Two-dimensional Difference Gel Electrophoresis Research Articles

Longitudinal study of circulating protein biomarkers in inflammatory bowel disease.

Crohn's disease (CD) and ulcerative colitis (UC) are the most common inflammatory bowel diseases (IBDs) occurring in humans. The major current diagnosis tool is colonoscopy, which is invasive and could lead to false diagnosis. The emergence of serological biomarkers enables the use of new diagnosis tools such as protein signatures for IBD diagnosis/management. Using 2D-DIGE coupled to mass spectrometry, our longitudinal study in a mouse model of colitis identified a signature of protein biomarkers for specific stages of disease.

Cantharidin, a protein phosphatase inhibitor, strongly upregulates detoxification enzymes in the Arabidopsis proteome

Cantharidin, a potent inhibitor of plant serine/threonine protein phosphatases (PPPs), is highly phytotoxic and dramatically affects the transcriptome in Arabidopsis. To investigate the effect of cantharidin on the Arabidopsis proteome, a combination of two-dimensional difference gel electrophoresis (2-D DIGE) and matrix-assisted laser desorption ionization time-of-flight (MALDI/TOF) mass spectrometry was employed for protein profiling. Multivariate statistical analysis identified 75 significant differential spots corresponding to 59 distinct cantharidin-responsive proteins, which were representative of different biological processes, cellular components, and molecular functions categories. The majority of identified proteins localized in the chloroplast had a significantly decreased presence, especially proteins involved in photosynthesis. Detoxification enzymes, especially glutathione-S-transferases (GSTs), were the most upregulated group (ca. 1.5- to 3.3-fold). Given that the primary role of GSTs is involved in the process of detoxification of both xenobiotic and endobiotic compounds, the induction of GSTs suggests that cantharidin promoted inhibition of PPPs may lead to defense-like responses through regulation of GST enzymes as well as other metabolic pathways.

Shigella flexneri infection in Caenorhabditis elegans: cytopathological examination and identification of host responses.

The Gram-negative bacterium Shigella flexneri is the causative agent of shigellosis, a diarrhoeal disease also known as bacillary dysentery. S. flexneri infects the colonic and rectal epithelia of its primate host and induces a cascade of inflammatory responses that culminates in the destruction of the host intestinal lining. Molecular characterization of host-pathogen interactions in this infection has been challenging due to the host specificity of S. flexneri strains, as it strictly infects humans and non-human primates. Recent studies have shown that S. flexneri infects the soil dwelling nematode Caenorhabditis elegans, however, the interactions between S. flexneri and C. elegans at the cellular level and the cause of nematode death are unknown. Here we attempt to gain insight into the complex host-pathogen interactions between S. flexneri and C. elegans. Using transmission electron microscopy, we show that live S. flexneri cells accumulate in the nematode intestinal lumen, produce outer membrane vesicles and invade nematode intestinal cells. Using two-dimensional differential in-gel electrophoresis we identified host proteins that are differentially expressed in response to S. flexneri infection. Four of the identified genes, aco-1, cct-2, daf-19 and hsp-60, were knocked down using RNAi and ACO-1, CCT-2 and DAF-19, which were identified as up-regulated in response to S. flexneri infection, were found to be involved in the infection process. aco-1 RNAi worms were more resistant to S. flexneri infection, suggesting S. flexneri-mediated disruption of host iron homeostasis. cct-2 and daf-19 RNAi worms were more susceptible to infection, suggesting that these genes are induced as a protective mechanism by C. elegans. These observations further our understanding of the processes involved in S. flexneri infection of C. elegans, which is immensely beneficial to the routine use of this new in vivo model to study S. flexneri pathogenesis.

