SDS-PAGE Gel Electrophoresis Research Articles

Abstract B11: Prostate apoptosis response-4 (PAR4) secretion in breast tumor and normal cell lines cultured in hypoxic conditions

Abstract The prostate apoptosis response-4 (PAR4) gene encodes a 38kDa proapoptotic protein with tumor cell selective action and/or cell sensitization to an additional proapoptotic insult. This selective apoptotic ability is associated with PAR4 nuclear translocation and phosphorylation state. PAR-4 acts through intrinsic and extrinsic apoptotic pathways. PAR-4 can also be secreted by normal/immortalized and cancer cells via the classical ER stress pathway mediated by GRP78. The previous study from our group provided evidence that MCF10A cells cultured in three-dimensional conditions display an increased expression of PAR4 in the center of the acini, where the cells are dying to lumen formation and where the O2 levels are expected to be lower. In the present study, we sought to evaluate the effects of hypoxia on PAR4 secretion in mammary epithelial cells and breast cancer cells. MCF10A (immortalized mammary epithelial cells) and the breast cancer cells MCF7, MDA-MB-231, and SKBR3 were cultured in normoxic and hypoxic (1% O2) conditions. The conditioned medium (CM) was concentrated using Amicon Ultra 10kDA filters and resolved by SDS-PAGE gel electrophoresis in parallel to cell lysate analysis. Cells maintained under hypoxic conditions for 6 h, 24 h, and 48 h showed no alteration in intracellular PAR4 protein levels in relation to cells maintained in normoxia. After 72 h we observed a decrease in intracellular PAR4 protein expression. The GRP78 protein expression did not change in the same time periods. However, Caspase-7 activation could be seen in 24 h, 48 h, and 72 h. Furthermore, we observed an increase (2.7 fold) in PAR4 protein expression in the CM after 72 h under hypoxia. Our results demonstrated for the first time that PAR4 secretion is modulated by hypoxia in normal and tumor breast cells. Further experimental and clinical studies are needed to determine the mechanism and role played by PAR4 secretion in cells' sensitivity to standard chemotherapeutics agents. Supported by FAPESP and CNPq. Citation Format: Simone Aparecida de Bessa Garcia, Irina Gueroldovna Brobovnitchaia, Flavia Rotea Mangone, Maria Aparecida Nagai. Prostate apoptosis response-4 (PAR4) secretion in breast tumor and normal cell lines cultured in hypoxic conditions [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B11.

Pro-inflammatory mechanism of lectin from Pinellia pedatisecta on macrophage

To investigate the mechanism of lectin from Pinellia pedatisecta(PPL) on macrophage-induced inflammation and its association with inflammatory corpuscles NLRP3. Lectin from P. pedatisecta was isolated and purified by gel chromatography, and its purity was analyzed by using SDS-PAGE gel electrophoresis. ELISA was used to investigate the effect of PPL on inflammatory cytokines released by macrophages, with IL-1β as indicators;and fluorescence probe DCFH-DA fluorometer was used to determine changes in active oxygen ROS of macrophages after application of lectin from P. pedatisecta.RAW264.7 cells were pre-treated with ROS inhibitor N-acetylcysteine (NAC) to investigate the effect on ROS and the release of inflammatory factor IL-1β from macrophages to research the relationship between them. The protein levels of NLRP3, Caspase-1 p20, ASC and TXNIP were determined by Western blot.The results showed that isolated and purified PPL could reach electrophoretic purity; PPL stimulated macrophages and induced the excessive release of ROS, leading to strong oxidative stress reaction, and the levels of intracellular inflammatory factorsIL-1β were significantly increased. NAC could inhibit PPL-induced ROS excessive production and significantly reduce the release of IL-1β. In addition, PPL could induce the increase in protein expression levels of Caspase-1 p20, NLRP3 and ASC, and significantly reduce TXNIP expression. The results showed that PPL could cause a strong oxidative stress response by stimulating macrophages, activate inflammatory corpuscles NLRP3, and result in large amount of IL-1β release. That is, PPL could lead to inflammatory cascade reaction by promoting the maturation and secretion of IL-1β through ROS-TXNIP-NLRP3-IL-1β signaling pathway.

