Generation of a Rat Monoclonal Antibody for Human PAICS, a de novo Purine Biosynthetic Enzyme.

Phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS) is a de novo purine biosynthetic enzyme. It has been found to be overexpressed in various types of cancer and is related to cell proliferation, invasion, the epithelial-mesenchymal transition, and efficient tumor growth. In this study, we describe a rat monoclonal antibody (mAb) 6A10, which was generated as an antigen of human PAICS. This mAb was generated to interact with the N-terminal region of human PAICS and was found to recognize endogenous PAICS enzymes in several cancer cells. Our results also indicated that it can recognize monkey and dog PAICS, which possess the same amino acid sequence in the antigenic region as human PAICS, but it does not recognize rat and mouse PAICS. Furthermore, our data indicated that this mAb is suitable for immunoprecipitation and immunoblotting use for several cancer cell lines. We, therefore, anticipate that mAb 6A10 will be useful for functional analyses of human PAICS in several cancers and for diagnosis of malignant transformation.

Generation of Rat Monoclonal Antibody for Human Nucleolin.

Nucleolin (NCL) is a multifunctional phosphoprotein that is mainly localized in the nucleolus, but it is also found in the nucleoplasm, cytoplasm, and cell membrane. The principal functions of NCL involve DNA and RNA metabolism, gene transcription and translation, ribosome biogenesis, and mRNA stability. It was also reported that the localization of human NCL (hNCL) is related to tumor malignancy. Therefore, analyzing the cellular dynamics of NCL could be useful. In this article, we describe rat monoclonal antibody (mAb) 6F9A6 that was generated against a hNCL peptide. This mAb recognizes endogenous human, monkey, dog, and mouse NCL and was shown to be useful in immunofluorescence staining, immunoprecipitation, and immunoblotting experiments in several cancer cell lines. We anticipate that the mAb 6F9A6 will be useful for functional analyses of hNCL in cancer cells.

Generation of Rat Monoclonal Antibodies Against Human Epidermal Growth Factor Receptor 2.

Human epidermal growth factor receptor 2 (HER2) is a transmembrane tyrosine kinase receptor without any known ligands and a member of the epidermal growth factor receptor (EGFR) family. It is a proto-oncogenic protein that, through signaling cascades, promotes cell proliferation and inhibits apoptosis in cancer cells through homo- and heterodimerization with other EGFR family receptors. Since several cancers, including breast cancer, overexpress HER2, it is a target of tumor therapy. Both trastuzumab and pertuzumab are recombinant humanized monoclonal antibodies (mAbs) used in clinical trials that target the extracellular domain (ECD) of HER2. Therefore, it is important to generate antibodies against various ECDs of HER2. In this study, we describe rat mAbs, which were generated against the ECD of human HER2. The human breast cancer cell line SK-BR-3 was subjected to immunofluorescence staining as it expresses HER2, and mAbs can detect both intact and endogenous HER2 within the cell line.

Antibodies against Noncatalytic B Subunit of Factor XIII Inhibit Activation of Factor XIII and Fibrin Crosslinking.

