Blue Membrane Research Articles

Molecular characterization of an iPSC line derived from a patient with cone‐rod dystrophy related to a mutation in the PROM1 gene

Aims/Purpose: The PROM1 gene encodes the protein Prominin‐1, which plays a crucial role in the morphogenesis of photoreceptor outer segments. The c.1354dupT mutation, associated with various inherited retinal diseases (IRD) phenotypes, leads to a premature stop codon. This study aimed to assess the silencing efficiency of reprogramming vectors and the expression of endogenous pluripotency genes in the iPSC line [DCB]‐FiPSC1‐Ep5F‐2.Methods: The iPSC line [DCB]‐FiPSC1‐Ep5F‐2 was generated from a patient with Cone‐rod dystrophy (CRD) linked to the c.1354dupT mutation in the PROM1 gene. iPSC colonies were stained using alkaline phosphatase (AP) blue membrane substrate. The c.1354dupT mutation was Sanger sequenced. To confirm the absence of episomal vector integration, quantitative PCR (qPCR) targeting a region common to all reprogramming vectors (within the EBNA1 gene) was conducted. Vector‐specific primer‐based quantitative reverse transcription PCR (qRT‐PCR) was used to assess the expression of reprogramming genes from the episomal vectors. Additionally, qRT‐PCR with gene‐specific primers was utilized to evaluate the expression of endogenous pluripotency genes. Untransfected fibroblasts served as the negative control, while fibroblasts transfected with the episomal vectors 72 h earlier were used as the positive control.Results: The iPSC line [DCB]‐FiPSC1‐Ep5F‐2 exhibited positive AP activity. The presence of the c.1354dupT mutation was confirmed. Furthermore, the number of copies of the EBNA1 gene was significantly lower in the generated iPSC line compared to the control. The relative mRNA expression of the reprogramming vectors was also significantly lower in the iPSC line, while the relative mRNA expression of the endogenous pluripotency genes was significantly higher than in the control.Conclusions: While further immunocytochemical characterization is necessary to fully ascertain iPSC pluripotency, the patient‐derived iPSC line [DCB]‐FiPSC1‐Ep5F‐2 shows promise as a disease model for personalized therapy development.

Layered Double Hydroxide‐Assisted Fabrication of Prussian Blue Membranes for Precise Molecular Sieving

AbstractPrussian Blue (PB), which was first discovered as robust blue‐colored pigment in the year 1706, has shown promising prospects in disease treatment, energy conversion, water splitting, and sensing. Relying on the uniform 3.2 Å‐sized pore channels as well as high stability in aqueous environments, in this study, we pioneered in situ preparation of polycrystalline PB membranes to justify their dye rejection and metal ion discrimination ability in aqueous environments. Among various factors, the introduction of calcined NiFe layered double hydroxide buffer layers on porous α‐Al2O3 substrates was found to play a paramount role in the formation of continuous polycrystalline PB membranes, thereby leading to excellent dye rejection efficiency (>99.0 %). Moreover, prepared PB membranes enabled discriminating different monovalent metal ions (e.g., Li+, Na+, and K+) depending on their discrepancy in Stokes diameters, showing great promise for lithium extraction from smaller‐sized metal ions.

Layered Double Hydroxide-Assisted Fabrication of Prussian Blue Membranes for Precise Molecular Sieving.

Prussian Blue (PB), which was first discovered as robust blue-colored pigment in the year 1706, has shown promising prospects in disease treatment, energy conversion, water splitting, and sensing. Relying on the uniform 3.2 Å-sized pore channels as well as high stability in aqueous environments, in this study, we pioneered in situ preparation of polycrystalline PB membranes to justify their dye rejection and metal ion discrimination ability in aqueous environments. Among various factors, the introduction of calcined NiFe layered double hydroxide buffer layers on porous α-Al2 O3 substrates was found to play a paramount role in the formation of continuous polycrystalline PB membranes, thereby leading to excellent dye rejection efficiency (>99.0 %). Moreover, prepared PB membranes enabled discriminating different monovalent metal ions (e.g., Li+ , Na+ , and K+ ) depending on their discrepancy in Stokes diameters, showing great promise for lithium extraction from smaller-sized metal ions.