Extensively Drug-Resistant Klebsiella pneumoniae Causing Nosocomial Bloodstream Infections in China: Molecular Investigation of Antibiotic Resistance Determinants, Informing Therapy, and Clinical Outcomes.

The rise in diversity of antimicrobial resistance phenotypes seen in Klebsiella pneumoniae is becoming a serious antibiotic management problem. We sought to investigate the molecular characteristics and clinical implications of extensively drug-resistant (XDR) K. pneumoniae isolated from different nosocomial bloodstream infections (BSIs) patients from July 2013 to November 2015. Even in combination treatment, meropenem did not protect against mortality of BSIs patients (P = 0.015). In contrast, tigecycline in combination with other antimicrobial agents significantly protected against mortality (P = 0.016). Antimicrobial susceptibility tests, molecular detection of antibiotic resistance determinants, conjugation experiments, multilocus sequence typing (MLST), S1-PFGE, Southern blot, SDS-PAGE, immunoblot analysis, and pulsed-field gel electrophoresis (PFGE) were used to characterize these isolates. These XDR K. pneumoniae strains were resistant to conventional antimicrobials except tigecycline and polymyxin B and co-harbored diverse resistance determinants. rmtB, blaKPC−2 as well as blaCTX−M−9 were located on a transferable plasmid of ~54.2 kb and the most predominant replicon type was IncF. 23 of the 35 isolates belonging the predominant clone were found to incorporate the globally-disseminated sequence type ST11, but others including a unique, previously undiscovered lineage ST2281 (allelic profile: 4-1-1-22-7-4-35) were also found and characterized. The porins OmpK35 and OmpK36 were deficient in two carbapenemase-negative carbapenem-resistant strains, suggesting decreased drug uptake as a mechanism for carbapenem resistance. This study highlights the importance of tracking hospital acquired infections, monitoring modes of antibiotic resistance to improve health outcomes of BSIs patients and to highlight the problems of XDR K. pneumoniae dissemination in healthcare settings.

Proteomic approach to investigate the impact of different dietary supplementation on lamb meat tenderness

The aim of this study was to evaluate the effect of dietary supplementation of linseed and/or quinoa on tenderness and on proteome of lamb meat. Thirty-two Italian Merino lambs were distributed into 4 groups with different diet: control (CO) with no supplemental fat, linseed (LS), quinoa (QS) and QS+LS diets. Meat obtained by lamb fed linseed showed the lowest values of WBSF (P<0.001), hardness (P<0.01), gumminess (P<0.01) and chewiness (P<0.01). Proteomic changes of myofibrillar and sarcoplasmic proteins were estimated with SDS-PAGE, Western Blot and Two-Dimensional Gel Electrophoresis. In linseed group proteomic analysis revealed a degradation of desmin and TnT proteins complex and a major number of spots and phosphorylation isoforms of fast MLC2 patterns. Meat obtained by lamb fed quinoa showed a minor effect on the instrumental evaluation of meat tenderness and a major number of spots ascribed to sarcoplasmic proteins and fMHC. Data suggest that dietary supplementation may act on meat tenderness and on proteolytic pattern of myofibrillar fraction.

Novel mononuclear Cu (II) terpyridine complexes: Impact of fused ring thiophene and thiazole head groups towards DNA/BSA interaction, cleavage and antiproliferative activity on HepG2 and triple negative CAL-51 cell line

Two mononuclear copper (II) terpyridine complexes namely, [Cu(Btptpy) (ClO4)](ClO4) 1, and [Cu(Bttpy) (ClO4)](ClO4) 2, (Btptpy (L1) = 4’-(Benzothiophene)-2,2’:6′,2″-terpyridine, Bttpy (L2) = 4’-(Benzylthiazolyl)-2,2’:6′,2″-terpyridine) have been synthesized and characterized. Single crystal X-ray diffraction shows that, both ligands belong to monoclinic crystal system with space group P21/c (L1) and P21/n (L2). Absorption spectral titration, DNA melting study, circular dichroism and viscosity measurement reveal that, complex 1 and 2 bind with DNA through intercalation. In addition, interaction between the two copper (II) complexes and bovine serum albumin (BSA) has been studied by fluorescence titration, circular dichroism and their protease activity has been investigated using SDS-PAGE gel electrophoresis. Agarose (AGE) and SDS-PAGE gel electrophoresis reveals both complexes have good nucleolytic and proteolytic property in the presence of additive hydrogen peroxide. Both complexes shows remarkable cytotoxic property against triple negative CAL-51 human breast cancer cell line and hepatocellular carcinoma (HepG2) cancer cell lines and bears very less cytotoxicity towards liver normal cell line (Changs). DCF-DA and TBRAS assay also supported that complex 1 and 2 induces elevated level of reactive oxygen species (ROS) and oxidative stress in cancer cells than normal cell line. Furthermore, FACS analysis confirms complex 1 and 2 brings apoptosis by growth phase cell cycle arrest.

