Tricine Gel Research Articles

Antifungal activities of leaf extracts from the Capsicum genus

The emergence of microorganisms resistant to antimicrobials has driven the search for new substances aimed at the control and prevention of pathogens. Plant antimicrobial peptides have biotechnological applications in the development of drugs and agricultural inputs for this purpose. The present work aims to evaluate the antifungal activity of extracts containing peptides obtained from the leaves of Capsicum plants. The accessions UENF 1626, 1632 and 1381 and the cultivar Carioquinha were used. The leaves were macerated, and then extraction was performed using a 60% methanol and dichloromethane solution in a 1:1 ratio. The hydromethanolic phase was separated and used for testing after evaporation of methanol. All extracts have a similar electrophoretic profile on tricine gel stained with Coomassie Blue, showing 2 bands of approximately 6.5 and 3.5 kDa. Inhibition of fungal growth with 24 h of culture was performed in liquid culture medium in 96-well plates with reading at 620 nm using extracts with a protein concentration of 200 mg mL-1. The filamentous fungi Fusarium solani, Fusarium lateritium, and Colletotrichum lindemuthianum and the yeasts Candida albicans, Candida buinensis, Candida tropicalis, Saccharomyces cerevisiae, Pichia membranifaciens and Kluyveromyces marxianus were used. The extract with the greatest antifungal potential was the leaves of Capsicum annuum cv. Carioquinha, inhibiting the growth of F. solani by 29%, C. lindemuthianum by 15%, Candida buinensis by 22% and K. marxianus by 24%. Therefore, the extract has peptides with biotechnological potential.

Identification, biochemical characterization and biological role of defense proteins from common bean genotypes seeds in response to Callosobruchus maculatus infestation

Common bean is a legume of significant socioeconomic importance and is cultivated worldwide. This crop is affected by several pests and diseases, which cause considerable economic losses and reduce yield. In recent years, several studies have demonstrated the role of proteins and peptides with activity against a wide range of insects and pathogens. The objective of this work was to identify defense proteins, such as antimicrobial peptides, protease and amylases inhibitors in common bean genotypes and evaluate the relationship of these proteins with Phaseolus vulgaris seed resistance to Callosobruchus maculatus infestation. Nineteen common bean genotypes were subjected to protein extraction, pH 5.4, and precipitation with ammonium sulfate at 70% saturation. The obtained extracts were separated by tricine gel electrophoresis. Experiments were carried out with natural seeds of common beans and artificial seeds (Vigna unguiculata seeds covered with seed coats of common beans) to evaluate the rate of oviposition and development of the insect species Callosobruchus maculatus. Lipid-transfer proteins were identified in nine genotypes whereas defensins were present in all genotypes. The inhibitory activity of α-amylases and trypsin and fungal development were determined in crude extracts (50 μg mL−1). The results also indicated that the extracts from all bean genotypes inhibited the activity of human salivary α-amylase and C. maculatus larval α-amylase. Except for the extracts of four genotypes, all other extracts inhibited trypsin activity. None of the extracts from the evaluated bean genotypes inhibited the growth of tested fungi. Natural seeds from all genotypes did not inhibit insect oviposition, however, the larvae did not survive after feeding on these seeds. Artificial seeds containing seed coat flour of all genotypes inhibited the oviposition of C. maculatus, indicating that the seed coat was also repellent to insect.

Characterization and antifungal activity of a plant peptide expressed in the interaction between Capsicum annuum fruits and the anthracnose fungus.

