K. Namikawa, Yumi Sato, T. Maruo, F. Sunaga, K. Sakaguchi, J. Suzuki
Due to the negative charges on their surface membrane, erythrocytes usually do not agglutinate each other in vivo. However, when mixed with 5% glucose solution in a test tube, some feline erythrocytes exhibit agglutination. To investigate the reasons for this phenomenon, we extracted erythrocyte membrane proteins from these cells and subjected them to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In samples in which agglutination did not occur, a band was seen at the 36-kDa position, whereas in samples showing agglutination, this band disappeared or has lower intensity. The 36-kDa position corresponds to glycophorin A in the human erythrocyte membrane, but no immunochemical cross-reaction with this band was seen. We therefore conducted mass spectrometry in order to investigate the composition, and found a partial amino acid sequence, His-Ile-Thr-Ser-Tyr-Pro-Glu-Thr-His-Glu-Gly. Furthermore, although no protein showing this sequence was found in any database, this protein was confirmed to be an acidic glycoprotein, therefore it is thought to be a glycophorin-like molecule in the feline erythrocyte membrane that could contribute to inhibition of agglutination.
{"title":"A study of an erythrocyte membrane protein that contributes to inhibition of agglutination of feline erythrocytes in glucose solution","authors":"K. Namikawa, Yumi Sato, T. Maruo, F. Sunaga, K. Sakaguchi, J. Suzuki","doi":"10.2198/JELECTROPH.54.9","DOIUrl":"https://doi.org/10.2198/JELECTROPH.54.9","url":null,"abstract":"Due to the negative charges on their surface membrane, erythrocytes usually do not agglutinate each other in vivo. However, when mixed with 5% glucose solution in a test tube, some feline erythrocytes exhibit agglutination. To investigate the reasons for this phenomenon, we extracted erythrocyte membrane proteins from these cells and subjected them to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In samples in which agglutination did not occur, a band was seen at the 36-kDa position, whereas in samples showing agglutination, this band disappeared or has lower intensity. The 36-kDa position corresponds to glycophorin A in the human erythrocyte membrane, but no immunochemical cross-reaction with this band was seen. We therefore conducted mass spectrometry in order to investigate the composition, and found a partial amino acid sequence, His-Ile-Thr-Ser-Tyr-Pro-Glu-Thr-His-Glu-Gly. Furthermore, although no protein showing this sequence was found in any database, this protein was confirmed to be an acidic glycoprotein, therefore it is thought to be a glycophorin-like molecule in the feline erythrocyte membrane that could contribute to inhibition of agglutination.","PeriodicalId":15059,"journal":{"name":"Journal of capillary electrophoresis","volume":"1 1","pages":"9-12"},"PeriodicalIF":0.0,"publicationDate":"2010-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"88706146","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2009-12-01DOI: 10.2198/JELECTROPH.53.51
J. Suzuki, Shogo Ujiie, Ryosuke Kira, K. Sakaguchi, Yoichi Kakuno, M. Honda
We examined the effect of diethylstilbestrol(DES) administration on newborn male rats by rapid micro two-dimensional polyacrylamide gel electrophoresis (RM2D-PAGE) under non-denaturing/denaturing conditions and a 10-17% second-dimensional gel. We began by visually analyzing the changes at each week over time (weeks 1-9) in plasma protein spots from the same individual DES-administered male rats. We found the tendency for mercaptoalbumin (reduced form) spots to increase in week 1 in DES-administered rats. We also analyzed the quantitative alterations in α2u-globulin (AUG), a biomarker of endocrine disruptors, and found a two-week delay in expression in DES-administered rats in comparison to those not given DES (control rats). We then investigated the reproducibility of these results by using samples taken from multiple male rats in weeks 5, 7 and 9 to perform electrophoretic analysis three times for each individual, and observed the changes in AUG. AUG production was not seen in week 5 in the control rats, but was present at weeks 7 and 9. In DES-administered rats, expression was not observed at week 5 or 7, but was present at week 9. In addition, we identified the spots of AUG by Western blotting after electrophoresis. We consequently confirmed that AUG production delayed by two weeks in DES-administered rats, in comparison with control rats, which suggests that the effect of either a decline in AUG synthesis function or its depletion results in stunting of the reproductive organs, which was confirmed through autopsy.
