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Several types of yeasts were isolated from wild flowers around Jangseong Lake in Jeollanam-do, Republic of Korea and identified by comparing the nucleotide sequences of the PCR amplicons for the D1/D2 variable domain of the 26S ribosomal DNA using Basic Local Alignment Search Tool (BLAST) analysis. In total, 60 strains from 18 species were isolated, and Pseudozyma spp. (27 strains), which included Pseudozyma rugulosa (7 strains) and Pseudozyma aphidis (6 strains), was dominant species. Among the 60 strains, Bullera coprosmaensis JS00600 represented a newly recorded yeast strain in Korea, and its microbiological characteristics were investigated. The yeast cell has an oval-shaped morphology measuring 1.4 × 1.7 µm in size. Bullera coprosmaensis JS00600 is an asporous yeast that exhibits no pseudomycelium formation. It grew well in vitamin-free medium as well as in yeast extract-malt extract broth and yeast extract-peptone-dextrose (YPD) broth, and it is halotolerant growing in 10% NaCl-containing YPD broth.

Six unrecorded yeasts, Cryptococcus festucosus 41-3, Cryptococcus heveanensis 56-4, Debaryomyces nepalensis 95- 4, Issatchenkia occidentalis 142-1, Dioszegia zsoltii 39-1, and Kwoniella europala 47-2 were screened from 108 yeasts isolated from flowers and fruits in orchards of Yesan-gun, Chungcheongnam-do, Korea. The morphological and cultural characteristics of these unrecorded yeasts were investigated. They had various shapes, including ellipsoidal, globose, and oval, and also had budding mode in vegetable reproduction, except I. occidentalis 142-1 (fission mode). K. europaea 47-2 only formed pseudomy- celium. D. zsoliti 39-1 did not grow in yeast extract-malt extract medium, potato dextrose medium, and vitamin-free medium. C. festucosus 41-3 grew well in 5% NaCl-containing yeast extract-peptone-dextrose medium and had a growth pH range of 7.0~ 10.0. Three unrecorded yeasts Ogataea polymorpha HB45-1, Rhodotonula hinnulla HB62-2, and Cryptococcus rajasthanensis HB80-4 were screened from 51 yeasts isolated from flowers in Hanbat arboretum in Daejeon city, Korea. They were globose in shape and did not form pseudomycelium. In addition, O. polymorpha HB45-1 and C. rajasthanensis HB80-4 had budding mode in vegetable reproduction. All of them grew well in vitamin-free medium and C. rajasthanesis HB80-4 also grew in 50% glucose and 5% NaCl-containing YPD medium.

Two unrecorded yeasts, Meyerozyma caribbica UL5-1 and Pichia silvicola UL6-1 were screened from 58 yeasts which were isolated from wild flowers in Ulleungdo in Gyeongsangbuk-do, Korea. The morphological and cultural characteristics of these unrecorded yeasts were investigated. Both yeasts were oval in shape and formed pseudomycelia. P. silvicola UL6-1 formed ascospore, but M. UL5-1 did not. P. silvicola UL6-1 and M. caribbica UL5-1 also grew in vitamin-free medium and 5% NaCl-containing yeast extract-peptone-dextrose medium. The two unrecorded yeasts assimilated glucose, galactose, xylose, cellobiose, trehalose, glycerol and sorbitol, and also fermented glucose, fructose and mannose. The supernatant of both M. caribbica UL5-1 and P. silvicola UL6-1 showed high antihypertensive angiotensin I-converting enzyme inhibitory activity of 84.2% and 82.6%, respectively. Cell-free extract of P. silvicola UL6-1 also showed very high anti-diabetic -glucosidase inhibitory activity (85.8%).

Yeasts are common inhabitants of the phyllosphere, but our knowledge of their diversity in various fruits and blossoms are limited. We collected different kinds of blossoms and unripened fruits from Hanbat arboretum, Daejeon, Korea at the year of 2013. Yeasts were isolated by plating of suspensions prepared for collected samples onto YPD medium containing antibiotics. BLAST searches were subsequently performed for the comparison of the partially determined sequences of D1/D2 domain of 26S rDNA. As a result, we isolated 57 yeast strains of 31 species from 29 different kinds of blossoms and 6 kinds of fruits samples. We found huge differences in yeast flora depending on the sample collection season.

Various yeasts from wild flowers of Ulleungdo in Gyeongsangbuk-do and Yokjido in Gyeongsangnam-do, Korea were isolated and identified by comparison of nucleotide sequences for PCR-amplified D1/D2 region of 26S rDNA using BLAST. Forty eight yeast strains of twenty two species and sixty yeast strains of twenty five species were isolated from wild flowers of Ulleungdo and Yokjido, respectively. Only seven species were overlapped from the two different islands areas: Cryptococcus albidus, Cryptococcus laurentii, Metschnikowia reukafii, Pichia scolyti, Rhodotorula glutinis, Rhodotorula graminis and Rhodotorula mucilaginosa. Among forty species from two different islands, other thirty three species were restricted to specific collection site suggesting that each area has distinctive yeast flora.

