Isoelectric Focusing Technique Research Articles

P0044 What are the similarities between primary immunodeficiency states and ulcerative colitis: damage to the microbiome, impaired mucosal secretion of proteins and short-chain fatty acids, or something else?

Abstract Background Primary immunodeficiency (PID), common variable immunodeficiency (CVID) are primary immunodeficiencies with disorder of the adaptive immune system, organ-specific pathology, as well as defects in the gut microbiome [1]. In patients with ulcerative colitis (UC) during relapse, similar changes. Methods The aim of the research is a comprehensive study of the structure of gut microbiota and mucosal protein profile (MPP) in patients with PID, CVID and UC in different phases of the disease course. The research included 19 patients with PID, 28 with CVID and 32 with UC (22 with relapse and 10 with persistent remission of the disease), control group - 20 healthy volunteers. Estimation of gut microbiota was performed by microbacteriological seeding faeces (MSF). Content of short-chain fatty acids (SCFA) in the feces as determined by gas-chromatography-mass spectrometry (GC-MS). Mucosal protein profile was based on isoelectric focusing techniques (SDS-PAGE, 2DGE). Mass spectrograms were obtained using MALDI-TOF-MS/MS (Bruker, USA). Molecular interaction and functional characteristics of proteins were studied using the STRING 10.0 databases. Results SCFA production showed a 6-, 9- and 11-fold decrease in propionic and butyric acids, mainly in patients with PID and UC: 0.2 + 0.1, 0.14 + 0.03 and 0.04 + 0.02, 0.08 + 0.02 mg/g, respectively (tab. 1). MSF showed a decrease in titers of E. coli, bifido- and lactobacilli on average 4.6 + 0.8 Lg. The conditionally pathogenic microbiota was represented by E. coli, hemolytic strains (n=34,104/g), Proteus spp. (n=48,105/g), Staphylococcus spp. (n=29,105–6/g), Candida alb. / glabrata (n=55,104–6/g) and Clostridium spp. (n = 38,104/g). Results of MPP (detection rate of 75% or more) of the colon mucosa in patients with PID and CVID were detected: 1, 2, 4 okkludin, kininogen 1, interleukin-1B, interleukin 8, B2-glycoprotein, heat shock protein 27, in patients with CVID - translational elongation factor, apolipoprotein C-III. In patients with UC - NF-kB, alipoprotein C-III, TNF-α, heat shock protein 27, interleukin-2 and 8 were presented. MPP patients with UC (stable remission phase): a-enolase, b-defensin-1, cathepsin D, prohibitin (tab. 2). Conclusion 1. In patients with PID, CVID and UC during relapse, markers of damage to the intestinal microbiome, a decrease in the production of SCFA and proteins characteristic of inflammation, apoptosis, proliferation, as well as proteins reflecting the activity of the autoimmune process, were identified. 2. During the period of drug remission in patients with UC, PID and CVID, specific components of altered MPP disappear, and a tendency towards restoration of the gut microbiota and SCFA production is recorded. References Jørgensen SF, Trøseid M, Kummen M, et al. Altered gut microbiota profile in common variable immunodeficiency associates with levels of lipopolysaccharide and markers of systemic immune activation. Mucosal Immunol. 2016; 9:1455-1465.

Isoelectric focusing technique enables protein detection beyond standard limits

The technique is an inexpensive and versatile means of enhancing existing detection methods.

Profiling of antioxidative proteolysate enzymatically hydrolysed from stone fish (Actinopyga lecanora)

Marine livings represent one of the richest sources of protein with valuable bioactives. The present work explores the antioxidative potential of stone fish, a sea cucumber species typically discarded as by-catch. Stone fish was enzymatically hydrolysed using papain, and the resulting proteolysate exhibited strong antioxidant activity in DPPH• radical scavenging (IC50 = 0.49 mg/mL), ABTS• (IC50 = 0.36 mg/mL) radical scavenging, and FRAP value (0.29 mM FeSO4) after 8 h of hydrolysis. Fractionation of proteolysate was then performed using three approaches namely ultrafiltration, reversed-phase high performance liquid chromatography, and isoelectric focusing techniques to profile and characterise the antioxidative proteolysate. Results indicated that papain-generated proteolysate from stone fish flesh possessed considerable amount of antioxidative peptides with molecular weight of approximately 2 kDa, low hydrophobicity (< 20%), and pI = 9.

