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81.
Lee D Lee J Park N Oh YK Kwon M Kim YB 《Journal of microbiology and biotechnology》2008,18(3):585-590
Human tropic Porcine Endogenous Retroviruses (PERVs) are the major concern in zoonosis for xenotransplantation because PERVs cannot be eliminated by specific pathogen-free breeding. Recently, a PERV A/C recombinant with PERV-C bearing PERV-A gp70 showed a higher infectivity (approximately 500-fold) to human cells than PERV-A. Additionally, the chance of recombination between PERVs and HERVs is frequently stated as another risk of xenografting. Overcoming zoonotic barriers in xenotransplantation is more complicated by recombination. To achieve successful xenotransplantation, studies on the recombination in PERVs are important. Here, we cloned and sequenced proviral PERV env sequences from pig gDNAs to analyze natural recombination. The envelope is the most important element in retroviruses as a pivotal determinant of host tropisms. As a result, a total of 164 PERV envelope genes were cloned from pigs (four conventional pigs and two miniature pigs). Distribution analysis and recombination analysis of PERVs were performed. Among them, five A/B recombinant clones were identified. Based on our analysis, we determined the minimum natural recombination frequency among PERVs to be 3%. Although a functional recombinant envelope clone was not found, our data evidently show that the recombination event among PERVs may occur naturally in pigs with a rather high possibility. 相似文献
82.
Crk-associated substrate (CAS) is a focal adhesion protein that is involved in integrin signaling and cell migration. CAS deficiency reduces the migration and spreading of cells, both of which are processes mediated by Rac activation. We examined the functions of v-Crk, the oncogene product of the CT10 virus p47gag-crk, which affects cell migration and spreading, membrane ruffling, and Rac activation in CAS-deficient mouse embryonic fibroblasts (CAS-/- MEFs). CAS-/- MEFs showed less spreading than did CAS+/+ MEFs, but spreading was recovered in mutant cells that expressed v-Crk (CAS-/-v-Crk MEF). We observed that the reduction in spreading was linked to the formation of membrane ruffles, which were accompanied by Rac activation. In CAS-/- MEFs, Rac activity was significantly reduced, and Rac was not localized to the membrane. In contrast, Rac was active and localized to the membrane in CAS-/-v-Crk MEFs. Lamellipodia protrusion and ruffle retraction velocities were both reduced in CAS-/- MEFs, but not in CAS-/-v-Crk MEFs. We also found that microinjection of anti-gag antibodies inhibited the migration of CAS-/-v-Crk MEFs. These findings indicate that v-Crk controls cell migration and membrane dynamics by activating Rac in CAS-deficient MEFs. 相似文献
83.
84.
High-level expression of an antimicrobial peptide histonin as a natural form by multimerization and furin-mediated cleavage 总被引:2,自引:0,他引:2
Kim JM Jang SA Yu BJ Sung BH Cho JH Kim SC 《Applied microbiology and biotechnology》2008,78(1):123-130
Direct expression of an antimicrobial peptide (AMP) in Escherichia coli causes several problems such as the toxicity of AMP to the host cell, its susceptibility to proteolytic degradation, and
decreased antimicrobial activity due to the additional residue(s) introduced after cleavage of AMPs from fusion partners.
To overcome these problems and produce a large quantity of a potent AMP histonin (RAGLQFPVGKLLKKLLKRLKR) in E. coli, an efficient expression system was developed, in which the toxicity of histonin was neutralized by a fusion partner F4 (a
truncated fragment of PurF protein) and the productivity was increased by a multimeric expression of a histonin gene. The
expression level of the fusion proteins reached a maximum with a 12-mer of a histonin gene. In addition, because of the RLKR
residues present at the C terminus of histonin, furin cleavage of the multimeric histonin expressed produces an intact, natural
histonin. The AMP activity of the histonin produced in E. coli was identical to that of a synthetic histonin. With our expression system, 167 mg of histonin was obtained from 1 l of E. coli culture. These results may lead to a cost-effective solution for the mass production of AMPs that are toxic to a host. 相似文献
85.
