• Journal of Life Science
• /
• v.26 no.11
• /
• pp.1259-1268
• /
• 2016

In this study, we investigated antimicrobial peptide from the acidified muscle extract of Mytilus coruscus, which mostly inhabits China, Japan, and Korea, to develop a natural product-derived antibiotics substitution in terms of its abuse and restriction. Antimicrobial peptide was purified by $C_{18}$ reversed-phase high-performance liquid chromatography and was detected as having a molecular mass of 6,701 Da by MALDI-TOF/MS. The N-terminal amino acid sequence of the purified peak was obtained from edman degradation, and 20 identified residues shown 100% identity with the N-terminus region of sperm-specific protein and protamine-like PL-II/PL-IV precursor of Mytilus californianus. We also identified 60 open-reading frame (ORF) encoding amino acids with 183 bp of purified peptide based on the obtained amino acid residues. The amino acid sequence of ORF showed 100% and the nucleotide sequence revealed 97.2% identity with the protamine-like PL-II/PL-IV precursor of Mytilus californianus. Synthesized antimicrobial peptide showed antimicrobial activity against gram-positive bacteria, including Bacillus cereus (minimal effective concentration [MEC], $20.8{\mu}g/ml$), Bacillus subtilis (MEC, $0.2{\mu}g/ml$), Streptococcus mutans (MEC, $0.2{\mu}g/ml$), gram-negative bacteria including Pseudomonas aeruginosa (MEC, $5.7{\mu}g/ml$), Escherichia coli (MEC, $2.6{\mu}g/ml$) and fungi, Candida albicans (MEC, $56.3{\mu}g/ml$). In addition, synthesized peptide showed stable activities under heat and salt conditions against gram-positive and gram-negative bacteria, but was inhibited by salt against only C. albicans. With these results, isolated peptide from M. coruscus could be an alternative agent to antibiotics for defending against pathogenic microorganisms, and helpful information to understand the innate immune system of marine invertebrates.

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

• Asian-Australasian Journal of Animal Sciences
• /
• v.29 no.9
• /
• pp.1239-1246
• /
• 2016

The breeding of yaks is highly seasonal, there are many crucial proteins involved in the reproduction control program, especially in follicular development. In order to isolate differential proteins between mature and immature follicular fluid (FF) of yak, the FF from yak follicles with different sizes were sampled respectively, and two-dimensional gel electrophoresis (2-DE) of the proteins was carried out. After silver staining, the Image Master 2D platinum software was used for protein analysis and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) was performed for differential protein identification. The expression level of transferrin and enolase superfamily member 1 (ENOSF1) was determined by Western blotting for verification analysis. The results showed that 2-DE obtained an electrophoresis map of proteins from mature and immature yak FF with high resolution and repeatability. A comparison of protein profiles identified 12 differently expressed proteins, out of which 10 of them were upregulated while 2 were downregulated. Western blotting showed that the expression of transferrin and ENOSF1 was enhanced with follicular development. Both the obtained protein profiles and the differently expressed proteins identified in this study provided experimental data related to follicular development during yak breeding seasons. This study also laid the foundation for understanding the microenvironment during oocyte development.

• Molecules and Cells
• /
• v.26 no.2
• /
• pp.175-180
• /
• 2008

$I{\kappa}B$ kinase (IKK), the pivotal kinase in signal-dependent activation of nuclear factor-${\kappa}B$ (NF-${\kappa}B$), is composed of multiple protein components, including IKK ${\alpha}/{\beta}/{\gamma}$ core subunits. To investigate the regulation of the IKK complex, we immunoaffinity purified the IKK complex, and by MALDI-TOF mass spectrometry identified a splice variant of zinc finger protein 268 (ZNF268) as a novel IKKinteracting protein. Both the full-length and the spliced form of the ZNF268 protein were detected in a variety of mammalian tissues and cell lines. The genes were cloned and expressed by in vitro transcription/translation. Several deletion derivatives, such as KRAB domain (KRAB) on its own, the KRAB/spacer/4-zinc fingers (zF4), and the spacer/4-zinc fingers (zS4), were ectopically expressed in mammalian cells and exhibited had different subcellular locations. The KRAB-containing mutants were restricted to the nucleus, while zS4 was localized in the cytosol. TNF-${\alpha}$-induced NF-${\kappa}B$ activation was examined using these mutants and only zS4 was found to stimulate activation. Collectively, the results indicate that a spliced form of ZNF268 lacking the KRAB domain is located in the cytosol, where it seems to play a role in TNF-${\alpha}$-induced NF-${\kappa}B$ activation by interacting with the IKK complex.

