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991.
The refolding kinetics of Escherichia coli trp aporepressor were monitored using stopped-flow far-ultraviolet circular dichroism and 8-anilino-1-naphthalene sulfonate fluorescence spectroscopy. Significant gains in secondary structure and the development of hydrophobic surface, respectively, were observed within the dead time of mixing (4-5 ms). These initial increases, or burst phase amplitudes, plotted as a function of final urea concentration, exhibited sigmoidal, coincident unfolding transition curves. The transition curves were fit to a two-state model, and the resulting free energies of folding in the absence of denaturant were found to be similar (approximately 3.3 kcal/mol). Three subsequent slow refolding phases exhibited relaxation times and amplitudes similar to those previously observed for tryptophan fluorescence [Gittelman, M. S., & Matthews, C. R. (1990) Biochemistry 29, 7011-7021]. These results support the proposals that a stable, monomeric intermediate is rapidly formed during the folding of trp aporepressor and that this species contains a significant amount of secondary structure and hydrophobic surface. This early intermediate is then processed through folding and association reactions that result in the formation of the remaining secondary, tertiary, and quaternary structure. 相似文献
992.
MC Thomas SP Ballantine SS Bethell S Bains P Kellam CJ Delves 《Canadian Metallurgical Quarterly》1998,37(33):11629-11636
In the opportunistic pathogen Pneumocystis carinii, dihydroneopterin aldolase function is expressed as the N-terminal portion of the multifunctional folic acid synthesis protein (Fas). This region encompasses two domains, FasA and FasB, which are 27% amino acid identical. FasA and FasB also share significant amino acid sequence similarity with bacterial dihydroneopterin aldolases. In the present study, this enzyme function has been overproduced as an independent monofunctional activity in Escherichia coli. Recombinant FasAB-Met23 (amino acids 23-290 of the predicted open reading frame) was purified and shown to contain dihydroneopterin aldolase activity. The native FasAB-Met23 is a tetramer of the 30-kDa subunit, demonstrating characteristics of an associating-dissociating equilibrium system in which only the multimeric form of the enzyme is active. Multiple sequence alignment of FasA and FasB with other dihydroneopterin aldolases highlights only three positions where the amino acid is invariable between all the predicted proteins. The role of these conserved amino acid residues in enzyme function was investigated using site-directed mutagenesis. Mutant FasAB-Met23 species were overproduced and purified to near homogeneity. Three FasA domain mutants and two FasB domain mutants had little or no detectable dihydroneopterin aldolase activity, implicating both FasA and FasB in the catalytic mechanism. We show that each mutant protein containing an inactivating amino acid substitution has lost its ability to form stable tetramers. 相似文献
993.
A Mariotti T Hassell D Jacobs CJ Manning AF Hefti 《Canadian Metallurgical Quarterly》1998,27(6):260-266
The responsiveness of human gingival fibroblast populations to cyclosporin A (CsA) and its principal metabolite, hydroxycyclosporine (M17), was evaluated in cell culture. Gingival fibroblasts exhibited a dose-dependent accumulation and bell-shaped distribution of dansylated CsA. A 100-fold excess of non-labeled CsA prevented the accumulation of the fluorescent probe in the fibroblasts. Both CsA (400 ng/ml) and M17 (100 ng/ml) stimulated mean gingival fibroblast cell number to 23.2% and 36.7% above controls, and reduced mean collagen production by 37.7% and 37.4% below controls, respectively; however, neither CsA nor M17 affected mean protein production in comparison to control cultures. Analyses of responses to CsA and M17 by ligand-accumulating and non-accumulating fibroblasts sorted out from the parent cultures did not provide consistent interstrain responses either by cells representing the upper quartile of fluorescence or cells representing the bottom quartiles of fluorescence. These data demonstrate that CsA is accumulated by gingival fibroblasts and that CsA and M17 are potent modulators of gingival fibroblast phenotype. 相似文献
994.
