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Volume 44, Issue 1
Displaying 1-8 of 8 articles from this issue
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Hitomi Mizuma, Nao Yamaguchi, Takashi Manabe2000 Volume 44 Issue 1 Pages 1-7
Published: March 15, 2000
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESSThe procedure to quantitate human plasma proteins separated by non-denaturing two-dimensional gel electrophoresis (2-DE) was examined using micro size gels and a commercially available software of image analysis. Varying volumes of a human plasma sample were loaded on micro 2-DE gels and the stained gels were subjected to image acquisition and image analysis. Plasma proteins were identified on the 2-DE gels referring to an “identification map, ” which has been prepared by blotting-immunochemical staining. Integrated density and loaded volume of plasma were linearly related for most of the spots, but saturation of integrated density was observed for albumin and some major plasma proteins. Various quantities (0.5-100μg) of purified human albumin were subjected to micro 2-DE and image analysis, and a quantity standard curve was prepared. The quantities and relative contents of plasma proteins, calculated from the standard curve as albumin equivalent values, generally coincided with those reported in literatures.View full abstractDownload PDF (3225K) -
Mayumi Imoto, Hyogo Sinohara, Ikunosuke Sakurabayashi, Kazuhiko Yamamo ...2000 Volume 44 Issue 1 Pages 9-14
Published: March 15, 2000
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESSWe encountered a case of IgD-type multiple myeloma in which we could not identify the type of light (L) chains of the IgD M-protein in the patient's serum by conventional immunoelectrophoresis or immunofixation electrophoresis. To clarify the cause of this failure, the immunochemical and structural properties of IgD in the patient's serum were characterized. The immunochemical reactivity of IgD M-protein against anti-λ anti- bodies was extremely low and the IgD-IgG complex was present in the patient's serum. The complex was dissociated in the presence of SDS, indicating that the association was formed via non-covalent binding. In a reassociation test, IgG prepared from normal subjects did not form a complex with the patient's IgD pretreated with a reducing agent, but formed a complex with IgD pretreated with papain. These results suggest that IgG formed a complex with the Fab of the patient's IgD.View full abstractDownload PDF (6994K) -
Shuichi Hashimoto, Toshiyuki Toen2000 Volume 44 Issue 1 Pages 15-19
Published: March 15, 2000
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESSTo detect sensitively alkaline phosphatase (ALP) activity in gels after nonreduced-type SDS- or native (0.1% Nonidet P-40)-polyacrylamide gel electrophoresis (PAGE) using glycine, effects of glycine and Zn2+ on its activity were analyzed by active staining with indigogenic dye. Both activities of ALP in SDS- and native-PAGEs with 192mM glycine decreased to one-half those of PAGEs with 40mM borate, respectively. Once the enzyme was inhibited by glycine, it could not be reactivated by the addition of Zn2+. On the contrary, when 192mM glycine was premixed with Zn2+, ALP activity in PAGE increased according to increment of zinc concentration and the maximal enzyme activity induced by 0.1mM Zn2+ was equal to or higher than that after PAGEs with borate. 65Zn complexes in the electrophoresis proteins moved to the only anode side in gels during PAGEs with glycine. By the experiment using Eriochrom Black T, it was found that complexing capacity of 200mM glycine disappeared by more than 0.1mM Zn2+. This study explained that glycine removes zinc from ALP, and that detection sensitivity of ALP activity in gels after PAGEs is strikingly enhanced by premixing glycine electrode buffers with zinc corresponding to one-two thousandth of glycine concentration.View full abstractDownload PDF (4366K) -
Youji Shimazaki, Mai Fujiwara, Takashi Manabe2000 Volume 44 Issue 1 Pages 21-25
Published: March 15, 2000
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESSWe have reported that the third component of complement (C3) is observed in the pattern of microscale non-denaturing two-dimensional electrophoresis (2-DE) after removal of IgG and IgA using protein A agarose and antibodies.1) For the investigation of constitutive polypeptides of the C3, the constituent polypeptides of human plasma proteins were analyzed using non-denaturing isoelectric focusing (IEF) in the first dimension and treated with mercaptoethanol/SDS, and then subjected to the second dimensional SDS electrophoresis (non-denaturing IEF/reduced-SDS PAGE). The α chain of C3 spot was detected in the pattern of non-denaturing IEF/reduced-SDS PAGE, but, the β chain of C3 spot was hidden by albumin spot. After albumin was removed using polyethylene glycol, both C3α and C3β spots were observed in the pattern of the non-denaturing IEF/reduced-SDS PAGE. The combining analysis of C3 in the non-denaturing 2-DE and the constituent polypeptides in the non-denaturing IEF/reduced-SDS PAGE can be useful for the analysis of C3 and its fragments during the activation of complements.View full abstractDownload PDF (4031K) -
Hirokazu Kouguchi, Yoshimasa Sagane, Toshihiro Watanabe, Tohru Ohyama2000 Volume 44 Issue 1 Pages 27-34
Published: March 15, 2000
