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21.
Fibronectin receptor functions in embryonic cells deficient in alpha 5 beta 1 integrin can be replaced by alpha V integrins. 总被引:7,自引:1,他引:6
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alpha 5 beta 1 integrin mediates cell adhesion to extracellular matrix by interacting with fibronectin (FN). Mouse lines carrying null mutations in genes encoding either the alpha 5 integrin subunit or FN have been generated previously. Both mutations are embryonic lethal with overlapping defects, but the defects of alpha 5-null embryos are less severe. Primary embryonic cells lacking alpha 5 beta 1 are able to adhere to FN, form focal contacts, migrate on FN, and assemble FN matrix. These results suggest the involvement of (an)other FN receptors(s). In this study, we examined functions of alpha 4 beta 1 and alpha V integrins in embryonic cells lacking alpha 5 beta 1. Our analysis of cells lacking both alpha 4 beta 1 and alpha 5 beta 1 showed that alpha 4 beta 1 is also not required for these FN-dependent functions. Using alpha V-specific blocking reagents, we showed that alpha V integrins are required for alpha 5-null cells, but not wild-type cells, to adhere and spread on FN. Our data also showed that, although the expression levels of alpha V integrins on the wild-type and alpha 5-null cells are similar, there is an increase in recruitment of alpha V integrins into focal contacts in alpha 5-null cells plated on FN, indicating that alpha V integrins can compensate functionally for the loss of alpha 5 beta 1 in focal contacts of alpha 5-null cells. Finally, our data suggested possible roles for alpha V integrins in replacing the role of alpha 5 beta 1 in FN matrix assembly in vitro and in FN-dependent embryonic functions in vivo. 相似文献
22.
Crystal structures of bovine chymotrypsin and trypsin complexed to the inhibitor domain of Alzheimer's amyloid beta-protein precursor (APPI) and basic pancreatic trypsin inhibitor (BPTI): engineering of inhibitors with altered specificities. 总被引:1,自引:0,他引:1
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A. J. Scheidig T. R. Hynes L. A. Pelletier J. A. Wells A. A. Kossiakoff 《Protein science : a publication of the Protein Society》1997,6(9):1806-1824
The crystal structures of the inhibitor domain of Alzheimer's amyloid beta-protein precursor (APPI) complexed to bovine chymotrypsin (C-APPI) and trypsin (T-APPI) and basic pancreatic trypsin inhibitor (BPTI) bound to chymotrypsin (C-BPTI) have been solved and analyzed at 2.1 A, 1.8 A, and 2.6 A resolution, respectively. APPI and BPTI belong to the Kunitz family of inhibitors, which is characterized by a distinctive tertiary fold with three conserved disulfide bonds. At the specificity-determining site of these inhibitors (P1), residue 15(I)4 is an arginine in APPI and a lysine in BPTI, residue types that are counter to the chymotryptic hydrophobic specificity. In the chymotrypsin complexes, the Arg and Lys P1 side chains of the inhibitors adopt conformations that bend away from the bottom of the binding pocket to interact productively with elements of the binding pocket other than those observed for specificity-matched P1 side chains. The stereochemistry of the nucleophilic hydroxyl of Ser 195 in chymotrypsin relative to the scissile P1 bond of the inhibitors is identical to that observed for these groups in the trypsin-APPI complex, where Arg 15(I) is an optimal side chain for tryptic specificity. To further evaluate the diversity of sequences that can be accommodated by one of these inhibitors, APPI, we used phage display to randomly mutate residues 11, 13, 15, 17, and 19, which are major binding determinants. Inhibitors variants were selected that bound to either trypsin or chymotrypsin. As expected, trypsin specificity was principally directed by having a basic side chain at P1 (position 15); however, the P1 residues that were selected for chymotrypsin binding were His and Asn, rather than the expected large hydrophobic types. This can be rationalized by modeling these hydrophilic side chains to have similar H-bonding interactions to those observed in the structures of the described complexes. The specificity, or lack thereof, for the other individual subsites is discussed in the context of the "allowed" residues determined from a phage display mutagenesis selection experiment. 相似文献
23.