Plant defensins are low molecular weight basic peptides ranging from 5 to 7 kDa, with capacity of inhibiting various pathogens, including fungi. They are present in different tissues of plants, including floral parts and fruits of Capsicum sp. The IIF48 extract, present in immature fruits of Capsicum annuum inoculated with C. gloeosporioides, was able to inhibit up to 100% growth ‘in vitro’ of the fungus Colletotrichum gloeosporioides. The main objective of this work was the purification and antifungal activity characterization of a defense-related plant defensin-like isolated of the IIF48 immature fruits extract. The IIF48 extract was subjected to HPLC purification and 13 fractions were obtained, followed by a tricine gel electrophoresis to obtain the protein profile. The different fractions were submitted to a growth inhibition assay against C. gloeosporioides fungus. Fraction 7 (F7) was the most active causing 73% inhibition. Because of the higher F7 activity and the presence of only a peptide of approximately 5 kDa this fraction was subjected to N-terminal sequencing. F7 fraction was carried out plasma membrane permeabilization assays, induction of intracellular ROS production analysis and investigated mitochondrial membrane potential. The F7 fraction showed significant inhibitory activity on the tested fungus, besides promoting membrane permeabilization, induction of endogenous ROS production in Colletotrichum cells and impairing mitochondrial functionality. The first 18 amino acid sequence of the F7 fraction peptide suggests homology to plant-like defensin and was named IIFF7Ca. We also concluded that IIFF7Ca peptide has an effective antimicrobial action against the fungus C. gloeosporioides.

Biochemical analysis of antimicrobial peptides in two different Capsicum genotypes after fruit infection by Colletotrichum gloeosporioides.

There are several phytosanitary problems that have been causing serious damage to the Capsicum crops, including anthracnose. Upon attack by certain pathogens, various protein molecules are produced, which are known as proteins related to pathogenesis (PR proteins), including antimicrobial peptides such as protease inhibitors, defensins and lipid transfer proteins (LTPs). The objective of this work is to identify antimicrobial proteins and/or peptides of two genotypes from Capsicum annuum fruits infected with Colletotrichum gloeosporioides. The fungus was inoculated into Capsicum fruits by the deposition of a spore suspension (106 conidia ml−1), and after 24 and 48 h intervals, the fruits were removed from the humid chamber and subjected to a protein extraction process. Protein analysis of the extracts was performed by tricine gel electrophoresis and Western blotting. The distinctive bands between genotypes in the electrophoresis profiles were subjected to mass spectrometry sequencing. Trypsin inhibition assays, reverse zymographic detection of protease inhibition and β-1,3-glucanase activity assays were also performed and extracts were also tested for their ability to inhibit the growth of C. gloeosporioides fungi ‘in vitro’. There were several low molecular weight proteins in all treated samples, and some treatments in which antimicrobial peptides such as defensin, lipid transfer protein (LTP) and protease inhibitor have been identified. It was shown that the green fruits are more responsive to infection, showing the production of antimicrobial peptides in response to injury and inoculation of the fungus, what did not occur in ripe fruits under any treatment.

Characterization of Capsicum annuum L. leaf and root antimicrobial peptides: antimicrobial activity against phytopathogenic microorganisms

This study aimed to detect and characterize antimicrobial proteins, especially antimicrobial peptides (AMPs) from leaves and roots of Capsicum annuum and to evaluate their inhibitory activities against different phytopathogenic fungi and the bacterium Xanthomonas euvesicatoria. Two methodologies were used for the extraction of peptides from leaves and roots of C. annuum: acid and ethanolic extraction. Extracts were subjected to reversed-phase chromatography on HPLC. The extraction and purification procedures were analysed by uni- and bi-dimensional electrophoresis in tricine gels. Our results show that alcoholic and acid extracts from both tissues can inhibit the growth of the phytopathogenics fungi C. lindemuthianum and C. gloeosporioides. The acid extracts from both tissues are active against X. euvesicatoria and only leaf extracts displayed specific inhibitory activity towards trypsin and α-amylase activity. The data compiled here aim to contribute to establish the multiplicity of potential uses of plant AMPs for the control of pests and pathogens of agricultural relevance.

