Early pregnancy results in th production of various signal molecules such as steroids, prostaglandins, and many protein factors. The proteins especially produced by the placenta have been used to detect pregnancy for many years in other species. More recently, pregnancy-specific protein B, which is a placental glycoprotein can be measured by RIA or proteomic methods in serum of pregnant cow. And 2D Fluorescence difference gel electrophoresis (DIGE) is an emerging technique for comparative proteomics, which improves the reproducibility and reliability of differential protein expression analysis between samples. For this reason, we are analyzed serum of bovine. The purpose of this study was to apply DIGE technique for identification of bovine pregnancy-specific proteins using bovine pregnant and non-pregnant serum samples. Serums of 2 pregnant Holstein dairy cattle at day 21 after AI and those of 2 non-pregnant were used in this study. The molecular weight and charge matched cyanine dyes enable pre-electrophoretic labeling of non-pregnancy and pregnant serum proteins which are then mixed and labeled with Cy2 were used as an internal standard. Two pools of proteins are labeled with Cy3 and Cy5 fluorescent dyes, respectively. Labeled proteins with Cy2, Cy3 and Cy5 mixed together and separated in same gel and then were detected by fluorescence image analyzer. The 2D DIGE analysis using fluorescence CyDye flour showed higher sensitivity and better reproducible results than conventional 2D gel electrophoresis. Approximately 1,500 protein spots were detected by 2D DIGE. The differentially expressed proteins were identified by MALDI-TOF Mass spectrometer. Total 16 protein spots differentially expressed in the pregnant serum were detected, among which 7 spots were up-regulated proteins identified as conglutinin precursor, modified bovine fibrinogen, IgG1 etc, and 6 spots were down-regulated proteins identified as hemoglobin, complement component 3, bovine fibrinogen, IgG2a etc. These results indicated that DIGE system could be advantageous for the analysis of serum proteomics diversified by physiological conditions.

A growing interest in ecotoxicological study is on the development of tools and technical resources to assess or determine the effects of a variety of stresses on ecosystems. As many chemicals are synthesized and used for the various purposes, it is inevitable circumstance for organisms to meet the stressors in the environment. Thus, it is important for us to understand the impacts of the stressors to organisms and is essential to equip with a fast detecting system to predict of their behaviors. The high throughput technology using proteomic and metabolomic techniques has been introduced to satisfy such requirements to study the potential adverse effects of some chemicals. Ecotoxicproteomics and Ecotoxicometabolomics are discussed in this talk in terms of their possible role in ecotoxicological studies.

An understanding of oocyte gene expression is a necessary for the study of biological development. Recently, Oocyte has been used in many techniques such as somatic cell nuclear transfer (SCNT), intracytoplasmic sperm injection (ICSI) and embryonic stem cell derivation. However, the molecular mechanism underlying porcine oocyte is still unclear. In this study, we present the description of the porcine oocyte proteome. Proteins within the isoelectric point ranges of 3.0 to 10.0 were analyzed separately using 2‐dimensional electrophoresis (2‐DE). About 450 spots were detected in 2‐ D gel of oocytes, stained with Coomassie blue. Subsequent excision of 227 spots from gels and MALDI‐TOF MS analysis allowed the identification of 85 proteins. Our results indicated the composite profiles of proteins in the porcine oocyte. Tubulin beta chain and meiosis‐specific nuclear structural protein 1 antibody was used to confirm those antibody expression levels in immature, mature and parthenogenetic embryo. Western blot analysis showed that expressions of those proteins increased during mature and parthenogenetic embryo. These protein profiles will make available important guides for the study of oocyte function and assist in functional analysis of the proteins.

The aim of this study was to evaluate the changes of protein patterns in granulosa cells and corpus luteum during the estrus cycle in bovine ovary by proteomics ^techniques. Our study was devided into five steps for follicular, ovulatory, early-lteal, midluteal and late-luteal. The protein was extracted from glanulosa cell and corpus luteum proteins by using M-PER Mammalian Protein Extraction Reagent. Proteins were refined by clean-up kit and quantified by Bradford method until total protein was 700 μg. Immobilized pH gradient (IPG) strip was used 18 cm and 3 11 NL. SDS-PAGE was used 10% acrylamide gel. The protein spots were visualized by Coomassie Brilliant Blue (CBB) staining, analyzed by MALDI mass spectrometry and searched on NCIBlnr. As the result, 61 spots of total 85 spots were repeated on follicular stage and 51 spots of total 114 spots were repeated on ovulatory stage. 40 spots of total 129 were repeated on early-luteal and 49 spots of total 104 spots were repeated on mid-luteal stage. Also 41 spots of total 60 spots were repeated on last-luteal stage. There were differences in the ovulation (follicular∼ovultory stage) in which the spots of follicular stage 19 was only and in ovulation stage was 10 spots. The difference between the luteinization (ovultory∼mid-luteal stage) was the spots counted in each stage. The spots of ovulatory stage was 1, early-luteal stage was 1 and in mid-luteal stage was 2. Eleven spots were found in mid-luteal stage and 2 spots were found in last-luteal stage. In conclusion, we confirmed that there were 7 spots in ovulation, 4 spots in luteinization and 2 spots in luteolysis. Spot No. 89-93 from ovulation were transferrin, and spot No.94 and 95 were HSP60. Spot No. 103 were Dusty PK, spot No. 135 were OGDC-E2, and spot No. 175, 176 were Rab GDI beta from luteinization. Spot No. 178 and 179 from luteolysis were vimentin.

