Podcasts about phosphatase

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Best podcasts about phosphatase

Latest podcast episodes about phosphatase

Authentic Biochemistry
Biochemical Mosaic I PA-phosphatase c.8. Membrane fusion and the dynamics of mitochondrial reduction vs. proliferation; the PA phosphatase in lipid metabolism. DJGPhD. 16.2.24 Authentic Biochemistry

Authentic Biochemistry

Play Episode Listen Later Feb 17, 2024 29:48


References Dr Guerra membrane lectures archive FASEB J. 2021 Jun; 35(6): e21620. Vivaldi, A. 1711. Concerto for 4 Violins in B minor RV 580 https://youtu.be/SY3Kxf7ZTeI?si=JLQXMtK06Y6Yfu_k --- Send in a voice message: https://podcasters.spotify.com/pod/show/dr-daniel-j-guerra/message Support this podcast: https://podcasters.spotify.com/pod/show/dr-daniel-j-guerra/support

Authentic Biochemistry
Biochemical Mosaic I. Phosphatidic acid Phosphatase. c.7. Mutational reversal of IDH1 leads to NADPH depletion concomitant with potent anti-PHD enantiomeric 2-hydroxyglutarate obtaining pseudohypoxia.

Authentic Biochemistry

Play Episode Listen Later Feb 16, 2024 30:00


References FEMS Microbiol Rev.1998. Oct;22(4):255-75 Discoveries(Craiova). 2017 Jul-Sep; 5(3): e77. J Cell Mol Med. 2015 Jul; 19(7): 1427–1440. Oncogene. 2017 Mar 23; 36(12): 1607–1618. Bach, JS. 1742. Kunst der Fuge , BWV 1080; Marta Czech https://youtu.be/p1Sq1HOYglU?si=2GMF7kf3dLW4rr2O Lennon and MCartney.1968. "Martha my Dear" https://youtu.be/RXawa90YU2s?si=dUPDtTdm4UqrgWit --- Send in a voice message: https://podcasters.spotify.com/pod/show/dr-daniel-j-guerra/message Support this podcast: https://podcasters.spotify.com/pod/show/dr-daniel-j-guerra/support

Authentic Biochemistry
Biochemical Mosaic I. The PA phosphatase c.6. Alteration of the TCA pathway intermediates and hypoxia/nutrient deficiency control HIF1α stability indirectly regulating IMM protein and lipid turnover.

Authentic Biochemistry

Play Episode Listen Later Feb 13, 2024 30:00


References Front. Oncol. 2017. 26 November.V.7. Oncogene. 2017 Mar 23; 36(12): 1607–1618. Schubert, F. 1819 Piano Quintet in A major, D. 667/ "Trout" https://youtu.be/syod5rkFdNY?si=5F3rexiou7dTifYg Hunter, R., Garcia , J. 1970 "Ripple" https://youtu.be/xofhZx9eWUQ?si=tpOAMzyW5qUy01P_ --- Send in a voice message: https://podcasters.spotify.com/pod/show/dr-daniel-j-guerra/message Support this podcast: https://podcasters.spotify.com/pod/show/dr-daniel-j-guerra/support

Authentic Biochemistry
The Biochemical Mosaic I. Phosphatidic Acid Phosphatase c.5. Tumor cells adapt bioenergetic fuel economy toward cell immortalization via re-crafting of mitochondrial IM lipid speciation.DJGPhD.12Feb24

Authentic Biochemistry

Play Episode Listen Later Feb 12, 2024 30:00


References Cell Metabolism 2019. 30: 720–734 Nature 2019. 575: 361–365 Squire, C. 1978. "Onward" Yes. --- Send in a voice message: https://podcasters.spotify.com/pod/show/dr-daniel-j-guerra/message Support this podcast: https://podcasters.spotify.com/pod/show/dr-daniel-j-guerra/support

Authentic Biochemistry
Biochemical Mosaic I. Phosphatidic Acid Phosphatase c.4 GLN utilization and an IMM protease regulate mitochondrial involvement in cancer growth. DJGPhD. Authentic Biochemistry 10Feb24

Authentic Biochemistry

Play Episode Listen Later Feb 11, 2024 30:00


References Cell Death & Disease 2022. volume 13.Article number: 444. Nature 2019. volume 575, pages 361–365 Scientific Reports 2021. volume 11, Article number: 16512 Experimental & Molecular Medicine 2020. volume 52, pages 1496–1516 Mozart, WA. 1788. Piano Trio in G Major, KV 564 https://youtu.be/oOEuVtr8sF0?si=3-9RK1XkGkSWne7I --- Send in a voice message: https://podcasters.spotify.com/pod/show/dr-daniel-j-guerra/message Support this podcast: https://podcasters.spotify.com/pod/show/dr-daniel-j-guerra/support

Authentic Biochemistry
Biochemical Mosaic Event Ontologies I. Phosphatidic Acid Phosphatase. c.3 Indirect consequences of mitochondrial proteomic variation potentially obtains GLN-linked Warburg glycolysis preventing ROS.

Authentic Biochemistry

Play Episode Listen Later Feb 9, 2024 29:48


References Cell Metabolism 2019. 30, 720–734 Redox Biology2013. Volume 1, Issue 1: 427-432 Nature 2019. volume 575. 361–365 Mozart, WA, 1779.Sinfonia Concertante Es-Dur, KV 364 https://youtu.be/iGDfj5uM_qA?si=bKNmj1r1o9zSMaA7 Lennon-McCartney. 1966. "Im Only Sleeping" https://youtu.be/sAG3m3p9UeI?si=oT6Luxnc6eFS_pEq --- Send in a voice message: https://podcasters.spotify.com/pod/show/dr-daniel-j-guerra/message Support this podcast: https://podcasters.spotify.com/pod/show/dr-daniel-j-guerra/support

Authentic Biochemistry
Biochemical Mosaic Event Ontologies I. Phosphatidic Acid Phosphatase. Autophagy commitment is linked to mTORC1 v. AMPK oppostional kinase networks maintaining proteostasis and lipid homeostasis.DJGPhD

Authentic Biochemistry

Play Episode Listen Later Feb 9, 2024 30:00


References Nature 2019.volume 575, pages 361–365 Cells 2022, 11(21), 3339 Guerra: Polypeptide, endomembranous lipid homeostasis, and bioenergetics lecture archives. --- Send in a voice message: https://podcasters.spotify.com/pod/show/dr-daniel-j-guerra/message Support this podcast: https://podcasters.spotify.com/pod/show/dr-daniel-j-guerra/support

Authentic Biochemistry
Biochemical Mosaic Event Ontologies I. Phosphatidic Acid Phosphatase. Prologomena c.1.

Authentic Biochemistry

Play Episode Listen Later Feb 7, 2024 30:00


References J Cell Biol. 2014 Mar 17; 204(6):919–929 Nature 2019. Volume 575, pages 361–365. Redox Biology 2013. Volume 1, Issue 1. Pages 427-432.Madrigali Guerrieri et Amorosi {Libro Ottavo} https://youtu.be/2TjkhWARBVE?si=-ZI_kVa5WZX_9qnB --- Send in a voice message: https://podcasters.spotify.com/pod/show/dr-daniel-j-guerra/message Support this podcast: https://podcasters.spotify.com/pod/show/dr-daniel-j-guerra/support

Empowered Patient Podcast
Developing Phosphatase Inhibitors to Treat Rare Neurodevelopmental Disorder with Andreas Grill DepYmed TRANSCRIPT

Empowered Patient Podcast

Play Episode Listen Later Nov 20, 2023


Andreas Grill, President and CEO of DepYmed, discusses protein tyrosine phosphatase-targeted drugs, a new class of drugs. With a focus on the specific enzyme PTP1B, DepYmed discovered orally bioavailable molecules that inhibit PTP1B, targeting the signal transaction pathway. They are initially testing to treat Rett syndrome, a rare disease with no current therapy while exploring the use of PTP1B inhibitors to treat inflammatory diseases, cancer, diabetes, and neurological diseases.   Andreas elaborates, "In DepYmed, we're focused on a specific enzyme. It's PTP1B. It's part of a family of enzymes called protein tyrosine phosphatases, and, in particular, we're looking at PTP1B. It's a metabolic regulatory enzyme that regulates signal transduction between cells and how cells communicate with each other. It's been worked on in the '90s and early 2000s. A couple of companies were working on the target, and they failed in the target, mainly because they couldn't create an orally bioavailable compound that would inhibit the PTP1B enzyme itself." "So that was one of the holy grails that we were able to find, where we were able to discover molecules that were orally bioavailable and would inhibit PTP1B. It was a game-changer when it came to the therapeutics around the target of PTP1B. Much of this work came out of Dr. Nicholas Tonks' laboratory out of Cold Spring Harbor Laboratory. We are in close collaboration with Nick and his team at Cold Spring Harbor, developing this new area of PTP1B inhibitors targeting the signal transaction pathway." #DepYmed #RettSyndrome #DPM1003 #PTP1B #Phosphatases #PhosphatasesInhibitors #RareDisease DepYmed.com Listen to the podcast here

Empowered Patient Podcast
Developing Phosphatase Inhibitors to Treat Rare Neurodevelopmental Disorder with Andreas Grill DepYmed

Empowered Patient Podcast

Play Episode Listen Later Nov 20, 2023 19:57


Andreas Grill, President and CEO of DepYmed, discusses protein tyrosine phosphatase-targeted drugs, a new class of drugs. With a focus on the specific enzyme PTP1B, DepYmed discovered orally bioavailable molecules that inhibit PTP1B, targeting the signal transaction pathway. They are initially testing to treat Rett syndrome, a rare disease with no current therapy while exploring the use of PTP1B inhibitors to treat inflammatory diseases, cancer, diabetes, and neurological diseases.   Andreas elaborates, "In DepYmed, we're focused on a specific enzyme. It's PTP1B. It's part of a family of enzymes called protein tyrosine phosphatases, and, in particular, we're looking at PTP1B. It's a metabolic regulatory enzyme that regulates signal transduction between cells and how cells communicate with each other. It's been worked on in the '90s and early 2000s. A couple of companies were working on the target, and they failed in the target, mainly because they couldn't create an orally bioavailable compound that would inhibit the PTP1B enzyme itself." "So that was one of the holy grails that we were able to find, where we were able to discover molecules that were orally bioavailable and would inhibit PTP1B. It was a game-changer when it came to the therapeutics around the target of PTP1B. Much of this work came out of Dr. Nicholas Tonks' laboratory out of Cold Spring Harbor Laboratory. We are in close collaboration with Nick and his team at Cold Spring Harbor, developing this new area of PTP1B inhibitors targeting the signal transaction pathway." #DepYmed #RettSyndrome #DPM1003 #PTP1B #Phosphatases #PhosphatasesInhibitors #RareDisease DepYmed.com Download the transcript here

Authentic Biochemistry
BioMedical Portrait V. c7. NETs and the combined activities of elastase,NOX2,MPO and IP3 PTEN phosphatase potentiate inflammation-linked pathologies of lung and pancreas. DJGPhD.31.10.23 and

Authentic Biochemistry

Play Episode Listen Later Nov 1, 2023 29:59


References J Zhejiang Univ Sci B. 2022 Jul 15; 23(7): 607–612 Biomaterials. 2020 Apr; 238: 119836. Int J Nanomedicine. 2023;18:5265-5287 Child Ballad 100. 1775. Pentangle. 1972.Willie O' Winsbury https://youtu.be/nwqP_yoszCE?si=C0NH51euOOTlPYn9 --- Send in a voice message: https://podcasters.spotify.com/pod/show/dr-daniel-j-guerra/message Support this podcast: https://podcasters.spotify.com/pod/show/dr-daniel-j-guerra/support

Lab Values Podcast (Nursing Podcast, normal lab values for nurses for NCLEX®) by NRSNG

Objective: Determine the significance and clinical use of  alkaline phosphatase in clinical practice   Lab Test Name: Alkaline Phosphatase – ALP   Description: Measures amount of ALP in circulation Located in several places in the body: Liver Intestines Biliary tract Bones Placenta Different isoenzymes of ALP are used to determine: Liver, bone, intestine and other cancers Bone turnover in postmenopausal women   Indications: Evaluation of ALP: Hepatobiliary disease Malignancies Bone disease Bone damage in renal patients   Normal Therapeutic Values: Normal – 40-130 U/L Collection:  Plasma separator tube   What would cause increased levels? Increased levels assessed in: Liver disease Bone disease Pregnancy Amyloidosis Lung cancer Pancreatic cancer Congestive heart failure Ulcerative colitis Hodgkin's disease Chronic renal failure Sarcoidosis   What would cause decreased levels? Hypophosphatasia (spelling error on existing outline on NURSING.com) Anemia Kwashiorkor Cretinism Hypothyroidism Zinc or magnesium deficiency Scurvy

PaperPlayer biorxiv cell biology
Inducible protein degradation as a strategy to identify Phosphoprotein Phosphatase 6 substrates in RAS-mutant colorectal cancer cells

PaperPlayer biorxiv cell biology

Play Episode Listen Later Mar 25, 2023


Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.25.534211v1?rss=1 Authors: Mariano, N. C., Rusin, S. F., Nasa, I., Kettenbach, A. N. Abstract: Protein phosphorylation is an essential regulatory mechanism that controls most cellular processes, including cell cycle progression, cell division, and response to extracellular stimuli, among many others, and is deregulated in many diseases. Protein phosphorylation is coordinated by the opposing activities of protein kinases and protein phosphatases. In eukaryotic cells, most serine/threonine phosphorylation sites are dephosphorylated by members of the Phosphoprotein Phosphatase (PPP) family. However, we only know for a few phosphorylation sites which specific PPP dephosphorylates them. Although natural compounds such as calyculin A and okadaic acid inhibit PPPs at low nanomolar concentrations, no selective chemical PPP inhibitors exist. Here, we demonstrate the utility of endogenous tagging of genomic loci with an auxin-inducible degron (AID) as a strategy to investigate specific PPP signaling. Using Protein Phosphatase 6 (PP6) as an example, we demonstrate how rapidly inducible protein degradation can be employed to identify dephosphorylation SITES and elucidate PP6 biology. Using genome editing, we introduce AID-tags into each allele of the PP6 catalytic subunit (PP6c) in DLD-1 cells expressing the auxin receptor Tir1. Upon rapid auxin-induced degradation of PP6c, we perform quantitative mass spectrometry-based proteomics and phosphoproteomics to identify PP6 substrates in mitosis. PP6 is an essential enzyme with conserved roles in mitosis and growth signaling. Consistently, we identify candidate PP6c-dependent phosphorylation sites on proteins implicated in coordinating the mitotic cell cycle, cytoskeleton, gene expression, and mitogen-activated protein kinase (MAPK) and Hippo signaling. Finally, we demonstrate that PP6c opposes the activation of large tumor suppressor 1 (LATS1) by dephosphorylating Threonine 35 (T35) on Mps One Binder (MOB1), thereby blocking the interaction of MOB1 and LATS1. Our analyses highlight the utility of combining genome engineering, inducible degradation, and multiplexed phosphoproteomics to investigate signaling by individual PPPs on a global level, which is currently limited by the lack of tools for specific interrogation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

