scholarly journals The Trk fused gene-product (Tfg) is part of a 600-700kDa CARMA1 complex

2019 ◽  
Author(s):  
Markus Grohmann ◽  
Tobit Steinmetz ◽  
Hans-Martin Jäck ◽  
Dirk Mielenz
Keyword(s):  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Peptide Mass Fingerprinting ◽  
Cell Receptor ◽  
Nuclear Factor Κb ◽  
Peptide Mass ◽  
Interaction Partners ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
Fused Gene ◽  
Blue Native

AbstractB cell receptor (BCR) mediated activation of nuclear factor κB (NF-κB) is key to humoral immunity. CARMA1 (CARD11) is essential for BCR mediated NF-κB activation by interacting with Bcl10 and MALT1. Besides these two main players, few interaction partners of the CARMA1 complex are known. Here we identified new interaction partners of CARMA1. We generated two rabbit antisera against mouse CARMA1 to immunopurify endogenous CARMA1 from lysates of mouse B cells. Nik-binding protein (NIBP), Ras-GAP SH3 binding protein 2 (G3BP1) and Trk-fused gene (Tfg) were identified by peptide mass fingerprinting in immunopurified CARMA1 complexes. The interaction of Tfg and CARMA1 was confirmed by co-immunoprecipitation and Blue native polyacrylamide gel electrophoresis using the anti CARMA1 and newly generated anti Tfg antibodies. This analysis revealed that CARMA1 formed complexes of 600-1000 kDa. Additionally, Tfg was found in complexes of 500-600 kDa which increased in size to ∼740 kDa upon overexpresssion.

scholarly journals Glycerol-3-Phosphate-Induced Catabolite Repression in Escherichia coli

2002 ◽  
Vol 184 (11) ◽  
pp. 3044-3052 ◽  
Author(s):  
Tanja Eppler ◽  
Pieter Postma ◽  
Alexandra Schütz ◽  
Uwe Völker ◽  
Winfried Boos
Keyword(s):  
Adenylate Cyclase ◽  
Catabolite Repression ◽  
Binding Protein ◽  
Tricarboxylic Acid Cycle ◽  
Polyacrylamide Gel Electrophoresis ◽  
Peptide Mass Fingerprinting ◽  
Phosphotransferase System ◽  
Peptide Mass ◽  
Dramatic Shift ◽  
Phosphorylated Sugars

ABSTRACT The formation of glycerol-3-phosphate (G3P) in cells growing on TB causes catabolite repression, as shown by the reduction in malT expression. For this repression to occur, the general proteins of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), in particular EIIAGlc, as well as the adenylate cyclase and the cyclic AMP-catabolite activator protein system, have to be present. We followed the level of EIIAGlc phosphorylation after the addition of glycerol or G3P. In contrast to glucose, which causes a dramatic shift to the dephosphorylated form, glycerol or G3P only slightly increased the amount of dephosphorylated EIIAGlc. Isopropyl-β-d-thiogalactopyranoside-induced overexpression of EIIAGlc did not prevent repression by G3P, excluding the possibility that G3P-mediated catabolite repression is due to the formation of unphosphorylated EIIAGlc. A mutant carrying a C-terminally truncated adenylate cyclase was no longer subject to G3P-mediated repression. We conclude that the stimulation of adenylate cyclase by phosphorylated EIIAGlc is controlled by G3P and other phosphorylated sugars such as d-glucose-6-phosphate and is the basis for catabolite repression by non-PTS compounds. Further metabolism of these compounds is not necessary for repression. Two-dimensional polyacrylamide gel electrophoresis was used to obtain an overview of proteins that are subject to catabolite repression by glycerol. Some of the prominently repressed proteins were identified by peptide mass fingerprinting. Among these were periplasmic binding proteins (glutamine and oligopeptide binding protein, for example), enzymes of the tricarboxylic acid cycle, aldehyde dehydrogenase, Dps (a stress-induced DNA binding protein), and d-tagatose-1,6-bisphosphate aldolase.

scholarly journals Novel Victorivirus from a Pakistani Isolate of Alternaria alternata Lacking a Typical Translational Stop/Restart Sequence Signature

