Abstract:Of the major components of the kynurenine pathway for the oxidative metabolism of tryptophan, most attention has focussed on the N-methyl-D-aspartate (NMDA) receptor agonist quinolinic acid, and the glutamate receptor blocker kynurenic acid. However, there is increasing evidence that the redox-active compound 3-hydroxyanthranilic acid may also have potent actions on cell function in the nervous and immune systems, and recent clinical data show marked changes in the levels of this compound, associated with changes in anthranilic acid levels, in patients with a range of neurological and other disorders including osteoporosis, chronic brain injury, Huntington's disease, coronary heart disease, thoracic disease, stroke and depression. In most cases, there is a decrease in 3-hydroxyanthranilic acid levels and an increase in anthranilic acid levels. In this paper, we summarise the range of data obtained to date, and hypothesise that the levels of 3-hydroxyanthranilic acid or the ratio of 3-hydroxyanthranilic acid to anthranilic acid levels, may contribute to disorders with an inflammatory component, and may represent a novel marker for the assessment of inflammation and its progression. Data are presented which suggest that the ratio between these two compounds is not a simple determinant of neuronal viability. Finally, a hypothesis is presented to account for the development of the observed changes in 3-hydroxyanthranilic acid and anthranilate levels in inflammation and it is suggested that the change of the 3HAA:AA ratio, particularly in the brain, could possibly be a protective response to limit primary and secondary damage.

The γH2AX focus assay represents a fast and sensitive approach for detection of one of the critical types of DNA damage – double-strand breaks (DSB) induced by various cytotoxic agents including ionising radiation. Apart from research applications, the assay has a potential in clinical medicine/pathology, such as assessment of individual radiosensitivity, response to cancer therapies, as well as in biodosimetry. Given that generally there is a direct relationship between numbers of microscopically visualised γH2AX foci and DNA DSB in a cell, the number of foci per nucleus represents the most efficient and informative parameter of the assay. Although computational approaches have been developed for automatic focus counting, the tedious and time consuming manual focus counting still remains the most reliable approach due to limitations of computational approaches. We suggest a computational approach and associated software for automatic focus counting that minimises these limitations. Our approach, while using standard image processing algorithms, maximises the automation of identification of nuclei/cells in complex images, offers an efficient way to optimise parameters used in the image analysis and counting procedures, optionally invokes additional procedures to deal with variations in intensity of the signal and background in individual images, and provides automatic batch processing of a series of images. We report results of validation studies that demonstrated correlation of manual focus counting with results obtained using our computational algorithm for mouse jejunum touch prints, mouse tongue sections and human blood lymphocytes as well as radiation dose response of γH2AX focus induction for these biological specimens.

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SummaryProteins in basement membrane (BM) are long‐lived and accumulate chemical modifications during aging; advanced glycation endproduct (AGE) formation is one such modification. The human lens capsule is a BM secreted by lens epithelial cells. In this study, we have investigated the effect of aging and cataracts on the AGE levels in the human lens capsule and determined their role in the epithelial‐to‐mesenchymal transition (EMT) of lens epithelial cells. EMT occurs during posterior capsule opacification (PCO), also known as secondary cataract formation. We found age‐dependent increases in several AGEs and significantly higher levels in cataractous lens capsules than in normal lens capsules measured by LC‐MS/MS. The TGFβ2‐mediated upregulation of the mRNA levels (by qPCR) of EMT‐associated proteins was significantly enhanced in cells cultured on AGE‐modified BM and human lens capsule compared with those on unmodified proteins. Such responses were also observed for TGFβ1. In the human capsular bag model of PCO, the AGE content of the capsule proteins was correlated with the synthesis of TGFβ2‐mediated α‐smooth muscle actin (αSMA). Taken together, our data imply that AGEs in the lens capsule promote the TGFβ2‐mediated fibrosis of lens epithelial cells during PCO and suggest that AGEs in BMs could have a broader role in aging and diabetes‐associated fibrosis.

NAD+ -dependent DNA ligases are essential enzymes in bacteria, with the most widely studied of this class of enzymes being LigA from Escherichia coli. NAD + -dependent DNA ligases comprise several discrete structural domains, including a BRCT domain at the C-terminus that is highly-conserved in this group of proteins. The over-expression and purification of various fragments of E. coli LigA allowed the investigation of the different domains in DNA-binding and ligation by this enzyme. Compared to the full-length protein, the deletion of the BRCT domain from LigA reduced in vitro ligation activity by 3-fold and also reduced DNA binding. Using an E. coli strain harbouring a temperature-sensitive mutation of ligA, the over-expression of protein with its BRCT domain deleted enabled growth at the non-permissive temperature. In gel-mobility shift experiments, the isolated BRCT domain bound DNA in a stable manner and to a wider range of DNA molecules compared to full LigA. Thus, the BRCT domain of E. coli LigA can bind DNA, but it is not essential for DNA nick-joining activity in vitro or in vivo. D

The dietary component SFN demonstrates an ability to protect human lens cells against oxidative stress and thus could potentially delay the onset of cataract.

Sequencing of the genomes of Mycobacterium tuberculosis H37Rv and Streptomyces coelicolor A3(2) identified putative genes for an NAD ؉ -dependent DNA ligase. We have cloned both open reading frames and overexpressed the protein products in Escherichia coli. In vitro biochemical assays confirm that each of these proteins encodes a functional DNA ligase that uses NAD ؉ as its cofactor. Expression of either protein is able to complement E. coli GR501, which carries a temperaturesensitive mutation in ligA. Thus, in vitro and in vivo analyses confirm predictions that ligA genes from M. tuberculosis and S. coelicolor are NAD ؉ -dependent DNA ligases. Proteins 2003;51:321-326.

SummaryThe nuclear lamina is essential for the proper structure and organization of the nucleus. Deregulation of A‐type lamins can compromise genomic stability, alter chromatin organization and cause premature vascular aging. Here, we show that accumulation of the lamin A precursor, prelamin A, inhibits 53BP1 recruitment to sites of DNA damage and increases basal levels of DNA damage in aged vascular smooth muscle cells. We identify that this genome instability arises through defective nuclear import of 53BP1 as a consequence of abnormal topological arrangement of nucleoporin NUP153. We show for the first time that this nucleoporin is important for the nuclear localization of Ran and that the deregulated Ran gradient is likely to be compromising the nuclear import of 53BP1. Importantly, many of the defects associated with prelamin A expression were significantly reduced upon treatment with Remodelin, a small molecule recently reported to reverse deficiencies associated with abnormal nuclear lamina.