DJ-1, a Parkinson's disease related protein, aggregates under denaturing conditions and co-aggregates with α-synuclein through hydrophobic interaction

DJ-1, a small ubiquitously expressed protein implicated in several pathways associated with Parkinson's disease pathogenesis, has been found to interact with α-synuclein and modulate its aggregation, yet the exact mechanisms remain unclear. The stability and aggregation properties of wild-type DJ-1 under denaturing conditions, such as low pH, high temperature, presence of denaturants were investigated. The interaction between DJ-1 and α-synuclein was tested by SDS-PAGE gel and native gel electrophoresis and by size-exclusion HPLC. Fibrillization was monitored by thioflavin T fluorescence assays and amorphous aggregation was followed by light scattering measurements. The morphology of aggregated species was observed by transmission electron microscopy and atomic force microscopy. Protein secondary structures were characterized by far-UV circular dichroism. DJ-1 fibrillization was first observed at low pH or by adding denaturants. Amorphous aggregates formed at neutral pH, and the aggregation was dramatically accelerated by elevated temperature and the presence of α-synuclein. Aggregation of DJ-1 were enhanced by heating and perturbed by the co-occurrence of α-synuclein but strong interactions between the two proteins were not found. Varying environmental factors led to different aggregation pathways of DJ-1 although a simulated physiological condition would not lead to fibrillization. DJ-1 co-aggregating with α-synuclein may result from weak hydrophobic interaction and DJ-1 exhibited chaperon-like activity in the initial time of α-synuclein aggregation at high temperature. This research on DJ-1 presented its aggregation behavior under denaturing conditions and interaction mechanism with α-synuclein that may help to decipher its potential neuroprotective or neurotoxic role in Parkinson's disease.

The Impacts of TRR14-Overexpression on Arabidopsis thaliana Growth and Photosynthetic Parameters

Background TRR14 protein is a small protein, a member of a multigene family in Arabidopsis which was found as the fi rst protein during screening seedlings for their resistant to the trehalose sugar. Objectives A number of TRR14-overexpressing plants were subjected to the characterization in the present research, among which, the associated morphological features and changes accompany growth pattern and photosynthesis related parameters. Materials and Methods TRR14 gene was isolated from Arabidopsis Thaliana and cloned into the pBin-35S vector. Recombinant vector was transferred to the Arabidopsis (Col-0) via Agrobacterium tumefaciens using the Floral Dipping method. Seeds from the TRR14 overexpressed (TRR14) and the Col-0 wild-type (WT) plants were shown on soil under long day conditions. Several measurements were then performed including determination of the fresh and dry weights, leaf area, chlorophyll a and b (Chl a and Chl b) content, Chl a/b ratio, total chlorophyll and carotenoids content, soluble and insoluble sugars content, total and soluble protein content, the Hill reaction rate, chlorophyll fl uorescence, as well as photorespiration rate. Meanwhile, the chloroplastic proteins were investigated by SDS-PAGE analysis. Results TRR14 plants showed a signifi cant increase in fresh and dry weights, leaf area, and total and soluble protein content along with a signifi cant decrease in the insoluble sugar contents was observed in comparison to the WT plants. Chl a, Chl b, total chlorophyll content, Chl a/b ratio, carotenoids content, Hill reaction rate, and chlorophyll fl uorescence didn’t show a signifi cant diff erence between TRR14 and WT plants. The SDS-PAGE gel electrophoresis of the chloroplastic proteins showed a thick band with a molecular mass of 25 kDa in TRR14-overexpressed plants, compared to the WT plants. Remarkably, photorespiration rate was decreased in TRR14 plants compared to WT plants. Conclusion The increased biomass of TRR14 transformed plants might be due to its ability in reducing photorespiration through concentrating CO2 in the leaf’s intercellular spaces.