Viruses ◽  
2019 ◽  
Vol 11 (6) ◽  
pp. 577 ◽  
Author(s):  
Atif Jamal ◽  
Yukiyo Sato ◽  
Sabitree Shahi ◽  
Wajeeha Shamsi ◽  
Hideki Kondo ◽  
...  
Keyword(s):  
Alternaria Alternata ◽  
Rna Silencing ◽  
Polyacrylamide Gel Electrophoresis ◽  
Peptide Mass Fingerprinting ◽  
Cryphonectria Parasitica ◽  
Fungal Species ◽  
Open Reading Frames ◽  
Fusion Product ◽  
Peptide Mass ◽  
Sequence Signature

The family Totiviridae currently contains five genera Totivirus, Victorivirus, Leishmavirus, Trichomonasvirus, and Giardiavirus. Members in this family generally have a set of two-open reading frame (ORF) elements in their genome with the 5′-proximal ORF (ORF1) encoding a capsid protein (CP) and the 3′-proximal one (ORF2) for RNA-dependent RNA polymerase (RdRp). How the downstream open reading frames (ORFs) are expressed is genus-specific. All victoriviruses characterized thus far appear to use the stop/restart translation mechanism, allowing for the expression of two separate protein products from bicitronic genome-sized viral mRNA, while the totiviruses use a −1 ribosomal frame-shifting that leads to a fusion product of CP and RdRp. We report the biological and molecular characterization of a novel victorivirus termed Alternaria alternata victorivirus 1 (AalVV1) isolated from Alternaria alternata in Pakistan. The phylogenetic and molecular analyses showed AalVV1 to be distinct from previously reported victoriviruses. AalVV1 appears to have a sequence signature required for the −1 frame-shifting at the ORF1/2 junction region, rather than a stop/restart key mediator. By contrast, SDS–polyacrylamide gel electrophoresis and peptide mass fingerprinting analyses of purified virion preparations suggested the expression of two protein products, not a CP-RdRp fusion product. How these proteins are expressed is discussed in this study. Possible effects of infection by this virus were tested in two fungal species: A. alternata and RNA silencing proficient and deficient strains of Cryphonectria parasitica, a model filamentous fungus. AalVV1 showed symptomless infection in all of these fungal strains, even in the RNA silencing deficient C. parasitica strain.

scholarly journals STIM1 enhances SR Ca2+ content through binding phospholamban in rat ventricular myocytes

2015 ◽  
Vol 112 (34) ◽  
pp. E4792-E4801 ◽  
Author(s):  
Guiling Zhao ◽  
Tianyu Li ◽  
Didier X. P. Brochet ◽  
Paul B. Rosenberg ◽  
W. J. Lederer
Keyword(s):  
Cellular Localization ◽  
Ventricular Myocytes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Heart Cells ◽  
Short Term ◽  
Rat Ventricular Myocytes ◽  
Adult Rat ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
Blue Native ◽  
Cellular Movement

In ventricular myocytes, the physiological function of stromal interaction molecule 1 (STIM1), an endo/sarcoplasmic reticulum (ER/SR) Ca2+ sensor, is unclear with respect to its cellular localization, its Ca2+-dependent mobilization, and its action on Ca2+ signaling. Confocal microscopy was used to measure Ca2+ signaling and to track the cellular movement of STIM1 with mCherry and immunofluorescence in freshly isolated adult rat ventricular myocytes and those in short-term primary culture. We found that endogenous STIM1 was expressed at low but measureable levels along the Z-disk, in a pattern of puncta and linear segments consistent with the STIM1 localizing to the junctional SR (jSR). Depleting SR Ca2+ using thapsigargin (2–10 µM) changed neither the STIM1 distribution pattern nor its mobilization rate, evaluated by diffusion coefficient measurements using fluorescence recovery after photobleaching. Two-dimensional blue native polyacrylamide gel electrophoresis and coimmunoprecipitation showed that STIM1 in the heart exists mainly as a large protein complex, possibly a multimer, which is not altered by SR Ca2+ depletion. Additionally, we found no store-operated Ca2+ entry in control or STIM1 overexpressing ventricular myocytes. Nevertheless, STIM1 overexpressing cells show increased SR Ca2+ content and increased SR Ca2+ leak. These changes in Ca2+ signaling in the SR appear to be due to STIM1 binding to phospholamban and thereby indirectly activating SERCA2a (Sarco/endoplasmic reticulum Ca2+ ATPase). We conclude that STIM1 binding to phospholamban contributes to the regulation of SERCA2a activity in the steady state and rate of SR Ca2+ leak and that these actions are independent of store-operated Ca2+ entry, a process that is absent in normal heart cells.

