Specific Binding Activity Research Articles

Copper binding in IscA inhibits iron-sulphur cluster assembly in Escherichia coli.

Among the iron-sulphur cluster assembly proteins encoded by gene cluster iscSUA-hscBA-fdx in Escherichia coli, IscA has a unique and strong iron binding activity and can provide iron for iron-sulphur cluster assembly in proteins in vitro. Deletion of IscA and its paralogue SufA results in an E. coli mutant that fails to assemble [4Fe-4S] clusters in proteins under aerobic conditions, suggesting that IscA has a crucial role for iron-sulphur cluster biogenesis. Here we report that among the iron-sulphur cluster assembly proteins, IscA also has a strong and specific binding activity for Cu(I) in vivo and in vitro. The Cu(I) centre in IscA is stable and resistant to oxidation under aerobic conditions. Mutation of the conserved cysteine residues that are essential for the iron binding in IscA abolishes the copper binding activity, indicating that copper and iron may share the same binding site in the protein. Additional studies reveal that copper can compete with iron for the metal binding site in IscA and effectively inhibits the IscA-mediated [4Fe-4S] cluster assembly in E. coli cells. The results suggest that copper may not only attack the [4Fe-4S] clusters in dehydratases, but also block the [4Fe-4S] cluster assembly in proteins by targeting IscA in cells.

Micromolar concentrations of rimonabant directly inhibits delta opioid receptor specific ligand binding and agonist-induced G-protein activity

What is knownThere is a growing number of evidence showing, that the cannabinoid receptor 1 (CB1) antagonist rimonabant has many non-cannabimimetic actions, such as affecting the opioid system. The direct effect of rimonabant on opioid receptors has been studied so far mainly on μ-opioid receptors. However recently the δ-opioid receptor (DOR) receives much more attention as before, due to its potential therapeutic applications, such as nociception or treatment for psychiatric disorders. ObjectivesTo investigate the direct effect of rimonabant on DOR specific ligand binding and on the DOR mediated G-protein activation. ResultsMicromolar concentrations of rimonabant directly inhibited the DOR specific agonist binding in radioligand competition binding experiments using Chinese hamster ovary cells stably transfected with mouse DOR (CHO-mDOR). However the inhibition occurred also in the subnanomolar range during DOR specific antagonist binding in similar experimental conditions. In functional [35S]GTPγS binding assays rimonabant significantly decreased the basal receptor activity in CHO-mDOR but also in parental CHO cell membranes. During DOR agonist stimulation, micromolar concentration of rimonabant attenuated the DOR G-protein activation and the potency of the activator ligand in [35S]GTPγS binding assays performed in CHO-mDOR, in wild type and also in CB1/CB2 double knock-out mouse forebrain membranes. Yet again this inhibitory action was DOR specific, since it did not occur during other specific GPCR agonist mediated G-protein activation. ConclusionRimonabant directly inhibited DOR function in the micromolar concentrations. The inhibitory actions indicate an antagonistic behavior towards DOR which was established by the followings: (i) rimonabant inhibited DOR antagonist binding more effectively than agonist binding, (ii) the inverse agonistic, agonistic effect of the compound can be excluded, and (iii) additionally according to previous findings the allosteric mechanism can also be foreclosed.

Generating novel recombinant prokaryotic lectins with altered carbohydrate binding properties through mutagenesis of the PA-IL protein from Pseudomonas aeruginosa

