High Molecular Weight Bands Research Articles

ANTIMICROBIAL ACTIVITY OF LECTIN ISOLATED FROM EGYPTIAN WHEAT CULTIVAR GIZA 171

In the current study crude lectin was isolated from Egyptian wheat seeds cultivarGiza 171 and it was characterized by several methods such as sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE), Native-PAGE, Iso-electric point evaluation, Fourier transforminfrared (FTIR) spectroscopy and amino acids composition. Furthermore, antibacterial and antifungalactivities were estimated against some gram positive, gram negative bacteria and pathogenic fungus.Based on Native-PAGE (unreduced) of wheat seeds lectin indicates one major band with a molecularweight of approximately 50 kDa. SDS-PAGE of wheat seeds lectin separated this high molecularweight band into 2 bands corresponding to smaller molecular weights (14,20 kDa). The iso-electricpoints were deduced from the protein pH-solubility curve. It is clear that the least soluble points wereobtained at pH 5.5. The content of the hydrophobic amino acids residues (proline, valine, isoleucine,leucine and phenyl alanine) valued 27.8% and this represent 31.1% of the total amino acids. Theminimum inhibitory concentrations (MIC) of lectin against L. monocytogenes and P. aeruginosa was800 μg/ml, 1600 μg/ml for B. subtillis and 3200 μg/ml against E. coli. Lectin inhibited mycelialgrowth of F. oxysporun and F. solani at a wide concentration range (250, 500 and 1000 μg/ml). Thisstudy has elucidated that the lectin isolated from wheat seeds cultivar Giza 171 have the potentantibacterial and antifungal activities against selected pathogenic bacteria and fungai.

Finding of bands of higher molecular weight than expected in three proteins in bovine preimplantation embryos

SummaryWe report here the existence of bands of higher molecular weight after western blot analysis in three proteins - Skp1, p27 and IκBα in bovine preimplantation embryos. This finding is specific to preimplantation embryos (from the 2-cell stage to the blastocyst stage) and not differentiated fibroblast cells in which these bands were of expected molecular weight. We suggest that these bands of higher molecular weight represent a complex of proteins that are characteristic of preimplantation embryos.

An App knock-in mouse inducing the formation of a toxic conformer of Aβ as a model for evaluating only oligomer-induced cognitive decline in Alzheimer's disease

Irie and colleagues identified a “toxic conformer”, which possesses a turn structure at positions 22-23, among various conformations of Aβ and have been reporting its potent oligomeric capacity and neurotoxicity. This toxic conformer was detected in the brains of AD patients and AD model mice (Tg2576 line), and passive immunization targeting this conformer ameliorated the cognitive dysfunction in an AD model. In this study, we developed a novel AD mouse model (AppNL-P-F/NL-P-F) with Swedish mutation (NL), Iberian mutation (F), and mutation (P) overproducing E22P-Aβ, a mimic of the toxic conformer, utilizing the knock-in technique that well recapitulates the Aβ pathology of AD patients in mice and avoids the artificial phenotype observed in transgenic-type model mice. We confirmed that AppNL-P-F/NL-P-F mice produce Aβ by ELISA and accumulate senile plaques by immunohistochemistry at eight months of age. In WB, we observed a potential trimer band and high molecular-weight oligomer bands without a monomeric band in the TBS-soluble fraction of AppNL-P-F/NL-P-F mice at six months of age. In the novel object recognition test, cognitive impairment was observed at six months of age in these mice. These findings suggest that the toxic conformer of Aβ induces cognitive dysfunction mediated by its oligomer formation in this mouse brain. AppNL-P-F/NL-P-F mice may be a useful model for evaluating Aβ oligomer-induced cognitive impairment in AD and will aid in exploring therapeutic targets for AD pathology.

Tough aged meat presents greater expression of calpastatin, which presents postmortem protein profile and tenderization related to Nellore steer temperament

Eighteen Nellore steers were classified as either excitable (high index) or calm (low index) temperament, and high or low WB Shear Force (SF) in Longissimus lumborum at 16 days postmortem, with a subset assigned to four groups: HTHS–high temperament and SF (n = 5); HTLS–high temperament and low SF (n = 4); LTHS–low temperament and high SF (n = 4); LTLS–low temperament and SF (n = 5). Only the second calpastatin peak was detected at 48 h in the all samples, and the calpastatin activity only differed (P < .05) between shear force groups. High WBSF groups had greater expression of CAST gene (P < .05) in the high and low temperament. Western blots showed a calpastatin fragmentation pattern very particular to each group with high molecular weight bands present in the HTHS. Temperament was not a determinant factor in postmortem proteolysis variation. However, greater WBSF aged meat from high temperament animals had greater calpastatin activity and less calpastatin fragments formed during the postmortem period analyzed.

