Using a recombinant bispecific antibody to block Na<Superscript>+</Superscript>-channel toxins protects against experimental scorpion envenoming

In recent years, several molecular engineering methods of designing bispecific antibodies in various formats have been developed. Tandem-scFvs comprising two scFvs fused together via a peptide are 55-kDa molecules, and are one of the most promising and most straightforward approaches to bispecific antibody production. We report an attempt to design more effective antivenoms to the Androctonus australis scorpion using murine scFvs as building blocks to create a unique bispecific molecule that neutralizes the potent neurotoxins AahI and AahII. The tandem-scFv was produced in recombinant bacteria, purified by immobilized metal ion affinity chromatography, and analyzed by polyacrylamide gel electrophoresis, Western blot, gel filtration, mass spectrometry, and direct and competitive radioimmunoassay. In vivo, it neutralized the binding of the AahI and AahII toxins to their receptor, and protected mice against experimental envenomation. The findings reported here highlight the potential of recombinant antibody fragments for protecting against scorpion venom toxicity. Received 8 September 2006; received after revision 10 November 2006; accepted 27 November 2006     

可溶性重组全长人PPARγ的表达、纯化及功能表征

目的 建立可溶性表达、纯化有活性的重组全长人PPARγ蛋白的方法.方法 构建pReceiver-B13-SUMO-PPARγ质粒转化至Escherichia coli BL21(DE3)细胞,进行诱导表达,优化细胞生长环境;利用亲和色谱法纯化可溶性重组全长人PPARγ蛋白;以罗格列酮为阳性配体,以GW9662为拮抗剂,采用分子排阻色谱-高效液相色谱法(SEC-HPLC)法测定重组全长人PPARγ蛋白与配体药物的结合活性.结果 在优化条件(16℃、IPTG浓度0.4 mM、诱导时间18～20 h)下能获得高水平、可溶性重组全长人PPARγ蛋白;经亲和色谱一次纯化,得到纯度约为90％的重组全长人PPARγ蛋白;该蛋白与罗格列酮特异性结合率约为70％,Kd为500nM.结论 本文所建立的方法能可溶性表达、纯化得到有活性、纯度较高的重组全长人PPARγ蛋白,为该蛋白功能研究和配体药物筛选奠定基础.     

Permeability of arthrodial membrane to water: A first measurement using in vivo techniques

Summary Despite differences in surface morphology and fine structure, the permeabilities of untreated scorpion sternite and pleural cuticle to water are similar (0.69 versus 0.79 g·cm^–2·h^–1·mm Hg^–1). Hexane applied to pleural membrane increased its permeability 9-fold, but neither hexane nor chloroform: methanol had much effect on sternite permeability. When sternite cuticle was treated with 1.0 N KOH followed by chloroform: methanol, permeability increased about three times over control values. In contrast, cockroach pronotum, which is 17 times more permeable than scorpion sternite, exhibited a marked increase in permeability when treated with just hexane. In both the scorpion pleuron and cockroach pronotum, disruption of the lipid barrier caused by rubbing is partially responsible for the higher permeabilities observed following solvent treatment.     

Biochemical characterization and bacterial expression of an odorant-binding protein from <Emphasis Type="Italic">Locusta migratoria</Emphasis>

Analysis of soluble proteins from different body parts of Locusta migratoria revealed a fast-migrating component in native electrophoresis, unique to antennae of both sexes. N-terminal sequence analysis and cloning identified this protein as a member of the insect odorant-binding proteins, carrying a well-conserved six-cysteine motif. Mass spectrometry analysis confirmed the occurrence of two distinct polypeptide species determined by nucleotide sequencing and demonstrated that the cysteine residues are paired in an interlocked fashion. The protein was expressed in a bacterial system with yields of about 10 mg/l of culture, mostly present as inclusion bodies. However, this recombinant product was solubilized after disulfide reduction. Air oxidation yielded a species with all disulfides spontaneously formed as in the native counterpart. Both native and recombinant proteins migrated as a dimer in gel filtration chromatography. Ligand binding was measured, using N-phenyl-1-naphthylamine as the fluorescent probe; the affinity of other ligands was measured in competitive binding assays. The protein exhibited great resistance to thermal denaturation even following prolonged treatment at 100 degrees C. A structural model for this dimeric species was generated on the basis of its sequence homology with Bombyx mori pheromone-binding protein, whose three-dimensional structure has been resolved as an unbound species and in complex with its physiological ligand. This is the first report of an odorant-binding protein identified and characterized from Orthoptera.     