Identification of putative fertility markers in seminal plasma of crossbred bulls through differential proteomics

Sub-fertility is a major problem in crossbred bulls leading to disintegration of breeding systems and huge economic loss. Identification of some potential biomarkers to determine the latent fertility of bulls accurately has long been the interest of researchers. In this study, we analyzed the proteome of seminal plasma (SP) from bulls with varying fertility to identify the fertility-associated proteins. The proteomic profile of high- and low-fertile bulls was compared by two-dimensional difference gel electrophoresis and differentially expressed proteins were identified through matrix-assisted laser desorption/ionization–time of flight/mass spectrometry. Out of the 18 differentially expressed proteins (P < 0.05), 9 were overexpressed in SP of high-fertile bulls and 9 were overexpressed in SP of low-fertile bulls. The differential expressions ranged from 1.5- to 5.5-fold between the two groups, where protection of telomeres-1 protein (POT1) was highly overexpressed (2.9-fold) in high-fertile group and prostaglandin E2 receptor EP3 (PTGER3) was highly abundant (5.5-fold) in low-fertile group. The protein interaction network was elucidated using STRING software tool, and the functional bioinformatics analysis was done using Blast2Go software. Most of the differentially expressed proteins were found to be involved in cellular processes and biological regulation with binding and catalytic function. It is inferred that the expression of certain proteins in the SP varied with bull fertility, and concurrent appraisal of their expression along with other fertility assays may help in determining bull fertility.

Proteasome inhibition in skeletal muscle cells unmasks metabolic derangements in type 2 diabetes.

Two-dimensional difference gel electrophoresis (2-D DIGE)-based proteome analysis has revealed intrinsic insulin resistance in myotubes derived from type 2 diabetic patients. Using 2-D DIGE-based proteome analysis, we identified a subset of insulin-resistant proteins involved in protein turnover in skeletal muscle of type 2 diabetic patients, suggesting aberrant regulation of the protein homeostasis maintenance system underlying metabolic disease. We then validated the role of the ubiquitin-proteasome system (UPS) in myotubes to investigate whether impaired proteasome function may lead to metabolic arrest or insulin resistance. Myotubes derived from muscle biopsies obtained from people with normal glucose tolerance (NGT) or type 2 diabetes were exposed to the proteasome inhibitor bortezomib (BZ; Velcade) without or with insulin. BZ exposure increased protein carbonylation and lactate production yet impaired protein synthesis and UPS function in myotubes from type 2 diabetic patients, marking the existence of an insulin-resistant signature that was retained in cultured myotubes. In conclusion, BZ treatment further exacerbates insulin resistance and unmasks intrinsic features of metabolic disease in myotubes derived from type 2 diabetic patients. Our results highlight the existence of a confounding inherent abnormality in cellular protein dynamics in metabolic disease, which is uncovered through concurrent inhibition of the proteasome system.

Combined polyethylene glycol and CaCl2 precipitation for the capture and purification of recombinant antibodies

A direct capture step was developed for the purification of recombinant antibodies from clarified culture supernatants. This method is a combination of CaCl2 and polyethylene glycol (PEG) precipitation. For separation of high molecular weight impurities, such as dsDNA and aggregates, CaCl2 precipitation was the method of choice, and PEG precipitation was used for monoclonal antibody (mAb) capturing to separate low molecular weight impurities, mainly host cell proteins (HCP). Precipitation tests were performed with five different CHO cell culture supernatants, and showed yields of at least 80–95%. Purity was determined by size exclusion chromatography, HCP ELISA, and two-dimensional differential in-gel electrophoresis (2D DIGE); the measured values from CaCl2/PEG precipitation were comparable to those from Protein A purification. These results suggest that this novel purification method could be used as the basis for a platform process to capture antibodies from a clarified culture supernatant. CaCl2/PEG precipitation provides enough selectivity and recovery to substantiate replacing the Staphylococcus Protein A affinity chromatography step of the antibody purification process.

Expression and clinical significance of AHSG and complement C3 in pancreatic ductal adenocarcinoma

