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111.
Lee K Song EH Kim HS Yoo JH Han HJ Jung MS Lee SM Kim KE Kim MC Cho MJ Chung WS 《The Journal of biological chemistry》2008,283(35):23581-23588
The mitogen-activated protein kinases (MAPKs) are key signal transduction molecules, which respond to various external stimuli. The MAPK phosphatases (MKPs) are known to be negative regulators of MAPKs in eukaryotes. We screened an Arabidopsis cDNA library using horseradish peroxidase-conjugated calmodulin (CaM), and isolated AtMKP1 as a CaM-binding protein. Recently, tobacco NtMKP1 and rice OsMKP1, two orthologs of Arabidopsis AtMKP1, were reported to bind CaM via a single putative CaM binding domain (CaMBD). However, little is known about the regulation of phosphatase activity of plant MKP1s by CaM binding. In this study, we identified two Ca(2+)-dependent CaMBDs within AtMKP1. Specific binding of CaM to two different CaMBDs was verified using a gel mobility shift assay, a competition assay with a Ca(2+)/CaM-dependent enzyme, and a split-ubiquitin assay. The peptides for two CaMBDs, CaMBDI and CaMBDII, bound CaM in a Ca(2+)-dependent manner, and the binding affinity of CaMBDII was found to be higher than that of CaMBDI. CaM overlay assays using mutated CaMBDs showed that four amino acids, Trp(453) and Leu(456) in CaMBDI and Trp(678) and Ile(684) in CaMBDII, play a pivotal role in CaM binding. Moreover, the phosphatase activity of AtMKP1 was increased by CaM in a Ca(2+)-dependent manner. Our results suggest that two important signaling pathways, Ca(2+) signaling and the MAPK signaling cascade, are connected in plants via the regulation of AtMKP1 activity. To our knowledge, this is the first report to show that the biochemical activity of MKP1 in plants is regulated by CaM. 相似文献
112.
Ma CJ 《Biotechnology letters》2008,30(5):961-965
When growth-phase cell suspension cultures of Capsicum annuum were treated with cellulase-elicitor preparation at 3 μg/ml, the level of capsidiol was transiently increased in the culture
media rather than in the cells reaching its maximum approx 24 h after treatment. With methyl jasmonate it took 18 h. Elicitor
treatment doubled phospholiphase A2 (PLA2) activity but simultaneous treatment with aristolochic acid, a PLA2 inhibitor, inhibited sesquiterpenoid accumulation as well as PLA2 activity. Mastoparan, a G protein activator, treatment also increased PLA2 activity and capsidiol production. Taken together, the present study shows that induction of capsidiol production in the
C. annuum is mediated by PLA2 activation. 相似文献
113.
Although hydrolases are used in several industrial processes, its industrial applications have some limitations in specific cases since some industrial processes are carried out at pH value which is different from optimum pH of the enzyme. Alkaline side optimum pH of hydrolases is always desirable, and it is proved difficult to achieve that by mutation only. Hence, molecular modeling was applied to select the promising mutants. The changes in electrostatic potential, which was calculated using Delphi, were compared to the changes in pH optimum of four hydolases and their mutants. The results showed that the change in electrostatic potential can be used as an indicator for selecting relevant candidates of mutation. Bacillus circulans xylanase was selected as a model system, and the promising mutants were picked up by the molecular modeling. Q167M and R73V, had a higher pH optimum than the wild type, while K175Q had a similar pH-activity profile of the wild type. 相似文献
114.
Seung Hwan Kim Seung Hoon Song Young Je Yoo 《Biotechnology and Bioprocess Engineering》2005,10(1):47-51
The bioelectrochemical reduction of nitrate in the presence of various mediators including methyl viologen and azure A was
studied using a 3-electrode voltammetric system. The catalytic potential for the reduction of the mediators was observed in
the reactor, which for methyl viologen and azure A were −0.74 V and −0.32 V, respectively, with respect to the potential of
Ag/AgCl reference electrode. This potential was then applied to a working electrode to reduce each mediator for enzymatic
nitrate reduction. Nitrite, the product of the reaction, was measured to observe the enzymatic nitrate reduction in the reaction
media. Methyl viologen was observed as the most efficient mediator among those tested, while azure A showed the highest electron
efficiency at the intrinsic reduction potential when the mediated enzyme reactions were carried out with the freely solubilized
mediator. The electron transfer of azure A with respect to time was due to the adhesion of azure A to the hydrophilic surface
during the reduction. In addition, the use of the adsorbed mediator on conductive activated carbon was proposed to inhibit
the change in the electron transfer rate during the reaction by maintaining a constant mediator concentration and active surface
area of the electrode. Azure A showed better than nitrite formation than methyl viologen when used with activated carbon. 相似文献
115.
Chul Min Kim Byoung Il Je Ja Choon Koo Hai Long Piao Soon Ju Park Joo Mi Jeon Min Kyoung Kim Sung Han Park Jin Young Park Eun Jin Lee Woo Sik Chung Kon Ho Lee Kyu Young Kang Sung-Ho Lee Chang-deok Han 《Journal of Plant Biology》2004,47(1):1-7
A maize transposable family, Ac/Ds, has been successfully utilized as a mutagenizing agent not only in monocot but also in
dicot. In order to develop insertional mutagenesis system in pepper, the mobility of Ac/Ds has been examined. In this study,
the excision of the elements was monitored via transient assay system with protoplasts. Two different systems were developed
and compared; one- and two-elements systems. In a one-element system, Ac alone was introduced into cells. As a two-element
system, Ac and Ds were cloned into a single vector and were expressed in protoplasts. Our data showed that both Ac and Ds
elements were highly mobile in pepper cells. This is the first report suggesting that Ac/Ds mediated gene tagging system could
be successfully operated in pepper. 相似文献
116.
