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1.
It is well-established that the binding of N-formyl peptides to the N-formyl peptide receptor on neutrophils can be described by a kinetic scheme that involves two ligand-bound receptor states, both a low affinity ligand-receptor complex and a high affinity ligand-receptor complex, and that the rate constants describing ligand-receptor binding and receptor affinity state interconversion are ligand-specific. Here we examine whether differences due to these rate constants, i.e. differences in the numbers and lifetimes of particular receptor states, are correlated with neutrophil responses, namely actin polymerization and oxidant production. We find that an additional receptor state, one not discerned from kinetic binding assays, is required to account for these responses. This receptor state is interpreted as the number of low affinity bound receptors that are capable of activating G proteins; in other words, the accumulation of these active receptors correlates with the extent of both responses. Furthermore, this analysis allows for the quantification of a parameter that measures the relative strength of a ligand to bias the receptor into the active conformation. A model with this additional receptor state is sufficient to describe response data when two ligands (agonist/agonist or agonist/antagonist pairs) are added simultaneously, suggesting that cells respond to the accumulation of active receptors regardless of the identity of the ligand(s).  相似文献   

2.
Abstract

Chemoattractant receptors represent a major subset of the G-protein coupled receptor (GPCR) family. One of the best characterized, the N-formyl peptide receptor (FPR), participates in host defense responses of neutrophils. The features of the ligand which regulate its interaction with the FPR are well-known. By manipulating these features we have developed new ligands to probe structural and mechanistic aspects of the peptide-receptor interaction. Three ligand groups have been developed: 1) ligands containing a Lys residue located in positions 2 through 7 that can be conjugated to FITC (N-formyl-Met1-Lys2-Phe3-Phe4, N-formyl-Met1-Leu2-Lys3-Phe4, N-formyl-Met1-Leu2-Phe3-Lys4, N-formyl-Met1-Leu2-Phe3-Phe4-Lys5, N-formyl-nLeu1-Leu2-Phe3-nLeu4-Tyr5-Lys6 and N-formyl-Met1-Leu2-Phe3-Phe4-Gly5-Gly6-Lys7; 2) fluorescent pentapeptide ligands (N-formyl-Met-X-Phe-Phe-Lys(FITC) where X = Leu, Ala, Val or Gly); and 3) small crosslinking ligands where the photoaffinity crosslinker 4-azidosalicylic acid (ASA) was conjugated to Lys in positions 3 and 4 and p-benzoyl-phenylalanine (Bpa) was located in position 2 in N-formyl-Met1-Bpa2-Phe3-Tyr4. The peptides were characterized according to activity and affinity in human neutrophils and cell lines transfected with FPR. All of the peptides were agonists, with parallel affinity and activity. In the first group, the peptide activity decreases as Lys is placed closer to the N-formyl group and the activity is improved by 1–3 orders of magnitude by conjugation with FITC. In the second group, the dissociation rate of the peptide from the receptor increases as position 2 is replaced by aliphatic amino acids with smaller alkyl groups. In the third group, crosslinking ligands remain biologically active, display nM affinity and covalently label the FPR.  相似文献   

3.
Stimulation of human neutrophils with the chemotactic N-formyl peptide causes production of oxygen radicals and conversion of monomeric actin (G-actin) to polymeric actin (F-actin). The effects of the binary botulinum C2 toxin on the amount of F-actin and on neutrophil cell responses were studied. Two different methods for analyzing the actin response were used in formyl peptide-stimulated cells: staining of F-actin with rhodamine-phalloidin and a transient right angle light scatter. Preincubation of neutrophils with 400 ng/ml component I and 1,600 ng/ml component II of botulinum C2 toxin for 30 min almost completely inhibited the formyl peptide-stimulated polymerization of G-actin and at the same time decreased the amount of F-actin in unstimulated neutrophils by an average of approximately 30%. Botulinum C2 toxin preincubation for 60 min destroyed approximately 75% of the F-actin in unstimulated neutrophils. Right angle light scatter analysis showed that control neutrophils exhibited the transient response characteristic of actin polymerization; however, after botulinum C2 toxin treatment, degranulation was detected. Single components of the binary botulinum C2 toxin were without effect on the actin polymerization response. Fluorescence flow cytometry and fluorospectrometric binding studies showed little alteration in N-formyl peptide binding or dissociation dynamics in the toxin-treated cells. However, endocytosis of the fluorescent N-formyl peptide ligand-receptor complex was slower but still possible in degranulating neutrophils treated with botulinum C2 toxin for 60 min. The half-time of endocytosis, estimated from initial rates, was 4 and 8 min in control and botulinum C2 toxin-treated neutrophils, respectively.  相似文献   