PaperPlayer biorxiv cell biology
The tyrosine phosphatase LAR acts as a receptor of the nidogen-tetanus toxin complex

PaperPlayer biorxiv cell biology

Play Episode Listen Later Feb 4, 2023


Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.03.526966v1?rss=1 Authors: Surana, S., Villarroel-Campos, D., Panzi, C., Novoselov, S. S., Richter, S., Zanotti, G., Schiavo, G. Abstract: Tetanus toxin is one of the most potent neurotoxins and is the causative agent of tetanus. This neurotoxin binds to the neuromuscular junction and, after internalisation into motor neurons, undergoes long-distance axonal transport and transcytosis into spinal cord inhibitory interneurons. Inside the cytoplasm of interneurons, the catalytic chain of the toxin blocks neurotransmitter release, leading to spastic paralysis. Whilst the effects of tetanus toxin intoxication have been extensively studied, the molecular composition of its receptor complex is still poorly understood. We have previously shown that the extracellular matrix proteins nidogens are essential for binding of the toxin to the neuromuscular junction. In this study, we show that the tyrosine phosphatase LAR interacts with the nidogen-tetanus toxin complex and enables its uptake into motor neurons. Binding of LAR to the toxin complex is mediated by its fibronectin III domains, which we have harnessed to inhibit tetanus toxin entry into motor neurons. Surprisingly, this function of LAR is independent of its role in regulating the neurotrophic activity of the TrkB receptor, which has previously been shown to augment the axonal transport of signalling endosomes containing tetanus neurotoxin. These findings identify a multi-subunit complex acting as a protein receptor for tetanus neurotoxin, and demonstrate a novel endocytic trafficking route for extracellular matrix proteins in neurons. Our study paves the way for dissecting the molecular mechanisms that control the recognition and uptake of physiological ligands and pathological proteins at the neuronal plasma membrane, as well as their targeting to the axonal retrograde pathway for long-distance transport within the nervous system. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

PaperPlayer biorxiv cell biology
Activation of Ca2+ phosphatase Calcineurin regulates Parkin translocation to mitochondria and mitophagy

PaperPlayer biorxiv cell biology

Play Episode Listen Later Feb 1, 2023


Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.01.31.526442v1?rss=1 Authors: Marchesan, E., Nardin, A., Mauri, S., Di Paola, S., Chinellato, M., von Stockum, S., Chakraborty, J., Herkenne, S., Basso, V., Schrepfer, E., Marin, O., Cendron, L., Medina, D. L., Scorrano, L., Ziviani, E. Abstract: Selective removal of dysfunctional mitochondria via autophagy is crucial for the maintenance of cellular homeostasis. This event is initiated by the translocation of the E3 ubiquitin ligase Parkin to damaged mitochondria, and it requires the Serine/Threonine-protein kinase PINK1. In a coordinated set of events, PINK1 operates upstream of Parkin in a linear pathway that leads to the phosphorylation of Parkin, Ubiquitin, and Parkin mitochondrial substrates, to promote ubiquitination of outer mitochondrial membrane proteins. Ubiquitin decorated mitochondria are selectively recruiting autophagy receptors,which are required to terminate the organelle via autophagy. In this work we show a previously uncharacterized molecular pathway that correlates the activation of the Ca2+-dependent phosphatase Calcineurin to Parkin-dependent mitophagy. Calcineurin downregulation or genetic inhibition prevents Parkin translocation to CCCP-treated mitochondria, and impairs stress-induced mitophagy, whereas Calcineurin activation promotes Parkin mitochondrial recruitment and basal mitophagy. Calcineurin interacts with Parkin, and promotes Parkin translocation in the absence of PINK1, but requires PINK1 expression to execute mitophagy in MEF cells. Genetic activation of Calcineurin in vivo boosts basal mitophagy in neurons, and corrects locomotor dysfunction and mitochondrial respiratory defects of a Drosophila model of impaired mitochondrial functions. Our study identifies Calcineurin as a novel key player in the regulation of Parkin translocation and mitophagy. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Evidence-Based GI: An ACG Publication and Podcast
Ozanimod, a Selective Selective Sphingosine-1-Phosphatase Receptor Modulator, for Moderate-Severe Ulcerative Colitis: Re-Thinking the Top-Down Treatment Algorithm for UC

Evidence-Based GI: An ACG Publication and Podcast

Play Episode Listen Later Dec 13, 2022 17:50


PaperPlayer biorxiv neuroscience
Regulatory imbalance between LRRK2 kinase, PPM1H phosphatase, and ARF6 GTPase disrupts the axonal transport of autophagosomes

PaperPlayer biorxiv neuroscience

Play Episode Listen Later Nov 14, 2022


Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.14.516471v1?rss=1 Authors: Dou, D., Smith, E. M., Evans, C. S., Boecker, C. A., Holzbaur, E. L. F. Abstract: Gain-of-function mutations in the LRRK2 gene cause Parkinson's disease (PD), increasing phosphorylation of RAB GTPases through hyperactive kinase activity. We found that LRRK2-hyperphosphorylated RABs disrupt the axonal transport of autophagosomes by perturbing the coordinated regulation of cytoplasmic dynein and kinesin motors. In iPSC-derived human neurons, knock-in of the strongly-hyperactive LRRK2-p.R1441H mutation caused striking impairments in autophagosome transport, inducing frequent directional reversals and pauses. Knock-out of the opposing Protein Phosphatase 1H (PPM1H) phenocopied the effect of hyperactive LRRK2. Overexpression of ADP-ribosylation factor 6 (ARF6), a GTPase that acts as a switch for selective activation of dynein or kinesin, attenuated transport defects in both p.R1441H knock-in and PPM1H knock-out neurons. Together, these findings support a model where a regulatory imbalance between LRRK2-hyperphosphorylated RABs and ARF6 induces an unproductive "tug-of-war" between dynein and kinesin, disrupting processive autophagosome transport. This disruption may contribute to PD pathogenesis by impairing the essential homeostatic functions of axonal autophagy. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

PaperPlayer biorxiv cell biology
SHIP2 controls matrix mineralization by regulation of the RhoA/ROCK pathway and remodeling of the actin cytoskeleton

PaperPlayer biorxiv cell biology

Play Episode Listen Later Oct 31, 2022


Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.30.514432v1?rss=1 Authors: Fradet, A., Fitzgerald, J. Abstract: Mutations in INPPL1, the gene coding for SH2 Domain-Containing Inositol 5'-Phosphatase 2 (SHIP2), cause Opsismodysplasia, a severe chondrodysplasia characterized by delayed bone maturation. The mechanism by which the loss of an inositol phosphatase causes a major skeletal developmental defect is unclear. To investigate the role of SHIP2 in mineralization, the INPPL1 gene was deleted in vitro in chondrocyte and osteoblast differentiation models and the effect of the loss of SHIP2 on cell differentiation, subsequent mineralization, and on actin cytoskeleton formation was investigated. The loss of SHIP2 does not impact differentiation but, consistent with the disease phenotype, induces a significant reduction in extracellular matrix mineralization in both cell types. Absence of SHIP2 also altered the actin cytoskeleton to increase cell adhesion and focal adhesion formation. Furthermore, inhibition of actin polymerization in SHIP2-deficient cells rescued the mineralization phenotype. RhoA/ROCK, Cdc42 and Rac1 are the three main RhoGTPases responsible for actin cytoskeleton regulation in bone cells. Specific inhibitors of these RhoGTPases were used to determine the pathways involved in SHIP2-mediated mineralization. Since only the ROCK pathway inhibitor rescued the mineralization phenotype, it is concluded that SHIP2 regulates actin cytoskeleton remodeling and consequently extracellular matrix mineralization by inhibiting the RhoA/ROCK pathway. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Authentic Biochemistry
Membrane Biochemistry 58. Cardiolipin biosynthesis involves a PC-linoleic acid requiring maturase and a unique phosphatidylglycerol phosphate phosphatase. DJGPhD.25.9.22.Authentic Biochemistry

Authentic Biochemistry

Play Episode Listen Later Sep 26, 2022 29:59


References Dr Guerra membrane lectures Cell Metab. 2020 Dec 1;32(6):981-995.e7 PNAS 2011.108 (29) 11860-11865 J. Dev. Biol. 2020, 8(2), 10. Chem & Phys of Lipids.2000. June. 106:1-29 --- Send in a voice message: https://anchor.fm/dr-daniel-j-guerra/message

consilium - der Pädiatrie-Podcast
#16 mit Prof. Dr. Corinna Grasemann: Vitamin D

consilium - der Pädiatrie-Podcast

Play Episode Listen Later Sep 8, 2022 47:22


Die Hauptaufgabe von Vitamin D ist, die Kalziumaufnahme in den Körper zu ermöglichen und damit einen ausreichenden Kalziumspiegel zu sichern, so Frau Prof. Dr. Corinna Grasemann, Leiterin des Zentrums für seltene Erkrankungen an der Universitätskinderklinik in Bochum. Zusammen mit Dr. Axel Enninger spricht sie über ein Thema, das wortwörtlich in aller Munde ist: Jeder redet über Vitamin D, dennoch bleibt das Gefühl, es gibt nur eine Menge Halbwissen dazu. Da wollen die beiden Experten nachdrücklich Aufklärung leisten! Erhalten Sie in diesem Podcast gesicherte Informationen rund um das Steroidhormon Vitamin D, seinen Partner Kalzium und… … wie Vitamin D und Kalzium zusammenspielen. (03:05) … warum der Darm entscheidet, wie viel Vitamin D wir brauchen. (05:10) … welche Formen von Vitamin D wichtig sind und was bestimmt werden sollte. (05:49) … weshalb die Normwerte für Vitamin D nicht aus der Luft gegriffen sind. (08:20) … wo gesunder Menschenverstand hilft, wenn es um die Sonnenexposition geht. (11:42) … wie Einstrahlwinkel, Kleidung und Sonnencreme auf die Vitamin-D-Bildung wirken. (14:34) … warum Rachitis auch heute noch ein Thema ist. (17:40) … wann hohe Werte der alkalischen Phosphatase nicht für eine „irre seltene Erkrankung“ sprechen. (23:47) … weshalb zu den Risikogruppen nicht nur chronisch Darmkranke, Migrantenkinder und Krebspatienten gehören. (25:50) … die „Dos“- und die „Don‘ ts“ von Prof. Dr. Corinna Grasemann (43:43) Weiterführende Links: Leitlinien der Deutschen Gesellschaft für Kinder- und Jugendmedizin (DGKJ): https://www.dgkj.de/fileadmin/user_upload/Stellungnahmen/1804_EKSN_VitaminDnach2temLj.pdf Patienten-Ratgeber: Verständliche Broschüre für Eltern und Betreuungspersonen https://www.infectopharm.com/fuer-patienten/patienten-ratgeber/vitamin-d/ consilium collegiale-Fortbildungen: https://www.infectopharm.com/fortbildungen CME-zertifizierter Podcast mit Prof. Höger: https://consilium.podigee.io/5-5-mit-prof-peter-hoger-psoriasis Fragen & Antworten-Hefte, Themenhefte u. v. m. können Sie anfordern unter: servicematerial@infectopharm.com Kontakte: Feedback zum Podcast? consilium@infectopharm.com Homepage zum Podcast: www.infectopharm.com/consilium/podcast/ Homepage InfectoPharm: www.infectopharm.com Disclaimer: Der consilium – Pädiatrie-Podcast dient der neutralen medizinischen Information und Fortbildung für Ärzte. Für die Inhalte sind der Moderator und die Gäste verantwortlich, sie unterliegen dem wissenschaftlichen Wandel des Faches. Änderungen sind vorbehalten.

Nourishing Liberty Podcast
Why You Should Care About Phosphatase (And What Is It Anyway?)

Nourishing Liberty Podcast

Play Episode Listen Later Aug 8, 2022 38:53


Here we go on about raw milk again! Liz delves into details about raw milk--from a culinary and adventure perspective and some of the practical chemistry. What IS the big deal about phosphatase? And why should you care? We answer this and many other questions. Rachel tells us a bit about why precious metals are important too. Join us to learn about our food systems, and how to grow and prepare your own food. We'll discuss tips and tricks for time management for families so YOU can fully participate in your food system from start to finish. We will talk about how to stay healthy and balanced in body, mind and spirit. Find the guide to local farms here Learn to grow your own food. Please like and subscribe and share this podcast with your friends.

Save My Thyroid
Alkaline Phosphatase and Hyperthyroidism

Save My Thyroid

Play Episode Listen Later Jul 28, 2022 6:48


In this Q & A episode Dr. Eric answers the question "How concerned should I be with an elevated alkaline phosphatase?"  This episode is mainly for those with hyperthyroidism, as it's common for people with different types of hyperthyroid conditions to have an elevated alkaline phosphatase.

Lab Values Podcast (Nursing Podcast, normal lab values for nurses for NCLEX®) by NRSNG

Get a free nursing lab values cheat sheet at NURSING.com/63labs   Objective: Determine the significance and clinical use of  alkaline phosphatase in clinical practice   Lab Test Name: Alkaline Phosphatase – ALP   Description: Measures amount of ALP in circulation Located in several places in the body: Liver Intestines Biliary tract Bones Placenta Different isoenzymes of ALP are used to determine: Liver, bone, intestine and other cancers Bone turnover in postmenopausal women   Indications: Evaluation of ALP: Hepatobiliary disease Malignancies Bone disease Bone damage in renal patients   Normal Therapeutic Values: Normal – 40-130 U/L Collection:  Plasma separator tube   What would cause increased levels? Increased levels assessed in: Liver disease Bone disease Pregnancy Amyloidosis Lung cancer Pancreatic cancer Congestive heart failure Ulcerative colitis Hodgkin's disease Chronic renal failure Sarcoidosis   What would cause decreased levels? Hypophosphatasia (spelling error on existing outline on NURSING.com) Anemia Kwashiorkor Cretinism Hypothyroidism Zinc or magnesium deficiency Scurvy

PCICS Podcast
Episode 08: Alkaline Phosphatase Infusion to Prevent Organ Injury After Circulatory Arrest

PCICS Podcast

Play Episode Listen Later Feb 3, 2021 14:07


Episode 08: Alkaline Phosphatase Infusion to Prevent Organ Injury After Circulatory Arrest Guest: Jesse Davidson, MD, MPH/MSPH. Host: David Werho, MD Dr. Jesse Davidson from Children's Hospital of Colorado discusses a piglet model for cardiopulmonary bypass and its use to test alkaline phosphatase to mitigate organ injury after cardiac surgery.