Several yeasts were isolated from flowers found in Gyonggi-do Province and Jeju island in Korea. They were then identified by a comparison of their PCR-amplified D1/D2 regions of 26S rDNA, internal transcribed spacer 1 and 2 inclusive of 5.8S rDNA, using the BLAST database. A total of fifty four yeast strains were isolated from wild flowers in Gyonggi-do and the genus Pseudozyma was noted as being dominant. A total of thirty two strains were isolated from Songaksan and Seongsan-ilchulbong in Jeju island and Sporobolomyces ruberrimus was seen to be dominant. The anti-gout xanthine oxidase inhibitory activities of the culture broths and cell-free extracts from eighty six yeast strains were then determined. The cell-free extracts of Pseudozyma hubeiensis 228-S-1 exhibited the highest xanthine oxidase inhibitory activity of 19.6%. The XOD inhibitor was also maximally produced when Pseudozyma hubeiensis 228-S-1 was cultured at for 36h in YEPD medium.

Several yeast colonies were isolated from wild flowers collected from East, West and South coast areas of Korea by plating of flower suspensions on the YPD plates containing antibiotics, streptomycin and ampicillin. Polymerase chain reactions (PCR) were performed for the amplification of D1/D2 region of 26S rDNA for those colonies. PCR-amplified nucleotide sequences were compared using BLAST for their identification. As results, 27 yeast strains belonged to 15 species were isolated from wild flowers collected at Donghae, where is located in eastern coast of Korea. Also, 34 strains belonged to 17 species were isolated from wild flowers of Daecheon, where is located in western coast of Korea. In addition, 22 strains belonged to 13 species were isolated from wild flowers collected at Wando, where is located in southern coast of Korea. Among those 45 species isolated from 3 different collection sites, only 4 species including Cryptococcus laurentii, Metschnikowia koreensis, Pseudozyma rugulosa, and Rhodotorula mucilaginosa were found from all 3 different collection sites. And 5 species including Cryptococcus aureus, Cryptococcus flavus, Hanseniaspora uvarum, Pichia guilliermondii, and Rhodosporidium fluviale were overlapped from the at least 2 different collection sites. Other 23 species were found only in a specific collection sites implying that each area has distinctive yeast flora.

Yeasts isolated from wild flowers of Gyejoksan in Daejeon city, Oseosan in Chungchungnamdo, and Baekamsan in Jeollabukdo, Korea were identified by comparison of nucleotide sequences for PCR-amplified D1/D2 region of 26S rDNA or internal transcribed spacer (ITS) 1 and 2 including 5.8S rDNA using BLAST. Twelve yeast strains of ten species and seventeen yeast strains of ten species were isolated from wild flowers of Gyejoksan and Oseosan, respectively. And thirty seven yeast strains of twenty four species were isolated from wild flowers of Baekamsan. Total thirty four yeast species were isolated from three different sample collection areas, but only nine species were overlapped from the at least two different sampling areas: Cryptococcus sp., Cryptococcus aureus, Cryptococcus flavescens, Cryptococcus flavus, Metschnikowia sp., Pseudozyma aphidis, Rhodotorula glutinis, Sporobolomyces carnicolor, and Sporobolomyces ruberrimus. Among them only Cryptococcus aureus was occurred from all three different collection sites. Other twenty five species were restricted to specific collection site suggesting that each area has distinctive yeast flora.

The goal of this study was to characterize the physicochemical properties, lactic acid bacteria and physiological functionality of Korean commercial Makgeolli. We collected 60 kinds of raw and pasteurized Makgeolli commercially available in Korea. Their physicochemical properties, lactic acid bacteria content and physiological functionalities were determined. In the commercial raw Makgeolli, ethanol and residual sugar content ranged from 3.6% to 9.6% and 0.20% to 4.52%, respectively. Furthermore, the raw Makgeolli contained 3.50 to 101.9 mg% of amino nitrogen. In the pasteurized Makgeolli, ethanol content ranged from 4.0% to 7.0% and their residual sugar content were 0.98% to 7.57%. The raw Makgeolli contained 2.0 ~1.2 CFU/mL of lactic acid bacteria. Among several functionalities, the antihypertensive angiotensin I-converting enzyme inhibitory activities of EDS-14 raw Makgeolli and PWR-12 pasteurized Makgeolli were very high, at 89.9% and 87.0% respectively. The other functionalities were seen to be below 30% or not detectable. The -Glucan contents of the raw and pasteurized Makgeolli were noted as average, at 14.1% and 14.6%, respectively.

딸기를 이용한 건강막걸리를 개발하고자, 딸기를 첨가하여 막걸리를 제조한 후 이들의 품질 특성을 조사하였다. 먼저 딸기막걸리 제조에 적합한 효모를 선발하기 위해 시판 알코올 발효효모와 배재대학교에서 분리 보관중인 알코올 발효효모를 이용하여 딸기막걸리를 제조한 결과 분리 효모인 Saccharomyces cerevisiae JSK...

