The dispensability of V-V pairing and the indispensability of V domain integrity in the IgG1 secretion process.

The C domain of IgG antibodies controls assembly and secretion, mediated by the molecular chaperone BiP via the endoplasmic reticulum protein quality control (ERQC) mechanism. However, it is not clear whether the variable domains are necessary for this process. Here, we generated IgG1 antibodies in which the V domain (V and/or V) was either removed or replaced, and then assessed expression, assembly, and secretion in HEK293 cells.

M13KO7 bacteriophage enables Potato Virus Y detection.

In this study, we confirmed the binding of M13KO7 to (PVY) using enzyme-linked immunosorbent assay. M13KO7 is a "bald" bacteriophage in which no recombinant antibody is displayed. M13KO7 is easy to propagate by using , making this method more reasonable in economic perspective.

MARCH5-dependent NLRP3 ubiquitination is required for mitochondrial NLRP3-NEK7 complex formation and NLRP3 inflammasome activation.

The NLRP3 inflammasome plays a key role in responding to pathogens, and endogenous damage and mitochondria are intensively involved in inflammasome activation. The NLRP3 inflammasome forms multiprotein complexes and its sequential assembly is important for its activation. Here, we show that NLRP3 is ubiquitinated by the mitochondria-associated E3 ligase, MARCH5.

Nanogel-mediated delivery of oncomodulin secreted from regeneration-associated macrophages promotes sensory axon regeneration in the spinal cord.

Preconditioning nerve injury enhances axonal regeneration of dorsal root ganglia (DRG) neurons in part by driving pro-regenerative perineuronal macrophage activation. How these macrophages influence the neuronal capacity of axon regeneration remains elusive. We report that oncomodulin (ONCM) is produced from the regeneration-associated macrophages and strongly influences regeneration of DRG sensory axons.

A Recombinant Ig Fragment (IgCw-γεκ) Comprising the Cγ-Cε and C Domains Is an Alternative Reagent to Human IgE.

Human IgE is useful for immunological assays, such as sensitization of FcεRI-positive cells and IgE measurement. In this study, we report the development of a recombinant Ig fragment, designated IgCw-γεκ, as an alternative reagent to human IgE. IgCw-γεκ (∼130 kDa) comprises two hybrid constant H chain regions (Cγ-Cε, each ∼53 kDa) and two constant κ L chains (C, each ∼12 kDa) and lacks a V domain.

Assembly and Folding Properties of Cytosolic IgG Intrabodies.

Intrabodies, antibodies expressed within cells, offer an interesting way to target intracellular molecules, making them potentially useful for biotechnology and medicine. However, it remains controversial whether full-size IgG intrabodies expressed in the reducing environment of the cytosol of mammalian cells are workable and structurally sound. Herein, we settle this issue with a systematic investigation of the structure and functionality of four chimeric IgG1s with distinct variable (V) domains but identical constant (C) domains.

Antigen-binding affinity and thermostability of chimeric mouse-chicken IgY and mouse-human IgG antibodies with identical variable domains.

Constant (C)-region switching of heavy (H) and/or light (L) chains in antibodies (Abs) can affect their affinity and specificity, as demonstrated using mouse, human, and chimeric mouse-human (MH) Abs. However, the consequences of C-region switching between evolutionarily distinct mammalian and avian Abs remain unknown. To explore C-region switching in mouse-chicken (MC) Abs, we investigated antigen-binding parameters and thermal stability of chimeric MC-6C407 and MC-3D8 IgY Abs compared with parental mouse IgGs and chimeric MH Abs (MH-6C407 IgG and MH-3D8 IgG) bearing identical corresponding variable (V) regions.

Applications of the immunoglobulin Cw fragment (IgC) composed of the constant regions of heavy and light (C and C) chains.

We report the production and application of a recombinant IgCw molecule, which is composed of only the constant domains of the heavy (C) and light (C) chains, lacking a variable (V) domain. We produced IgCw, especially human IgCw-γκ (98 kDa), composed of two human Cγ chains (37 kDa each) and two Cκ chains (12 kDa each), using HEK293F cell culture. We found that the yield of IgCw-γκ protein was ∼20 mg/L, which was comparable to that of full-size IgG; it bound to Fcγ receptor-positive cells with a low background noise on Fcγ receptor-negative cells; and IgCw-γκ can be used as a reference for measurement of Ig concentration.

Cytosolic Internalization of Anti-DNA Antibodies by Human Monocytes Induces Production of Pro-inflammatory Cytokines Independently of the Tripartite Motif-Containing 21 (TRIM21)-Mediated Pathway.