Clones of Chinese hamster ovary (CHO) cells were isolated by single-step selection for resistance to killing Concanavalin A (ConA) and certain cellular and membrane properties were examined. The ConA-resistant isolates were only about 2-fold more resistant than wild type cells to the selecting lectin, but exhibited pleiotropic temperature-sensitivity for growth, markedly altered morphology and adherence, and significant difference in susceptibility to other agents such as colchicine. Two revertants to full temperature-resistance were isolated from different ConA-resistant mutants. One revertant clone had reacquired wild type sensitivity to ConA while the other revertant remained ConA-resistant. The two series of wild typed, ConA-resistant, and temperature revertant clones were analyzed for altered mobility of cell surface glycoproteins using lactoperoxidase/125I and galactose oxidase/(3H) borohydride labelling procedures. The ConA-resistant clones showed increased mobility on polyacrylamide gels of three classes of labelled proteins, in the molecular weight ranges 225,000, 200,000, and 130,000 daltons. These changes persisted in the temperature-revertant that remained ConA-resistant, while two of the altered protein closses were restored to wild type mobility in the revertant that regained ConA-sensitivity. Cell hybridization experiments indicated that the temperature-sensitivity phenotypes of different ConA-resistant isolates are recessive and noncomplementing, implying that the same gene is affected in each case. The reversions to temperature resistance appear to be recessive suppressor mutation in different genes. 相似文献
24.
Rats withdrawn from continuous morphine infusion showed reliable occurrence of withdrawal body shakes. This sign of narcotic withdrawal was inhibited by the neuroleptic drug, (+) butaclamol. (?) Butaclamol was inactive. (+) Butaclamol activity was not antagonized by naloxone (5 mg/kg). The anti-withdrawal mechanism of (+) butaclamol is discussed in terms of effects on dopamine and narcotic receptors.The butyrophenone neuroleptic, haloperidol, has been used successfully to reduce signs of narcotic withdrawal in laboratory animals (1–4) and human addicts (5). Other neuroleptics of the butyrophenone type also show anti-withdrawal action (6, 7). The mechanism of action of these neuroleptics in blocking narcotic withdrawal is not understood. Butaclamol is a new neuroleptic drug that is available in two enantiomers and only (+) butaclamol possesses neuroleptic activity (8–10). Because of its demonstrated stereo-specificity in producing its pharmacological action, we employed this drug to establish specificity of action of neuroleptics in blocking narcotic withdrawal. 相似文献
25.
C3 component of complement secreted by established cell lines 总被引:11,自引:0,他引:11
The hamster cell line NIL8 secretes the C3 component of complement as well as collagenous molecules and fibronectin (LETS protein). The C3 is found in the culture medium as a disulfidebonded complex of two polypeptides of 130,000 daltons (alpha) and 65,000 daltons (beta). The secreted C3 can be quantitatively cleaved to C3b and further cleaved by C3 inactivators. The activation of C3 to C3b is promoted by zymosan or by antibody-coated erythrocytes, demonstrating participation in both the classical and alternative complement pathways. The availability of this culture system has enabled us to show that C3 is synthesized as a 185,000 dalton precursor (proC3) which is biologically inactive and becomes cleaved to active C3. Some other established cell lines (NIL1 and BALB/c 3T3) also secrete C3, but some others do not. 相似文献
26.
Effect of paracrystalline protein surface layers on predation by Bdellovibrio bacteriovorus. 总被引:6,自引:5,他引:1
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We determined that paracrystalline protein surface arrays (S layers) protected gram-negative eubacteria from predation by Bdellovibrio bacteriovorus. Aquaspirillum serpens VHA and MW5 and Aquaspirillum sinuosum were resistant to predation by B. bacteriovorus 6-5-S when fully covered by their S layers. The S layer of Aeromonas salmonicida A449 protected the cells from predication by B. bacteriovorus 109J. A predacious, plaque-forming vibrio that lysed an S-layer- variant of Caulobacter crescentus but was not predacious on the parental strain which possessed an S layer was isolated from raw sewage. Since S layers are stable components of many bacterial surfaces in nature, they can provide this protective function in both aquatic and terrestrial habitats where Bdellovibrio spp. are found. 相似文献
27.
