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111.
Eukaryotic ribonuclease (RNase) H2 consists of one catalytic and two accessory subunits. Several single mutations in any one of these subunits of human RNase H2 cause Aicardi-Goutières syndrome. To examine whether these mutations affect the complex stability and activity of RNase H2, three mutant proteins of His-tagged Saccharomyces cerevisiae RNase H2 (Sc-RNase H2*) were constructed. Sc-G42S*, Sc-L52R*, and Sc-K46W* contain single mutations in Sc-Rnh2Ap*, Sc-Rnh2Bp*, and Sc-Rnh2Cp*, respectively. The genes encoding the three subunits were coexpressed in Escherichia coli, and Sc-RNase H2* and its derivatives were purified in a heterotrimeric form. All of these mutant proteins exhibited enzymatic activity. However, only the enzymatic activity of Sc-G42S* was greatly reduced compared to that of the wild-type protein. Gly42 is conserved as Gly10 in Thermococcus kodakareansis RNase HII. To analyze the role of this residue, four mutant proteins, Tk-G10S, Tk-G10A, Tk-G10L, and Tk-G10P, were constructed. All mutant proteins were less stable than the wild-type protein by 2.9-7.6 degrees C in T(m). A comparison of their enzymatic activities, substrate binding affinities, and CD spectra suggests that the introduction of a bulky side chain into this position induces a local conformational change, which is unfavorable for both activity and substrate binding. These results indicate that Gly10 is required to make the protein fully active and stable. 相似文献
112.
113.
Arinobu T Hattori H Iwai M Ishii A Kumazawa T Suzuki O Seno H 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2002,776(1):107-113
Haloperidol and its two metabolites, reduced haloperidol and 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP) in human plasma and urine were analyzed by HPLC-MS using a new polymer column (MSpak GF-310), which enabled direct injection of crude biological samples without pretreatment. Recoveries of haloperidol and reduced haloperidol spiked into plasma were 64.4-76.1% and 46.8-50.2%, respectively; those for urine were 87.3-99.4% and 94.2-98.5%, respectively; those of CPHP for both samples were not less than 92.7%. The regression equations for haloperidol, reduced haloperidol and CPHP showed good linearity in the ranges of 10-800, 15-800 and 400-800 ng/ml, respectively, for both plasma and urine. Their detection limits were 5, 10 and 300 ng/ml, respectively, for both samples. Thus, the present method was sensitive enough for detection and determination of high therapeutic and toxic levels for haloperidol and its metabolites present in biological samples. 相似文献
114.
Shigenori Tanaka Takanori Nakamura Takashi Morita Sadaaki Iwanaga 《Biochemical and biophysical research communications》1982,105(2):717-723
Exposure of amebocytes to bacterial endotoxins (lipopolysaccharides, LPS) results in the activation of the coagulation system, which consists of several protein components. During the separation of these components, a potent anticoagulant, named tentatively anti-LPS factor, which inhibits the endotoxin-mediated coagulation reaction, was found in both amebocytes from the hemolymphs of and . The principle purified partially from amebocyte lysate had a molecular weight less than 10,000, as judged with the ordinary gelfiltration experiment. It inhibited specifically the activation of factor B, which has recently been characterized to be a coagulation factor highly sensitive to LPS, but it did not inhibit the activities of the active factor B and the active clotting enzyme separated from the lysate. The inhibitory activity of anti-LPS factor disappeared almost completely by the treatments with pronase-P and subtilisin, suggesting its polypeptide-like substance, but it resisted to a boiling treatment. A possible site of the anticoagulant action on the coagulation system was discussed. 相似文献
115.
Mitotic events from prophase to telophase are defined by morphology or movement of chromatin, nuclear envelope, centrosomes and spindles. Live-cell imaging is useful for characterizing the whole chromosome segregation process in the living state. In this study, we constructed three quadruple-fluorescent MDA435 cell lines in which chromatin, kinetochores, nuclear envelope and either inner centromere, microtubules or centrosomes/spindles were differentially visualized with cyan, green, orange and red fluorescent proteins (ECFP, EGFP, mKO and DsRed). Each mitotic stage of the individual cells could be identified by capturing live-cell images without the requirement of fixing or staining steps. In addition, we obtained four-color time-lapse images of one cell line, MDA-Auro/imp/H3/AF, from prophase to metaphase and from early anaphase to telophase. These quadruple-fluorescent cell lines will be useful for precisely analyzing the mitotic events from prophase through to telophase in single cells in the future. 相似文献
116.
