Search

Application of antibodies in most therapeutic area is limited to extracellular or membranous targets because of their impermeability of membrane. For the purpose of biotechnological and therapeutic application, developing intracellular localizing antibody is the invaluable research field. A new recombinant single-chain variable fragment of an anti-dsDNA monoclonal antibody G2-6, IgG of which has been previously shown to have a cell-penetrating activity, was engineered and produced for the use as a delivery vehicle of biomolecule(s). The penetrating capacity of single-chain variable fragment in three mammalian cell lines, L929, NIH/3T3, and COS-7 was analyzed using flow cytometry and confocal microscopy. The results demonstrated that the single-chain variable fragment can effectively internalize all three cell lines, although the internalization level varied. It was also shown that the internalization was time- and concentration-dependent. Moreover, the single-chain variable fragment was located in...

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces cancer cell death with minimal damage to normal cells, however, some cancer cells are resistant to TRAIL. TRAIL resistance may be overcome by agonistic antibodies to TRAIL receptors. In this study, we report the toxic effects of a novel recombinant agonistic human anti&ndash, TRAIL receptor 1 (DR4) monoclonal antibody Fab fragment, DR4-4, on various TRAIL-resistant and -sensitive cancer cell lines. The mechanisms of DR4-4 Fab&ndash, induced cell death in a human T cell leukemia cell line (Jurkat) were investigated using cell viability testing, immunoblotting, immunoassays, flow cytometry, and morphological observation. DR4-4 Fab&ndash, induced caspase-independent necrosis was observed to occur in Jurkat cells in association with p38 mitogen-activated protein kinase activation, cellular FLICE (FADD-like IL-1&beta, converting enzyme)-inhibitory protein degradation, decreased mitochondrial membrane potential, and increased mitochondrial reactive oxygen species production. Increased cytotoxic effects of DR4-4 Fab were observed in combination with TRAIL or &gamma, irradiation. Our results indicate that the novel DR4-4 Fab might overcome TRAIL-resistance and induce death in leukemia cells via cellular mechanisms different from those activated by TRAIL. DR4-4 Fab may have application as a potential therapeutic antibody fragment in single or combination therapy for cancer.

An asymmetric small molecule composed of anthracene and alkylated thienothiophene (2-(anthracen-2-yl)-5-hexylthieno[3,2-b]thiophene) was synthesized via the Suzuki coupling reaction. The thermal stability, photochemical properties, and morphological characteristics were investigated using thermogravimetric analysis, differential scanning calorimetry, cyclic voltammetry, UV–visible spectroscopy, and X-ray diffraction techniques. The compound demonstrated a good thermal stability of 5% at a decomposition temperature of 336 °C. The solution-processed single-crystal transistor, devised with 2-(anthracen-2-yl)-5-hexylthieno[3,2-b]thiophene, exhibited high performance with a hole mobility of 0.1 cm2/Vs. Furthermore, by utilizing the high mobility of 2-(anthracen-2-yl)-5-hexylthieno[3,2-b]thiophene, we demonstrated its unprecedented high photoresponsivity of 3370 A/W, a finding that can be attributed to the very efficient photoconductive behavior of a single crystal of 2-(anthracen-2-yl)-5-hexylthieno[3,2-b]thiophene.