Optimization of Extraction and Isolation of Proteins from Eggshells

The eggshells of the common chicken (Gallus gallus domesticus) are a large waste product of the food industry; thus, developing methods for harvesting bioactive proteins from eggshells is of great economic interest. Eggshells are composed of a calcium carbonate mineral structure along with up to a 3 to 5 % organic fraction by mass (Nys et al., 2004, General Paleontology, 3: 549–562). While the mineral fraction has already found widespread use in agriculture as a fertilizer and more recently as a catalyst for biodiesel production, the protein fraction, has yet to be fully utilized. Before eggshells can be utilized, however, the egg shell inner and outer membranes must be removed. For large scale membrane separations, several industrial processes involving the use of pressurized gases, multiphase floatation tanks or liquid cavitation are available. However, these methods are not practical for small scale exploratory preparations.For small‐scale protein extractions from egg shells, various incubation solutions for membrane removal were assessed using individual eggshells. Raw eggshells were incubated in solutions of 1 M HCl, 1 % SDS, 50 mM EDTA and reverse osmosis‐treated water. The ease of which membranes could be separated from eggshells manually was assessed. Once a membrane removal procedure was established, the optimal method was used for further experimentation. Membrane‐less eggshells were then rinsed with a 1 % SDS solution. The remaining shell was then dissolved in a minimum volume of 10 % acetic acid solution. After dialysis of the resulting solution, micro‐chromatography techniques were employed to isolate specific fractions of protein. Bradford assays were performed between each step to assess total protein content, while SDS‐PAGE using 12% and tricine gels were performed to evaluate the sizes of proteins recovered.Removing the entire membrane at once is important as it greatly reduces the potential of leaving behind small portions of membrane protein, which would contaminate the extract. Several published protocols for separating inner and outer egg shell membranes have called for chemical mixtures of 1 M HCl, weak EDTA solutions or SDS. However, prolonged exposure causes the membrane itself to weaken and tear, causing small fragments of membrane to remain attached to the shell. After comparing the results of the varying incubation solutions, simply using a large quantity of water proved to be the most effective. Using aqueous solutions hydrates the membranes, causing them to separate from the shell more easily and reduces tearing. Using water has the additional benefit of reducing chemical waste. Following this step an SDS wash was used to lightly remove any remaining contamination.Once isolated from the shell, the total protein fraction was enriched for phosphoproteins using micro‐chromatographic techniques involving Ni2+‐affinity resin and/or hydroxyapatite chromatography. Additionally, ammonium sulfate fractionation was used to divide the total protein fraction into discrete protein fractions by ammonium sulfate cuts of 40%, 60% and 80%.Support or Funding InformationThis research was supported by a University of Wisconsin‐Stout Student Research Grant to RR.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Diffusion, Frowning and Smiling of Low Molecular Weight Protein Bands: A Simple, Rapid and Efficient Solution

A modified method of SDS-polyacrylamide gel electrophoresis for the efficient separation of low molecular weight proteins (ranging 1.4–10 kDa, containing di-sulfide bonds) without frowning, smiling and diffusion is described. SDS-polyacrylamide and tricine gel electrophoresis are considered as standard approaches for low molecular weight protein separation, despite frowning, smiling and diffusion problems. These methods are inefficient and confusing as Coomassie brilliant blue stained low molecular weight protein bands are difficult to identify since proteins get diffused and, it is difficult to identify as proteins. The method developed offers a solution to the existing problem and is more suitable for the efficient separation of low molecular weight proteins with high resolution (with di-sulfide bonds).

A pilot proteomic study of protein markers in autism spectrum disorder

Autism spectrum disorder (ASD) diagnosis is increasing, with 1/88 children believed to be affected by the disorder, with a most recent survey suggesting numbers as high as 1/50. Treatment and understanding of ASD causes is a pressing health concern. ASD protein biomarkers may provide clues about ASD cause. Protein biomarkers for ASDs could be used for ASD diagnosis, subtyping, treatment monitoring, and identifying therapeutic targets. Here, we analyzed the sera from seven children with ASD and seven matched controls using Tricine gel electrophoresis (Tricine-PAGE) and LC-MS/MS. Overall, we found increased levels of apolipoproteins ApoA1 and ApoA4, involved in cholesterol metabolism and of serum paraoxanase/arylesterase 1, involved in preventing oxidative damage, in the sera of children with ASD, compared with their matched controls. All three proteins are predicted to interact with each other and are parts of high-density lipoproteins. Further studies are needed to validate these findings in larger subject numbers.

