Liquid Isoelectric Focusing Research Articles

Horse meat tenderization in relation to post-mortem evolution of the myofibrillar sub-proteome

The ageing process after animal slaughter enhances tenderness and influences the value of meat. Horse meat is becoming more popular but lacks standardized ageing practices that should be supported by a better understanding of post-mortem muscle biochemistry. Steaks from Longissimus Thoracis et Lumborum (LTL) of eight Hispano-Bretón horses were aged for 0, 7, 14 and 21 days and myofibrillar proteins were resolved by one dimensional gel electrophoresis (1-DE). Ten protein bands were found to change (p ≤ 0.05) among ageing periods. Most changes were observed between days 0 and 14, suggesting that tenderization occurred primary during the first two weeks. Liquid isoelectric focusing (OFFGEL) technology was applied to better resolve myofibrillar sub-proteome and evidenced fourteen protein bands that changed (p ≤ 0.05) between 0 and 21 days. Three of them were protein fragments coming from troponins T and I and from creatine kinase. Identified molecules could be further studied as potential markers for horse meat tenderness.

A straightforward gel-free proteomics pipeline assisted by liquid isoelectric focusing (OFFGEL) and mass spectrometry analysis to study bovine meat proteome.

Bovine sarcoplasmic sub-proteome was studied through a straightforward gel-free pipeline supported by liquid isoelectric focusing (OFFGEL) protein fractionation coupled to liquid chromatography-mass spectrometry (LC-MS) analysis. Full-MS and data-dependent MS/MS analyses were simultaneously performed by a conventional three-dimensional ion-trap addressing targeted quantitative and untargeted qualitative research, respectively. There were unambiguously identified 47 proteins distributed along 12 OFFGEL fractions assayed. Regarding intermediate- and high-abundant peptides, bulky quantitative data processing performed by MZmine 2 freeware yielded a satisfactory linearity and coefficient of variation with r2 in the 0.95-0.99 range and about 25%, respectively. Up to 41 peptides from 20 identified proteins were relatively quantified throughout OFFGEL fractions. This reliable, flexible and affordable gel-free proteomic approach could be readily implemented by industry to improve quality assessment of protein-based food products.

A Wide Spectrum Study of α-Globin Chain Variants: Cases from the UK

Over many years, cases of suspected α-globin chain variants were collected from different parts of the UK. The suspicion was based on the clinical picture, high performance liquid chromatography (HPLC) variant percentage, retention time (RT) and isoelectric focusing (IEF). DNA sequencing and the restriction enzyme EaeI were used for definitive diagnosis. One hundred and forty-eight variants were confirmed on one or both of the two α-globin genes (HBA2, HBA1). These cases were identified as 46 different α-globin chain variants. The most common variants were Hb J-Meerut [HBA2: c.362C>A (or HBA1)] (10.1%) and Hb Q-India (HBA1: c.193G>C) (8.1%), followed by Hb J-Paris-I [HBA2: c.38C>A (or HBA1)] and Hb Manitoba II (HBA1: c.309C>A) (7.4% for each). Other α variants were detected at lower frequencies. Two novel alleles were also detected: Hb Walsgrave [α116(GH4)Glu→Val (HBA2: c.350A>T)] and Hb Coombe Park [α127(H10)Lys→Glu (HBA2: c.382A>G)]. The majority of the ethnic origin was Indian. The positive predictive value for α variant identification by HPLC-RT analysis was 65.9%, 41.9% by IEF, and using both RT and IEF, the value was 72.1%. The number of variants was higher in HBA1 than in HBA2 genes and in exons 1 and 2 than in exon 3. There was no clustering of mutations in consecutive codons. This study, the characterization of a wide spectrum of α-globin chain variants, can facilitate the presumptive diagnosis of these variants prior to screening by a panel of amplification refractory mutation system-polymerase chain reaction (ARMS-PCR), and a definitive diagnosis by DNA sequencing.