BACKGROUND: Coagulation factor XIII (FXIII) is a proenzyme of plasma transglutaminase. It comprises two catalytic A subunits (FXIII-A) and two carrier B subunits (FXIII-B). We previously reported that alloantibodies against FXIII-B could promote FXIII clearance in a patient with congenital FXIII-B deficiency who had received infusions of plasma-derived human FXIII (A2B2 heterotetramer). OBJECTIVES: We aimed to investigate whether anti-FXIII-B antibodies affect the catalytic function of FXIII. METHODS: FXIII activation and fibrin crosslinking were examined in the presence of patient plasma, isolated patient IgG, or rat anti-FXIII-B monoclonal antibodies. RESULTS: Alloantibody levels were increased by repeated infusions of plasma-derived A2B2 heterotetramer, which enhanced binding to the functionally important FXIII-B sushi domains. The patient plasma strongly inhibited cleavage of the FXIII-A activation peptide, amine incorporation, and fibrin crosslinking in normal plasma. Furthermore, anti-FXIII-B alloantibodies blocked the formation of the complex of FXIII-B with FXIII-A, and fibrinogen. Rat monoclonal antibodies against the 10th sushi domain of FXIII-B inhibited the incorporation of FXIII-B to fibrin, FXIII activation (i.e., cleavage of FXIII-A activation peptide), and ultimately fibrin crosslinking in normal plasma, independent of their effect on heterotetramer assembly with FXIII-A. Alloantibody binding to the A2B2 heterotetramer blocked the access of thrombin to the FXIII-A cleavage site, as indicated by the reaction of the alloantibodies to the A2B2 heterotetramer and FXIII-B, but not to FXIII-A. CONCLUSION: Anti-FXIII-B antibodies binding to the A2B2 heterotetramer and FXIII-B inhibited FXIII activation and its crosslinking function despite being directed against its noncatalytic subunit (FXIII-B).

Novel Immunochromatographic Test for Anti-factor XIII B Subunit Autoantibodies to Diagnose Autoimmune Acquired Factor XIII Deficiency.

Autoimmune factor XIII (FXIII) deficiency (AiF13D) is an acquired life-threatening bleeding disorder due to anti-FXIII autoantibodies (autoAbs). We previously established an immunochromatographic test (ICT) for detection of anti-FXIII-A subunit (FXIII-A) autoAbs. Conversely, the detection of anti-FXIII-B subunit (FXIII-B) autoAbs is currently performed in a limited number of medical facilities through time-consuming and expensive laboratory tests, such as dot-blotting analysis and enzyme-linked immunosorbent assay (ELISA). Accordingly, in this study, we generated eight rat monoclonal antibodies (mAbs) against human FXIII-B using the rat lymph node method. By employing an ELISA, two mAbs, 2G12B10 and 8H12B9, were selected considering the distance between the recognition regions of each mAb (the 6th and 9th-10th Sushi domain, respectively) and the strength of their reactivity. Using this mAb combination, we prototyped an ICT to detect anti-FXIII-B autoAbs and distinguish between AiF13D and "nonimmune" acquired FXIII deficiency (acF13D), and tested it with 22 healthy controls, 23 acF13D patients, 15 AiF13D patients without anti-FXIII-B autoAbs, and 8 AiF13D patients with anti-FXIII-B autoAbs. Receiver operating characteristic curve analyses of ICTs for anti-FXIII-B autoAbs were performed and revealed a precision similar to dot-blot analysis. Human anti-FXIII-A mAbs were also generated from a single patient with AiF13D using a new cDNA cloning method, and their binding properties were characterized. Consequently, anti-FXIII-A immunoglobulin G preparations were established as potentially permanent positive controls of ICT for anti-FXIII-A antibodies. Combining the previously developed ICT for anti-FXIII-A autoAbs and the novel ICT for anti-FXIII-B autoAbs may reduce false negatives and lead to appropriate diagnosis and treatment.

Generation of Rat Monoclonal Antibody for Mouse Nucleolin by Immunization of Ferroptosis-Induced Hepa 1-6 Cells.

Nucleolin is a multifunctional phosphoprotein that is ubiquitously distributed in the nucleus, nucleoplasm, cytoplasm, and cell membrane. The principal functions of nucleolin involve DNA and RNA metabolism, gene transcription and translation, ribosome biogenesis, and mRNA stability. Ferroptosis is a type of regulated cell death that is characterized by the iron-dependent accumulation of lipid peroxidation products. In a previous study, we produced monoclonal antibodies (mAbs) against lysates prepared from ferroptosis-induced Hepa 1-6 cells. In this study, we describe one of those rat mAbs, 4B5, which was generated against mouse nucleolin. This mAb was useful in immunofluorescence staining, immunoblotting, and immunoprecipitation experiments, and was confirmed to recognize endogenous nucleolin in mouse cell lines and tissues. We anticipate that mAb 4B5 will be useful for functional analyses of nucleolin.

