• Reproductive and Developmental Biology
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• v.32 no.4
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• pp.255-261
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• 2008

The early diagnosis of bovine pregnancy is an essential component of successful reproductive planning on farms, because lack of bovine pregnancy over the long term results in reproductive failure and low milk yield-the latter of which is a special concern on dairy farms. This study was designed to identify early pregnancy-specific whey proteins in bovine, by comparing milk samples collected from cattle during pregnancy (Days 30 and 50) and from non-pregnant cattle. In this study, differentially expressed proteins in five pregnant and five non-pregnant Holstein dairy cattle were investigated and compared, using proteomics analysis. The first dimension was applied to a pH $3.0{\sim}10.0$ strip, by loading a 2-mg milk protein sample. After the second-dimension separation was performed, the gels were stained with colloidal Coomassie brilliant blue. The stained gels were scanned and the images were analyzed, to detect variations in protein spots between non-pregnant and pregnant cattle milk protein spots, using ImageMaster, this was followed by analysis with MALDI TOF-MS. Analysis of the 2-DE gel image resulted in a total of approximately $500{\sim}600$ protein spots, of which 12 spots were differentially expressed, six spots were up-regulated, and four spots were down-regulated; two spots were identified as pregnancy-specific proteins. These proteins were identified as lactoferrin, NA-DH dehydrogenase subunit 2, albumin, serum albumin precursor and transferrin. Our results via 2-D PAGE analysis revealed composite profiles of several milk proteins related to early bovine pregnancy, implying the possible use of these milk proteins in the early detection of bovine pregnancy.

• Korean Journal of Food Science and Technology
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• v.40 no.4
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• pp.394-399
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• 2008

In this study, peptides were isolated from Crassostrea gigas using an ultrasonification process at $40^{\circ}C$. The yield of the peptides was greater than 34%, and their cytotoxicity was found to be less than 22.8% against several cell lines that were treated with the extracts at a dose of 1.0 mg/mL. In addition, the tyrosinase inhibitory and melanin synthesis of the peptides isolated from Crassostrea gigas were also evaluated to determine if they could be used as a potential cosmetic agent. The peptides were found to significantly inhibit the melanin synthesis of the clone M-3 cell line by up to 62.7%. The inhibitory activities of the tyrosinase were observed 34.51% in ascorbic acid, 42.49% in extract with the ultrasonification at $40^{\circ}C$ and 35.37% in $40^{\circ}C$ extract at 1.0 mg/mL concentration, respectively. Finally, when samples were treated with the peptide extracts at a concentration of 0.6 mg/mL, PGE2 expression was significantly decreased. Taken together, these results indicate that Crassostrea gigas may be a source of cosmetic agents capable of improving physiological hyperpigmenting and immuno-modulating skin disorders.

• Reproductive and Developmental Biology
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• v.34 no.3
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• pp.263-270
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• 2010

Luteal cells produce progesterone that supports pregnancy. Steroidogenesis requires coordination of the anabolic and catabolic pathways of lipid metabolism. In the present study, the corpus luteum (CL) in early pregnancy established from luteal phase and pregnant phase was analyzed. The first study determined progesterone changes in the bovine CL at day 19 (early maternal recognition period) and day 90 in mid-pregnancy and compared them to the CL from day 12 of the estrous cycle. CL alternation was tested using two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). Comparing CL from luteal phase to those from pregnant phase counterparts, significant changes in expression level were found in 23 proteins. Of these proteins 17 were not expressed in pregnant phase CL but expressed in luteal phase counterpart, whereas, the expression of the other 6 proteins was limited only in pregnant phase CL. Among these proteins, vimentin is considered to be involved in regulation of post-implantation development. In particular, vimentin may be used as marker for CL development during pregnancy because the expression level changed considerably in pregnant phase CL tissue compared with its luteal phase counterpart. Data from 2-DE suggest that protein expression was disorientated in mid pregnancy from luteal phase, but these changes was regulated with progression of pregnancy. These findings demonstrate CL development during mid-pregnancy from luteal phase and suggest that alternations of specific CL protein expression may be involved in maintenance of pregnancy.

