10 research outputs found
Metabolism of Cryptic Peptides Derived from Neuropeptide FF Precursors: The Involvement of Insulin-Degrading Enzyme
The term âcryptomeâ refers to the subset of cryptic peptides with bioactivities that are often unpredictable and very different from the parent protein. These cryptic
peptides are generated by proteolytic cleavage of proteases, whose identification in vivo can be very challenging. In this work, we show that insulin-degrading enzyme (IDE) is able to degrade specific amino acid sequences present in the neuropeptide pro-NPFFA (NPFF precursor), generating some cryptic peptides that are also observed after incubation with rat brain cortex homogenate. The reported experimental findings support the increasingly accredited hypothesis, according to which, due to its wide substrate selectivity, IDE is involved in a wide variety of physiopathological processes
The Forty-Sixth Euro Congress on Drug Synthesis and Analysis: Snapshot
The 46th EuroCongress on Drug Synthesis and Analysis (ECDSA-2017) was arranged within the celebration of the 65th Anniversary of the Faculty of Pharmacy at Comenius University in Bratislava, Slovakia from 5-8 September 2017 to get together specialists in medicinal chemistry, organic synthesis, pharmaceutical analysis, screening of bioactive compounds, pharmacology and drug formulations; promote the exchange of scientific results, methods and ideas; and encourage cooperation between researchers from all over the world. The topic of the conference, Drug Synthesis and Analysis, meant that the symposium welcomed all pharmacists and/or researchers (chemists, analysts, biologists) and students interested in scientific work dealing with investigations of biologically active compounds as potential drugs. The authors of this manuscript were plenary speakers and other participants of the symposium and members of their research teams. The following summary highlights the major points/topics of the meeting
The Study of Derivatization Prior MALDI MSI AnalysisâCharge Tagging Based on the Cholesterol and Betaine Aldehyde
Mass spectrometry imaging is a powerful tool for analyzing the different kinds of molecules in tissue sections, but some substances cannot be measured easily, due to their physicochemical properties. In such cases, chemical derivatization could be applied to introduce the charge into the molecule and facilitate its detection. Here, we study cholesterol derivatization with betaine aldehyde from tissue slices and evaluate how different sample preparation methods influence the signal from the derivatization product. In this study, we have tested different solutions for betaine aldehyde, different approaches to betaine aldehyde deposition (number of layers, deposition nozzle height), and different MALDI matrices for its analysis. As a result, we proved that the proposed approach could be used for the analysis of cholesterol in different tissues
Mammalian Oocyte Analysis by MALDI MSI with Wet-Interface Matrix Deposition Technique
Oocytes are a special kind of biological material. Here, the individual variability of a single cell is important. It means that the opportunity to obtain information about the lipid content from the analysis of a single cell is significant. In our study, we present a method for lipid analysis based on the MALDI-based mass spectrometry imaging (MSI) approach. Our attention was paid to the sample preparation optimization with the aid of a wet-interface matrix deposition system (matrix spraying). Technical considerations of the sample preparation process, such as the number of matrix layers and the position of the spraying nozzle during the matrix deposition, are presented in the article. Additionally, we checked if changing the 2,5-dihydroxybenzoic acid (DHB) and 9-Aminoacridine (9AA) matrix concentration and their solvent composition may improve the analysis. Moreover, the comparison of paraformaldehyde-fixed versus nonfixed cell analysis was performed. We hope that our approach will be helpful for those working on lipid analyses in extraordinary material such as a single oocyte. Our study may also offer clues for anybody interested in single-cell analysis with the aid of MALDI mass spectrometry imaging and the wet-interface matrix deposition method
Metabolism of Cryptic Peptides Derived from Neuropeptide FF Precursors: The Involvement of Insulin-Degrading Enzyme
IDE Degrades Nociceptin/Orphanin FQ through an Insulin Regulated Mechanism
