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1.
Evidence that diphtheria toxin and modeccin enter the cytosol from different vesicular compartments 总被引:11,自引:6,他引:5
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Inhibition of protein synthesis in Vero cells was measured at different periods of time after treatment with diphtheria toxin and the related plant toxin modeccin. Diphtheria toxin acted much more rapidly than modeccin. Cells were protected against both toxins with antiserum as well as with agents like NH4Cl, procaine, and the ionophores monensin, FCCP, and CCCP, which increase the pH of intracellular vesicles. Antiserum, which is supposed to inactivate toxin only at the cell surface, protected only when it was added within a short period of time after modeccin. Compounds that increase the pH of intracellular vesicles, protected even when added after 2 h, indicating that modeccin remains inside vesicles for a considerable period of time before it enters the cytosol. After addition of diphtheria toxin to the cells, compounds that increase the pH of intracellular vesicles protected only approximately to the same extent as antitoxin. This indicates that after endocytosis diphtheria toxin rapidly enters the cytosol. At 20 degrees C, the cells were more strongly protected against modeccin than against diphtheria toxin. The residual toxic effect of diphtheria toxin at 20 degrees C could be blocked with NH4Cl whereas this was not the case with modeccin. This indicates that at 20 degrees C the uptake of diphtheria toxin occurs by the normal route, whereas the uptake of modeccin occurs by a less efficient route than that dominating at 37 degrees C. The results indicate that after endocytosis diphtheria toxin rapidly enters the cytosol from early endosomes with low pH (receptosomes). Modeccin enters the cytosol much more slowly, possibly after fusion of the endocytic vesicles with another compartment. 相似文献
2.
When Vero cells were incubated with TPA (12-O-tetradecanoylphorbol 13-acetate) and related tumor promoters, their ability to bind diphtheria toxin in a functional way was rapidly reduced to less than 1% of the normal value. Upon further incubation with TPA, the cells recovered their ability to bind the toxin, apparently because they became resistant to TPA. Treatment with Na3VO4 reduced the ability of the cells to bind diphtheria toxin to approximately the same extent as treatment with TPA, but the reduction required longer time to develop and it persisted upon prolonged incubation with Na3VO4. ATP depletion of the cells prevented the reduction in binding capability. Such treatment also prevented the reduction in toxin binding induced by treatment with salicylate or fluoride. Treatment with TPA, fluoride, vanadate, and salicylate altered the ability of the cells to carry out anion transport and interfered with their ability to regulate the transport. The results indicate that the binding sites for diphtheria toxin on Vero cells are modulated by TPA, Na3VO4, salicylate, and fluoride by a process which requires ATP. The possibility is discussed that the modulation consists in phosphorylation of the toxin binding sites, which may be identical with, or closely linked to, the anion antiporter in the cells. 相似文献
3.
Entry of diphtheria toxin-protein A chimeras into cells 总被引:6,自引:0,他引:6
I H Madshus H Stenmark K Sandvig S Olsnes 《The Journal of biological chemistry》1991,266(26):17446-17453
Fusion proteins consisting of diphtheria toxin and a duplicated Fc-binding domain of protein A were made in vitro after amplification of the DNA template by the polymerase chain reaction. The fusion proteins bound avidly to Vero cells coated with antibodies. A fusion protein containing full-length diphtheria toxin was toxic at lower concentrations than diphtheria toxin alone, apparently due to more efficient binding. The enzymatic part of the fusion protein was translocated across the surface membrane upon exposure to low pH. Like authentic diphtheria toxin, the fusion protein formed cation selective channels at low pH. Excess amounts of unlabeled diphtheria toxin inhibited formation of pronase-protected fragments derived from radiolabeled fusion protein. Furthermore, conditions that down-regulate the diphtheria toxin receptors reduced the sensitivity of the cells to the fusion protein, supporting the notion that authentic diphtheria toxin receptors are required. At temperatures below 18 degrees C the toxicity of the fusion protein was strongly reduced, whereas there was no temperature block for authentic diphtheria toxin. Brefeldin A protected Vero cells against the fusion protein but not against diphtheria toxin. The results indicate that the diphtheria toxin receptor is required for efficient toxin translocation even under conditions where the toxin is bound by an alternate binding moiety, and they suggest that the intracellular routing of the fusion protein is different from that of diphtheria toxin. 相似文献
4.