Peptidomimetic analogues of an Arg-Trp-x-x-Trp motif responsible for interaction of translocase MraY with bacteriophage ϕX174 lysis protein E

Translocase MraY is the target for bacteriophage ϕX174 lysis protein E, which interacts via a protein–protein interaction mediated by Phe-288 and Glu-287 of E. coli MraY, and an Arg-Trp-x-x-Trp motif on protein E, also found in several cationic antimicrobial peptides. Analogues of Arg-Trp-octyl ester, found previously to show antimicrobial activity, were tested for antimicrobial activity, with Lys-Trp-oct (MIC50P. fluorescens 5 µg/mL) and Arg-Trp-decyl ester (MIC50P. fluorescens 3 µg/mL) showing enhanced antimicrobial activity. Synthesis and testing of α-helix peptidomimetic analogues for this motif revealed improved antibacterial activity (MIC50E. coli 4–7 µg/mL) for analogues containing two aromatic substituents, mimicking the Arg-Trp-x-x-Trp motif, and MraY inhibition (IC50 140 µM) by one such peptidomimetic. Investigation of mechanism of action using the Alamar Blue membrane permeabilisation assay revealed bacteriostatic and bacteriocidal mechanisms in different members of this set of compounds, raising the possibility of more than one biological target. The observed antimicrobial activity and MraY inhibition shown by peptidomimetic compounds confirms that this site could be targeted by drug-like molecules.

Facilitated transport of ammonia in ultra-thin Prussian Blue membranes with potential-tuned selectivity

Here we report on tunable ammonia transport through ultrathin Prussian Blue (PB) membranes facilitated with protonic carrier. Continuous gas-tight 15–50 nm Prussian Blue layers were prepared by cyclic electrodeposition on porous gold/anodic aluminum oxide supports. The membranes reveal fast ammonium transport with NH3 permeance exceeding 0.3 m3(STP) m-2 bar-1 h-1, ultimate ideal NH3/H2 selectivity attaining 40 and NH3/N2 selectivity over 100. In mixed-gas separation thermodynamically-favored sorption leads to additional NH3/H2 selectivity rise to ~70. Facilitation of NH3 transport occurs upon its conversion to NH4+ maintaining the electroactivity of the selective layer and reversibly tuned by electrochemical framework loading. Engagement of the protonation step is supported with Raman spectroscopy and close values of NH3 diffusion coefficients measured in gas permeation experiments and NH4+ diffusion coefficient determined by electrochemical impedance spectroscopy in liquid phase. The impact of water vapors partial pressure as backward protonic carrier on NH3 transport efficiency is exposed and discussed.

Symptoms: Blue Tympanic Membrane and Conductive Hearing Loss

Symptoms: Blue Tympanic Membrane and Conductive Hearing Loss

Blue membranes: Sulfonated copper(II) phthalocyanine tetrasulfonic acid based composite membranes for DMFC and low relative humidity PEMFC

