Evaluation of the allergenicity of ω5-gliadin-deficient Hokushin wheat (1BS-18) in a wheat allergy rat model

We previously developed Hokushin wheat line as a hypoallergenic wheat lacking ω5-gliadin (1BS-18), a major allergen for wheat-dependent exercise-induced anaphylaxis. However, the allergenicity of 1BS-18 has not been understood completely. In this study, we evaluated the allergenicity of 1BS-18 such as anaphylactic elicitation ability and sensitization ability using rats sensitized with ω5-gliadin or glutens prepared from Hokushin (Hokushin gluten) or 1BS-18 (1BS-18 gluten). Rats were sensitized by intraperitoneal administration of ω5-gliadin, Hokushin gluten or 1BS-18 gluten. Immunoglobulin E-mediated systemic anaphylaxis was evaluated by measuring changes in rectal temperature for 30 min after intravenous challenge with ω5-gliadin or the test glutens in unsensitized rats or rats sensitized with ω5-gliadin or the test glutens. In ω5-gliadin-sensitized rats, intravenous challenge with ω5-gliadin or Hokushin gluten significantly decreased the rectal temperature at 30 min after challenge while challenge with 1BS-18 gluten did not reduce the rectal temperature. Furthermore, intravenous challenge with ω5-gliadin significantly decreased the rectal temperature in rats sensitized with Hokushin gluten or 1BS-18 gluten. However, the reduced degree observed in 1BS-18 gluten-sensitized rats was smaller than that in Hokushin gluten-sensitized rats. In conclusion, 1BS-18 elicited no allergic reaction in ω5-gliadin-sensitized rats and had less sensitization ability for ω5-gliadin than that of Hokushin wheat.     

Adapter-Directed Display: A Modular Design for Shuttling Display on Phage Surfaces

A novel adapter-directed phage display system was developed with modular features. In this system, the target protein is expressed as a fusion protein consisting of adapter GR1 from the phagemid vector, while the recombinant phage coat protein is expressed as a fusion protein consisting of adapter GR2 in the helper phage vector. Surface display of the target protein is accomplished through specific heterodimerization of GR1 and GR2 adapters, followed by incorporation of the heterodimers into phage particles. A series of engineered helper phages were constructed to facilitate both display valency and formats, based on various phage coat proteins. As the target protein is independent of a specific phage coat protein, this modular system allows the target protein to be displayed on any given phage coat protein and allows various display formats from the same vector without the need for reengineering. Here, we demonstrate the shuttling display of a single-chain Fv antibody on phage surfaces between multivalent and monovalent formats, as well as the shuttling display of an antigen-binding fragment molecule on phage coat proteins pIII, pVII, and pVIII using the same phagemid vectors combined with different helper phage vectors. This adapter-directed display concept has been applied to eukaryotic yeast surface display and to a novel cross-species display that can shuttle between prokaryotic phage and eukaryotic yeast systems.     

Expression of hepatitis B virus surface antigen P31 gene in Escherichia coli

A hepatitis B virus surface antigen (HBsAg) P31-coding DNA was constructed from a DNA fragment of the plasmid pHBr330 containing the entire hepatitis B virus (HBV) adr DNA and a chemically synthesized adaptor. The P31 gene was inserted into an expression vector, pTRP771, having an Escherichia coli tryptophan operon (trp) promoter to give a recombinant plasmid pTRP P31-R. The distance between the Shine-Dalgarno (SD) sequence and the initiation codon of P31 gene was adjusted to 9 bp. The expression level of HBsAg by E. coli 294[pTRP P31-R] was significantly elevated, in contrast to that of HBsAg by E. coli 294[pTRP SS-6]. Western blotting analysis has shown that E. coli[pTRP P31-R] synthesizes a specific polypeptide P31 of about 31 kDal, which reacts with anti-HBsAg antibody. The binding studies with polyalbumins from various species have also suggested that HBsAg P31 specifically binds to polymerized human serum albumin.     