“A2 milk” authentication using isoelectric focusing and different PCR techniques

Genetic variants of milk proteins have attracted great interest for decades as they are related to important issues such as the composition and technological properties of milk. More recently, an “A1/A2 hypothesis” was developed saying that β-casein variant A1 may be a dietary risk factor for cardiovascular diseases, type 1 diabetes, sudden infant death syndrome and neurological disorders due to the release of β-casomorphin-7, whereas no evidence for such adverse effects was assumed for β-casein A2. Thus, the aim of this study was to adapt and establish analytical methods for the identification of genetic variants of β-casein using isoelectric focusing of milk proteins as well as appropriate PCR techniques. Allele-specific polymerase chain reaction (AS-PCR) proved to be a reliable method for differentiating most common β-casein variants (A1, A2, B, C), amplification-created restriction site (ACRS)-PCR using three different restriction enzymes allowed also the detection of variant A3, and the restriction fragment length polymorphism (RFLP)-PCR method enabled the reliable discrimination between A2 (homozygote/heterozygote) and non-A2 animals. Since traces of β-casein A1 were also found in commercial “A2 milk” in Austria, the authentication of such expensive dairy products is urgently recommended, both by genotyping of all dairy cows at farm level (to confirm that all cows are homozygous β-casein A2A2) and by screening commercial products on the market (to confirm the absence of β-casein variants A1, B, and C in dairy products labelled “A2 milk”) to protect consumers from this unexpected fraud.

Rapid Determination of Full and Empty Adeno-Associated Virus Capsid Ratio by Capillary Isoelectric Focusing.

Adeno-associated virus (AAV) is one of the most promising gene transfer vector types featuring long-term gene expression and low toxicity. The lack of pathogenicity and the availability of many serotypes augmented the applicability of AAV virions in gene therapy applications. The recombinant AAV capsid includes the therapeutic protein-coding transgene as well as a promoter to initiate translation and a poly A sequence portion for stabilization. Current AAV manufacturing technologies, however, cannot guarantee the generation of only full capsids, i.e., including the entire required genome. Partially filled and empty capsids are also part of the product, decreasing in this way the efficacy and safety upon clinical translation. Therefore, rapid, accurate and QC friendly analysis of the full and empty capsid ratio is of high importance during AAV vector manufacturing and release testing. In this paper, an automated capillary isoelectric focusing technique is introduced, readily applicable in the biopharmaceutical industry for fast and efficient determination of the full and empty capsid ratio. The method also reveals information about the proportion of partially filled capsids. For higher resolution (<0.1 pI unit), mixtures of wide and narrow range ampholytes were utilized. The isoelectric point and peak area percentage reproducibility (RSD) of the mixed ampholyte assay were as low as 1.67% and 2.45 %, respectively, requiring only 65 nL of sample volume per injection.

P081 The structure of the proteomic profile and gut microbiota in patients with primary immunodeficiency, common variable immunodeficiency and inflammatory bowel disease

