Identification of a human monoclonal Fab with neutralizing activity against H3N2 influenza A strain from a newly constructed human Fab library

A combinatorial Fab library was constructed in pComb3H phagemid vectors, using RNA from peripheral blood lymphocytes of a healthy volunteer who had recovered from an influenza A virus infection. The library contained approximately 1.3 x 10(8)E. coli transformants. Bio-panning was carried out against an influenza vaccine containing components of influenza A/New Caledonia/20/99 (H1N1), A/Panama/2007/99 (H3N2), and B/Shandong/7/97 for the enrichment of phages displaying human Fab specific to the viral proteins. E. coli transformed with IF1A11, 1 of 94 randomly selected clones, displayed a human Fab antibody molecule (FabIF1A11) with efficient neutralizing activity against H3N2 influenza A virus strains. The purified FabIF1A11 demonstrated neutralizing activity against A/Okayama/6/01 (H3N2) and A/Kitakyushu/159/93 (H3N2) with 50% plaque reduction neutralization titers of 0.11 microg/ml (2.2 nM) and 1.4 microg/ml (28 nM) respectively. However, FabIF1A11 did not show neutralizing activity against the influenza A virus strain A/USSR/77 (H1N1) or the influenza B virus strain B/Kanagawa/73, even at a concentration of 20 microg/ml (400 nM). The Kd of FabIF1A11 was calculated as 3.6 x 10(-9) M. FabIF1A11 was estimated to recognize a conformational epitope on the hemagglutinin of A/Okayama/6/01 (H3N2). The human monoclonal Fab product FabIF1A11 may have potential as a therapeutic or short-term prophylactic molecule for humans with influenza A H3N2 infection.     

Cysteine-to-serine shuffling using a Saccharomyces cerevisiae expression system improves protein secretion: case of a nonglycosylated mutant of miraculin, a taste-modifying protein

PURPOSE OF WORK: Soluble protein expression is an important first step during various types of protein studies. Here, we present the screening strategy of secretable mutant. The strategy aimed to identify those cysteine residues that provoke protein misfolding in the heterologous expression system. Intentional mutagenesis studies should consider the size of the library and the time required for expression screening. Here, we proposed a cysteine-to-serine shuffling mutation strategy (CS shuffling) using a Saccharomyces cerevisiae expression system. This strategy of site-directed shuffling mutagenesis of cysteine-to-serine residues aims to identify the cysteine residues that cause protein misfolding in heterologous expression. In the case of a nonglycosylated mutant of the taste-modifying protein miraculin (MCL), which was used here as a model protein, 25% of all constructs obtained from CS shuffling expressed MCL mutant, and serine mutations were found at Cys47 or Cys92, which are involved in the formation of the disulfide bond. This indicates that these residues had the potential to provoke protein misfolding via incorrect disulfide bonding. The CS shuffling can be performed using a small library and within one week, and is an effective screening strategy of soluble protein expression.     

Dominance of Emerging G9 and G12 Genotypes and Polymorphism of VP7 and VP4 of Rotaviruses from Bhutanese Children with Severe Diarrhea Prior to the Introduction of Vaccine

A prospective study was performed to determine the molecular characteristics of rotaviruses circulating among children aged <5 years in Bhutan. Stool samples were collected from February 2010 through January 2011 from children who attended two tertiary care hospitals in the capital Thimphu and the eastern regional headquarters, Mongar. The samples positive for rotavirus was mainly comprised genotype G1, followed by G12 and G9. The VP7 and VP4 genes of all genotypes clustered mainly with those of neighboring countries, thereby indicating that they shared common ancestral strains. The VP7 gene of Bhutanese G1 strains belonged to lineage 1c, which differed from the lineages of vaccine strains. Mutations were also identified in the VP7 gene of G1 strains, which may be responsible for neutralization escape strains. Furthermore, we found that lineage 4 of P[8] genotype differed antigenically from the vaccine strains, and mutations were identified in Bhutanese strains of lineage 3. The distribution of rotavirus genotypes varies among years, therefore further research is required to determine the distribution of rotavirus strain genotypes in Bhutan.     

