The Experts below are selected from a list of 15 Experts worldwide ranked by ideXlab platform
Peter H. Cheung - One of the best experts on this subject based on the ideXlab platform.
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Structure and function of C-CAM1. The first immunogLobuLin domain is required for interceLLuLar adhesion.
Journal of Biological Chemistry, 1993Co-Authors: Peter H. Cheung, Karen Earley, Xiaohong Zhang, Paula MillironsAbstract:Abstract CeLL-CAM105 proteins (aLso caLLed C-CAM) are epitheLiaL ceLL adhesion moLecuLes of the immunogLobuLin (Ig) superfamiLy. The sequences of C-CAM are highLy homoLogous to those of human carcinoembryonic antigen (CEA)-famiLy proteins. In previous studies using bacuLoviraL vectors, we showed that expression of the L-Form ceLL-CAM105 (aLso caLLed C-CAM1) in insect ceLLs resuLted in ceLL aggregation (Cheung, P. H., Thompson, N. L., EarLey, K., CuLic, O., Hixson, D., and Lin, S. H. (1993) J. BioL. Chem. 268, 6139-6146). This resuLt indicates that the insect-ceLL system is suitabLe for studying the adhesion function of C-CAM. Since C-CAM1 contains four extraceLLuLar Ig-domains, the structuraL features directLy responsibLe for C-CAM1 adhesion function were investigated by site-directed deLetion and expression in the bacuLovirus/insect ceLL system. ResuLts from these studies indicated that the first Ig domain Located in the NH2-terminaL of C-CAM pLays a cruciaL roLe in interceLLuLar adhesion. Site-directed deLetion producing mutants Lacking the second, third, or fourth Ig domains had no effect on the adhesion function. In addition, adhesion function was retained when both the third and fourth Ig domains were deLeted, aLthough the adhesion activity was reduced to haLf that in controL ceLLs. However, simuLtaneous deLetion of the second, third, and fourth domains aboLished adhesion, suggesting that these domains affect the accessibiLity of the binding site LocaLized in the first domain. In our previous studies, we showed that the cytopLasmic domains of C-CAM pLay a significant roLe in the isoForms' adhesion activity since expression of a C-CAM isoForm containing onLy 6 instead of 71 amino acids intraceLLuLarLy faiLed to show the adhesion phenotype (Cheung, P. H., CuLic, O., Qiu, Y., EarLey, K., Thompson, N., Hixson, D. C., and Lin, S.-H. (1993) Biochem. J. 295, in press). These resuLts together suggest that both the cytopLasmic domain and the first N-terminaL Ig-Like domain are required for C-CAM-mediated ceLL adhesion activity.
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ConFormation Dependence of Antipeptide Antibodies: Characterization of CeLL-CAM105 IsoForm-Specific Antipeptide Antibodies Using Proteins Expressed in Insect CeLLs with BacuLoviraL Vectors
Archives of Biochemistry and Biophysics, 1993Co-Authors: Peter H. Cheung, Karen Earley, Tzyy Chyau LiangAbstract:Abstract CeLL-CAM105 proteins are hepatocyte adhesion moLecuLes of the immunogLobuLin superfamiLy. The two isoForms, L-Form and S-Form, are highLy homoLogous. In their extraceLLuLar domains, onLy 16 amino acid substitutions are found scattered in the first immunogLobuLin domain of 105 amino acids. Peptide sequences containing these differences are seLected for production of antibodies. IsoForm specificities of these antibodies were evaLuated with proteins expressed in the bacuLovirus-insect ceLL system. In immunobLot and immunoprecipitation experiments, anti-C1 antibody, which was generated against a peptide sequence found onLy in the cytopLasmic domain of the L-Form, reacted onLy with the L-Form ceLL-CAM105 protein. Anti-N antibody, which was generated against a pentadecapeptide of the L-Form, reacted with both the S- and L-isoForms. The Lack of isoForm specificity of anti-N is not surprising because there is onLy one amino acid substitution in this pentadecapeptide sequence. In contrast, S3, a S-Form-specific pentadecapeptide with four amino acid substitutions was abLe to eLicit antibody, anti-S3, specific for the S-isoForm. With the commonLy used immunoprecipitation procedures, onLy anti-C1 was abLe to precipitate ceLL-CAM105 from the Liver membrane. Anti-N and anti-S3 couLd precipitate the proteins onLy after the Liver membrane sampLes had been boiLed in the presence of denaturing agents. Hydropathy anaLysis of these peptides reveaLed that both peptides N and S3 are more hydrophobic than peptide C1, suggesting that the peptide fragments N and S3 are probabLy not Located on the surface of the protein. This may expLain why boiLing of the protein sampLe was necessary before anti-N and anti-S3 couLd precipitate the protein. The present study demonstrates that it is possibLe to produce isoForm-specific antibodies for highLy homoLogous proteins. Furthermore, we show that speciaL sampLe treatment may be required to expose the antigenic sites.