Abstract Background Infringement of intestinal microbiota in patients with primary immunodeficiency (PID), common variable immunodeficiency (CVID) and ulcerative colitis (UC) continues to be studied. The aim of the research is a comprehensive study of the structure of gut microbiota and mucosal protein profile (MPP) in patients with PID, CVID and UC in different phases of the disease course. Methods The research included 15 patients with PID, 22 CVID and 46 UC (28 with relapse and 18 with persistent remission of the disease), control group—20 healthy volunteers. Estimation of gut microbiota was performed by bacteriological seeding, hydrogen breath test (HBT) with lactulose. Content of short-chain fatty acids (SCFAs) and microbial lipid markers (MLM) in faeces and colon mucosa was determined by gas–liquid chromatographic and gas chromatography–mass spectrometry (GC–MS). MPP was based on isoelectric focusing techniques (SDS–PAGE, 2DGE). Mass spectrograms were obtained using MALDI-TOF-MS/MS (Bruker, USA). Results HBT showed a 4.5-, 8- and 11-fold increase in hydrogen production on a 150th-minute study. SCFA showed a 6-, 9- and 11-fold decrease in propionic and butyric acids, mainly in patients with PID and UC: 0.2 + 0.1, 0.14 + 0.03 and 0.04 + 0.02 mg/g, respectively. Microbiological analysis showed a decrease in titers of E. coli, bifido- and lactobacilli on average 4.6 + 0.8 Lg. The conditionally pathogenic microflora was represented by E. coli, lactose-negative strains (n = 51,105/g) and haemolytic strains (n = 34,104/g, Proteus spp. (n = 48,105/g), Staphylococcus spp. (n = 29,105–6/g), Candida (n = 51,104–6/g) and Clostridium spp. (n = 38,104/g) in stool culture. MLN GS-MS results showed a 5.5-, 3- and 6-fold increase in total bacterial load in patients with PID, CVID and UC, respectively, which was represented by resident anaerobic microflora: Streptococcus mutants, Clostridium difficile, Candida albicans. Results of MPP (detection rate of 75% or more) of the colon mucosa in patients with PID were detected: 1, 2, 4 okkludin, kininogen 1, interleukin-1B, interleukin 8, B2-glycoprotein, heat shock protein 27, in patients with CVID—translational elongation factor, apolipoprotein C-III. In patients with UC—NF-kB, alipoprotein C-III, TNF-α, interleukin-2 and 8 were presented. Conclusion In patients with PID, CVID and UC markers of colon excess growth and significant decrease in SCFA are recorded during the relapse period. MPP in patients with PID, CVID and UC was characterised by proteins characteristic of inflammation, apoptosis, proliferation and proteins reflecting the activity of the autoimmune process. Patients with UC in the remission have recovery trend of gut microbiota, production of SCFA, disappearance of specific components of altered MPP.

Neonatal Screening for Lubumbashi Drepacytosis: Feasibility and Suitable Performance

Sickle cell anemia is a blood disorder linked to an abnormality in the structure of hemoglobin. The objective of this study was to determine the frequency of the sickle cell gene on the basis of early detection of hemoglobin using the isoelectric focusing technique in maternities in Lubumbashi. Our descriptive cross-sectional study consisted of newborns and infants up to 6 months of age. The capillary venous blood was collected on the finger for the rapid and direct test and on blotting paper for the electrophoresis of the hemoglobin of a few drops and the hemoglobin typing techniques performed by the isoelectric focusing technique. Statistical analyses were done using R version 2.0 software. We recruited 165 children, including (79 boys and 86 girls) who were at most 6 months old and all in good condition (100%). For demographic characteristics, there was no statistically significant difference between males and females. We received almost all the tribes of the DRC in our study; the Luba of Katanga and those of Kasai were on the fringes and represented respectively (32.1% and 29.1%). (20.6%) newborns with RDT and electrophoresis had an abnormality of hemoglobin in the homozygous or heterozygous state with (10.9% SS; 9.7% AS; and 0.6%). Despite the large proportion in the group of children without tare after TRD and electrophoresis, our study did not show statistically significant differences. There is a significant correlation between the electrophoresis of hemoglobin between sex and age (coefficients of correlation r = 0.99; p

Characterization of a Novel Porcine CSN2 Polymorphism and Its Distribution in Five European Breeds.

