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141.
Shimada M Chen X Cvrk T Hilfiker H Parfenova M Segre GV 《The Journal of biological chemistry》2002,277(35):31774-31780
The human parathyroid hormone (PTH) receptor (hPTH1R), containing a 9-amino acid sequence of rhodopsin at its C terminus, was transiently expressed in COS-7 cells and solubilized with 0.25% n-dodecyl maltoside. Approximately 18 microg of hPTH1R were purified to homogeneity per mg of crude membranes by single-step affinity chromatography using 1D4, a monoclonal antibody to a rhodopsin epitope. The N terminus of the hPTH1R is Tyr(23), consistent with removal of the 22-amino acid signal peptide. Comparisons of hPTH1R by quantitative immunoblotting and Scatchard analysis revealed that 75% of the receptors in membrane preparations were functional; there was little, if any, loss of functional receptors during purification. The binding affinity of the purified hPTH1R was slightly lower than membrane-embedded hPTH1R (K(d) = 16.5 +/- 1.3 versus 11.9 +/- 1.9 nm), and the purified receptors bound rat [Nle(8,21),Tyr(34)]PTH-(1-34)-NH(2) (PTH-(1-34)), and rat [Ile(5),Trp(23),Tyr(36)]PTHrP-(5-36)-NH(2) with indistinguishable affinity. Maximal displacement of (125)I-PTH-(1-34) binding by rat [alpha-aminoisobutyric acid (Aib)(1,3),Nle(8),Gln(10),Har(11),Ala(12),Trp(14),Arg(19),Tyr(21)]PTH-(1-21)-NH(2) and rat [Aib(1,3),Gln(10),Har(11),Ala(12),Trp(14)]PTH-(1-14)-NH(2) of 80 and 10%, respectively, indicates that both N-terminal and juxtamembrane ligand binding determinants are functional in the purified hPTH1R. Finally, PTH stimulated [(35)S]GTP gamma S incorporation into G alpha(s) in a time- and dose-dependent manner, when recombinant hPTH1R, G alpha(s)-, and beta gamma-subunits were reconstituted in phospholipid vesicles. The methods described will enable structural studies of the hPTH1R, and they provide an efficient and general technique to purify proteins, particularly those of the class II G protein-coupled receptor family. 相似文献
142.
Zhang L Hosoi M Fukuzawa M Sun H Rawlings RR Weight FF 《The Journal of biological chemistry》2002,277(48):46256-46264
Ethanol can potentiate serotonin type 3 (5-HT(3)) receptor-mediated responses in various neurons and in cells expressing 5-HT(3A) receptors. However, the molecular basis for alcohol modulation of 5-HT(3) receptor function has not been determined. Here we report that point mutations of the arginine at amino acid 222 in the N-terminal domain of the 5-HT(3A) receptor can alter the EC(50) value of the 5-HT concentration-response curve. Some point mutations at amino acid 222 resulted in spontaneous opening of the 5-HT(3A) receptor channel and an inward current activated by ethanol in the absence of agonist. Among these mutant receptors, the amplitude of the current activated by ethanol in the absence of agonist was correlated with the amplitude of the current resulting from spontaneous channel openings, suggesting that the sensitivity of the receptor to ethanol in the absence of agonist is, at least in part, dependent on the preexisting conformational equilibrium of the receptor protein. On the other hand, point mutations that conferred greater sensitivity to ethanol potentiation of agonist-activated responses were less sensitive or insensitive to ethanol in the absence of agonist. For these receptors, the magnitude of the potentiation of agonist-activated responses by ethanol was inversely correlated with the EC(50) values of the 5-HT concentration-response curves, suggesting that these mutations may modulate ethanol sensitivity of the receptor by altering the EC(50) value of the receptor. Thus, distinct molecular processes may determine the sensitivity of 5-HT(3A) receptors to ethanol in the absence and presence of agonist. 相似文献
143.
