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 $35^{\circ}C$, 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 $Ca^{2+},\;Zn^{2+},\;and\;Mg^{2+}$ 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.
In this study, we have cloned a novel cDNA encoding for a papain-family cysteine protease from the Uni-ZAP XR cDNA library of the polychaete, Periserrula leucophryna. This gene was expressed in Escherichia coli using the T7 promoter system, and the protease was characterized after partial purification. First, the partial DNA fragment (498 bp) was amplified from the total RNA via RT-PCR using degenerated primers derived from the conserved region of cysteine protease. The full-length cDNA of cysteine protease (PLCP) was prepared via the screening of the Uni-ZAP XR cDNA library using the $^{32}P-labeled$ partial DNA fragment. As a result, the PLCP gene was determined to consist of a 2591 bp nucleotide sequence (CDS: 173-1024 bp) which encodes for a 283-amino acid polypeptide, which is itself composed of an 59-residue signal sequence, a 6-residue propeptide, a 218-residue mature protein, and a long 3'-noncoding region encompassing 1564 bp. The predicted molecular weights of the preproprotein and the mature protein were calculated as 31.8 kDa and 25 kDa, respectively. The results of sequence analysis and alignment revealed a significant degree of sequence similarity with other eukaryotic cysteine proteases, including the conserved catalytic triad of the $Cys^{90},\;His^{226},\;and\;Asn^{250}$ residues which characterize the C1 family of papain-like cysteine protease. The nucleotide and amino acid sequences of the novel gene were deposited into the GenBank database under the accession numbers, AY390282 and AAR27011, respectively. The results of Northern blot analysis revealed the 2.5 kb size of the transcript and ubiquitous expression throughout the entirety of the body, head, gut, and skin, which suggested that the PLCP may be grouped within the cathepsin F-like proteases. The region encoding for the mature form of the protease was then subcloned into the pT7-7 expression vector following PCR amplification using the designed primers, including the initiation and termination codons. The recombinant cysteine proteases were generated in a range of 6.3 % to 12.5 % of the total cell proteins in the E. coli BL21(DE3) strain for 8 transformants. The results of SDS-PAGE and Western blot analysis indicated that a cysteine protease of approximately 25 kDa (mature form) was generated. The optimal pH and temperature of the enzyme were determined to be approximately 9.5 and $35^{\circ}C$, respectively, thereby indicating that the cysteine protease is a member of the alkaline protease group. The evaluation of substrate specificity indicated that the purified protease was more active towards Arg-X or Lys-X and did not efficiently cleave the substrates with non-polar amino acids at the P1 site. The PLCP evidenced fibrinolytic activity on the plasminogen-free fibrin plate test.
Hyalophora cecropia attacin-like antibacterial gene was isolated from Bombyx mori induced with nonpathogenic bacteria. It was expressed in Spodopfera frugiperda 9 (Sf9 cells using baculovirus expression vector system (BEVS), and examined its antibacterial activity. With a cDNA library constructed from fifthinstar B. mori injected with Escherichia coli(4 X IOhcellsllarva), differential screening was performed using naive and induced mRNA probes. BmInc6 clone was screened by partial nucleotide sequence and GenBank database analysis. A complete nucleotide sequence of Bmlnc6 cDNA was determined (GenBank, AF005384). Its insert size was 852 bp and had open reading frame that started translation at position 35 and stopped at 679. And its putative polyadenylational signal existed at 812 bp. The number of amino acid deduced from Bmlnc6 cDNA was 214 and hydropathy analysis showed that this peptide was hydrophilic. This peptide deduced by BmInc6 was named nuecin. When the nuecin gene was expressed in Sf9 cells using BEVS, about 950 bp of the transcripts was detected. In addition, SDS-PAGE analysis showed that the molecular weights of intracellular expressed protein and the mature protein secreted to culture media were approximately 23 and 20 kDa, respectively. The antibacterial activity of nuecin against E. coli and Bacillus subtilis was significantly high, demonstrating that nuecin had a wider antibacterial spectrum with gram negative and positive bacteria than attacin.
