Affinity purification of plasmid DNA directly from crude bacterial cell lysates

We have shown previously that a sequence-specific DNA-binding protein based on the Lac repressor protein can isolate pre-purified DNA efficiently from simple buffer solution but our attempts to purify plasmids directly from crude starting materials were disappointing with unpractically low DNA yields. We have optimized tbe procedure and present a simple affinity methodology whereby plasmid DNA is purified directly by mixing two crude cell lysates, one cell lysate containing the plasmid and the other the protein affinity ligand, without the need for treatment by RNaseA. After IMAC chromatography, high purity supercoiled DNA is recovered in good yields of 100-150 μg plasmid per 200 mL shake flask culture. Moreover, the resulting DNA is free from linear or open-circular plasmid DNA, genomic DNA, RNA, and protein, to the limits of our detection. Furthermore, we show that lyophilized affinity ligand can be stored at room temperature and re-hydrated for use when required.

Affinity adsorption of plasmid DNA

The development of a protein-mediated dual functional affinity adsorption of plasmid DNA is described in this work. The affinity ligand for the plasmid DNA comprises a fusion protein with glutathione-S-transferase (GST) as the fusion partner with a zinc finger protein. The protein ligand is first bound to the adsorbent by affinity interaction between the GST moeity and gluthathione that is covalently immobilized to the base matrix. The plasmid binding is then enabled via the zinc finger protein and a specific nucleotide sequence inserted into the DNA. At lower loadings, the binding of the DNA onto the Fractogel, Sepharose, and Streamline matrices was 0.0078 ± 0.0013, 0.0095 ± 0.0016, and 0.0080 ± 0.0006 mg, respectively, to 50 μL of adsorbent. At a higher DNA challenge, the corresponding amounts were 0.0179 ± 0.0043, 0.0219 ± 0.0035, and 0.0190 ± 0.0041 mg, respectively. The relatively constant amounts bound to the three adsorbents indicated that the large DNA molecule was unable to utilize the available zinc finger sites that were located in the internal pores and binding was largely a surface adsorption phenomenon. Utilization of the zinc finger binding sites was shown to be highest for the Fractogel adsorbent. The adsorbed material was eluted with reduced glutathione, and the eluted efficiency for the DNA was between 23% and 27%. The protein elution profile appeared to match the adsorption profiles with significantly higher recoveries of bound GST-zinc finger protein.

Large-scale interlaboratory study to develop, analytically validate and apply highly multiplexed, quantitative peptide assays to measure cancer-relevant proteins in plasma

There is an increasing need in biology and clinical medicine to robustly and reliably measure tens-to-hundreds of peptides and proteins in clinical and biological samples with high sensitivity, specificity, reproducibility and repeatability. Previously, we demonstrated that LC-MRM-MS with isotope dilution has suitable performance for quantitative measurements of small numbers of relatively abundant proteins in human plasma, and that the resulting assays can be transferred across laboratories while maintaining high reproducibility and quantitative precision. Here we significantly extend that earlier work, demonstrating that 11 laboratories using 14 LC-MS systems can develop, determine analytical figures of merit, and apply highly multiplexed MRM-MS assays targeting 125 peptides derived from 27 cancer-relevant proteins and 7 control proteins to precisely and reproducibly measure the analytes in human plasma. To ensure consistent generation of high quality data we incorporated a system suitability protocol (SSP) into our experimental design. The SSP enabled real-time monitoring of LC-MRM-MS performance during assay development and implementation, facilitating early detection and correction of chromatographic and instrumental problems. Low to sub-nanogram/mL sensitivity for proteins in plasma was achieved by one-step immunoaffinity depletion of 14 abundant plasma proteins prior to analysis. Median intra- and inter-laboratory reproducibility was <20%, sufficient for most biological studies and candidate protein biomarker verification. Digestion recovery of peptides was assessed and quantitative accuracy improved using heavy isotope labeled versions of the proteins as internal standards. Using the highly multiplexed assay, participating laboratories were able to precisely and reproducibly determine the levels of a series of analytes in blinded samples used to simulate an inter-laboratory clinical study of patient samples. Our study further establishes that LC-MRM-MS using stable isotope dilution, with appropriate attention to analytical validation and appropriate quality c`ontrol measures, enables sensitive, specific, reproducible and quantitative measurements of proteins and peptides in complex biological matrices such as plasma.