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

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

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

• KSBB Journal
• /
• v.26 no.6
• /
• pp.517-522
• /
• 2011

In our previous study, we isolated and characterized a 1-deoxynojirimycin (DNJ)-producing bacterium, Bacillus subtilis MORI, from chungkookjang, a Korean traditional food. B. subtilis MORI was subjected to ${\gamma}$-irradiation and the resulting bacteria were screened for increased DNJ production. A mutant was identified that produced 7.6 times more DNJ and named B. subtilis MORI 3K-85. In this study, the protein profiles of both strains were compared by one-dimensional and two-dimensional gel electrophoresis (1-DE and 2-DE, respectively) under both native and denaturing conditions. The 1-DE native-PAGE and 1-DE SDS-PAGE analyses identified 5 and 7 bands, respectively, that were found at higher concentrations in B. subtilis MORI 3K-85 than in B. subtilis MORI. Similarly, 2-DE analyses identified 20 protein spots which were found at higher concentrations in B. subtilis MORI 3K-85. The peptide mass profiles of these 20 proteins were analyzed by MALDI-TOF and compared with peptide sequences of B. subtilis and B. amyloliquefaciens in the MASCOT database. This screening suggested that three dehydrogenases, an aldolase, a synthetase, an isomerase, a reductase, and a peroxidase are elevated in B. subtilis MORI 3K-85. Based on this data, one or more of the elevated 8 enzymes might be related to the DNJ biosynthetic pathway.

• Molecular & Cellular Toxicology
• /
• v.1 no.2
• /
• pp.116-123
• /
• 2005

The application of high pressure on cellular morphology, proliferation and protein expression of Jurkat cells (human T lymphocyte cell line) has been extensively investigated. In the present study, we manufactured a novel pressure chamber that modulates 5% $CO_{2}$, temperature and pressure (up to 3 ATA). Jurkat cells was incubated 2 ATA pressure and analyzed cellular morphology and growth using an electron microscopy and MTT assay. The cells showed the morphological changes in the cell surface, which appeared to cause a severe damage in cell membrane. The growth rate of the cells under 2 ATA pressure decreased as cultured time got increased. Furthermore, a long term exposure of high pressure on Jurkat cells may act as one of the important cellular stresses that leads to inducing cell death. Cellular proteomes were separated by 2-dimensional electrophoresis with pH 3-10 ranges of IPG Dry strips. And many proteins showed significant up-and-down expressions with hyperbaric pressure. Out of all, 10 spots were identified significantly using matrix-assisted laser desorption/ionization-time of fight (MALDI-TOF) mass spectrometry. We and found that 9 protein expressions were decreased and one protein, heat shock protein HSP 60, was increased in Jurkat cells under 2 ATA. Identified proteins were related to lipid metabolism and signal transduction.

• Archives of Pharmacal Research
• /
• v.29 no.3
• /
• pp.224-234
• /
• 2006

We employed human SK-MEL-28 cells as a model system to identify cellular proteins that accompany N-(4-methyl)phenyl-O-(4-methoxy)phenyl-thionocarbamate (MMTC)-induced apoptosis based on a proteomic approach. Cell viability tests revealed that SK-MEL-28 skin cancer cells underwent more cell death than normal HaCaT cells in a dose-dependent manner after treatment with MMTC. Two-dimensional electrophoresis in conjunction with matrixassisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry analysis or computer matching with a protein database further revealed that the MMTC-induced apoptosis is accompanied by increased levels of caspase-1, checkpoint suppressor-1, caspase-4, NF-kB inhibitor, AP-2, c-Jun-N-terminal kinase, melanoma inhibitor, granzyme K, G1/S specific cyclin D3, cystein rich protein, Ras-related protein Rab-37 or Ras-related protein Rab-13, and reduced levels of EMS (oncogene), ATP synthase, tyrosine-phosphatase, Cdc25c, 14-3-3 protein or specific structure of nuclear receptor. The migration suppressing effect of MMTC on SK-MEL-28 cell was tested. MMTC suppressed the metastasis of SK-MEL-8 cells. It was also identified that MMTC had little angiogenic effect because it did not suppress the proliferation of HUVEC cell line. These results suggest that MMTC is a novel chemotherapeutic and metastatic agents against the SK-MEL-28 human melanoma cell line.