Coomassie Staining Research Articles

Acid‐Mediated Dissociation of Haemophilus ducreyi Cytolethal Distending Toxin Subunits Upon Cell Entry

Haemophilus ducreyi is a Gram-negative bacterium responsible for the sexually transmitted disease chancroid. It releases a heterotrimeric cytolethal distending toxin (CDT) that contains a catalytic subunit (CdtB) which has a DNAse activity and two binding components (CdtA and CdtC) responsible for entry into the cell. Our hypothesis predicts that the endocytosed holotoxin disassembles through two steps, where CdtA is dissociated from CdtB/CdtC subunits in the late endosomes due to the low pH of this compartment. Afterward, CdtC is released in the endoplasmic reticulum. This allows CdtB to enter the nucleus, causing DNA damage and cell cycle arrest. To study the mechanism of CdtA dissociation, each Cdt subunit was expressed in bacteria transformed with Cdt expression plasmids. Subunit purification was confirmed using SDS-PAGE with Coomassie stain. Afterward, circular dichroism studies tested the structural stabilities of each Cdt subunit at extracellular pH (7.4), early endosome pH (6.3), and late endosome pH (5.2). These structural studies showed that CdtB and CdtC do not undergo significant secondary structure changes at acidic pH. Acid-induces changes were observed with the CdtA subunit, causing its unfolding, aggregation and precipitation at pH 5.2. Additional studies assembled the holotoxin from the individual subunits and introduced it to different pH levels to evaluate the association and dissociation of the subunits using size exclusion chromatography. The results showed that the active holotoxin disassembles at a pH of 5.2, suggesting that the toxin dissociates in the acidified endosomal compartments due to the unfolding of CdtA. Knowledge of the CDT dissociation mechanism could be used to find treatments for chancroid that would prevent toxin function by blocking its disassembly.

Analysis of Periodontitis Biomarker Expression in Gingival Crevicular Fluids

Background: Periodontal disease, also known as gum disease, is a major dental inflammatory disease with a very high prevalence; it is the main cause of tooth loss. Therefore, diagnostic biomarkers that can monitor gum inflammation are important for oral healthcare. Since the gingival crevicular fluid (GCF) adequately reflects changes in the periodontal environment, they have become a target for the development of effective diagnostic biomarkers for periodontitis. In the present study, the level of the target molecules suggested as diagnostic biomarkers for periodontitis were analyzed in GCF samples collected from healthy individuals and periodontitis patients. In addition, useful targets for the diagnosis of periodontitis were evaluated. Methods: GCF samples were collected from healthy individuals and periodontitis patients using absorbent paper points. SDS-PAGE and Coomassie staining were performed for protein analysis. The protein concentrations of GCF specimens were determined using the Bradford method. The levels of the target molecules appropriate for diagnosing periodontal disease were measured by ELISA, according to the manufacturer’s protocol. Results: The protein concentration of GCF collected from periodontitis patients was 3.72 fold higher than that in an equal volume of GCF collected from healthy individuals. ELISA analysis showed that the level of interukin-6 (IL-6), IL-8, metalloproteinases 2 (MMP-2), MMP-9, tumor necrosis factor-alpha (TNF-α), azurocidin, and odontogenic ameloblast-associated protein (ODAM) were higher in the GCF samples from the periodontitis patients than in those from the healthy individuals. However, the level of IL-6 and TNF-α were relatively low (＞ 5 pg/ml). The prostaglandin E2 (PGE2) levels were not significantly different between the two GCF samples. Conclusion: These results indicate that IL-8, MMP-2, MMP-9, azurocidin, and ODAM are potentially useful diagnostic biomarkers for periodontitis; combining multiple biomarkers will improve the diagnostic accuracy of periodontitis.

