for this multivariate response phenotype that involves trans effects modulating expression of genes following heat shock, including HSF1 and UBQLN1. Conclusion This study defines gene expression following heat shock for a cohort of individuals, establishing insights into the biology of the heat shock response and hypotheses for how variation in this may be modulated by underlying genetic diversity.
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    Open Access
    Desensitization of Gonadotropin-releasing Hormone Action in αT3-1 Cells Due to Uncoupling of Inositol 1,4,5-Trisphosphate Generation and Ca 2+ Mobilization
    (1996) McArdle, Craig A; Willars, Gary B; Fowkes, Robert C; Nahorski, Stefan R; Davidson, James S; Forrest-Owen, Wyn
    Gonadotropin-releasing hormone (GnRH) acts via a G-protein coupled receptor on gonadotropes to increase cytosolic Ca2+ and stimulate gonadotropin secretion. Sustained exposure causes desensitization of these effects, but the GnRH receptor has no C-terminal tail and does not undergo rapid (<5 min) desensitization. Nevertheless, pretreatment of alphaT3-1 cells with GnRH reduced the spike Ca2+ response to GnRH and decreased the GnRH effect on inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) by 30-50%. Ca2+-free medium with or without thapsigargin also decreased GnRH-stimulated Ins(1,4,5)P3 generation, implying that attenuation of the Ca2+ response underlies the Ins(1,4,5)P3 reduction rather than vice versa. Intracellular Ca2+ pool depletion cannot explain desensitization of the Ca2+ response because pool depletion and repletion were faster (half-times, <1 min) than the onset of and recovery from desensitization (half-times 10-20 min and 4-6 h). Moreover, 1-h GnRH pre-treatment attenuated the spike Ca2+ response to GnRH but not that to ionomycin, and brief GnRH exposure in Ca2+-free medium reduced the response to ionomycin more effectively in controls than in desensitized cells. GnRH pretreatment also attenuated the Ca2+ response to PACAP38. This novel form of desensitization does not reflect uncoupling of GnRH receptors from their immediate effector system but rather a reduced efficiency of mobilization by Ins(1,4,5)P3 of Ca2+ from an intact intracellular pool.
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    DNA hypermethylation in sodium butyrate-treated WI-38 fibroblasts
    (1986) PARKER, M Iqbal; de Haa, Judy B; Gevers, Wieland
    Sodium butyrate is very often used to alter gene expression in cultured cells. In this study, we examined the effects of this compound on various cellular events in WI-38 human embryonic lung fibroblasts in culture. During a 16-20-h treatment at sodium butyrate concentrations of between 5 and 20 mM, no adverse effects on cell morphology were observed. However, cell division and DNA synthesis were reversibly inhibited, the latter by 85, 80, and 70% at sodium butyrate concentrations of 5, 10, and 20 mM, respectively. Although overall protein synthetic activity was not significantly affected, RNA synthesis decreased to 76% of the control values at a sodium butyrate concentration of 5 mM. Butyrate treatment also caused hypermethylation of DNA cytosines as determined by differential digestion by MspI/HpaII restriction endonucleases and by high performance liquid chromatography analysis of the DNA. The 5-methylcytosine content of the DNA in untreated WI-38 fibroblasts was 2.94 +/- 0.46% of total cytosine residues, while in cultures treated with 5, 10, and 20 mM sodium butyrate, these values were 5.76 +/- 0.28, 5.91 +/- 0.37, and 6.8 +/- 0.44%, respectively. An interesting feature is that this hypermethylation occurred in DNA which was synthesized in the presence of sodium butyrate (newly synthesized) as well as in DNA which had been synthesized before butyrate administration (pre-existing DNA). The hypermethylated state was conserved only in the former situation, since the methylcytosines were rapidly lost in the subsequent generation in the latter case. It would therefore appear that methylcytosines are maintained after cell replication only if they are generated on newly synthesized DNA.