Fruit fly is one of the most important pests for vegetables and crops worldwide. Since 1895, four species of fruit flies has invaded into Hawaii. In 2000, a group of scientists from Hawaii has initiated and implemented an area wide pest management program to suppress fruit fly population in Hawaii. Six techniques developed within the program has been transferred to many countries that have the fruit fly problem. Four techniques (monitoring, sanitation, bait spray, and male annihilation) are readily done by farmers. The other two techniques (sterile insect release and augmentative parasitoid release) involve mass fruit fly stock. Sterile insect technique (SIT) used in sterile insect release requires continuous mass rearing. Current mass rearing system has been satisfactory for rearing need. However, there are problems such as pesticide contamination of supporting material, spent diet management, labor intensive, and space issue. USDA-Agricultural Research Service looked for alternatives. In 2004, a novel fruit fly liquid diet has been developed. The core of this diet is using an inert substance (sponge cloth) to replace biological supporting material for mill feed (wheat product). During this diet development process, we have observed that fruit fly performance changes associate with the change of diet components. One of the most significant components is wheat germ oil. Larval diet supplemented with wheat germ oil (WGO) causes physiological reactions, such as increased fecundity and fertility, in some insects. Although the impact of WGO on insect physiology is important, the mechanisms of these actions are poorly understood. In this presentation, we will confirm our hypothesis that the addition of WGO to medium developed for larval oriental fruit flies modulates gene expression in the corresponding adults and further to identify when and how these gene expressed during different life cycle stages. We separately reared larvae of Bactrocera dorsalis on diets lacking or supplemented with WGO, and analyzed for expressed proteins in the resulting adult males and females by 2D-electrophoresis. Analysis of the gels revealed significant changes in expression levels of >70 proteins, 64 of which were identified by mass spectrometric analysis on MALDI-TOF/TOF. Apparent changes in expression levels for 6 of these proteins were confirmed by quantitative real-time PCR, showing that the changes in mRNA expression were reflected in changes in protein expression. These findings support the hypothesis that one mechanism of WGO actions in insect nutrition is the modulation of gene expression. Our goal is to identify molecular markers that serve as early indicators of the quality of insect culture media. Markers of deficient culture media will increase the efficiency of developing optimal systems for mass rearing beneficial insects and some pest species because decisions on culture media quality can be made without waiting through one or several life cycles.

Because of the irreversible nature of periodontal disease, early diagnosis is an important aspect of management of patients with periodontal disease. Human saliva is an attractive medium for disease diagnosis because its collection is noninvasive and simple. Analysis of saliva may be especially beneficial in the determination of current periodontal status and serve as means for the screening of periodontal disease. In the present study, we investigated potential biochemical markers in whole saliva samples for the screening of periodontal disease using proteomics technique. We enrolled five subjects each from four different groups on the basis of measures of periodontal health (healthy group, gingivitis group, chronic periodontitis group and aggressive periodontitis group). Eleven proteins in whole saliva samples were identified as differentially expressed proteins between the healthy and periodontal disease groups using 2-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight / time-of-flight mass spectrophotometry (MADLI-TOF/TOF MS) approaches. Although the diagnostic value of oral fluid has been recognized for some time and potential biomarkers of periodontal disease have been identified in saliva, this, to our knowledge, is one of the first studies to examine large-scale proteomic profiling to identify the extent of periodontal destruction. Thus, this work provides an important framework for future efforts aimed at understanding salivary responses to periodontal destruction and predicting the future disease progression.

Upon oviposition, parasitoid wasps inject their eggs along with venom, teratocytes and polydnavirus (PDV) on the host. Among these parasitic factors, PDVs are known to suppress the host immune system and utilize the host translational mechanisms allowing the juvenile parasitoid to develop. Polydnavirusencoded genes can selectively inhibit host translation and still use the translation machinery of the host to synthesize their own proteins. In this study, we utilize a proteomic approach involving two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) that couples isoelectric focusing (IEF) and SDS-PAGE to resolve complex protein mixtures that results from the parastization of Cotesia plutellae on the lepidopteran host, Plutella xylostella. We specifically analyze the changes in protein synthesis using this technique after treatment of HTIFs that has been previously identified on C. plutellae. The difference in protein profile due to parasitization was confirmed by in vitro translation assay using rabbit reticulosyte lysate.

Cyclosporin A (CsA) has been used clinically as an immunosuppressive drug to prevent organ transplant rejection and in basic research as a mitochondrial permeability blocker. It has been reported that CsA has a protective role in severed neurons and a neurotrophic effect in neuronal cells. However, the molecular mechanisms underlying the stimulation of neuronal cell proliferation by CsA have not yet been elucidated. In our current study, we investigated CsA responsive proteins in PC12 cells using a systematic proteomic approach. The viability of these cells following CsA treatment increased in a dose- and time-dependent manner. Proteins in the CsA-treated PC12 cells were profiled by two-dimensional gel electrophoresis (2-DE) and identified by matrix-assisted laser desorption ionization time-of flight (MALDI-TOF) and electrospray ionization quadupole time-of-flight mass spectrometries (EIQ-TOFMS). This differential expression analysis showed significant changes for 10 proteins (6 up-regulated and 4 down-regulated) upon CsA treatment that were related to cell proliferation, metabolism and the stress response. These proteomics data further our understanding of the proliferation mechanisms of PC12 cells exposed to CsA and demonstrate that our methodology has potential to further elucidate the mechanisms and pathways involved.