Anti-DNA autoantibodies are a hallmark of systemic lupus erythematosus (SLE). A subset of anti-DNA IgG autoantibodies is cell-internalizable; thus they can enter living cells in the form of free IgG. However, the contribution made by the Fc region of internalized free-form IgG to the cytokine response has not been studied, despite the recent discovery of tripartite motif-containing 21 (TRIM21), a cytosolic Fc receptor involved in immune signaling.

Heparan sulfate proteoglycans (HSPGs) and chondroitin sulfate proteoglycans (CSPGs) function as endocytic receptors for an internalizing anti-nucleic acid antibody.

A subset of monoclonal anti-DNA autoantibodies enters a variety of living cells. Here, we aimed to identify the endocytic receptors recognized by an internalizing anti-nucleic acid autoantibody, the 3D8 single-chain variable fragment (scFv). We found that cell surface binding and internalization of 3D8 scFv were inhibited markedly in soluble heparan sulfate (HS)/chondroitin sulfate (CS)-deficient or -removed cells and in the presence of soluble HS and CS.

The catalytic activity of a recombinant single chain variable fragment nucleic acid-hydrolysing antibody varies with fusion tag and expression host.

The antigen-binding properties of single chain Fv antibodies (scFvs) can vary depending on the position and type of fusion tag used, as well as the host cells used for expression. The issue is even more complicated with a catalytic scFv antibody that binds and hydrolyses a specific antigen. Herein, we investigated the antigen-binding and -hydrolysing activities of the catalytic anti-nucleic acid antibody 3D8 scFv expressed in Escherichia coli or HEK293f cells with or without additional amino acid residues at the N- and C-termini.

Distribution of radon concentrations in child-care facilities in South Korea.

This study was conducted to provide fundamental data on the distribution of radon concentrations in child day-care facilities in South Korea and to help establish radon mitigation strategies. For this study, 230 child-care centers were randomly chosen from all child-care centers nationwide, and alpha track detectors were used to examine cumulative radon exposure concentrations from January to May 2015. The mean radon concentration measured in Korean child-care centers is approximately 52 Bq m, about one-third of the upper limit of 148 Bq m, which is recommended by South Korea's Indoor Air Quality Control in Public Use Facilities, etc.

The regulation of TIM-3 transcription in T cells involves c-Jun binding but not CpG methylation at the TIM-3 promoter.

Tim-3 is an immunomodulatory protein that is expressed constitutively on monocytes but is induced in activated T cells. The mechanisms involved in the regulation of TIM-3 transcription are poorly understood. In the present study, we investigated whether methylation of the TIM-3 promoter is involved in regulatingTIM-3 transcription in T cells, and identified a transcription factor that regulates TIM-3 transcription by associating with the TIM-3 minimal promoter region.

Antitumor effects of calgranulin B internalized in human colon cancer cells.

Calgranulin B is a small, calcium-binding protein expressed in neutrophils that is secreted into the tumor microenvironment in cancer cases. We previously showed that calgranulin B levels are increased in the stools of colorectal cancer patients. In patient tumor tissues, calgranulin B protein levels correlated with the presence of stromal inflammatory cells surrounding tumor cells, and calgranulin B promoter methylation was observed in both paired human tissues and colon cancer cell lines.

Functional stability of 3D8 scFv, a nucleic acid-hydrolyzing single chain antibody, under different biochemical and physical conditions.

3D8 single-chain Fv (scFv) is a catalytic nucleic acid antibody with anti-viral activity against a broad spectrum of viruses. Here we investigated the functional stability of 3D8 scFv to provide a basis for engineering a 3D8 scFv derivative and for developing stable formulations with improved stability and potential use as an anti-viral agent. The stability of 3D8 scFv was assessed by measuring its DNA-hydrolyzing activity under different biochemical and physical conditions using a fluorescence resonance energy transfer (FRET)-based method.

The chromatin remodeller RSF1 is essential for PLK1 deposition and function at mitotic kinetochores.

Accumulation of PLK1 at kinetochores is essential for chromosome alignment and segregation; however, the mechanism underlying PLK1 recruitment to kinetochores remains unresolved. The chromatin remodeller RSF1 tightly associates with centromere proteins, but its mitotic function is unknown. Here we show that RSF1 localizes at mitotic kinetochores and directly binds PLK1.

In-Cell RNA Hydrolysis Assay: A Method for the Determination of the RNase Activity of Potential RNases.