Chorein,the protein responsible for chorea-acanthocytosis,interacts with β-adducin and β-actin

Chorea-acanthocytosis (ChAc) is an autosomal, recessive hereditary disease characterized by striatal neurodegeneration and acanthocytosis, and caused by loss of function mutations in the vacuolar protein sorting 13 homolog A (VPS13A) gene. VPS13A encodes chorein whose physiological function at the molecular level is poorly understood. In this study, we show that chorein interacts with β-adducin and β-actin. We first compare protein expression in human erythrocyte membranes using proteomic analysis. Protein levels of β-adducin isoform 1 and β-actin are markedly decreased in erythrocyte membranes from a ChAc patient. Subsequent co-immunoprecipitation (co-IP) and reverse co-IP assays using extracts from chorein-overexpressing human embryonic kidney 293 (HEK293) cells, shows that β-adducin (isoforms 1 and 2) and β-actin interact with chorein. Immunocytochemical analysis using chorein-overexpressing HEK293 cells demonstrates co-localization of chorein with β-adducin and β-actin. In addition, immunoreactivity of β-adducin isoform 1 is significantly decreased in the striatum of gene-targeted ChAc-model mice. Adducin and actin are membrane cytoskeletal proteins, involved in synaptic function. Expression of β-adducin is restricted to the brain and hematopoietic tissues, corresponding to the main pathological lesions of ChAc, and thereby implicating β-adducin and β-actin in ChAc pathogenesis.     

Validation of the organic electrochemical transistor for in vitro toxicology

Background

The gastrointestinal epithelium provides a physical and biochemical barrier to the passage of ions and small molecules; however this barrier may be breached by pathogens and toxins. The effect of individual pathogens/toxins on the intestinal epithelium has been well characterized: they disrupt barrier tissue in a variety of ways, such as by targeting tight junction proteins, or other elements of the junctions between adjacent cells. A variety of methods have been used to characterize disruption in barrier tissue, such as immunofluorescence, permeability assays and electrical measurements of epithelia resistance, but these methods remain time consuming, costly and ill-suited to diagnostics or high throughput toxicology.

Methods

The advent of organic electronics has created a unique opportunity to interface the worlds of electronics and biology, using devices such as the organic electrochemical transistor (OECT), whose low cost materials and potential for easy fabrication in high throughput formats represent a novel solution for assessing epithelial tissue integrity.

Results

In this study, OECTs were integrated with gastro-intestinal cell monolayers to study the integrity of the gastrointestinal epithelium, providing a very sensitive way to detect minute changes in ion flow across the cell layer due to inherent amplification by the transistor.

Major conclusions

We validate the OECT against traditional methods by monitoring the effect of toxic compounds on epithelial tissue. We show a systematic characterization of this novel method, alongside existing methods used to assess barrier tissue function.

General significance

The toxic compounds induce a dramatic disruption of barrier tissue, and the OECT measures this disruption with increased temporal resolution and greater or equal sensitivity when compared with existing methods. This article is part of a Special Issue entitled Organic Bioelectronics — Novel Applications in Biomedicine.     

Isolation of a novel neutralizing antibody fragment against human vascular endothelial growth factor from a phage-displayed human antibody repertoire using an epitope disturbing strategy

Following the clinical success of Bevacizumab, a humanized monoclonal antibody that blocks the interaction between vascular endothelial growth factor (VEGF) and its receptors, the search for new neutralizing antibodies targeting this molecule has continued until now. We used a human VEGF variant containing three mutations in the region recognized by Bevacizumab to direct antibody selection towards recognition of other epitopes. A total of seven phage-displayed antibody fragments with diverse binding properties in terms of inter-species cross-reactivity and sensitivity to chemical modifications of the antigen were obtained from a human phage display library. All of them were able to recognize not only the selector mutated antigen, but also native VEGF. One of these phage-displayed antibody fragments, denominated 2H1, was shown to compete with the VEGF receptor 2 for VEGF binding. Purified soluble 2H1 inhibited in a dose dependent manner the ligand-receptor interaction and abolished VEGF-dependent proliferation of human umbilical vein endothelial cells. Our epitope disturbing strategy based on a triple mutant target antigen was successful to focus selection on epitopes different from a known one. Similar approaches could be used to direct phage isolation towards the desired specificity in other antigenic systems.     

