共查询到20条相似文献,搜索用时 15 毫秒
1.
《MABS-AUSTIN》2013,5(5):443-452
Manufacturing processes for therapeutic monoclonal antibodies (mAbs) have evolved tremendously since the first licensed mAb product (OKT3) in 1986. The rapid growth in product demand for mAbs triggered parallel efforts to increase production capacity through construction of large bulk manufacturing plants as well as improvements in cell culture processes to raise product titers. This combination has led to an excess of manufacturing capacity, and together with improvements in conventional purification technologies, promises nearly unlimited production capacity in the foreseeable future. The increase in titers has also led to a marked reduction in production costs, which could then become a relatively small fraction of sales price for future products which are sold at prices at or near current levels. The reduction of capacity and cost pressures for current state-of-the-art bulk production processes may shift the focus of process development efforts and have important implications for both plant design and product development strategies for both biopharmaceutical and contract manufacturing companies. 相似文献
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
13.
14.
《Biochimica et Biophysica Acta (BBA)/Molecular and Cell Biology of Lipids》2017,1862(8):786-791
The identification and quantification of lipids from plant tissues have become commonplace and many researchers now incorporate lipidomics approaches into their experimental studies. Plant lipidomics research continues to involve technological developments such as those in mass spectrometry imaging, but in large part, lipidomics approaches have matured to the point of being accessible to the novice. Here we review some important considerations for those planning to apply plant lipidomics to their biological questions, and offer suggestions for appropriate tools and practices. This article is part of a Special Issue entitled: BBALIP_Lipidomics Opinion Articles edited by Sepp Kohlwein. 相似文献
15.
Ubiquilin proteins are conserved across all eukaryotes and function in the regulation of protein degradation. We found that ubiquilin functions to regulate macroautophagy and that the protein is also a substrate of chaperone-mediated autophagy.Key words: autophagy, cell death, LC3, protein turnover, ubiquitinUbiquilin proteins are present in all eukaryotes and appear to function in protein degradation pathways. Humans contain four ubiquilin genes each encoding a separate protein. The proteins are approximately 600 amino acids in length and share extensive homology with one another. They are characterized by an N-terminal sequence that is very similar to ubiquitin, called the ubiquitin-like domain (UBL), followed by a longer, more variable central domain, and terminate with a conserved 50-amino-acid sequence called a ubiquitin-associated domain (UBA). This structural organization is characteristic of proteins that function to deliver ubiquitinated proteins to the proteasome for degradation. In accordance with this function, the UBL domain of ubiquilin binds subunits of the proteasome, and its UBA domain binds to polyubiquitin chains that are typically conjugated onto proteins that are marked for destruction. Indeed, we recently showed that ubiquilin is recruited to the endoplasmic reticulum where it binds and promotes the degradation of misfolded proteins to the proteasome during ER-associated degradation (ERAD).Remarkably, ubiquilin was also recently reported to be involved in macroautophagy. The finding was based on colocalization of ubiquilin with autophagosomal marker LC3 in cells, and because overexpression of ubiquilin-1 suppresses and silencing of its expression enhances, starvation-induced cell death. In our recently published paper we describe our evidence linking ubiquilin to autophagy. We demonstrate that ubiquilin is indeed present in different structures associated with macroautophagy and that it is required for a critical step in autophagosome formation. Additionally, we also demonstrate that ubiquilin is a substrate of chaperone-mediated autophagy. The findings suggest that ubiquilin might play an important, and perhaps a crucial, role in dictating the pathway of protein degradation in cells.In previous studies we found that ubiquilin proteins expressed in normal growing HeLa cells are very stable with a rate of turnover in excess of 20 h. Because most long-lived proteins are degraded by autophagy, we felt it was important to distinguish whether ubiquilin localization in autophagosomes was simply related to the expected route of degradation of the protein or whether it was related to some special function in autophagy. Accordingly, our experiments were designed to distinguish between these two possibilities.Using double immunofluorescence microscopy we found that endogenous ubiquilin and LC3 proteins are present in puncta in HeLa cells. To ensure this was not an artifact of the staining procedure, we cotransfected HeLa cells with ubiquilin-1 and LC3 expression constructs that were tagged with either mRFP or GFP proteins and again found that the two expressed proteins are colocalized in puncta, irrespective of which tag was fused to the proteins. Further evidence supporting ubiquilin localization to autophagosomes was obtained by showing strong enrichment of ubiquilin proteins upon purification of autophagosomes from mouse liver and by the strong immunogold staining of the protein in autophagosomes in mouse brains in a transgenic mouse model of Alzheimer disease.To determine if ubiquilin localization to autophagosomes is mediated by interaction with LC3 we conducted immunoprecipitation experiments to examine whether the two proteins coimmunoprecipitate with each other. Indeed, our results showed that the two proteins coimmunoprecipitate with one another, indicating that they bind together in a complex. However, we did not detect any strong binding between bacterially expressed forms of the proteins, suggesting that the interaction between the proteins in cells might be mediated by a bridging factor(s).We next used a pH-sensitive tandem-tagged mCherry-GFP-LC3 