Vascular endothelial growth factor is an autocrine growth factor, signaling through neuropilin-1 in non-small cell lung cancer

Background The VEGF pathway has become an important therapeutic target in lung cancer, where VEGF has long been established as a potent pro-angiogenic growth factor expressed by many types of tumors. While Bevacizumab (Avastin) has proven successful in increasing the objective tumor response rate and in prolonging progression and overall survival in patients with NSCLC, the survival benefit is however relatively short and the majority of patients eventually relapse. The current use of tyrosine kinase inhibitors alone and in combination with chemotherapy has been underwhelming, highlighting an urgent need for new targeted therapies. In this study, we examined the mechanisms of VEGF-mediated survival in NSCLC cells and the role of the Neuropilin receptors in this process. Methods NSCLC cells were screened for expression of VEGF and its receptors. The effects of recombinant VEGF and its blockade on lung tumor cell proliferation and cell cycle were examined. Phosphorylation of Akt and Erk1/2 proteins was examined by high content analysis and confocal microscopy. The effects of silencing VEGF on cell proliferation and survival signaling were also assessed. A Neuropilin-1 stable-transfected cell line was generated. Cell growth characteristics in addition to pAkt and pErk1/2 signaling were studied in response to VEGF and its blockade. Tumor growth studies were carried out in nude mice following subcutaneous injection of NP1 over-expressing cells. Results Inhibition of the VEGF pathway with anti-VEGF and anti-VEGFR-2 antibodies or siRNA to VEGF, NP1 and NP2 resulted in growth inhibition of NP1 positive tumor cell lines associated with down-regulation of PI3K and MAPK kinase signaling. Stable transfection of NP1 negative cells with NP1 induced proliferation in vitro, which was further enhanced by exogenous VEGF. In vivo, NP1 over-expressing cells significantly increased tumor growth in xenografts compared to controls. Conclusions Our data demonstrate that VEGF is an autocrine growth factor in NSCLC signaling, at least in part, through NP1. Targeting this VEGF receptor may offer potential as a novel therapeutic approach and also support the evaluation of the role of NP1 as a biomarker predicting sensitivity or resistance to VEGF and VEGFR-targeted therapies in the clinical arena.

Differential interaction between the C2B domain of synaptotagmin-I and the Drosophila stonedA and stonedB proteins

The stoned locus in Drosophila encodes two proteins StonedA (STNA) and StonedB (STNB), both of which have been suggested to act as adaptins in mediating synaptic vesicle recycling. A combination of immunological, genetic and biochemical studies have shown an interaction of STNA and STNB with the C2B domain of Synaptotagmin-I (SYT-1), an integral synaptic vesicle protein that mediates Ca2+-dependent exocytosis, as well as endocytosis. The C2B domain of SYT-1 contains an AP-2 binding site that controls the size of recycled vesicles, and a C-terminal tryptophan-containing motif that acts as an internalization signal. Investigation of SYT-1 mutations in Drosophila has shown that altering the Ca2+ binding region of the C2B domain, results in a reduction in the rate of vesicle recycling, implicating this region in SYT-I endocytosis. In this poster, we report the molecular dissection of the interactions between the STNA and STNB proteins and the C2B domain of SYT-1. Deletion of the AP-2 binding site decreased the binding of both STNA and STNB. However, C-terminal deletions of the C2B domain significantly increased STNB binding. In contrast, the same C-terminal deletions reduced the affinity of the C2B domain for STNA. The possible interactions of both STNB and STNA with the Ca2+ binding region of SYT-1 will be also investigated.

