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Noriega, Guillermo O., Adela A. Juknat, and Alcira M. del C. Batlle. "Non-Essential Activation of Rat Liver Porphobilinogen-Deaminase by Folic Acid." Zeitschrift für Naturforschung C 47, no. 5-6 (June 1, 1992): 416–19. http://dx.doi.org/10.1515/znc-1992-0616.
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This report demonstrates the ability of folic acid to activate rat liver porphobilinogen-deaminase (PBG -D). Lineweaver-Burk analysis revealed an increase in Vmax (38%) without affecting the Km. In the concentration range assayed, secondary replots of 1/Δslope and 1/Δintersect versus 1/[folic acid] yielded straight lines, indicating the binding of a single molecule of activator to the enzyme PBG-D , with a KA = 1.66 mᴍ. Results presented here show that folic acid acts as a non-essential activator (α = 1; β = 1.6). The activating effect of folic acid has been observed employing the 35-70% ammonium sulphate precipitated fraction, desalted by dialysis or gel filtration, whereas no action was detected when other partially purified PBG -D preparations were utilized as the enzyme source, suggesting either the presence of sites saturated for the activator, or the existence of a different structural protein conformation, or both.
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Gai, Lili, Yuting Zhu, Chun Zhang, and Xianfang Meng. "Targeting Canonical and Non-Canonical STAT Signaling Pathways in Renal Diseases." Cells 10, no. 7 (June 27, 2021): 1610. http://dx.doi.org/10.3390/cells10071610.
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Signal transducer and activator of transcription (STAT) plays an essential role in the inflammatory reaction and immune response of numerous renal diseases. STATs can transmit the signals of cytokines, chemokines, and growth factors from the cell membrane to the nucleus. In the canonical STAT signaling pathways, upon binding with their cognate receptors, cytokines lead to a caspase of Janus kinases (JAKs) and STATs tyrosine phosphorylation and activation. Besides receptor-associated tyrosine kinases JAKs, receptors with intrinsic tyrosine kinase activities, G-protein coupled receptors, and non-receptor tyrosine kinases can also activate STATs through tyrosine phosphorylation or, alternatively, other post-translational modifications. Activated STATs translocate into the nucleus and mediate the transcription of specific genes, thus mediating the progression of various renal diseases. Non-canonical STAT pathways consist of preassembled receptor complexes, preformed STAT dimers, unphosphorylated STATs (U-STATs), and non-canonical functions including mitochondria modulation, microtubule regulation and heterochromatin stabilization. Most studies targeting STAT signaling pathways have focused on canonical pathways, but research extending into non-canonical STAT pathways would provide novel strategies for treating renal diseases. In this review, we will introduce both canonical and non-canonical STAT pathways and their roles in a variety of renal diseases.
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Das-Panja, Kaberi, Vidya Jonnalagadda, and Sobhanaditya Jonnalagadda. "Orthophosphate is a non-essential activator of Vigna radiata flavokinase." IUBMB Life 47, no. 4 (April 1999): 547–54. http://dx.doi.org/10.1080/15216549900201583.
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Künnapuu, Jaana, Honey Bokharaie, and Michael Jeltsch. "Proteolytic Cleavages in the VEGF Family: Generating Diversity among Angiogenic VEGFs, Essential for the Activation of Lymphangiogenic VEGFs." Biology 10, no. 2 (February 23, 2021): 167. http://dx.doi.org/10.3390/biology10020167.
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Specific proteolytic cleavages turn on, modify, or turn off the activity of vascular endothelial growth factors (VEGFs). Proteolysis is most prominent among the lymphangiogenic VEGF-C and VEGF-D, which are synthesized as precursors that need to undergo enzymatic removal of their C- and N-terminal propeptides before they can activate their receptors. At least five different proteases mediate the activating cleavage of VEGF-C: plasmin, ADAMTS3, prostate-specific antigen, cathepsin D, and thrombin. All of these proteases except for ADAMTS3 can also activate VEGF-D. Processing by different proteases results in distinct forms of the “mature” growth factors, which differ in affinity and receptor activation potential. The “default” VEGF-C-activating enzyme ADAMTS3 does not activate VEGF-D, and therefore, VEGF-C and VEGF-D do function in different contexts. VEGF-C itself is also regulated in different contexts by distinct proteases. During embryonic development, ADAMTS3 activates VEGF-C. The other activating proteases are likely important for non-developmental lymphangiogenesis during, e.g., tissue regeneration, inflammation, immune response, and pathological tumor-associated lymphangiogenesis. The better we understand these events at the molecular level, the greater our chances of developing successful therapies targeting VEGF-C and VEGF-D for diseases involving the lymphatics such as lymphedema or cancer.
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Inoue, Kazushi, and Elizabeth A. Fry. "Aberrant Expression of Cyclin D1 in Cancer." Signal Transduction Insights 4 (January 2015): STI.S30306. http://dx.doi.org/10.4137/sti.s30306.
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Cyclin D1 binds and activates cyclin-dependent kinases 4/6 (Cdk4/6) to phosphorylate the retinoblastoma (RB) family proteins, relieving E2F/DPs from the negative restraint of RB proteins and histone deacetylases (HDACs). The cyclin D-Cdk4/6 complexes activate cyclin E/Cdk2 through titration of the Cdk inhibitors p21Cip1/p27Kip1. Cyclin E/Cdk2 further phosphorylates RBs, thereby activating E2F/DPs, and cells enter the S-phase of the cell cycle. Cyclin D-Cdk4/6 also phosphorylates MEP50 subunit of the protein arginine methyltransferase 5 (PRMT5), which cooperates with cyclin D1 to drive lymphomagenesis in vivo. Activated PRMPT5 causes arginine methylation of p53 to suppress expression of proapoptotic and antiproliferative target genes, explaining the molecular mechanism for tumorigenesis. Cyclin D1 physically interacts with transcription factors such as estrogen receptor, androgen receptor, and Myb family proteins to regulate gene expression in Cdk-independent fashion. Dmp1 is a Myb-like protein that quenches the oncogenic signals from activated Ras or HER2 by inducing Arf/p53-dependent cell cycle arrest. Cyclin D1 binds to Dmp1 to activate both Arf and Ink4a promoters to induce cell cycle arrest or apoptosis in non-transformed cells to prevent them from neoplastic transformation. Dmp1deficiency significantly accelerates mouse mammary tumorigenesis with reduced apoptosis and increased metastasis. Cyclin D1 interferes with ligand activation of PPARγ involved in cellular differentiation; it also physically interacts with HDACs and p300 to repress gene expression. It has also been shown that cyclin D1 accelerates tumorigenesis through transcriptional activation of miR-17/20 and Dicer1 which, in turn, represses cyclin D1 expression. Identification of cyclin D1-binding proteins/promoters will be essential for further clarification of its biological activities.
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Jastrzebska, Beata, Yaroslav Tsybovsky, and Krzysztof Palczewski. "Complexes between photoactivated rhodopsin and transducin: progress and questions." Biochemical Journal 428, no. 1 (April 28, 2010): 1–10. http://dx.doi.org/10.1042/bj20100270.
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Activation of GPCRs (G-protein-coupled receptors) leads to conformational changes that ultimately initiate signal transduction. Activated GPCRs transiently combine with and activate heterotrimeric G-proteins resulting in GTP replacement of GDP on the G-protein α subunit. Both the detailed structural changes essential for productive GDP/GTP exchange on the G-protein α subunit and the structure of the GPCR–G-protein complex itself have yet to be elucidated. Nevertheless, transient GPCR–G-protein complexes can be trapped by nucleotide depletion, yielding an empty-nucleotide G-protein–GPCR complex that can be isolated. Whereas early biochemical studies indicated formation of a complex between G-protein and activated receptor only, more recent results suggest that G-protein can bind to pre-activated states of receptor or even couple transiently to non-activated receptor to facilitate rapid responses to stimuli. Efficient and reproducible formation of physiologically relevant, conformationally homogenous GPCR–G-protein complexes is a prerequisite for structural studies designed to address these possibilities.
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Vainchenker, William, Stefan N. Constantinescu, and Isabelle Plo. "Recent advances in understanding myelofibrosis and essential thrombocythemia." F1000Research 5 (April 19, 2016): 700. http://dx.doi.org/10.12688/f1000research.8081.1.
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The classicBCR-ABL-negative myeloproliferative neoplasms (MPNs), a form of chronic malignant hemopathies, have been classified into polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). ET and PMF are two similar disorders in their pathogenesis, which is marked by a key role of the megakaryocyte (MK) lineage. Whereas ET is characterized by MK proliferation, PMF is also associated with aberrant MK differentiation (myelodysplasia), leading to the release of cytokines in the marrow environment, which causes the development of myelofibrosis. Thus, PMF is associated with both myeloproliferation and different levels of myelodysplastic features. MPNs are mostly driven by mutated genes called MPN drivers, which abnormally activate the cytokine receptor/JAK2 pathway and their downstream effectors. The recent discovery ofCALRmutations has closed a gap in our knowledge and has shown that this mutated endoplasmic reticulum chaperone activates the thrombopoietin receptor MPL and JAK2. These genetic studies have shown that there are two main types of MPNs: JAK2V617F-MPNs, including ET, PV, and PMF, and the MPL-/CALR-MPNs, which include only ET and PMF. These MPN driver mutations are associated with additional mutations in genes involved in epigenetics, splicing, and signaling, which can precede or follow the acquisition of MPN driver mutations. They are involved in clonal expansion or phenotypic changes or both, leading to myelofibrosis or leukemic transformation or both. Only a few patients with ET exhibit mutations in non-MPN drivers, whereas the great majority of patients with PMF harbor one or several mutations in these genes. However, the entire pathogenesis of ET and PMF may also depend on other factors, such as the patient’s constitutional genetics, the bone marrow microenvironment, the inflammatory response, and age. Recent advances allowed a better stratification of these diseases and new therapeutic approaches with the development of JAK2 inhibitors.
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Marschke, Keith, Deepa Rungta, Daniela A. Slavin, Jennifer Sanders, Steven L. Roach, Jason C. Pickens, Yixing Shen, et al. "Discovery of Non-Peptidyl Small-Molecule Human GCSF Receptor Agonists for the Potential Treatment of Neutropenia,." Blood 118, no. 21 (November 18, 2011): 3391. http://dx.doi.org/10.1182/blood.v118.21.3391.3391.