Ion fluxes associated with translocation of diphtheria toxin across the surface membrane of Vero cells were studied. When cells with surface-bound toxin were exposed to low pH to induce toxin entry, the cells became permeable to Na+, K+, H+, choline+, and glucosamine+. There was no increased permeability to Cl-, SO4(-2), glucose, or sucrose, whereas the uptake of 45Ca2+ was slightly increased. The influx of Ca2+, which appears to be different from that of monovalent cations, was reduced by several inhibitors of anion transport and by verapamil, Mn2+, Co2+, and Ca2+, but not by Mg2+. The toxin-induced fluxes of N+, K+, and protons were inhibited by Cd2+. Cd2+ also protected the cells against intoxication by diphtheria toxin, suggesting that the open cation-selective channel is required for toxin translocation. The involvement of the toxin receptor is discussed. 相似文献
5.
Effect of potassium depletion of cells on their sensitivity to diphtheria toxin and pseudomonas toxin 总被引:7,自引:0,他引:7
When Vero cells were depleted of potassium, the cells were protected against diphtheria toxin. Potassium depletion of Vero cells strongly reduced the binding of the toxin to cell surface receptors. Likewise, potassium depleted L-cells were protected against pseudomonas toxin. Diphtheria toxin binding was completely restored upon addition of potassium to the cells. This restoration was not prevented by inhibition of protein synthesis by cycloheximide. When cells were depleted of potassium in the presence of metabolic inhibitors, and then treated with diphtheria toxin, protein synthesis was reduced to the same extent as in cells with normal intracellular level of potassium. The results indicate that potassium depletion of Vero cells reduces the ability of the cells to bind diphtheria toxin by an ATP requiring process, and that binding, endocytosis and transfer of diphtheria fragment A across the membrane may occur at low intracellular levels of potassium. 相似文献
6.
Requirement of specific receptors for efficient translocation of diphtheria toxin A fragment across the plasma membrane 总被引:8,自引:0,他引:8
The role of specific receptors in the translocation of diphtheria toxin A fragment to the cytosol and for the insertion of the B fragment into the cell membrane was studied. To induce nonspecific binding to cells, toxin was either added at low pH, or biotinylated toxin was added at neutral pH to cells that had been treated with avidin. In both cases large amounts of diphtheria toxin became associated with the cells, but there was no increase in the toxic effect. There was also no increase in the amount of A fragment that was translocated to the cytosol, as estimated from protection against externally added Pronase E. In cells where specific binding was abolished by treatment with 12-O-tetradecanoyl-phorbol 13-acetate, trypsin, or 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid, unspecific binding did not induce intoxication or protection against protease. This was also the case in untreated L cells, which showed no specific binding of the toxin. When Vero cells with diphtheria toxin bound to specific receptors were exposed to low pH, the cells were permeabilized to K+, whereas this was not the case when the toxin was bound nonspecifically at low pH or via avidin-biotin. The data indicate that the cell-surface receptor for diphtheria toxin facilitates both insertion of the B fragment into the cell membrane and translocation of the A fragment to the cytosol. 相似文献
7.
《The Journal of cell biology》1980,87(3):828-832
At neutral pH, NH4Cl and chloroquine protected cells against diphtheria toxin. A brief exposure of the cells to low pH (4.5-5.5) at 37 degrees completely abolished this protection. When, to cells preincubated with diphtheria toxin and NH4Cl, neutralizing amounts of anti-diphtheria toxin were added before the pH was lowered, the toxic effect was considerably reduced, but it was not completely abolished. A much stronger toxic effect was seen when antibodies were added immediately after incubation at low pH. Upon a short incubation with diphtheria toxin at low pH, the rate of protein synthesis in the cells decreased much faster than when the normal pH was maintained. The data suggest that, at low pH, diphtheria toxin (or its A fragment) penetrates directly through the surface membrane of the cell. The possibility is discussed that, when the medium has a neutral pH, the entry of diphtheria toxin involves adsorptive endocytosis and reduction of the pH in the vesicles possibly by fusion with lysosomes. Low pH did not facilitate the entry of the closely related toxins abrin, ricin, and modeccin. 相似文献
8.