Methionine Sulfoxide Formation by Cigarette Smoke is Associated with the Degradation of Plasma Proteins

Background: High level of protein carbonyls have been found in plasma proteins in smokers compared to non-smokers. While oxidation may directly interfere with activity, the extent to which oxidation affects protein turnover is less clear.Objectives: To determine levels of oxidized serum proteins cleared in the urine of smokers and non-smokers with focus on methionine sulfoxide (MSO) formation in Human Serum Albumin (HSA) and to determine the effect of methionine oxidation on the turnover of HSA.Method: 100 mL of urine were obtained from smoker and non-smokers. proteins were concentrated by reducing the sample size to 1.5% of the original volume. 200 µL of the concentrate then were separated by SDS-page gel electrophoresis. The band with intact HSA was cut out and the remainder of the gel was cut into four different pieces. Gel sections then were digested with trypsin. Levels of MSO in the resulting peptides were assessed by LC-MS/MS and data analysis was performed using the Skyline software package.Results: A group comparison between non-smokers (control) and smokers showed a slight increase in the levels of MSO found in intact HSA of smokers relative to non-smokers. Regions of gels with proteins of lower mass than intact HSA showed that degraded fragments of HSA in urine of both smokers and non-smokers have higher levels of MSO than are found in intact HSA.Conclusions: HSA in smokers has statistically significant higher levels of MSO than HSA in non-smokers. However, the higher levels of oxidation in smokers are concentrated in partially degraded HSA. At the moment it is not possible to say unequivocally whether oxidized HSA is more likely to be cleaved and cleared, if cleaved protein is more likely to be oxidized before clearance, or both.

Changes in physical, chemical and functional properties of whey protein isolate (WPI) and sugar beet pectin (SBP) conjugates formed by controlled dry-heating

A Maillard type reaction in the dry state was utilized to create conjugates between whey protein isolate (WPI) and sugar beet pectin (SBP) to achieve improved functional properties including solubility, colloidal stability and oil-in-water emulsion stability. To optimize the reaction conditions, mixtures of WPI and SBP at varying weight ratios, 3:1, 2:1 and 1:1, were heated at 60 °C, 79% RH for 72 h. Changes in the physical properties, i.e. solubility, and chemical compositions, i.e. free sulfhydryl and amine contents were assessed using chemical assays. Bradford assay results demonstrated that the protein solubility of WPI increased significantly, ∼20% by reacting with lower levels of SBP (3:1 and 2:1) whereas the total powder solubility was not affected until an equal ratio of SBP was used, and the reduction was only ∼15%. The free sulfhydryl (SH) and primary (NH2) and secondary amine (NH) contents of WPI were also mitigated by conjugating with SBP. The formation of the covalent conjugates between WPI and SBP was confirmed by SDS-PAGE gel electrophoresis through staining for both proteins and glycoproteins. The UV–VIS spectroscopy showed significant changes in the molecular electronic transition states of both WPI and the feruloyl moieties of SBP upon interaction and conjugation. Measurements of ζ-potential, particles size distribution and average particle size demonstrated that the WPI-SBP conjugates prepared at the weight ratio of 3:1 were most effective at stabilizing oil-in-water emulsions than other ratios used in this work.

Covalent bonding of grafted polymer brushes of poly(poly(ethylene glycol) monomethacrylate) on surface of silicon quantum dots and the activation of the end hydroxyls