To analyze serum proteins from pancreatic carcinoma patients, pancreatic benign tumor patients, chronic pancreatitis patients and normal controls to discover potential and specific biomarkers. Serum samples were collected from 40 pancreatic carcinoma patients, 10 pancreatic benign tumor patients, 10 chronic pancreatitis patients and 40 cancer-free controls from May 2009 to April 2011. The samples were compared with two-dimensional differential gel electrophoresis (2D-DIGE) and differentially expressed proteins were further identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). Then, two up-regulated proteins were further validated by real-time polymerase chain reaction (PCR), Western blot analysis and immunohistochemistry (IHC) from transcriptional and proteinic aspects. We identified 12 differently expressed proteins in pancreatic carcinoma group compared with normal control group, including complement component C3, hemopexin, alpha-2-HS-glycoprotein, apolipoprotein H, serotransferrin, haptoglobin, apolipoprotein E, transthyretin, serum amyloid P-component, vitronectin, prothrombin and isoform 2 of Ig mu chain C region. High level of C3 and AHSG were detected in cancerous tissues by real-time PCR, Western blot and immunohistochemisty. Western blot revealed that gray ratios of C3 and AHSG were 0.11 ± 0.01 and 0.26 ± 0.02 respectively. The Immunohistochemical results showed that positive rate of C3 and AHSG were 72.5% and 82.5% in cancerous group versus 32.5% and 25% respectively in normal control. C3 and AHSG may become pancreatic carcinoma-related biomarkers.

Proteomic changes of alveolar lining fluid in illnesses associated with exposure to inhaled non-infectious microbial particles.

BackgroundHyperresponsiveness to inhaled non-infectious microbial particles (NIMPs) has been associated with illnesses in the airways. Hypersensitivity pneumonitis (HP) is considered to be the prototype for these NIMPs-related diseases; however, there is no consensus on the definitions or diagnostic criteria for HP and the spectrum of related illnesses.Methods and FindingsIn order to identify the possible diagnostic markers for illnesses associated with NIMPs in alveolar lining fluid, we performed a proteomic analysis using a two-dimensional difference gel electrophoresis on bronchoalveolar lavage (BAL) fluid from patients with exposure to NIMPs in the context of damp building-related illness (DBRI) or conditions on the borderline to acute HP, designated here as agricultural type of microbial exposure (AME). Samples from patients with HP and sarcoidosis (SARC) were included for reference. Results were compared to results of healthy subjects (CTR). Western blot was used for validation of potential marker proteins from BAL fluid and plasma. Protein expression patterns suggest a close similarity between AME and HP, while DBRI was similar to CTR. However, in DBRI the levels of the inflammation associated molecules galectin-3 and alpha-1-antitrypsin were increased. A novel finding emerging from this study was the increases of semenogelin levels in BAL fluid from patients with AME, HP and SARC. Histone 4 levels were increased in AME, HP and SARC. Elevated plasma levels of histone 2B were detected in HP and SARC, suggesting it to be a potential blood indicator for inflammatory diseases of the lungs.ConclusionsIn this study, the proteomic changes in bronchoalveolar lavage of DBRI patients were distinct from other NIMP exposure associated lung diseases, while changes in AME overlapped those observed for HP patient samples. Some of the proteins identified in this study, semenogelin and histone 4, could function as diagnostic markers for differential diagnosis between DBRI and HP-like conditions.

The day/night proteome in the murine heart.

Circadian rhythms are essential to cardiovascular health and disease. Temporal coordination of cardiac structure and function has focused primarily at the physiological and gene expression levels, but these analyses are invariably incomplete, not the least because proteins underlie many biological processes. The purpose of this study was to reveal the diurnal cardiac proteome and important contributions to cardiac function. The 24-h day-night murine cardiac proteome was assessed by two-dimensional difference in gel electrophoresis (2D-DIGE) and liquid chromatography-mass spectrometry. Daily variation was considerable, as ∼7.8% (90/1,147) of spots exhibited statistical changes at paired times across the 24-h light- (L) dark (D) cycle. JTK_CYCLE was used to investigate underlying diurnal rhythms in corresponding mRNA. We next revealed that disruption of the L:D cycle altered protein profiles and diurnal variation in cardiac function in Langendorff-perfused hearts, relative to the L:D cycle. To investigate the role of the circadian clock mechanism, we used cardiomyocyte clock mutant (CCM) mice. CCM myofilaments exhibited a loss of time-of-day-dependent maximal calcium-dependent ATP consumption, and altered phosphorylation rhythms. Moreover, the cardiac proteome was significantly altered in CCM hearts, especially enzymes regulating vital metabolic pathways. Lastly, we used a model of pressure overload cardiac hypertrophy to demonstrate the temporal proteome during heart disease. Our studies demonstrate that time of day plays a direct role in cardiac protein abundance and indicate a novel mechanistic contribution of circadian biology to cardiovascular structure and function.