An Additional Case of Hb Saint Nazaire [β103(G5)Phe→Ile; HBB: c.310T>A] Leading to Moderate Erythrocytosis in a French Family

We report two members of a French family who are carriers of a rare hemoglobin (Hb) variant leading to erythrocytosis: Hb Saint Nazaire [β103(G5)Phe→Ile; HBB: c.310T>A]. The proband is a 38-year-old woman referred to our institution for a moderate but persistent polycythemia without any clinical consequence. As her mother had a similar blood count, a diagnosis of a Hb variant with high oxygen affinity was proposed. The variant was difficult to detect by capillary electrophoresis (CE) and not distinguishable by high performance liquid chromatography (HPLC) and isoelectric focusing. Finally, a heterozygous mutation on the HBB gene corresponding to Hb Saint Nazaire was identified. This case report illustrates that this rare cause of erythrocytosis can be easily under or misdiagnosed unless several Hb separation techniques are used.

Behi-haragiaren samurtasuna aztergai: estrategia proteomiko berri baten garapena

Samurtasuna behi-haragiaren kalitatean eragiten duen ezaugarri garrantzitsua da, kontsumitzaileak produktua aukeratzeko orduan gehien balioesten duenen artean dagoena. Hori dela eta, muskulua haragi bihurtu bitarteko prozesu biokimikoaren azterketak berebiziko garrantzia dauka. Izan ere, ontze-prozesu honetan garatzen da neurri handi batean amaierako produktuaren samurtasuna. Prozesu honetan proteinek daukaten garrantzizko papera laburbilduko da artikulu honetan, bai eta teknika proteomikoek haragiaren kalitatea aztertzeko eta optimizatzeko eskaintzen dituzten aukerak ere, haragi-industriari begira probetxuzkoak izan daitezkeenak. Zentzu honetan, gure taldeak garatutako estrategia proteomiko berria aurkeztuko da, isoelektrofokatze likidoa (OFFGEL) eta elektroforesia oinarri dituena. Maine Anjou behi arrazako animalien haragian burututako esperimentuan samurtasunaren adierazgarri diren 7 banda identifikatu eta neurtu dira, muskuluaren proteoma ikertzeko metodologia berritzaile honen potentziala agerian utziz.

Search for proteomic biomarkers related to bovine pre-slaughter stress using liquid isoelectric focusing (OFFGEL) and mass spectrometry

Proteome changes derived from animals that have suffered pre-slaughter stress are a fact. In this study, Proteomic analysis was carried out on 20 bovine loin samples from Asturiana de los Valles and crossbreds cattle previously classified as normal and DFD meat at 24 h post-mortem using pH measurements. Sarcoplasmic sub-proteome of Longissimus thoracis at 24 h post-mortem was fractionated by the use of liquid isoelectric focusing (OFFGEL) in the pH range 3–10, followed by SDS-PAGE analysis of each retrieved fraction. The protein fractionation profile showed high reproducibility along the different sample groups. Five protein bands showed significant differences (p < 0.05) between the two groups, allowing discrimination between them. Proteins present in these bands, which were identified by LC-MS, were actin, phosphoglucomutase-1, alpha-crystallin B, heat shock protein beta-6 and heat shock protein beta-1. SignificanceThe significance of this study relies on the optimization of OFFGEL fractionation as a promising technology to search for reliable biomarkers of pre-slaughter stress. This method separates proteins along different liquid fractions according to their isoelectric point; the obtained fractions can be further characterized by SDS-PAGE or directly identified by LC-MS. This achievement stands out as an alternative to the use of 2-DE electrophoresis in protein separation and analysis.

Use of liquid isoelectric focusing (OFFGEL) on the discovery of meat tenderness biomarkers

Protein biomarkers of meat tenderness are known to be of primary importance for the prediction of meat quality, and hence, industry profitability. Proteome analysis was performed on meat from 8 Main Anjou beef cattle, previously classified as tender or tough meats by Warner Bratzler shear force measurements. Myofibrillar fraction of Longissimus thoracis muscle was separated by a novel fractionation approach based on liquid isoelectric focusing (OFFGEL) and further analyzed by SDS-PAGE and liquid chromatography coupled to tandem mass spectrometry.Obtained OFFGEL fraction profiles were reproducible allowing the comparison of both meat qualities and revealing 7 protein bands capable to discriminate between tender and tough samples. The proteins present in these bands were troponin T, Heat Shock protein beta-1, creatine kinase, actin, troponin C, myosins 1 and 2 and myozenin-1. The latter protein has not been previously reported as a marker of meat tenderness. SignificanceThis study introduces an innovative proteomic approach for the study of muscle proteome. The fact of obtaining fractions in liquid state after OFFGEL fractionation allows for a faster analysis of proteins by mass spectrometry, being an interesting alternative to more classical proteomic approaches based on two dimensional gel electrophoresis (2-DE).