Serological study of Ia antigens determined at the I-A and I-E sub-regions of the H-E complex in the H-2 complex of mice

Immune serum B10.S (7R) anti-B10.S (9R)(anti I-JEkCd) contained as expected an anti-Ia7 antibody. A series of weaker but reproducible extra-reactions might recognize Ia3 specificity coded at the I-A subregion of the H-2 complex. Results with recombinant haplotypes confirmed this mapping. Such a reactivity could be interpreted as an interlocus cross-reaction (I-E/I-A) since the immunization was induced against an I-E subregion product. Another interpretation was possible: the immune serum would thus contain an antibody recognizing Ia7 (on the E alpha k Ia chain) and another antibody recognizing an antigenic determinant carried by the E beta k Ia chain. The latter antibody might recognize by cross-reaction as specificity carried by the A beta chain of various haplotypes (H-2b,k,q).     

The scorpine family of defensins: gene structure,alternative polyadenylation and fold recognition

Small cationic antimicrobial peptides (SCAMPs) as effectors of animal innate immunity provide the first defense against infectious pathogens. This class of molecules exists widely in invertebrate hemolymph and vertebrate skin secretion, but animal venoms are emerging as a new rich resource. Scorpine is a unique scorpion venom defensin peptide that has an extended amino-terminal sequence similar to cecropins. From the African scorpion Opistophthalmus carinatus venom gland, we isolated and identified several cDNAs encoding four new homologs of scorpine (named opiscorpines 1–4). Importantly, we show for the first time the existence of multiple opiscorpine mRNAs with variable 3 untranslated regions (UTRs) in the venom gland, which may be generated by alternative usage of polyadenylation signals. The complete opiscorpine gene structure including its promoter region is determined by genomic DNA amplification. Two large introns were found to be located within the 5 UTR and at the boundary of the mature peptide-coding region. Such a gene structure is distinct, when compared with other scorpion venom peptide genes. However, a comparative promoter analysis revealed that both opiscorpine and scorpion venom neurotoxins share a similar promoter organization. Sequence analysis and structural modeling allow us to group the scorpines and scorpion long-chain K-channel toxins together into one family that shares a similar fold with two distinct domains. The N-terminal cecropin-like domain displaying a clear antimicrobial activity implies that the scorpine family represents a group of real naturally occurring hybrids. Based on the phylogenetic analysis, a possible cooperative interaction between the N and C domains is elucidated, which provides an evolutionary basis for the design of a new class of anti-infectious drugs.Received 5 April 2004; accepted 17 May 2004     

Production and detection of antibody against estradiol receptor in the calf uterus. Interaction with the estrogen receptor from the hen oviduct]

The antibodies against estrogen receptor were obtained after injecting Rabbits with a cytoplasmic receptor fraction isolated from Calf uterus. The estrogen receptor was partially proteolysed by the action of trypsin and subsequently purified by affinity chromatography (purification 4,000 to 10,000 fold, to a purity of 5-20%). The affinity of the antibody for the proteolysed receptor is KD approximately 1 nM and serum titres have reached values of approximately 50 nM. The values remained constant after the third injection. Preliminary results indicate that the antibody has approximately the same affinity for "native" cytoplasmic estrogen receptor from Calf uterus, as well as for the "trypsinized" forms of estrogen receptor isolated from Calf uterine cytosol and Hen oviduct nuclei.     

Recombinant anti-P protein autoantibodies isolated from a human autoimmune library: reactivity,specificity and epitope recognition

The ribosomal P proteins are specific and important autoantigens in patients affected by systemic lupus erythematosus. In this study, we describe for the first time the selection and characterization of recombinant human monoclonal anti-P protein (auto)-antibody fragments from an autoimmune patient-derived phage display antibody library. The selected recombinant anti-P antibodies specifically recognize the P proteins in immunofluorescence assays on HEp-2 cells and in immunoblotting assays, and they immunoprecipitate the P proteins under native conditions. Using both anti-P-positive patient sera and the selected recombinant anti-P antibodies, the immunodominant epitope was determined and shown to be located at the C-terminal end of the P proteins (amino acids 111-115). Inhibition of in vitro protein translation demonstrated that interaction of the monoclonal patient-derived anti-P antibodies with their native epitope functionally inhibits the activity of the P proteins on the ribosome, confirming the notion that patient autoantibodies are often directed to the functional centre of their autoantigenic target.     