Di-(2-Ethylhexyl) Phthalate Promotes Release of Tissue Factor-Bearing Microparticles From Macrophages via the TGFβ1/Smad/PAI-1 Signaling Pathway

BackgroundPlasminogen activator inhibitor type 1 promotes formation of endothelial microparticles with procoagulant activity. However, it remains unclear whether di-(2-ethylhexyl) phthalate, a peroxisome proliferator-activated receptor α agonist, influences microparticle formation. Materials and MethodsThe effect of di-(2-ethylhexyl) phthalate on release of tissue factor-bearing microparticles was investigated using human M1 macrophages. ResultsExposure of M1 macrophages to di-(2-ethylhexyl) phthalate significantly upregulated expression of plasminogen activator inhibitor type 1, whereas incubation of macrophages with small interfering RNA for peroxisome proliferator-activated receptor α attenuated it. Di-(2-ethylhexyl) phthalate significantly increased the tissue factor protein level in culture supernatants of M1 macrophages, but not M2 macrophages. After purification of proteins by centrifugal filtration, western blotting detected 2 high molecular weight bands of tissue factor-bearing microparticles in culture supernatants of M1 macrophages. The upper band showed binding to factor VIIa and tissue factor pathway inhibitor, unlike the lower band. This suggested heterogeneity of the procoagulant activity of tissue factor-bearing microparticles, presumably dependent upon encryption/decryption of tissue factor. Phosphatidylserine contributes to tissue factor decryption, and western blotting revealed that the density of phosphatidylserine was reduced in the upper tissue factor band compared with the lower band. Di-(2-ethylhexyl) phthalate also upregulated transforming growth factor-β1 protein production by M1 macrophages. Moreover, silencing of Smad2, Smad3 or Smad4 attenuated plasminogen activator inhibitor type 1 expression and tissue factor-release from macrophages after di-(2-ethylhexyl) phthalate stimulation. ConclusionsDi-(2-ethylhexyl) phthalate promotes formation of tissue factor-bearing microparticles in human M1 macrophages via the transforming growth factor-β1/Smad/ plasminogen activator inhibitor type 1 signaling pathway.

Redox of Cysteines and Protein Folding of Snap-25

The SNARE proteins SNAP-25, syntaxin, and VAMP play critical roles in neuronal exocytosis by providing the four helical regions (SNARE domains) that form a coiled-coil complex required for neurotransmitter release. This complex is continually formed and unwound as exocytotic vesicles fuse and are recycled. Using multiple techniques, we observe clear structural changes in SNAP-25B due to temperature, pH, ionic strength, methanol, and oxidation state. Here we focus on redox state and structural changes due to temperature and methanol. Using Circular Dichroism to measure the secondary structure of SNAP-25B in low ionic strength solutions, we observe an increase in alpha-helical structure when methanol is added, or the temperature is lowered, similar to the helical shift observed when SNAP-25 forms a complex with syntaxin and VAMP. Methanol (30%) or heating (34C) also breaks up large aggregates of SNAP-25B, as observed by decreased opacity in the CD signal and a loss of high molecular weight bands in non-denaturing gels. The four cysteines in SNAP-25B are thought to be palmitoylated in vivo and can be modified by addition of iodoacetic acid (IAA). Using non-denaturing gels, we observe a shift of mobility of SNAP-25B depending on the redox potential of the reaction solution. We previously showed that these cysteines are oxidized by micromolar Cu2+ by measuring the number of cysteines accessible for labeling with a Biotin-maleimide tag technique developed in our lab (Woodbury et al., 2011. Anal. Biochem. 417:165-173). These data support the hypothesis that SNAP-25B structure and cysteine accessibility are sensitive to environmental changes, which may modulate SNAP-25 palmitoylation and neurosecretion in vivo.