PH Fractionation and identification of proteins: Comparing column chromatofocusing versus liquid isoelectric focusing techniques

In proteomic investigations, a number of different separation techniques can be applied to fractionate whole cell proteomes into more manageable fractions for subsequent analysis. In this work, utilizing HPLC and mass spectrometry for protein identification, two different fractionation methods were compared and contrasted to determine the potential of each method for the simple and reproducible fractionation of a bacterial proteome. Column-based chromatofocusing and liquid-based isoelectric focusing both utilized pH gradients to produce similar results in terms of the numbers of proteins successfully identified (402 and 378 proteins) and the consistency of proteins identified from one experiment to the next (<10% change). However, there was limited overlap in the protein sets with <50% of the proteins identified as common between the sets of proteins identified by the different systems. In addition to the numbers of proteins identified and consistency of those identified, the reduced monetary costs of experimentation and increased assay flexibility produced by using isoelectric focusing was considered in order to adopt a system best suited for comparative proteomic projects.

The Frequency of 1,4-Benzoquinone-Lysine Adducts in Cytochrome c Correlate with Defects in Apoptosome Activation

Electrophile-mediated post-translational modifications (PTMs) are known to cause tissue toxicities and disease progression. These effects are mediated via site-specific modifications and structural disruptions associated with such modifications. 1,4-Benzoquinone (BQ) and its quinone-thioether metabolites are electrophiles that elicit their toxicity via protein arylation and the generation of reactive oxygen species. Site-specific BQ-lysine adducts are found on residues in cytochrome c that are necessary for protein-protein interactions, and these adducts contribute to interferences in its ability to facilitate apoptosome formation. To further characterize the structural and functional impact of these BQ-mediated PTMs, the original mixture of BQ-adducted cytochrome c was fractionated by liquid isoelectric focusing to provide various fractions of BQ-adducted cytochrome c species devoid of the native protein. The fractionation process separates samples based on their isoelectric point (pI), and because BQ adducts form predominantly on lysine residues, increased numbers of BQ adducts on cytochrome c correlate with a lower protein pI. Each fraction was analyzed for structural changes, and each was also assayed for the ability to support apoptosome-mediated activation of caspase-3. Circular dichroism revealed that several of the BQ-adducted cytochrome c species maintained a slightly more rigid structure in comparison to native cytochrome c. BQ-adducted cytochrome c also failed to activate caspase-3, with increasing numbers of BQ-lysine adducts corresponding to a greater inability to activate the apoptosome. In summary, the specific site of the BQ-lysine adducts, and the nature of the adduct, are important determinants of the subsequent structural changes to cytochrome c. In particular, adducts at sites necessary for protein-protein interactions interfere with the proapoptotic function of cytochrome c.

Discovery of a Novel and Rich Source of Gluten-Degrading Microbial Enzymes in the Oral Cavity