Alkaline phosphatase binds to collagen; a hypothesis on the mechanism of extravesicular mineralization in epiphyseal cartilage

Summary Affinity chromatography on Sepharose 4B-collagen gels was used to test the affinity of alkaline phosphatase for collagen. Results indicate that 1) alkaline phosphatase of preosseous cartilage binds to collagen probably by electrostatic interactions, 2) this interaction is inhibited by proteoglycan subunits. These results suggest that, in vivo, the formation of a collagen-alkaline phosphatase complex may be a step of the process leading to cartilage calcification.This investigation was supported by research grants of C.N.R. (C.T. 82.02437.04), of Ministero della Pubblica Istruzione and of the University of Trieste.To whom correspondence and reprint requests should be addressed.     

Aspects of measurement and analysis of regulatory peptides

Summary Although almost all methods of mass measurement of regulatory peptides still depend on the high affinity antibody, the traditional Yalow and Berson radioimmunoassay technique is becoming outdated. Pure monoclonal antibodies allow excess antibody two site assay techniques with a variety of different labels (preferentially non-radioactive) of great sensitivity and speed. The large amounts of particular monoclonal antibodies available allow several different laboratories to use the same reagents and have increased comparability. Unfortunately many regulatory peptides exist in multiple molecular forms and attention must be paid to antibody region specificity. Improved methods of extraction of regulatory peptides from plasma tissue allow more accurate quantitation. New techniques for rapid high resolution chromatography make distinction of different molecular forms much easier than hitherto. Better education in techniques and/or attention to inter-assay standards are necessary to improve the comparability of regulatory peptide measurement in the future.     

Study by a rapid mixing method of the interaction between a fluorescent cholinergic agonist and membrane fragments rich in cholinergic receptors from Torpedo marmorata]

The analysis by stopped-flow of the interaction of fluorescent agonist (C5DACho1) with the acetylcholine receptor in its membrane-bound form reveals several kinetic steps: a fast one, in the millisecond range, associated with the binding of C5DACho1 to a high affinity state and a "medium" and "slow" one, the last one representing possibly an isomerisation of the receptor molecule towards the high affinity state.     

Affinity chromatographic preparation of arterial heavy meromyosin subfragment-1

Summary Heavy meromyosin subfragment-1 (HMM S-1) was prepared by papain digestion of arterial myosin or actomyosin and was purified by agarose-ATP affinity chromatography. Proteolysis of crude arterial myosin suspensions was preceded by solubilization. HMM-S-1 thus obtained consisted mainly of a 90,000 dalton polypeptide and fully retained the K⁺- and Ca²⁺-ATPase of the parent myosin. Its affinity to agarose-ATP was comparable to that of skeletal muscle HMM S-1.The support of this work by an EMBO short time fellowship (to R. L.) and by the Deutsche Forschungsgemeinschaft (No. SFB 90, B 7) and the excellent technical assistance byM. Troschka andC. Köhler are gratefully acknowledged.     

Involvement of a surface concanavalin A-binding glycoprotein in the adhesion ofTrichomonas vaginalis to substrates

Summary Treatment ofTrichomonas vaginalis with EDTA removes their ability to adhere to glass surfaces and changes their affinity to Concanavalin A (ConA) by a different distribution of their surface structures. Filtrates of the EDTA-treatedTrichomonas passed through affinity chromatography columns (ConA bound to Sepharose 4B0 separate into 2 fractions, one fraction was bound to the ConA-Sepharose beads, the other was not. The Con A-bound fraction appears to be a glycoprotein which restores in a specific way the ability of the EDTA-treated protozoa to adhere to glass. We want to thank Dr.P. M. Comoglio for the donation of the FITC-labelled immunoglobulin fraction of anti-ConA rabbit antiserum. This work was supported by a grant from the Italian National Research Council (C.N.R.)     

Targeted polymeric micelles for delivery of poorly soluble drugs

Polymeric micelles (micelles formed by amphiphilic block copolymers) demonstrate a series of attractive properties as drug carriers, such as high stability both in vitro and in vivo and good biocompatibility, and can be successfully used for the solubilization of various poorly soluble pharmaceuticals. These micelles can also be used as targeted drug delivery systems. The targeting can be achieved via the enhanced permeability and retention effect (into the areas with the compromised vasculature), by making micelles of stimuli-responsive amphiphilic block copolymers, or by attaching specific targeting ligand molecules to the micelle surface. Immunomicelles prepared by coupling monoclonal antibody molecules to p-nitrophenylcarbonyl groups on the water-exposed termini of the micelle corona-forming blocks demonstrate high binding specificity and targetability. Immunomicelles prepared with cancer-specific monoclonal antibody 2C5 specifically bind to different cancer cells in vitro and demonstrate increased therapeutic activity in vivo. This new family of pharmaceutical carriers can be used for the solubilization and targeted delivery of poorly soluble drugs to various pathological sites in the body.     

