INFLUENCE OF AGE AND MICROCLIMATE ON THE PHOTOCHEMISTRY OF RHODODENDRON MAXIMUM LEAVES II. CHLOROPLAST STRUCTURE AND PHOTOSYNTHETIC LIGHT RESPONSE

The influence of age on chloroplast structure and photosynthetic light response of Rhododendron maximum L. was studied in three different microhabitats. The three microhabitats constituted a gradient of low, intermediate, and high irradiance levels. The most dramatic change in chloroplast structure with increasing age was the proliferation of the number and size of plastoglobuli. The magnitude and age specific rate of chloroplast occlusion by plastoglobuli increased in habitats with higher irradiance. Photosynthetic responses to light differed among the age categories of leaves. Light saturated photosynthesis and quantum yield decreased as leaves aged. However, in high light environments the rate of reduction of quantum yield or light saturated photosynthetic rate was more rapid than in the low light environment. The quantity of plastoglobuli increased in association with reduced light reaction capacity. The presence and abundance of plastoglobuli in R. maximum chloroplasts and their association with reduced photosynthetic performance indicates that the photosynthetic apparatus of the R. maximum chloroplast is sensitive to photodestruction by high irradiance: commonly a winter phenomenon in these environments.     

Preventing oxidation of cellular XRCC1 affects PARP-mediated DNA damage responses

Poly(ADP-ribose) polymerase-1 (PARP-1) binds intermediates of base excision repair (BER) and becomes activated for poly(ADP-ribose) (PAR) synthesis. PAR mediates recruitment and functions of the key BER factors XRCC1 and DNA polymerase β (pol β) that in turn regulate PAR. Yet, the molecular mechanism and implications of coordination between XRCC1 and pol β in regulating the level of PAR are poorly understood. A complex of PARP-1, XRCC1 and pol β is found in vivo, and it is known that pol β and XRCC1 interact through a redox-sensitive binding interface in the N-terminal domain of XRCC1. We confirmed here that both oxidized and reduced forms of XRCC1 are present in mouse fibroblasts. To further understand the importance of the C12–C20 oxidized form of XRCC1 and the interaction with pol β, we characterized cell lines representing stable transfectants in Xrcc1^?/? mouse fibroblasts of wild-type XRCC1 and two mutants of XRCC1, a novel reduced form with the C12–C20 disulfide bond blocked (C12A) and a reference mutant that is unable to bind pol β (V88R). XRCC1-deficient mouse fibroblasts are extremely hypersensitive to methyl methanesulfonate (MMS), and transfected wild-type and C12A mutant XRCC1 proteins similarly reversed MMS hypersensitivity. However, after MMS exposure the cellular PAR level was found to increase to a much greater extent in cells expressing the C12A mutant than in cells expressing wild-type XRCC1. PARP inhibition resulted in very strong MMS sensitization in cells expressing wild-type XRCC1, but this sensitization was much less in cells expressing the C12A mutant. The results suggest a role for the oxidized form of XRCC1 in the interaction with pol β in (1) controlling the PAR level after MMS exposure and (2) enabling the extreme cytotoxicity of PARP inhibition during the MMS DNA damage response.     

Interaction between DNA Polymerase β and BRCA1

The breast cancer 1 (BRCA1) protein is a tumor suppressor playing roles in DNA repair and cell cycle regulation. Studies of DNA repair functions of BRCA1 have focused on double-strand break (DSB) repair pathways and have recently included base excision repair (BER). However, the function of BRCA1 in BER is not well defined. Here, we examined a BRCA1 role in BER, first in relation to alkylating agent (MMS) treatment of cells and the BER enzyme DNA polymerase β (pol β). MMS treatment of BRCA1 negative human ovarian and chicken DT40 cells revealed hypersensitivity, and the combined gene deletion of BRCA1 and pol β in DT40 cells was consistent with these factors acting in the same repair pathway, possibly BER. Using cell extracts and purified proteins, BRCA1 and pol β were found to interact in immunoprecipitation assays, yet in vivo and in vitro assays for a BER role of BRCA1 were negative. An alternate approach with the human cells of immunofluorescence imaging and laser-induced DNA damage revealed negligible BRCA1 recruitment during the first 60 s after irradiation, the period typical of recruitment of pol β and other BER factors. Instead, 15 min after irradiation, BRCA1 recruitment was strong and there was γ-H2AX co-localization, consistent with DSBs and repair. The rapid recruitment of pol β was similar in BRCA1 positive and negative cells. However, a fraction of pol β initially recruited remained associated with damage sites much longer in BRCA1 positive than negative cells. Interestingly, pol β expression was required for BRCA1 recruitment, suggesting a partnership between these repair factors in DSB repair.     

Copper accumulation in the soluble and particulate fractions of renal cortex in the streptozotocin-diabetic rat

The accumulation and subcellular distribution of copper in the kidney of streptozotocin-diabetic rats were investigated. Male Sprague-Dawley rats received streptozotocin (50 mg/kg body wt on two consecutive days) intraperitoneally and were fed either commercial or purified diet. The concentrations of copper, zinc, iron, and manganese present in intact kidney, renal cortex, and renal medulla were compared at various times. Chow-fed diabetic rats had a renal copper concentration 2.6 times greater than age-matched controls after 2 weeks. The concentration of zinc was only 30% higher in diabetic kidney than in control tissue, whereas the iron and manganese concentrations were similar for both groups. The additional complement of renal copper was localized entirely in the cortex and was significantly reduced by oral treatment with penicillamine, a copper chelating agent. When diabetic rats were fed purified diet (15-20 ppm Cu), the quantity of copper accumulated in the renal cortex increased from 2.3 to 8.7-fold higher than in control tissue from 1 to 4 weeks, respectively, after injection with streptozotocin. Copper levels in. both the soluble and particulate (165, 000g pellet) fractions of diabetic renal cortex were similarly increased at each time. Gel filtration Chromatographic analysis of the cytosol showed that all of the copper accumulated in the soluble fraction was associated with metallothionein. The distribution of excess copper in the particulate fraction was determined by differential centrifugation. The additional quantity of metal was localized in the crude nuclear fraction of renal cortex in the diabetic rat. Further analysis revealed that the lysosomal fraction from 3-weeek diabetic rats had a copper level 16-fold higher than in the controls. The possibility that accumulation of excessive levels of copper in the streptozotocin-diabetic kidney may contribute to the development of diabetic nephropathy is discussed.