BackgroundProkaryotic lectins offer significant advantages over eukaryotic lectins for the development of enhanced glycoselective tools. Amenability to recombinant expression in Escherichia coli simplifies their production and presents opportunities for further genetic manipulation to create novel recombinant prokaryotic lectins (RPLs) with altered or enhanced carbohydrate binding properties. This study explored the potential of the α-galactophilic PA-IL lectin from Pseudomonas aeruginosa for use as a scaffold structure for the generation of novel RPLs. MethodSpecific amino acid residues in the carbohydrate binding site of a recombinant PA-IL protein were randomly substituted by site-directed mutagenesis. The resulting expression clones were then functionally screened to identify clones expressing rPA-IL proteins with altered carbohydrate binding properties. ResultsThis study generated RPLs exhibiting diverse carbohydrate binding activities including specificity and high affinity for β-linked galactose and N-acetyl-lactosamine (LacNAc) displayed by N-linked glycans on glycoprotein targets. Key amino acid substitutions were identified and linked with specific carbohydrate binding activities. Ultimately, the utility of these novel RPLs for glycoprotein analysis and for selective fractionation and isolation of glycoproteins and their glycoforms was demonstrated. ConclusionsThe carbohydrate binding properties of the PA-IL protein can be significantly altered using site-directed mutagenesis strategies to generate novel RPLs with diverse carbohydrate binding properties. General significanceThe novel RPLs reported would find a broad range of applications in glycobiology, diagnostics and in the analysis of biotherapeutics. The ability to readily produce these RPLs in gram quantities could enable them to find larger scale applications for glycoprotein or biotherapeutic purification.

A Review on Metallic Silver Nanoparticles

Nanotechnology refers to the branch of science and engineering dedicated to materials, having dimensions in the order of 100th of nm or less. The term being new, but has been widely used for the development of more efficient technology. In recent years, nanotechnology has been embraced by industrial sectors due to its applications in the field of electronic storage systems, biotechnology, magnetic separation and pre concentration of target analytes, targeted drug delivery and vehicles for gene and drug delivery. Consequently, with wide range of applications available, these particles have potential to make a significant impact to the society. In general, nanoparticles used in the field of biotechnology range in particle size between 10 and 500 nm, seldom exceeding 700 nm. The nanosize of these particles allows various communications with biomolecules on the cell surfaces and within the cells in way that can be decoded and designated to various biochemical and physiochemical properties of these cells. Similarly, its potential application in drug delivery system and noninvasive imaging offered various advantages over conventional pharmaceutical agents. More specific targeting systems are designed to recognize the targeted cells such as cancer cells. This can be achieved by conjugating the nanoparticle with an appropriate ligand, which has a specific binding activity with respect to the target cells. In addition, nanoparticles provide a platform to attach multiple copies of therapeutic substance on it and hence increase the concentration of therapeutic and diagnostic substances at the pathological site. Also, the concentration and dynamics of the active molecule can be varied by controlling the particle size of nanoparticles (>3–5 nm).

Muscarinic Receptor Binding of the Novel Radioligand, [3H]Imidafenacin in the Human Bladder and Parotid Gland

The aim of the current study was to demonstrate highly specific and direct binding activity of tritium ([(3)H])-labeled imidafenacin for labeling muscarinic receptors in human bladder and parotid gland. Specific binding of [(3)H]imidafenacin in human tissues was saturable, reversible, and of high affinity. The Kd value for specific [(3)H]imidafenacin binding in the human bladder was approximately 3 times higher than that in the parotid gland. Unlabeled imidafenacin as well as the clinically used antimuscarinic agents, oxybutynin, tolterodine, and solifenacin, competed with [(3)H]imidafenacin for binding sites in the human bladder and parotid gland in a concentrationdependent manner, which indicated pharmacological specificity of [(3)H]imidafenacin binding sites. The Ki for imidafenacin in the human bladder approximately corresponded to pharmacological potency for the competitive blockade of carbachol-induced contractions of bladder, indicating a close correlation between binding affinity of imidafenacin to bladder muscarinic receptors and its pharmacological effects in the bladder. In conclusion, the current study is the first to provide direct evidence to demonstrate that imidafenacin bound muscarinic receptors in the human bladder, supporting its clinical relevance as a therapeutic agent for overactive bladder. [(3)H]Imidafenacin may also be a useful radioligand for labeling the M3 subtype of muscarinic receptors with higher selectivity than other radioligands.