Total 50 species, 80 strains: Eighty strains of yeasts were isolated from wild flowers of Daejeon city and its surroundings in Korea. Isolated yeasts were identified by comparison of their PCR-amplified 18s rDNA V3 region or internal transcribed spacer-2 (ITS-2) genes with BLAST database. Among them, 32 species, 54 strains of yeasts were isolated and identified from wild flowers of Daejeon city, and Cryptococcus species were dominant yeasts. Furthermore, 18 species, 26 strains of yeasts were isolated and identified from wild flowers of Geumsan-gun and Nonsan-gun, Chungcheongnam-do in Korea, and also Candida species and Pseudozyma species were dominant yeasts.

In this study, we investigated the microbial flora changes in Gugija-Liriope tuber Makgeolli during fermentation and storage periods. We brewed Gugija-Liriope tuber Makgeolli for a week through twostage fermentations and stored the fermentation broth for a month at or . We collected the samples periodically and analyzed microbial flora changes using viable cell counts and PCR-denaturing gradient gel electrophoresis (DGGE). Yeast viable cells were seen to have decreased to 13% of pre-storage levels after storage for 15 days at ; however significant changes were not observed during storage at . Prolongation of storage time dramatically decreased the availability of viable cells. Yeast viable cell numbers had decreased to 38% of pre-storage levels at and 4.8% at after storage for 30 days. The results of the DGGE profile for yeast showed that Saccharomyces cerevisiae and Saccharomyces sp. were the predominant strains at the beginning of fermentation and throughout the whole period of storage. Viable cell counts for total bacteria had decreased to 36% of pre-storage levels after storage for 15 days but did not significantly change for the full 30 days of storage at . Similarly, viable cell counts for bacteria had decreased to 5% while viable cell numbers did not significantly change for the full 30 days at . Viable cell counts for lactic acid bacteria were performed and the results were similar to those for total bacteria. The results of the DGGE profile for bacteria showed that Weissella cibaria was the predominant strain at the beginning of fermentation. However it had disappeared by the end of fermentation, and Lactobacillus fermentum and Pediococcus acidilactici became the predominant species during storage.

We measured physiological functionalities, including antihypertensive angiotensin I-converting enzyme inhibitory activity and immun-stimulating β-glucan content for sixty kinds of Makgeolli that is commercially available from the market. As a result, we selected R-12 commercial raw Makgeolli, with a high content of immuno-stimulating β-glucan, and R-14 commercial raw Makgeolli, exhibiting high antihypertensive activity. Due to the similarities in their overall physicochemical properties and raw materials used for fermentation, we compared the microbial flora in order to investigate the reason for the differences in their functionalities. Nested PCR and denaturing gradient gel electrophoresis for yeasts and bacteria were performed for analysis of microbial diversity of two different kinds of Makgeolli (i.e., R-12, R-14), which showed immuno-stimulating β-glucan content and exhibited a very high level of antihypertensive activity, respectively. Analysis of the 18S rDNA amplicon revealed a major presence of the yeast strain Pichia burtonii in every Makgeolli sample. Analysis of the 16S rDNA amplicon revealed a predominance of lactic acid bacteria, and the most frequent lactic acid bacteria were Lactobacillus ingluviei, L. fermentum, and L. harbinensis, and Lactobacillus sp. Among these, L. harbinensis was detected only in R-12 and L. ingluviei was found only in R-14. Different functionalities from the individual commercially available Makgeolli may be attributed to actions of different microbial flora during fermentation.

12-O-tetradecanoylphorbol-13-acetate (TPA) has been shown to induce transcriptional activation of human manganese superoxide dismutase (MnSOD) mRNA in human lung carcinoma cells, A549, mediated by a protein kinase C (PKC)-dependent activation of cAMP-responsive element-binding protein (CREB)-1/ATF-1-like factors. In this study, we showed that MnSOD protein expression was elevated in response to TPA or TNF-α, but not to hydrogen peroxide treatment. TPA-induced generation of reactive oxygen species (ROS) was blocked by pretreatment of the PKC inhibitor BIM and NADPH oxidase inhibitor DPI. Small interfering RNA (siRNA) experiments indicated that knocking down the NADPH oxidase components e.g. Rac1, p22phox, p67phox, and NOXO1 in A549 cells impaired TPA-induced MnSOD expression. To identify the PKC isozyme involved, we used a sod2 gene response reporter plasmid, pSODLUC-3340-I2E-C, capable of sensing the effect of TNF-α and TPA, to monitor the effects of PKC isozyme-specific inhibitors and siRNA-induced knockdown of specific PKC isozyme. Our data indicate that TPA-induced MnSOD expression was independent of p53 and most likely mediated by PKC-α-, and -ϵ-dependent signaling pathways. Furthermore, siRNA-induced knock-down of CREB and Forkhead box class O (FOXO) 3a led to a reduction in TPA-induced MnSOD gene expression. Together, our results revealed that TPA up-regulates, in part, two PKC-dependent transcriptional pathways to induce MnSOD expression. One pathway involves PKC-α catalyzed phosphorylation of CREB and the other involves a PKC-mediated the PP2A catalyzed dephosphorylation of Akt at Ser473 which in turn leads to FOXO3a Ser253 dephosphorylation and its activation.