A technique of covalent bonding of grafted polymer brushes of poly(poly(ethylene glycol) monomethacrylate) (P(PEGMA)) on the surface of silicon quantum dots (SiQDs) and activation of the end hydroxyls with N-hydroxysuccinimide (NHS) esters were described in this work in detail. Firstly, a Si/SiQDs slice was disposed with hydrofluoric acid solution to produce surface Si-Hx (x=1, 2 or 3) species, which combined with vinyl group of 1-undecenol covalently gives terminal hydroxyls. Such hydroxyls were initialized and were involved in atom transfer radical polymerization to construct grafted polymer brushes of P(PEGMA). Hence the density of the surface hydroxyls was enhanced extremely on account of the end hydroxyls of the side chains of the polymer brushes. Then carboxyl groups were introduced via terminal esterification with succinic anhydride, which were activated subsequently with NHS giving NHS esters under rather mild conditions. The reactivity of the terminal NHS esters was demonstrated by SDS-PAGE gel electrophoresis and fluorescence imaging. The SiQDs were demonstrated by transmission electron microscopy. The stepwise modifications on SiQDs were characterized by infrared and X-ray photoelectron spectra. The photoluminescence of the SiQDs was remained during the chemical modifications, proved by the measurements of fluorescence spectra.

Evaluation of Silver Nanoparticles Toxicity against Toxic Black Mold Stachybotrys chartarum

Stachybotrys chartarum is very common in buildings and homes and will grow anywhere indoors where there is moisture. Therefore, in the current study it was isolated from walls with excessive moisture covered with susceptible paint. As an alternative to synthetic fungicides, the use of silver nanoparticles (AgNPs) as antifungal agents has become more widespread. Ag-NPs exhibited a potent antifungal activity against S. chartarum, similar to the antifungal activity of chemical fungicide Carbendazim. Synergistic action was reported when AgNPs was added to chemical fungicide. The inhibition zone in case of 5 ppm of Carbendazim was 12 mm but the addition of 25 ppm, 50 ppm, 75 ppm and 100 ppm of AgNPs increases the inhibition zone to 20 mm, 26 mm, 34 mm and 36 mm respectively. Nine bands of DNA with different molecular weights 8900 bp, 7700 bp, 4600 bp, 2200 bp, 1100 bp, 900 bp, 750 bp, 500 bp and 300 bp were detected in S. chartarum at 50 ppm of AgNPs while one band was detected in untreated fungus with molecular weight 9300 bp indicating that the AgNPs causes DNA fragmentation. SDS-PAGE gel electrophoresis was carried out to monitor the change in gene expression of S. chartarum exposed to AgNPs where the protein bands (15 bands) appeared in control and treated S. chartarum except band number 3 with molecular weight 15.0 KD was detected only in control and shifted to 16.0 KD in treated fungus. Finally, AgNPs application to building materials and walls could effectively protect indoor environments from mould development.

Uptake and reduction of Se(IV) in two heterotrophic aerobic Pseudomonads strains isolated from boreal bog environment.

Selenite (Se(IV), SeO32−) uptake and the effect of selenite supplement on protein synthesis was investigated in two Pseudomonas sp. strains isolated from a boreal bog. These aerobic bacteria efficiently reduced Se(IV) with intracellular reduced Se0 observed in the cytoplasm under dark aerobic conditions. The proteome analysis of Se(IV) supplement and temperature responses by SDS-PAGE gel electrophoresis showed variations in the protein expression on the 40–60 kDa regions following these stress factors, probably through enzymes associated to oxidative stress or temperature adaptation. NO3−/NO2−/SO42− addition enhanced Se(IV) uptake in both bacteria, but Se(IV) uptake sustained also under sulphur and nitrogen starvation. Our findings suggest two different transport mechanisms for Se(IV) uptake in these Pseudomonas sp. strains; a low affinity transport system up-regulated by NO3−/NO2−/SO42− and a distinct Se(IV)O32− regulated transport system. Following transport, Se(IV) is reduced in the cytoplasm, forming Se0 granules, visible in TEM and verified using EDX.

Characterization of Chemically-Induced Bacterial Ghosts (BGs) Using Sodium Hydroxide-Induced Vibrio parahaemolyticus Ghosts (VPGs)

