Mascot Search Research Articles

Differential protein expression in compatible and incompatible pollen-pistil interactions in almond [Prunus dulcis (Miller) D. A. Webb] by 2D-DIGE and HPLC-MS/MS

SummarySelf-incompatibility (SI) in almond [Prunus dulcis (Miller) D.A.Webb] is of the gametophytic-type and is controlled by the S-locus, which is highly polymorphic and includes two tightly-linked genes expressed in the pistil (as an SRNase) and in the pollen [as an S haplotype-specific F-box (SFB) protein]. Experimental evidence indicates that other proteins are also needed for the functioning of self-incompatibility. However, to date, S-RNase and SFB are the only components of the SI system identified in almond. Moreover, the cause(s) of a lack of S-RNase activity in selfcompatible almonds remain(s) unclear. This work aimed to identify the other proteins involved in the self- (in)compatible response in almond by comparative quantitative analysis of the differential expression of proteins following incompatible and compatible pollinations. Four self-incompatible almond cultivars were self-pollinated or cross-pollinated, and a homozygous self-compatible almond selection was self-pollinated. Proteins were extracted from the pollinated pistils and analysed by two-dimensional difference gel electrophoresis (2D-DIGE) and high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Searches were performed against the NCBI database using the MASCOT and SEQUEST search engines and Proteome Discoverer v1.4 software. A total of 43 proteins, some with a key role in pollen-pistil interactions, were identified as being differentially expressed following incompatible or compatible pollination. These results provide the first profiles of differential protein expression during self-(in)compatibility in almond.

Increased cell proliferation and differential protein expression induced by low-level Er:YAG laser irradiation in human gingival fibroblasts: proteomic analysis.

Erbium-doped yttrium aluminum garnet (Er:YAG) laser treatment has demonstrated favorable wound healing effect after periodontal therapy. One of the reasons may be the positive biological effect of the low-level laser on the irradiated tissues, although the mechanism remains unclear. The aim of this study was to investigate the effect of low-level Er:YAG laser irradiation on cell proliferation and laser-induced differential expression of proteins in human gingival fibroblasts (HGFs) by proteomic analysis. In the first experiment, HGFs were exposed to low-level Er:YAG laser irradiation and the laser-induced cell proliferation and damage were evaluated on day 3. In the second experiment, proteomic analysis was performed on day 1 after irradiation. The peptides prepared from HGFs were analyzed by a hybrid ion trap-Fourier transform mass spectrometer, Mascot search engine, and UniProtKB database. A significant increase in cell proliferation without cell damage after irradiation was observed. Among the total identified 377 proteins, 59 proteins, including galectin-7, which was associated with the process of wound healing, were upregulated and 15 proteins were downregulated in laser-treated HGFs. In the third experiment, the increase in messenger RNA (mRNA) and protein expression of galectin-7 in the irradiated HGFs was validated by various analytical techniques. In addition, the effect of recombinant human galectin-7 on the modulation of HGFs proliferation was confirmed. The results indicate that low-level Er:YAG laser irradiation can promote HGF proliferation and induce a significant change in protein expression and the upregulation of galectin-7 expression may partly contribute to the increase in cell proliferation.

Multifunctional peptides derived from an egg yolk protein hydrolysate: isolation and characterization.

An egg yolk protein by-product following ethanol extraction of phospholipids (YP) was hydrolyzed with pepsin to produce and identify novel peptides that revealed antioxidant, ACE inhibitory and antidiabetic (α-glucosidase and DPP-IV inhibitory) activities. The peptic hydrolysate of YP was fractionated by ion-exchange chromatography and reversed-phase high-pressure liquid chromatography. Isolated peptides were identified using mass spectrometry (MALDI-ToF) and the Mascot Search Results database. Four peptides of MW ranging from 1,210.62 to 1,677.88 Da corresponded to the fragments of Apolipoprotein B (YINQMPQKSRE; YINQMPQKSREA), Vitellogenin-2 (VTGRFAGHPAAQ) and Apovitellenin-1 (YIEAVNKVSPRAGQF). These peptides were chemically synthesized and showed antioxidant, ACE inhibitory or/and antidiabetic activities. Peptide YIEAVNKVSPRAGQF exerted the strongest ACE inhibitory activity, with IC50 = 9.4 µg/mL. The peptide YINQMPQKSRE showed the strongest DPPH free radical scavenging and DPP-IV inhibitory activities and its ACE inhibitory activity (IC50) reached 10.1 µg/mL. The peptide VTGRFAGHPAAQ revealed the highest α-glucosidase inhibitory activity (IC50 = 365.4 µg/mL). A novel nutraceutical effect for peptides from an egg yolk hydrolysate was shown.