Zyxin modulates the transmigration of Haemophilus influenzae to the central nervous system

The mechanism by which Haemophilus influenzae causes meningitis is unclear. Previously, we established murine meningitis by intranasal instillation of H. influenzae as a cell-bound organism (CBO). In this study, we aimed to identify the molecules associated with inhibiting the transmigration of cells across the blood–brain barrier (BBB). Two-dimensional difference gel electrophoresis and protein identification by mass spectrometry were used for proteomic analysis. Analysis of the membranous extract from a tumor necrosis factor (TNF)-α-treated human brain microvascular endothelial cell (HBMEC) monolayer revealed 41 differentially expressed proteins. Zyxin, which is thought to be essential for tight cell-to-cell junctions, decreased 1.8-fold in TNF-α-treated HBMECs. In addition, zyxin transcript levels decreased 1.5-fold in cells derived from TNF-α-treated HBMECs. Intranasal instillation of CBOs in zyxin-deficient mice resulted in a significant higher mortality rate than in wild-type mice. Transmigration of CBOs across a HBMEC monolayer pretreated with TNF-α (1 ng/mL), interleukin (IL)-1β (10 ng/mL), or lipopolysaccharide (LPS; 10 ng/mL) was assayed by counting CBOs that migrated from an upper chamber into a lower chamber. HBMEC pretreated with TNF-α exhibited significantly greater migration (P < 0.01) than did control cells or cells treated with IL-1β or LPS. Our findings highlight that zyxin is an important protein protecting the tight junction of the BBB against cell transmigration across the BBB. Finally, TNF-α produced in respiratory infection when the primary infection reached the BBB caused decreased zyxin levels in BBB cell membranes. Furthermore, H. influenzae reaching the BBB as CBOs could transmigrate into cerebrospinal fluid across the zyxin-decreased BBB.

Expression of Hemopexin in Acute Rejection of Rat Liver Allograft Identified by Serum Proteomic Analysis

Acute rejection (AR) and acceptance of allograft after liver transplantation (LTx) remain critical issues that need addressing to improve prognosis. We therefore performed rat orthotopic LTx and proteomic analyses to screen for immune response-related biomarkers in sera. Markers identified were validated at the mRNA and/or protein levels, and the molecules of interest were functionally explored. Compared with syngeneic controls, signs of AR as well as spontaneous acceptance were observed in hematoxylin and eosin-stained sections of liver allografts. In accordance with the severity of AR, 30 protein spots displaying significant changes in abundance were identified using two-dimensional differential gel electrophoresis. Ultimately, 14 serum proteins were sequenced and five spots of interest were identified as hemopexin (HPX). Expression of HPX was significantly and inversely associated with the severity of AR at both the mRNA and protein levels. In vitro, Mt-1, Ho-1, Fth, Ifn-γ, and Il-17 transcripts were significantly upregulated in lysates of lymphocytes stimulated with HPX, whereas Il-10 markedly was remarkably downregulated. Interferon-γ, IL-10, and IL-17 proteins in the supernatant of HPX-stimulated lymphocytes were significantly altered in keeping with the mRNA level. Our data facilitated the generation of a proteomic profile to enhance the understanding of rat liver AR. In view of finding that the HPX serum level is negatively associated with the severity of AR of rat liver allograft, we propose that in vitro treatment with HPX regulates cytokine expression in rat lymphocytes.

Long-term supranutritional supplementation with selenate decreases hyperglycemia and promotes fatty liver degeneration by inducing hyperinsulinemia in diabetic db/db mice.