Production of functional rat liver PSP protein in Escherichia coli

An efficient Escherichia coli expression system for the production of a perchloric acid-soluble protein (PSP) has been constructed. Complementary DNA encoding PSP was inserted into an inducible bacterial expression vector pGEX-4T-1. After the plasmid introduced into E. coli was expressed by isopropyl 1-thio-β-D-galactopyranoside (IPTG), the recombinant product was purified by glutathione-Sepharose 4B affinity chromatography. The purified product showed the expected NH₂-terminal sequence, but the translation inhibitory activity of this product was 10 times lower compared with that of authentic PSP isolated from rat liver. Received 8 October 1998; received after revision 6 November 1998; accepted 6 November 1998     

Self-labeling of human polymorphonuclear leucocyte myeloperoxidase with 125iodine.

In order to obtain a radioimmunoassay (RIA) technique for the measurement of human plasma myeloperoxidase (MPO), we purified the enzyme from polymorphonuclear granulocytes (neutrophils), and compared three methods of labeling it with 125Iodine:chloramine T, lactoperoxidase, and an original technique of 'self labeling' based on the ability of the enzyme to oxidize and bind 125I in the presence of H2O2. The chloramine T technique produced a degraded protein, as well shown by a high non-specific binding of tracer to antibody. The lactoperoxidase technique did not succeed in labeling MPO with an adequate specific activity. In contrast, the self-labeling method gave a stable tracer with a specific activity of 23 microCi/micrograms MPO (85 MBq), a satisfactory level of immunoreactivity, and a low-specific binding (less than or equal to 3%). After labeling, purification of tracer was achieved by gel filtration chromatography in phosphate buffer (0.05 M; pH7) to which 0.1% poly-L-lysine was added. The labeled molecule remained stable for 40 days and could be used for RIA with a polyclonal antibody raised in rabbits.     

Production of passive cutaneous anaphylaxis (PCA) and reversed PCA by rat IgE antibody in the mouse

Although IgE antibody is generally characterized as a homocytotropic antibody, it has been well known for some time that mouse IgE antibody causes potent sensitization of rat skin for PCA. The present study clearly shows the reciprocal cross-sensitization of mouse skin with rat IgE molecules. PCA and RPCA were produced by rat IgE antibody in an inbred mouse strain. DS/Shi, though not in C3H/HeShi, C57BL/6JShi and BALB/cCrj strains. Sensitization of DS/Shi mouse skin for PCA with rat IgE antibody was comparable in sensitivity with that of rat skin, but lasted only for a short term in comparison with the long persistence in rat skin.     

High affinity binding of 5-hydroxytryptamine to some membrane preparations from cattle brain. Relationship to lysergic acid diethylamide (LSD)]

5 hydroxytryptamine binds to crude brain membrane preparations with two different affinities (KD = 1 to 2 X 10(-9) M for the highest, 1 to 2 X 10(-8) M for the lowest). LSD also binds with two affinities (KD = 3 to 4 X 10(-9) M and KD = 2 to 3 X 10(-8) M). Subcellular distribution of these sites shows that binding involves the two binding affinities in microsomal membranes but solely the high affinity binding sites are present in purified synaptosomal membranes. High affinity sites for 5 HT and for LSD are different as no direct competitive inhibition is observed in that case. On microsomal membranes, direct relationship occurs between low affinity binding for 5 HT and high affinity binding for LSD.     

Surface display of a single-domain antibody library on Gram-positive bacteria

Combinatorial protein engineering for selection of proteins with novel functions, such as enzymes and affinity reagents, is an important tool in biotechnology, drug discovery, and other biochemical fields. Bacterial display is an emerging technology for isolation of new affinity proteins from such combinatorial libraries. Cells have certain properties that are attractive for directed evolution purposes, in particular the option to use quantitative flow-cytometric cell sorting for selection of binders. Here, an immune library of around 10⁷ camelid single-domain antibody fragments (Nanobodies) was displayed on both the Gram-positive bacterium Staphylococcus carnosus and on phage. As demonstrated for the first time, the antibody repertoire was found to be well expressed on the bacterial surface and flow-cytometric sorting yielded a number of Nanobodies with subnanomolar affinity for the target protein, green fluorescent protein (GFP). Interestingly, the staphylococcal output repertoire and the binders from the phage display selection contained two slightly different sets of clones, containing both unique as well as several similar variants. All of the Nanobodies from the staphylococcal selection were also shown to enhance the fluorescence of GFP upon binding, potentially due to the fluorescence-based sorting principle. Our study highlights the impact of the chosen display technology on the variety of selected binders and thus the value of having alternative methods available, and demonstrates in addition that the staphylococcal system is suitable for generation of high-affinity antibody fragments.