Evidence for Base Excision Repair of Oxidative DNA Damage in Chloroplasts of Arabidopsis thaliana

Chloroplasts are the sites of photosynthesis in plants, and they contain their own multicopy, requisite genome. Chloroplasts are also major sites for production of reactive oxygen species, which can damage essential components of the chloroplast, including the chloroplast genome. Compared with mitochondria in animals, relatively little is known about the potential to repair oxidative DNA damage in chloroplasts. Here we provide evidence of DNA glycosylase-lyase/endonuclease activity involved in base excision repair of oxidized pyrimidines in chloroplast protein extracts of Arabidopsis thaliana. Three base excision repair components (two endonuclease III homologs and an apurinic/apyrimidinic endonuclease) that might account for this activity were identified by bioinformatics. Transient expression of protein-green fluorescent protein fusions showed that all three are targeted to the chloroplast and co-localized with chloroplast DNA in nucleoids. The glycosylase-lyase/endonuclease activity of one of the endonuclease III homologs, AtNTH2, which had not previously been characterized, was confirmed in vitro. T-DNA insertions in each of these genes were identified, and the physiological and biochemical phenotypes of the single, double, and triple mutants were analyzed. This mutant analysis revealed the presence of a third glycosylase activity and potentially another pathway for repair of oxidative DNA damage in chloroplasts.Reactive oxygen species (ROS)² are inevitable by-products of metabolism in all aerobic organisms (1). Plants and algae are especially prone to photo-oxidative stress because of ROS generated during oxygenic photosynthesis. Several types of ROS are generated at various sites in the photosynthetic electron transport chain in chloroplasts, and their production is enhanced by such factors as excess or varying light intensities and extremes of temperature, drought, nutrient deficiencies, and herbicides (2). These ROS can damage many chloroplast constituents, including lipids, proteins, pigments, and the multicopy genome.Plants have evolved numerous mechanisms to deal with photo-oxidative stress, including dissipation of excess light energy, synthesis of antioxidant molecules and scavenging enzymes, and targeted repair (2). DNA repair of oxidized bases, such as thymine glycol (TG) or 8-oxoguanine, can be hypothesized as an important element of chloroplast photoprotection. Although there is considerable overlap in both the types of DNA lesions caused by different insults and the targeting of different DNA repair mechanisms, base excision repair (BER) is considered to be the main repair pathway for oxidative DNA damage, at least in the nucleus and mitochondrion (3, 4).BER repairs single damaged bases (because of oxidation, deamination, alkylation, etc.) in DNA by removing them, breaking the phosphodiester backbone, excising the sugar residue at the abasic site, and filling the gap (reviewed in Refs. 5, 6). BER begins with a DNA glycosylase or glycosylase-lyase. There are many types of glycosylases in any given organism and across taxa, and they are distinguishable by their substrate specificity, whether they are monofunctional (glycosylase activity only) or bifunctional (glycosylase plus apurinic/apyrimidinic (AP) lyase activities; see below), by the phylogenetic family in which they reside, and/or by conserved structural characteristics (reviewed in Refs. 6–8). The glycosylases involved in BER of oxidative DNA damage can be roughly divided into those that target either oxidized purines or oxidized pyrimidines (4, 9). For example, TG is a common type of oxidized pyrimidine, which is removed primarily by endonuclease III (Nth), endonuclease VIII (Nei), or their homologs (10). TG is only poorly mutagenic, but it strongly blocks polymerases, inducing cell cycle arrest and potentially cell death if it is not removed.After an appropriate glycosylase cleaves the N-glycosyl bond attaching a damaged base to deoxyribose, leaving an abasic site, the sugar-phosphate backbone is nicked. Bifunctional glycosylases also have an AP lyase activity that cleaves on the 3′ side of the AP site. However, the site still requires the function of a separate AP endonuclease that cuts on the 5′ side of the AP site to remove the 3′-deoxyribose residue at the nick site (11) before repair can continue. In the case of a monofunctional glycosylase, an AP endonuclease nicks the strand on the 5′ side of the AP site. Escherichia coli has two unrelated AP endonucleases, exonuclease III (Xth) and endonuclease IV (Nfo). In humans Ape1/Ref-1 is an Xth homolog, and in yeast Apn1p is an Nfo homolog (5, 12). Following generation of the AP site and nicking of the backbone, the gap is filled by a polymerase in either a short or long patch and then sealed by a ligase.BER of oxidative DNA lesions such TG has been studied intensively in E. coli, yeast, and mammals, whereas comparatively little is known about BER in plants. For example, only two genes involved in BER of oxidized pyrimidines have been characterized previously in the model plant Arabidopsis thaliana (13, 14), and their localization within the plant cell is unknown. An Nth homolog in Arabidopsis, AtNTH1 (At2g31450), has the expected bifunctional glycosylase-lyase activity in vitro (14). The ARP gene (At2g41460) in Arabidopsis encodes an enzyme with AP endonuclease activity (13).Here we present the results of experiments conducted to address whether there is BER of oxidized pyrimidines in the Arabidopsis chloroplast. Chloroplast protein extracts were assayed for glycosylase-lyase/endonuclease activity. The chloroplast localization of ARP, AtNTH1, and AtNTH2, a second Arabidopsis homolog of Nth, was tested experimentally, and the predicted activity of AtNTH2 was confirmed in vitro. In addition, an analysis of T-DNA insertion mutants affecting each of these three BER genes was performed.     