• Journal of the Korean Society of Food Science and Nutrition
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• v.39 no.11
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• pp.1647-1653
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• 2010

Glycosaminoglycan (GAG) was purified from polysaccharide extracted from sea hare muscle on DEAE-Sepharose column and investigated for the functional groups, distribution of sugars, composition of disaccharide and structure of GAG. Purified GAG was composed of disaccharide above 55% of total sugar. Purified GAG showed amide I peak in 1648/cm and C-O stretch peak as properties of carbohydrate, amino acid peak in 1457/cm, and peak in 866/cm as properties of monosaccharide by FT-IR. Fucose, N-acetylgalactosamine, N-acetylglucosamine, glucose, galactose, mannose and xylose were found in MALDI-TOF MS/MS spectra of hydrolysates by chondroitin sulfate ABC lyase and heparanase I. Purified GAG was expected to be heparan sulfate including N-acetylgalactosamine and N-acetylglucosamine above 70% of total sugar. The structure of GAG was supposed as GlyUA(2S)-GlcNS and GlyUA-GlcNS(6S) with O-linkage on protein core.

• Microbiology and Biotechnology Letters
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• v.33 no.4
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• pp.295-301
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• 2005

About seven hundred bacterial strains were collected from Jeot-Kal, a Korean traditional fermented fishes, in various Korean districts. One of the strains designated JKK238 has its ability to antagonize in vitro the growth of a wide variety of plant pathogenic fungi responsible for diseases of economical importance. The JKK238 strain was isolated from Oh-Jeot, a kind of fermented shrimps, of Kangkyeung in Korea, and was identified as Bacillus subtilis based on its physiological characteristics, fatty acids compositions of cellular wall, and 16S rDNA sequence analysis. We isolated simply antimicrobial lipopeptides (AMLP) by $25\%$ ammonium sulfate precipitation of 3 days-old tryptic soy broth cultures of the JKK238 strain. Further analysis of AMLP revealed that B. subtilis JKK238 produces a wide variety of antifungal lipopeptide isomers from the iturin, fengycin and surfactin families simultaneously. Above results indicate that the JKK238 strain can be added to the limited number B. subtilis strains reported to co-produce the three kinds of lipopeptide families.

• Proceedings of the Korean Journal of Food and Nutrition Conference
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• 2000.05a
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• pp.1-3
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• 2000

A kind of traditional herbal prescription, Sip-Jeon-Dae-Bo-Tang (TJ-48), has been reported to improve the general condition of cancer patients receiving chemotherapy and /or radiation therapy, and to accelerate hematopoietic recovery from bone marrow injury by mitomycin C. In the present studies, we found that hot-water extract from Atractylodes lancea DC. rhizomes contributed mainly to intestinal immune modulating activity of TJ-48 on Peyer's patch cells mediated-hematopoietic response. After the fractionation, ALR-5 II a-1-1, 5 II b-2-2 and 5 II c-3-1 were further purified from crude polysaccharide fraction. Chemical analyses of each fraction indicated that ALR-5 II a-1-1 mainly contained arabinogalactan fraction whereas ALR-5 II b-2-2 and 5 II c-3-1 mostly comprised pectic polysaccharide fractions as the active polysaccharide ingredients. In order to analyze the essential structure of the activity, ALR-5 II a-1-1 was treated by sequential enzymatic digestion using exo-${\alpha}$-L-arabinofuranosidase and exo-${\beta}$-D-(1\longrightarrow3)-galactanase. Based upon the results of chemical and MALDI-TOF-MS analyses and activity on the digested fractions, the galactosyl side chains consisting of 6-linked Galf and Galp over tetrasaccharide in ALR-5 II a-1-1 might be responsible for the potent intestinal immune modulating activity. To characterize moiety of ALR-5 II c-3-1 for the expression of activity, endo-${\alpha}$-D-(1\longrightarrow4)-polygal acturonase (GL-PGase) purified from dried leaves of Panax ginseng digested ALR-5 II c-3-1. The results of structural analyses and activity on the digested fractions showed that PG-2, which structurally resembles to rhamnogalacturonan II (RG II), and PG-3 (galacturono-oligosaccharides) contained potent intestinal immune modulating activity. Further purification of the other acidic fraction (ALR-5 II b-2-2) indicated that ALR-5 II b-2-2Bb showed that the most potent activity. ALR-5 II b-2-2Bb also contained the unusual component sugars characteristics in RG- II as well as PG-2 derived from ALR-5 II c-3-1, but it could not be digested with GL-PGase. The present studies of relationship between structures and intestinal immune modulating activity of the active polysaccharides purified from A. lancea DC. rhizomes suggested that neutral galactosyl chains consisting mainly of (1\longrightarrow6)-linked Galf and Galp, and RG- II -like moiety with unique component sugars, such as 2-Me-Xyl, 2-Me-Fuc, Api, AceA, Kdo and Dha should play an important role in the potent intestinal immune modulating action of A. lancea DC. rhizomes.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.6
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• pp.880-884
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• 2001