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Abstract Abstract 3391 Granulocyte colony stimulating factor (GCSF) is the essential cytokine for the regulation of neutrophilic granulocytes. Binding of GCSF to its receptor (GCSFR) triggers receptor dimerization, leading to activation of JAK1 and JAK2, phosphorylation of GCSFR, STAT3, STAT5, and Ras/mitogen-activated protein kinase (MAPK), and results in proliferation and differentiation of granulocytic cells. Recombinant human GCSF (rhGCSF) is used successfully to alleviate chemotherapy-induced neutropenia, neutropenia associated with hematopoietic stem cell transplantation, and severe chronic neutropenia. A small molecule oral GCSFR agonist may offer a safer and more convenient alternative to the current injectable rhGCSF therapy. Whereas a previous effort to identify small-molecule mimetics of GCSF found SB-247464 that selectively activated the murine GCSFR, no small-molecule human GCSF mimetics have been developed. Recently, we have discovered a series of novel non-peptidyl small molecules that selectively activate human GCSFR (hGCSFR) function, and may provide a significant innovation in the treatment of neutropenia. In cells transiently transfected with an hGCSFR expression vector and a STAT3-responsive luciferase reporter, a lead compound, LG7455, activates luciferase expression with an efficacy of 50% relative to rhGCSF, and potency (EC50) of 100 nM. LG7455 also activates luciferase expression in cells transfected with hGCSFR and a STAT5-responsive luciferase reporter (65%, 40 nM EC50). The activity of LG7455 is dependent on the expression of hGCSFR, and LG7455 is not active in luciferase assays when human thrombopoietin receptor (hTPOR) or erythropoietin receptor (hEPOR) is expressed. In UT-7 cells made responsive to GCSF by stable transfection of hGCSFR (UTP-hGCSFR), LG7455 stimulated cell growth and increased the phosphorylation of STAT3 and STAT5. LG7455 did not increase growth of TPO- or EPO-responsive UT-7 cells. In CD34 positive human bone marrow hematopoietic cells (BM-HCs), LG7455, increased the percentage of cells positive for the granulocyte-specific marker CD15 (FUT4). The effect of LG7455 in BM-HCs was additive to the effect of rhGCSF. LG7455 is active in luciferase assays with expressed cynomolgus monkey GCSFR, but not mouse, guinea pig or rabbit GCSFR. Similar to what has been demonstrated for small-molecule human TPOR agonists such as eltrombopag, the activity of LG7455 is dependent on a specific residue in the hGCSFR transmembrane domain. When histidine 627 (His-627) in hGCSFR is changed to asparagine present at a similar location in the mouse GCSFR (Asp-602), unlike rhGCSF, LG7455 is no longer active. LG7455 is active, however, on mouse GCSFR with Asp-602 replaced by His. In radioligand-binding experiments using UTP-hGCSFR cells, LG7455 did not displace [125I]rhGCSF, however binding of [125I]rhGCSF was augmented in a concentration dependent manner consistent with allosteric receptor modulation. These data demonstrate that LG7455 is a novel small-molecule selective hGCSFR agonist that activates the receptor in a manner distinct from GCSF and similar to the mechanism of small-molecule hTPOR agonists. Further optimization of the LG7455 chemical series should provide orally-available molecules to treat neutropenia with improved safety and convenience compared to current injectable rhGCSF. Disclosures: Marschke: Ligand Pharmaceuticals: Employment. Rungta:Ligand Pharmaceuticals: Employment. Slavin:Ligand Pharmaceuticals: Employment. Sanders:Ligand Pharmaceuticals: Employment. Roach:Ligand Pharmaceuticals: Employment. Pickens:Ligand Pharmaceuticals: Employment. Shen:Ligand Pharmaceuticals: Employment. van Oeveren:Ligand Pharmaceuticals: Employment. Hong:Ligand Pharmaceuticals: Employment. Sun:Ligand Pharmaceuticals: Employment. Bissonnette:Ligand Pharmaceuticals: Employment. Syka:Ligand Pharmaceuticals: Employment. Zhi:Ligand Pharmaceuticals: Employment.
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Działo, Edyta, Karolina Tkacz, and Przemysław Błyszczuk. "Crosstalk between TGF-β and WNT signalling pathways during cardiac fibrogenesis." Acta Biochimica Polonica 65, no. 3 (July 24, 2018): 341–49. http://dx.doi.org/10.18388/abp.2018_2635.
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Cardiac fibrosis is referred to as an excessive accumulation of stromal cells and extracellular matrix proteins in the myocardium. Progressive fibrosis causes stiffening of the cardiac tissue and affects conduction of electrical impulses, leading to heart failures in a broad range of cardiac conditions. At the cellular level, activation of the cardiac stromal cells and myofibroblast formation are considered as hallmarks of fibrogenesis. At the molecular level, transforming growth factor β (TGF-β) is traditionally considered as a master regulator of the profibrotic processes. More recently, the WNT signalling pathway has also been found to be implicated in the development of myocardial fibrosis. In this review, we summarize current knowledge on the involvement of TGF-β and WNT downstream molecular pathways to cardiac fibrogenesis and describe a crosstalk between these two profibrotic pathways. TGF-β and WNT ligands bind to different receptors and trigger various outputs. However, a growing body of evidence points to cross-regulation between these two pathways. It has been recognized that in cardiac pathologies TGF-β activates WNT/β-catenin signalling, which in turn stabilizes the TGF-β/Smad response. Furthermore both, the non-canonical TGF-β and non-canonical WNT signalling pathways, activate the same mitogen-activated protein kinases (MAPKs): the extracellular signal-regulated kinase (Erk), the c-Jun N-terminal kinases (JNKs) and p38. The cross-talk between TGF-β and WNT pathways seems to play an essential role in switching on the genetic machinery initiating profibrotic changes in the heart. Better understanding of these mechanisms will open new opportunities for development of targeted therapeutic approaches against cardiac fibrosis in the future.
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Guertin, Michael J., Xuesen Zhang, Scott A. Coonrod, and Gordon L. Hager. "Transient Estrogen Receptor Binding and p300 Redistribution Support a Squelching Mechanism for Estradiol-Repressed Genes." Molecular Endocrinology 28, no. 9 (September 1, 2014): 1522–33. http://dx.doi.org/10.1210/me.2014-1130.
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Proper gene regulation is essential for proper organismal development and appropriate responses to external stimuli. Specialized factors, termed master regulators, are often responsible for orchestrating the molecular events that result from signaling cascades. Master regulators coordinate the activation and repression of specific gene classes. Estrogen receptor α (ER) precipitates the signaling cascade that results from endogenous or exogenous estrogen hormones. ER is a classic transcriptional activator and the mechanisms by which ER coordinates gene activation are well characterized. However, it remains unclear how ER coordinates the immediate repression of genes. We integrated genomic transcription, chromosome looping, transcription factor binding, and chromatin structure data to analyze the molecular cascade that results from estradiol (E2)-induced signaling in human MCF-7 breast cancer cells and addressed the context-specific nature of gene regulation. We defined a class of genes that are immediately repressed upon estrogen stimulation, and we compared and contrasted the molecular characteristics of these repressed genes vs activated and unregulated genes. The most striking and unique feature of the repressed gene class is transient binding of ER at early time points after estrogen stimulation. We also found that p300, a coactivator and acetyltransferase, quantitatively redistributes from non-ER enhancers to ER enhancers after E2 treatment. These data support an extension of the classic physiological squelching model, whereby ER hijacks coactivators from repressed genes and redistributes the coactivators to ER enhancers that activate transcription.
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Fortune, Joanne E., Ming Y. Yang, and Wanzirai Muruvi. "In vitro and in vivo regulation of follicular formation and activation in cattle." Reproduction, Fertility and Development 23, no. 1 (2011): 15. http://dx.doi.org/10.1071/rd10250.
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The establishment of a stockpile of non-growing, primordial follicles and its gradual depletion through activation of primordial follicles are essential processes for female fertility. However, the mechanisms that regulate follicle formation, the activation of primordial follicles to begin growth and the primary-to-secondary follicle transition are poorly understood, especially in domestic animals and primates. The authors’ laboratory is engaged in studying early stages of follicular development in cattle and this review summarises the progress to date. Bovine follicles begin to form in fetal ovaries around the beginning of the second trimester of pregnancy (about Day 90), but the first activated, primary follicles do not appear until after Day 140. Bovine fetal ovaries produce steroids and production is highest during the first trimester. In vitro, oestradiol and progesterone inhibit follicle formation and acquisition by newly formed follicles of the capacity to activate. Meiotic arrest of the oocyte in the diplotene stage of first prophase does not occur until after follicle formation and is correlated with acquisition of the capacity to activate. This may explain the gap between follicle formation and appearance of the first activated follicles. Once capacity to activate has been acquired, it seems likely that activation in vivo is controlled by the balance between stimulators and inhibitors of activation. Insulin and kit ligand stimulate and anti-Müllerian hormone (AMH) inhibits activation in vitro. Few bovine follicles transition from the primary to the secondary stage in vitro, but this transition is increased by medium supplements, testosterone and vascular endothelial growth factor (VEGF).
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Reimer, Raylene A. "Meat hydrolysate and essential amino acid-induced glucagon-like peptide-1 secretion, in the human NCI-H716 enteroendocrine cell line, is regulated by extracellular signal-regulated kinase1/2 and p38 mitogen-activated protein kinases." Journal of Endocrinology 191, no. 1 (October 2006): 159–70. http://dx.doi.org/10.1677/joe.1.06557.
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Glucagon-like peptide-1 (GLP-1) is a potent insulin secretagogue released from L-cells in the intestine. Meat hydrolysate (MH) is a powerful activator of GLP-1 secretion in the human enteroendocrine NCI-H716 cell line, but the mechanisms involved in nutrient-stimulated GLP-1 secretion are poorly understood. The objective of this study was to characterize the intracellular signalling pathways regulating MH- and amino acid-induced GLP-1 secretion. Individually, the pharmacological inhibitors, SB203580 (inhibitor of p38 mitogen-activated protein kinase (MAPK)), wortmannin (inhibitor of phosphatidyl inositol 3-kinase) and U0126 (inhibitor of mitogen activated or extracellular signal-regulated protein kinase (MEK1/2) upstream of extracellular signal-regulated kinase (ERK)1/2) all inhibited MH-induced GLP-1 secretion. Further examination of the MAPK pathway showed that MH increased the phosphorylation of ERK1/2, but not p38 or c-Jun N-terminal kinase over 2–15 min. Incubation with SB203580 resulted in a decrease in phosphorylated p38 MAPK and a concomitant increase in the phosphorylation of ERK1/2. Phosphorylation of ERK1/2 was augmented by co-incubation of MH with SB203580. Inhibitors of protein kinase A and protein kinase C did not inhibit MH-induced GLP-1 secretion. In contrast to non-essential amino acids, essential amino acids (EAAs) increased GLP-1 secretion and similar to MH, activated ERK1/2. However, they also activated p38-suggesting type of protein may affect GLP-1 secretion. In conclusion, there appears to be a crosstalk between p38 and ERK1/2 MAPK in the human enteroendocrine cell with the activation of ERK1/2 common to both MH and EAA. Understanding the cellular pathways involved in nutrient-stimulated GLP-1 secretion has important implications for the design of new treatments aimed at increasing endogenous GLP-1 release in type-2 diabetes and obesity.
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de Souza Almeida Matos, Ana Laura, Jonathan S. Oakhill, José Moreira, Kim Loh, Sandra Galic, and John W. Scott. "Allosteric regulation of AMP-activated protein kinase by adenylate nucleotides and small-molecule drugs." Biochemical Society Transactions 47, no. 2 (April 18, 2019): 733–41. http://dx.doi.org/10.1042/bst20180625.