A chimeric toxin to study the role of the 21 kDa GTP binding protein rho in the control of actin microfilament assembly. 总被引:11,自引:1,他引:10
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We have developed a new tool for studying the role of rho in actin stress fibre formation. Clostridium botulinum exoenzyme C3 which affects actin microfilament assembly by ADP-ribosylation of p21 rho was genetically fused in various ways to diphtheria toxin (DT). The resulting chimeric toxins were tested on Vero cells. Chimeras of C3 and both the A and B fragments of diphtheria toxin had reduced cell binding activities but were apparently able to penetrate into Vero cells by the same mechanism as DT. Upon exposure to low pH, DC3B, a fusion protein of C3 and DT B fragment, had a high affinity for the DT receptor, but was apparently not able to translocate to the cytosol upon acidification. In spite of this, addition of picomolar concentrations of DC3B to the growth medium caused disruption of the cell microfilament system associated with vinculin and blocked cell growth efficiently, indicating that the C3 part of DC3B reached the cytosol, albeit by a different mechanism than that of whole diphtheria toxin. The chimeric DC3B toxin was also applied to Vero cells infected by Listeria monocytogenes, a pathogenic bacterium that uses an unknown mechanism of actin polymerization to move rapidly in the cytosol. DC3B inhibited the bacterially induced microfilament assembly indicating that L. monocytogenes utilizes a cellular rho dependent mechanism in this process. 相似文献
9.
Interactions between diphtheria toxin entry and anion transport in Vero cells. II. Inhibition of anion antiport by diphtheria toxin 总被引:1,自引:0,他引:1
When cells with surface-bound diphtheria toxin were exposed to pH 4.5, the toxin became shielded against lactoperoxidase-catalyzed radioiodination, indicating that the toxin was inserted into the membrane. Cells thus treated had strongly reduced ability to take up 36Cl-, 35SO4(2-), and [14C]SCN-. The reduction of chloride uptake was strongest at neutral pH, whereas that of sulfate was strongest at acidic pH. Lineweaver-Burk plots indicated that the toxin treatment reduced the Jmax but not the Km for the anions. The toxin also inhibited the NaCl-stimulated efflux of 35SO4(2-), indicating that the toxin inhibits the antiporter. No inhibition was found when toxin-treated cells were not exposed to low pH, whereas exposure to pH 4.5 for 20 s induced close to maximal inhibition. Half-maximal inhibition was obtained after exposure to pH 5.4. The concentration of diphtheria toxin required to obtain maximal inhibition (0.3 micrograms/ml) was sufficient to ensure close to maximal toxin binding to the cells. Even in ATP-depleted cells and in the absence of permeant anions, low pH induced inhibition of anion antiport in toxin-treated Vero cells. There was no measurable inhibition of anion antiport in cells with little or no ability to bind the toxin. 相似文献
10.
Low pH-induced release of diphtheria toxin A-fragment in Vero cells. Biochemical evidence for transfer to the cytosol 总被引:13,自引:0,他引:13
When Vero cells with surface-bound 125I-labeled, nicked diphtheria toxin were exposed to pH 4.5, two polypeptides of Mr 20,000 and 25,000 became protected against externally applied Pronase E. The 20-kDa polypeptide appears to be the toxin A-fragment, whereas the 25-kDa polypeptide must be derived from the B-fragment. Permeabilization of the cells with saponin allowed efflux of the 20-kDa fragment to occur, whereas most of the 25-kDa polypeptide remained associated with the cells. A number of compounds and conditions which protect cells against diphtheria toxin prevented the protection against Pronase E. Protection of the 25-kDa polypeptide occurred even when the transmembrane proton gradient (delta pH) was dissipated by acidification of the cytosol, whereas protection and release of the A-fragment were prevented under these conditions. Electrical depolarization and ATP depletion of the cells did not inhibit protection and release of the A-fragment. The data indicate that delta pH is required for the transfer of the A-fragment to the cytosol, whereas the insertion of part of the B-fragment into the membrane occurs at low pH, even in the absence of a delta pH. 相似文献
11.