Rumen microbiology research has undergone several evolutionary steps: the isolation and nutritional characterization of readily cultivated microbes; followed by the cloning and sequence analysis of individual genes relevant to key digestive processes; through to the use of small subunit ribosomal RNA (SSU rRNA) sequences for a cultivation-independent examination of microbial diversity. Our knowledge of rumen microbiology has expanded as a result, but the translation of this information into productive alterations of ruminal function has been rather limited. For instance, the cloning and characterization of cellulase genes in Escherichia coli has yielded some valuable information about this complex enzyme system in ruminal bacteria. SSU rRNA analyses have also confirmed that a considerable amount of the microbial diversity in the rumen is not represented in existing culture collections. However, we still have little idea of whether the key, and potentially rate-limiting, gene products and (or) microbial interactions have been identified. Technologies allowing high throughput nucleotide and protein sequence analysis have led to the emergence of two new fields of investigation, genomics and proteomics. Both disciplines can be further subdivided into functional and comparative lines of investigation. The massive accumulation of microbial DNA and protein sequence data, including complete genome sequences, is revolutionizing the way we examine microbial physiology and diversity. We describe here some examples of our use of genomics- and proteomics-based methods, to analyze the cellulase system of Ruminococcus flavefaciens FD-1 and explore the genome of Ruminococcus albus 8. At Illinois, we are using bacterial artificial chromosome (BAC) vectors to create libraries containing large (>75 kbases), contiguous segments of DNA from R. flavefaciens FD-1. Considering that every bacterium is not a candidate for whole genome sequencing, BAC libraries offer an attractive, alternative method to perform physical and functional analyses of a bacterium's genome. Our first plan is to use these BAC clones to determine whether or not cellulases and accessory genes in R. flavefaciens exist in clusters of orthologous genes (COGs). Proteomics is also being used to complement the BAC library/DNA sequencing approach. Proteins differentially expressed in response to carbon source are being identified by 2-D SDS-PAGE, followed by in-gel-digests and peptide mass mapping by MALDI-TOF Mass Spectrometry, as well as peptide sequencing by Edman degradation. At Ohio State, we have used a combination of functional proteomics, mutational analysis and differential display RT-PCR to obtain evidence suggesting that in addition to a cellulosome-like mechanism, R. albus 8 possesses other mechanisms for adhesion to plant surfaces. Genome walking on either side of these differentially expressed transcripts has also resulted in two interesting observations: i) a relatively large number of genes with no matches in the current databases and; ii) the identification of genes with a high level of sequence identity to those identified, until now, in the archaebacteria. Genomics and proteomics will also accelerate our understanding of microbial interactions, and allow a greater degree of in situ analyses in the future. The challenge is to utilize genomics and proteomics to improve our fundamental understanding of microbial physiology, diversity and ecology, and overcome constraints to ruminal function.

• Journal of Microbiology and Biotechnology
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• v.18 no.8
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• pp.1386-1392
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• 2008