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Abstract The AMP (adenosine 5′-monophosphate)-activated protein kinase (AMPK) is a key regulator of cellular and whole-body energy homeostasis that co-ordinates metabolic processes to ensure energy supply meets demand. At the cellular level, AMPK is activated by metabolic stresses that increase AMP or adenosine 5′-diphosphate (ADP) coupled with falling adenosine 5′-triphosphate (ATP) and acts to restore energy balance by choreographing a shift in metabolism in favour of energy-producing catabolic pathways while inhibiting non-essential anabolic processes. AMPK also regulates systemic energy balance and is activated by hormones and nutritional signals in the hypothalamus to control appetite and body weight. Failure to maintain energy balance plays an important role in chronic diseases such as obesity, type 2 diabetes and inflammatory disorders, which has prompted a major drive to develop pharmacological activators of AMPK. An array of small-molecule allosteric activators has now been developed, several of which can activate AMPK by direct allosteric activation, independently of Thr172 phosphorylation, which was previously regarded as indispensable for AMPK activity. In this review, we summarise the state-of-the-art regarding our understanding of the molecular mechanisms that govern direct allosteric activation of AMPK by adenylate nucleotides and small-molecule drugs.
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Kim, Jeong Ah, Sungwoo Park, Linda Fetters, Sandrah P. Eckel, Masayoshi Kubo, and Barbara Sargent. "Quantifying Infant Exploratory Learning." Journal of Motor Learning and Development 10, no. 1 (April 1, 2022): 167–83. http://dx.doi.org/10.1123/jmld.2021-0029.
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Exploration is considered essential to infant learning, but few studies have quantified infants’ task exploration. The purpose of this study was to quantify how infants explored task space with their feet while learning to activate a kick-activated mobile. Data were analyzed from fifteen 4-month-old infants who participated in a 10-min mobile task on 2–3 consecutive days. Infants learned that their vertical leg movements above a systematically increased threshold height activated the mobile. Five kinematic variables were analyzed: (a) exploration space volume, (b) exploration path length, (c) duration of time in the region of interest around the threshold that activated the mobile, (d) task-specific vertical variance of kicks, and (e) non-task-specific horizontal variance of kicks. The infants increased their general spatial exploration, volume, and path, and the infants adapted their exploration by maintaining their feet within the region of interest, although the task-specific region increased in height as the threshold increased. The infants used task-specific strategies quantified by the increased variance of kicks in the vertical direction and no change in the horizontal variance of kicks. Quantifying infants’ task exploration may provide critical insights into how learning emerges in infancy and enable researchers to more systematically describe, interpret, and support learning.
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Geletu, Mulu, Zaid Taha, Patrick T. Gunning, and Leda Raptis. "PI3k and Stat3: Oncogenes that are Required for Gap Junctional, Intercellular Communication." Cancers 11, no. 2 (February 1, 2019): 167. http://dx.doi.org/10.3390/cancers11020167.
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Gap junctional, intercellular communication (GJIC) is interrupted in cells transformed by oncogenes such as activated Src. The Src effector, Ras, is required for this effect, so that Ras inhibition restores GJIC in Src-transformed cells. Interestingly, the inhibition of the Src effector phosphatidyl-inositol-3 kinase (PI3k) or Signal Transducer and Activator of Transcription-3 (Stat3) pathways does not restore GJIC. In the contrary, inhibition of PI3k or Stat3 in non-transformed rodent fibroblasts or epithelial cells or certain human lung carcinoma lines with extensive GJIC inhibits communication, while mutational activation of PI3k or Stat3 increases GJIC. Therefore, it appears that oncogenes such as activated Src have a dual role upon GJIC; acting as inhibitors of communication through the Ras pathway, and as activators through activation of PI3k or Stat3. In the presence of high Src activity the inhibitory functions prevail so that the net effect is gap junction closure. PI3k and Stat3 constitute potent survival signals, so that their inhibition in non-transformed cells triggers apoptosis which, in turn, has been independently demonstrated to suppress GJIC. The interruption of gap junctional communication would confine the apoptotic event to single cells and this might be essential for the maintenance of tissue integrity. We hypothesize that the GJIC activation by PI3k or Stat3 may be linked to their survival function.
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Ni, Shuang, Hami Heal, Jie Xu, Robert Paulson, and Pamela H. Correll. "The Activation of Stat3 by the Stk Receptor Tyrosine Kinase Requires Src Kinases and Gab2, and Is Essential for Friend Virus Induced, Epo-Independent Growth of Primary Erythroid Cells." Blood 104, no. 11 (November 16, 2004): 820. http://dx.doi.org/10.1182/blood.v104.11.820.820.
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Abstract Friend virus-induced erythroleukemia is a model for the study of the multi-stage nature of carcinogenesis. The early stage of the disease is characterized by a polyclonal expansion of infected erythroid precursor cells, and the later stage is marked by the emergence of fully transformed cells in the spleen, blood, bone marrow and liver, leading to the development of erythroleukemia. Previous findings have demonstrated that a naturally occurring, N-terminally truncated form of the stk receptor tyrosine kinase (Sf-stk) provides signals necessary for the polycolonal expansion of infected cells. Signal transducer and activator of transcription (Stat) proteins play an important role in normal and malignant hematopoiesis. Moreover it has been shown that growth factors can induce Stat3 in a Src family kinase (SFK)-dependent manner. Here we show that dominant negative Stat3 and c-Src significantly inhibit the BFU-e and CFU-e colony number induced by friend virus. In addition, the non-specific SFK inhibitor PP1 also abrogated Epo-independent colony formation. Moreover we found that the inhibition of SFKs by PP1 suppresses Stat3 tyrosine phosphorylation activated by Sf-stk. To elucidate the signaling mechanism by which Sf-stk activates Stat3, we mutated the docking site tyrosines to phenylalanine. Our results indicated that the mutation of Y436F eliminates Sf-stk-induced Stat3 tyrosine phosphorylation. Studies from our lab have further demonstrated that tyrosine 436 of Sf-stk is essential for the cytokine-independent growth of primary erythroblasts induced by Friend virus, by providing a docking site for Grb2, which recruits Gab2 to induce erythroleukemia. To further determine the relationship between Gab2 and Stat3, we generated fusion proteins, in which Sf-stk lacking the docking site tyrosines is fused to Gab2 or Gab1. The Sf-stk/Gab2, but not Sf-stk/Gab1, fusion protein supported Epo-independent colony formation and induced Stat3 tyrosine phosphorylation. In conclusion, these results suggest that SFKs, Gab2 and Stat3 are all important in Friend virus induced erythroleukemia mediated by Sf-stk, and that Gab2 and SFKs lie upstream of Stat3 in this signaling pathway.
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Yang, Dun, Hong Lu, Yong Hong, Timothy M. Jinks, Patricia A. Estes, and James W. Erickson. "Interpretation of X Chromosome Dose at Sex-lethalRequires Non-E-Box Sites for the Basic Helix-Loop-Helix Proteins SISB and Daughterless." Molecular and Cellular Biology 21, no. 5 (March 1, 2001): 1581–92. http://dx.doi.org/10.1128/mcb.21.5.1581-1592.2001.
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ABSTRACT For Drosophila melanogaster flies, sexual fate is determined by the X chromosome number. The basic helix-loop-helix protein product of the X-linked sisterlessB(sisB or scute) gene is a key indicator of the X dose and functions to activate the switch gene Sex-lethal (Sxl) in female (XX), but not in male (XY), embryos. Zygotically expressed sisB and maternal daughterless (da)proteins are known to form heterodimers that bind E-box sites and activate transcription. We examined SISB-Da binding atSxl by using footprinting and gel mobility shift assays and found that SISB-Da binds numerous clustered sites in the establishment promoter SxlPe . Surprisingly, most SISB-Da sites at SxlPe differ from the canonical CANNTG E-box motif. These noncanonical sites have 6-bp CA(G/C)CCG and 7-bp CA(G/C)CTTG cores and exhibit a range of binding affinities. We show that the noncanonical sites can mediate SISB-Da-activated transcription in cell culture. P-element transformation experiments show that these noncanonical sites are essential for SxlPe activity in embryos. Together with previous deletion analysis, the data suggest that the number, affinity, and position of SISB-Da sites may all be important for the operation of the SxlPe switch. Comparisons with other dose-sensitive promoters suggest that threshold responses to diverse biological signals have common molecular mechanisms, with important variations tailored to suit particular functional requirements.
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HACKENG, Tilman M., Guido TANS, Stefan J. KOPPELMAN, Philip G. de GROOT, Jan ROSING, and Bonno N. BOUMA. "Protein C activation on endothelial cells by prothrombin activation products generated in situ: meizothrombin is a better protein C activator than α-thrombin." Biochemical Journal 319, no. 2 (October 15, 1996): 399–405. http://dx.doi.org/10.1042/bj3190399.
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The conversion of protein C into activated protein C (APC) by the thrombin-thrombomodulin complex on the surface of endothelial cells initiates an essential negative feedback reaction on blood coagulation. APC, together with its non-enzymic cofactor protein S, inactivates factors Va and VIIIa, the non-enzymic protein cofactors of the prothrombinase and intrinsic tenase complex, by proteolytic degradation. In this study we report that prothrombin activation products, generated by the prothrombinase complex on the surface of quiescent endothelial cells, are able to activate protein C. Subsequent inactivation of factor Va by the APC that was formed decreased the rate of prothrombin activation, thus demonstrating in vitro the negative feedback loop on coagulation factor activation. The anticoagulant feedback reaction of APC on the prothrombinase complex was stimulated 3–4-fold by the addition of protein S but not by thrombin-cleaved protein S or by protein S complexed with C4b-binding protein. Stimulation of endothelial cells with 50 pM tumour necrosis factor (TNF) or 500 pM interleukin 1 (IL-1) resulted in a 70% decrease in activation of protein C by exogenously added α-thrombin, which seemed to be due to down-regulation of thrombomodulin activity on the surface of endothelial cells. However, when prothrombin activation products generated in situ were allowed to activate protein C, stimulation of endothelial cells with TNF and IL-1 resulted in only a 25% decrease in activation of protein C. Stimulation with TNF or IL-1 did not affect the ability of endothelial cells to support prothrombinase activity. We investigated whether the differences in extent of protein C activation by exogenously added α-thrombin and by prothrombin activation products generated in situ were due to meizothrombin formed during prothrombin activation. Previous reports from our groups revealed that meizothrombin is generated as a transient intermediate during prothrombin activation on phospholipid vesicles and endothelial cells. Here we show that meizothrombin is at least a 6-fold better activator of protein C on the surface of endothelial cells than is α-thrombin. These results demonstrate that meizothrombin, formed during the initial phase of prothrombin activation, efficiently down-regulates both its own formation and that of thrombin.
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WACHTEL, Marco, Karl FREI, Elisabeth EHLER, Christian BAUER, Max GASSMANN, and Sergio M. GLOOR. "Extracellular signal-regulated protein kinase activation during reoxygenation is required to restore ischaemia-induced endothelial barrier failure." Biochemical Journal 367, no. 3 (November 1, 2002): 873–79. http://dx.doi.org/10.1042/bj20020746.
