Because phosphorylation of the infectious bronchitis virus (IBV) nucleocapsid (N) protein may regulate its multiple roles in viral replication, the dynamics of N phosphorylation were examined. In the infected cell, N was the only viral protein that was phosphorylated as shown by 32P-orthophosphate labeling and Western blot analysis and with IBV specific polyclonal chicken antibody. Using pulse-labeling with 32Porthophosphate, the IBV N protein was found to be phosphorylated in the virion, as well as at all times during infection of Vero cells. One-hour pulse-chase analysis followed by immunoprecipitation of IBV N using rabbit anti-IBV N polyclonal antibody showed that the phosphate on the protein did not fall below 70% of the maximum and remained stable. The small but reproducible drop in phosphorylation could modulate the various functions of the N protein in the infected cell. Simultaneous labeling with 32Porthophosphate and 3H-leucine of infected CEK cells indicated a 3.5-fold increase in the ratio of the 32P:3H counts per minute (cpm) on the virion N protein as compared to the 32P:3H cpm ratio of the N protein from lysates at 7 h p.i. The 32P:3H cpm ratio of the N protein from virion from infected-Vero cell lysates was 10.5X more than the 32P:3H cpm ratio of the N protein obtained at 7 h p.i. It has been shown that the N proteins from the measles and rabies viruses form helical nucleocapsid-like structures when expressed in bacteria (Schoehn et al., 2001; Warnes et al., 1995). The ability of E. coli expressed IBV N protein to form helical-nucleocapsid-like structures was investigated using transmission electron microscopy. Full-length, purified histidine-tagged IBV N protein formed nucleocapsid-like structures when expressed in bacteria. Because E. coli -expressed histidine-tagged fragments of the IBV N protein did not form helical nucleocapsid-like structures, the full-length protein is probably required for assembly of these structures. The highly conserved IBV N protein was also used as a diagnostic tool in an ELISA for detecting anti-IBV antibody in chicken serum using a specialized microwave called the BIOWAVE. The BIOWAVE improves the processing time for an ELISA.

Thesis (Ph. D.)--Ohio State University, 2003. / Title from first page of PDF file. Document formatted into pages; contains x, 92 p.: ill. Includes abstract and vita. Advisor: Clay B. Marsh, Dept. of Veterinary Biosciences. Includes bibliographical references (p. 84-92).

published_or_final_version / Physiology / Master / Master of Philosophy

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

No description available.

No description available.

Capacitation is essential for fertilization of ovulated oocytes. Capacitation is correlated with activation of a signal transduction pathway leading to protein tyrosine phosphorylation, an essential prerequisite for fertilization. Oviductin has been shown to bind to the acrosomal cap and the equatorial segment region of the sperm head. In light of findings reported in previous studies, we hypothesized that estrus stage-specific oviductin (EOV) enhances tyrosine phosphorylation. Immunofluorescent detection by light and confocal microscopy and immunogold labeling by electron microscopy and surface replica techniques were used to localize tyrosine phosphorylated proteins to the equatorial segment region and midpiece after incubation in medium in the presence or absence of EOV. In the presence of EOV, an increase in tyrosine phosphorylation in the equatorial segment region was observed as early as 5 minutes after incubation. On prolonging incubation in medium containing EOV immunostaining further increased, indicative of increased levels of tyrosine phosphorylation of sperm proteins as capacitation proceeds. Regardless of the presence or absence of EOV, phosphotyrosine expression was observed along the tail, specifically at the midpiece. However, this reactivity was enhanced in the presence of EOV. Western blot analysis of NP-40 extractable and non-extractable sperm proteins confirmed these observations. NP-40 extractable sperm proteins (25, 37, 44kDa) and non-extractable sperm proteins (70, 83, 90kDa) showed increased intensity when sperm were capacitated in the presence of EOV after 5-, 60-, 120- and 180-minutes of capacitation. Mass spectrophotometric analysis identified enolase, ATP-specific succinyl CoA, succinate CoA ligase, zona pellucida binding protein, heat shock protein 90, aconitase and hexokinase as proteins that undergo enhancement in tyrosine phosphorylation in the presence of EOV. The proteins identified are known to be involved in specific functions including cellular metabolism, molecular chaperoning and normal sperm development. In summary, the present investigation has provided new evidence showing that sperm capacitated in vitro in the presence of EOV display an enhanced expression of tyrosine phosphorylation compared to sperm incubated in capacitating medium alone. These results indicate that inclusion of oviductin in media used for in vitro fertilization (IVF) may improve success rates of IVF by enhancing the signaling pathways involved in sperm capacitation. / Thesis (Master, Anatomy & Cell Biology) -- Queen's University, 2009-01-30 15:38:54.594

