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991.
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993.
To test the hypothesis that substrate-induced steric compression between His 57 and Asp 102 at the active site of chymotrypsin can increase the basicity of His 57, we have synthesized thecis- andtrans-isomers of 2-bromo-3-(N-tritylimidazole)-2-propenoic acid and 2-chloro-3-(N-tritylimidazole)-2-propenoic acid and compared selected properties with those ofcis-andtrans-urocanic acids. Thecis-isomers display low field1H NMR signals at 17 ppm in dimethylsulfoxide, similar tocis-urocanic acid; whereas thetrans-isomers do not show strong hydrogen bonds. Increasing the size of the C2 substituent (H < Cl < Br) in thecis-isomers increases the pKaof the imidazolium group from 6.78 for H to 7.81 and 9.10 for Cl and Br, respectively; whereas the pKas of thetransisomers are all 6.0 ± 0.1. The results indicate that thecis-urocanic acid derivatives with large substituents at C2 act as proton sponges in water, and they support the concept that steric compression in the catalytic triad of chymotrypsin can increase the basicity of His 57. 相似文献
994.
Inositol monophosphatase (EC 3.1.3.25) plays a pivotal role in the biosynthesis of di-myo-inositol-1,1′-phosphate, an osmolyte found in hyperthermophilic archaea. Given the sequence homology between the MJ109 gene product of Methanococcus jannaschii and human inositol monophosphatase, the MJ109 gene was cloned and expressed in Escherichia coli and examined for inositol monophosphatase activity. The purified MJ109 gene product showed inositol monophosphatase activity with kinetic parameters (Km = 0.091 ± 0.016 mM; Vmax = 9.3 ± 0.45 μmol of Pi min−1 mg of protein−1) comparable to those of mammalian and E. coli enzymes. Its substrate specificity, Mg2+ requirement, Li+ inhibition, subunit association (dimerization), and heat stability were studied and compared to those of other inositol monophosphatases. The lack of inhibition by low concentrations of Li+ and high concentrations of Mg2+ and the high rates of hydrolysis of glucose-1-phosphate and p-nitrophenylphosphate are the most pronounced differences between the archaeal inositol monophosphatase and those from other sources. The possible causes of these kinetic differences are discussed, based on the active site sequence alignment between M. jannaschii and human inositol monophosphatase and the crystal structure of the mammalian enzyme.The sole pathway for myo-inositol biosynthesis is the cyclization of glucose-6-phosphate to inositol-1-phosphate (I-1-P) by I-1-P synthase (EC 5.5.1.4) and the dephosphorylation of I-1-P by inositol monophosphatase (I-1-Pase; EC 3.1.3.25) (7–9, 12, 16, 24). This de novo pathway provides the ultimate source of free inositol for the cell. It is also a key enzyme involved in second-message signal transduction processes in mammalian and plant cells (2, 24, 28, 37). In phosphoinositide signaling (2, 37), I-1-Pase recycles the water-soluble phospholipase C phospholipid degradation products, inositol phosphates, to myo-inositol and helps to maintain a moderate inositol pool. Its inhibition by millimolar concentrations of lithium (19) has made it a putative target of lithium therapy for manic depression (34).Di-myo-inositol-1,1′-phosphate (DIP), a novel inositol phosphate compound found in hyperthermophilic archaea, including Pyrococcus woesei (43), Pyrococcus furiosus (41), Methanococcus igneus (11), and Thermotoga maritima (36), is used for osmotic balance at high growth temperatures. In order to understand what regulates its accumulation in cells, the DIP biosynthetic pathway must be well characterized in vitro. Based on 13C-labeling studies and assays of crude protein extracts from M. igneus (10), a pathway was proposed that converts glucose-6-phosphate to I-1-P (step 1), hydrolyzes some of the I-1-P to myo-inositol (step 2), and activates I-1-P to CDP-inositol (CDP-I) (step 3) for a final reaction (step 4) whereby CDP-I is coupled to myo-inositol, generating DIP and CMP (Fig. (Fig.1).1). Activities for I-1-P synthase, I-1-Pase, and DIP synthase in the DIP biosynthetic pathway have been detected in crude protein extracts of M. igneus (10). Phosphatase activities are ubiquitous in cells, and the observed activity in M. igneus could be due to a specific I-1-Pase activity or a nonspecific phosphatase. For mammalian and plant cells, I-1-Pases are all lithium sensitive and are inhibited at millimolar concentrations of Li+ (14, 15, 19, 30, 42). The partially purified phosphatase in M. igneus exhibited substrate specificity for dl-I-1-P over other sugar phosphates (10). It had an absolute requirement for Mg2+, a characteristic of all specific I-1-Pases studied thus far, and was also partially inhibited by Li+, though at a much higher concentration (160 mM for 50% activity inhibition) than reported for I-1-Pases from other cells. While this was suggestive of a specific I-1-Pase, the same protein fractions demonstrated considerable activity toward p-nitrophenylphosphate (pNPP), a very poor substrate for mammalian enzymes (1, 14). These preliminary characterizations of phosphatase activity suggested that archaeal I-1-Pases might be different from mammalian and plant enzymes. Open in a separate windowFIG. 