Polymer electrolyte membranes (PEMs) consisting of copper(II)phthalocyanine tetrasulfonic acid tetrasodium salt (CuPCSA) and disulfonated poly(arylene ether sulfone) (SES0005) are prepared. The TEM analysis results prove the incorporation of CuPCSA as nanoparticles into the composite membranes. Catalytic activity of CuPCSA towards peroxide degradation is shown by CV. Addition of CuPCSA increases the dimensional stability in contact with water (18% vs. 43% linear swelling for pristine SES0005). Addition of 10wt% CuPCSA (SES0005-IM10) increases the proton conductivity four fold to 16.8mScm−1 at 120°C and 50% relative humidity (rh). Activation energy decreases with CuPCSA content, reducing the conductivity's temperature dependence. Membranes were tested in low and medium temperature PEM fuel cells at 65 and 120°C, respectively, at 50% rh. In the LT-PEMFC, 40µm thick SES0005-IM10 and Nafion 212 based MEAs exhibited current densities of 470 and 454mAcm−2 at 0.7V, respectively. In the MT-PEMFC, SES0005-IM10 based MEAs demonstrated a current density of 405mAcm−2 at 0.5V, 2.4 folds more than pristine membrane based MEAs. In the DMFC, SES0005-IM10 enabled a peak power density of 153mWcm−2 at 70°C and 1M methanol feed, 20% higher than Nafion 212, 38% higher than mPES60.

Blue fluorogenic probes for cell plasma membranes fill the gap in multicolour imaging

Newly synthesized blue membrane probes, due to high brightness, large Stokes shift and fluorogenic response, overcome the problem of cell auto-fluorescence and enable multicolor cellular imaging with common green and red markers.

An aryleneethynylene fluorophore for cell membrane staining

The use of an amphiphilic aryleneethynylene fluorophore as a plasma membrane marker in fixed and living mammalian cells and liposome model systems is demonstrated. We show here that the optical properties of the novel dye are almost independent on pH, in the range 5.0–8.0. Spectroscopic characterization performed on unilamellar liposomes ascertained that the fluorescence intensity of the aryleneethynylene fluorophore greatly increases after incorporation in lipidic membranes. Experiments performed on different mammalian cells demonstrated that the novel membrane marker exhibits fast staining and a good photostability that make it a suitable tool for live cell imaging. Importantly, the aryleneethynylene fluorophore was also shown to be a fast and reliable blue membrane marker in classical multicolor immunofluorescence experiments. This study adds new important findings to the recent exploitation of the wide class of aryleneethynylene molecules as luminescent markers for biological investigations.

A new immunohistochemistry‐based assay SBrC5 classifies invasive breast cancer subtypes ‐profiling five biomarkers in one single test

Obtaining expression status of ER, PR and Her2 is essential for guiding treatment and predicting outcome for breast cancers. It has been reported that by adding cytokeratin 5/6 and EGFR to the above biomarkers, a five‐biomarker breast cancer panel had been validated for identifying the basal‐like subtype with superior prognostic values than the triple negative subtype1. The current study aims to evaluate a new immunohistochemical assay, named Subtyping Breast Cancers 5‐in‐1 Assay (SBrC5) for classifying breast cancer subtypes. A cocktail of ER, PR, Her2, cytokeratin 5/ 6 and EGFR antibodies (SBrC5 Cocktail) and the MultiVision Detection Kit (two colors, red and blue) were used. The assay was performed on a breast cancer tissue microarray of 75 cases. Both color and cellular localization of the staining was used for the result interpretation. There were three staining clusters: 1) Red Nuclei indicated ER or PR positive; 2) Red Membrane indicated Her2 expression; and 3) Blue Membrane or Cytoplasm indicated EGFR or cytokeratine 5/6 positivity respectively. The concordance for the assay when compared to the individual tests for each biomarker were 94.1%, 93.8% and 92.4% for Her2, combined ER and PR, and combined cytokeratine 5/6 and EGFR respectively. It is concluded that the SBrC5 assay is able to provide sufficient information for classifying breast cancer subtypes with only a single IHC test.

