Novel monoclonal antibody recognition of oxidative DNA damage adduct, deoxycytidine-glyoxal

Glyoxal, a reactive aldehyde, is a decomposition product of lipid hydroperoxides, oxidative deoxyribose breakdown, or autoxidation of sugars, such as glucose. It readily forms DNA adducts, generating potential carcinogens such as glyoxalated deoxycytidine (gdC). A major drawback in assessing gdC formation in cellular DNA has been methodologic sensitivity. We have developed an mAb that specifically recognizes gdC. Balb/c mice were immunized with DNA, oxidatively modified by UVC/hydrogen peroxide in the presence of endogenous metal ions. Although UVC is not normally considered an oxidizing agent, a UVC/hydrogen peroxide combination may lead to glyoxalated bases arising from hydroxyl radical damage to deoxyribose. This damaging system was used to induce numerous oxidative lesions including glyoxal DNA modifications, from which resulted a number of clones. Clone F3/9/H2/G5 showed increased reactivity toward glyoxal-modified DNA greater than that of the immunizing antigen. ELISA unequivocally showed Ab recognition toward gdC, which was confirmed by gas chromatography-mass spectrometry of the derivatized adduct after formic acid hydrolysis to the modified base. Binding of Ab F3/9 with glyoxalated and untreated oligomers containing deoxycytidine, deoxyguanosine, thymidine, and deoxyadenosine assessed by ELISA produced significant recognition (p 0.0001) of glyoxal-modified deoxycytidine greater than that of untreated oligomer. Additionally, inhibition ELISA studies using the glyoxalated and native deoxycytidine oligomer showed increased recognition for gdC with more than a 5-fold difference in IC50 values. DNA modified with increasing levels of iron (II)/EDTA produced a dose-dependent increase in Ab F3/9 binding. This was reduced in the presence of catalase or aminoguanidine. We have validated the potential of gdC as a marker of oxidative DNA damage and showed negligible cross-reactivity with 8-oxo-2'-deoxyguanosine or malondialdehyde-modified DNA as well as its utility in immunocytochemistry. Formation of the gdC adduct may involve intermediate structures; however, our results strongly suggest Ab F3/9 has major specificity for the predominant product, 5-hydroxyacetyl-dC.

The fall and rise of tear albumin levels:a multifactorial phenomenon

Albumin in tears is used as a diagnostic marker of ocular insult and inflammation, but whether its presence in tears is responsive or part of an adaptive reaction remains unresolved. A review of the literature on tear albumin concentration emphasizes that variables such as collection method, stimulus, assay technique, and disease state influence the quoted values to different extents. Influence of assay technique is negligible in comparison to variation in sampling conditions. Ocular disease increases albumin concentrations but not in a specific manner. The literature review also highlighted that little systematic research has been carried out on the daily cycle of tear albumin levels. In order to remedy this shortcoming, we investigated variations in tear albumin concentration during the waking day. The concentration of albumin in 400 tear samples collected from 13 subjects was assessed at 2-hourly intervals throughout the waking day. Highest daytime albumin concentrations were obtained within 10 minutes of waking, with a mean concentration of >50 ± 22 µg/ml. Albumin levels were at their lowest, but most consistent, 2-6 hours post-waking. This pattern was followed by a progressive increase in albumin concentration during the latter part of the day. Although individual subject-to-subject concentration differences were observed, this distinctive pattern of diurnal variation was found in all subjects. The results presented suggest a regulated, not random, pattern of variation within the period of study. © 2013 Elsevier Inc. All rights reserved.

Albumin in tears

Albumin is not endogenous to the tear film and is present as a product of plasma leakage. It is used as a diagnostic marker of ocular insult and inflammation. Tear albumin is, however, poorly understood, with large variations in reported concentrations between studies. There is also no authoritative information on whether its presence in tears is responsive or part of an adaptive reaction.The presented research aimed to resolve the disparities in published tear albumin concentrations and investigate the role of albumin in the tear film. Collation and evaluation of the available literature identified collection method, stimulus, assay technique, and disease state as factors able to influence quoted tear albumin to different extents. Difference in sampling technique exhibited the largest variations in mean tear albumin concentrations. Review of the literature also highlighted that little systematic investigations of the daily cycle of tear albumin levels, and subject-to-subject-variation, had been carried out. In order to remedy this shortcoming, variations in tear albumin concentration were investigated in 13 subjects throughout the waking day. Results identified a time period where albumin levels are relatively stable (2-6 hours post-waking). This was designated a suitable baseline for the determinations of tear albumin concentrations and subject-to-subject comparisons. Significantly, a previously unrecognised progressive increase in albumin concentration during the latter part of the day was also identified in the population. This increase suggests that albumin may play a more active and dynamic role in the ocular environment than is commonly perceived. To facilitate the collection of additional tear albumin data, tear sampling and point-of-care analysis in contact lens clinics were investigated. Two instruments were evaluated and were found to be suitable for the analysis of tear albumin in commercial institutions. Collectively, the described research has provided new insight into tear albumin and a strong foundation for further studies.

Light scattering study of human serum albumin in pre-denaturation:relation to dynamic transition in water at 42°C

Protein functional motions are ultimately connected to water dynamics. The goal of this study is to link the conformational dynamics of albumin to a dynamic transition taking place at ∼ 42°C in water. We report the results of dynamic light scattering measurements of albumin aqueous solution in the temperature interval 20-65°C. The processing of the experimental data produced the temperature dependence of the macromolecular hydrodynamic radius. We demonstrate that the growth of the macromolecular size in this temperature range can be divided into two stages that are connected to the dynamical properties of water. © 2012 Elsevier B.V. All rights reserved.