BackgroundCeliac disease is a T cell mediated-inflammatory enteropathy caused by the ingestion of gluten in genetically predisposed individuals carrying HLA-DQ2 or HLA-DQ8. The immunogenic gliadin epitopes, containing multiple glutamine and proline residues, are largely resistant to degradation by gastric and intestinal proteases. Salivary microorganisms however exhibit glutamine endoprotease activity, discovered towards glutamine- and proline-rich salivary proteins. The aim was to explore if gliadins can serve as substrates for oral microbial enzymes.Methodology/Principal FindingsProteolytic activity in suspended dental plaque was studied towards a) gliadin-derived paranitroanilide(pNA)-linked synthetic enzyme substrates b) a mixture of natural gliadins and c) synthetic highly immunogenic gliadin peptides (33-mer of α2-gliadin and 26-mer of γ-gliadin). In addition, gliadin zymography was conducted to obtain the approximate molecular weights and pH activity profiles of the gliadin-degrading oral enzymes and liquid iso-electric focusing was performed to establish overall enzyme iso-electric points. Plaque bacteria efficiently hydrolyzed Z-YPQ-pNA, Z-QQP-pNA, Z-PPF-pNA and Z-PFP-pNA, with Z-YPQ-pNA being most rapidly cleaved. Gliadin immunogenic domains were extensively degraded in the presence of oral bacteria. Gliadin zymography revealed that prominent enzymes exhibit molecular weights >70 kD and are active over a broad pH range from 3 to 10. Liquid iso-electric focusing indicated that most gliadin-degrading enzymes are acidic in nature with iso-electric points between 2.5 and 4.0.Conclusions/SignificanceThis is the first reported evidence for gluten-degrading microorganisms associated with the upper gastro-intestinal tract. Such microorganisms may play a hitherto unappreciated role in the digestion of dietary gluten and thus protection from celiac disease in subjects at risk.

Role of Peroxiredoxin 1 and Peroxiredoxin 4 in Protection of Respiratory Syncytial Virus-Induced Cysteinyl Oxidation of Nuclear Cytoskeletal Proteins

The respiratory epithelium plays a central role in innate immunity by secreting networks of inflammatory mediators in response to respiratory syncytial virus (RSV) infection. Previous proteomic studies focusing on the host cellular response to RSV indicated the existence of a nuclear heat shock response and cytoplasmic depletion of antioxidant proteins in model type II-like airway epithelial cells. Here, we increased the depth of nuclear proteomic interrogation by using fluorescence difference labeling followed by liquid isoelectric focusing prefractionation/two-dimensional gel electrophoresis (2-DE) to identify an additional 41 proteins affected by RSV infection. Surprisingly, we found inducible oligomers and shifts in isoelectric points for peroxiredoxin 1 (Prdx-1), Prdx-3, and Prdx-4 isoforms without changes in their total abundance, indicating that Prdxs were being oxidized in response to RSV. To address the role of Prdx-1 and Prdx-4 in RSV infection, isoforms were selectively knocked down by small interfering RNA (siRNA) transfection. Cells lacking Prdx-1, Prdx-4, or both showed increased levels of reactive oxygen species formation and a higher level of protein carbonylation in response to RSV infection. Using a novel saturation fluorescence labeling 2-DE analysis, we showed that 15 unique proteins had enhanced oxidative modifications of at least >1.2-fold in the Prdx knockdowns in response to RSV, including annexin A2 and desmoplakin. Our results suggest that Prdx-1 and Prdx-4 are essential for preventing RSV-induced oxidative damage in a subset of nuclear intermediate filament and actin binding proteins in epithelial cells.

277. Equine chorionic gonadotrophin isoform composition in commercial products compared with isoform composition in pregnant mare plasma

It is well documented that there is considerable batch to batch variation in the activity of commercial preparations of gonadotrophins. These products are used in a variety of assisted reproductive procedures in the livestock industry, consequently this high degree of variation between products and batches adds to the already considerable between animal variations in response to the treatment. Equine chorionic gonadotrophin (eCG) is a heterodimeric glycoprotein hormone secreted by the placental endometrial cups during the first third of gestation in the horse. Plasma is harvested from pregnant mares between 40–90 days of gestation and the eCG isolated and used to formulate commercial preparations. Previous research has shown that eCG like the other gonadotrophins is a highly heterogeneous molecule with significant differences in bioactivity between isoforms. The aim of this study was to determine whether significant differences in isoform composition exist between various commercial preparations of eCG (n = 15), and how this compares with the isoform composition found in plasma (n = 23). Concentrations of eCG were determined using a competitive eCG ELISA. Liquid phase iso-electric focusing was used to fractionate plasma and the commercial preparations into 10 pH ranges from pH 3.0 to pH 10.0. Data from the 10 fractions were grouped into acidic (pH 3.0–5.1), intermediate (pH 5.2–7.9), or basic (pH 8.0–10.0) isoform categories for analysis. Immunoactivity between commercial eCG products ranged from 44% to 362% of stated bioactivity. Iso-electric focusing showed that the majority of the immunoactivity (92%) of the commercial preparations was found in the acidic fractions (pH 3.0–5.1), and in particular in the pH range 3.0–3.8. This contrasted starkly with isoform profiles found in pregnant mare plasma samples which showed a much greater spread across all 3 pH ranges. In summary, the isolation processes of commercial eCG preparations appears to selectively favour the acidic isoforms of eCG.