Flow cytometric determination of ferroptosis using a rat monoclonal antibody raised against ferroptotic cells.

Ferroptosis, a type of iron-dependent necrotic cell death, is specifically associated with increased lipid peroxidation. The dysfunction of the glutathione (GSH) production via the starvation of cysteine or the inhibition of phospholipid hydroperoxide glutathione peroxidase (GPX4) typically results in the accumulation of lipid peroxidation products and, consequently, the development of ferroptosis. We recently reported on the production of a rat monoclonal antibody, referred to as FerAb, against mouse-derived Hepa 1-6 cells that had been cultivated in cystine-deprived medium. Immunocytological analyses by means of fluorescence microscopy revealed that FerAb binds to fixed ferroptotic cells regardless of the species from which they were obtained, but not to apoptotic cells. We report herein on an in-depth characterization of the reactivity of FerAb with respect to unfixed cells by means of flow cytometry. The binding of FerAb to the cells was stimulated by incubating the cells in cystine deprived culture medium or treatment with RSL3, a GPX4 inhibitor, while treatment with staurosporine, an apoptosis inducer, had no effect on its binding to the cells. Supplementation with ferrostatin-1, a ferroptosis inhibitor, effectively suppressed the binding of FerAb to cells that had been cultivated in cystine-deprived medium or treated with RSL3, further confirming the specific binding of FerAb to ferroptotic cells. Thus, FerAb combined with a flow cytometry can be used to distinguish ferroptotic cells from living cells or apoptotic cells without the need for fixation. Applications of this combined technique will enable the quantitative evaluation of ferroptotic cells under a variety of patho-physiological conditions and will contribute to our understanding of the roles of ferroptosis in the body as well as cultured cells.

Design, synthesis, and evaluation of the self-assembled antimicrobial peptides based on the ovalbumin-derived peptide TK913.

The preparation, self-assembly, and antimicrobial activity of peptides based on TK913 is described. TK9Z4 incorporating a Pro-Pro motif exhibited self-assembly but no cytotoxicity. However, peptide TKZ3 (obtained by changing the amino acid sequence of TK9Z4) showed morphological changes at different concentrations, potent antimicrobial activity, low cytotoxicity, and trypsin resistance. Accordingly, TKZ3 is proposed as new AMP derived from ovalbumin-derived peptides.

Nitric oxide protects against ferroptosis by aborting the lipid peroxidation chain reaction.

Ferroptosis is a type of iron-dependent necrotic cell death, which is typically triggered by the depletion of intracellular glutathione (GSH), which is associated with increased lipid peroxidation. Nitric oxide (NO) is a highly reactive gaseous radical mediator with anti-oxidation properties that terminates lipid peroxidation reactions. In the current study, we report the anti-ferroptotic action of NOC18, an NO donor that spontaneously releases NO, in cells under various ferroptotic conditions in vitro. Our results indicate that, when mouse hepatoma Hepa 1-6 cells are incubated with NOC18, cell death induced by various ferroptotic stimuli such as cysteine (Cys) starvation, the inhibition of glutathione peroxidase 4 (GPX4) and treatment with tertiary-butyl hydroperoxide (TBHP) is significantly reduced. Treatment with NOC18 failed to improve the decrease in the levels of Cys or GSH and the accumulation of ferrous iron upon ferroptotic stimuli. The fluorescent intensity of C11-BODIPY581/591, a probe that is used to detect lipid peroxidation products, was increased somewhat by treatment with NOC18 under conditions of Cys starvation, and the accumulation of lipid peroxidation end-products, as evidenced by the levels of 4-hydroxynonenal, were effectively suppressed. The pre-incubation of TBHP with NOC7, a short-lived NO donor completely eliminated its ability to trigger ferroptosis. These collective results indicate that NO exerts a cytoprotective action against various ferroptotic stimuli by aborting the lipid peroxidation chain reaction.