scholarly journals Staphylococcal Superantigen-Like Protein 5 Inhibits Matrix Metalloproteinase 9 from Human Neutrophils

2010 ◽  
Vol 78 (7) ◽  
pp. 3298-3305 ◽  
Author(s):  
Saotomo Itoh ◽  
Eri Hamada ◽  
Go Kamoshida ◽  
Kana Takeshita ◽  
Teruaki Oku ◽  
...  
Keyword(s):  
Matrix Metalloproteinase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Peptide Mass Fingerprinting ◽  
Matrix Metalloproteinase 9 ◽  
Basement Membranes ◽  
Human Neutrophils ◽  
Surface Plasmon Resonance Analysis ◽  
Major Band ◽  
Peptide Mass ◽  
Metalloproteinase 9

ABSTRACT Staphylococcal superantigen-like proteins (SSLs) constitute a family of exoproteins exhibiting structural similarities to superantigens and enterotoxins but no superantigenic activity. In this article, we present evidence that SSL5 specifically binds to matrix metalloproteinase 9 (MMP-9) and inhibits its enzymatic activity. When human neutrophil cell lysate was applied to recombinant His-tagged SSL5 conjugated to Sepharose, the bound fraction gave a major band of approximately 100 kDa in SDS-polyacrylamide gel electrophoresis. This protein was identified as the proform of MMP-9 (proMMP-9) by peptide mass fingerprinting analysis. The recombinant SSL5-Sepharose also bound to proMMP-9 secreted by interleukin 8 (IL-8)-stimulated neutrophils and HT1080 fibrosarcoma cells. Surface plasmon resonance analysis revealed that recombinant SSL5 bound to proMMP-9 with rather high affinity (dissociation constant [K D] = 1.9 nM). Recombinant SSL5 was found to effectively inhibit MMP-9-catalyzed hydrolysis of gelatin and a synthetic fluorogenic peptide in a noncompetitive manner (K i = 0.097 nM), as assessed by zymography and the fluorescence quenching method. Finally, the transmigration of neutrophils across Matrigel basement membranes in response to N-formyl-methionyl-leucyl-phenylalanine (FMLP) was suppressed by the presence of recombinant SSL5. We discuss possible roles that SSL5 may play in immune evasion of staphylococci by inhibiting MMP and interfering with leukocyte trafficking.

scholarly journals Tropomyosin micelles are the major white components in the boiled soup of shellfish

2022 ◽  
Author(s):  
Takashi Akihiro ◽  
Ryou Yasui ◽  
Shinji Yasuhira ◽  
Ken-ich Matsumoto ◽  
Yasuhiro Tanaka ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Trichloroacetic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Laser Desorption ◽  
Peptide Mass Fingerprinting ◽  
Hot Water ◽  
Tandem Mass ◽  
Intense Band ◽  
Peptide Mass

Abstract Basket clam soup, a popular Asian dish, is prepared by boiling clams in hot water. The soup is generally cloudy and considered more delicious as cloudiness increases. However, the identity of the whitening ingredients and their relationship with taste remain unclear. In this study, we aimed to identify the components that contribute to the white color of the boiled soup. The white component was precipitated with trichloroacetic acid and reacted positively with ninhydrin, indicating the presence of proteins. The proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and an intense band was observed at 33 kDa. Peptide mass fingerprinting of this band using matrix-assisted laser desorption/ionisation-time-of-flight tandem mass spectrometry revealed the protein to be tropomyosin. Basket clam tropomyosin expressed and purified from Escherichia coli turned the extracted solution white, confirming that tropomyosin contributed to the white color of clam soup.

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