Bacillus subtilis chitinase identified by matrix-assisted laser desorption/ionization time-of flight/time of flight mass spectrometry has insecticidal activity against Spodoptera litura Fab.

An extracellular chitinase was identified and purified (CS1 and CS2) from Bacillus subtilis. The 16S rRNA sequencing was submitted in GenBank (accession numbers KC336487 and KC412256). The purified crude enzymes were identified through matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF MS) analysis. The peptide sequences were matched with chitinase sequences. The peak m/z with 1297. 592 and 3094.570 mascot search resulted sequence was blasted with NCBI protein sequences and confirmed that it is a chitinase enzyme. The effects of chitinase on gut enzymes lactate dehydrogenase, acid phosphatase, alkaline phosphatase and adenosine triphosphatase of the tobacco cutworm Spodoptera litura larvae were investigated. At all concentrations tested, chitinase decreased the activities of these gut enzymes relative to the control. When chitinase treated leaves were fed to larvae in bioassays, gut tissue and gut enzymes were affected. The histological study clearly shows the chitinase treated larval gut, peritrophic membrane and epithelial cells were affected significantly. Chitinase isolated from B. subtilis has effectively reduced the gut enzyme activity and growth of S. litura. The chitin based bioformulation may serve as an effective biocide against the polyphagous pest like S. litura.

A Systematic Proteome Study of Seed Storage Proteins from Two Soybean Genotypes

ABSTRACT Soybean seed is a good source of plant protein in human consumables such as baby formula and protein concentrate. The seeds contain an abundance of storage proteins, namely β-conglycin and glycinin that account for ~ 70-80% of the total seed protein content. Proteome profiling has been proved to be an efficient way that can help us to investigate the seed storage proteins. In the present study, the seeds were removed from the pods and the cotylendonary tissues were separated from the testa for proteome analysis in order to investigate the seed storage proteins. A systematic proteome profiling was conducted through one-dimensional gel electrophoresis followed by MALDI-TOF-TOF mass spectrometry in the seeds (cotyledonary tissue) of soybean genotypes. Two dimensional gels stained with CBB, a total of 10 proteins were identified and analyzed using MASCOT search engine according to the similarity of sequences with previously characterized proteins along with the UniProt database. A total of ten proteins such as glycinin Gy4 precursor, glycinin G3 precursor, glycinin G1 precursor, glycinin chain A2B1a precursor, glycinin chain A2B1a precursor were identified in our investigation. However, the glycinin subunit may be considered to play important roles in soybean breeding and biochemical characterization. In addition, the improved technique will be useful to dissect the genetic control of glycinin expression in soybean.

A Proteomic Analysis of Human Follicular Fluid: Comparison between Younger and Older Women with Normal FSH Levels

The follicular fluid (FF) is produced during folliculogenesis and contains a variety of proteins that play important roles in follicle development and oocyte maturation. Age-related infertility is usually considered as a problem that can be solved by assisted reproduction technology. Therefore, the identification of novel biomarkers that are linked to reproductive aging is the subject of this study. FF was obtained from healthy younger (20–32 years old) and older (38–42 years old) women undergoing intracytoplasmic sperm injection (ICSI) due to male factor infertility. The FF was analyzed by two-dimensional gel electrophoresis (2-DE). The power of two-dimensional gel electrophoresis and the identification of proteins were exploited using matrix-assisted laser desorption-ionization time-of-flight/time-of-flight (MALDI-TOF-TOF) mass spectrometry. Twenty three protein spots showed reproducible and significant changes in the aged compared to the young group. Of these, 19 protein spots could be identified using MALDI-TOF-TOF-MS. As a result of MASCOT search, five unique downregulated proteins were identified in the older group. These were identified as serotransferrin, hemopexin precursor, complement C3, C4 and kininogen. A number of protein markers were found that may help develop diagnostic methods of infertility.

