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|Title:||Immunological Studies of Chicken Brain: Isolation, Purification, and Localization of a Neural Specific Protein CNA-1|
|Authors:||Redshaw, Douglas James|
|Department:||Medical Sciences (Growth and Development)|
|Keywords:||Medical Sciences;Medical Sciences|
|Abstract:||<p>The differentiation of any cell leads to the synthesis of certain proteins which are responsible for its specific function. In the nervous system this includes such functions as the conductance of action potentials, synaptic transmission and the establishment of specific connections. The developmental biology of proteins unique to the nervous system is therefore of great importance when studying the relationship of proteins to physiological function.</p> <p>Although some neural specific components of adult chicken brain and their ontogeny in the chicken embryo have been previously described by others, they have not been extensively characterized. The present investigation was undertaken in order to enumerate, characterize and study the embryological appearance of antigens specific to the adult chicken brain that can be demonstrated by rabbit anti-brain sera. Particular interest was given to the isolation and purification of one such neural specific antigen in hopes of further elucidating its role in adult function and embryonic development.</p> <p>Antiserum produced in response to a saline soluble extract of adult chicken brain (ABE), when absorbed with adult liver, serum and kidney extracts, yielded a neural specific antiserum (AABS). This polyvalent antiserum was capable of demonstrating at least eight adult neural specific antigens within ABE during immunoelectrophoret analysis. The sequential appearance of seven of these antigens during embryonic development (D2 to D11 of incubation) was established. An eighth antigen could not be detected within the embryonic brain extracts and was assumed to be associated with posthatching neural development. By immunochemical criteria, the majority of the neural specific antigens, but not all, were demonstrated to be restricted to avian brain. Similarly the majority of these antigens, but not all, were demonstrated to be protein in nature since reactions with trypsin and chymotrypsin resulted in loss of antigenicity.</p> <p>Separation of the antigens within ABE, based on their net ionic charge and molecular weight, was attempted with each resulting fraction being analyzed by immunoelectrophoresis. One neural antigen (CNA-1), possessing alpha-1 globulin mobility was isolated from the other neural specific antigens. This thesis further describes a procedure developed for the purification of CNA-1, utilizing ammonium sulphate fractionation, ion exchange chromatography, gel filtration chromatography and preparative polyacrylamide gel electrophoresis. Highly sensitive and quantitative immunochemical techniques (crossed and fused rocket immunoelectrophoresis) were employed in monitoring the purification steps.</p> <p>Studies on the purified antigen revealed it to be homogeneous by a number of criteria, having a native molecular weight of 65,000 daltons as determined by molecular exclusion chromatography and possessing two identical subunits (MW 30,000) as determined by SDS polyacrylamide gel electrophoresis. The structure of CNA-1 was concluded to be protein on the basis of its sensitivity to trypsin and chymotrypsin.</p> <p>Immunochemical studies utilizing a monovalent antiserum to CNA-1 revealed the protein to be avian specific and not present in brain extracts of mammalian species. Also both a high (> 1,500,000 daltons) and low (65,000 daltons) molecular weight component which shared the CNA-1 antigenic determinant were observed, indicating possible aggregation of the protein during the isolation procedure.</p> <p>CNA-1 was first detectable by the seventh day of incubation and continued to accumulate during the period of embryonic neural development in which both neurogenesis and synaptogenesis are known to occur. Within the adult chicken cerebellum, CNA-1 was specifically localized to the cell surfaces of particular neuronal cell types (i.e. Purkinje cells, granule cells and neurons of the deep cerebellar nuclei) by immunohistochemical techniques. Glial elements were not labelled.</p> <p>Organ specific brain antigens have been described by different investigators. The antigen described within this thesis appears to be completely different from all others described in the literature as revealed by a study of its physical and chemical properties. The function and role of CNA-1 on the cell surface remains to be elucidated. Its localization on specific cell types suggests that it may be associated with inhibitory synaptic function. For this reason, further study of CNA-1 may prove to be important in unravelling some of the questions concerning cell-cell interaction and neuronal specificity during development of the central nervous system.</p>|
|Appears in Collections:||Open Access Dissertations and Theses|
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