Pores Formation in Porcine Sperm Incubated in Streptolysin O and Its Post Thawing Viability after Trehalose Treatment

Background: Streptolysin O (SLO), a pore-forming protein in plasma membrane (PM), has been used to internalize a variety of molecules (DNA and RNA) in cells. In sperm, however, SLO has only been used to release acrosomal contents. Its possible use as biotechnology in the cryopreservation of pig semen. However, porcine sperm are very sensitive to the freeze-thaw process. The study aimed to evaluate the pore formation in the PM, the addition of trehalose and the post cryopreservation viability of porcine spermatozoa using SLO.Methods: Research period was spring 2017- summer 2018. Thirty ejaculates from five mature boars were used. Semen was incubated in SLO 0.6 IU/ml and trehalose (added at 100, 200 and 400 ìM). Semen diluted in commercial diluent as control group. Presence of pores was checked by scanning electronic microscopy. To evaluate sperm membrane integrity and functional status the Coomassie stain with HOST test and the Chlortetracycline test were used. Result: It was found that SLO could form pores in the sperm cell membrane The addiction of 200 ìM trehalose to the freezing medium have different effects on the quality of boar sperm, showing highest motility and viability during the cooling process.

Comprehensive analysis of the isozyme composition of laccase derived from Japanese lacquer tree, Toxicodendron vernicifluum

We performed an analysis using isoelectric focusing to comprehensively clarify the isozyme composition of laccase derived from Japanese lacquer tree, Toxicodendron vernicifluum. When water extracts of acetone powder obtained from lacquer were subjected to isoelectric focusing, five bands within pI 7.35–9.30 and nine bands within pI 3.50–5.25 were detected using Coomassie staining. Similarly, laccase activity staining using guaiacol showed five bands within pI 7.35–9.30 and three bands within pI 3.50–4.25. However, laccase activity staining using gallic acid showed remarkable staining within pI 3.50–5.85, whereas staining was very weak within pI 7.35–9.30. When the water extracts of acetone powder were fractionated into the fractions containing bands within pI 7.35–9.30 and pI 3.50–5.85 by SP-Sepharose column chromatography, the former had a blue color and the latter a yellow color. The laccase activity was measured for each of the fractions in buffer solution in the pH range of 2.5–8.0. When syringaldazine, guaiacol, and 2,6-dimethoxyphenol were used as substrates, the yellow fraction showed considerably higher activity than the blue fraction for pH 5.5–7.5. When 3-methylcatechol and 4-methylcatechol were used as substrates, the yellow fraction showed higher activity for pH 4.5–6.5, and the blue fraction showed higher activity for pH 7.0–8.0. When 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) was used as the substrate, both fractions showed maximum activity at optimum pH of 3.0–4.0. Conventionally, in research on blue laccase derived from lacquer, the non-blue fraction corresponding to the yellow fraction lower than pI 6 has been removed during the purification process and thus has not been analyzed. Our results indicated that yellow laccase was present in the non-blue components of lacquer and that it may play a role in urushiol polymerization with previously reported blue laccase.

Effects of fermentation with Bacillus natto on the allergenicity of peanut

In the present study, we evaluated the immunoreactivity reduction of raw peanut pulp (RPP) by using a combination protocol of autoclave and fermentation with Bacillus natto. Assessing through an indirect ELISA, we found that the method reduced more than 77.3% of the IgE reactivity in peanut protein preparations. Further analyses with SDS-PAGE and Coomassie staining revealed that sequential autoclave and fermentation for different time caused gradually degradation of high molecular weight peanut proteins. Results from Circular dichroism and Ultraviolet absorption spectroscopies indicated that the autoclave and fermentation induced extensive proteolysis, unfolding and conformational changes, and reduction of α-helices, which all affected epitopes formation significantly. The effects of the method on peanut protein structure were further proved by the changes of surface hydrophobicity, assessed by ANS fluoresence probe, and by the alteration of free sulfhydryl groups, measured by SH-interacting DTNB. Interestingly, during the process of fermentation for 48 h, the immunoreactivity reduction of RPP was positively correlated with the content of free sulfhydryl group and negatively correlated with UV absorption intensity at 210 nm. Therefore, autoclaving assisted fermentation with Bacillus natto was an effective procedure to diminish the allergens in raw peanuts.