{"title":"Analysis of plasma protein in newborn rats treated with diethylstilbestrol by using rapid micro two-dimensional electrophoresis","authors":"J. Suzuki, Shogo Ujiie, Ryosuke Kira, K. Sakaguchi, Yoichi Kakuno, M. Honda","doi":"10.2198/JELECTROPH.53.51","DOIUrl":"https://doi.org/10.2198/JELECTROPH.53.51","url":null,"abstract":"We examined the effect of diethylstilbestrol(DES) administration on newborn male rats by rapid micro two-dimensional polyacrylamide gel electrophoresis (RM2D-PAGE) under non-denaturing/denaturing conditions and a 10-17% second-dimensional gel. We began by visually analyzing the changes at each week over time (weeks 1-9) in plasma protein spots from the same individual DES-administered male rats. We found the tendency for mercaptoalbumin (reduced form) spots to increase in week 1 in DES-administered rats. We also analyzed the quantitative alterations in α2u-globulin (AUG), a biomarker of endocrine disruptors, and found a two-week delay in expression in DES-administered rats in comparison to those not given DES (control rats). We then investigated the reproducibility of these results by using samples taken from multiple male rats in weeks 5, 7 and 9 to perform electrophoretic analysis three times for each individual, and observed the changes in AUG. AUG production was not seen in week 5 in the control rats, but was present at weeks 7 and 9. In DES-administered rats, expression was not observed at week 5 or 7, but was present at week 9. In addition, we identified the spots of AUG by Western blotting after electrophoresis. We consequently confirmed that AUG production delayed by two weeks in DES-administered rats, in comparison with control rats, which suggests that the effect of either a decline in AUG synthesis function or its depletion results in stunting of the reproductive organs, which was confirmed through autopsy.","PeriodicalId":15059,"journal":{"name":"Journal of capillary electrophoresis","volume":"53 1","pages":"51-56"},"PeriodicalIF":0.0,"publicationDate":"2009-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"73996304","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2009-12-01DOI: 10.2198/JELECTROPH.53.63
Chisato Harada, H. Tajima, S. Hirohashi, T. Kondo
A vacuum-driven antibody-reaction system enables high-throughput western blotting, where the antibodies pass through protein-transferred membrane and antibody-antigen reaction is completed within 30 min. With the vacuum-based device, two protein detection methods compatible with by laser scanning were compared; one with enhanced chemiluminescence reaction and the other using fluorescence-labeled antibody. Considering sensitivity, quantitative capability, reproducibility and labor intensiveness, we concluded that fluorescence-based detection is more suitable in combination with the vacuum-driven antibody-reaction system and a laser scanner.
{"title":"Toward high performance western blotting using vacuum-driven system and laser scanner; comparison between two signal detection methods based on chemiluminescent and immunofluorescent imaging","authors":"Chisato Harada, H. Tajima, S. Hirohashi, T. Kondo","doi":"10.2198/JELECTROPH.53.63","DOIUrl":"https://doi.org/10.2198/JELECTROPH.53.63","url":null,"abstract":"A vacuum-driven antibody-reaction system enables high-throughput western blotting, where the antibodies pass through protein-transferred membrane and antibody-antigen reaction is completed within 30 min. With the vacuum-based device, two protein detection methods compatible with by laser scanning were compared; one with enhanced chemiluminescence reaction and the other using fluorescence-labeled antibody. Considering sensitivity, quantitative capability, reproducibility and labor intensiveness, we concluded that fluorescence-based detection is more suitable in combination with the vacuum-driven antibody-reaction system and a laser scanner.","PeriodicalId":15059,"journal":{"name":"Journal of capillary electrophoresis","volume":"8 1","pages":"63-66"},"PeriodicalIF":0.0,"publicationDate":"2009-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"82463273","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2009-12-01DOI: 10.2198/JELECTROPH.53.57
T. Odake, T. Saheki, K. Kamezawa, Kozue Miyata, H. Takiguchi, H. Hotta, Onda Koki, Yasuaki Kojima, Jinxiang Li, K. Tsunoda