117.
Satoru Fujihiro Ryusuke Higuchi Shin Hisamatsu Shigenori Sonoki 《Applied microbiology and biotechnology》2009,82(5):853-860
The white-rot fungus T. versicolor UAMH 8272 produced two groups of laccases, each of which included several isoforms showing different isoelectric points (pI). Group 1 and group 2 laccases, respectively, displayed higher pI 5–6 and lower pI 3–4. Of the four cloned full-length laccase cDNAs, Lac 1 and Lac 4 were expressed in the heterologous protein expression
system using Aspergillus oryzae. The measured pI of each Lac 1 and Lac 4 expressed in A. oryzae was lower than that of pI predicted from the amino acid composition. With this regard, isoelectric focusing of Lac 1 showed the presence of multiple
protein bands in the 3.0–4.0 pI range, although the predicted pI value of Lac 1 was 4.7. Similarly, Lac 4 exhibited a pI value which was lower than that predicted (3.6 vs. 4.3, respectively). In all tested hydroxyPCBs, higher chlorinated hydroxyPCBs
were less susceptible to in vitro degradation by laccase than lower chlorinated hydroxyPCBs. Although Lac 4 showed a generally
higher activity than Lac 1, the two laccases were characterized by quite different substrate specificity toward two hydroxy-tetrachlorobiphenyl
congeners. Two metabolites were obtained from the metabolism of hydroxy-pentachlorobiphenyl: a ten chlorine-substituted dimer
with a C–O bond, and one with a C–C bond. 相似文献
118.
Maurizio Pellecchia Gerhard Wider Hideo Iwai Kurt Wüthrich 《Journal of biomolecular NMR》1997,10(2):193-197
A novel 2D NMR experiment, 2D HE(NE)HGHH, is presented for the assignment ofarginine side chain 1H and 15N resonances inuniformly 15N-labeled proteins. Correlations between1H, 1Hand 1H are established on the basis of3J(15N,1H) heteronuclear scalarcoupling constants, and sequence-specific assignments are obtained by overlapof these fragments with 1H chemical shiftsobtained by assignment procedures starting from the polypeptide backbone.Since guanidino protons exchange quite rapidly with the bulk water, the 2DHE(NE)HGHH pulse scheme has been optimized to avoid saturation and dephasingof the water magnetization during the course of the experiment. As anillustration, arginine side chain assignments are presented for two uniformly15N-labeled proteins of 7 and 23 kDa molecular weight. 相似文献
119.
N Iwai M Matsunaga K Ogawa A Matsumori H Yoshida E Ohta C Kawai 《Biochemical and biophysical research communications》1987,143(1):288-293
Cos 7 cells transfected with human atrial natriuretic polypeptide (hANP) gene with SV40 enhancer and replication origin sequences expressed hANP gene. The expressed RNA was indistinguishable from native hANP mRNA and the transcribed protein seemed to be properly processed to alpha-hANP and beta-hANP. This system provides a useful approach to investigate the processing of hANPs and the structure-function relationship of amino acid sequences of hANPs. 相似文献
120.
T cell subsets responsible for clearance of Sendai virus from mouse lungs determined by adoptive transfer of immune spleen cell fractions to infected nude mice. T cells with antiviral activity developed in spleens by 7 days after intranasal infection. Spleen cell fractions depleted of Lyt-2+, Lyt-1+, or L3T4+ cells showed antiviral activity in vivo, although the degree of the activity was lower than that of control whole spleen cells. The antiviral activity of the Lyt-2+ cell-depleted fraction was consistently higher than that of L3T4+ (Lyt-1+)-depleted cells. In vitro cytotoxic activity against Sendai virus-associated, syngeneic lipopolysaccharide-blast cells was detected in stimulated cells from intraperitoneally immunized mice but was lost after depletion of Lyt-2+ cells. Multiple injection of anti-Sendai virus antibody into infected nude mice had no effect on lung virus titer. These results indicate that L3T4+ (Lyt-1+) and Lyt-2+ subsets are cooperatively responsible for efficient clearance of Sendai virus from the mouse lung. 相似文献