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51.
Mitsuo Satoh Shinji Hosoi Seiji Sato 《In vitro cellular & developmental biology. Plant》1990,26(11):1101-1104
Summary The protease activity in serum-free conditioned medium of chinese hamster ovary (CHO) cells was measured using peptidyl (or
aminoacyl)-4-methylcoumaryl-7-amides (MCAs) as the substrates. Aminopeptidase increased in level as amounts of nonviable cells
increased during cultivation in serum-free medium, indicating that the activity seems to be originated from intracellular
proteases. The activity toward Boc-Leu-Arg-Arg-MCA, which was strongly inhibited by p-chloromercuribenzonate and N-ethylmaleimide,
was the strongest among those toward peptidyl-MCAs in the conditioned medium within 48 h-cultivation in serum-free medium.
In contrast to the case of aminopeptidase activity, the endopeptidase activity decreased in level after 48 h-cultivation although
amounts of nonviable cells increases. Thus, CHO cells continuously secrete the cysteine proteases.
This work was supported by the management of the Research Association for Biotechnology as a part of the R&D of Basic Technology
for Future Industries sponsored by NEDO (New Energy and Industrial Technology Development Organization). 相似文献
52.
Y. Satoh K. Hatakeyama K. Kohama M. Kobayashi Y. Kurusu H. Yukawa 《Journal of industrial microbiology & biotechnology》1990,5(2-3):159-165
Summary Electroporation allowed transformation of intact cells ofBrevibacterium flavum MJ-233. The two plasmids used for electroporation were pCRY2 (6.3 kilobases) and pCRY3 (8.2 kilobases). Both plasmids contain the chloramphenicol-resistance gene and the autonomous replication origin inB. flavum MJ-233. The efficiency of electrotransformation was optimal with cells harvested at the middle log phase of growth, and was imporved by the addition of 1.0U/ml of penicillin G to the culture medium. The optimum yield of transformants per g DNA was 5×104 when the cell suspension was pulsed at a cell density of 1×1010/ml and at a DNA amount of 1.0g. 相似文献
53.
T Mio T Yabe M Sudoh Y Satoh T Nakajima M Arisawa H Yamada-Okabe 《Journal of bacteriology》1996,178(8):2416-2419
The CHS2 and CHS3 genes of Candida albicans were disrupted. The double disruptant was still viable. Assessment of chitin and of calcofluor white resistance shows that CHS1 is responsible for septum formation and CHS3 is responsible for overall chitin synthesis otherwise. There were only small differences in virulence to immunocompromised mice of homozygous chs2 delta amd chs3 delta null mutants. 相似文献
54.
Identification of the sequence on NS4A required for enhanced cleavage of the NS5A/5B site by hepatitis C virus NS3 protease. 总被引:9,自引:7,他引:2 下载免费PDF全文
Y Shimizu K Yamaji Y Masuho T Yokota H Inoue K Sudo S Satoh K Shimotohno 《Journal of virology》1996,70(1):127-132
In addition to NS3 protease, the NS4A protein is required for efficient cleavage of the nonstructural protein region of the hepatitis C virus polyprotein. To investigate the function and the sequence of NS4A required for the enhancement of NS3 protease activity, we developed an in vitro NS3 protease assay system consisting of three purified viral elements: (i) a recombinant NS3 protease which was expressed in Escherichia coli as a maltose-binding protein-NS3 fusion protein (MBP-NS3), (ii) synthetic NS4A fragments, and (iii) a synthetic peptide substrate which mimics the NS5A/5B junction. We showed that the NS3 protease activity of MBP-NS3 was enhanced in a dose-dependent manner by 4A18-40, which is a peptide composed of amino acid residues 18 to 40 of NS4A. The optimal activity was observed at a 10-fold molar excess of 4A18-40 over MBP-NS3. The coefficient for proteolytic efficiency, kcat/Km, of NS3 protease was increased by about 40 times by the addition of a 10-fold molar excess of 4A18-40. Using a series of truncations of 4A18-40, we estimated that amino acid residues 22 to 31 in NS4A (SVVIVGRIIL) constituted the core sequence for the effector activity. Single-substitution experiments with 4A21-34, a peptide composed of amino acid residues 21 to 34 of NS4A, suggested the importance of several residues (Val-23, Ile-25, Gly-27, Arg-28, Ile-29, and Leu-31) for its activity. In addition, we found that some single-amino-acid substitutions in 4A21-34 were able to inhibit the enhancement of NS3 protease activity by 4A18-40. This approach has potential as a novel strategy for inhibiting the NS3 protease activity important for hepatitis C virus proliferation. 相似文献
55.
The ascidian egg contains muscle and endoderm determinants that play critical roles in the specification of muscle and endoderm cells, respectively. Endoderm cells of the ascidian embryo express alkaline phosphatase (AP) as a tissue-specific enzyme. We obtained egg fragments from the unfertilized eggs of Ciona savignyi by means of centrifugal force. The largest fragment (red fragments) contained the egg nucleus while other small fragments (black, clear and brown fragments) were anucleate. When inseminated, only red fragments developed into partial embryos, which showed only epidermis cell differentiation and, very rarely, AP activity. When red fragments were fused with other fragments, only black fragments promoted AP expression, suggesting that endoderm determinants were concentrated in the black fragments. A lower dose (1500 J/m2 ) of ultraviolet (UV) light did not eliminate the AP-promoting ability of black fragments, while this dose significantly repressed the ability to promote the expression of the muscle-marker. A higher dose (4500 J/m2 ) of UV light markedly reduced the AP-promoting activity of black fragments. These results suggest that factors for endodermal AP development are inactivated by UV irradiation, but are more resistant than muscle determinants. 相似文献
56.
