Podcasts about subunit

En este episodio, Dany y Osmi comentaron el nuevo Ep de New Jeans “Get Up”, el comeback de verano de oh my girl “Summer Comes”, mas el debut de la subunidad de Monsta X Shownu y Hyungwoom, y el debut de unidad japonesa de Twice “Misamo” kpop news y mas --- Send in a voice message: https://podcasters.spotify.com/pod/show/k-chismografo/message

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.28.538778v1?rss=1 Authors: Li, J., Veeraraghavan, P., Young, S. M. Abstract: Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.28.537363v1?rss=1 Authors: Gao, R., Schneider, A. M., Mulloy, S. M., Lee, A. M. Abstract: Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.06.535882v1?rss=1 Authors: Ciotta, G., Singh, S., Gupta, A., Torres, D. C., Fu, J., Choudhury, R., Chu, W. K., Choudhary, C., Gahurova, L., Al-Fatlawi, A., Schroeder, M., Aasland, R., Poetsch, A., Anastassiadis, K., Stewart, A. F. Abstract: SETD1A is the histone 3 lysine 4 (H3K4) methyltransferase central to the mammalian version of the highly conserved eight subunit Set1 complex (Set1C) that apparently conveys H3K4 trimethylation (H3K4me3) onto all active Pol II promoters. Accordingly, mouse embryonic stem cells (ESCs) die when SETD1A is removed. We report that death is accompanied by loss of expression of DNA repair genes and accumulating DNA damage. BOD1L and BOD1 are homologs of the yeast Set1C subunit, Shg1, and subunits of the mammalian SETD1A and B complexes. We show that the Shg1 homology region binds to a highly conserved central a-helix in SETD1A and B. Like mutagenesis of Shg1 in yeast, conditional mutagenesis of Bod1l in ESCs promoted increased H3K4 di- and tri-methylation but also, like loss of SETD1A, loss of expression of DNA repair genes, increased DNA damage and cell death. In contrast to similar losses of gene expression, the converse changes in H3K4 methylation implies that H3K4 methylation is not essential for expression of the DNA repair network genes. Because BOD1L becomes highly phosphorylated after DNA damage and acts to protect damaged replication forks, the SETD1A complex and BOD1L in particular are key nodes for the DNA damage repair network. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.13.527949v1?rss=1 Authors: Ruan, J., Xie, H., Quan, X., Zhang, Y., Zhang, D., Wang, W., Hu, X., Tao, Y., Zhao, B., Lu, D., Jia, J.-M. Abstract: Proper brain function depends on intact neurovascular coupling (NVC). Through NVC, local cerebral blood flow (CBF) is rapidly increased in activated cerebral regions to meet the corresponding demands of neural activities. NVC has been believed to support advanced cerebral functions such as complex emotional homeostasis. However, few studies provide causal link evidence with specific molecular and cellular identities. Here, using RNAscope, cell-type specific conditional knockout (cKO) perturbation in BL6/C57 mice, laser speckle contrast imaging, and mouse behavior assays, we demonstrated that the N-methyl-D-aspartic acid (NMDA) receptor noncore subunit NR2D is only expressed in cerebral arteriole smooth muscle cells (aSMCs) but not in peripheral aSMCs. Both aSMC-NR2D cKO and aSMC-NR2D+NMDA receptor subunit 1 (NR1) double cKO mice displayed a severe decline in functional hyperemia induced by whisker stimulation. In addition, the NR2D pathway was independent of previously demonstrated COX-2-PGE2-dependent mechanism and high-fat diet (HFD)-induced impairments in NVC. Caldesmon is a potential downstream protein in the NR2D pathway for NVC regulation. Notably, we demonstrated that NVC deficits caused abnormalities in complex emotional behaviors, such as anxiety-like behaviors (exhibiting increased defecation and urine), reduced curiosity and decreased fear instinct (displaying less fear of aggressive CD1 mice). Furthermore, fear-response gene c-FOS expression in the amygdala neurons was attenuated in aSMC-NR2D knockout mice after response to acute stresses. In contrast, its expression level remained unchanged in the non-fear-related cerebral regions such as the piriform cortex and hippocampus. These findings demonstrated that inadequate functional hyperemia impaired cerebral complex emotional functions and suggested a co-existence of anxiety and fearlessness in single individuals. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

In this episode of the PRS Global Open Keynotes Podcast, Dr. David Chong for the Royal Children's Hospital Melbourne discusses repair of Tessier craniofacial clefts 3 and 4 using the anatomical subunit principle. This episode discusses the following PRS Global Open article: “The Anatomical Subunit Approach to Managing Tessier Numbers 3 and 4 Craniofacial Clefts” by Aaron C. Van Slyke, Jonathan Burge, Ria Bos, Gary Parker and David K. Chong, Read the article for free on PRSGlobalOpen.com: https://bit.ly/Tessier3and4 Dr. David Chong is a board-certified plastic surgeon and Associate Professor of Surgery at the University of Melbourne. He is also the current President of the Australian Cleft Lip and Palate Association. Your host, Dr. Damian Marucci, is a board-certified plastic surgeon and Associate Professor of Surgery at the University of Sydney in Australia. #PRSGlobalOpen #KeynotesPodcast #PlasticSurgery

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.12.13.520293v1?rss=1 Authors: Gross, A., Ghillebert, R., Schuetter, M., Reinartz, E., Rowland, A., Graef, M. Abstract: Cells convert complex metabolic information into stress-adapted autophagy responses. Canonically, multilayered protein kinase networks converge on the conserved Atg1/ULK kinase complex (AKC) to induce non-selective and selective forms of autophagy in response to metabolic changes. Here, we show that, upon phosphate starvation, the metabolite sensor Pho81 interacts with the adaptor subunit Atg11 at the AKC via an Atg11/FIP200 interaction motif to modulate pexophagy by virtue of its conserved phospho-metabolite sensing SPX domain. Notably, we find core AKC components Atg13 and Atg17 are dispensable for phosphate starvation-induced autophagy revealing significant compositional and functional plasticity of the AKC. Our data indicate that, instead of functioning as a selective autophagy receptor, Pho81 compensates for partially inactive Atg13 during pexophagy when TORC1 remains active under phosphate starvation. Our work shows Atg11/FIP200 adaptor subunits not only bind selective autophagy receptors but also modulator subunits that convey metabolic information directly to the AKC for autophagy regulation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

An RSV vaccine, if approved, may reduce disease burden in older adults. In this recap of the most common questions from a symposium at the Family Medicine Experience 2022 conference, listen as we discuss when an RSV vaccine may be approved and how to overcome barriers to the implementation of vaccines for older adults in primary care settings.Hear answer to questions such as:When might potential RSV vaccines be available, and when might the Advisory Committee on Immunization Practices make recommendations on their use?How will the vaccine roll out? Who would receive the vaccine first?If approved, what will the RSV vaccination schedule be?How can we overcome vaccine hesitancy?PresentersJohn J. Russell, MD, FAAFPClinical Professor of Family and Community MedicineSidney Kimmel College of MedicineThomas Jefferson UniversityPhiladelphia, PennsylvaniaChair, Department of Family and Community MedicineAbington-Jefferson HealthAbington, PennsylvaniaLaura Hurley, MD, MPHAssociate Professor of MedicineDivision of General Internal MedicineDepartment of MedicineUniversity of Colorado Anschutz Medical CampusAurora, ColoradoPrimary Care PhysicianDivision of General Internal MedicineDepartment of MedicineDenver HealthDenver, ColoradoPamela Rockwell, DO, FAAFPProfessorFamily MedicineUniversity of Michigan Medical SchoolAnn Arbor, MichiganView the full program at:https://bit.ly/3Wt87GI

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.24.513629v1?rss=1 Authors: Wang, Y.-J., Di, X.-J., Han, D.-Y., Nashmi, R., Henderson, B. J., Moss, F. J., Mu, T.-W. Abstract: Protein homeostasis (proteostasis) deficiency is recognized as a contributing factor to many neurodegenerative, neurological, and metabolic diseases. However, how the proteostasis network orchestrates the folding and assembly of multi-subunit membrane proteins is not completely understood. In this investigation, we focus on characterizing the biogenesis pathway of a multi-subunit neuroreceptor, the gamma-aminobutyric acid type A (GABAA) receptor. Previous proteomics studies identified Hsp47 (Gene: SERPINH1), a heat shock protein in the endoplasmic reticulum lumen, as the most enriched GABAA receptor-interacting chaperone. Here, we show that Hsp47 enhances neuronal GABAA receptor functional surface expression, acting after Binding immunoglobulin Protein (BiP), to preferentially bind the folded conformation of GABAA receptors. Therefore, Hsp47 promotes the subunit-subunit interaction, the receptor assembly process, and the anterograde trafficking of GABAA receptors. These Hsp47 properties are also extended to other Cys-loop receptors, including nicotinic acetylcholine receptors. Therefore, in addition to its known function as a collagen chaperone, this work establishes that Hsp47 also plays a critical and general role in the maturation of multi-subunit neuroreceptors. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.12.511771v1?rss=1 Authors: Castellano-Pozo, M., Sioutas, G., Barroso, C., Lopez-Jimenez, P., Jaso-Tamame, A. L., Crawley, O., Shao, N., Page, J., Martinez-Perez, E. Abstract: The cohesin complex plays essential roles in chromosome segregation, 3D genome organisation, and DNA damage repair through its ability to modify DNA topology. In higher eukaryotes, meiotic chromosome function, and therefore fertility, requires cohesin complexes containing meiosis-specific kleisin subunits: REC8 and RAD21L in mammals and REC-8 and COH-3/4 in C. elegans. How these complexes perform the multiple functions of cohesin during meiosis and whether this involves different modes of DNA binding or dynamic association with chromosomes is poorly understood. Combining time-resolved methods of protein removal with live imaging and exploiting the temporospatial organisation of the C. elegans germline, we show that REC-8 complexes provide sister chromatid cohesion (SCC) and DNA repair, while COH-3/4 complexes control higher-order chromosome structure. High-abundance COH-3/4 complexes associate dynamically with individual chromatids in a manner dependent on cohesin loading (SCC-2) and removal (WAPL-1) factors. In contrast, low-abundance REC-8 complexes associate stably with chromosomes, tethering sister chromatids from S-phase until the meiotic divisions. Our results reveal that kleisin identity determines the function of meiotic cohesin by controlling the mode and regulation of cohesin-DNA association, and are consistent with a model in which SCC and DNA looping are performed by variant cohesin complexes that coexist on chromosomes. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Konstantin Batygin, an oenophilist and author of "Codex, a Gardener's Bible," visits Lexman to discuss his fascination with ginsengs, which he claims to have Bedeviled for years. Batygin discusses the scientific mysteries of thesubunit and glueyness.

Most people are focused on the mRNA vaccines ( Pfizer-BioNTech & Moderna) or the viral-vector vaccines, like J&J, but many researchers and companies are working on protein subunit vaccines for COVID-19. Dr. Petrovsky and his company, Vaxine, created a protein subunit vaccine for COVID-19 that was tested in randomized controlled trials in Iran and just recently approved for use there. ( I believe it is the first protein subunit vaccine approved for use anywhere.) In this podcast he will discuss how it was made, efficacy and safety results, possible side effects, where it is currently available, if it is easily updated and more. In the second half of the podcast he shares his experience of the political situation in Australia. I can't comment on that, because I don't know exactly what is happening in Australia, but it sounds concerning. Dr. Petrovsky is a doctor, vaccine researcher and director of endocrinology at Flinders Medical Centre in Australia. He is vice-president and secretary-general of the international immunomics society. He is the founder and chairman of Vaxine, a company funded by the NIH to create new vaccine technology. Dr. Petrovsky has developed vaccines for influenza, hepatitis b, malaria, rabies and more. He is author of over 90 papers and book chapters. To contact Dr. Eeks, do so through bloomingwellness.comTo  listen to her comedic parody/audiobook on the sleazy side of the wellness industry, check out Yours in Wellness, Krystal Heeling on Amazon.Or you can listen to it from Google Play or Walmart!Or follow her on Instagram here.Twitter here.Or Facebook here.Subcribe to her newsletter here!