The significance of soil environment is gradually increased due to the soil and underwater contamination caused by petroleum leak accidents. It requires a high cost and long period for the purification of soil compared with other environmental matrix such as water and air. For this reason, it has been embroiled in a legal conflict to find the pollution source and charge of cleanup. In this study, we analyzed the physical properties and typical additives of jet fuel to search a method that can distinguish kerosene and jet fuel contamination. In particular, the chemical marker in kerosene was visualized by the developer and the additives in jet fuel, such as antioxidant and metal deactivator were detected by GC-MS. This study could be used to judge petroleum source at soil contaminant accident sites.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers tumor-specific apoptosis. However, some tumors and cancer cell lines are resistant to TRAIL. Here, the effect of the non-steroidal anti-inflammatory drug aspirin on sensitization of human cervical cancer cells to TRAIL and the underlying mechanism(s) of the effect were explored. Combination treatment with aspirin and TRAIL markedly enhanced apoptotic cell death, as assessed by lactate dehydrogenase (LDH) assay and analysis of cell cycle sub-G1 phase. The two agents together activated the several caspases and mitochondrial signaling pathway. Whereas Mcl-1 protein level was increased and extracellular signal-related kinase (ERK)1/2 was activated in cells treated with TRAIL alone, combination treatment dramatically inhibited ERK1/2 activation and down-regulated Mcl-1 protein level. An inhibitor of ERK1/2 activation, PD98059, also augmented TRAIL-induced apoptosis. Combination treatment with PD98059 and TRAIL showed the activation of caspases and mitochondrial pathway, and the down-regulation of Mcl-1 level. These results suggest that cancer cells can be sensitized to TRAIL-induced apoptosis by pre-treatment with aspirin via suppression of ERK1/2 activation. These findings provide a basis for further exploring the potential applications of this combination approach for the treatment of cancer, including cervical cancer.

This study was undertaken to produce a Fab fragment of a human monoclonal antibody reactive to oxidized and carbamylated low-density lipoprotein (oxLDL and cLDL) using phage display technology. An analysis of DNA sequences of this Fab, termed plaque 15,16-46 Fab, revealed that the rearranged VH was highly mutated. Complementarity-determining regions of the VH showed a very high R/S ratio and contained many positively charged amino acids. In direct binding and competitive ELISA, the Fab reacted strongly with both MDA-LDL and Cu-oxLDL forms of oxLDL, and also showed high affinity for cLDL. Immunofluorescence and immunohistochemical analyses showed that this Fab positively stained atherosclerotic aortic plaques in ApoE−/− mice as well as those in patients with atherosclerosis. The Fab also showed positive staining in placental decidua from patients with preeclampsia. It is suggested that the plaque 15,16-46 Fab against oxLDL and cLDL might possibly be applicable for developing a diagnostic reagent f...

Anti-DNA autoantibodies are one of the frequently found autoantibodies in systemic lupus erythematosus patient sera. RNA aptamers for the monoclonal G6-9 anti-DNA autoantibody were selected from a random pool of RNA library. Binding affinity of the best aptamer is around 2nM, which is at least 100-fold higher than that of cognate DNA antigen to the autoantibody. Aptamer binds specifically to the G6-9 autoantibody but not to other similar autoantibodies. Minimal binding motif of the aptamer was mapped, providing a hint for a natural epitope of the autoantibody. DNA binding to the G6-9 autoantibody is shown to be efficiently inhibited by the aptamer. Such binding property of the RNA aptamer could be used not only as a modulator for the pathogenic anti-DNA autoantibody, but also as a useful biochemical reagent for elucidating a fine specificity of the autoantibody-nucleic acid interaction.

A well-characterized recombinant anti-idiotype to an anti-DNA antibody can be useful for studies of the regulation of anti-DNA-producing B cells. Using a hybridoma technique, a monoclonal anti-idiotypic antibody, designated O2F3, was obtained, and its scFv gene was constructed. O2F3 single chain Fv (scFv) was produced against an idiotope of a monoclonal anti-DNA antibody, 3D8, that was obtained from an autoimmune-prone mouse, MRL-lpr/lpr. Here we describe the production and in vitro characterization of the O2F3 scFv, and compare it with its parent monoclonal antibody, O2F3 IgM. To characterize O2F3 scFv and O2F3 IgM, we generated recombinant 3D8 fragments, including 3D8 scFv, 3D8 VH, and 3D8 VL, that were used as antigens in several assays. ELISA and Western blot analysis showed that both O2F3 scFv and O2F3 IgM recognized a conformational determinant formed by the association of the variable region heavy and light chains of the 3D8 antibody, suggesting that O2F3 scFv retained a similar binding pattern to its parent O2F3 antibody. The idiotope recognized by O2F3 was shown by competitive ELISA to be outside of the DNA binding site of the 3D8 antibody. This characterized O2F3 scFv could be applied for the regulation of anti-DNA antibody production and the manipulation of recombinant antibody-based proteins to which toxins, enzymes, and chemical agents can be connected.

