encoding AGIF that inhibits the process of adipogenesis as described [13], and exposed at -70% to an X-ray film with an intensifying screen.
Tumor necrosis factor (TNF) is implicated in wasting syndromes and insulin resistance in chronic infection and obese-linked diabetes. TNF (10 ng/ml) inhibited adipocyte differentiation of 3T3-L1 cells, and in these TNF treated cells little insulin-stimulated glucose uptake was observed. Treatment of 3T3-L1 cells with troglitazone (1-10 microM) partially prevented this inhibitory effect of TNF on adipogenesis, and enhanced expression of C/EBP alpha and GLUT4, even in the presence of TNF. Troglitazone also prevented the inhibitory effects of interleukin-1, interleukin-6, and leukemia inhibitory factor, but not of transforming growth factor beta on adipocyte differentiation of 3T3-L1 cells. These effects might contribute to the antidiabetic effect of troglitazone in obese diabetic animals.
The lysate from intact chloroplasts mechanically isolated from primary leaves of 9 day old seedlings of wheat (Triticum aestivum L. var Aoba) was incubated in the pH range of 5.5 to 8.5 at 370C for 5 hours. Proteolytic activity against ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) was estimated by disappearance of the large subunit of Rubisco or the appearance of its degradation products. Although the activity in lysates was weak, the products were detected by applying Westem blotting. The degradation products were similar to those obtained when Rubisco was incubated with the lysate of vacuoles isolated from like leaves. Although some of the products were similar to those from vacuole lysates, many were clearly different after incubation of Rubisco with trypsin, V-8 protease, or reactive oxygen (hydroxy radical). Lysates of chloroplasts, pretreated with thermolysin at 40C for 30 minutes, had no proteolytic activity against Rubisco after incubation at 370C for 5 hours. These results show that the proteolytic activity against Rubisco found in lysates of our mechanically isolated chloroplasts was mostly due to the contamination of vacuolar proteases adhering to the outer envelope of the chloroplasts during their isolation.Rubisco2 (EC 4.1.1.39) is responsible for the primary step of CO2 fixation in C3 plants and exists in chloroplasts as a stromal protein. Rubisco content in leaves is proportional to the rate of photosynthesis throughout the life span of leaves. Thus, the degradation of Rubisco in leaves is an important factor in regulating the rate of leaf photosynthesis, but the mechanism of degradation is not known yet.Proteolytic activity against Rubisco has been found in the chloroplasts prepared from the protoplasts ofwheat and barley mature leaves (4, 13). In those studies, the activity was relatively strong and could be assessed by measuring a loss of LSU. There is also a report that mechanically isolated chloroplasts from wheat leaves contained weak proteolytic activity against Rubisco, which could be detected by employing immunoblotting (8). ' 1215We postulated that the proteolytic activity against Rubisco in chloroplast lysates might be due to a contamination of proteases derived from vacuoles during isolation of chloroplasts. This was based on similar pH dependence and similar responses to protease inhibitors (2). The purpose of the research reported in this paper was to clarify whether proteolytic activity against Rubisco exists in chloroplasts in vivo, or is derived from other sources during isolation. The degradation products resulting from incubation of chloroplast lysates with Rubisco were compared to those of proteases from vacuoles, using Western blotting with purified anti-LSU antibody. The possible adhering of vacuolar proteases during isolation was examined by treatment of intact chloroplasts with thermolysin, which eliminated proteins attached to the outer envelope. MATERIALS AND METHODS Plant MaterialWheat (Triticum aestivum L. var Aoba) seeds were planted o...
Secretory capacities including folding and assembly are believed to be limiting factors in the establishment of mammalian cell lines producing high levels of recombinant therapeutic proteins. To achieve industrial success, it is also important to improve protein folding, assembly, and secretory processes in combination with increasing transcription and translation. Here, we identified the expression of CHOP/Gadd153 and GRP78, which are unfolded protein response (UPR)-related genes, correlated with recombinant antibody production in stable CHO cells. Subsequently, CHOP overexpression resulted in increasing recombinant antibody production in some mammalian cell lines, and in addition a threefold further enhancement was obtained by combining expression with UPR-related genes or ER chaperones in transient assays. Overexpression of CHOP had no effect on the biochemical characteristics of the product. These results suggest overexpression of CHOP and its combinations may be an effective method to efficiently select a single cell line with a high level of antibody production in the development of cell lines for manufacturing.
Apoptosis, or programmed cell death, is involved in many biological processes including embryogenesis, development of the immune system, elimination of virus-infected cells, and maintenance of tissue homeostasis.1) Binding of Fas ligand (FasL) to Fas induces apoptosis. Fas is a type-I membrane protein and belongs to the tumor necrosis factor (TNF) receptor family.2,3) FasL, a member of the TNF family, is synthesized as a type-II membrane protein and digested proteolytically to a soluble form. 4,5) Mice with mutations of Fas and/or FasL develop massive lymphadenopathy and autoimmune diseases, which indicates that the Fas-FasL system plays an important role in the elimination of autoreactive lymphoid cells. 6)The mouse monoclonal antibody HFE7A (m-HFE7A), raised against human Fas, binds to both human and mouse Fas. 7,8) Administration of m-HFE7A to mice induces apoptosis in thymocytes. However, in mice, m-HFE7A shows no sign of the hepatotoxicity observed with the hamster antimurine Fas monoclonal antibody Jo2. 9) Moreover, administration of m-HFE7A to mice prevents the injury of the liver induced by Jo2. Therefore, m-HFE7A may provide a useful treatment for autoimmune diseases such as rheumatoid arthritis and fulminant hepatitis.A major limitation in the use of murine antibodies for human therapy is the human anti-mouse antibody response, resulting in potentially harmful reactions and the rapid clearance of circulating antibody. 10,11) To reduce the immunogenicity of murine antibodies for human therapy, chimeric antibodies were initially developed.12) Chimeric antibodies consist of the mouse variable domains and the human constant domains. Even chimeric antibodies are still capable of eliciting a significant immune response.13) An approach to overcome the limitation in the clinical use of murine antibodies is humanization of murine antibodies.14) This technique involves transplantation of the murine residues in the complementarity-determining regions (CDRs), which are important for antigen binding, into a relevant human antibody. However, direct grafting of only the CDR residues has sometimes decreased the binding affinity. 15,16) Therefore, in addition to the CDR residues, some murine residues at the key positions in the framework region must also be incorporated into the human framework to maintain the proper CDR conformation. For this process, information on the three-dimensional structure is essential to minimize the number of murine residues to be transferred and avoid the human antimouse antibody response.In this report, we describe humanization of m-HFE7A by the CDR grafting method for the treatment of the human diseases. Five versions of humanized HFE7A (h-HFE7A) induced the same degree of apoptosis in WR19L12a cells as chimeric HFE7A (chi-HFE7A) does. To further probe the structural basis on antibody humanization, we selected one of five h-HFE7As, h-HFE7A-d, since the d version has the smallest number of residues transferred from m-HFE7A among five h-HFE7As and determined the crystal structure of the h-H...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.