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1.
NatB is an N-terminal acetyltransferase consisting of a catalytic Nat5 subunit and an auxiliary Mdm20 subunit. In yeast, NatB acetylates N-terminal methionines of proteins during de novo protein synthesis and also regulates actin remodeling through N-terminal acetylation of tropomyosin (Trpm), which stabilizes the actin cytoskeleton by interacting with actin. However, in mammalian cells, the biological functions of the Mdm20 and Nat5 subunits are not well understood. In the present study, we show for the first time that Mdm20-knockdown (KD), but not Nat5-KD, in HEK293 and HeLa cells suppresses not only cell growth, but also cellular motility. Although stress fibers were formed in Mdm20-KD cells, and not in control or Nat5-KD cells, the localization of Trpm did not coincide with the formation of stress fibers in Mdm20-KD cells. Notably, knockdown of Mdm20 reduced the expression of Rictor, an mTORC2 complex component, through post-translational regulation. Additionally, PKCαS657 phosphorylation, which regulates the organization of the actin cytoskeleton, was also reduced in Mdm20-KD cells. Our data also suggest that FoxO1 phosphorylation is regulated by the Mdm20-mTORC2-Akt pathway in response to serum starvation and insulin stimulation. Taken together, the present findings suggest that Mdm20 acts as a novel regulator of Rictor, thereby controlling mTORC2 activity, and leading to the activation of PKCαS657 and FoxO1. 相似文献
2.
Matuo Y Nishi N Muguruma Y Yoshitake Y Masuda Y Nishikawa K Wada F 《Cytotechnology》1988,1(4):309-318
Among several detergents, a zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), was found to be least cytotoxic for cultured mammalian cells. CHAPS improved the activity recovery and elution profile of crude and purified fibroblast growth factors (FGFs) during chromatographies. Diluted preparations of FGFs were stabilized by CHAPS against the loss during storage. Amino acid sequence analysis was not disturbed by CHAPS. CHAPS was removable by reversed-phase high-performance liquid chromatography. These results indicate that CHAPS is useful as a non-cytotoxic stabilizing agent in purification of various kinds of bioactive polypeptides.Abbreviations -MEM
Alpha Modification of Eagle's Minimal essential medium
- CHAPS
3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate
- CHAPSO
3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propane sulfonate
- CS
Calf Serum
- EGF
Epidermal Growth Factor
- FGF
Fibroblast Growth Factor
- HPLC
High-Performance Liquid Chromatography
- NGF
Nerve Growth Factor
- NOG
1-O-n-octyl--D-glucopyranoside
- NP-40
Nonidet P-40
- PBS
Phosphate-Buffered Saline
- SB 12
3-(dodecylmethylammonio)-1-propane sulfonate
- SDS
Sodium Dodecyl Sulfate
- TGF- and
Transforming Growth Factor type and 相似文献
3.
Isolation and Characterization of an Escherichia coli ruv Mutant Which Forms Nonseptate Filaments After Low Doses of Ultraviolet Light Irradiation 总被引:34,自引:17,他引:17
Two ultraviolet light (UV)-sensitive mutants have been isolated from Escherichia coli K-12. These mutants, designated RuvA(-) and RuvB(-), were controlled by a gene located close to the his gene on the chromosome map. They were sensitive to UV (10- to 20-fold increase) and slightly sensitive to gamma rays (3-fold increase). Host cell reactivation, UV reactivation and genetic recombination were normal in these mutants. Irradiation of the mutants with UV resulted in the production of single-strand breaks in deoxyribonucleic acid, which was repaired upon incubation in a growth medium. After UV irradiation, these mutants resumed deoxyribonucleic acid synthesis at a normal rate, as did the parent wild-type bacteria, and formed nonseptate, multinucleate filaments. From these results we concluded that the mutants have some defect in cell division after low doses of UV irradiation, similar to the lon(-) or fil(+) mutant of E. coli. The ruv locus was divided further into ruvA and ruvB with respect to nalidixic acid sensitivity and the effect of minimal agar or pantoyl lactone on survival of the UV-irradiated cell. The ruvB(-)mutant was more sensitive to nalidixic acid than were ruvA(-) and the parent strain. There was a great increase in the surviving fraction of the UV-irradiated ruvB(-) mutant when it was plated on minimal agar or L agar containing pantoyl lactone. No such increase in survival was observed in the ruvA(-) mutant. 相似文献
4.
Yuhsi Matuq Pamela S. Adams Nozomu Nishi Hidetaro Yasumitsu John W. Crabb Robert J. Matusik Wallace L. McKeehan 《In vitro cellular & developmental biology. Plant》1989,25(6):581-584
Summary Rat prostate extracts contain an abundant 20–22 kilodalton heparin-binding protein with near identical chromatographic properties,
but only 0.2–1% of the mitogenic activity, of bovine brain heparin-binding growth factor-1 (acidic fibroblast growth factor).