ปัจจุบันการระบาดของโควิด-19 ทำได้เราได้เห็นถึงเทคโนโลยีการผลิตวัคซีนที่น่าทึ่ง มีการผลิตวัคซีนหลากหลายประเภทออกมาเพื่อหยุดยั้งการระบาดของโรค ไม่ว่าจะเป็น Inactivated vaccine Viral vector vaccine Subunit vaccine หรือ mRNA vaccine แต่รู้หรือไม่เทคโนโลยีการผลิตวัคซีนบางชนิดมีมาก่อนที่จะเกิดการระบาดครั้งใหญ่ และบางเทคโนโลยีก็ต่อยอดมาจากเทคโนโลยีอื่น เช่น ยีนบำบัด (gene therapy) มาฟังเกร็ดน่ารู้เกี่ยวกับวัคซีน ทั้งความเป็นมา หลักการทำงาน และความพิเศษของเทคนิคการผลิตวัคซีนแต่ละชนิด พร้อมอัปเดตวัคซีนไทยที่น่าจับตามอง กับ ผู้ช่วยศาสตราจารย์ ดร.ป๋วย อุ่นใจ อาจารย์ประจำภาควิชาชีววิทยา คณะวิทยาศาสตร์ มหาวิทยาลัยมหิดล นักสื่อสารวิทยาศาสตร์ผู้มากความสามารถ หนึ่งในผู้เขียนหนังสือ Vaccine War สมรภูมิวัคซีนโควิด-19 ผู้แปลหนังสือ เมื่อโลกติดเชื้อ ฉบับกระชับ บรรณาธิการหนังสือ ไวรัส ฉบับกระชับ คอลัมน์นิสต์ประจำมติชนสุดสัปดาห์ กรุงเทพธุรกิจ ทั้งยังฝากผลงานไว้บนนิตยสาร อาทิ สาระวิทย์ สารคดี Update Science World รวมถึง Facebook page … Continue reading "เกร็ดน่ารู้วัคซีนโควิด – 19" The post เกร็ดน่ารู้วัคซีนโควิด – 19 appeared first on SIMPLE SCIENCE.

Uma das coisas que o fandom vem pedindo há anos, é uma sub unit da Moonbyul e da Solar. Recebemos uma surpresa recentemente. O relançamento de "Promise U" que faz parte do projeto REVIBE. Mas, as recentes aparições delas em programas, seriam indícios de algum lançamento oficial está por vir?

The bros took groups, songs, and a whole lot of randomness to make one of the best Kpop games out there! Check out the super subunit random game, where any group can be made into a subunit to perform any song! Who did JT and Fog pick to make their elite cover concert? Find out this week on episode 78! #TOHB #KPK See acast.com/privacy for privacy and opt-out information.

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.12.379867v1?rss=1 Authors: Matsuda, S., Yuzaki, M. Abstract: Long-term potentiation (LTP) and depression (LTD) of excitatory neurotransmission are believed to be the neuronal basis of learning and memory. Both processes are primarily mediated by neuronal activity-induced transport of postsynaptic AMPA-type glutamate receptors (AMPARs). While AMPAR subunits and their specific phosphorylation sites mediate differential AMPAR trafficking, LTP and LTD could also occur in a subunit-independent manner. Thus, it remains unclear whether and how, certain AMPAR subunits with phosphorylation sites are preferentially recruited to or removed from synapses during LTP and LTD. Here, we show that phosphorylation of the membrane-proximal region (MPR), which only occurs in GluA1 AMPAR subunits, mediates the subunit-dependent endosomal transport of AMPARs during LTD. AP-2 and AP-3, adaptor protein complexes necessary for clathrin-mediated endocytosis and late endosomal/lysosomal trafficking, respectively, are reported to be recruited to AMPARs by binding to the AMPAR auxiliary subunit, stargazin (STG), in an AMPAR subunit-independent manner. However, the association of AP-3, but not AP-2, with STG was indirectly inhibited by the phosphomimetic mutation in the MPR of GluA1. Thus, although AMPARs containing the phosphomimetic mutation at the MPR of GluA1 were endocytosed by a LTD-inducing stimulus, they were quickly recycled back to the cell surface in hippocampal neurons. These results could explain how the phosphorylation status of GluA1-MPR plays a dominant role in subunit-independent STG-mediated AMPAR trafficking during LTD. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.03.319137v1?rss=1 Authors: Smith, R. S., Florio, M., Akula, S., Neil, J., Wang, Y., Hill, R. S., Goldman, M., Mullally, C., Reed, N., Bello-Espinosa, L., Sarnat, L. F., Monteiro, F., Erasmo, C., Pinto e Vairo, F., Morava, E., Barkovich, A. J., Gonzalez-Heydrich, J., Brownstein, C., McCarroll, S., Walsh, C. A. Abstract: Osmotic equilibrium and membrane potential in animal cells depend on concentration gradients of sodium (Na+) and potassium (K+) ions across the plasma membrane, a function that is catalyzed by the Na,K-ATPase 3 subunit. In vertebrates, four paralogous genes, ATP1A1-4, encode distinct alpha subunit isoforms ( 1- 4), three of which ( 1, 2, 3; are expressed in the brain, and two ( 1, 3) predominantly in neurons. The 3 isoform, encoded by ATP1A3, is critical to neuronal physiology, and a growing spectrum of neurological diseases are associated with ATP1A3 pathogenic variants, with ages of onset ranging from early childhood to adulthood. Here, we describe ATP1A3 variants encoding dysfunctional 3 subunits in children affected by polymicrogyria, a developmental malformation of the cerebral cortex characterized by abnormal folding and laminar organization. To gain cell-biological insights into the spatiotemporal dynamics of prenatal ATP1A3 expression, we established a transcriptional atlas of ATP1A3 expression during cortical development using mRNA in situ hybridization and transcriptomic profiling of ~125,000 individual cells with single-cell RNA sequencing (Drop-Seq) from various areas of the midgestational human neocortex. We find that fetal expression of ATP1A3 is restricted to a subset of excitatory neurons carrying transcriptional signatures of neuronal activity and maturation characteristic of the developing subplate. Furthermore, by performing Drop-Seq on ~52,000 nuclei from four different areas of an infant human neocortex, we show that ATP1A3 expression persists throughout early postnatal development, not only within excitatory neurons across all cortical layers, but also and more predominantly in inhibitory neurons, with specific enrichment in fast-spiking basket cells. In addition, we show that ATP1A3 expression, both in fetal and postnatal neurons, tends to be higher in frontal cortical areas than in occipital areas, in a pattern consistent with the rostro-caudal maturation gradient of the human neocortex. Finally, we discover distinct co-expression patterns linking catalytic and {beta} ; subunit isoforms (ATP1A1,2,3) and auxiliary isoforms (ATP1B1,2,3), suggesting the ATPase pump may form non-redundant, cell-type specific alpha-beta combinations. Together, the importance of the developmental phenotypes and dynamic expression patterns of ATP1A3 point to a key role for 3 in the development and function of human cortex. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.30.320408v1?rss=1 Authors: Sobti, M., Walshe, J. L., Zeng, Y. C., Ishmukhametov, R., Stewart, A. G. Abstract: F1Fo ATP synthase functions as a biological rotary generator that makes a major contribution to cellular energy production. Proton flow through the Fo motor generates rotation of the central stalk, inducing conformational changes in the F11 motor that catalyzes ATP production via flexible coupling. Here we present a range of cryo-EM structures of E. coli ATP synthase in different rotational and inhibited states observed following a 45 second incubation with 10 mM MgATP. The structures generated describe multiple changes that occur following addition of MgATP, with the inhibitory C-terminal domain of subunit {epsilon} ({epsilon}CTD) disassociating from the central stalk to adopt a condensed ''down'' conformation. The transition to the {epsilon}CTD down state increases the torsional flexibility of the central stalk allowing its foot to rotate by ~50{degrees}, with further flexing in the peripheral stalk enabling the c-ring to rotate by two sub-steps in the Fo motor. Truncation mutants lacking the second helix of the {epsilon}CTD suggest that central stalk rotational flexibility is important for F1Fo synthase function. Overall this study identifies the potential role played by torsional flexing within the rotor and how this could be influenced by the {epsilon} subunit. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.21.306357v1?rss=1 Authors: Ma, J., Su, D., Huang, X., Liang, Y., Ma, Y., Liang, P., Zheng, S. Abstract: Less than a year after its emergence, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected over 22 million people worldwide with a death toll approaching 1 million. Vaccination remains the best hope to ultimately put this pandemic to an end. Here, using Trimer-Tag technology, we produced both wild-type (WT) and furin site mutant (MT) S-Trimers for COVID-19 vaccine studies. Cryo-EM structures of the WT and MT S-Trimers, determined at 3.2 Angstrom and 2.6 Angstrom respectively, revealed that both antigens adopt a tightly closed conformation and their structures are essentially identical to that of the previously solved full-length WT S protein in detergent. These results validate Trimer-Tag as a platform technology in production of metastable WT S-Trimer as a candidate for COVID-19 subunit vaccine. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.07.286831v1?rss=1 Authors: Cheng, R. C., Krishnamoorti, A., Berka, V., Durham, R. J., Jayaraman, V., Maduke, M. Abstract: CLC transporters catalyze the exchange of chloride ions for protons across cellular membranes. As secondary active transporters, CLCs must alternately allow ion permeation to the extracellular and intracellular sides of the membrane, adopting outward-facing and inward-facing conformational states. Here, we use single-molecule Forster resonance energy transfer (smFRET) to monitor the conformational state of CLC-ec1, an E. coli homolog for which high-resolution structures of occluded and outward-facing states are known. Since each subunit within the CLC homodimer contains its own transport pathways for chloride and protons, we developed a labeling strategy to follow conformational change within a subunit, without crosstalk from the second subunit of the dimer. Using this strategy, we evaluated smFRET efficiencies for labels positioned on the extracellular side of the protein, to monitor the status of the outer permeation pathway. When [H+] is increased to enrich the outward-facing state, the smFRET efficiencies for this pair decrease. In a triple-mutant CLC-ec1 that mimics the protonated state of the protein and is known to favor the outward-facing conformation, the lower smFRET efficiency is observed at both low and high [H+]. These results confirm that the smFRET assay is following the transition to the outward-facing state and demonstrate the feasibility of using smFRET to monitor the relatively small (~1 Angstrom) motions involved in CLC transporter conformational change. Using the smFRET assay, we show that the conformation of the partner subunit does not influence the conformation of the subunit being monitored by smFRET, thus providing evidence for the independence of the two subunits in the transport process. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.06.240010v1?rss=1 Authors: Elmasri, M., Hunter, D., Winchester, G., Aziz, W., Bates, E., Moolenaar Van Der Does, D., Karachaliou, E., Sakimura, K., Penn, A. C. Abstract: Dominant mutations in the human gene GRIN2A, encoding NMDA receptor (NMDAR) subunit GluN2A, make a significant and growing contribution to the catalogue of published single-gene epilepsies. Understanding the disease mechanism in these epilepsy patients is complicated by the surprising diversity of effects that the mutations have on NMDARs. We have examined the cell-autonomous impact of 5 severe GluN2A mutations by measuring NMDAR-mediated synaptic currents (NMDAR-EPSCs) in CA1 pyramidal neurons following rescue with human GluN2A mutants. Surprisingly, prolonged NMDAR-EPSC current decay and smaller peak amplitudes were common features of both gain- and loss-of-function mutants despite there being drastic differences between their effects on receptor function and enrichment at synapses. Modelling of NMDARs with mutant properties in CA1 neurons indicates that mutant NMDARs may contribute to broadening of depolarizations during bursts of high-frequency synaptic activity. Overall, the implication is that similar therapeutic approaches may be more widely applicable to patients with GRIN2A-related disorders irrespective of their molecular defect. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.26.222125v1?rss=1 Authors: Hernandez, G., Poirier, E., Lebied, K., Kouwenhoven, W. M., Levesque, D., Rompre, P.-P. Abstract: Background: Glutamate relays the reward signal from the dorsal raphe (DR) to ventral tegmental area (VTA) dopamine (DA) neurons. However, its role is complex and not clearly understood. We measured NMDA receptors subunits expression in limbic brain areas and studied the effects of VTA down-regulation of GluN2C subunit-containing NMDA receptor on the reward signal that arises from DR electrical stimulation. Methods: Via qPCR, we identified the relative composition of the different GluN2 NMDA receptors subunits in several brain areas. Then we used fluorescent in situ hybridization (FISH) to evaluate the colocalization of Grin2c and Tyrosine hydroxylase (Th) mRNA in VTA neurons. To assess the role of GluN2C in reward pursuit; we downregulated this receptor using small interfering RNA (siRNA) in rats self-stimulating for electrical pulses delivered to the DR. To delineate further the specific role of GluN2C in relaying the reward signal, we pharmacologically altered the function of VTA NMDA receptors by either bilaterally microinjecting the NMDA receptor antagonist PPPA or the glutamate transporter inhibitor (GLT-1), Dhk. Results: We identified that GluN2C is the most abundant NMDA receptor subunit expressed in the VTA. FISH revealed that a substantial number of TH+ neurons colocalize with Grin2C. The siRNA manipulation produced a selective down-regulation of GluN2C and a significant reduction in reward pursuit. Interestingly, PPPA and DHK respectively enhanced and suppressed reward pursuit, but only in rats that received the non-active RNA sequence. Conclusion: The present results suggest that VTA Glutamate neurotransmission relays the reward signal initiated by DR stimulation by acting on GluN2C NMDA receptors. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.06.19.161240v1?rss=1 Authors: Meserve, J. H., Nelson, J. C., Marsden, K. C., Hsu, J., Echeverry, F. A., Jain, R. A., Wolman, M. A., Pereda, A. E., Granato, M. Abstract: The acoustic startle response is an evolutionary conserved avoidance behavior. Disruptions in startle behavior, in particular startle magnitude, are a hallmark of several human neurological disorders. While the neural circuitry underlying startle behavior has been studied extensively, the repertoire of genes and genetic pathways that regulate this locomotor behavior has not been explored using an unbiased genetic approach. To identify such genes, we took advantage of the stereotypic startle behavior in zebrafish larvae and performed a forward genetic screen coupled with whole genome analysis. This identified mutants in eight genes critical for startle behavior, including two genes encoding proteins associated with human neurological disorders, Dolichol kinase (Dolk), a broadly expressed regulator of the glycoprotein biosynthesis pathway, and the potassium Shaker-like channel subunit Kv1.1. We demonstrate that Kv1.1 acts independently of supraspinal inputs to regulate locomotion, suggesting its site of action is within spinal circuitry. Moreover, we show that Kv1.1 protein is mis-localized in dolk mutants, suggesting they act in a common genetic pathway to regulate movement magnitude. Combined, our results identify a diverse set of eight genes all associated with human disorders that regulate zebrafish startle behavior and reveal a previously unappreciated role for Dolk and Kv1.1 in regulating movement magnitude via a common genetic pathway. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.06.11.145466v1?rss=1 Authors: Epstein, M., Maxwell, S., Piggot, T. J., Beeson, D., Bermudez, I., Biggin, P. C. Abstract: Muscle nicotinic acetylcholine receptors are a class of heteropentameric ligand-gated cation channels with constituent subunits adopting a fixed stoichiometric arrangement. The specific amino acid residues that govern subunit ordering are however, only partially understood. By integrating all-atom molecular dynamics simulations, bioinformatics, two-electrode voltage clamp electrophysiology and 125I--bungarotoxin assays of chimeric nAChR subunits, we identify residues across the extracellular, transmembrane and extended M4 helix of the {delta} subunit that make structural signatures that contribute to intransigent assembly rules. Furthermore, functional differences observed in 2{delta}2{beta} receptors can be rationalized by changes in dynamical behavior that manifest themselves at the agonist binding site. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.05.22.109462v1?rss=1 Authors: Wilson, C. S., Dohare, P., Orbeta, S., Ferland, R. J., Sah, R., Scimemi, A., Mongin, A. A. Abstract: The leucine-rich repeat-containing family 8 member A (LRRC8A) is an essential subunit of the volume-regulated anion channel (VRAC). VRAC is indispensable for cell volume regulation but its broader physiological functions remain a matter of investigation. Astrocyte-targeted Lrrc8a deletion in the nervous system reduces neuronal excitability, impairs synaptic plasticity and memory, and protects against ischemic damage. Here we show that deletion of LRRC8A in all brain cells, using NestinCre -driven Lrrc8afl/fl excision, is lethal. Mice devoid of brain LRRC8A are born close to the expected Mendelian ratio and develop without overt histological abnormalities through the postnatal week 6. Nevertheless, they die in the adolescence, between 5 to 8 weeks of age, with a seizure-like phenotype. Consistent with seizures, we found disruptions in cell excitability, GABAergic signaling, and astrocytic production of the GABA precursor glutamine, all of which might contribute to animal mortality. This work provides the first evidence of a critical role for VRAC in control of brain excitability during brain maturation. Copy rights belong to original authors. Visit the link for more info

