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91.
92.
93.
Longman E Kreusel K Tendler SB Fiebrig I King K Adair J O'Shea P Ortega A Garcia de la Torre J Harding SE 《European biophysics journal : EBJ》2003,32(5):503-510
A modified crystallohydrodynamic approach introduced in 2001 is applied to two human IgG4 constructs from mouse IgG1. The constructs were point mutants of the chimeric antibody molecule cB72.3(4): cB72.3(4A), devoid of inter-chain disulfide bridging, and cB72.3(4P), which has full inter-chain bridging. As before, the known crystallographic structures for the Fab and Fc domains were combined with the measured translational frictional ratios to obtain an estimate for the apparent time-averaged hydration of the domains and hence for that of the intact molecule. The original approach was modified with the hydrated dimensions of the domains being applied, rather than the anhydrous crystallographic dimensions, for assessing the inter-domain orientations using the algorithms HYDROSUB and SOLPRO. Both chimeric IgG4 molecules were found to have open, rather than compact, structures, in agreement with the previous study on wild-type human IgG4. The insertion of a frictionless connector between the domains was necessary, however, for representing the cB72.3(4A) chimera. It therefore appears that the inter-chain disulfide bonds act as physical constraints in the cB72.3(4P) chimera, forcing the antibody domains together and producing a less elongated structure than that of cB72.3(4A). The open structures produced for the two IgG4 chimeras showed similarity to those structures identified for murine IgG1 and IgG2a molecules through X-ray crystallography.Presented at the conference for Advances in Analytical Ultracentrifugation and Hydrodynamics, 8–11 June 2002, Grenoble, France 相似文献
94.
Phagosomes contain class II MHC (MHC-II) and form peptide:MHC-II complexes, but the source of phagosomal MHC-II molecules is uncertain. Phagosomes may acquire nascent MHC-II or preexisting, recycling MHC-II that may be internalized from the plasma membrane. Brefeldin A (BFA) was used to deplete nascent MHC-II in murine macrophages to determine the relative contributions of nascent and recycling MHC-II molecules to phagocytic Ag processing. In addition, biotinylation of cell-surface proteins was used to assess the transport of MHC-II from the cell surface to phagosomes. BFA inhibited macrophage processing of latex bead-conjugated Ag for presentation to T cells, suggesting that nascent MHC-II molecules are important in phagocytic Ag processing. Furthermore, detection of specific peptide:MHC-II complexes in isolated phagosomes confirmed that BFA decreased formation of peptide:MHC-II complexes within phagosomes. Both flow organellometry and Western blot analysis of purified phagosomes showed that about two-thirds of phagosomal MHC-II was nascent (depleted by 3 h prior treatment with BFA) and primarily derived from intracellular sites. About one-third of phagosomal MHC-II was preexisting and primarily derived from the plasma membrane. BFA had little effect on phagosomal H2-DM or the degradation of bead-associated Ag. Thus, inhibition of phagocytic Ag processing by BFA correlated with depletion of nascent MHC-II in phagosomes and occurred despite the persistent delivery of plasma membrane-derived recycling MHC-II molecules and other Ag-processing components to phagosomes. These observations suggest that phagosomal Ag processing depends primarily on nascent MHC-II molecules delivered from intracellular sites, e.g., endocytic compartments. 相似文献
95.
96.
alpha-crystallin protects glucose 6-phosphate dehydrogenase against inactivation by malondialdehyde 总被引:2,自引:0,他引:2
The present work investigates the effect of malondialdehyde (MDA) binding on the enzymic activity and on some structural properties of glucose 6-phosphate dehydrogenase (G6PD). We studied whether alpha-crystallin could protect the enzyme against MDA damage, and if so, by what mechanism. We also studied whether alpha-crystallin could renature G6PD denatured by MDA. alpha-Crystallin was prepared from bovine lenses by gel chromatography. MDA was freshly prepared and incubated with G6PD with or without alpha-crystallin. The results show that MDA reacted with G6PD non-enzymically causing inactivation at concentrations lower than those used previously on structural proteins. The modified enzyme became fluorescent. alpha-Crystallin, acting as a molecular chaperone, specifically protected the enzyme against inactivation by MDA. The enzyme was not reactivated by alpha-crystallin, but it was stabilised and protected against further denaturation. Complex formation between alpha-crystallin and the modified enzyme was demonstrated by immunoprecipitation. G6PD was very susceptible to MDA and we have shown for the first time that alpha-crystallin is able to protect the enzyme against this damage. 相似文献
97.
