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981.
Shen MH Kim JS Sapkota K Park SE Choi BS Kim S Lee HH Kim CS Chun HS Ryoo CI Kim SJ 《Journal of microbiology and biotechnology》2007,17(8):1271-1283
A fibrinolytic protease (PoFE) was purified from the cultured mycelia of the edible oyster mushroom Pleurotus ostreatus, using a combination of various chromatographies. The purification protocol resulted in an 876-fold purification of the enzyme, with a final yield of 6.5%. The apparent molecular mass of the purified enzyme was estimated to be 32 kDa by SDS-PAGE, fibrin-zymography, and size exclusion using FPLC. The optimal reaction pH value and temperature were pH 6.5 and 35 degrees C, respectively. PoFE effectively hydrolyzed fibrinogen, preferentially digesting the A alpha-chain and the B beta-chain over the gamma-chain. Enzyme activity was enhanced by the addition of Ca2+, Zn2+, and Mg2+ ions. Furthermore, PoFE activity was potently inhibited by EDTA, and it was found to exhibit a higher specificity for the chromogenic substrate S-2586 for chymotrypsin, indicating that the enzyme is a chymotrypsin-like metalloprotease. The first 19 amino acid residues of the N-terminal sequence were ALRKGGAAALNIYSVGFTS, which is extremely similar to the metalloprotease purified from the fruiting body of P. ostreatus. In addition, we cloned the PoFE protein, encoding gene, and its nucleotide sequence was determined. The cDNA of cloned PoFE is 867 nucleotides long and consists of an open reading frame encoding 288 amino acid residues. Its cDNA showed a high degree of homology with PoMEP from P. ostreatus fruiting body. The mycelia of P. ostreatus may thus represent a potential source of new therapeutic agents to treat thrombosis. 相似文献
982.
Balan S Choi JW Godwin A Teo I Laborde CM Heidelberger S Zloh M Shaunak S Brocchini S 《Bioconjugate chemistry》2007,18(1):61-76
The covalent conjugation of a functionalized poly(ethylene glycol) (PEG) to multiple nucleophilic amine residues results in a heterogeneous mixture of PEG positional isomers. Their physicochemical, biological, and pharmaceutical properties vary with the site of conjugation of PEG. Yields are low because of inefficient conjugation chemistry and production costs high because of complex purification procedures. Our solution to these fundamental problems in PEGylating proteins has been to exploit the latent conjugation selectivity of the two sulfur atoms that are derived from the ubiquitous disulfide bonds of proteins. This approach to PEGylation involves two steps: (1) disulfide reduction to release the two cysteine thiols and (2) re-forming the disulfide by bis-alkylation via a three-carbon bridge to which PEG was covalently attached. During this process, irreversible denaturation of the protein did not occur. Mechanistically, the conjugation is conducted by a sequential, interactive bis-alkylation using alpha,beta-unsaturated beta'-monosulfone functionalized PEG reagents. The combination of (a) maintaining the protein's tertiary structure after disulfide reduction, (b) the mechanism for bis-thiol selectivity of the PEG reagent, and (c) the steric shielding of PEG ensure that only one PEG molecule is conjugated at each disulfide bond. PEG was site-specifically conjugated via a three-carbon bridge to 2 equiv of the tripeptide glutathione, the cyclic peptide hormone somatostatin, the tetrameric protein l-asparaginase, and to the disulfides in interferon alpha-2b (IFN). SDS-PAGE, mass spectral, and NMR analyses were used to confirm conjugation, thiol selectivity, and connectivity. The biological activity of the l-asparaginase did not change after the attachment of four PEG molecules. In the case of IFN, a small reduction in biological activity was seen with the single-bridged IFN (without PEG attached). A significantly larger reduction in biological activity was seen with the three-carbon disulfide single-bridged PEG-IFNs and with the double-bridged IFN (without PEG attached). The reduction of the PEG-IFN's in vitro biological activity was a consequence of the steric shielding caused by PEG, and it was comparable to that seen with all other forms of PEG-IFNs reported. However, when a three-carbon bridge was used to attach PEG, our PEG-IFN's biological activity was found to be independent of the length of the PEG. This property has not previously been described for PEG-IFNs. Our studies therefore suggest that peptides, proteins, enzymes, and antibody fragments can be site-specifically PEGylated across a native disulfide bond using three-carbon bridges without destroying their tertiary structure or abolishing their biological activity. The stoichiometric efficiency of this approach also enables recycling of any unreacted protein. It therefore offers the potential to make PEGylated biopharmaceuticals as cost-effective medicines for global use. 相似文献
983.