A gene (sll0158) putatively encoding a glycogen branching enzyme (GBE, E.C. 2.4.1.18) was cloned from Synechocystis sp. PCC6803, and the recombinant protein expressed and characterized. The PCR-amplified putative GBE gene was ligated into a pET-21a plasmid vector harboring a T7 promoter, and the recombinant DNA transformed into a host cell, E. coli BL21(DE3). The IPTG-induced enzymes were then extracted and purified using Ni-NTA affinity chromatography. The putative GBE gene was found to be composed of 2,310 nucleotides and encoded 770 amino acids, corresponding to approx. 90.7 kDa, as confirmed by SDS-PAGE and MALDI-TOF-MS analyses. The optimal conditions for GBE activity were investigated by measuring the absorbance change in iodine affinity, and shown to be pH 8.0 and $30^{\circ}C$ in a 50 mM glycine-NaOH buffer. The action pattern of the GBE on amylose, an $\alpha$-(1,4)-linked linear glucan, was analyzed using high-performance anion-exchange chromatography (HPAEC) after isoamylolysis. As a result, the GBE displayed $\alpha$-glucosyl transferring activity by cleaving the $\alpha$-(1,4)-linkages and transferring the cleaved maltoglycosyl moiety to form new $\alpha$-(1,6)-branch linkages. A time-course study of the GBE reaction was carried out with biosynthetic amylose (BSAM; $M_p{\cong}$8,000), and the changes in the branch-chain length distribution were evaluated. When increasing the reaction time up to 48 h, the weight- and number-average DP ($DP_w$ and $DP_n$) decreased from 19.6 to 8.7 and from 17.6 to 7.8, respectively. The molecular size ($M_p$, peak $M_w{\cong}2.45-2.75{\times}10^5$) of the GBE-reacted product from BSAM reached the size of amylose (AM) in botanical starch, yet the product was highly soluble and stable in water, unlike AM molecules. Thus, GBE-generated products can provide new food and non-food applications, owing to their unique physical properties.

• Reproductive and Developmental Biology
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• v.29 no.2
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• pp.83-91
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• 2005

Somatic cell nuclear transfer in cattle has limited efficiency in terms of production of live offspring due to high incidence of fetal failure after embryo transfer to recipients. Such low efficiency of cloning could possibly arise from abnormal and poorly developed placenta. In the present study the placental proteome in late pregnancy established from in vitro fertilization (IVF) and nuclear transfer (NT) was analysed. Proteome alternation was tested using two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI- TOF). Comparing placenta from NT embryos to those from IVF counterparts, significant changes in expression level were found in 18 proteins. Of these proteins 12 were not expressed in NT placenta but expressed in IVF counterpart, whereas the expression of the other 6 proteins was limited only in NT placenta. Among these proteins, cytokeratin 8 and vimentin are considered to be involved in regulation of post-implantation development. In particular, cytokeratin 8 and vimentin may be used as makers for placental development during pregnancy because their expression levels changed considerably in NT placental tissue compared with its IVF counterpart. Data from 2-DE suggest that protein expression was disorientated in late pregnancy from NT, but this distortion was eliminated with progression of pregnancy. These findings demonstrate abnormal placental development during late pregnancy from NT and suggest that alterations of specific placental protein expression may be involved in abnormal function of placenta.

• Journal of Microbiology and Biotechnology
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• v.25 no.2
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• pp.152-161
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• 2015

CodY is a highly conserved protein in low G+C gram-positive bacteria that regulates genes involved in sporulation and stationary-phase adaptation. Bacillus thuringiensis is a grampositive bacterium that forms spores and parasporal crystals during the stationary phase. To our knowledge, the regulatory mechanism of CodY in B. thuringiensis is unknown. To study the function of CodY protein in B. thuringiensis, BMB171codY^- was constructed in a BMB171 strain. A shuttle vector containing the ORF of cry1Ac10 was transformed into BMB171 and BMB171codY^-, named BMB171cry1Ac and BMB171codY^-cry1Ac, respectively. Some morphological and physiological changes of codY mutant BMB171codY^-cry1Ac were observed. A comparative proteomic analysis was conducted for both BMB171codY^-cry1Ac and BMB171cry1Ac through two-dimensional gel electrophoresis and MALDI-TOF-MS/MS analysis. The results showed that the proteins regulated by CodY are involved in microbial metabolism, including branched-chain amino acid metabolism, carbohydrate metabolism, fatty acid metabolism, and energy metabolism. Furthermore, we found CodY to be involved in sporulation, biosynthesis of poly-β-hydroxybutyrate, growth, genetic competence, and translation. According to the analysis of differentially expressed proteins, and physiological characterization of the codY mutant, we performed bacterial one-hybrid and electrophoretic mobility shift assay experiments and confirmed the direct regulation of genes by CodY, specifically those involved in metabolism of branched-chain amino acids, ribosomal recycling factor FRR, and the late competence protein ComER. Our data establish the foundation for in-depth study of the regulation of CodY in B. thuringiensis, and also offer a potential biocatalyst for functions of CodY in other bacteria.