Full textAbstract:
During an ischaemic insult, oedema formation occurs as a consequence of increased vascular permeability. To study mechanisms leading to vascular barrier failure, endothelial cells were exposed to ischaemia (1% O2 in serum- and glucose-free medium) for 5h. In in vitro conditions, ischaemia increased paracellular permeability, disassembled actin stress fibres, displaced focal adhesion kinase (FAK) from focal adhesions and enhanced cytoskeletal association of occludin. Reoxygenation restored paracellular barrier function, actin organization and FAK distribution. The mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase (ERK) was rapidly activated after 30min, strongly inhibited after 5h of continuous ischaemia and reactivated 3 times more than control during reoxygenation. Inhibition of ERK activation during reoxygenation with U0126, an inhibitor of the ERK activator, MAPK/ERK kinase 1/2, prevented both barrier restoration and stress-fibre formation, but did not prevent recruitment of FAK to focal contacts. Under normoxic conditions, ERK inhibition led to barrier failure and disassembly of stress fibres only in the absence of serum. These results demonstrate that ERK activity is essential to rebuild a disrupted endothelial barrier after ischaemia and to maintain barrier function in cells exposed to non-ischaemic stress.
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Bonif, Marianne, Marie-Alice Meuwis, Pierre Close, Valérie Benoit, Karen Heyninck, Jean-Paul Chapelle, Vincent Bours, et al. "TNFα- and IKKβ-mediated TANK/I-TRAF phosphorylation: implications for interaction with NEMO/IKKγ and NF-κB activation." Biochemical Journal 394, no. 3 (February 24, 2006): 593–603. http://dx.doi.org/10.1042/bj20051659.
Full textAbstract:
Pro-inflammatory cytokines trigger signalling cascades leading to NF-κB (nuclear factor-κB)-dependent gene expression through IKK [IκB (inhibitory κB) kinase]-dependent phosphorylation and subsequent degradation of the IκB proteins and via induced phosphorylation of p65. These signalling pathways rely on sequentially activated kinases which are assembled by essential and non-enzymatic scaffold proteins into functional complexes. Here, we show that the pro-inflammatory cytokine TNFα (tumour necrosis factor α) promotes TANK [TRAF (TNF receptor-associated factor) family member associated NF-κB activator] recruitment to the IKK complex via a newly characterized C-terminal zinc finger. Moreover, we show that TANK is phosphorylated by IKKβ upon TNFα stimulation and that this modification negatively regulates TANK binding to NEMO (NF-κB essential modulator). Interestingly, reduced TANK expression by RNA interference attenuates TNFα-mediated induction of a subset of NF-κB target genes through decreased p65 transactivation potential. Therefore the scaffold protein TANK is required for the cellular response to TNFα by connecting upstream signalling molecules to the IKKs and p65, and its subsequent IKKβ-mediated phosphorylation may be a mechanism to terminate the TANK-dependent wave of NF-κB activation.
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Ma, Shuai, Cheng Cao, Shiyou Che, Yuejiao Wang, Dongxue Su, Shuai Liu, Wenchen Gong, et al. "PHF8-promoted TOPBP1 demethylation drives ATR activation and preserves genome stability." Science Advances 7, no. 19 (May 2021): eabf7684. http://dx.doi.org/10.1126/sciadv.abf7684.
Full textAbstract:
The checkpoint kinase ATR [ATM (ataxia-telangiectasia mutated) and rad3-related] is a master regulator of DNA damage response. Yet, how ATR activity is regulated remains to be investigated. We report here that histone demethylase PHF8 (plant homeodomain finger protein 8) plays a key role in ATR activation and replication stress response. Mechanistically, PHF8 interacts with and demethylates TOPBP1 (DNA topoisomerase 2-binding protein 1), an essential allosteric activator of ATR, under unperturbed conditions, but replication stress results in PHF8 phosphorylation and dissociation from TOPBP1. Consequently, hypomethylated TOPBP1 facilitates RAD9 (RADiation sensitive 9) binding and chromatin loading of the TOPBP1-RAD9 complex to fully activate ATR and thus safeguard the genome and protect cells against replication stress. Our study uncovers a demethylation and phosphorylation code that controls the assembly of TOPBP1-scaffolded protein complex, and provides molecular insight into non-histone methylation switch in ATR activation.
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Speijer, Han, José W. P. Govers-Riemslag, Robert F. A. Zwaal, and Jan Rosing. "Platelet Procoagulant Properties Studied with Snake Venom Prothrombin Activators." Thrombosis and Haemostasis 57, no. 03 (1987): 349–55. http://dx.doi.org/10.1055/s-0038-1651132.
Full textAbstract:
SummaryPurified snake venom prothrombin activators were used to probe the procoagulant properties of platelet membranes. Human platelets were able to stimulate prothrombin activation by the venom activators from Oxyuranus scutellatus and Notechis scutatus, while the prothrombin activator from Echis carinatus was not affected by the presence of platelets. The prothrombinconverting activity of platelets was further studied with the venom activator from Oxyuranus scutellatus and with the factor Xa-Va complex as prothrombin activating enzymes. Stimulation of platelets with collagen, collagen plus thrombin or with the Ca-ionophore A23187 resulted in a considerable increase of platelet prothrombin converting activity probed with the factor Xa-Va complex as well as with the prothrombin activator from Oxyuranus scutellatus. The stimulatory effect of activated platelets on the rates of prothrombin activation by Oxyuranus scutellatus was similar to that determined for factor Xa-Va-catalyzed prothrombin activation. Compared to non-stimulated platelets, platelets stimulated with thrombin plus collagen exposed 20-times more procoagulant sites for as well the factor Xa-Va complex, as for the venom activator from Oxyuranus scutellatus. The actual number of procoagulant sites per platelet determined with the factor Xa-Va complex was in close agreement with the number of sites determined with the venom activator. Also the time course of appearance of procoagulant activity during platelet stimulation by collagen plus thrombin was comparable for both activator complexes. Phospholipase A2 treatment of stimulated platelets resulted in an almost complete loss of their ability to stimulate prothrombin activation by the enzyme from Oxyuranus scutellatus or by factor Xa-Va complex. The findings presented in this paper suggest: a) that the factor Xa-Va complex and the prothrombin activator from Oxyuranus scutellatus recognize the same procoagulant sites on both stimulated and unstimulated platelets and b) that negatively-charged phospholipids are essential components of these procoagulant sites.
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Peluso, Heather, Julie A. Caffrey, and Stephen M. Milner. "4G/4G PAI-1 gene variant in a patient with non-healing ulcers." Journal of Epidemiological Research 2, no. 1 (November 11, 2015): 91. http://dx.doi.org/10.5430/jer.v2n1p91.
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Plasminogen activator inhibitor is a serine protease inhibitor from the serpin gene family that modulates fibrin clot breakdown.PAI-1 irreversibly inhibits tissue plasminogen activator (t-PA) and urokinase plasminogen activator (u-PA) from activatingplasminogen. PAI-1 also inhibits integrin-vitronectin and vitronectin-vitronectin interactions that are essential for cell migration,adhesion, and angiogenesis. We describe a patient, who developed chronic non-healing ulcers after minimal trauma to severalareas of his body. Genetic testing revealed the 4G/4G homozygous genotype for the polymorphism in the promoter regionof the PAI-1 gene. Increased PAI-1 activity prevents the breakdown of the fibrin clot and cell migration to remodel damagedtissue. A combination of poor clot fibrinolysis and cell recruitment to the site of injury may explain our patient’s non-healingulcers following minor traumatic injury. Early treatment with excision and skin grafting may benefit patients presenting withnon-healing ulcers and the homozygous 4G/4G PAI-1 variant. To our knowledge, there have been no reports in the literatureassociating PAI-1 overexpression and chronic non-healing wounds.
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Lin, Qiong, Jian Wang, Chandra Childress, and Wannian Yang. "The activation mechanism of ACK1 (activated Cdc42-associated tyrosine kinase 1)." Biochemical Journal 445, no. 2 (June 27, 2012): 255–64. http://dx.doi.org/10.1042/bj20111575.
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ACK [activated Cdc42 (cell division cycle 42)-associated tyrosine kinase; also called TNK2 (tyrosine kinase, non-receptor, 2)] is activated in response to multiple cellular signals, including cell adhesion, growth factor receptors and heterotrimeric G-protein-coupled receptor signalling. However, the molecular mechanism underlying activation of ACK remains largely unclear. In the present study, we demonstrated that interaction of the SH3 (Src homology 3) domain with the EBD [EGFR (epidermal growth factor receptor)-binding domain] in ACK1 forms an auto-inhibition of the kinase activity. Release of this auto-inhibition is a key step for activation of ACK1. Mutation of the SH3 domain caused activation of ACK1, independent of cell adhesion, suggesting that cell adhesion-mediated activation of ACK1 is through releasing the auto-inhibition. A region at the N-terminus of ACK1 (Leu10–Leu14) is essential for cell adhesion-mediated activation. In the activation of ACK1 by EGFR signalling, Grb2 (growth-factor-receptor-bound protein 2) mediates the interaction of ACK1 with EGFR through binding to the EBD and activates ACK1 by releasing the auto-inhibition. Furthermore, we found that mutation of Ser445 to proline caused constitutive activation of ACK1. Taken together, our studies have revealed a novel molecular mechanism underlying activation of ACK1.
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Wang, Xinjiang, Junru Wang, and Xuejun Jiang. "MdmX Protein Is Essential for Mdm2 Protein-mediated p53 Polyubiquitination." Journal of Biological Chemistry 286, no. 27 (May 13, 2011): 23725–34. http://dx.doi.org/10.1074/jbc.m110.213868.
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Genetic evidence has implicated both Mdm2 and MdmX as essential in negative regulation of p53. However, the exact role of MdmX in this Mdm2-dependent protein degradation is not well understood. Most, if not all, previous Mdm2 studies used GST-Mdm2 fusion proteins in the in vitro assays. Here, we show that the p53 polyubiquitination activity of GST-Mdm2 is conferred by the GST tag and non-GST-tagged Mdm2 only catalyzes monoubiquitination of p53 even at extremely high concentrations. We further demonstrate that MdmX is a potent activator of Mdm2, facilitating dose-dependent p53 polyubiquitination. This activation process requires the RING domains of both MdmX and Mdm2 proteins. The polyubiquitination activity of Mdm2/MdmX is Mdm2-dependent. Unlike Mdm2 or MdmX overexpression alone, co-overexpression of MdmX and Mdm2 consistently triggered p53 degradation in cells. Moreover, cellular polyubiquitination of p53 was only observable in the cytoplasm where both Mdm2 and MdmX are readily detectable. Importantly, RNAi knockdown of MdmX increased levels of endogenous p53 accompanied by reduced p53 polyubiquitination. In conclusion, our work has resolved a major confusion in the field derived from using GST-Mdm2 and demonstrated that MdmX is the cellular activator that converts Mdm2 from a monoubiquitination E3 ligase to a polyubiquitination E3 ligase toward p53. Together, our findings provide a biochemical basis for the requirement of both Mdm2 and MdmX in the dynamic regulation of p53 stability.