To develop new functional traditional rice wines using Gugija and Liriope tuber as raw materials, screenings of optimal fungal nuruk and alcohol fermentative yeast for brewing of Gugija-L. tuber traditional rice wine were performed with investigation of optimal fermentation condition. Finally, we selected commercial SJ nuruk and Saccharomyces cerevisiae C-2 as optimal nuruk and yeast for Gugija-L. tuber traditional rice wine. Furthermore, a new antihypertensive and anti-gout Gugija-L. tuber traditional rice wine was produced when 3% of Jangmyong Gugija and L. tuber No.1 were added into cooked rice and then fermented at 25oC for 5 days with SJ nuruk and S. cerevisiae C-2.

oC for 7 days. Among several alcohol fermentation yeasts, ethanol contents was the highest in pear Yakju made by S. cerevisiae K-7 and also showed high ethanol content in pear Yakju which was made by commercial S. cerevisiae C-2. Therefore, we selected S. cerevisiae K-7 and S. cerevisiae C-2 as suitable yeasts for brewing of KTPY. Maximal ethanol production (10.4%) was obtained when cooked non-glutinous rice (100 g) and nuruk (30 sp/g) were mixed and added into pear juice (600 ml) with S. cerevisiae K-7 (5%) and fermented at 25 o C for 7 days and also its antihypertensive angiotensin I-converting enzyme (ACE) inhibitory activity was 57.2%. Addition of antihypertensive starchy materials into the mash was not affected in ACE inhibitory activity and total acceptability of KTPY.

To produce bioactive compound enriched yeast using medicinal Gugiga (Lycium chinensis Mill), several edible Saccharomyces species were cultured in Gugija extracts added yeast extract, peptone and dextrose medium (GE - YEPD medium) at 30℃ for 24 hr, and their growth were determined. Growth of Saccharomyces cerevisiae K-7 and Sacchromyces cerevisiae ACTC 7904 were better than those of the other yeasts. Two yeasts were selected and then determined their some physiological functionalities after cultivated the yeasts in the GE - YEPD medium and compared those grown on YEPD medium. Antihypertensive angiotensin I-converting enzyme (ACE) inhibitory activity of S. cerevisiae K-7 grown on GE - YEPD medium was about 20% higher than that grown on YEPD medium. Superoxide dismutase-like activity of S. cerevisiae ACTC 7904 was also about 12% more high. However, the other physiological functionalities were almost same or lower. Optimal addition concentration of Gugija extract was 10%, and maximally growth and ACE inhibitory activity of S. cerevisiae K-7 were shown when the strain was cultured in 10% Gugija extracts containing YEPD medium at 30℃ for 12 hr.

우리나라 야생화들의 효모 종 다양성을 확립하고 이들을 고부가가치의 건강산업에 응용하기 위한 자료를 얻고자 한국의 제주도, 울릉도, 욕지도와 선유도 등 4개의 섬지역 산들과 계족산, 오서산, 백암산과 덕유산 등 4개의 내륙지역산에서 개화한 야생화들을 2012년에서 2014년 사이에 수집하여 효모 134종 289균주를 분리 동정하였다. 분리한 289균주 중에서 Cryptococcus속 균주들이 가장 많이 분리되었고 Cryptococcus aureus를 포함하는 23균주들이 섬과 내륙 산지역에서 공통적으로 분리 동정되었다. 또한 단일 균주로는 Metschnikowia reukaufii가 전체 균주 중 10.3%인 30균주로 가장 많이 분리되었다. 분리한 균주들을 yeast peptone dextrose (YPD) 배지에서 24시간 배양하여 각각의 배양상등액과 무세포추출물을 제조한 후 이들의 주요 생리기능성을 측정한 결과, Candida sp. 78-J-2의 배양상등액이 22.5%의 항산화활성, Metschnikowia reukaufii SY44-6의 배양상등액이 49.6%의 항통풍성 xanthine oxidase 저해활성과 38.4%의 미백성 tyrosinase 저해활성을 보였다.

