Slow repair of lipid peroxidation-induced DNA damage at p53 mutation hotspots in human cells caused by low turnover of a DNA glycosylase

Repair of oxidative stress- and inflammation-induced DNA lesions by the base excision repair (BER) pathway prevents mutation, a form of genomic instability which is often observed in cancer as ‘mutation hotspots’. This suggests that some sequences have inherent mutability, possibly due to sequence-related differences in repair. This study has explored intrinsic mutability as a consequence of sequence-specific repair of lipid peroxidation-induced DNA adduct, 1, N⁶-ethenoadenine (εA). For the first time, we observed significant delay in repair of ϵA at mutation hotspots in the tumor suppressor gene p53 compared to non-hotspots in live human hepatocytes and endothelial cells using an in-cell real time PCR-based method. In-cell and in vitro mechanism studies revealed that this delay in repair was due to inefficient turnover of N-methylpurine-DNA glycosylase (MPG), which initiates BER of εA. We determined that the product dissociation rate of MPG at the hotspot codons was ≈5–12-fold lower than the non-hotspots, suggesting a previously unknown mechanism for slower repair at mutation hotspots and implicating sequence-related variability of DNA repair efficiency to be responsible for mutation hotspot signatures.     

Delivery-Corrected Imaging of Fluorescently-Labeled Glucose Reveals Distinct Metabolic Phenotypes in Murine Breast Cancer

When monitoring response to cancer therapy, it is important to differentiate changes in glucose tracer uptake caused by altered delivery versus a true metabolic shift. Here, we propose an optical imaging method to quantify glucose uptake and correct for in vivo delivery effects. Glucose uptake was measured using a fluorescent D-glucose derivative 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-deoxy-D-glucose (2-NBDG) in mice implanted with dorsal skin flap window chambers. Additionally, vascular oxygenation (SO₂) was calculated using only endogenous hemoglobin contrast. Results showed that the delivery factor proposed for correction, “R_D”, reported on red blood cell velocity and injected 2-NBDG dose. Delivery-corrected 2-NBDG uptake (2-NBDG₆₀/R_D) inversely correlated with blood glucose in normal tissue, indicating sensitivity to glucose demand. We further applied our method in metastatic 4T1 and nonmetastatic 4T07 murine mammary adenocarcinomas. The ratio 2-NBDG₆₀/R_D was increased in 4T1 tumors relative to 4T07 tumors yet average SO₂ was comparable, suggesting a shift toward a “Warburgian” (aerobic glycolysis) metabolism in the metastatic 4T1 line. In heterogeneous regions of both 4T1 and 4T07, 2-NBDG₆₀/R_D increased slightly but significantly as vascular oxygenation decreased, indicative of the Pasteur effect in both tumors. These data demonstrate the utility of delivery-corrected 2-NBDG and vascular oxygenation imaging for differentiating metabolic phenotypes in vivo.     

Determination on the binding of thiadiazole derivative to human serum albumin: a spectroscopy and computational approach

4-[3-acetyl-5-(acetylamino)-2,3-dihydro-1,3,4-thiadiazole-2-yl]phenyl benzoate from the family of thiadiazole derivative has been newly synthesized. It has good anticancer activity as well as antibacterial and less toxic in nature, its binding characteristics are therefore of huge interest for understanding pharmacokinetic mechanism of the drug. The binding of thiadiazole derivative to human serum albumin (HSA) has been investigated by studying its quenching mechanism, binding kinetics and the molecular distance, r between the donor (HSA) and acceptor (thiadiazole derivative) was estimated according to Forster’s theory of non-radiative energy transfer. The Gibbs free energy (ΔG), enthalpy (ΔH) and entropy (ΔS) changes of temperature-dependent K_b was calculated, which explains that the reaction is spontaneous and exothermic. The microenvironment of HSA have also been studied using synchronous fluorescence spectroscopy, and the feature of thiadiazole derivative-induced structural changes of HSA have been carried using Fourier transform infrared spectroscopy and the Molecular modelling simulations explore the hydrophobic and hydrogen bonding interactions.     

Expression of lysyl oxidase from cDNA constructs in mammalian cells: The propeptide region is not essential to the folding and secretion of the functional enzyme

Rat aortic lysyl oxidase cDNA was expressed under a metallothionein promoter in Chinese hamster ovary cells using a dihydrofolate reductase selection marker. One methotrexate-resistant cell line, LOD-06, generated by transfecting with full-length cDNA, yielded lysyl oxidase proteins consistent with the 50 kDa proenzyme and a 29 kDa mature catalyst. A second cell line, LOD32–2, was generated by transfection with a truncated cDNA lacking sequences which code for the bulk of the propeptide region. Both cell lines secreted apparently identical, 29 kDa forms of mature lysyl oxidase each of which catalyzed the deamination of human recombinant tropoelastin and alkylamines, consistent with the known specificity of lysyl oxidase. The secreted enzyme forms were inhibited by chemical inhibitors of lysyl oxidase activity, including β-aminopropionitrile, phenylhydrazine, ethylenediamine, α,α′-dipyridyl, and diethyl-dithiocarbamate. Sensitivity to these agents is consistent with the presence of copper and carbonyl cofactors in the expressed enzymes, characteristic of lysyl oxidase purified from connective tissues. These results indicate the lack of essentiality of the deleted proprotein sequence for the proper folding, generation of catalytic function, and secretion of lysyl oxidase. © 1995 Wiley-Liss, Inc.     