We have used single and multiple site-directed mutagenesis, and molecular modeling, to identify critical residues in the DNA binding site of MAb 2C10, an IgG anti-dsDNA autoantibody from an MRL/lpr lupus mouse. Simultaneous replacement of four Arg residues in the CDR3H abolished binding activity. With one exception, replacement of any one of these Arg residues reduced the activity to 20-50% of the unmutated scFv activity. Arg to Asp replacements had a slightly greater effect than Arg to Ala replacements. In the one exceptional case, replacement of Arg99 with Ala actually increased DNA binding five-fold and replacement by Asp had little effect. Mutation of Phe32 and Asn35 to A1a in CDRIH decreased DNA binding, whereas replacement of Arg31 with A1a had negligible effect. Ala substitution of any one of a cluster of Asp residues in CDR1L increased DNA binding three to six-fold, confirming previous findings that the L-chain of MAb 2C10 is not favorable for DNA binding. The L-chain does participate in shaping the selectivity of antigen binding, and mutation of CDR3L residue Asp92 or Asn93 caused a decrease in DNA binding activity. Directed mutagenesis, consistent with a molecular model, indicates that: several CDR amino acids contribute to DNA binding, without one residue dominating; both VH and VL CDR3 domains contribute to specificity of binding whereas the CDR1L hinders DNA binding. The results suggest a significant role for electrostatics in the interaction of DNA with MAb 2C10.

Twenty‐one monoclonal anti‐DNA autoantibodies were produced by fusing spleen cells from an autoimmune MRL//pr mouse with SP2/0 myeloma cells. Hybridomas generated by the fusions were chosen for cloning on the basis of DNA binding by supernatant antibody. Each monoclonal antibody was purified to homogeneity and analyzed for the heavy and light chain isotypes and the binding specificity for single‐stranded DNA, double‐stranded DNA, and RNA. Sequence specificities and isoelectric points of the antibodies were also examined. All of the antibodies were IgG and tended to bind to both single‐stranded and double‐stranded DNA with a preference for the double‐stranded form. Some of them also bound to RNA. Isoelectric points of the antibodies were shown to be high. The antibodies described in this report have characteristics of pathogenic anti‐DNA antibodies

In the present study, we investigated the substrate specificity of catalytic activity of a cytotoxic anti-DNA monoclonal autoantibody, G1-5, which was obtained from an MRL- lpr/lpr mouse by hybridoma technology. The antibody catalyzed hydrolysis of single- and double-stranded DNA with a higher substrate specificity for thymine than adenine by either β-glycosidic or phosphodiester bond cleavage. The hydrolysis rate ( k cat ) showed maximum at acidic pH conditions, suggesting that the catalytic site of the antibody contains essential carboxylic group(s). Treatment of cells with the antibody promoted cell death and induced the activation of caspases. The cell death induced by the antibody was inhibited by the pan-caspase inhibitor. Furthermore, the antibody binds to cell membrane and penetrates into the cells. Our results suggest that the cell death is initiated by antibodies penetrating to cells and nucleus, hydrolyzing considerable amount of DNA, and mediating the caspase-dependent apoptotic pathway.

Oxidized low-density lipoprotein (oxLDL) is a key autoantigen in atherosclerosis. The genetic structures and pathogenic roles of autoantibodies against this protein remain to be established. In this study, we cloned several monoclonal IgG autoantibody Fab fragments specific for oxLDL from peripheral blood lymphocytes of atherosclerosis patients, using phage display technology. The sequences of their variable regions were determined at the cDNA level. The closest germline counterparts for the heavy chains belonged to the VH3 or VH1 family. The sequences and lengths of complementarity-determining regions (CDR)3-VH were diverse, and frequent mutations of positively charged amino acids (particularly arginine) over entire VH and VL sequences were observed. It is proposed that anti-oxLDL autoantibody formation is driven by antigens. Among the Fabs, P2-8 and P3-175 bound to both MDA-LDL and Cu-oxLDL, and inhibited the uptake of oxLDL by macrophages, suggesting the epitope(s) recognized by the Fabs is a part of ligands on oxLDL that is involved in uptake by macrophage scavenger receptor. These human autoantibody Fabs require detailed investigation to ascertain their potential as agents for clinical applications.