Cell-mediated reduction of the interfragment disulfide in nicked diphtheria toxin. A new system to study toxin entry at low pH 总被引:6,自引:0,他引:6
When 125I-labeled nicked diphtheria toxin bound to Vero cells was exposed to pH less than 5.0, a small fraction was reduced to yield A- and B-fragments. The pH required for reduction correlates well with that required to induce intoxication, and the amount of A-fragment released was of the same order as that required to intoxicate the cells. Conditions that protect cells against intoxication, such as acidification of the cytosol, treatment with anion transport inhibitors, or treatment with anti-diphtheria toxin antibodies, prevented the reduction of the interfragment disulfide in cell-bound toxin. In vitro, thioredoxin reduced nicked diphtheria toxin only at pH 5.0 and lower, and the reduction was inhibited by anti-toxin antibodies. This indicates that a conformational change in the toxin, necessary for reduction by the thioredoxin system, is prevented by the antibodies. Reduction by glutathione and cysteine was most efficient at neutral pH and was not inhibited by anti-toxin. The results are consistent with the possibility that cell-mediated reduction of the interfragment disulfide is a measure of the entry of fragment A into the cytosol. 相似文献
12.
Evidence that membrane phospholipids and protein are required for binding of diphtheria toxin in Vero cells 总被引:1,自引:0,他引:1
Treatment with phospholipase C strongly protected monkey kidney (Vero) cells against diphtheria toxin and reduced the ability of the cells to bind 125I-labelled toxin. Treatment with phospholipase D and with trypsin also protected the cells, although to a lesser extent. Phospholipase A2 had no protective effect. Phospholipase C also protected fetal hamster kidney cells against the toxin. After removal of the enzymes, as well as after treatment of the cells with 4-acetamide 4'-isothiocyanostilbene 2,2'-disulfonic acid, diphtheria toxin binding capability was restored slowly, apparently by a process requiring protein synthesis, since cycloheximide blocked the restoration. The data indicate that both phospholipids and protein are involved in the binding sites for diphtheria toxin. 相似文献
13.
R Iwamoto H Senoh Y Okada T Uchida E Mekada 《The Journal of biological chemistry》1991,266(30):20463-20469
A monoclonal antibody that blocks the binding of diphtheria toxin to Vero cells was isolated by immunizing mice with Vero cell membrane. The antibody inhibits the binding of diphtheria toxin and also CRM197, a mutant form of diphtheria toxin, to Vero cells, and consequently inhibits the cytotoxicity of diphtheria toxin. This antibody does not directly react with the receptor molecule of diphtheria toxin (DTR14.5). Immunoprecipitation and immunoblotting studies revealed that this antibody binds to a novel membrane protein of 27 kDa (DRAP27). When diphtheria toxin receptor was passed through an affinity column made with this antibody, the receptor was trapped only in the presence of DRAP27. These results indicate that DRAP27 and DTR14.5 closely associate in Vero cell membrane and that the inhibition of the binding of diphtheria toxin to the receptor is due to the binding of the antibody to the DRAP27 molecule. Binding studies using 125I-labeled antibody showed that there are many more molecules of DRAP27 on the cell surface than diphtheria toxin-binding sites. However, there is a correlation between the sensitivity of a cell line to diphtheria toxin and the number of DRAP27 molecules on the cell surface, suggesting that DRAP27 is involved in the entry of diphtheria toxin into the target cell. 相似文献
14.
Entry of prebound diphtheria toxin at low pH occurred rapidly in the presence of isotonic NaCl, NaBr, NaSCN, NaI, and NaNO3, but not in the presence of Na2SO4, 2-(N-morpholino)ethanesulfonic acid neutralized with Tris, or in buffer osmotically balanced with mannitol. SCN- was the most efficient anion to facilitate entry. Uptake studies with radioactively labeled anions showed that SCN- was transported into cells 3 times faster than Cl-, while the entry of SO2-4 occurred much more slowly. The anion transport inhibitors 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid and piretanide inhibited entry at low pH even in the presence of permeant anions. When cells with bound toxin were exposed to low pH in the absence of permeant anions, then briefly exposed to neutral pH and subsequently exposed to pH 4.5 in the presence of isotonic NaCl, toxin entry was induced. The data indicate that efficient anion transport at the time of exposure to low pH is required for entry of surface-bound diphtheria toxin into the cytosol. Since insertion of diphtheria toxin into the membrane occurs even in the absence of permeant anions, the results indicate that low pH is required not only for insertion of fragment B into the membrane, but also for the subsequent entry of fragment A into the cytosol. 相似文献
15.