High Sensitivity Protein Gel Electrophoresis Label Compatible with Mass-Spectrometry.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a widely utilized technique for macromolecule and protein analysis. While multiple methods exist to visualize the separated protein bands on gels, one of most popular methods of staining the proteins is with Coomassie dye. A more recent approach is to use Bio-Rad stain-free technology for visualizing protein bands with UV light and achieve similar or greater sensitivity than that of Coomassie dye. Here, we developed a method to further amplify the sensitivity of stain-free gels using carboxyfluorescein succinimidyl ester (CFSE) staining. We compared our novel method using foetal bovine serum samples with Coomassie dye, standard stain-free gels, and silver staining. Our results show that while silver staining remains a gold-standard method in terms of sensitivity; CFSE staining of samples prior to use with stain-free gels results in a 10–100-fold increase in sensitivity over Coomassie staining and the standard stain-free method. Our method offers a sensitivity similar to that of silver staining which is compatible with downstream mass spectrometry, and therefore more advantageous for further retrieval and analysis of macromolecules in bands.

Effects Of High-Load Versus High-Volume Resistance Training On Muscle Sarcoplasmic, Actin, And Myosin Protein Concentrations

PURPOSE: Our laboratory has recently shown high-volume resistance training (RT) can elicit increases in skeletal muscle sarcoplasmic protein concentrations, while also causing a dilution of contractile protein concentrations (i.e., sarcoplasmic hypertrophy). The purpose of this study was to evaluate effects of 6 weeks of high-load (HL) and high-volume (HV) training on skeletal muscle sarcoplasmic and contractile protein concentrations. METHODS: Trained college-aged males (n = 15; training age = 7 ± 3 yrs; mean 1RM squat relative to bodyweight = 1.9 ± 0.4 kg) performed 6 weeks of unilateral lower-body RT, with one leg performing HV training and the contralateral leg performing HL training using leg press and leg extension. Participants underwent a period of passive recovery lasting 10 days following the training intervention. Vastus lateralis biopsies were obtained from both legs prior to the start of training (PRE), 72 hours following the last training day (POST), and 1 week following POST testing (POSTPR). Sarcoplasmic protein content was determined following differential centrifugation using bicinchoninic assays, and actin and myosin concentrations were quantified using SDS-PAGE and Coomassie staining. RESULTS: Significant main effects of time (p = 0.022) and condition (p=0.002) were observed and condition by time approached significance for sarcoplasmic protein concentrations (p = 0.088). There were no significant interactions or main effects for actin or myosin concentrations. CONCLUSIONS: Contrary to our prior data, sarcoplasmic, actin and myosin concentrations remained unaffected with HV training. However, interesting trends were observed for sarcoplasmic protein concentrations and these will be further interogated.

Consumer and health-related traits of seed from selected commercial and breeding lines of industrial hemp, Cannabis sativa L.

Global food security and sustainability can be enhanced with increased production and use of hemp seed ( Cannabis sativa L.) that not only provides healthy sources of protein and oil, but the whole plant can be used for fiber, especially new applications including high-performance textiles. There is limited knowledge of the variation in hemp seed for health and consumer traits, such as the complex plant polysaccharides comprising dietary fiber. To fill this gap, seed from 20 hemp cultivars and advanced breeding lines were analyzed for a variety of traits including complex carbohydrates, protein, lipids, heart percent, phytate and lignin. The analyses revealed different polysaccharides in hull and heart fractions, with hulls varying in cellulose (22.0–36.7%) and xylan (5.7–17.1%) whereas heart fractions contained xyloglucan and pectin, and comprised different proportions of heart and hull. Staining revealed that cellulose and lignin are localized in hull not heart tissue. Protein content of hemp seed ranged from 19.5 to 26.9% and Coomassie staining showed that cells of hemp hearts contain numerous protein bodies but few starch granules. Analysis of selected lines confirmed that hemp hearts are low in starch (<2%). Lipid content varied from 26.6 to 37.8%, with the omega-6:3 ratio varying from 2.1 to 4.9 with seven lines having a ‘desirable’ omega-6:3 ratio of less than or equal to three. This research provides breeders with additional traits to consider for maximizing consumer satisfaction and human health. • Hemp cultivars and breeding lines have variation for omega-3 α-linolenic acid. • Compositional analysis of heart (kernel) and hull (husk) fractions of hemp seed. • Hemp seed contain non-cellulosic dietary fiber including xylan, xyloglucan & pectin. • Hemp hearts are low in starch (<2%) and soluble sugars. • Hemp proteins are found in protein bodies.