Prefractionation of Proteome by Liquid Isoelectric Focusing Prior to Two-Dimensional Liquid Chromatography Mass Spectrometric Identification

Due to the complexity of proteomes, developing methods of sample fractionation, separation, concentration, and detection have become urgent to the identification of large numbers of proteins, as well as the acquisition of those proteins in low abundance. In this work, liquid isoelectric focusing (LIEF) combined with 2D-LC-MS/MS was applied to the proteome of Saccharomyces cerevisiae. This yielded a total of 1795 proteins that were detected and identified by 30 fractions of protein prefractionation. Categorization of these hits demonstrated the ability of this technology to detect and identify proteins rarely seen in proteome analysis without protein fractionation. LIEF-2D-LC-MS/MS also produced improved resolution of low-abundance proteins. Furthermore, we analyzed the characteristics of proteins obtained by LIEF-2D-LC-MS/MS. 1103 proteins with CAI under 0.2 were identified, allowing us to specifically obtain detailed biochemical information on these kind proteins. It was observed that LIEF-2D-LC-MS/MS is useful for large-scale proteome analysis and may be specifically applied to systems with wide dynamic ranges.

Different molecules at the surface of stimulated T cells induce IL-1beta, tumour necrosis factor and IL-1RA in human monocytes

Imbalance in cytokine homeostasis is thought to play an important part in the pathogenesis of chronic inflammatory diseases such as rheumatoid arthritis. We demonstrated that T cells might exert a pathological effect through direct cellular contact with monocyte-macrophages, inducing a massive upregulation of IL-1β and tumour necrosis factor (TNF) [1]. We showed that this mechanism that might be relevant to chronic inflammation is specifically inhibited by high-density lipoproteins (HDL) [2]. Like many other stimuli, besides proinflammatory cytokines, the contact-mediated activation of monocytes induces the production of cytokine inhibitors such as IL-1Ra. HDL inhibited the production of IL-1β and TNF but not that of IL-1Ra induced in monocytes activated by membranes isolated from stimulated T cells to mimic cellular contact. This was also the case in peripheral blood mononuclear cells stimulated by either phytoheamagglutinin or tetanus toxoid. Similarly, IL-1Ra mRNA expression was not inhibited contrary to IL-1β and TNF mRNA. This demonstrates that different molecules at the surface of stimulated HUT-78 cells are involved in the induction of IL-1β, TNF and IL-1Ra in monocytes, IL-1β and TNF being activated by HDL-specific ligand(s). Separation of CHAPS-solubilized membrane molecules by liquid isoelectric focusing showed that two activity peaks were present; one activating IL-1β, TNF and IL-1Ra production, the other inducing the production of IL-1Ra in the absence of IL-1β and TNF. Further isolation of these two types of factor by gel filtration demonstrated that factor(s) inducing IL-1β, TNF and IL-1Ra displayed a Mr around 40,000 kDa, whereas factors inducing IL-1Ra only displayed Mr around 70,000 kDa and 30,000 kDa. Thus different factors are expressed at the surface of stimulated T cells that differentially trigger the production of proinflammatory and anti-inflammatory factors, and are differently affected by HDL.

