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41.
A 54 kDa cysteine protease purified from the crude extract of Neodiplostomum seoulense adult worms 总被引:4,自引:0,他引:4
As a preliminary study for the explanation of pathobiology of Neodiplostomum seoulense infection, a 54 kDa protease was purified from the crude extract of adult worms by sequential chromatographic methods. The crude extract was subjected to DEAE-Sepharose Fast Flow column, and protein was eluted using 25 mM Tris-HCl (pH 7.4) containing 0.05, 0.1, 0.2 and 0.4 M NaCl in stepwise elution. The 0.2 M NaCl fraction was further purified by Q-Sepharose chromatography and protein was eluted using 20 mM sodium acetate (pH 6.4) containing 0.05, 0.1, 0.2 and 0.3 M NaCl, respectively. The 0.1M NaCl fraction showed a single protein band on SDS-PAGE carried out on a 7.5-15% gradient gel. The proteolytic activities of the purified enzyme were specifically inhibited by L-trans-epoxy-succinylleucylamide (4-guanidino) butane (E-64) and iodoacetic acid. The enzyme, cysteine protease, showed the maximum proteolytic activity at pH 6.0 in 0.1 M buffer, and degraded extracellular matrix proteins such as collagen and fibronectin with different activities. It is suggested that the cysteine protease may play a role in the nutrient uptake of N. seoulense from the host intestine. 相似文献
42.
The Arabidopsis dwarf1 mutant is defective in the conversion of 24-methylenecholesterol to campesterol in brassinosteroid biosynthesis 总被引:6,自引:0,他引:6
Choe S Dilkes BP Gregory BD Ross AS Yuan H Noguchi T Fujioka S Takatsuto S Tanaka A Yoshida S Tax FE Feldmann KA 《Plant physiology》1999,119(3):897-908
Since the isolation and characterization of dwarf1-1 (dwf1-1) from a T-DNA insertion mutant population, phenotypically similar mutants, including deetiolated2 (det2), constitutive photomorphogenesis and dwarfism (cpd), brassinosteroid insensitive1 (bri1), and dwf4, have been reported to be defective in either the biosynthesis or the perception of brassinosteroids. We present further characterization of dwf1-1 and additional dwf1 alleles. Feeding tests with brassinosteroid-biosynthetic intermediates revealed that dwf1 can be rescued by 22alpha-hydroxycampesterol and downstream intermediates in the brassinosteroid pathway. Analysis of the endogenous levels of brassinosteroid intermediates showed that 24-methylenecholesterol in dwf1 accumulates to 12 times the level of the wild type, whereas the level of campesterol is greatly diminished, indicating that the defective step is in C-24 reduction. Furthermore, the deduced amino acid sequence of DWF1 shows significant similarity to a flavin adenine dinucleotide-binding domain conserved in various oxidoreductases, suggesting an enzymatic role for DWF1. In support of this, 7 of 10 dwf1 mutations directly affected the flavin adenine dinucleotide-binding domain. Our molecular characterization of dwf1 alleles, together with our biochemical data, suggest that the biosynthetic defect in dwf1 results in reduced synthesis of bioactive brassinosteroids, causing dwarfism. 相似文献
43.
Kim SU Lee KM Park SK Yoo KY Noh DY Choe KJ Ahn SH Hirvonen A Kang D 《Journal of biochemistry and molecular biology》2004,37(5):582-585
To evaluate the potential association between the GSTP1 genotype and the development of breast cancer, a hospital based case-control study was conducted on Korean women. The study population consisted of 171 histologically confirmed incident breast cancer cases and 171 age-matched controls with no present or previous history of cancer. PCR-RFLP was used for the GSTP1 genotyping and statistical evaluations were performed using an unconditional logistic regression model. Postmenopausal women with the GSTP1 Val allele were found to have a reduced risk of breast cancer (OR = 0.3, 95 % CI = 0.10-0.74). A significant interaction was observed between the GSTP1 genotype and alcohol consumption (p for interaction = 0.01); compared with never-drinking women with Ile/Ile genotype, ever-drinking women with the GSTP1 Val allele had almost a three-fold risk of breast cancer (OR = 2.9, 95 % CI = 1.05-7.85), whereas never-drinking women with Val allele had half this risk (OR = 0.5, 95 % CI = 0.27-0.93). Our findings suggest that the GSTP1 polymorphism influences individual susceptibility to breast cancer in the Korean women and this effect may be modified by alcohol consumption. 相似文献
44.