Amino terminal amino acid sequence (met-met-thr-asp-lys-asn-leu-lys-lys-lys-ile-glu-gly-asn-trp-arg-thr-val-tyr-leu-ala-ala-ser-?-val-glu-lys-ile-asn-glu-gly-ser-pro)
and immunochemical analysis revealed that the protein is identical to the androgen-dependent protein “probasin”.
This work was supported in part by NCI grant CA37589 (W. L. M., J. W. C.) and the Medical Research Council of Canada (R. J.
M.). 相似文献
5.
Łukasz Wejnerowski Ewa Poniecka Jakub Buda Piotr Klimaszyk Agnieszka Piasecka Marcin Krzysztof Dziuba Gianmarco Mugnai Nozomu Takeuchi Krzysztof Zawierucha 《Journal of phycology》2023,59(5):939-949
Cryoconite, the dark sediment on the surface of glaciers, often aggregates into oval or irregular granules serving as biogeochemical factories. They reduce a glacier's albedo, act as biodiversity hotspots by supporting aerobic and anaerobic microbial communities, constitute one of the organic matter (OM) sources on glaciers, and are a feeder for micrometazoans. Although cryoconite granules have multiple roles on glaciers, their formation is poorly understood. Cyanobacteria are ubiquitous and abundant engineers of cryoconite hole ecosystems. This study tested whether cyanobacteria may be responsible for cryoconite granulation as a sole biotic element. Incubation of Greenlandic, Svalbard, and Scandinavian cyanobacteria in different nutrient availabilities and substrata for growth (distilled water alone and water with quartz powder, furnaced cryoconite without OM, or powdered rocks from glacial catchment) revealed that cyanobacteria bind mineral particles into granules. The structures formed in the experiment resembled those commonly observed in natural cryoconite holes: they contained numerous cyanobacterial filaments protruding from aggregated mineral particles. Moreover, all examined strains were confirmed to produce extracellular polymeric substances (EPS), which suggests that cryoconite granulation is most likely due to EPS secretion by gliding cyanobacteria. In the presence of water as the only substrate for growth, cyanobacteria formed mostly carpet-like mats. Our data empirically prove that EPS-producing oscillatorialean cyanobacteria isolated from the diverse community of cryoconite microorganisms can form granules from mineral substrate and that the presence of the mineral substrate increases the probability of the formation of these important and complex biogeochemical microstructures on glaciers. 相似文献
6.
Chemical change involved in the oxidative reductive depolymerization of hyaluronic acid 总被引:3,自引:0,他引:3
H Uchiyama Y Dobashi K Ohkouchi K Nagasawa 《The Journal of biological chemistry》1990,265(14):7753-7759
The oxidative reductive depolymerization (ORD) of hyaluronate has been investigated. A solution of hyaluronate (Mr 4.07 x 10(5] in phosphate buffer (pH 7.2) was incubated in the presence of Fe2+ for 24 h at 37 degrees C under an oxygen atmosphere to yield depolymerized hyaluronate (ORD fragments; an average Mr of 2,600). The ORD fragments contain 21 and 24% less hexosamine and uronic acid, respectively, but no olefinic linkage. They were exhaustively digested with chondroitinase AC-II. The resulting oligosaccharides and monosaccharides were separated by gel filtration and ion-exchange chromatography, and their structures were determined by proton and carbon-13 NMR, fast atom bombardment mass spectrometry, and chromatographic techniques combined with chemical modifications. The following structures derived from the reducing ends of the ORD fragments were identified: 4,5-unsaturated GlcA(beta 1----3)-N-acetyl-D-glucosaminic acid (where GlcA- represents glucuronosyl-) (21%), 4,5-unsaturated GlcA(beta 1----3)GlcNAc(beta 1----3)-D-arabo-pentauronic acid (24%), and N-acetyl-D-glucosamine (51%). The following structures derived from the nonreducing ends were identified: L-threo-tetro-dialdosyl-(1----3)GlcNAc (a tentative structure, 8%), N-acetylhyalobiuronic acid (20%), and N-acetyl-D-glucosamine (45%). The results indicate that the ORD reaction of hyaluronate proceeds essentially by random destruction of unit monosaccharides due to oxygen-derived free radicals, followed by secondary hydrolytic cleavage of the resulting unstable glycosidic substituents. 相似文献
7.
8.
