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1.
Yutaro Yamagata Yukiko Muramoto Sho Miyamoto Keiko Shindo Masahiro Nakano Takeshi Noda 《Microbiology and immunology》2019,63(5):164-171
Defective interfering (DI) influenza viruses carry a large deletion in a gene segment that interferes with the replication of infectious virus; thus, such viruses have potential for antiviral therapy. However, because DI viruses cannot replicate autonomously without the aid of an infectious helper virus, clonal DI virus stocks that are not contaminated with helper virus have not yet been generated. To overcome this problem, we used reverse genetics to generate a clonal DI virus with a PB2 DI gene, amplified the clonal DI virus using a cell line stably expressing the PB2 protein, and confirmed its ability to interfere with infectious virus replication in vitro. Thus, our approach is suitable for obtaining purely clonal DI viruses, will contribute to the understanding of DI virus interference mechanisms and can be used to develop DI virus‐based antivirals. 相似文献
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3.
A single point mutation confers tetrodotoxin and saxitoxin insensitivity on the sodium channel II 总被引:22,自引:0,他引:22
A single point mutation of the rat sodium channel II reduces its sensitivity to tetrodotoxin and saxitoxin by more than three orders of magnitude. The mutation replaces glutamic acid 387 with a glutamine and has only slight effects on the macroscopic current properties, as measured under voltage-clamp in Xenopus oocytes injected with the corresponding cDNA-derived mRNA. 相似文献
4.
H Miyata H Hayashi S Suzuki N Noda A Kobayashi H Fujiwake M Hirano N Yamazaki 《Biochemical and biophysical research communications》1989,163(1):500-505
Isolated rat heart myocytes were loaded with both the Ca2+ sensitive fluorescent probe fura-2/AM and the fluorescent pH indicator 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF/AM). Changes in [Ca2+]i and pHi were measured simultaneously using digitized video fluorescence microscopy. In measurement of [Ca2+]i and pHi, the ratios of dual-loaded cells were not different from single-loaded cells. Using this method, [Ca2+]i and pHi in myocytes were 48 +/- 7 nM and 7.17 +/- 0.05. It is concluded that [Ca2+]i and pHi could be measured simultaneously in isolated myocyte using dual-loading of fura-2 and BCECF. 相似文献
5.
Ryutaro Utsumi Manjiro Noda Makoto Kawamukai Tohru Komano 《FEMS microbiology letters》1988,50(2-3):217-221
Abstract An adenylate cyclase gene ( cya ) mutant was mutagenized and an adenosine 3,5-cyclic monophosphate (cAMP)-requiring mutant (KM8161) was obtained on Davis minimal medium containing glucose in the presence or absence of cAMP. KM8161 also required N -acetylglucosamine for its growth instead of cAMP. Furthermore, the mutant could use neither glucosamine nor N -acetylglucosamine as the carbon source. These results indicate that the cAMP-requiring property is due to multiple mutations of a few genes involved in amino sugar metabolism in addition to cya . By genetic analysis of KM8161, one gene, which was tentatively termed cidA and located near 2 min on the chromosomal map, proved to be defective. Reversion of cidA mutation in KM8161 resulted in recovery of not only the cAMP-requiring phenotype but also non-utilization of amino sugars. When both cAMP and N -acetylglucosamine or glucosamine were added to the culture medium for KM8161, only N -acetylglucosamine could be utilized as the carbon source. These studies s strongly suggest that the cidA or cya mutation in KM8161 causes deficiency in different stages of amino sugar metabolism and the regulatory circuit of growth by cAMP is mediated via control of N -acetylglucosamine metabolism. 相似文献
6.
Progression of the phenotype of transformed cells after growth stimulation of cells by a human papillomavirus type 16 gene function. 总被引:1,自引:1,他引:0
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Alteration of the growth properties of the established murine fibroblast cell lines NIH 3T3 and 3Y1 was studied in monolayer cultures and in cells suspended in semisolid medium after introduction of a cloned human papillomavirus type 16 (HPV16) DNA. HPV 16 DNA stimulated both cell lines to grow beyond their saturation densities in monolayer cultures without any apparent morphological changes or tendency to pile up. These cells were also stimulated to grow in soft agar. Since essentially all the cells that received the viral gene were stimulated to grow, the growth-stimulatory activity of HPV16 appeared to be due to the direct effect of a viral gene function. The NIH 3T3 cells showed an additional change in growth properties upon prolonged incubation of dense monolayers of cells containing the HPV16 DNA; morphologically recognizable dense foci appeared at a frequency of about 10(-3). These cells, when cloned from the foci, grew more rapidly in soft agar than the parental cells and were morphologically transformed. In other words, there were two sequential steps in cell transformation induced by HPV16. Practically all the viral DNAs were present in the cells as large rearranged multimers and were integrated into host chromosomal DNA. There was no obvious difference in the state of viral DNA in the cells of the original clone or the three subclones derived from it as dense foci. There was no difference in the amount or the number of viral RNA species expressed in the cells at these two stages. The secondary changes in the growth properties of NIH 3T3 cells appear to be due to some cellular alterations. 相似文献
7.