Influence of Heparin Mimetics on Assembly of the FGF·FGFR4 Signaling Complex

Fibroblast growth factor (FGF) signaling regulates mammalian development and metabolism, and its dysregulation is implicated in many inherited and acquired diseases, including cancer. Heparan sulfate glycosaminoglycans (HSGAGs) are essential for FGF signaling as they promote FGF·FGF receptor (FGFR) binding and dimerization. Using novel organic synthesis protocols to prepare homogeneously sulfated heparin mimetics (HM), including hexasaccharide (HM₆), octasaccharide (HM₈), and decasaccharide (HM₁₀), we tested the ability of these HM to support FGF1 and FGF2 signaling through FGFR4. Biological assays show that both HM₈ and HM₁₀ are significantly more potent than HM₆ in promoting FGF2-mediated FGFR4 signaling. In contrast, all three HM have comparable activity in promoting FGF1·FGFR4 signaling. To understand the molecular basis for these differential activities in FGF1/2·FGFR4 signaling, we used NMR spectroscopy, isothermal titration calorimetry, and size-exclusion chromatography to characterize binding interactions of FGF1/2 with the isolated Ig-domain 2 (D2) of FGFR4 in the presence of HM, and binary interactions of FGFs and D2 with HM. Our data confirm the existence of both a secondary FGF1·FGFR4 interaction site and a direct FGFR4·FGFR4 interaction site thus supporting the formation of the symmetric mode of FGF·FGFR dimerization in solution. Moreover, our results show that the observed higher activity of HM₈ relative to HM₆ in stimulating FGF2·FGFR4 signaling correlates with the higher affinity of HM₈ to bind and dimerize FGF2. Notably FGF2·HM₈ exhibits pronounced positive binding cooperativity. Based on our findings we propose a refined symmetric FGF·FGFR dimerization model, which incorporates the differential ability of HM to dimerize FGFs.     

Proteolytic processing of the vitellogenin precursor in the boll weevil,Anthonomus grandis

The soluble proteins of the eggs of the coleopteran insect Anthonomus grandis Boheman, the cotton boll weevil, consist almost entirely of two vitellin types with M_rs of 160,000 and 47,000. We sequenced their N-terminal ends and one internal cyanogen bromide fragment of the large vitellin and compared these sequences with the deduced amino acid sequence from the vitellogenin gene. The results suggest that both the boll weevil vitellin proteins are products of the proteolytic cleavage of a single precursor protein. The smaller 47,000 M vitellin protein is derived from the N-terminal portion of the precursor adjacent to an 18 amino acid signal peptide. The cleavage site between the large and small vitellins at amino acid 362 is adjacent to a pentapeptide sequence containing two pairs of arginine residues. Comparison of the boll weevil sequences with limited known sequences from the single 180,000 M_r honey bee protein show that the honey bee vitellin N-terminal exhibits sequence homology to the N-terminal of the 47,000 M_r boll weevil vitellin. Treatment of the vitellins with an N-glycosidase results in a decrease in molecular weight of both proteins, from 47,000 to 39,000 and from 160,000 to 145,000, indicating that about 10–15% of the molecular weight of each vitellin consists of N-linked carbohydrate. The molecular weight of the deglycosylated large vitellin is smaller than that predicted from the gene sequence, indicating possible further proteolytic processing at the C-terminal of that protein. © 1993 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Synthesis and in vitro anticancer and antitubercular activity of diarylpyrazole ligated dihydropyrimidines possessing lipophilic carbamoyl group

A series of dihydropyrimidine derivatives were synthesized by utilizing Biginelli reaction and evaluated for their in vitro anticancer activity against MCF-7 human breast cancer (HBC) cell line using sulforhodamine B (SRB) assay and antitubercular activity against Mycobacterium tuberculosis (MTB) H(37)Rv using Microplate Alamar Blue Assay (MABA). Compounds 13p, 13t were exhibited 70.6% and 63.7% of HBC cell growth inhibition at 10 μM concentration. Interestingly compound 13p was also found to be the most potent in the series against MTB H(37)Rv with MIC value of 0.125 μg/mL.     

