The Phototoxic Effect of Water-Soluble Porphyrins on Human Clear Cell Renal Cell Carcinoma Line Сaki-1

Biophysics - Two tetrapyridine porphyrins, cationic porphyrin P4 (TMPyP4) and its amphiphilic derivative porphyrin P1 with carboxyl groups, and their zinc-containing analogs ZnP4 and ZnP1 were...     

Ethidium Probing of the Parallel Double- and Four-stranded Structures Formed by the Telomeric DNA Sequences dG(GT)4G and d(GT)5

Abstract

Oligonucleotides 3′-d(GT)₅-(CH₂CH₂O)₃-d(GT)₅-3′ (parGT), containing GT repeats present in the telomeric DNA from Saccharomyces cerevisiae, had been demonstrated to form bimolecular structure, GT-quadruplex (qGT) [O. F. Borisova et al. FEBS Letters 306, 140–142 (1992)]. Four d(GT)₅ strands of the GT-quadruplex are parallel and form five G-quartets while thymines are bulged out. The four GT repeats when flanked by guanines, 3′-dG(TG)₄G-(CH₂CH₂O)₃- dG(GT)₄G-3′ (hp-GT), had been shown to form a novel parallel-stranded (ps) double helix with G·G and T·T base pairs (hp-GT ps-DNA) [A. K. Shchyolkina et al. J. Biomol. Struct. Dynam. 18, 493–503 (2001)]. In the present study the intercalator ethidium bromide (Et) was used for probing the two structures. The mode of Et binding and its effect on thermostability of qGT and hp-GT were compared. The quantum yield (q) and the fluorescence lifetime (τ) of Et:qGT (q = 0.15 ±0.01 and τ = 24 ±1 ns) and Et:hp-GT (q = 0.10 ± 0.01 and τ = 16.5 ± 1 ns) indicative of intercalation mode of Et binding were determined. Et binding to qGT was found to be cooperative with corresponding coefficient ω = 3.9 ± 0.1 and the binding constant K= (6.4 ± 0.1)·10M^?1. The maximum number of Et molecules intercalating into GT-quadruplex is as high as twice the number of inerspaces between G-quartets (eight in our case). The data conform to the model of Et association with GT-quadruplex suggested earlier [O. F. Borisova et al. Mol. Biol. (Russ) 35, 732–739 (2001)]. The anticooperative type of Et binding was observed in case of hp- GT ps-DNA, with the maximum number of bound Et molecules, N = 4 ÷ 5, and the association constant K = (1.5 ± 0.1)·10⁵ M^?1. Thermodynamic parameters of formation of Et:qGT and EtBr:hp-GT complexes were calculated from UV thermal denaturation profiles.     

Complexes of antiparallel telomeric G-quadruplex d(TTAGGG)4 with carboxymethyl tetracationic porphyrins

Porphyrins are a chemical class that is widely used in drug design. Cationic porphyrins may bind to DNA guanine quadruplexes. We report the parameters of the binding of 5,10,15,20-tetrakis(N-carboxymethyl-4-pyridinium) porphyrin (P1) and 5,10,15,20-tetrakis(N-etoxycarbonylmethyl-4-pyridinium) porphyrin (P2) to antiparallel telomeric G-quadruplex formed by d(TTAGGG)₄ sequence (TelQ). The binding constants (K _i ) and the number of binding sites (N _j ) were determined from absorption isotherms generated from the absorption spectra of complexes of P1 and P2 with TelQ. Compound P1 demonstrated a high affinity to TelQ (K _i = (40 ± 6) × 10⁶ M^?1, N ₁ = 1; K ₂ = (5.4 ± 0.4) × 10⁶ M^?1, N ₂ = 2). In contrast, the binding constants of P2-TelQ complexes (K ₁ = (3.1 ± 0.2) × 10⁶ M^?1, N ₁ = 1; K ₂ = (1.2 ± 0.2) × 10⁶ M^?1, N ₂ = 2) were one order of magnitude smaller than the corresponding values for P2-TelQ complexes. Measurements of the quantum yield and fluorescence lifetime of the drug’s TelQ complexes revealed two types of binding sites for P1 and P2 on the quadruplex oligonucleotide. We concluded that strong complexes can result from the interaction of the porphyrins with TTA loops whereas the weaker complexes are formed with G-quartets. The altered TelQ conformation detected by the circular dichroism spectra of P1-TelQ complexes can be explained by the disruption of the G-quartet. We conclude that peripheral carboxy groups contribute to the high affinity of P1 for the antiparallel telomeric G-quadruplex.     