Implications of glycoprotein VI for theranostics

Glycoprotein VI (GPVI), a membrane glycoprotein solely expressed in platelets and megakaryocytes, plays a critical role in thrombus formation due to collagen/GPVI-mediated platelet activation and adhesion. Recent studies have shown that surface expression of GPVI on circulating platelets is enhanced in acute cardiovascular diseases such as myocardial infarction and ischaemic stroke. Increased GPVI levels are associated with poor clinical outcome and are an early indicator for imminent myocardial infarction in patients with chest pain. The soluble form of the dimeric GPVI fusion protein (sGPVI-Fc) binds with high affinity to collagen and atherosclerotic plaque tissue. Non-invasive imaging studies with radiolabelled sGPVI-Fc show specific binding activity to vascular lesions in vivo. Further, sGPVI-Fc has been developed as a new therapeutic platelet-based strategy for lesion-directed antithrombotic therapy. This review summarises the potential of GPVI for diagnostic and therapeutic options based on novel non-invasive molecular imaging modalities to ameliorate care of patients with cardiovascular diseases.

Two-Dimensional Electrophoresis-Based Selection of Aptamers Against an Unidentified Protein in a Tissue Sample

A fully automated two-dimensional electrophoresis (2DE) system was employed for DNA aptamer selection against an unidentified protein in a mouse liver tissue extract as a model target. A 2DE-based systematic evolution of ligands by exponential enrichment (2DE-SELEX) was demonstrated for aptamer selection against a single protein spot that was separated on a nitrocellulose membrane. After four iterative 2DE-SELEX cycles, the oligonucleotide pool was sequenced and aptamer sequences were identified. A blotting assay showed that an identified aptamer with a stable stem–loop structure had specific binding activity against the target protein. The 2DE-SELEX was shown to be promising for the development of aptamers against unidentified proteins in complex samples for proteomic analysis and biomarker discovery. Supplemental materials are available for this article. Go to the publisher's online edition of Analytical Letters to view the supplemental file.

Enhanced membrane protein expression by engineering increased intracellular membrane production

BackgroundMembrane protein research is frequently hampered by the low natural abundance of these proteins in cells and typically relies on recombinant gene expression. Different expression systems, like mammalian cells, insect cells, bacteria and yeast are being used, but very few research efforts have been directed towards specific host cell customization for enhanced expression of membrane proteins. Here we show that by increasing the intracellular membrane production by interfering with a key enzymatic step of lipid synthesis, enhanced expression of membrane proteins in yeast is achieved.ResultsWe engineered the oleotrophic yeast, Yarrowia lipolytica, by deleting the phosphatidic acid phosphatase, PAH1, which led to massive proliferation of endoplasmic reticulum (ER) membranes. For all eight tested representatives of different integral membrane protein families, we obtained enhanced protein accumulation levels and in some cases enhanced proteolytic integrity in the ∆pah1 strain. We analysed the adenosine A2AR G-protein coupled receptor case in more detail and found that concomitant induction of the unfolded protein response in the ∆pah1 strain enhanced the specific ligand binding activity of the receptor. These data indicate an improved quality control mechanism for membrane proteins accumulating in yeast cells with proliferated ER.ConclusionsWe conclude that redirecting the metabolic flux of fatty acids away from triacylglycerol- and sterylester-storage towards membrane phospholipid synthesis by PAH1 gene inactivation, provides a valuable approach to enhance eukaryotic membrane protein production. Complementary to this improvement in membrane protein quantity, UPR co-induction further enhances the quality of the membrane protein in terms of its proper folding and biological activity. Importantly, since these pathways are conserved in all eukaryotes, it will be of interest to investigate similar engineering approaches in other cell types of biotechnological interest, such as insect cells and mammalian cells.

Automated Analysis of (123)I-beta-CIT SPECT Images with Statistical Probabilistic Anatomical Mapping.