Conventional procedures to assay RNA degradation by a protein with ribonuclease (RNase) activity require a step to isolate intact RNA molecules, which are used as a substrate. Here, we established a novel "In-cell RNA hydrolysis assay" in which RNAs within cells are used as a substrate for the RNA-hydrolyzing protein, thereby avoiding the need to prepare intact RNA molecules. In this method, the degree of RNA degradation is indicated by the fluorescence intensity of RNA molecules released from fixed and permeabilized cells following treatment with the potential RNase.

Generation of a chickenized catalytic anti-nucleic acid antibody by complementarity-determining region grafting.

In contrast to a number of studies on the humanization of non-human antibodies, the reshaping of a non-human antibody into a chicken antibody has never been attempted. Therefore, nothing is known about the animal species-dependent compatibility of the framework regions (FRs) that sustain the appropriate conformation of the complementarity-determining regions (CDRs). In this study, we attempted the reshaping of the variable domains of the mouse catalytic anti-nucleic acid antibody 3D8 (m3D8) into the FRs of a chicken antibody (“chickenization”) by CDR grafting, which is a common method for the humanization of antibodies.

Regulation of the subcellular shuttling of Sgo1 between centromeres and chromosome arms by Aurora B-mediated phosphorylation.

A minor fraction of cohesin complexes at chromosome arms is not removed by the prophase pathway, and maintained until metaphase and enriched at centromeres. Sgo1 localizes to chromosome arms from prophase to metaphase, and is indispensable for removing cohesin complexes from chromosome arms. However, it has not been established how the chromosome arm localization of Sgo1 leads to the establishment of cohesion on chromosomes.

A putative pH-dependent nuclear localization signal in the juxtamembrane region of c-Met.

The C-terminal fragment of the c-Met receptor tyrosine kinase is present in the nuclei of some cells irrespective of ligand stimulation, but the responsible nuclear localization signal (NLS) has not been previously reported. Here, we report that two histidine residues separated by a 10-amino-acid spacer (H1068-H1079) located in the juxtamembrane region of c-Met function as a putative novel NLS. Deletion of these sequences significantly abolished the nuclear translocation of c-Met, as did substitution of the histidines with alanines.

A nucleic-acid hydrolyzing single chain antibody confers resistance to DNA virus infection in hela cells and C57BL/6 mice.

Viral protein neutralizing antibodies have been developed but they are limited only to the targeted virus and are often susceptible to antigenic drift. Here, we present an alternative strategy for creating virus-resistant cells and animals by ectopic expression of a nucleic acid hydrolyzing catalytic 3D8 single chain variable fragment (scFv), which has both DNase and RNase activities. HeLa cells (SCH07072) [corrected] expressing 3D8 scFv acquired significant resistance to DNA viruses.

Functional consequences of complementarity-determining region deactivation in a multifunctional anti-nucleic acid antibody.

Many murine monoclonal anti-DNA antibodies (Abs) derived from mice models for systemic lupus erythematosus have additional cell-penetration and/or nucleic acid-hydrolysis properties. Here, we examined the influence of deactivating each complementarity-determining region (CDR) within a multifunctional anti-nucleic acid antibody (Ab) that possesses these activities, the catalytic 3D8 single chain variable fragment (scFv). CDR-deactivated 3D8 scFv variants were generated by replacing all of the amino acids within each CDR with Gly/Ser residues.

Cellular internalization mechanism and intracellular trafficking of filamentous M13 phages displaying a cell-penetrating transbody and TAT peptide.

Cellular internalization of bacteriophage by surface-displayed cell penetrating peptides has been reported, though the underlying mechanism remains elusive. Here we describe in detail the internalization mechanism and intracellular trafficking and stability of filamentous M13 phages, the cellular entry of which is mediated by surface-displayed cell-penetrating light chain variable domain 3D8 VL transbody (3D8 VL-M13) or TAT peptide (TAT-M13). Recombinant 3D8 VL-M13 and TAT-M13 phages were efficiently internalized into living mammalian cells via physiologically relevant, energy-dependent endocytosis and were recovered from the cells in their infective form with the yield of 3D8 VL-M13 being higher (0.

T Cell Immunoglobulin Mucin Domain (TIM)-3 Promoter Activity in a Human Mast Cell Line.

T cell immunoglobulin mucin domain (TIM)-3 is an immunomodulatory molecule and upregulated in T cells by several cytokines. TIM-3 also influences mast cell function but its transcriptional regulation in mast cells has not been clarified. Therefore, we examined the transcript level and the promoter activity of TIM-3 in mast cells.