Mannose Ligands for Mannose Receptor Targeting.

The mannose receptor (MR, CD 206) is an endocytic receptor primarily expressed by macrophages and dendritic cells, which plays a critical role in both endocytosis and antigen processing and presentation. MR carbohydrate recognition domains (CRDs) exhibit a high binding affinity for branched and linear oligosaccharides. Furthermore, multivalent mannose presentation on the various templates like peptides, proteins, polymers, micelles, and dendrimers was proven to be a valuable approach for the selective and efficient delivery of various therapeutically active agents to MR. This review provides a detailed account of the most relevant and recent aspects of the synthesis and application of mannosylated bioactive formulations for MR-mediated delivery in treatments of cancer and other infectious diseases. It further highlights recent findings related to the necessary structural features of the mannose-containing ligands for successful binding to the MR.

Effect of Prolonged Photoperiod on Light-Dependent Photosynthetic Reactions in Cannabis.

Industrial hemp is a fast-growing, short-day plant, characterized by high biomass yields and low demands for cultivation. To manipulate growth, hemp is usually cultivated under prolonged photoperiods or continuous light that could cause photooxidative damage and adjustments of photosynthetic reactions. To determine the extent of changes in photosynthetic response caused by prolonged light exposure, we employed chlorophyll a fluorescence measurements accompanied with level of lipid peroxidation (TBARS) and FT-IR spectroscopy on two Cannabis cultivars. Plants were grown under white (W) and purple (P) light at different photoperiods (16/8, 20/4, and 24/0). Our results showed diverse photosynthetic reactions induced by the different light type and by the duration of light exposure in two cultivars. The most beneficial condition was the 16/8 photoperiod, regardless of the light type since it brought the most efficient physiological response and the lowest TBARS contents suggesting the lowest level of thylakoid membrane damage. These findings indicate that different efficient adaptation strategies were employed based on the type of light and the duration of photoperiod. White light, at both photoperiods, caused higher dissipation of excess light causing reduced pressure on PSI. Efficient dissipation of excess energy and formation of cyclic electron transport around PSI suggests that P20/4 initiated an efficient repair system. The P24/0 maintained functional electron transport between two photosystems suggesting a positive effect on the photosynthetic reaction despite the damage to thylakoid membranes.

Possible Role of Extracellular Vesicles in Hepatotoxicity of Acetaminophen.

We examined proteomic profiles of rat liver extracellular vesicles (EVs) shed following treatment with a sub-toxic dose (500 mg/kg) of the pain reliever drug, acetaminophen (APAP). EVs representing the entire complement of hepatic cells were isolated after perfusion of the intact liver and analyzed with LC-MS/MS. The investigation was focused on revealing the function and cellular origin of identified EVs proteins shed by different parenchymal and non-parenchymal liver cells and their possible role in an early response of this organ to a toxic environment. Comparison of EV proteomic profiles from control and APAP-treated animals revealed significant differences. Alpha-1-macroglobulin and members of the cytochrome P450 superfamily were highly abundant proteins in EVs shed by the normal liver. In contrast, proteins like aminopeptidase N, metalloreductase STEAP4, different surface antigens like CD14 and CD45, and most members of the annexin family were detected only in EVs that were shed by livers of APAP-treated animals. In EVs from treated livers, there was almost a complete disappearance of members of the cytochrome P450 superfamily and a major decrease in other enzymes involved in the detoxification of xenobiotics. Additionally, there were proteins that predominated in non-parenchymal liver cells and in the extracellular matrix, like fibronectin, receptor-type tyrosine-protein phosphatase C, and endothelial type gp91. These differences indicate that even treatment with a sub-toxic concentration of APAP initiates dramatic perturbation in the function of this vital organ.

Changes in the proteome of extracellular vesicles shed by rat liver after subtoxic exposure to acetaminophen.