Population-based statistical probabilistic anatomical maps have been used to generate probabilistic volumes of interest for analyzing perfusion and metabolic brain imaging. We investigated the feasibility of automated analysis for dopamine transporter images using this technique and evaluated striatal binding potentials in Parkinson's disease and Wilson's disease. We analyzed 2β-Carbomethoxy-3β-(4-(123)I-iodophenyl)tropane((123)I-beta-CIT) SPECT images acquired from 26 people with Parkinson's disease (M:F = 11:15, mean age = 49 ± 12years), 9 people with Wilson's disease (M: F = 6:3, mean age = 26 ± 11years) and 17 normal controls (M:F = 5:12, mean age = 39 ± 16years). A SPECT template was created using striatal statistical probabilistic map images. All images were spatially normalized onto the template, and probability-weighted regional counts in striatal structures were estimated. The binding potential was calculated using the ratio of specific and nonspecific binding activities at equilibrium. Voxel-based comparisons between groups were also performed using statistical parametric mapping. Qualitative assessment showed that spatial normalizations of the SPECT images were successful for all images. The striatal binding potentials of participants with Parkinson's disease and Wilson's disease were significantly lower than those of normal controls. Statistical parametric mapping analysis found statistically significant differences only in striatal regions in both disease groups compared to controls. We successfully evaluated the regional (123)I-beta-CIT distribution using the SPECT template and probabilistic map data automatically. This procedure allows an objective and quantitative comparison of the binding potential, which in this case showed a significantly decreased binding potential in the striata of patients with Parkinson's disease or Wilson's disease.

Optimized and enhanced DNA plasmid vector based in vivo construction of a neutralizing anti-HIV-1 envelope glycoprotein Fab

Monoclonal antibody preparations have demonstrated considerable clinical utility in the treatment of specific malignancies, as well as inflammatory and infectious diseases. Antibodies are conventionally delivered by passive administration, typically requiring costly large-scale laboratory development and production. Additional limitations include the necessity for repeat administrations, and the length of in vivo potency. Therefore, the development of methods to generate therapeutic antibodies and antibody like molecules in vivo, distinct from an active antigen-based immunization strategy, would have considerable clinical utility. In fact, adeno-associated viral (AAV) vector mediated delivery of immunoglobulin genes with subsequent generation of functional antibodies has recently been developed. As well, anon-viral vector mediated nucleic acid based delivery technology could permit the generation of therapeutic/prophylactic antibodies in vivo, obviating potential safety issues associated with viral vector based gene delivery. This delivery strategy has limitations as well, mainly due to very low in vivo production and expression of protein from the delivered gene. In the study reported here we have constructed an “enhanced and optimized” DNA plasmid technology to generate immunoglobulin heavy and light chains (i.e., Fab fragments) from an established neutralizing anti-HIV envelope glycoprotein monoclonal antibody (VRC01). This “enhanced” DNA (E-DNA) plasmid technology includes codon/RNA optimization, leader sequence utilization, as well as targeted potentiation of delivery and expression of the Fab immunoglobulin genes through use of “adaptive” in vivo electroporation. The results demonstrate that delivery by this method of a single administration of the optimized Fab expressing constructs resulted in generation of Fab molecules in mouse sera possessing high antigen specific binding and HIV neutralization activity for at least 7 d after injection, against diverse HIV isolates. Importantly, this delivery strategy resulted in a rapid increase (i.e., in as little as 48 h) in Fab levels when compared with protein-based immunization. The active generation of functional Fab molecules in vivo has important conceptual and practical advantages over conventional ex vivo generation, purification and passive delivery of biologically active antibodies. Further study of this technique for the rapid generation and delivery of immunoglobulin and immunoglobulin like molecules is highly relevant and timely.