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CeLL-CAM105 isoForms with different adhesion functions are coexpressed in aduLt rat tissues and during Liver deveLopment.
Journal of Biological Chemistry, 1993Co-Authors: Peter H. Cheung, Nancy L Thompson, Karen Earley, Ognjen Culic, Douglas C. HixsonAbstract:Abstract The rat hepatocyte ceLL adhesion moLecuLe ceLL-CAM105 has recentLy been shown to be composed of at Least two isoForms. Expression of the two isoForms in different tissues and during fetaL Liver deveLopment in rats was studied by RNase protection using a probe which couLd specificaLLy and simuLtaneousLy detect both isoForms. This probe reveaLed protected fragments of expected Lengths for the L-Form and the S-Form in RNA sampLes isoLated from various aduLt rat tissues. High LeveLs of the L-Form and S-Form messages were detected in Liver and intestine, moderate LeveLs were detected in Lung, and weak signaLs were detected in muscLe, kidney, and spLeen. In Liver deveLopment studies, the messages for ceLL-CAM105 showed a major increase on the first day after birth compared to the fetaL stage, and both isoForm messages were proportionaLLy increased. These resuLts indicate that both ceLL-CAM105 isoForms may have function(s) reLated to hepatocyte differentiation. To study the adhesion function of ceLL-CAM105 isoForms, fuLL-Length cDNAs for these isoForms were expressed in insect ceLLs. The insect ceLLs expressing the L-Form ceLL-CAM105 were found to aggregate. However, expression of S-Form ceLL-CAM105 did not support ceLL aggregation. These resuLts indicate that L-Form, but not S-Form, ceLL-CAM105 directLy mediates the ceLL adhesion function.
Tzyy Chyau Liang - One of the best experts on this subject based on the ideXlab platform.
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ConFormation Dependence of Antipeptide Antibodies: Characterization of CeLL-CAM105 IsoForm-Specific Antipeptide Antibodies Using Proteins Expressed in Insect CeLLs with BacuLoviraL Vectors
Archives of Biochemistry and Biophysics, 1993Co-Authors: Peter H. Cheung, Karen Earley, Tzyy Chyau LiangAbstract:Abstract CeLL-CAM105 proteins are hepatocyte adhesion moLecuLes of the immunogLobuLin superfamiLy. The two isoForms, L-Form and S-Form, are highLy homoLogous. In their extraceLLuLar domains, onLy 16 amino acid substitutions are found scattered in the first immunogLobuLin domain of 105 amino acids. Peptide sequences containing these differences are seLected for production of antibodies. IsoForm specificities of these antibodies were evaLuated with proteins expressed in the bacuLovirus-insect ceLL system. In immunobLot and immunoprecipitation experiments, anti-C1 antibody, which was generated against a peptide sequence found onLy in the cytopLasmic domain of the L-Form, reacted onLy with the L-Form ceLL-CAM105 protein. Anti-N antibody, which was generated against a pentadecapeptide of the L-Form, reacted with both the S- and L-isoForms. The Lack of isoForm specificity of anti-N is not surprising because there is onLy one amino acid substitution in this pentadecapeptide sequence. In contrast, S3, a S-Form-specific pentadecapeptide with four amino acid substitutions was abLe to eLicit antibody, anti-S3, specific for the S-isoForm. With the commonLy used immunoprecipitation procedures, onLy anti-C1 was abLe to precipitate ceLL-CAM105 from the Liver membrane. Anti-N and anti-S3 couLd precipitate the proteins onLy after the Liver membrane sampLes had been boiLed in the presence of denaturing agents. Hydropathy anaLysis of these peptides reveaLed that both peptides N and S3 are more hydrophobic than peptide C1, suggesting that the peptide fragments N and S3 are probabLy not Located on the surface of the protein. This may expLain why boiLing of the protein sampLe was necessary before anti-N and anti-S3 couLd precipitate the protein. The present study demonstrates that it is possibLe to produce isoForm-specific antibodies for highLy homoLogous proteins. Furthermore, we show that speciaL sampLe treatment may be required to expose the antigenic sites.
