William W. Bachovchin

[10] IgA-specific prolyl endopeptidases: Serine type

Publisher Summary This chapter describes IgA-specific serine endopeptidases. IgA proteinases are a group of endopeptidases produced by medically important bacteria in the genera Streptococcus , Neisseria , and Haemophilus . The enzymes are secreted to the extracellular environment and all have pronounced substrate specificity for human IgA 1 immunoglobulins, one of the two IgA isotypes that are the dominant form of antibody in human secretions. The IgA proteinases of the gram-negative bacteria Neisseria gonorrhoeae , Neisseria meningitidis , and Haemophilus influenzae are encoded by a single chromosomal iga gene. There is no known gene regulation, and the enzyme is constitutively expressed in vitro . Enzyme secretion to the culture medium takes place throughout the logarithmic phase of bacterial growth. IgA proteinases cleave both serum and secretory human IgA l proteins. The polymeric immunoglobulin receptor covalently bound to mucosal dimeric IgA does not interfere with cleavage. IgA proteinase activity is determined using human IgA 1 substrate, the preferred source being the monoclonal paraproteins in plasma of patients with multiple myeloma. These proteins are at extremely high concentration and are easy to purify.

Perfusion of cell spheroids for study by NMR spectroscopy

Abstract Many anchorage-dependent types of cells can be cultured in suspension as small clumps of defined size known as spheroids. These spheroids are particularly suited for study with NMR spectroscopy because, unlike suspensions of single cells, they can be perfused without being carried along with the perfusate, thus eliminating the need for filters or gels. We have successfully maintained human breast cancer cells (MCF-7) and Chinese hamster cells (V 79) in a physiological steady state as spheroids for more than 80 hours in a 10 mm NMR tube using a simple and inexpensive perfusion setup which requires no modification of NMR instrumentation. Although we were primarily interested in obtaining 31P NMR spectra, cell mass was sufficiently high that natural abundance 13C NMR spectra were also readily obtained.