Complete genome sequence of the orange-red pigmented, radioresistant Deinococcus proteolyticus type strain (MRP(T))

Deinococcus proteolyticus (ex Kobatake et al. 1973) Brook and Murray 1981 is one of currently 47 species in the genus Deinococcus within the family Deinococcaceae. Strain MRP(T) was isolated from feces of Lama glama and possesses extreme radiation resistance, a trait is shares with various other species of the genus Deinococcus, with D. proteolyticus being resistant up to 1.5 Mrad of gamma radiation. Strain MRP(T) is of further interest for its carotenoid pigment. The genome presented here is only the fifth completed genome sequence of a member of the genus Deinococcus (and the forth type strain) to be published, and will hopefully contribute to a better understanding of how members of this genus adapted to high gamma- or UV ionizing-radiation. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 2,886,836 bp long genome with its four large plasmids of lengths 97 kbp, 132 kbp, 196 kbp and 315 kbp harbors 2,741 protein-coding and 58 RNA genes and is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

A ginger derivative,zingerone—a phenolic compound—induces ROS‐mediated apoptosis in colon cancer cells (HCT‐116)

Zingerone (ZO), an active phenolic agent derived from Zingiber officinale (Ginger), has many pharmacological properties such as antioxidant, antiangiogenic, and antitumor. However, its potential value in cancer and the mechanism by which ZO wields its therapeutic effects remain obscure. Therefore, in this current study, we explored the effects of ZO on suppressing cell proliferation and enhancing apoptosis in colon cancer cells (HCT116). Our results indicated that ZO significantly enhances the production of reactive oxygen species, lipid peroxidation (thiobarbituric acid reactive substance [TBARS]), and loss of cell viability; and reduces mitochondrial membrane potential and antioxidant levels (SOD, CAT, and GSH) in ZO‐treated HCT116 cells in a dose‐dependent (2.5, 5, and 10 µM) manner. Furthermore, ZO induces oxidative stress‐mediated apoptosis as evidenced by apoptotic morphological changes predicted by AO/EtBr, Hoechst staining and further confirmed by comet assay. Moreover, immunoblotting techniques showed that ZO treatment effectively enhances Bax, caspase‐9, and caspase‐3 expressions and decreases the expression of Bcl‐2 in colon cancer cells. Together, our results evidenced that the antitumor effects of ZO reduce cell proliferation and stimulate apoptosis through modulating pro‐ and antiapoptotic molecular events in HCT116 colon cancer cells. Therefore, based on our findings, ZO may be used as a therapeutic agent for the treatment of colon cancer.     

Mycobacterium senegalense from bovines in Eastern Nigeria

M ohan , K. 1985. Mycobacterium senegalense from bovines in Eastern Nigeria. Journal of Applied Bacteriology 59 , 277–281.
Detailed characteristics of three mycobacterial strains sharing important properties of Mycobacterium senegalense are described. Their physiological properties were compared with those of a typical M. senegalense strain described by Chamoiseau (1979), six strains of M. senegalense and one typical strain of M. fortuitum from the culture collection of Institute of Microbiology, Rutgers. The three Nigerian strains exhibited minor variations in their physiological properties when compared with other strains of M. senegalense . Unlike the strain of Chamoiseau the Nigerian strains did not utilize benzoate or citrate. The strains were also different from the other six strains of M. senegalense by utilizing trehalose and in failing to produce acid in mannitol. Unlike earlier isolates of M. senegalense the Nigerian strains were not from cases of bovine farcy but from cases with pathological manifestations of pulmonary tuberculosis. They appeared to be intermediate strains between M. senegalense and M. fortuitum . These results raise doubts on the justification for giving specific rank to M. senegalense .     

Cloning,expression, and molecular dynamics simulations of a xylosidase obtained from Thermomyces lanuginosus

The aim of this study was to clone, express, and characterize a β-xylosidase (Tlxyn1) from the thermophilic fungus Thermomyces lanuginosus SSBP in Pichia pastoris GS115 as well as analyze optimal activity and stability using computational and experimental methods. The enzyme was constitutively expressed using the GAP promoter and secreted into the medium due to the alpha-mating factor secretion signal present on the expression vector pBGPI. The 1276 bp gene consists of an open reading frame that does not contain introns. A 12% SDS–PAGE gel revealed a major protein band at an estimated molecular mass of 50 kDa which corresponded to zymogram analysis. The three-dimensional structure of β-xylosidase was predicted, and molecular dynamics simulations at different ranges of temperature and pH were performed in order to predict optimal activity and folding energy. The results suggested a strong conformational temperature and pH dependence. The recombinant enzyme exhibited optimal activity at pH 7 and 50°C and retained 80% activity at 50°C, pH 7 for about 45 min. This is the first report of the cloning, functional expression, and simulations study of a β-xylosidase from Thermomyces species in a fungal host.     

Studies on the production and application of cellulase from Trichoderma reesei QM-9414

High yielding mutant strain, Trichoderma reesei QM-9414, was employed for the cellulase enzyme production. Enzyme production conditions (pH, inoculum age and concentration, and organic supplements) were optimized. The ability of partially purified enzyme to hydrolyze various regionally abundant lignocellulosic raw materials was studied. Enzymatic hydrolysis conditions (temperature, pH, enzyme and substrate concentrations) were optimized. Temperature 50v°C, pH 4.5, enzyme concentration 40 FPU/g substrate and substrate concentration 2.5% were found to be optimum for the maximum yields of sugars. #-glucosidase supplementation was found to increase both the sugar yield and hydrolysis rate, and shorten the reaction time significantly.