Purification and Some Properties of Debranching Enzyme of Germinating Rice Endosperm

A debranching enzyme was extracted from the endosperm of germinating rice seeds and purified through three steps, namely cyclohexaamylose-coupled Sepharose 6B, Ultrogel AcA-44 and Bio-Gel P-150 column chromatography. This disc-electrophoretically homogeneous enzyme showed a specific activity of 43 units/mg of protein (30°C) with a pH optimum of 5.5. The isoelectric point was 4.9, unlike that (pI 3.5) of debranching enzyme of ungerminated rice seeds. Our enzyme hydrolyzed pullulan rapidly, and glutinous rice starch and waxy corn starch moderately. The enzyme was also able to act on phytoglycogen and glycogen unlike debranching enzymes originating in some plants.     

An Inducible Copper-Thiolate Complex in the Fern, Athyrium yokoscense: Involvement in Copper-Tolerance of the Fern

Two peaks of Cu-containing material were isolated from the solublecytoplasmic fraction of roots of Athyrium yokoscense, a fernwith a tolerance for heavy metals, by gel filtration on Bio-GelP-30 and Sephadex G-25. Peak I (apparent molecular weight 9.5kDa) was only observed in the case of ferns growing on Cu-contaminatedsoil. Peak II (apparent molecular weight 2 kDa) was found inextracts of ferns from both Cu-contaminated and uncontaminatedsoil. Peak I was induced in the fern collected from an uncontaminatedarea by cultivation in Cu-contaminated soil for 14 months. Theinduced Cu-binding complex present in peak I, therefore, maybe involved in mechanisms of Cu-tolerance in the fern. The complexwas purified by column chromatography on DEAE-cellulofine andsubsequent high-performance liquid chromatography. The purifiedcomplex contained a high percentage (26.8%) of cysteine, typicalof metal-binding peptides from plants. (Received June 1, 1988; Accepted September 12, 1988)     

Highly efficient method of preparing human catalytic antibody light chains and their biological characteristics

The ultimate goal of catalytic antibody research is to develop new patient therapies that use the advantages offered by human catalytic antibodies. The establishment of a high-throughput method for obtaining valuable candidate catalytic antibodies must be accelerated to achieve this objective. In this study, based on our concept that we can find antibody light chains with a high probability of success if they include a serine protease-like catalytic triad composed of Ser, His, and Asp on a variable region of the antibody structure, we amplified and cloned DNAs encoding human antibody light chains from germline genes of subgroup II by seminested PCR using two primer sets designed for this purpose. Seven DNA fragments encoding light chains in 17 clones were derived from germline gene A18b, 6 DNA fragments from A3/A19, 2 DNA fragments from A17, and a clone DNA fragment from A5 and O11/O1. All light chains expressed in Escherichia coli and highly purified under nondenaturing conditions exhibited amidolytic activity against synthetic peptides. Some of the light chains exhibited unique features that suppressed the infectious activity of the rabies virus. Furthermore, the survival rate of mice in which a lethal level of the rabies virus was coinoculated directly into the brain with light chain 18 was significantly improved. In the case of humans, these results demonstrate that high-throughput selection of light chains possessing catalytic functions and specificity for a target molecule can be attained from a light-chain DNA library amplified from germline genes belonging to subgroup II.     

Characterization of heme-coordinating histidyl residues of cytochrome b5 based on the reactivity with diethylpyrocarbonate: a mechanism for the opening of axial imidazole rings

We investigated the reactivity of heme-coordinating imidazole with diethylpyrocarbonate using a soluble domain of cytochrome b(5). Analyses with various spectroscopic methods including MALDI-TOF-MS indicated that two axial His residues (His44 and His68) of cytochrome b(5) were protected from the modification by several factors, i.e., limited steric exposure of the axial imidazole to the solvent, the Fe-N(epsilon2) coordination bond, and protonation of the N(delta1) position by forming a hydrogen bond with its immediate surroundings. However, once N-carbethoxylation at the N(epsilon2) position of the axial His residues occurred with a higher concentration of diethylpyrocarbonate, displacement of heme prosthetic group from the protein moiety continued. Simultaneously, it facilitated the second N-carbethoxylation to take place at the N(epsilon1) position of the same imidazole ring, leading to a bis-N-carbethoxylated derivative and further to a ring-opened derivative. A similar mechanism seemed in operation for one non-axial His residue (His85), in which the N(delta1) atom works as a hydrogen acceptor in a strong hydrogen-bond and the other N(epsilon2) atom is in a protonated form, resulting in a formation of the ring-opened derivative upon treatment with a higher concentration of diethylpyrocarbonate. These results suggested that the use of diethylpyrocarbonate for MALDI-TOF-MS analysis might provide a unique method to characterize the protonation state of His residues and the strength of their hydrogen-bondings at the active site of enzymes.     