4.
5.
6.
Ammonia is a bacterial metabolite which is commonly used to alter cytoplasmic and lysosomal pH of eukaryotic cells. Here we examine its effect on external N-formyl peptide receptors of human neutrophils. Ammonia does not affect the number of N-formyl peptide receptors on the cell surface, nor the association of the ligand-receptor complex with the cytoskeleton. However, ammonia causes a marked decrease in the affinity of the chemotactic peptide receptor for its ligand. The Kd of untreated cell for the chemotactic peptide was 0.65 +/- 0.06 nM, whereas that of ammonia treated cells was 1.02 +/- 0.10 nM (Mean +/- SEM, N = 6). These results suggest that ammonia can affect external as well as internal cellular components. Since ammonia is used to alter lysosomal and cytoplasmic pH, and is a metabolite of common bacterial pathogens, these results bear directly on its use in cell biology and on its potential as a virulence factor.  相似文献   

7.
The kinetics of receptor up-regulation was examined in isolated neutrophils and in whole blood by flow cytometry during cell activation. Stimulation of neutrophils prepared without exposure to LPS with chemoattractants induced fast up-regulation of N-formyl peptide receptors and C receptor type 3 (CR3). Biphasic N-formyl peptide binding curves were detected for saturating concentrations of N-formyl peptide at 37 degrees C. The bulk of the rapid binding during the first 30 to 60 s is attributed to already expressed binding sites whereas the slow binding over the next 3 to 4 min represents a time course of receptor up-regulation. Support for this interpretation comes from conditions under which the number of binding sites and the progress of the binding curves were affected. Cells treated with LPS, which caused expression of internal N-formyl peptide receptors, exhibited rapid, monophasic binding curves with increased total binding. In LPS-untreated, calcium-depleted cells, N-formyl peptide receptor up-regulation was inhibited and rapid, monophasic binding to a smaller total number of expressed sites was observed. Cytochalasin B enhanced the total number of available N-formyl peptide receptors in LPS-untreated but not LPS-treated cells. In both cases binding was rapid and monophasic suggesting that receptors were either fully or rapidly up-regulated. Although not studied in real-time, C receptor type 3 up-regulation was similar to N-formyl peptide receptor up-regulation in response to LPS, or stimulation by N-formyl peptide, C product C5a, leukotriene B4, and platelet-activating factor in isolated cells and in whole blood. After stimulation with formyl peptide, LPS, or C product 5a, the release of vitamin B12-binding protein paralleled up-regulation of receptors. These data indicate that untreated cells up-regulate N-formyl peptide receptors during cell response at a rate of approximately 10,000/min in a calcium-dependent manner whereas LPS-treated cells already express the bulk of their receptors. In cytochalasin B-treated, degranulating cells 30,000 to 50,000 receptors were up-regulated within a minute.  相似文献   

8.
Current cancer therapies exploit either differential metabolism or targeting to specific individual gene products that are overexpressed in aberrant cells. The work described herein proposes an alternative approach--to specifically target combinations of cell-surface receptors using heteromultivalent ligands ("receptor combination approach"). As a proof-of-concept that functionally unrelated receptors can be noncovalently cross-linked with high avidity and specificity, a series of heterobivalent ligands (htBVLs) were constructed from analogues of the melanocortin peptide ligand ([Nle(4), dPhe(7)]-α-MSH) and the cholecystokinin peptide ligand (CCK-8). Binding of these ligands to cells expressing the human Melanocortin-4 receptor and the Cholecystokinin-2 receptor was analyzed. The MSH(7) and CCK(6) were tethered with linkers of varying rigidity and length, constructed from natural and/or synthetic building blocks. Modeling data suggest that a linker length of 20-50 ? is needed to simultaneously bind these two different G-protein coupled receptors (GPCRs). These ligands exhibited up to 24-fold enhancement in binding affinity to cells that expressed both (bivalent binding), compared to cells with only one (monovalent binding) of the cognate receptors. The htBVLs had up to 50-fold higher affinity than that of a monomeric CCK ligand, i.e., Ac-CCK(6)-NH(2). Cell-surface targeting of these two cell types with labeled heteromultivalent ligand demonstrated high avidity and specificity, thereby validating the receptor combination approach. This ability to noncovalently cross-link heterologous receptors and target individual cells using a receptor combination approach opens up new possibilities for specific cell targeting in vivo for therapy or imaging.  相似文献   

