Immunochemical recognition of A2E, a pigment in the lipofuscin of retinal pigment epithelial cells.
(2007)
Journal - Proceedings of the National Academy of Sciences of the United States of America (United States )
Abstract :
The autofluorescent lipofuscin pigment A2E accumulates in retinal pigment epithelial cells with age and is particularly abundant in some retinal disorders. To generate a polyclonal antibody that recognizes this pyridinium bisretinoid molecule, we immunized rabbits with bovine serum albumin (BSA) conjugates in which the protein was linked to the A2E molecule via its pyridinium ethanolamine moiety. Analysis by matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF MS) of the A2E-BSA conjugate indicated the presence of five intact A2E molecules covalently linked to BSA, thus deeming it a suitable antigen for immunization. By immunocytochemical staining, the rabbit polyclonal antibody recognized A2E that had accumulated in cultured cells, whereas dot-blot analysis revealed binding to both A2E and A2E-rabbit serum albumin (A2E-RSA) conjugate but no cross-reactivity with various retinoids. Preimmune serum was nonreactive. In fluorescence spectroscopy studies, antibody-A2E binding was evidenced by a fluorescence increase and by a blue-shift in the emission maximum consistent with a change in A2E milieu upon antibody binding. The changes in fluorescence emission upon antibody binding could reflect several processes including restrictions on trans-cis isomerization and intersystem crossing of photo-excited A2E.
| ISSN : | 0027-8424 |
|---|
| Mesh Heading : | Animals Cattle Cells, Cultured Circular Dichroism Fluorescent Antibody Technique, Indirect Humans Immunohistochemistry Lipofuscin Models, Chemical Molecular Structure Pigment Epithelium of Eye Pyridinium Compounds Rabbits Retina Retinoids Serum Albumin, Bovine Spectrometry, Fluorescence Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Spectrophotometry, Ultraviolet chemistry cytology chemistry biosynthesis chemistry immunology metabolism |
|---|
| Mesh Heading Relevant : | metabolism metabolism metabolism cytology metabolism |
|---|
Inhibition of protein kinase C by dequalinium analogues: structure-activity studies on head group variations.
(2006)
Journal - Bioorganic & medicinal chemistry (England )
Abstract :
New dequalinium analogues and related heteroaromatic systems were synthesized and evaluated for inhibition of protein kinase Calpha. In vitro assays with recombinant human PKCalpha showed that the number of the aromatic ring head groups as well as their electron-richness, are critical factors that determine potency. The inhibitory strengths of the synthesized compounds are shown to correlate well with Mulliken charges on the head group ring nitrogen atoms making it possible to design likely candidate molecules having improved protein kinase Calpha inhibitory activity.
| ISSN : | 0968-0896 |
|---|
| Mesh Heading : | Dequalinium Drug Design Electrons Humans Hydrocarbons, Aromatic Protein Kinase C-alpha Recombinant Proteins Structure-Activity Relationship pharmacology chemistry |
|---|
| Mesh Heading Relevant : | analogs & derivatives antagonists & inhibitors |
|---|
Immunochemical recognition of A2E, a pigment in the lipofuscin of retinal pigment epithelial cells
(2007)
Journal - Proceedings of the National Academy of Sciences of the United States of America
Abstract :
The autofluorescent lipofuscin pigment A2E accumulates in retinal pigment epithelial cells with age and is particularly abundant in some retinal disorders. To generate a polyclonal antibody that recognizes this pyridinium bisretinoid molecule, we immunized rabbits with bovine serum albumin (BSA) conjugates in which the protein was linked to the A2E molecule via its pyridinium ethanolamine moiety. Analysis by matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF MS) of the A2E–BSA conjugate indicated the presence of five intact A2E molecules covalently linked to BSA, thus deeming it a suitable antigen for immunization. By immunocytochemical staining, the rabbit polyclonal antibody recognized A2E that had accumulated in cultured cells, whereas dot-blot analysis revealed binding to both A2E and A2E-rabbit serum albumin (A2E–RSA) conjugate but no cross-reactivity with various retinoids. Preimmune serum was nonreactive. In fluorescence spectroscopy studies, antibody-A2E binding was evidenced by a fluorescence increase and by a blue-shift in the emission maximum consistent with a change in A2E milieu upon antibody binding. The changes in fluorescence emission upon antibody binding could reflect several processes including restrictions on trans-cis isomerization and intersystem crossing of photo-excited A2E.
| ISSN : | 0027-8424 |
|---|
| Keywords : | age-related macular degeneration (AMD),antibody,immunocytochemistry |
|---|