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 Biochemical and Genetic Analysis of 4-Hydroxypyridine Catabolism in Arthrobacter sp. Strain IN13

2020 ◽  
Vol 8 (6) ◽  
pp. 888
Author(s):  
Justas Vaitekūnas ◽  
Renata Gasparavičiūtė ◽  
Jonita Stankevičiūtė ◽  
Gintaras Urbelis ◽  
Rolandas Meškys
Keyword(s):  
Recombinant Expression ◽  
Expression Profiles ◽  
Peptide Mass Fingerprinting ◽  
Sole Source ◽  
Open Reading Frames ◽  
Expression Of Genes ◽  
Peptide Mass ◽  
Genes Encoding ◽  
Protein Expression Profiles ◽  
Reading Frames

N-Heterocyclic compounds are widely spread in the biosphere, being constituents of alkaloids, cofactors, allelochemicals, and artificial substances. However, the fate of such compounds including a catabolism of hydroxylated pyridines is not yet fully understood. Arthrobacter sp. IN13 is capable of using 4-hydroxypyridine as a sole source of carbon and energy. Three substrate-inducible proteins were detected by comparing protein expression profiles, and peptide mass fingerprinting was performed using MS/MS. After partial sequencing of the genome, we were able to locate genes encoding 4-hydroxypyridine-inducible proteins and identify the kpi gene cluster consisting of 16 open reading frames. The recombinant expression of genes from this locus in Escherichia coli and Rhodococcus erytropolis SQ1 allowed an elucidation of the biochemical functions of the proteins. We report that in Arthrobacter sp. IN13, the initial hydroxylation of 4-hydroxypyridine is catalyzed by a flavin-dependent monooxygenase (KpiA). A product of the monooxygenase reaction is identified as 3,4-dihydroxypyridine, and a subsequent oxidative opening of the ring is performed by a hypothetical amidohydrolase (KpiC). The 3-(N-formyl)-formiminopyruvate formed in this reaction is further converted by KpiB hydrolase to 3-formylpyruvate. Thus, the degradation of 4-hydroxypyridine in Arthrobacter sp. IN13 was analyzed at genetic and biochemical levels, elucidating this catabolic pathway.

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.

scholarly journals Homotaurine Metabolized to 3-Sulfopropanoate in Cupriavidus necator H16: Enzymes and Genes in a Patchwork Pathway

2009 ◽  
Vol 191 (19) ◽  
pp. 6052-6058 ◽  
Author(s):  
Jutta Mayer ◽  
Alasdair M. Cook
Keyword(s):  
Ralstonia Eutropha ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Peptide Mass Fingerprinting ◽  
Sole Source ◽  
Cupriavidus Necator ◽  
Semialdehyde Dehydrogenase ◽  
Peptide Mass ◽  
Cupriavidus Necator H16 ◽  
Sulfoacetaldehyde Acetyltransferase

ABSTRACT Homotaurine (3-aminopropanesulfonate), a natural product and an analogue of GABA (4-aminobutyrate), was found to be a sole source of nitrogen for Cupriavidus necator (Ralstonia eutropha) H16, whose genome sequence is known. Homotaurine nitrogen was assimilated into cell material, and the quantitative fate of the organosulfonate was sulfopropanoate, which was recovered in the growth medium. The first scalar reaction was shown to be inducible homotaurine:2-oxoglutarate aminotransferase, which released 3-sulfopropanal from homotaurine. This aminotransferase was purified to homogeneity and characterized. Peptide mass fingerprinting yielded locus tag H16_B0981, which was annotated gabT, for GABA transaminase (EC 2.6.1.19). Inducible, NAD(P)+-coupled 3-sulfopropanal dehydrogenase, which yielded 3-sulfopropanoate from 3-sulfopropanal, was also purified and characterized. Peptide mass fingerprinting yielded locus tag H16_B0982, which was annotated gabD1, for succinate-semialdehyde dehydrogenase (EC 1.2.1.16). GabT and GabD1 were each induced during growth with GABA, and cotranscription of gabTD was observed. In other organisms, regulator GabC or GabR is encoded contiguous with gabTD: candidate GabR′ was found in strain H16 and in many other organisms. An orthologue of the GABA permease (GabP), established in Escherichia coli, is present at H16_B1890, and it was transcribed constitutively. We presume that GabR′PTD are responsible for the inducible metabolism of homotaurine to intracellular 3-sulfopropanoate. The nature of the exporter of this highly charged compound was unclear until we realized from the sodium dodecyl sulfate-polyacrylamide gel electrophoresis data that sulfoacetaldehyde acetyltransferase (EC 2.3.3.15; H16_B1872) was strongly induced during growth with homotaurine and inferred that the sulfite exporter encoded at the end of the gene cluster (H16_B1874) has a broad substrate range that includes 3-sulfopropanoate.