This time we tackle the latest releases leading to the end of the year, with new music from IU, AOA, EXO, WJSN, Twice, Crush, Sejeong. The meat of the episode is however, the Korean adventures of No Kpop No Life Texan subunit composed by @ElevenGreenEggs and @david_theworld. They explained their experience visiting different cities, their food discoveries, language struggles, watching some nugus busking, SM Museum and LOONA (and specially Olivia Hye’s) fan landmark, “Cafe Rosy”. We close by discussing the sad but expected Momoland lineup changes and the underwhelming award shows to start the holiday season. 1:08 IU – Blueming and Above the time 8:50 WJSN – As you Wish 18:52 AOA – Come see me 27:08 EXO – Obsession 37:10 Crush – From midnight to sunrise 41:00 Twice - &Twice 44:46 Sejeong – Tunnel 45:20 Blue.D – Nobody (ft. Mino) 48:53 CSVC – Just 4 U… 53:05 J.Y.P. – Fever (ft. Bibi) 59:00 NKNL TX subunit korean adventures 1:50:40 LOONA’s Orbit 2.0 fankit 1:52:00 News: Momoland’s lineup shake up 2:14:04 News: MAMA and other Meaningless Asian Music Awards 2:26:20 Best of the year awards teaser Social links: Twitter: https://twitter.com/nknlpodcast Spotify: https://open.spotify.com/show/4T4aS4bRc1Cq4Iftn08TWZ Youtube: https://www.youtube.com/channel/UCdyD386Vzn4y-y1Nv0I7Lvw Apple Podcasts: https://podcasts.apple.com/podcast/id1474831936 Anchor: https://anchor.fm/nokpopnolife/ Panel: @llehutyERA @david_theworld, @doulmagus, @ElevenGreenEggs, @roasoneTM Editing: @david_theworld

We now have two vaccinations to protect against herpes zoster — a live-attenuated vaccine (Zostavax) and the new recombinant subunit vaccine (Shingrix). While the live-attenuated vaccine has been available for more than a decade and a CDC-recommended vaccine in older adults, only one in three eligible patients have received it.  Based on the results of two recently published studies, the new recombinant subunit vaccine appears to provide substantially improved efficacy and duration. Guest Author:  Katherine Montag Schafer, PharmD, BCACP Music by Good Talk