Karen Earley - One of the best experts on this subject based on the ideXlab platform.
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Structure and function of C-CAM1. The first immunogLobuLin domain is required for interceLLuLar adhesion.
Journal of Biological Chemistry, 1993Co-Authors: Peter H. Cheung, Karen Earley, Xiaohong Zhang, Paula MillironsAbstract:Abstract CeLL-CAM105 proteins (aLso caLLed C-CAM) are epitheLiaL ceLL adhesion moLecuLes of the immunogLobuLin (Ig) superfamiLy. The sequences of C-CAM are highLy homoLogous to those of human carcinoembryonic antigen (CEA)-famiLy proteins. In previous studies using bacuLoviraL vectors, we showed that expression of the L-Form ceLL-CAM105 (aLso caLLed C-CAM1) in insect ceLLs resuLted in ceLL aggregation (Cheung, P. H., Thompson, N. L., EarLey, K., CuLic, O., Hixson, D., and Lin, S. H. (1993) J. BioL. Chem. 268, 6139-6146). This resuLt indicates that the insect-ceLL system is suitabLe for studying the adhesion function of C-CAM. Since C-CAM1 contains four extraceLLuLar Ig-domains, the structuraL features directLy responsibLe for C-CAM1 adhesion function were investigated by site-directed deLetion and expression in the bacuLovirus/insect ceLL system. ResuLts from these studies indicated that the first Ig domain Located in the NH2-terminaL of C-CAM pLays a cruciaL roLe in interceLLuLar adhesion. Site-directed deLetion producing mutants Lacking the second, third, or fourth Ig domains had no effect on the adhesion function. In addition, adhesion function was retained when both the third and fourth Ig domains were deLeted, aLthough the adhesion activity was reduced to haLf that in controL ceLLs. However, simuLtaneous deLetion of the second, third, and fourth domains aboLished adhesion, suggesting that these domains affect the accessibiLity of the binding site LocaLized in the first domain. In our previous studies, we showed that the cytopLasmic domains of C-CAM pLay a significant roLe in the isoForms' adhesion activity since expression of a C-CAM isoForm containing onLy 6 instead of 71 amino acids intraceLLuLarLy faiLed to show the adhesion phenotype (Cheung, P. H., CuLic, O., Qiu, Y., EarLey, K., Thompson, N., Hixson, D. C., and Lin, S.-H. (1993) Biochem. J. 295, in press). These resuLts together suggest that both the cytopLasmic domain and the first N-terminaL Ig-Like domain are required for C-CAM-mediated ceLL adhesion activity.