An expression vector was constructed to overproduce a maltose binding protein (MBP)-esterase fusion protein in Escherichia coli. Soluble fusion protein was separated by centrifugation after cell disruption. The fusion protein was partially purified with amylose resin. The higher concentration of fusion protein (above 2 mg/ml) did not show any activity but about 0.3 mg/ml of fusion protein had the highest activity (142 U/ml). It is due to the difficulty of contact between substrate and active site of enzyme in compact form at high concentration. The fusion protein over-expressed could not be separated into MBP and esterase by the action of protease ‘Factor Xa’. The esterase could be cleaved from MBP fusion protein by the treatment of SDS with the Factor Xa, and the resulting esterase activity was increased to 34% after cleavage. 相似文献
117.
Induction of endothelial apoptosis by 4-hydroxyhexenal. 总被引:1,自引:0,他引:1
Ji Young Lee Jeong Hwan Je Dae Hyun Kim Sang Woon Chung Yani Zou Nam Deuk Kim Mie Ae Yoo Hyung Suck Baik Byung Pal Yu Hae Young Chung 《European journal of biochemistry》2004,271(7):1339-1347
Lipid peroxidation and its products such as 4-hydroxy-2-nonenal (HNE) and 4-hydroxyhexenal (HHE) are known to affect redox balance during aging and various degenerative processes, including vascular dysfunction. Deterioration of the endothelial cells that line the vascular wall is known to be an underlying cause of vascular dysfunction. At present, little is known about the mechanism by which HHE induces endothelial cell death (i.e. apoptosis), although HNE-induced apoptotic cell death has been reported. The aim of this study was to determine whether apoptosis induced by HHE in endothelial cells involves peroxynitrite (ONOO(-)). Our results show that in endothelial cells HHE triggers apoptotic cell death by inducing apoptotic Bax coupled with a decrease in anti-apoptotic Bcl-2. Results show that HHE induces reactive oxygen species (ROS), nitric oxide, and ONOO(-) generation, leading to redox imbalance. Furthermore, the antioxidant N-acetyl cysteine, ROS scavenger, and penicillamine, an ONOO(-) scavenger, were found to block HHE-mediated apoptosis. We used confocal laser microscopy to estimate the ability of these inhibitors to attenuate HHE-induced intracellular ONOO(-) levels thus confirming the oxidative mediation of apoptosis in endothelial cells. These findings strongly suggest that accumulated HHE triggers reactive species-mediated endothelial apoptosis, leading to vascular dysfunction as well as vascular aging. During aging, increased lipid peroxidation and its associated production of HHE may exacerbate the weakened redox balance, leading to various chronic degenerative processes including vascular dysfunction. 相似文献
118.
119.
Yeon Ho Je Jin Hee Chang Mi Hyang Kim Jong Yul Roh Byung Rae Jin David R. O''''Reilly 《Biotechnology letters》2001,23(21):1809-1817
A system is described for the rapid generation of Bombyx mori nucleopolyhedrovirus (BmNPV)-based expression vectors. A series of novel BmNPV genomes, that include a mini-F replicon and therefore can be maintained in Escherichia coli, have been generated. These genomes lack a portion of the essential ORF1629 gene and cannot replicate independently in insect cells. However, they can be used as parental genomes for the generation of expression vectors by cotransfection with a transfer plasmid that includes an intact ORF1629. Only recombinant viruses that have acquired the ORF1629 gene from the transfer vector, and have therefore also acquired the foreign gene of interest, can replicate after cotransfection. Parental genomes with and without a polyhedrin gene are described, enabling the generation of occlusion-positive and occlusion-negative recombinant viruses. Occlusion-positive expression vectors enable the oral infection of B. mori larvae and can therefore be used for the mass production of a foreign protein in infected insects. 相似文献
120.
Haeshin Lee Ji Hoon Jeong Je Hoon Lee Tae Gwan Park 《Biotechnology and Bioprocess Engineering》2001,6(4):269-273
This study presents a new formulation method for improving DNA transfection efficiency using a fusogenic peptide and polyethylene
glycol grafted polyethylenimine. Succinimidyl succinate polyethylene glycol (PEG-SSA) was conjugated with polyethylenimine
(PEI). PEI is well known for a good endosomal escaping and DNA condensing agent. The positively charged synthetic fusogenic
peptide, KALA, was coated on the negatively charged PEG-g-PEI/DNA and PEI/DNA complexes. The KALA/PEI/DNA complexes exhibited
aggregation behavior at higher KALA coating amounts with an effective diameter of around 1,000 nm. However, the KALA/PEG-g-PEI/DNA
complexes were 100–300 nm in size with a surface zeta-potential (ζ) value of about +20 mV. The conjugated PEG molecules suppressed
any KALA-mediated inter-particle aggregation, and thereby improved the transfection efficiency. Consequently, the transfection
efficiency of the KALA/PEG-g-PEI/DNA complexes was obtained by utilizing both the fusogenic activity of KALA and the steric
repulsion effect of PEC. 相似文献