Identification of a perchloric acid-soluble protein (PSP)-like ribonuclease from Trichomonas vaginalis

A perchloric acid-soluble protein (PSP), named here tv-psp1, was identified in Trichomonas vaginalis. It is expressed under normal culture conditions according to expressed sequence tag (EST) analysis. On the other hand, Tv-PSP1 protein was identified by mass spectrometry with a 40% of identity to human PSP (p14.1). Polyclonal antibodies against recombinant Tv-PSP1 (rTv-PSP1) recognized a single band at 13.5kDa in total protein parasite extract by SDS-PAGE and a high molecular weight band analyzed by native PAGE. Structural analysis of Tv-PSP1, using dynamic light scattering, size exclusion chromatography, and circular dichroism spectroscopy, showed a trimeric structure stable at 7M urea with 38% α-helix and 14% β-sheet in solution and a molecular weight of 40.5kD. Tv-PSP1 models were used to perform dynamic simulations over 100ns suggesting a stable homotrimeric structure. Tv-PSP1 was located in the nucleus, cytoplasm, and hydrogenosomes of T. vaginalis, and the in silico analysis by Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) showed interactions with RNA binding proteins. The preliminary results of RNA degradation analysis with the recombinant Tv-PSP1 showed RNA partial deterioration suggesting a possible putative ribonuclease function.

Connective tissue growth factor contributes to joint homeostasis and osteoarthritis severity by controlling the matrix sequestration and activation of latent TGFβ

ObjectivesOne mechanism by which cartilage responds to mechanical load is by releasing heparin-bound growth factors from the pericellular matrix (PCM). By proteomic analysis of the PCM, we identified connective tissue...

Characterization of Some Extremely Halophilic Bacteria Isolated from Salt Marshes of Gamasa Egypt

Halophiles can be classified as extreme, moderate and slight halophiles. Many extremophiles produce unique biotechnological and commercial materials. Salinity stress is one of the major factors negatively affecting bacterial growth. Cellular biochemical changes could be monitored via protein pattern in response to different NaCl concentrations. So our work aimed to do a comparison among salt-tolerant bacteria in their response to different NaCl concentrations via halotolerance test, antimicrobial susceptibility, amino acid profile and protein banding pattern. From haloterance test of eleven bacterial isolates, STB5, STB8 and Shigella dysenteriae were more tolerance to NaCl. From 16S rRNA gene sequencing, the two isolates STB5 and STB8 were identified as Halomonas caseinilytica and Paraliobacillus quinghaiensis. From 32used antibiotics only 5 antibiotics, cefotaxime, ampicillin, penicillin G, ceftazidime and aztreonam, showed resistance. From SDS PAGE, the appearance or absence of high or low molecular weight bands may be directly associated with the bacterial response to salt. Protein profile of tested halophilic bacteria under different concentrations of NaCl% (0, 5% and 20%) showed variable polymorphism in Halomonas caseinilytica MG199079, Paraliobacillus quinghaiensis MG250199and Shigella dysenteriae. Environment-specific patterns of amino acids profile were observed. Based on the obtained results, it could be concluded that the three organisms Halomonas caseinilytica MG199079, Paraliobacillus quinghaiensis MG250199and Shigella dysenteriae, are extreme halophilic bacteria, they were different in of amino acids accumulation protein profile and in different concentration of NaCl.

Application of a recombinant laccase-chlorogenic acid system in protein crosslink and antioxidant properties of the curd

Milk protein crosslink through the action of enzymes represents a feasible strategy to impart new functionalities to cheese. In this work we reported the effects of a laccase mediator system (LMS) on protein crosslink and antioxidant property of curd. The crosslinking activity of a purified recombinant laccase Ery4 and a commercial enzyme preparation (cLC), with three mediators was firstly evaluated in milk and then applied before curd manufacture. Only Ery4-LMS significantly increased curd weight compared to that of the control sample. SDS-PAGE revealed that similar high molecular weight bands produced by both LMSs in milk were also retained in curds. The antioxidant activity recorded in curds with Ery4-LMS was the highest among all samples both before and after gastro-pancreatic digestion. This is the first time that a CGA-based LMS is used in manufacture of curd with improved antioxidant properties. These results open new perspectives for dairy applications.

Enzymes in Commercial Cellulase Preparations Bind Differently to Dioxane Extracted Lignins.