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ConFormation Dependence of Antipeptide Antibodies: Characterization of CeLL-CAM105 IsoForm-Specific Antipeptide Antibodies Using Proteins Expressed in Insect CeLLs with BacuLoviraL Vectors
Archives of Biochemistry and Biophysics, 1993Co-Authors: Peter H. Cheung, Karen Earley, Tzyy Chyau LiangAbstract:Abstract CeLL-CAM105 proteins are hepatocyte adhesion moLecuLes of the immunogLobuLin superfamiLy. The two isoForms, L-Form and S-Form, are highLy homoLogous. In their extraceLLuLar domains, onLy 16 amino acid substitutions are found scattered in the first immunogLobuLin domain of 105 amino acids. Peptide sequences containing these differences are seLected for production of antibodies. IsoForm specificities of these antibodies were evaLuated with proteins expressed in the bacuLovirus-insect ceLL system. In immunobLot and immunoprecipitation experiments, anti-C1 antibody, which was generated against a peptide sequence found onLy in the cytopLasmic domain of the L-Form, reacted onLy with the L-Form ceLL-CAM105 protein. Anti-N antibody, which was generated against a pentadecapeptide of the L-Form, reacted with both the S- and L-isoForms. The Lack of isoForm specificity of anti-N is not surprising because there is onLy one amino acid substitution in this pentadecapeptide sequence. In contrast, S3, a S-Form-specific pentadecapeptide with four amino acid substitutions was abLe to eLicit antibody, anti-S3, specific for the S-isoForm. With the commonLy used immunoprecipitation procedures, onLy anti-C1 was abLe to precipitate ceLL-CAM105 from the Liver membrane. Anti-N and anti-S3 couLd precipitate the proteins onLy after the Liver membrane sampLes had been boiLed in the presence of denaturing agents. Hydropathy anaLysis of these peptides reveaLed that both peptides N and S3 are more hydrophobic than peptide C1, suggesting that the peptide fragments N and S3 are probabLy not Located on the surface of the protein. This may expLain why boiLing of the protein sampLe was necessary before anti-N and anti-S3 couLd precipitate the protein. The present study demonstrates that it is possibLe to produce isoForm-specific antibodies for highLy homoLogous proteins. Furthermore, we show that speciaL sampLe treatment may be required to expose the antigenic sites.
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CeLL-CAM105 isoForms with different adhesion functions are coexpressed in aduLt rat tissues and during Liver deveLopment.
Journal of Biological Chemistry, 1993Co-Authors: Peter H. Cheung, Nancy L Thompson, Karen Earley, Ognjen Culic, Douglas C. HixsonAbstract:Abstract The rat hepatocyte ceLL adhesion moLecuLe ceLL-CAM105 has recentLy been shown to be composed of at Least two isoForms. Expression of the two isoForms in different tissues and during fetaL Liver deveLopment in rats was studied by RNase protection using a probe which couLd specificaLLy and simuLtaneousLy detect both isoForms. This probe reveaLed protected fragments of expected Lengths for the L-Form and the S-Form in RNA sampLes isoLated from various aduLt rat tissues. High LeveLs of the L-Form and S-Form messages were detected in Liver and intestine, moderate LeveLs were detected in Lung, and weak signaLs were detected in muscLe, kidney, and spLeen. In Liver deveLopment studies, the messages for ceLL-CAM105 showed a major increase on the first day after birth compared to the fetaL stage, and both isoForm messages were proportionaLLy increased. These resuLts indicate that both ceLL-CAM105 isoForms may have function(s) reLated to hepatocyte differentiation. To study the adhesion function of ceLL-CAM105 isoForms, fuLL-Length cDNAs for these isoForms were expressed in insect ceLLs. The insect ceLLs expressing the L-Form ceLL-CAM105 were found to aggregate. However, expression of S-Form ceLL-CAM105 did not support ceLL aggregation. These resuLts indicate that L-Form, but not S-Form, ceLL-CAM105 directLy mediates the ceLL adhesion function.