Gene regulation by translational inhibition is determined by Dicer partnering proteins

MicroRNAs (miRNAs) are small regulatory RNAs produced by Dicer proteins that regulate gene expression in development and adaptive responses to the environment1,​2,​3,​4. In animals, the degree of base pairing between a miRNA and its target messenger RNA seems to determine whether the regulation occurs through cleavage or translation inhibition1. In contrast, the selection of regulatory mechanisms is independent of the degree of mismatch between a plant miRNA and its target transcript5. However, the components and mechanism(s) that determine whether a plant miRNA ultimately regulates its targets by guiding cleavage or translational inhibition are unknown6. Here we show that the form of regulatory action directed by a plant miRNA is determined by DRB2, a DICER-LIKE1 (DCL1) partnering protein. The dependence of DCL1 on DRB1 for miRNA biogenesis is well characterized7,​8,​9, but we show that it is only required for miRNA-guided transcript cleavage. We found that DRB2 determines miRNA-guided translational inhibition and represses DRB1 expression, thereby allowing the active selection of miRNA regulatory action. Furthermore, our results reveal that the core silencing proteins ARGONAUTE1 (AGO1) and SERRATE (SE) are highly regulated by miRNA-guided translational inhibition. DRB2 has been remarkably conserved throughout plant evolution, raising the possibility that translational repression is the ancient form of miRNA-directed gene regulation in plants, and that Dicer partnering proteins, such as human TRBP, might play a similar role in other eukaryotic systems.

Adaptive aneuploidy protects against thiol peroxidase deficiency by increasing respiration via key mitochondrial proteins

Aerobic respiration is a fundamental energy-generating process; however, there is cost associated with living in an oxygen-rich environment, because partially reduced oxygen species can damage cellular components. Organisms evolved enzymes that alleviate this damage and protect the intracellular milieu, most notably thiol peroxidases, which are abundant and conserved enzymes that mediate hydrogen peroxide signaling and act as the first line of defense against oxidants in nearly all living organisms. Deletion of all eight thiol peroxidase genes in yeast (∆8 strain) is not lethal, but results in slow growth and a high mutation rate. Here we characterized mechanisms that allow yeast cells to survive under conditions of thiol peroxidase deficiency. Two independent ∆8 strains increased mitochondrial content, altered mitochondrial distribution, and became dependent on respiration for growth but they were not hypersensitive to H2O2. In addition, both strains independently acquired a second copy of chromosome XI and increased expression of genes encoded by it. Survival of ∆8 cells was dependent on mitochondrial cytochrome-c peroxidase (CCP1) and UTH1, present on chromosome XI. Coexpression of these genes in ∆8 cells led to the elimination of the extra copy of chromosome XI and improved cell growth, whereas deletion of either gene was lethal. Thus, thiol peroxidase deficiency requires dosage compensation of CCP1 and UTH1 via chromosome XI aneuploidy, wherein these proteins support hydroperoxide removal with the reducing equivalents generated by the electron transport chain. To our knowledge, this is the first evidence of adaptive aneuploidy counteracting oxidative stress.

Incorporation of human recombinant tropoelastin into silk fibroin membranes with the view to repairing Bruch’s membrane

Bombyx mori silk fibroin membranes provide a potential delivery vehicle for both cells and extracellular matrix (ECM) components into diseased or injured tissues. We have previously demonstrated the feasibility of growing retinal pigment epithelial cells (RPE) on fibroin membranes with the view to repairing the retina of patients afflicted with age-related macular degeneration (AMD). The goal of the present study was to investigate the feasibility of incorporating the ECM component elastin, in the form of human recombinant tropoelastin, into these same membranes. Two basic strategies were explored: (1) membranes prepared from blended solutions of fibroin and tropoelastin; and (2) layered constructs prepared from sequentially cast solutions of fibroin, tropoelastin, and fibroin. Optimal conditions for RPE attachment were achieved using a tropoelastin-fibroin blend ratio of 10 to 90 parts by weight. Retention of tropoelastin within the blend and layered constructs was confirmed by immunolabelling and Fourier-transform infrared spectroscopy (FTIR). In the layered constructs, the bulk of tropoelastin was apparently absorbed into the initially cast fibroin layer. Blend membranes displayed higher elastic modulus, percentage elongation, and tensile strength (p < 0.01) when compared to the layered constructs. RPE cell response to fibroin membranes was not affected by the presence of tropoelastin. These findings support the potential use of fibroin membranes for the co-delivery of RPE cells and tropoelastin.