Acellular bacterial ghosts (BGs) are empty non-living bacterial cell envelopes, commonly generated by controlled expression of the cloned lysis gene E of bacteriophage PhiX174. In this study, Vibrio parahaemolyticus ghosts (VPGs) were generated by chemically-induced lysis and the method is based on minimum inhibitory concentration (MIC) of sodium hydroxide (NaOH), acetic acid, boric acid, citric acid, maleic acid, hydrochloric acid, and sulfuric acid. The MIC values of the respective chemicals were 3.125, 6.25, <50.0, 25.0, 6.25, 1.56, and 0.781 mg/mL. Except for boric acid, the lysis efficiency reached more than 99.99% at 5 min after treatment of all chemicals. Among those chemicals, NaOH-induced VPGs appeared completely DNA-free, which was confirmed by quantitative real-time PCR. Besides, lipopolysaccharides (LPS) extracted from the NaOH-induced VPGs showed no distinctive band on SDS-PAGE gel after silver staining. On the other hand, LPS extracted from wild-type bacterial cells, as well as the organic acids-induced VPGs showed triple major bands and LPS extracted from the inorganic acids-induced VPGs showed double bands. It suggests that some surface structures in LPS of the NaOH-induced VPGs may be lost, weakened, or modified by the MIC of NaOH. Nevertheless, Limulus amoebocyte lysate assay revealed that there is no significant difference in endotoxic activity between the NaOH-induced VPGs and wild-type bacterial cells. Macrophages exposed to the NaOH-induced VPGs at 0.5 × 106 CFU/mL showed cell viability of 97.9%, however, the MIC of NaOH did not reduce the cytotoxic effect of wild-type bacterial cells. Like Escherichia coli LPS, the NaOH-induced VPGs are an excellent activator of pro-inflammatory cytokines (IL-1β and iNOS), anti-inflammatory cytokine (IL-10), and dual activities (IL-6) in the stimulated macrophage cells. On the other hand, the induction of TNF-α mRNA was remarkable in the macrophages exposed with wild-type cells. Scanning electron microscopy showed the formation of trans-membrane lysis tunnel structures in the NaOH-induced VPGs. SDS-PAGE and agarose gel electrophoresis also confirmed that cytoplasmic proteins and genomic DNA released from the VPGs to culture medium through the lysis tunnel structures. Taken together, all these data indicate that the NaOH-induced VPGs show the potency of a safe, economical, and effective inactivated bacterial vaccine candidate.

Identifikasi Kerang Kapah Di Pantai Timur Pulau Tarakan

We identified the species of “kerang kapah” (hard clam) using the Gel Electrophoresis SDS Page, inorder to clarify the species of kerang kapah those found living along the East Coast of TarakanIsland. The study was conducted from July to December 2014. The kerang kapah were collectedfrom the several parts of East Coast Tarakan Island. Eight group samples of kerang kapah were used for Gel Electrophoresis SDS Page and scanned using the IMAGE software. Generally, thekerang kapah those found at the shouth part of East Coast of Tarakan Island have very similarmorphologies and shell colors, making species identification difficult. Four species of kerang kapahwere identified. Three species belong to Veneridae and one species belong to Corbiludae. Thespecies of Veneridae were Meretrix meretrix, Meretrix lyrata, and Meretrix sp. The species karangkapah belong to Corbiludae is Polymesoda erusa.Keywords: kerang kapah, meretrix, polymesoda, gel electrophoresis.

Thiol oxidation and di-tyrosine formation in human plasma proteins induced by inflammatory concentrations of hypochlorous acid