The Influence of Brightness on Functional Assessment by mfERG: A Study on Scaffolds Used in Retinal Cell Transplantation in Pigs

To determine the effect of membrane brightness on multifocal electroretinograms (mfERGs), we implanted poly lactic-co-glycolic acid (PLGA) membranes in the subretinal space of 11 porcine eyes. We compared membranes with their native shiny white color with membranes that were stained with a blue dye (Brilliant Blue). Histological and electrophysiological evaluation of the overlying retina was carried out 6 weeks after implantation. Histologically, both white and blue membranes degraded in a spongiform manner leaving a disrupted outer retina with no preserved photoreceptor segments. Multifocal ERG revealed the white membranes to have a significantly higher P1-amplitude ratio than the blue (P = 0.027), and a correlation between brightness ratio and P1-amplitude ratio was found (r = 0.762). Based on our findings, we conclude that bright subretinal objects can produce normal mfERG amplitude ratios even when the adjacent photoreceptors are missing. Functional assessment with mfERG in scaffold implant studies should therefore be evaluated with care.

P5-11-04: A New Immunohistochemistry-Based Assay SBrC5 Classifies Invasive Breast Cancer Subtypes – Profiling Five Biomarkers in One Single Test.

Abstract Background: Obtaining expression status of estrogen receptor (ER), progesterone receptor (PR) and Her2 is essential for guiding treatment and predicting outcome for breast cancers. By adding cytokeratin 5/6 and EGFR to the above mentioned three common biomarkers, a five-biomarker breast cancer panel has been validated in previous studies for identifying the basal-like subtype with superior prognostic values than the triple negative subtype1. The current study aimed to evaluate a new immunohistochemical assay, named Subtyping Breast Cancers 5-in-1 Assay (abbreviated as SBrC5, or “the assay” in this poster), for classifying breast cancer subtypes. Material and Methods: A cocktail of ER, PR, Her2, cytokeratin 5/6 and EGFR antibodies (SBrC5 Cocktail) and the MultiVision Detection Kit (TFS Cat. #: TL-012-MARH) were used in generating the data for this poster. The Multivision Detection Kit is a double staining system with two visualizing colors, red and blue. The assay was performed on a breast cancer tissue microarray containing 75 cases in duplicate. Both color and cellular localization of the staining generated from the assay was used for the result interpretation. There were three staining clusters: 1) Red Nuclei indicated ER or PR positive; 2) Red Membrane indicated Her2 expression; and 3) Blue Membrane or Cytoplasm indicated EGFR or cytokeratine 5/6 positivity respectively. The same breast cancer tissue microarray was also subjected to individual IHC tests of ER, PR, Her2, cytokeratin 5/6 and EGFR. The concordance between the assay and the individual tests was analyzed. Results: The assay SBrC5 was found to have excellent effectiveness in detecting ER or PR positivity, Her2 over expression, and cytokeratine 5/6 or EGFR positivity. The concordance for the assay when compared to the individual tests for each biomarker were 94.1%, 93.8% and 92.4% for Her2, combined results of ER and PR, and combined results of cytokeratine 5/6 and EGFR respectively. When a similar comparison was made, the sensitivity was 87.9%, 94.3% and 73.7%; the specificity was 100%, 93.1% and 100%. Discussion: This study has demonstrated that a single test of the assay SBrC5 was able to accomplish the collective task of the individual IHC tests of the five-biomarker breast cancer panel. The assay is able to provide sufficient information for classifying breast cancer subtypes with only a single IHC test. Thus it makes it possible to obtain subtypes information in biopsy settings where the tumor tissue obtained is often insufficient for multiple tests required to accomplish such task by existing methods. Reference: Maggie C.U. Cheang, David Voduc, Chris Bajdik, et al. Clin Cancer Res March 1, 2008 14; 1368. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-11-04.