DNA methylation presents distinct binding sites for human transcription factors

DNA methylation, especially CpG methylation at promoter regions, has been generally considered as a potent epigenetic modification that prohibits transcription factor (TF) recruitment, resulting in transcription suppression. Here, we used a protein microarray-based approach to systematically survey the entire human TF family and found numerous purified TFs with methylated CpG (mCpG)-dependent DNA-binding activities. Interestingly, some TFs exhibit specific binding activity to methylated and unmethylated DNA motifs of distinct sequences. To elucidate the underlying mechanism, we focused on Kruppel-like factor 4 (KLF4), and decoupled its mCpG- and CpG-binding activities via site-directed mutagenesis. Furthermore, KLF4 binds specific methylated or unmethylated motifs in human embryonic stem cells in vivo. Our study suggests that mCpG-dependent TF binding activity is a widespread phenomenon and provides a new framework to understand the role and mechanism of TFs in epigenetic regulation of gene transcription. DOI:http://dx.doi.org/10.7554/eLife.00726.001.

Evaluation of Phage Display Discovered Peptides as Ligands for Prostate-Specific Membrane Antigen (PSMA)

The aim of this study was to identify potential ligands of PSMA suitable for further development as novel PSMA-targeted peptides using phage display technology. The human PSMA protein was immobilized as a target followed by incubation with a 15-mer phage display random peptide library. After one round of prescreening and two rounds of screening, high-stringency screening at the third round of panning was performed to identify the highest affinity binders. Phages which had a specific binding activity to PSMA in human prostate cancer cells were isolated and the DNA corresponding to the 15-mers were sequenced to provide three consensus sequences: GDHSPFT, SHFSVGS and EVPRLSLLAVFL as well as other sequences that did not display consensus. Two of the peptide sequences deduced from DNA sequencing of binding phages, SHSFSVGSGDHSPFT and GRFLTGGTGRLLRIS were labeled with 5-carboxyfluorescein and shown to bind and co-internalize with PSMA on human prostate cancer cells by fluorescence microscopy. The high stringency requirements yielded peptides with affinities KD∼1 µM or greater which are suitable starting points for affinity maturation. While these values were less than anticipated, the high stringency did yield peptide sequences that apparently bound to different surfaces on PSMA. These peptide sequences could be the basis for further development of peptides for prostate cancer tumor imaging and therapy.

Mode of binding of RNA polymerase α subunit to the phased A-tracts upstream of the phospholipase C gene promoter of Clostridium perfringens

Three phased A5-6-tracts lie upstream of the promoter of plc encoding the α-toxin (phospholipase C) of Clostridium perfringens. The α subunits of C. perfringens RNA polymerase bind directly to the phased A-tracts via the C-terminal domain of the α subunit (αCTD). To identify the amino acid residues involved in the binding of C. perfringens α subunits to the phased A-tracts, 27 amino acid residues in C. perfringens αCTD were substituted with alanine. The affinities of the mutated α subunits for the phased A-tracts were examined by gel shift assays and surface plasmon resonance (SPR). The SPR analyses revealed that the phased A-tracts themselves facilitated a complex formation between the phased A-tracts and C. perfringens α subunits [Kd was 6.1 (±0.3) × 10−8 M], and that Arg261, Asn264, Gly292 and Lys294 in C. perfringens αCTD were critical for the binding to the phased A-tracts. The topology of these amino acid residues on the predicted structure of C. perfringens αCTD indicated a contact path with the phased A-tracts that was similar to that of Escherichia coli αCTD with the upstream (UP) element. On the other hand, SPR analyses at different temperatures (15, 25 and 37 °C) indicated that the affinity of the C. perfringens α subunits for the phased A-tracts increased in a low-temperature-dependent manner, whereas that of the E. coli α subunit for the UP element did not. This suggests that the phased A-tracts may not simply be a subset of the UP element, and that they show specific binding activity with the RNA polymerase α subunit.