Generation of Rat Monoclonal Antibody for Human IQGAP1 by Immunization of Three-Dimensional-Cultured Cancer Cells.

The scaffold protein IQ motif containing GTPase activating protein 1 (IQGAP1) is an adherens junction component in the epithelial tissue that binds many signaling and structural molecules to regulate biological processes. It is known that IQGAP1 is overexpressed in some tumors. In this study, we produced rat monoclonal antibodies (mAbs) through immunization of the lysate from three-dimensional (3D)-cultured DLD-1 cells to elucidate a characteristic feature of a tumor. In cancer research, 3D-cultured cancer cells are used as an intermediate model between in vitro cancer cell line cultures and in vivo tumors. Our results showed that mAb 7E11 recognized increasing antigen in the lysate of 3D-cultured cells comparing with two-dimensional-cultured cells, and its antigen is the human IQGAP1. Furthermore, we indicated that mAb 7E11 was used in immunoblotting, immunoprecipitation, and immunofluorescence staining. Therefore, it may be useful in the analysis of human cancer.

Characterization of a rat monoclonal antibody raised against ferroptotic cells.

Ferroptosis is regulated, non-apoptotic cell death in which ferrous iron and lipid peroxidation products play essential roles. While the ferroptotic pathway is now becoming unveiled, it is difficult to determine its involvement in situ because no unique marker for ferroptotic cells is known. In this study, we report on raising a rat monoclonal antibody against mouse-derived Hepa 1-6 cells that had been cultivated in cystine-deprived media. Binding of the resulting antibody, designated as FerAb, increased during advancing ferroptosis which was caused, not only by cystine deprivation but also treatment with erastin or RSL3, while apoptotic cell death induced by a staurosporine treatment had no effect on the binding. The FerAb was found to bind to 4-hydroxy-2-nonenal (HNE)-modified bovine serum albumin, but no specific protein was detected in ferroptotic cells in an immunoblot analysis. These results indicate that non-proteinaceous, HNE-like structural moiety was part of the antigen for FerAb, although the binding profiles of FerAb to ferroptotic cells were different from those of the currently available anti-HNE antibody. Immunocytological detection revealed inhomogenous staining within cells and partial co-localization with peripheral mitochondria and other cellular components. FerAb was found to be applicable for ferroptotic cells in other mouse cells and cultured human cells that were examined. Thus, the properties of the rat monoclonal antibody FerAb established in this study promise to be useful for the characterization of ferroptotic cell death.

Generation of Rat Monoclonal Antibody for Cytokeratin 18 by Immunization of Three-Dimensional-Cultured Cancer Cells.

Cytokeratin (CK) 18 is an intermediate filament protein that plays a major functional role in the integrity and mechanical stability of cells. Since both CK8 and CK18 are major components of simple epithelia, in the context of tumors, they are expressed in most carcinomas, and have been studied as diagnostic and prognostic markers in tumor pathology. CK18 is also cleaved by some caspases during apoptosis. Three-dimensional (3D)-cultured cancer cells are useful for cancer research as an intermediate model between in vitro cancer cell line cultures and in vivo tumors. In this study, we produced rat monoclonal antibodies (mAbs) through immunization of the lysate from 3D-cultured DLD-1 cells to elucidate a characteristic feature of a tumor, and our results showed that mAb 2H7 recognized human CK18. Furthermore, we indicated that mAb 2H7 was useful for immunoblotting, immunoprecipitation, and immunofluorescence staining. Therefore, it may be useful as a diagnostic tool for evaluating malignancy.

A simple method for isolation and culture of primary hepatocytes from Salvelinus leucomaenis (White-spotted Charr).