A miniaturized two-dimensional gel electrophoresis (2-DE) system was constructed with a multifilament-supporting (MFS) gel as a first-dimensional gel for isoelectric focusing (IEF). The MFS gel was 1 mm in diameter, had a multifilament yarn of acrylic as a gel-support, and could provide low-concentration (2.5%T) polyacrylamide with sufficient mechanical strength, resulting in fast IEF, and clear 2-D spots due to the relatively large pore and improved transfer efficiency from the first-dimensional gel to the second-dimensional one of especially high-molecular-weight (HMW) proteins. In this study, by using the MFS gel of 4 cm in length as a miniaturized IEF gel, three colored proteins were separated in 8 min, which was about twice faster than using the MFS gel of standard length (7 cm). The second-dimensional gel was also miniaturized to be 55 mm wide, 40 mm long and 0.75 mm thick. The low-concentration MFS gel after IEF was easily transferred onto the top of the miniaturized second-dimensional gel with a tweezers. By using the miniaturized 2-DE system, the HMW size marker was separated in 30 min by IEF and in 25 min by native-polyacrylamide gel electrophoresis (Native-PAGE), which was at least twice faster than by using the standard size 2-DE system using MFS gel. This miniaturized 2-DE system could be expected as a useful separation method for fast protein diagnosis and screening.
{"title":"Miniaturized two-dimensional gel electrophoresis of high-molecular-weight proteins using low-concentration multifilament-supporting gel for isoelectric focusing","authors":"T. Odake, T. Saheki, K. Kamezawa, Kozue Miyata, H. Takiguchi, H. Hotta, Onda Koki, Yasuaki Kojima, Jinxiang Li, K. Tsunoda","doi":"10.2198/JELECTROPH.53.57","DOIUrl":"https://doi.org/10.2198/JELECTROPH.53.57","url":null,"abstract":"A miniaturized two-dimensional gel electrophoresis (2-DE) system was constructed with a multifilament-supporting (MFS) gel as a first-dimensional gel for isoelectric focusing (IEF). The MFS gel was 1 mm in diameter, had a multifilament yarn of acrylic as a gel-support, and could provide low-concentration (2.5%T) polyacrylamide with sufficient mechanical strength, resulting in fast IEF, and clear 2-D spots due to the relatively large pore and improved transfer efficiency from the first-dimensional gel to the second-dimensional one of especially high-molecular-weight (HMW) proteins. In this study, by using the MFS gel of 4 cm in length as a miniaturized IEF gel, three colored proteins were separated in 8 min, which was about twice faster than using the MFS gel of standard length (7 cm). The second-dimensional gel was also miniaturized to be 55 mm wide, 40 mm long and 0.75 mm thick. The low-concentration MFS gel after IEF was easily transferred onto the top of the miniaturized second-dimensional gel with a tweezers. By using the miniaturized 2-DE system, the HMW size marker was separated in 30 min by IEF and in 25 min by native-polyacrylamide gel electrophoresis (Native-PAGE), which was at least twice faster than by using the standard size 2-DE system using MFS gel. This miniaturized 2-DE system could be expected as a useful separation method for fast protein diagnosis and screening.","PeriodicalId":15059,"journal":{"name":"Journal of capillary electrophoresis","volume":"77 1","pages":"57-61"},"PeriodicalIF":0.0,"publicationDate":"2009-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"88560341","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2009-09-01DOI: 10.2198/JELECTROPH.53.45
J. Suzuki
We previously confirmed that crude streptolysin O (SLO) consists of heterogeneous components with different isoelectric points (pIs) and hemolytic efficiency, as determined by the slope of the titration curve. The two preparations of SLO having different pI distributions were further characterized in this research. The pIs of the major components of the two SLO preparations are around pH 6 (acidic) and 7.5 (neutral). Although the neutral SLO was shown to be more hydrophobic than the acidic SLO based on the behavior on hydrophobic interaction chromatography, the capacities of the two types to bind to erythrocytes did not differ. On the other hand, the neutral SLO destroyed liposomes to some extent, whereas the acidic SLO did not, even at concentrations of 100 HD50 (50% hemolytic dose), suggesting that the mode of membrane damage by the two types of SLO are different. Furthermore, electron microscopic observation indicated that the acidic SLO preferably formed an irregular-shaped complex and semicircular pores on erythrocyte membranes, in contrast to the complete pore formation by the neutral SLO. Thus, we clarified that the two hemolytic forms of SLO have different pore formation efficiencies based on liposome membrane damage and electron micrographs of ghosts.
