Eosinophils and basophils are now recognized as critical cells in the pathophysiology of bronchial asthma and other hypersensitivity diseases. Because of their potent biological activities, cationic eosinophil granule proteins are implicated as mediators of eosinophil functions. However, little information is available regarding basophil granule proteins. Prior studies under the auspices of this grant characterized the structure and function of the principal granule proteins including the major basic protein (MBP), eosinophil peroxidase (EPO), eosinophil-derived neurotoxin (EDN), and eosinophil cationic protein (ECP). Here, we identify three new lines of investigation. First, MBP is produced as a pro-molecule (proMBP) and converted to 14 kDa MBP. Preliminary experiments indicate that cathepsin L has proMBP converting activity, and we identify experiments to test this hypothesis. Second, the three-dimensional structures of 14 kDa MBP and proMBP will be determined by X-ray crystallography. The 14 kDa MBP have already been crystallized, and a native dataset at 2.8 angstrom has been obtained. However, 14 kDa MBP crystals are often twinned, and it may be necessary to express and crystallize proMBP in order to solve the structure of 14 kDa MBP. Third, experiments to identify novel basophil granule proteins are described. Basophils from a patient with basophilic leukemia were obtained by cytapheresis and granules isolated. High performance liquid chromatography of granule lysates revealed the presence of numerous proteins and the partial sequences of eight were obtained. We propose to stimulate umbilical cord mononuclear cells to differentiate to eosinophil/basophil hybrids and to produce a cDNA library from these hybrids. The hybrids will be compared to IL-5 stimulated umbilical cord cells by differential display analyses and by sequencing of randomly selected cDNA clones. Using degenerate oligonucleotides and novel DNA sequences from differential display and from sequencing of randomly selected clones, we will identify full-length cDNA clones and express the novel basophil proteins with the goal of producing antibody specific for the basophil. Overall, these studies will provide important new information regarding the mechanism of conversion of proMBP to MBP, the three dimensional structure of eosinophil granule proteins and the existence of novel basophil-associated proteins.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI009728-30
Application #
6124327
Study Section
Special Emphasis Panel (ZRG2-IVP (02))
Program Officer
Plaut, Marshall
Project Start
1978-05-01
Project End
2001-11-30
Budget Start
1999-12-01
Budget End
2000-11-30
Support Year
30
Fiscal Year
2000
Total Cost
$287,136
Indirect Cost
Name
Mayo Clinic, Rochester
Department
Type
DUNS #
City
Rochester
State
MN
Country
United States
Zip Code
55905
Behroozian, Adam A; Chludzinski, Jeffrey P; Lo, Eugene S et al. (2013) Detection of mixed populations of Clostridium difficile from symptomatic patients using capillary-based polymerase chain reaction ribotyping. Infect Control Hosp Epidemiol 34:961-966
Natarajan, Mukil; Walk, Seth T; Young, Vincent B et al. (2013) A clinical and epidemiological review of non-toxigenic Clostridium difficile. Anaerobe 22:1-5
Feglo, Patrick; Adu-Sarkodie, Yaw; Ayisi, Lord et al. (2013) Emergence of a novel extended-spectrum-?-lactamase (ESBL)-producing, fluoroquinolone-resistant clone of extraintestinal pathogenic Escherichia coli in Kumasi, Ghana. J Clin Microbiol 51:728-30
Wagner, Lori A; Wang, Shuping; Wayner, Elizabeth A et al. (2013) Developing and mature human granulocytes express ELP 6 in the cytoplasm. Hum Antibodies 22:21-9
Waslawski, Sheila; Lo, Eugene S; Ewing, Sarah A et al. (2013) Clostridium difficile ribotype diversity at six health care institutions in the United States. J Clin Microbiol 51:1938-41
Walk, Seth T; Micic, Dejan; Jain, Ruchika et al. (2012) Clostridium difficile ribotype does not predict severe infection. Clin Infect Dis 55:1661-8
Gu, Qihai; Lim, Michelle E; Gleich, Gerald J et al. (2009) Mechanisms of eosinophil major basic protein-induced hyperexcitability of vagal pulmonary chemosensitive neurons. Am J Physiol Lung Cell Mol Physiol 296:L453-61
Verbout, Norah G; Jacoby, David B; Gleich, Gerald J et al. (2009) Atropine-enhanced, antigen challenge-induced airway hyperreactivity in guinea pigs is mediated by eosinophils and nerve growth factor. Am J Physiol Lung Cell Mol Physiol 297:L228-37
Plager, Douglas A; Davis, Mark D P; Andrews, Amy G et al. (2009) Eosinophil ribonucleases and their cutaneous lesion-forming activity. J Immunol 183:4013-20
Wagner, Lori A; Christensen, Clarissa J; Dunn, Diane M et al. (2007) EGO, a novel, noncoding RNA gene, regulates eosinophil granule protein transcript expression. Blood 109:5191-8