The matrix metalloproteinase (MMP) stromelysin-3 (ST3) has long been implicated to play an important role in extracellular matrix (ECM) remodeling and cell fate determination during normal and pathological processes. ...The matrix metalloproteinase (MMP) stromelysin-3 (ST3) has long been implicated to play an important role in extracellular matrix (ECM) remodeling and cell fate determination during normal and pathological processes. However like other MMPs, the molecular basis of ST3 function in vivo remains unclear due to the lack of information on its physiological substrates. Furthermore, ST3 has only weak activities toward all tested ECM proteins. Using thyroid hormone-dependent Xenopus laevis metamorphosis as a model, we demonstrated previously that ST3 is important for apoptosis and tissue morphogenesis during intestinal remodeling. Here, we used yeast two-hybrid screen with mRNAs from metamorphosing tadpoles to identify potential substrate of ST3 during development. We thus isolated the 37 kd laminin receptor precursor (LR). We showed that LR binds to ST3 in vitro and can be cleaved by ST3 at two sites distinct from where other MMPs cleave. Through peptide sequencing, we determined that the two cleavage sites are in the extracellular domain between the transmembrane domain and laminin binding sequence. Furthermore, we demon strated that these cleavage sites are conserved in human LR. These results together with high levels of human LR and ST3 expression in carcinomas suggest that LR is a likely in vivo substrate of ST3 and that its cleavage by ST3 may alter cell-extracellular matrix interaction, thus, playing a role in mediating the effects of ST3 on cell fate and behavior ob- served during development and pathogenesis.展开更多
The 67KD laminin receptor (LN-R ) that binds laminin (LN) is involved in the metastasis cascade. Using immunohistochemical technique, in situ hybridization and reverse transcription polymerase chain reaction(RT-PCR), ...The 67KD laminin receptor (LN-R ) that binds laminin (LN) is involved in the metastasis cascade. Using immunohistochemical technique, in situ hybridization and reverse transcription polymerase chain reaction(RT-PCR), we studied LN-R protein and RNA levels in 30 cases of human hepatocellular carcinoma (HCC) to further understand its role in the metastasis of HCC. In our 14 cases of HCC with metastasis, its positive rates were 71. 4 %, 57. 1%, 85.7% respectively, whereas its positive expression in 16 cases without metastasis were 31.3 %, 18. 8 %, 50. 0 % respectively. The significant difference was found between these two groups. The results suggest that the 67KD LN-R expression plays a very important role in the metastasis of HCC.展开更多
Objective: To prepare monoclonal antibody (McAb) against the Integrin a6 extracellular domain and identify its biological activities. Methods: Fusion-protein of integrin a6 extracellular domain (GST-IAGED) was express...Objective: To prepare monoclonal antibody (McAb) against the Integrin a6 extracellular domain and identify its biological activities. Methods: Fusion-protein of integrin a6 extracellular domain (GST-IAGED) was expressed in E.coli. JM109 and used for immunizing BALB/C mice. The spleen cells from immunized mice were fused with SP2/0 cells and selectively cultured with HAT medium. ELISA and immunocytochemistry staining were used to select hybridomas. Results: One strain of hybridoma cells that secreted specific monoclonal antibody against integrin a6 extracellular domain was indentified. The immunoglobulin subclass of the McAb was IgG1. Conclusion: The McAb against the extracellular domain of integrin a6 was successfully prepared by using GST-IA6ED fusion protein expressed by E.Coli. And the McAb had positive reaction with human hepatocarcinoma cells-BEL-7402.展开更多
The variation of membrane surface and lateral diffusion of membrane protein was studied after the interaction of laminin with its membrane receptor in mouse macrophages. A pattern of membrane surface which showed smal...The variation of membrane surface and lateral diffusion of membrane protein was studied after the interaction of laminin with its membrane receptor in mouse macrophages. A pattern of membrane surface which showed smaller and bigger peaks was obtained by scanning tunneling microscope(STM), looking like the domains of lipid groups and proteins in the model of fluid mosaic biomembraoe. Some even more higher and wider peaks projected out from the membrane surface in STM im-age after the interacting of laminin with membrane receptor were probably, tile complexes of laminin and membraue receptor. Furthermore. the deeceasad lateral diffusion coofficeent value(D^_) was obtained by Huorescence recovery after photobleaching (FRAP) after the laminin was reacted with membrane receptor. This phenomenon provides an evidence that the complexes of laminin and its membrane receptor were located on the membrane of macrophages. So we could consider that the laminin is combined with membrane receptor leading to the variation in the properties of membrane surface.展开更多