Commercial fungal cellulases used in biomass-to-biofuels processes can be grouped into three general classes: native, augmented, and engineered. Colorimetric assays for general glycoside hydrolase activities showed distinct differences in enzyme binding to lignin for each enzyme activity. Native cellulase preparations demonstrated low binding of endo- and exocellulases, high binding of xylanase, and moderate binding for β-D-glucosidases. Engineered cellulase formulations exhibited low binding of exocellulases, very strong binding of endocellulases and β-D-glucosidase, and mixed binding of xylanase activity. The augmented cellulase had low binding of exocellulase, high binding of endocellulase and xylanase, and moderate binding of β-D-glucosidase activities. Bound and unbound activities were correlated to general molecular weight ranges of proteins as measured by loss of proteins bands in bound fractions on SDS-PAGE gels. Lignin-bound high molecular weight bands correlated to binding of β-D-glucosidase activity. Whereas β-D-glucosidases demonstrated high binding in many cases, they have been shown to remain active. Bound low molecular weight bands correlated to xylanase activity binding. Contrary to other literature, exocellulase activity did not show strong lignin binding. The variation in enzyme activity binding between these three classes of cellulases preparations indicates that it is possible to alter the binding of specific glycoside hydrolase activities during the enzyme formulation process. It remains unclear whether or not loss of endocellulase activity to lignin binding is problematic for biomass conversion.

Temperature‐Induced Changes to Aquaporin Localization in the Freeze‐Tolerant Cope's Gray Treefrog

Cope's gray treefrogs alter their physiology in response to cold as part of their freeze tolerance mechanism. One such change involves the accumulation, both extracellularly and intracellularly, of cryoprotective glycerol, which likely crosses cellular membranes using glycerol‐permeable channels called aquaglyceroporins (GLPs). Three GLPs (HC‐3, HC‐7, and HC‐9) have been identified in the Cope's gray treefrog, representing homologs to the mammalian AQP3, AQP7, and AQP9, respectively.Regulation of transmembrane glycerol diffusion depends on GLP expression in the plasma membrane resulting from changes to transcriptional and translational output of the GLPs and/or by changes in protein trafficking to the plasma membrane. We have previously reported that GLP mRNA and protein expression are modulated during cold acclimation and freezing; however, localization of GLP proteins in the plasma membrane has not been studied thoroughly. We hypothesized that changes in acclimation temperature would alter the localization of the GLPs HC‐3 and HC‐9 in the plasma membrane and confirmed this with immunofluorescence, plasma membrane fractionation, and biotinylation.For HC‐9 in liver, quantitation of protein expression (by immunoblot) showed a 4‐ to 6‐fold increase in the higher molecular weight band of frozen (−2.5°C for 24 hours) and thawed (5°C for 24 hours after freezing) over warm (20°C) treefrogs. In immunohistochemistry images, HC‐9 signal appeared to be localized to the plasma membrane of hepatocytes of warm‐ and cold‐acclimated animals but dispersed intracellularly in frozen treefrog hepatocytes. In preliminary studies, fractionation of cellular protein (Biovision, Milpitas, CA) indicated that between freezing and thawing, the nominal (~34 kDa) molecular weight band of HC‐9 diminishes in the plasma membrane fraction while the (putatively) glycosylated high molecular weight band of HC‐9 increases.For HC‐3 in erythrocytes, protein expression is enhanced by cold acclimation and by incubation in medium containing glycerol. We incubated erythrocytes taken from warm‐acclimated animals at 4°C for 48 hours and biotinylated the surface proteins. In immunoblots of the biotinylated fraction probed with anti‐HC‐3 primary antibody, the high molecular weight (glycosylated) band was enriched and the nominal (~32 kDa) molecular weight band was diminished. These results suggest that water and glycerol permeability during cold acclimation and freezing can be modulated by protein trafficking, which may be influenced by the post‐translational modification of glycosylation.Support or Funding InformationSupported by NSF IOS‐1121457.

Regulation of the Sigma‐1 Receptor Quaternary Structure by Ligand Binding

Since the initial proposal of sigma receptors four decades ago, extensive studies have shown that the sigma‐1 receptor (σ1R) is a unique membrane protein that may serve as a molecular chaperone. In earlier attempts to purify sigma receptors from native tissues using [3H](+)SKF10047 or [3H]azido‐DTG as affinity ligands, the labeled protein complexes appeared to be &gt; 150 kDa under non‐denaturing conditions. Recent breakthrough on the crystal structure of σ1R revealed a homotrimer, with each protomer containing a single transmembrane domain and a cytoplasmic ligand‐binding domain. Whether ligand binding can affect the quaternary structure of σ1R has not been fully investigated. We devised a novel biochemical method to examine the multimeric status of σ1R. Epitope‐tagged σ1R from transfected cells was solubilized using mild detergents, electrophoretically separated under non‐denaturing conditions, and detected by immunoblotting. The majority of σ1R appeared as high molecular weight (MW) bands (apparent MW &gt; 132 kDa), suggesting a high‐order quaternary organization of multimers. Pretreatment with σ1R agonists, such as (+)‐pentazocine and DTG, decreased σ1R high‐MW bands, while increased σ1R lower‐MW bands that were presumably dimers and monomers. Conversely, pretreatment with σ1R antagonists, such as BD1063 and haloperidol, exhibited opposite effects on σ1R multimerization. These results demonstrate that the quaternary structure of σ1R is regulated by binding of agonists or antagonists in a distinct manner. As σ1R has been shown to alter the function of various membrane proteins through dynamic interactions, we hypothesize that σ1R agonists promote the formation of σ1R monomers and facilitate their modulation of interacting partners.Support or Funding InformationButler University Faculty Startup Fund and Holcomb Research Award.