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    Open Access
    Glutamate 301 of the mouse gonadotropin-releasing hormone receptor confers specificity for arginine 8 of mammalian gonadotropin-releasing hormone
    (1994) Flanagan, C A; Becker, I I; Davidson, J S; Wakefield, I K; Zhou, W; Sealfon, S C; Millar, R P
    The Arg residue at position 8 of mammalian GnRH is necessary for high affinity binding to mammalian GnRH receptors. This requirement has been postulated to derive from an electrostatic interaction of Arg8 with a negatively charged receptor residue. In order to identify such a residue, 8 conserved acidic residues of the mouse GnRH receptor were mutated to isosteric Asn or Gln. Mutant receptors were tested for decreased preference for Arg8-containing ligands by ligand binding and inositol phosphate production. One of the mutants, in which the Glu301 residue was mutated to Gln, exhibited a 56-fold decrease in apparent affinity for mammalian GnRH. The mutant receptor also exhibited decreased affinity for [Lys8]GnRH, but its affinity for [Gln8]GnRH was unchanged compared with the wild type receptor. The apparent affinity of the mutant receptor for the acidic analogue, [Glu8]GnRH, was increased more than 10-fold. The mutant receptor did not, therefore, distinguish mammalian GnRH from analogues with amino acid substitutions at position 8 as effectively as the wild type receptor. This loss of discrimination was specific for the residue at position 8, because the mutant receptor did distinguish mammalian GnRH from analogues with favorable substitutions at positions 5, 6, and 7. These findings show that Glu301 of the GnRH receptor plays a role in receptor recognition of Arg8 in the ligand and are consistent with an electrostatic interaction between these 2 residues.
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    Poxvirus Protein N1L Targets the I-κB Kinase Complex, Inhibits Signaling to NF-κB by the Tumor Necrosis Factor Superfamily of Receptors, and Inhibits NF-κB and IRF3 Signaling by Toll-like Receptors
    (2004) DiPerna, Gary; Stack, Julianne; Bowie, Andrew G; Boyd, Annemarie; Kotwal, Girish; Zhang, Zhouning; Arvikar, Sheila; Latz, Eicke; Fitzgerald, Katherine A; Marshall, William L
    Poxviruses encode proteins that suppress host immune responses, including secreted decoy receptors for pro-inflammatory cytokines such as interleukin-1 (IL-1) and the vaccinia virus proteins A46R and A52R that inhibit intracellular signaling by members of the IL-1 receptor (IL-1R) and Toll-like receptor (TLR) family. In vivo, the TLRs mediate the innate immune response by serving as pathogen recognition receptors, whose oligomerized intracellular Toll/IL-1 receptor (TIR) domains can initiate innate immune signaling. A family of TIR domain-containing adapter molecules transduces signals from engaged receptors that ultimately activate NF-kappaB and/or interferon regulatory factor 3 (IRF3) to induce pro-inflammatory cytokines. Data base searches detected a significant similarity between the N1L protein of vaccinia virus and A52R, a poxvirus inhibitor of TIR signaling. Compared with other poxvirus virulence factors, the poxvirus N1L protein strongly affects virulence in vivo; however, the precise target of N1L was previously unknown. Here we show that N1L suppresses NF-kappaB activation following engagement of Toll/IL-1 receptors, tumor necrosis factor receptors, and lymphotoxin receptors. N1L inhibited receptor-, adapter-, TRAF-, and IKK-alpha and IKK-beta-dependent signaling to NF-kappaB. N1L associated with several components of the multisubunit I-kappaB kinase complex, most strongly associating with the kinase, TANK-binding kinase 1 (TBK1). Together these findings are consistent with the hypothesis that N1L disrupts signaling to NF-kappaB by Toll/IL-1Rs and TNF superfamily receptors by targeting the IKK complex for inhibition. Furthermore, N1L inhibited IRF3 signaling, which is also regulated by TBK1. These studies define a role for N1L as an immunomodulator of innate immunity by targeting components of NF-kappaB and IRF3 signaling pathways.