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1.
An amphibian egg recovered from the body cavity is enclosed by a coelomic egg envelope. Upon transport down the oviduct, the envelope is converted to the vitelline envelope. The coelomic and vitelline envelopes are distinct in terms of sperm penetrability, ultrastructural morphology, and radioiodination profiles. In this study, the macromolecular compositions of these two envelopes were determined. Isolated envelopes were compared by one- and two-dimensional gel electrophoresis, peptide mapping, and radiolabeling. A protein with a molecular weight of 57,000 (57K) was present in the vitelline envelope but was absent in the coelomic envelope. A glycoprotein with a molecular weight of 43K in the coelomic envelope was converted to a component with a molecular weight of 41K in the vitelline envelope. The 43K-molecular weight component of the coelomic envelopes could be radioiodinated by lactoperoxidase but no labeling of the 41K-molecular weight component occurred in the vitelline envelope. Peptide mapping using limited proteolysis established that the 43K-molecular weight component of the coelomic envelope was a precursor to the 41K-molecular weight component of the vitelline envelope. These molecular alterations may underlie the ultrastructural and physiological changes occurring in these envelopes.  相似文献   

2.
Interacting egg envelope and sperm surface components were identified for Xenopus laevis using blotting methods. Sperm were extracted with sodium dodecyl sulfate (SDS), the extracted proteins separated by gel electrophoresis and blotted, and the blots treated with 125I-labeled heat solubilized envelopes. The converse experiment was also performed where envelope components were separated by gel electrophoresis, blotted, and the blots treated with 125I-labeled sperm components. Blotted sperm components with apparent molecular weights of 14K, 19K, 25K, and 35K selectively bound the solubilized envelopes. All of the envelope binding components were found to be localized on the sperm surface by radioiodinating intact sperm using Iodo-Gen. The blotted egg envelope component with an apparent molecular weight of 37K selectively bound to solubilized sperm components, and this binding was due to the protein moiety of the glycoprotein. 125I-labeled heat solubilized envelopes from unfertilized and fertilized eggs showed the same pattern of binding to blotted sperm components. Selected sulfated carbohydrates (fucoidan, dextran sulfate, and heparin, but not chondroitin sulfate) inhibited fertilization and binding of 125I-labeled heat solubilized envelopes to blotted sperm extract. Thus, the binding of heat solubilized envelopes to electrophoretically separated and blotted sperm proteins may reflect cellular interactions.  相似文献   

3.
It has been recently shown that, in several genera of annelids, including Chaetopterus, fertilizing sperm attach to and fuse with egg microvilli which penetrate the vitelline envelope. This suggests that the annelid vitelline envelope may have no direct or obligatory role in normal fertilization. The present study was undertaken to investigate the involvement of the vitelline envelope in fertilization in Chaetopterus experimentally, by examining the fertilization of vitelline envelope-free eggs quantitatively and qualitatively. Brief exposure of the eggs to isotonic sucrose-EDTA removed the vitelline envelope as determined by both phase-contrast and electron microscopy, rendered the eggs more sensitive to polyspermy and substantially reduced the binding of supernumerary sperm to eggs but did not decrease fertilizability as determined by sperm dilution assay and did not make the eggs more sensitive to cross-fertilization. The events of fertilization were examined by electron microscopy and found to be very similar in vitelline envelope-free eggs to those in intact eggs. We conclude that the vitelline envelope in Chaetopterus has binding sites for sperm but that it has no obligatory role in fertilization and is primarily involved in the prevention of polyspermy.  相似文献   

4.
The vitelline envelope (VE) is an extremely thin, acellular, proteinaceous coat that surrounds the extracellular surface of sea urchin eggs. Despite previous studies on VE composition, structure and function, our understanding of the envelope is still incomplete at the molecular level. We have isolated VE components from intact, unfertilized Strongylocentrotus purpuratus eggs by reduction with alkaline dithiothreitol-sea water solutions and have characterized the macromolecules by SDS-PAGE. There were eight major glycoprotein bands, including two high molecular weight components at 265 and 300 kDa, and several minor components. We have revealed, by lectin blot analysis, that most components contain mannose, while a subset of glycoproteins contain fucose and N -acetylglucosamine; galactose and sialic acid were also detected. The components in the VE preparations were compared with cell surface complex preparations by immunoblot analysis, using antisera against a VE preparation, a 305 kDa electrophoretically purified VE glycoprotein and an extracellular portion of the sea urchin egg recombinant 350 kDa sperm receptor. Serum against the recombinant sperm receptor reacted with a component of ∼350 kDa on blots, but did not react with the 300 kDa component found in VE preparations. Therefore, we suggest these two glycoproteins are not the same.  相似文献   