57.
Studies of the Inheritance of Human Ribosomal DNA Variants Detected in Two-Dimensional Separations of Genomic Restriction Fragments 总被引:5,自引:0,他引:5 下载免费PDF全文
R. Kuick J. I. Asakawa J. V. Neel M. Kodaira C. Satoh D. Thoraval I. L. Gonzalez S. M. Hanash 《Genetics》1996,144(1):307-316
We have investigated the variation in human ribosomal DNA repeat units as revealed in two-dimensional electrophoretic separations of genomic restriction fragments that were end-labeled at NotI cleavage sites. The transcribed portion of the ribosomal DNA results in ~20 labeled fragments visible on each gel as multicopy spots. We have mapped these spots to the sequences responsible for their appearance on the gels, based on their migration positions and direct sequencing of spots, and describe several previously unreported sources of variation. By studying mother/father/child families we gained information on how much of the between-repeats variation is due to differences between and within repeat arrays on homologous chromosomes. Two instances in which a child exhibited more copies of a particular fragment than were present in the parents are described and hypothesized to be due to events such as multiple unequal sister-chromatid exchanges or gene conversions. 相似文献
58.
Two Polypeptides Inducible by Low Levels of CO2 in Soluble Protein Fractions from Chlorella regularis Grown at Low or High pH 总被引:1,自引:0,他引:1
Previous studies suggested that certain protein(s) other thancarbonic anhydrase might play an important role in the facilitatedtransport of dissolved inorganic carbon (DIC) from the mediumto the site of CO2 fixation by ribulose-1,5-bisphosphate carboxylase/oxygenasein the unicellular green alga Chlorella regularis adapted tolow-CO2 (ordinary air) conditions [Shiraiwa et al. (1991) Jpn.J. Phycol. 39: 355; Satoh and Shiraiwa (1992) Research in Photosynthesis,Vol. III, p. 779]. The proteins that might be involved in thisfacilitated transport of DIC were investigated by pulse-labelingof induced proteins with 35S-sulfate during adaptation of cellsgrown under high-CO2 conditions to low CO2. Analysis by SDS-PAGErevealed that synthesis of two polypeptides, with molecularmasses of 98 and 24 kDa, respectively, was induced under low-CO2conditions. The 24-kDa polypeptide was induced at pH 5.5 butnot at pH 8.0, whereas the 98-kDa polypeptide was induced atboth pH 5.5 and pH 8.0. The possible role of these polypeptidesin the facilitated transport of DIC in Chlorella regularis isdiscussed. (Received October 30, 1995; Accepted February 26, 1996) 相似文献
59.
Itoh Shigeru; Iwaki Masayo; Tomo Tatsuya; Satoh Kimiyuki 《Plant & cell physiology》1996,37(6):833-839
Transient absorbance changes of the primary electron donor chlorophylla (P680) and acceptor pheophytin a (H) were measured at 77 Kby nanosecond laser spectroscopy in the D1-D2-cytochrome b559photosystem II reaction center complex containing dibromomethylisopropylbenzoquinone (DBMIB). After the laser excitation of the reactioncenter in the presence of DBMIB, only the P680+-(DBMIB-) statewas detected. P680+ mainly decayed with a t1/e of 11 ms. Inthe absence of DBMIB, the excitation produced the P680+H- radicalpair. The radical pair produced the triplet state (P680T) witha t1/e of 50 ns, and P680T then decayed with a t1/e of 2.1 ms.It was concluded that H- was oxidized by DBMIB in a time rangefaster than the detecting time resolution (3.5 ns) even at 77K. The rapid oxidation of H- by DBMIB was also confirmed bythe suppression of delayed fluorescence with a decay t1/e of50 ns. The P680+(DBMIB-)/P680(DBMIB) difference spectrum exhibiteda Qy, band with a peak at 682 nm with a shoulder at 673 nm.The spectral shape was almost temperature insensitive between77 and 265 K. The feature of this spectrum in the wavelengthrange between 330 and 720 nm was compared with that of P680T/P680or H-/H at 77 K. (Received May 8, 1996; Accepted June 24, 1996) 相似文献
60.
Koike Hiroyuki; Yoneyama Koichi; Kashino Yasuhiro; Satoh Kazuhiko 《Plant & cell physiology》1996,37(7):983-988
Interactions of externally added plastoquinone (PQ) derivatives(PQ0-PQ3) with the photosystem II (PSII) acceptor side wereinvestigated in PSII membrane fragments prepared from spinachby measuring the photoreduction rates of PQ derivatives at variousPQ concentrations, and the following results were obtained. From the kinetic analysis, all the PQ derivatives (PQ0-PQ3)except PQ3 were shown to accept electrons at two sites (theQB site and the PQ site) as in the case of Synechococcus vulcanusPSII particles with benzoquinone derivatives [Satoh et al. (1995)Plant Cell Physiol. 36: 597]. Affinities of PQ derivatives at the QB site increased as thelength of the isoprene side chain got longer, while those atthe PQ site were not very much different for all the PQ derivativestested in this study. The inhibitory effect of DCMU was noncompetitive, and, therefore,the affinity of PQ3 for the PQ site was determined while thatfor the QB site could not be estimated presumably due to itsfairly high affinity to the site. Based on the results obtained using PQ derivatives, the mechanismof interaction of an authentic PQ, PQ9, at the QB site is discussed. (Received May 2, 1996; Accepted July 24, 1996) 相似文献