Abstract TMP120: Autoantigen(s) Trigger a Robust Immune Response in Cerebral Cavernous Malformations

Introduction: Previous studies have reported robust inflammatory cell infiltration, selective synthesis of IgG, B-cell clonal expansion, and deposition of immune complexes and complement within Cerebral Cavernous Malformation (CCM) lesions. Furthermore,B-cell depletion has been shown to reduce the maturation of CCM in murine models. We hypothesize that specific autoantigen(s) within the lesional milieu trigger the pathogenetic immune responses in CCMs. This study aims to identify those putative autoantigen(s) using recombinant antibodies (rAbs) derived from plasma cells found in surgical human CCM lesions. Methods: CD138 + plasma cells were laser captured from fresh frozen surgically resected human CCM lesions. Clonally expanded immunoglobulin heavy- and light-chain variable region pairs were cloned into IgG expression vectors and expressed as monoclonal antibodies. Purified rAbs were assayed by immunofluorescence with CCM lesion tissue and normal brain tissue sections. rAbs assayed by immunocytochemistry with human primary cell line were used to further define the staining pattern. The cell lysates were immunoprecipitated with rAb, after protein purification by SDS-PAGE, and analyzed by Mass spectrometry. Results: In normal brain tissue, rAbs stained endothelial cells with limited staining of glial cells. In CCM lesional tissue, rAbs stained endothelial cells, glial cells as well as structures in the acellular matrix adjacent to caverns. In cultured Human Brain Microvascular Endothelial Cells (HBMECs) and Human Astrocytes (HAs), rAbs co-localized with cytoplasmic components. After HBMEC and HA cell lysates were immunoprecipitated with rAb, a Coomassie Stain detected bands of approximately 50 kDa. Conclusions: Our results suggest that autoantigen(s) in human CCM lesions are cytoplasmic components present in lesional tissue as well as in normal brain tissue. Molecular level identification of the triggering antigen is still ongoing by mass spectrometry. Identification of the autoantigen(s) in the lesional milieu might explain the propensity of lesion development from leaky endothelium in the neuroglial parenchyma. Characterization of the autoantigen triggers will open new venues for therapy or vaccine in this disease.

Characterization of the Active Components of the Multimerized sTNFRIIAdiponectin Fusion Protein Showing Both TNFα-Antagonizing and Glucose Uptake-Promoting Activities.

The sTNFRII-adiponectin fusion protein previously showed strong TNFα antagonistic activity. However, the fusion protein exists as mixture of different multimers. The aim of the present study was to characterize its active components. In this study, the fusion protein was isolated and purified by Ni-NTA affinity and gel exclusion chromatography, and further identified by Coomassie staining and western blotting. The TNFα antagonistic and glucose uptake-promoting activities were determined in vitro. The glucose detection kit and 2- NBDG (2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose) were used to measure their effects on glucose metabolism (including glucose consumption and glucose uptake in HepG2 and H9C2 cells). The effect of the fusion protein on glucose uptake was also examined in free fatty acid (FFA)- induced insulin resistance cell model. The sTNFRII-adiponectin fusion protein was found to exist in three forms: 250 kDa (hexamer), 130 kDa (trimer), and 60 kDa (monomer), with the final purity of 90.2%, 60.1%, and 81.6%, respectively. The fusion protein could effectively antagonize the killing effect of TNFα in L929 cells, and the multimer was found to be superior to the monomer. In addition, the fusion protein could increase glucose consumption without impacting the number of cells (HepG2, H9C2 cells) in a dosedependent manner. Mechanistically, glucose uptake was found to be enhanced by the translocation of GLUT4. However, it could not improve glucose uptake in the cell model of insulin resistance. In summary, the active components of the fusion protein are hexamers and trimers. The hexamer and trimer of sTNFRII-adiponectin fusion protein had both TNFα-antagonizing and glucose uptake-promoting activities, although neither of them could improve glucose uptake in the cell model of insulin resistance.

Skeletal Muscle Myofibrillar Protein Abundance Is Higher in Resistance-Trained Men, and Aging in the Absence of Training May Have an Opposite Effect.