9.
BACKGROUND: Fluorescently labeled ligands and flow cytometric methods allow quantification of receptor-ligand binding. Such methods require calibration of the fluorescence of bound ligands. Moreover, binding of unlabeled ligands can be calculated based on their abilities to compete with a labeled ligand. In this study, calibration parameters were determined for six fluorescently labeled N-formyl peptides that bind to receptors on neutrophils. Two of these ligands were then used to develop and validate competitive binding protocols for determining binding constants of unlabeled ligands. METHODS: Spectrofluorometric and flow cytometric methods for converting relative flow cytometric intensities to number of bound ligand/cell were extended to include peptides labeled with fluorescein, Bodipy, and tetramethylrhodamine. The validity of flow cytometric competitive binding protocols was tested using two ligands with different fluorescent properties that allowed determination of rate constants both directly and competitively for one ligand, CHO-NLFNYK-tetramethylrhodamine. RESULTS: Calibration parameters were determined for six fluorescently-labeled N-formyl peptides. Equilibrium dissociation constants for these ligands varied over two orders of magnitude and depended upon the peptide sequence and the molecular structure of the fluorescent tag. Kinetic rate constants for CHO-NLFNYK-tetramethylrhodamine determined directly or in competition with CHO-NLFNYK-fluorescein were statistically identical. CONCLUSIONS: Combination of spectrofluorometric and flow cytometric methods allows convenient calculation of calibration parameters for a series of fluorescent ligands that bind to the same receptor site. Competitive binding protocols have been independently validated.  相似文献   

10.
《The Journal of cell biology》1989,109(6):2791-2799
Most ligand-receptor interactions result in an immediate generation of various second messengers and a subsequent association of the ligand- receptor complex to the cytoskeleton. Depending on the receptor involved, this linkage to the cytoskeleton has been suggested to play a role in the termination of second messenger generation and/or the endocytic process whereby the ligand-receptor complex is internalized. We have studied how the binding of chemotactic peptide-receptor complexes to the cytoskeleton of human neutrophils is accomplished. As much as 76% of the tritiated formylmethionyl-leucyl-phenylalanine (fMet- Leu-[3H]Phe) specifically bound to intact cells, obtained by a 30-s stimulation with 20 nM fMet-Leu-[3H]Phe, still remained after Triton X- 100 extraction. Preincubating intact cells with dihydrocytochalasin B (dhCB) or washing the cytoskeletal preparation with a high concentration of potassium, reduced the binding of ligand-receptor complexes to the cytoskeleton by 46% or more. Inhibition of fMet-Leu- Phe-induced generation of second messengers by ADP-ribosylating the alpha-subunit of the receptor-coupled G-protein with pertussis toxin, did not reduce the binding of ligand-receptor complexes to the cytoskeleton. However, using guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) to prevent the dissociation of the fMet-Leu-Phe-associated G- protein within electrically permeabilized cells, led to a pronounced reduction (62%) of the binding between ligand-receptor complexes and the cytoskeleton. In summary, in human neutrophils the rapid association between chemotactic peptide-receptor complexes and the cytoskeleton is dependent on filamentous actin. This association is most likely regulated by the activation and dissociation of the fMet- Leu-Phe-associated G-protein.  相似文献   