Simple SummaryPolymorphisms in genes encoding major milk proteins have been intensely studied, and are used, worldwide, in marker-assisted selection in dairy species: αS1-casein in goats—associated with milk protein content and flavor; κ-casein and β-lactoglobulin in cattle—associated with milk quantity and quality; bovine β-casein—associated with human health, etc. This aspect has scarcely been investigated in pigs. Using an electrophoretic technique, we previously identified a novel porcine β-casein (encoded by CSN2) polymorphism. Here, we fully characterize it at protein and DNA level, propose a genotyping protocol, and investigate its distribution in five European porcine breeds. In brief, a G/A point mutation in position 647 of the porcine CSN2 cDNA leads to an arginine/glutamine substitution in position 201 of the protein. This mutation can be typed via a StyI PCR-RFLP assay. The frequency of the G allele was 0.965 in the investigated Landrace population (number of individuals genotyped n = 67), one in the Pietrain (n = 40), 0.705 in the Large White (n = 36), 0.885 in the Bazna (n = 13), and 0.555 in the Mangalita population (n = 11). Considering that milk protein content still varies widely within (and between) porcine breeds, this and/or other similar polymorphisms may have implications for the dynamics of piglet growth during suckling.Here, we describe a novel porcine β-casein (CNS2) polymorphism, initially identified using the isoelectric focusing (IEF) technique, and provide its distribution in five European breeds. Porcine CSN2 cDNA samples, from sows identified using IEF as carriers of polymorphic variants, were sequenced, and based on the sequence alignments, a genotyping assay was developed. The distribution of the polymorphism was investigated by genotyping 167 sows. Population genetic indexes were computed using POPGENE32 version 1.32. Sequence alignments revealed that the mutation which caused the different β-casein IEF migration profiles was c.647G>A, a substitution located in exon 7, which modifies the amino acid from position 201 of the mature protein from arginine to glutamine. The frequency of the G allele was 0.965 in the investigated Landrace population (number of individuals genotyped n = 67), one in the Pietrain population (n = 40), 0.705 in the Large White population (n = 36), 0.885 in the Bazna population (n = 13), and 0.555 in the Mangalita population (n = 11). For all breeds, except Pietrain (monomorphic), the genotype distribution was in accordance with the Hardy–Weinberg equilibrium. Given that β-casein is the most important protein in sows’ milk, a polymorphism like the one described here may prove interesting for marker-assisted selection.

Varietal identification of Thai hot chilli using seed protein analysis by ultrathin layer isoelectric focusing technique

Varietal identification of Thai hot chilli using seed protein analysis by ultrathin layer isoelectric focusing technique

Low-molecular-mass nitrophenol-based compounds suitable for the effective tracking of pH gradient in isoelectric focusing

Low-molecular-mass isoelectric point (pI) markers are a reasonable alternative to commonly used peptide and protein pI markers in the field of isoelectric focusing. We present a comparative study dealing with characterization of 44 nitrophenol-based compounds synthesized by the Mannich reaction which pI values cover a pH range from 3.2 to 10.4. The values of pIs of all the presented nitrophenol-based compounds were precisely determined by capillary isoelectric focusing technique (cIEF) when a standard deviation of measurement reached a value of a few hundredths of a pH unit (n ≥ 3). Differences have been found between in silico analysis of acid-base properties of the investigated compounds and the experimental cIEF data especially at the basic pH region. The data from the acidic pH range showed better correlation between these methods. Only three compounds were excluded from the group of pI markers because they contained impurities originating from the synthesis or long-term storage. Based on the presented results we identified 41 nitrophenol-based pI markers (NPIMs) which can fulfill requirements of the most applications in a field of isoelectric focusing analyses.

Isoelectric Focusing to Quantify Rhodopsin Phosphorylation in Mouse Retina.

Rhodopsin is a G-protein coupled receptor (GPCR) that mediates vision under dim light. Upon light exposure, rhodopsin is phosphorylated at multiple serine and threonine sites at its carboxyl-terminus by rhodopsin kinase (GRK1). This, in turn, reduces its ability to activate the visual G-protein transducin. Binding of light-activated, phosphorylated rhodopsin by arrestin (ARR1) fully terminates the catalytic activity of rhodopsin. Quantification of the levels of the differentially phosphorylated rhodopsin species provides definitive information about the role of phosphorylated rhodopsin in visual functions. Isoelectric Focusing (IEF) is a technique which is used to separate ampholytic components, such as proteins, based on their isoelectric point (pI). It is a useful technique used to distinguish protein isoforms and post-translational modifications such as phosphorylation, glycosylation, deamination, and acetylation, due to their effects on the protein's pI. Isoelectric Focusing can provide high resolution of differentially phosphorylated forms of a protein. Though other techniques such as kinase activity assays, phospho-specific antibodies, western blot, enzyme-linked immunosorbent assays (ELISA), radiolabeling and mass spectrometry are used to detect and quantify protein phosphorylation, IEF is a simple and cost-effective method to quantify rhodopsin phosphorylation, as it can readily detect individual phosphorylated forms. Here we provide a detailed protocol for determining phosphorylated rhodopsin species using the Isoelectric Focusing technique.