Screening and identification of peptides that bind human midkine selected from phage display library

Objective To screen human midkine (h-midkine) inhibitory peptides from the phage display peptide library.Methods Recombinant human midkine (rh-midkine) protein was used to search for the specific peptides by panning.The specific binding activities of positive clones to target protein were examined by phage enzyme linked immunosorbent assay (ELISA).The effects of binding peptides on HepG2 cells proliferation were analyzed by methyl thiazol tetrazolium (MTT) method.Results Thenty positive clones were obtained after three rounds of selection.Three linear peptides and three cycle peptides,more frequently occurring in the affinity-selected phage population and higher specificity to target protein,were chemically synthesized.The synthetic peptides produced inhibition of the HepG2 cells proliferation to different degrees,the MP-3 (CTSTAMDNC) has the strongest inhibitory effect for the HepG2 proliferation (P ＜ 0.01),the inhibition rate is about 40.7％.Conclusion Four peptides that can specifically bind to MK protein have been screened successfully,and these four peptides show suppression effects on the proliferation in tumor cells. Key words: Midkine ; Phage display ; Peptide

The red clover necrotic mosaic virus capsid protein N-terminal amino acids possess specific RNA binding activity and are required for stable virion assembly

The red clover necrotic mosaic virus (RCNMV) bipartite RNA genome is packaged into two virion populations containing either RNA-1 and RNA-2 or multiple copies of RNA-2 only. To understand this distinctive packaging scheme, we investigated the RNA-binding properties of the RCNMV capsid protein (CP). Maltose binding protein-CP fusions exhibited the highest binding affinities for RNA probes containing the RNA-2 trans-activator or the 3′ non-coding region from RNA-1. Other viral and non-viral RNA probes displayed CP binding but to a much lower degree. Deletion of the highly basic N-terminal 50 residues abolished CP binding to viral RNA transcripts. In planta studies of select CP deletion mutants within this N-terminal region revealed that it was indispensable for stable virion formation and the region spanning CP residues 5–15 is required for systemic movement. Thus, the N-terminal region of the CP is involved in both producing two virion populations due to its RNA binding properties and virion stability.

Phages bearing affinity peptides to severe acute respiratory syndromes-associated coronavirus differentiate this virus from other viruses

BackgroundTransmission of SARS-associated coronavirus (SARS-CoV) is now well controlled, nevertheless, it is important to develop effective methods to identify this virus from other pathogens. ObjectivesThe purpose of this study was to identify potential ligands and develop a novel diagnostic test to SARS-CoV using phage display technology. Study designThe SARS-CoV spike 1 (S1) protein containing the receptor binding region (RBD) was used as an immobilized target followed by incubation with a 12-mer phage display random peptide library. After four rounds of biopanning, 10 monoclonal phages with specific binding activity to the S1-RBD protein were obtained and subjected to binding and diagnostic assays. ResultsDNA sequencing showed that two phage displayed peptides HHKTWHPPVMHL (phage-H) and SQWHPRSASYPM (phage-S) that were specific ligands to the S1 protein. Moreover, the selected phage-H and phage-S were capable of differentiating SARS-CoV from other coronaviruses in indirect enzyme-linked immunosorbent assays. ConclusionThe peptides identified in this study are useful reagents for detection of SARS-CoV.