Zn-responsive proteome profiling and time-dependent expression of proteins regulated by MTF-1 in A549 cells.

Zinc plays a critical role in many biological processes. However, it is toxic at high concentrations and its homeostasis is strictly regulated by metal-responsive transcription factor 1 (MTF-1) together with many other proteins to protect cells against metal toxicity and oxidative stresses. In this paper, we used high-resolution two-dimensional gel electrophoresis (2DE) to profile global changes of the whole soluble proteome in human lung adenocarcinoma (A549) cells in response to exogenous zinc treatment for 24 h. Eighteen differentially expressed proteins were identified by MALDI TOF/TOF and MASCOT search. In addition, we used Western blotting and RT-PCR to examine the time-dependent changes in expression of proteins regulated by MTF-1 in response to Zn treatment, including the metal binding protein MT-1, the zinc efflux protein ZnT-1, and the zinc influx regulator ZIP-1. The results indicated that variations in their mRNA and protein levels were consistent with their functions in maintaining the homeostasis of zinc. However, the accumulation of ZIP-1 transcripts was down-regulated while the protein level was up-regulated during the same time period. This may be due to the complex regulatory mechanism of ZIP-1, which is involved in multiple signaling pathways. Maximal changes in protein abundance were observed at 10 h following Zn treatment, but only slight changes in protein or mRNA levels were observed at 24 h, which was the time-point frequently used for 2DE analyses. Therefore, further study of the time-dependent Zn-response of A549 cells would help to understand the dynamic nature of the cellular response to Zn stress. Our findings provide the basis for further study into zinc-regulated cellular signaling pathways.

Identification of active site residues of Fenugreek β-amylase: Chemical modification and in silico approach

The amino acid sequence of Fenugreek β-amylase is not available in protein data bank. Therefore, an attempt has been made to identify the catalytic amino acid residues of enzyme by employing studies of pH dependence of enzyme catalysis, chemical modification and bioinformatics. Treatment of purified Fenugreek β-amylase with EDAC in presence of glycine methyl ester and sulfhydryl group specific reagents (IAA, NEM and p-CMB), followed a pseudo first-order kinetics and resulted in effective inactivation of enzyme. The reaction with EDAC in presence of NTEE (3-nitro-l-tyrosine ethylester) resulted into modification of two carboxyl groups per molecule of enzyme and presence of one accessible sulfhydryl group at the active site, per molecule of enzyme was ascertained by titration with DTNB. The above results were supported by the prevention of inactivation of enzyme in presence of substrate. Based on MALDI-TOF analysis of purified Fenugreek β-amylase and MASCOT search, β-amylase of Medicago sativa was found to be the best match. To further confirm the amino acid involved in catalysis, homology modelling of β-amylase of M. sativa was performed. The sequence alignment, superimposition of template and target models, along with study of interactions involved in docking of sucrose and maltose at the active site, led to identification of Glu187, Glu381 and Cys344 as active site residues.

Proteomic profiling of the signal crayfish Pacifastacus leniusculus egg and spermatophore

Proteins of the signal crayfish Pacifastacus leniusculus egg and spermatophore were identified using in-gel digestion, mass spectrometry, and Mascot search. Forty-one and one-hundred-fifty proteins were identified in egg and spermatophore, respectively. The proteins were classified into nine categories including cell defence, cell signaling, cytoskeleton, DNA related activity, metabolism and energy production, protease and protease inhibitor, respiration, transportation, and others and unknown. Twenty-two proteins were found in both egg and spermatophore. The respiration and cytoskeleton groups are the most diverse categories in the protein profiles of the egg and spermatophore, respectively. No protein was assigned to DNA related activity and cell defence categories in the protein profile of the crayfish egg. Differences between protein profiles of the crayfish egg and spermatophore show different functional priorities for each of gametes. Several proteins having possible roles in gametogenesis, capacitation, acrosome reaction, and fertilization were identified. This proteomic profile of signal crayfish gametes provides a basis for further investigation of functional roles of the identified proteins in aspects of reproduction such as capacitation and fertilization.

