Improvements in turkey reproductive efficiency is a very desirable goal for the turkey industry. The ability to maintain turkey hens in the egg-laying (LAY) stage and produce one additional egg per hen a year is estimated to save the turkey industry approximately $1.5 million dollars per year. Overall protein expression generated by tissues of the hypothalamic-hypophyseal complex, namely the anterior pituitary, of the mature turkey hen have a profound impact on reproductive cycling (Scanes, 2000). One of the key physiological factors produced by the anterior pituitary and shown to play a significant role in the regulation of egg laying is the protein prolactin (Prl). The objectives for this study are to examine the overall protein expression patterns from turkey hen pituitary tissue during the nonphotostimulated (NPS), photostimulated (PS), and egg laying (LAY) stages. Attempts to isolate transcription factors that regulate the expression of Prl using an affinity chromatography technique or southwestern screening of a bacteriophage expression library were not successful. A global analysis of protein expression, using two-dimensional polyacrylamide gels (2D gels), was conducted using whole cell, cytoplasmic and nuclear protein extracts from pituitary tissue collected during the NPS, PS and LAY reproductive stages. Approximately 1,046 proteins ranging in pI from 4.6-8.2 and molecular weights between 100 kDa-6kDa were resolved. Protein expression patterns were replicated and verified using pituitaries harvested from NPS, PS and LAY stage turkey hens from another laboratory. Proteins showing considerable changes (563 proteins increased in expression and 98 proteins decreased in expression from the NPS to the LAY stage) in their expression between the reproductive stages were grouped in analysis sets for future identification. These proteins may prove to be important to the reproductive cycling of the turkey hen and warrant future investigation. The results of this study contribute to the overall understanding of the role that the pituitary, as a critical part of the hypothalamic-hypophyseal complex, plays in turkey hen reproductive cycling. / Master of Science

The baculovirus expression vector system (BEVS) is an effective way to produce recombinant proteins for biopharmaceuticals. However baculovirus stocks are stored in subzero temperatures to maintain virus stability, and fetal bovine serum is commonly used in the storage solution. In an effort to lower transportation and storage costs, a storage formulation that can effectively store the baculovirus in above frozen temperatures without the use of FBS would be beneficial. In this study, DMSO, ethylene glycol, glycerol, sucrose, sorbitol, sucrose-phosphate, and sucrose-phosphate-glutamate were added to baculovirus stock at various concentrations to determine the most effective stabilizer for virus storage at 4°C. Of the seven additives studied, 1 M sorbitol most effectively preserved baculovirus stock over a period of 47 weeks stored in 4°C. Formulations that include sucrose, L-arginine, and Pluronic F68 were created to determine their effectiveness on virus stability in a freeze-dried state stored at room temperature. In a lyophilized state, 0.5 M sucrose maintained baculovirus stock stability after 5 weeks of storage. Lyophilized stocks not containing sucrose were no longer infective after 5 weeks. / Master of Science

No description available.

The recent increase in availability of next generation sequencing methodologies has led to extensive analysis of the genome of Clostridium difficile. In contrast, protein expression analysis, crucial to the elucidation of mechanisms of disease, has severely lagged behind. In this study, in-depth proteomic analysis of three strains of varying virulence, demonstrated previously in an animal model, has been undertaken against a background of the sequenced genomes. Strain B-1 is a historic, virulent, ribotype 005 clone, strain A represents the emerging hypervirulent 027 ribotype, while strain Tra5/5, ribotype 001, is of low virulence. To undertake a comprehensive overview of the expressed proteome, both 1D and 2D gel electrophoresis were used to separate and display the protein content of each isolate. This was coupled to MALDI-TOF and LC-MS/MS mass spectrometry for protein identification. A total of 888 different proteins were characterised by comparative analysis of isolates grown in parallel for 64 hours on blood agar. Of these, only 38% were shared between all isolates. An additional 350, 243 and 398 proteins were detected from broth cultures, and the use of a hexapeptide bead library, designed to capture low abundance proteins, led to the detection of a further 148, 127, and 171 proteins in strains A, B-1 and Tra5/5 respectively. Relative differential expression was investigated using Differential In Gel Electrophoresis (DIGE), and five proteins were shown to have a statistically higher concentration in strain A, twelve in strain B-1 and eight in strain Tra5/5. A number of these were surface proteins, with selected S-layer proteins found to be up-regulated in each strain, and the flagellar protein, FliC, up-regulated in both A and B-1. Furthermore, differential post-translation modification events were seen in flagellar and S-layer proteins. In-vivo expression of these proteins was mapped using Western blotting. Immunodetection of the majority of these, including FliC and the high molecular weight S-layer protein, were conserved between the three strains, but a notable series of immunoreactive protein spots were present in strains A and Tra5/5 but not B-1, most likely corresponding to an additional S-layer protein present in the genomes these strains, but not that of B-1. Protein expression differences for a number of previously proposed virulence proteins were evident between strains, including toxin B, sporulation, flagella and the S-layer proteins, metabolic enzymes, stress response proteins and ABC transporters. This study strongly supports the view that the virulence of Clostridium difficile is multifactorial, and that a number of related factors, although not directly required for pathogenicity, may serve to modulate the virulence of individual strains.