Glycosylated Chromogranin A in Heart Failure: Implications for Processing and Cardiomyocyte Calcium Homeostasis.

Chromogranin A (CgA) levels have previously been found to predict mortality in heart failure (HF), but currently no information is available regarding CgA processing in HF and whether the CgA fragment catestatin (CST) may directly influence cardiomyocyte function. CgA processing was characterized in postinfarction HF mice and in patients with acute HF, and the functional role of CST was explored in experimental models. Myocardial biopsies from HF, but not sham-operated mice, demonstrated high molecular weight CgA bands. Deglycosylation treatment attenuated high molecular weight bands, induced a mobility shift, and increased shorter CgA fragments. Adjusting for established risk indices and biomarkers, circulating CgA levels were found to be associated with mortality in patients with acute HF, but not in patients with acute exacerbation of chronic obstructive pulmonary disease. Low CgA-to-CST conversion was also associated with increased mortality in acute HF, thus, supporting functional relevance of impaired CgA processing in cardiovascular disease. CST was identified as a direct inhibitor of CaMKIIδ (Ca2+/calmodulin-dependent protein kinase IIδ) activity, and CST reduced CaMKIIδ-dependent phosphorylation of phospholamban and the ryanodine receptor 2. In line with CaMKIIδ inhibition, CST reduced Ca2+ spark and wave frequency, reduced Ca2+ spark dimensions, increased sarcoplasmic reticulum Ca2+ content, and augmented the magnitude and kinetics of cardiomyocyte Ca2+ transients and contractions. CgA-to-CST conversion in HF is impaired because of hyperglycosylation, which is associated with clinical outcomes in acute HF. The mechanism for increased mortality may be dysregulated cardiomyocyte Ca2+ handling because of reduced CaMKIIδ inhibition.

Engineering Periodic shRNA for Enhanced Silencing Efficacy.

RNA interference (RNAi) provides a versatile therapeutic approach via silencing of specific genes, particularly undruggable targets in cancer and other diseases. However, challenges in the delivery of small interfering RNA (siRNA) have hampered clinical translation. Polymeric or periodic short hairpin RNAs (p-shRNAs)-synthesized by enzymatic amplification of circular DNA-are a recent development that can potentially address these delivery barriers by showing improved stability and complexation to enable nanoparticle packaging. Here, we modify these biomacromolecules via structural and sequence engineering coupled with selective enzymatic digestion to generate an open-ended p-shRNA (op-shRNA) that is cleaved over ten times more efficiently to yield siRNA. The op-shRNA induces considerably greater gene silencing than p-shRNA in multiple cancer cell lines up to 9 days. Furthermore, its high valency and flexibility dramatically improve complexation with a low molecular weight polycation compared to monomeric siRNA. Thus, op-shRNA provides an RNAi platform that can potentially be packaged and efficiently delivered to disease sites with higher therapeutic efficacy.

First Report of White clover mosaic virus and Turnip mosaic virus Mixed Infection on Garlic Mustard in Pennsylvania

