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Abstract. Although a series of proteins in the brain have been shown to be qualitatively or quantitatively dysregulated following morphine administration, a systematic proteomic study has not been carried out so far. We therefore aimed to show the effect of morphine on protein levels in the rat brain. For this purpose rats were given a morphine base in subcutaneously placed pellets and subsequently the cerebral cortex, hippocampus and striatum were taken for proteomic studies after three days. Extracted proteins were run on twodimensional gel electrophoresis, scanned and quantified by specific software. Proteins with significantly different levels were analysed by mass spectrometry (MALDI-TOF-TOF). Twenty-six proteins were found to be differentially expressed and were unambiguously identified. Dysregulated proteins were from several protein pathways and cascades including signaling, metabolic, protein handling, antioxidant and miscellaneous classes. These findings represent an initial approach to the generation of a 'morphinome' and may form the basis for further protein chemical studies as a valuable analytical tool. Moreover, the study reveals morphine-regulated proteins in different brain areas and indicates the pathways involved following morphine administration in the rat, the main species for pharmacological studies in the field.

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BackgroundThe highly pathogenic avian influenza viruses of the H5 subtype, such as the H5N1 viral strains or the novel H5N8 and H5N2 reassortants, are of both veterinary and public health concern worldwide. To combat these viruses, monoclonal antibodies (mAbs) against H5 hemagglutinin (HA) play a significant role. These mAbs are effective diagnostic and therapeutic agents and powerful tools in vaccine development and basic scientific research. The aim of this study was to obtain diagnostically valuable mAbs with broad strain specificity against H5-subtype AIVs.ResultsWe applied the hybridoma method to produce anti-HA mAbs. The cloning and screening procedures resulted in the selection of 7 mouse hybridoma cell lines and their respective antibody clones. Preliminary immunoreactivity studies showed that these newly established mAbs, all of the IgG1 isotype, had high specificity and broad-range activities against the H5 HAs. However, these studies did not allow for a clear distinction among the selected antibodies and mAb-secreting hybridoma clones. To differentiate the analyzed mAbs and determine the exact number of hybridoma clones, peptide mapping of the Fc and Fab fragments was performed using a Matrix-Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF/TOF) mass spectrometer. Detailed analyses of the acquired MS and MS/MS spectra confirmed that the Fc fragments constituted highly conserved species- and isotype-immunoglobulin components, whereas the Fab fragments exhibited considerable variation in the sequences that determine antibody specificity. This approach enabled unambiguous characterization of the selected mAbs according to their peptide composition. As a result, 6 different clones were distinguished.ConclusionsOur work provided a unique panel of anti-H5 HA mAbs, which meets the demand for novel, high-specificity analytical tools for use in serologic surveillance. Applications of these mAbs in areas other than diagnostics are also possible. Moreover, we demonstrated for the first time that peptide mapping of antibody fragments with mass spectrometry is an efficient method for the differentiation of antibody clones and relevant antibody-producing cell lines. The method may be successfully used to characterize mAbs at the protein level.Electronic supplementary materialThe online version of this article (10.1186/s12985-017-0886-2) contains supplementary material, which is available to authorized users.

This review provides an outline of the most important proteomic applications in the study of neurodegenerative disorders including Alzheimer's (AD), Parkinson's (PD), Huntington's (HD), and prion diseases, and also discusses advances in cancer and addiction. One of the scopes is to illustrate the potential of proteomics in the biomarkers discovery of these diseases. Finally, this article comments the advantages and drawbacks of the most commonly used techniques and methods for samples preparation.

BackgroundGene overlapping is a frequent phenomenon in microbial genomes. Excluding so-called “trivial overlapping”, there are significant implications of such genetic arrangements, including regulation of gene expression and modification of protein activity. It is also postulated that, besides gene duplication, the appearance of overlapping genes (OGs) is one of the most important factors promoting a genome’s novelty and evolution. OGs coding for in-frame proteins with different functions are a particularly interesting case. In this study we identified and characterized two in-frame proteins encoded by OGs on plasmid pIGRK from Klebsiella pneumoniae, a representative of the newly distinguished pHW126 plasmid family.ResultsA single repR locus located within the replication system of plasmid pIGRK encodes, in the same frame, two functional polypeptides: a full-length RepR protein and a RepR’ protein (with N-terminal truncation) translated from an internal START codon. Both proteins form homodimers, and interact with diverse DNA regions within the plasmid replication origin and repR promoter operator. Interestingly, RepR and RepR’ have opposing functions – RepR is crucial for initiation of pIGRK replication, while RepR’ is a negative regulator of this process. Nevertheless, both proteins act cooperatively as negative transcriptional regulators of their own expression.ConclusionsRegulation of the initiation of pIGRK replication is a complex process in which a major role is played by two in-frame proteins with antagonistic functions. In-frame encoded Rep proteins are uncommon, having been described in only a few plasmids. This is the first description of such proteins in a plasmid of the pHW126 family.

Astrocytes are supportive cells, necessary for ensure optimal environment for neural cells functioning. They are involved in extracellular K+ level regulation and neurotransmitters removal. They are also dependent for myelination and synapses formation. They may make a contribution in signal propagation in the central nervous system, for example, through Ca2+ signaling. With the use of neonatal pure astrocyte cell culture, we investigated changes in astrocyte's proteomes under the influence of morphine. We found 10 major proteins, which show different expression between physiological cell culture and morphine treatment. With 2D gel electrophoresis and nanoLC-ESI-MS/MS, we identified proteins and characterized their potential role in morphine dependence. Observed differences were also confirmed by Western blotting. Our data suggests a role for astrocytes in the formation of the morphine dependence at the molecular level. This finding may support interpretation of causes of morphine dependence formation based only on behavioral data.

Using primary neuronal cell culture assays, combined with 2-D gel electrophoresis and capillary LC-MS, we identified differences in proteomes between control and morphine-treated cells. Statistically significant differences were observed among 26 proteins. Nineteen of them were up-regulated, while seven were down-regulated in morphine-treated cell populations. The identified proteins belong to classes involved in energy metabolism, associated with oxidative stress, linked with protein biosynthesis, cytoskeletal ones, and chaperones. The detected proteins demand further detailed studies of their biological roles in morphine addiction. It is crucial to confirm observed processes in vivo in order to reveal the nature and importance of the biological effect of proteome changes after morphine administration. Further investigations may lead to the discovery of new proteome-based effects of morphine on living organisms.