Tissue-specific activation of the osmotin gene by ABA,C2H4 and NaCl involves the same promoter region

The gene encoding the antifungal protein osmotin is induced by several hormonal and environmental signals. In this study, tissue-specific and inducer-mediated expression of the reporter gene -glucuronidase (uidA) fused to different fragment lengths of the osmotin promoter was evaluated in transgenic tobacco (Nicotiana tabacum). The region of the promoter between –248 to –108 (Fragment A) was found to be essential and sufficient for inducer (abscisic acid (ABA), C₂H₄ and NaCl)-mediated expression of the reporter gene. Expression of the reporter gene was developmentally regulated and increased with maturity of leaves, stem and flowers. Expression also was tissue-specific being most highly expressed in epidermis and vascular parenchyma of the stem. The regulators ABA, C₂H₄ and NaCl exhibited tissue-specific induction of this promoter. The promoter was specifically responsive to C₂H₄ in flowers at virtually all stages of development, but not responsive in these tissues to ABA or NaCl. Conversely, ABA and NaCl were able to induce reporter gene activity using promoter Fragment A in specific tissues of root where C₂H₄ was unable to induce activity. Further dissection of the promoter Fragment A into fragments containing either the conserved GCC element (PR); PR/AT; or G/AT sequences, and subsequent testing of these fragments fused to GUS in transgenic plants was performed. These experiments revealed that the promoter fragment containing PR element alone, although required, was barely able to allow responsiveness to C₂H₄. However, significant C₂H₄-induced activity was obtained with a promoter fragment containing the AT and PR elements together.     

Comparison of the N-linked glycopeptides of DQw1 and DR1 molecules

HLA class II molecules have been isolated from a [3H]mannose-labeled GM3104 B lymphoblastoid cell line with the phenotype DQw1, DR1. The DQw1 molecules were purified by affinity to 77-34 IgG specifically reactive with the DQw1 specificity. The DR1 molecules were separated into two subsets, DR1a (70 to 80%) and DR1b (20 to 30%), by sequential affinity to 21r5-IgG and 21w4-IgG Sepharose. The alpha- and beta-chains of [3H]mannose-labeled DQw1, DR1a, and DR1b molecules were separated by SDS-PAGE and were recovered by electrophoretic elution. The isolated chains were digested with pronase and the glycopeptides were fractionated by sequential lectin chromatography on immobilized concanavalin A (Con A), Lens culinaris (Lens), and Ricinus communis agglutinin type I (RCA). The N-linked glycopeptides derived from the alpha-chains of DQw1, DR1a, or DR1b showed similar profiles on Con A Sepharose: 45% unbound (ConA I), 25% weakly bound (ConA II), and 30% tightly bound (ConA III). The glycopeptides derived from the beta-chains of DQw1 or DR1 molecules were found almost exclusively (80%) in the fraction unbound to Con A Sepharose, with only 11% and 9% in ConA II and ConA III fractions, respectively. The observation that most of the binding to Con A is associated with the alpha-chain glycopeptides suggests that binding of membrane-associated class II molecules to that lectin must be mediated by the alpha-chains. Binding to Lens Sepharose was higher for beta-(50%) than for alpha-(15%) chain glycopeptides, suggesting that within the intact glycoproteins, the beta-chains are responsible for the interaction with Lens. The ConA I fractions derived from the alpha-chain glycopeptides of either DQw1 or DR1 molecules were separated on RCA-agarose as follows: 60% unbound, 17% retarded, and 20% bound and eluted with 0.1 M galactose. The ConA I fractions derived from the beta-chain glycopeptides of either subset of class II molecules also had a similar profile on RCA-agarose: 70% unbound, 16% retarded, and 10% bound and eluted specifically. After removal of sialic acid residues, all of the ConA I fractions of alpha- and beta-chains bound to RCA-agarose. A high degree of similarity was observed between the corresponding glycopeptides of the three subsets of class II molecules and between the complex N-linked structures of alpha- and beta-chains. Minor variations were observed between DR1a and DR1b glycopeptides which appear greater than those observed between DR1 and DQw1 glycopeptides.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effect of a "bisecting" N-acetylglucosaminyl group on the binding of biantennary, complex oligosaccharides to concanavalin A, Phaseolus vulgaris erythroagglutinin (E-PHA), and Ricinus communis agglutinin (RCA-120) immobilized on agarose