Comparative proteomic analysis on anti-fibrotic effect of N-acetyl-seryl-aspartyl-lysyl-proline in rats with silicosis

To perform a comparative proteomic analysis for identification of pulmonary proteins related to the progression of silicosis and anti-fibrotic effect of N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP). Bronchial instillation of SiO₂powder (for 4 or 8 weeks) was applied in rats to establish a silicosis model. Ac-SDKP treatment was performed before (prevention group) or after (treatment group) SiO₂instillation. The control group was treated by bronchial instillation of sodium chloride solution of the same volume as SiO₂powder for 4 or 8 weeks. Proteins in lung tissue were separated by two-dimensional gel electrophoresis and stained with colloidal Coomassie brilliant blue. The gel images were scanned with the Lab Scan III system and analyzed with Imagemaster 6.0. The protein spots with significant differences between two groups (i.e., P value was less than 0.05 in One-way ANOVA) and with a change in volume over 30% were defined as differential proteins. Comparison was performed between the silicosis group and control group after 4 or 8 weeks, between the Ac-SDKP treatment group and silicosis group after 8 weeks, and between the Ac-SDKP prevention group and silicosis group after 8 weeks. The differentially expressed proteins were subjected to in-gel digestion with trypsin and MALDI-TOF-MS and Mascot search engine analysis to identify these proteins. Thirty-three differential proteins were identified. In comparison with the control group (4 weeks), the silicosis group (4 weeks) had 17 up-regulated proteins and 11 down-regulated proteins. In comparison with the control group (8 weeks), the silicosis group (8 weeks) had 16 up-regulated proteins and 12 down-regulated proteins. In comparison with the silicosis group (8 weeks), the Ac-SDKP treatment group had 5 up-regulated proteins and 6 down-regulated proteins, and the Ac-SDKP prevention group had 8 up-regulated proteins and 10 down-regulated proteins. Critical regulatory proteins related to silicotic fibrosis and anti-silicotic effect of Ac-SDKP have been identified. These proteins may play an important role in proliferation, apoptosis, inflammation, epithelial-mesenchymal transition, and signal transduction in silicosis.

Comparative proteomic analysis of renal tissue in IgA nephropathy with iTRAQ quantitative proteomics.

Immunoglobulin (Ig) A nephropathy (IgAN) is the most common form of glomerulonephritis. In clinical practice, it is difficult to monitor the repeating relapse in patients suffering from IgAN, which usually occurs within 10 years of end-stage renal disease. In order to identify and quantify the total protein content in the renal tissue of patients with IgAN, isobaric tags for relative and absolute quantification (iTRAQ) technology was performed. iTRAQ coupled with multiple chromatographic fractionation and tandem mass spectrometry was used to analyze the total protein of normal renal tissue in IgAN and healthy patients. The individual proteins were identified by the Mascot search engine and any that were differentially expressed were monitored. A total of 574 different proteins were identified, and 287 proteins were up- or downregulated by >1 fold alteration in levels. The results showed that iTRAQ-based quantitative proteomic technology for the identification and relative quantitation of the renal tissue proteome is efficiently applicable. The differential expression of the proteome profiles for IgAN patients was determined. Further studies using large cohorts of patient samples with long-term clinical follow-up data should be conducted to evaluate the usefulness of the pathogenesis and novel biomarker candidates of IgAN, which may develop a novel technique for the diagnosis of IgAN.