DNA topoisomerase II is an essential enzyme in eukaryotes and is required for many aspects of DNA metabolism including DNA replication, recombination, chromosome segregation and chromosome condensation. It is also a major component of the nuclear scaffold. Topoisomerase II from lower eukaryotes has been shown to be phosphorylated in vivo and this phosphorylation leads to a modulation of activity. However, unlike these lower eukaryotes, human topoisomerase II exists as two closely related, but genetically distinct, isozymes which have markedly different expression and localization patterns. Topoisomerase IIα is a 170kDa protein and topoisomerase IIβ is 180kDa. This study set out to analyse the phosphorylation of these specific isozymes and understand how this leads to the regulation of their distinct biological functions. In order to undertake this study, two polyclonal anti-topoisomerase II antibodies were generated and a series of other polyclonal and monoclonal antibodies characterized. Furthermore, the α isozyme of human topoisomerase II was purified to near homogeneity from cultured HeLa cells. A kinase activity with the biochemical characteristics of casein kinase II co-purified with and could phosphorylate the purified topoisomerase Hot protein. The α and β isozymes of human topoisomerase II were both shown to be phosphoproteins in vivo. The α isozyme is phosphorylated predominantly on serine residues but with a minor proportion of phosphothreonine. Both the α isozyme and a stable ISOkDa fragment of the β isozyme are phosphorylated in vitro by casein kinase II and the catalytic subunit of PKA (cAMP-dependent protein kinase). The α isozyme can also be phosphorylated in vitro by Ca²⁺-dependent and -independent isozymes of protein kinase C and the cell cycle-regulated p34^cdc2 kinase. Two-dimensional tryptic phosphopeptide mapping suggested that the pattern of phosphorylation of human topoisomerase Ha protein in vivo is complex with phosphorylation occurring on multiple residues. Comparison with in vitro maps suggested that casein kinase II and PKA could account for most of the phosphorylation seen in vivo. Using a one- dimensional phosphopeptide mapping approach, a major site of phosphorylation in vivo appeared to be within the C-terminal 20kDa, and that casein kinase II, PKA and PKC may all phosphorylate this region. Phosphorylation of human topoisomerase Hoc protein by casein kinase II, PKA and PKC all led to a stimulation of activity as measured by plasmid relaxation and decatenation. In contrast, dephosphorylation led to a marked decrease in activity of the enzyme. The dephosphorylated enzyme could be reactivated by casein kinase II but not PKA phosphorylation. These data suggest that phosphorylation plays a crucial role in the control of DNA tertiary structure in human cells via regulation of the activity of topoisomerase II proteins.

The application of the proteomic approach has facilitated efforts directed toward the mapping of the entire Arabidopsis cell wall proteome. Proteins were sequentially extracted from purified cell walls using 0.2 M CaC1(_2) followed by a urea buffer. The extracts were resolved via large format two dimensional polyacrylamide gel electrophoresis (2-D PAGE) and were visualised via Coomassie brilliant blue staining. The aim was to identify and characterise as many cell wall proteins as possible, with the hope of identifying novel cell wall proteins. Out of 325 spots visualised on the 2-D polyacrylamide gel, 144 gave a positive protein identification representing 104 different proteins. The identified proteins were divided into 3 categories. The first category included proteins that have been previously identified as plant cell wall proteins. The second category was designated to include novel cell wall proteins (hypothetical proteins) and the third category was made up of proteins, which had recognised functions, but had never hitherto been known to be secreted to the extracellular matrix. Among the identified novel cell wall proteins there were several that shared high homology with protein kinases. These proteins possessed all the characteristics of secreted polypeptides, such as the cleavable N-terminal signal peptide, and were found to lack both the transmembrane domain and the endoplasmic reticulum retention tetrapeptides (HDEL and KDEL). These observations suggested that, as in animal cells, plant cells had extracellular protein kinase activity (phosphorylation). This was supported by the recent discovery that plant cells secrete ATP to the extracellular matrix (Thomas et al., 2000). Verification of the occurrence of extracellular protein kinase activity was further strengthened by the identification of phosphorylated bona fide cell wall proteins and stress responses caused by the depletion extracellular ATP.

