scholarly journals Auxin Metabolite Profiling in Isolated and Intact Plant Nuclei

2021 ◽  
Vol 22 (22) ◽  
pp. 12369
Author(s):  
Vladimír Skalický ◽  
Tereza Vojtková ◽  
Aleš Pěnčík ◽  
Jan Vrána ◽  
Katarzyna Juzoń ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Flow Cytometry ◽  
Mass Spectrometry Analysis ◽  
Auxin Signaling ◽  
Auxin Homeostasis ◽  
Spectrometry Analysis ◽  
New Horizons ◽  
New Information ◽  
Nuclear Isolation ◽  
Cellular Control

The plant nucleus plays an irreplaceable role in cellular control and regulation by auxin (indole-3-acetic acid, IAA) mainly because canonical auxin signaling takes place here. Auxin can enter the nucleus from either the endoplasmic reticulum or cytosol. Therefore, new information about the auxin metabolome (auxinome) in the nucleus can illuminate our understanding of subcellular auxin homeostasis. Different methods of nuclear isolation from various plant tissues have been described previously, but information about auxin metabolite levels in nuclei is still fragmented and insufficient. Herein, we tested several published nucleus isolation protocols based on differential centrifugation or flow cytometry. The optimized sorting protocol leading to promising yield, intactness, and purity was then combined with an ultra-sensitive mass spectrometry analysis. Using this approach, we can present the first complex report on the auxinome of isolated nuclei from cell cultures of Arabidopsis and tobacco. Moreover, our results show dynamic changes in auxin homeostasis at the intranuclear level after treatment of protoplasts with free IAA, or indole as a precursor of auxin biosynthesis. Finally, we can conclude that the methodological procedure combining flow cytometry and mass spectrometry offers new horizons for the study of auxin homeostasis at the subcellular level.

scholarly journals PgTI, the First Bioactive Protein Isolated from the Cactus Pilosocereus gounellei, is a Trypsin Inhibitor with Antimicrobial Activity

2019 ◽  
pp. 1-11
Author(s):  
Cláudio Alberto Alves Da Rocha Filho ◽  
Poliana Karla Amorim ◽  
Thâmarah de Albuquerque Lima ◽  
Pollyanna Michelle da Silva ◽  
Maiara Celine de Moura ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Flow Cytometry ◽  
Antimicrobial Activity ◽  
Trypsin Inhibitor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Mass Spectrometry Analysis ◽  
Minimal Bactericidal Concentration ◽  
Trypsin Inhibitor Activity ◽  
Minimal Inhibitory Concentrations ◽  
Spectrometry Analysis

Aims: This work aimed to isolate, characterize and evaluate the antimicrobial activity of a trypsin inhibitor (PgTI) from the stem of Pilosocereus gounellei. Place and Duration of Study: Departamento de Bioquímica, Universidade Federal de Pernambuco between March 2013 and October 2018. Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro between June and July 2018. Methodology: PgTI was isolated from P. gounellei stem extract by gel filtration and ion exchange chromatographies. The inhibitor was characterized by isoelectric focusing, polyacrylamide gel electrophoresis, tryptic digestion followed by mass spectrometry analysis and for stability towards heating. Antibacterial and antifungal activities were investigated through broth microdilution assays. Viability of the microbial cells was also evaluated by flow cytometry analysis using thiazol orange and propidium iodide. Results: PgTI appeared as a single polypeptide band of 37.1 kDa and isoelectric point (pI) 5.88. The inhibition constant (Ki) for bovine trypsin was 14 nM and mass spectrometry analysis of PgTI did not reveal similarities with other plant proteins. Trypsin inhibitor activity was stable at temperatures up to 50ºC. PgTI inhibited growth of Gram-positive and Gram-negative bacteria (minimal inhibitory concentrations (MIC) from 7.5 to150 µg/mL) with bactericidal activity only against Escherichia coli (minimal bactericidal concentration: 75.0 µg/mL). PgTI also inhibited the growth of Candida krusei (MIC of 60 µg/mL). Flow cytometry confirmed that PgTI did not affect the viability of E. coli and C. krusei cells at the MIC. Conclusion: This is the first report on a bioactive protein purified from P. gounellei, which provides biotechnological value to this cactus.

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.

scholarly journals N-Terminomics Strategies for Protease Substrates Profiling

Molecules ◽  
2021 ◽  
Vol 26 (15) ◽  
pp. 4699
Author(s):  
Mubashir Mintoo ◽  
Amritangshu Chakravarty ◽  
Ronak Tilvawala
Keyword(s):  
Mass Spectrometry ◽  
Protease Activity ◽  
Viral Infections ◽  
Mass Spectrometry Analysis ◽  
Post Translational Modification ◽  
Spectrometry Analysis ◽  
Physiological Conditions ◽  
Inflammatory Conditions ◽  
Biological Mixtures

Proteases play a central role in various biochemical pathways catalyzing and regulating key biological events. Proteases catalyze an irreversible post-translational modification called proteolysis by hydrolyzing peptide bonds in proteins. Given the destructive potential of proteolysis, protease activity is tightly regulated. Dysregulation of protease activity has been reported in numerous disease conditions, including cancers, neurodegenerative diseases, inflammatory conditions, cardiovascular diseases, and viral infections. The proteolytic profile of a cell, tissue, or organ is governed by protease activation, activity, and substrate specificity. Thus, identifying protease substrates and proteolytic events under physiological conditions can provide crucial information about how the change in protease regulation can alter the cellular proteolytic landscape. In recent years, mass spectrometry-based techniques called N-terminomics have become instrumental in identifying protease substrates from complex biological mixtures. N-terminomics employs the labeling and enrichment of native and neo-N-termini peptides, generated upon proteolysis followed by mass spectrometry analysis allowing protease substrate profiling directly from biological samples. In this review, we provide a brief overview of N-terminomics techniques, focusing on their strengths, weaknesses, limitations, and providing specific examples where they were successfully employed to identify protease substrates in vivo and under physiological conditions. In addition, we explore the current trends in the protease field and the potential for future developments.

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