Increasing fruit and vegetable consumption is an important health behaviour. Parental and other psychosocial influences on children's fruit and vegetable consumption are poorly understood. The contribution of a variety of psychosocial and environmental factors to consumption of fruit and vegetables by children aged 9-11 years was explored. Ninety-two mothers and children (48 girls and 44 boys) were recruited via urban primary health-care practices. Socio-economic and educational level, nutritional knowledge and health- and diet-related beliefs and attitudes were assessed in mothers and children by questionnaires and semistructured interviews. Mothers> diets were measured by a food frequency questionnaire, while children's diets were assessed by 3-day diaries (N=80). The pattern of influence of the various measures on fruit and vegetable consumption was compared with that on children's confectionery intake. The children's intakes of macronutrients were typical for the U.K. (37% fat, 50% carbohydrate and 13% protein by energy; 12 g/day fibre), while median fruit, fruit juice and vegetable intake amounted to about 2.5 servings/day. Univariate correlations and subsequent multiple regression analyses revealed quite different influences on the three food types. Independent predictors of children's fruit intake included mothers> nutritional knowledge (beta=0.37), mothers> frequency of fruit consumption (beta=0.30) and mothers> attitudinal conviction that increasing fruit and vegetable consumption by their children could reduce their risk of developing cancer (beta=0.27; multiple r2=0.37,p<0.0001). Children's vegetable consumption was independently explained by the child's liking for commonly eaten vegetables (beta=0.36) and the mother's belief in the importance of disease prevention when choosing her child's food (beta=-0.27 r2=0.20,p<0.001). Children's confectionery consumption was predicted by the mother's liking for confectionery (beta=0.32) and the children's concern for health in choosing what to eat (beta=-0.26 r2=0.16, p<0.005). Children's consumption of fruit and vegetables are related to different psychosocial and environmental factors. Promotion of this behaviour may require attention to nutritional education and child feeding strategies of parents. 相似文献
995.
SF Vervoordeldonk K Doumaid EB Remmerswaal IJ ten Berge JM Wilmink LP de Waal CJ Boog 《Canadian Metallurgical Quarterly》1998,102(4):1004-1009
Renal allograft survival is prolonged after pretransplantation blood transfusion. The aim of this study was to test retrospectively the development and persistence of microchimaerism after pretransplantation blood transfusion and to assess whether the type of blood transfusion (partially matched [= sharing of at least one HLA-B and one HLA-DR antigen between blood donor and recipient] versus mismatched) influences the (continued) presence of donor-type cells. A sensitive nested PCR technique based on HLA-DRB1 allele-specific amplification using sequence-specific primers (detection level: one donor cell among 10(5) recipient cells) for detection of donor cells was implemented in our laboratory. We studied 21 patients for microchimaerism in the peripheral blood compartment, following blood transfusion. Our preliminary data show that microchimaerism was detectable up to 8 weeks after blood transfusion. In all patients receiving a partially matched blood transfusion, donor-type cells were detected in the first week after transfusion, in 7/8 patients 2-4 weeks after transfusion, and in some patients up to 8 weeks after transfusion. After mismatched transfusion a tendency to shorter duration of microchimaerism was observed. 相似文献
996.
R Saouaf CJ Grassi GG Hartnell H Wheeler JN Suojanen 《Canadian Metallurgical Quarterly》1998,53(8):579-586
OBJECTIVE: To assess the combination of duplex Doppler ultrasound (DUS) and complete carotid magnetic resonance angiography (MRA) for the non-invasive imaging of carotid disease and their effect on outcomes. Determine inter-reader agreement of carotid MRA. MATERIALS AND METHODS: One-hundred and ten carotid bifurcations were evaluated using DUS, 2D and 3D time-of-flight MRA from the aortic arch to the Circle of Willis in 55 patients. Percentage stenoses were determined by two blinded readers using standardized criteria. Clinical follow-up was by chart review. RESULTS: Correlation of Doppler and MRA was excellent (r=0.903, P<0.001). Inter-reader agreement (K) for MRA was good: internal carotid artery (ICA) (0.750), external carotid artery (ECA) (0.674) and common carotid artery (CCA) (0.410). Differences in CCA readings were due to minor differences in categorizing lesions as CCA versus ICA or ECA. MRA and Doppler detected nine occluded ICAs. Two DUS occlusions had ICA flow by MRA; one due to a reconstituted precavernous ICA, one a near occluded vessel. Five patients (9%) had surgical management modified by MRA with four not having surgery: three distal ICA/Siphon occlusions and one less severe stenosis by MRA. One tandem lesion not visualized by DUS was surgically significant. Nine aortic arch abnormalities had no surgical impact, possibly due to small sample size. Of 41 endarterectomies, there were no complications from errors of diagnosis. CONCLUSION: Carotid MRA correlates well with DUS with good inter-reader agreement. MRA confirms Doppler findings, expands anatomical information and identifies tandem lesions from the aortic arch to the Circle of Willis which can affect surgical management. This approach to carotid artery imaging appears to have no negative effect on surgical outcome. 相似文献
997.