Cronobacter sakazakii and Salmonella enterica Typhimurium are hazardous pathogens, especially for ready-toeat foods. For control of pathogens, the virulent bacteriophages were isolated, identified, and applied to infant formula milk and vegetable juice. The phages were isolated from swine feces and identified by morphology and molecular characteristics. ES2 phage for C. sakazakii and ST2 phage for S enterica Typhimurium were identified as Myoviridae and Siphoviridae, respectively. Their burst sizes were $52{\pm}5PFU/cell$ for ES2 phage and $21{\pm}3PFU/cell$ for ST2 phage after latent period of 30-40 minutes. ST2 phage showed higher heat stability at $60^{\circ}C$ than ES2 phage. ES2 phage held the growth of C. sakazakii untill 6 hr afterwhich the number decreased when applied to the infant formula milk and vegetable juice. ST2 phage also showed growth inhibition so that the number of S. enterica Typhimurium decreased. Therefore, virulent bacteriophages might be an agent for the growth inhibition of C. sakazakii and S. enterica Typhimurium in such the ready-to-eat foods.
Most traditional newspaper publishers provide online editions to counter the competition of online news providers. However, the relationship between the online and print editions of the same newspaper has not been clearly defined. Some see the online newspaper as a substitute, while others consider it a complement. A 2002 NAA online newspaper consumer survey indicated that one-third of its respondents said they were now using the print newspaper less. Others have argued that the online edition will not wipe out print consumption, and may even complement it. While the print edition offers particular advantages such as portability, less eye strain, and the tactile experience of a printed page, the online edition also offers specific advantages such as access to breaking news, continually updated information, access to old archives, etc. All these factors would tend to lower the degree of interchangeability between the products. However, recent empirical studies show that the online edition is a substitute for rather than a complement of the print edition. Still, to some print readers, the online edition provides additional value. In this paper, by capturing the two different aspects of online editions the substitute aspect and the additional value added aspect as well as other available online alternatives, we develop an analytical model to derive the optimal production and distribution strategies of both online and print editions. Confronting the "free versus fee" issue, we show that it is optimal to provide an online version of the print newspaper for free to non-print subscribers. However, the amount of free news content that the publishers need to put on the Web depends on the available alternatives on the online market. The "fee" and "free" options both have merits and demerits as well. If the publisher charges for the online version of the print newspaper, she can generate revenue from the fee charged to online readers. However, doing so will limit the size of the online audience and further reduce online advertising revenue. At the same time, by providing a high-quality online version and charging for it, the price of the print newspaper must stay low in order to lure high valued readers. On the contrary, if the publisher provides an online version of the print newspaper for free, she can obtain a larger audience for the online version. At the same time, by providing a low-quality online newspaper, the publisher can increase the print newspaper price to get more revenue from high valued offline readers, although no revenue is incoming from online version readers. Through systematic measuring of all the pros and cons, our analysis shows that the optimal option is not "fee" but "free."
The digital cartoons market is looking for a new growth momentum as the radical increases of the demands and markets about the mobile contents with portable instrument popularization. The conventional digital cartoons markets which are based on web-toon, page viewer cartoons and e-paper cartoons have been studied various fields to overcome some limitations such as the traditional cartoons had. The mobile cartoons which have been changed more and more, have some canvas limitations due to the mobile screen size. These limitations lead to the communication problems between the cartoonists and the subscribers and resulting some obstacles of mobile cartoons activations. In this paper, we developed a authoring tool applied the Screen Flow method to overcome inefficiency of conventional authoring methods. This proposed method can reflect the cartoonists' during the process of authoring mobile cartoons, thereafter we studied about the authoring method of mobile cartoons and its effects. For the convenience of users creating and distributing content in a way has been studied.
An isolate of Cucumber mosaic cucumovirus(CMV) was isolated from Hydrangea macrophylla for. otaksa(Sieb. et Zucc. ) Wils. showing mosaic symptoms, and designated as Hm-CMV. Hm-CMV was characterized by the tests of host range, physical properties, serological properties, RNA and coat protein compositions, and reverse transcription and polymerase chain reaction (RT-PCR) analysis. Twelve species in 4 families were used in the host range test of Hm-CMV and could be differentiated from Y-CMV used as a control CMV by the ringspot and line pattern on inoculated leaves of several tobacco plants. Thevirus produced local lesions on inoculated leaves of Chenopodium amarticolor, C. quinoa and Vigna unguiculata. The physical properties of the virus were as follows; thermal inactivation point(TIP) was 60$\^{C}$, dilution end point (DEP) was 10$\^$-3/, and longevity in vitro (LIP) was 3∼4 days. Hm-CMV was serologically identical to Y-CMV. SDS-polyaciylamide gel electrophoresis(SDS-PAGE) showed one major protein band of about 28 kDa. In RNA or dsRNA analysis, Hm-CMV consisted of four RNA or dsRNA species, but satellite RNA was not detected. In RT-PCR using CMV-common primer and CMV subgroup I-specific primer, bothe amplified expected size of about 490 bp and 200 bp DNA fragments from Hm-CMV, respectively. Restriction enzyme analysis of the 490 bp RT-PCR products using EcoR I and Msp I showed that Hm-CMV belonged to CMV subgroup I. However, Hm-CMV could be differentiated from other CMV subgroup I isolates by RNA fingerprinting by arbitrarily primed polymerase chain reaction (RAP-PCR).