{"title":"Characterization of acidic and neutral streptolysin O","authors":"J. Suzuki","doi":"10.2198/JELECTROPH.53.45","DOIUrl":"https://doi.org/10.2198/JELECTROPH.53.45","url":null,"abstract":"We previously confirmed that crude streptolysin O (SLO) consists of heterogeneous components with different isoelectric points (pIs) and hemolytic efficiency, as determined by the slope of the titration curve. The two preparations of SLO having different pI distributions were further characterized in this research. The pIs of the major components of the two SLO preparations are around pH 6 (acidic) and 7.5 (neutral). Although the neutral SLO was shown to be more hydrophobic than the acidic SLO based on the behavior on hydrophobic interaction chromatography, the capacities of the two types to bind to erythrocytes did not differ. On the other hand, the neutral SLO destroyed liposomes to some extent, whereas the acidic SLO did not, even at concentrations of 100 HD50 (50% hemolytic dose), suggesting that the mode of membrane damage by the two types of SLO are different. Furthermore, electron microscopic observation indicated that the acidic SLO preferably formed an irregular-shaped complex and semicircular pores on erythrocyte membranes, in contrast to the complete pore formation by the neutral SLO. Thus, we clarified that the two hemolytic forms of SLO have different pore formation efficiencies based on liposome membrane damage and electron micrographs of ghosts.","PeriodicalId":15059,"journal":{"name":"Journal of capillary electrophoresis","volume":"7 1","pages":"45-50"},"PeriodicalIF":0.0,"publicationDate":"2009-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"85634786","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2009-09-01DOI: 10.2198/JELECTROPH.53.39
K. Tsuzaki, K. Kotani, Kazunori Yamada, Y. Sano, Yukiyo Matsuoka, Kentaro Okazaki, Toshiyuki Yamada, A. Shimatsu, N. Sakane
To prevent the development of atherosclerotic diseases, the Japanese guideline for prevention of atherosclerotic cardiovascular diseases (released from Japan Atherosclerosis Society in 2007) is clinically referred. In addition, the importance of evaluating not only the level but also the quality of low-density lipoprotein (LDL) has been advocated. Small dense LDL (sdLDL) is regarded as representation of the quality of LDL. With the LipoprintTM LDL subfraction system, utilizing non-denaturing polyacrylamide gel electrophoresis method which can evaluate several sdLDL-related parameters such as the area percentage of sdLDL, mean LDL particle size (mean LDL-PS), relative mobility in the form of the relative flotation rate (Rf), and LDL size-based phenotypic patterns. We attempted to assess the Japanese guideline for prevention of atherosclerotic cardiovascular diseases in terms of sdLDL-related parameters. We studied with 46 asymptomatic and cardiovascular disease-free dyslipidemic subjects (23 men, aged 30-58 years olds; 23 women, 31-58 years olds), who were within the primary prevention level. From the increased number of categories in the guideline, the following trends were observed; the smaller LDL-PS (p=0.002), the larger of area percentage of sdLDL (p=0.042), an increase in Rf (p=0.068), and an increase in the presence of pattern B (atherogenic phenotype) (p=0.031). These results suggested that, with the LipoprintTM system, the usage of Japanese guideline may clinically reflect well the sdLDL-related parameters, in particular the mean LDL-PS, the area percentage of sdLDL and the pattern B.