Personalized vaccine for advanced HCC.

TPS527 Background: The chronic inflammation and viral infection associated with HCC combined with liver tolerogenic mechanisms creates a profoundly immunosuppressive microenvironment. Modulating the microenvironment with checkpoint blockade benefits ~20% of patients. The non-responders and those with poor liver function are an unmet need. Checkpoint blockade requires effector cells to be resident within tumors. However, the majority of HCC lesions lack effector cells. Therefore, a strategy for amplifying the tumor-specific immune response, while counteracting the immunosuppressive mechanisms may provide an improved immunotherapy. Endogenous heat shock proteins (HSP) chaperone tumor neoantigens. A concentrated composition of calreticulin, hsp70, hsp90 and gr94/gp96 (CRCL) is purified from biopsy samples. In addition, a bioengineered allograft (BAG) derived from healthy blood donors with a Th1 memory phenotype, CD3/CD28-coated microbeads and high expression of CD40L has potent immunomodulatory and counter-regulatory properties. The combination of CRCL with BAG is being evaluated as a strategy to amplify tumor-specific immunity and counter-regulate the immunosuppressive microenvironment. Methods: An open-label Phase II clinical trial evaluating the safety and efficacy of CRCL+BAG vaccine in Child-Pugh A/B advanced/metastatic HCC w/o Sorafenib. Approximately 6-10 cores of tumor are collected at baseline. The tumor is lysed and CRCL purified using an isoelectric focusing technique. Subjects are primed with multiple intradermal injections of BAG cells alone to increase the titer of allo-specific Th1/Tc1 cells. Next, multiple intradermal injections of BAG+CRCL are administered to elicit increased titers of tumor-specific Th1/Tc1 cells. Subsequent intravenous infusions of BAG cells activate allo- and tumor-specific memory cells, enabling their infiltration into tumor lesions. The BAG allo-rejection response creates a type I cytokine storm which serves to down-regulate suppressor circuits. Response is evaluated by mRECIST and experimental biomarkers. Longitudinal CT scans with concurrent biopsies as well as multiple collections of PBMC and plasma permits immunomonitoring of these immune mechanisms. Clinical trial information: NCT02409524.

Impact of signal peptide and transmembrane segments on expression and biochemical properties of a lipase from Bacillus sphaericus 205y

A total of 97 amino acids, considered as the signal peptide and transmembrane segments were removed from 205y lipase gene using polymerase chain reaction technique that abolished the low activity of this enzyme. The mature enzyme was expressed in Escherichia coli using pBAD expression vector, which gave up to a 13-fold increase in lipase activity. The mature 205y lipase (without signal peptide and transmembrane; −SP/TM) was purified to homogeneity using the isoelectric focusing technique with 53% recovery. Removing of the signal peptide and transmembrane segments had resulted in the shift of optimal pH, an increase in optimal temperature and tolerance towards more water-miscible organic solvents as compared to the characteristics of open reading frame (ORF) of 205y lipase. Also, in the presence of 1mM inhibitors, less decrease in the activity of mature 205y lipase was observed compared to the ORF of the enzyme. Protein structure modeling showed that 205y lipase consisted of an α/β hydrolase fold without lid domain. However, the transmembrane segment could effect on the enzyme activity by covering the active site or aggregation the protein.

Analysis of Factor D Isoforms in Malpuech-Michels-Mingarelli-Carnevale Patients Highlights the Role of MASP-3 as a Maturase in the Alternative Pathway of Complement.