The Mitochondrial Intermembrane Space Oxireductase Mia40 Funnels the Oxidative Folding Pathway of the Cytochrome c Oxidase Assembly Protein Cox19

Mia40-catalyzed disulfide formation drives the import of many proteins into the mitochondria. Here we characterize the oxidative folding of Cox19, a twin CX9C Mia40 substrate. Cox19 oxidation is extremely slow, explaining the persistence of import-competent reduced species in the cytosol. Mia40 accelerates Cox19 folding through the specific recognition of the third Cys in the second helical CX9C motif and the subsequent oxidation of the inner disulfide bond. This renders a native-like intermediate that oxidizes in a slow uncatalyzed reaction into native Cox19. The same intermediate dominates the pathway in the absence of Mia40, and chemical induction of an α-helical structure by trifluoroethanol suffices to accelerate productive folding and mimic the Mia40 folding template mechanism. The Mia40 role is to funnel a rough folding landscape, skipping the accumulation of kinetic traps, providing a rationale for the promiscuity of Mia40.

Proteolytic Activity of the MMGP1 Antifungal Peptide Derived from Marine Metagenome

An antifungal peptide, MMGP1 with direct cell penetrating property was recently reported from marine metagenome. The peptide showed efficient in vitro proteolytic activity, which could be associated with its antifungal activity. The proteolytic activity of MMGP1 was confirmed by tricine SDS-PAGE and gel filtration chromatography. Liquid chromatography-mass spectrometry analysis of MMGP1 treated bovine serum albumin (BSA), RNaseA and casein substrates revealed that the peptide does not have common cleavage position and it cleaves the substrates non-specifically at all peptide bonds. The proteolytic activity of MMGP1 was enhanced in the presence of Mn2+. Molecular docking studies revealed that the predicted active site residues of MMGP1 could interact with BSA, RNaseA and casein.

Characterization of the Staphylococcus aureus rRNA Methyltransferase Encoded by orfX, the Gene Containing the Staphylococcal Chromosome Cassette mec (SCCmec) Insertion Site

The gene orfX is conserved among all staphylococci, and its complete sequence is maintained upon insertion of the staphylococcal chromosome cassette mec (SCCmec) genomic island, containing the gene encoding resistance to β-lactam antibiotics (mecA), into its C terminus. The function of OrfX has not been determined. We show that OrfX was constitutively produced during growth, that orfX could be inactivated without altering bacterial growth, and that insertion of SCCmec did not alter gene expression. We solved the crystal structure of OrfX at 1.7 Å and found that it belongs to the S-adenosyl-L-methionine (AdoMet)-dependent α/β-knot superfamily of SPOUT methyltransferases (MTases), with a high structural homology to YbeA, the gene product of the Escherichia coli 70 S ribosomal MTase RlmH. MTase activity was confirmed by demonstrating the OrfX-dependent methylation of the Staphylococcus aureus 70 S ribosome. When OrfX was crystallized in the presence of its AdoMet substrate, we found that each monomer of the homodimeric structure bound AdoMet in its active site. Solution studies using isothermal titration calorimetry confirmed that each monomer bound AdoMet but with different binding affinities (K(d) = 52 ± 0.4 and 606 ± 2 μm). In addition, the structure shows that the AdoMet-binding pocket, formed by a deep trefoil knot, contains a bound phosphate molecule, which is the likely nucleotide methylation site. This study represents the first characterization of a staphylococcal ribosomal MTase and provides the first crystal structure of a member of the α/β-knot superfamily of SPOUT MTases in the RlmH or COG1576 family with bound AdoMet.

The Native Structure and Composition of the Cruciferin Complex in Brassica napus

Globulins are an important group of seed storage proteins in dicotyledonous plants. They are synthesized during seed development, assembled into very compact protein complexes, and finally stored in protein storage vacuoles (PSVs). Here, we report a proteomic investigation on the native composition and structure of cruciferin, the 12 S globulin of Brassica napus. PSVs were directly purified from mature seeds by differential centrifugations. Upon analyses by blue native (BN) PAGE, two major types of cruciferin complexes of ∼ 300-390 kDa and of ∼470 kDa are resolved. Analyses by two-dimensional BN/SDS-PAGE revealed that both types of complexes are composed of several copies of the cruciferin α and β polypeptide chains, which are present in various isoforms. Protein analyses by two-dimensional isoelectric focusing (IEF)/SDS-PAGE not only revealed different α and β isoforms but also several further versions of the two polypeptide chains that most likely differ with respect to posttranslational modifications. Overall, more than 30 distinct forms of cruciferin were identified by mass spectrometry. To obtain insights into the structure of the cruciferin holocomplex, a native PSV fraction was analyzed by single particle electron microscopy. More than 20,000 images were collected, classified, and used for the calculation of detailed projection maps of the complex. In contrast to previous reports on globulin structure in other plant species, the cruciferin complex of Brassica napus has an octameric barrel-like structure, which represents a very compact building block optimized for maximal storage of amino acids within minimal space.