Ren Q Barber HK Crawford GL Karim ZA Zhao C Choi W Wang CC Hong W Whiteheart SW 《Molecular biology of the cell》2007,18(1):24-33
Platelet secretion is critical to hemostasis. Release of granular cargo is mediated by soluble NSF attachment protein receptors (SNAREs), but despite consensus on t-SNAREs usage, it is unclear which Vesicle Associated Membrane Protein (VAMPs: synaptobrevin/VAMP-2, cellubrevin/VAMP-3, TI-VAMP/VAMP-7, and endobrevin/VAMP-8) is required. We demonstrate that VAMP-8 is required for release from dense core granules, alpha granules, and lysosomes. Platelets from VAMP-8-/- mice have a significant defect in agonist-induced secretion, though signaling, morphology, and cargo levels appear normal. In contrast, VAMP-2+/-, VAMP-3-/-, and VAMP-2+/-/VAMP-3-/- platelets showed no defect. Consistently, tetanus toxin had no effect on secretion from permeabilized mouse VAMP-3-/- platelets or human platelets, despite cleavage of VAMP-2 and/or -3. Tetanus toxin does block the residual release from permeabilized VAMP-8-/- platelets, suggesting a secondary role for VAMP-2 and/or -3. These data imply a ranked redundancy of v-SNARE usage in platelets and suggest that VAMP-8-/- mice will be a useful in vivo model to study platelet exocytosis in hemostasis and vascular inflammation. 相似文献
984.
Conditions were evaluated for optimum cryopreservation of primary chicken embryo kidney (CEK) cells. The recovery of viable
CEK cells was best (50.8% viability) when the concentration of dimethyl sulfoxide (DMSO) in the freezing medium was 20% (v/v).
The viability of primary CEK cells was not influenced by the concentration of calf serum in the freezing medium, the duration
of storage at −70°C before storage in liquid nitrogen, cell concentration, or the method of addition or dilution of DMSO.
Thawed cells recovered and grew in complete growth medium similarly to cells freshly isolated from kidney, and influenza viruses
produced plaques in the monolayer. The cryopreservation procedures described here may facilitate maintenance of a standard
stock of primary CEK cells for laboratories where preparation of primary CEK cells is not an option. 相似文献
985.
Rogers JV Choi YW Richter WR Rudnicki DC Joseph DW Sabourin CL Taylor ML Chang JC 《Journal of applied microbiology》2007,103(4):1104-1112
AIMS: To evaluate the decontamination of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surface materials using formaldehyde gas. METHODS AND RESULTS: B. anthracis, B. subtilis, and G. stearothermophilus spores were dried on seven types of indoor surfaces and exposed to approx. 1100 ppm formaldehyde gas for 10 h. Formaldehyde exposure significantly decreased viable B. anthracis, B. subtilis, and G. stearothermophilus spores on all test materials. Significant differences were observed when comparing the reduction in viable spores of B. anthracis with B. subtilis (galvanized metal and painted wallboard paper) and G. stearothermophilus (industrial carpet and painted wallboard paper). Formaldehyde gas inactivated>or=50% of the biological indicators and spore strips (approx. 1x10(6) CFU) when analyzed after 1 and 7 days. CONCLUSIONS: Formaldehyde gas significantly reduced the number of viable spores on both porous and nonporous materials in which the two surrogates exhibited similar log reductions to that of B. anthracis on most test materials. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide new comparative information for the decontamination of B. anthracis spores with surrogates on indoor surfaces using formaldehyde gas. 相似文献
986.
Lim SJ Lee YJ Park DH Lee E Choi MK Park W Chun KH Choi HG Cho JS 《Apoptosis : an international journal on programmed cell death》2007,12(2):423-431
Sulindac sulfone (also known as exisulind) and its chemical derivatives are promising anticancer agents capable of inducing
apoptosis in a variety of malignant cell types with minimal toxicity to normal cells. Here, we tested the ability of alpha-tocopheryl
succinate (TOS), another promising anticancer agent, to sensitize colon cancer cells to exisulind-induced apoptosis. We found
that sub-apoptotic doses of TOS greatly enhanced exisulind-induced growth suppression and apoptosis in the HCT116, LoVo and
SNU-C4 human colon cancer cell lines. Our results revealed that this was accounted for primarily by an augmented cleavage
of poly(ADP-ribose) polymerase (PARP) and enhanced activation of caspase-8, -9 and -3. Pretreatment with z-VAD-FMK (a pan-caspase inhibitor), z-IETD-FMK (a caspase-8 inhibitor) or z-LEHD-FMK (a caspase-9 inhibitor) blocked TOS and exisulind cotreatment-induced PARP cleavage and apoptosis. Furthermore,
TOS/exisulind cotreatment induced JNK phosphorylation, while pretreatment with SP600151 (a JNK inhibitor) partially blocked
cotreatment-induced caspase-dependent PARP cleavage and apoptosis. Taken together, these findings indicate that TOS sensitizes
human colon cancer cells to exisulind-induced apoptosis. Apoptotic synergy induced by exisulind plus TOS seems likely to be
mediated through a mechanism involving activation of caspases and JNK.
S.-J. Lim, Y.-J. Lee both authors are contributed equally to this study. 相似文献
987.