Factor D (FD), which is also known as adipsin, is regarded as the first-acting protease of the alternative pathway (AP) of complement. It has been suggested that FD is secreted as a mature enzyme that does not require subsequent activation. This view was challenged when it was shown that mice lacking mannose-binding lectin (MBL)-associated serine protease-1 (MASP-1) and MASP-3 contain zymogenic FD (pro-FD), and it is becoming evident that MASP-3 is implicated in pro-FD maturation. However, the necessity of MASP-3 for pro-FD cleavage has been questioned, because AP activity is still observed in sera from MASP-1/3-deficient Malpuech-Michels-Mingarelli-Carnevale (3MC) patients. The identification of a novel 3MC patient carrying a previously unidentified MASP-3 G665S mutation prompted us to develop an analytical isoelectric focusing technique that resolves endogenous FD variants in complex samples. This enabled us to show that although 3MC patients predominantly contain pro-FD, they also contain detectable levels of mature FD. Moreover, using isoelectric focusing analysis, we show that both pro-FD and FD are present in the circulation of healthy donors. We characterized the naturally occurring 3MC-associated MASP-3 mutants and found that they all yielded enzymatically inactive proteins. Using MASP-3-depleted human serum, serum from 3MC patients, and Masp1/3-/- mice, we found that lack of enzymatically active MASP-3, or complete MASP-3 deficiency, compromises the conversion of pro-FD to FD. In summary, our observations emphasize that MASP-3 acts as an important maturase in the AP of complement, while also highlighting that there exists MASP-3-independent pro-FD maturation in 3MC patients.

Characterization and production of multifunctional cationic peptides derived from rice proteins.

Food proteins have been identified as a source of bioactive peptides. These peptides are inactive within the sequence of the parent protein and must be released during gastrointestinal digestion, fermentation, or food processing. Of bioactive peptides, multifunctional cationic peptides are more useful than other peptides that have specific activity in promotion of health and/or the treatment of diseases. We have identified and characterized cationic peptides from rice enzymes and proteins that possess multiple functions, including antimicrobial, endotoxin-neutralizing, arginine gingipain-inhibitory, and/or angiogenic activities. In particular, we have elucidated the contribution of cationic amino acids (arginine and lysine) in the peptides to their bioactivities. Further, we have discussed the critical parameters, particularly proteinase preparations and fractionation or purification, in the enzymatic hydrolysis process for producing bioactive peptides from food proteins. Using an ampholyte-free isoelectric focusing (autofocusing) technique as a tool for fractionation, we successfully prepared fractions containing cationic peptides with multiple functions.

Capillary isoelectric focusing with whole column imaging detection (iCIEF): A new approach to the characterization and quantification of salivary α-amylase

Saliva is an easily collected biological fluid with potentially important diagnostic value. While gel electrophoresis is generally used for salivary analysis, we employed the capillary isoelectric focusing technique to allow for a rapid, automated mode of electrophoresis. Capillary isoelectric focusing coupled with UV whole column imaging detection (iCIEF) was used to develop a robust protocol to characterize salivary α-amylase collected from various glands. Notably, three sample preparation methods were examined: ultrafiltration, gel-filtration, and starch affinity interaction with salivary amylase. Salivary α-amylase separated into two major peaks before sample treatment; while both filtration methods and starch affinity interaction of salivary amylase enhanced the resolution of isozymes, desalting with gel-filtration displayed the best recovery and the highest resolution of isozymes. Good agreement existed between the observed isoelectric points and the values reported in the literature. In addition, a high level of precision was apparent, and the relative standard deviation for replicates was less than 0.5% for pIs (peak positions) and below 10% for peak area. Furthermore, saliva secreted from the parotid gland proved to have a higher amylase content compared to either secretions from the submandibular/sublingual complex, or whole saliva, as well as amylase enhancement under stimulation. The results suggest that the iCIEF technique can be used to accurately resolve and quantitate amylase isozymes in a rapid and automated fashion, and that gel-filtration should be applied to saliva samples beforehand to allow for optimal purification and characterization.

Purification and Characterization of Nitric Oxide Inhibitory Peptides from Actinopyga lecanora Through Enzymatic Hydrolysis