Lange UE Backfisch G Delzer J Geneste H Graef C Hornberger W Kling A Lauterbach A Subkowski T Zechel C 《Bioorganic & medicinal chemistry letters》2002,12(10):1379-1382
Solid-phase synthesis and SAR of integrin alpha(V)beta3-receptor antagonists containing a urea moiety as non-basic guanidine mimetic are described. The most potent compounds exhibited IC(50) values towards alpha(V)beta3 in the nanomolar range and high selectivity versus related integrins like alpha(IIb)beta3. For selected examples efficacy in functional cellular assays is demonstrated. 相似文献
144.
Critical residues for structure and catalysis in short-chain dehydrogenases/reductases 总被引:15,自引:0,他引:15
Filling C Berndt KD Benach J Knapp S Prozorovski T Nordling E Ladenstein R Jörnvall H Oppermann U 《The Journal of biological chemistry》2002,277(28):25677-25684
Short-chain dehydrogenases/reductases form a large, evolutionarily old family of NAD(P)(H)-dependent enzymes with over 60 genes found in the human genome. Despite low levels of sequence identity (often 10-30%), the three-dimensional structures display a highly similar alpha/beta folding pattern. We have analyzed the role of several conserved residues regarding folding, stability, steady-state kinetics, and coenzyme binding using bacterial 3beta/17beta-hydroxysteroid dehydrogenase and selected mutants. Structure determination of the wild-type enzyme at 1.2-A resolution by x-ray crystallography and docking analysis was used to interpret the biochemical data. Enzyme kinetic data from mutagenetic replacements emphasize the critical role of residues Thr-12, Asp-60, Asn-86, Asn-87, and Ala-88 in coenzyme binding and catalysis. The data also demonstrate essential interactions of Asn-111 with active site residues. A general role of its side chain interactions for maintenance of the active site configuration to build up a proton relay system is proposed. This extends the previously recognized catalytic triad of Ser-Tyr-Lys residues to form a tetrad of Asn-Ser-Tyr-Lys in the majority of characterized short-chain dehydrogenases/reductase enzymes. 相似文献
145.
Kristen U Bischoff N Lisboa S Schirmer E Witt S Quader H 《Alternatives to laboratory animals : ATLA》2002,30(3):323-329
Tobacco pollen tubes were used as a standard in vitro system to investigate cell growth aberrations caused by some of the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) programme chemicals and other toxic compounds. Changes in cytoskeletal pattern were observed in the tube cells by using tubulin immunofluorescence and rhodamin-phalloidin fluorescence for the localisation of microtubules and actin filaments, respectively. Four different types of cell malformation were found: screw-like growth, isodiametric tip swelling, hook formation, and pollen grain enlargement. We suggest that these malformations resulted from an interference by the chemicals with the cytosolic calcium gradient which controls tip growth and the orientation of the pollen tube. The results may contribute to a general understanding of toxicity-based cell malformations. 相似文献
146.
Makie T Harada M Kinukawa N Toyoshiba H Yamanaka T Nakamura T Sakamoto M Nose Y 《International journal of biometeorology》2002,46(1):38-41
We carried out a statistical study of the influence of meteorological and day-of-the-week factors on the intrinsic emergency patients transported to hospitals by ambulance. Multiple piecewise linear regression analysis was performed on data from 6,081 emergency admissions for 1 year between April 1997 and March 1998 in Fukuoka, Japan. The response variable was the daily number of emergency patients admitted with three types of disease: cerebrovascular, respiratory and digestive diseases. The results showed that the number of emergency patients admitted daily with cerebrovascular disease was significantly associated with temperature on the day of admission and whether the day was Sunday. As it became colder than 12 degrees C, emergency admissions of patients with cerebrovascular disease increased drastically, reaching a plateau at 4 degrees C. On the 3rd and 7th days after the temperature fell below 10 degrees C, the daily admission of patients with respiratory disease significantly increased. We also observed a weak association between emergency admissions of patients suffering from digestive disease and rising barometric pressure on the day of admission. 相似文献
147.