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Bowman, Teresa V., Rosannah C. Cameron, Kathryn S. Potts, Mia McKinstry, Varun Gupta, and Xiaoying Bai. "Sf3b1 Regulation of Jak/Stat Signaling Is Essential for Hematopoietic Stem Cell Formation." Blood 132, Supplement 1 (November 29, 2018): 1268. http://dx.doi.org/10.1182/blood-2018-99-118158.
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Abstract Hematopoietic stem cells (HSCs) maintain the hematopoietic system throughout the lifetime of an organism. During embryonic development, HSCs emerge through an endothelial-to-hematopoietic transition (EHT) from specialized hemogenic endothelial (HE) cells in the dorsal aorta. HSC fate specification depends on gene expression, which is the culmination of coordinated transcription, RNA splicing, and translation. Although transcriptional regulation of HSC fate choice is well studied, the regulatory role of RNA splicing in this process is poorly understood. Using zebrafish loss-of-function mutants for the spliceosomal component splicing factor 3b, subunit 1 (sf3b1), we identified that impaired splicing hindered HSC production. Surprisingly, we found that this constitutive splicing factor selectively regulates the fate of hemogenic endothelium while leaving the identity of closely-related non-hemogenic endothelium unperturbed. To identify Sf3b1-regulated transcripts important in EHT, we performed RNA-sequencing on purified kdrl:gfp+ endothelial cells from sf3b1 mutant and wild-type siblings at 24 hpf. Approximately 900 genes were mis-spliced, 144 of which were differentially expressed. Ingenuity Pathway Analysis identified Janus Kinase (Jak)/Signaling Transducer and Activator of Transcription (Stat) signaling, in particular Stat3, as one of the top perturbed pathways in the mis-spliced gene set. Stat3 is a transcription factor activated in response to several cytokine and inflammatory signals. To determine if altered splicing of stat3 was critical for HSC formation, we injected antisense splice-blocking morpholinos (MO) targeting the Sf3b1-sensitive stat3 exon19 into wild-type and sf3b1 heterozygous embryos, which normally generate equivalent levels of HSCs. We observed an impairment of HSC production in stat3 morpholino-injected sf3b1 heterozygotes, but not wild-type siblings, indicating a synthetic lethal interaction between sf3b1 and stat3. We also found that overexpression of a constitutively active form of Stat3 significantly suppressed the HSC defects in sf3b1 homozygous mutants. Together, these data indicate that Sf3b1-mediated splicing regulation of the Jak/Stat pathway is critical for HSC emergence. Disclosures No relevant conflicts of interest to declare.
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Sundaram, Kumaran, Srinivasan Shanmugarajan, D. Sudhaker Rao, and Sakamuri V. Reddy. "Mutant p62P392L Stimulation of Osteoclast Differentiation in Paget's Disease of Bone." Endocrinology 152, no. 11 (August 30, 2011): 4180–89. http://dx.doi.org/10.1210/en.2011-1225.
Full textAbstract:
Paget's disease of the bone (PDB) is an autosomal dominant trait with genetic heterogeneity, characterized by abnormal osteoclastogenesis. Sequestosome 1 (p62) is a scaffold protein that plays an important role in receptor activator of nuclear factor κB (RANK) signaling essential for osteoclast (OCL) differentiation. p62P392L mutation in the ubiquitin-associated (UBA) domain is widely associated with PDB; however, the mechanisms by which p62P392L stimulate OCL differentiation in PDB are not completely understood. Deubiquitinating enzyme cylindromatosis (CYLD) has been shown to negatively regulate RANK ligand-RANK signaling essential for OCL differentiation. Here, we report that CYLD binds with the p62 wild-type (p62WT), non-UBA mutant (p62A381V) but not with the UBA mutant (p62P392L) in OCL progenitor cells. Also, p62P392L induces expression of c-Fos (2.8-fold) and nuclear factor of activated T cells c1 (6.0-fold) transcription factors critical for OCL differentiation. Furthermore, p62P392L expression results in accumulation of polyubiquitinated TNF receptor-associated factor (TRAF)6 and elevated levels of phospho-IκB during OCL differentiation. Retroviral transduction of p62P392L/CYLD short hairpin RNA significantly increased TRAP positive multinucleated OCL formation/bone resorption activity in mouse bone marrow cultures. Thus, the p62P392L mutation abolished CYLD interaction and enhanced OCL development/bone resorption activity in PDB.
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Keller, P., N. Vollaard, J. Babraj, D. Ball, D. A. Sewell, and J. A. Timmons. "Using systems biology to define the essential biological networks responsible for adaptation to endurance exercise training." Biochemical Society Transactions 35, no. 5 (October 25, 2007): 1306–9. http://dx.doi.org/10.1042/bst0351306.
Full textAbstract:
We predict that RNA level regulation is as diverse and powerful as protein level regulation when considering physiological adaptation. Non-coding RNA molecules, such as miRNAs (microRNAs), have emerged as a powerful mechanism for post-transcriptional regulation of mRNA. In an effort to define the role of miRNA in human skeletal-muscle biology, we have initiated profiling of muscle RNA before and after endurance exercise training. The robust molecular phenotype of muscle is established using unbiased analysis strategies of the raw data, reflecting the statistical power of gene ontology and network analysis. We can thus determine the structural features of the skeletal-muscle transcriptome, identify discrete networks activated by training and utilize bioinformatics predictions to establish the interaction between non-coding RNA modulation and Affymetrix expression profiles.
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Fujita, Morihisa, Takehiko Yoko-o, Michiyo Okamoto, and Yoshifumi Jigami. "GPI7Involved in Glycosylphosphatidylinositol Biosynthesis Is Essential for Yeast Cell Separation." Journal of Biological Chemistry 279, no. 50 (September 27, 2004): 51869–79. http://dx.doi.org/10.1074/jbc.m405232200.
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GPI7is involved in adding ethanolaminephosphate to the second mannose in the biosynthesis of glycosylphosphatidylinositol (GPI) inSaccharomyces cerevisiae. We isolatedgpi7mutants, which have defects in cell separation and a daughter cell-specific growth defect at the non-permissive temperature.WSC1,RHO2,ROM2,GFA1, andCDC5genes were isolated as multicopy suppressors ofgpi7-2mutant. Multicopy suppressors could suppress the growth defect ofgpi7mutants but not the cell separation defect. Loss of function mutations of genes involved in the Cbk1p-Ace2p pathway, which activates the expression of daughter-specific genes for cell separation after cytokinesis, bypassed the temperature-sensitive growth defect ofgpi7mutants. Furthermore, deletion ofEGT2, one of the genes controlled by Ace2p and encoding a GPI-anchored protein required for cell separation, ameliorated the temperature sensitivity of thegpi7mutant. In this mutant, Egt2p was displaced from the septal region to the cell cortex, indicating thatGPI7plays an important role in cell separation via the GPI-based modification of daughter-specific proteins inS. cerevisiae.
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Hardie, D. Grahame. "Energy sensing by the AMP-activated protein kinase and its effects on muscle metabolism." Proceedings of the Nutrition Society 70, no. 1 (November 11, 2010): 92–99. http://dx.doi.org/10.1017/s0029665110003915.
Full textAbstract:
The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status, and a regulator of energy balance at both the cellular and whole body levels. Although ubiquitously expressed, its function is best understood in skeletal muscle. AMPK contains sites that reversibly bind AMP or ATP, with an increase in cellular AMP:ATP ratio (signalling a fall in cellular energy status) switching on the kinase. In muscle, AMPK activation is therefore triggered by sustained contraction, and appears to be particularly important in the metabolic changes that occur in the transition from resistance to endurance exercise. Once activated, AMPK switches on catabolic processes that generate ATP, while switching off energy-requiring processes not essential in the short term. Thus, it acutely activates glucose uptake (by promoting translocation of the transporter GLUT4 to the membrane) and fatty acid oxidation, while switching off glycogen synthesis and protein synthesis (the later via inactivation of the mammalian target-of-rapamycin pathway). Prolonged AMPK activation also causes some of the chronic adaptations to endurance exercise, such as increased GLUT4 expression and mitochondrial biogenesis. AMPK contains a glycogen-binding domain that causes a sub-fraction to bind to the surface of the glycogen particle, and it can inhibit glycogen synthesis by phosphorylating glycogen synthase. We have shown that AMPK is inhibited by exposed non-reducing ends in glycogen. We are working on the hypothesis that this ensures that glycogen synthesis is rapidly activated when glycogen becomes depleted after exercise, but is switched off again as soon as glycogen stores are replenished.
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Bilder, D., Y. Graba, and M. P. Scott. "Wnt and TGFbeta signals subdivide the AbdA Hox domain during Drosophila mesoderm patterning." Development 125, no. 9 (May 1, 1998): 1781–90. http://dx.doi.org/10.1242/dev.125.9.1781.
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Hox genes have large expression domains yet control the formation of fine pattern elements at specific locations. We have examined the mechanism underlying subdivision of the abdominal-A (abdA) Hox domain in the visceral mesoderm. AbdA directs formation of an embryonic midgut constriction at a precise location within the broad and uniform abdA expression domain. The constriction divides the abdA domain of the midgut into two chambers, the anterior one producing the Pointed (Pnt) ETS transcription factors and the posterior one the Odd-paired (Opa) zinc finger protein. Transcription of both pnt and opa is activated by abdA but the adjacent non-overlapping patterns are not due to mutual opa-pnt regulation. Near the anterior limit of the abdA domain, two signals, Dpp (a TGFbeta) and Wg (a Wnt), are produced, in adjacent non-overlapping patterns, under Hox control in mesoderm cells. The two signals are known to regulate local mesodermal cell fates and to signal to the endoderm. We find that, in addition, they precisely subdivide the abdA domain: Wg acts upon anterior abdA domain cells to activate pnt transcription, while Dpp is essential in the same region to prevent abdA from activating opa transcription. pnt activation is required to determine the appropriate numbers of mesodermal cells in the third midgut chamber.
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Hagerty, Laura, Douglas H. Weitzel, Jenica Chambers, Christopher N. Fortner, Matthew H. Brush, David Loiselle, Hiroshi Hosoya, and Timothy A. J. Haystead. "ROCK1 Phosphorylates and Activates Zipper-interacting Protein Kinase." Journal of Biological Chemistry 282, no. 7 (December 8, 2006): 4884–93. http://dx.doi.org/10.1074/jbc.m609990200.
Full textAbstract:
Zipper-interacting protein kinase (ZIPK) regulates Ca2+-independent phosphorylation of both smooth muscle (to regulate contraction) and non-muscle myosin (to regulate non-apoptotic cell death) through either phosphorylation and inhibition of myosin phosphatase, the myosin phosphatase inhibitor CPI17, or direct phosphorylation of myosin light chain. ZIPK is regulated by multisite phosphorylation. Phosphorylation at least three sites Thr-180, Thr-225, and Thr-265 has been shown to be essential for full activity, whereas phosphorylation at Thr-299 regulates its intracellular localization. Herein we utilized an unbiased proteomics screen of smooth muscle extracts with synthetic peptides derived from the sequence of the regulatory phosphorylation sites of the enzyme to identify the protein kinases that might regulate ZIPK activity in vivo. Discrete kinase activities toward Thr-265 and Thr-299 were defined and identified by mass spectrometry as Rho kinase 1 (ROCK1). In vitro, ROCK1 showed a high degree of substrate specificity toward native ZIPK, both stoichiometrically phosphorylating the enzyme at Thr-265 and Thr-299 as well as bringing about activation. In HeLa cells, coexpression of ZIPK with ROCK1 altered the ROCK-induced phenotype of focused stress fiber pattern to a Rho-like phenotype of parallel stress fiber pattern. This effect was also dependent upon phosphorylation at Thr-265. Our findings provide a new regulatory pathway in smooth muscle and non-muscle cells whereby ROCK1 phosphorylates and regulates ZIP kinase.