Creatine kinase is a member of the phosphagen kinase family, which catalyzes the reversible phosphoryl transfer reaction that occurs between ATP and creatine to produce ADP and phosphocreatine. Here, three structural aspects of human-brain-type-creatine-kinase (hBB-CK) were identified by X-ray crystallography: the ligand-free-form at 2.2 Å; the ADP-Mg2+, nitrate, and creatine complex (transition-state-analogue complex; TSAC); and the ADP-Mg2+-complex at 2.0 Å. The structures of ligand-bound hBB-CK revealed two different monomeric states in a single homodimer. One monomer is a closed form, either bound to TSAC or the ADP-Mg2+-complex, and the second monomer is an unliganded open form. These structural studies provide a detailed mechanism indicating that the binding of ADP-Mg2+ alone may trigger conformational changes in hBB-CK that were not observed with muscle-type-CK. 相似文献
86.
Polydiacetylenes (PDAs), which possess unique properties that allow them to change color in response to environmental changes such as variations in pH, temperature, and molecular binding, have been widely investigated as signal transducers in biosensor applications. Most PDA-based sensors reported to date have been evaluated largely on the basis of their ability to detect purified samples, however, and their specificity has rarely been tested. In this study, novel PDAs fabricated on polyvinylidene fluoride (PVDF) strips by photoreaction of composite diacetylene self-assemblies were developed as biosensors, and nonspecific binding to off-target biomolecules was assessed. A mixed PDA surface containing biotin and ethanolamide bound the target, i.e., streptavidin, more specifically than did biotin alone. The optimized PDA biosensor exhibited approximately 2850-fold higher selectivity for streptavidin relative to bovine serum albumin controls. A PDA biosensor that was further prepared showed distinctive signals for the urine of diabetic patients compared to urine samples from healthy/non-diabetic person due to the concentration of microalbuminuria. To our knowledge, this is the first strip-type biosensor fabricated with PDAs and the first PDA-based biosensor that can effectively overcome the problem of nonspecific binding. 相似文献
87.
88.
Jong-Chul Park Min Sub Lee Dong Hee Lee Bong Joo Park Dong-Wook Han Masakazu Uzawa Kosuke Takatori 《Applied microbiology》2003,69(4):2405-2408
Seawater used in mariculture has been suspected of being a potential source of infection. In this study, the lethal effects of low-amperage electric treatment on microorganisms were examined in natural seawater and in seawater inoculated with Vibrio parahaemolyticus. In both cases, bacteria including V. parahaemolyticus in seawater were completely eliminated in 100 ms by a 0.5-A, 12-V direct current. Electron microscopic investigation of the electrically treated bacteria revealed substantial structural damage at the cellular level. In conclusion, our results indicate that low-amperage electric treatment is effective for rapid inactivation of microorganisms in seawater. 相似文献
89.
Dong Up Song Young Do Jung Kee Oh Chay Min A Chung Kee Hyung Lee Sung Yeul Yang Boo Ahn Shin Bong Whan Ahn 《Archives of biochemistry and biophysics》2002,397(2):424-429
Tea catechins and other flavonoids have been shown to potentially protect against chronic cardiovascular diseases such as coronary heart disease and atherosclerosis. In this study, 6-month-old female Sprague-Dawley rats were fed green tea extract (50 mg/100 ml in drinking water) up to the age of 22 months, and the age-associated changes in Maillard-type fluorescence and carbonyl groups in the aortic and skin collagen were compared with those occurring in the water-fed control animals. Collagen-linked Maillard-type fluorescence was found to increase in both the aortic and skin tissues as animals aged. The age-associated increase in the fluorescence in the aortic collagen was remarkably inhibited by the green tea extract treatment, while that occurring in the skin collagen was not significantly inhibited by the treatment. The collagen carbonyl content also increased in both the aortic and skin tissues as animals aged. In contrast with the case of Maillard-type fluorescence, however, the age-associated increase in the carbonyl content was not inhibited by the green tea extract treatment either in the aortic or skin collagen. These results suggest that the inhibition of AGE formation in collagen is an important mechanism for the protective effects of tea catechins against cardiovascular diseases. 相似文献
90.
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