In an Experimental Rat model for endometrial carcinoma (EC) a minimal region of recurrent deletion/allelic loss at the neighborhood of the Tp53 gene has been identified. A similar observation of deletion at the homologous position on human chromosome 17 unassociated with TP53 mutation has been reported in several human cancer types. Thus an important tumor suppressor activity located close to, but distinct of TP53 is suggested. Detailed molecular analysis of this candidate region in a tumor model suggested Myo1c (myosin 1C) and Inpp5k (inositol polyphosphate-5-phosphatase K), also known as Skip (skeletal muscle and kidney enriched inositol polyphosphate phosphatase), as the best candidates. These two genes are suggested to be involved in glucose metabolism through PI3K/AKT signaling and neither of them has earlier been reported as a tumor suppressor gene. The present work aimed to investigate the potential correlation of MYO1C and/or INPP5K proteins with components of PI3K/AKT signaling pathway involved in cell growth and survival. Cells were transfected with increasing amounts of MYO1C- or INPP5K- gene expression constructs and protein extracts of the cells were subjected to Western Blot analysis for 13 important components of the signaling pathway: p110β\α\δ, p85, pAkt308&473, 14-3-3β, PTEN, Akt, pErk, Erk, Ras, p4EBP1 and 4EBP1. The analysis showed dose-dependent changes in the expression levels of several of these proteins, and the observed changes for the most part were directed towards negative regulation of cell proliferation and survival. The presented data further extended the initial hypothesis for potential tumor suppressor activities of MYO1C and INPP5K proteins through PI3K/AKT pathway.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a transmembrane protein that functions as an ion channel. Mutations in this protein cause Cystic Fibrosis. For this reason, it is important to study the structure and function of CFTR. In this study, constructs of CFTR (C-terminii), a CFTR-interacting protein and full-length CFTR were cloned, expressed and purified for structural and functional studies. The purified C-terminal polypeptides of CFTR were soluble and shown to interact with NHERF1 PDZ 1 (a CFTR-interacting protein). The CFTR C-terminus and NHERF1 PDZ 1 domain were co-expressed and co-purified. The purified complex showed a strong interaction that might induces a conformational change. Site-directed mutation of the C-terminus of CFTR was performed in order to examine the effect of removing a potentially flexible amino acid (Arginine) on protein crystallization. Pull-down assay experiments with full-length CFTR demonstrated an interaction between CFTR (in DDM detergent) and NHERF1 PDZ 1(+). No interaction was observed for CFTR in LPG (a relatively denaturing detergent) and NHERF1, implying that the interaction between the PDZ motive of CFTR and NHERF1 requires a stable folded structure for both proteins. In addition, full-length CFTR in DDM has been studied by electron microscopy and Single Particle Analysis in the presence of NHERF1 PDZ 1(+). A 3D structure was generated for the CFTR-NHERF1 PDZ 1(+) complex at a resolution of ~ 18 A. This 3D structure showed a new open conformation of CFTR (V shape). In comparable studies with CFTR alone, a 3D structure was generated at a resolution of 27 A and this structure showed a closed state as previously reported. This new data suggest a possible role for NHERF1 in terms of CFTR channel gating or activation.

