scholarly journals The N -linking glycosylation system from Actinobacillus pleuropneumoniae is required for adhesion and has potential use in glycoengineering

Open Biology ◽  
2017 ◽  
Vol 7 (1) ◽  
pp. 160212 ◽  
Author(s):  
Jon Cuccui ◽  
Vanessa S. Terra ◽  
Janine T. Bossé ◽  
Andreas Naegeli ◽  
Sherif Abouelhadid ◽  
...  
Keyword(s):  
Outer Membrane Proteins ◽  
Actinobacillus Pleuropneumoniae ◽  
Mass Spectrometry Analysis ◽  
Respiratory Pathogen ◽  
Transcriptional Unit ◽  
Biotechnological Application ◽  
Spectrometry Analysis ◽  
Porcine Pleuropneumonia ◽  
Repeat Units ◽  
Potential Use

Actinobacillus pleuropneumoniae is a mucosal respiratory pathogen causing contagious porcine pleuropneumonia. Pathogenesis studies have demonstrated a major role for the capsule, exotoxins and outer membrane proteins. Actinobacillus pleuropneumoniae can also glycosylate proteins, using a cytoplasmic N -linked glycosylating enzyme designated NGT, but its transcriptional arrangement and role in virulence remains unknown. We investigated the NGT locus and demonstrated that the putative transcriptional unit consists of rimO , ngt and a glycosyltransferase termed agt. From this information we used the A. pleuropneumoniae glycosylation locus to decorate an acceptor protein, within Escherichia coli, with a hexose polymer that reacted with an anti-dextran antibody. Mass spectrometry analysis of a truncated protein revealed that this operon could add up to 29 repeat units to the appropriate sequon. We demonstrated the importance of NGT in virulence, by creating deletion mutants and testing them in a novel respiratory cell line adhesion model. This study demonstrates the importance of the NGT glycosylation system for pathogenesis and its potential biotechnological application for glycoengineering.

Analysis of biofluids by paper spray-MS in forensic toxicology

Bioanalysis ◽  
2020 ◽  
Vol 12 (15) ◽  
pp. 1087-1102
Author(s):  
Ana Luiza Freitas de Assis Linhares ◽  
Mauricio Yonamine
Keyword(s):  
Drugs Of Abuse ◽  
Forensic Toxicology ◽  
Ambient Ionization ◽  
Sample Volume ◽  
Mass Spectrometry Analysis ◽  
Sample Treatment ◽  
Biological Matrices ◽  
Paper Spray ◽  
Spectrometry Analysis ◽  
Potential Use

Direct ambient ionization techniques have been developed with the aim to reduce the complexity of mass spectrometry analysis by minimizing sample preparation and chromatographic separation. In this context, paper spray-MS (PS-MS) is an innovative approach that provides faster and cheaper analysis of biofluids by the addition of the sample directly to a paper. In forensic toxicology, the analytical workflow for the detection and quantification of drugs of abuse is onerous, including sample treatment, extraction and clean up, especially regarding complex biological matrices. PS-MS allows the detection of analytes of toxicological interest in blood, plasma and urine using low sample volume. This review aims to discuss the potential use, advances and challenges of PS-MS in forensic toxicology.

scholarly journals Design and Characterization of a Novel Tool for the Antigenic Enrichment of Actinobacillus pleuropneumoniae Outer Membrane

Pathogens ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 1014
Author(s):  
Fabio Antenucci ◽  
Armen Ovsepian ◽  
Agnieszka Wrobel ◽  
Hanne Cecilie Winther-Larsen ◽  
Anders Miki Bojesen
Keyword(s):  
Outer Membrane ◽  
Acyl Carrier Protein ◽  
Actinobacillus Pleuropneumoniae ◽  
Mass Spectrometry Analysis ◽  
Economic Losses ◽  
Bacterial Strains ◽  
Spectrometry Analysis ◽  
Fast Screening ◽  
Chimeric Construct ◽  
Fluorescent Tag