Dr. Carolyn Lam:               Welcome to Circulation on the Run, your weekly podcast summary and backstage pass to the journal and its editors. I'm Dr. Carolyn Lam, associate editor from the National Heart Center, and Duke National University of Singapore.                                                 In just a moment, we will take a deep dive into the issue of age and its association with outcomes of primary prevention ICDs in patients with non-ischemic systolic heart failure.                                                 Yes, a long-awaited discussion from the Danish trial. That, in just a moment. First, here's your summary of this week's Journal.                                                 The first original paper provides evidence of a true association between disturbed genetic imprinting and Preeclampsia. This paper is from co-first authors, Dr. Zadora, and Dr. Singh, and co-corresponding authors, Dr. Izsvak, from the Max Delbrück Center for Molecular Medicine; Dr. Hurst, from the University of Bath; and Dr. Dechend, from the Experimental and Clinical Research Center of Berlin.                                                 These authors performed an unbiased analysis of genome-wide molecular data on raw characterized patient material, from normal controls, and patients with  Preeclampsia, and identified DLX-5 as an imprinted target gene, with novel placental function in Preeclampsia. Due to loss of imprinting, DLX5 was upregulated in 69% of placentas from Preeclampsia patients. Levels of DLX5 correlated with the classical Preeclampsia markers.                                                 DLX5 was expressed in human, but not in urine trophoblast, underlying the known human specificity of Preeclampsia. Finally, DLX5-induced overexpression if trophoblasts faithfully modeled Preeclampsia in a cell culture system. In summary, this paper shows that disturbed imprinting is common, and may play a causal role in Preeclampsia.                                                 The next study affirms that stenosis severity is better discriminated using coronary invasive physiologic indices, than using coronary angiographic assessment. First author, Dr. Lee, corresponding author Dr. Koo, colleagues of Seoul National University Hospital, studied 115 patients with left anterior descending artery stenosis, who underwent both ammonia positron emission tomography, or PET, an invasive physiologic measurement.                                                 Myocardial blood flow measured using PET, and invasively measured coronary pressures, were used to calculate microvascular resistance, and stenosis resistance. They found that both fractional flow reserve, or FFR, and instantaneous weight free ratio, or IFR, decreased as angiographic stenosis severity, resistance, and pressure gradient increased, and hyperemic myocardial blood flow decreased.                                                 When the presence of myocardial ischemia was defined by both low hyperemic myocardial blood flow, and low coronary flow reserve, the diagnostic accuracy of FFR and IFR did not differ, regardless of cutoff values for hyperemic myocardial blood flow, and CFR. However, at any given stratum of a given stenosis, physiologic classification of stenosis severity using FFR or IFR showed better discrimination of a unique relationship between absolute myocardial blood flow, and pressure gradient, than anatomic classification using angiographic percentage.                                                 In summary, by demonstrating coronary physiologic responses to coronary stenosis, these authors showed that stenosis severity is better discriminated, using invasive physiologic indices, than using angiographic assessment.                                                 The next paper identifies a previously unknown angiogenic growth factor that can be enhanced therapeutically to repair the heart after myocardial infarction. This novel growth factor is endoplasmic reticulum membrane complex, Subunit 10, or EMC10, which the authors previously identified by bioinformatic secretome analysis in bone marrow cells.                                                 In the current paper, from co-first authors Dr. [Rabel 00:04:35], and [Krof Clengobill 00:04:37], and corresponding author Dr. Wollert, from Hanover Medical Center, and colleagues, the authors investigated the angiogenic potential of EMC10, and its mouse homologue, in cultured endo fetal cells and infarcted heart explants. They found that EMC10 and its mouse homologue signal a virus, small GTAPases; p21-activated kinase; and p38 mitogen-activated protein kinase, to promote endothelial cell migration.                                                 In mice with acute myocardial infarction, bone marrow derived monocytes and macrophages produced EMC10 endogenously, to enhance infarct vascularization, tissue repair, and heart function. Furthermore, subcutaneous treatment with recombinant EMC10 for one week, after myocardial infarction, augmented infarct vascularization and repair, and led to a sustained improvement in heart function and survival.                                                 The next study is the first prospective randomized trial of screening for atrial fibrillation, with a smartphone-based, single-lead, electrocardiographic system in 1,001 patients, aged 65 years and above, with a CHA2DS2-VASc score of two and above, and without a history of atrial fibrillation.                                                 In this paper, from first and corresponding author Dr. Halcox, from Swansea University Medical School, in the United Kingdom, and colleagues, patients were randomized, either to biweekly electrocardiographic recordings with the iPhone device, or to routine over a 12-month period.                                                 The smartphone-based electrocardiographic approach was at least three times more likely to identify incident atrial fibrillation, than routine care, and at a cost of just over $10,000 per case identified, and was judged to be a highly acceptable approach in this group of patients. These results support consideration of evaluation in an appropriately-powered, event-driven randomized trial, to confirm the clinical and cost effectiveness of such an approach to stroke prevention in atrial fibrillation.                                                 Well, that wraps it up for your summaries. Now for our feature discussion. The Danish trial really created a huge splash last year, when it was reported that a primary prevention ICD in patients with non-ischemic systolic heart failure, may not actually reduce all cause mortality. Something that we had, perhaps, taken for granted, and in fact, entered our guidelines.                                                 Now, however, there was a pre-specified subgroup analysis at the time, that suggested a possible age-dependent association, between ICD and mortality, in the Danish trial. This week, we are so pleased to be discussing an in-depth analysis of the association between age and outcomes in the Danish trial.                                                 I'm so pleased to have the first author of today's featured paper, Dr. Marie Bayer Elming, of Copenhagen, Denmark, as well as Dr. Sana Al-Khatib, who's not only an associate editor of circulation, but also the author of an accompanying, and she is from Duke, Durham, North Carolina. Welcome, ladies! Dr. Bayer Elming:              Thank you. Happy to be here. Dr. Sana Al-Khatib:          Thank you so much. Dr. Carolyn Lam:               Sana, could you start by framing why this paper is so important, and why we've been looking forward in anticipation to these results? Dr. Sana Al-Khatib:          Absolutely. As you know, data on the outcomes of primary prevention ICDs in patients with non-ischemic cardiomyopathy started emerging in the early 2000s, or so. Then in 2005, the sudden cardiac deaths and heart failure trial was published, that included a large number of patients with non-ischemic cardiomyopathy, and absolutely showed survival benefits from primary prevention ICDs in those patients. Of course, there were also patients with ischemic cardiomyopathy.                                                 But really, that trial formed the basis of the guidelines, recommendations, that have informed our practice for the last 12 years, that basically tell us that we should consider implanting a primary prevention ICD in patients with non-ischemic cardiomyopathy, who have an EF of 35% or less, who have Class II or III heart failure symptoms. As long as they are on optimal care at the end, they have a reasonable life expectancy.                                                 So that's what's we've been doing for years, and then, the Danish trial was published this past year, that really called into question the prior findings, and the current practice. Because Danish, as you stated, showed no survival benefit with primary prevention ICDs, but there are many aspects about the trial that people need to pay attention to, to put the results in perspective.                                                 The fact that 58% of patients in the trial, in those arms, received cardiac resynchronization therapy ... the fact that the trial required that patients have an elevated NTproBMB level, to be considered for enrollment ... that may have biased the results toward a higher risk of non-sudden cardiac deaths, so on, so forth.                                                 I think what was really interesting, and caught people's attention, when the paper was published, was this subgroup analysis that showed that younger patients may benefit more than older patients. I think, many of us, Carolyn, were really awaiting the results of a more dedicated analysis, looking at age in Danish, and Dr. Elming and her colleagues did a great job looking at this very closely in their paper, and showed great results, and probably will let Dr. Elming share those results with us. Dr. Carolyn Lam:               Yes, absolutely, Sana. Actually, I just wanted to echo how surprised everyone was, and the immediate thing was, "Oh, my goodness. What do we do with the guidelines?" Maybe we should get back to that later, and Marie, please share with us, what did you do, and what did you find this time? Dr. Bayer Elming:              The reason why we did this study was that, in this main Danish trial, age was the only one of the 13 pre-specified subgroups that had a significant treatment by a subgroup interaction. This suggested that a younger patient might have a survival benefit from ICD ... the implication, even though the overall study was neutral. So we wanted to further investigate this relationship between age and effective ICD implantation.                                                 What we did was to look at the relation between age and effective ICD, and we found that there was this linear relation, for each year of younger age, that was associated with a reduction, a 3% reduction in the hazard ratio, for the benefit of ICD.                                                 Also, we did this selection impact curve, which is a bit technical, but what it does is to describe the expected survival for the population, on as a whole, for the different age cutoffs for ICD treatments.                                                 So, if we take into account, both the patients receiving an ICD, and those who did not, we could see why we would get the highest survival for the population as a whole. What we found was that, when no one in the population received an ICD, around 70% would survive.                                                 If everyone in the population received an ICD, only 72% would survive, but if we chose 70 years as the age cutoff ... so, patients younger than 70 years received an ICD, and patients older than 70 years did not receive an ICD, we got the highest survival for the population, and 75% would survive. Dr. Carolyn Lam:               Thank you, Marie. What important results. So, maybe, still consider ICDs for primary prevention ... in our non-ischemic systolic heart failure, patients were less than 70 years old. Is it as simple as that, Sana? You wrote a beautiful editorial. Tell us, what are the clinical implications? Dr. Sana Al-Khatib:          This is an important question. Danish was an important trial, but in my mind, it truly doesn't refute the role of primary prevention ICDs in patients with non-ischemic cardiomyopathy. As I mentioned earlier, the majority of patients enrolled in Danish received a CRT device. And so, you end up questioning, what does that actually mean, for those patients who are not eligible for cardiac resynchronization therapy?                                                 So, I actually believed that, and as you know, Carolyn, and maybe Marie knows, as well, there have been several meta analyses that have been published, combining data on patients with non-ischemic cardiomyopathy only, and excluding patients with cardiac resynchronization therapy from Danish, that have actually now shown, consistently, a significant improvement in survival, with a primary prevention ICD ... including one that was done by our group.                                                 So, no, I don't think that, based on the results, we should say, "No, we shouldn't be offering primary prevention ICDs to patients with non-ischemic cardiomyopathy," and this beautiful analysis that was done by Marie and her group actually shows that, at least for those patients who are 70 years of age and younger, I think we should absolutely continue to consider them for the therapy, and offer them the therapy, if they're appropriate candidates.                                                 Then, of course, if the patients are older than 70,, and they meet criteria for cardiac resynchronization therapy, I think it will be important for us to be talking to the patients about ... is the RTD with a defibrillator, versus a CRTP only, with a pacemaker, and talking about the pros and cons, and everything else? But in those patients who are older than 70, who don't meet criteria for CRT, I think more research is needed, to really understand the role of primary prevention ICDs in those patients. We definitely need more data there. Dr. Bayer Elming:              I definitely agree that, of course, for the patients older than 70 years were not candidates for CRT treatment. These patients, we do not know very much about 'em, and this study that we did, do not answer that question. Based on the Danish study, and this further analysis of the age inspection, the guidelines in Denmark also state that patients younger than, we say, 68 years, because that was the age cutoff used in the '08 Danish trial, you should definitely think of giving patients with non-ischemic cardiomyopathy an ICD.                                                 But for the older patients, it depends on a variety of co-factors, such as co-morbidity, or frailty, and it should be an individual assessment of the patient. So, I agree with you, Sana. Dr. Carolyn Lam:               That's wonderful. Hey, just one more question. Sana, I'd like you to put on your AE hat, now, and sort of think with me. In circulation, we don't ... well, we're careful about publishing subgroup analyses, so to speak, right, of results. You articulated, in your editorial, reasons why this, perhaps subgroup analysis, may be different from others. Could you elaborate on that a bit? Dr. Bayer Elming:              Yeah, and absolutely, that's a great question. As you pointed out, I mean, you really ... the conventional wisdom in clinical research is to be careful, interpreting subgroup analyses. I think there are some strengths in this particular analysis, as Marie stated: "Here's what we specified." The other thing is, I believe that Marie and her group then came, and did their very robust statistical methods, and really, probably most importantly, if you look at their findings, they actually really align well, and support their main conclusion.                                                 For example, looking at the fact that older patients had the higher presence of co-morbidities, that they had a higher level of [Co-BMP 00:17:00], they had had a longer duration of heart failure ... I mean, all those things most likely had an impact on their mode of death, really making it more likely for those patients to succumb to non-sudden cardiac death. I think the whole story makes a lot of sense. Dr. Bayer Elming:              If I can elaborate a bit on this, I think one of the important findings from the study is that we show that mode of death varied according to age. So, the rates of sudden cardiac death were almost similar, between the younger and the older part of the population. But the rates of non-sudden death were almost twice as high in the older part of the population. This is a really good explanation why the ICD implantations have less impact in the older patients. Dr. Carolyn Lam:               Yeah, because ICDs would definitely not be expected to reduce non-sudden cardiac deaths. Really, really, well put. Oh, thank you so much, Marie. We're so proud to be publishing your beautiful paper, as well as your editorial, Sana, and thank you for this great conversation.                                                 Well, listeners, I'm sure you enjoyed that as much as I did. Thank you for joining us this week, and don't forget to tune in next week.

Live Strictlyfishwrap Science Radio Hour field report recorded on April 30th, 2016. Apologies for any mispronunciation of names. Reporter: Skylar Bayer Photo credit: Kevin Job Tracks: Run Fish Run by Aaron Nigel Smith & Aya World Productions

Join Snow from Snow White and the Asian Pear and Tracy, sometimes known as Darling, from fanserviced-b as they dive deep into the most extreme ways to shop for kbeauty without leaving your home. Sites we mention that have English language, global shipping wings: English 11ST Global Gmarket qoo10 Global Interpark free shipping page Proxy... Read More » The post Episode 7: Hardcore Kbeauty Shopping with the Snow-Darling Subunit appeared first on The Snailcast.

Alexander Mankin –called Shura --of the University of Illinois, Chicago, and Michael Jewett at Northwestern University talk with Jeff Fox about their recent success in joining the 30S and 50S bacterial ribosomal subunits into a single, functioning entity and the implications of that work in terms of making specialized proteins and for studying antibiotics that target the ribosome.  This story was featured in the October 2015 issue of Microbe magazine. Visit microbeworld.org/mmp for more.

Background Non-coding single nucleotide polymorphisms within the nicotinic acetylcholine receptor alpha 4 subunit gene (CHRNA4) are robustly associated with various neurological and behavioral phenotypes including schizophrenia, cognition and smoking. The most commonly associated polymorphisms are located in exon 5 and segregate as part of a haplotype. So far it is unknown if this haplotype is indeed functional, or if the observed associations are an indirect effect caused by linkage disequilibrium with not yet identified adjacent functional variants. We therefore analyzed the functional relevance of the exon 5 haplotype alleles. Results Using voltage clamp experiments we were able to show that the CHRNA4 haplotype alleles differ with respect to their functional effects on receptor sensitivity including reversal of receptor sensitivity between low and high acetylcholine concentrations. The results indicate that underlying mechanisms might include differences in codon usage bias and changes in mRNA stability. Conclusions Our data demonstrate that the complementary alleles of the CHRNA4 exon 5 haplotype are functionally relevant, and might therefore be causative for the above mentioned associations.

Fri, 20 Jul 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/14587/ https://edoc.ub.uni-muenchen.de/14587/1/Vukajlovic_Marija.pdf Vukajlovic, Marija

Fibrinogen is a target of autoimmune reactions in rheumatoid arthritis (RA). Fibrin(ogen) derivatives are involved in inflammatory processes and the generation of a stable fibrin network is necessary for sufficient inflammation control. As the density and stability of fibrin networks depend on complex interactions between factor XIIIA (F13A) and fibrinogen genotypes, the authors studied whether these genotypes were related to C-reactive protein (CRP) levels during acute-phase reactions.