Diverse modes of 5′‐[4‐(aminoiminomethyl)phenyl]‐[2,2′‐bifuran]‐5‐carboximidamide (DB832) interaction with multi‐stranded DNA structures

The modes of binding of 5′‐[4‐(aminoiminomethyl)phenyl]‐[2,2′‐Bifuran]‐5‐carboximidamide (DB832) to multi‐stranded DNAs: human telomere quadruplex, monomolecular R‐triplex, pyr/pur/pyr triplex consisting of 12 T*(T·A) triplets, and DNA double helical hairpin were studied. The optical adsorption of the ligand was used for monitoring the binding and for determination of the association constants and the numbers of binding sites. CD spectra of DB832 complexes with the oligonucleotides and the data on the energy transfer from DNA bases to the bound DB832 assisted in elucidating the binding modes. The affinity of DB832 to the studied multi‐stranded DNAs was found to be greater (K_ass ≈ 10⁷M^?1) than to the duplex DNA (K_ass ≈ 2 × 10⁵M^?1). A considerable stabilizing effect of DB832 binding on R‐triplex conformation was detected. The nature of the ligand tight binding differed for the studied multi‐stranded DNA depending on their specific conformational features: recombination‐type R‐triplex demonstrated the highest affinity for DB832 groove binding, while pyr/pur/pyr TTA triplex favored DB832 intercalation at the end stacking contacts and the human telomere quadruplex d[AG₃(T₂AG₃)₃] accommodated the ligand in a capping mode. Additionally, the pyr/pur/pyr TTA triplex and d[AG₃(T₂AG₃)₃] quadruplex bound DB832 into their grooves, though with a markedly lesser affinity. DB832 may be useful for discrimination of the multi‐sranded DNA conformations and for R‐triplex stabilization. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 8–20, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Conformational variability of recombination R-triplex formed by the mammalian telomeric sequence

Alignment of three nucleic acids strands, in which the third strand is identical to one of the DNA duplex strands, occurs in various cellular systems. In the case of telomeric t-loops, recognition between the DNA duplex and the homologous single strand is likely to be mediated by proteins through formation of the transient recombination-type R-triplex. Earlier, using 2-aminopurine as a fluorescent reporting base, we evaluated the thermodynamic characteristics of intramolecular R-triplex formed by a mixed nucleotide sequence. Here, we used this approach to explore a propensity of the telomeric TTAGGG repeat to form the R-triplex. The circular dichroism spectral changes detected upon formation of the R-triplex suggest that this process is accompanied by specific conformational changes in DNA, including a local destabilization of the target duplex next to a GGG run revealed by the fluorescence of the reporting 2-aminopurine base. Surprisingly, stability of the R-triplex formed by telomeric sequence depends strikingly on the counter ion, being higher for Na⁺ than for Li⁺. Taken together these findings indicate a significant conformational variability of telomeric DNA in the context of recombination-type R-triplex, a phenomenon of possible biological relevance.     

Development of the EpiOcular(TM) eye irritation test for hazard identification and labelling of eye irritating chemicals in response to the requirements of the EU cosmetics directive and REACH legislation

The recently implemented 7th Amendment to the EU Cosmetics Directive and the EU REACH legislation have heightened the need for in vitro ocular test methods. To address this need, the EpiOcular(TM) eye irritation test (EpiOcular-EIT), which utilises the normal (non-transformed) human cell-based EpiOcular tissue model, has been developed. The EpiOcular-EIT prediction model is based on an initial training set of 39 liquid and 21 solid test substances and uses a single exposure period and a single cut-off in tissue viability, as determined by the MTT assay. A chemical is classified as an irritant (GHS Category 1 or 2), if the tissue viability is ≤ 60%, and as a non-irritant (GHS unclassified), if the viability is > 60%. EpiOcular-EIT results for the training set, along with results for an additional 52 substances, which included a range of alcohols, hydrocarbons, amines, esters, and ketones, discriminated between ocular irritants and non-irritants with 98.1% sensitivity, 72.9% specificity, and 84.8% accuracy. To ensure the long-term commercial viability of the assay, EpiOcular tissues produced by using three alternative cell culture inserts were evaluated in the EpiOcular-EIT with 94 chemicals. The assay results obtained with the initial insert and the three alternative inserts were very similar, as judged by correlation coefficients (r2) that ranged from 0.82 to 0.96. The EpiOcular-EIT was pre-validated in 2007/2008, and is currently involved in a formal, multi-laboratory validation study sponsored by the European Cosmetics Association (COLIPA) under the auspices of the European Centre for the Validation of Alternative Methods (ECVAM). The EpiOcular-EIT, together with EpiOcular's long history of reproducibility and proven utility for ultra-mildness testing, make EpiOcular a useful model for addressing current legislation related to animal use in the testing of potential ocular irritants.     