The DNA-recognizing autoantibodies were prepared in milligram scale and their catalytic activities were investigated using various standard substrates for hydrolysis of natural biomolecules such as DNA, carbohydrates, and proteins. Only phosphatase and glycosidase activity was found and no peptidase, sulfatase, or esterase activity was detected in most of anti-DNA monoclonal autoantibodies we tested. Antibody G1-2 showed the highest catalytic activities and its enzymatic characteristics were further investigated. The antibody showed phosphatase activity with sub-millimolar substrate specificity and 10(4)-10(5) rate enhancements. However, Ab G1-2 showed low micro-molar specificity with p-nitrophenyl-beta-D-N-acetylglucosamide with 10(4)-10(5) rate enhancements. Both of the catalytic activities showed pH maximum at 4-5, suggesting that the carboxylate(s) in antigen-binding site is involved in the catalytic mechanism. Chemical protection of carboxylate(s) with diazoacetamide showed much reduced activity of the Ab, confirming that the catalytic activity comes from carboxylate(s) in the Ag-binding region. The activities of phosphatase and glycosidase were thoroughly inhibited by DNA with almost identical K(i) values. These data suggest that DNA-binding site(s) is the enzymatic active site of the catalytic Abs. Capabilities of the DNA recognition might make it possible to confer the Ab the catalytic activity of phosphate and glycosidic bond hydrolysis, which can be the main cause of DNA cleavage.

The light chain of 2C10, an anti-double stranded DNA (dsDNA) autoantibody, is not favorable for DNA binding and it was suggested that the light chain might modulate the specificity of the antibody in DNA binding. We studied several mutant scFvs expressing mutated VL and normal VH of 2C10 to explore the role of the light chain in determining the fine specificity of the antibody, which we define as the preferential binding to a specific sequence of bases or a helical conformation compared to dsDNA from calf thymus. The wild-type Fab and scFv of 2C10 bind to poly(dA–dC)·(dG–dT) better than to dsDNA. However, in the absence of the light chain domain, the VH domain bound dsDNA better than poly(dA–dC)·(dG–dT), indicating the possible involvement of the light chain in determining the fine specificity in DNA binding. The mutations we studied were located in either CDR1L or CDR3L of the antibody. The CDR1 mutants, D28A, D30A, D31A, and D32A have been previously shown to cause an increase in the affinity of 2C10 scFv to DNA. The fine specificity of 2C10 was not affected by the CDR1 mutants which bound to poly(dA–dC)·(dG–dT) better than dsDNA. However, CDR3L mutants, D92A and N93A, which had been shown to be involved in direct interaction with DNA, preferred dsDNA to poly(dA–dC)·(dG–dT) in their binding. Our results indicate that the fine specificity of 2C10 in DNA binding is modulated primarily by Asp at 92 and Asn at 93 in CDR3L. The effects of CDR1L mutations indicate that this region affects only the affinity but not the fine specificity of 2C10.

Background: Anti-cardiolipin antibody (Anti-CL Ab) is one of the various antiphospholipid antibodies (Anti-PL Abs) and found in the plasma of patients with systemic lupus erythematosus (SLE), atherosclerosis, and other infectious diseases. While anti-PL Abs found in the sera of patients with infectious diseases bind directly to CL, binding of anti-PL Abs to CL circulating in the sera of patients with autoimmune diseases is mediated by glycoprotein 1 (). The purpose of this study is to investigate the effect of on the antigen binding assay of anti-CL Abs present in the sera of patients with atherosclerosis, which has been known as one of autoimmune diseases. Methods: ELISA was performed with sera containing anti-CL Abs from three patients with atherosclerosis in the presence or absence of or FBS. Results: Reactivity of anti-CL Abs to CL was increased in the presence of or FBS in a dose dependent manner. Conclusion: or FBS could be used as co-factor in CL ELISA with anti-CL Abs present in the sera of patients with atherosclerosis. It is suggested that anti-CL Abs found in atherosclerosis patients are similar in terms of antigen binding property to those circulating in the patients with autoimmune diseases, not to infectious diseases.