Mass mapping of cancer cell lysates using two-dimensional liquid separations, electrospray-time of flight-mass spectrometry, and automated data processing

Intact protein masses from immortal, nontransformed MCF10A, a human breast epithelial cell line, and its malignant derivative MCF10CA1a.cl1 have been mapped using a combination of all-liquid separations and automated data interpretation. Preparative liquid isoelectric focusing combined with nonporous silica reverse-phase high-performance liquid chromatography allows efficient separation of a large number of proteins in complex mixtures such as whole-cell lysates. Molecular weight determination of these proteins is achieved using electrospray-time of flight-mass spectrometry, however, manual data analysis for these separations is both complex and time-consuming. Protein mass mapping can be significantly enhanced by automating deconvolution functions typically performed manually, with resulting reductions in hands-on analysis time from 20-30 h per chromatogram to approximately 15 min. This reduction in analysis time allows for rapid screening of cancer cell lines for potential biomarkers over a wider pI range than would otherwise be possible.

The antigenic and catalytically active formamidase of Paracoccidioides brasiliensis: protein characterization, cDNA and gene cloning, heterologous expression and functional analysis of the recombinant protein

Paracoccidioides brasiliensis is a well-characterized pathogen of humans. To identify proteins involved in the fungus–host interaction, P. brasiliensis yeast proteins were separated by liquid isoelectric focusing, and fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Immunoreactive bands were detected with pooled sera of patients with P. brasiliensis infection. A protein species with a molecular mass of 45 kDa was subsequently purified to homogeneity by preparative gel electrophoresis. The amino acid sequence of four endoproteinase Lys-C-digested peptides indicated that the protein was a formamidase (FMD) (E.C. 3.5.1.49) of P. brasiliensis. The complete cDNA and a genomic clone ( Pbfmd) encoding the isolated FMD were isolated. An open reading frame predicted a 415-amino acid protein. The sequence contained each of the peptide sequences obtained from amino acid sequencing. The Pbfmd gene contained five exons interrupted by four introns. Northern and Southern blot analysis suggested that there is one copy of the gene in P. brasiliensis and that it is preferentially expressed in mycelium. The complete coding cDNA was expressed in Escherichia coli to produce a recombinant fusion protein with glutathione S-transferase (GST). The purified recombinant protein was recognized by sera of patients with proven paracoccidioidomycosis and not by sera of healthy individuals. The recombinant 45-kDa protein was shown to be catalytically active; FMD activity was detected in P. brasiliensis yeast and mycelium.

Comprehensive proteome analysis of ovarian cancers using liquid phase separation, mass mapping and tandem mass spectrometry: a strategy for identification of candidate cancer biomarkers.

A two-dimensional (2-D) liquid phase separation method, liquid isoelectric focusing followed by nonporous reversed-phase high performance liquid chromatography (HPLC), was used to separate proteins from human ovarian epithelial whole cell lysates. HPLC eluent was interfaced on-line to an electrospray ionization (ESI) time of flight (TOF) mass spectrometer to obtain accurate intact protein molecular weights (Mr). 2-D protein expression maps were generated displaying protein isoelectric point (pI) versus intact protein Mr. Resulting 2-D images effectively displayed quantitative differential protein expression in ovarian cancer cells versus non-neoplastic ovarian epithelial cells. Protein peak fractions were collected from the HPLC eluent, enzymatically digested, and analyzed by matrix-assisted laser desorption/ionization (MALDI) TOF-mass spectrometry (MS) peptide mass fingerprinting and by MALDI-quadrupole TOF tandem mass spectrometry peptide sequencing. Interlysate comparisons of differential protein expression between two ovarian adenocarcinoma cell lines, ES2 and MDAH-2774, and ovarian surface epithelial cells was performed. Five pI fractions from each sample were selected for comparative study and over 300 unique proteins were positively identified from the 2-D liquid expression maps using MS, which covered around 60% of proteins detected by on-line ESI-TOF-MS. This represents one of the most comprehensive proteomic analyses of ovarian cancer samples to date. Protein bands with significant up- or down-regulation in one cell line versus another as viewed in the 2-D expression maps were identified. This strategy may prove useful in identifying novel ovarian cancer marker proteins.