Chemical derivatization combined with capillary LC or MALDI-TOF MS for trace determination of lipoic acid in cosmetics and integrated protein expression profiling in human keratinocytes

Lipoic acid (LA) is an essential cofactor in mitochondrial enzymes and an ideal antioxidant in prokaryotic and eukaryotic cells. Capillary liquid chromatography coupled with ultraviolet detection (CapLC–UV) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) are two environmentally friendly methods for determining LA. In this study, a pre-column microwave-assisted derivatization with 4-bromomethyl-6,7-dimethoxycoumarin enhanced the UV absorbance of LA and was monitored at 345nm by CapLC–UV. Gradient separation was performed using a reversed-phase C18 column with a mobile phase consisting of acetonitrile–0.1% formic acid solution. The ionization of LA was increased, and the LA derivative was detected by MALDI-TOF MS at m/z 683 with an α-cyano-4-hydroxycinnamic acid matrix. The linear response ranged from 0.1 to 40μM with a correlation coefficient of 0.999. The CapLC–UV and MALDI-TOF MS had detection limits of 5 and 4fmol, respectively. These methods effectively detected LA in dietary supplements and cosmetics. Cellular proteomes of a human keratinocyte cell line (HaCaT) irradiated with UV radiation were also compared with and without LA treatment. The cellular proteomes were identified by nanoultra performance LC with LTQ Orbitrap system after trypsin digestion. Protein identification was performed by simultaneous peptide sequencing and MASCOT search. The analysis revealed changes in several proteins, including CDC42, TPI1, HNRPA2B1, PRDX1, PTGES3 and MYL6.

The midgut of Aedes albopictus females expresses active trypsin-like serine peptidases

BackgroundAedes albopictus is widely distributed across tropical and sub-tropical regions and is associated with the transmission of several arboviruses. Although this species is increasingly relevant to public health due its ability to successfully colonize both urban and rural habitats, favoring the dispersion of viral infections, little is known about its biochemical traits, with all assumptions made based on studies of A. aegypti. In previous studies we characterized the peptidase profile of pre-imaginal stages of A. albopictus and we reported the first proteomic analysis of the midgut from sugar-fed females of this insect species.MethodsIn the present work, we further analyzed the peptidase expression in the midgut of sugar-fed females using 1DE-substrate gel zymography, two-dimensional electrophoresis (2DE), mass spectrometry (MS), and protein identification based on similarity.ResultsThe combination of zymography, in solution assays using fluorescent substrates and 2DE-MS/MS allowed us to identify the active serine peptidase “fingerprint” in the midgut of A. albopictus females. Zymographic analysis revealed a proteolytic profile composed of at least 13 bands ranging from ~25 to 250 kDa, which were identified as trypsin-like serine peptidases by using specific inhibitors of this class of enzymes. Concomitant use of the fluorogenic substrate Z-Phe-Arg-AMC and trypsin-like serine protease inhibitors corroborated the zymographic findings. Our proteomic approach allowed the identification of two different trypsin-like serine peptidases and one chymotrypsin in protein spots of the alkaline region in 2DE map of the A. albopictus female midgut. Identification of these protein coding genes was achieved by similarity to the A. aegypti genome sequences using Mascot and OMSSA search engines.ConclusionThese results allowed us to detect, identify and characterize the expression of active trypsin-like serine peptidases in the midgut of sugar-fed A. albopictus females. In addition, proteomic analysis allowed us to confidently assign the expression of two trypsin genes and one chymotrypsin gene to the midgut of this mosquito. These results contribute to the gene annotation in this species of unknown genome and represent a small but important step toward the protein-level functional and localization assignment of trypsin-like serine peptidase genes in the Aedes genus.

Antiproliferative protein from the culture supernatant of Lentinula edodes C91-3 mycelia.