The voltage-gated calcium channel subunit (128 plays a fundamental role in propagation of excitatory signals associated with release of glutamate and neuropeptides substance P (SP) and calcitonin gene-related protein (CGRP). It can be selectively inhibited by gabapentinoids. Hence, investigation of the alpha(2)delta subunit may predict the efficacy of gabapentinoid therapy in neuropathic pain. Since sensory processing underlies significant age-related changes, this study was conducted in order to elucidate the role of the alpha(2)delta subunit in the sensory transmission during canine development. Dorsal root ganglia (DRG) were harvested from four spinal segments of 16 puppies and 10 adult dogs without a history of neurological signs, pain, spinal disease or orthopedic disorders. alpha(2)delta-Subunit expression and coexpression with SP and CGRP was evaluated immunohistochemically regarding the number of immunopositive ganglion cells, staining intensity and subcellular distribution. All tested ganglia were immunopositive for alpha(2)delta. Cell counts and expression levels were significantly lower in pups than in adult dogs (p < 0.05). In the cervical segments of both groups, the number and percentage of innmunopositive neurons was significantly higher than in lumbar DRG (p < 0.05). Multilabeling studies in all tested animals confirmed the coexpression of alpha(2)delta and pain peptides SP and CGRP. This anatomical study for the first time documents the involvement of alpha(2)delta subunits in sensory signal processing in dogs. The proportion of positive neurons and the intracellular expression levels show a net increase from early postnatal life to adulthood. A significant portion of alpha(2)delta-positive cells in the dogs exhibited C- and A delta-phenotypes compatible with nociceptive neurons. The coexpression of alpha(2)delta, SP and CGRP imply that these neurons are involved with peptidergic nociception. The cervicolumbar gradient of alpha(2)delta expression in adults reflects functional differences in between forelimbs and hind limbs. These data will facilitate translational studies on neuropathic pain states in this species such as common canine nerve entrapment syndromes. Copyright (C) 2012 S. Karger AG, Basel

Audio PodcastAired date: 9/7/2011 3:00:00 PM Eastern Time

Video Podcast (CC)Aired date: 9/7/2011 3:00:00 PM Eastern Time

Thu, 20 May 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/11908/ https://edoc.ub.uni-muenchen.de/11908/1/Becirovic_Elvir.pdf Becirovic, Elvir

Background and Purpose: Oxidative stress has been proposed as a major contributing factor for vascular disease, that acts independently from its participation in predisposing disorders such as diabetes and arterial hypertension. A functionally relevant C242T polymorphism of the CYBA gene encoding the NAD(P)H oxidase p22(phox) subunit, is supposed to lead to an abnormal reduction in the generation of reactive oxygen species in vascular smooth-muscle and endothelial cells. Methods: We investigated the p22(phox) C242T single-nucleotide polymorphism by polymerase chain reaction in consecutive patients with ischemic stroke or transient ischemic attack under the age of 50 (n = 161) and in population-based control subjects (n = 136). Results: Homozygosity for the T variant was associated with an enhanced risk for cerebral ischemia (odds ratio 3.85, confidence interval 1.39-10.64) after adjusting for classical risk factors. Risk for cerebral ischemia was not increased in heterozygous subjects. Conclusion: The p22(phox) C242T single-nucleotide polymorphism is associated with stroke risk. This finding supports the hypothesis that oxidative stress may contribute to stroke pathogenesis. Copyright (C) 2008 S. Karger AG, Basel.

In this study a structure–function analysis has been employed to analyze transcriptional regulation through the Mediator subunit MED25. A relationship could be established between predicted structural domains and functional characteristics of this protein. Most critically the region responsible for interaction of MED25 with the Mediator was identified. Immunoprecipitation experiments demonstrated that the so–called VWA domain (von–Willebrand A domain, amino acids 1–290) is both sufficient and required for this contact. Site–directed mutagenesis indicates that this binding reaction involves the non–conserved loop SR2, which is protruding from this domain. Based on the results of this analysis a model was proposed, in which the primary contact is established by ionic forces and is further stabilized by hydrophobic interactions. The previously identified ACID domain was reported to bind to VP16. Targeted mutagenesis of four different motifs in this region impaired not only transcriptional activation through MED25 but also led to reduced binding to VP16. In particluar a lysine–rich motif is also present in two domains of PTOV1, a close homolog of MED25. Noteworthy, K518 is not conserved in the PTOV1_B domain, which in contrast to PTOV1_A and the ACID domain of MED25 does not bind to VP16. This led to the hypothesis that K518 is critically involved in the binding of VP16 to MED25. Furthermore it could be demonstrated that MED25 contains an intrinsic transcriptional activation capacity, which is localized in the region 290–715. This indicates additional recruitment of other factors to promoters through this region. Together with the Mediator binding VWA–domain and the VP16–interaction domain this region might facilitate transcriptional activation. A genome–wide screen showed downregulation of c–Jun and FosB following overexpression of MED25. Interestingly, expression of GSK3β, a downstream target of which is cyclin D1, seems to be stimulated by MED25. Together with the finding that overexpression of MED25 leads to activation of a p21 reporter, this raises the possibility that MED25 is involved in cell cycle control. An overlap has been discovered by comparison of MED25 target genes and genes identified previously as target for the viral activator EBNA2. The close homology between the activation domains of EBNA2 and VP16 implies a common mechanism of transcriptional activation by these two viral proteins through MED25. The involvement of MED25 in gene activation by viral activators might indicate a role for this Mediator subunit in viral transcription.

Precursor protein targeting toward surfaces of organelles is assisted by different cytosolic chaperones. The Toc translocon recognizes precursor proteins and facilitates their translocation across the outer envelope of chloroplasts. Toc64 is a subunit of the chloroplast protein import machinery. This work focuses on topological and functional properties of Toc64. The topological prediction of the protein by different programs revealed that Toc64 contains three transmembrane domains, which has been confirmed by the obtained biochemical an experimental results. It was demonstrated that the TPR domain of Toc64 is cytosolic exposed, whereas a second domain of about 30 kDa is exposed to the intermembrane space and protected by the chloroplast outer envelope, which is a part of the amidase and charged regions. Functional analysis demonstrated that Toc64 is a bi-functional preprotein receptor. First, the cytosolic exposed TPR is the docking site for Hsp90 bound precursor proteins. The Hsp90 is recognised by the clamp type TPR of Toc64. Hence, a novel mechanism in which chaperones are recruited for a specific targeting event by a membrane-inserted receptor is outlined. Second, the intermembrane space exposed domain allows the association of Toc64 with the Toc complex and is involved in precursor protein recognition and translocation across the intermembrane space. This domain also participates in the formation of the intermembrane space complex, which involves Toc12, isHsp70 and Tic22.

2. Summary In Drosophila melanogaster the transcriptional activity of the male X chromosome is upregulated to compensate for the reduced dosage of X-linked genes as compared to the two X chromosomes in females. This process is mediated by the Dosage Compensation Complex (DCC), a ribonucleoprotein complex consisting of five proteins (MSL1, MSL2, MSL3, MOF and MLE) and two non-coding RNAs (roX1 and roX2). The DCC preferentially localizes on the X chromosomes in males where it doubles its transcription rate. Two enzymes are associated with the DCC: the acetyltransferase MOF, specific for the lysine 16 of H4 (H4-K16), and the DNA/RNA helicase MLE. Genetic experiments demonstrated that both activities are required for dosage compensation in male flies. However, the weak association of MLE to the DCC has complicated its biochemical analysis and, so far, the involvement of MLE RNA helicase in dosage compensation has only been demonstrated genetically. Using different in vivo and in vitro approaches the physical and functional interactions of MLE with the other MSL proteins and with the roX RNAs was addressed. Monoclonal antibodies, specifically recognizing MLE, were raised in rats, offering a new tool for MLE characterization. By coexpression of the DCC subunits in SF9 cells, a recombinant complex containing MSL1-2-3, MOF, MLE and the roX2 RNA was reconstituted and purified. A specific integration of roX2 into the DCC could be observed only in the absence of MLE. Non specific RNA binding properties seemed instead associated to MLE RNA helicase. Moreover, the purified MSL complex did not affect the ATPase activity of MLE in the presence or absence of roX2 RNA. In vitro, MLE showed a preferential association with MSL1 and MSL2 and MLE interaction with both MSL proteins were not RNA mediated. In view of these results we suggest that binding to roX2 is not the main determinant for MLE integration into the DCC complex and protein-protein interactions might instead contribute to its proper recruitment to the X chromosome. MLE is a member of the DEAD-box RNA helicase family and it shares with the other members the same domain organization. In addition to a central ATPase/helicase domain, two predicted N-terminal double strand (ds) RNA-binding motifs (dsRBM1 and dsRBM2) and a predicted C-terminal single strand (ss) RNA/DNA-binding domain (RGG-box) are also present in MLE protein. These domains have been extensively characterized in RHA, human ortholog of MLE, and their RNA binding properties confirmed. However, it is not known how MLE binds RNA and how the different RNA binding modules contribute to stimulate its enzymatic activities. A preferential binding of MLE to dsRNA compared to ssRNA was shown by binding assays. In addition, changes in the affinity of MLE for both ssRNA and dsRNA were observed in the presence of different nucleotides. Deletion mutants of MLE were produced and purified from insect cells in order to address the contribution of the different RNA binding domains to MLE enzymatic activities. By transient expression in Drosophila cells of the same deletion mutants fused to GFP, the effects of individual domains on MLE recruitment to the X chromosome were also determined. The results show that unlike RHA, the dsRB1 and the RGG domains are dispensable for MLE RNA binding and unwinding, whereas dsRB2 seems to play the major role in coordinating both activities. However, the enzymatic activities alone are not sufficient to properly target MLE to the X chromosome. These results provide new data on the functional properties of MLE RNA helicase that may help to elucidate its molecular mechanisms of action.

ATP synthase is one of the major photosynthetic complexes that represents one of the smallest molecular motors known in nature. The rotating γ subunit is a key feature of this enzyme. It contains features specific for the chloroplast ATP synthase. In this work the γ subunit has been functionally analyzed in Arabidopsis thaliana. - The nuclear gene atpC1 encoding the γ subunit of the plastid ATP synthase has been inactivated by T-DNA insertion mutagenesis. In the seedling-lethal dpa1 mutant the absence of detectable amounts of the γ subunit destabilizes the entire ATP synthase complex and consequently photophosphorylation is abolished. However, in vivo protein labelling analysis suggests that assemαβ bly of the ATP synthase and subunits into the thylakoid membrane still occurs in dpa1. Further effects of the mutation include an increased light sensitivity of the plants and an altered photosystem II activity. A high non-photochemical quenching develops with increasing actinic light intensity. It has been shown that a high proton gradient is responsible for most quenching (qE). The photoprotective role of qE was further demonstrated in the double mutant dpa1 x psbS in which PsbS, essential factor for qE, is missing. - The expression of a second gene copy, atpC2, is unaltered in dpa1 and is not sufficient to compensate for the lack of atpC1 expression. The two proteins, AtpC1 and AtpC2, share less similarity than AtpC1 of Arabidopsis with γ subunits of other plant species suggesting that the γ subunits so far isolated in other plant species are AtpC1 orthologs. It has been established that AtpC2 is also imported into the chloroplast. Therefore, it is likely that the chloroplast ATP synthase complexes contain both atpC1 and atpC2 encoded γ subunits. However, the atpC2 gene is expressed more than hundred times at a lower level than atpC1 and array data show the differential and tissue specific expression of the two genes. The function of AtpC2 could not be revealed by inactivating the gene. Overexpression of atpC2 in dpa1 generated viable lines with an ATP synthase complex containing only γ2, although wild type phenotype is not completely restored. The second part of this work regarded the optimization of conditions for plastid transformation in Arabidopsis thaliana. An efficient and fast regeneration system from cotyledon protoplasts was established for Arabidopsis thaliana accessions C24, Columbia, and Wassilewskija. Culture conditions and media compositions were optimized for the development of protoplasts embedded in thin alginate layers. The protocol is reproducible, efficient, extremely fast, and regenerated plants are fertile. Thus, this cotyledon-based system could prove useful for studying plant cell and molecular biology in A. thaliana. - The sul gene appeared to be a potential novel candidate as selectable marker for plastid transformation. However, genetic and molecular studies demonstrated that sul can not be used for this purpose. On the other hand a new function of sul appeared. The gene could be the missing marker for mitochondria transformation in higher plants.