Resistance training generally increases skeletal muscle hypertrophy, whereas aging is associated with a loss in muscle mass. Interestingly, select studies suggest that aging, as well as resistance training, may lead to a reduction in the abundance of skeletal muscle myofibrillar (or contractile) protein (per mg tissue). Proteomic interrogations have also demonstrated that aging, as well as weeks to months of resistance training, lead to appreciable alterations in the muscle proteome. Given this evidence, the purpose of this small pilot study was to examine total myofibrillar as well as total sarcoplasmic protein concentrations (per mg wet muscle) from the vastus lateralis muscle of males who were younger and resistance-trained (denoted as YT, n = 6, 25 ± 4 years old, 10 ± 3 self-reported years of training), younger and untrained (denoted as YU, n = 6, 21 ± 1 years old), and older and untrained (denoted as OU, n = 6, 62 ± 8 years old). The relative abundances of actin and myosin heavy chain (per mg tissue) were also examined using SDS-PAGE and Coomassie staining, and shotgun proteomics was used to interrogate the abundances of individual sarcoplasmic and myofibrillar proteins between cohorts. Whole-body fat-free mass (YT > YU = OU), VL thickness (YT > YU = OU), and leg extensor peak torque (YT > YU = OU) differed between groups (p < 0.05). Total myofibrillar protein concentrations were greater in YT versus OU (p = 0.005), but were not different between YT versus YU (p = 0.325). The abundances of actin and myosin heavy chain were greater in YT versus YU (p < 0.05) and OU (p < 0.001). Total sarcoplasmic protein concentrations were not different between groups. While proteomics indicated that marginal differences existed for individual myofibrillar and sarcoplasmic proteins between YT versus other groups, age-related differences were more prominent for myofibrillar proteins (YT = YU > OU, p < 0.05: 7 proteins; OU > YT = YU, p < 0.05: 11 proteins) and sarcoplasmic proteins (YT = YU > OU, p < 0.05: 8 proteins; OU > YT&YU, p < 0.05: 29 proteins). In summary, our data suggest that modest (~9%) myofibrillar protein packing (on a per mg muscle basis) was evident in the YT group. This study also provides further evidence to suggest that notable skeletal muscle proteome differences exist between younger and older humans. However, given that our n-sizes are low, these results only provide a preliminary phenotyping of the reported protein and proteomic variables.

In vitro Glutamylation Inhibition of Ubiquitin Modification and Phosphoribosyl-Ubiquitin Ligation Mediated by Legionella pneumophila Effectors.

Glutamylation is a posttranslational modification where the amino group of a free glutamate amino acid is conjugated to the carboxyl group of a glutamate side chain within a target protein. SidJ is a Legionella kinase-like protein that has recently been identified to perform protein polyglutamylation of the Legionella SdeA Phosphoribosyl-Ubiquitin (PR-Ub) ligase to inhibit SdeA's activity. The attachment of multiple glutamate amino acids to the catalytic glutamate residue of SdeA by SidJ inhibits SdeA's modification of ubiquitin (Ub) and ligation activity. In this protocol, we will discuss a SidJ non-radioactive, in vitro glutamylation assay using its substrate SdeA. This will also include a second reaction to assay the inhibition of SdeA by using both modification of free Ub and ligation of ADP-ribosylated Ubiquitin (ADPR-Ub) to SdeA's substrate Rab33b. Prior to the identification and publication of SidJ's activity, no SdeA inhibition assays existed. Our group and others have demonstrated various methods to display inhibition of SdeA's activity. The alternatives include measurement of ADP-ribosylation of Ub using radioactive NAD, NAD hydrolysis, and Western blot analysis of HA-Ub ligation by SdeA. This protocol will describe the inhibition of both ubiquitin modification and the PR-Ub ligation by SdeA using inexpensive standard gels and Coomassie staining.

Dynamic Stretching of Fibrillar Collagen Enhances Cross-linking by Transglutaminas