Laminin receptor(LN-R, Mr=70,000) isolated from murine Lewis lung carcinoma was shown to be a single band on SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide gel electrophoresis) after reduction. The present study Indi...Laminin receptor(LN-R, Mr=70,000) isolated from murine Lewis lung carcinoma was shown to be a single band on SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide gel electrophoresis) after reduction. The present study Indicates that LN-R was a kind of glycoproteins by periodic add Schiff staining. It’s pI was 4. 93. LN-R preperation was associated with phospholipid and neutral glycolipid by TLC (thin layer chromatography) or HPTLC (high performance thin layer chromtography) of chloroform-methanol extracts of LN-R, respectively, but without acidic glycolipid. Binding of LN-R with its ligand, Lamlnin, depended on the presence of Ca+ + , Mg++. LN-R may bind to actin.展开更多
Objective To investigate the relationship between expression of type Ⅳ collagenase (Ⅳ col ) andthe invasion of laminin-receptors (LNR) in human colorectal carcinoma cells. Methods: The expression ofLNR and Ⅳ col in...Objective To investigate the relationship between expression of type Ⅳ collagenase (Ⅳ col ) andthe invasion of laminin-receptors (LNR) in human colorectal carcinoma cells. Methods: The expression ofLNR and Ⅳ col in human colorectal carcinoma(HCC) cell lines were detected quantitatively with different potential metastasis. By using human amniotic membrane(HAM) invasion model, we observed the expressionof Ⅳ col and invasion in both HCC cell lines affected by LNR monoclonal antibodies. Results: High metastaticpotential cell line Lovo expressed higher levels of LNR and Ⅳ col than HT29, a low metastatic potential HCCcell line. The number of cells adhered to HAM of Lovo were more than that of HT29. When the cells of thetwo HCC cell lines blocked by LNR antibodies, the expression of Ⅳcol were decreased, and the cells adheredto HAM expressed Ⅳ col were also decreased. Conclusion: LNR might affect the regulation of expression ofⅣ col in tumor cells by cellular signal communication, and then affect the invasion and metastasis of tumor.展开更多
The validity of 99mTc-YIGSR,a novel receptor radio-tracer,in imaging the Ehrlich ascites tumor was evaluated.YIGSR,a pentapeptide of laminin,was labeled with 99mTc by using a bifunc-tional chelator S-Acetly-NH3-MAG3.T...The validity of 99mTc-YIGSR,a novel receptor radio-tracer,in imaging the Ehrlich ascites tumor was evaluated.YIGSR,a pentapeptide of laminin,was labeled with 99mTc by using a bifunc-tional chelator S-Acetly-NH3-MAG3.The MIBI was labeled with 99mTc by following the kit instruc-tion.The mice of tumor group were intravenously injected 1-2 mCi of 99mTc-YIGSR or 99mTc-MIBI via caudal vein,immobilized and imaged under a Gamma camera.The same procedure was per-formed in mice of blockade group,in which the unlabeled YIGSR was previously injected to block the receptor-recognition sites,and inflammation group serving as control.The reverse-phase Sep-Pak C18 chromatogram was found to have an essentially complete conjugation between YIGSR and S-Acetly-NH3-MAG3.The conjugated YIGSR could be radio-labeled successfully with 99mTc at room temperature and neutral pH,with a radio-labeling yield of 62%.Without the chelator S-Acetly-NH3-MAG3,the YIGSR was labeled with 99mTc at an efficiency of 4%.The imagological study revealed obvious tumor accumulation of 99mTc-YIGSR 15 min after the injection,and the up-take peaked after 3 h with a tumor-to-muscle ratio(T/M) of 11.36.The radio-tracer was slowly cleared up and resulted in a T/M of 3.01 at the 8th h after the injection.As for blocked group,the tu-mor uptake of radiotracer was significantly lower,with the highest T/M being 4.61 after 3 h and 0.89 after 8 h.The T/M was 3.72 at the 3rd h and 1.29 at the 8th h after the 99mTc-YIGSR injection in the inflammatory group.The T/M was significantly higher in tumor group than in inflammatory group or control group(P<0.001).In the 99mTc-MIBI group,the T/M was 1.40 at the 3rd h and 0.55 at the 8th h after the injection,which showed a significant difference as compared with 99mTc-YIGSR(P<0.001).It is concluded that YIGSR can be successfully radiolabelled by using S-Acetly-NH3-MAG3.99mTc-YIGSR has many advantages in tumor imaging,such as quick and clear visualization,high sensitivity and specificity,and satisfactory target/non-target ratio(N/NT).It promises to be tumor ra-dio-tracer.展开更多