Izsvák Z Khare D Behlke J Heinemann U Plasterk RH Ivics Z 《The Journal of biological chemistry》2002,277(37):34581-34588
Sleeping Beauty (SB) is the most active Tc1/mariner-like transposon in vertebrate species. Each of the terminal inverted repeats (IRs) of SB contains two transposase-binding sites (DRs). This feature, termed the IR/DR structure, is conserved in a group of Tc1-like transposons. The DNA-binding region of SB transposase, similar to the paired domain of Pax proteins, consists of two helix-turn-helix subdomains (PAI + RED = PAIRED). The N-terminal PAI subdomain was found to play a dominant role in contacting the DRs. Transposase was able to bind to mutant sites retaining the 3' part of the DRs; thus, primary DNA binding is not sufficient to determine the specificity of the transposition reaction. The PAI subdomain was also found to bind to a transpositional enhancer-like sequence within the left IR of SB, and to mediate protein-protein interactions between transposase subunits. A tetrameric form of the transposase was detected in solution, consistent with an interaction between the IR/DR structure and a transposase tetramer. We propose a model in which the transpositional enhancer and the PAI subdomain stabilize complexes formed by a transposase tetramer bound at the IR/DR. These interactions may result in enhanced stability of synaptic complexes, which might explain the efficient transposition of Sleeping Beauty in vertebrate cells. 相似文献
148.
In contrast to canonical mRNAs, translation of leaderless mRNA has been previously reported to continue in the presence of the antibiotic kasugamycin. Here, we have studied the effect of the antibiotic on determinants known to affect translation of leadered and leaderless mRNAs. Kasugamycin did not affect the Shine-Dalgarno (SD)-anti-SD (aSD) interaction or the function of translation initiation factor 3 (IF3). Thus, the preferential translation of leaderless mRNA in the presence of kasugamycin can neither be attributed to an expanding pool of 30S subunits with a "blocked" aSD nor to a lack of action of IF3, which has been shown to discriminate against translation initiation at 5'-terminal start codons. Using toeprinting, we observed that on leaderless mRNA 70S in contrast to 30S translation initiation complexes are comparatively resistant to the antibiotic. These results taken together with the known preference of 70S ribosomes for 5'-terminal AUGs lend support to the hypothesis that translation of leaderless mRNAs may as well proceed via an alternative initiation pathway accomplished by intact 70S ribosomes. 相似文献
149.
Shinjoh M Tomiyama N Miyazaki T Hoshino T 《Bioscience, biotechnology, and biochemistry》2002,66(11):2314-2322
The D-sorbitol dehydrogenase gene, sldA, and an upstream gene, sldB, encoding a hydrophobic polypeptide, SldB, of Gluconobacter suboxydans IFO 3255 were disrupted in a check of their biological functions. The bacterial cells with the sldA gene disrupted did not produce L-sorbose by oxidation of D-sorbitol in resting-cell reactions at pHs 4.5 and 7.0, indicating that the dehydrogenase was the main D-sorbitol-oxidizing enzyme in this bacterium. The cells did not produce D-fructose from D-mannitol or dihydroxyacetone from glycerol. The disruption of the sldB gene resulted in undetectable oxidation of D-sorbitol, D-mannitol, or glycerol, although the cells produced the dehydrogenase. The cells with the sldB gene disrupted produced more of what might be signal-unprocessed SldA than the wild-type cells did. SldB may be a chaperone-like component that assists signal processing and folding of the SldA polypeptide to form active D-sorbitol dehydrogenase. 相似文献
150.
Helias?A.?Udo?de?HaesEmail author Olivier?Jolliet Greg?Norris Konrad?Saur 《The International Journal of Life Cycle Assessment》2002,7(4):192-195
The conclusions about the development of the content of the LC Initiative are the following:
相似文献
– | A specific niche for the Life Cycle Initiative has developed, compared with the role of SETAC, the International Society of Industrial Ecology (ISIE) and ISO. |
– | The aims of the initiative have step by step been extended, by bringing the initiative at a world level, by including both LCI and LCIA, and by including a program on Life Cycle Management (LCM). |
– | In the LCM program due attention is to be given to other tools and approaches than quantitative LCA which are relevant for life-cycle thinking in general, and also to the other two dimensions of sustainability, i.e. the social and economic dimensions. |
– | A number of important questions regarding the scope of the initiative and the methodological set-up have been in-depth discussed, thus resulting in a clear basis for the technical content of work to come. |
– | Three definition studies will now be implemented which will define the work program for the three programs of the initiative; these studies will be finalised by the end of 2002. |