ABSTRACTActinopyga lecanora, commonly known as sea cucumber, is a rich protein source. This marine protein source was hydrolyzed using six proteases to generate anti-inflammatory hydrolysates and bioactive peptides. Bromelain hydrolysates after 1 h hydrolysis exhibited the highest nitric oxide (NO) inhibitory activity reflecting anti-inflammatory activity. A sequence of two fractionation methods was employed to fractionate the peptides based on their hydrophobicity using a semi-preparative RP-HPLC and isoelectric points using isoelectric focusing technique. Based on these fractionation methods, basic peptides with relatively higher hydrophobicity provided higher NO-inhibitory activity than did acidic peptides. Furthermore, using Q-TOF mass spectrometry; 12 peptide sequences were successfully identified. The inhibitory effect of the purified peptides from A. lecanora on NO production by lipopolysaccharide (LPS)-stimulated RAW 264.7 cells was investigated. The three identified bioactive peptides, namely LREMLSTMCTARGA, AVGPAGPRG and VAPAWGPWPKG, exhibited the highest NO-inhibitory activity with values of 76.3, 66.6 and 69.9%, respectively. These results revealed that A. lecanora could be used as an economical protein source for the production of high-value bioactive peptides with potent anti-inflammatory activity using RAW 264.7 cell lines as model. These peptides may be useful ingredients in food and pharmaceutical applications.

Coupling Electrochemistry with Probe Electrospray Ionization Mass Spectrometry.

A new coupling of electrochemistry with mass spectrometry (MS) using probe electrospray ionization (PESI) is presented. Due to the high salt tolerance of PESI, the detection of electrochemical reaction products in room-temperature ionic liquids (RTILs) is realized for the first time. Furthermore, PESI-MS allows the analysis of electrochemical reaction products on different or multiple electrode surfaces. In addition, peptides and proteins fractionated through isoelectric focusing (IEF) in the presence of an external electric field can also be directly analyzed by using PESI-MS, suggesting a new and rapid characterization means for the IEF technique. This study reveals the versatility of EC/PESI-MS, which could have an impact in electrochemistry and bioanalysis fields.

Impact of post mortem aging of pork on changes in the isoelectric point of the proteins and tenderness

The aim of the study was to evaluate the post mortem proteolysis of centrifugal drip protein derived from pork. The varied course of the process of meat tenderization in muscles using the isoelectric focusing (IEF) technique was observed. The experimental material was the longissimus lumborum et thoracis muscle excised from 24 pigs of known origin, breeding and rearing conditions. Meat of normal quality (RFN) was examined in this study. On the basis of shear force values, the experimental muscles were divided into 4 groups of different courses of tenderization: A – meat remaining tough during the entire 6-day period of post-slaughter changes; B – meat characterized by a typical tenderization process, i.e. tough 48 h after slaughter and tender after 6 days of storage; C – tender or relatively tender meat on both dates of examination; D – meat which was the toughest 48 hours after slaughter and the most tender 144 hours after slaughter. Proteins for electrophoretic analysis on the agarose gel of the centrifugal drip fraction were submitted after 48 h and 144 h post mortem. The separation of the proteins was performed in a horizontal layout, using an FBE-3000 apparatus and an ECPS power pack (Pharmacia). On every path of the proteins separation, four ranges of pH value were evaluated, namely: 4.0 ÷ 6.0 pI, 6.1 ÷ 7.2 pI, 7.3 ÷ 7.7 pI and &gt; 7.7 pI. Meat tenderness on the second day after slaughter was influenced by proteins, in which their pI was in the range of 6.1÷ 7.2 and 4.0 ÷ 6.0. From the evaluation which was carried out in the immunoblotting analysis of proteins using troponin T (9D) antibodies it was demonstrated that 48 h after slaughtering the most intense reactions were observed in the range of pI of 5.5 ÷ 5.9, which is the pH range corresponding to the isoelectric point of troponin T. However, after 144 h post mortem the most intense reaction was demonstrated in the range of pI of 6.1÷ 7.7. This indicates the breakdown of troponin T and an increase of the number of degradation products of this protein occurring while the meat tenderization process progresses. The degradation products of troponin T identified by IEF can be an indicator of meat tenderization.

Current concepts on intrathecal humoral immune response and diagnostic importance of oligoclonal immunoglobulins in multiple sclerosis

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system of probable autoimmune origin. In this review of literature, the authors present a constantly broadening list of potential antigens, including myelin and non-myelin structures, that cause the autoimmune reaction. Determination of antibodies to any antigen has not sufficient specificity and sensitivity for the use in routine laboratory practice. Oligoclonal immunoglobulins determined with isoelectric focusing (IEF) technique are currently considered as the main immunological MS markers. The sensitivity and specificity of IEF are 90 and 86%, respectively. The authors have considered the additional markers such as oligoclonal IgM and MRZ-response.