Alfalfa mosaic alfamoviruses(AIMV) were isolated from infected potato (Solanum tuberosum) and azuki bean (Paseolus angularis) in Korea. Two AIMV isolated from potatoes were named as strain KR (AIMV-KR1 and KR2) and AIMV isolated from azuki bean was named as strain Az (AIMV-Az). Each isolated AIMV strain was characterized by using their host ranges, symptom developments, serological relations and nucleotide sequence analysis of coat protein (CP) gene. Strains KR1, KR2, and Az were readily transmitted to 20 of 22 inoculated plant species including bean, cowpea, tomato, tobacco, and potato. AIMV-KR1 and KR2 produced the typical symptoms like chlorotic or necrotic spots in Chenopodium quinoa and Solanum tuberosum cv. Superior. AIMV-Az caused bright yellow mosaic symptom and leaf malformation in Nicotiana glauca, which were different from the common mosaic symptom caused by AIMV-KR1 and KR2. Electron microscope observation of purified virus showed bacilliform virions containing a single-stranded plus-strand RNAs of 3.6, 2.6, 2.0 and 0.9 kbp in length, respectively, similar in size and appearance to those of Alfamovirus. In SDS-PAGE, the coat protein of the two viruses formed a consistent band that estimated to be about 24kDa. The CP genes of the AIMV strains, KR1, KR2, and Az have been amplified by RT-PCR using the specific primers designed to amplify CP gene from viral RNA-3, cloned and sequenced. Computer aided analysis of the amplified cDNA fragment sequence revealed the presence of a single open reading frame capable of encoding 221 amino acids. The nucleotide and peptide sequence of viral CP gene showed that strain KR1, KR2, and Az shared highest nucleotide sequence identities with AIMV strain 425-M at 97.7%, 98.2%, and 97.2%, respectively. CP gene sequences of two strains were almost identical compared with each other. Altogether, physical, serological, biological and molecular properties of the purified virus.
The use of antihemophilic factor IX complex has been associated with a variety of thrombotic complications, the major cause of which was the contamination of thrombogenic proteins such as vitamin K-dependent clotting factors II, VII, and X. In order to produce a commercial factor IX (GreenNine VF) free from thrombogenic potential, industrial-scale production process for high-purity factor IX from human plasma has been developed. The purification process contains cryo-precipitation, DEAE-sephadex A-50 anion-exchange chromatography, DEAE-toyopearl 650M anion-exchange column chromatography, heparin-sepharose 6FF affinity column chromatography, and CM-sepharose FF cation-exchange column chromatography. Also the process includes two viral inactivation and removal procedures, solvent/detergent treatment and nanofiltration using Viresolve NFP filter. The purification yield was 35.4%. The specific activity in the purified concentrate was 190.8 IU/mg which exceeded that in the factor IX complex (FacNine) by a factor of 48. The activities of factor II, VII, and X were not detected in GreenNine VF. SDS-PAGE analysis showed that GreenNine VF had the highest purity in comparison with commercially available high purity factor IX concentrates, Mononine, Octanyne, Berinin HS, and Immunine STIM plus 600. One batch size of the production was 2,400 vials of 250 IU product or 1,200 vials of 500 IU product from 1,600 L cryo-poor plasma.
By SDS-polyacrylamide gel elcctrophoresis (SDS-PAGE) and immunoblot analysis that a protein with 66,000 daltons in size was recognized by P. aeruginosa anti-exotoxin A from P. sp. PY002. The yields of exotoxin A in P. sp. PY002 culture were influenced by the concentration of iron in the culture media. Increasing of the exotoxin A production and siderophore production was made slight increasing in the MKB medium. On the other hand, to clone the gene encoding the exoloxin A genomic library of P. sp. PY002 was constructed in pBluescript SK(+). From this library a exotoxin A homologous gene was isolated by immunological hybridization method using P. aemginosa anti-exoloxin A as a probe. Two putative clones were isolated and designated pETA23 and pETA42. Thc restriction analysis ol pETA42 demonstrated that thc 1760 bp insert contained one NcoI, PvuII, SstI, Kpnl and EcoRI site and three SmaI and HaeD sites.
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