Receptor-mediated internalization and degradation of diphtheria toxin by monkey kidney cells. 总被引:15,自引:0,他引:15
R B Dorland J L Middlebrook S H Leppla 《The Journal of biological chemistry》1979,254(22):11337-11342
The receptor-mediated internalization and degradation of radiolabeled diphtheria toxin by cultured monkey kidney cells was studied. The ability of a number of enzymes and chemicals to remove cell surface-bound toxin was tested; the combination of pronase and inositol hexaphosphate (PIHP) proved most effective. Using PIHP, the kinetics of toxin-cell association at 37 degrees C was resolved into two compounds: surface binding and internalization. The PIHP assay also allowed estimation of the half-time of toxin internalization (about 25 min). An assay involving precipitation of culture supernatants with trichloroacetic acid was developed and used to measure the rate of degradation and excretion of cell-associated toxin. Agents which markedly inhibited toxin internalization similarly prevented degradation, implying an intracellular location for the degradative process. The primary radioactive product excreted by Vero cells was monoiodotyrosine. The extent and rate of toxin degradation indicated lysosomal involvement. Finally, agents which blocked internalization or degradation, or both, (e.g. antibody and concanavalin A), protected cells from the cytotoxin action of diphtheria toxin, suggesting that these processes are necessary for expression of biological effect. 相似文献
16.
Monoclonal antibody against diphtheria toxin. Effect on toxin binding and entry into cells 总被引:7,自引:0,他引:7
S Hayakawa T Uchida E Mekada M R Moynihan Y Okada 《The Journal of biological chemistry》1983,258(7):4311-4317
A number of monoclonal antibodies against diphtheria toxin were isolated. Some of their properties were determined. Antibody 2 reacts with the region of between 30 and 45 kDa from the NH2 terminus of toxin. Antibody 7 reacts with the COOH-terminal 17-kDa region of toxin. These two antibodies show sharp contrasts in their effects on toxin action in cultured cells. When antibody 2 or 7 and toxin were mixed, incubated at 37 degrees C, and then added to sensitive Vero cells, antibody 7 blocked toxin action, but antibody 2 did not. When antibody 2 or 7 was added to cells to which toxin had been prebound at 4 degrees C, and the cells were then shifted to 37 degrees C, antibody 7 did not block toxin action, but antibody 2 inhibited intoxication. Antibody 7 blocked binding of 125I-toxin to cells and did not block degradation of toxin associated with cells. Antibody 2 did not block binding of 125I-toxin to cells, and was able to bind to cells in the presence of toxin. The results obtained from the effect of antibody 2 on degradation of 125I-toxin associated with cells resemble those seen with amines, which block toxin action but do not inhibit binding of toxin to cells. These facts show that antibody 2 does not block binding of toxin to cell surfaces, but blocks the entry of toxin into the cytosol at a step after binding of toxin to the receptor. Antibodies 14 and 15 react with fragment A of diphtheria toxin, but have no effect on any activity of toxin. The other monoclonal antibodies have effects on toxin binding and entry intermediate between those of 2 and 7. 相似文献
17.
In the present study, we compared the abilities of ricin and diphtheria toxin to induce apoptosis in Vero cells. The cytolysis and DNA fragmentation by ricin paralleled its protein synthesis inhibitory activity. However, unlike ricin, diphtheria toxin could induce neither cytolysis nor DNA fragmentation in Vero cells up to very high concentration, in spite of the fact that Vero cells were even more sensitive to protein synthesis inhibition by diphtheria toxin than ricin. Interestingly, coexistence of brefeldin A (BFA) and okadaic acid (OA) significantly enhanced diphtheria toxin-mediated cytolysis and DNA fragmentation without affecting the activity of protein synthesis inhibition. Ammonium chloride almost completely abolished the ability of diphtheria toxin to induce apoptosis in the presence of BFA and OA as well as the protein synthesis inhibitory activity. The mutant CRM 197, which does not catalyze the ADP ribosylation of elongation factor-2 (EF-2), failed to induce apoptosis in Vero cells even in the presence of BFA and OA. Thus, translocation of diphtheria toxin into the cytosol and subsequent enzymatic inactivation of EF-2 may be necessary steps to induce apoptosis. Taken together our results suggest that protein synthesis inhibition by toxins is not sufficient to induce apoptosis, and underlying mechanisms of apoptosis induction may be distinct between ricin and diphtheria toxin. Since a morphological change in the Golgi complex was observed in Vero cells treated with BFA and OA, modulation of the Golgi complex by these reagents may be partly responsible for enhanced apoptosis induction by diphtheria toxin. 相似文献
18.
