No abstract

Gold nanorods of different aspect ratios are prepared using the growth-directing surfactant, cetyltrimethylammonium bromide (CTAB), which forms a bilayer on the gold nanorod surface. Toxicological assays of CTAB-capped nanorod solutions with human colon carcinoma cells (HT-29) reveal that the apparent cytotoxicity is caused by free CTAB in solution. Overcoating the nanorods with polymers substantially reduces cytotoxicity. The number of nanorods taken up per cell, for the different surface coatings, is quantitated by inductively coupled plasma mass spectrometry on washed cells; the number of nanorods per cell varies from 50 to 2300, depending on the surface chemistry. Serum proteins from the biological media, most likely bovine serum albumin, adsorb to gold nanorods, leading to all nanorod samples bearing the same effective charge, regardless of the initial nanorod surface charge. The results suggest that physiochemical surface properties of nanomaterials change substantially after coming into contact with biological media. Such changes should be taken into consideration when examining the biological properties or environmental impact of nanoparticles.

The human 3-methyladenine DNA glycosylase [alkyladenine DNA glycosylase (AAG)] catalyzes the first step of base excision repair by cleaving damaged bases from DNA. Unlike other DNA glycosylases that are specific for a particular type of damaged base, AAG excises a chemically diverse selection of substrate bases damaged by alkylation or deamination. The 2.1-Å crystal structure of AAG complexed to DNA containing 1,N 6 -ethenoadenine suggests how modified bases can be distinguished from normal DNA bases in the enzyme active site. Mutational analyses of residues contacting the alkylated base in the crystal structures suggest that the shape of the damaged base, its hydrogen-bonding characteristics, and its aromaticity all contribute to the selective recognition of damage by AAG. DNA bases are chemically reactive and readily undergo deamination and alkylation on the inevitable exposure to reactive cellular metabolites and environmental toxicants (1-4). Alkylation occurs at many different positions of DNA, producing a variety of lesioned bases (4, 5) that can block replication or interfere with other enzymatic activities templated by DNA. Hypoxanthine is an abundant deaminated base, and it too corrupts the DNA template. Remarkably, human cells appear to produce a single enzyme, alkyladenine DNA glycosylase [AAG (3-methyladenine DNA glycosylase, ANPG, or MPG)], which recognizes and removes hypoxanthine plus a variety of alkylated bases that include 3-methyladenine, 7-methylguanine, and 1,N 6 -ethenoadenine (A; refs. 6 -13). AAG cleaves the N-glycosylic bond joining the damaged base to the DNA backbone, and the resulting abasic nucleotide is excised and replaced with a normal nucleotide by the sequential action of an endonuclease, a polymerase, and DNA ligase (14). The high selectivity for damaged vs. normal bases is essential because normal bases are present in vast excess. AAG can distinguish alternations in both adenine and guanine and can recognize changes present in both the major and minor grooves of DNA. We set out to determine how AAG achieves selectivity for chemically diverse substrates.We previously reported a 2.7-Å crystal structure of AAG complexed to DNA containing a transition-state mimic of the glycosylase reaction, the pyrrolidine abasic nucleotide (pyr; PDB ID code 1bnk; refs. 15 and 16). In the AAG͞pyr-DNA complex, the pyr ring is f lipped into the proposed active site by intercalation of the Tyr-162 side chain into the minor groove of the DNA (15). A bound water molecule in the active site is aligned for a back-side attack of the abasic sugar, but the pyr inhibitor lacks a base, and we could not deduce how AAG recognizes alkylated bases in preference to normal bases. Structures of several other DNA N-glycosylases complexed to their DNA substrates have been reported (17)(18)(19)(20). These enzymes are selective for one type of damaged DNA base and, correspondingly, their active site structures are tailor made for specific interactions with these substrates. For example, uracil DNA glycosylase f lips ur...

The chemical methylating agents methylmethane sulfonate (MMS) and N-methyl-N′-nitro-Nnitrosoguanidine (MNNG) have been used for decades as classical DNA damaging agents. These agents have been utilized to uncover and explore pathways of DNA repair, DNA damage response, and mutagenesis. MMS and MNNG modify DNA by adding methyl groups to a number of nucleophilic sites on the DNA bases, although MNNG produces a greater percentage of O-methyl adducts. There has been substantial progress elucidating direct reversal proteins that remove methyl groups and base excision repair (BER), which removes and replaces methylated bases. Direct reversal proteins and BER thus counteract the toxic, mutagenic and clastogenic effects of methylating agents. Despite recent progress, the complexity of DNA damage responses to methylating agents is still being discovered. In particular, there is growing understanding of pathways such as homologous recombination, lesion bypass, and mismatch repair that react when the response of direct reversal proteins and BER is insufficient. Furthermore, the importance of proper balance within the steps in BER has been uncovered with the knowledge that DNA structural intermediates during BER are deleterious. A number of issues complicate elucidating the downstream responses when direct reversal is insufficient or BER is imbalanced. These include inter-species differences, cell-type specific differences within mammals and between cancer cell lines, and the type of methyl damage or BER intermediate encountered. MMS also carries a misleading reputation of being a 'radiomimetic,' i.e., capable of directly producing strand breaks. This review focuses on the DNA methyl damage caused by MMS and MNNG for each site of potential methylation to summarize what is known about the repair of such damage and the downstream responses and consequences if not repaired.

The DNA binding properties of a series of imidazole-containing and C-terminus-modified analogues 4-7 of distamycin are described. These analogues contain one to four imidazole units, respectively. Data from the ethidium displacement assay showed that these compounds bind in the minor groove of DNA, with the relative order of binding constants of 6 (Im3) > 7 (Im4) > 5 (Im2) > 4 (Im1). The reduced binding constants of these compounds for poly(dA-dT) relative to distamycin, while they still interact strongly with poly(dG-dC), provided evidence of GC sequence acceptance. The preferences for GC-rich sequences by these compounds were established from a combination of circular dichroism (CD) titration, proton nuclear magnetic resonance (1H-NMR), and methidiumpropylethylenediaminetetraacetate-iron(II) [MPE.Fe-(II)] footprinting studies. In the CD studies, these compounds produced significantly larger DNA-induced ligand bands with poly(dG-dC) than poly(dA-dT) at comparable ligand concentrations. 1H-NMR studies of the binding of 5 to d-[CATGGCCATG]2 provided further evidence of the recognition of GC sequences by these compounds, and suggested that the ligand was located on the underlined sequence in the minor groove with the C-terminus oriented over the T residue. MPE footprinting studies on a GC-rich BamHI/SalI fragment of pBR322 provided unambiguous evidence for the GC sequence selectivity for some of these compounds. Compounds 4 and 7 produced poor footprints on the gels; however, analogues 5 and 6 gave strong footprints.(ABSTRACT TRUNCATED AT 250 WORDS)