Daiquiris and Dermatology
Alkaline Phosphatase Tests for Liver Disease

Daiquiris and Dermatology

Play Episode Listen Later Dec 18, 2020 2:47


This episode covers alkaline phosphatase tests for liver disease! --- This episode is sponsored by · Anchor: The easiest way to make a podcast. https://anchor.fm/app

Circulation on the Run
Circulation December 1, 2020 Issue

Circulation on the Run

Play Episode Listen Later Nov 30, 2020 25:06


This week's episode features author Torbjørn Omland and Senior Guest Editor Vera Bittner as they discuss the artile "Growth Differentiation Factor-15 Provides Prognostic Information Superior to Established Cardiovascular and Inflammatory Biomarkers in Unselected Patients Hospitalized with COVID-19." TRANSCRIPT BELOW: Dr. Carolyn Lam: Welcome to Circulation on the Run, your weekly podcast summary, and backstage pass to the journal and its editors. We are your co-hosts, I'm Dr. Carolyn Lam, associate editor from the National Heart Center and Duke National University of Singapore. Dr. Greg Hundley: I'm Dr. Greg Hundley, Director of the Pauley Heart Center at VCU Health in Richmond, Virginia. Well, Carolyn our feature this week gets into inflammatory biomarkers in patients that have been hospitalized with COVID-19, but before we get to that, how about we grab a cup of coffee and work through some of the papers in the issue. Would you like to go first? Dr. Carolyn Lam: Absolutely. With both the coffee and the papers. So great, for this first paper, have you thought about concentric versus eccentric cardiac hypertrophy? We traditionally associate them with pressure versus volume overload respectively in cardiovascular disease, both though conferring an increased risk of heart failure. These contrasting forms of hypertrophy are characterized by asymmetric growth of the cardiac myocytes in mainly width or length respectively. However, the molecular mechanisms determining myocyte preferential growth in width versus length remain poorly understood. Dr. Carolyn Lam: That is until today's paper, and it is from Dr. Kapiloff from Stanford University, and Dr. Rosenfeld from UCSD, School of Medicine and their colleagues, and what they did was used primary adult rat ventricular myocytes, as well as Adeno associated virus mediated gene delivery in mice, to define a regulatory pathway controlling pathological myocyte hypertrophy, and they found that asymmetric cardiac myocyte hypertrophy is modulated by serum response factor phosphorylation, constituting an epigenomic switch balancing the growth in width versus length of adult ventricular myocytes In vitro, and In vivo. Dr. Carolyn Lam: Serum response factor phosphorylation was bi-directionally regulated at signalosomes organized by the scaffold protein muscle, A kinase anchoring protein beta. This newly identified molecular switch controlled a transcriptional program responsible for modulating changes in cardiomyocyte morphology that occurs secondary to pathological stressors. Dr. Greg Hundley: Very nice, Carolyn. So switches controlling this transcriptional program. Tell us a little bit, and bring us back to the clinical relevance of this and starting with that concentric versus eccentric hypertrophy? Dr. Carolyn Lam: I thought you may ask. The identification of a molecular mechanism regulating that asymmetric cardiomyocyte growth, really provides a new target for the inhibition of pathological cardiac hypertrophy. Studies in mice using these Adeno associated virus based gene therapies to modulate that signalosome, really provided proof of concept for translational potential in the treatment of pathological cardiac remodeling and prevention of heart failure. Dr. Greg Hundley: Oh, wow. Very nice, Carolyn. Well, my first paper comes to us from Professor Dirk Westermann from Hamburg, and focuses on cardiogenic shock patients, and veno-arterial ECMO, the results from the international multicenter cohort study. So Carolyn this study evaluated data from 686 consecutive patients with cardiogenic shock treated with VA ECMO with or without left ventricular unloading using an Impella, and they conducted this at 16 tertiary care centers across four countries. They examined the association between left ventricular unloading and 30 day mortality. Dr. Carolyn Lam: Huh, so what did they find? Dr. Greg Hundley: Okay. Carolyn. Well, left ventricular unloading was used in 337 of the 686 patients enrolled, and after propensity matching 255 patients with left ventricular unloading were compared with the 255 patients without left ventricular unloading. In the match cohort, left ventricular unloading was associated with lower 30 day mortality without differences in the various subgroups. However, complications occurred more frequently in patients with left ventricular unloading, like severe bleeding, which happened in 38.4% versus only 17.9% in those without unloading. There was also access-related ischemia and renal replacement therapy. Dr. Greg Hundley: So Carolyn, the take-home message from this International multi-center cohort study, is that left ventricular unloading is associated with lower mortality, and cardiogenic shock patients treated with VA ECMO, despite higher complication rates. In the absence of randomized trial data these findings support the use of left ventricular unloading and cardiogenic shock patients treated with VA ECMO, and call for further validation, ideally in a randomized controlled trial. Dr. Carolyn Lam: Very nice. Well for my next paper, Greg, it's all about desmin. Now we know that mutations in the human desmin gene caused myopathies and cardiomyopathies. Well, today's authors, Dr. Hermann and Schroeder from University Hospital Erlangen in Germany and Dr. Lilienbaum from University of Paris and France and their colleagues, report an adolescent patient who underwent cardiac transplantation, due to restrictive cardiomyopathy caused by a heterozygous R406W desmin mutation. Sections of the explanted heart were analyzed with antibodies specific to 406W-desmin, and to intercalated disc proteins. Effects of this mutation on the molecular properties of desmin were then addressed by cell transfection and In vitro assembly experiments. They further generated these desmin mutation knock-in mice haboring the orthologous form of the human, R406W-desmin. Dr. Greg Hundley: So Carolyn, what did they find? Dr. Carolyn Lam: Well, they demonstrated a novel pathomechanism in which cardiotoxic R406W-desmin, could adapt dual functional status with the abilities to integrate into the indogenous intermediate filament network, and to cause formation a protein aggregates. This R406W-desmin modified the extra sarcomeric cytoskeleton, such that desmin filaments were not anchored to desmosomes anymore. Thereby destroying the structural, and functional integrity of intercalated discs. Dr. Greg Hundley: What are the clinical implications? Dr. Carolyn Lam: Well, since these cardiotoxic desmin mutations could affect the integrity of intercalated discs, thereby inducing conduction defects and malignant arrhythmias, they suggest early implantation of pacemaker, or cardioverter defibrillator devices, may be considered to prevent certain cardiac death in patients with these mutations. Furthermore, state-of-the-art basic molecular risk stratification of desmin mutations may encompass a multidisciplinary experimental approach as exemplified by the approach taken here, which comprises assessment of the tissue pathology in conjunction with genome analysis and desmin assembly studies as well as patient mimicking cell and animal models for the In vivo validation of these mutations. Dr. Greg Hundley: Well, fantastic, Carolyn. Well, my next paper comes to us from Dr. Ravi Shah from the Massachusetts General Hospital. This study evaluated 2,330 white and black young adults, average age of 32 years, in the Coronary Artery Risk Development in Young Adults, or the cardiac study, to identify metabolite profiles associated with an adverse cardiovascular disease phenom that included, myocardial structure and function, fitness, vascular calcification, and then also mechanisms, and other cardiovascular outcomes that would occur over the next two decades. Statistical learning methods, including elastic nets and principal component analysis, and Cox regression generated parsimonious metabolite based risk scores, validated in over 1800 individuals in the Framingham Heart Study. Dr. Carolyn Lam: Wow. What did they show, Greg? Wow, that's a lot of work. Dr. Greg Hundley: Yeah. So Carolyn, the authors found two multiparametric metabolite-based scores linked independently to vascular, and myocardial health. With metabolites included in each score specifying microbial metabolism, hepatic steatosis, oxidative stress, nitric oxide modulation, and finally collagen metabolism. Over nearly 25 year median follow-up, and cardia, this metabolite based vascular score, and the myocardial score, and the third and fourth decade of life were associated with clinical cardiovascular disease. Importantly, the authors replicated these findings in 1,898 individuals in the Framingham Heart Study followed over two decades, such that young adults with poor metabolite based health scores had higher hazard ratios of future cardiovascular disease related events. Dr. Carolyn Lam: Oh wow. Greg, what an elegant study with both development and validation cohort evaluating the metabolome. Dr. Greg Hundley: Yes. Carolyn. So metabolic signatures of myocardial, and vascular health in young adulthood specify known novel pathways of metabolic dysfunction, relevant to cardiovascular disease associated with outcomes in two independent cohorts. So these data suggests that efforts to include precision measures of metabolic health in risk stratification to interrupt cardiovascular disease at an early at stage, are warranted. Dr. Carolyn Lam: Wow. So interesting. Other very interesting articles in today's issue, there's an In Depth article by Dr. Angiolillo entitled, “The Antithrombotic Therapy for Atherosclerotic Cardiovascular Disease Risk Mitigation in Patients with Coronary Artery Disease and Diabetes.” There's also Research Letters, one by Dr. Sultan on, “The Longterm Outcomes of Primary Cardiac Lymphoma” and one by Dr. Wang on, “Loss of Phosphatase and Tensin Homolog Promotes Cardiomyocyte Proliferation and Cardiac Repair Following Myocardial Infarction.” Dr. Greg Hundley: Great, Carolyn. Well, I've got a couple other articles in this issue as well. One is by Professor Ganesan Karthikeyan who has an On My Mind piece entitled an “Alternative Hypothesis to explain Disease Progression in Rheumatic Heart Disease.” Dr. Stuart Chen has an ECG challenge entitled, “Alternating QRS Duration and a Normal T-waves. What is the mechanism?” Then finally, Carolyn, a series of Letters to the Editor, one by Dr. Peterzan and the other by Dr. Mehmood regarding the prior published article, entitled “Cardiac Energetics in Patients with Aortic Stenosis and Preserved Ejection Fraction.” Well, Carolyn, how about we get onto that feature article and learn more about inflammatory biomarkers in hospitalized patients with COVID-19? Dr. Carolyn Lam: Yes. Let's go. Greg. Biomarkers are really playing an increasingly important role in cardiovascular disease, and even in the current COVID 19 pandemic, there's been a lot of news about how biomarkers such as traponin may be prognostic, and in fact, we're all wondering about maybe even newer biomarkers. In fact, today's feature discussion does bring to light one of the newest, and in fact, this is the first publication on the role of Growth Differentiation Factor 15 or GDF-15 in COVID-19. We're so pleased to be discussing this with the corresponding author, Dr. Torbjørn Omland from University of Oslo, in Norway, as well as our senior guest editor, Dr. Vera Bittner from University of Alabama at Birmingham. So welcome both. Tobjorn, could you tell us a little bit about GDF-15 and what made you look at it, and what did you find? Dr. Torbjørn Omland: Yeah, so GDF-15, that's a very interesting biomarker. It's considered a biomarker of biological aging cellular stress, and perhaps also the inflammation, and tests being studied within the cardiovascular field for some years now, and it has been shown to be a strong prognostic indicator across the cardiovascular spectrum, actually. So it is a new biomarker in one sense, but there are some data already in the cardiovascular field. Dr. Carolyn Lam: Not in COVID. So this is the first study to really look at its prognostic value in COVID 19. So congratulations Torbjorn, and if I may also to the first author, Dr. Peter Meer, a good friend as well, but please, could you tell us about your study and what you found? Dr. Torbjørn Omland: Yes. So when the COVID pandemic hit Norway in the spring, we thought that we should plan a prospective biomarker study. So we had to really fast track approval by the IRB and so forth, and we're able to actually cover most of the patients that were hospitalized in our hospital, Akershus University hospital, which is right outside of Oslo, and it's a pretty large hospital by Norwegian standards. It covers about 11% of the Norwegian population. Dr. Torbjørn Omland: So in that period, when we were including, we had 136 patients hospitalized with confirmed COVID 19, and we have biobank bank samples from 123 of these, and then there have been reports from retrospective studies, first from China, that seemed to suggest that markers like cardiac troponin, Anti-Troponin T, and Ferritin were associated with outcome, but those studies were prone to selection bias in that the measurements were performed in the most sick patients. So in this study we included all patients and then we thought we should examine a broad panel of biomarkers, and that included Interleukin 6, CRP, Procalcitonin, Ferritin, and the D-dimer Cardiac troponin, and N-terminal pro B, and GDF-15. Dr. Carolyn Lam: Wow. Thank you, Torbjorn. Even before you carry on with the results, can I just say having visited your hospital in pre-COVID days, I can only imagine what a work of love this was to do it prospectively. Any particular experiences to talk about, to get a fast-track even in the midst of to perform a well done prospective study, that must have taken a lot. Dr. Torbjørn Omland: Yes. But it's also interesting in that the whole sort of ablation on Norway was very much into this from the highest political level. Also, the decision that the older research on COVID should be prepared to retire, then the IRB had an eight hour and deadline for them to approve or not approve the study. So that's went surprisingly smoothly, I must say. Dr. Carolyn Lam: Wow, that's great. So what did you find? Dr. Torbjørn Omland: Yeah, so we found that among these biomarkers, several seem to predict outcome, and the primary end point of this study was to combined end-point of the hospitalization in the ICU, or death. We found that also markers like cardio traponin, BNP, ferritin, and the D-dimer and so forth, in univariable analysis, were very associated with outcome, but when we perform a more comprehensive, mostly variable modeling, then the prognostic value of some of these markers disappeared. In contrast, for GDF-15, it seemed to perform very strongly, both on the baseline sample, and interestingly also it increased in those reaching the primary end-point during the hospitalization. So it provided a very strong and independent information also when we adjusted for clinical risk scores, like the NEWS score. So that was a very pleasant surprise to see that there was one marker that's actually performed so well. The other marker that's also performed well was Ferritin. Dr. Carolyn Lam: Very interesting, and so the new score being the National Early Warning Score. Thank you. Verra, I really love to bring in your thoughts. I mean, could you take us behind the scenes with the editors? What did you think when you saw this paper? Dr. Vera Bittner: As you know, I mean, a lot of journals have been inundated by COVID papers, and so this one stuck out to us, because it's the first time that we had seen that anybody linked GDF-15 to a COVID population, even though it has been out in the literature for ACS, and in my prognostication, and in a healthy populations, and in chronic coronary disease populations, heart failure, and so on. So this is the first time that we've seen it applied there. Dr. Vera Bittner: Then I would echo some of the things that Torbjorn said, that we were also impressed, that it was prospective, because when you look at some of the other biomarker studies, what was prognostic in one with then not shake out the other one, because either different variables were included in the models, because the population's differed. So to have something that was representative of the population that was actually admitted to this, Norwegian Academic Hospital, stood out to us. So we're excited to get this paper basically for circulation, and hope that it also will be impetus for future research. Dr. Carolyn Lam: Thank you so much for sharing that end for helping us publish such a beautiful paper. Did you have some questions for two of your own? Dr. Vera Bittner: Yeah. So what stuck out to me is that you had this a whole crew of biomarkers, and then when you looked ultimately at the final model, there were two that were standing out, that was ferritin, and it was the GDF-15, and then when I looked at your graph, it looks like not only did these biomarkers measure different contrasts, but their time-course also seemed to be different, and so I was just wondering whether you had thought about, maybe using these to joint the model outcome, and whether we might even be able to get more information that way. Dr. Torbjørn Omland: I think that's an excellent suggestion, and as you correctly pointed out, they do have different sort of profiles and ferritin being an acute phase reactant, having various sort of dramatic early rise whereas we see that GDF-15 increased progressively during the course of hospitalization in the most severe patients. I think when combining them, is actually a great IMT that we should look further into. Dr. Carolyn Lam: Very nice. Torbjorn, if I could, I've got a couple of questions too. So 123 patients, 35 of whom had the primary outcome, right? So that may be sort of seen as, is this too small? and they're all hospitalized patients. So could I ask, what do you predict maybe seen in a larger population or outside of Norway or in a non-hospitalized population? Dr. Torbjørn Omland: So as you say, we were early with this report, but since it was submitted, there has been a couple of smaller studies that seemed to confirm our results. So that is reassuring, but of course we would like to have studied this in logical patients. We are in touch with the other biobank samples that could possibly confirm the data. So that's one obvious step. Then it's very interesting, as you say, could we sort of expand this to also apply to non-hospitalized patients? I think that it would be a very interesting hypothesis to test, and I think there's still a pretty good rationale for this. Dr. Torbjørn Omland: It's interesting that the insoluble group actually showed a correlation that when the soluble ST2 concentrations and GDF-15. So there might be that those with more susceptibility to COVID infections, actually, I thought that, that is actually reflected by GDF-15 concentrations, but the challenge is how to sort of get a representative non-hospitalized population, but interestingly, I was approached by some of the hospital staff that actually are in contact with general practitioners, and wanted sort of implement this test also for this group. Dr. Carolyn Lam: So Verra, we're really grateful that Allan Jaffe was working with you in managing this beautiful paper, and if you don't mind me cheekily paraphrasing that you said you might channel him, if you could, what would the channeled Allan Jaffe perhaps say about what's needed in this whole biomarkers fear in COVID-19? Dr. Vera Bittner: Hopefully, many. A channeling element is obviously difficult, because he is such an incredible expert on biomarkers that I can't even pretend to be able to see, that you might be thinking, but it seems to me that one thing that we could all agree on is that it would be really exciting if something like the: get with the guidelines COVID registry, could decide to measure this marker perspectively in the participating hospitals, for example. Dr. Vera Bittner: Then be able to look at this in a much, much larger population. I mean, especially with different ethnic backgrounds as well. I mean, I noticed actually to my surprise that, this Norwegian study how to fairly high proportion of Asians in the sample, but that may not be the ethnic distribution that we might see in different regions of the US, or different regions of the world. So it would be really nice to incorporate the measurement of this biomarker in much larger datasets. So things can be explored a bit further. Dr. Carolyn Lam: That's excellent, and Torbjorn, if you could channel Allan. What would you say? Dr. Torbjørn Omland: That's a difficult path, but absolutely just to me what Verra said. Then I think the importance of prospective studies in the COVID biomarker field, I think is our at most importance. Dr. Carolyn Lam: I think on behalf of both Torbjorn and I, and in fact everyone in circulation. Thank you, Verra for the amazing work that you and your team do for circulation as well. Thank you so much for making the time to share your thoughts today and thank you for that beautiful, beautiful paper both of you. Thank you. (singing). Listeners you've been listening to Circulation on the Run. Thank you for joining us from Greg and I. Don't forget to tune in again next week. Dr. Greg Hundley: This program is copyright, the American Heart Association 2020.  