Serum levels of soluble receptor for advanced glycation end products and of S100 proteins are associated with inflammatory, autoantibody, and classical risk markers of joint and vascular damage in rheumatoid arthritis

Introduction: The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily of cell surface receptor molecules. High concentrations of three of its putative proinflammatory ligands, S100A8/A9 complex (calprotectin), S100A8, and S100A12, are found in rheumatoid arthritis (RA) serum and synovial fluid. In contrast, soluble RAGE (sRAGE) may prevent proinflammatory effects by acting as a decoy. This study evaluated the serum levels of S100A9, S100A8, S100A12 and sRAGE in RA patients, to determine their relationship to inflammation and joint and vascular damage. Methods: Serum sRAGE, S100A9, S100A8 and S100A12 levels from 138 patients with established RA and 44 healthy controls were measured by ELISA and compared by unpaired t test. In RA patients, associations with disease activity and severity variables were analyzed by simple and multiple linear regressions. Results: Serum S100A9, S100A8 and S100A12 levels were correlated in RA patients. S100A9 levels were associated with body mass index (BMI), and with serum levels of S100A8 and S100A12. S100A8 levels were associated with serum levels of S100A9, presence of anti-citrullinated peptide antibodies (ACPA), and rheumatoid factor (RF). S100A12 levels were associated with presence of ACPA, history of diabetes, and serum S100A9 levels. sRAGE levels were negatively associated with serum levels of C-reactive protein (CRP) and high-density lipoprotein (HDL), history of vasculitis, and the presence of the RAGE 82Ser polymorphism. Conclusions: sRAGE and S100 proteins were associated not just with RA inflammation and autoantibody production, but also with classical vascular risk factors for end-organ damage. Consistent with its role as a RAGE decoy molecule, sRAGE had the opposite effects to S100 proteins in that S100 proteins were associated with autoantibodies and vascular risk, whereas sRAGE was associated with protection against joint and vascular damage. These data suggest that RAGE activity influences co-development of joint and vascular disease in rheumatoid arthritis patients.

Induction of a Th1 immune response and suppression of IgE via immunotherapy with a recombinant hybrid molecule encapsulated in liposome-protamine-DNA nanoparticles in a model of experimental allergy

Liposome-protamine-DNA nanoparticles (LPD) are safe, effective, and non-toxic adjuvants that induce Th1-like immune responses. We hypothesized that encapsulation of allergens into liposomes could be an appropriate option for immunotherapy. The present study evaluated the immunotherapeutic potential of a recombinant hybrid molecule (rHM) encapsulated in LPD nanoparticles in a murine model of Chenopodium album allergy. BALB/c mice were sensitized with the allergen in alum, and the immunotherapy procedure was performed by subcutaneous injections of LPD-rHM, rHM, or empty LPD at weekly intervals. Sensitized mice developed a Th2-biased immune response characterized by strong specific IgG1 and IgE production, IL-4, and the transcription factor GATA3 in spleen cell cultures. Treatment with the LPD-rHM resulted in a reduction in IgE and a marked increase in IgG2a. The LPD-rHM induced allergen-specific responses with relatively high interferon-gamma production, as well as expression of the transcription factor T-bet in stimulated splenocytes. In addition, lymphoproliferative responses were higher in the LPD-rHM-treated mice than in the other groups. Removal of the nanoparticles from the rHM resulted in a decrease in the allergen's immunogenicity. These results indicate that the rHM complexed with LPD nanoparticles has a marked suppressive effect on the allergic response and caused a shift toward a Th1 pathway.

