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121.
A Thr (or Ser) residue on the I-helix is a highly conserved structural feature of cytochrome P450 enzymes. It is believed to be indispensable as a proton delivery shuttle in the oxygen activation process. Previous work showed that P450cin (CYP176A1), which contains an Asn instead of the conserved Thr, is fully functional in the catalytic oxidation of cineole [D.B. Hawkes, G.W. Adams, A.L. Burlingame, P.R. Ortiz de Montellano, J.J. De Voss, J. Biol. Chem. 277 (2002) 27725-27732]. To determine whether the substitution of Asn for Thr is specific or general, the conserved Thr252 in P450cam (CYP101) was mutated to generate the T252N, T252N/V253T, and T252A mutants. Steady-state kinetic analysis of the oxidation of camphor by these mutants indicated that the T252N and T252N/V253T mutants have comparable turnover numbers but higher Km values relative to the wild-type enzyme. Spectroscopic binding assays indicate that the higher Km values reflect a decrease in the camphor binding affinity. Non-productive H2O2 generation was negligible with the T252N and T252N/V253T mutants, but, as previously observed, was dominant in the T252A mutant. Our results, and a structure model based on the crystal structures of the ferrous dioxygen complexes of P450cam and its T252A mutant, suggest that Asn252 can stabilize the ferric hydroperoxy intermediate, preventing premature release of H2O2 and enabling addition of the second proton to the distal oxygen to generate the catalytic ferryl species. 相似文献
122.
Hong S Heo J Lee S Heo S Kim SS Lee YD Kwon M Hong S 《Biochemical and biophysical research communications》2008,377(3):935-940
We have analyzed the importance of substrate methylation by S-adenosylmethionine-dependent methyltransferases for neuronal differentiation of P19 embryonal carcinoma cells. We show that treatment of cells with methyltransferase inhibitor adenosine dialdehyde (AdOx) interferes with neuronal differentiation. Retinoic acid (RA) and AdOx co-treated cells had a decreased number of neurites and a flattened morphology compared with cells differentiated by RA. Also, the amount of neuronal class III tubulin (Tuj1) decreased from 76% to 9.6% with AdOx-treatment. Gene expression levels of wnt-1, brn-2, neuroD, and mash-1 were also down-regulated by AdOx-treatment. But AdOx-treatment did not up-regulate BMP-4 and GFAP genes. Treatment of RA decreased E-cadherin expression during neuronal differentiation. However, in AdOx/RA co-treated cells, E-cadherin expression was restored to the control level. Also, mRNA expression of N-cadherin decreased with AdOx-treatment. Taken together, these data show that methylation reactions might influence the cell-fate decision and neuronal differentiation of P19 cells. 相似文献
123.
Heo KS Kim DU Kim L Nam M Baek ST Park SK Park Y Myung CS Hwang SO Hoe KL 《Biochemical and biophysical research communications》2008,368(1):126-131
Native LDL may be a mitogenic stimulus of VSMC proliferation in lesions where endothelial disruption occurs. Recent studies have demonstrated that the mitogenic effects of LDL are accompanied by Erk1/2 activation via an unknown G-protein-coupled receptor (GPCR). In this article, we report that LDL translocated PKCβII and PKCθ from cytosol to plasma membrane, and inhibition of PKCβII and PKCθ decreased LDL effects via the deactivation of Erk1/2. Moreover, pertussis toxin, but not cholera toxin or heparin, inhibited LDL-induced translocation of PKCβII and PKCθ, suggesting that Gi protein plays a role in LDL effects. Of LPA, S1P, and LDL, whose signaling is conveyed via Gi/o proteins, only LDL induced translocation of PKCβII and PKCθ. Inhibition of PKCβII or PKCθ, as well as of Erk1/2 and GPCR, decreases LDL-induced upregulation of Egr-1, which is critical for cell proliferation. This is the first report, to our knowledge, that the participation of PKCθ in VSMC proliferation is unique. 相似文献
124.
125.
Thi Nguyen Quynh Kozai Toyoki Heo Jeongwook Xuan Thai Du 《Plant Cell, Tissue and Organ Culture》2001,66(3):217-225
Effects of two ventilation methods (forced and natural) and two photosynthetic photon fluxes (PPF, 150 and 250 μmol m−2 s−1) on the photoautotrophic growth of in vitro cultured coffee (Coffea arabusta) plantlets were investigated. Number of air exchanges was 2.7, 5.9 and 3.9 h−1 for forced low rate, forced high rate and natural ventilation, respectively. Single node cuttings of in vitro cultured coffee plantlets were cultured on Florialite, a mixture of vermiculite and cellulose fibers with high air porosity,
emerged in liquid half strength basal MS medium, without sucrose, vitamins and plant growth regulators. The study included
40 days in the in vitro stage and 10 days in the ex vitro stage. Mean fresh and dry weights, leaf area, shoot and root lengths and net photosynthetic rate per plantlet were significantly
greater in forced high rate treatments compared with those in natural and forced low rate treatments. PPF had a distinct effect
on shoot length suppression and root elongation of coffee plantlets in forced high rate treatments. The control of carbon
dioxide concentration inside the culture box according to the plant demand when growing was easy with the forced ventilation
method in photoautotrophic micropropagation.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
126.