PaperPlayer biorxiv biochemistry
High catalytic rate of the cold-active Vibrio alkaline phosphatase depends on a hydrogen bonding network involving a large interface loop

PaperPlayer biorxiv biochemistry

Play Episode Listen Later Oct 27, 2020


Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.27.357921v1?rss=1 Authors: Hjorleifsson, J. G., Helland, R., Magnusdottir, M., Asgeirsson, B. Abstract: The role of surface loops in mediating communication through residue networks is still a relatively poorly understood part of cold-adaptation of enzymes, especially in terms of their quaternary interactions. Alkaline phosphatase (AP) from the psychrophilic marine bacterium Vibrio splendidus (VAP) is characterized by an analogous large surface loop in each monomer, referred to as the large-loop, that hovers over the active site of the other monomer. It presumably has a role in VAP high catalytic efficiency that accompanies extremely low thermal stability. We designed several different mutagenic variants of VAP with the aim of removing inter-subunit interactions at the dimer interface. Breaking the inter-subunit contacts from one residue in particular (Arg336) caused diminished temperature stability of the catalytically potent conformation and a drop in catalytic rate by a half. The relative B-factors of the R336L crystal structure, compared to the wild-type, confirmed increased surface flexibility in a loop on the opposite monomer, but not in the large-loop. Contrary to expectations, the observed reduction in stability with an expected increase in dynamic mobility resulted in reduced catalytic rate. This contradicts common theories explaining high catalytic rates of enzyme from cold-adapted organisms as being due to reduced internal cohesion bringing increased dynamic flexibility to catalytic groups. The large-loop increases the area of the interface between the subunits through its contacts and may facilitate an alternating structural cycle demanded by a half-of-sites reaction mechanism through stronger ties, as the dimer oscillates between high affinity (active) or low phosphoryl-group affinity (inactive). Copy rights belong to original authors. Visit the link for more info

PaperPlayer biorxiv biophysics
Elucidation and control of low and high active populations of alkaline phosphatase molecules for quantitative digital bioassay

PaperPlayer biorxiv biophysics

Play Episode Listen Later Oct 18, 2020


Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.18.336891v1?rss=1 Authors: Ueno, H., Kato, M., Minagawa, Y., Hirose, Y., Noji, H. Abstract: Alkaline phosphatase (ALP), a homo-dimeric enzyme has been widely used in various bioassays as disease markers and enzyme probes. Recent advancements of digital bioassay revolutionized ALP-based diagnostic assays as seen in rapid growth of digital ELISA and the emerging multiplex profiling of single-molecule ALP isomers. However, the intrinsic heterogeneity found among ALP molecules hampers the ALP-based quantitative digital bioassays. This study aims quantitative analysis of single-molecule activities of ALP from Escherichia coli and reveals the static heterogeneity in catalytic activity of ALP with two distinct populations: half-active and fully active portions. Digital assays with serial buffer exchange uncovered single-molecule Michaelis-Menten kinetics of ALP; half-active molecules have halved values of the catalytic turnover rate, kcat, and the rate constant of productive binding, kon, of the fully active molecules. These findings suggest that half-active ALP molecules are heterogenic dimers composed of inactive and active monomer units, while fully active ALP molecules comprise two active units. Static heterogeneity was also observed for ALP with other origins: calf intestine or shrimp, showing how the findings can be generalized across species. Cell-free expression of ALP with disulfide bond enhancer and spiked zinc ion resulted in homogenous population of ALP of full activity, revealing that inactive monomer units of ALP are deficient in disulfide bond formation and zinc ion coordination, and also offering the way to prepare homogenous and active populations of ALP for quantitative digital bioassays of ALP. Copy rights belong to original authors. Visit the link for more info

PaperPlayer biorxiv biochemistry
Development and validation of nanobodies specific to the oncogenic phosphatase Protein Tyrosine Phosphatase 4A3 (PTP4A3 or PRL-3)

PaperPlayer biorxiv biochemistry

Play Episode Listen Later Oct 3, 2020


Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.02.311787v1?rss=1 Authors: Smith, C. N., Wei, M., Williamson, Z. A., Chernyavskyay, Y., Chow, K. M., Hersh, L. B., Korotkov, K. V., Blackburn, J. S. Abstract: Protein Tyrosine Phosphatase 4A3 (PTP4A3 or PRL-3) is an oncogenic dual specificity phosphatase that drives tumor metastasis, promotes cancer cell survival, has been linked to poor patient prognosis in a variety of tumor types. The mechanisms by which PRL-3 promotes tumor progression are not well understood, which is in part due to lack of tools to study this protein. The development of small molecule inhibitors that are specific to PRL-3 has proven difficult, as the PRL family is >80% homologous. As research tools, antibodies directed against PRL-3 have been more successful, although they are limited to certain assay types. To address this, we have designed, purified, and tested alpaca-derived PRL-3 single domain antibodies, or nanobodies. We have identified 7 unique nanobodies that bind to PRL-3 in ELISA with no activity towards PRL-1 and PRL-2. Anti-PRL-3 nanobodies immunoprecipitated PRL-3 from cell lysates and were used to define PLR-3 localization in human colon cancer cells in immunofluorescence cell staining experiments. These anti-PRL-3 nanobodies represent an important expansion of the tool set used for the study of PRL-3, and can be used to better define the role of PRL-3 in cancer progression while serving as a useful first step in the development of biologics to target PRL-3 in the clinic. Copy rights belong to original authors. Visit the link for more info

PaperPlayer biorxiv biochemistry
Insulin inhibits protein phosphatase 2A to impair β-adrenergic receptor function

PaperPlayer biorxiv biochemistry

Play Episode Listen Later Sep 19, 2020


Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.17.302448v1?rss=1 Authors: Sahu, A., Sun, Y., Mukherjee, S., Witherow, C., Stenson, K., Tesmer, J. J. G., Mohan, M., Naga Prasad, S. N. Abstract: Insulin impairs {beta}2-adrenergic receptor ({beta}2AR) function through G protein-coupled receptor kinase 2 (GRK2) by phosphorylation but less is known about dephosphorylation mechanisms mediated by protein phosphatase 2A (PP2A). Pharmacologic or genetic inhibition of phosphoinositide 3-kinase {gamma} (PI3K{gamma}) unexpectedly resulted in significant reduction of insulin-mediated {beta}2AR phosphorylation. Interestingly, {beta}2AR-associated phosphatase activity was inhibited by insulin but was reversed by knock-down of PI3K{gamma} showing negative regulation of PP2A by PI3K{gamma}. Co-immunoprecipitation and surface plasmon resonance studies using purified proteins showed that GRK2 and PI3K{gamma} form a complex and could be recruited to {beta}2ARs as GRK2 interacts with insulin receptor substrate following insulin treatment. Consistently, {beta}-blocker pretreatment did not reduce insulin-mediated {beta}2AR phosphorylation indicating agonist- and G{beta}{gamma}-independent non-canonical regulation of receptor function. Mechanistically, PI3K{gamma} inhibits PP2A activity at the {beta}AR complex by phosphorylating an intracellular inhibitor of PP2A (I2PP2A). Knock-down or CRISPR ablation of endogenous I2PP2A unlocked PP2A inhibition mediating {beta}2AR dephosphorylation showing an unappreciated acute regulation of PP2A in mediating insulin-{beta}2AR cross-talk. Copy rights belong to original authors. Visit the link for more info

PaperPlayer biorxiv biophysics
Determination of the molecular reach of the protein tyrosine phosphatase SHP-1

PaperPlayer biorxiv biophysics

Play Episode Listen Later Sep 5, 2020


Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.04.283341v1?rss=1 Authors: Clemens, L., Kutuzov, M., Bayer, K. V., Goyette, J., Allard, J., Dushek, O. Abstract: Immune receptor signalling proceeds by the binding of enzymes to their cytoplasmic tails before they catalyse reactions on substrates within reach. Studies of binding and catalysis have led to a binding- induced allosteric activation model for enzymes, such as SHP-1, whose catalytic activity increases upon recruitment to inhibitory receptors, such as PD-1. However, the impact of molecular reach is poorly understood. Here, we use surface plasmon resonance to measure binding, catalysis, and molecular reach for tethered SHP-1 reactions. We find a molecular reach for SHP-1 (13.0 nm) that is smaller than a maximum stretch estimate (20.4 nm) but larger than an estimate from crystal structure of the active conformation (5.3 nm), suggesting that SHP-1 explores a spectrum of active conformations. The molecular reach confines SHP-1 to a small membrane-proximal volume near its substrates leading membrane recruitment to increase its activity by a 1000-fold increase in concentration with a smaller 2-fold activity increase by PD-1 binding-induced allostery. Lastly, we use mathematical modelling to quantify the degree of co-clustering required for PD-1-SHP-1 complexes to reach their substrates. Copy rights belong to original authors. Visit the link for more info

PaperPlayer biorxiv biophysics
Peptide-based Inhibitors of Protein-Protein Interactions of Src Homology 2 Domain-Containing Phosphatase 2

PaperPlayer biorxiv biophysics

Play Episode Listen Later Aug 28, 2020


Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.28.271809v1?rss=1 Authors: Bobone, S., Pannone, L., Biondi, B., Solman, M., Flex, E., Canale, V., Calligari, P., De Faveri, C., Gandini, T., Quercioli, A., Torini, G., Venditti, M., Lauri, A., Fasano, G., Hoeksma, J., Santucci, V., Cattani, G., Bocedi, A., Carpentieri, G., Tirelli, V., Sanchez, M., Peggion, C., Formaggio, F., Den Hertog, J., Martinelli, S., Bocchinfuso, G., Tartaglia, M., Stella, L. Abstract: Mutations of PTPN11, the gene coding for the Src homology 2 domain-containing phosphatase 2 (SHP2), cause childhood leukemias and developmental disorders. SHP2 inhibitors targeting the catalytic site or an allosteric pocket lack specificity or are ineffective on pathogenic variants. In addition, several data indicate that increased association with cognate proteins, through its SH2 domains, rather than enhanced catalytic activity, is the main effect of mutations causing hyperactivation of SHP2-mediated signaling. We developed peptide-based molecules with low nM affinity to the N-SH2 domain and high specificity. These molecules bind to pathogenic variants of SHP2 with an affinity up to 20 times higher than to the wild-type protein, in contrast to allosteric inhibitors, and were able to revert the effects of a pathogenic SHP2 mutation in zebrafish embryos. Our results provide a novel route for SHP2-targeted therapies and a tool to investigate the role of protein-protein interactions in the function of SHP2. Copy rights belong to original authors. Visit the link for more info

PaperPlayer biorxiv genetics
Protein Phosphatase 2A-B56 maintains the meiotic spindle, kinetochore attachments and cohesion by antagonizing Aurora B Drosophila Oocytes

PaperPlayer biorxiv genetics

Play Episode Listen Later Aug 3, 2020


Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.01.232512v1?rss=1 Authors: Jang, J. K., Gladstein, A., Das, A., Cisco, Z., McKim, K. Abstract: Meiosis in female oocytes lack centrosomes, the major microtubule-organizing center, which may make them especially vulnerable to aneuploidy. In the acentrosomal oocytes of Drosophila, meiotic spindle assembly depends on the chromosomal passenger complex (CPC). Aurora B is the catalytic component of the CPC while the remaining subunits regulate its localization. Using an inhibitor of Aurora B activity, Binucleine 2, we found that continuous Aurora B activity is required to maintain the oocyte spindle during meiosis I. Furthermore, the necessity of a kinase for spindle regulation suggests that spindle dynamics is regulated by phosphatases. Our result have shown that the protein complex Protein Phosphatase 2A (PP2A) opposes CPC activity, probably by dephosphorylating spindle associated proteins such as the Kinesins. PP2A exists in two varieties, B55 and B56. While both antagonize Aurora B, they typically exhibit different localization and function. B55 has only minor roles in meiosis I spindle function. The B56 subunit is encoded by two partially redundant paralogs in the Drosophila genome, wdb and wrd. Knocking down both B56 subunits showed they are critical for multiple functions during meiosis I, including maintaining sister centromere and arm cohesion, end-on microtubule attachments, and the metaphase I arrest in oocytes. We found that WDB recruitment to the centromeres depends on BubR1, MEI-S332, and kinetochore protein SPC105R. However, only SPC105R is required for cohesion maintenance during meiosis I. We propose that SPC105R promotes cohesion maintenance by recruiting two proteins that further recruit PP2A, MEI-S332, and the Soronin homolog Dalmatian. Copy rights belong to original authors. Visit the link for more info