A. M. Paton - One of the best experts on this subject based on the ideXlab platform.
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Growth and physioLogicaL characteristics of BaciLLus subtiLis L-Forms
Journal of Applied Microbiology, 1993Co-Authors: E.j. Allan, Amijee F, Tyson Rh, J.a. Strang, C. M. Innes, A. M. PatonAbstract:The specific growth rate of stabLe BaciLLus subtiLis L-Forms was sLower (mu = 0.127) than the ceLL-waLLed Form (mu = 0.219) when measured by opticaL density (O.D.). However, the L-Form growth rate increased (mu = 0.288) when determined by viabLe ceLL counts. L-Forms of B. subtiLis appear to enter a phase of rapid ceLL division, foLLowed by a period of ceLL enLargement which is associated with an increase in the number of ceLLs with vacuoLes and granuLes. Thus, maximum viabLe L-Form numbers and DNA content were attained at approx. 30h, before maximum protein content was achieved (46h) and before maximum O.D. was observed at 71 h. Measurements showed that L-Form ceLL size increased even after ceLL division had stopped. O.D. was therefore inaccurate for assessment of L-Form growth. L-Forms were sensitive to osmotic shock and unLike the ceLL-waLLed organisms from which they were derived, were resistant to peniciLLin, indicating a Loss of peptidogLycan. The L-Forms were simiLar to ceLL-waLLed Forms in that antibiotic(s) and proteases were produced.
E.j. Allan - One of the best experts on this subject based on the ideXlab platform.
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An investigation of enumeration and DNA partitioning in BaciLLus subtiLis L-Form bacteria
Journal of Applied Microbiology, 1994Co-Authors: Rosemary N. Waterhouse, E.j. Allan, Firoz Amijee, Valerie J. Undrill, L. Anne GloverAbstract:R.N. WATERHOUSE, E.J. ALLAN, F. AMIJEE, V.J. UNDRILL AND L.A. GLOVER. 1994. CeLL numbers of two morphogenic Forms of BaciLLus subtiLis (the ceLL-waLLed parentaL and the derived stabLe ceLL waLL-deficient L-Form) have been compared by two methods: DNA hybridization (i.e. deduced genome numbers) and viabLe ceLL counts (i.e. number of coLony-Forming units (cfu)). The DNA hybridization method was shown to be a reLiabLe and reproducibLe method for estimating genome numbers. Comparison of different L-Form popuLations showed that the two methods of enumeration gave different vaLues, with the deduced genome numbers much higher (by severaL orders of magnitude) than ceLL numbers deduced from viabLe ceLL counts. In contrast, when a cuLture of the ceLL-waLLed Form was enumerated, the discrepancy between the two methods was Low (by a factor of about 6) The combination of a high number of L-Form genomes detected by DNA hybridization and a reLativeLy Low number of cfu was thought to be a consequence of a diminished co-ordination between the DNA repLication and ceLL division processes in L-Form bacteria. This suggestion was further substantiated by assessing the stabiLity of pLasmid pPL608 in a transFormed B. subtiLis L-Form ceLL Line, where even in the presence of continued kanamycin seLection, 25% of the popuLation Lost kanamycin resistance. The resuLts are discussed with particuLar reference to ceLL division in ceLL waLL-deficient, stabLe L-Form bacteria.
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Growth and physioLogicaL characteristics of BaciLLus subtiLis L-Forms
Journal of Applied Microbiology, 1993Co-Authors: E.j. Allan, Amijee F, Tyson Rh, J.a. Strang, C. M. Innes, A. M. PatonAbstract:The specific growth rate of stabLe BaciLLus subtiLis L-Forms was sLower (mu = 0.127) than the ceLL-waLLed Form (mu = 0.219) when measured by opticaL density (O.D.). However, the L-Form growth rate increased (mu = 0.288) when determined by viabLe ceLL counts. L-Forms of B. subtiLis appear to enter a phase of rapid ceLL division, foLLowed by a period of ceLL enLargement which is associated with an increase in the number of ceLLs with vacuoLes and granuLes. Thus, maximum viabLe L-Form numbers and DNA content were attained at approx. 30h, before maximum protein content was achieved (46h) and before maximum O.D. was observed at 71 h. Measurements showed that L-Form ceLL size increased even after ceLL division had stopped. O.D. was therefore inaccurate for assessment of L-Form growth. L-Forms were sensitive to osmotic shock and unLike the ceLL-waLLed organisms from which they were derived, were resistant to peniciLLin, indicating a Loss of peptidogLycan. The L-Forms were simiLar to ceLL-waLLed Forms in that antibiotic(s) and proteases were produced.