{"title":"The parameters related to small dense low-density lipoprotein evaluated by non-denaturing polyacrylamide gel electrophoresis method in dyslipidemic subjects : with reference to the Japanese guideline for prevention of atherosclerotic cardiovascular diseases","authors":"K. Tsuzaki, K. Kotani, Kazunori Yamada, Y. Sano, Yukiyo Matsuoka, Kentaro Okazaki, Toshiyuki Yamada, A. Shimatsu, N. Sakane","doi":"10.2198/JELECTROPH.53.39","DOIUrl":"https://doi.org/10.2198/JELECTROPH.53.39","url":null,"abstract":"To prevent the development of atherosclerotic diseases, the Japanese guideline for prevention of atherosclerotic cardiovascular diseases (released from Japan Atherosclerosis Society in 2007) is clinically referred. In addition, the importance of evaluating not only the level but also the quality of low-density lipoprotein (LDL) has been advocated. Small dense LDL (sdLDL) is regarded as representation of the quality of LDL. With the LipoprintTM LDL subfraction system, utilizing non-denaturing polyacrylamide gel electrophoresis method which can evaluate several sdLDL-related parameters such as the area percentage of sdLDL, mean LDL particle size (mean LDL-PS), relative mobility in the form of the relative flotation rate (Rf), and LDL size-based phenotypic patterns. We attempted to assess the Japanese guideline for prevention of atherosclerotic cardiovascular diseases in terms of sdLDL-related parameters. We studied with 46 asymptomatic and cardiovascular disease-free dyslipidemic subjects (23 men, aged 30-58 years olds; 23 women, 31-58 years olds), who were within the primary prevention level. From the increased number of categories in the guideline, the following trends were observed; the smaller LDL-PS (p=0.002), the larger of area percentage of sdLDL (p=0.042), an increase in Rf (p=0.068), and an increase in the presence of pattern B (atherogenic phenotype) (p=0.031). These results suggested that, with the LipoprintTM system, the usage of Japanese guideline may clinically reflect well the sdLDL-related parameters, in particular the mean LDL-PS, the area percentage of sdLDL and the pattern B.","PeriodicalId":15059,"journal":{"name":"Journal of capillary electrophoresis","volume":"124 1","pages":"39-43"},"PeriodicalIF":0.0,"publicationDate":"2009-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"79395552","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2009-06-01DOI: 10.2198/JELECTROPH.53.27
H. Kajiwara, A. Imamaki, Masatoshi Nakamura, K. Mita, Q. Xia, M. Ishizaka
Proteomic analysis of silkworm hemolymph at the fifth-instar day-3 larva stage was performed by combination of two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. Prominent spots on 2D-gel were analyzed after trypsinization and proteins were identified by Mascot and Sequest using the amino acid sequence data obtained from the genome sequence of silkworm. The gel images were tracked from the fourth-instar day-1 larva stage to the stage before adult moth for twenty-one days. The results of mass spectrometry analysis and changes in gel images are summarized in the silkworm proteome database (http://kaiko2ddb.dna.affrc.go.jp/cgi-bin/search_2DDB.cgi).
{"title":"Proteome analysis of silkworm 2. Hemolymph","authors":"H. Kajiwara, A. Imamaki, Masatoshi Nakamura, K. Mita, Q. Xia, M. Ishizaka","doi":"10.2198/JELECTROPH.53.27","DOIUrl":"https://doi.org/10.2198/JELECTROPH.53.27","url":null,"abstract":"Proteomic analysis of silkworm hemolymph at the fifth-instar day-3 larva stage was performed by combination of two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. Prominent spots on 2D-gel were analyzed after trypsinization and proteins were identified by Mascot and Sequest using the amino acid sequence data obtained from the genome sequence of silkworm. The gel images were tracked from the fourth-instar day-1 larva stage to the stage before adult moth for twenty-one days. The results of mass spectrometry analysis and changes in gel images are summarized in the silkworm proteome database (http://kaiko2ddb.dna.affrc.go.jp/cgi-bin/search_2DDB.cgi).","PeriodicalId":15059,"journal":{"name":"Journal of capillary electrophoresis","volume":"95 1","pages":"27-31"},"PeriodicalIF":0.0,"publicationDate":"2009-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"76844526","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2009-06-01DOI: 10.2198/JELECTROPH.53.33
H. Kajiwara, A. Imamaki, Masatoshi Nakamura, K. Mita, Q. Xia, M. Ishizaka
Proteins in the Malpighian tubes of silkworms were proteomically analyzed by combination of two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. Several proteins were identified using peptide mass fingerprinting by mass spectrometry and subsequent MS/MS analysis. In this analysis, the electrophoresis patterns of the Malpighian tubes were compared from the fourth-instar day-1 larva stage to the stage before adult moth. The results of mass spectrometry analysis and changes in gel images are summarized in the silkworm proteome database (http://kaiko2ddb.dna.affrc.go.jp/cgi-bin/search_2DDB.cgi).