There are conflicting reports on the link between the micronutrient selenium and the prevalence of diabetes. To investigate the possibility that selenium acts as a “double-edged sword” in diabetes, cDNA microarray profiling and two-dimensional differential gel electrophoresis coupled with mass spectrometry were used to determine changes in mRNA and protein expression in pancreatic and liver tissues of diabetic db/db mice in response to dietary selenate supplementation. Fasting blood glucose levels increased continuously in db/db mice administered placebo (DMCtrl), but decreased gradually in selenate-supplemented db/db mice (DMSe) and approached normal levels after termination of the experiment. Pancreatic islet size was increased in DMSe mice compared with DMCtrl mice, resulting in a clear increase in insulin production and a doubling of plasma insulin concentration. Genes that encode proteins involved in key pancreatic β-cell functions, including regulation of β-cell proliferation and differentiation and insulin synthesis, were found to be specifically upregulated in DMSe mice. In contrast, apoptosis-associated genes were downregulated, indicating that islet function was protected by selenate treatment. Conversely, liver fat accumulation increased in DMSe mice together with significant upregulation of lipogenic and inflammatory genes. Genes related to detoxification were downregulated and antioxidant enzymatic activity was reduced, indicating an unexpected reduction in antioxidant defense capacity and exacerbation of fatty liver degeneration. Moreover, proteomic analysis of the liver showed differential expression of proteins involved in glucolipid metabolism and the endoplasmic reticulum assembly pathway. Taken together, these results suggest that dietary selenate supplementation in db/db mice decreased hyperglycemia by increasing insulin production and secretion; however, long-term hyperinsulinemia eventually led to reduced antioxidant defense capacity, which exacerbated fatty liver degeneration.

Identification of reduced circulating haptoglobin concentration as a biomarker of the severity of pulmonary embolism: a nontargeted proteomic study.

Risk stratification of patients with pulmonary embolism (PE) may identify patients at high risk of early death who may benefit from more intensive surveillance or aggressive therapy. Nontargeted proteomics may identify biomarkers useful for the risk stratification of patients with acute symptomatic pulmonary embolism (PE). We studied 6 patients presenting with low-risk PE and 6 patients presenting with intermediate (n = 3) or high-risk (n = 3) PE. Two-dimensional difference gel electrophoresis was used to compare their plasma protein abundances. Candidate protein markers were identified by matrix assisted laser desorption ionization time-of-flight mass spectrometry. A panel of four biomarkers (haptoglobin, hemopexin, α2-macroglobulin, and Ig α1-chain C region) showed differences in plasma abundance among patients with acute symptomatic PE of different severity. Haptoglobin and hemopexin were decreased, whereas α2-macroglobulin and Ig α1-chain C region were increased, in patients with high or intermediate-risk PE compared with low-risk PE patient. In a separate clinical population consisting of 104 adults with acute PE, serum haptoglobin concentrations had an 85% chance of correctly identifying patients with high-risk PE according to receiving operating characteristics curve analysis. Moreover, serum haptoglobin concentrations ≤1 g/l showed an 80% sensitivity and a 96% specificity for the diagnosis of high-risk PE. Nontargeted proteomics identified protein biomarkers for the severity of PE that are involved in iron metabolism pathways and acute-phase response. Among them, reduced serum haptoglobin concentrations show a high accuracy for the biochemical detection of high-risk PE.

Phagocytosis of nano-sized titanium dioxide triggers changes in protein acetylation

Nano-sized titanium dioxide (nTiO2) is one of the most produced engineered nanomaterials and therefore carries a high risk for workplace exposure. In several nanosafety studies, exposure to nTiO2 has been shown to trigger inflammation in mice lung and to cause oxidative stress. Here, cytoplasmic proteome changes in human monocyte derived macrophages were investigated with two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry to evaluate the adverse cellular effects after exposure to different types of TiO2 nanoparticles (NPs). Both studied TiO2 NPs (rutile TiO2 with or without silica coating) evoked similar proteome alterations. The identified proteins were linked to metabolic homeostasis, cytoskeleton remodeling and oxidative stress. The abundances of chloride intracellular channel protein 1 and cathepsin D changed only after exposure to nTiO2 as compared to a coarse particle analog. Enrichment analysis revealed that 70% of the proteins with changed intensities contained known acetylation sites, and it was possible to confirm a significant induction of cytoplasmic protein acetylation after nTiO2 exposure. The course of the events during phagocytosis could account for the observed membrane maintenance, metabolic and cytoskeletal protein expression changes. Lysine acetylation of cytoplasmic proteins in macrophages is emerging as a major cell regulation mechanism after nTiO2 exposure. While the amount of nanosafety research conducted in recent years has been constantly increasing, proteomics has not yet been utilized widely in this field. In addition, reversible protein post-translational modifications (PTMs) such as acetylation and phosphorylation have not been investigated in-depth in nanomaterial exposed cells. Proteome changes observed in nanomaterial exposed macrophages revealed active phagocytosis of the particles and provided new insights into underlying mechanisms of biological responses to nTiO2 exposures. Moreover, reversible protein acetylation might be a major cellular regulation event occurring in nanomaterial exposed cells.