文摘The matrix metalloproteinase (MMP) stromelysin-3 (ST3) has long been implicated to play an important role in extracellular matrix (ECM) remodeling and cell fate determination during normal and pathological processes. However like other MMPs, the molecular basis of ST3 function in vivo remains unclear due to the lack of information on its physiological substrates. Furthermore, ST3 has only weak activities toward all tested ECM proteins. Using thyroid hormone-dependent Xenopus laevis metamorphosis as a model, we demonstrated previously that ST3 is important for apoptosis and tissue morphogenesis during intestinal remodeling. Here, we used yeast two-hybrid screen with mRNAs from metamorphosing tadpoles to identify potential substrate of ST3 during development. We thus isolated the 37 kd laminin receptor precursor (LR). We showed that LR binds to ST3 in vitro and can be cleaved by ST3 at two sites distinct from where other MMPs cleave. Through peptide sequencing, we determined that the two cleavage sites are in the extracellular domain between the transmembrane domain and laminin binding sequence. Furthermore, we demon strated that these cleavage sites are conserved in human LR. These results together with high levels of human LR and ST3 expression in carcinomas suggest that LR is a likely in vivo substrate of ST3 and that its cleavage by ST3 may alter cell-extracellular matrix interaction, thus, playing a role in mediating the effects of ST3 on cell fate and behavior ob- served during development and pathogenesis.
文摘The 67KD laminin receptor (LN-R ) that binds laminin (LN) is involved in the metastasis cascade. Using immunohistochemical technique, in situ hybridization and reverse transcription polymerase chain reaction(RT-PCR), we studied LN-R protein and RNA levels in 30 cases of human hepatocellular carcinoma (HCC) to further understand its role in the metastasis of HCC. In our 14 cases of HCC with metastasis, its positive rates were 71. 4 %, 57. 1%, 85.7% respectively, whereas its positive expression in 16 cases without metastasis were 31.3 %, 18. 8 %, 50. 0 % respectively. The significant difference was found between these two groups. The results suggest that the 67KD LN-R expression plays a very important role in the metastasis of HCC.
基金This work was supported by a grand from the Natural Science Foundation of Beijing (No. 7022006).
文摘Objective: To prepare monoclonal antibody (McAb) against the Integrin a6 extracellular domain and identify its biological activities. Methods: Fusion-protein of integrin a6 extracellular domain (GST-IAGED) was expressed in E.coli. JM109 and used for immunizing BALB/C mice. The spleen cells from immunized mice were fused with SP2/0 cells and selectively cultured with HAT medium. ELISA and immunocytochemistry staining were used to select hybridomas. Results: One strain of hybridoma cells that secreted specific monoclonal antibody against integrin a6 extracellular domain was indentified. The immunoglobulin subclass of the McAb was IgG1. Conclusion: The McAb against the extracellular domain of integrin a6 was successfully prepared by using GST-IA6ED fusion protein expressed by E.Coli. And the McAb had positive reaction with human hepatocarcinoma cells-BEL-7402.
基金a great from the Nationa Natural Science Foundation,
文摘The variation of membrane surface and lateral diffusion of membrane protein was studied after the interaction of laminin with its membrane receptor in mouse macrophages. A pattern of membrane surface which showed smaller and bigger peaks was obtained by scanning tunneling microscope(STM), looking like the domains of lipid groups and proteins in the model of fluid mosaic biomembraoe. Some even more higher and wider peaks projected out from the membrane surface in STM im-age after the interacting of laminin with membrane receptor were probably, tile complexes of laminin and membraue receptor. Furthermore. the deeceasad lateral diffusion coofficeent value(D^_) was obtained by Huorescence recovery after photobleaching (FRAP) after the laminin was reacted with membrane receptor. This phenomenon provides an evidence that the complexes of laminin and its membrane receptor were located on the membrane of macrophages. So we could consider that the laminin is combined with membrane receptor leading to the variation in the properties of membrane surface.
