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1.
In the present experiment, a new slot comb was designed in order to form a wide and sloped sample well on the stacking gel of electrophoresis. Using this slot comb, a gradient of the reagent layer of interest can be easily formed transversely on the gel that is perpendicular to the direction of electrophoresis. Thus, the protein sample overlaid on the agent migrates across the gradient layer during electrophoresis to produce a continuous electrophoretic band reflecting the interaction between the protein and reagent. This new slot comb (tentatively called slope comb) was applied to the following two experiments. In the first experiment, in combination with this comb and a reducing agent, 2-mercaptoethanol, the reducing steps of cross-linked axonemal proteins with o-iodosobenzoic acid (OIBA) were analyzed electrophoretically, enabling visualization of the reducing pattern of each axonemal protein in a single experiment. The results obtained indicate that alpha and beta tubulins are cross-linked differently by OIBA. In the second experiment, the formation of the Ca2+ gradient layer using this slope comb could electrophoretically differentiate the Ca2+ sensitivity of three Ca(2+)-binding proteins. 相似文献
2.
Y S Ho P J Sheffield J Masuyama H Arai J Li J Aoki K Inoue U Derewenda Z S Derewenda 《Protein engineering》1999,12(8):693-700
Platelet-activating factor acetylhydrolases (PAF-AHs) are unique PLA2s which hydrolyze the sn-2 ester linkage in PAF-like phospholipids with a marked preference for very short acyl chains, typically acetyl. The recent solution of the crystal structure of the alpha(1) catalytic subunit of isoform Ib of bovine brain intracellular PAF-AH at 1.7 A resolution paved the way for a detailed examination of the molecular basis of substrate specificity in this enzyme. The crystal structure suggests that the side chains of Thr103, Leu48 and Leu194 are involved in substrate recognition. Three single site mutants (L48A, T103S and L194A) were overexpressed and their structures were solved to 2.3 A resolution or better by X-ray diffraction methods. Enzyme kinetics showed that, compared with wild-type protein, all three mutants have higher relative activity against phospholipids with sn-2 acyl chains longer than an acetyl. However, for each of the mutants we observed an unexpected and substantial reduction in the V(max) of the reaction. These results are consistent with the model in which residues Leu48, Thr103 and Leu194 indeed contribute to substrate specificity and in addition suggest that the integrity of the specificity pocket is critical for the expression of full catalytic function, thus conferring very high substrate selectivity on the enzyme. 相似文献
3.
M. Masuyama 《Biometrical journal. Biometrische Zeitschrift》1991,33(6):725-733
A mathematical model of the linearized growth curve and its physiological interpretation by the introduction of proper age, which is proportional to the chronological age, are presented here. In the second phase, but not in the first phase, this constant of proportionality is highly correlated with the age at menarche. 相似文献
4.
In the present study, anti-metastatic effect of Z-100 on the spontaneous pulmonary metastases of Lewis lung carcinoma (3LL)
was examined in an attempt to regulate suppressor T cells. When Z-100 (10 mg/kg) was daily injected i.p. after 3LL inoculation,
survival rate of these mice was increased significantly (p<0.05). In addition, the number of pulmonary metastatic colonies of 3LL in Z-100-treated mice were significantly decreased
by 38% at 21 days, as compared with that of control mice (p<0.05). Along with the decrease of pulmonary metastases, suppressor cell activity was also gradually reduced in these mice,
as compared with that of control mice. When splenic suppressor cells (5×107 cells) from 3LL-bearing mice were adoptively transferred into normal mice (recipients) just before inoculation of 3LL, the
development of pulmonary metastases in recipients was significantly accelerated. However, splenocytes from 3LL-bearing mice
treated with Z-100 did not affect the development of pulmonary metastasis. The potential to accelerate the metastasis of splenic
mononuclear cells from 3LL-bearing mice was decreased significantly by the treatment with anti-Thy 1.2 monoclonal antibody
(mAb), anti-Lyt 2.2 mAb or anti-CD11b mAb followed by complement. IL-4 activity in the sera of 3LL-bearing mice was detected
15 days after tumor inoculation (13 pg/ml) and gradually increased (18 pg/ml) 20 days after tumor inoculation. However, when
Z-100 (10 mg/kg) was daily injected i.p., IL-4 activity in sera was decreased significantly, and the IL-4 activity was not
detected in these mice on day 20. These results suggest that Z-100 could inhibit the pulmonary metastases in 3LL-bearing mice
through the inhibition of suppressor T cell activity and a possible candidate of its effector molecule, IL-4. 相似文献
5.
K Nakamura E Masuyama T Suzaki Y Shigenaka 《Archives of biochemistry and biophysics》1982,214(1):172-179
Axonemes of sperm flagella were prepared from the annelid, Tylorrhynchus heterochaetus. Dialysis of the axonemes against 1 mm Tris-HCl buffer (pH 8.3)-0.1 mm EDTA-0.1 mm dithiothreitol (Tris-EDTA solution) caused disintegration of typical 9 + 2 microtubules into each doublet, resulting in extraction of one-third of the protein and almost all ATPase activity. Agarose polyacrylamide gel electrophoresis of the extract showed the presence of three kinds of dyneins actively stained for ATPase (designated as bands I, II, and III) and two non-ATPase proteins (bands IV, V). The polypeptide components of each dynein molecule and intact axoneme were analyzed by subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis to obtain the following results: (1) In the highmolecular-weight region, the intact axonemes yield two major polypeptides with molecular weights of 365,000 and 345,000 (designated as bands A and B, respectively) and three minor polypeptides, 310,000, 290,00, and 270,00 (C1, C2, C3). (2) All three dyneins contain A-band polypeptide as a common polypeptide component. In addition, band I dynein and band II dynein also contain B and C1 polypeptides, and C3 polypeptide, respectively, as high-molecular-weight components. (3) Band III dynein also contains four polypeptides in the lower molecular-weight region, which migrate similarly with those of 21 S dynein from sea urchin sperm flagella or 18 S dynein from Chlamydomonas. 相似文献
6.