Induction of Betalain Pigmentation in Hairy Roots of Red Beet under Different Radiation Sources 总被引:5,自引:0,他引:5
The effects of aluminum on lipid peroxidation and activities of antioxidative enzymes were investigated in detached rice leaves treated with 0 to 5 mM AlCl3 at pH 4.0 in the light. AlCl3 enhanced the content of malondialdehyde but not the content of H2O2. Superoxide dismutase activity was reduced by AlCl3, while catalase and glutathione reductase activities were increased. Peroxidase and ascorbate peroxidase activities were increased only after prolonged treatment, when toxicity occurred. The results give evidence that Al treatment caused oxidative stress and in turn, it caused lipid peroxidation. 相似文献
127.
三氧化二砷对食管癌细胞增殖和热休克蛋白70表达的影响 总被引:2,自引:0,他引:2
目的:研究三氧化二砷(As2O3)对食管癌细胞增殖和热休克蛋白70(HSP70)表达的影响。方法:通过相差显微镜、流式细胞术、免疫细胞化学染色和免疫印迹分析等方法观察As2O3对人食管癌细胞株EC1的作用效果和作用机制。结果:与对照组相比,经2μmol/L和5μmol/Las2O3作用的细胞出现明显的生长抑制,G2/M期细胞比例增加;2μmol/Las2O3作用48h后经Ecl细胞HSP70(heat shock protein70)及HSC70(heat shock cognate protein70)表达均增加。结论:As2O3诱导食管癌细胞G2/M期阻滞抑制细胞增殖和生长;HSP70的升高是细胞对As2O3作用后出现的应激反应,并与细胞周期阻滞相关。 相似文献
128.
Purification of recombinant human epidermal growth factor secreted from the methylotrophic yeast Hansenula polymorpha. 总被引:3,自引:0,他引:3
Joo-Hyung Heo Hye Soon Won Hyun Ah Kang Sang-Ki Rhee Bong Hyun Chung 《Protein expression and purification》2002,24(1):117-122
The gene encoding human epidermal growth factor (hEGF) was expressed as a fusion protein with the Saccharomyces cerevisiae-derived prepro alpha-factor leader in the methylotrophic yeast Hansenula polymorpha. The recombinant hEGF(1-53), when secreted by H. polymorpha, rapidly cleaved to hEGF(1-52) by carboxy-terminal proteolysis, resulting in the accumulation of C-terminal-truncated hEGF(1-52) in the culture medium. To solve this problem, we constructed a H. polymorpha mutant in which the KEX1 gene coding for carboxypeptidase ysc(alpha) was disrupted. The extent of C-terminal proteolysis of hEGF was significantly reduced when this kex1 disruptant was used as a host strain. After 24 h of shake-flask culture, most of the hEGF secreted by the kex1 disruptant remained intact, whereas more than 90% of the hEGF secreted by the wild-type was C-terminally cleaved. The recombinant hEGF was purified to >98% purity by two sequential steps of preparative scale anion exchange chromatography and reverse-phase HPLC. The authenticity of purified hEGF was confirmed by HPLC, N-terminal amino acid sequencing, and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy analyses. 相似文献
129.
To maintain embryogenic cell lines ofPimpinella brachycarpa, we suspension-cultured friable and rapidly growing yellowish calli in an MS liquid medium containing 0.2 ~ 2,4-D and 0.5pM BAP. Efficient somatic embryogenesis was achieved when selected cells were then transferred to an MS medium (0.2% gelrite)
that contained 0.2gM 2,4-D, 0.5 uM BAP, and 10.0 laM TDZ (thidiazuron). These cells were cultured at 27°C under continuous illumination (21.5
I~E m-2 s-l). Embryogenic calli expanded about four-fold, and developed into pale yellow calli. Somatic embryogenesis was initiated only
from glossy and nodular-type calli. After two more weeks of culture, globular embryos appeared on the surface of calli grown
in the MS medium that contained 10.0 /aM TDZ only, or in combination with 0.5 gM NAA. Experimenting with 2,4-D, an auxin,
to promote embryogenic calli resulted in excessive browning and death. We overcame this problem by growing glossy embryogenic
and nodular calli on media that contained 10.0 gM TDZ. Calli that were not treated with TDZ turned dark brown and were not
viable. Up to 74% of the calli showed somatic embryos when the medium was supplemented with 10.0 uM TDZ and 0.5 uM NAA. Embryos
from these TDZ-induced, somatic embryogenic calli grew efficiently, forming multiple shoots and developing into normal plants.
Therefore, efficient differentiation of suspension-cultured cell clusters into embryogenic calli, along with treatment of
subsequent somatic embryos by TDZ, suggests that TDZ probably helps in establishing the optimum cytokinin-auxin ratio required
for induction and expression of somatic embryogenesis. 相似文献
130.