{"title":"Proteome analysis of silkworm 3. Malpighian tube","authors":"H. Kajiwara, A. Imamaki, Masatoshi Nakamura, K. Mita, Q. Xia, M. Ishizaka","doi":"10.2198/JELECTROPH.53.33","DOIUrl":"https://doi.org/10.2198/JELECTROPH.53.33","url":null,"abstract":"Proteins in the Malpighian tubes of silkworms were proteomically analyzed by combination of two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. Several proteins were identified using peptide mass fingerprinting by mass spectrometry and subsequent MS/MS analysis. In this analysis, the electrophoresis patterns of the Malpighian tubes were compared from the fourth-instar day-1 larva stage to the stage before adult moth. The results of mass spectrometry analysis and changes in gel images are summarized in the silkworm proteome database (http://kaiko2ddb.dna.affrc.go.jp/cgi-bin/search_2DDB.cgi).","PeriodicalId":15059,"journal":{"name":"Journal of capillary electrophoresis","volume":"1 1","pages":"33-38"},"PeriodicalIF":0.0,"publicationDate":"2009-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"79358959","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2009-06-01DOI: 10.2198/JELECTROPH.53.19
H. Kajiwara, A. Imamaki, Masatoshi Nakamura, K. Mita, Q. Xia, M. Ishizaka
Proteome analysis of silkworm (Bombyx mori L.) fat bodies was performed on the fifth-instar day-3 larva stage and the expression of proteins separated on two-dimensional polyacrylamide gels was tracked from the fourth-instar day-1 larva stage to the stage before adult moth. Proteins on the 2D-gels were excised manually and treated with 4-vinylpyridine for alkylation. Each spot was analyzed by capillary high-performance liquid chromatography coupled with ion-trap mass spectrometry after proteolysis using trypsin. In the previous report1), the amino acid sequence database obtained from the Drosophila genome was used for the protein identification. This analysis used the amino acid sequence data elucidated from the silkworm genome. Additionally, protein expression in fat bodies was compared with that at other stages and in other tissues described in the silkworm proteome database (http://kaiko2ddb.dna.affrc.go.jp/cgi-bin/search_2DDB.cgi). Several unidentified proteins from the previous report1) were identified with high Mascot scores. Induction and reduction of proteins during metamorphosis were observed as changes in spot concentration on sequential 2D-gels on the web.