Neutralization of bitis parviocula (Ethiopian mountain adder) venom by the south african institute of medical research (SAIMR) antivenom

The Ethiopian mountain adder (Bitis parviocula) is a viperid known only from a few locations in southwestern Ethiopia. a total of 30 µg of B. arietans and B. parviocula venoms were run on a 10-20% Tricine gel. To assay lethality dose fifty (LD50), five groups of eight mice for each venom were used. Hemorrhagic activity for crude venom was tested. Fibrinogenolytic activity of crude venom was measured using (2.5 mg/mL) of fibrinogen solution and (0.03 mg/mL) of crude venom. Gelatinase activity of the venom was tested on a Kodak X-OMAT TM film. Crude venoms of B. parviocula and B. arietans were tested for their abilities to affect clotting time, clotting rate and platelet function on whole human blood. The (SAIMR) antivenom was confirmed in this study to neutralize the lethal activity of venom from Bitis parviocula. The ED50s of SAIMR antivenom on B. parviocula and B. arietans neutralized half of 18.2 and 66.7 mg of venom, respectively. The hemorrhagic activities (MHDs) of B. parviocula and B. arietans were 0.88 and 1.7 µg, respectively. Bitis arietans and B. parviocula venoms degradated α and β chains at different times. The γ chains remained unaffected. Bitis parviocula venom did not exhibit gelatinase activity, while B. arietans had a MGD of 6.9 µg. At 3 mg/mL, the crude venoms of B. parviocula and B. arietans did not significantly affect clotting time or clotting rate. The SAIMR antivenom is very effective in neutralizing the venom of B. parviocula and should be considered in treating envenomations by these snakes.

Crystal Structure of the E2 Domain of Amyloid Precursor Protein-like Protein 1 in Complex with Sucrose Octasulfate

Missense mutations in the amyloid precursor protein (APP) gene can cause familial Alzheimer disease. It is thought that APP and APP-like proteins (APLPs) may play a role in adhesion and signal transduction because their ectodomains interact with components of the extracellular matrix. Heparin binding induces dimerization of APP and APLPs. To help explain how these proteins interact with heparin, we have determined the crystal structure of the E2 domain of APLP1 in complex with sucrose octasulfate (SOS). A total of three SOS molecules are bound to the E2 dimer. Two SOSs are bound inside a narrow intersubdomain groove, and the third SOS is bound near the two-fold axis of the protein. Mutational analyses show that most residues interacting with SOS also contribute to heparin binding, although in varying degrees; a deep pocket, defined by His-376, Lys-422, and Arg-429, and an interfacial site between Lys-314 and its symmetry mate are most important in the binding of the negatively charged polysaccharide. Comparison with a lower resolution APP structure shows that all key heparin binding residues are conserved and identically positioned, suggesting that APLP1 and APP may bind heparin similarly. In transfected HEK-293 cells, mutating residues responsible for heparin binding causes little change in the proteolysis of APP by the secretases. However, mutating a pair of conserved basic residues (equivalent to Arg-414 and Arg-415 of APLP1) immediately adjacent to the heparin binding site affects both the maturation and the processing of APP.

Tissue-specific Distribution and Dynamic Changes of 5-Hydroxymethylcytosine in Mammalian Genomes