A Gram-positive, rod- or coccoid-shaped andN-hexanoyl-l-homoserine lactone-degrading bacterial strain, A2-4T, was isolated from a soil in Korea, and its taxonomic position was investigated by using a polyphasic approach. Strain A2-4Tgrew optimally at pH 7.0–8.0 and 30 °C without NaCl. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain A2-4Tis most closely related to members of the genusNocardioides. Strain A2-4Tpossessed chemotaxonomic properties indicative of members of the genusNocardioides; the cell-wall peptidoglycan type was based onll-diaminopimelic acid, MK-8(H4) was the predominant menaquinone and iso-C16 : 0was the predominant fatty acid. The DNA G+C content was 72.1 mol%. The 16S rRNA gene sequence of strain A2-4Twas 98.3–99.1 % similar to those of the type strains ofNocardioides simplex,Nocardioides aromaticivoransandNocardioides nitrophenolicusand 93.8–96.3 % similar to those of the type strains of otherNocardioidesspecies. Strain A2-4Tcould be distinguished from the three phylogenetic relatives,N. nitrophenolicus,N. aromaticivoransandN. simplex, by DNA–DNA relatedness (25–42 %) and by differences in some phenotypic characteristics. On the basis of the phenotypic, phylogenetic and genetic data, the strain represents a novel species of the genusNocardioides, for which the nameNocardioides kongjuensissp. nov. is proposed. The type strain is A2-4T(=KCTC 19054T=JCM 12609T).

Deer antlers are the only mammalian organs capable of repeated regeneration. Although antlers are known to develop from pedicles, which arise from antlerogenic cells of cranial periosteum, their developmental process is not fully elucidated. For example, while endocrine and environmental factors influence the antler development, it is still unclear which signaling pathways are involved in the transduction of such stimuli. To study the developmental process of antlers and identify proteins functioning in their growth, we have established proteome maps of red deer (Cervus elaphus) antlers. With two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry, we analyzed more than 800 protein spots and identified approximately 130 individual proteins derived from the growing tip of antlers. The overall profile of the antler proteome was dissimilar to those of other types of tissue. Also comparison of proteomes derived from proximal bony tissue and the growing tip of antlers revealed substantial differences. Moreover several cell growth or signaling-related proteins are expressed exclusively in the growing tip, suggesting that these proteins function in the growth and differentiation of antlers. Currently, using the antler proteome maps, we are actively searching for the regulatory factor(s) that may control the antler development.

A bacterial strain WJ-98 found to produce active extracellular keratinase was isolated from the soil of a poultry factory. It was identified asParacoccus sp. based on its 16S rRNA sequence analysis, morphological and physiological characteristics. The optimal culture conditions for the production of keratinase byParacoccus sp. WJ-98 were investigated. The optimal medium composition for keratinase production was determined to be 1.0% keratin, 0.05% urea and NaCl, 0.03% K2HPO4, 0.04% KH2PO4, and 0.01% MgCl2·6H2O. Optimal initial pH and temperature for the production of keratinase were 7.5 and 37°C, respectively. The maximum keratinase production of 90 U/mL was reached after 84 h of cultivation under the optimal culturing conditions. The keratinase fromParacoccus sp. WJ-98 was partially purified from a culture broth by using ammonium sulfate precipitation, ion-exchange chromatography on DEAE-cellulose, followed by gel filtration chromatography on Sephadex G-75. Optimum pH and temperature for the enzyme reaction were pH 6.8 and 50°C, respectively and the enzymes were stable in the pH range from 6.0 to 8.0 and below 50°C. The enzyme activity was significantly inhibited by EDTA, Zn2+ and Hg2+. Inquiry into the characteristics of keratinase production from these bacteria may yield useful agricultural feed processing applications.

Among 80 types of yeast isolated from wild flowers in Daejeon, Korea, two species that have not yet been identified by phylogenetic analysis of the internal transcribed spacer-2 (ITS2) genes and 26S rDNA sequences were identified as Candida sp. 44-C-1 and Cryptococcus sp. 9-D-1. Neither of the newly identified species formed ascospores, while Candida sp. 44-C-1 formed pseudomycelium and Cryptococcus sp. 9-D-1 did not.

We report morphological and physiological characteristics of newly isolated six kinds of yeasts which are new in Korean mycoflora. The yeasts were isolated from wild flowers in Daejeon city, Korea and identified by molecular analysis of the amplified internal transcribed space rDNA sequence or partial 18S rDNA sequence. Species of isolates were identified as Kuraishia capsulate, Lodderomyces elongisporus, Pseudozyma antarctica, Starmerella bombicola.