reporter that is used to monitor maturation of autophagosomes to autolysosomes to determine whether ubiquilin is present during the different steps of macroautophagy. Indeed, we found that anti-ubiquilin staining is present throughout the different structures involved in the process, and interestingly, we also noted that the structures are enriched for K48- and K63-ubiquitin linkages. Because ubiquilin contains a UBA domain that binds ubiquitin chains we examined whether proteins containing K48- and K63-ubiquitin linkages coimmunoprecipitate with ubiquilin. Indeed, our immunoblots indicated that proteins containing both of these types of linkages coprecipitate with ubiquilin, consistent with the idea that ubiquilin might target proteins with diverse ubiquitin linkages for degradation by autophagy.To determine if ubiquilin is required for autophagy, we knocked down the ubiquilin-1 and -2 proteins in HeLa cells (which mainly express these two ubiquilin isoforms) by siRNA transfection and examined if loss of the proteins altered LC3-I and LC3-II levels. Interestingly, we found that ubiquilin knockdown over a 72 h time period is associated with a progressive increase in LC3-I levels and a concomitant decrease in LC3-II levels. Furthermore, ubiquilin knockdown led to an ∼45% reduction in the number of cells containing five or more autophagosomes. Based on these results we propose that ubiquilin is required for maturation of LC3-I to LC3-II, which we speculate might be related to the requirement of the protein in macroautophagy.We next asked if ubiquilin protein is consumed during autophagy. We examined this by treating HeLa cells with puromycin to induce protein misfolding and macroautophagy. Immunoblot analysis of the protein lysates examined at 2 h intervals over a 7 h period of exposure to puromycin revealed a direct correlation between stimulation of macroautophagy and a time-dependent decrease in the ubiquilin and LC3-II protein levels. The time-dependent decline in the proteins is inhibited by treatment of cells with two different autophagy inhibitors, 3-methyladenine and bafilomycin A1. The results suggest that ubiquilin protein is consumed during macroautophagy.The consumption of ubiquilin during macroautophagy prompted us to examine if ubiquilin might also be involved in chaperone-mediated autophagy (CMA), which involves the active transport of proteins into lysosomes. Support for this idea arose because ubiquilin proteins contain two sequences that conform to a pentapeptide motif involved in CMA. An in vitro CMA assay using recombinant GST-ubiquilin-1 fusion protein and purified lysosomes confirmed ubiquilin is an active CMA substrate. The results suggested that ubiquilin can be consumed by two different types of autophagy, macroautophagy and CMA. We speculate that this dual mode of consumption may provide a potential switch whereby changes in ubiquilin levels beyond a certain threshold might trigger execution of either macroautophagy or CMA. The idea that such a switch exists stems from previous work that showed inhibition of CMA can lead to activation of macroautophagy and vice versa.Several intriguing new questions emerge from this and previous works, including what exact function ubiquilin serves in autophagy, particularly in the execution of macroautophagy and CMA. Is there a signal that instructs ubiquilin to choose between its known functions in autophagy and ERAD or is the choice random? What role do its different domains play in these processes? The answers to these questions are likely to be important because in previous studies we showed that overexpression of ubiquilin protects cells against potentially toxic mutant huntingtin proteins containing polyglutamine expansions. In our new work we also found that ubiquilin overexpression protects cells against starvation-induced cell death caused by mutations in presenilin-2 proteins. The underlying conclusion from these studies is that ubiquilin appears to play important roles in regulating protein degradation pathways that are likely to have important implications in cell survival. Clearly, understanding ubiquilin function in different protein degradation pathways could lead to novel approaches to prevent diseases associated with protein misfolding. 相似文献
16.
17.
《Autophagy》2013,9(7):979-980
Ubiquilin proteins are conserved across all eukaryotes and function in the regulation of protein degradation. We found that ubiquilin functions to regulate macroautophagy and that the protein is also a substrate of chaperone-mediated autophagy. 相似文献
18.
19.
《Ecological Engineering》2005,24(4):391-401
The Pantanal, a large and still rather pristine wetland in the center of the South American continent, is becoming increasingly threatened by large development programs. Agroindustries and reservoirs for hydroelectric power generation in the catchment area modify discharge pattern and sediment load of the tributaries, plans for canalization of the Paraguay River (hidrovia) are putting in risk the natural flood regime of large areas inside the Pantanal, and attract industries with high potential for environmental pollution, economic pressure on the traditional cattle ranchers accelerates the transformation of natural vegetation into pasture, etc. These activities negatively affect habitat and species diversity and scenic beauty but also the hydrological buffer capacity of the Pantanal. The article summarizes the ecological conditions of the Pantanal, discusses commercial and non-commercial values of the area, describes constraints for the development of intensive agriculture and cattle ranching, and discusses development alternatives. Considering the low density of human population inside the Pantanal, it can be concluded that development pressure on the Pantanal arises mostly from pressure groups outside the area that will also mostly benefit from the economic return of the development projects. Low density of human population would still allow the application of economically viable and environmentally friendly development alternatives that maintain and sustainably manage one of the largest wetlands in the world. 相似文献