White-spotted charr (Salvelinus leucomaenis, S. I.) is an anadromous cold water-adapted fish, distributed in the Far East. We have previously reported the complete mitochondrial DNA sequences of white-spotted chars (S. l. imbrius and S. l. pluvius) in Japan. In general, fish hepatocytes are useful for cellular and biochemical studies of fish. In this study, we isolated hepatocytes from the liver of white-spotted charr and used basic methods, such as enzyme digestion and low centrifugation, to analyze the molecular mechanisms involved in specific cellular responses. The isolated hepatocytes could be cultured at 5-20 °C but not 37 °C. The morphology of hepatocytes was altered in a temperature-dependent manner. The properties of hepatocyte were similar to those of living fish. Moreover, the proliferation rate and damage of isolated hepatocytes depended on the concentration of fetal bovine serum in the culture medium. Taken together, this study demonstrates that this simple method for isolation and culture of hepatocytes from white-spotted charr may be useful for other biochemical and cellular studies.

Antimicrobial activity and secondary structure of a novel peptide derived from ovalbumin.

A novel antimicrobial peptide derived from ovalbumin has been discovered. First, the peptide fragment RKIKVYLPRMK (TK9.1) was identified based on computerized predictions of the secondary structure of peptides in a protein data bank. Using HeliQuest, the sequence was developed into RKIKRYLRRMI (TK9.1.3), which was synthesized using Fmoc-solid phase peptide synthesis, and found to have strongly antimicrobial activity against Gram-positive and Gram-negative bacteria, and fungi but not cytotoxic to HeLa cells and hemolysis in mouse red blood cells. Although ovalbumin itself does not have an antibacterial activity, our results suggest that it may supply the organisms that consume it with antimicrobial peptides, in support of their immunodefence.

Generation and Application of Rat Monoclonal Antibodies Specific for a Human Blood Coagulation Protein: von Willebrand Factor.

von Willebrand factor (VWF) is a glycoprotein that plays a central role in the initiation of blood coagulation. VWF performs two important functions: it acts as a molecular bridge between platelets and as a carrier for coagulation factor VIII (FVIII). von Willebrand disease (VWD) and acquired von Willebrand syndrome (AVWS) are caused by the absence of and/or abnormality in VWF. The pathophysiology of VWD and AVWS is complex and, therefore, it is difficult to diagnose them by conducting the same laboratory tests in all patients. To develop useful monoclonal antibodies (mAbs) for the diagnosis of VWD and AVWS, rat mAbs against human VWF were generated. Immunoblotting analysis revealed that mAbs recognized the reduced and nonreduced VWF protein and endogenous VWF in normal human plasma. Furthermore, we developed a highly sensitive monoclonal antibody-based sandwich enzyme-linked immunosorbent assay technique. In conclusion, the development of VWF-specific mAbs would be useful in the diagnosis of VWD and AVWS.

Three populations of adult Leydig cells in mouse testes revealed by a novel mouse HSD3B1-specific rat monoclonal antibody.

Leydig cells play a pivotal function in the synthesis of a male sex steroid, testosterone. The ability of the steroid production is dependent on the expression of the steroidogenic genes, such as HSD3B (3ß-hydroxysteroid dehydrogenase/Δ5- Δ4 isomerase). It has been established that two different types of Leydig cells, fetal Leydig cells (FLCs) and adult Leydig cells (ALCs), are developed in mammalian testes. FLCs and ALCs are characterized by different sets of marker gene expression. In the case of mouse Leydig cells, Hsd3b1 (Hsd3b type 1) is expressed both in FLCs and ALCs whereas Hsd3b6 (Hsd3b type 6) is expressed in ALCs but not in FLCs. However, because the antibodies established so far for HSD3B were unable to distinguish between the HSD3B1 and HSD3B6 isoforms, it remained unclear whether both of them are expressed in every ALC. Therefore, in the present study, we generated a rat monoclonal antibody specific for mouse HSD3B1. Intriguingly, this monoclonal antibody together with an antibody specific for HSD3B6 identified three populations of ALCs based on the expression levels of these HSD3Bs.

Oxidized unsaturated fatty acids induce apoptotic cell death in cultured cells.