HomePlant DiseaseVol. 100, No. 4First Report of White clover mosaic virus and Turnip mosaic virus Mixed Infection on Garlic Mustard in Pennsylvania PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of White clover mosaic virus and Turnip mosaic virus Mixed Infection on Garlic Mustard in PennsylvaniaK. Zhao, P. Margaria, and C. RosaK. ZhaoSearch for more papers by this author, P. MargariaSearch for more papers by this author, and C. RosaSearch for more papers by this authorAffiliationsAuthors and Affiliations K. Zhao P. Margaria C. Rosa , Pennsylvania State University, University Park, PA 16802. Published Online:10 Feb 2016https://doi.org/10.1094/PDIS-09-15-1083-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat In spring 2015, 12 weedy garlic mustard (Alliaria petiolata) plants with mosaic symptoms were found in a garden in State College, PA. The same symptoms are seen on many weedy garlic mustards in the area each spring, as they were in previous years at this location. Incidence was ∼10% in the garden, with 2 of ∼20 groups of 4 to 6 plants infected. Quality of RNA extracted from garlic mustard was poor, so symptomatic leaves were used for mechanical transmission to Nicotiana benthamiana. Seven days after mechanical inoculation, plants showed necrotic spots and leaf deformation on the systemic leaves. Double-stranded RNA was extracted from symptomatic N. benthamiana leaves and electrophoresis was performed on 6% polyacrylamide gel to detect dsRNA, produced in plants infected with RNA viruses (Valverde et al. 1990). A band of high molecular weight confirmed the presence of dsRNA. Total RNA was then extracted from the N. benthamiana plants using Spectrum Plant Total RNA Kit (Sigma-Aldrich). A tobravirus or a potyvirus were suspected as possible causal agents, based on literature search for the potyvirus and on the presence a tobravirus infected ornamental plant in the same garden. Two pairs of degenerate primers (Chen et al. 2001; Jones et al. 2008) were used separately in one tube reverse-transcription PCR (RT-PCR) and 835-bp and 654-bp amplicons were cloned into pGEM-T Easy Vector (Promega) and sequenced by Sanger sequencing. The sequences were deposited as GenBank Accession Nos. KT799530 and KT799528, respectively. BLAST results showed that the first sequence had 96% nucleotide identity with the RNA dependent RNA polymerase (RdRp) of a previously reported White clover mosaic virus (WClMV) sequence (AB669182). The second sequence had 95% nucleotide identity to a Turnip mosaic virus (TuMV) sequence across the NIb and coat protein genes (AB252117). Two newly designed primer pairs W_1F (5′-CAGAACGAGCCATCCGCAGAA), W_1500R (5′-TCAGTGCAGTCAGCATTATGAG) and TuMV_F (5′-TCTTGGACGAAGCATGGAGC), TuMV_R (5′-TCGGACTGCCTCTTTGCCTG) were used to further confirm the presence in N. benthamiana of WClMV and TuMV, respectively. A 1,457-bp amplicon was obtained by using W_1F and W_1500R and sequenced. This sequence (KT799531) had 95 to 96% nucleotide identity with several WClMV RdRp sequences (AB056720, AB669182, and X16636). A 577-bp amplicon was obtained by using the primer pair specific to TuMV sequence. The sequenced amplicon (KT788529) showed 97% nucleotide identity to a TuMV sequence spanning the P3 and 6K1 protein (AB701705). Due to the poor quality of RNA we could not test the original or other garlic mustard plants for the presence of the two viruses, but all weeds had uniform symptoms appearance and severity. To our knowledge, White clover mosaic virus and Turnip mosaic virus have not been previously reported in garlic mustard plants in Pennsylvania. Other potyviruses including TuMV have been reported in garlic mustard and Brassicaceae while WClMV to our knowledge was never reported in Brassicaceae or in garlic mustard before. TuMV is a member of the Potyviridae family, is transmitted by aphids in a nonpersistent manner, and causes a variety of symptoms in many Brassica spp.(Tomlinson and Ward 1978) The presence on garlic mustard of a strain of TuMV very similar to a strain already reported in Europe on arugula (Eruca or Brassica vesicaria subsp. sativa) could indicate that this virus can be transmitted between cultivated and not cultivated Brassicaceae and that wild Brassicaceae can potentially serve as virus reservoir (Lockhart 2012). The mode of transmission of WClMV is not known and reports of its transmission by contact or by dodder (Cuscuta spp.) (probably under experimental conditions) have been controversial; therefore, it is hard to speculate if this virus can move from crops to natural environment and vice versa. This is the first report of infection of garlic mustard with TuMV and WClMV, which have the potential to reduce the fitness of this plant species that is considered invasive in North America.

