Kimberly A. Conlon

Light-induced proteolysis of myosin heavy chain by Rose Bengal-conjugated antibody complexes

The specific light-induced, non-enzymatic digestion of chicken skeletal muscle myosin heavy chain by xanthene dye-conjugated antibodies is reported. The xanthene dye Rose Bengal was conjugated to either a mouse monoclonal anti-myosin primary specific antibody or to goat anti-mouse IgG secondary antibodies. Under our experimental conditions, visible light induced the non-enzymatic breakdown of myosin heavy chains when chicken skeletal muscle myosin either directly formed a complex with Rose Bengal-conjugated anti-myosin antibodies or indirectly formed a complex with anti-myosin antibody-Rose Bengal-conjugated secondary antibodies. The rate of the photochemical reaction depended on irradiation time and temperature. Although SDS-PAGE and immunoblot analyses showed that fragments migrating below the myosin heavy chain polypeptide predominated, these analyses also showed higher molecular mass polypeptides were generated.

Antifading agents for confocal fluorescence microscopy.

Publisher Summary The chapter discusses strategies to reduce specimen-associated background fluorescence and the properties and performance of several antifading agents during the acquisition of multiple optical sections. Discussions are focused on indirect immunofluorescence, and other in situ localization methods are also discussed. Currently, single- and dual labeling immunofluorescence, imaging of green fluorescent protein, and fluorescent in situ hybridization (FISH) are the most widely used applications of the confocal laser scanning microscope (CLSM). Single- or dual-labeling immunofluorescence may involve either a direct or an indirect method. The greatest advantage of the CLSM is its ability to discriminate between signals originating from in-focus and out-of-focus optical planes to produce digital images that can be merged pixel by pixel to generate perfectly registered two- and three-dimensional renditions. The chapter also discusses specimen preparation, antifading agents, and acquisition of fading fluorescent images. The reduction of specimen-associated background fluorescence is performed immediately after paraformaldehyde fixation of the sample.

Oxidation of guanine in liver and lung DNA of prematurely aging OXYS rats

Immunofluorescence assay was applied for determination of 8-oxoguanine (8-oxoG) in DNA. The 8-oxoG content in liver and lung DNA of 2-and 18-month-old Wistar rats was compared with that of prematurely aging OXYS rats. It was shown that for rats of both strains, 8-oxoG content in lung DNA compared with liver DNA was 1.7–2.0-fold and 1.3–1.7-fold higher for 2-and 18-month-old rats, respectively. However, the degree of oxidative damage in liver DNA of OXYS rats was 2.4-(p < 0.01) and 1.5-fold (p < 0.05) higher for 2-and 18-month-old animals, respectively, than that in liver DNA of Wistar rats. Oxidation of guanine in lung DNA of OXYS rats was 2-(p < 0.01) and 1.7-fold (p < 0.05) higher for 2-and 18-month-old animals, respectively, than that in lung DNA of Wistar rats. The data indicate that elevated DNA oxidative damage in various organs of OXYS rats may be an important factor of accelerated again and progression of age-related diseases—cataract, macular dystrophy, hypertension, osteoporosis, cognitive and behavioral dysfunctions, and also lung and liver pathologies.

Site-directed photochemical disruption of the actin cytoskeleton by actin-binding Rose Bengal-conjugates

The in situ light-induced, non-enzymatic digestion of cytoskeletal actin by a xanthene dye conjugated to heavy meromyosin, anti-actin antibodies and/or anti-myosin antibodies is reported. The dye Rose Bengal was conjugated to either anti-actin antibodies, anti-myosin antibodies or heavy meromyosin. Under our experimental conditions, visible light induced the non-enzymatic breakdown of cytoskeletal actin when mammalian tissue culture cells were probed either with Rose Bengal-conjugated anti-actin and/or anti-myosin antibodies. Similar results were obtained when tissue culture cells were probed with Rose Bengal-conjugated heavy meromyosin before irradiation with visible light. The in situ photochemical reaction depended on the presence of actin-binding Rose Bengal-conjugates.

A method for direct cross-linking of DNA bases containing aromatic amino groups to proteins.

A one step method to cross-link DNA bases containing aromatic amino groups directly to proteins was developed. No chemical modification of the base is required prior to conjugation, which is performed at neutral pH. Work focused on 8-oxoguanine and the carrier protein, bovine serum albumin. Conjugates were stable after sodium dodecyl sulfate (SDS)-induced protein denaturation and were characterized by UV spectroscopy, enzyme linked immunosorbent assay (ELISA), SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analyses. This method is a viable alternative to existing procedures for generating DNA base-protein conjugates for antibody characterization and affinity purification.