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1.
We show that expanded bed protein A affinity chromatography using Streamline rProtein A media is an efficient method for purifying a recombinant humanized monoclonal antibody from unclarified Chinese hamster ovary cell culture fluid and that it provides purification performance comparable to using a packed bed. We determined that the dynamic capacity of the expanded bed media is related to flow rate (measured in column volumes per hour) by a power function, which allows a high capacity at a low flow rate. At 250 cm h-1 with a 25 cm bed height (10 column volumes h-1), the dynamic capacity is 30 g l-1. The yield and purity (measured by the amount of host cell proteins, DNA, SDS-PAGE, and turbidity) of the antibody purified by expanded bed is comparable to the yield and purity obtained on a standard packed bed method using Prosep A media. 相似文献
2.
3.
Chemical modification of bovine prothrombin fragment 1 in the presence of Tb3+ ions. Sequence studies on 3-gamma-MGlu-fragment 总被引:1,自引:0,他引:1
G A Zapata P Berkowitz C M Noyes J S Pollock D W Deerfield L G Pedersen R G Hiskey 《The Journal of biological chemistry》1988,263(17):8150-8156
Chemical modification of the gamma-carboxyglutamyl (Gla) residues of bovine prothrombin fragment 1 using the formaldehyde-morpholine method in the presence of 100 Kappm Tb3+ ions at pH 5.0 provided a modified protein containing 3 gamma-methyleneglutamyl residues (gamma-MGlu) and 7 Gla residues (bovine 3-gamma-MGlu-fragment 1). The modified protein bound the same number of Ca2+ ions as the native protein (six to seven), exhibited 28Mg2+-binding properties identical to native fragment 1 (five Mg2+ ions bound), exhibited the metal ion-promoted quenching of the intrinsic fluorescence in a manner similar to the native protein, but did not bind to phosphatidylserine (PS)/phosphatidylcholine (PC) vesicles in the presence of Ca2+ ions. Modification of the bovine protein using [14C]formaldehyde-morpholine provided a 14C-labeled 3-gamma-MGlu-fragment 1 suitable for sequence analysis. Edman sequencing of the peptides released by a tryptic digest of the reduced and carboxymethylated bovine [14C]3-gamma-MGlu-fragment 1 indicated that Gla residues at positions 7, 8, and 33 had been converted to [14C]gamma-methyleneglutamyl residues. In addition Lys97 was found to contain a 14C label. Similar analysis of the human [14C]3-gamma-MGlu-fragment 1 indicated that Gla residues at positions 7 and 32 were major modification sites and that Gla residues at positions 6 and 14 were partially modified. Lysine 96 was also modified in the human protein. The incorporation of a 14C label at Lys97 in bovine 3-gamma-MGlu-fragment 1 protein is not responsible for the loss of Ca2+-promoted binding to PS/PC vesicles. We suggest that Gla residues 7, 8, and 33 are elements of the first Ca2+-binding site; occupancy of this site establishes the Ca2+-specific conformation which is essential for the Ca2+-promoted interaction of the bovine protein with PS/PC vesicles. These studies also suggest that the loss of Gla residues at positions 7 and 32 prevents the formation of the initial Ca2+-binding site in the human protein. 相似文献
4.
Payant V; Abukashawa S; Sasseville M; Benkel BF; Hickey DA; David J 《Molecular biology and evolution》1988,5(5):560-567
Nuclear DNA was extracted from each of the eight species comprising the
Drosophila melanogaster species subgroup. Southern hybridization of this
DNA by using a molecular probe specific for the alpha-amylase coding region
showed that the duplicated structure of the amylase locus, first found in
D. melanogaster, is conserved among all species of the melanogaster
subgroup. Evidence is also presented for the concerted evolution of the
duplicated genes within each species. In addition, it is shown that the
glucose repression of amylase gene expression, which has been extensively
studied in D. melanogaster, is not confined to this species but occurs in
all eight members of the species subgroup. Thus, both the duplicated gene
structure and the glucose repression of Drosophila amylase gene activity
are stable over extended periods of evolutionary time.
相似文献
5.
Summary The aim of the present study was to analyze the nature of lymphoid and non-lymphoid cellular components occurring in distinct histological compartments of the splenic white pulp of the turtle, Mauremys caspica, in order to define their possible correlations with those of the spleen of higher vertebrates, principally mammals. The white pulp of M.caspica consisted of 3 clearly distinguishable regions: (1) the periateriolar lymphoid sheath, and (2) the inner and (3) the outer zones of the periellipsoidal lymphoid sheath. Reticular cells intimately associated with reticular fibres constituted an extensive meshwork in the periarteriolar lymphoid sheath which housed principally Ig-negative lyphoid cells, mature and immature plasma cells, and interdigitating cells. A few Ig-positive cells were also present in the peripheral region of the periarteriolar lymphoid sheath. The inner and outer zones of the periellipsoidal lymphoid sheath were separated by a discontinuous layer of reticular cell processes. In the inner zone, surface Ig-positive lymphoid cells predominated as well as dendritic cells, resembling ultrastructurally the mammalian follicular dendritic cells, although no germinal centres were found in the turtle spleen. Macrophages, some cytoplasmic Ig-positive cells, and Ig-negative lymphoid cells appeared in the outer zone of the periellipsoidal lymphoid sheath. These results allow us to speculate on a phylogenetic relationship between the periarteriolar lymphoid sheath and the inner and the outer zones of the periellipsoidal lymphoid sheath of the spleen of M. caspica and the periarteriolar lymphoid tissue, the lymphoid follicles and the marginal zone, respectively, of the mammalian splenic white pulp. 相似文献
6.