Fluorescence Spectroscopy Reveals a 2:1 Stoichiometry for Dimeric Cardiac Calcium Pump (SERCA2a) Bound to a Phospholamban Monomer

Previous in vitro studies have suggested that the cardiac calcium ATPase (SERCA) can form functional homo‐dimers. To determine whether SERCA dimerizes in live cells, we fused Cerulean (Cer) or yellow fluorescent protein (YFP) to the N‐terminus of canine SERCA2a. We measured 18% maximum fluorescence resonance energy transfer (FRETmax) from Cer‐SERCA to YFP‐SERCA, suggesting an interaction between SERCA protomers. To determine whether the measured FRET was due to a specific interaction, we co‐transfected cells with unlabeled SERCA to compete for binding with Cer‐SERCA and YFP‐SERCA. FRETmax progressively decreased with increasing expression of unlabeled SERCA to a minimum of 5%. Competition with increasing molar ratios of unlabeled phospholamban (PLB) did not reduce the measured FRET or the apparent affinity of the SERCA dimer. In order to determine whether PLB phosphorylation status alters SERCA dimerization, we induced phosphorylation of PLB at serine 16 with PKA activation by Forskolin. PLB phosphorylation or phosphomimetic mutation (S16E) did not alter the maximum SERCA‐SERCA FRET or the apparent dissociation constant (Kd) of the SERCA dimer. The apparent affinity of the dimer did not vary with the presence or absence of calcium, ATP, or with the addition of the SERCA inhibitor, thapsigargin. The SERCA‐SERCA interaction was further supported by co‐immunoprecipitation experiments in cells co‐transfected with YFP‐SERCA and myc‐tagged SERCA. Immunoprecipitation with an anti‐mycTag antibody co‐immunoprecipitated YFP‐SERCA, as detected by western blot. Rabbit left ventricular myocytes were isolated and permeabilized in the presence of a benzophenone‐4‐maleimide crosslinker. Western blot analysis revealed two bands at approximately 110 and 200 kD, consistent with the expected molecular weights of a monomer and dimer, respectively. PLB was also detected in these higher molecular weight bands. We also quantified FRET from GFP‐SERCA to mCherry‐PLB using time correlated single photon counting to determine the GFP excited state lifetime. We observed a biexponential fluorescence decay consistent with two populations of donors with distinct lifetimes. The long lifetime was similar to that of a GFP‐alone control. The shorter lifetime was consistent with a high FRET population. Interestingly, the FRET efficiency of this species was 45%, approximately double the FRETmax value obtained from steady‐state FRET measurements. The population of the high FRET species approached, but did not exceed, 50% of the total, even at PLB to SERCA ratios as high as 40:1. This observation is consistent with a complex of two SERCA protomers in which only one of the pumps engages in FRET with a bound PLB.

Conjugation of ampicillin and enrofloxacin residues with bovine serum albumin and raising of polyclonal antibodies against them.

Aim:The aim of this study is to test the potency of bovine serum albumin (BSA) conjugated ampicillin (AMP) and enrofloxacin (ENR) antigens in eliciting an immune response in rats using indirect competitive enzyme-linked immunosorbent assay (icELISA).Materials and Methods:AMP and ENR antibiotics were conjugated with BSA by carbodiimide reaction using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) as a cross-linker. The successful conjugation was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Sprague-Dawley rats were immunized with the conjugates and blood samples were collected serially at 15 days time interval after first immunization plus first booster, second booster, third booster, and the fourth sampling was done 1½ month after the third booster. The antibody titres in the antisera of each antibiotic in all the four immunization cycles (ICs) were determined by an icELISA at various serum dilutions ranging from 1/100 to 1/6400.Results:Analysis of antibiotic-BSA conjugates by sodium dodecyl sulfate polyacrylamide gel electrophoresis and coomassie blue staining revealed high molecular weight bands of 85 kDa and 74 kDa for AMP-BSA and ENR-BSA respectively when compared to 68 kDa band of BSA. Both the antibiotic conjugates elicited a good immune response in rats but comparatively the response was more with AMP-BSA conjugate than ENR-BSA conjugate. Maximum optical density 450 value of 2.577 was recorded for AMP-BSA antisera, and 1.723 was recorded for ENR-BSA antisera at 1/100th antiserum dilution in third IC.Conclusion:AMP and ENR antibiotics proved to be good immunogens when conjugated to BSA by carbodiimide reaction with EDC as crosslinker. The polyclonal antibodies produced can be employed for detecting AMP and ENR residues in milk and urine samples.

