ISSN:
0886-1544
Keywords:
conservative nucleoskeletal epitopes
;
in situ cross-reacting antibodies
;
immunofluorescence microscopy
;
Life and Medical Sciences
;
Cell & Developmental Biology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Medicine
Notes:
The vegetative giant nucleus of the unicellular and uninucleate marine green alga Acetabularia is a depot of conservative epitopes, homologous to antigenic determinants of vertebrate actin, myosin, actinin, and vinculin. The epitopes appear at the nucleolar surface (actin, actinin, vinculin determinants), in the caryoplasm (actin, myosin determinants), along “caryoplasmic” filaments (actin determinants), and in “nuclear envelope plus perinuclear bodies” (actin, myosin, actinin, vinculin determinants). The structure homologies of the nuclear antigenic determinants to those of the vertebrate muscular and/or microfilamental proteins were deduced from in situ cross-reaction of anti-chicken actin (cross-reaction also with rabbit actin), anti-chicken alpha actinin, anti-chicken vinculin, and anti-bovine myosin (cross-reaction also with chicken myosin), respectively, by indirect immunofluorescence microscopy. Artifacts which arise from the binding of contaminating unspecific markers or from unspecific adherence of specific ones to the algal nucleus have been overcome by the use of both polyclonal and/or monoclonal immunoglobulins as primary markers and different types of second markers each conjugated with fluorescein isothiocyanate (FITC). Fluorescein staining of primary markers was performed either with fluorescent anti-immunoglobulin antibodies in a one-step (AIA-FITC) or highly sensitive two-step procedure (AIA/AIA-FITC), covalently labeled F(ab′)2 specific for either Fc or F(ab′)2 (the latter anti-fragment antibody excluded both possible interactions between nuclear “lectins” and glycosidic residues of Fc and staining of glycosidic nuclear antigens by AIAs or anti-Fc specific for the glycosidic part of the immunoglobulin antigen) or fluorescent complex “protein A-biotin-avidin” (PABA-FITC, a highly sensitive nonimmunoglobulin second reagent). Three of four different AIA-FITC preparations tested alone and also “F(ab′)2 anti-Fc” showed reactivities with the nucleoli and the nuclear envelope. This indicates the presence of glycosidic determinants at the sites of reaction. Each of the other fluorescent markers, including AIA/AIA-FITC, reacted with the primary marker only, although they were different in sensitivity.The staining patterns of nuclear actin epitopes differed in certain details if primary marker (monoclonal against polyclonal), second marker (AIA-FITC against PABA-FITC), or nuclear preparation (degree of nuclear flattening by the cover slide and salt condition) were changed. It suggests that type and number of actin epitopes, valence, affinity, and number of anti-actin clones, but also subclass or class specificity of the second marker and accessibility of the nuclear actin determinant(s), were involved. The nuclear actin and myosin epitopes stained most intensively in a “high salt” environment (100 mM PBS, 50 mM/1 KC1; pH 7.2) if compared to “low salt staining.” This indicates local concentrations and/or accessibility of antigenic determinants which were hidden in “low salt” (1.5 mN/1 Na2HPO4, 8mM/1 KH2PO4, 2.7 mM/1KCl, 137 mM/1 KCl, 137 mM/1 NaCl; pH 7.4) conditions.
Additional Material:
4 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/cm.970070110
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