Polyunsaturated fatty acids are oxidized by non­enzymatic or enzymatic reactions. The oxidized products are multifunctional. In this study, we investigated how oxidized fatty acids inhibit cell proliferation in cultured cells. We used polyunsaturated and saturated fatty acids, docosahexaenoic acid (DHA; 22:6), eicosapentaenoic acid (EPA; 20:5), linoleic acid (LA; 18:2), and palmitic acid (16:0). Oxidized fatty acids were produced by autoxidation of fatty acids for 2 days in the presence of a gas mixture (20% O2 and 80% N2). We found that oxidized polyunsaturated fatty acids (OxDHA, OxEPA and OxLA) inhibited cell proliferation much more effectively compared with un­oxidized fatty acids (DHA, EPA and LA, respectively) in THP­1 (a human monocytic leukemia cell line) and DLD­1 (a human colorectal cancer cell line) cells. In particular, OxDHA markedly inhibited cell proliferation. DHA has the largest number of double bonds and is most susceptible to oxidation among the fatty acids. OxDHA has the largest number of highly active oxidized products. Therefore, the oxidative levels of fatty acids are associated with the anti­proliferative activity. Moreover, caspase­3/7 was activated in the cells treated with OxDHA, but not in those treated with DHA. A pan­caspase inhibitor (zVAD­fmk) reduced the cell death induced by OxDHA. These results indicated that oxidized products from polyunsaturated fatty acids induced apoptosis in cultured cells. Collectively, the switch between cell survival and cell death may be regulated by the activity and/or number of oxidized products from polyunsaturated fatty acids.

Induction of oxidative stress by anticancer drugs in the presence and absence of cells.

Reactive oxygen species (ROS) are generated in the cell through multiple mechanisms. Intracellular ROS are rapidly detoxified by various enzymatic and non-enzymatic mechanisms; however, disruption of the oxidant-antioxidant balance causes oxidative stress and elicits cell damage. The oxidative stress induced by chemotherapy is known to cause side effects in patients with cancer. However, few studies have examined whether anticancer drugs induce oxidative stress in cancer cells. Furthermore, the precise mechanism by which anticancer drugs induce the generation of ROS remains unclear. In the present study, to investigate whether anticancer drugs induce oxidative stress, DLD-1 human colorectal cancer cells were treated with 20 different anticancer drugs and then stained with CellROX® ROS detection reagent. Furthermore, an oxygen radical absorbance capacity assay in the presence of copper was performed to estimate the oxidative activities of the anticancer drugs in the absence of cells. The data of the present study using assay methods in the presence and absence of cells suggest that nimustine, actinomycin D, doxorubicin, mitomycin C, mitoxantrone, carmofur, gemcitabine, mercaptopurine, camptothecin, paclitaxel, vinblastine, and vinorelbine are able to induce oxidative stress.

Application of Monoclonal Antibodies Against Mouse Dermokine.

Dermokine is one of the most highly expressed proteins in differentiating keratinocytes. Mouse dermokine has been reported to be encoded by 22 exons, and its expression leads to three transcripts, ß, γ, and α, which are transcribed from two different transcriptional start sites. The α isoform represents the carboxyl-terminal domain of the ß isoform, whereas the γ isoform lacks this domain. To reveal the distributions and expression levels of each isoform in mice, we generated rat monoclonal antibodies against dermokine-ß/γ and dermokine-ß/α. In immunofluorescence studies, the expression levels of dermokine in the cytosol of the cultured mouse keratinocytes were significantly elevated by high levels of extracellular calcium. In Western blot analyses, the expression levels of dermokine-ß and dermokine-α were increased in the presence of high calcium. Finally, we developed a monoclonal antibody-based sensitive sandwich enzyme-linked immunosorbent assay (ELISA) and showed that the secreted dermokine-ß into the culture medium from mouse keratinocytes was significantly increased in a manner dependent on the extracellular calcium concentration. These dermokine-specific antibodies have allowed us to gain new insights into the role of each dermokine isoform in cutaneous homeostasis.