Cytosine residues in the vertebrate genome are enzymatically modified to 5-methylcytosine, which participates in transcriptional repression of genes during development and disease progression. 5-Methylcytosine can be further enzymatically modified to 5-hydroxymethylcytosine by the TET family of methylcytosine dioxygenases. Analysis of 5-methylcytosine and 5-hydroxymethylcytosine is confounded, as these modifications are indistinguishable by traditional sequencing methods even when supplemented by bisulfite conversion. Here we demonstrate a simple enzymatic approach that involves cloning, identification, and quantification of 5-hydroxymethylcytosine in various CCGG loci within murine and human genomes. 5-Hydroxymethylcytosine was prevalent in human and murine brain and heart genomic DNAs at several regions. The cultured cell lines NIH3T3 and HeLa both displayed very low or undetectable amounts of 5-hydroxymethylcytosine at the examined loci. Interestingly, 5-hydroxymethylcytosine levels in mouse embryonic stem cell DNA first increased then slowly decreased upon differentiation to embryoid bodies, whereas 5-methylcytosine levels increased gradually over time. Finally, using a quantitative PCR approach, we established that a portion of VANGL1 and EGFR gene body methylation in human tissue DNA samples is indeed hydroxymethylation.

Proprotein Convertase PC7 Enhances the Activation of the EGF Receptor Pathway through Processing of the EGF Precursor

Although the processing profile of the membrane-bound epidermal growth factor precursor (pro-EGF) is tissue-specific, it has not been investigated at the cellular level nor have the cognate proteinases been defined. Among the proprotein convertases (PCs), only the membrane-bound PC7, the most ancient and conserved basic amino acid-specific PC family member, induces the processing of pro-EGF into an ∼115-kDa transmembrane form (EGF-115) at an unusual VHPR(290)↓A motif. Because site-directed mutagenesis revealed that Arg(290) is not critical, the generation of EGF-115 by PC7 is likely indirect. This was confirmed by testing a wide range of protease inhibitors, which revealed that the production of EGF-115 is most probably achieved via the activation by PC7 of a latent serine and/or cysteine protease(s). EGF-115 is more abundant at the cell surface than pro-EGF and is associated with a stronger EGF receptor (EGFR) activation, as evidenced by higher levels of phosphorylated ERK1/2. This suggests that the generation of EGF-115 represents a regulatory mechanism of juxtacrine EGFR activation. Thus, PC7 is distinct from the other PCs in its ability to enhance the activation of the cell surface EGFR.

Plasmid-encoded OXA-48 carbapenemase in Escherichia coli from Israel

Sir, Carbapenem resistance among Enterobacteriaceae in Israel emerged in 2004 and was observed mainly in Klebsiella pneumoniae, but also in Enterobacter species and Escherichia coli. Since its emergence, carbapenem resistance in these species, both in clinical and colonizing isolates, has been rendered by the production of plasmid-mediated K. pneumoniae carbapenemase (KPC). In late 2007, a woman in her early thirties previously diagnosed with acute lymphoblastic leukaemia, was admitted to the haematology ward in Tel Aviv Sourasky Medical Center. The patient had arrived in Israel from Jordan in order to undergo chemotherapy and, later, bone marrow transplantation. During the first month after transplantation the patient was intermittently febrile and was treated with various antimicrobials, including piperacillin/tazobactam, amikacin, ciprofloxacin, vancomycin, imipenem and voriconazole. Two months after admission, while being treated with imipenem and voriconazole, the patient suffered from fever, dyspnoea and renal failure, and was transferred to the intensive care unit. During that period a carbapenem-resistant E. coli strain (E. coli 1736) was isolated from a Hickman catheter, leading to removal of the catheter and further treatment with ceftazidime and colistin. Treatment with these antibacterial agents cleared the OXA-48-producing E. coli, yet unfortunately the patient died 3 months later from a systemic Pseudomonas infection. E. coli 1736 was multidrug resistant, showing resistance to penicillins, piperacillin/tazobactam, aminoglycosides, quinolones and carbapenems, but susceptibility to all cephalosporins, aztreonam, tigecycline and colistin (Table 1). Analytical isoelectric focusing (IEF) performed on crude enzyme preparations revealed the presence of two b-lactamases with pIs of 5.4 and 7.2 (data not shown). PCR screening for the presence of b-lactamases in E. coli 1736 indicated the presence of blaTEM-1 and blaOXA-48 genes corresponding to the pIs of the b-lactamases visualized by IEF. Plasmid analysis of E. coli 1736 revealed four plasmids, three of around ≤50 kb in size and a larger plasmid of around 100 kb. Plasmid DNA was purified and transformed into E. coli DH10B. Transformant colonies that were screened positive for blaOXA-48 by PCR harboured the 50 kb plasmid. Southern analysis of plasmid DNA derived from these transformants using a blaOXA-48-labelled probe demonstrated the presence of blaOXA-48 on the acquired 50 kb plasmid. Acquisition of this plasmid increased the MICs of imipenem, meropenem and ertapenem without conferring full resistance (Table 1). PCR mapping of the genetic environment surrounding blaOXA-48 was performed in collaboration with the laboratory of Professor P. Nordmann (Hospital de Bicetre, Paris, France). blaOXA-48 was found to be located inside Tn1999.2, similar to the structure described for other enteric strains, such as the E. coli strain from Turkey and the K. pneumoniae strain from Lebanon. E. coli 1736 was not resistant to all b-lactam antibiotics as reported for other OXA-48-producing strains, yet it presented a high level of resistance to the commonly used carbapenems (MICs ≥16 mg/L), higher than usually seen in KPC-producing E. coli strains isolated in our hospital. These high carbapenem MICs suggested the presence of additional resistance mechanisms together with OXA-48 carbapenemase. Outer membrane protein (OMP) produced by E. coli 1736 was determined by PCR and sequencing of ompA, ompC and ompF genes. Further OMP analysis was performed by protein extraction and separation on Tris–Tricine gels using SDS-PAGE followed by mass spectrometry (performed in the Biological Mass Spectrometry Facility at the Weizmann Institute of Science). Both methods indicated the absence of at least one major porin, OmpC. Until the isolation of E. coli 1736, carbapenem resistance in E. coli in our country was exclusively attributed to the Ambler class A carbapenemase KPC. This is the first identified Enterobacteriaceae isolate in our country possessing a carbapenem-hydrolysing oxacillinase. To seek the possible origin of this strain we performed multilocus sequence typing (MLST; http://www.pasteur.fr/recherche/genopole/PF8/mlst/EColi. html), which genotyped the strain as sequence type (ST) 2, an E. coli ST that has never been recorded previously in our Research letters