We purified and isolated a novel protein (LFP(91-3)A2) with antitumor effect from Lentinula edodes C(91-3) liquid mycelial culture supernatant. LFP(91-3)A2 was purified by (NH4)2SO4 precipitation, ion-exchange chromatography (DEAE-cellulose) and gel filtration chromatography (Sephacryl S-200HR). SDS-PAGE and MALDI-TOF/MS analysis Mascot search showed LFP(91-3)A2 is a new protein with apparent molecular weight of 26 kDa. The effect on tumor cell proliferation was assessed by using MTT assay in vitro, and the LFP(91-3)A2 reduced tumor cell growth obviously in a dose dependent manner (5-15 μg/mL) (p < 0.05), while it exhibited no toxic effect on normal chick embryo fibroblasts. The antiproliferative mechanism of LFP(91-3)A2 was found to be associated with inducing cell apoptosis by flow cytometry analysis and transmission electron microscopy. The LFP(91-3)A2 is a novel protein from Lentinula edodes with tumor-suppressive activity via inducing apoptosis of tumor cells without toxicity on normal cells and may be beneficial to natural products in clinical treatment.

Proteomic analysis of thyroid tissue in Graves' disease and toxic multinodular goiter

Introduction Graves’ disease (GD) and toxic multinodular goiter (MNG) are two common disorders which are known to have different etiologies and pathogenesis. We searhed for the molecular pathways that may underline the differences between these two devastating conditions. Methods / Design Difference Gel Electrophoresis (DIGE) was performed with the pools of protein extracts. Thyroid tissue samples from toxic multinodular guatr patiens and Graves’ disease patients (12 of each) were used for protein extraction. After 2D separation, gels were imaged to reveal differantially expressed proteins which were identified by Matrixassisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF/TOF) and MASCOT search. The identified proteins were classified on the basis of their functions in metabolic pathways by using PANTHER. In addition, Ingenuity Pathways Analysis (IPA) was performed to reveal the interconnections between relevant metabolic and conanical pathways. Results A total of 330±20 proteins spots were revealed on the gels. Fifteen regulated spots were identified and classified based on their molecular function and biological process that they are involved. Conclusion Although our findings are preliminary, they hold importance by providing the first comprehensive comparative proteomics data for GD and toxic MNG.

Potential of a Funalia trogii laccase enzyme as an anticancer agent

Funalia trogii extract has been reported to have antitumour properties, selectively killing cancer cells without affecting normal cells in vitro; the extract has also been found to be effective in reducing tumour growth in vivo. The present study was carried out to identify the bioactive constituents in the extract; in particular, the enzyme components (laccase and peroxidase). Results confirmed that laccase (molecular size 65 kDa) was one of the bioactive agents present in the extract. Native gels were stained for protein and the excised bands were analysed using Mascot and Blastp search tools. Laccase was confirmed to be one of the protein bands; however, the identity of the second band could not be confirmed. Also, the role of enzyme peroxidase could not be established in the cytotoxicity of the F. trogii extract. In conclusion, the results indicate that laccase may be one of the potential bioactive components of F. trogii extract, and further studies are required to understand its course of action in conferring cytotoxic properties. The ability to produce these enzymes under liquid fermentation conditions in relatively pure form lends itself for large scale industrial production.

Comparative Protein Composition of the Brains of PACAP-Deficient Mice Using Mass Spectrometry-Based Proteomic Analysis

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a widespread neuropeptide acting as a neurotransmitter, neuromodulator, or neurotrophic factor. The diverse biological actions provide the background for the variety of deficits observed in mice lacking endogenous PACAP. PACAP-deficient mice display several abnormalities, such as sudden infant death syndrome (SIDS)-like phenotype, decreased cell protection, and increased risk of Parkinson's disease. However, the molecular and proteomic background is still unclear. Therefore, our aim was to investigate the differences in peptide and protein composition in the brains of PACAP-deficient and wild-type mice using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometric (MS)-based proteomic analysis. Brains from PACAP-deficient mice were removed, and different brain areas (cortex, hippocampus, diencephalon, mesencephalon, brainstem, and cerebellum) were separated. Brain pieces were weighed, homogenized, and further processed for electrophoretic analysis. Our results revealed several differences in diencephalon and mesencephalon. The protein bands of interest were cut from the gel, samples were digested with trypsin, and the tryptic peptides were measured by matrix-assisted laser desorption ionization time of flight (MALDI TOF) MS. Results were analyzed by MASCOT Search Engine. Among the altered proteins, several are involved in metabolic processes, energy homeostasis, and structural integrity. ATP-synthase and tubulin beta-2A were expressed more strongly in PACAP-knockout mice. In contrast, the expression of more peptides/proteins markedly decreased in knockout mice, like pyruvate kinase, fructose biphosphate aldolase-A, glutathione S-transferase, peptidyl propyl cis-trans isomerase-A, gamma enolase, and aspartate amino transferase. The altered expression of these enzymes might partially account for the decreased antioxidant and detoxifying capacity of PACAP-deficient mice accompanying the increased vulnerability of these animals. Our results provide novel insight into the altered biochemical processes in mice lacking endogenous PACAP.