Mediators released from the granules of antigen-activated mast cells contribute to allergies, inflammation and diseases such as asthma. One of the major models used to study mucosal mast cells is the RBL-2H3 mast cell line. While there has been considerable research on the initial signalling events following IgE receptor (Fc??RI) cross-linking, the movement of granules to sites of exocytosis is poorly understood. Understanding the mechanisms that control granule movement to and fusion with the plasma membrane could provide novel targets for improved asthma and allergy therapeutics. To this end, an isolated intact population of granules from the RBL-2H3 mast cell provides a powerful research tool and as such the primary aim of this work was to isolate intact granules from the RBL-2H3 mast cell. Using iso-osmotic Percoll gradients we have isolated an intact granule population from RBL-2H3 mast cells. This granule population contained three granule markers: ??- hexosaminidase, serotonin and chymase. Triton X-100 pre-lysis resulted in loss of granule markers from this main peak, indicating that the isolated granules are in fact intact. Further analysis of the granule population showed that it is free from bulk contamination with other organelles and plasma membrane. The granules were estimated to have a density of 1.055 ??? 1.092g/mL, significantly less dense than that of rat peritoneal mast cell granules (1.2g/mL; [1]). Using an intact versus lysed approach, granule-associated proteins and phosphoproteins, from unactivated RBL-2H3 cells, were determined. Nine unknown granule-associated proteins were found using silver staining of gradient fractions separated on a SDSPAGE gel. In addition, four unknown serine or threonine granule-associated phosphoproteins were found. Molecular weight comparison suggested overlap in some of the unknown proteins and phosphoproteins. Probing for protein kinase C (PKC) isoforms confirmed previous results suggesting that a small population of PKC?? localised to the granules [2], and extended these results to include a population of PKC??I. The serine/threonine phosphatase PP1 does not appear to be granule associated. However, there was a small loss of PP2A from the granules (upon lysis), suggesting that perhaps a subpopulation of PP2A is granule-associated. The main granule peak represents a secretion competent population as Fc??RI-mediated activation of the cells resulted in a significant loss of granule markers from this peak. At the peak rate of antigen-induced secretion a number of changes occur in the phosphorylation of granule-associated phosphoproteins. In addition to an increase in the phosphorylation of three of the phosphoproteins seen in resting mast cell granules, eight new proteins were seen. Whether these proteins are granule-associated is currently unknown. PKC?? was found to translocate away from the granules at the peak rate of secretion, perhaps representing an important control mechanism in granule exocytosis. None of the tested PKC isoforms were found to translocate to the granules, providing little clue as to the identity of the kinase that may be involved in these phosphorylation events. However, as PKC??I is granule-associated and does not translocate off the granules, it would suggest that this kinase might be important for some of the observed phosphorylations. Overall the studies in this thesis show for the first time a rapid gradient-based method for the isolation of intact granules from unactivated and activated RBL-2H3 mast cells. These granules were used to determine granule-associated proteins and phosphoproteins, as well as to investigate changes that occur during the secretory process. In addition, the results show that a number of proteins have increased serine/threonine phosphorylation at the peak rate of antigen-stimulated secretion. This implies that phosphorylation is likely to play a role in the control of granule exocytosis. The identity of these proteins deserves further investigation. Thus, isolated intact RBL- 2H3 mast cell granules provide a powerful research tool to further investigate the mechanism and control of granule exocytosis.