Characterizing circular peptides in mixtures: sequence fragment assembly of cyclotides from a violet plant by MALDI-TOF/TOF mass spectrometry

Cyclotides are a very abundant class of plant peptides that display significant sequence variability around a conserved cystine-knot motif and a head-to-tail cyclized backbone conferring them with remarkable stability. Their intrinsic bioactivities combined with tools of peptide engineering make cyclotides an interesting template for the design of novel agrochemicals and pharmaceuticals. However, laborious isolation and purification prior to de novo sequencing limits their discovery and hence their use as scaffolds for peptide-based drug development. Here we extend the knowledge about their sequence diversity by analysing the cyclotide content of a violet species native to Western Asia and the Caucasus region. Using an experimental approach, which was named sequence fragment assembly by MALDI-TOF/TOF, it was possible to characterize 13 cyclotides from Viola ignobilis, whereof ten (vigno 1-10) display previously unknown sequences. Amino acid sequencing of various enzymatic digests of cyclotides allowed the accurate assembly and alignment of smaller fragments to elucidate their primary structure, even when analysing mixtures containing multiple peptides. As a model to further dissect the combinatorial nature of the cyclotide scaffold, we employed in vitro oxidative refolding of representative vigno cyclotides and confirmed the high dependency of folding yield on the inter-cysteine loop sequences. Overall this work highlights the immense structural diversity and plasticity of the unique cyclotide framework. The presented approach for the sequence analysis of peptide mixtures facilitates and accelerates the discovery of novel plant cyclotides

Organelle Isolation for Proteomics: Mitochondria from Peripheral Blood Mononuclear Cells

Mitochondria play key roles in many cell functions including energy production, fatty acid metabolism, pyrimidine biosynthesis, calcium homeostasis, and aging. They also regulate crucial signaling cascades such as apoptosis and oxidative stress. The proteome is often used to investigate the functional correlations on protein levels. Based upon the human, genome there is estimated 2000 to 2500 associated mitochondrial proteins, however, just over 600-800 have been identified at the protein level. For this reason, mitochondria contain a great number of proteins that have yet to be identified and characterized. The identification of these proteins can help in discovery of biological process. This protocol focuses on step-by-step procedure of mitochondrial proteome extraction from peripheral blood mononuclear cell (PBMC) mitochondria. The isolation and preparation procedures described here require 6 hours approximately

Cyclotides Isolated from an Ipecac Root Extract Antagonize the Corticotropin Releasing Factor Type 1 Receptor

Cyclotides are plant derived, cystine-knot stabilized peptides characterized by their natural abundance, sequence variability and structural plasticity. They are abundantly expressed in Rubiaceae, Psychotrieae in particular. Previously the cyclotide kalata B7 was identified to modulate the human oxytocin and vasopressin G protein-coupled receptors (GPCRs), providing molecular validation of the plants’ uterotonic properties and further establishing cyclotides as valuable source for GPCR ligand design. In this study we screened a cyclotide extract derived from the root powder of the South American medicinal plant ipecac (Carapichea ipecacuanha) for its GPCR modulating activity of the corticotropin-releasing factor type 1 receptor (CRF1R). We identified and characterized seven novel cyclotides. One cyclotide, caripe 8, isolated from the most active fraction, was further analyzed and found to antagonize the CRF1R. A nanomolar concentration of this cyclotide (260 nM) reduced CRF potency by ∼4.5-fold. In contrast, caripe 8 did not inhibit forskolin-, or vasopressin-stimulated cAMP responses at the vasopressin V2 receptor, suggesting a CRF1R-specific mode-of-action. These results in conjunction with our previous findings establish cyclotides as modulators of both classes A and B GPCRs. Given the diversity of cyclotides, our data point to other cyclotide-GPCR interactions as potentially important sources of drug-like molecules

Non-thermal plasma radiation-induced changes in antibiotic susceptibility and protein profile of Staphylococcus aureus

Background and Objectives: Plasma radiation is a widely used technique for sterilization or decontamination in various industries, as well as in some healthcare settings such as dentistry. The primary aim of this study was to assess the potential of plasma radiation to create a new population of Staphylococcus aureus cells with distinct characteristics that could lead to novel healthcare challenges. Materials and Methods: A homemade non-thermal plasma apparatus was applied and the effects of plasma treatment on S. aureus ATCC25923 was assessed. Plasma radiation was applied under controlled conditions to ensure that some bacterial cells remained viable. The treatment was repeated 10 times, with each round followed by a recovery phase to collect any surviving bacterial cells. To assess the potential changes in the bacterial population, we examined the antibiotic susceptibility pattern, micro-structural characteristics using scanning electron microscopy (SEM), and total protein profile using the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) technique. Results: The experimental results revealed slight variations in the antibiotic susceptibility patterns of certain cell wall agents (imipenem, cephalothin, and cefepime), as well as in the MALDI-TOF spectra. However, no changes were observed in the SEM images. Conclusion: The insufficient application of non-thermal plasma in bacterial decontamination may lead to physiological changes that could enrich or select certain subpopulations of S. aureus

Optimization of ultrasonic assisted extraction of fatty acids from Borago Officinalis L. flower by central composite design

In the present study, the ultrasonic assisted extraction (UAE) of essential oils and fatty acids from Borago officinalis L. flower was developed by using n-hexane as extracting solvent. The obtained extracts were compared by hydrodistillation. Four parameters such as temperature, time, power of ultrasonic, and the ratio of extracting solvent volume to the weight of the plant were optimized using a central composite design after a full factorial design. Based on direct observation and analysis, the highest yields for UAE were obtained at a temperature of 48 °C, an extraction time of 30 min, minimum power of ultrasonic and in the ratio of extracting solvent volume to weight of plant 36:1 mL/g. The chemical compositions of the UAE extract were identified by GC–MS after derivation. The extraction yield base on ultrasonic assisted extraction varied in the range of 0.12–1.04% (w/w)

Identification and primary characterization of a plant antimicrobial peptide with remarkable inhibitory effects against antibiotic resistant bacteria

On the basis of a primary screening scheme by using plant seed methanol extract against a collection of human pathogenic bacteria, Medicago sativa L. seeds were chosen as the best potential resource against tested Gram positive bacteria. Then an agar-overlay method using fully separated proteins on sodium dodecyl sulphate-polyacryliamide gel electrophoresis (SDS-PAGE) gels was used for initial determination and primary characterization of active putative defensins in the plant seeds. Clear and remarkable zones of inhibition in a region corresponding to peptides slightly larger than 6 kDa were recorded for Methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococcus faecium (VRE) strains and yeast but a smaller inhibition zone with several colonies for tested Gram negative strain of Escherichia coli. Further characterization experiments using two-dimensional gel electrophoresis and subsequent agar-overlay assay against both strains confirmed the peptide nature of the active substance. Also gel filtration separation using Sephadex G-25 superfine and subsequent antibacterial assays could confirm the presence of a low molecular weight anti-MRSA and anti-VRE peptide in the total water soluble proteins obtained from M. sativa L. seeds which also showed high level of thermo stability.Key words: Medicago sativa L., methicillin resistant bacteria, vancomycin resistant bacteria, plant defensins

Silica microspheres from rice husk: A good opportunity for chromatography stationary phase

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