Production and isolation of recombinant proteins are costly and work-intensive processes, especially in immunology when tens or hundreds of potential immunogens need to be purified for testing. Here we propose an alternative method for fast screening of immunogen candidates, based on genetic engineering of recombinant bacterial strains able to express and expose selected antigens on their outer membrane. In Actinobacillus pleuropneumoniae, a Gram-negative porcine pathogen responsible for extensive economic losses worldwide, we identified a conserved general secretion pathway (GSP) domain in the N-terminal part of the outer membrane protein ApfA (ApfA stem: ApfAs). ApfAs was used as an outer membrane anchor, to which potential immunogens can be attached. To enable confirmation of correct positioning, ApfAs, was cloned in combination with the modified acyl carrier protein (ACP) fluorescent tag ACP mini (ACPm) and the putative immunogen VacJ. The chimeric construct was inserted in the pMK-express vector, subsequently transformed into A. pleuropneumoniae for expression. Flow cytometry, fluorescence imaging and mass spectrometry analysis were employed to demonstrate that the outer membrane of the transformed strain was enriched with the chimeric ApfAs-ACPm-VacJ antigen. Our results confirmed correct positioning of the chimeric ApfAs-ACPm-VacJ antigen and supported this system’s potential as platform technology enabling antigenic enrichment of the outer membrane of A. pleuropneumoniae.

scholarly journals Evaluation of the chemical defense fluids of Macrotermes carbonarius and Globitermes sulphureus as possible household repellents and insecticides

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
S. Appalasamy ◽  
M. H. Alia Diyana ◽  
N. Arumugam ◽  
J. G. Boon
Keyword(s):  
Mass Spectrometry ◽  
Gas Chromatography ◽  
Periplaneta Americana ◽  
Mass Spectrometry Analysis ◽  
Gas Chromatography Mass Spectrometry ◽  
Controlled Environment ◽  
Spectrometry Analysis ◽  
Chromatography Mass Spectrometry ◽  
Potential Use ◽  
Macrotermes Carbonarius

AbstractThe use of chemical insecticides has had many adverse effects. This study reports a novel perspective on the application of insect-based compounds to repel and eradicate other insects in a controlled environment. In this work, defense fluid was shown to be a repellent and insecticide against termites and cockroaches and was analyzed using gas chromatography-mass spectrometry (GC–MS). Globitermes sulphureus extract at 20 mg/ml showed the highest repellency for seven days against Macrotermes gilvus and for thirty days against Periplaneta americana. In terms of toxicity, G. sulphureus extract had a low LC50 compared to M. carbonarius extract against M. gilvus. Gas chromatography–mass spectrometry analysis of the M. carbonarius extract indicated the presence of six insecticidal and two repellent compounds in the extract, whereas the G. sulphureus extract contained five insecticidal and three repellent compounds. The most obvious finding was that G. sulphureus defense fluid had higher potential as a natural repellent and termiticide than the M. carbonarius extract. Both defense fluids can play a role as alternatives in the search for new, sustainable, natural repellents and termiticides. Our results demonstrate the potential use of termite defense fluid for pest management, providing repellent and insecticidal activities comparable to those of other green repellent and termiticidal commercial products.

Two glutathione transferase isoforms isolated from juvenile cysts ofTaenia crassiceps: identification, purification and characterization

2017 ◽  
Vol 92 (6) ◽  
pp. 687-695 ◽  
Author(s):  
G. Maldonado ◽  
G. Nava ◽  
A. Plancarte
Keyword(s):  
Glutathione Transferase ◽  
Taenia Solium ◽  
Single Step ◽  
Glutathione Transferases ◽  
Mass Spectrometry Analysis ◽  
Purification And Characterization ◽  
Spectrometry Analysis ◽  
Enzyme Subunits ◽  
Molecular Masses ◽  
Potential Use