Category:Basic Sciences/BiologicsIntroduction/Purpose:New approaches to improve tendon repair after injury are an active area of research. Critical properties of tendons are governed by the production and assembly of fibrillar collagens. Cross-linking of fibrillar collagen is a primary factor in determining the function and mechanical properties of the collagen fibers comprising Enzymatic cross-linking by lysyl oxidase in the telopeptide domain of collagen I and III is one determinant of collagen fibril assembly and is the best characterized biochemical cross-link. Transglutaminase catalyzes the modification of lysine residues that in turn form an n-e-glutamyl lysine bond between proteins in the extracellular space. We hypothesize that transglutaminase-dependent modification of collagen in tendons is also a principal determinant of tendon strength and function and is dependent upon tension.Methods:3-D collagen gels were generated from acid solubilized type I collagen with telopeptides (Advanced BioMatrix). Collagen gels were plated and loaded into a MechanoCulture FX apparatus (CellScale). Gels were subjected to a 10% stretch for 24 hrs at 37°C at 2hz (dynamic) or no stretch, static controls. Gels exposed to enzymatic cross-linking were incubated with either 2.4 ng of recombinant Transglutaminase 2 (Axxora) in a 10 mM Ca2+ solution. Inhibition and labeling of transglutaminase substrates was performed by incubation of collagen gels with 0.2 mM aminopentyl biotinamide in DMSO. Soluble collagen was separated from insoluble collagen by centrifugation at 10,000G. Insoluble fractions were boiled in SDS-Laemmli buffer prior to separation by SDS-PAGE. Collagen in soluble and insoluble fractions was evaluated by Coomassie stain whereas transglutaminase modification was detected via western blot using streptavidin conjugated horse radish peroxidase to detect biotinylated proteins.Results:Evaluation of collagen gels subjected to dynamic versus static stretch revealed minor differences in insoluble collagen incorporation in the two conditions. Notably, higher molecular weight cross-linked forms of collagen appeared to be higher in dynamic versus static gels. In the presence of transglutaminase, differences in higher molecular weight cross-linked forms of collagen, beta-bands, were also detected. Finally, incorporation of biotinylated transglutaminase substrate into collagen alpha bands was enriched in dynamic versus static cultures. Hence, preliminary results support a differential role for transglutaminase modification in collagen under cyclic tension versus static conditions.Conclusion:A better understanding of the role of dynamic stretching and differential tension in the regulation of collagen cross- link formation is predicted to contribute to improved strategies to treat injured tendons.

An Inexpensive Staining Alternative for Gelatin Zymography Gels.

Zymography is a widely used electrophoretic method to determine proteolytic activities in samples from various sources. The method is based on copolymerizing a suitable protein substrate within a sodium dodecyl sulfate-polyacrylamide gel. Following electrophoretic separation of the protease containing samples and a suitable incubation period, degradation of the substrate can be visualized through staining with Coomassie blue. Sites of proteolysis become visible as white bands on a dark blue background. However, this staining protocol requires considerable amounts of ethanol and acetic acid to remove unbound dye molecules. In this report, we describe a new staining protocol using Ponceau S which offers substantial advantages in terms of assay usability and cost reduction, especially when performing large quantities of zymograms or in resource-limited settings. Fast and reproducible staining of zymograms with our protocol is demonstrated, and reliable quantitation of proteolytic activity in comparison to the standard Coomassie staining procedure is shown.

Abstract 3008: PSMA glycosylation and aggressive prostate cancer progression

Abstract Introduction: Prostate cancer has a heterogeneous spectrum of clinical outcomes with phenotypes ranging from indolent asymptomatic cases to very aggressive metastatic and lethal forms. Our long term objective is to identify molecules and characterize mechanisms for PCa progression to aggressive lethal disease. PSMA, a membrane bound glycoprotein is significantly over-expressed in more aggressive and metastatic prostate carcinomas with a negative correlation with patient prognosis. The role of its glycans in disease progression and in the development of biomarkers for the disease and potential therapeutics are currently unknown. Aberrant glycosylation of glycoproteins has been observed in many malignancies, and these changes influence disease progression. Our rationale is that differential glycosylation of PSMA correlates with PCa aggressive disease progression. In this study, we characterize N-linked glycosylation profiles in disease stratified PCa cells and prostate proximal fluids show differential expression of specific PSMA conjugated glycan structures that correlates with aggressive phenotypes. Methods/Results: PSMA was isolation from PCa cell lysates and post-DRE urines by column based affinity chromatography followed by SDS-PAGE and Western Blot and Coomassie staining. The fractionated PSMA protein band was excised and processed for proteomic and glycomic analysis. Isolated PSMA was trypsin digested and treated with PNGaseF and other glycosidases to cleave of N-linked glycans from the peptides. Isolated glycans were subjected to permethylation before MALDI-TOF-MS and high resolution, high mass accuracy and high sensitivity LC/MS/MS on an Orbitrap Fusion Lumos Tribrid MS instrument with multiple fragmentation capabilities (CID, HCD, ETD and EThcD) for glycan identification and quantitation. Validation of differential glycan expression was further performed by targeted glycan based MS and lectin based biochemical technologies. Conclusions: Our preliminary studies show that there is differential expression of PSMA N-linked glycans with disease severity in prostate cancer cells and post DRE urine samples. Further analyses are currently underway using an extensive cohort of post DRE urine samples and additional studies are focusing on uncovering the mechanisms that are modulated by PSMA glycosylation that are responsible for PCa disease progression. These could assist in the development of novel diagnostic and therapeutic strategies for the disease. Citation Format: Tanya C. Burch, Stephen S. Mackay, Ian O. Oduor, Joseph J. Otto, Raymond S. Lance, Dean A. Troyer, Oliver John Semmes, Julius O. Nyalwidhe. PSMA glycosylation and aggressive prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3008.