5.
Egg envelope conversion following fertilization in Bufo japonicus   总被引:4,自引:0,他引:4  
The envelope of the Bufo japonicus egg becomes impenetrable to sperm following fertilization. Electrophoretic analysis of envelopes showed that two glycoprotein components with apparent molecular weights of 65,000 and 61,000 were hydrolyzed during fertilization to 62,000 and 58,000, respectively. These two envelope components were structurally related as shown by peptide mapping and deglycosylation studies. Hardening of the envelope following egg activation was also observed, as detected by an increase in the envelope melting temperature. The involvement of proteolytic activities in the envelope hydrolysis and hardening reactions was demonstrated using protease inhibitors, and was verified for the hydrolysis reaction by observing a loss of mass in deglycosylated envelope components obtained before and after fertilization. A low ionic strength medium (less than 50 mM) was required for both the hardening and hydrolysis reactions. Envelopes from eggs activated in a high ionic strength medium were resistant to lysin from sperm, indicating that neither hydrolysis nor hardening was necessary to block lysin activity on the envelope. Both envelope hydrolysis and hardening could be effected in the absence of sperm (i.e., when eggs were activated by electric shock) and after egg jelly had been removed, indicating that neither sperm nor jelly factors were required for the envelope modifications. In addition, when eggs were activated in the presence of NH4Cl to suppress cortical granule exocytosis, envelope hardening and hydrolysis were still observed, indicating that a cortical granule-derived factor may not be involved.  相似文献   

6.
An isolated surface complex consisting of the vitelline layer, plasma membrane, and attached secretory vesicles has been examined for its ability to bind sperm and to form the fertilization envelope. Isolated surface complexes (or intact eggs) fixed in glutaraldehyde and then washed in artificial sea water are capable of binding sperm in a species-specific manner. Sperm which bind to the isolated surface complex exhibit the acrosomal process only when they are associated with the exterior surface (vitelline layer) of the complex. Upon resuspension of the unfixed surface complex in artificial sea water, a limiting envelope is formed which, based on examination of thin sections and negatively stained surface preparations, is structurally similar to the fertilization envelope formed by the fertilized egg. These results suggest that the isolated egg surface complex retains the sperm receptor, as well as integrated functions for the secretion of components involved in assembly of the fertilization envelope.  相似文献   

7.
Vitelline envelopes are composed of glycoproteins that participate in sperm-egg interactions during the initial stages of fertilization. In Xenopus laevis, the vitelline envelope is composed of at least 4 glycoproteins (ZPA, ZPB, ZPC, and ZPX). A sperm binding assay involving the covalent coupling of envelope glycoproteins to silanized glass slides was developed. In our assay, sperm bound to the egg envelopes derived from oviposited eggs but not activated eggs. The majority of the egg envelope ligand activity for sperm binding was derived from the complex N-linked oligosaccharides of ZPC. This sperm binding involved N-acetylglucosamine and fucose residues, as binding was abolished after treatment with cortical granule beta-N-acetylglucosaminidase and commercial beta-N-acetylglucosaminidases and was reduced by 44% after treatment with alpha-fucosidase. Although both the envelope glycoproteins ZPA and ZPC possessed independent ligand activity, ZPC was the major ligand for sperm binding (75%). Mixing of isolated ZPA, ZPB, and ZPC in a ratio of 1:4:4 (equal to that in the egg envelope) resulted in sperm binding that was greater than that of the sum of the separate components. The egg glycoproteins acted in synergy to increase sperm binding. Thus, ZPC possessed both independent and hetero-oligomeric-dependent ligand activities for sperm binding.  相似文献   