Mamoru Tamura Nozomu Oshino Britton Chance Ian A. Silver 《Archives of biochemistry and biophysics》1978,191(1):8-22
The optical characteristics of hemoglobin-free perfused rat heart have been examined in detail. Ethyl hydrogen peroxide is found to convert myoglobin into “ferryl compound” in the perfused heart, as is also seen in vitro. After pretreatment with ethyl hydrogen peroxide, a typical mitochondrial absorption spectrum, similar to that of isolated rat heart mitochondria, is obtained in perfused heart. The overall absorption spectrum of the heart obtained by the aerobic to anaerobic transition is a superposition of the mitochondrial spectrum on that of myoglobin. By comparing these spectra, it is found that measurement of cytochrome a + a3 at 605–620 nm is possible in spite of the absorbance change due to the oxygenation-deoxygenation of myoglobin, whereas the wavelength pairs for cytochrome c at 550-540 nm, cytochrome b at 562–575 nm and cytochrome a + a3 at 445–450 nm can not be used in the heart because of interference from the absorption change of myoglobin. The partial pressure of O2 (P50) which is required for half maximal deoxygenation (or oxygenation) of myoglobin in perfused heart is found to be 2.4 mm Hg at room temperature and the Hill constant, n, is 1.1; these values are similar to those of myoglobin purified from rat heart. The steady-state O2 titration has been performed by using absorbancy changes of myoglobin and cytochrome a + a3 as intracellular O2 indicators. In the perfused heart, the percentage change of oxygenation-deoxygenation of myoglobin parallels the oxidation-reduction of cytochrome a + a3, while the mixture of purified myoglobin and isolated mitochondria shows a deviation, reflecting the difference of O2 affinities between myoglobin and cytochrome a + a3. The results indicate that there may be an O2 gradient between cytosolic and mitochondrial compartments in the hemoglobin-free perfused heart. The absorption changes of myoglobin and of cytochrome a + a3 can be measured in a single contraction-relaxation cycle. A triple beam method was introduced to eliminate the effect of light scattering changes in these measurements. The results demonstrated that myoglobin is more oxygenated during the systolic and diastolic periods and deoxygenated in the resting period, whereas cytochrome a + a3 is more reduced in systole and diastole and oxidized in the resting state. Changing the perfusion conditions greatly alters the time course of the events which occur during the contraction-relaxation cycle of the perfused heart. 相似文献
9.
Daniel Evans-Yamamoto Franois D Rouleau Piyush Nanda Koji Makanae Yin Liu Philippe
C Desprs Hitoshi Matsuo Motoaki Seki Alexandre K Dub Diana Ascencio Nozomu Yachie Christian
R Landry 《Nucleic acids research》2022,50(9):e54
Barcode fusion genetics (BFG) utilizes deep sequencing to improve the throughput of protein–protein interaction (PPI) screening in pools. BFG has been implemented in Yeast two-hybrid (Y2H) screens (BFG-Y2H). While Y2H requires test protein pairs to localize in the nucleus for reporter reconstruction, dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA) allows proteins to localize in broader subcellular contexts and proves to be largely orthogonal to Y2H. Here, we implemented BFG to DHFR-PCA (BFG-PCA). This plasmid-based system can leverage ORF collections across model organisms to perform comparative analysis, unlike the original DHFR-PCA that requires yeast genomic integration. The scalability and quality of BFG-PCA were demonstrated by screening human and yeast interactions for >11 000 bait-prey pairs. BFG-PCA showed high-sensitivity and high-specificity for capturing known interactions for both species. BFG-Y2H and BFG-PCA capture distinct sets of PPIs, which can partially be explained based on the domain orientation of the reporter tags. BFG-PCA is a high-throughput protein interaction technology to interrogate binary PPIs that exploits clone collections from any species of interest, expanding the scope of PPI assays. 相似文献
10.
Kosuke Kurosawa Seiji Sugita Ko Ishibashi Sunao Hasegawa Yasuhito Sekine Nanako O. Ogawa Toshihiko Kadono Sohsuke Ohno Naohiko Ohkouchi Yoichi Nagaoka Takafumi Matsui 《Origins of life and evolution of the biosphere》2013,43(3):221-245
Cyanide compounds are amongst the most important molecules of the origin of life. Here, we demonstrate the importance of mid-size (0.1–1 km in diameter) hence frequent meteoritic impacts to the cyanide inventory on the early Earth. Subsequent aerodynamic ablation and chemical reactions with the ambient atmosphere after oblique impacts were investigated by both impact and laser experiments. A polycarbonate projectile and graphite were used as laboratory analogs of meteoritic organic matter. Spectroscopic observations of impact-generated ablation vapors show that laser irradiation to graphite within an N2-rich gas can produce a thermodynamic environment similar to that produced by oblique impacts. Thus, laser ablation was used to investigate the final chemical products after this aerodynamic process. We found that a significant fraction (>0.1 mol%) of the vaporized carbon is converted to HCN and cyanide condensates, even when the ambient gas contains as much as a few hundred mbar of CO2. As such, the column density of cyanides after carbon-rich meteoritic impacts with diameters of 600 m would reach ~10 mol/m2 over ~102 km2 under early Earth conditions. Such a temporally and spatially concentrated supply of cyanides may have played an important role in the origin of life. 相似文献