Antibodies specific for u.v.-induced DNA damage were raised in rabbits, and used to study damage and repair of nuclear DNA in nude mouse and human skin in vivo by immuno-fluorescence and immunoperoxidase techniques. Purification of the antibodies by affinity chromatography strongly reduced unspecific background staining. In situ denaturation of nuclear DNA with 70 mM NaOH in 70% ethanol increased the sensitivity of the assay approximately 10-fold. Absorption experiments indicated that the specificity of the antibodies was primarily directed against pyrimidine dimers in single stranded DNA. Immunofluorescence and immunoperoxidase staining were essentially equally sensitive and positive responses using these techniques were already apparent in epidermal cell nuclei after 0.5 minimal erythemal dose (MED) of u.v. light. At higher doses, such as 2 MED, the staining was strong in all the epidermal layers and could also be observed in dermis. Even so, removal of antibody binding sites was well under way at 4-5 h post-irradiation and essentially complete after 24 h. Visible light increased the rate of repair, indicating the involvement of a photoreactivation enzyme in human skin in vivo.

Abstract. The putative virulence factors of Aeromonas salmonicida, the aetiological agent of furunculosis in salmonids, are candidates for protective antigens in effective vaeeines against furunculosis. In this report, the authors have compared the immunogenieily of eell‐associated and extracellular antigens of A. salmonicida in Atlantic salmon, Salmo salar L., to that in rabbit. The animals were immunized with formalin‐killed whole cells and formalin‐inactivated extracellular products (ECP), either separately or in combination. The ability of the antigens to induce antibody production was studied by elisa and Western blotting techniques. These results confirm previous reports that far more structures are immunogenie in rabbit compared to the antibody responses elicited in salmon. However, in both species, some antigens were dominant, including a caseinolytic protease in addition to the A‐protein and high and low MW LPS.

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. The predominant cell surface protein (A‐protein) of Aeromonas salmonicida has been purified by a method utilizing a glycine/hydrochloridc extraction from whole cells and HPLC/ion exchanger (DEAE) columns. This procedure yielded two LPS‐frec molecules (a 40‐ and a 50‐kDa form) both shown to contain A‐protein determinants. The former appears to be a digest product of the latter, as a serine protease produced by A. salmonicida was shown to process the 50‐kDa form into a 40‐kDa molecule in vitro. The A‐layer protein was shown to contain one isoform, although multiple isoelectric forms appeared as preparative artifacts, probably due to deamidation. The A‐layer protein and LPS arc the most significant surface antigens recognized by the Atlantic salmon B‐lymphocytes or antibodies. Immunological studies of LPS‐free and LPS‐containing A‐protein preparations were undertaken to test whether the two components behave like antigenie competitors or whether the LPS moiety could adjuvant the antibody response against the A‐protein. The latter was shown to be the case.

Extracellular haemolytie activities of Aeromonas salmonicida ssp. salmonicida to salmon red blood cells were shown to be due to different forms of the mcmbranc-activc enzyme glyccrophospholipid:choicstcrol acyltransferase {GCAT). About 10% of the total haemolytie activity was due to a high molecular mass complex of LPS and GCAT (mol. mass > 1000kDa), containing 35-50% neutral sugars and 1-5-2-0% protein. Some haemolytie activity (30-40% of total), corresponding to 5O-70kDa by gel filtration, also contained GCAT-aetivity and may represent aggregated forms of GCAT. However, about 50% or more of the haemol>tic activity was due to a protein of 26kDa free GCAT. Rabbit antibodies to GCAT neutralized the haemolytie activity of both GCAT and GCAT-LPS. A transposon-produced serine protease negative mutant of the same A. salmonicida strain showed reduced haemolytie -activity. The mutant produced a 38-kDa GCAT preform of low haemolytie activity. The proform was processed by autogenous serine protease to a highly haemolytie 26-kDa molecule with pi 6-3. similar to GCAT of the parent strain. The weakly haemolytie GCAT-LPS analogue of the mutant strain did not contain detectable amounts of the 26-kDa molecule and was not activated by proteases.