To investigate adrenomedullary radiopharmaceuticals for positron emission tomography (PET), we have developed no-carrier-added m-(omega-[18F]fluoroalkyl)benzylguanidines. m-(omega-[18F]Fluoroalkyl)benzylguanidines were prepared in two steps starting from N,N'-bis(tert-butyloxycarbonyl)-N' '-(omega-methanesulfonyloxyalkyl)benzylguanidines in 20-30% radiochemical yields (decay corrected for 100 min) and with high radiochemical purity (>97%) and shown to be stable (>90%) in an in vitro metabolic stability assay. The binding of m-(3-[18F]fluoropropyl)benzylguanidine ((18F]3) to SK-N-SH human neuroblastoma cells was temperature dependent, and binding levels at 4 degrees C were reduced to half of that at 37 degrees C, which was similar to the reduction rate observed for [123I]MIBG. Tissue distribution studies in mice showed the highest uptake in the adrenals (%ID/g = 27.2 +/- 5.0%) with relatively high uptake in the myocardium (%ID/g = 9.3 +/- 0.5%). The results suggest that this radiotracer holds promise as a useful adrenomedullary radiopharmaceutical for PET imaging. 相似文献
45.
Choe MM Sporn PH Swartz MA 《American journal of physiology. Lung cellular and molecular physiology》2003,285(2):L427-L433
Recent studies have shown that mechanical forces on airway epithelial cells can induce upregulation of genes involved in airway remodeling in diseases such as asthma. However, the relevance of these responses to airway wall remodeling is still unclear since 1). mechanotransduction is highly dependent on environment (e.g., matrix and other cell types) and 2). inflammatory mediators, which strongly affect remodeling, are also present in asthma. To assess the effects of mechanical forces on the airway wall in a relevant three-dimensional inflammatory context, we have established a tissue culture model of the human airway wall that can be induced to undergo matrix remodeling. Our model contains differentiated human bronchial epithelial cells characterized by tight junctions, cilia formation, and mucus secretion atop a collagen gel embedded with human lung fibroblasts. We found that addition of activated eosinophils and the application of 50% strain to the same system increased the epithelial thickness compared with either condition alone, suggesting that mechanical strain affects airway wall remodeling synergistically with inflammation. This integrated model more closely mimics airway wall remodeling than single-cell, conditioned media, or even two-dimensional coculture systems and is relevant for examining the importance of mechanical strain on airway wall remodeling in an inflammatory environment, which may be crucial for understanding and treating pathologies such as asthma. 相似文献
46.
Certain pathogenic trypanosomatids are highly dependent on glycolysis for ATP production, and hence their glycolytic enzymes, including glycerol-3-phosphate dehydrogenase (GPDH), are considered attractive drug targets. The ternary complex structure of Leishmania mexicana GPDH (LmGPDH) with dihydroxyacetone phosphate (DHAP) and NAD(+) was determined to 1.9A resolution as a further step towards understanding this enzyme's mode of action. When compared with the apo and binary complex structures, the ternary complex structure shows an 11 degrees hinge-bending motion of the C-terminal domain with respect to the N-terminal domain. In addition, residues in the C-terminal domain involved in catalysis or substrates binding show significant movements and a previously invisible five-residue loop region becomes well ordered and participates in NAD(+) binding. Unexpectedly, DHAP and NAD(+) appear to form a covalent bond, producing an adduct in the active site of LmGPDH. Modeling a ternary complex glycerol 3-phosphate (G3P) and NAD(+) with LmGPDH identified ten active site residues that are highly conserved among all GPDHs. Two lysine residues, Lys125 and Lys210, that are presumed to be critical in catalysis, were mutated resulting in greatly reduced catalytic activity. Comparison with other structurally related enzymes found by the program DALI suggested Lys210 as a key catalytic residue, which is located on a structurally conserved alpha-helix. From the results of site-directed mutagenesis, molecular modeling and comparison with related dehydrogenases, a catalytic mechanism of LmGPDH and a possible evolutionary scenario of this group of dehydrogenases are proposed. 相似文献
47.