Abstract 4474: Cellular effects of fluorescent quinolinium drugs on normal lymphoblast and A431 carcinoma cells

Abstract This study aims to evaluate the cellular effects of amino benzazolo [3,2- a] quinolinium drugs on normal lymphoblast cells and compare them to effects on A431 epitheloid carcinoma cells. The drugs under study are amino containing heterocyclic compounds possessing a positive charge that could facilitate their interaction with cell organelles specially mitochondria. The fluorescent properties could also facilitate observation of their cellular binding location. These compounds have also been demonstrated to have mutagenic and DNA binding capacity on A431 cancer cells. A431 and TK6 cells in culture where exposed for 48 hours to determine the IC50 dose, mitochondrial membrane permeability, ROS generation and fluorescent microscopy analysis of intracellular binding. IC50 was determined by trypan blue exclusion and mitochondrial membrane permeability applying the Nucleo counter 3000, JC-1 assay. For ROS determination the fluorescent dye 2,7-dichlorofluorescein diacetate (DCFH-DA) was applied. Determination of intracellular binding was performed applying confocal microscopy. In the TK6 normal lymphoblast cells the observed IC50 concentrations were as follow: ABQ38=310µM, and ABQ95=270µM. On A431 IC50s were as follows: ABQ38 = 32 µM and ABQ95 = 36 µM. Overall, normal TK6 demonstrated higher tolerance to the tested ABQS than A 431. Preliminary results on ROS generation indicated a stronger ROS release on A431 than on TK6 lymphoblast. Analysis of the effects of ABQS on the mitochondrial permeability where also consistent with the cytotoxicity observed, demonstrating higher levels of membrane permeability on treated tumor cells than on normal. Microscopy images of ABQS treated cells revealed location of these fluorescent compounds in organelles such as mitochondria and DNA. DNA fragmentation analyses are being implemented. This study presents preliminary evidence of an overall higher tolerance of normal TK6 lymphoblast to the tested fluorescent drugs in comparison to treated A431. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4474. doi:10.1158/1538-7445.AM2011-4474

Abstract 1342: Effects of the plasticizer metabolite 2 ethyl-1-hexanol on human lymphoblast cells

Abstract The study main objective was to determine the toxicity of 2-ethyl-1-hexanol (EH) and to compare them to its precursor di-ethylhexyl phthalate (DEHP) on the TK6 human lymphoblast cell line. EH is an organic alcohol produced in cells as result of the biotransformation of DEHP. Release of EH from lung cancer cells was also reported recently. Environmental human exposure to the parent DEHP can result through interaction with personal care products, paint lacquer, inks, rubber, dry cleaning and plasticizers such as PVC. Analyzed effects in this study included doses that inhibit cell growth (IC50), damage to the mitochondrial membrane, generation of Reactive Oxygen Species (ROS), and activation of caspase 3 and 7. TK6 cells were cultured on 12.5cm2 flasks with modified RPMI 1640 culture media with 10% FBS, and incubated at 37°C with 5% CO2. IC50 was determined by trypan blue exclusion and mitochondrial membrane permeability applying the Mito PT JC-1 assay from Immunochemistry. For ROS determination the fluorescent dye 2,7-dichlorofluorescein diacetate (DCFH-DA) was applied. For caspases 3 and 7 analysis the Magic RedTM assay from Immunochemistry was applied. Results indicated that IC50s of DEHP and EH on TK6 cells were 234 μM and 75nM respectively. Cells where then exposed to the identified IC50 for 48 hours to measure effects on mitochondria and ROS generation. ROS results indicated that EH generated an average of 96.31 Fluorescence Standard Units (FSU), the negative control Dimethyl Sulphoxide (DMSO) generated 58.22 FSU, the compound used as positive control cis-Di ammine platinum (II) dichloride (Cisplatin) generated 1309.67 FSU and for DEHP the ROS analysis resulted on 793.27 FSU. The level of ROS generated between the parent and this metabolite are not consistent with the degree of toxicity observed. Determination and comparison of the mitochondrial membrane permeabilization as well as Caspases 3 and 7-activation assays of EH are still developing. The preliminary results, however indicates a wide difference in citotoxicity between the parent compound and its metabolite clearly supporting the need for prevention strategies reducing exposure to these controversial plasticizer and therefore potential health effects. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1342. doi:10.1158/1538-7445.AM2011-1342