Audio-only streams of our videos
Anne Bertolotti Part 2: Benefits of Phosphatase Inhibition for Neurodegenerative Diseases

Audio-only streams of our videos

Play Episode Listen Later Jun 29, 2019 30:07


Bertolotti’s lab has had a long time interest in understanding protein folding and the role of misfolded proteins in neurodegenerative disease. In her second talk, Bertolotti explains how her lab found that selectively inhibiting the dephosphorylation of eIF2⍺, a translation initiation factor, led to a reduction in protein synthesis. Decreasing protein synthesis allowed cells to “catch up” with the degradation of misfolded proteins that may accumulate as a result of cell stress. Her lab went on to show that a selective small molecule phosphatase inhibitor had therapeutic effects in a mouse model of Charcot-Marie-Tooth disease; a disease that results from the accumulation of misfolded protein in the ER.  This exciting result suggested that targeted inhibition of protein phosphatases may have therapeutic potential for neurodegenerative diseases.

iBiology Videos
Anne Bertolotti Part 2: Benefits of Phosphatase Inhibition for Neurodegenerative Diseases

iBiology Videos

Play Episode Listen Later Jun 29, 2019 30:12


Bertolotti's lab has had a long time interest in understanding protein folding and the role of misfolded proteins in neurodegenerative disease. In her second talk, Bertolotti explains how her lab found that selectively inhibiting the dephosphorylation of eIF2⍺, a translation initiation factor, led to a reduction in protein synthesis. Decreasing protein synthesis allowed cells to “catch up” with the degradation of misfolded proteins that may accumulate as a result of cell stress. Her lab went on to show that a selective small molecule phosphatase inhibitor had therapeutic effects in a mouse model of Charcot-Marie-Tooth disease; a disease that results from the accumulation of misfolded protein in the ER.  This exciting result suggested that targeted inhibition of protein phosphatases may have therapeutic potential for neurodegenerative diseases.

iBiology Videos
Anne Bertolotti Part 3: A Platform to Identify Selective Protein Phosphatase Inhibitors

iBiology Videos

Play Episode Listen Later Jun 29, 2019 34:14


Bertolotti describes a platform developed by her lab that has allowed them to rationally identify selective protein phosphatase inhibitors. Using this platform her lab identified a novel small molecule phosphatase inhibitor that blocks the accumulation of misfolded proteins in the cytosol or nucleus and showed the therapeutic effects of the molecule in a model of Huntington's disease.

Audio-only streams of our videos
Anne Bertolotti Part 3: A Platform to Identify Selective Protein Phosphatase Inhibitors

Audio-only streams of our videos

Play Episode Listen Later Jun 29, 2019 34:10


Bertolotti describes a platform developed by her lab that has allowed them to rationally identify selective protein phosphatase inhibitors. Using this platform her lab identified a novel small molecule phosphatase inhibitor that blocks the accumulation of misfolded proteins in the cytosol or nucleus and showed the therapeutic effects of the molecule in a model of Huntington’s disease.

Christian Natural Health
Alkaline Phosphatase Levels

Christian Natural Health

Play Episode Listen Later Mar 29, 2019 6:03


Today's podcast comes from this article, Low Alkaline Phosphatase

PCICS Podcast
Episode 08: Alkaline phosphatase infusion to prevent organ injury after circulatory arrest

PCICS Podcast

Play Episode Listen Later Mar 1, 2019 14:08


Dr. Jesse Davidson from Children's Hospital of Colorado discusses a piglet model for cardiopulmonary bypass and its use to test alkaline phosphatase to mitigate organ injury after cardiac surgery. Guest: Jesse Davidson, MD, MPH/MSPH. Host: David Werho, MD

PCICS Podcast
Episode 08: Alkaline phosphatase infusion to prevent organ injury after circulatory arrest

PCICS Podcast

Play Episode Listen Later Mar 1, 2019 14:08


Dr. Jesse Davidson from Children's Hospital of Colorado discusses a piglet model for cardiopulmonary bypass and its use to test alkaline phosphatase to mitigate organ injury after cardiac surgery. Guest: Jesse Davidson, MD, MPH/MSPH. Host: David Werho, MD

Lab Values Podcast (Nursing Podcast, normal lab values for nurses for NCLEX®) by NRSNG

The post Alkaline Phosphatase appeared first on NURSING.com.

Science Signaling Podcast
Science Signaling Podcast, 19 March 2013

Science Signaling Podcast

Play Episode Listen Later Mar 18, 2013 13:46


Vitamin E promotes dephosphorylation of the kinase Akt to suppress the growth of prostate cancer cells.

Medizin - Open Access LMU - Teil 21/22
The Endothelial Tyrosine Phosphatase SHP-1 Plays an Important Role for Vascular Haemostasis in TNF alpha-Induced Inflammation In Vivo

Medizin - Open Access LMU - Teil 21/22

Play Episode Listen Later Jan 1, 2013


Introduction. Inflammation and endothelium-derived superoxides are important pathomechanisms in atherothrombotic diseases. We could previously show that the tyrosine phosphatase SHP-1 acts as a negative regulator in endothelial superoxide production. In this study we investigated the influence of SHP-1 on platelet-endothelium interaction and arterial thrombosis in TNF alpha-induced endothelial inflammation in vivo. Methods. Arteriolar thrombosis and platelet rolling in vivo were investigated in C57BL/6 mice using intravital microscopy in the dorsal skinfold chamber microcirculation model. Results. Inhibition of SHP-1 by the specific pharmacological inhibitor sodium stibogluconate did not significantly enhance platelet-endothelium interaction in vivo under physiological conditions but led to an augmented fraction of rolling platelets in TNF alpha-induced systemic inflammation. Accordingly, ferric-chloride-induced arteriolar thrombus formation, which was already increased by SHP-1 inhibition, was further enhanced in the setting of TNF alpha-induced inflammation. Platelet aggregation in vitro as well as ex vivo was not influenced by SHP-1-inhibition. In cultured endothelial cells, sodium stibogluconate increased TNF alpha-induced surface expression of p-selectin and von Willebrand factor. Additionally, TNF alpha increased SHP-1 activity and protein expression. Conclusions. The endothelial tyrosine phosphatase SHP-1 plays an important role for vascular hemostasis in vivo, which is crucial in TNF alpha-induced endothelial inflammation where it may serve as an autoinhibitory molecule to prevent excess inflammatory response and thrombus formation.

Science Signaling Podcast
Science Signaling Podcast, 21 December 2010

Science Signaling Podcast

Play Episode Listen Later Dec 20, 2010 14:01


Targeted removal of individual enzymes elicits changes throughout the entire network of kinases and phosphatases in yeast.

Tierärztliche Fakultät - Digitale Hochschulschriften der LMU - Teil 04/07
Einfluss von Omega-3-Fettsäuren aus Algen auf das Fettsäuremuster und auf Knochenparameter beim Pony

Tierärztliche Fakultät - Digitale Hochschulschriften der LMU - Teil 04/07

Play Episode Listen Later Jul 17, 2009


In der vorliegenden Arbeit wurde geprüft, wie sich der Anteil an Ω-3-Fettsäuren im Gesamtfettsäuremuster von Pferden nach Fütterung von Algen, die reich an Ω-3-Fettsäuren sind, verändert. Neben Fischöl gelten Mikroalgen als Quelle für die beiden Ω-3-Fettsäuren Eicosapentaensäure (EPA) und Docosahexaensäure (DHA). Positive Effekte von Ω-3-Fettsäuren auf den Knochen werden diskutiert. Aufgrund dessen wurde in der hier durchgeführten Studie erstmals die Wirkung von Ω-3-Fettsäuren auf den Knochenstoffwechsel des Pferdes untersucht. Während des insgesamt 24-wöchigen Versuchs wurden dem Futter von 4 Ponies über einen Zeitraum von 12 Wochen DHA-reiche Mikroalgen (0.32g/kg KM) zugesetzt. Im Serum wurden das Gesamtfettsäuremuster und die Knochenformationsmarker, gesamtalkalische Phosphatase und Osteocalcin bestimmt. Im Urin wurden die Knochenresorptionsmarker Collagen-Crosslinks (Pyridinolin und Desoxypyridinolin) und der Calciumgehalt untersucht. Im Serum der Tiere konnte nach Zusatz der Algen zum Futter ein deutlicher Anstieg der beiden Ω-3-Fettsäuren EPA und DHA auf das 13-fache bzw. das 23-fache nachgewiesen werden. 6 Wochen nach Absetzen der Algensupplementierung wurde die DHA-Konzentration des Ausgangswertes fast wieder erreicht. Die Verabreichung der Omega-Algen führte weder bei den Markern der Knochenformation noch bei den Markern der Knochenresorption zu signifikanten Veränderungen. Es lassen sich lediglich Tendenzen feststellen. Eventuell ist eine längere Verabreichung oder eine höhere Dosierung nötig, um eine Veränderung des Knochenstoffwechsels zu erzielen.

Science Signaling Podcast
Science Signaling Podcast, 28 October 2008

Science Signaling Podcast

Play Episode Listen Later Oct 27, 2008 8:57


Rune Linding explains how combining NetPhorest with a genetic screen enables researchers to gain insight into the relationships among the identified players.

Medizin - Open Access LMU - Teil 15/22
Inhibition of the tyrosine phosphatase SHP-2 suppresses angiogenesis in vitro and in vivo

Medizin - Open Access LMU - Teil 15/22

Play Episode Listen Later Jan 1, 2008


Endothelial cell survival is indispensable to maintain endothelial integrity and initiate new vessel formation. We investigated the role of SHP-2 in endothelial cell survival and angiogenesis in vitro as well as in vivo. SHP-2 function in cultured human umbilical vein and human dermal microvascular endothelial cells was inhibited by either silencing the protein expression with antisense-oligodesoxynucleotides or treatment with a pharmacological inhibitor (PtpI IV). SHP-2 inhibition impaired capillary-like structure formation (p < 0.01; n = 8) in vitro as well as new vessel growth ex vivo (p < 0.05; n = 10) and in vivo in the chicken chorioallantoic membrane (p < 0.01, n = 4). Additionally, SHP-2 knock-down abrogated fibroblast growth factor 2 (FGF-2)-dependent endothelial proliferation measured by MTT reduction ( p ! 0.01; n = 12). The inhibitory effect of SHP-2 knock-down on vessel growth was mediated by increased endothelial apoptosis ( annexin V staining, p ! 0.05, n = 9), which was associated with reduced FGF-2-induced phosphorylation of phosphatidylinositol 3-kinase (PI3-K), Akt and extracellular regulated kinase 1/2 (ERK1/2) and involved diminished ERK1/2 phosphorylation after PI3-K inhibition (n=3). These results suggest that SHP-2 regulates endothelial cell survival through PI3-K-Akt and mitogen-activated protein kinase pathways thereby strongly affecting new vessel formation. Thus, SHP-2 exhibits a pivotal role in angiogenesis and may represent an interesting target for therapeutic approaches controlling vessel growth. Copyright (C) 2007 S. Karger AG, Basel.

Tierärztliche Fakultät - Digitale Hochschulschriften der LMU - Teil 03/07
Untersuchung des Knochenstoffwechsels anhand der Knochenmarker Knochenspezifische Alkalische Phosphatase und Pyridinolin am intakten und ovariohysterektomierten Osteoporosemodell Minipig

Tierärztliche Fakultät - Digitale Hochschulschriften der LMU - Teil 03/07

Play Episode Listen Later Jul 20, 2007


Ziel dieser Studie war es, am Tiermodell Minipig eine eventuelle Zyklusabhängigkeit des Knochenstoffwechsels zu zeigen und im weiteren Verlauf der Studie die Auswirkung der Ovariohysterektomie bei diesem Großtiermodell auf den Knochenstoffwechsel anhand von ausgewählten Knochenmarkern zu untersuchen. Die Bestimmung von biochemischen Knochenmarkern hat sich als Osteoporose-Monitoring zur Analyse des Knochenstoffwechsels weit verbreitet. Bei Frauen konnte in diesem Zusammenhang eine Abhängigkeit vom Zyklus nachgewiesen werden. Für diese Untersuchung wurden 16 weibliche intakte Minipigs nach Gewicht randomisiert in drei Versuchsgruppen eingeteilt. Alle Tiere erhielten ein bedarfsgerechtes Futtermittel, wobei der Calciumgehalt in zwei Gruppen 0,69% und in der dritten Gruppe 0,9% betrug. Die Tiere einer Gruppe wurden nach 65 Wochen ovariohysterektomiert (OHX) und noch weitere 8 Wochen betrachtet. Während des gesamten Versuchszeitraums wurden zu verschiedenen Zeitpunkten Blutproben entnommen. In diesen Proben wurden zur Kontrolle des Zyklusstands die Hormone Progesteron und Östradiol und zur Beurteilung des Knochenstoffwechsels die Marker Knochenspezifische Alkalische Phosphatase (BAP) und Pyridinolin (PYD) bestimmt. Die Entwicklung der Knochenmarker BAP und PYD zeigte vor Ovariohysterektomie einen Zusammenhang mit dem altersbedingten Wachstum, wobei bei Pyridinolin ein starker Effekt im Zusammenhang mit der Calciumreduktion zu beobachten war. Dies kann auf einen Anstieg von Parathormon und Calcitriol bei gleichzeitiger Aktivierung der Osteoklasten zurückzuführen sein. Ein eindeutiger Zusammenhang zwischen Zyklus und Knochenstoffwechsel konnte nicht belegt werden. Es ist zu überlegen, ob dieser Zusammenhang eventuell in einem Versuch mit größerer Tierzahl und häufigeren Blutentnahmen über einen längeren Zeitraum gezeigt werden könnte. Nach Ovariohysterektomie konnte der high turnover des Knochenstoffwechsels in einem deutlichen Anstieg der BAP-Konzentration in Woche 4 post OHX gezeigt werden. Bei der Bestimmung von Pyridinolin zeigte sich zu den jeweiligen Probezeitpunkten kein Effekt der OHX. Diese Studie bestätigt den Nutzen des Minipigs als Osteoporosemodell im Hinblick auf die Verwendung des Knochenformationsmarker Knochenspezifische Alkalische Phosphatase, um dynamische Knochenturnovervorgänge darzustellen.

Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 02/06
The role of Src-homology 2 domain containing tyrosine phosphatase 2 in growth factor dependent endothelial signalling and angiogenesis

Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 02/06

Play Episode Listen Later May 24, 2007


Endothelial cell survival is indispensable to maintain endothelial integrity and initiate new vessel formation. We investigated the role of SHP-2 in proliferation, survival and sprouting of human microvascular- and umbilical vein endothelial cells (HMEC, HUVEC) using antisense oligonucleotides (AS-ODN) and a pharmacological SHP-2 inhibitor (PtpI IV). Knock-down of SHP-2 decreased bFGF and PDGF dependent endothelial cell proliferation (p

Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 02/06
Structure and mechanism of the RNA polymerase II CTD phosphatase Scp1 and large-scale preparation of the RNA polymerase II-TFIIF complex

Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 02/06

Play Episode Listen Later May 29, 2006


TFIIF is the only general transcription factor that has been implicated in the preinitiation complex assembly, open complex formation, initiation and transcription elongation. In addition, TFIIF stimulates Fcp1, a central phosphatase needed for recycling of RNA polymerase II (Pol II) after transcription by dephosphorylation of the Pol II C-terminal domain (CTD). This thesis reports the X-ray structure of the small CTD phosphatase Scp1, which is homologous to the Fcp1 catalytic domain. The structure shows a core fold and an active center similar to phosphotransferases and –hydrolases that solely share a DXDX(V/T) signature motif with Fcp1/Scp1. It was further demonstrated that the first aspartate in the signature motif undergoes metalassisted phosphorylation during catalysis, resulting in a phosphoaspartate intermediate that was structurally mimicked with the inhibitor beryllofluoride. Specificity may result from CTD binding to a conserved hydrophobic pocket between the active site and an insertion domain that is unique to Fcp1/Scp1. Fcp1 specificity may additionally arise from phosphatase recruitment near the CTD via the Pol II subcomplex Rpb4/7, which is shown to be required for Fcp1 binding to the polymerase in vitro. Until now, the main impediment in the high resolution crystallographic studies of TFIIF in complex with Pol II and other members of transcription machinery was unavailability of soluble, stoichiometric TFIIF complex in sufficient amounts. This thesis reports on the development of the overexpression system in yeast and a purification protocol that enabled for the first time to isolate milligram amounts of a pure and soluble, 15-subunit (~0,7 MDa) stoichiometric Pol IITFIIF complex. Such complex together with the promoter DNA, RNA, TBP and TFIIB assembles in vitro into the yeast initially transcribing complex, which can now be studied structurally.

Tierärztliche Fakultät - Digitale Hochschulschriften der LMU - Teil 02/07
IRAG als funktionales Element der NO/cGMP Signalkaskade im Gastrointestinaltrakt

Tierärztliche Fakultät - Digitale Hochschulschriften der LMU - Teil 02/07

Play Episode Listen Later Feb 10, 2006


Die cGMP-abhängige Proteinkinase (cGKI) vermittelt den relaxierenden Effekt der NO/cGMP Signalkaskade im glatten Muskel. Die Phosphorylierung des IP3-Rezeptor assoziierten cGMP-Kinase Substrats (IRAG) ist ein Prozess, der in diesem Mechanismus involviert ist. Um dieses Modell genauer zu verifizieren, wurde die cGMP-abhängige Relaxation in Mäusen, die ein modifiziertes IRAG expremieren, untersucht. Bei der IRAGD12/D12 Maus handelt es sich um eine Deletionsmutante, bei der die Interaktionsstelle von IRAG mit dem IP3-Rezeptor Typ I zerstört wurde, was dazu führt, dass IRAG nicht mit dem IP3-Rezeptor Typ I interagieren kann. Diese Mäuse zeigen eine normale Futteraufnahme, der Kotabsatz ist aber signifikant geringer als bei Wildtypmäusen. Eine Röntgenkontrastuntersuchung mit Hilfe von Bariumsulfat offenbarte eine deutliche Verlängerung der Darmpassagezeit, einen Megaoesophagus sowie ein Megacolon. In situ-Erhebungen an der eröffneten Bauchhöhle bestätigen diese Befunde. Gründe für diese Veränderungen könnten Funktionsstörungen in der glatten Muskulatur sein. Zur Stützung dieser Vermutung wurden die cGMP-abhängigen Effekte an der glatten Muskulatur des Darmtraktes untersucht. Die Untersuchung des Hormon induzierten Tonus im Jejunum ergab keinen signifikanten Unterschied in der cGMP-Wirkung zwischen den Wildtyp- und den IRAGD12/D12 Mäusen. Der CCh induzierte Tonus im Colon der Wildtypmäuse wird im Gegensatz zu den IRAGD12/D12 Mäusen durch 8 Br- cGMP um ca. 90% reduziert. Bei den IRAGD12/D12 Mäusen bewirkt 8 Br cGMP nur eine sehr geringe Relaxation (16%) des Hormon induzierten Tonus am Längsmuskel des Colons. Eine Vorinkubation mit dem Phosphatase-Hemmstoff Calyculin A in Präparaten von Wildtypmäusen hebt den relaxierenden Effekt von 8-Br-cGMP im glatten Muskel des Dünndarms auf, im glatten Muskel des Colons findet dagegen keine Aufhebung statt. Die Ergebnisse zeigen, dass IRAG eine entscheidende Bedeutung für die cGMP/cGKI-vermittelte Relaxation im Colon, aber nicht im Jejunum hat. Eine cGMP/cGKI-vermittelte Aktivierung einer Phosphatase kann als möglicher Mechanismus der cGMP abhängigen Relaxation im Jejunum in Frage kommen. Eine mögliche Phosphatase könnte hierbei die Myosin leichte Ketten Phosphatase (MLCP) sein. Es ist aber nach wie vor unklar, ob diese Ergebnisse Grund für die geringe Lebenserwartung der IRAGD12/D12 Mäuse sind. Um die Ursachen dafür zweifelsfrei feststellen zu können, bedarf es weiterführender Untersuchungen.

Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 04/19
Die Bedeutung von Genveränderungen bei Tyrosinphosphatasen

Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 04/19

Play Episode Listen Later Nov 10, 2005


In der vorliegenden Arbeit wurde die Tyrosinphosphatase hVH-5 nach Genveränderungen untersucht. Als Mitglied der Familie der dual-spezifischen Phosphatasen ist hVH-5 (homologue of vaccinia virus H1 phosphatase gene clone 5) an der Signaltransduktion der Zelle durch Dephosphorylierung von stress-aktivierter Proteinkinase (SAPK) und p38 beteiligt. Aufgrund seiner Rolle als potentielles Tumorsuppressorgen können Genveränderungen oder Fehlregulationen von hVH-5 zur Entstehung von Tumoren beitragen. Wie bereits bekannt ist, zählt die Lokalisation dieses Gens auf Chromosom 11p15.5 zu häufig beobachteten Loss Of Heterozygosity (LOH)-Regionen bei Krebserkrankungen, u.a auch bei Brustkrebs. Im Rahmen dieser Arbeit wurde eine Expressionsanalyse der hVH-5 Phosphatase in Normalgewebe und Brustkrebszelllinien durchgeführt. Es konnte gezeigt werden, dass hVH-5 nicht nur, wie schon bekannt, in humanem Gehirn, Herz und Skelettmuskel transkribiert wird, sondern eine Trankription darüber hinaus auch noch in 10 weiteren humanen Geweben sowie in allen untersuchten Brustkrebszelllinien nachgewiesen werden konnte. Um ein zeitsparendes und effektives Mutationsscreening zu ermöglichen, wurde eine neue Methode, Conformation Sensitive Gel Electrophoresis (CSGE), etabliert. Dadurch konnte bereits im ersten Exon dieser Phosphatase eine Heteroduplexbildung als Hinweis auf eine Genveränderung sichtbar gemacht werden. Mittels Sequenzierungs- sowie Restriktions-Fragment-Längen-Polymorphismus-Analyse konnte die exakte Nukleotidsequenz bestimmt werden. Es wurde festgestellt, dass es sich hierbei um eine bisher unbekannte Gensequenz eines prozessierten Pseudogens der hVH-5 Phosphatase handelt. Prozessierte Pseudogene sind dadurch definiert, dass sie typischerweise durch einen Verlust des Promotors transkriptionell inaktiv sind. Eine Datenbankrecherche (Sanger Center) ermöglichte die Lokalisation des Pseudogens auf Chromosom 10q22.2. Phylogenetische Analysen führten zu dem Ergebnis, dass das Gen vor ca. 6,8 Mio. Jahren entstanden sein müsste. Es konnte gezeigt werden, dass das in dieser Arbeit beschriebene Pseudogen von hVH-5 (ψhVH-5) entgegen der Definition eines Pseudogens in gesunden menschlichen Geweben transkribiert wird. Diese Tatsache deutet auf eine evtl. funktionelle Bedeutung dieses Gens hin. Weiterhin konnte ein möglicher Zusammenhang zwischen der Transkription des Pseudogens und der Entstehung von Brustkrebs nicht ausgeschlossen werden, da dieses in drei von 14 untersuchten Brustkrebszelllinien nicht transkribiert wird. Aus der Übersetzung der Nukleinsäuresequenz von ψhVH-5 in Aminosäuren resultierte ein Peptid von 8.8 kDa, welches sich stark vom hVH-5 Wildtyp unterscheidet und keinerlei funktionelle Domänen aufweist. Es konnte weder ein Nachweis des transient überexprimierten Proteins noch eines in vivo translatierten Proteins erbracht werden. Die Akkumulation der zahlreichen Mutationen im Pseudogen verglichen mit dem Wildtyp deutet stark darauf hin, dass dieses Gen in der Evolutionsgeschichte aufgrund fehlender funktioneller Bedeutung zumindest zeitweise keinem Selektionsdruck unterlag oder aber sich zu einem selbständigen Gen mit noch unbekannter Funktion weiterentwickelte. In der vorliegenden Arbeit konnten Hinweise dafür gebracht werden, dass ψhVH-5 sich möglicherweise derart entwickelt hat, um durch andere Regulationsmechanismen, beispielsweise solche, die für nicht-kodierende RNAs bekannt sind, mit dem Wildtypgen der hVH-5 Phosphatase zu interagieren.

Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 02/19
Therapeutische Angiogenese bei chronischer Myokardischämie - Applikation von FGF-2 mittels selektiver druckregulierter Retroinfusion im tierexperimentellen Modell am Schwein

Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 02/19

Play Episode Listen Later May 13, 2004


Die Applikation vaskulärer Wachstumsfaktoren zur Stimulation des Kollateral- (therapeutische Arteriogenese) und des Kapillarwachstums (therapeutische Angiogenese) stellt einen möglichen neuen Ansatz in der Behandlung der koronaren Herzerkrankung dar. Der klinische Einsatz vaskulärer Wachstumsfaktoren ist derzeit aber vor allem durch ein klinisch verfügbares sicheres und effektives Applikationsverfahren limitiert. Die selektive synchronisierte druckregulierte Retroinfusion (SSR) von Koronarvenen ist ein klinisch etabliertes Herzkatheterverfahren und erlaubt eine effektive Applikation von Medikamenten und Genvektoren in ischämisches Myokardgewebe. In der vorliegenden Arbeit wurde deshalb die Auswirkung der Retroinfusion des vaskulären Wachstumsfaktors FGF-2 in die Koronarvene auf das Kollateralwachstum (Arteriogenese), das Kapillarwachstum (Angiogenese), den myokardialen Blutfluss und die kontraktile Myokardfunktion bei chronischer, experimenteller Myokardischämie (Schwein) untersucht, und mit der intrakoronaren Applikation von FGF-2 verglichen. Eine hochgradige koronararterielle Stenose, mit Progression zur Komplettokklusion bis zum Tag 28 der Untersuchung, wurde durch Implantation eines Reduktionsstent-Graft in die LAD induziert. Nach 7 Tagen wurde eine 30 minütige Retroinfusion (anteriore Herzvene) ohne (Kontrollgruppe A, n=7) und unter Zugabe von 150µg FGF-2 (Gruppe B, n=7) durchgeführt, und mit der antegraden Infusion (30 min) von FGF-2 in die Koronararterie (LAD) verglichen (Gruppe C, n=7). 28 Tage nach Implantation des Reduktionsstent erfolgte die Bestimmung der Anzahl der Kollateralarterien (post-mortem Angiographie) und der Kapillardichte (Histologie, Färbung für alkalische Phosphatase). Der regionale myokardiale Blutfluss (fluoreszierende Mikrosphären) und die kontraktile Myokardfunktion (Sonomikrometrie) wurden unter Ruhebedingungen und Bedingungen mit gesteigertem Sauerstoffbedarf (rechts-atriales Pacing) gemessen. Am Versuchende, 28 Tage nach Implantation des Reduktionsstent, konnte die Retroinfusion von FGF-2 in die Koronarvene (Gruppe B), verglichen mit den unbehandelten Kontrolltieren (Gruppe A) und der antegraden Applikation von FGF-2 in die Koronararterie (Gruppe C), eine signifikante Zunahme der Kollateralarterien (5,2±1,1 vs. 2,95±0,4 vs. 3,3±0,3, p

Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 02/19
Charakterisierung von humanen mesenchymalen Stammzellen und Zellen der osteoblastären Differenzierungskaskade

Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 02/19

Play Episode Listen Later Mar 3, 2004


Mit zunehmendem Wissen über die Komplexität der Osteogenese wurde die Aussagekraft einzelner osteoblasten-assoziierter Marker in den letzten Jahren immer mehr in Frage gestellt. Denn der Nachweis einzelner Marker erlaubt weder eine eindeutige Identifikation undifferenzierter hMSC noch eine Abgrenzung von hMSC zu anderen Reifungsstufen der osteoblastären Kaskade. Das Ziel dieser Untersuchung war es daher, über die Erstellung eines immunzytochemischen Färbeprofils mehrerer Marker gleichzeitig, undifferenzierte hMSC eindeutig zu identifizieren und sie gegenüber hOB und evtl. weiteren Differenzierungsstufen der osteoblastären Kaskade abzugrenzen. Durch die Erstellung eines immunzytochemischen Färbeprofils ist es möglich, heterogene Zellpopulationen auf Einzelzellniveau zu charakterisieren und sie voneinander zu unterscheiden. Diese Untersuchung stellt einen sehr erfolgversprechenden Ansatz dar, undifferenzierte humane mesenchymale Stammzellen (hMSC) zu identifizieren und sie von Zellen der osteoblastären Differenzierungskaskade sowie anderen Zelltypen abgrenzen zu können. Insgesamt konnte in dieser Arbeit gezeigt werden, dass humane MSC und humane Osteoblasten jeweils spezifische Färbeprofile aufweisen, die eine eindeutige Diskriminierung der beiden Zellpopulationen erlauben. Für eine spezifische Unterscheidung auf Einzelzellnineau ist eine intensive Suche nach spezifischen Markern oder die Kombination mehrerer Marker notwendig. Von den in dieser Arbeit ausgewählten Proteinen ergaben Bone Sialoprotein, Osteocalcin, Decorin sowie Kollagen-IV und Kollagen-X unterschiedliche Färbemuster. Kollagen-IV und –X waren besonders in hMSC positiv, weswegen sie sich für eine Erkennung unreifer osteoblastärer Zellen anbieten. Bone Sialoprotein, Osteocalcin und Decorin eignen sich dagegen für einen Nachweis reifer osteoblastärer Zellen, denn sie waren nur in hOB positiv. Osteocalcin und Decorin erlaubten außerdem eine Abgrenzung differenzierter hOB. Die Proteine Versican und Matrixmetalloproteinase-2 erscheinen darüber hinaus für eine Unterscheidung von osteoblastären und fibroblastären Zellen nützlich zu sein. Die klassischen osteoblastären Marker alkalische Phosphatase, Kollagen-I, Osteopontin oder Osteonectin waren für eine Unterscheidung von hMSC und hOB nicht geeignet, da sie in beiden Zellpopulationen gleiche Ergebnisse lieferten. Insbesondere der Wert der alkalische Phosphatase als immunzytochemischer Marker der osteoblastären Kaskade wird in dieser Untersuchung in Frage gestellt. Eine osteogene Stimulation von hMSC mit Dexamethason, b-Glyzerophosphat und Askorbinsäure bewirkte eine deutliche Veränderung der Morphologie, der Wachstumseigenschaften und des Färbeprofils. Allerdings konnte durch eine osteogene Stimulation kein immunzytochemischer Phänotyp von hMSC induziert werden, welcher identisch mit dem der hOB war. Daher ist es unwahrscheinlich, dass eine Stimulation in vitro mit den oben aufgeführten Zusätzen die komplizierten Verhältnisse der Osteogenese in vivo nachahmen kann.

Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 02/19
Untersuchungen zur Wechselbeziehung von Struktur und Funktion von Transkriptionsaktivatoren am PHO5-Promotor in Saccharomyces cerevisiae

Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 02/19

Play Episode Listen Later Nov 13, 2003


Im Rahmen dieser Arbeit wurden zwei Fragen zur Wechselbeziehung von Struktur und Funktion von Transkriptionsaktivatoren am PHO5-Promotor in Saccharomyces cerevisiae bearbeitet. Im ersten Teil der Arbeit wurden zwei Deletionsmutanten des Transkriptionsfaktors Pho4 hergestellt und charakterisiert. Dazu wurden aus der PHO4-Sequenz mit Hilfe einer PCR-gestützten Methode jeweils die für die Aminosäuren 97 bis 106 sowie 101 bis 110 kodierenden Sequenzen entfernt. Mit Hilfe von geeigneten Expressionsvektoren wurden beide Deletionsmutanten in S. cerevisiae exprimiert. Das Ausmaß der Transkriptionsaktivierung durch die Konstrukte wurde durch Messung der Aktivität der saueren Phosphatase bestimmt. Beide Mutanten bewirkten eine deutliche Aktivierung der Transkription am PHO5-Promotor. Daraus kann geschlossen werden, dass die hier deletierten Abschnitte von PHO4 keinen wesentlichen Anteil an der Transkriptionsaktivierung durch Pho4 haben. Zwischenzeitlich wurde der Nachweis einer minimalen transkriptionsaktivierenden Domäne von Pho4 publiziert. Die minimale transaktivierende Domäne besteht aus den Aminosäuren 79 bis 99. Dieser Abschnitt ist notwendig und ausreichend für die Öffnung der Chromatinstruktur des Promotors und die Aktivierung der Transkription. Die hier beschriebene Herstellung und Charakterisierung der Deletionsmutanten von PHO4 leistete einen wesentlichen Beitrag zur Eingrenzung der transaktivierenden Domäne von Pho4. Der zweite Teil der Arbeit befasste sich mit der Wirkung des als GAGA-Faktor (GAF) bezeichneten Transkriptionsfaktors aus Drosophila melanogaster am PHO5-Promotor von S. cerevisiae. Dazu wurden chimäre Konstrukte aus dem GAF und der DNA-bindende Domäne des Transkriptionsaktivators Pho4 hergestellt. Zudem wurden auch Fusionskonstrukte mit jeweils nur einer Hälfte des GAF und der DNA-bindende Domäne des Pho4 erzeugt. Diese Konstrukte wurden in S. cerevisiae exprimiert. Hier zeigte sich, dass die Verbindung aus dem gesamten GAF und der DNA-bindenden Domäne von Pho4, vermutlich aufgrund eines gestörten intrazellulären Transports oder aufgrund einer Instabilität des Proteins, keine Aktivität aufwies. Dagegen waren die Konstrukte, welche jeweils nur eine Hälfte des GAF enthielten, in der Lage, eine Öffnung der Chromatinstruktur und eine Aktivierung der Transkription am PHO5-Promotor zu bewirken.Um zu untersuchen, welche Veränderungen die chimären Konstrukte durch ihr Angreifen am PHO5-Promotor an der Chromatinstruktur des Promotors verursachen, wurden spezielle experimentelle Verfahren angewendet. Diese nutzen die unterschiedliche Zugänglichkeit der DNA für Enzyme bei geöffneter oder geschlossener Chromatinstruktur. Die Untersuchungen zeigten eine gleichartige Öffnung der Chromatinstruktur am PHO5-Promotor durch beide Konstrukte. Die gleichartige Öffnung der Chromatinstruktur korrelierte dabei nicht mit dem unterschiedlichen Umfang der Transkriptionsaktivierung von PHO5. Aufgrund der fehlenden Korrelation von Chromatinöffnung und Transkriptionsaktivierung stehen diese Ergebnisse im deutlichen Widerspruch zu der von vielen Autoren vertretenen Hypothese, der GAF würde die Transkription indirekt, nur durch die Öffnung repressiver Chromatinstrukturen aktivieren (Derepression). Vielmehr weisen diese Ergebnisse darauf hin, das der GAF auch als klassischer Transaktivator eine direkte Aktivierung der Transkription bewirken kann. Die Aktivierung der Transkription durch beide Hälften des GAF in den chimären Konstrukten war ein überraschendes Ergebnis. Es zeigt, dass GAF neben der glutaminreichen Domäne eine weitere Domäne enthält, die in vivo eine Aktivierung der Transkription bewirken kann. Diese Erkenntnis ist grundsätzlich neu und in der Literatur bisher nicht beschrieben worden.

Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 02/19
Einfluß der Chromatinumgebung auf die Genregulation durch den Transkriptionsfaktor Sin4 aus der Hefe Saccharomyces cerevisiae

Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 02/19

Play Episode Listen Later Oct 9, 2003


Nach der Aufklärung der Basenabfolge des Genoms von Saccharomyces cerevisiae ist die Funktion der 30.000-40.000 Gene und insbesondere das Zusammenspiel der Regulation der einzelnen Gene ein zentrales Thema der Molekularbiologie. Die DNA eukaryonter Zellen liegt durch Bindungen an Histon-Proteine im Zellkern als Chromatin vor. Die Chromatinstruktur dient nicht nur der Komprimierung der DNA auf engstem Raume, sondern hat auch starke Auswirkungen auf die Funktion der DNA. So müssen Gene bei ihrer Aktivierung durch Veränderung ihrer Chromatinstruktur, die bis zur Ablösung der Histone führen kann, den für die Transkription benötigten Enzymen und Faktoren erst zugänglich gemacht werden. Das PHO5-Gen der Hefe Saccharomyces cerevisiae stellt ein sehr gut untersuchtes Modell dar, bei dem Veränderungen der Chromatinstruktur genau untersucht und mit dem funktionellen Zustand des Gens korreliert worden sind. PHO5 kodiert für eine saure Phosphatase, die bei Verbrauch der Phosphatreserven der Zelle in den periplasmatischen Raum sezerniert wird, um aus dort eventuell vorhandenen organischen Phosphatverbindungen Phosphat zu gewinnen. Ist im Medium genügend Phosphat vorhanden, ist PHO5 reprimiert. In diesem Zustand ist die Chromatinstruktur des PHO5-Promotors durch vier dicht aufeinander folgende Nukleosomen gekennzeichnet, wodurch der Promotor Enzymen und regulatorischen Proteinen allgemein schlecht zugänglich ist. Nur zwischen dem zweiten und dem dritten Nukleosom ist die dichte Anordnung der Nukleosomen durch einen etwa 70 bp langen gut zugänglichen Bereich unterbrochen. In dieser sogenannten hypersensitiven Region bindet bei Phosphatmangel der aktivierende Transkriptionsfaktor Pho4 gemeinsam mit dem Faktor Pho2 an ein UAS-Element und induziert die PHO5-Expression. Dabei lösen sich die vier Nukleosomen vom DNA-Strang ab. Sin4 ist ein Transkriptionsfaktor der Hefe Saccharomyces cerevisiae, der auf mehrere Promotoren zumeist reprimierenden Einfluss ausübt. Ausgangspunkt der hier vorliegenden Arbeit war der Befund, dass in Abwesenheit von Sin4 die Gegenwart der prokaryontischen lacZ Sequenz stromaufwärts des PHO5-Promotors zu einer Derepression des PHO5-Gens führt, und zwar in Gegenwart von Phosphat, also unter eigentlich reprimierenden Bedingungen. Dieser Effekt wurde ursprünglich bei der Verwendung der kodierenden Sequenz von lacZ als dem PHO5-Promotor nachgeschalteten Reporter-Gen in sin4-Hefezellen entdeckt. Eine Frage der hier vorliegenden Arbeit galt der Ursache der Derepression von PHO5 durch die lacZ kodierenden DNA-Sequenz. Dazu interessierte uns, ob die Derepression ein spezielles Phänomen der lacZ-Sequenz ist oder ob es sich hierbei eher um eine allgemeine Eigenschaft von DNA-Fragmenten handelt. Außerdem interessierte uns, ob die Herkunft der DNA aus prokaryonten oder eukaryonten Zellen eine Rolle spielen könnte. Dazu wurde jeweils eine große Anzahl zufällig ausgewählter DNA-Fragmente einer Länge zwischen 900bp und 1200bp aus den Genomen der Hefe Saccharomyces cerevisiae und der Bakterien Escherichia coli und Micrococcus lysodeikticus an entsprechender Stelle vor den PHO5-Promotor integriert. Die so konstruierten Plasmide wurden in einen Hefestamm transformiert, in dem das SIN4-Gen zerstört worden war. Insgesamt wurden 400 Klone mit integrierten Hefe-DNA-Fragmenten, 300 Klone mit integrierten M. lysodeikticus-DNA-Fragmenten und 14 Klone mit integrierten E. coli-DNA-Fragmenten untersucht. Die Bestimmung der Phosphatase-Aktivitäten der einzelnen Klone ergab für fast alle Plasmide mit integrierten E. coli- und M. lysodeikticus-DNA-Fragmenten eine erhöhte Aktivität trotz phosphatreichen Mediums. Im Gegensatz dazu zeigten die wenigsten Plasmide mit integrierten Hefe-DNA-Fragmenten eine Erhöhung der PHO5-Expression unter denselben Bedingungen. Von den insgesamt 400 getesteten Plasmiden wiesen nur neun eine gesteigerte PHO5-Expression auf. In allen Fällen, also für alle E. coli-, M. lysodeikticus- und Hefe-DNA-Fragmente, wurde nur in Abwesenheit von Sin4 eine erhöhte Phosphatase-Aktivität gemessen. Bei seiner Anwesenheit wurden in phosphatreichem Medium nie gesteigerte Aktivitäten beobachtet. Diese Ergebnisse zeigen deutlich, dass die hier beobachtete Derepression typischerweise eine Eigenschaft prokaryonter DNA ist. Nur ein Bruchteil der eukaryonten DNA-Fragmente aus dem Hefe-Genom führt zu einer Derepression der Promotoraktivität, während dies nahezu alle prokaryonten DNA-Fragmente aus Escherichia coli- bzw. Micrococcus lysodeikticus tun. Um die neun Hefe-DNA-Fragmente, die zu einer Aktivierung des PHO5-Promotors führten, auf eventuelle Besonderheiten zu untersuchen, wurden ihre DNA-Sequenzen bestimmt und analysiert. Außerdem wurden noch zwei E. coli-DNA-Fragmente sequenziert, die zu keiner gesteigerten PHO5-Expression geführt haben. Diese sehr eindeutigen Ergebnisse werfen Fragen nach dem zugrunde liegenden Mechanismus auf. Eventuelle DNA-Methylierungen oder kryptische Promotoren schieden als Erklärung des Phänomens aus. Unterschiede des G-C-Gehalts der einzelnen DNA-Fragmente könnten besonders für die prokaryonte DNA teilweise eine Erklärung liefern. Die beiden prokaryonten Genome haben mit 51% bzw.72% einen wesentlich höheren G-C-Gehalt als das Hefegenom mit 38%. Besonders die beiden E. coli-DNA-Fragmente, die zu keiner gesteigerten PHO5-Expression führten, besitzen einen wesentlich geringeren G-C-Gehalt als der Durchschnitt des gesamten E. coli-Genoms (44,7% bzw. 38,0% im Vergleich zu 51%). Eukaryonte DNA besitzt in ihrer Sequenz im Gegensatz zu der aus Prokaryonten eine gewisse Periodizität, die sich etwa alle 10,5bp wiederholt und die Ausbildung von Nukleosomen erleichtert. Das Fehlen dieser Periodizität in prokaryonter DNA könnte sich ebenfalls auswirken, z.B. über eine labile Chromatinstruktur, die sich auch auf den benachbarten PHO5-Promotor auswirkt und dadurch eine Dereprimierung von PHO5 in sin4-Zellen auslöst. Die Dereprimierung des PHO5-Promotors durch die wenigen Hefe-DNA-Fragmente trotz reprimierender Bedingungen könnten aufgrund anderer Mechanismen zustande zu kommen. Die neun Hefe-DNA-Fragmente, die zu einer Aktivierung des PHO5-Promotors führten, zeigten auch keinen vom Hefegenom abweichenden G-C-Gehalt. Es ist auffällig, dass alle 9 DNA-Fragmente intergenische Bereiche enthalten. In diesen Bereichen gibt es oft regulatorische Elemente, die häufig in hypersensitiven Regionen gefunden werden. Hypersensitive Regionen sind nicht in Nukleosomen gepackt und könnten dadurch auch die umgebene Chromatinstruktur beeinflussen. Unabhängig von den mechanistischen Überlegungen zeigen diese Untersuchungen, dass die Aktivität eines Promotors von der Umgebung beeinflusst werden kann und dass daher der Einsatz von heterologen Reportergenen mit Vorsicht betrachtet werden muss.

Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 01/06
Charakterisierung der Rolle und Funktion der Protein-Tyrosin-Phosphatase Meg2

Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 01/06

Play Episode Listen Later Jun 17, 2003


In dieser Arbeit wurde die biologische Funktion der PTP-Meg2 in der zellulären Signaltransduktion untersucht. Analysen mittels c-DNA-Filter, „Real Time PCR” und Immunblot zeigen eine ubiquitäre Expression der PTP-Meg2 auf ähnlichem, jedoch geringem Niveau in fast allen untersuchten Krebszelllinien unterschiedlicher Gewebeherkunft, wobei die Expressions-stärke nicht in direktem Zusammenhang mit krebsrelevanten Eigenschaften wie Invasivität und Metastasierung steht. Die induzierte Differenzierung von MCF 7-Zellen durch Natriumbutyrat steigert die Meg2-Expression um das 5-fache, wogegen die Differenzierung von SW948- und SK-N- SH-Zellen mit TPA bzw. Retinolsäure die Meg2-Expression reprimiert. Zellfraktionierung und Immunfluoreszenz zeigen eine primär zytosolische, aber partiell auch vesikuläre bzw. strukturierte Lokalisation der PTP-Meg2, für welche die CRALBP-Domäne der PTP-Meg2 mitverantwortlich ist. Untersuchungen der endogenen Meg2-Aktivität nach Immunpräzipitation und in vitro Phosphatasetests zeigen eine erhöhte Phosphataseaktivität nach Stimulation von Zellen mit FCS, EGF und LPA, wogegen TPA stark inhibierenden wirkt. Aktivitätsstudien mit GST-Meg2-Fusionsproteinen zeigen, dass die CRALBP-Domäne die Meg2-Phosphataseaktivität negativ reguliert. Im Protein-Lipid-Overlay interagiert PTP-Meg2 mit PI(3)P, PI(4)P, PI(5)P und Phosphatidylserin. Eine Interaktion mit PI(4)P führt zu einer erhöhten Meg2 Aktivität. Pervanadat-Stimulation von Zellen führt zu einer Tyrosinphosphorylierung sowie einer Mobilitätsänderung der PTP-Meg2, was auch mit einer katalytisch inaktiven Meg2-Mutante beobachtet wurde. PTP-Meg2 interagiert in vitro und in Koexpressionsstudien mit dem EGF-Rezeptor in Abhängigkeit von dessen Aktivierung. Eine physiologische Relevanz konnte nicht gezeigt werden. Die Depletion der PTP-Meg2 durch spezifische siRNA führt zu einer erhöhten Tyrosinphosphorylierung einiger, noch zu identifizierender Proteine. PTP-Meg2 vermindert, die inaktive PTP-Meg2CS-Mutante erhöht die durch v-ErbB und EGF-Rezeptor, nicht aber die durch HER2 und v-Ki-Ras induzierte Transformation von NIH3T3-Zellen im Focusbildungstest. Zudem bewirkt PTP-Meg2CS, mit Ausnahme der v-Ki-Ras infizierten Zellen, eine leicht erhöhte ERK1/2-Aktivität. Ferner stimuliert PTP-Meg2 die Migration von NIH3T3-Zellen im Wundheilungsexperiment. Ein Einfluss auf die basale und durch Stimuli induzierte Proliferation von Zellen in Wachstumstests wurde nicht beobachtet. Ein durch siRNA-vermittelter Meg2- „knockdown“ führte zur Induktion bzw. Repression der Expression von Genen, wie z.B. einiger Liganden, Caveolin-2, Nck und Rock, was auf eine Beteiligung der PTP-Meg2 an der Regulation von Signalwegen kleiner GTPasen bzw. von endo- sowie exocytotischen Prozessen schließen lässt.

Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 01/06
Kooperative Wechselwirkungen von Transkriptionsfaktoren und Histonen mit Promotorelementen der Phosphatasegene PHO5 und PHO8 in Saccharomyces cerevisiae

Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 01/06

Play Episode Listen Later Jul 25, 2001


In der vorliegenden Arbeit wurde die Wechselwirkung zwischen Transkriptionsfaktoren und Elementen der Chromatinstruktur bei der Regulation zweier Promotoren des Phosphatase-systems der Hefe Saccharomyces cerevisiae untersucht. Als Modellsystem wurden die Promotoren der Gene PHO5 und PHO8 gewählt, die für eine saure bzw. eine alkalische Phosphatase kodieren und durch Phosphatmangel induziert werden. Während der Induktion findet eine charakteristische Chromatin-Umordnung am Promotor statt, die sich bei PHO5 über einen Bereich von vier Nukleosomen ausdehnt, bei PHO8 jedoch signifikant geringer ist. Für die transkriptionelle Aktivierung sind insbesondere zwei Transkriptionsfaktoren nötig: das bHLH-Protein Pho4 und das Homöodomänenprotein Pho2. Der PHO5-Promotor besitzt zwei Pho4-Bindestellen, die den regulatorischen Elementen UASp1 und UASp2 entsprechen. Während UASp1 in einem hypersensitiven Bereich zwischen den Nukleosomen liegt, ist UASp2 intranukleosomal lokalisiert. Mutagenese einer der beiden Bindestellen führte zu einer zehnfachen Abnahme der Promotoraktivität, während Mutagenese beider Stellen die Induzierbarkeit des Promotors völlig aufhob. Um die Bedeutung der Lokalisation der UAS-Elemente im Chromatin zu analysieren, wurde ein Operator für den a2-Repressorkomplex oberhalb des PHO5-Promotors eingebaut. Dieser Repressorkomplex bildet im Kontext bestimmter Promotoren eine zum a2-Operator benachbarte repressive Chromatinstruktur mit basenpaargenauer Nukleosomenposition aus. Im PHO5-Promotor führte der Einbau dieses Operators zur Repression der Promotoraktivität und einer leicht verminderten Chromatinzugänglichkeit. Demnach kann der a2-Operator transkriptionshemmende Strukturen initiieren, wobei die Repression durch verstärkte Chromatinkondenation und möglicherweise durch die Rekrutierung von reprimierenden Mediatorproteinen des RNA-PolymeraseII-Holoenzyms vermittelt wird. Durch Deletionen von Bereichen zwischen dem a2-Operator und den UAS-Elementen konnten Nukleosomen wie das Nukleosom -2 zwar stabilisiert, aber nicht gezielt verschoben werden. Rekonstitutionsexperimente mit einem 180 Bp-DNA-Fragment, das den Bereich des Nukleosoms -2 enthielt, zeigten zwar einen gewissen Beitrag der Sequenz zur Histon-DNA-Bindung, dieser allein kann jedoch keinesfalls die Positionierung erklären, vielmehr scheinen Wechselwirkungen der Histone mit anderen Chromatinkomponenten entscheidenden Anteil zu haben. Zusammenfassung E -144-Der Mechanismus der Chromatin-Umordnung am PHO5-Promotor durch die Transkriptions-faktoren Pho4 und Pho2 wurde zunächst durch in vitro Experimente unter Verwendung von rekombinantem Pho2-Protein analysiert. Es konnten mehrere Pho2-Bindestellen verschie-dener Affinität im PHO5-Promotor gefunden werden. Eine der hochaffinen Pho2-Binde-stellen überlappt größtenteils mit der Pho4-Bindestelle UASp1. Die kooperative DNA-Bindung der beiden Proteine an ihre überlappenden Bindestellen resultierte in einem hochaffinen ternären Komplex. Auch am UASp2-Element, bei dem zwei Pho2-Bindestellen eine Pho4-Bindestelle flankieren, findet eine kooperative Bindung von Pho2 und Pho4 an die DNA statt. Durch Mutation der mittels in vitro-Footprints entdeckten Pho2-Bindestellen konnte gezeigt werden, daß diese zur Promotoraktivität beitragen. Sie sind nicht nur wichtig, um Pho2 an den Promotor zu rekrutieren, sondern ermöglichen auch die kooperative DNA-Bindung mit Pho4 über direkte Protein-Protein-Wechselwirkung zwischen Pho2 und Pho4. Eine Pho2-Interaktionsdomäne von Pho4 ist essentiell für die Aktivierung des PHO5-Promotors, wie durch Deletionsanalyse demonstriert wurde. Die kooperative DNA-Bindung dieser Faktoren scheint demnach sehr wichtig für die Transkriptionsregulation des PHO5-Gens zu sein. Getrennte Untersuchungen von UASp1 und UASp2 in einem CYC1-Promotor-Kontext zeigen einen eindrucksvollen Unterschied zwischen den zwei UAS-Elementen und verdeutlichen die duale Rolle von Pho2 in der Aktivierung des PHO5-Promotors. Es ist in entscheidender Weise für die Rekrutierung von Pho4 zur UASp1-Stelle nötig und verstärkt darüber hinaus das Pho4-Aktivierungspotential, während es an der UASp2-Stelle eher nur das Pho4-Aktivierungspotential erhöht. Trotz der koordinierten Regulation beider Promotoren ist der PHO8- fast 10mal schwächer als der PHO5-Promotor. Von den beiden Pho4-Bindestellen am PHO8-Promotor, welche früher in vitro bestimmt worden waren, ist nur eine in vivo funktional. Der Austausch des inaktiven PHO8-UASp1-Elements durch das UASp1-Element des PHO5-Promotors erhöht das Ausmaß der Chromatinöffnung im Bereich der Nukleosomen -3 und -2 und ergibt einen zweifachen Anstieg der Promotoraktivität. Im Gegensatz dazu verhindert der Austausch der hochaffinen UASp2-Stelle durch die entsprechende UASp2-Stelle von PHO5 die Chromatin-umordnung und Promotoraktivierung, obwohl eine effiziente Bindung von Pho4 an dieser Stelle besteht. Diese Daten zeigen, daß eine quantitative Bindung von Pho4 an ein UAS-Element ohne irgendeine Chromatin-Umordnung und Promotoraktivierung möglich ist. Die Deletion der Promotorregion, die normalerweise von den Nukleosomen -3 und -2 bedeckt wird, ergibt einen zweifachen Anstieg der Promotoraktivität, was die repressive Rolle dieser Zusammenfassung -145-Nukleosomen anzeigt. Die gute Korrelation zwischen Promotoraktivität und Ausmaß der Chromatin-Umordnung impliziert, daß für das Ausmaß der PHO8-Induktion im Vergleich zu PHO5 die Qualität der Histon-DNA-Wechselwirkung eine Rolle spielt, da auch bei Einführung des PHO5-UASp1-Elements in den PHO8-Promotor keine vollständige Chromatinöffnung beobachtet wird. Obwohl Pho4 in Pho2-unabhängiger Weise am PHO8-Promotor bindet und Chromatin remoduliert, ist Pho2 dennoch an der Promotoraktivität durch Erhöhen des Aktivierungspotentials von Pho4 beteiligt, ähnlich wie am UASp2-Element des PHO5-Promotors. Die Ergebnisse dieser Arbeit haben die Rolle des Homöoproteins Pho2 bei der Induktion des PHO5- und PHO8-Promotors aufgeklärt und unterstreichen die enorme Bedeutung des kooperativen Bindens der Transkriptionsfaktoren Pho4 und Pho2. Zum anderen haben sie das Wechselspiel zwischen Transkriptionsfaktoren und der Chromatinstruktur am Beispiel dieser Promotoren besser definiert.

Medizin - Open Access LMU - Teil 11/22
Immunocytochemical Phenotyping of Disseminated Tumor Cells in Bone Marrow by uPA Receptor and CK18: Investigation of Sensitivity and Specificity of an Immunogold/Alkaline Phosphatase Double Staining Protocol

Medizin - Open Access LMU - Teil 11/22

Play Episode Listen Later Jan 1, 1997


Phenotyping of cytokeratin (CK) 18-positive cells in bone marrow is gaining increasing importance for future prognostic screening of carcinoma patients. Urokinase-type plasminogen activator receptor (uPA-R) is one example of a potential aggressive marker for those cells. However, a valid and reliable double staining method is needed. Using monoclonal antibodies against uPA-R and CK18, we modified an immunogold/alkaline phosphatase double staining protocol. UPA-R/CK18-positive tumor cell controls exhibited black uPA-R staining in 15–80 of cases and red CK18 staining in almost 100 of tumor cells. Isotype- and cross-matched controls were completely negative. Bone marrow from healthy donors was always CK18-negative. Reproducibility of CK18-positive cell detection was estimated in a series of specimens from 61 gastric cancer patients comparatively stained with the single alkaline phosphatase-anti-alkaline phosphatase (APAAP) and our double staining method (106 bone marrow cells/patient). In four cases, double staining could not reproduce CK18-positive cells. In 34 cases it revealed fewer or equal numbers, and in 23 cases more CK18-positive cells than the APAAP method. Overall quantitative analysis of detected cell numbers (838 in APAAP, range 1–280 in 106; double staining 808, range 0–253) demonstrated relative reproducibility of APAAP results by double staining of 97. Correlation of results between both methods was significant (p

Medizin - Open Access LMU - Teil 07/22
Determination of islet cell antibodies using an ELISA system with a preparation of rat insulinoma (RIN A2) cells

Medizin - Open Access LMU - Teil 07/22

Play Episode Listen Later Jan 1, 1989


An enzyme-linked immunosorbent assay (ELISA) was established for the detection of islet cell antibodies in human sera. The antigen was prepared from rat insulinoma (RIN A2) cells. Cells were dissociated in lysis buffer and the lysate was centrifuged at 100,000 x g. The supernatant was used to coat microtiter ELISA plates (10 micrograms protein/ml in PBS pH 7.2). Non-specific binding sites on the plates were blocked with 2% PBS-BSA. Human test sera were preabsorbed on separate plates using 2% PBS-BSA and incubated on precoated plates at an optimal dilution of 1/10 in 60 mM PBS for 60 min at 37 degrees C. Phosphatase-labeled anti-human IgG serum and phosphatase substrate were applied and the reaction was stopped by adding 3 M NaOH. Out of 90 sera from type I diabetic patients, 47 (52.2%) reacted in the new ELISA whereas none of 15 type II diabetics, 50 sera containing non-islet specific antibodies or 100 normal controls were positive. In the same group of patients, ICA were positive in 63.3%. When both, the ELISA and conventional ICA testing were applied, the number of positives was increased to 83%. The ICA-ELISA with the above described antigen preparation provides a well standardized and reproducible test method which is highly specific for type I diabetes. It may therefore be useful for large screening procedures.

Medizin - Open Access LMU - Teil 02/22
Membrane effects on hepatic microsomal glucose-6-phosphatase

Medizin - Open Access LMU - Teil 02/22

Play Episode Listen Later Jan 1, 1975


Wed, 1 Jan 1975 12:00:00 +0100 https://epub.ub.uni-muenchen.de/7130/1/7130.pdf Gratzl, Manfred ddc:610, Medizin

PaperPlayer biorxiv neuroscience
Inhibition of Striatal-Enriched Protein Tyrosine Phosphatase (STEP) Activity Reverses Behavioral Deficits in a Rodent Model of Autism.

PaperPlayer biorxiv neuroscience

Play Episode Listen Later Jan 1, 1970


Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.04.17.047597v1?rss=1 Authors: Chatterjee, M., Singh, P., Xu, J., Lombroso, P., Kurup, P. Abstract: Autism spectrum disorders (ASDs) are highly prevalent childhood illnesses characterized by impairments in communication, social behavior, and repetitive behaviors. Studies have found aberrant synaptic plasticity and neuronal connectivity during the early stages of brain development and have suggested that these contribute to an increased risk for ASD. STEP is a protein tyrosine phosphatase that regulates synaptic plasticity and is implicated in several cognitive disorders. Here we test the hypothesis that STEP may contribute to some of the aberrant behaviors present in the VPA-induced mouse model of ASD. In utero VPA exposure of pregnant dams results in autistic-like behavior in the pups, which is associated with a significant increase in the STEP expression in the prefrontal cortex. The elevated STEP protein levels are correlated with increased dephosphorylation of STEP substrates GluN2B, Pyk2 and ERK, suggesting upregulated STEP activity. Moreover, pharmacological inhibition of STEP rescues the sociability, repetitive and abnormal anxiety phenotypes commonly associated with ASD. These data suggest that STEP may play a role in the VPA model of ASD and that STEP inhibition may have a potential therapeutic benefit in this model. Copy rights belong to original authors. Visit the link for more info