1Proposed DIP biosynthetic pathway. Glucose-6-phosphate is converted to I-1-P (step 1), some of which is hydrolyzed to myo-inositol (step 2), and I-1-P is activated to CDP-I (step 3) for a final reaction in which CDP-I is coupled to myo-inositol (step 4), generating DIP and CMP.Methanococcus jannaschii was the first archaeon whose complete genomic sequence was determined (6). Of all the archaea with sequenced genomes, it is the closest to M. igneus. MJ109 encodes a 252-amino-acid protein that is highly homologous to both I-1-Pase and extragenic suppressor (the suhB gene product) (6). The latter gene product cloned in E. coli also has I-1-Pase activity (29). The putative identification of the MJ109 gene product as an I-1-Pase prompted us to express the gene product in E. coli and to examine its specific activity toward a variety of phosphate esters. The protein produced in this fashion clearly has I-1-Pase activity and shows several striking differences from plant and mammalian I-1-Pase activities. 相似文献
995.
Rotavirus 2/6 Viruslike Particles Administered Intranasally with Cholera Toxin, Escherichia coli Heat-Labile Toxin (LT), and LT-R192G Induce Protection from Rotavirus Challenge 总被引:1,自引:0,他引:1
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Christine M. ONeal John D. Clements Mary K. Estes Margaret E. Conner 《Journal of virology》1998,72(4):3390-3393
We have shown that rotavirus 2/6 viruslike particles composed of proteins VP2 and VP6 (2/6-VLPs) administered to mice intranasally with cholera toxin (CT) induced protection from rotavirus challenge, as measured by virus shedding. Since it is unclear if CT will be approved for human use, we evaluated the adjuvanticity of Escherichia coli heat-labile toxin (LT) and LT-R192G. Mice were inoculated intranasally with 10 μg of 2/6-VLPs combined with CT, LT, or LT-R192G. All three adjuvants induced equivalent geometric mean titers of rotavirus-specific serum antibody and intestinal immunoglobulin G (IgG). Mice inoculated with 2/6-VLPs with LT produced significantly higher titers of intestinal IgA than mice given CT as the adjuvant. All mice inoculated with 2/6-VLPs mixed with LT and LT-R192G were totally protected (100%) from rotavirus challenge, while mice inoculated with 2/6-VLPs mixed with CT showed a mean 91% protection from challenge. The availability of a safe, effective mucosal adjuvant such as LT-R192G will increase the practicality of administering recombinant vaccines mucosally. 相似文献
996.
Abstract: A common feature of Alzheimer's disease pathology is an abundance of activated glia, indicative of an inflammatory reaction in the brain. The relationship between glial activation and neurodegeneration is not known, although several cytokines and inflammatory mediators produced by activated glia have the potential to initiate or exacerbate the progression of neuropathology. As β-amyloid (Aβ) is one of several stimuli that can activate glia, it is important to determine how Aβ-induced glial activation is influenced by other proteins present in the plaque, such as apolipoprotein E (apoE). We examined the effect of native preparations of apoE on activation of rat cortical astrocyte cultures by Aβ1–42. The apoE source was conditioned medium from human embryonic kidney 293 cells stably transfected with human apoE3 or apoE4 cDNA. By morphological criteria, apoE inhibited Aβ-induced astrocyte activation in three experimental paradigms: apoE pretreatment blocked subsequent Aβ-induced activation, Aβ aged in the presence of apoE did not activate astrocytes, and apoE addition to activated astrocytes transiently reversed the activated phenotype. No apoE isoform selectivity was observed. The effect of apoE appears to be specific to Aβ, as apoE did not attenuate cyclic AMP-induced astrocyte activation. These data suggest that apoE may modulate the ability of Aβ to induce inflammatory responses in the brain. 相似文献
997.