文摘Laminin receptor(LN-R, Mr=70,000) isolated from murine Lewis lung carcinoma was shown to be a single band on SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide gel electrophoresis) after reduction. The present study Indicates that LN-R was a kind of glycoproteins by periodic add Schiff staining. It’s pI was 4. 93. LN-R preperation was associated with phospholipid and neutral glycolipid by TLC (thin layer chromatography) or HPTLC (high performance thin layer chromtography) of chloroform-methanol extracts of LN-R, respectively, but without acidic glycolipid. Binding of LN-R with its ligand, Lamlnin, depended on the presence of Ca+ + , Mg++. LN-R may bind to actin.
文摘Objective To investigate the relationship between expression of type Ⅳ collagenase (Ⅳ col ) andthe invasion of laminin-receptors (LNR) in human colorectal carcinoma cells. Methods: The expression ofLNR and Ⅳ col in human colorectal carcinoma(HCC) cell lines were detected quantitatively with different potential metastasis. By using human amniotic membrane(HAM) invasion model, we observed the expressionof Ⅳ col and invasion in both HCC cell lines affected by LNR monoclonal antibodies. Results: High metastaticpotential cell line Lovo expressed higher levels of LNR and Ⅳ col than HT29, a low metastatic potential HCCcell line. The number of cells adhered to HAM of Lovo were more than that of HT29. When the cells of thetwo HCC cell lines blocked by LNR antibodies, the expression of Ⅳcol were decreased, and the cells adheredto HAM expressed Ⅳ col were also decreased. Conclusion: LNR might affect the regulation of expression ofⅣ col in tumor cells by cellular signal communication, and then affect the invasion and metastasis of tumor.
基金This project was supported by a grant from the National Natural Sciences Foundation of China (No 30400170)
文摘The validity of 99mTc-YIGSR,a novel receptor radio-tracer,in imaging the Ehrlich ascites tumor was evaluated.YIGSR,a pentapeptide of laminin,was labeled with 99mTc by using a bifunc-tional chelator S-Acetly-NH3-MAG3.The MIBI was labeled with 99mTc by following the kit instruc-tion.The mice of tumor group were intravenously injected 1-2 mCi of 99mTc-YIGSR or 99mTc-MIBI via caudal vein,immobilized and imaged under a Gamma camera.The same procedure was per-formed in mice of blockade group,in which the unlabeled YIGSR was previously injected to block the receptor-recognition sites,and inflammation group serving as control.The reverse-phase Sep-Pak C18 chromatogram was found to have an essentially complete conjugation between YIGSR and S-Acetly-NH3-MAG3.The conjugated YIGSR could be radio-labeled successfully with 99mTc at room temperature and neutral pH,with a radio-labeling yield of 62%.Without the chelator S-Acetly-NH3-MAG3,the YIGSR was labeled with 99mTc at an efficiency of 4%.The imagological study revealed obvious tumor accumulation of 99mTc-YIGSR 15 min after the injection,and the up-take peaked after 3 h with a tumor-to-muscle ratio(T/M) of 11.36.The radio-tracer was slowly cleared up and resulted in a T/M of 3.01 at the 8th h after the injection.As for blocked group,the tu-mor uptake of radiotracer was significantly lower,with the highest T/M being 4.61 after 3 h and 0.89 after 8 h.The T/M was 3.72 at the 3rd h and 1.29 at the 8th h after the 99mTc-YIGSR injection in the inflammatory group.The T/M was significantly higher in tumor group than in inflammatory group or control group(P<0.001).In the 99mTc-MIBI group,the T/M was 1.40 at the 3rd h and 0.55 at the 8th h after the injection,which showed a significant difference as compared with 99mTc-YIGSR(P<0.001).It is concluded that YIGSR can be successfully radiolabelled by using S-Acetly-NH3-MAG3.99mTc-YIGSR has many advantages in tumor imaging,such as quick and clear visualization,high sensitivity and specificity,and satisfactory target/non-target ratio(N/NT).It promises to be tumor ra-dio-tracer.