한국 전통 생 막걸리의 최적 저장 조건을 확립하기 위하여 먼저 전통 생 막걸리를 제조한 후 $4^{\circ}C$ 와 $20^{\circ}C$ 에 저장하면서 효모와 총 세균, 젖산균 수의 변화를 조사하였다. 효모는 $20^{\circ}C$ 에서 30일 저장 후 89%가 감소 되었으나, $4^{\circ}C$ 저장에서는 큰 변화가 없었다. 총 세균수는 $20^{\circ}C$ 에서 30일 저장시 98.0%가 감소 되었고 젖산균 역시 99%가 감소되었다. 그러나 $4^{\circ}C$ 저장에서는 65%미만의 낮은 감소를 보였다. 위의 결과와 관능검사 결과 등을 종합하였을 때 본 연구에서 제조한 생 막걸리는 $4^{\circ}C$ 와 $20^{\circ}C$ 에서 15일까지도 큰 품질저하 없이 음용할 수 있는 상태로 저장이 가능한 것으로 판단된다. 【In order to optimize storage conditions of Korean traditional Makgeolli, we brewed Korean traditional Makgeolli for 1 week with two-stage fermentations and investigated changes viable cell counts of yeasts and bacteria during storage for 1 month at $4^{\circ}C$ and $20^{\circ}C$ . Yeast viable cell counts were decreased to 89.0% after storage for 30 days at $20^{\circ}C$ , however, those were not significantly changed at $4^{\circ}C$ storage. Bacteria cell counts were decreased to 59.0% of initial cell counts for 30 days at $4^{\circ}C$ . In the storage at $20^{\circ}C$ , bacteria were significantly decreased to 98.0% of initial cell counts after storage for 30 days. Lactic acid bacteria were also similar to those of total bacteria cell counts at $4^{\circ}C$ storage, however, 99% of lactic acid bacteria were decreased at $20^{\circ}C$ storage for 30 days.】

To explore the oxygen response regulators involved in thiol peroxidase gene (tpx) expression in Escherichia coli, we constructed a single-copy tpx-lacZ operon fusion and monitored tpx-lacZ expression in various genetic backgrounds. Expression of the tpx-lacZ fusion was increased 4-fold by aerobic growth. Anaerobic expression of tpx-lacZ in either (delta)arcA or delta(fnr) strains was 2.5-fold depressed compared with that of the wild-type strain. The results of immunoblotting experiments also demonstrated that ArcA and Fnr regulatory proteins repressed thiol peroxidase gene expression during anaerobic growth. Inspection of the tpx promoter region revealed putative binding sites for ArcA and Fnr. It thus appears that ArcA and Fnr function as repressors by blocking the binding of RNA polymerase to the tpx promoter in E. coli under anaerobic growth conditions.

E. coli thiol peroxidase (Tpx) linked to the thioredoxin as an in vivo thiol regenerating system acts as an antioxidant enzyme removing peroxides and H 2 O 2 . In order to elucidate the mechanism regulating tpx gene expression in E. coli in response to oxygen stress, we made 5′ progressive deletions of upstream region from tpx gene, and fused to lacZ gene. LacZ activity was increased 6-fold by oxygen stress and inverted repeat sequence located between −47 and −33 nt was proven to be essential for the oxygen response of tpx promoter. Primer extension experiment and analysis of upstream sequence revealed transcription start point, −10, and −35 regions, which are in good agreements with the consensus sequences recognized by Eσ 70 . Northern hybridization showed that expression of tpx gene is regulated at the transcriptional level. DNA binding assays using inverted repeat sequence including −35 region provides preliminary evidence that expression of tpx requires additional transcriptional factor in response to oxygen stress.

Three different molecular masses (24, 22, and 20 kDa) of antioxidant proteins were purified in Escherichia coli. These proteins exhibited the preventive effects against the inactivation of glutamine synthetase activity and the cleavage of DNA by a metal-catalyzed oxidation system capable of generating reactive oxygen species. Their antioxidant activities were supported by a thiol-reducing equivalent such as dithiothreitol. Analysis of the amino-terminal amino acid sequences and the immunoblots between 24- and 22-kDa proteins indicates that the 24-kDa protein is an intact form of the 22-kDa protein that was previously identified 22-kDa subunit (AhpC) of E. coli alkyl hydroperoxide reductase (AhpC/AhpF). We isolated and sequenced an E. coli genomic DNA fragment that encodes 20-kDa protein. Comparison of the deduced amino acid sequence of the 20-kDa protein with that of AhpC revealed no sequence homology. A search of a data bank showed that the 20-kDa protein is a new type of antioxidant enzyme. The synthesis of this novel 20-kDa protein was increased in response to oxygen stress during growth. The 20-kDa protein resides mainly in the periplasmic space of E. coli, whereas the 24-kDa AhpC resides mainly in the matrix. The 20-kDa protein was functionally linked to the thioredoxin as an in vivo thiol-regenerating system and exerted a peroxidase activity. This 20-kDa protein is thus named "thiol peroxidase," which could act as an antioxidant enzyme removing peroxides or H2O2 within the catalase- and peroxidase-deficient periplasmic space of E. coli.

The complete cDNA encoding human thiol-specific antioxidant protein (PRP) was isolated from a human brain cDNA library in the lambda Zap expression vector. An open reading frame (ORF) was identified and found to encode a polypeptide of 197 aa with a M(r) of 21,729. The cDNA contained 98 bp of 5'-untranslated sequence (UTR) and 259 bp of 3'-UTR containing a poly(A) signal, AATAAA. Expression of the human PRP cDNA in Escherichia coli yielded a functionally active protein. The observed local sequence homologies between human PRP and other homologous proteins whose functions have not yet been defined give important insight into elucidating the biochemical function of a new protein family which has highly conserved regions containing cysteine.