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESSClostridium botulinum type C strain Stockholm (C-St) produced two types of progenitor toxins (M and L). The purified L toxin consisted of neurotoxin (NT), nontoxic-nonhemagglutinin (NTNHA) and hemagglutinin (HA) components which could be separated into four subcomponents, HA-33, HA-17, HA-22-23 and HA-55. On the other hand, purified M toxin lacked HA components. We isolated all components from the progenitor L toxin by following chromatographic methods. The L toxin was separated into an NT and NTNHA/HAs complex via Mono Q anion exchange column chromatography. Using gel filtration with a TSK-gel HW-55S column in the presence of 6M guanidine hydrochloride, the NTNHA/HAs complex derived from the L toxin could be separated into three fractions, NTNHA single component, HA-55/33 mixture and HA-22-23/17 mixture. Then, the HA-55/33 mixture was separated into each component using Mono S cation exchange column chromatography in the presence of 8M urea. The HA-22-23/17 mixture was also separated from each other with Superose 12 gel filtration column chromatography in the presence of 8M urea. The separated NT, NTNHA and HA subcomponents were homogenous on SDS-PAGE, and were identified to be C-St progenitor toxin gene products based on their N-terminal amino acid sequence analysis. This study presents for the first time an attempt to isolate components from the progenitor toxin. Although reassociation of the isolated components was also attempted via mixing of NTNHA, HA-55, HA-33, HA-22-23 and HA-17, HA activity of the mixture was lower than that of parent NTNHA/HAs complex.View full abstractDownload PDF (6011K) -
Mayumi Imoto, Hyogo Sinohara, Toshiyuki Akiyama, Itaru Furuta2000 Volume 44 Issue 1 Pages 35-37
Published: March 15, 2000
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESSSensitivity and specificity of commercial anti-human free light (L) chains antibodies (DAKO Co. and Binding Site Co.) for detection of Bence Jones protein (BJP) were compared with those of conventional anti-human L chains antibodies (DAKO Co.), and the usefulness and problems of these antibodies were investigated. When BJP positive serum and urine were used, sensitivity of anti-human free L chains antibodies against BJP was lower than that of conventional anti-human L chains antibodies in many cases. The anti-human free L chains antibodies also reacted with not only free L chains but also with intact Igs, i.e., L chains linked to H chains. Therefore, care must be taken when the antibodies are used in a specific sensitive method such as immunoblotting.View full abstractDownload PDF (4775K) -
Shiro Iijima, Saeko Shimura, Atsuko Tsuchida, Miyako Kimura, Kiyoko Sh ...2000 Volume 44 Issue 1 Pages 39-42
Published: March 15, 2000
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESSSEPARAX-SP membrane is used generally as supporting medium for serum protein fractionation in many clinical laboratories, but it has not been used for isoelectric focusing (IEF). Although the exclusive cellulose acetate membrane for IEF is a SEPARAX-EF membrane, we studied usefulness of SEPARAX-SP membrane as supporting medium for cellulose acetate membrane IEF as comparison with SEPARAX-EF membrane. Serum sample was applied and electrophoresed in according to our method. After electrophoresis, all cellulose acetate membranes were stained with Coomassie brilliant blue G-250. The both membranes showed clear and similar isoelectrophoretic patterns of serum protein. Furthermore, we confirmed that isoelectric focusing can be carried out on six layers of SEPARAX-SP membranes as same as SEPARAX-EF membrane. All six SEPARAX-SP membranes were showed clear isoelectrophoretic patterns of serum protein, but detected serum protein concentration was decreased in lower layers of SEPARAX-SP membrane. Therefore, we examined the distribution of protein among the six layers of SEPARAX-SP membrane and compared to those of SEPARAX-EF membrane. The ratio of protein distribution between top membrane and bottom membrane was 2:1 on SEPARAX-SP membrane against to the ratio of 30:1 on SEPARAX-EF membrane. Furthermore, protein distribution of six layers on SEPARAX-SP membrane was more uniform than that on SEPARAX-EF membrane. These results indicated that SEPARAX-SP membrane was useful supporting medium for cellulose acetate membrane IEF.View full abstractDownload PDF (4462K) -
Yuriko Kurihara, Kiyoko Shiba, Kazunori Yokoyama, Sachiko Kamei2000 Volume 44 Issue 1 Pages 43-46
Published: March 15, 2000
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESSWe measured total protein (TP) and immunoglobulin levels in cyst fluid taken from 19 patients who had postoperative maxillary cysts (POMC). In the 19 cases, TP, IgG, IgA, and IgM levels were 7990±3558 (mean±1 SD), 1890±1085, 648±1085, and 664±1489mg/dl, respectively. Immunoglobulin correlations for IgG and IgA, IgG and IgM, and IgA and IgM were 0.706, 0.654, and 0.951, respectively. Using cellulose acetate membrane electrophoresis, we also examined the glycosaminoglycan fractions in POMC fluid. Four (21.1%) of the 19 samples showed a separation-impossible fraction of glycosaminoglycans, while 15 samples (78.9%) showed a separation-possible fraction. By digestion with pronase, most samples could be separated into 3 bands: an unknown band, a hyaluronic acid band, and a chondroitin sulfate A band (CSA). The unknown bands of samples having a separation-impossible fraction and of those having a separation-possible fraction were 32.4±3.5% (mean±1 SD) and 58.4±9.6%. The hyaluronic acid bands in the two fractions were 56.0±12.9% and 30.7±8.1%, and the CSA bands were 5.7±8.0% and 8.7±4.3%. On the unknown and CSA bands, samples having a separation-possible fraction were higher than those having a separation-impossible fraction. On the hyaluronic acid band, samples having a separation-impossible fraction were higher than those having a separation-possible fraction, but there were no significant differences.View full abstractDownload PDF (2400K)
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