Entry of diphtheria toxin into cells: possible existence of cellular factor(s) for entry of diphtheria toxin into cells was studied in somatic cell hybrids and hybrid toxins 总被引:1,自引:1,他引:0
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Ehrlich ascites tumor cells were found to be very insensitive to diphtheria toxin. We formed 37 hybrids from Ehrlich tumor cells and diphtheria toxin-sensitive human fibroblasts. The effects of diphtheria toxin on protein synthesis in those hybrids were examined. The hybrids were divided into three groups on the basis of toxin sensitivity. Group A hybrids were as sensitive to diphtheria toxin as human fibroblasts, Group C were as resistant as Ehrlich tumor cells, and Group B had intermediate sensitivity. Group A hybrids had diphtheria toxin-binding sites but Group B and C had no detectable binding sites. Elongation factor-2 of all the hybrids was susceptible to ADP-ribosylation by fragment A of diphtheria toxin. Cells of Group A and B became more sensitive to CRM 45 (cross-reacting material 45 of diphtheria toxin) after they were exposed to low pH (pH = 4.5). The resistance of Group C to CRM 45 was not affected by the same treatment. Group A and B hybrids and human fibroblasts had similar sensitivities to a hybrid toxin composed of wheat germ agglutinin and fragment A of diphtheria toxin, but Group C and Ehrlich tumor cells were resistant to this hybrid toxin. All the hybrids and Ehrlich tumor cells were more sensitive to a hybrid toxin composed of wheat germ agglutinin and subunit A of ricin than were human fibroblasts. On subcloning of Group B hybrids, one Group C hybrid was obtained, but no Group A hybrid. These facts suggest that Ehrlich ascites tumor cells differ from human fibroblasts in the expression of a factor(s) that is involved in entry of fragment A of diphtheria toxin into the cytoplasm after the toxin binds to its surface receptors. 相似文献
19.
Synthesis of a cytotoxic insulin cross-linked to diphtheria toxin fragment A capable of recognizing insulin receptors 总被引:4,自引:0,他引:4
Insulin has been cross-linked via a disulfide bond to the diphtheria toxin fragment A which is catalytically active in ADP-ribosylating elongation factor-2 but does not retain binding sites for toxin receptors. The purified conjugate proved to be cytotoxic to mouse Swiss/3T3 cells which are toxin resistant but express insulin receptors. This cytotoxicity coincided with a decrease in protein synthesis and with drastic morphology changes. In contrast, IN-2 cells, which are insulin-nonresponsive variants derived from mouse cells, were resistant to the conjugate. Thus, the conjugate (a chimeric insulin) appears to mediate entry of the toxic fragment A into 3T3 cells through insulin receptors. 相似文献
20.
A new mRNA coding for the heparin-binding EGF-like growth factor (HB-EGF) was found in Vero cells. The corresponding cDNA had C-156 in place of T, which resulted in a loss of the NheI site and a substitution of Leu-33 with Pro in the HB-EGF precursor. The known and new forms of the precursor were accordingly termed L and P. A possible conformational change in the corresponding propeptide region were assumed to affect processing of soluble secreted HB-EGF. The L and P mRNAs are differently expressed in various cell lines and have the identical 5'-untranslated sequences. Possibly, they are transcribed from one promoter and then alternatively spliced. Stimulation of resting Vero cells with tetraphorbol ester (TPA) substantially increased production of the L form, decreased production of the P form, and did not affect expression of the total HB-EGF mRNA. This was associated with an increase in binding of the diphtheria toxin, suggesting that the L HB-EGF precursor acts as its receptor. 相似文献