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Ouabain-insensitive salt and water movements in duck red cells. II. The role of chloride in the volume response 下载免费PDF全文
This paper describes the effect of external chloride on the typical swelling response induced in duck red cells by hypertonicity or norepinephrine. Lowering chloride inhibits swelling and produces concomitant changes in net movements of sodium and potassium in ouabain-treated cells, which resemble the effect of lowering external sodium or potassium. Inhibition is the same whether chloride is replaced with gluconate or with an osmotic equivalent of sucrose. Since changes in external chloride also cause predictable changes in cell chloride, pH, and water, these variables were systematically investigated by varying external pH along with chloride. Lowering pH to 6.60 does not abolish the response if external chloride levels are normal, although the cells are initially swollen due to the increased acidity. Cells deliberately preswollen in hypotonic solutions with appropriate ionic composition can also respond to norepinephrine by further swelling. These results rule out initial values of cell water, chloride, and pH as significant variables affecting the response. Initial values of the chloride equilibrium potential do have marked effect on the direction and rate of net water movement. If chloride is lowered by replacement with the permeant anion, acetate, E(Cl) is unchanged and a normal response to norepinephrine, which is inhibited by furosemide, is observed. Increasing internal sodium by the nystatin technique also inhibits the response. A theory is developed which depicts that the cotransport carrier proposed in the previous paper (W.F. Schmidt and T.J. McManus. 1977b. J. Gen. Physiol. 70:81-97) moves in response to the net electrochemical potential difference driving sodium and potassium across the membrane. Predictions of this theory fit the data for both cations and anions. 相似文献
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Michael J Gramer Ewald TJ van den Bremer Muriel D van Kampen Amitava Kundu Peter Kopfmann Eric Etter David Stinehelfer Justin Long Tom Lannom Esther H Noordergraaf Jolanda Gerritsen Aran F Labrijn Janine Schuurman Patrick HC van Berkel Paul WHI Parren 《MABS-AUSTIN》2013,5(6):962-973
The manufacturing of bispecific antibodies can be challenging for a variety of reasons. For example, protein expression problems, stability issues, or the use of non-standard approaches for manufacturing can result in poor yield or poor facility fit. In this paper, we demonstrate the use of standard antibody platforms for large-scale manufacturing of bispecific IgG1 by controlled Fab-arm exchange. Two parental antibodies that each contain a single matched point mutation in the CH3 region were separately expressed in Chinese hamster ovary cells and manufactured at 1000 L scale using a platform fed-batch and purification process that was designed for standard antibody production. The bispecific antibody was generated by mixing the two parental molecules under controlled reducing conditions, resulting in efficient Fab-arm exchange of >95% at kg scale. The reductant was removed via diafiltration, resulting in spontaneous reoxidation of interchain disulfide bonds. Aside from the bispecific nature of the molecule, extensive characterization demonstrated that the IgG1 structural integrity was maintained, including function and stability. These results demonstrate the suitability of this bispecific IgG1 format for commercial-scale manufacturing using standard antibody manufacturing techniques. 相似文献
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Two novel gene orders and the role of light-strand replication in rearrangement of the vertebrate mitochondrial genome 总被引:22,自引:8,他引:14
Macey JR; Larson A; Ananjeva NB; Fang Z; Papenfuss TJ 《Molecular biology and evolution》1997,14(1):91-104
Two novel mitochondrial gene arrangements are identified in an agamid
lizard and a ranid frog. Statistical tests incorporating phylogeny indicate
a link between novel vertebrate mitochondrial gene orders and movement of
the origin of light-strand replication. A mechanism involving errors in
light-strand replication and tandem duplication of genes is proposed for
rearrangement of vertebrate mitochondrial genes. A second mechanism
involving small direct repeats also is identified. These mechanisms
implicate gene order as a reliable phylogenetic character. Shifts in gene
order define major lineages without evidence of parallelism or reversal.
The loss of the origin of light-strand replication from its typical
vertebrate position evolves in parallel and, therefore, is a less reliable
phylogenetic character. Gene junctions also evolve in parallel. Sequencing
across multigenic regions, in particular transfer RNA genes, should be a
major focus of future systematic studies to locate novel gene orders and to
provide a better understanding of the evolution of the vertebrate
mitochondrial genome.
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Braunstein M Griffin TJ IV Kriakov JI Friedman ST Grindley ND Jacobs WR 《Journal of bacteriology》2000,182(10):2732-2740
Secreted and cell envelope-associated proteins are important to both Mycobacterium tuberculosis pathogenesis and the generation of protective immunity to M. tuberculosis. We used an in vitro Tn552'phoA transposition system to identify exported proteins of M. tuberculosis. The system is simple and efficient, and the transposon inserts randomly into target DNA. M. tuberculosis genomic libraries were targeted with Tn552'phoA transposons, and these libraries were screened in M. smegmatis for active PhoA translational fusions. Thirty-two different M. tuberculosis open reading frames were identified; eight contain standard signal peptides, six contain lipoprotein signal peptides, and seventeen contain one or more transmembrane domains. Four of these proteins had not yet been assigned as exported proteins in the M. tuberculosis databases. This collection of exported proteins includes factors that are known to participate in the immune response of M. tuberculosis and proteins with homologies, suggesting a role in pathogenesis. Nine of the proteins appear to be unique to mycobacteria and represent promising candidates for factors that participate in protective immunity and virulence. This technology of creating comprehensive fusion libraries should be applicable to other organisms. 相似文献
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