8.
The sea urchin egg vitelline envelope (VE) is composed of eight major glycopolypeptides that are heavily mannosylated and contain fucose and N-acetylglucosamine moieties based on lectin staining. In the present study, the macromolecular composition of the VE and the potential role of a purified VE glycoprotein in initial gamete binding was investigated. The VE components were solubilized from the surface of intact, dejellied eggs with dithiothreitol in divalent cation-free seawater, and analyzed using native, reduced electrophoresis and immunoblotting. Three major VE glycoproteins, VE-A, VE-B and VE-C, and one minor component, VE-D, were identified with antisera against whole VE preparations and against glutaraldehyde-fixed, unfertilized eggs. The electrophoretically purified glycoproteins resolved into a common subunit doublet and one unique subunit each of decreasing size on blots of sodium dodecylsulfate polyacrylamide gels. Lectin affinity chromatography was used for analysis and purification of reduced VE components; a glycoprotein eluted from Con A columns with methyl-mannoside comigrated with VE-B when analyzed by immunoblotting. Whole VE preparations and VE-B obtained from Con A columns were found to inhibit fertilization when preincubated with sperm, thus directly establishing a role for VE-B in gamete binding.  相似文献   

9.
During activation of amphibian eggs, cortical granule exocytosis causes elaborate ultrastructural changes in the vitelline envelope. These changes involve modifications in the structure of the vitelline envelope and formation of a fertilization envelope (FE) that can no longer be penetrated by sperm. In Bufo arenarum, as the egg traverses the oviduct, the vitelline envelope is altered by a trypsin-like protease secreted by the oviduct, which induces an increased susceptibility of the vitelline envelope to sperm lysins. Full-grown oocytes of B. arenarum, matured in vitro by progesterone, are polyspermic, although cortical granule exocytosis seems to occur within a normal chronological sequence. These oocytes can be fertilized with or without trypsin treatment, suggesting that the vitelline envelope is totally sperm-permeable. Vitelline envelopes without trypsin treatment cannot retain either gp90 or gp96. This suggests that these glycoproteins are involved in the block to polyspermy and that trypsin treatment of matured in vitro oocytes before insemination is necessary to enable vitelline envelopes to block polyspermy. The loss of the binding capacity in vitelline envelopes isolated from B. arenarum oocytes matured in vitro with trypsin treatment and activated by electric shock suggests that previous trypsin treatment is a necessary step for sperm block to occur. When in vitro matured oocytes were incubated with the product of cortical granules obtained from in vitro matured oocytes (vCGP), vitelline envelopes with trypsin treatment were able to block sperm entry. These oocytes exhibited the characteristic signs of activation. These results support the idea that B. arenarum oocytes can be activated by external stimuli and suggest the presence of unknown oocyte surface receptors linked to the activation machinery in response to fertilization. Electrophoretic profiles obtained by SDS-PAGE of solubilized vitelline envelopes from oocytes matured in vitro revealed the conversion of gp40 (in vitro matured oocytes, without trypsin treatment) to gp38 (ascribable to trypsin activity or cortical granule product activity, CGP) and the conversion of gp70 to gp68 (ascribable to trypsin activity plus CGP activity). Taking into account that only the vitelline envelopes of in vitro matured oocytes with trypsin treatment and activated can block sperm entry, we may suggest that the conversion of gp70 to gp68 is related to the changes associated with sperm binding.  相似文献   

10.
The extracellular matrix of Xenopus laevis eggs was analyzed at fixed intervals after prick-activation using quick-freeze, deep-etch, rotary-shadow electron microscopy. This technique revealed that the modifications of the matrix seen at fertilization do not occur simultaneously, but that instead there is an orderly progression of alterations at activation. The first modification, conversion of the vitelline envelope (VE) to the altered vitelline envelope (VE), occurs within 2 to 3 min after activation. Intermediate stages of the VE to VE transformation can be visualized traveling around the egg in a wave-like fashion. Upon completion of the wave, the loosely woven outer surface of the VE, believed to be the prefertilization layer, remains unaltered. Subsequent formation of the fertilization (F) layer at this VE-jelly interface occurs between 4 and 8 min postactivation. Finally, between 10 and 15 min postactivation, the smooth (S) layer forms on the tips of the microvilli and surrounds the entire egg.  相似文献   