AbstractWe identified and characterized the first two glutathione transferases (GSTs) isolated from juvenile cysts ofTaenia crassiceps(EC 2.5.1.18). The two glutathione transferases (TcGST1 and TcGST2) were purified in a single-step protocol using glutathione (GSH)-sepharose chromatography in combination with a GSH gradient. The specific activities of TcGST1 and TcGST2 were 26 U mg−1and 19 U mg−1, respectively, both at 25°C and pH 6.5 with 1-chloro-2,4-dinitrobenzene (CDNB) and GSH as substrates. TheKm(CDNB)andKcat(CDNB)values for TcGST1 and TcGST2 (0.86 μmand 62 s−1; 1.03 μmand 1.97 s−1, respectively) andKm(GSH)andKcat(GSH)values for TcGST1 and TcGST2 (0.55 μmand 11.61 s−1; 0.3 μmand 32.3 s−1, respectively) were similar to those reported for mammalian and helminth GSTs. Mass spectrometry analysis showed that eight peptides from each of the two parasite transferases were a match for gi|29825896 glutathione transferase (Taenia solium), confirming that both enzymes are GSTs. The relative molecular masses were 54,000 ± 0.9 for the native enzymes and 27,500 ± 0.5 for the enzyme subunits. Thus, TcGST1 and TcGST2 are dimeric proteins. Optimal TcGST1 and TcGST2 activities were observed at pH 8.5 in the range of 20–55°C and pH 7.5 at 35–40°C, respectively. TcGST1 and TcGST2 were inhibited by cibacron blue (CB), bromosulphophthalein (BST), rose bengal (RB), indomethacin and haematin (Hm) with 50% inhibitory concentrations (IC50) in the μmrange. TcGST1 was inhibited in a non-competitive manner by all tested inhibitors with the exception of indomethacin, which was uncompetitive. The discovery of these new GSTs facilitates the potential use ofT. crassicepsas a model to investigate multifunctional GSTs.

Spectrophotometry Measurement of Polynuclear Aromatic Hydrocarbons in Hamilton Harbour Sediments

1991 ◽  
Vol 26 (1) ◽  
pp. 1-16 ◽  
Author(s):  
T.P. Murphy ◽  
H. Brouwer ◽  
M.E. Fox ◽  
E. Nagy
Keyword(s):  
Aromatic Hydrocarbons ◽  
Total Concentration ◽  
Coal Tar ◽  
Sediment Cores ◽  
Polynuclear Aromatic Hydrocarbons ◽  
Mass Spectrometry Analysis ◽  
Gas Chromatography Mass Spectrometry ◽  
Near Surface ◽  
Spectrometry Analysis ◽  
Hamilton Harbour

Abstract Eighty-one sediment cores were collected to determine the extent of coal tar contamination in a toxic area of Hamilton Harbour. Over 800 samples were analyzed by a UV spectrophotometric technique that was standardized with gas chromatography/mass spectrometry analysis. The coal tar distribution was variable. The highest concentrations were near the Stelco outfalls and the Hamilton-Wentworth combined sewer outfalls. The total concentration of the 16 polynuclear aromatic hydrocarbons (PAHs) in 48,300 m3 of near-surface sediments exceeded 200 µg/g.

scholarly journals Effect of Ocular Hypertension on D-β-Aspartic Acid-Containing Proteins in the Retinas of Rats

2019 ◽  
Vol 2019 ◽  
pp. 1-8 ◽  
Author(s):  
Takashi Kanamoto ◽  
Takashi Tachibana ◽  
Yasushi Kitaoka ◽  
Toshio Hisatomi ◽  
Yasuhiro Ikeda ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Ocular Hypertension ◽  
Aspartic Acid ◽  
Atp Synthase ◽  
Wistar Rats ◽  
Protein Band ◽  
Mass Spectrometry Analysis ◽  
Liquid Chromatography Mass Spectrometry ◽  
Spectrometry Analysis ◽  
Sds Page

Purpose. To investigate the effect of ocular hypertension-induced isomerization of aspartic acid in retinal proteins. Methods. Adult Wistar rats with ocular hypertension were used as an experimental model. D-β-aspartic acid-containing proteins were isolated by SDS-PAGE and western blot with an anti-D-β-aspartic acid antibody and identified by liquid chromatography-mass spectrometry analysis. The concentration of ATP was measured by ELISA. Results. D-β-aspartic acid was expressed in a protein band at around 44.5 kDa at much higher quantities in the retinas of rats with ocular hypertension than in those of normotensive rats. The 44.5 kDa protein band was mainly composed of α-enolase, S-arrestin, and ATP synthase subunits α and β, in both the ocular hypertensive and normotensive retinas. Moreover, increasing intraocular pressure was correlated with increasing ATP concentrations in the retinas of rats. Conclusion. Ocular hypertension affected the expression of proteins containing D-β-aspartic acid, including ATP synthase subunits, and up-regulation of ATP in the retinas of rats.

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