EK Outwater ES Siegelman P Chiowanich AM Kilger CJ Dunton A Talerman 《Canadian Metallurgical Quarterly》1998,208(2):463-469
A complex of four proteins isolated from neuronal membranes has ligand binding sites for N-methyl-d-aspartate (NMDA) receptor agonists and antagonists and forms NMDA-activated ion channels upon reconstitution into lipid membranes. In this study, the cDNA of a subunit of this complex containing binding sites for the competitive antagonists of NMDA receptors was cloned. The cDNA clone coded for a protein of 719 amino acids (78.9 kDa). The expressed protein had binding activity for the agonists l-[3H]glutamate and [3H]glycine, the antagonist (+/-)-[3H]-(E)-2-amino-4-propyl-5-phosphonopentanoic acid ([3H]CGP 39653), but not the ion channel inhibitors. The cloned cDNA had no homology to other cloned cDNAs. Northern blot analyses indicated high expression of an 3.8 kb poly(A+) RNA in brain, but not in other tissues. These findings indicate that proteins that have recognition sites for NMDA receptor activators and inhibitors and that differ from the well-characterized NMDA receptor proteins NR1-3 are expressed in mammalian brain. 相似文献
998.
CJ Visser 《Canadian Metallurgical Quarterly》1998,28(5):1273-1284
Not one post, core, margin, impression material, cement, or final restoration can be used in all clinical situations. This article does not discuss the merits and shortcomings of the numerous restorative concepts and techniques that exist, but rather has concentrated on those that the author believes are valid and applicable today. If one third or more of the anatomic crown remains, or if this is achieved by crown lengthening, a post may not be necessary; however, a crown restoration should definitely be considered. Veterinary dentistry cannot limit those variables that occur daily in clinical practice. Veterinarians must learn to work with these variables and spend less time trying to find the one that applies to all cases. When the basic concepts of how to retain the various restorative components and how to protect remaining tooth structure are understood, the ability to answer numerous questions that arise during the restorative process is facilitated and results in final restorations that are based on sound design principles. 相似文献
999.
WM Aarts R Willemze RJ Bende CJ Meijer ST Pals CJ van Noesel 《Canadian Metallurgical Quarterly》1998,92(10):3857-3864
Primary cutaneous B-cell lymphomas are B-cell non-Hodgkin's lymphomas that arise in the skin. The major subtypes discerned are follicle center cell lymphomas, immunocytomas (marginal zone B-cell lymphomas), and large B-cell lymphomas of the leg. In this study, we analyzed the variable heavy chain (VH) genes of 7 of these lymphomas, ie, 4 follicle center cell lymphomas (diffuse large-cell lymphomas) and 3 immunocytomas. We show that all these lymphomas carry heavily mutated VH genes, with no obvious bias in VH gene usage. The low ratios of replacement versus silent mutations observed in the framework regions of 5 of the 7 lymphomas suggest that the structure of the B-cell antigen receptor was preserved, as in normal B cells that are selected for antibody expression. Moreover, evidence for ongoing mutation was obtained in 3 immunocytomas and in one lymphoma of large-cell type. In addition, in 1 immunocytoma, both IgG- and IgA-expressing clones were found, indicative of isotype switching. Our data provide insight into the biology of primary cutaneous B-cell lymphomas and may be of significance for their classification. 相似文献
1000.