scholarly journals Identification, Molecular Cloning, and Evaluation of Potential Use of Isocitrate Dehydrogenase II of Mycobacterium bovis BCG in Serodiagnosis of Tuberculosis

2002 ◽  
Vol 9 (4) ◽  
pp. 846-851 ◽  
Author(s):  
W. Florio ◽  
D. Bottai ◽  
G. Batoni ◽  
S. Esin ◽  
M. Pardini ◽  
...  
Keyword(s):  
Mycobacterium Bovis ◽  
Isocitrate Dehydrogenase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Peptide Mass Fingerprinting ◽  
Igg Antibodies ◽  
Healthy Individuals ◽  
Recombinant Gene Expression ◽  
Serum Dilution ◽  
Peptide Mass ◽  
Potential Use

ABSTRACT Diagnosis of tuberculosis is time-consuming and requires infrastructures which are often not available in countries with high incidences of the disease. In the present study, an 82-kDa protein antigen was isolated by affinity chromatography and was identified by peptide mass fingerprinting as isocitrate dehydrogenase II, which is encoded by the icd2 gene of Mycobacterium bovis BCG. The icd2 gene of BCG was cloned by PCR, and the product of recombinant gene expression was purified and analyzed by two-dimensional polyacrylamide gel electrophoresis. The recombinant protein, named rICD2, was tested for its recognition by immunoglobulin G (IgG) antibodies from the sera of 16 patients with tuberculosis (TB) and 23 healthy individuals by Western blotting. The results showed that rICD2 is recognized by IgG antibodies from the sera of all TB patients tested at serum dilutions of ≥1:640. At a serum dilution of 1:1,280, the sensitivity was 50% and the specificity was 86.9%. These results indicate that rICD2 might represent a candidate for use in a new assay for the serodiagnosis of TB.

scholarly journals Proteomic Analysis of Azole Resistance in Candida albicans Clinical Isolates

2004 ◽  
Vol 48 (7) ◽  
pp. 2733-2735 ◽  
Author(s):  
Massoumeh Z. Hooshdaran ◽  
Katherine S. Barker ◽  
George M. Hilliard ◽  
Harald Kusch ◽  
Joachim Morschhäuser ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Proteomic Analysis ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Peptide Mass Fingerprinting ◽  
Ergosterol Biosynthesis ◽  
Azole Resistance ◽  
Biosynthesis Pathway ◽  
Two Dimensional ◽  
Peptide Mass

ABSTRACT Changes in protein expression within a matched set of Candida albicans isolates representing the acquisition of azole resistance were examined by two-dimensional polyacrylamide gel electrophoresis and peptide mass fingerprinting. Proteins differentially expressed in association with azole resistance included Grp2p, Ifd1p, Ifd4p, Ifd5p, and Erg10p, a protein involved in the ergosterol biosynthesis pathway.

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

2021 ◽  
Author(s):  
Ishida Hideki ◽  
Takshi Akihiro ◽  
Ryo Yasui ◽  
Shinji Yasuhira ◽  
Ken-ich Matsumoto ◽  
...  
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.

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.

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