Purification of the major group 1 allergen from bahia grass pollen, Pas n 1

Background Group 1 grass pollen allergens are glycoproteins of the β-expansin family. They are a predominant component of pollen and are potent allergens with a high frequency of serum IgE reactivity in grass pollen-allergic patients. Bahia grass is distinct from temperate grasses and has a prolonged pollination period and wide distribution in warmer climates. Here we describe the purification of the group 1 pollen allergen, Pas n 1, from Bahia grass (Paspalum notatum), an important subtropical aeroallergen source. Methods Pas n 1 was purified from an aqueous Bahia grass pollen extract by ammonium sulphate precipitation, hydrophobic interaction and size exclusion chromatography, and assessed by one- and two-dimensional gel electrophoresis, immunoblotting and ELISA. Results Pas n 1 was purified to a single 29-kDa protein band containing two dominant isoforms detected by an allergen-specific monoclonal antibody and serum IgE of a Bahia grass pollen-allergic donor. The frequency of serum IgE reactivity with purified Pas n 1 in 51 Bahia grass pollen-allergic patients was 90.6%. Serum IgE reactivity with purified Pas n 1 was highly correlated with serum IgE reactivity with Bahia grass pollen extract and recombinant Pas n 1 (r = 0.821 and 0.913, respectively). Conclusions Pas n 1 is a major allergen reactive at high frequency with serum IgE of Bahia grass pollen-allergic patients. Purified natural Pas n 1 has utility for improved specific diagnosis and immunotherapy for Bahia grass pollen allergy.

Pharmacology in one semester [Version 3, Semester 1, 2014]

- For use in Introductory Units/Courses to Biomedical/Science Students - For use with Allied Health Students who are taking pharmacology as a Unit/Course or a part Unit/Course

Vaccination of koalas (Phascolarctos cinereus) with a recombinant chlamydial major outer membrane protein adjuvanted with poly I:C, a host defense peptide and polyphosphazine, elicits strong and long lasting cellular and humoral immune responses

Chlamydial infections are wide spread in koalas across their range and a solution to this debilitating disease has been sought for over a decade. Antibiotics are the currently accepted therapeutic measure, but are not an effective treatment due to the asymptomatic nature of some infections and a low efficacy rate. Thus, a vaccine would be an ideal way to address this infectious disease threat in the wild. Previous vaccine trials have used a three-dose regimen; however this is very difficult to apply in the field as it would require multiple capture events, which are stressful and invasive processes for the koala. In addition, it requires skilled koala handlers and a significant monetary investment. To overcome these challenges, in this study we utilized a polyphosphazine based poly I:C and a host defense peptide adjuvant combined with recombinant chlamydial major outer membrane protein (rMOMP) antigen to induce long lasting (54 weeks) cellular and humoral immunity in female koalas with a novel single immunizing dose. Immunized koalas produced a strong IgG response in plasma, as well as at mucosal sites. Moreover, they showed high levels of C. pecorum specific neutralizing antibodies in the plasma as well as vaginal and conjunctival secretions. Lastly, Chlamydia-specific lymphocyte proliferation responses were produced against both whole chlamydial elementary bodies and rMOMP protein, over the 12-month period. The results of this study suggest that a single dose rMOMP vaccine incorporating a poly I:C, host defense peptide and polyphosphazine adjuvant is able to stimulate both arms of the immune system in koalas, thereby providing an alternative to antibiotic treatment and/or a three-dose vaccine regime.

Cloning and gastrointestinal expression of rat hephaestin: Relationship to other iron transport proteins

The membrane-bound ceruloplasmin homolog hephaestin plays a critical role in intestinal iron absorption. The aims of this study were to clone the rat hephaestin gene and to examine its expression in the gastrointestinal tract in relation to other genes encoding iron transport proteins. The rat hephaestin gene was isolated from intestinal mRNA and was found to encode a protein 96% identical to mouse hephaestin. Analysis by ribonuclease protection assay and Western blotting showed that hephaestin was expressed at high levels throughout the small intestine and colon. Immunofluorescence localized the hephaestin protein to the mature villus enterocytes with little or no expression in the crypts. Variations in iron status had a small but nonsignificant effect on hephaestin expression in the duodenum. The high sequence conservation between rat and mouse hephaestin is consistent with this protein playing a central role in intestinal iron absorption, although its precise function remains to be determined.