11.
Stimulation of human neutrophils with the chemoattractant N-formyl peptide caused rapid polymerization of F-actin as detected by right angle light scatter and 7-nitrobenz-2-oxa-1,3-diazol (NBD)-phallacidin staining of F-actin. After labeling neutrophils with 32P, exposure to N-formyl peptide induced a fast decrease of phosphatidylinositol 4-bisphosphate (PIP)2, a slow increase of phosphatidic acid, and a rapid rise of phosphatidylinositol 4-trisphosphate (PIP3). Formation of PIP3 as well as actin polymerization was near maximal at 10 s after stimulation. Half-maximal response and PIP3 formation at early time points resulted from stimulation of neutrophils with 0.01 nM N-formyl peptide or occupation of about 200 receptors. Sustained elevation of PIP3, prolonged right angle light scatter response, and F-actin formation required higher concentrations of N-formyl peptide, occupation of thousands of receptors, and high binding rates. When ligand binding was interrupted with an antagonist, F-actin rapidly depolymerized, transient light scatter response recovered immediately, and elevated [32P]PIP3 levels decayed toward initial values. However, recovery of [32P]PIP2 was not influenced by the antagonist. Based on the parallel time courses and dose response of [32P] PIP3, the right angle light scatter response, and F-actin polymerization, PIP3 is more likely than PIP2 to be involved in modulation of actin polymerization and depolymerization in vivo.  相似文献   

12.
Family 1a GPCRs are thought to bind small molecule ligands in a pocket comprising sequences from non-contiguous transmembrane helices. In this study, receptor-ligand binding determinants were defined by building a series of complex chimeras where multiple sequences were exchanged between related G-protein coupled receptors. Regions of P2Y(1), P2Y(2) and BLT(1) predicted to interact with nucleotide and leukotriene ligands were identified and receptors were engineered within their transmembrane helices to transpose the ligand binding site of one receptor on to another receptor. Ligand-induced activation of chimeras was compared with wild-type receptor activation in a yeast reporter gene assay. Binding of ligand to a P2Y(2)/BLT(1) chimera confirmed that the ligand binding determinants of BLT(1) are located in the upper regions of the helices and extracellular loops of this receptor and that they had been successfully transferred to a receptor that normally binds unrelated ligands.  相似文献   

13.
We investigated the interaction of the N-formyl peptide receptor (NFPR) with G proteins in infected Sf9 insect cells expressing the recombinant NFPR. Recombinant receptor expression of up to 27 pmol/mg protein was achieved in these cells. The receptor was recognized by an antiserum raised against an NFPR carboxyl-terminal peptide, and displayed specific and saturable binding of the formyl peptide ligand fMet-Leu-[3H]Phe. Scatchard analysis of the binding data yielded a dissociation constant of approximately 62 nM, a binding affinity of 60- to 120-fold lower than that of the high affinity sites in neutrophils and in transfected mammalian cell lines expressing the NFPR. That this low binding affinity was due to a lack of receptor coupling to G protein was suggested by the failure of guanine nucleotides to regulate receptor affinity and by the lack of formyl peptide-stimulated GTPase activity in these cells. Furthermore, immunoblotting with an anti-G(i) antibody and ADP-ribosylation experiments indicated that the approximately 40-kDa G(i) alpha subunit, which couples to the NFPR in neutrophils, is not present in Sf9 cell membranes. Thus, the current study provides for the first time evidence that a major G protein is absent in the Sf9 insect cells. Potential applications of the Sf9 system for in vitro reconstitution of the NFPR-G protein interaction are discussed.  相似文献   

14.
Peptide ligands bind the CRF(1) receptor by a two-domain mechanism: the ligand's carboxyl-terminal portion binds the receptor's extracellular N-terminal domain (N-domain) and the ligand's amino-terminal portion binds the receptor's juxtamembrane domain (J-domain). Little quantitative information is available regarding this mechanism. Specifically, the microaffinity of the two interactions and their contribution to overall ligand affinity are largely undetermined. Here we measured ligand interaction with N- and J-domains expressed independently, the former (residues 1-118) fused to the activin IIB receptor's membrane-spanning alpha-helix (CRF(1)-N) and the latter comprising residues 110-415 (CRF(1)-J). We also investigated the effect of nonpeptide antagonist and G-protein on ligand affinity for N- and J-domains. Peptide agonist affinity for CRF(1)-N was only 1.1-3.5-fold lower than affinity for the whole receptor (CRF(1)-R), suggesting the N-domain predominantly contributes to peptide agonist affinity. Agonist interaction with CRF(1)-J (potency for stimulating cAMP accumulation) was 12000-1500000-fold weaker than with CRF(1)-R, indicating very weak direct agonist interaction with the J-domain. Nonpeptide antagonist affinity for CRF(1)-J and CRF(1)-R was indistinguishable, indicating the compounds bind predominantly the J-domain. Agonist activation of CRF(1)-J was fully blocked by nonpeptide antagonist, suggesting antagonism results from inhibition of agonist-J-domain interaction. G-protein coupling with CRF(1)-R (forming RG) increased peptide agonist affinity 92-1300-fold, likely resulting from enhanced agonist interaction with the J-domain rather than the N-domain. Nonpeptide antagonists, which bind the J-domain, blocked peptide agonist binding to RG, and binding of peptide antagonists, predominantly to the N-domain, was unaffected by R-G coupling. These findings extend the two-domain model quantitatively and are consistent with a simple equilibrium model of the two-domain mechanism: (1) The N-domain binds peptide agonist with moderate-to-high microaffinity, substantially increasing the local concentration of agonist and so allowing weak agonist-J-domain interaction. (2) Agonist-J-domain interaction is allosterically enhanced by receptor-G-protein interaction and inhibited by nonpeptide antagonist.  相似文献   