To identify changes in extracellular vesicles (EVs) secreted by the liver following drug-induced liver injury (DILI), rats were treated with a subtoxic dose (500 mg/kg) of the analgesic drug, acetaminophen (APAP). EVs were collected by liver perfusion of sham and APAP-treated rats. Changes in EVs morphology were examined by transmission electron microscopic analysis of negatively stained vesicles. Results from morphometric analysis of EVs revealed striking differences in their size and distribution. Proteome composition of EVs collected by liver perfusion was determined by mass spectrometry using methods of sample preparation that enabled better detection of both highly hydrophobic proteins and proteins with complex post-translational modifications. The collection of EVs after liver perfusion is an approach that enables the isolation of EVs shed not only by isolated hepatocytes, but also by the entire complement of hepatic cells. EVs derived after DILI had a lower content of alpha-1-macroglobulin, ferritin, and members of cytochrome 450 family. Fibronectin, aminopeptidase N, metalloreductase STEAP4, integrin beta, and members of the annexin family were detected only in APAP-treated samples of EVs. These results show that the present approach can provide valuable insights into the response of the liver following drug-induced liver injury.

Proteomic analysis of pyridoxal oxime derivatives treated Listeria monocytogenes reveals down-regulation of the main virulence factor, Listeriolysin O.

Proteomic analysis of foodborne pathogen Listeria monocytogenes after treatment with three disinfectants based on ammonium salts of pyridoxal oxime (POD) reveal perturbation of cellular processes. These inhibitors caused disturbance in the synthesis of plasma membrane proteins and cell wall proteoglycans. Some of key proteins and proteoglycans from these two groups that are important for bacterial growth are down-regulated. Additionally, we demonstrated that the main bacterial toxin Listeriolysin O (LLO) is significantly down-regulated after treatment with each of three investigated inhibitors. These investigations confirm already postulated mechanism of action of POD-based inhibitors that results in disturbance of key cell surface proteins and proteoglycans in Gram-positive bacteria. Additionally, the use of some proteins such as LLO, as potential biomarker candidates of food poisoning with this bacterium is discussed.

Soft agar-based selection of spontaneously transformed rat prostate epithelial cells with highly tumorigenic characteristics.

The critical molecular and cellular mechanisms involved in the development and progression of prostate cancer remain elusive. In this report, we demonstrate that normal rat prostate epithelial cells (PEC) undergo spontaneous transformation at high passage (p > 85) evidenced by the acquisition of anchorage independent growth when plated on soft agar and tumorigenicity when injected into immunodeficient mice. In addition, we also report the discovery of a minor subpopulation of spontaneously transformed PEC derived from high passage PEC with the ability to migrate through a layer of 1% agar and form expanding colonies on the underlying plastic substratum. Comparison of these soft agar invasive (SAI) cells with low (p < 35), mid (p36-84) and high passage (p > 85) PEC identified marked differences in cell morphology, proliferation and motility. The SAI subpopulation was more tumorigenic than the high passage anchorage independent cultures from which they were isolated, as manifested by a decreased latency period and an increase in the size of tumors arising in immunodeficient mice. In contrast, low and mid passage cells were unable to grow on soft agar and failed to form tumors when injected into immunodeficient mice. Screening with antibody-based signaling arrays identified several differences in the altered expression levels of signaling proteins between SAI-derived cells and low or high passage PEC, including the up-regulation of EGFR and MAPK-related signaling pathways in SAI-selected cells. In summary, these studies suggest that the SAI assay selects for a novel, highly tumorigenic subpopulation of transformed cells that may represent an early step in the progression of slow growing prostatic carcinomas into more rapidly growing and aggressive tumors.

Sample preparation in foodomic analyses.

Representative sampling and adequate sample preparation are key factors for successful performance of further steps in foodomic analyses, as well as for correct data interpretation. Incorrect sampling and improper sample preparation can be sources of severe bias in foodomic analyses. It is well known that both wrong sampling and sample treatment cannot be corrected anymore. These, in the past frequently neglected facts, are now taken into consideration, and the progress in sampling and sample preparation in foodomics is reviewed here. We report the use of highly sophisticated instruments for both high-performance and high-throughput analyses, as well as miniaturization and the use of laboratory robotics in metabolomics, proteomics, peptidomics, and genomics.