Abstract P6-04-11: Quantitative Differences among Spectral Counts Based on LC-MS/MS Spectra Accurately Cluster Basal, Her2-Overexpressing and Luminal A Breast Carcinomas

Abstract Characterization of breast cancer subtypes at the protein-level is largely unexplored and is a powerful approach to identify novel biomarkers that may have diagnostic and prognostic utility. Shotgun proteomics analyzes mixtures at the peptide-level to generate MS/MS spectra that are then used to identify the peptides and proteins from which they were derived. We developed a method in our laboratory utilizing SDS-PAGE followed by in-gel digestion coupled with reversed-phase (RP) liquid chromatography-tandem mass spectrometry (RP-LC-MS/MS) for proteomic analysis of LCM-acquired cells. This method was then applied to a clinical sample set consisting of basal, Her2-overexpressing and luminal A frozen tumor tissues as determined by prior microarray studies. Three tumors per group were dissected in triplicate, for a total of 27 samples. Each dissection consisted of approximately 10,000 cells, corresponding to 3-5 μg protein. Following LCM, the thermoplastic membranes containing the captured cells were peeled from the cap, suspended in SDS loading buffer and gently heated for 10 minutes. The solubilized proteins were electrophoresed 2 cm into a 10-20% Tricine gel followed by in-gel trypsin digestion and peptide extraction. Replicate LC-MS/MS analysis of the tryptic peptides from each of the 27 samples was performed on a Thermo LTQ XL ion trap mass spectrometer. The resultant tandem mass spectra were searched against the human IPI database using the Myrimatch search algorithm and the results filtered using IDPicker. A spectral counting approach was applied to the data-dependent data to compare quantitative differences among the identified proteins. A total of 91,646 spectra were confidently identified, corresponding to 1671 protein groups across all biological and technical replicates. An average of 545, 543, and 610 protein groups were identified in the basal, ERBB2, and luminal A subtypes, respectively, from an equivalent of approximately 800 cells. Spectral count differences between datasets were analyzed using both a Quasi-likelihood Poisson regression model, developed in-house, and the Limma package in Bioconductor. Using a False Discovery Rate < 0.05, a total of 90 proteins showed statistically significant differences in expression among the breast tumor subtypes. Hierarchical cluster analysis generated a heat map of the protein expression patterns. Novel proteins as well as proteins corresponding to genes previously shown by gene array studies to be specific to each tumor type, including NES, ERBB2 and LGALS3BP, were represented among the differentially expressed proteins. These results demonstrate the feasability of this method to identify differential expression of protein biomarkers among breast cancer subtypes. These biomarkers should yield new insights into the pathogenesis of breast cancer and could be rapidly adapted into clinical diagnostics to guide therapeutic decisions for individual patients. The possible role in breast cancer for the novel proteins identified is currently under investigation. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-04-11.