A thiol-specific antioxidant protein (Protector Protein, PRP) was purified from human red blood cells (RBC). The PRP exists as a predominant protein in human RBC, which showed distinct thiol-specific antioxidant activities in the presence of dithiothreitol (DTT) as a reducing equivalent. The human RBC PRP (HRPRP) completely inhibited visible absorption spectral changes of oxyhemoglobin, DNA cleavage, and the peroxidation of RBC membrane by a nonenzymatic Fe3+/O2/thiol mixed-function oxidation system capable of generating hydroxyl radical. These observations suggest that HRPRP could act as a new type of antioxidant protein to maintain the RBC integrity by scavenging reactive oxygen species.

12-O-tetradecanoylphorbol-13-acetate (TPA) has been shown to induce transcriptional activation of human manganese superoxide dismutase (MnSOD) mRNA in human lung carcinoma cells, A549, mediated by a protein kinase C (PKC)-dependent activation of cAMP-responsive element-binding protein (CREB)-1/ATF-1-like factors. In this study, we showed that MnSOD protein expression was elevated in response to TPA or TNF-α, but not to hydrogen peroxide treatment. TPA-induced generation of reactive oxygen species (ROS) was blocked by pretreatment of the PKC inhibitor BIM and NADPH oxidase inhibitor DPI. Small interfering RNA (siRNA) experiments indicated that knocking down the NADPH oxidase components e.g. Rac1, p22(phox), p67(phox), and NOXO1 in A549 cells impaired TPA-induced MnSOD expression. To identify the PKC isozyme involved, we used a sod2 gene response reporter plasmid, pSODLUC-3340-I2E-C, capable of sensing the effect of TNF-α and TPA, to monitor the effects of PKC isozyme-specific inhibitors and siRNA-induced knockdown of specific PKC isozyme. Our data indicate that TPA-induced MnSOD expression was independent of p53 and most likely mediated by PKC-α-, and -ε-dependent signaling pathways. Furthermore, siRNA-induced knock-down of CREB and Forkhead box class O (FOXO) 3a led to a reduction in TPA-induced MnSOD gene expression. Together, our results revealed that TPA up-regulates, in part, two PKC-dependent transcriptional pathways to induce MnSOD expression. One pathway involves PKC-α catalyzed phosphorylation of CREB and the other involves a PKC-mediated the PP2A catalyzed dephosphorylation of Akt at Ser(473) which in turn leads to FOXO3a Ser(253) dephosphorylation and its activation.

Missense mutations of SOD1 are linked to familial amyotrophic lateral sclerosis (FALS) through a yet-to-be identified toxic-gain-of-function. One of the proposed mechanisms involves enhanced aggregate formation. However, a recent study showed that dual transgenic mice overexpressing both G93A and CCS copper chaperone (G93A/CCS) exhibit no SOD1-positive aggregates yet show accelerated FALS symptoms with enhanced mitochondrial pathology compared to G93A mice. Using a dicistronic mRNA to simultaneously generate hSOD1 mutants, G93A, A4V and G85R, and hCCS in AAV293 cells, we revealed: (i) CCS is degraded primarily via a macroautophagy pathway. It forms a stable heterodimer with inactive G85R, and via its novel copper chaperone-independent molecular chaperone activity facilitates G85R degradation via a macroautophagy-mediated pathway. For active G93A and A4V, CCS catalyzes their maturation to form active and soluble homodimers. (ii) CCS reduces, under non-oxidative conditions, yet facilitates in the presence of H(2)O(2), mitochondrial translocation of inactive SOD1 mutants. These results, together with previous reports showing FALS SOD1 mutants enhanced free radical-generating activity, provide a mechanistic explanation for the observations with G93A/CCS dual transgenic mice and suggest that free radical generation by FALS SOD1, enhanced by CCS, may, in part, be responsible for the FALS SOD1 mutant-linked aggregation, mitochondrial translocation, and degradation.

Direct expression of lactoferricin, an antimicrobial peptide, is lethal to Escherichia coli. For the efficient production of lactoferricin in E. coli, we developed an expression system in which the gene for the lysine- and arginine-rich cationic lactoferricin was fused to an anionic peptide gene to neutralize the basic property of lactoferricin, and successfully overexpressed the concatemeric fusion gene in E. coli. The lactoferricin gene was linked to a modified magainin intervening sequence gene by a recombinational polymerase chain reaction, thus producing an acidic peptide-lactoferricin fusion gene. The monomeric acidic peptide-lactoferricin fusion gene was multimerized and expressed in E. coli BL21(DE3) upon induction with isopropyl-beta-D-thiogalactopyranoside. The expression levels of the fusion peptide reached the maximum at the tetramer, while further increases in the copy number of the fusion gene substantially reduced the peptide expression level. The fusion peptides were isolated and cleaved to generate the separate lactoferricin and acidic peptide. About 60 mg of pure recombinant lactoferricin was obtained from 1 L of E. coli culture. The purified recombinant lactoferricin was found to have a molecular weight similar to that of chemically synthesized lactoferricin. The recombinant lactoferricin showed antimicrobial activity and disrupted bacterial membrane permeability, as the native lactoferricin peptide does.