A brief review of the genetic studies on ribonuclease P (RNase P) fromEscherichia coli is presented. Temperature-sensitive mutants ofE. coli defective in tRNA processing were isolated by screening cells which were unable to synthesize a suppressor tRNA at restrictive temperature. Structural analysis of accumulated tRNA precursors showed that the isolated mutants were defective in RNase P activity. Analyses of the mutants revealed that the enzyme is essential for the synthesis of all tRNA molecules in cells and that the enzymes consists of two subunits. Analyses of the isolated mutants revealed a possible domain structure of the RNA subunit of the enzyme.Abbreviations
E. coli
Escherichia coli
- RNase P
ribonuclease P 相似文献
7.
Toshihiko Sudo Xiaoxian Zhao Yoko Wakamatsu Miki Shibahara Nobuhiko Nomura Tadaatsu Nakahara Akemi Suzuki Yoshiro Kobayashi Chunyuan Jin Takehide Murata Kazunari K. Yokoyama 《Cytotechnology》2000,33(1-3):259-264
Four analogs of succinoyl trehalose lipid-3 (STL-3)with saturated even-number or odd-number carbonchains, and unsaturated or halogenated fatty acidswere examined for their ability to inhibit the growthand induce the differentiation of HL-60 humanpromyelocytic leukemia cells. The optimalconcentration of STL-3 at which such activities wererecognized was closed to the critical micelleconcentration of STL-3. Analog of STL-3 witheven-number or odd-number carbon chain and unsaturatedfatty acids strongly inhibited growth and induced thedifferentiation of HL-60 cells, as evaluated in termsof nitroblue tetrazilium-reducing activity and theappearance of the CD36 antigen. An analog of STL-3with halogenated fatty acids significantly inhibitedproliferation but only induced the differentiation ofHL-60 cells. Our results indicate that the effects ofSTL-3 and its analogs on HL-60 cells depend on thestructure of the hydrophobic moiety of STL-3.These authors contributed equally to this work 相似文献
8.
Sugawara K Nakamura H Kanai M Iwata S Kawai Y Taki Y Yodoi J Takabayashi A 《Redox report : communications in free radical research》2002,7(3):165-169
Surgical stress is difficult to evaluate quantitatively. It has been reported that mitochondrial membrane potential (delta psi(m)) in the peripheral blood lymphocytes (PBLs) is decreased by surgical stress. Thioredoxin (TRX), a small protein with redox-active dithiol/disulfide in the active site, is induced by a variety of oxidative stresses and secreted from the cells. Accumulating evidence shows that plasma levels of TRX are elevated in oxidative stress-associated disorders. In the present study, we examined plasma levels of TRX in cases undergoing operations for gastrointestinal cancer. Plasma levels of TRX were significantly elevated on the first postoperative day compared with the pre-operative levels. The changes in the plasma TRX levels tended to show an inverse relationship with the changes in delta psi(m) in PBLs, which shows a significant decrease caused by surgical stress. Plasma TRX levels as well as delta psi(m) in PBLs are valuable markers to evaluate surgical stress. 相似文献
9.
Yamanaka K Saito Y Yamamori T Urano Y Noguchi N 《The Journal of biological chemistry》2011,286(28):24666-24673
24(S)-Hydroxycholesterol (24S-OHC) produced by cholesterol 24-hydroxylase expressed mainly in neurons plays an important physiological role in the brain. Conversely, it has been reported that 24S-OHC possesses potent cytotoxicity. The molecular mechanisms of 24S-OHC-induced cell death have not yet been fully elucidated. In this study, using human neuroblastoma SH-SY5Y cells and primary cortical neuronal cells derived from rat embryo, we characterized the form of cell death induced by 24S-OHC. SH-SY5Y cells treated with 24S-OHC exhibited neither fragmentation of the nucleus nor caspase activation, which are the typical characteristics of apoptosis. 24S-OHC-treated cells showed necrosis-like morphological changes but did not induce ATP depletion, one of the features of necrosis. When cells were treated with necrostatin-1, an inhibitor of receptor-interacting serine/threonine kinase 1 (RIPK1) required for necroptosis, 24S-OHC-induced cell death was significantly suppressed. The knockdown of RIPK1 by transfection of small interfering RNA of RIPK1 effectively attenuated 24S-OHC-induced cell death. It was found that neither SH-SY5Y cells nor primary cortical neuronal cells expressed caspase-8, which was regulated for RIPK1-dependent apoptosis. Collectively, these results suggest that 24S-OHC induces neuronal cell death by necroptosis, a form of programmed necrosis. 相似文献
10.
Tsukio Masegi Satoshi Nakamura Masami Fukuoka Kazuo Kitai Yataro Ichikawa Naoki Watanabe Yoshiro Niitsu 《Biotechnology letters》1993,15(11):1107-1110
Summary We prepared various TNF- derivatives by protein engineering techniques. Mutant 471, in which 7 N-terminal amino acids were deleted and Pro8Ser9Asp10 was replaced by ArgLysArg, had a 8-fold higher antitumor activity against mouse L929 cells than wild-type TNF-. The additional substitution of Ala156 or Leu157 by more hydrophobic amino acids enhanced the activity of mutant 471. These results suggested that the combinational mutations in the N- and C-terminal regions of TNF- are effective for the improvement of antitumor activity. 相似文献