Abstract 3746: Proteomic analysis of laser capture microdissected cells from basal, Her 2 positive and luminal A human breast tumors

Abstract The analysis of tissue is often confounded by the heterogenous nature, small size of biopsies, and focal presence of tumors. The use of laser capture microdissection (LCM) allows for isolation of nearly pure cell populations enhancing specificity of subsequent analyses. A method developed in our laboratory facilitating the removal of adhered cells utilizing SDS-PAGE followed by in-gel digestion and tandem mass spectrometry was applied to LCM-acquired cells from Basal, Her 2 positive and Luminal A breast tumor tissues. Cells were LCM-acquired from frozen tissue sections corresponding to Basal, Her 2 positive or Luminal A human breast tumor tissue. The sample set consisted of 3 tumors per subgroup with 3 caps per tumor, for a total of 27 caps each containing approximately 10,000 cells corresponding to 3-5 μg protein. The thermoplastic membranes containing the captured cells were peeled from the cap and suspended in SDS loading buffer and gently heated for 10 minutes. The solubilized proteins were electrophoresed 2 cm into a 10-20% Tricine gel followed by in-gel trypsin digestion and peptide extraction. Tryptic peptides were analyzed by reversed-phase (RP) liquid chromatography-tandem mass spectrometry (LC-MS-MS) on a Thermo Orbitrap instrument. Following LC/MS/MS analysis of the LCM digests, tandem spectra were searched against the human IPI database using the search algorithm, Myrimatch, and the search results filtered using IDPicker. A spectral counting approach was applied to the data-dependent data to compare quantitative differences of proteins identified. A total of 91646 spectra were confidently identified which corresponded to 1671 protein groups across all biological and technical replicates. An average of 545, 543, and 610 protein groups were identified in the basal, Her 2 positive, and Luminal A subtypes, respectively, from an equivalent of roughly 800 cells. Spectral count differences between datasets between normal and tumor samples were analyzed using both a Quasi-likelihood Poisson regression model, developed in-house, in addition to using the Limma package in Bioconductor. Using the criteria of a False Discovery Rate (FDR) < 0.05, a total of 90 proteins showed statistically significant differences in expression among the breast tumor subtypes. A hierarchical clustering of these proteins was performed, creating a heat map representing the gene expression patterns of these proteins. As expected, ERBB2 was identified in the Her 2 positive tumor tissue samples; however, this protein was not identified in either the Basal or Luminal A samples. The proteins identified which were found to distinguish each tissue subtype are currently being investigated for their biological relevance in breast cancer pathways and as possible prognostic and predictive biomarkers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3746.

Simple purification of human antimicrobial peptide dermcidin (MDCD-1L) by intein-mediated expression in E.coli

Among human antimicrobial peptides (hAMPs), DCD-1L has a broad spectrum of antimicrobial activity over a wide pH range and in high salt concentrations. It offers a promising alternative to conventional antibiotics. The 458-bp-long dermcidin cDNA was amplified by PCR using a human fetal cDNA library as a template. The 147-bp fragment of the MDCD-1L gene encoding an additional methionine residue was subcloned into the pTYB11 vector. Recombinant MDCD-1L was expressed as an intein fusion protein in E. coli, and then purified by affinity chromatography using chitin beads. A small peptide with a molecular mass of about 5 kDa was detected by tricine gel electrophoresis. The recombinant MDCD-1L peptide was purified from the gel and its amino acid sequence was determined by nanoLC-ESI-MS/MS analysis. The initiating amino acid, methionine, remained attached to the N-terminal region of recombinant MDCD-1L. Purified MDCD-1L showed antimicrobial activity against a Micrococcus luteus test strain.