Detailed clinical, neuroradiological, histological, biochemical, and genetic investigations were undertaken in a child suffering from Leigh syndrome. The clinical symptoms started at age five months and led to a severe progressive neurodegenerative disorder causing epilepsy, psychomotor retardation, and tetraspasticity. Biochemical measurement of skeletal muscle showed a severe decrease in mitochondrial complex II. Sequencing of SDHA revealed compound heterozygosity for a nonsense mutation in exon 4 (W119X) and a missense mutation in exon 3 (A83V), both absent in normal controls. In six additional patients---five with Leigh or Leigh-like syndrome and one with neuropathy and ataxia associated with isolated deficiency of complex II---mutations in SDHA were not detected, indicating genetic heterogeneity.

Background: The proteasome system has a pivotal role in the control of the immune response, which suggests that it might be involved in the pathogenesis of autoimmune disorders.Objective: To investigate the expression profile of selected proteasomal genes in human peripheral blood mononuclear cells in patients with a variety of autoimmune diseases compared with healthy subjects.Methods: Real time quantitative RT-PCR was used to analyse the mRNA expression pattern of the proteasome activator subunits PA28textgreeka and PA28textgreekb and of constitutive proteasome and interferon-textgreekg-inducible immunoproteasome subunits in peripheral blood mononuclear cells. Simultaneously, protein expression of selected proteasome subunits was quantified by immunoblotting.Results: Under systemic inflammatory conditions the proteasome subunits LMP2 (textgreekb1i), LMP7 (textgreekb5i), MECL1 (textgreekb2i), and PA28textgreeka were expressed abundantly at the protein level in the vast majority of systemic autoimmune disorders. However, simultaneous mRNA and protein quantification showed a characteristic proteasome expression signature in primary Sjögren's syndrome. At the transcript level, the interferon-textgreekg-responsive subunits LMP2 (textgreekb1i), MECL1 (textgreekb2i), and the proteasome activator subunit PA28textgreeka were markedly up regulated. In contrast, LMP2 (textgreekb1i) deficiency was evident at the protein level, indicating deregulation of proteasome expression in Sjögren's syndrome.Conclusions: These data provide evidence for a regulatory defect in the proteasome system in human autoimmune disorders, pointing to a unique role for LMP2 (textgreekb1i) in the pathogenesis of primary Sjögren's syndrome.

The Mediator of transcriptional regulation is the central coactivator that enables a response of RNA polymerase II to activators and repressors. It is conserved from yeast to human and consists of 25 subunits in yeast that are organized in four modules called head, middle, tail, and CDK8/Cyclin C module. Despite its central role in transcription the functional mechanism remains enigmatic. To overcome the lack of detailed structural data on the Mediator a recombinant expression system was established that allows large-scale purifications of Mediator head module subcomplexes. It has been shown that via limited proteolysis assays and multicistronic expression the problems of insolubility and low expression rates of Mediator subunits can be overcome, paving the way for structural studies on subcomplexes of the Mediator head module. First data indicated that a reconstitution of the complete head module is within close reach. Large-scale copurification data led to a detailed interaction map of subunits and subcomplexes from within the head module and towards the middle module. The second part of this work describes the structure solution of a subunit in the CDK8/Cyclin C module – Cyclin C. Cyclin C binds the cyclin-dependent kinases CDK8 and CDK3, which regulate mRNA transcription and the cell cycle, respectively. The crystal structure of Cyclin C reveals two canonical five-helix repeats and a specific N-terminal helix. In contrast to other cyclins, the N-terminal helix is short, mobile, and in an exposed position that allows for interactions with proteins other than the CDKs. A model of the CDK8/Cyclin C pair reveals two regions in the interface with apparently distinct roles. A conserved region explains promiscuous binding of cyclin C to CDK8 and CDK3, and a non-conserved region may be responsible for discrimination of CDK8 against other CDKs involved in transcription. A conserved and Cyclin C-specific surface groove may recruit substrates near the CDK8 active site. Activation of CDKs generally involves phosphorylation of a loop at a threonine residue. In CDK8, this loop is longer and the threonine is absent suggesting an alternative mechanism of activation is discussed based on a CDK8-Cyclin C model.

RNA polymerase II (Pol II) is the central enzyme, that synthetizes all mRNA in eukaryotic cells. In this work, I solved the structure of the complete, initiation-competent 12-subunit yeast RNA polymerase II at 3.8 Å. I also solved the structure of the Pol II subcomplex of Rpb4/7 alone at 2.3 Å resolution. These structures reveal the details of Pol II assembly from 12 subunits and give important insights into the initiation of transcription. The refined, atomic model of the complete 12-subunit Pol II enabled homology modeling of the two other nuclear RNA polymerases. In Pol I and Pol III, 65 % and 77 % of the Pol II fold are conserved, respectively. Together with a recent structure of a Pol II elongation complex, these results show that the basic mechanism of transcription applies also to the two other nuclear RNA polymerases

BRG1 is a conserved subunit of the SWI/SNF family of ATP dependent chromatin remodeling complexes. These complexes play an important role in the transcription of various genes by making promoters accessible to the transcription machinery. Mutations in BRG1 have been connected to various cancers. In addition, a BRG1 knock-out in mice is lethal at the periimplantation stage, while BRG1 heterozygote mice are predisposed to exencephaly and tumors of epithelial origin, showing the importance of BRG1 in normal development and disease. In this study, I used Xenopus laevis to study the role of BRG1 because this system allows manipulation of endogenous protein levels by the use of antisense oligonucleotide mediated knock-down as well as interference analysis at early stages of development by overexpression of wild type and dominant negative protein variants. Since BRG1 is conserved among all vertebrates, I initially studied the role of BRG1 in Xenopus development by overexpression of wild type and dominant negative human BRG1. Overexpression of dominant negative human BRG1 gave a ventralized phenotype suggesting a role of BRG1 in dorsal-ventral patterning. The specificity of phenotypes was confirmed by using wild type human BRG1. On the other hand, overexpression of wild type and dominant negative variants of human BRM showed no developmental phenotypes. Prompted by these results, a frog brg1 cDNA was cloned by searching the Xenopus laevis EST database, using human BRG1 as a query. In addition, monoclonal antibodies specific to xBRG1 were raised and characterized. The expression pattern of Xbrg1 was found to be ubiquitous until gastrula stage and is tissue specific from neurula stage onwards. A Xenopus homologue of INI1, a subunit of SWI/SNF chromatin-remodeling complex, was cloned using database search. The expression pattern of Xini1 was found to be similar to Xbrg1. Using site directed mutagenesis, a dominant negative construct of xBRG1 was made by mutating the conserved lysine into arginine (K770R). Loss and gain of function studies showed that BRG1 is involved in AP axis formation during Xenopus development. The gain of function studies were done by overex-pressing wild type and dominant negative xBRG1, while loss of function studies were done using highly specific antisense morpholino oligos. Specificity of morpholino treatment was further proven by the rescue of ventralized phenotypes of morphant embryos by overexpression of human BRG1. It was found that BRG1 knock-down affects several tissues as assessed by in-situ hybridization using tissue specific markers. To determine the molecular explanation for these pleiotropic effects, several genes involved in early patterning of Xenopus embryo during organizer formation were analyzed. The analysis was done using whole mount in-situ hybridization, revealing the spatial gene expression pattern. This analysis revealed that BRG1 mostly affects WNT signaling dependent genes required for dorsal mesoderm formation while leaving pan-mesodermal genes unaffected. Furthermore the genetic interaction of BRG1 with the WNT pathway was confirmed by epistasis experiments showing that overexpression of β-CATENIN can rescue the xBrg1 antisense morpholino oligos dependent ventralized phenotypes as well as formation of secondary axis by overexpression of β-CATENIN could be prevented by BRG1 knock-down. Since the whole embryo represents a complex situation whereby many signaling pathways interact with each other and influence the outcome, the animal cap system was used to analyze the effect of BRG1 on various signaling pathways by analyzing corresponding direct target genes. Animal cap assays showed that the effect of BRG1 is signal specific. Moreover, among the affected signaling pathways, BRG1 knock-down affected only specific genes. These results showed that the BRG1 effect is gene and signal specific. The importance of WNT signaling has also been shown in cancer as well as in haematopoietic and embryonic stem cell self renewal. Given the importance of the WNT signaling, the role of BRG1 on the WNT signaling pathway was further investigated. Treatment of animal cap cells with various doses of Wnt8 mRNA showed the differential requirement of the WNT signal for maximal stimulation of direct target genes. The direct target genes of the WNT pathway showed various degrees of reduction in their maximal stimulation upon BRG1 protein knock-down. The requirement of BRG1 for proper stimulation of the WNT target genes was further confirmed by overexpression of xBRG1 under sub-optimal conditions of WNT stimulation. A major conclusion from these experiments is that BRG1 protein defines signaling thresholds for WNT-mediated activation of target genes. This implies that chromatin remodeling complexes are part of the machinery, which translates inductive signals into spatial gene expression domains.