In this study, we assessed the oxidative damage occurring in plasma proteins when human blood was exposed to inflammatory concentrations of hypochlorous acid (HOCl). We used specific thiol labelling and Western blot analyses to determine protein thiol oxidation, as well as analytical gel filtration HPLC coupled to fluorescence detection to explore formation of high molecular weight (HMW) protein aggregates. Thiol-containing proteins oxidized by HOCl were identified by redox proteomics. Mass spectrometry (MS) analysis was performed to elucidate the protein composition of HMW aggregates. α1-antitrypsin, transthyretin, and haptoglobin showed thiol oxidation at HOCl concentrations higher than those causing complete oxidation of albumin. At the highest HOCl concentrations, formation of carbonylated and di-tyrosine cross-linked HMW protein aggregates also occurred. MS analysis identified fibrinogen, complement C3 and apolipoprotein A-I as components of HMW protein aggregates. These results could be relevant for human diseases characterized by inflammatory conditions in which myeloperoxidase and HOCl are involved. Biological significanceIn this study we evaluated the oxidative damage occurring on plasma proteins when reconstituted human blood was exposed to inflammatory concentrations of hypochlorous acid (HOCl). Pathophysiological concentrations of HOCl are able to induce different modifications on plasma proteins such as carbonylation, sulfhydryl oxidation and formation of high molecular weight (HMW) protein aggregates characterized by di-tyrosine fluorescence. There are two relevant aspects emerging from this paper. The first one consists on identifying low abundant proteins undergoing sulfhydryl oxidation by biotin-maleimide derivatization followed by MALDI-TOF mass spectrometry. This approach suggests three low-abundant proteins undergoing HOCl-induced oxidation: transthyretin, α1-antitrypsin, and haptoglobin. In addition, we analysed HMW protein aggregates forming after HOCl exposure. These aggregates are characterized by carbonylation, intra- and/or intermolecular di-tyrosine bridges. After their isolation from SDS-PAGE gel electrophoresis, using electrospray tandem mass spectrometry coupled to reversed-phase nanoscale capillary liquid chromatography, we identified some protein constituents of these HMW aggregates such as α, β, γ fibrinogen chains, apolipoprotein A-I and complement C3. In particular, our work highlights how fibrinogen is an important constituent of HOCl-induced HMW protein aggregates validating the mass spectrometry result with additional experiments. Further investigations are required in order to evaluate the possibility to use carbonylated and di-Tyr cross-linked HMW protein aggregates as (early) biomarkers for disease progression in inflammatory conditions in which myeloperoxidase and HOCl are involved.

UV-B Radiation Stress Causes Alterations in Whole Cell Protein Profile and Expression of Certain Genes in the Rice Phyllospheric Bacterium Enterobacter cloacae.

Among the different types of UV radiation, UV-B radiation (280-315 nm) has gained much attention mainly due to its increasing incidence on the Earth’s surface leading to imbalances in natural ecosystems. This study deals with the effects of UV-B radiation on the proteome and gene expression in a rice phyllospheric bacterium, Enterobacter cloacae. Of the five bacteria isolated from rice leaves, E. cloacae showed the highest level of resistance to UV-B and total killing occurred after 8 h of continuous exposure to UV-B. Reactive oxygen species were induced by UV-B exposure and increased with increasing duration of exposure. Protein profiling by SDS-PAGE and 2-dimensional gel electrophoresis (2-DE) revealed major changes in the number as well as expression of proteins. Analysis of 2-DE gel spots indicated up/down-regulation of several proteins under the stress of UV-B radiation. Thirteen differentially expressed proteins including two hypothetical proteins were identified by MALDI-TOF MS and assigned to eight functional categories. Both the hypothetical proteins (gi 779821175 and gi 503938301) were over-expressed after UV-B irradiation; gi 503938301 was characterized as a member of FMN reductase superfamily whereas gi 779821175 seems to be a structural protein as it did not show any functional domain. That the expression of certain proteins under UV-B stress is indeed up-regulated was confirmed by qRT-PCR. Transcript analysis of selected gene including genes of hypothetical proteins (cp011650 and cp002886) showed over-expression under UV-B stress as compared to untreated control cultures. Although this study deals with a limited number of proteins, identification of differentially expressed proteins reported herein may prove useful in future studies especially for assessing their significance in the protection mechanism of bacteria against UV-B radiation stress.

RETRACTED: Production and Characterization of Recombinant Rat Non-collagen Domain of α3 Chain of Type IV Collagen α3 (IV) NC1 Antigen

Glomerulonephritis disease is characterized by inflammation of glomeruli or small blood vessels in the kidney which causes kidney diseases. Glomerulonephritis disease deposits the anti-GBM auto antibody in the glomerular basement membrane. The type IV collagen is the main component of glomerular basement membrane that has α3 chain of type (IV) collagen of non-collagenous domain which contains N-terminal 7S domain, a triple helical collagenous domain, and a C- terminal non-collagenous glomerular domain (NC1). The amino terminal of α3 (IV) NC1 that induces the experimental autoimmuno glomerulonephritis (EAG) in rat model has been identified. The recombinant rat α3 (IV)NC1 antigen has nine amino acid span that is consistent with antibody or T cell epitope which is induced in EAG. The research is carried out on the recombinant rat α3 (IV) NC1 production, purification, quantification, and characterization. The circulation of Anti-GBM antibody in glomerular basement membrane can be measured by the ELISA assay. In addition, the recombinant rat antigen is secreted in HEK293 cell supernatant which is purified by Anti-FLAG M2 monoclonal IgG antibody affinity column. In addition, it is characterized and quantified by SDS-PAGE gel electrophoresis and Western blotting techniques.