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Chernov-Rogan, Tania, Tianbo Li, Gang Lu, Henry Verschoof, Kuldip Khakh, Steven W. Jones, Maureen H. Beresini, et al. "Mechanism-specific assay design facilitates the discovery of Nav1.7-selective inhibitors." Proceedings of the National Academy of Sciences 115, no. 4 (January 8, 2018): E792—E801. http://dx.doi.org/10.1073/pnas.1713701115.
Full textAbstract:
Many ion channels, including Nav1.7, Cav1.3, and Kv1.3, are linked to human pathologies and are important therapeutic targets. To develop efficacious and safe drugs, subtype-selective modulation is essential, but has been extremely difficult to achieve. We postulate that this challenge is caused by the poor assay design, and investigate the Nav1.7 membrane potential assay, one of the most extensively employed screening assays in modern drug discovery. The assay uses veratridine to activate channels, and compounds are identified based on the inhibition of veratridine-evoked activities. We show that this assay is biased toward nonselective pore blockers and fails to detect the most potent, selective voltage-sensing domain 4 (VSD4) blockers, including PF-05089771 (PF-771) and GX-936. By eliminating a key binding site for pore blockers and replacing veratridine with a VSD-4 binding activator, we directed the assay toward non–pore-blocking mechanisms and discovered Nav1.7-selective chemical scaffolds. Hence, we address a major hurdle in Nav1.7 drug discovery, and this mechanistic approach to assay design is applicable to Cav3.1, Kv1.3, and many other ion channels to facilitate drug discovery.
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Cramm, R., A. Büsch, and K. Strube. "NO-dependent transcriptional activation of gene expression in Ralstonia eutropha H16." Biochemical Society Transactions 34, no. 1 (January 20, 2006): 182–84. http://dx.doi.org/10.1042/bst0340182.
Full textAbstract:
The σ54-dependent transcriptional regulator NorR of Ralstonia eutropha H16 activates gene expression in response to nitric oxide (NO). The N-terminal domain of NorR is thought to be involved in signal perception. A C112S exchange within this domain abolished promoter activation by the mutated protein, indicating that Cys112 is essential for the signalling mechanism of NorR. The DNA region recognized by NorR contains three copies of a conserved element termed the NorR-box. Alteration of bases within any of the NorR-boxes resulted in a significant decrease in promoter activation. Therefore all three boxes have to be recognized by NorR to activate its target promoter. NorR controls expression of an operon that encodes a redox-active non-haem-iron protein NorA and an NO reductase NorB. NorA exerts a negative effect on signal-dependent promoter activation by NorR. Optical spectroscopy of purified NorA indicates that the reduced protein can react with NO to form a ferrous nitrosyl adduct. Hence, NO binding by NorA opens up the possibility that NorA and NorR compete for NO in the cytoplasm.
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Zhang, Hua, Bruce Levine, Nga Voong, Alan S. Wayne, Carl H. June, and Crystal L. Mackall. "Artificial Antigen Presenting Cells (aAPC) Expanded NK-Mediated Enhanced Lysis of Allogeneic Tumor Cells Regardless of HLA Class I/KIR Mismatch and Its Implication of Use in Eradicating Acute Lymphoblastic Leukemia (ALL)." Blood 114, no. 22 (November 20, 2009): 3023. http://dx.doi.org/10.1182/blood.v114.22.3023.3023.
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Abstract Abstract 3023 Poster Board II-999 NK Killer cell immunoglobulin-like receptors (KIRs) and their human leukocyte antigen (HLA) ligands play critical roles in maintaining natural killer (NK) cell tolerance, while providing surveillance against pathogens and malignant transformation. Natural killer (NK) cells have been explored as tools for adoptive anti-tumor or leukemia immunotherapy and current models hold that a mismatch or absence of KIR ligands on target cells is essential for efficient NK cell mediated cytolysis. However, new approaches are now available to activate NK cells and the role for KIR mediated signaling in regulating cytotoxicity of activated NK cells has not been well studied. In this study, aAPCs comprising IL15Ra+K562 cells engineered to express 4-1BBL activated and expanded peripheral NK cells in the presence of exogenous IL15 up to 1000-fold in 3 weeks. Compared to resting NK cells, 4-1BBL/IL15-activated NK cells upregulated TRAIL and NKp30, 44, 46 expression, and showed significantly enhanced cytotoxicity against a multitude of tumor targets including K562, Daudi, Ewing's tumors, osteosarcoma, as well as autologous tumors (50%-90% killing vs. 0%-8% with non-activated NK cells). Meanwhile we could detect little to no influence of KIR signaling in regulating cytotoxicity by aAPC activated NK cells, since sorted CD158a+ and CD158b+ activated NK cells showed similar killing of tumor cells expressing HLA group C1 (CD158b ligand) and/or C2 (CD158a ligand) antigens. In contrast, killer activating receptors (KARs) were indispensable for the cytolysis of solid pediatric tumors by aAPC-activated NK cells, since the killing was significantly inhibited by fusion proteins binding to the ligands of NKG2D, NK p30, p44, p46, p80 (KARs). About 20-40% inhibition of the killing was accomplished when all four activating receptors were blocked, though other activating receptors have not been well defined. Although acute lymphoblastic leukemia (ALL) blasts were refractory to fresh NK cytotoxicity, 4-1BBL/IL15 activated NK cells demonstrated higher lytic activities (20%-50%) against ALL blasts from either patients or cell lines. ALL blast lysis could be completely or partially inhibited by KAR-blocking fusion proteins, indicating that expression levels of KAR ligands vary among ALL cases and other solid tumors. We conclude that KIR ligand mismatch or absence is not essential for effective NK cytotoxicities on either solid tumors or ALL when fully activated NK cells are utilized. This suggests that adoptive therapy with autologous aAPC-activated NK cells may prove effective in some clinical settings, such as ALL, AML, or certain solid tumors. Further studies to assess the impact of KAR ligand expression on aAPC-activated NK killing of ALL blasts are in progress. Percentage of Activated NK Killings vs. Fresh NK's with/without KAR-Ig Fusion Proteins Activated NK (E:T=2.5:1) Fresh NK (E:T=25:1) -KAR-Ig Fc +KAR-Ig Fc SB tumor (Ewing's) 48% 30% 0.5% HOS (Osteo sarcoma) 63% 36% 0.7% Daudi (B. lymphoma) 78% 46% 0.2% REH (ALL) 54% 8% 3% Disclosures No relevant conflicts of interest to declare.
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van der Velden, Gisela J., Monique A. Vink, Ben Berkhout, and Atze T. Das. "Tat has a dual role in simian immunodeficiency virus transcription." Journal of General Virology 93, no. 10 (October 1, 2012): 2279–89. http://dx.doi.org/10.1099/vir.0.044511-0.
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Tat has a pivotal role in human and simian immunodeficiency virus (HIV and SIV) replication because it stimulates transcription by binding to the trans-activator response (TAR) element. In addition, several other Tat functions have been proposed. Most studies have focused on HIV-1 Tat and much less is known about SIV Tat. An SIVmac239 variant was constructed previously in which the Tat–TAR transcription mechanism is functionally replaced by the doxycycline-inducible Tet-On gene expression mechanism (SIV-rtTA). In this study, SIV-rtTA variants were used to analyse the functions of SIV Tat. It was shown that Tat-minus SIV-rtTA variants replicated efficiently in PM1 T-cells, ruling out an additional essential Tat function. Nevertheless, replication was suboptimal in other cells, and evolutionary pressure to repair Tat expression was documented. It was demonstrated that SIV-rtTA required Tat for optimal gene expression, despite the absence of the Tat–TAR axis. This Tat effect was lost upon replacement of the long terminal repeat promoter region by a non-related promoter. These results indicate that Tat can activate SIV transcription via TAR RNA and U3 DNA elements but has no other essential function in replication in cultured cells. The experiments were limited to cell lines and PBMCs, and did not exclude an accessory Tat function under specific conditions or in vivo.
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Miller, M. Clarke, Jacqueline D. Fetherston, Carol L. Pickett, Alexander G. Bobrov, Robert H. Weaver, Edward DeMoll, and Robert D. Perry. "Reduced synthesis of the Ybt siderophore or production of aberrant Ybt-like molecules activates transcription of yersiniabactin genes in Yersinia pestis." Microbiology 156, no. 7 (July 1, 2010): 2226–38. http://dx.doi.org/10.1099/mic.0.037945-0.
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Synthesis of the siderophore yersiniabactin (Ybt) proceeds by a mixed nonribosomal peptide synthetase/polyketide synthase mechanism. Transcription of ybt genes encoding biosynthetic and transport functions is repressed under excess iron conditions by Fur, but is also activated by Ybt via the transcriptional regulator YbtA. While mutations in most biosynthetic genes and ybtA negate transcription activation from the regulated promoters, three biosynthetic mutations do not reduce this transcriptional activation. Here we show that two of these mutants, one lacking the putative type II thioesterase (TE) YbtT and the other with a mutation in the TE domain of HMWP1, produce reduced levels of authentic Ybt that are capable of signalling activity. Alanine substitutions in two residues of YbtT that are essential for catalytic activity in other type II TEs reduced the ability of Yersinia pestis to grow under iron-chelated conditions. The third mutant, which lacks the salicylate synthase YbtS, did not make authentic Ybt but did produce a signalling molecule. Finally, a Δpgm strain of Y. pestis, which lacks essential Ybt biosynthetic genes, also produced a signalling molecule that can activate transcription of ybt genes. The non-Ybt signal molecules from these two mutants are likely separate compounds. While these compounds are not biologically relevant to normal Ybt regulation, a comparison of the structures of Ybt and other signalling molecules will help in determining the chemical structures recognized as a Ybt signal.
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Gonzalez, Angel, Angela Restrepo, and Luz E. Cano. "Role of iron in the nitric oxide-mediated fungicidal mechanism of IFN-gamma-activated murine macrophages against Paracoccidioides brasiliensis conidia." Revista do Instituto de Medicina Tropical de São Paulo 49, no. 1 (February 2007): 11–16. http://dx.doi.org/10.1590/s0036-46652007000100003.