11.
Direct isolation of the sea urchin egg vitelline envelope with intact sperm receptors is difficult because the envelope is firmly attached to the egg plasma membrane. We now report a method for producing an inseminated egg preparation in Strongylocentrotus purpuratus (using soybean trypsin inhibitor [STI] and Ca2+, Mg2+-free seawater) that contains an elevated vitelline envelope (VE*-STI). The VE*-STI is devoid of cortical granule material, and supernumerary sperm do not detach postinsemination, suggesting that the VE*-STI contains active sperm receptors. VE*-STIs contain a 305-kD polypeptide and additional components that range from 225 to 31 kD, whereas the 305-kD polypeptide was considerably reduced in VE*s. Electrophoresis of sperm receptor hydrolase digests of VE*-STIs showed that the 305-kD polypeptide and several other envelope polypeptides are protease substrates. Univalent Fab fragments against VE*s, VE*-STIs, and 305 and 225-kD polypeptides blocked sperm binding and fertilization in an Fab concentration-dependent manner. The 305 and 225-kD polypeptides were localized in the VE*-STI using indirect immunofluorescence. Enzyme-linked immunosorbent assays showed that the 305 and 225-kD polypeptides share determinants, suggesting that the 225-kD polypeptide may be derived from the 305-kD polypeptide by the proteolysis that occurs at the cell surface during fertilization. Fab fragments against S purpuratus VE*-STI antigens neither bound to nor blocked homologous sperm binding and fertilization of Lytechinus variegatus eggs. Cross fertilizability occurred to the extent of 5% or less between L variegatus and S purpuratus, therefore, we conclude that the 305 kD-polypeptide isolated from S purpuratus is a species-specific vitelline envelope sperm receptor.  相似文献   

12.
鲍配子识别蛋白的研究   总被引:4,自引:0,他引:4  
配子相互作用的生化机制对于进一步阐明生殖过程具有重要作用,它是深入了解细胞内识别的理想体系。精卵细胞相互作用包括一系列的步骤,开始于精子与卵细胞外被的接触,终止于两性细胞的融合及精子核进入卵细胞质中,而精卵细胞的识别具有建立于各自性细胞表面成分基础上的种的特异性,鲍则是研究精卵识别的好材料。鲍精子在发生顶体反应时释放出两种蛋白质——细胞溶素(1ysin)和18ku糖蛋白(spl8),其中的细胞溶素与其卵黄膜上的受体紧密结合,并利用非酶反应在卵黄膜上穿一个小孔,整个精子则从此孔穿过卵黄膜与卵细胞融合;spl8释放后则覆盖到精子细胞膜表面,起到溶解卵细胞脂质体的作用,即spl8介导精、卵细胞膜的融合。鲍卵细胞膜上存在细胞溶素受体,它是大的不分支的糖蛋白分子,占据了卵黄膜30%的组分,可以专一性地与细胞溶素相结合。这些配子识别蛋白共同进化且速度很快,其中细胞溶素和18ku糖蛋白通过正向选择进化,而细胞溶素受体进行协同进化。  相似文献   

13.
Previous studies have shown that the Xenopus laevis egg envelope exists in three forms with differing ultrastructural, macromolecular, and sperm penetrability properties. The coelomic envelope (CE) is derived from eggs released from the ovary into the body cavity of the female, the vitelline envelope (VE) from eggs which have passed through the oviduct, and the fertilization envelope (FE) from fertilized eggs. In the present study, the physicochemical characteristics of these three envelope types were differentiated. Investigation of envelope solubility, deformability, sulfhydryl reactivity, and hydrophobic dye and ferritin binding capacity demonstrated that profound physicochemical changes occur in envelope conversions CE----VE----FE. The physical strength of the envelopes, as evidenced by deformability studies, ranked FE greater than CE greater than VE. These differences were not accountable by differences in the number of disulfide bonds, although the CE sulfhydryl groups were significantly less accessible than those in the VE or FE. All three envelope forms were hydrophilic in nature, exhibiting little ability to bind 1-anilino-8-naphthalenesulfonic acid. The CE bound greater amounts of ferritin in comparison to the VE and FE, indicating the presence of a basic domain, presumably in the 43-kDa glycoprotein, which is lost upon proteolysis to 41 kDa during the CE----VE conversion. The envelope integrity of all three forms was maintained by both noncovalent and covalent (disulfide) bonds. Measurements of the effect of pH on envelope solubilization indicated the involvement of an ionizable group with pKa of 8.0 in maintaining envelope structure.  相似文献   