Nitrate is a ubiquitous drinking water contaminant with potential adverse effects on human health. However, little is known about nitrate toxicity at the cellular and molecular level. The purpose of this study was to examine the effects of environmentally relevant concentrations of nitrate on cytotoxicity and protein expression in human cell lines. To determine if tissue-specific responses occurred, a human hepatoma cell line (HepG2) and a human embryonic kidney cell line (HEK293) were used. Both potassium and ammonium salts of nitrate were used to determine salt-specific toxicity. Test concentrations of nitrate varied from 1 μg/L to 5000 mg/L. Cells were exposed to a nitrate salt for 24, 48, or 72 hours and then examined for effects on viability (using the Neutral Red assay) or proliferation (using the BrdU ELISA assay). To determine the effects of nitrate on protein expression, levels of PCNA, Hsp70, Hsc70, and VEGF protein were monitored using Western blotting in HepG2 and HEK293 cells exposed to KNO3 or NH4NO3 for 24 hours.<p>Nitrate was cytotoxic to both cell types at high concentrations, with EC50 values between 1557 mg/L (approximately) 5852mg/L for viability, and ~2.5 mg/L 3631 mg/L for proliferation. Several EC50 values were not calculable based on the available data, but appeared to be far greater than 5000 mg/L. Ammonium nitrate was generally more toxic than potassium nitrate, and increasing exposure time generally resulted in greater toxicity. The HepG2 and HEK293 cells displayed similar responses for most assays, except the 24 hour KNO3 Neutral Red assay. Here, HEK293 viability increased with increasing KNO3 concentrations, while HepG2 viability decreased. The reason for this finding is unknown, but may involve cell-specific homeostatic mechanisms. A hormetic-like effect was observed in both cell types in several of the proliferation assays; the biological significance of this effect remains unknown.<p>No significant changes in protein expression were observed under these experimental conditions. Some subtle trends were present, such as a slight increase in Hsp70 expression with increasing nitrate concentration in both cell types. In HepG2 cells, PCNA expression increased slightly with increasing nitrate concentrations; however, the opposite effect was observed in HEK293 cells. This may be due to transcriptional or translational regulation.<p>In summary, environmentally relevant concentrations of nitrate did not appear to evoke significant cytotoxicity or changes in protein expression. Cell viability and proliferation effects were observed at higher concentrations of nitrate. Private water supplies may contain nitrate concentrations above the EC50 values in these experiments. More research is required to determine if this poses a direct threat to human health.

No description available.

Nitrate is a ubiquitous drinking water contaminant with potential adverse effects on human health. However, little is known about nitrate toxicity at the cellular and molecular level. The purpose of this study was to examine the effects of environmentally relevant concentrations of nitrate on cytotoxicity and protein expression in human cell lines. To determine if tissue-specific responses occurred, a human hepatoma cell line (HepG2) and a human embryonic kidney cell line (HEK293) were used. Both potassium and ammonium salts of nitrate were used to determine salt-specific toxicity. Test concentrations of nitrate varied from 1 μg/L to 5000 mg/L. Cells were exposed to a nitrate salt for 24, 48, or 72 hours and then examined for effects on viability (using the Neutral Red assay) or proliferation (using the BrdU ELISA assay). To determine the effects of nitrate on protein expression, levels of PCNA, Hsp70, Hsc70, and VEGF protein were monitored using Western blotting in HepG2 and HEK293 cells exposed to KNO3 or NH4NO3 for 24 hours.<p>Nitrate was cytotoxic to both cell types at high concentrations, with EC50 values between 1557 mg/L (approximately) 5852mg/L for viability, and ~2.5 mg/L 3631 mg/L for proliferation. Several EC50 values were not calculable based on the available data, but appeared to be far greater than 5000 mg/L. Ammonium nitrate was generally more toxic than potassium nitrate, and increasing exposure time generally resulted in greater toxicity. The HepG2 and HEK293 cells displayed similar responses for most assays, except the 24 hour KNO3 Neutral Red assay. Here, HEK293 viability increased with increasing KNO3 concentrations, while HepG2 viability decreased. The reason for this finding is unknown, but may involve cell-specific homeostatic mechanisms. A hormetic-like effect was observed in both cell types in several of the proliferation assays; the biological significance of this effect remains unknown.<p>No significant changes in protein expression were observed under these experimental conditions. Some subtle trends were present, such as a slight increase in Hsp70 expression with increasing nitrate concentration in both cell types. In HepG2 cells, PCNA expression increased slightly with increasing nitrate concentrations; however, the opposite effect was observed in HEK293 cells. This may be due to transcriptional or translational regulation.<p>In summary, environmentally relevant concentrations of nitrate did not appear to evoke significant cytotoxicity or changes in protein expression. Cell viability and proliferation effects were observed at higher concentrations of nitrate. Private water supplies may contain nitrate concentrations above the EC50 values in these experiments. More research is required to determine if this poses a direct threat to human health.

Xenograpsus testudinatus is a dominant species in shallow hydrothermal vents off Kueishan Island, Taiwan. But we know little about crab populations in vent areas. In this study, stable isotope and one dimension protein expression profiles were studied to evaluate the differences of X. testudinatus from different habitats, i.e. white and yellow vents. Results indicated food sources between the two habitats were different in 2009, but similar in 2010. The ranges of £_15N value of X. testudinatus were broad which might result from abundant food in white vent. Furthermore, zooplankton might not be the major food source of X. testudinatus. Significant difference in protein expression profiles between habitats indicate that they adapt to different habitats in small scales with migration between habitats. And, crabs may have other habitats outside vent areas. After culture in laboratory for 12 hours, crab protein expression profiles were different from their original ones, indicating their acclimation to a new environment is relative fast.