对家蚕(Bombyx mori L.)第5龄第3天幼虫进行了脂肪体蛋白质组学分析，并从第4龄第1天幼虫到成蛾前，用二维聚丙烯酰胺凝胶分离蛋白的表达情况进行了跟踪。手工切除2d凝胶上的蛋白质并用4-乙烯基吡啶进行烷基化处理。每个斑点经胰蛋白酶水解后，采用毛细管高效液相色谱-离子阱质谱联用分析。在之前的报道1)中，我们使用果蝇基因组中获得的氨基酸序列数据库进行蛋白质鉴定。利用家蚕基因组的氨基酸序列数据进行分析。此外，将脂肪体中的蛋白质表达与蚕蛋白质组数据库(http://kaiko2ddb.dna.affrc.go.jp/cgi-bin/search_2DDB.cgi)中描述的其他阶段和其他组织中的蛋白质表达进行了比较。从先前的报道中鉴定出几个未确定的蛋白质(1)具有较高的Mascot分数。在变形过程中，蛋白质的诱导和减少被观察到，在网络上连续的2d凝胶上，斑点浓度的变化。
{"title":"Proteome analysis of silkworm 1. Fat body","authors":"H. Kajiwara, A. Imamaki, Masatoshi Nakamura, K. Mita, Q. Xia, M. Ishizaka","doi":"10.2198/JELECTROPH.53.19","DOIUrl":"https://doi.org/10.2198/JELECTROPH.53.19","url":null,"abstract":"Proteome analysis of silkworm (Bombyx mori L.) fat bodies was performed on the fifth-instar day-3 larva stage and the expression of proteins separated on two-dimensional polyacrylamide gels was tracked from the fourth-instar day-1 larva stage to the stage before adult moth. Proteins on the 2D-gels were excised manually and treated with 4-vinylpyridine for alkylation. Each spot was analyzed by capillary high-performance liquid chromatography coupled with ion-trap mass spectrometry after proteolysis using trypsin. In the previous report1), the amino acid sequence database obtained from the Drosophila genome was used for the protein identification. This analysis used the amino acid sequence data elucidated from the silkworm genome. Additionally, protein expression in fat bodies was compared with that at other stages and in other tissues described in the silkworm proteome database (http://kaiko2ddb.dna.affrc.go.jp/cgi-bin/search_2DDB.cgi). Several unidentified proteins from the previous report1) were identified with high Mascot scores. Induction and reduction of proteins during metamorphosis were observed as changes in spot concentration on sequential 2D-gels on the web.","PeriodicalId":15059,"journal":{"name":"Journal of capillary electrophoresis","volume":"9 1","pages":"19-26"},"PeriodicalIF":0.0,"publicationDate":"2009-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"88631712","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
K. Kanamori, M. Kameko, H. Kitamura, Junichi Nishimaki, K. Shiba, Kenji Sato
We measured the urinary excretion of retinol-binding protein (RBP), β 2 -microglobulin ( β 2 -m), albumin, and N-acetyl- β -D-glucosaminidase as well as serum glucose, RBP, β 2 -m, hemoglobin A1c, and creatinine in type 2 diabetes mellitus 158 patients. In urinary RBP, there was a significant difference between the patients who had nephropathy and those without complications. In the cases with nephropathy, urinary RBP had a significantly high value, but no significant difference was observed in the case with retinopathy. There were no significant correlations between urinary RBP and serum RBP. The index of correlation was 0.351. According The main for all three had molecular urinary RBP4, kDa for cases retinopathy, a and an light-colored band seen at 55 kDa, the possibility of early detection of retinopathy by examining these bands.
{"title":"The usefulness of electrophoretic analysis of urinary retinol-binding protein and retinol-binding protein 4 in type 2 diabetes mellitus patients","authors":"K. Kanamori, M. Kameko, H. Kitamura, Junichi Nishimaki, K. Shiba, Kenji Sato","doi":"10.2198/JELECTROPH.53.7","DOIUrl":"https://doi.org/10.2198/JELECTROPH.53.7","url":null,"abstract":"We measured the urinary excretion of retinol-binding protein (RBP), β 2 -microglobulin ( β 2 -m), albumin, and N-acetyl- β -D-glucosaminidase as well as serum glucose, RBP, β 2 -m, hemoglobin A1c, and creatinine in type 2 diabetes mellitus 158 patients. In urinary RBP, there was a significant difference between the patients who had nephropathy and those without complications. In the cases with nephropathy, urinary RBP had a significantly high value, but no significant difference was observed in the case with retinopathy. There were no significant correlations between urinary RBP and serum RBP. The index of correlation was 0.351. According The main for all three had molecular urinary RBP4, kDa for cases retinopathy, a and an light-colored band seen at 55 kDa, the possibility of early detection of retinopathy by examining these bands.","PeriodicalId":15059,"journal":{"name":"Journal of capillary electrophoresis","volume":"18 1","pages":"7-12"},"PeriodicalIF":0.0,"publicationDate":"2009-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"85197726","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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