15.
G-protein coupled receptors (GPCRs) comprise a large family of membrane proteins and attract pharmaceutical interest as therapeutic targets. Two examples of class B GPCRs that are involved in metabolic diseases are the Parathyroid hormone receptor 1 (PTHR1) and the Glucagon-like-peptide-1 receptor (GLP-1R) which play central roles in osteoporosis and diabetes mellitus type II, respectively. Class B GPCRs are characterised by a large extracellular N-terminal domain with a typical disulfide bridge pattern. This domain is responsible for the binding of peptide hormone ligands. Here we report the recombinant expression of these ligands in natural and several modified forms for their use in functional assays, NMR analyses or affinity purification of receptor/ligand complexes for crystallisation. Applying the SUMO system, low cost expression of soluble fusion-proteins is achieved. Moreover, via the SUMO cleavage site, the authentic N-terminal sequence which is essential for ligand-receptor interactions can be obtained. Purification of the peptide by RP-HPLC results in >98% pure preparations. The strategy can also be adopted for many other purposes, especially if small peptides are needed at either large amounts or with specific features like isotope, affinity or fluorescent labels. Furthermore, for the growing demand for therapeutic peptides, this method could represent a straightforward production process.  相似文献   

16.
A new superior photoaffinity ligand for the N-formyl peptide receptor was prepared by derivatization of N-formyl-Met-Leu-Phe-Lys with a commercially available heterobifunctional crosslinking agent. The product, N-formyl-Met-Leu-Phe-N epsilon-(2-(p-azidosalicylamido)ethyl-1,3'- dithiopropionyl)-Lys was readily synthesized and radiolabelled, and had increased specificity and stability as compared to previously used photoaffinity ligands. The ligand rapidly associated with the receptor with high affinity (Kd = 0.28 nM). Once bound, it was virtually non-dissociable (in the absence of photolysis) in an experimental time-frame (t1/2 (off) = 154 min). The covalent incorporation of the photoaffinity ligand into the receptor upon irradiation was complete within 5 min, minimizing cell damage, and the efficiency of covalent incorporation was approx. 40%. The derivative had enhanced biological activity, having an ED50 for superoxide anion production of 0.23 nM, 27-fold lower than its parent peptide. This derivative of the N-formyl peptide was useful for up to 3 months after radiolabelling, showing a progressive decline in specific activity during storage in the dark at 4 degrees C. The enhanced stability, reproducibility and solubility of the photoaffinity ligand is expected to aid in the purification of the N-formyl peptide receptor and will prove a useful tool for analysing receptor-mediated processes.  相似文献   