Insulin-degrading enzyme (IDE) was applied to catalyze hydrolysis of Nociceptin/Orphanin 1-16 (OFQ/N) to show the involvement of the enzyme in degradation of neuropeptides engaged in pain transmission. Moreover, IDE degradative action towards insulin (Ins) was inhibited by the OFQ/N fragments, suggesting a possible regulatory mechanism in the central nervous system. It has been found that OFQ/N and Ins affect each other degradation by IDE, although in a different manner. Indeed, while the digestion of OFQ/N is significantly affected by the presence of Ins, the kinetic profile of the Ins hydrolysis is not affected by the presence of OFQ/N. However, the main hydrolytic fragments of OFQ/N produced by IDE exert inhibitory activity towards the IDE-mediated Ins degradation. Here, we present the results indicating that, besides Ins, IDE cleaves neuropeptides and their released fragments act as inhibitors of IDE activity toward Ins. Having in mind that IDE is present in the brain, which also contains Ins receptors, it cannot be excluded that this enzyme indirectly participates in neural communication of pain signals and that neuropeptides involved in pain transmission may contribute to the regulation of IDE activity. Finally, preliminary results on the metabolism of OFQ/N, carried out in the rat spinal cord homogenate in the presence of various inhibitors specific for different classes of proteases, show that OFQ/N proteolysis in rat spinal cord could be due, besides IDE, also to a cysteine protease not yet identified
Nanohydrogels Based on Self-Assembly of Cationic Pullulan and Anionic Dextran Derivatives for Efficient Delivery of Piroxicam
Advances in the Study of AptamerâProtein Target Identification Using the Chromatographic Approach
Ever since the development of the
process known as the systematic
evolution of ligands by exponential enrichment (SELEX), aptamers have
been widely used in a variety of studies, including the exploration
of new diagnostic tools and the discovery of new treatment methods.
Aptamersâ ability to bind to proteins with high affinity and
specificity, often compared to that of antibodies, enables the search
for potential cancer biomarkers and helps us understand the mechanisms
of carcinogenesis. The blind spot of those investigations is usually
the difficulty in the selective extraction of targets attached to
the aptamer. There are many studies describing the cell SELEX for
the prime choice of aptamers toward living cancer cells or even whole
tumors in the animal models. However, a dilemma arises when a large
number of proteins are being identified as potential targets, which
is often the case. In this article, we present a new analytical approach
designed to selectively target proteins bound to aptamers. During
studies, we have focused on the unambiguous identification of the
molecular targets of aptamers characterized by high specificity to
the prostate cancer cells. We have compared four assay approaches
using electrophoretic and chromatographic methods for âfishing
outâ aptamer protein targets followed by mass spectrometry
identification. We have established a new methodology, based on the
fluorescent-tagged oligonucleotides commonly used for flow-cytometry
experiments or as optic aptasensors, that allowed the detection of
specific aptamerâprotein interactions by mass spectrometry.
The use of atto488-labeled aptamers for the tracking of the formation
of specific aptamerâtarget complexes provides the possibility
of studying putative protein counterparts without needing to apply
enrichment techniques. Significantly, changes in the hydrophobic properties
of atto488-labeled aptamerâprotein complexes facilitate their
separation by reverse-phase chromatography combined with fluorescence
detection followed by mass-spectrometry-based protein identification.
These comparative results of several methodological approaches confirmed
the universal applicability of this method to studying aptamerâprotein
interactions with high sensitivity, showing superior properties compared
with pull-down techniques
HemorphinsâFrom Discovery to Functions and Pharmacology
During the last three decades, a variety of different studies on bioactive peptides that are opioid receptor ligands, have been carried out, with regard to their isolation and identification, as well as their molecular functions in living organisms. Thus, in this review, we would like to summarize the present state-of-the art concerning hemorphins, methodological aspects of their identification, and their potential role as therapeutic agents. We have collected and discussed articles describing hemorphins, from their discovery up until now, thus presenting a very wide spectrum of their characteristic and applications. One of the major assets of the present paper is a combination of analytical and pharmacological aspects of peptides described by a team who participated in the initial research on hemorphins. This review is, in part, focused on the analysis of endogenous opioid peptides in biological samples using advanced techniques, description of the identification of synthetic/endogenous hemorphins, their involvement in pharmacology, learning, pain and other function. Finally, the part regarding hemorphin analogues and their synthesis, has been added