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Iron is an essential growth element of virtually all microorganisms and its restriction is one of the mechanisms used by macrophages to control microbial multiplication. Paracoccidioides brasiliensis, the agent of paracoccidioidomycosis, an important systemic mycosis in Latin America, is inhibited in its conidia-to-yeast conversion in the absence of iron. We studied the participation of iron in the nitric oxide (NO)-mediated fungicidal mechanism against conidia. Peritoneal murine macrophages activated with 50U/mL of IFN-gamma or treated with 35 µM Deferoxamine (DEX) and infected with P. brasiliensis conidia, were co-cultured and incubated for 96 h in the presence of different concentrations of holotransferrin (HOLO) and FeS0(4). The supernatants were withdrawn in order to assess NO2 production by the Griess method. The monolayers were fixed, stained and observed microscopically. The percentage of the conidia-to-yeast transition was estimated by counting 200 intracellular propagules. IFN-gamma-activated or DEX-treated Mthetas presented marked inhibition of the conidia-to-yeast conversion (19 and 56%, respectively) in comparison with non-activated or untreated Mthetas (80%). IFN-gamma-activated macrophages produced high NO levels in comparison with the controls. Additionally, when the activated or treated-macrophages were supplemented with iron donors (HOLO or FeSO4), the inhibitory action was reversed, although NO production remained intact. These results suggest that the NO-mediated fungicidal mechanism exerted by IFN-gamma-activated macrophages against P. brasiliensis conidia, is dependent of an iron interaction.
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Tu, Tony C., Nicholas K. Brown, Tae-Jin Kim, Joanna Wroblewska, Xuanming Yang, Xiaohuan Guo, Seoyun Hyunji Lee, Vinay Kumar, Kyung-Mi Lee, and Yang-Xin Fu. "CD160 is essential for NK-mediated IFN-γ production." Journal of Experimental Medicine 212, no. 3 (February 23, 2015): 415–29. http://dx.doi.org/10.1084/jem.20131601.
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NK-derived cytokines play important roles for natural killer (NK) function, but how the cytokines are regulated is poorly understood. CD160 is expressed on activated NK or T cells in humans but its function is unknown. We generated CD160-deficient mice to probe its function. Although CD160−/− mice showed no abnormalities in lymphocyte development, the control of NK-sensitive tumors was severely compromised in CD160−/− mice. Surprisingly, the cytotoxicity of NK cells was not impaired, but interferon-γ (IFN-γ) secretion by NK cells was markedly reduced in CD160−/− mice. Functionally targeting CD160 signaling with a soluble CD160-Ig also impaired tumor control and IFN-γ production, suggesting an active role of CD160 signaling. Using reciprocal bone marrow transfer and cell culture, we have identified the intrinsic role of CD160 on NK cells, as well as its receptor on non-NK cells, for regulating cytokine production. To demonstrate sufficiency of the CD160+ NK cell subset in controlling NK-dependent tumor growth, intratumoral transfer of the CD160+ NK fraction led to tumor regression in CD160−/− tumor-bearing mice, indicating demonstrable therapeutic potential for controlling early tumors. Therefore, CD160 is not only an important biomarker but also functionally controls cytokine production by NK cells.
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Weiler, Elmar, Farhad Khalil-Manesh, and Harvey C. Gonick. "Effects of Lead and a Low-Molecular-Weight Endogenous Plasma Inhibitor on the Kinetics of Sodium—Potassium-Activated Adenosine Triphosphatase and Potassium-Activated p-Nitrophenylphosphatase." Clinical Science 79, no. 2 (August 1, 1990): 185–92. http://dx.doi.org/10.1042/cs0790185.
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1. Lead, ouabain and an endogenous plasma inhibitor were all found to be potent inhibitors of purified hog cerebral cortex sodium—potassium-activated adenosine triphosphatase and potassium-stimulated p-nitrophenylphosphatase. 2. The kinetic characteristics of inhibition of both enzymes by lead and the endogenous plasma inhibitor differed in several respects. For sodium—potassium-activated adenosine triphosphatase, lead and the endogenous plasma inhibitor were non-competitive inhibitors with respect to potassium; lead was competitive with respect to sodium, whereas the endogenous plasma inhibitor had no effect; lead was competitive with respect to magnesium adenosine triphosphate, whereas the endogenous plasma inhibitor was uncompetitive. For potassium-activated p-nitrophenylphosphatase, both lead and the endogenous plasma inhibitor were competitive with respect to potassium; lead showed a mixed type of inhibition with respect to p-nitrophenylphosphate, whereas the endogenous plasma inhibitor was non-competitive. 3. Lead and the endogenous plasma inhibitor exhibited synergistic inhibitory activity on sodium—potassium-activated adenosine triphosphatase. 4. These results suggest that lead could play a contributory role in the pathogenesis of essential hypertension via an additive inhibition of vascular smooth muscle sodium—potassium-activated adenosine triphosphatase.
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Kim, Mihwa, Liza Morales, Ik-Soon Jang, Yong-Yeon Cho, and Dae Kim. "Protein Tyrosine Phosphatases as Potential Regulators of STAT3 Signaling." International Journal of Molecular Sciences 19, no. 9 (September 11, 2018): 2708. http://dx.doi.org/10.3390/ijms19092708.
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The signal transducer and activator of transcription 3 (STAT3) protein is a major transcription factor involved in many cellular processes, such as cell growth and proliferation, differentiation, migration, and cell death or cell apoptosis. It is activated in response to a variety of extracellular stimuli including cytokines and growth factors. The aberrant activation of STAT3 contributes to several human diseases, particularly cancer. Consequently, STAT3-mediated signaling continues to be extensively studied in order to identify potential targets for the development of new and more effective clinical therapeutics. STAT3 activation can be regulated, either positively or negatively, by different posttranslational mechanisms including serine or tyrosine phosphorylation/dephosphorylation, acetylation, or demethylation. One of the major mechanisms that negatively regulates STAT3 activation is dephosphorylation of the tyrosine residue essential for its activation by protein tyrosine phosphatases (PTPs). There are seven PTPs that have been shown to dephosphorylate STAT3 and, thereby, regulate STAT3 signaling: PTP receptor-type D (PTPRD), PTP receptor-type T (PTPRT), PTP receptor-type K (PTPRK), Src homology region 2 (SH-2) domain-containing phosphatase 1(SHP1), SH-2 domain-containing phosphatase 2 (SHP2), MEG2/PTP non-receptor type 9 (PTPN9), and T-cell PTP (TC-PTP)/PTP non-receptor type 2 (PTPN2). These regulators have great potential as targets for the development of more effective therapies against human disease, including cancer.
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Wang, Mong-Lien, Yi-Fan Hsu, Chih-Hsuan Liu, Ya-Ling Kuo, Yi-Chen Chen, Yi-Chen Yeh, Hsiang-Ling Ho, Yu-Chung Wu, Teh-Ying Chou, and Cheng-Wen Wu. "Low-Dose Nicotine Activates EGFR Signaling via α5-nAChR and Promotes Lung Adenocarcinoma Progression." International Journal of Molecular Sciences 21, no. 18 (September 17, 2020): 6829. http://dx.doi.org/10.3390/ijms21186829.
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Nicotine in tobacco smoke is considered carcinogenic in several malignancies including lung cancer. The high incidence of lung adenocarcinoma (LAC) in non-smokers, however, remains unexplained. Although LAC has long been less associated with smoking behavior based on previous epidemiological correlation studies, the effect of environmental smoke contributing to low-dose nicotine exposure in non-smoking population could be underestimated. Here we provide experimental evidence of how low-dose nicotine promotes LAC growth in vitro and in vivo. Screening of nicotinic acetylcholine receptor subunits in lung cancer cell lines demonstrated a particularly high expression level of nicotinic acetylcholine receptor subunit α5 (α 5-nAChR) in LAC cell lines. Clinical specimen analysis revealed up-regulation of α 5-nAChR in LAC tumor tissues compared to non-tumor counterparts. In LAC cell lines α 5-nAChR interacts with epidermal growth factor receptor (EGFR), positively regulates EGFR pathway, enhances the expression of epithelial-mesenchymal transition markers, and is essential for low-dose nicotine-induced EGFR phosphorylation. Functionally, low-dose nicotine requires α 5-nAChR to enhance cell migration, invasion, and proliferation. Knockdown of α 5-nAChR inhibits the xenograft tumor growth of LAC. Clinical analysis indicated that high level of tumor α 5-nAChR is correlated with poor survival rates of LAC patients, particularly in those expressing wild-type EGFR. Our data identified α 5-nAChR as an essential mediator for low-dose nicotine-dependent LAC progression possibly through signaling crosstalk with EGFR, supporting the involvement of environmental smoke in tumor progression in LAC patients.
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Rocha, Kathy, Gwenny Fuhler, Joseph Johnson, Justine Clark, Gisela Caceres, Lubomir Sokol, and Alan F. List. "Membrane Raft Localization Is Critical for Erythropoietin Receptor Signaling." Blood 114, no. 22 (November 20, 2009): 2506. http://dx.doi.org/10.1182/blood.v114.22.2506.2506.
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Abstract Abstract 2506 Poster Board II-483 Erythropoietin (EPO) is the principal regulator of erythroid progenitor proliferation, differentiation, and survival. Upon ligand engagement, the EPO-receptor (R) homodimerizes to activate the tyrosine kinases, Janus kinase-2 (JAK-2) and Lyn, which in turn phosphorylate the signal transducer and activator of transcription (STAT)-5. Although recent investigations have identified key negative regulators of the EPO-R signal, little is known about the membrane localization and dynamic control of receptor signal fidelity. Here we show a critical role for membrane raft microdomains in the creation of signaling platforms that are essential for signal integrity. Using UT-7 cells, we showed that recombinant human EPO (rhuEPO) stimulation rapidly induced raft formation and aggregation. Confocal microscopy quantitation of the raft ganglioside GM-1 fluorescence showed that raft aggregates increased from a mean of 4.3 ± 1.4 (SE) per cell to 25.6 ± 3.2 aggregates per cell after cytokine stimulation (p≤0.001), accompanied by a greater than 3-fold increase in cluster size (mean stimulated to untreated aggregate size ratio: 3.33 ± 0.11, p≤0.001). Isolation and immunoblot analysis of detergent insoluble raft fractions showed that the EPO-R translocated to lipid rafts after EPO stimulation of UT-7 cells. Confocal microscopy confirmed translocation of the EPO-R into membrane rafts in EPO-stimulated UT-7 cells and normal erythroid bursts. Receptor recruitment into rafts was accompanied by Jak2, Lyn and STAT5 loading into membrane raft fractions upon EPO stimulation. Treatment with methyl-β-cyclodextrin (MBCD) to deplete raft cholesterol and disrupt raft integrity extinguished EPO-induced STAT5 phosphorylation in UT-7 cells and human bone marrow progenitors. Similarly, membrane cholesterol-sequestration by nystatin markedly reduced EPO induced STAT5 phosphorylation in UT-7 cells. MBCD pretreatment of these cells prior to stimulation with phorbol 12-myristate 13-acetate (PMA) did not alter mitogen-activated protein kinase (MAPK) phosphorylation, indicating preservation of non-receptor, non-raft signal integrity despite depletion of cholesterol rich microdomains. These data establish a critical role for membrane raft microdomains in the recruitment and physical assembly of the EPO-R and its signaling intermediates into discrete platforms necessary to optimize signal integrity Disclosures: No relevant conflicts of interest to declare.