17.
Activation of neutrophils by N-formyl chemotactic peptides   总被引:3,自引:0,他引:3  
The response of neutrophils to N-formyl peptides is mediated via a specific 50,000- to 60,000-dalton (Mr) receptor. Real-time kinetic analysis indicated that most of the cellular responses elicited by this ligand began within 5-10 s of addition to the cells at 37 C. Of three possible biochemical changes measured that could serve as transducers or second messengers, two, i.e., increases in cyclic AMP (cAMP) and intracellular free Ca2+, occurred within 5 s of stimulus addition. In contrast, internalization of the ligand by cells showed a latency time of 20-30 s, which indicates that it probably plays no role in triggering later responses. Using pulse binding techniques that allow the level of a given response to be measured as a function of the measured level of surface receptor occupancy, we found that O2- production required up to 30% receptor occupancy to elicit 50% of maximal response. In contrast, secretion, cAMP changes, Ca2+ changes, and membrane potential changes required less than 5% occupancy. Within 5 s, occupied receptors were converted at the cell surface to a slowly dissociating form. The receptors, exhibiting apparent higher affinity, were transiently associated with the cell cytoskeleton as defined by their conversion to a Triton X-100-insoluble form. Internalized receptor-ligand complexes were transported, in large part, to the Golgi apparatus. Further analyses of the mechanism of stimulation of leukocytes have been performed with monoclonal antibodies directed against the neutrophil surface. Data with these antibodies, which are not directed to the N-formyl peptide receptor, reveal that some modulated the N-formyl peptide-mediated responses and other antibodies initiated responses of the cells.  相似文献   

18.
Pertussis toxin (PT) inhibits polymorphonuclear leukocyte (PMN) function by ADP-ribosylating and inactivating guanine nucleotide binding proteins (G-proteins) that transduce activation by chemoattractants such as N-formyl peptides (FP). Studies of PMN activation during the time course of PT treatment yielded these results. 1) Responses were differentiated based on their sensitivity to PT treatment. Suboptimal PT treatment that resulted in 50% inhibition of the FP-induced actin-associated right angle light scatter response resulted in greater than 90% inhibition of oxidant production. Exhaustive PT treatment was required to completely inhibit the right angle light scatter response. This is consistent with previous observations that, relative to oxidant production, actin polymerization requires 100-fold fewer active N-formylpeptide receptors to elicit the response. This differential sensitivity to PT treatment has important implications for studies that use pertussis toxin to determine if the neutrophil G-protein is involved in the signaling of responses. If inhibition of oxidant production is used as the only indicator of the effectiveness of PT treatment, significant cytoskeletal changes may still be activated in these cells. Inhibition of actin polymerization is a much more rigorous indicator of complete G-protein inhibition by PT. 2) Analysis of FP-induced actin polymerization and cytosolic calcium elevation using flow cytometry, which measures individual cell responses, revealed that PT treatment resulted in the conversion of PMN from a responding to a non-responding population. In contrast, in control PMN, submaximal doses of FP caused submaximal stimulation of all the cells. The all-or-none effect of PT may result from heterogeneous insertion of the A-promoter of PT into the cell or it may result from a sharp threshold of coupled G-proteins required to transduce the responses.  相似文献   

19.
The N-formyl peptide receptor mediates a number of host defensive responses of human neutrophils that result in chemotaxis, secretion of hydrolytic enzymes, and superoxide generation. Inappropriate activation or defective regulation of these responses can result in pathogenic states responsible for inflammatory disease. The receptor is a 50 to 70-kD, integral plasma membrane glycoprotein with intracellular and surface localization. Its abundance in the membrane is regulated by membrane flow and recycling processes. Cytoskeletal interactions are believed to control its organization in the plane of the membrane and interaction with other proteins. The receptor's most important interaction is with guanyl nucleotide binding proteins that serve as signal transduction partners ultimately leading to activation of effector responses. Because the interaction of the receptor with G proteins is necessary for transduction, control of this interaction may be at the root of understanding the molecular control of responses in these cells. This review briefly summarizes some of the molecular properties, dynamics, and interactions of this receptor system in human neutrophils and discusses how these characteristics may pertain to the activation and control of superoxide generation.  相似文献   

20.
A number of new synthetic nociceptin ligands were studied in receptor binding and functional tests in rat brain membranes and in cloned systems. Ligand binding experiments were performed with three different radioprobes developed in our lab. The nociceptin derivatives exhibited high affinity in competition experiments. Receptor-mediated G-protein activation was determined in [35S]GTPgS binding assays. Among the new structures examined, Ac-RYYRIK-ol was found to be only a weak stimulator by itself, whereas this compound inhibited receptor-mediated G-protein activation. These data suggest that Ac-RYYRIK-ol is a high affinity peptide antagonist for the nociceptin receptor.
Acknowledgements:  Supported by the Hungarian Scientific Research Fund OTKA T-035211, T-033078, T-030841, and the Ministry of Education, NKFP 1/027 Hungary.  相似文献   

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