Serum amyloid A is increased in children with abusive head trauma: a gel-based proteomic analysis.

Abusive head trauma (AHT) is the leading cause of death from traumatic brain injury in infants and young children. Identification of mild AHT (Glasgow Coma Scale score: 13-15) is difficult because children can present with nonspecific symptoms and with no history of trauma. Two-dimensional difference gel electrophoresis combined with mass spectrometry was used to compare the serum protein profile of children with mild AHT and age-matched controls. Protein changes were confirmed by western blots. Western blots were performed using serum from children with mild, moderate, and severe AHT to assess the effect of injury severity on protein intensity. The protein identified--serum amyloid A (SAA)--was then measured by enzyme-linked immunosorbent assay. Using serum from 18 mild AHT cases and 20 controls, there were ~1,000 protein spots; 2 were significantly different between groups. Both spots were identified as SAA. There was no relationship between protein levels and injury severity. SAA concentrations measured by enzyme-linked immunosorbent assay were increased in cases vs. controls. SAA may be a potential biomarker to identify children with mild AHT who present for medical care without a history of trauma and who might otherwise not be recognized as needing a head computed tomography.

Molecular mechanism of acute radiation enteritis revealed using proteomics and biological signaling network analysis in rats.

Radiation enteritis (RE) has emerged as a significant complication that can progress to severe gastrointestinal disease and the mechanisms underlying its genesis remain poorly understood. The aim of this study was to identify temporal changes in protein expression potentially associated with acute inflammation and to elucidate the mechanism underlying radiation enteritis genesis. Male Sprague-Dawley rats were irradiated in the abdomen with a single dose of 10Gy to establish an in vivo model of acute radiation enteritis. Two-dimensional fluorescence difference gel electrophoresis, matrix-assisted laser desorption/ionization time-of-flight spectrometer (MALDI-TOF) tandem mass spectrometry, and peptide mass fingerprinting were used to determine differentially expressed proteins between normal and inflamed intestinal mucosa. Additionally, differentially expressed proteins were evaluated by KO Based Annotation System to find the biological functions associated with acute radiation enteritis. Intensity changes of 86 spots were detected with statistical significance (ratio≥1.5 or≤1.5, P<0.05). Sixty one of the 86 spots were identified by MALDI-TOF/TOF tandem mass spectrometry. These radiation-induced proteins with biological functions showed that the FAS pathway and glycolysis signaling pathways were significantly altered using the KOBAS tool. Our results reveal an underlying mechanism of radiation-induced acute enteritis, which may help clarify the pathogenesis of RE and point to potential targets for therapeutic interventions.

Examination of the leaf proteome during flooding stress and the induction of programmed cell death in maize.