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Walker, William H. "Non-classical actions of testosterone and spermatogenesis." Philosophical Transactions of the Royal Society B: Biological Sciences 365, no. 1546 (May 27, 2010): 1557–69. http://dx.doi.org/10.1098/rstb.2009.0258.
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Testosterone is essential to maintain spermatogenesis and male fertility. In the absence of testosterone stimulation, spermatogenesis does not proceed beyond the meiosis stage. After withdrawal of testosterone, germ cells that have progressed beyond meiosis detach from supporting Sertoli cells and die, whereas mature sperm cannot be released from Sertoli cells resulting in infertility. The classical mechanism of testosterone action in which testosterone activates gene transcription by causing the androgen receptor to translocate to and bind specific DNA regulatory elements does not appear to fully explain testosterone regulation of spermatogenesis. This review discusses two non-classical testosterone signalling pathways in Sertoli cells and their potential effects on spermatogenesis. Specifically, testosterone-mediated activation of phospholipase C and calcium influx into Sertoli cells is described. Also, testosterone activation of Src, EGF receptor and ERK kinases as well as the activation of the CREB transcription factor and CREB-mediated transcription is reviewed. Regulation of germ cell adhesion to Sertoli cells and release of mature sperm from Sertoli cells by kinases regulated by the non-classical testosterone pathway is discussed. The evidence accumulated suggests that classical and non-classical testosterone signalling contribute to the maintenance of spermatogenesis and male fertility.
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Zhao, Mengmeng, Kun Song, Wenzhuo Hao, Lingyan Wang, Girish Patil, Qingmei Li, Lingling Xu, et al. "Non-proteolytic ubiquitination of OTULIN regulates NF-κB signaling pathway." Journal of Molecular Cell Biology 12, no. 3 (September 3, 2019): 163–75. http://dx.doi.org/10.1093/jmcb/mjz081.
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Abstract NF-κB signaling regulates diverse processes such as cell death, inflammation, immunity, and cancer. The activity of NF-κB is controlled by methionine 1-linked linear polyubiquitin, which is assembled by the linear ubiquitin chain assembly complex (LUBAC) and the ubiquitin-conjugating enzyme UBE2L3. Recent studies found that the deubiquitinase OTULIN breaks the linear ubiquitin chain, thus inhibiting NF-κB signaling. Despite the essential role of OTULIN in NF-κB signaling has been established, the regulatory mechanism for OTULIN is not well elucidated. To discover the potential regulators of OTULIN, we analyzed the OTULIN protein complex by proteomics and revealed several OTULIN-binding proteins, including LUBAC and tripartite motif-containing protein 32 (TRIM32). TRIM32 is known to activate NF-κB signaling, but the mechanism is not clear. Genetic complement experiments found that TRIM32 is upstream of OTULIN and TRIM32-mediated NF-κB activation is dependent on OTULIN. Mutagenesis of the E3 ligase domain showed that the E3 ligase activity is essential for TRIM32-mediated NF-κB activation. Further experiments found that TRIM32 conjugates polyubiquitin onto OTULIN and the polyubiquitin blocks the interaction between HOIP and OTULIN, thereby activating NF-κB signaling. Taken together, we report a novel regulatory mechanism by which TRIM32-mediated non-proteolytic ubiquitination of OTULIN impedes the access of OTULIN to the LUBAC and promotes NF-κB activation.
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Geisler, Caroline E., Kendra E. Miller, Susma Ghimire, and Benjamin J. Renquist. "The Role of GPR109a Signaling in Niacin Induced Effects on Fed and Fasted Hepatic Metabolism." International Journal of Molecular Sciences 22, no. 8 (April 13, 2021): 4001. http://dx.doi.org/10.3390/ijms22084001.
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Signaling through GPR109a, the putative receptor for the endogenous ligand β-OH butyrate, inhibits adipose tissue lipolysis. Niacin, an anti-atherosclerotic drug that can induce insulin resistance, activates GPR109a at nM concentrations. GPR109a is not essential for niacin to improve serum lipid profiles. To better understand the involvement of GPR109a signaling in regulating glucose and lipid metabolism, we treated GPR109a wild-type (+/+) and knockout (−/−) mice with repeated overnight injections of saline or niacin in physiological states characterized by low (ad libitum fed) or high (16 h fasted) concentrations of the endogenous ligand, β-OH butyrate. In the fed state, niacin increased expression of apolipoprotein-A1 mRNA and decreased sterol regulatory element-binding protein 1 mRNA independent of genotype, suggesting a possible GPR109a independent mechanism by which niacin increases high-density lipoprotein (HDL) production and limits transcriptional upregulation of lipogenic genes. Niacin decreased fasting serum non-esterified fatty acid concentrations in both GPR109a +/+ and −/− mice. Independent of GPR109a expression, niacin blunted fast-induced hepatic triglyceride accumulation and peroxisome proliferator-activated receptor α mRNA expression. Although unaffected by niacin treatment, fasting serum HDL concentrations were lower in GPR109a knockout mice. Surprisingly, GPR109a knockout did not affect glucose or lipid homeostasis or hepatic gene expression in either fed or fasted mice. In turn, GPR109a does not appear to be essential for the metabolic response to the fasting ketogenic state or the acute effects of niacin.
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MATSUURA, Kazuya, Yoshiyuki TAMADA, Yoshihiro DEYASHIKI, Yoshiyuki MIYABE, Masayuki NAKANISHI, Isao OHYA, and Akira HARA. "Activation of human liver 3α-hydroxysteroid dehydrogenase by sulphobromophthalein." Biochemical Journal 313, no. 1 (January 1, 1996): 179–84. http://dx.doi.org/10.1042/bj3130179.
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Human liver contains at least two isoenzymes (DD2 and DD4) of 3α-hydroxysteroid/dihydrodiol dehydrogenase. The NADP(H)-linked oxidoreductase activities of DD4 were activated more than 4-fold by sulphobromophthalein at concentrations above 20 μM and under physiological pH conditions. Sulphobromophthalein did not stimulate the activities of DD2 and human liver aldehyde reductase, which are functionally and/or structurally related to DD4. No stimulatory effect on the activity of DD4 was observed with other organic anions such as Indocyanine Green, haematin and Rose Bengal. The binding of sulphobromophthalein to DD4 was instantaneous and reversible, and was detected by fluorescence and ultrafiltration assays. The activation by sulphobromophthalein decreased the activation energy in the dehydrogenation reaction for the enzyme, and increased both kcat. and Km values for the coenzymes and substrates. Kinetic analyses with respect to concentrations of NADP+ and (S)-(+)-indan-1-ol indicated that sulphobromophthalein was a non-essential activator of mixed type showing a dissociation constant of 2.6 μM. Thus, the human 3α-hydroxysteroid dehydrogenase isoenzyme has a binding site specific to sulphobromophthalein, and the hepatic metabolism mediated by this isoenzyme may be influenced when this drug is administered.
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Richter, HE, T. Albrektsen, and N. Billestrup. "The role of signal transducer and activator of transcription 5 in the inhibitory effects of GH on adipocyte differentiation." Journal of Molecular Endocrinology 30, no. 2 (April 1, 2003): 139–50. http://dx.doi.org/10.1677/jme.0.0300139.
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GH inhibits primary rat preadipocyte differentiation and expression of late genes required for terminal differentiation. Here we show that GH-mediated inhibition of fatty acid-binding protein aP2 gene expression correlates with the activation of the Janus kinase-2/signal transducer and activator of transcription (STAT)-5 signalling pathway. Within minutes of treatment, GH induced the tyrosine phosphorylation, nuclear localization and DNA binding of STAT5. Importantly, there was no evidence that STAT5 acted via an interaction with peroxisome proliferator-activated receptor gamma. To further understand the mechanism of STAT5 action, we reconstituted the inhibition of aP2 in a non-adipogenic cell line. Using this system, we showed that the ability of GH to inhibit a 520 bp aP2 reporter was largely dependent upon the presence of either STAT5A or STAT5B. Mutant analysis confirmed that the tyrosine phosphorylation of STAT5 was essential for this signalling. However, STAT5's C-terminal transactivation domain was fully dispensable for this inhibition. Taken together, these data confirm a key regulatory role of STAT5 in adipose tIssue and point to STAT5 as the repressing modulator of GH-mediated inhibition in primary preadipocytes.
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Pillai, Beena, Vinaya Sampath, Nimisha Sharma, and Parag Sadhale. "Rpb4, a Non-essential Subunit of Core RNA Polymerase II ofSaccharomyces cerevisiaeIs Important for Activated Transcription of a Subset of Genes." Journal of Biological Chemistry 276, no. 33 (May 29, 2001): 30641–47. http://dx.doi.org/10.1074/jbc.m010952200.
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Koenen, Mascha, Stephan Culemann, Sabine Vettorazzi, Giorgio Caratti, Lucien Frappart, Wolfgang Baum, Gerhard Krönke, Ulrike Baschant, and Jan P. Tuckermann. "Glucocorticoid receptor in stromal cells is essential for glucocorticoid-mediated suppression of inflammation in arthritis." Annals of the Rheumatic Diseases 77, no. 11 (July 11, 2018): 1610–18. http://dx.doi.org/10.1136/annrheumdis-2017-212762.
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BackgroundGlucocorticoid (GC) therapy is frequently used to treat rheumatoid arthritis due to potent anti-inflammatory actions of GCs. Direct actions of GCs on immune cells were suggested to suppress inflammation.ObjectivesDefine the role of the glucocorticoid receptor (GR) in stromal cells for suppression of inflammatory arthritis.MethodsBone marrow chimeric mice lacking the GR in the hematopoietic or stromal compartment, respectively, and mice with impaired GR dimerisation (GRdim) were analysed for their response to dexamethasone (DEX, 1 mg/kg) treatment in serum transfer-induced arthritis (STIA). Joint swelling, cell infiltration (histology), cytokines, cell composition (flow cytometry) and gene expression were analysed and RNASeq of wild type and GRdim primary murine fibroblast-like synoviocytes (FLS) was performed.ResultsGR deficiency in immune cells did not impair GC-mediated suppression of STIA. In contrast, mice with GR-deficient or GR dimerisation-impaired stromal cells were resistant to GC treatment, despite efficient suppression of cytokines. Intriguingly, in mice with impaired GR function in the stromal compartment, GCs failed to stimulate non-classical, non-activated macrophages (Ly6Cneg, MHCIIneg) and associated anti-inflammatory markers CD163, CD36, AnxA1, MerTK and Axl. Mice with GR deficiency in FLS were partially resistant to GC-induced suppression of STIA. Accordingly, RNASeq analysis of DEX-treated GRdim FLS revealed a distinct gene signature indicating enhanced activity and a failure to reduce macrophage inflammatory protein (Mip)-1α and Mip-1β.ConclusionWe report a novel anti-inflammatory mechanism of GC action that involves GR dimerisation-dependent gene regulation in non-immune stromal cells, presumably FLS. FLS control non-classical, anti-inflammatory polarisation of macrophages that contributes to suppression of inflammation in arthritis.