Doktorarbeit zur Untersuchung der grossen Untereinheit des RNA polymerase II

Bei der akuten myeloischen Leukämie (AML) stellen die unkontrollierte Proliferation und Reifungsblockade myeloider Vorläuferzellen, Expansion dieser Zellen in das periphere Blut, extramedulläre Manifestationen und verminderte Elimination der Leukämiezellen durch das Immunsystem grundlegende Pathomechanismen dar. Diese Vorgänge werden über ein komplexes Zusammenspiel von Zytokinen und Adhäsionsmolekülen reguliert. In dieser Arbeit wurde daher mittels Durchflußzytometrie die Expression von Zytokinrezeptoren, Adhäsions- und kostimulatorischen Molekülen in Knochenmarks(KM-) Proben von 103 AML-Patienten bei Diagnosestellung und acht gesunden Probanden untersucht. Zytokinrezeptoren weisen bei der normalen Hämopoese ein reifegradabhängiges und linienspezifisches Expressionsmuster auf. Es wurden daher zum einen Zytokinrezeptoren ausgewählt, die schon in der frühen Hämopoese exprimiert werden, wie der SCF-R (CD117), FL-R (CD135), IL-3-R (CD123) und zum andern Zytokinrezeptoren, die erst in späteren Differenzierungsstadien der monozytären Zelllinie (v.a. GM-CSF-R; CD116) und der granulozytären Zelllinie (v.a. G-CSF-R, CD114) exprimiert werden. Die gp130-Subunit (CD130) stellt die signaltransduzierende Untereinheit von IL-6, IL-11, LIF etc. dar und wirkt synergistisch auf allen Stufen der Hämopoese mit. Die untersuchten Adhäsionsmoleküle wurden in drei Gruppen unterteilt: a) Adhäsionsmoleküle, die den Kontakt zur KM-Matrix oder zu sich selbst beeinflussen: VLA-2 (CD49b), VLA-3 (CD49c) und die erst kürzlich auf hämopoetischen Zellen gefundenen Adhäsionsmoleküle PRR-1 und PRR-2. b) Adhäsionsmoleküle, die den Kontakt zum Endothel fördern: LFA-1 (CD11a), Mac-1 (CD11b), L-Selektin (CD62L) und UPA-R (CD87) c) kostimulatorische Moleküle, die eine Rolle bei der Interaktion der Leukämiezellen mit immunkompetenten Zellen spielen: ICAM-1 (CD54), LFA-3 (CD58), B7-1 (CD80), B7-2 (CD87) und NCAM (CD58). Eine KM-Probe wurde als positiv gewertet, wenn mehr als 20% der Blasten im Auswertefenster den entsprechenden Marker exprimierten. Ergebnisse: Der durchschnittliche Anteil Zytokinrezeptoren exprimierender Zellen war in KM-Proben von AML-Patienten deutlich höher als in KM-Proben von gesunden Probanden. Einzige Ausnahme bildete die gp130-Subunit, die nur auf durchschnittlich 4% der AML-Blasten exprimiert wurde, während durchschnittlich 23% der Zellen in gesunden KM-Proben die gp130-Subunit exprimierten. Bei den Adhäsionsmolekülen zeigte sich im Vergleich zu den gesunden KM-Proben bei der AML ein höherer Anteil von Zellen, die kostimulatorische und Endothel-Kontakt-fördernde Moleküle exprimierten, während der Anteil von Zellen, die das Stroma-Kontakt-fördernde ß1-Integrin VLA-2 exprimierten, vermindert war. VLA-3 konnte dagegen in keinem der untersuchten AML-Fälle und der gesunden KM-Proben als positiv gewertet werden. Innerhalb der AML-Subtypen konnte ein reifegrad– und linienabhängiges (monozytäres, granulozytäres) Verteilungsmuster der Zytokinrezeptoren festgestellt werden: Blasten unreifer Leukämien (M0; M1) exprimierten bevorzugt SCF-R und FL-R. Blasten von AML-Subtypen, die der granulozytären Differenzierungslinie zugeordnet werden (M2, M3), exprimierten v.a. G-CSF-R. Blasten monozytärer Leukämien (M4, M5) exprimierten v.a. GM-CSF-R und FL-R. Der IL-3-R wurde in fast allen AML-KM-Proben auf einem Großteil der Blasten exprimiert. Den größten Anteil positiver Zellen für Adhäsions- und kostimulatorische Moleküle (Integrine, B7-2, NCAM, UPA-R) wiesen die monozytären Leukämien auf. B7-1 wurde v.a. auf Blasten des FAB-Typs M3 exprimiert. L-Selektin, ICAM-1 und PRR-1/PRR-2 zeigten eine variable Expression innerhalb aller FAB-Typen. In der Gruppe der sekundären Leukämien waren signifikant mehr Fälle Mac-1-positiv als in der Gruppe der primären Leukämien (p = 0.074, Qui2-Test). Ansonsten zeigten sich zwischen primären und sekundären Leukämien keine signifikanten Unterschiede. Wichtig für die Entscheidung über Art und Intensität der Therapie bei der AML ist das Abschätzen der Prognose eines Patienten bei Diagnosestellung. Bislang werden Patienten v.a. anhand zytogenetischer Untersuchungen von Karyotypanomalien in Prognosegruppen eingeteilt. Da aber nur ca. 50-60% der AML-Patienten chromosomale Veränderungen aufweisen, besteht ein Bedarf an Karyotyp-unabhängigen Prognosekriterien. Zytogenetische Analysen wurden bei allen AML-KM-Proben durchgeführt und die Expression der Marker sowohl mit den zytogenetischen Risikogruppen als auch mit dem tatsächlichen klinischen Verlauf der Patienten korreliert. In die klinische Auswertung wurden nur Patienten (n = 55) eingeschlossen, die nach dem Therapieprotokoll der German AML-Cooperative-Group behandelt worden waren. In der zytogenetisch günstigen Prognosegruppe zeigten sich im Vergleich zur zytogenetisch ungünstigen Prognosegruppe signifikant mehr G-CSF-R-positive Zellen (p = 0.027, T-Test), signifikant weniger L-Selektin-positive Fälle (p = 0.037, Qui2-Test) und signifikant mehr Mac-1- und PRR-1-positive Fälle (p = 0.005; p = 0.009; Qui2-Test). Diese Marker zeigten aber keine signifikanten Unterschiede bezüglich Remissionrate und progressfreier Überlebenswahrscheinlichkeit der Patienten. Dies läßt sich auf die zum Teil geringe Fallzahl und die kurze Beobachtungsdauer von im Mittel 11 Monaten nach Remission erklären. Andere Marker zeigten dagegen keine Korrelation mit den zytogenetischen Risikogruppen, dagegen aber mit dem tatsächlichen klinischen Verlauf der Patienten: VLA-2-, NCAM-, UPA-R-positive Leukämien zeigten eine signifikant niedrigere Remissionsrate (p = 0.049, p = 0.03, p = 0.03, Qui2-Test). Patienten, in deren KM-Proben >85% der Blasten den FL-R oder >45,5% den SCF-R exprimierten, wiesen eine signifikant niedrigere Wahrscheinlichkeit für progressfreies Überleben auf, ebenso wie Patienten, in deren KM-Proben >60,5% der Blasten ICAM-1-, >15% B7-1-, >65% B7-2- und >8% NCAM-positiv waren. NCAM korrelierte als einziger Marker negativ sowohl mit der Remissionsrate, als auch mit der progressfreien Überlebenswahrscheinlichkeit, allerdings nicht mit der Einteilung in zytogenetische Risikogruppen. Auch für die übrigen Marker konnten Cut-off-Werte für den Anteil Marker-positiver Blasten ermittelt werden, bei denen aus dem Vergleich der entstandenen Gruppen ein deutlicher Unterschied in der Dauer der progressfreien Überlebenszeit hervorging. Diese Unterschiede waren allerdings aufgrund der geringen Fallzahl nicht signifikant, so dass sich eine eindeutige prognostische Aussagen nicht treffen ließ. Dabei wiesen Patienten mit einem höheren Anteil von G-CSF-R-, GM-CSF-R- und einem niedrigeren Anteil von IL-3-R-exprimierenden Blasten eine längere progressfreie Überlebenszeit auf. Patienten mit sehr hohem Anteil PRR-2- oder mit geringem Anteil PRR-1-positiver Blasten tendierten zu einer eher kürzeren progressfreien Überlebenszeit. Umgekehrt wies eine niedrige Expression von Endothel-Kontakt fördernden Oberflächenmolekülen, wie z.B. L-Selektin, Mac-1 und UPA-R auf eine schlechte Prognose hinsichtlich der Dauer des progressfreien Überlebens hin. Therapeutische Konsequenzen: Die in dieser Arbeit aufgezeigten Zusammenhänge zwischen der Expression bestimmter Oberflächenmarker und dem klinischen Verlauf der Patienten helfen, die Prognoseeinschätzung von Patienten - über die Zytogenetik hinaus - weiter zu spezifizieren: So stellt die NCAM-positive Leukämie eine eigene Entität mit prognostisch schlechtem Verlauf unabhängig vom Karyotyp dar. Bei UPA-R- und/oder VLA-2-positiven AML-Fällen sollten aufgrund der verminderten Remissionswahrscheinlichkeit intensivere therapeutische Induktionstherapien eingeleitet werden. Für die Remissionsdauer ist sowohl die hohe Expression kostimulatorischer Moleküle, als auch die hohe Expression von Zytokinrezeptoren, die v.a. auf Stammzellebene wirksam sind und die die Expression von diesen kostimulatorischen Molekülen fördern, prognostisch ungünstig. Diese Patienten sollten bei intensiver Konsolidierungstherapie engmaschig kontrolliert werden und die Indikation zur Knochenmarkstransplantation sollte frühzeitig gestellt weren. In der Zytokintherapie werden G-CSF und GM-CSF regelmäßig in der Klinik zur Verkürzung der Neutropeniephase nach Chemotherapie eingesetzt. Dagegen konnte mit dem Einsatz von G-CSF und GM-CSF als Priming-Medikamente bisher noch kein eindeutiger klinischer Benefit für die Patienten erzielt werden. Die in dieser Arbeit vorgestellten Ergebnisse einer linienspezifischen und reifegradabhängigen Expression der Zytokinrezeptoren legen nahe, dass G-CSF als Primingmedikament v.a. bei granulozytär-differenzierten AML-Subtypen und GM-CSF eher bei monozytär-differenzierten AML-Subtypen eingesetzt werden sollte. In der Supportivtherapie, bei der die Stimulation von AML-Blasten nicht mehr gewünscht ist, sollten G- und GM-CSF genau umgekehrt eingesetzt werden. Da eine hohe Expression von FL-R und SCF-R mit einer schlechten Prognose für die Dauer des progressfreien Überlebens korrelierte, kann sich eine Stimulation dieser Rezeptoren durch die Gabe von SCF und FL in der Supportivtherapie eher ungünstig auswirken, ebenso wie beim Priming, da auch gesunde Stammzellen stimuliert und damit sensibler gegen Zytostatika werden. Darüber hinaus geben diese Ergebnisse auch Hinweise auf mögliche pathobiologische Bedeutungen und damit verbundener neuer therapeutischer Strategien bei der AML: So kann die erhöhte FL-R-Expression - wie bei der Tandemduplikation des FL-R auch - zu einer erhöhten, prognostisch ungünstigen Phophorylierung von Tyrosinkinasen führen. Auch der SCF-R aktiviert intrazellulär Tyrosinkinasen. Neue Medikamente, wie z.B. Tyrosinkinase-Inhibitoren, oder Dexamethason, das die FL-R-Expression auf den AML-Blasten herunterreguliert, könnten bei diesen AML-Patienten neue benefit-bringende therapeutische Möglichkeiten darstellen. Ebenso scheint die Immunantwort bei AML-Patienten trotz, oder vielleicht sogar gerade bei Expression von kostimulatorischen Molekülen vermindert zu sein, was die Gabe von immunstimulierenden Medikamenten, wie rIL-2 oder CTLA-4-Inhibitoren im Bereich der Immuntherapie sinnvoll erscheinen lässt. So leistet diese Arbeit nicht nur einen Beitrag zur Diagnostik, Prognose und Biologie der AML, sondern entwickelt in Zusammenschau mit bereits publizierten Daten neue, therapeutische Möglichkeiten für die Behandlung der AML.

We have isolated a full-length mouse cDNA encoding a lysine-rich protein of 1,131 amino acids with a calculated molecular mass of 126 kDa. The protein binds in a sequence-unspecific manner to DNA, is localized exclusively in the nucleus, and contains a putative ATP binding site and a stretch of 80 amino acids with homology to the carboxy terminus of prokaryotic DNA ligases. On the basis of the following facts, we conclude that the isolated cDNA encodes the 140-kDa subunit of mouse replication factor C (mRFC140). (i) The sequence around the ATP binding site shows significant homology to three small subunits of human replication factor C. (ii) Polyclonal antibodies raised against the protein encoded by this cDNA cross-react with the 140-kDa subunit of purified human replication factor C (hRFC140) and recognize in mouse cell extracts an authentic protein with an apparent molecular mass of 130 kDa. (iii) Sequence comparison with a human cDNA isolated by using tryptic peptide sequence information from purified hRFC140 revealed 83% identity of the encoded proteins. The mRFC140 gene is ubiquitously expressed, and two mRNAs approximately 5.0 and 4.5 kb long have been detected. The gene was mapped by in situ hybridization to mouse chromosome 5, and its human homolog was mapped to chromosome 4 (p13-p14).

Sat, 1 Jan 1994 12:00:00 +0100 https://epub.ub.uni-muenchen.de/3781/1/92.pdf Oesterhelt, Dieter; Zinth, Wolfgang; Lauterwasser, Christoph; Holzapfel, Wolfgang; Finkele, Ulrich; Stilz, Hans Ulrich

Sat, 1 Jan 1994 12:00:00 +0100 http://epub.ub.uni-muenchen.de/4055/ http://epub.ub.uni-muenchen.de/4055/1/038.pdf Cheng, Qi; Cant, Charles; Moll, Thomas; Hofer-Warbinek, Renate; Wagner, Ernst; Birnstiel, Max L.; Bach, Fritz H.; Martin, Rainer de Cheng, Qi; Cant, Charles; Moll, Thomas; Hofer-Warbinek, Renate; Wagner, Ernst; Birnstiel, Max L.; Bach, Fritz H. und Martin, Rainer de (1994): NK-kappa B subunit-specific regulation of the I kappa B alpha promoter. In: Journal of Biological Chemistry, Vol. 269: pp. 13551-13557. Chemie und Pharmazie

Sat, 1 Jan 1994 12:00:00 +0100 http://epub.ub.uni-muenchen.de/3217/ http://epub.ub.uni-muenchen.de/3217/1/019.pdf Faust, Daniela M.; Boshart, Michael; Schütz, Günther; Weiss, Mary C. Faust, Daniela M.; Boshart, Michael; Schütz, Günther und Weiss, Mary C. (1994): Constancy of Expression of the Protein Kinase A Regulatory Subunit Rla in Hepatoma Cell Lines of Different Phenotypes. In: Cell Growth and Differentiation, Vol. 5: pp. 47-53.