Use of dry-moist heat effects to improve the functionality, immunogenicity of whole wheat flour and its application in bread making

Whole wheat flour samples having protein content of 8.9% and 10.6% were subjected to dry and moist heat conditions to improve the functionality. Dry heat treated flours (DHTF) had higher values of falling number and SDS sedimentation values when compared to moist heat treated flour (MHTF). MHTF showed decrease in water absorption from 75.4 to 56.7%, increase in dough development time from 3.3 to 11.9 min, increase in peak viscosity and cold paste viscosity from 467 to 778 BU and 678 to 1017 BU respectively when compared to untreated flour. MHTF lost its elasticity, SDS-page gel electrophoresis indicated the change especially in the region of gliadin and ELISA indicated 41% reduction in immunogenicity against gliadin. The specific volume of breads prepared from MHTF was significantly lower whereas the crumb firmness value was higher than breads from untreated flours. Breads from treated flours also showed reduction in immunogenicity against gliadin.

Production and Characterization of Recombinant Rat Non-Collagen Domain of &lt;i&gt;α&lt;/i&gt;3 Chain of Type IV Collagen &lt;i&gt;α&lt;/i&gt;3 (IV) NC1 Antigen

The glomerulonephritis disease is characterized by inflammation of glomeruli or small blood vessels in the kidney that causes kidney diseases. The reason of glomerulonephritis disease is to deposit the anti-GBM auto antibody in the glomerular basement membrane. The type IV collagen is the main component of glomerular basement membrane that has α3 chain of type (IV) collagen of non-collagenous domain which contains N-terminal 7S domain, a triple helical collagenous domain and C-terminal non-collagenous glomerular domain (NC1). The amino terminal of α3 (IV) NC1 that induces the Experimental Autoimmuno Glomerulonephritis (EAG) in rat model has been identified. The recombinant rat α3 (IV) NC1 antigen has nine amino acid spans that are consistent with antibody or T cell epitope that induces in EAG. The research is carried out on the recombinant rat α3 (IV) NC1 production, purification, quantification, and characterization. The circulation of anti-GBM antibody in glomerular basement membrane can be measured by the ELISA assay. In addition, the recombinant rat antigen is secreted in HEK293 cell supernatant that is purified by Anti-FLAG M2 monoclonal IgG antibody affinity column and characterized and quantified by SDS-PAGE gel electrophoresis and Western blotting techniques.

Screening and identification of SUMP-proteins in sub-acute treatment with diazinon.

Small ubiquitin-like modifiers (SUMOs) are a family of ubiquitin-related, proteins that are involved in a wide variety of signaling pathways. SUMOylation, as a vital post translational modification, regulate protein function in manycellular processes. Diazinon (DZN), an organophosphate insecticide, causses oxidative stress and subsequently programmed cell death in different tissues. The aim of this study was to evaluate the role and pattern of SUMO modificationas a defense mechanism against stress oxidative, in the heart tissuesof the DZN treated rats. Diazinon (15 mg/kg/day), corn oil (control) were administered via gavageto male Wistar rats for four weeks. SUMO1 antibody was covalently crosslinked to protein A/G agarose. heart tissue lysate were added to agarosebeads, After isolation of target proteins(SUMO1- protein)SDS-PAGE gel electrophoresis was performed. Protein bands were identified using MALDI-TOF/TOF and MASCOT). Fold change of (DZN/Ctrl) separated proteins was evaluated using UVband software (UVITEC, UK). Our result showed that subacute exposure to DZN increased SUMOylationoffour key proteins involved in the metabolic process including; Acyl-CoA dehydrogenase, creatine kinase, glyceraldehyde-3-phosphate dehydrogenase and ATP synthase, in the heart tissue of animals. A probability value of less than 0.05 was considered significant (P<0.05). It seems that protein SUMOylation provides a safeguard mechanism against DZN Toxicity.