Fluorescence-based optimization of human bitter taste receptor expression in Saccharomyces cerevisiae

Human TAS2 receptors (hTAS2Rs) perceive bitter tastants, but few studies have explored the structure-function relationships of these receptors. In this paper, we report our trials on the large-scale preparations of hTAS2Rs for structural analysis. Twenty-five hTAS2Rs were expressed using a GFP-fusion yeast system in which the constructs and the culture conditions (e.g., the signal sequence, incubation time and temperature after induction) were optimized by measuring GFP fluorescence. After optimization, five hTAS2Rs (hTAS2R7, hTAS2R8, hTAS2R16, hTAS2R41, and hTAS2R48) were expressed at levels greater than 1 mg protein/L of culture, which is a preferable level for purification and crystallization. Among these five bitter taste receptors, hTAS2R41 exhibited the highest detergent solubilization efficiency of 87.1% in n-dodecyl-β-d-maltopyranoside (DDM)/cholesteryl hemisuccinate (CHS). Fluorescence size-exclusion chromatography showed that hTAS2R41 exhibited monodispersity in DDM/CHS without aggregates, suggesting that hTAS2R41 is a good target for future crystallization trials.     

Interleukin-21 triggers both cellular and humoral immune responses leading to therapeutic antitumor effects against head and neck squamous cell carcinoma

BACKGROUND: Interleukin-21 (IL-21) plays important roles in the regulation of T, B, and natural killer (NK) cells. We hypothesized that the cytokine may provide a novel immunotherapy strategy for cancer by stimulating both Th1 and Th2 immune responses. In this context, antitumor immunity induced by IL-21 was examined in mice bearing subcutaneous head and neck squamous cell carcinomas (HNSCC). METHODS: A plasmid vector encoding murine IL-21 was injected intravenously into mice with pre-established HNSCC tumors, either alone or in combination with a vector construct expressing IL-15. Cytotoxic T lymphocyte (CTL) and NK killing activities were evaluated by chrome release assays, while HNSCC-specific antibody was examined by flow cytometry and ELISA. RESULTS: Significant antitumor effects were obtained by repeated transfection with either the IL-21 or the IL-15 gene. Co-administration of both cytokine genes resulted in increased suppression of tumor growth, significantly prolonging the survival periods of the animals. Thirty percent of the tumor-bearing mice that received the combination therapy survived for more than 300 days, completely rejecting rechallenge with the tumor at a distant site. IL-21 induced significant elevation of HNSCC-specific CTL activity, while IL-21 and IL-15 augmented NK activity in an additive manner. IL-21 gene transfer also promoted the production of tumor-specific IgG. CONCLUSIONS: In vivo transduction of the IL-21 gene elicits powerful antitumor immunity, including both humoral and cellular arms of the immune response, and results in significant suppression of pre-established HNSCC. Co-transfer of the IL-15 gene further improved the therapeutic outcome, mainly by augmenting NK tumoricidal activity. The biological effects of IL-21 may be in sharp contrast to those of conventional Th1 and Th2 cytokines, suggesting intriguing implications of this cytokine for the classical concept of Th1 vs. Th2 paradigm.     

Induction of atherosclerosis in Brown Norway rats by immunization with ovalbumin

A study was carried out to establish an animal model that would be suitable for evaluating the role of the diet in immune cell-mediated atherogenesis. Brown Norway rats were initially treated with hypervitamin D2 for 4 days and then fed on an atherogenic diet for 3 months, during which period the rats were either immunized with ovalubumin plus Al(OH)3 (OVA group) or with Al(OH)3 alone (control group) every 3 weeks. Aortic lesions were mainly composed of foam cells, the lesions evaluated by the intimal thickness of the ascending aorta being more severe in the OVA group than in the control group. The OVA group, in comparison with the control group, showed prominently increased serum levels of OVA-specific IgG and rat chymase, an indicator of mast cell degranulation. The intimal thickness was positively correlated with the level of chymase. Immunization had no effect on the serum lipid levels. These results support the hypothesis that mast cells play a role in the early stage of atherosclerosis and suggest that this animal model could be useful for evaluating the role of the diet in immune-related atherogenesis.