Simple Generation of Human Induced Pluripotent Stem Cells Using Poly-β-amino Esters As the Non-viral Gene Delivery System

Reprogramming of somatic cells to induced pluripotent stem (iPS) cells can be achieved by the delivery of a combination of transcription factors, including Oct4, Sox2, Klf4, and c-Myc. Retroviral and lentiviral vectors are commonly used to express these four reprogramming factors separately and obtain reprogrammed iPS cells. Although efficient and reproducible, these approaches involve the time-consuming and labor-intensive production of retroviral or lentiviral particles together with a high risk of working with potentially harmful viruses overexpressing potent oncogenes, such as c-Myc. Here, we describe a simple method to produce bona fide iPS cells from human fibroblasts using poly-β-amino esters as the transfection reagent for the delivery of a single CAG-driven polycistronic plasmid expressing Oct4, Sox2, Klf4, c-Myc, and a GFP reporter gene (OSKMG). We demonstrate for the first time that poly-β-amino esters can be used to deliver a single polycistronic reprogramming vector into human fibroblasts, achieving significantly higher transfection efficiency than with conventional transfection reagents. After a protocol of serial transfections using poly-β-amino esters, we report a simple methodology to generate human iPS cells from human fibroblasts avoiding the use of viral vectors.

881 A SYNTHETIC POLYVALENT LIGAND FOR α5β1 INTEGRIN ACTIVATES COMPONENTS OF THE UROLTHELIAL CARCINOMA CELL RESPONSE TO BCG

INTRODUCTION AND OBJECTIVES: Prior work has demonstrated that BCG binds to, and crosslinks, 5 1 integrins present on the surface of urothelial carcinoma cells (UCC). Antibody mediated crosslinking of 5 1 integrins can reproduce signal transduction, gene transactivation, and phenotypic changes similar to those observed in response to BCG. The purpose of the present study was to evaluate the effect of a synthetic polyvalent ligand for 5 1 on these elements of the tumor response to BCG. METHODS: The consensus 5 1 integrin binding tri-peptide “RGD” was linked to a MAP8 backbone to result in an octavalent construct targeting 5 1 integrin. Purified RGD-MAP8 was then used in dose response experiments to determine its effect on signaling pathway activation (NFKB, NF2, and CEBP), gene expression (p21, IL6, IL8, CXCL1, CXCL2, CCL20), and phenotypic changes (trypan blue exclusion, HMGB1 release) in UCC associated with BCG treatment. The effect of the construct was tested in the human UCC lines 253J and T24. The scrambled sequence GRD-MAP8 served as a negative control with BCG exposure serving as a positive control. RESULTS: The RGD-MAP8 construct activated NFKB, NF2, and CEBP signaling pathways in the 2 cell lines, in a sequence specific, dose-response manner, to maximum average values of 89%, 60% and 42% of BCG exposed positive controls respectively. p21 expression in response to RDG-MAP8 averaged 70% of positive controls. Although the synthetic construct transactivated cytokine gene expression in 2/5 and 5/5 target genes in the 253J and T24 cell line respectively, expression levels averaged only 18% of BCG treated control values. RGD-MAP8 exposure resulted in loss of trypan blue membrane integrity with peak values of 65% of BCG controls. In contrast to BCG exposure, RGD-MAP8 treatment did not result in HMGB1 release. None of the endpoints demonstrated a response to the scrambled sequence control. CONCLUSIONS: Treatment of UCC with a synthetic, polyvalent compound designed to crosslink cell surface 5 1 integrin activates intracellular signaling pathways and transactivates the expression of some BCG responsive genes. However, the magnitude of both signal induction and gene transactivation was less than observed in response to BCG. Moreover, some genes appeared unresponsive to RGD-MAP8 and no HMGB1 release (necrosis marker) was observed. These results suggest that while 5 1 integrin crosslinking contributes to the BCG response, it alone is insufficient to duplicate the full spectrum of BCG induced changes in UCC biology.