2.9 A resolution structure of an anti-dinitrophenyl-spin-label monoclonal antibody Fab fragment with bound hapten 总被引:5,自引:0,他引:5
The crystal structure of the Fab fragment of the murine monoclonal anti-dinitrophenyl-spin-label antibody AN02 complexed with its hapten has been solved at 2.9 A resolution using a novel molecular replacement method. Prior to translation searches, a large number of the most likely rotation function solutions were subjected to a rigid body refinement against the linear correlation coefficient between intensities of observed and calculated structure factors. First, the overall orientation of the search model and then the orientations and positions of the four Fab domains (VH, VL, CH1 and CL) were refined. This procedure clearly identified the correct orientation of the search model. The refined search model was then subjected to translation searches which unambiguously determined the enantiomer and position in the unit cell of the crystal. The successful search model was refined 2.5 A crystal structure of the Fab fragment of HyHel-5 from which non-matching residues in the variable domains had been removed. HyHel-5 is a murine monoclonal antibody whose heavy and light chains are of the same subclass (gamma 1, kappa, respectively) as AN02. After molecular replacement the structure of the AN02 Fab has been refined using simulated annealing in combination with model building and conjugate gradient refinement to a current crystallographic R-factor of 19.5% for 12,129 unique reflections between 8.0 and 2.9 A. The root-mean-square (r.m.s.) deviation from ideal bond lengths is 0.014 A, and the r.m.s. deviation from ideal bond angles is 3.1 degrees. The electron density reveals the hapten sitting in a pocket formed by the loops of the complementarity determining region. The dinitrophenyl ring of the hapten is sandwiched between the indole rings of Trp96 of the heavy-chain and Trp91 of the light-chain. The positioning of the hapten and general features of the combining site are in good agreement with the results of earlier nuclear magnetic resonance experiments. 相似文献
28.
X-ray crystal structure of the protease inhibitor domain of Alzheimer's amyloid beta-protein precursor 总被引:4,自引:0,他引:4
Alzheimer's amyloid beta-protein precursor contains a Kunitz protease inhibitor domain (APPI) potentially involved in proteolytic events leading to cerebral amyloid deposition. To facilitate the identification of the physiological target of the inhibitor, the crystal structure of APPI has been determined and refined to 1.5-A resolution. Sequences in the inhibitor-protease interface of the correct protease target will reflect the molecular details of the APPI structure. While the overall tertiary fold of APPI is very similar to that of the Kunitz inhibitor BPTI, a significant rearrangement occurs in the backbone conformation of one of the two protease binding loops. A number of Kunitz inhibitors have similar loop sequences, indicating the structural alteration is conserved and potentially an important determinant of inhibitor specificity. In a separate region of the protease binding loops, APPI side chains Met-17 and Phe-34 create an exposed hydrophobic surface in place of Arg-17 and Val-34 in BPTI. The restriction this change places on protease target sequences is seen when the structure of APPI is superimposed on BPTI complexed to serine proteases, where the hydrophobic surface of APPI faces a complementary group of nonpolar side chains on kallikrein A versus polar side chains on trypsin. 相似文献
29.
Expression and secretion in Aspergillus nidulans and Aspergillus niger of a cell surface glycoprotein from the cattle tick, Boophilus microplus, by using the fungal amdS promoter system.
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A cell surface glycoprotein (Bm86) from cells of the digestive tract of the cattle tick Boophilus microplus, which has been shown to elicit a protective immunological response in vaccinated cattle, was expressed and secreted in the filamentous fungi Aspergillus nidulans and Aspergillus niger by using the fungal amdS promoter system. The cloned gene coded for the Bm86 secretory signal and all of the Bm86 mature polypeptide except for the hydrophobic carboxy-terminal segment. High levels of Bm86 mRNA were detected in the transformed cells. Bm86 polypeptide was secreted from the cells in a soluble form and it was glycosylated, probably to a similar extent to the native glycoprotein. The recombinant product had an apparent molecular mass of 83 to 87 kilodaltons, whereas that predicted from the amino acid sequence was 69 kilodaltons. The Bm86 was expressed at levels of up to 1.8 mg/liter, or approximately 6% of secreted protein under the growth conditions used. No intracellular Bm86 was detected. A general relationship was observed between transformants containing a high number of copies of the expression plasmid and high expression levels. 相似文献
30.
A mutation, adjacent to gene amdS, defining the site of action of positive-control gene amdR in Aspergillus nidulans 总被引:6,自引:0,他引:6
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M J Hynes 《Journal of bacteriology》1980,142(2):400-406
In Aspergillus nidulans the acetamidase enzyme is inducible by omega-amino acids, sources of acetyl-coenzyme A, and benzoate. The amdR (or intA) gene is a positive-control gene involved in omega-amino acid induction only. A cis-acting mutation amdI93 located in a complex controlling region adjacent to the acetamidase structural gene was found to abolish induction by omega-amino acids but not induction by other sources of induction. As predicted, this mutation was epistatic to constitutive amdR alleles but did not affect the expression of mutations in other regulatory genes. 相似文献