The photochemical activities of phycoerythrocyanin α-subunits from Mastigocladus laminosus separated by isoelectric focusing were tested by irradiating at 500, 550, 577 and 600 nm. Two types of photoreversible photochromic responses have been characterized by absorption and absorption difference spectroscopy. Type I is the well-known absorption shift from 571 to 506 nm. Type II is a new response characterized by a line-broadening of the 570 nm absorption.

Complementary DNAs encoding three novel and distinct beta subunits (CaB2a, CaB2b and CaB3) of the high voltage activated (L-type) calcium channel have been isolated from rabbit heart. Their deduced amino acid sequence is homologous to the beta subunit originally cloned from skeletal muscle (CaB1). CaB2a and CaB2b are splicing products of a common primary transcript (CaB2). Northern analysis and specific amplification of CaB2 and CaB3 specific cDNAs by polymerase chain reactions showed that CaB2 is predominantly expressed in heart, aorta and brain, whereas CaB3 is most abundant in brain but also present in aorta, trachea, lung, heart and skeletal muscle. A partial DNA sequence complementary to a third variant of the CaB2 gene, subtype CaB2c, has also been cloned from rabbit brain. Coexpression of CaB2a, CaB2b and CaB3 with alpha 1heart enhances not only the expression in the oocyte of the channel directed by the cardiac alpha 1 subunit alone, but also effects its macroscopic characteristics such as drug sensitivity and kinetics. These results together with the known alpha 1 subunit heterogeneity, suggest that different types of calcium currents may depend on channel subunit composition.

Human chorionic gonadotropin (hCG) is a clinically relevant marker of trophoblastic and nontrophoblastic malignancies. In the present studies, in addition to determining serum hCG, we investigated the presence and properties of hCG immunoreactivity in ascites of patients with nontrophoblastic malignant tumors and, for comparison, in ascites caused by cirrhotic liver disease. Total hCG immunoreactivity [hCG (+hCG-ß)] was found to be elevated above the reference value (>5 IU/liter) in the serum of 2 of 20 patients with cirrhosis and 11 of 20 patients with malignant tumors. For comparison, in ascites, hCG (+hCG-ß) concentrations were frequently higher than in the corresponding serum samples and exceeded 10 IU/liter in 0 of 20 cirrhotic samples and in 16 of 20 malignant samples. In order to elucidate the nature of the hCG immunoreactive material, all samples were then assessed by immunoradiometric assays specific for the intact hCG molecule (holo-hCG) and the free hCG-ß subunit, respectively. In the holo-hCG assay, elevated values were detected in 0 of 20 (0 of 20) cirrhotic ascites (serum) samples and 0 of 20 (1 of 20) malignant ascites (serum) samples. In the free hCG-ß assay, on the other hand, no positive results were obtained in the ascites or serum of 20 patients with liver cirrhosis; however, 8 of 20 serum samples and 16 of 20 ascites samples derived from tumor patients were positive. In accord with the immunological data, gel chromatographical studies of malignant ascites revealed the abundance of free hCG-ß subunit rather than that of holo-hCG. In contrast to malignancy-related ascites, in ascites of patients receiving hCG injections for treatment of infertility, holo-hCG was more abundant than free hCG-ß immunoreactivity. Incubation experiments of purified holo-hCG in ascites for 24 h at -20, 20, or 37°C showed no substantial dissociation of the hCG molecule and release of free hCG-ß immunoreactivity, thus arguing against production of free hCG-ß by degradation of holo-hCG and in favor of its tumor-related secretion. In conclusion, hCG-ß immunoreactivity is frequently elevated in malignancy-related ascites and appears to be related to the presence of free ß subunit of hCG rather than that of the intact hCG molecule. Interestingly, hCG-ß determination in ascites proved to be clearly superior to serum measurement in discriminating between tumor and cirrhosis. Thus, hCG-ß might be a useful marker of malignancy-related ascites and should be prospectively assessed for possible clinical use in comparison with other well-established parameters, such as cytology and protein determination. For this purpose, according to our results, only assays that exhibit a high sensitivity for free hCG-ß subunit appear to be suitable.

Tue, 1 Jan 1991 12:00:00 +0100 http://epub.ub.uni-muenchen.de/8687/ http://epub.ub.uni-muenchen.de/8687/1/Mueller_Guenter_8687.pdf Müller, Günter; Bandlow, Wolfhard Müller, Günter und Bandlow, Wolfhard (1991): Two lipid-anchored cAMP-binding proteins in the yeast Saccharomyces cerevisiae are unrelated to the R subunit of cytoplasmic protein kinase A. In: European Journal of Biochemistry, Vol. 202, Nr. 2: pp.

The tissue-specific extinguisher locus TSE1, a dominant negative regulator of transcription in somatic cell hybrids, acts via a cAMP response element (CRE) to repress activity of a hepatocyte-specific enhancer. Guided by the antagonism between TSE1 and cAMP-mediated signal transduction, we identified the regulatory subunit Rlα of protein kinase A (PKA) as the product of the TSE1 locus. The evidence derives from concordant expression of Tlα mRNA and TSE1 genetic activity, high resolution mapping of the RIα gene and TSE1 on human chromosome 17, and the ability of a transfected RIα cDNA to generate a phenocopy of TSE1-mediated extinction. The mechanism of TSE1/RIα-mediated extinction involves repression of basal PKA activity, reduced phosphorylation of CREB at Ser-133, and a corresponding reduction of in vivo protein binding at the target CRE.

Sat, 25 Aug 1990 12:00:00 +0100 https://epub.ub.uni-muenchen.de/7603/1/Neupert_Walter_7603.pdf Tropschug, Maximilian; Neupert, Walter; Müller, Harald A.; Harmey, Matthew A.; Rassow, Joachim

Complementary DNAs for the γ subunit of the calcium channel of rabbit skeletal muscle were isolated on the basis of peptide sequences derived from the purified protein. The deduced primary structure is without homology to other known protein sequences and is consistent with the γ subunit being an integral membrane protein.

Sun, 1 Jan 1989 12:00:00 +0100 http://epub.ub.uni-muenchen.de/3643/ http://epub.ub.uni-muenchen.de/3643/1/001.pdf Ruth, Peter; Rohrkasten, A.; Biel, Martin; Bosser, E.; Regulla, S.; Meyer, H. E.; Flockerzi, Veit; Hofmann, Franz Ruth, Peter; Rohrkasten, A.; Biel, Martin; Bosser, E.; Regulla, S.; Meyer, H. E.; Flockerzi, Veit und Hofmann, Franz (1989): Primary structure of the beta subunit of the DHP-sensitive calcium channel from skeletal muscle. In: Science, Vol. 245: pp. 1115-1118.

Transport of cytoplasmically synthesized precursor proteins into or across the inner mitochondrial membrane requires a mitochondrial membrane potential. We have studied whether additional energy sources are also necessary for protein translocation. Reticulocyte lysate (containing radiolabelled precursor proteins) and mitochondria were depleted of ATP by pre-incubation with apyrase. A membrane potential was then established by the addition of substrates of the electron transport chain. Oligomycin was included to prevent dissipation of Δψ by the action of the F0F1-ATPase. Under these conditions, import of subunit β of F1-ATPase (F1β) was inhibited. Addition of ATP or GTP restored import. When the membrane potential was destroyed, however, the import of F1β was completely inhibited even in the presence of ATP. We therefore conclude that the import of F1β depends on both nucleoside triphosphates and a membrane potential.

Subunit 9 (dicyclohexylcarbodiimide binding protein, 'proteolipid') of the mitochondrial F1F0-ATPase is a nuclearly coded protein in Neurospora crassa. It is synthesized on free cytoplasmic ribosomes as a larger precursor with an NH2-terminal peptide extension. The peptide extension is cleaved off after transport of the protein into the mitochondria. A processing activity referred to as processing peptidase that cleaves the precursor to subunit 9 and other mitochondrial proteins is described and characterized using a cell-free system. Precursor synthesized in vitro was incubated with extracts of mitochondria. Processing peptidase required Mn2+ for its activity. Localization studies suggested that it is a soluble component of the mitochondrial matrix. The precursor was cleaved in two sequential steps via an intermediate-sized polypeptide. The intermediate form in the processing of subunit 9 was also seen in vivo and upon import of the precursor into isolated mitochondria in vitro. The two cleavage sites in the precursor molecule were determined. The data indicate that: (a) the correct NH2-terminus of the mature protein was generated, (b) the NH2-terminal amino acid of the intermediate-sized polypeptide is isoleucine in position-31. The cleavage sites show similarity of primary structure. It is concluded that processing peptidase removes the peptide extension from the precursor to subunit 9 (and probably other precursors) after translocation of these polypeptides (or the NH2-terminal part of these polypeptides) into the matrix space of mitochondria.

Isolated yeast mitochondria were able to take up Neurospora ATPase subunit 9 in vitro although the homologous yeast protein is synthesized within the mitochondria and inserted into the membrane from the matrix side (Tzagoloff, A., and Meagher, P. (1972) J. Biol. Chem. 247, 594- 603). The transfer of the protein was dependent on an energized mitochondrial inner membrane. It was accompanied by proteolytic processing of the precursor to the mature protein with the correct NH2 terminus as determined by Edman degradation of the transferred protein. The possibility is discussed that there are common features in the uptake machinery neither specific for one species nor specific for individual precursor proteins in the same species.

A new protein kinase of the cAMP independent type was found to be bound to the outer envelope membrane of spinach chloroplasts. While stimulated by Mg2+ and inhibited by ADP, the enzyme showed no response to conventional protein substrates and was essentially independent of pH in the physiological (pH 7 to 8) range. The new protein kinase phosphorylated the mature form of the small subunit of ribulose 1,5- bisphosphate carboxylase/oxygenase and, to a lesser extent, an unidentified 24-kDa polypeptide, both of which were bound to the outer envelope membrane. The results suggest that phosphorylation of cytoplasmically synthesized protein constituents of chloroplasts is involved in their transport through the chloroplast envelope membrane barrier.

Subunit 9 of mitochondrial ATPase (Su9) is synthesized in reticulocyte lysates programmed with Neurospora poly A-RNA, and in a Neurospora cell free system as a precursor with a higher apparent molecular weight than the mature protein (Mr 16,400 vs. 10,500). The RNA which directs the synthesis of Su9 precursor is associated with free polysomes. The precursor occurs as a high molecular weight aggregate in the postribosomal supernatant of reticulocyte lysates. Transfer in vitro of the precursor into isolated mitochondria is demonstrated. This process includes the correct proteolytic cleavage of the precursor to the mature form. After transfer, the protein acquires the following properties of the assembled subunit: it is resistant to added protease, it is soluble in chloroform/methanol, and it can be immunoprecipitated with antibodies to F1-ATPase. The precursor to Su9 is also detected in intact cells after pulse labeling. Processing in vivo takes place posttranslationally. It is inhibited by the uncoupler carbonylcyanide m- chlorophenylhydrazone (CCCP). A hypothetical mechanism is discussed for the intracellular transfer of Su9. It entails synthesis on free polysomes, release of the precursor into the cytosol, recognition by a receptor on the mitochondrial surface, and transfer into the inner mitochondrial membrane, which is accompanied by proteolytic cleavage and which depends on an electrical potential across the inner mitochondrial membrane.