Sara Dodd Graham A. Place R. L. Hall Stephen E. Harding 《European biophysics journal : EBJ》1998,28(1):38-47
We have used two different approaches to determine hydrodynamic parameters for mucins secreted by guinea-pig tracheal epithelial
cells in primary culture. Cells were cultured under conditions that promote mucous cell differentiation. Secreted mucins were
isolated as the excluded fraction from a Sepharose CL-4B gel filtration column run under strongly dissociating conditions.
Biochemical analysis confirmed the identity of the high molecular weight material as mucins. Analytical ultracentrifugation
was used to study the physical properties of the purified mucins. The weight average molecular mass (M
w
) for three different preparations ranged from 3.3×106 to 4.7×106 g/mol (corresponding to an average structure of 1 – 2 subunits), and the sedimentation coefficient from 25.5 to 35 S. Diffusion
coefficients ranging from 4.5×10–8 to 6.4×10–8 cm2/s were calculated using the Svedberg equation. A polydispersity index (M
z
/M
w
) of ∼1.4 was obtained. Diffusivity values were also determined by image analysis of mucin granule exocytosis captured by
videomicroscopy. The time course of hydration and dissolution of mucin was measured and a relationship is presented which
models both phases, each with first order kinetics, in terms of a maximum radius and rate constants for hydration and dissolution.
A median diffusivity value of 8.05×10–8 cm2/s (inter-quartile range = 1.11×10–7 to 6.08×10–8 cm2/sec) was determined for the hydration phase. For the dissolution phase, a median diffusivity value of 6.98×10–9 cm2/s (inter-quartile range = 1.47×10–8 to 3.25×10–9 cm2/sec) was determined. These values were compared with the macromolecular diffusion coefficients (D
20,w
) obtained by analytical ultracentrifugation. When differences in temperature and viscosity were taken into account, the resulting
D
37,g
was within the range of diffusivity values for dissolution. Our findings show that the physicochemical properties of mucins
secreted by cultured guinea-pig tracheal epithelial cells are similar to those of mucins of the single or double subunit type
purified from respiratory mucus or sputum. These data also suggest that measurement of the diffusivity of dissolution may
be a useful means to estimate the diffusion coefficient of mucins in mucus gel at the time of exocytosis from a secretory
cell.
Received: 10 March 1998 / Accepted: 27 March 1998 相似文献
98.
Deficiency of transporter for antigen presentation (TAP) in tumor cells allows evasion of immune surveillance and increases tumorigenesis. 总被引:4,自引:0,他引:4
A K Johnsen D J Templeton M Sy C V Harding 《Journal of immunology (Baltimore, Md. : 1950)》1999,163(8):4224-4231
Proteins involved in class I MHC (MHC-I) Ag processing, such as the TAP, are deficient in some human tumor cells. This suggests that antitumor responses by CD8 T cells provide selection pressure to favor outgrowth of cells with defective processing of tumor Ags. Nonetheless, this evidence is only correlative, and controlled in vivo experiments have been lacking to demonstrate that TAP deficiency promotes survival of tumor cells. To explore the role of Ag processing defects in tumor progression, matched panels of TAP1-positive and TAP1-negative tumor cell lines were generated from a parental transformed murine fibroblast line. Inoculation of C57BL/6 mice with TAP1-negative cells produced large and persistent tumors. In contrast, TAP1-positive cells did not generate lasting tumors, although small tumors were detected transiently and regressed spontaneously. Both TAP1-positive and TAP1-negative cells produced tumors in athymic mice, confirming that TAP-dependent differences in tumorigenicity were due to T cell-dependent immune responses. Inoculation of C57BL/6 mice with mixtures of TAP1-positive and TAP1-negative cells produced tumors composed exclusively of TAP1-negative cells, indicating in vivo selection for cells with TAP deficiency. Thus, loss of TAP function allows some tumor cells to avoid T cell-dependent elimination, resulting in selection for tumor cells with deficient Ag processing. 相似文献
99.
100.
P W Wilson J Rogers M Harding V Pohl G Pattyn D E Lawson 《Journal of molecular biology》1988,200(4):615-625