Dual-Tracer PET Using Generalized Factor Analysis of Dynamic Sequences

With single-photon emission computed tomography, simultaneous imaging of two physiological processes relies on discrimination of the energy of the emitted gamma rays, whereas the application of dual-tracer imaging to positron emission tomography (PET) imaging has been limited by the characteristic 511-keV emissions. To address this limitation, we developed a novel approach based on generalized factor analysis of dynamic sequences (GFADS) that exploits spatio-temporal differences between radiotracers and applied it to near-simultaneous imaging of 2-deoxy-2-[(18)F]fluoro-D-glucose (FDG) (brain metabolism) and (11)C-raclopride (D2) with simulated human data and experimental rhesus monkey data. We show theoretically and verify by simulation and measurement that GFADS can separate FDG and raclopride measurements that are made nearly simultaneously. The theoretical development shows that GFADS can decompose the studies at several levels: (1) It decomposes the FDG and raclopride study so that they can be analyzed as though they were obtained separately. (2) If additional physiologic/anatomic constraints can be imposed, further decomposition is possible. (3) For the example of raclopride, specific and nonspecific binding can be determined on a pixel-by-pixel basis. We found good agreement between the estimated GFADS factors and the simulated ground truth time activity curves (TACs), and between the GFADS factor images and the corresponding ground truth activity distributions with errors less than 7.3 ± 1.3%. Biases in estimation of specific D2 binding and relative metabolism activity were within 5.9 ± 3.6% compared to the ground truth values. We also evaluated our approach in simultaneous dual-isotope brain PET studies in a rhesus monkey and obtained accuracy of better than 6% in a mid-striatal volume, for striatal activity estimation. Dynamic image sequences acquired following near-simultaneous injection of two PET radiopharmaceuticals can be separated into components based on the differences in the kinetics, provided their kinetic behaviors are distinct.

Abstract 2663: SPECT/CT analysis of distribution of intracranially infused interleukin-13 Pseudomonas exotoxin, a targeted agent for interleukin-13 receptor expressing brain tumors.

Abstract Interleukin-13 Pseudomonas exotoxin (IL-13PE), a powerful targeted agent for IL-13 receptor α2 (IL-13Rα2) - positive tumors is being tested for safety and effectiveness in glioblastoma multiforme (GBM) clinical trials. However, distribution of IL-13PE after intra-cranial convection-enhanced delivery (CED) has not been determined. To study this, we radiolabeled IL-13PE with 125I and determined its binding and biological activity in vitro and distribution in a murine model of GBM. 125IL-13PE maintained its specific binding activity to IL-13Rα2 positive U251 glioblastoma tumor cells but not to T98G cells, as these cells did not express IL-13Rα2. 125IL-13PE was as cytotoxic to glioblastoma cells as non-radiolabeled IL-13PE, which killed U251 tumor cells in a concentration dependent manner but not T98G cells. Both binding and cytotoxic activities were blocked by a 100-fold excess of IL-13, which indicated that IL-13PE binding was specific. Athymic nude mice with intracranially implanted U251 tumors were given stereotactic intratumoral injections of 125I-IL-13PE by either a bolus injection or a 3-day-CED. Animals were imaged by micro-SPECT/CT at various time points. SPECT/CT imaging revealed retention of 125I-IL-13PE in U251 tumors. CED showed significantly higher volumes of distribution and maintained detectable drug levels for longer periods of time compared to bolus injection. SPECT/CT images of whole brains corroborated with the autoradiography results performed on sectioned brain. These results indicate that IL-13PE can be successfully radiolabled with 125I without the loss of binding and cytotoxic activity and that 125I-IL-13PE administered by CED showed higher volumes of distribution than the bolus route. Thus, future clinical studies with 125I-IL-13PE or other agents should consider the CED mode of delivery for better distribution in intracranial environments. Volume of distribution of 125I-IL-13PE after bolus or CED administration into mouse brain Injection parameters SPECT Time Vi (μl)* Vd (mm3)** Vd:Vi ratio Bolus - day 1 10 minutes 5 10.2 ± 0.4 204.7 ± 7.0 Bolus - day 7 10 minutes 5 0.1 ± 0.02 2.0 ± 0.4 CED - day 1 3 days 100 120.4 ± 21.8 120.4 ± 21.6 CED - day 7 3 days 100 81.7 ± 6.9 81.7 ± 6.7 *Vi: Volume of injection **Vd: Volume of distribution Citation Format: Akiko Suzuki, Pamela Leland, Peter L. Choyke, Tomio Inoue, Bharat H. Joshi, Raj K. Puri. SPECT/CT analysis of distribution of intracranially infused interleukin-13 Pseudomonas exotoxin, a targeted agent for interleukin-13 receptor expressing brain tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2663. doi:10.1158/1538-7445.AM2013-2663