Richard S. Jope Ling Song Carol A. Grimes Mary A. Pacheco Ginny E. Dilley Xiaohua Li Herbert Y. Meltzer †James C. Overholser Craig A. Stockmeier 《Journal of neurochemistry》1998,70(2):763-771
Abstract: Comparisons of the activity of the G protein-mediated phosphoinositide signal transduction system and of G protein levels were made in two regions of frontal cortex from eight schizophrenic, alcohol-dependent, and control subjects. G protein-mediated phosphoinositide hydrolysis was measured by stimulating cortical membranes incubated with [3H]phosphatidylinositol with 0.3–10 µM guanosine 5′-O-(3-thio)triphosphate (GTPγS). In frontal cortex areas 8/9, GTPγS-induced phosphoinositide hydrolysis was 50% greater in schizophrenic than control or alcohol-dependent subjects, whereas there were no differences among these groups of subjects in the response to GTPγS in frontal cortex area 10. Agonists for dopaminergic, cholinergic, purinergic, serotonergic, histaminergic, and glutamatergic receptors coupled to the phosphoinositide signaling system increased [3H]phosphatidylinositol hydrolysis in a GTPγS-dependent manner. Responses to most agonists were similar in all three subject groups in both cortical regions, with the largest difference being a 40% greater response to dopaminergic receptor stimulation in frontal cortex 8/9 from schizophrenic subjects. Measurements of the levels of phospholipase C-β, and of α-subunits of Gq, Go, Gi1, Gi2, and Gs, made by immunoblot analyses revealed no differences among the groups of subjects except for increased Gαo in schizophrenic subjects and increased Gαo and Gαi1 in alcohol-dependent subjects. These results demonstrate that schizophrenia is associated with increased activity of the phosphoinositide signal transduction system and increased levels of Gαo, whereas the phosphoinositide system was unaltered in alcohol dependence, but Gαo and Gαi1 were increased. 相似文献
998.
Glucocorticoid-Dependent Action of Neural Crest Factor AP-2: Stimulation of Phenylethanolamine N-Methyltransferase Gene Expression 总被引:3,自引:3,他引:0
999.
1000.
Timothy W. McNellis Mary Beth Mudgett Karen Li Takashi Aoyama Diana Horvath NamHai Chua Brian J. Staskawicz 《The Plant journal : for cell and molecular biology》1998,14(2):247-257
Pathogenic strains of Pseudomonas syringae pv. tomato carrying the avrRpt2 avirulence gene specifically induce a hypersensitive cell death response in Arabidopsis plants that contain the complementary RPS2 disease resistance gene. Transient expression of avrRpt2 in Arabidopsis plants having the RPS2 gene has been shown to induce hypersensitive cell death. In order to analyze the effects of conditional expression of avrRpt2 in Arabidopsis plants, transgenic lines were constructed that contained the avrRpt2 gene under the control of a tightly regulated, glucocorticoid-inducible promoter. Dexamethasone-induced expression of avrRpt2 in transgenic lines having the RPS2 gene resulted in a specific hypersensitive cell death response that resembled a Pseudomonas syringae-induced hypersensitive response and also induced the expression of a pathogenesis-related gene (PR1). Interestingly, high level expression of avrRpt2 in a mutant rps2–101C background resulted in plant stress and ultimately cell death, suggesting a possible role for avrRpt2 in Pseudomonas syringae virulence. Transgenic RPS2 and rps2 plants that contain the glucocorticoid-inducible avrRpt2 gene will provide a powerful new tool for the genetic, physiological, biochemical, and molecular dissection of an avirulence gene-specified cell death response in both resistant and susceptible plants. 相似文献