Choi MR Groot M Drexler HC 《Apoptosis : an international journal on programmed cell death》2007,12(11):2025-2035
RhoGDI2, a cytosolic regulator of Rho GTPase, is cleaved during apoptosis in a caspase-3 dependent fashion. By using 2D-gel
electrophoresis, mass spectrometry and Western blotting we investigate in this paper the functional consequences of RhoGDI2
processing. We can show that loss of the N-terminal 19 amino acids results in a shift of the isoelectric point of the truncated
RhoGDI2 (NΔ19) to a more basic value due to the removal of 9 acidic amino acids from the N-terminus, which may be responsible
for enhanced retention of the N-terminally truncated protein within the nuclear compartment. Fusion of the p53 nuclear export
signaling sequence MFRELNEALELK to NΔ19 (NΔ19NES) abolished its apoptosis promoting properties, while overexpression of NΔ19
significantly increased the susceptibility to apoptosis induction by the proteasome inhibitor PSI and by staurosporine. These
results suggest that cleavage of RhoGDI2 by caspase-3 is not a functionally irrelevant bystander effect of caspase activation
during apoptosis, but rather expedites progression of the apoptotic process. 相似文献
988.
Regulation of cyclin-dependent kinase inhibitor p21<Superscript>WAF1/CIP1</Superscript> by protein kinase Cδ-mediated phosphorylation 总被引:1,自引:0,他引:1
Oh YT Chun KH Park BD Choi JS Lee SK 《Apoptosis : an international journal on programmed cell death》2007,12(7):1339-1347
Cyclin-dependent kinase (CDK) inhibitor p21WAF1/CIP1(-/-)-null mice have an increased incidence of tumor formation. Here, we demonstrate that p21WAF1/CIP1 is unstable in HeLa cells treated with siRNA duplexes that target PKCδ. PKCδ phosphorylates p21WAF1/CIP1 at a serine residue (146Ser) located in its C-terminal domain. In cells treated with 12-O-tetradecanoylphorbol 13-acetate, the levels of both p21WAF1/CIP1 and its 146Ser-phosphorylated form increased significantly. We also show that a substitution, resulting from a single nucleotide polymorphism
(SNP) at 149Asp found in certain cancer patients, strongly compromises PKCδ-mediated phosphorylation at 146Ser and results in cells that are relatively resistant to TNFα-induced apoptosis. Thus, post-translational phosphorylation
of p21WAF1/CIP1 is important from an apoptotic cell death, and may also have patho-physiological relevance for the development of human cancer. 相似文献
989.
AIMS: Enhancement of algicidal activity by immobilization of algicidal bacteria antagonistic to Stephanodiscus hantzschii. METHODS AND RESULTS: In laboratory studies, A diatom-lysing bacterium, Pseudomonas fluorescens HYK0210-SK09 showed strong algicidal activity against S. hantzschii, but a natural mesocosm study revealed that this bacterium failed to fully control natural blooms of Stephanodiscus at the low water temperatures that favour these blooms. Here, we sought to develop an effective immobilization strategy for enhancing the algicidal activity of HYK0210-SK09 in the natural setting. Bacterium HYK0210-SK09 was immobilized with various carriers including agar, alginate, polyurethane and cellulose sponge. The bacterial cells immobilized with cellulose sponge (CIS) induced more rapid and complete lysis of S. hantzschii than other carriers, and had a higher packing ability than polyurethane. Furthermore, CIS-immobilized cells showed higher lysis of S. hantzschii at the same concentrations as that of free cells (< or =1 x 10(7) cells ml(-1)), and had especially strong algicidal activity at the low temperatures (<10 degrees C). Based on these laboratory studies, we assessed the possible application of HYK0210-SK09 cells in the field by performing a mesocosm study during the winter season. The CIS-immobilized cells with species-specific activity towards the genera Stephanodiscus showed extremely high algicidal activity (up to 95%) against a bloom of Stephanodiscus hantzschii even at low water temperatures, because of high cell packing and subsequent cell protection against low temperatures and predators, whereas free cells showed negligible algicidal activities under these conditions. CONCLUSION: Immobilizing cells of HYK0210-SK09 in CIS foam, rather than in the other matrices tested, could achieve more efficient control of Stephanodiscus blooms and showed a significant algicidal activity on in vitro and in vivo blooms, even at low water temperature. SIGNIFICANCE AND IMPACT OF THE STUDY: Collectively, these results indicate that CIS of algicidal bacteria may form an important strategy for effective management of Stephanodiscus blooms at low water temperatures. 相似文献
990.
Choi YJ Lee EM Park JM Lee KM Han SH Kim JK Lee SH Song HJ Choi MS Kim IS Park KH Jang WJ 《Microbiology and immunology》2007,51(3):307-312
This study revealed the presence of various rickettsial agents in mites from wild rodents collected in Southern Jeolla Province, Korea, by nested polymerase chain reaction (PCR) and sequence analysis of a partial citrate synthase and rickettsia outer membrane protein B genes. Rickettsial agents closely related to the Rickettsia species TwKM02, R. australis, and the Rickettsia species Cf15 were successfully identified in this study, for the first time in Korea, and R. japonica, R. akari, R. conorii, R. felis, and R. typhi were also detected, as previously described. The data presented in this paper extend knowledge on the geographic distribution of SFG rickettsiae in eastern Asia and it may necessary to consider the role of mites in rickettsial transmission. 相似文献