Abstract 3775: p53 is differentially regulated in proliferative and invasive melanoma cells

Abstract Melanoma is one of the few cancer types where p53 is not often mutated. Cancers which have not mutated TP53, often attenuate p53 activity by affecting upstream regulators or downstream effectors of p53. As melanoma progresses, it by passes senescence, proliferates, and switches from a proliferative phenotype to a more invasive phenotype. We have found that invasive melanomas, characterized by high levels of the pro-invasive Wnt molecule, Wnt5A, express active p53. Conversely, melanomas with a more proliferative phenotype, characterized by increased MITF signaling and low Wnt5A expression, do not express active p53. We show that the expression of p53 appears to be controlled by Wnt5A, and this may occur via CAMKII. By Western blot, we have observed that more proliferative melanoma cells express a high molecular weight p53 (∼250 kDa). This high molecular weight p53 does not break down on an SDS-PAGE gel. Knocking down p53 in proliferative and invasive melanoma cells, using siRNA, decreases the high molecular weight band in the proliferative cells, and the 53 kDa band in the more invasive cells. When we over express HA-tagged p53 in proliferative cells, we see multiple bands for p53 by Western blot, including the high molecular weight bands, suggesting that p53 is being post-translationally modified in these cells. We confirmed that ubiquitination is involved in the formation of the high molecular weight p53, by treating proliferative cells with SMER3, an E3 ligase inhibitor. Following two hours of treatment, the high molecular weight p53 band decreased and this decrease persists for 16 h. Next, we transfected an E6 ligase into invasive melanoma cells, which express active p53. Expression of the E6 ligase increased the expression of the high molecular weight p53 band (250 kDa), but these cells also continued to express active p53 (53 kDa). To determine if ubiquitination is targeting p53 for degradation in proliferative cells, we treated invasive and proliferative cells with MG132, a proteasome inhibitor. Following treatment with MG132, p53 (53 kDa) increased in invasive cells, suggesting that p53 is degraded via the proteasome in these cells. However, when we treated proliferative cells with MG132 we did not see an increase in the high molecular weight band, suggesting that p53 is not being degraded via the proteasome in these cells. By immunofluorescence, we found that p53 is in the cytoplasm of proliferative cells, suggesting that ubiquitination may be playing a role in sequestration of p53 in proliferative cells. In invasive cells, p53 remains largely nuclear, and we demonstrate that this plays critical roles in response to therapy, counter-intuitively driving resistance, by allowing cells to undergo an adaptive stress response that we term “pseudosenescence” Citation Format: Marie R. Webster, Subhasree Basu, Amanpreet Kaur, Jessica Appleton, Maureen E. Murphy, Ashani T. Weeraratna. p53 is differentially regulated in proliferative and invasive melanoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3775. doi:10.1158/1538-7445.AM2015-3775

Role of insulin and insulin‐like‐growth factor (IGF) receptors in renal proximal tubule (PT) phosphorus handling

The renal PT is involved in the control of phosphorus (P) homeostasis via regulated reabsorption by the sodium phosphate cotransporter (NaPi‐2 or Slc34a1). Previous work suggests NaPi‐2 activity is increased by insulin and/or IGF; however, the mechanisms remain obscure. Our aim was to determine whether PT‐cell‐select knockout (KO) of both the insulin receptor (IR) and the IGF1 receptor (IGFR) would impact NaPi‐2 regulation and phosphorus homeostasis. Mice with both IR and IGF1R genes floxed were crossed for 2 generations with mice with Cre‐recombinase driven by the PT‐select γ‐glutamyl transferase promoter. No differences due to genotype were observed in food intake, body weight, or growth rate in either male (M) or female (F) mice. Similarly, 24‐hour urine volumes and P excretion did not differ. In contrast, plasma P levels were significantly lower in adult KO (6‐9 months) as compared to wild‐type (WT) mice (mg/dl): 12.9 ± 1.2 (WTM), 16.5 ± 2.3 (WTF), 10.2 ± 0.3 (KOM), 8.9 ± 1.3 (KOF), p = 0.014, 2‐way ANOVA for genotype, with no significant effect of sex. Western blotting of whole kidney homogenates revealed no differences due to genotype or sex for the major (84 kDa) band of NaPi‐2; however, a higher molecular weight band on the NaPi‐2 blots (~250 kDa) was markedly reduced in the KO, as well as in female mice. Band densities were (% WTM): 100 ± 7 (WTM), 32 ± 11 (WTF), 16 ± 9 (KOM), 1.6 ± 0.5 (KOF), p &lt;0.001 for genotype and sex. We speculate that this band may represent NaPi‐2 bound to regulatory proteins. Our data support a role for insulin and/or IGF1 receptor‐mediated action in the regulation of renal P reabsorption. Additional studies will be needed to determine the role and cellular actions of each individual receptor.