Management of Cystic Lymphangioma in the Head and Neck Region

Cystic lymphangiomas of the head and neck are very common and cannot be completely removed by surgical excision because of frequent complications. In this article, we report on 28 cases of huge cystic lymphangiomas in the head and neck region, which were treated successfully by endoscopic cautery in combination with postoperative intratumoral negative pressure. The cysts were 11 to 20 cm in diameter as diagnosed by ultrasonography and computed tomography scan. The 45-degree × 4-mm endoscopes were placed into the cysts through 1- to 2 cm-long hidden incisions to visualize the lymphangiomas, which were under the blue translucent mucous membranes. We cauterized the walls of the tumor cavities with 6% to 10% iodine twice and used topical negative-pressure therapy for 7 to 10 days after suture of the small incisions. This endoscopic approach can be successfully and safely performed by an experienced surgeon who complies with oncologic principles.

Acetylcholinesterase biosensor based on prussian blue-modified electrode for detecting organophosphorous pesticides

In this study, a novel acetylcholinesterase (AChE) biosensor was developed based on dual-layer membranes (chitosan membrane and prussian blue membrane) modifying glassy carbon electrode (GCE). A chitosan membrane was used for immobilizing AChE through glutaraldehyde cross-linking attachment to recognize pesticides selectively. A prussian blue (PB) membrane was electrodeposited on the surface of GCE to enhance electron transfer. Before the detection, the chitosan enzyme membrane was quickly fixed on the surface of PB/GCE with O-ring to prepare an amperometric AChE-PB/GCE sensor for organophosphorus (OP) pesticides. The electrochemical behaviour of AChE-PB/GCE was studied, and the results showed that the chitosan membrane as carrier can absorb a large amount of enzyme, and PB has a significant synergistic effect towards enzymatic catalysis. As a result of these two important enhancement factors, the proposed biosensor exhibited extreme sensitivity to OP pesticides compared to the other kinds of AChE biosensor. The influences of phosphate buffer pH, substrate concentration, incubation time of pesticide on the response of the fabricated biosensor were investigated. Under optimum conditions, the inhibition rates of these pesticides were proportional to their concentrations in the range of 0.01–10 μg l −1, 0.05–10 μg l −1, 0.03–5 μg l −1, and 0.05–10 μg l −1, respectively. The detection limits were found to be 2.5 ng l −1 for dichlorvos, 15 ng l −1for omethoate, 5 ng l −1 for trichlorfon and 10 ng l −1 for phoxim. Moreover, the biosensor exhibited good reproducibility and stability, and it was suitable for trace detection of OP pesticide residue.

Melittin-regenerated purple membrane

We have investigated the character of melittin-regenerated purple membrane. Adding melittin to blue membrane causes the color transition and partial regeneration of the photocycle and the proton pump. The reconstitution of bacteriorhodopsin by melittin is proved to be charge-dependent. In studying the location of melittin binding on the blue membrane, we suggest that melittin anchors on the membrane through both hydrophobic and electrostatic interactions. The electrostatic interaction is dominant. The binding sites for the electrostatic interaction should be on the surface of the membrane.

Unfolding study of native bacteriorhodopsin under acidic condition

In this study, we measured the structural properties of “blue membrane”, which is specific formation of a purple membrane (PM) upon acid titration or removal of cations, by the force curve measurement mode of an atomic force microscopy. The PM fragments were immobilized on a glass substrate and force curve measurements were carried out on the fragments under neutral (pH 7.2) and acidic (pH 2.4) condition. The results revealed that peak positions of the unfolding spectra obtained under acidic condition were shifted to the shorter extension region than those at neutral pH and that the relative position of only the first peak was changed by about 5 nm. These results suggest the possibility that the specific secondary structure is formed at the site from its C-terminus to helices F in acidified PM.