48.
Choe JY Nelson SW Arienti KL Axe FU Collins TL Jones TK Kimmich RD Newman MJ Norvell K Ripka WC Romano SJ Short KM Slee DH Fromm HJ Honzatko RB 《The Journal of biological chemistry》2003,278(51):51176-51183
A highly constrained pseudo-tetrapeptide (OC252-324) further defines a new allosteric binding site located near the center of fructose-1,6-bisphosphatase. In a crystal structure, pairs of inhibitory molecules bind to opposite faces of the enzyme tetramer. Each ligand molecule is in contact with three of four subunits of the tetramer, hydrogen bonding with the side chain of Asp187 and the backbone carbonyl of residue 71, and electrostatically interacting with the backbone carbonyl of residue 51. The ligated complex adopts a quaternary structure between the canonical R- and T-states of fructose-1,6-bisphosphatase, and yet a dynamic loop essential for catalysis (residues 52-72) is in a conformation identical to that of the T-state enzyme. Inhibition by the pseudo-tetrapeptide is cooperative (Hill coefficient of 2), synergistic with both AMP and fructose 2,6-bisphosphate, noncompetitive with respect to Mg2+, and uncompetitive with respect to fructose 1,6-bisphosphate. The ligand dramatically lowers the concentration at which substrate inhibition dominates the kinetics of fructose-1,6-bisphosphatase. Elevated substrate concentrations employed in kinetic screens may have facilitated the discovery of this uncompetitive inhibitor. Moreover, the inhibitor could mimic an unknown natural effector of fructose-1,6-bisphosphatase, as it interacts strongly with a conserved residue of undetermined functional significance. 相似文献
49.
A functional interaction between chromogranin B and the inositol 1,4,5-trisphosphate receptor/Ca2+ channel 总被引:2,自引:0,他引:2
Thrower EC Choe CU So SH Jeon SH Ehrlich BE Yoo SH 《The Journal of biological chemistry》2003,278(50):49699-49706
Chromogranins A and B (CGA and CGB) are high capacity, low affinity calcium (Ca2+) storage proteins found in many cell types most often associated with secretory granules of secretory cells but also with the endoplasmic reticulum (ER) lumen of these cells. Both CGA and CGB associate with inositol 1,4,5-trisphosphate receptor (InsP3R) in a pH-dependent manner. At an intraluminal pH of 5.5, as found in secretory vesicles, both CGA and CGB bind to the InsP3R. When the intraluminal pH is 7.5, as found in the ER, CGA totally dissociates from InsP3R, whereas CGB only partially dissociates. To investigate the functional consequences of the interaction between the InsP3R and CGB monomers or CGA/CGB heteromers, purified mouse InsP3R type I were fused to planar lipid bilayers and activated by 2 microM InsP3. In the presence of luminal CGB monomers or CGA/CGB heteromers the InsP3R/Ca2+ channel open probability and mean open time increased significantly. The channel activity remained elevated when the pH was changed to 7.5, a reflection of CGB binding to the InsP3R even at pH 7.5. These results suggest that CGB may play an important modulatory role in the control of Ca2+ release from the ER. Furthermore, the difference in the ability of CGA and CGB to regulate the InsP3R/Ca2+ channel and the variability of CGA/CGB ratios could influence the pattern of InsP3-mediated Ca2+ release. 相似文献
50.