1. Two mutants of the sodium channel II have been expressed inXenopus oocytes and have been investigated using the patch-clamp technique. In mutant E387Q the glutamic acid at position 387 has been replaced by glutamine, and in mutant D384N the aspartic acid at position 384 has been replaced by asparagine.2. Mutant E387Q, previously shown to be resistant to block by tetrodotoxin (Noda et al. 1989), has a single-channel conductance of 4 pS, that can be easily measured only using noise analysis. At variance with the wild-type, the openchannel current-voltage relationship of mutant E387Q is linear over a wide voltage range even under asymmetrical ionic conditions.3. Mutant D384N has a very low permeability for any of the following ions: Cl–, Na+, K+, Li+, Rb+, Ca2+, Mg2+, NH4
+ , TMA+, TEA+. However, asymmetric charge movements similar to the gating currents of the Na+-selective wild-type are still observed.4. These results suggest that residues E387 and D384 interact directly with the pathway of the ions permeating the open channel.Abbreviations TTX
tetrodotoxin; Na+, sodium; K+, potassium;
- NFR
normal frog Ringer
- HEPES
N-2-hydroxylethyl piperazine-N-2-ethanesulfonic acid
- EGTA
ethyleneglycol-bis(-amino-ethyl ether) N,N,N',N'-tetra acetic acid
- TEA
tetraethylammonium
- TMA
tetramethylammonium;I
g
, gating current; , single-channel conductance 相似文献
8.
M Takahashi T Shimizu K Ooi H Noda T Nasu G Sakaguchi 《Japanese journal of medical science & biology》1988,41(1):21-26
Bacteriological examinations were performed on the first case of infant botulism in Japan (an infant boy aged 79 days at onset of illness). Clostridium botulinum type A toxin and organisms were detected continually in the stools of the infant for at least 31 days and 39 days, respectively. The highest levels of the toxin and of the population of the organisms, 7.8 X 10(4) LD50/g and 1.3 X 10(6) colony forming units (cfu)/g, were detected in the stool specimen taken on the 20th day of illness. Type A organisms were detected also in the honey fed to the infant before onset of illness, teats of his feeding bottle, soil specimens taken at the house entry and the vacuum-cleaner dust. Fecal excretion of the toxin and organisms was no longer detected from the 68th day of illness and he recovered. 相似文献
9.
Type-beta transforming growth factor inhibits proliferation and expression of alkaline phosphatase in murine osteoblast-like cells 总被引:9,自引:0,他引:9
TGF-beta modulates growth and differentiation in many cell types. MC3T3E1 is a clonal non-transformed murine bone cell line which differentiates in culture. We tested the effect of porcine TGF-beta on the proliferation and differentiation of MC3T3E1 cells in monolayer cultures by following cell number, and alkaline phosphatase activity. TGF-beta treatment (2 ng/ml) altered the shape of MC3T3E1 cells from cuboidal to elongated/spindle-shape. TGF-beta inhibited the growth of MC3T3E1 by up to 40% (P less than 0.02) in a dose-dependent manner with half maximal inhibition at 1 ng/ml. Growth inhibition depended on serum concentration, maximal inhibition occurring at 2% serum. Expression of alkaline phosphatase, which peaks in vitro when the cells reach confluence, was strongly inhibited by TGF-beta, in a dose-dependent manner with half maximal inhibition at around 0.05 ng/ml and complete inhibition at 2 ng/ml. Alkaline phosphatase inhibition was irreversible after 24 hours exposure to TGF-beta. 相似文献
10.
N Chino K Yoshizawa-Kumagaye Y Noda T X Watanabe T Kimura S Sakakibara 《Biochemical and biophysical research communications》1986,141(2):665-672
To obtain antiparallel and parallel dimers of alpha-human atrial natriuretic peptide (alpha-hANP), two fully protected peptides I and II having the same amino acid sequence as alpha-hANP with different protective groups at the cysteinyl residues were synthesized, the former having Acm and Npys and the latter MeBzl and Acm. Equivalent amounts of peptides I and II were mixed and subjected to HF deprotection. Next, the first disulfide bond was linked between the remaining Npys group in I and the liberated SH group in II to form a monodisulfide dimer. The second disulfide bond was formed within the newly formed dimer between the remaining Acm groups by treatment with iodine, giving an antiparallel dimer. The parallel dimer of alpha-hANP was synthesized similarly starting from the protected peptide II. These dimers could be clearly segregated on HPLC. The retention time on HPLC of the antiparallel dimer was identical with that of natural beta-hANP. Both dimers showed biological activities as high as one third to one sixth of alpha-hANP in smooth muscle spasmolytic activity, and almost the same level of natriuretic activity as alpha-hANP at a high dose (10 nmol/kg) but about one fifth the activity at a low dose (1 nmol/kg). In these assay systems, the antiparallel dimer showed a slower onset and a tendency of longer duration than alpha-hANP. 相似文献