14.
The eggs of Xenopus laevis are surrounded by investment layers of egg jelly that interact with the sperm immediately prior to fertilization. Components of these egg jelly layers are necessary for the fertilization of the egg by incoming sperm. Eggs which are stripped of their jelly layers are refractile to fertilization by sperm, but the addition of solubilized jelly promotes fertilization. We have shown previously that the egg jelly layers are composed of a fibrous network of glycoconjugates which loosely hold smaller diffusible components. Extracts of these diffusible components were prepared by incubation of freshly ovulated eggs in high-salt buffers for 12 h at 4 degrees C. This diffusible component extract, when incubated with sperm, promoted the sperm's ability to fertilize dejellied eggs in a dose-dependent manner. In contrast, the high-molecular-weight "structural" glycoconjugates of jelly that remain after extraction of the diffusible components did not increase fertilization efficiency of dejellied eggs nor did nonspecific proteins, carbohydrate polymers, or organic polymers. The diffusible components, analyzed by SDS-PAGE, consisted of a mixture of proteins from 4 to 180 kDa. The protein responsible for fertilization rescue appeared to be <50 kDa and appeared to self-aggregate or to bind to larger proteins. This protein component was required during sperm binding to the egg, its action required an intact egg vitelline envelope, and its action was independent of large soluble polymers such as Ficoll.  相似文献   

15.
A quantitative assay was developed to study the interaction of Xenopus laevis sperm and eggs. Using this assay it was found that sperm bound in approximately equal numbers to the surface of both hemispheres of the unfertilized egg, but not to the surface of the fertilized egg. To understand the molecular basis of sperm binding to the egg vitelline envelope (VE), a competition assay was used and it was found that solubilized total VE proteins inhibited sperm-egg binding in a concentration-dependent manner. Individual VE proteins were then isolated and tested for their ability to inhibit sperm binding. Of the seven proteins in the VE, two related glycoproteins, gp69 and gp64, inhibited sperm-egg binding. Polyclonal antibody was prepared that specifically recognized gp69 and gp64. This gp69/64 specific antibody bound to the VE surface and blocked sperm binding, as well as fertilization. Moreover, agarose beads coated with gp69/64 showed high sperm binding activity, while beads coated with other VE proteins bound few sperm. Treatment of unfertilized eggs with crude collagenase resulted in proteolytic modification of only the gp69/64 components of the VE, and this modification abolished sperm-egg binding. Small glycopeptides generated by Pronase digestion of gp69/64 also inhibited sperm-egg binding and this inhibition was abolished by treatment of the glycopeptides with periodate. Based on these observations, we conclude that the gp69/64 glycoproteins in the egg vitelline envelope mediate sperm-egg binding, an initial step in Xenopus fertilization, and that the oligosaccharide chains of these glycoproteins may play a critical role in this process.  相似文献   

16.
Prior studies from this laboratory have identified a proteoglycan-like component of high molecular weight from the surface of the egg of the sea urchin Strongylocentrotus purpuratus that serves as a receptor for sperm. In the present study, a glycoconjugate has been isolated from uncrosslinked fertilization envelopes prepared from eggs activated by treatment with ionophore. Based on its high molecular weight (greater than 5 X 10(6)) and its ability to inhibit fertilization by acrosome-reacted sperm, this glycoconjugate has the properties of the previously described sperm receptor. Components of the fertilization envelope of lower molecular weight (less than 10(6)) showed little or no ability to inhibit fertilization.  相似文献   

17.
Formation and structure of the fertilization envelope in Xenopus laevis   总被引:19,自引:0,他引:19  
This paper reports the morphological events that occur when the vitelline envelope (VE) of an unfertilized egg of Xenopus laevis is transformed into the fertilization envelope (FE) surrounding the zygote. The VE is about 1 μm thick and is composed of an interlacing network of small filaments. The FE is constructed from the VE plus an electron-dense layer (fertilization layer), about 2–6 μm thick, on the outer surface of the VE, i.e., at the interface between the VE and the innermost jelly-coat layer. The fertilization layer is a stable component of the FE and is not removed by mercaptan solutions used to dejelly eggs. The events of FE formation were observed in the light and electron microscopes after dejellied eggs were activated by pricking. The FE is established when material from the cortical granules is extruded into the perivitelline space. The cortical granule material passes through the VE as the envelope lifts away from the egg surface. Some cortical granule material deposits in the interstices of the VE, but most of it forms the fertilization layer on the outer surface of the envelope. The cortical reaction is completed about 8–9 min after addition of sperm when eggs are fertilized in vitro.  相似文献   