Foodomics and Food Safety: Where We Are.

The power of foodomics as a discipline that is now broadly used for quality assurance of food products and adulteration identification, as well as for determining the safety of food, is presented. Concerning sample preparation and application, maintenance of highly sophisticated instruments for both high-performance and high-throughput techniques, and analysis and data interpretation, special attention has to be paid to the development of skilled analysts. The obtained data shall be integrated under a strong bioinformatics environment. Modern mass spectrometry is an extremely powerful analytical tool since it can provide direct qualitative and quantitative information about a molecule of interest from only a minute amount of sample. Quality of this information is influenced by the sample preparation procedure, the type of mass spectrometer used and the analyst's skills. Technical advances are bringing new instruments of increased sensitivity, resolution and speed to the market. Other methods presented here give additional information and can be used as complementary tools to mass spectrometry or for validation of obtained results. Genomics and transcriptomics, as well as affinity-based methods, still have a broad use in food analysis. Serious drawbacks of some of them, especially the affinity-based methods, are the cross-reactivity between similar molecules and the influence of complex food matrices. However, these techniques can be used for pre-screening in order to reduce the large number of samples. Great progress has been made in the application of bioinformatics in foodomics. These developments enabled processing of large amounts of generated data for both identification and quantification, and for corresponding modeling.

Proteomic analysis of food borne pathogens following the mode of action of the disinfectants based on pyridoxal oxime derivatives.

A comprehensive proteomic analysis of food borne pathogens after treatment with disinfectants based on ammonium salts of pyridinium oxime was performed. Changes in proteomes of the Gram-positive bacterium Bacillus subtilis and the Gram-negative one, Escherichia coli, were evaluated. Up and down-regulated proteins in these bacteria after growth under the inhibition with four different disinfectants based on chloride and bromide salts of pyridinium oxime were identified and their cellular localizations and functions were determined by gene ontology searching. Proteome changes presented here demonstrate different mechanisms of action of these disinfectants. In the Gram-positive food pathogen Bacillus subtilis, the inhibitory substances seem to act mainly at the cell surface and cause significant alterations of membrane and cell surface proteins. On the other hand, intracellular proteins were more affected in the Gram-negative pathogen Escherichia coli. This research is a contribution to the investigation of the virulence and pathogenicity of food borne bacteria and their survival under stress conditions, and can also lead the way for further development of new inhibitors of microbial growth and studies of mechanism of their actions.

IgG and IgM glycosylation patterns in patients undergoing image-guided tumor ablation.

BACKGROUND: Image-guided tumor ablation is a technique whereby needle-like applicators are placed directly into solid tumors under guidance typically with computed tomography or ultrasound. Changes in IgG and IgM antibody glycosylation were studied during ablation-induced immune response to cancer, and the use of glycosylation as a biomarker for diagnosis, prognosis and disease treatment was examined. METHODS: Plasma from 27 tumor patients was collected immediately before, after and for 6 months following ablation. IgG and IgM antibodies were isolated by use high-throughput chromatography, and analyzed by hydrophilic liquid chromatography. Thorough identification of glycan structures in each chromatography peak was performed by nano-liquid chromatography electrospray ionization mass spectrometry. RESULTS: Although antibody glycosylation was found to vary with cancer type, discernable patterns of change based on the successful treatment of tumors by ablation were not identified. One patient with renal clear cell carcinoma and poor disease outcome had unexpectedly high amount of oligomannose IgG glycans during the whole period of monitoring. In contrast, IgM antibodies did not follow the same pattern. CONCLUSIONS: These findings suggest that glycosylation patterns are indicative of an immune system that is unable to prevent different types of cancer, rather than products of the immunostimulatory response to the ablation of tumor itself. Analyses of the outcome effect suggested that IgG glycosylation and IgM glycosylation are not associated with tumor ablation. GENERAL SIGNIFICANCE: Present work opens a new way for parallel determination of glycosylation changes of both IgG and IgM antibodies by use of high-throughput methods, and their future use as biomarkers for disease diagnosis and prognosis. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.