The effect of a "bisecting" 2-acetamido-2-deoxy-beta-D-glucopyranosyl group, linked (1----4) to the beta-D-mannopyranosyl group of asparagine-linked complex and hybrid oligosaccharides, on the binding of [14C]acetylated glycopeptides to columns of immobilized concanavalin A (Con A), Phaseolus vulgaris erythroagglutinin (E-PHA), and Ricinus communis agglutinin-120 (RCA-120) was investigated. The presence of this "bisecting" GlcNAc group caused significant inhibition of the binding to ConA-agarose of biantennary complex glycopeptides in which the two branches are terminated at their nonreducing ends by two GlcNAc groups, or by a Gal and a GlcNAc group, or by two Gal groups, or by a Man and a GlcNAc group. Binding of biantennary, complex glycopeptides to E-PHA-agarose required a "bisecting" GlcNAc group, a Gal group at the nonreducing terminus of the alpha-D-Man-p-(1----6) branch, and a terminal or internal GlcNAc residue linked beta-(1----2) to the alpha-D-Manp-(1----3) branch. Binding to RCA-120-agarose occurred only when at least one nonreducing terminal Gal group was present, and increased as the proportion of terminal Gal groups increased; the presence of a "bisecting" GlcNAc group caused either enhancement or inhibition of these binding patterns. It is concluded that a "bisecting" GlcNAc group affects the binding of glycopeptides to all three lectin columns.     

Cloning, sequencing, and characterization of Escherichia coli thioesterase II.

The gene (tesB) encoding Escherichia coli thioesterase II, a low-abundance enzyme of unknown physiological function which can hydrolyze a broad range of acyl-CoA thioesters, has been localized by transposon mutagenesis, cloned and sequenced. A two-cistron construct containing both the lac and tesB promoters was used successfully to overexpress the 286-residue polypeptide. The recombinant enzyme constituted up to 25% of the soluble proteins of E. coli and was readily purified to homogeneity as a tetramer of approximately 120,000 Da. Amino-terminal sequence analysis and electrospray ionization mass spectrometry confirmed the identity of the thioesterase and revealed that the amino-terminal formyl-methionine had been removed yielding a subunit species of average molecular mass 31,842 Da. The protein does not contain the GXSXG motif found characteristically in animal thioesterases which function as chain-terminating enzymes in fatty acid synthesis and exhibits no sequence similarity with these or any other known proteins. Activity of the recombinant enzyme was inhibited by iodoacetamide and diethylpyrocarbonate. The carboxamidomethylated residue was identified as histidine 58, and a role for this amino acid in catalysis is suggested. E. coli strains having a large deletion within the genomic tesB gene grew normally but retained a low level of thioesterase activity toward decanoyl-CoA. This residual activity indicates the presence of an additional decanoyl-CoA hydrolase in E. coli. Over-expression of the recombinant enzyme, under control of the lac promoter, did not alter the fatty acids synthesized by E. coli at any stage of cell growth and the physiological role of this enzyme remains an enigma.     

Expression of N-acetylglucosaminyltransferase III in hepatic nodules during rat liver carcinogenesis promoted by orotic acid

The activity of N-acetylglucosaminyltransferase III, which adds a "bisecting" GlcNAc in beta 1,4 linkage to the beta-linked Man of the core of Asn-linked oligosaccharides (Narasimhan, S. (1982) J. Biol. Chem. 257, 10235-10242), was determined in hepatic nodules of rats initiated by administration of a single dose of carcinogen 1,2-dimethylhydrazine.2HCl (100 mg/kg, intraperitoneal) 18 h after partial hepatectomy and promoted by feeding a diet supplemented with 1% orotic acid for 32-40 weeks. N-Acetylglucosaminyltransferase III was assayed using glycopeptide GlcNAc beta 1,2Man alpha 1,6(GlcNAc beta 1,2Man alpha 1,3)Man beta 1, 4GlcNAc beta 1,4GlcNAc-Asn as substrate and, as enzyme sources, microsomal membranes of the hepatic nodules, surrounding liver, regenerating liver, and age- and sex-matched control liver. The nodules had significant N-acetylglucosaminyltransferase III activity (0.78-2.18 nmol GlcNAc transferred/h/mg of protein), while the surrounding liver, the regenerating liver (24 h after partial hepatectomy), and the control liver had negligible activity (0.02-0.03 nmol/h/mg of protein). Product isolated from a large scale N-acetylglucosaminyltransferase III incubation with hepatic nodules as enzyme source showed the presence of the bisecting GlcNAc residue by 500 MHz proton NMR spectroscopy. Concomitant with the appearance of N-acetylglucosaminyltransferase III activity in the preneoplastic nodules, the activities of N-acetylglucosaminyltransferase I and II were decreased in these membranes when compared to those from surrounding liver, regenerating liver, and control liver. These results suggest that N-acetylglucosaminyltransferase III is induced at the preneoplastic stage in liver carcinogenesis promoted by orotic acid and are consistent with the reported presence of bisecting GlcNAc residues in the Asn-linked oligosaccharides of rat and human hepatoma gamma-glutamyl transpeptidase and their absence in enzyme from normal liver of rats and humans (Kobata, A., and Yamashita, K. (1984) Pure Appl. Chem. 56, 821-832).