Improved expression and purification of sigma 1 receptor fused to maltose binding protein by alteration of linker sequence

Sigma 1 receptor (S1R) is a eukaryotic membrane protein that functions as an inter-organelle signaling modulator and chaperone. Here we report an improved expression of S1R in Escherichia coli as a fusion to maltose binding protein (MBP) and a high-yield purification. Variants with linking amino acid sequences consisting of 0–5 alanine residues between MBP and S1R were created and tested in several E. coli expression strains in order to determine the best combination of construct and host for production of active MBP-S1R. Among the linker variations, the protein containing a 4-Ala linker exhibited superior expression characteristics (MBP-4A-S1R); this construct was most productively paired with E. coli B834-pRARE2 and a chemically defined growth and expression medium. A 3-step purification was developed, including extraction from the E. coli membrane fraction using a mixture of Triton X-100 and n-dodecyl-beta-D-maltopyranoside identified by screening constrainted by retention of binding function, and purification by amylose affinity and gel filtration chromatographies. This procedure yields ∼3.5mg of purified fusion protein per L of bacterial culture medium. Purified MBP-4A-S1R showed a 175-fold purification from the starting cellular lysate with respect to specific ligand binding activity, and is stable during concentration and freeze–thaw cycling.

Resveratrol Induces Long-Lasting IL-8 Expression and Peculiar EGFR Activation/Distribution in Human Keratinocytes: Mechanisms and Implications for Skin Administration

Anti-inflammatory and skin tumour preventing effects of resveratrol have been extensively studied pre-clinically and resveratrol has been proposed for clinical investigations. To provide a basis or/and limitations for topical administration to human skin, molecular mechanisms underlying resveratrol effects towards normal human epidermal keratinocytes (NHEK) were evaluated. NHEK were challenged by either resveratrol alone or by its combination with TNFalpha or TGFalpha, and time-dependent molecular events were monitored. Interleukin 8 (IL-8) expression and its mRNA stability, ERK1/2, p65/RelA, and EGFR phosphorylation were determined. Intracellular distribution of EGFR/P-EGFR was measured in the membrane, cytoplasmic, and nuclear fractions. Specific DNA binding activity of NFκB (p65/RelA) and AP-1(c-Fos), NHEK proliferation, and molecular markers of apoptosis/cell cycle were detected. Resveratrol induced delayed, long-lasting and steadily growing IL-8 gene and protein over-expression as well as enhanced EGFR phosphorylation, both abrogated by the EGFR kinase inhibitor PD168393. However, resveratrol did not act as a phosphatase inhibitor. ERK phosphorylation was transiently inhibited at early time-points and activated at 6–24 h. Accordingly, c-Fos-specific DNA binding was increased by resveratrol. Cellular distribution of EGFR/P-EGFR was shifted to membrane and nucleus while cytosolic levels were reduced concomitant with enhanced degradation. Notwithstanding high nuclear levels of EGFR/P-EGFR, spontaneous and TGFalpha-triggered cell proliferation was strongly suppressed by resveratrol mainly through cell cycle arrest.Conclusions/SignificanceResveratrol synergized with TNFα in the induction of delayed, long-lasting IL-8 expression through sustained EGFR-ERK axis activation. The time course indicates that resveratrol metabolites could be implicated. Topical administration of Resv to psoriatic patients over-expressing TNFα, IL-8 and EGFR-ERK in the skin should be cautiously considered. Since high nuclear levels of EGFR correspond to increased risk of tumorigenesis, chronic resveratrol application to the skin may be potentially dangerous. Wound healing acceleration by resveratrol could not be envisaged due to its anti-proliferative effects towards normal keratinocytes.