Primary Effusion Lymphoma: Secretome Analysis Reveals Novel Candidate Biomarkers with Potential Pathogenetic Significance

Primary Effusion Lymphoma: Secretome Analysis Reveals Novel Candidate Biomarkers with Potential Pathogenetic Significance

Identification of commensal flora-associated antigen as a pathogenetic factor of autoimmune pancreatitis

ObjectivesAutoimmune pancreatitis (AIP) is a chronic fibro-inflammatory disease of the pancreas constituting, in part, a recently defined nosological entity of IgG4-related systemic sclerosing diseases. The pathogenetic factors of AIP have not been fully elucidated. We previously established a mouse model of AIP using chronic exposure to a commensal bacteria, Escherichia coli. MethodsTo determine the pathogenetically relevant antigen of E. coli, the outer membrane fractions of E. coli were subjected to two-dimensional gel electrophoresis followed by immunoblotting against sera from the AIP model. Immunoreactive spots were determined using MALDI TOF/MS and Mascot search. The recombinant protein of the identified antigen was examined for their ability to induce AIP-like disorder in C57BL/6 mice. Furthermore, the antibody titer against that antigen was determined in AIP patients. ResultsOne representative spot reacting with sera from E. coli-inoculated mice was identified as FliC from E. coli, based on the results of TOF/MS. The repeated inoculation of recombinant FliC in C57BL/6 mice induced AIP-like pancreatitis and higher titers of anti-lactoferrin and anti-carbonic anhydrase II. Sera from patients with AIP had the highest antibody titer, while those from patients with pancreatic diseases other than AIP had a higher antibody titer against E. coli and FliC, compared with pancreatic disease-free controls. ConclusionsFliC from E. coli may pathogenetically generate an AIP-like inflammation status. A reconsideration of the importance of commensal bacteria as an environmental factor(s) capable of inducing autoimmunity could provide insight to overcoming AIP.

Proteomic screening of molecular targets of crocin

BackgroundTraditional drug discovery approaches are mainly relied on the observed phenotypic changes following administration of a plant extract, drug candidate or natural product. Recently, target-based approaches are becoming more popular. The present study aimed to identify the cellular targets of crocin, the bioactive dietary carotenoid present in saffron, using an affinity-based method.MethodsHeart, kidney and brain tissues of BALB/c mice were homogenized and extracted for the experiments. Target deconvolution was carried out by first passing cell lysate through an affinity column prepared by covalently attaching crocin to agarose beads. Isolated proteins were separated on a 2D gel, trypsinized in situ and identified by MALDI-TOF/TOF mass spectrometry. MASCOT search engine was used to analyze Mass Data.ResultsPart of proteome that physically interacts with crocin was found to consist of beta-actin-like protein 2, cytochrome b-c1 complex subunit 1, ATP synthase subunit beta, tubulin beta-3 chain, tubulin beta-6 chain, 14-3-3 protein beta/alpha, V-type proton ATPase catalytic subunitA, 60 kDa heat shock protein, creatine kinase b-type, peroxiredoxin-2, cytochrome b-c1 complex subunit 2, acetyl-coA acetyltransferase, cytochrome c1, proteasome subunit alpha type-6 and proteasome subunit alpha type-4.ConclusionThe present findings revealed that crocin physically binds to a wide range of cellular proteins such as structural proteins, membrane transporters, and enzymes involved in ATP and redox homeostasis and signal transduction.