18.
During fertilization of the Xenopus laevis egg, the egg envelope is converted so that further sperm contact with the egg is prevented. In this study two envelope conversion reactions were investigated, envelope hardening and limited hydrolysis of two structurally related envelope glycoproteins. Both of these reactions were shown to be sensitive to protease inhibitors. In an attempt to identify egg proteases involved in envelope conversion, the medium around activated dejellied eggs was collected and analyzed. The exudate was able to convert isolated envelopes and, when the exudate was analyzed using peptide substrates, two major activities were found, one with a preference for cleavage after argininyl peptide bonds and one with a preference for phenylalaninyl peptide bonds. Analysis of exudate using SDS-polyacrylamide gel electrophoresis with gelatin cast into the gel showed two bands of proteolytic activity, one at Mr 45,000 that was identified as the trypsin-like activity and one at Mr 30,000 that was identified as the chymotrypsin-like activity. When cortical granule exocytosis was suppressed using ammonium chloride, release of the two exudate proteases was also suppressed. Studies of the envelope conversion reactions using protease inhibitors indicated that the chymotrysin-like protease was involved in envelope conversion once it had been activated by the trypsin-like protease.  相似文献   

19.
At fertilization, the glycocalyx (vitelline layer) of the sea urchin egg is transformed into an elevated fertilization envelope by the association of secreted peptides and the formation of intermolecular dityrosine bonds. Dityrosine cross-links are formed by a secreted ovoperoxidase that exists in a Ca2+-stabilized complex with proteoliaisin in the fertilization envelope. By using purified proteins, we now show that proteoliaisin is necessary and sufficient to link ovoperoxidase to the egg glycocalyx. Specifically, we have found that ovoperoxidase can associate with the vitelline layer only when complexed with proteoliaisin; proteoliaisin binds to the vitelline layer independently of its association with ovoperoxidase; proteolytic modification of the vitelline layer is not required for this interaction to occur; the binding of proteoliaisin to the vitelline layer is mediated by the synergistic action of the two major seawater divalent cations, Ca2+ and Mg2+; the number of proteoliaisin-binding sites on the vitelline layer of unfertilized eggs is equivalent to the amount of proteoliaisin secreted at fertilization; and the binding of ovoperoxidase to the vitelline layer, via proteoliaisin, permits the in vitro cross-linking of these two in vivo substrates. The association of purified ovoperoxidase and proteoliaisin with the vitelline layer of unfertilized eggs reconstitutes part of the morphogenesis of the fertilization envelope.  相似文献   

20.
To analyze sperm surface molecules involved in sperm–egg envelope binding in Xenopus laevis, heat‐solubilized vitelline envelope (VE) dot blotted onto a polyvinylidene difluoride (PVDF) sheet was incubated with a detergent extract of sperm plasma membrane (SP‐ML). The membrane components bound to the VE were detected using an antibody library against sperm plasma membrane components, and a hybridoma clone producing a monoclonal antibody (mAb) 16A2A7 was identified. This mAb was used in a Far Western blotting experiment in which VE was separated by electrophoresis, and then transferred to a PVDF strip that was incubated with SP‐ML. It was found that SP‐ML binds to the VE component gp37 (Xenopus homolog of mammalian ZP1). The antigens reactive to mAb 16A2A7 showed apparent molecular weights of 65–130 and 20–30 kDa, and were distributed relatively evenly over the entire sperm surface. Periodate oxidation revealed that both the pertinent epitope on the sperm surface and the ligands of VE gp37 were sugar moieties. VE gp37 was exposed on the VE surface, and the mAb 16A2A7 dose‐dependently inhibited sperm binding to VE. The sperm membrane molecules reactive with mAb 16A2A7 also reacted with mAb 2A3D9, which is known to recognize the glycoprotein SGP in the sperm plasma membrane and is involved in interactions with the egg plasma membrane, indicating that the sperm membrane glycoprotein has a bifunctional role in Xenopus fertilization. Mol. Reprod. Dev. 77: 728–735, 2010. © 2010 Wiley‐Liss, Inc.  相似文献   

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