Effect of Oral Spray on Dental Plaque Bacteria and Oral Epithelial Cells

Background: Good oral health is important for systemic body health and quality of life. Spray oral cleansers are increasingly preferred because of their convenience of carrying and the ease of oral hygiene management. In addition, many kinds of oral cleanser products containing various ingredients with antibacterial, washing, and moisturizing effects are being manufactured. However, concerns about the safety and side effects of oral sprays are increasing, and there is very little information regarding the use and care of oral sprays is available to consumers. This study aimed to investigate the effects of oral spray on oral bacteria and tissue to elucidate the factors that need to be considered when using oral sprays. Methods: The effects of oral spray on the growth of dental plaque bacteria was assessed using disk diffusion assays. Cytotoxicity and morphological changes in oral epithelial cells were observed by microscopy. The effects of oral spray on dental plaque growth were also confirmed on specimens from permanent incisors of bovines by Coomassie staining. Results: The pH of spray products, such as Perioe Dental Cooling, Cool Sense, and Dentrix, were 3.65, 3.61, and 6.15, respectively. All tested spray products showed strong toxicity to dental plaque bacteria and oral epithelial cells. Compared with those on the control, dental plaque bacteria deposits on the enamel surface increased following the use of oral spray. Conclusion: Three types of oral spray, namely Perioe Dental Cooling, Cool Sense, and Dentrix, strongly inhibited the growth of dental plaque bacteria and oral epithelial cells. The oral spray ingredient enhanced dental plaque growth on the enamel surface. Users should be informed of precautions when using oral sprays and the need for oral hygiene after its use.

SAT-558 Tsh Modulation Of Bone Biology - Further Evidence From A Recombinant Tsh-β Variant.

We have previously shown that TSH has modulatory influences on osteoblasts and osteoclasts and can act as a bone protection ligand. We have also shown that a novel TSH-β variant protein is produced locally by murine and human bone marrow macrophages (Endocrinology 154:4919-26, 2013 and Endocrinology 157:3658-67, 2016). Furthermore, we have also shown by molecular modelling that both TSH-β and TSH-βv can bind to the native TSH receptor. In order to characterize the biological activity of this TSH-β variant we have inserted the gene sequence together with a FLAG tag into the P-secTag2 vector which was then cloned into mammalian HEK-293 cells. Western blot analysis identified 17KD and a 12KD proteins corresponding to the wild type TSH-β and the TSH-βv nucleotide sequence in cell culture lysates and, less so in supernatants, by using a FLAG monoclonal antibody and a TSH-β polyclonal antibody. Protein bands of ~34 KD and 24 KD were observed only in the absence of DTT suggesting dimeric forms of TSH-β and TSH-βv respectively. Detection by Coomassie staining followed by sequence analysis using mass spectrometry confirmed the identity of these proteins after column purification. Furthermore, cyclic AMP responses were generated by both supernatant preparations with an 8-fold increase in luciferase activity with TSH-β and a 4-fold increase with TSH-βv. These data demonstrate the inherent bioactivity of single TSH-β chains which was mirrored by TSH-βv. Local paracrine action by macrophage derived TSH-βv on osteoblasts and osteoclasts is, therefore, most likely and raises the possibility of this variant form having significant osteoprotective activity.