Binding of a Naja naja venom acidic phospholipase A2 cognate complex to membrane-bound vimentin of rat L6 cells: Implications in cobra venom-induced cytotoxicity

An acidic phospholipase A₂ enzyme (NnPLA₂-I) interacts with three finger toxins (cytotoxin and neurotoxin) from Naja naja venom to form cognate complexes to enhance its cytotoxicity towards rat L6 myogenic cells. The cytotoxicity was further enhanced in presence of trace quantity of venom nerve growth factor. The purified rat myoblast cell membrane protein showing interaction with NnPLA₂-I was identified as vimentin by LC-MS/MS analysis. The ELISA, immunoblot and spectrofluorometric analyses showed greater binding of NnPLA₂-I cognate complex to vimentin as compared to the binding of individual NnPLA₂-I. The immunofluorescence and confocal microscopy studies evidenced the internalization of NnPLA₂-I to partially differentiated myoblasts post binding with vimentin in a time-dependent manner. Pre-incubation of polyvalent antivenom with NnPLA₂-I cognate complex demonstrated better neutralization of cytotoxicity towards L6 cells as compared to exogenous addition of polyvalent antivenom 60–240 min post treatment of L6 cells with cognate complex suggesting clinical advantage of early antivenom treatment to prevent cobra venom-induced cytotoxicity. The in silico analysis showed that 19–22 residues, inclusive of Asp48 residue, of NnPLA₂-I preferentially binds with the rod domain (99–189 and 261–335 regions) of vimentin with a predicted free binding energy (ΔG) and dissociation constant (K_D) values of ?12.86 kcal/mol and 3.67 × 10^?10 M, respectively; however, NnPLA₂-I cognate complex showed greater binding with the same regions of vimentin indicating the pathophysiological significance of cognate complex in cobra venom-induced cytotoxicity.     

Functional dissection of transmembrane domains of human TAP-like (ABCB9)

An ABC transporter, TAP-Like (TAPL), was dissected into its amino-terminal transmembrane domain and the following core domain. When these domains were transiently expressed as tagged proteins with a His6- or Myc-epitope tag, the amino-terminal ones (Met¹-Lys¹⁸²) could not associate with each other, or with the full-length transporter (Met¹-Ala⁷⁶⁶). However, both the core domain (Arg¹⁴¹-Ala⁷⁶⁶) and full-length protein mutually interacted. The amino-terminal domain (Met¹-Arg¹⁴¹) as well as the full-length transporter fused with fluorescent protein GFP was sorted to lysosomal membranes upon their stable expression, as visualized by means of fluorescent microscopy, while the core domain (Arg¹⁴¹-Ala⁷⁶⁶) was broadly distributed in the intra-cellular membranes. These results suggest that the sorting signal for lysosomes is present within the amino-terminal transmembrane domain (Met¹-Arg¹⁴¹) of the TAPL molecule.     

N-terminal signal sequence is required for cellular trafficking and hyaluronan-depolymerization of KIAA1199

Recently, we disclosed that KIAA1199-mediated hyaluronan (HA) depolymerization requires an acidic cellular microenvironment (e.g. clathrin-coated vesicles or early endosomes), but no information about the structural basis underlying the cellular targeting and functional modification of KIAA1199 was available. Here, we show that the cleavage of N-terminal 30 amino acids occurs in functionally matured KIAA1199, and the deletion of the N-terminal portion results in altered intracellular trafficking of the molecule and loss of cellular HA depolymerization. These results suggest that the N-terminal portion of KIAA1199 functions as a cleavable signal sequence required for proper KIAA1199 translocation and KIAA1199-mediated HA depolymerization.