BackgroundMaize is a major economic crop worldwide, with substantial crop loss attributed to flooding. During a stress response, programmed cell death (PCD) can be an effective way for plants better adapt. To identify flooding stress related PCD proteins in maize leaves, proteomic analysis was performed using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mass spectrometry.ResultsComparative proteomics was combined with physiological and biochemical analysis of maize leaves under flooding stress. Fv/Fm, qP, qN and relative water content (RWC) were found to be altered in response to flooding stress, with an increase in H2O2 content noted in vivo. Furthermore, DNA ladder detection indicated that PCD had occurred under flooding treatment. The maize leaf proteome was analyzed via 2D-DIGE gel, with a total of 32 differentially expressed spots isolated, 31 spots were successfully identified via MALDI-TOF/TOF MS which represent 28 proteins. The identified proteins were related to energy metabolism and photosynthesis, PCD, phytohormones and polyamines. To better characterize the role of translationally controlled tumor protein (TCTP) in PCD during a stress response, mRNA expression was examined in different plants by stress-induced PCD. These included heat stress induced rice protoplasts, Tobacco Mosaic Virus infected tobacco leaves and dark induced rice and Arabidopsis thaliana leaves, all of which showed active PCD, and TCTP expression was increased in different degrees. Moreover, S-adenosylmethionine synthase 2 (SAMS2) and S-adenosylmethionine decarboxylase (SAMDC) mRNA expression were also increased, but ACC synthase (ACS) and ACC oxidase (ACO) mRNA expression were not found in maize leaves following flooding. Lastly, ethylene and polyamine concentrations were increased in response to flooding treatment in maize leaves.ConclusionsFollowing flooding stress, the photosynthetic systems were damaged, resulting in a disruption in energy metabolism, with the noted photosynthetic decline also possibly attributed to ROS production. The observed PCD could be regulated by TCTP with a possible role for H2O2 in TCTP induction under flooding stress. Additionally, increased SAMS2 expression was closely associated with an increased polyamine synthesis during flooding treatment.

Effects of laser treatment on the expression of cytosolic proteins in the synovium of patients with osteoarthritis.

Low level laser therapy (LLLT) has been developed for non-invasive treatment of joint diseases. We have previously shown that LLLT influenced synovial protein expression in rheumatoid arthritis (RA). The aim of this study was to assess the effects of laser irradiation on osteoarthritic (OA) synovial protein expression. The synovial membrane samples removed from the knees of 6 OA patients were irradiated ex vivo using near infrared diode laser (807-811 nm; 25 J/cm(2) ). An untreated sample taken from the same patient served as control. Synovial protein separation and identification were performed by two-dimensional differential gel electrophoresis and mass spectrometry, respectively. Eleven proteins showing altered expression due to laser irradiation were identified. There were three patients whose tissue samples demonstrated a significant increase (P < 0.05) in mitochondrial heat shock 60 kD protein 1 variant 1. The expression of the other proteins (calpain small subunit 1, tubulin alpha-1C and beta 2, vimentin variant 3, annexin A1, annexin A5, cofilin 1, transgelin, and collagen type VI alpha 2 chain precursor) significantly decreased (P < 0.05) compared to the control samples. A single diode laser irradiation of the synovial samples of patients with osteoarthritis can statistically significantly alter the expression of some proteins in vitro. These findings provide some more evidence for biological efficacy of LLLT treatment, used for osteoarthritis.

Increased plasma lactoferrin levels in leucocytapheresis therapy in patients with rheumatoid arthritis.

The aim of this study was to clarify the mechanism of leucocytapheresis (LCAP) in patients with RA. Protein profiles of blood samples from two patients with RA obtained via LCAP column inlet and outlet lines were analysed by two-dimensional fluorescence difference gel electrophoresis and mass spectrometry. The lactoferrin (LTF) levels of peripheral and circulating blood samples from seven patients obtained via the LCAP column blood circuit were then determined by ELISA. Peripheral blood samples from 14 patients with RA were exposed to unwoven polyester fibre filters and the LTF level was determined. In addition, morphological changes in neutrophils after exposure to the filter were examined by optical microscopy, electronic microscopy and LTF immunostaining. LTF levels were increased in both samples from the LCAP column outlet and peripheral blood at the end of LCAP treatment. Furthermore, peripheral blood samples exposed to the filter revealed a decreased number of neutrophils and an increased level of LTF. Morphological analysis of the exposed neutrophils showed vacuolization of the cytoplasm and degranulation of LTF-positive granules. These data suggest that LTF stored in the granules of neutrophils is released from the neutrophils caught in the LCAP column. Because LTF has been reported to have multiple anti-inflammatory properties, increased levels of LTF may contribute to the clinical effect of LCAP in patients with RA.

AB0475 Increased Plasma Lactoferrin Levels in Leukocytapheresis Therapy in Patients with Rheumatoid Arthritis

ObjectivesTo clarify the mechanism of leukocytapheresis (LCAP) in patients with rheumatoid arthritis (RA).MethodsProtein profiles of blood samples from 2 patients with RA obtained via LCAP column inlet and outlet lines...