The cytoarchitecture of the lymphohaemopoietic masses occurring in the "meninx primitiva" of the stingray Dasyatis akajei (Elasmobranchii, Chondricthyes) has been analyzed by light and scanning and transmission electron microscopy. Lymphohaemopoietic aggregates showing similar morphologies occurred along all the central nervous system, but they were more frequent in the telencephalon, diencephalon, and mesencephalon. In each aggregate, the granulopoietic tissue appeared in a fibroblastic stroma surrounding the large blood vessels, and the lymphoid components were present in a reticular network. Developing and mature eosinophils and heterophils--as well as lymphocytes, monocytes, macrophages, and plasma cells--are the main free cells present in these meningeal aggregates. The remarkable intimate association between macrophages and lymphoid cells to form close cell clusters suggests some immunological capacity for the meningeal lymphohaemopoietic tissue. According to their capacities, presence of lymphoid tissue, and histological organization, the meningeal lymphohemopoietic aggregates of Dasyatis akajei resemble other lymphomyeloid aggregates associated with cranium and choroid plexuses in Holocephali and Ganoidei. The phylogenetical relationships of these aggregates with mammalian bone marrow are discussed. 相似文献
7.
A 23-year-old man developed thrombocytopenic purpura at the end of the second week of the clinical evolution of hepatitis A confirmed by viral markers. The bone marrow of this patient showed megakaryocytic hyperplasia. Circulating in his serum immune complexes were demonstrated by solid phase conglutinin enzymo-immunoassay. Platelet-reactive serum factors were also detected by an indirect immunofluorescence test using fresh donor platelets as targets. The evolution of both the hepatitis and the purpura were benign with no therapy other than bedrest. Platelet count normalized within five weeks of the onset of purpura, and IgM antibodies against hepatitis A virus as well as circulating immune complexes dropped to normal levels. It is postulated that the thrombocytopenia of this case was caused by nonspecific deposition of immune complexes at the platelet surface. 相似文献
8.
Experimental populations were examined for temporal changes of gametic disequilibria between allozyme loci (Lap and Pept-1) and gene arrangements of the O chromosome of Drosophila subobscura (O
st and O
3+4+7) under several environmental conditions. In the foundation of the experimental populations a genetic perturbation was carried out in order to test the relevance of the current hypotheses used to explain the allozyme-inversion associations observed in natural populations. Differential changes of gametic disequilibria were detected over generations under the different environmental conditions. Mere mechanical or stochastic factors cannot explain the results and natural selection is probably the major agent generating the detected gametic associations. The observations are interpreted as a proof of coadaptation of D. subobscura inversions. 相似文献
9.
K. S. Kumarasinghe F. Zapata G. Kovacs D. L. Eskew S. K. A. Danso 《Plant and Soil》1986,90(1-3):293-299
Summary The symbiotic association of the water fernAzolla with the blue-green algaAnabaena azollae can fix 30–60 kg N ha–1 per rice cropping season. The value of this fixed N for rice production, however, is only realized once the N is released from theAzolla biomass and taken up by the rice plants. The availability of N applied asAzolla or as urea was measured in field experiments by two15N methods. In the first,Azolla caroliniana (Willd.) was labelled with15N in nutrient solution and incorporated into the soil at a rate of 144 kg N ha–1. The recovery ofAzolla-N in the above ground parts of rice [Oryza sativa (L) cv. Nucleoryza] was found to be 32% vs. 26% for urea applied at a rate of 100 kg N/ha; there was no significant difference in recovery. In the second, 100 kg N/ha of15N-urea was applied separately or in combination with either 250 or 330 kg N ha–1 of unlabelledAzolla. At the higher rate, the recovery ofAzolla-N was significantly greater than that of urea. There was a significant interaction when both N sources were applied together, which resulted in a greater recovery of N from each source in comparison to that source applied separately. Increasing the combined urea andAzolla application rate from 350 kg N ha–1 to 430 kg N ha–1 increased the N yield but had no effect on the dry matter yield of rice plants. The additional N taken up at the higher level of N application accumulated to a greater extent in the straw compared to the panicles. Since no assumptions need to be made about the contribution of soil N in the method using15N-labelledAzolla, this method is preferable to the15N dilution technique for assessing the availability ofAzolla-N to rice. Pot trials usingAzolla stored at –20°C or following oven-drying showed that both treatments decreased the recovery of N by one third in comparison to freshAzolla. 相似文献
10.