Plasma membrane isolation using immobilized concanavalin A magnetic beads.

Isolation of highly purified plasma membranes is the key step in constructing the plasma membrane proteome. Traditional plasma membrane isolation method takes advantage of the differential density of organelles. While differential centrifugation methods are sufficient to enrich for plasma membranes, the procedure is lengthy and results in low recovery of the membrane fraction. Importantly, there is significant contamination of the plasma membranes with other organelles. The traditional agarose affinity matrix is suitable for isolating proteins but has limitation in separating organelles due to the density of agarose. Immobilization of affinity ligands to magnetic beads allows separation of affinity matrix from organelles through magnets and could be developed for the isolation of organelles. We have developed a simple method for isolating plasma membranes using lectin concanavalin A (ConA) magnetic beads. ConA is immobilized onto magnetic beads by binding biotinylated ConA to streptavidin magnetic beads. The ConA magnetic beads are used to bind glycosylated proteins present in the membranes. The bound membranes are solubilized from the magnetic beads with a detergent containing the competing sugar alpha methyl mannoside. In this study, we describe the procedure of isolating rat liver plasma membranes using sucrose density gradient centrifugation as described by Neville. We then further purify the membrane fraction by using ConA magnetic beads. After this purification step, main liver plasma membrane proteins, especially the highly glycosylated ones and proteins containing transmembrane domains could be identified by LC-ESI-MS/MS. While not described here, the magnetic bead method can also be used to isolate plasma membranes from cell lysates. This membrane purification method should expedite the cataloging of plasma membrane proteome.

High-throughput fractionation of human plasma for fast enrichment of low- and high-abundance proteins.

BACKGROUND: Fast, cost-effective and reproducible isolation of IgM from plasma is invaluable to the study of IgM and subsequent understanding of the human immune system. Additionally, vast amounts of information regarding human physiology and disease can be derived from analysis of the low abundance proteome of the plasma. In this study, methods were optimized for both the high-throughput isolation of IgM from human plasma, and the high-throughput isolation and fractionation of low abundance plasma proteins. MATERIALS AND METHODS: To optimize the chromatographic isolation of IgM from human plasma, many variables were examined including chromatography resin, mobile phases, and order of chromatographic separations. Purification of IgM was achieved most successfully through isolation of immunoglobulin from human plasma using Protein A chromatography with a specific resin followed by subsequent fractionation using QA strong anion exchange chromatography. Through these optimization experiments, an additional method was established to prepare plasma for analysis of low abundance proteins. This method involved chromatographic depletion of high-abundance plasma proteins and reduction of plasma proteome complexity through further chromatographic fractionation. RESULTS: Purification of IgM was achieved with high purity as confirmed by SDS-PAGE and IgM-specific immunoblot. Isolation and fractionation of low abundance protein was also performed successfully, as confirmed by SDS-PAGE and mass spectrometry analysis followed by label-free quantitative spectral analysis. DISCUSSION: The level of purity of the isolated IgM allows for further IgM-specific analysis of plasma samples. The developed fractionation scheme can be used for high throughput screening of human plasma in order to identify low and high abundance proteins as potential prognostic and diagnostic disease biomarkers.

Sample displacement chromatography as a method for purification of proteins and peptides from complex mixtures.

Sample displacement chromatography (SDC) in reversed-phase and ion-exchange modes was introduced approximately twenty years ago. This method takes advantage of relative binding affinities of components in a sample mixture. During loading, there is a competition among different sample components for the sorption on the surface of the stationary phase. SDC was first used for the preparative purification of proteins. Later, it was demonstrated that this kind of chromatography can also be performed in ion-exchange, affinity and hydrophobic-interaction mode. It has also been shown that SDC can be performed on monoliths and membrane-based supports in both analytical and preparative scale. Recently, SDC in ion-exchange and hydrophobic interaction mode was also employed successfully for the removal of trace proteins from monoclonal antibody preparations and for the enrichment of low abundance proteins from human plasma. In this review, the principals of SDC are introduced, and the potential for separation of proteins and peptides in micro-analytical, analytical and preparative scale is discussed.