Phospholipase D (PLD) is distributed widely in eukaryotes and prokaryotes, and is found in all mammalian cells. The mammalian enzyme is activated by a large variety of hormones, neurotransmitters, growth factors, and cytokines, and has been implicated in the control of cell growth, secretion, and the actin cyloskelelon. Alternatively spliced forms of Iwo mammalian PLD isozymes (PLD1 and PLD2) have been cloned. The PLD1 isoforms are directly regulated by classic protein kinase C (PKC) isozymes and members of the families off the two small GTPases ARF and Rho. The PLD2 isoforms show high basal activity and respond minimally, if at all, to PKC or the small GTPases. Subsequently, alternatively spliced PLD1 isozyme (PLD la) was cloned from human promyelocytic (HL-60) cells, and its properties were demonstrated to be almost identical to those of PLD1b. A different gene product (PLD2) was cloned from a mouse embryonic cDNA library and shown to be less subject to regulation in vitro. Since then, PLD isoforms have been cloned from other mammalian tissues. In general, their properties are similar or identical to those of the initially cloned enzymes.

A complementary DNA (cDNA) clone that encodes inositol 1,4,5-trisphosphate 3-kinase was isolated from a rat brain cDNA expression library with the use of monoclonal antibodies. This clone had an open reading frame that would direct the synthesis of a protein consisting of 449 amino acids and with a molecular mass of 49,853 daltons. The putative protein revealed a potential calmodulin-binding site and six regions with amino acid compositions (PEST regions) common to proteins that are susceptible to calpain. Expression of the cDNA in COS cells resulted in an approximately 150-fold increase in inositol 1,4,5-trisphosphate 3-kinase activity of these cells.

We have isolated cDNA fragments that were originated from P450 1A1 in human brain by polymerase chain reaction (PCR) analysis. Their size matched up to what we expected based on the reported P450 1A1 mRNA sequence of human breast carcinoma cell line MCF-7. To confirm proper PCR products, they were subcloned and their nucleotide sequences were determined. This result showed that PCR products were actually derived from P450 1A1 mRNA in human brain. We demonstrated for the first time that human brain contains a P450 1A1-dependent metabolism system which seems to be important for understanding the metabolism of endogenous and exogenous compounds.

A novel thioredoxin-linked thiol peroxidase (Px) from Escherichia coli has been reported previously (M. K. Cha, H. K. Kim, and I. H. Kim, J. Biol. Chem. 270:28635-28641, 1995). In an attempt to perform physiological and biochemical characterizations of the thiol Px, a thiol Px null (tpx) mutant and a functional-residue mutant of thiol Px were produced. The tpx mutant was viable in aerobic culture but grew more slowly than the wild-type cells. The difference in growth rate became more pronounced when oxidative-stress-inducing reagents, such as peroxides and paraquat, were added to the cultures. The viability of the individual tpx mutant under oxidative stress was much lower than that of wild-type cells. tpx mutants growing aerobically respond to paraquat with a sixfold greater induction of Mn-superoxide dismutase than that of the wild-type cells. The deduced amino acid sequence of the thiol Px was found to be from 42 to 72% identical to the sequences of proteins from Haemophilus influenzae (ToxR regulon), Vibrio cholerae (ToxR regulon), and three kinds of streptococci (coaggregation-mediating adhesins), suggesting that they all belong to a new thiol Px family. Alignment of the amino acid sequences of the thiol Px family members showed that one cysteine, which corresponds to Cys-94 in E. coli thiol Px, is perfectly conserved. The substitution of serine for this cysteine residue resulted in complete loss of Px activity. These results suggest that the members of the thiol Px family, including E. coli thiol Px, have a functional cysteine residue and function in vivo as peroxidases.

PDGF binding to its receptor promotes the association with and stimulates the phosphorylation of PLC-gamma 1 at tyrosine and serine residues. Also, PDGF induces an increase in the hydrolysis of inositol phospholipids by PLC. How PDGF activates PLC was investigated by substituting phenylalanine for tyrosine at PLC-gamma 1 phosphorylation sites 771, 783, and 1254 and expressing the mutant enzymes in NIH 3T3 cells. Phenylalanine substitution at Tyr-783 completely blocked the activation of PLC by PDGF, whereas mutation at Try-1254 inhibited and mutation at Tyr-771 enhanced the response. Like the wild type, PLC-gamma 1 substituted with phenylalanine at Tyr-783 became associated with the PDGF receptor and underwent phosphorylation at serine residues in response to PDGF. These results suggest that PLC-gamma 1 is the PLC isozyme that mediates PDGF-induced inositol phospholipid hydrolysis, that phosphorylation on Tyr-783 is essential for PLC-gamma 1 activation. These results provide direct evidence that growth factor receptors activate the function of intracellular protein by tyrosine phosphorylation.