Purification of the Porcine rubulavirus attachment protein by liquid isoelectric focusing

Porcine rubulavirus (PoRV) is an emerging virus responsible for meningoencephalitis, respiratory distress, and reproductive alterations in pigs. The hemagglutinin–neuraminidase (HN) glycoprotein is the most exposed and antigenic of the virus proteins. HN plays central roles in PoRV infection; i.e., it recognizes sialic acid-containing cell receptors that mediate virus attachment and penetration; in addition, its neuraminidase (sialic acid hydrolysis) activity has been proposed to be a virulence factor. So, HN is an ideal target for therapeutic treatment and prevention of this viral infection. This work describes a simple, fast, and sensitive method to purify the active form of HN protein based on its isoelectric point. HN was purified at a pH of 4.4, at which a single protein band of 66 kDa was observed on SDS–PAGE. Pure HN showed a maximal enzymatic activity at pH 3.5 and 37 °C using bovine fetuin as substrate. However, it retains circa 80% of its activity at a wide temperature range from 30 to 55 °C. We also describe improvements of neuraminidase determination method, which permits analysis in a microplate spectrophotometer, thereby increasing the sensitivity and reducing the costs of valuable reagents and biological samples.

Different molecules at the surface of stimulated T cells induce IL-1α, tumor necrosis factor and IL-1 receptor antagonist in human monocytes

An imbalance in cytokine homeostasis is thought to play an important part in the pathogenesis of chronic inflammatory diseases such as rheumatoid arthritis and multiple sclerosis. We demonstrated that T cells might exert a pathological effect through direct cellular contact with monocyte-macrophages, inducing a massive upregulation of IL-1 and tumor necrosis factor (TNF). More recently, we showed that this mechanism, likely to be of relevance to chronic inflammation, is specifically inhibited by high-density lipoproteins (HDL). Like many other stimuli besides proinflammatory cytokines, the contact-mediated activation of monocytes induces the production of cytokine inhibitors such as IL-1 receptor antagonist (IL-1Ra). We observed that HDL inhibited the production of IL-1β and TNF but not that of IL-1Ra induced in monocytes after activation by membranes isolated from stimulated T cells to mimic cellular contact. This was also observed in peripheral blood mononuclear cells stimulated by either phytohemagglutinin or tetanus toxoid, in which the presence of isolated HDL inhibited the production of IL-1β and TNF while that of IL-1Ra remained unchanged. This effect was confirmed by activating isolated monocytes with CHAPS-solubilized membranes from stimulated T cells, suggesting that HDL interacted with a specific factor expressed at the surface of T cells and not unspecifically with isolated membranes. Similarly, IL-1Ra mRNA expression was not inhibited, contrary to IL-1β and TNF mRNA. This demonstrates that different molecules at the surface of stimulated HUT-78 cells are involved in the induction of IL-1β, TNF and IL-1Ra in monocytes, IL-1β and TNF being activated by one or more HDL-specific ligands. Separation of CHAPS-solubilized membrane molecules by liquid isoelectric focusing revealed two activity peaks: one activating IL-1β, TNF and IL-1Ra production; the other inducing the production of IL-1Ra in the absence of IL-1β and TNF. Thus different factors are expressed at the surface of stimulated T cells that are differently affected by HDL and differentially trigger the production of proinflammatory and antiinflammatory factors.

Preparative liquid isoelectric focusing (Rotofor IEF) based Se-speciation of Se-enriched Agaricus bisporus

Preparative isoelectric focusing (Rotofor) was introduced to carry out the prefractionation of a selenised edible mushroom Agaricus bisporus sample containing 1037 ± 31 (SD) μg Se g−1. The water-soluble part of the sample was separated by the free solution apparatus into 20 fractions on the base of the pI values of the sample components. The separation was followed by the determination of total Se, soluble protein (by Bradford method) and Se-speciation of the fractions, summed up in 14 pools according to the actual individual pH values. Proteolytic sample preparation was chosen prior to the speciation analysis carried out with the help of a HPLC-UV-HG-AFS system, applying cation and anion exchange chromatographies. Achieving 19% column recovery, the presence of Se(IV), selenomethionine, selenocystine and methylselenocysteine was detected, the latter for the first time in a mushroom sample.