Morphology, Protein Profile and the Level of Infestation of Bedbugs (Hempitera: Cimicidae) in the Halls of Residence of a Higher Institution in Lagos.

Morphology, protein profile and the level of Bedbug infestations were carried out at a University in Lagos, Nigeria after an outcry of high infestation by the students in halls of residence. Three bedbugswere each collected from nine halls of residence for morphological characterization of adults and to carry out protein profile analysis usingSodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Coomassie staining method. Level of infestation of Bedbugs was carried out in two female halls A and Busing blood smeared on the walls. All bedbugs found were Cimex hemipterus. 29.8% of the rooms in Hall Bhad no bedbug infestation, 23.9%, 20.2%, 15.4% and 10.6% had low, average, high and very high levels of bedbug infestation respectively while in A hall, it was 21.4%, 30.5%, 24.4%, 14.5% and 9.2% respectively. In A hall, 4.6%, 16.8% and 0.8% had one, two and three out of the four mattresses in the room infested with bedbugs respectively while 56.5% had all the four mattresses infested with bedbugs. In B hall, 1.6%, 16.0% and 0% had one, two and three mattresses infested with bedbugs respectively while the remaining 52.7% had all the four mattresses in the room infested with bedbugs. There was a significant relationship between the level of infestation and the number of mattresses infested in each hall (P &lt; 0.01).The protein profile analysis of bedbugs did not show the protein bands clearly because of the low soluble protein content of Cimex hemipterus and the detection limit of Coomassie stain.

Beneficial effects of Coomassie staining on proteomic analysis employing PAGE separation followed with whole-gel slicing, in-gel digestion and quantitative LC-MS/MS

Proteomic analysis by combining PAGE separation, gel slicing and slice-by-slice in-gel digestion and LC-MS/MS has been frequently reported in recent years. Since the MS analysis would provide identities and quantities of the proteins along the whole lane, gel visualization by dye staining could be skipped to save time and labor. In this work, we examined the effects of CBB R-250 staining on the performance of the method and the results showed actually better results were obtained with CBB staining than without, both in nondenaturing PAGE and SDS-PAGE. A primary examination was firstly performed with excised gel bands of purified proteins and the LC-MS/MS results showed that almost all the proteins were detected with higher sequence coverages and quantities from the stained bands than from the unstained. Then a proteomic sample of rat heart soluble proteins was examined for the complete workflow. The sample was separated by both nondenaturing PAGE and SDS-PAGE and the gels were divided to halves for CBB staining and fixation without staining, respectively. Multi-blade cutters were used to simultaneously cut lanes and then slice each lane into about forty squares of the same size. All the gel pieces were analyzed in standard procedures of in-gel digestion, peptide extraction and label-free quantitative LC-MS/MS. The results showed more proteins, about 40% in nondenaturing PAGE and 18% in SDS-PAGE, were detected from the CBB-stained lanes than from the unstained ones. Examination on the detected quantities and square numbers of individual proteins also confirmed about the better detection with CBB staining. As the data showed the detection of proteins with lower molecular masses (e.g. <30 kDa) were more benefited by the staining, we speculate the dye binding might help retaining of the proteins in the gel matrix.

Detection of Posttranslational Modifications by Fluorescent Staining of Two-Dimensional Gels.

Posttranslational modifications (PTMs) are key to the regulation of functional activities of proteins. Quantitative and qualitative information about PTM stages of proteins is crucial for the discovery of disease biomarkers. Fluorescent dyes specifically staining protein PTMs such as phosphorylation and glycosylation enable the specific detection of protein regulations taking place with respect to these modifications. Activity and molecular interactions of many proteins are determined by their extent of phosphorylation. In our search for biomarkers of neurodegenerative diseases such as multiple sclerosis (MS), using an animal model, experimental autoimmune encephalomyelitis (EAE), we have applied the phosphorylation-specific fluorescent dye, ProQ Diamond, to study changes taking place in the phosphoproteome. Subsequent colloidal Coomassie staining of the same gels detects the changes at the whole proteome level. We have detected many changes taking place in the CNS tissue of the EAE animals at the whole proteome as well as at the phosphoproteome level resulting in valuable insights into the pathophysiological mechanism of EAE and MS.