Patterns of DNA Barcode Variation in Canadian Marine Molluscs

Background

Molluscs are the most diverse marine phylum and this high diversity has resulted in considerable taxonomic problems. Because the number of species in Canadian oceans remains uncertain, there is a need to incorporate molecular methods into species identifications. A 648 base pair segment of the cytochrome c oxidase subunit I gene has proven useful for the identification and discovery of species in many animal lineages. While the utility of DNA barcoding in molluscs has been demonstrated in other studies, this is the first effort to construct a DNA barcode registry for marine molluscs across such a large geographic area.

Methodology/Principal Findings

This study examines patterns of DNA barcode variation in 227 species of Canadian marine molluscs. Intraspecific sequence divergences ranged from 0–26.4% and a barcode gap existed for most taxa. Eleven cases of relatively deep (>2%) intraspecific divergence were detected, suggesting the possible presence of overlooked species. Structural variation was detected in COI with indels found in 37 species, mostly bivalves. Some indels were present in divergent lineages, primarily in the region of the first external loop, suggesting certain areas are hotspots for change. Lastly, mean GC content varied substantially among orders (24.5%–46.5%), and showed a significant positive correlation with nearest neighbour distances.

Conclusions/Significance

DNA barcoding is an effective tool for the identification of Canadian marine molluscs and for revealing possible cases of overlooked species. Some species with deep intraspecific divergence showed a biogeographic partition between lineages on the Atlantic, Arctic and Pacific coasts, suggesting the role of Pleistocene glaciations in the subdivision of their populations. Indels were prevalent in the barcode region of the COI gene in bivalves and gastropods. This study highlights the efficacy of DNA barcoding for providing insights into sequence variation across a broad taxonomic group on a large geographic scale.     

Expression of miRNAs in ovine fetal gonads: potential role in gonadal differentiation

Background  

Gonadal differentiation in the mammalian fetus involves a complex dose-dependent genetic network. Initiation and progression of fetal ovarian and testicular pathways are accompanied by dynamic expression patterns of thousands of genes. We postulate these expression patterns are regulated by small non-coding RNAs called microRNAs (miRNAs). The aim of this study was to identify the expression of miRNAs in mammalian fetal gonads using sheep as a model.     

Molecular evolution of mitochondrial 12S RNA and cytochrome b sequences in the pantherine lineage of Felidae

DNA sequence comparisons of two mitochondrial DNA genes were used to infer phylogenetic relationships among 17 Felidae species, notably 15 in the previously described pantherine lineage. The polymerase chain reaction (PCR) was used to generate sequences of 358 base pairs of the mitochondrial 12S RNA gene and 289 base pairs of the cytochrome b protein coding gene. DNA sequences were compared within and between 17 felid and five nonfelid carnivore species. Evolutionary trees were constructed using phenetic, cladistic, and maximum likelihood algorithms. The combined results suggested several phylogenetic relationships including (1) the recognition of a recently evolved monophyletic genus Panthera consisting of Panthera leo, P. pardus, P. onca, P. uncia, P. tigris, and Neofelis nebulosa; (2) the recent common ancestry of Acinonyx jubatus, the African cheetah, and Puma concolor, the American puma; and (3) two golden cat species, Profelis temmincki and Profelis aurata, are not sister species, and the latter is strongly associated with Caracal caracal. These data add to the growing database of vertebrate mtDNA sequences and, given the relatively recent divergence among the felids represented here (1-10 Myr), allow 12S and cytochrome b sequence evolution to be addressed over a time scale different from those addressed in most work on vertebrate mtDNA.      

Plutella australiana (Lepidoptera,Plutellidae), an overlooked diamondback moth revealed?by?DNA?barcodes

The genus Plutella was thought to be represented in Australia by a single introduced species, Plutella xylostella (Linnaeus), the diamondback moth. Its status as a major pest of cruciferous crops, and the difficulty in developing control strategies has motivated broad-ranging studies on its biology. Prior genetic work has generally supported the conclusion that populations of this migratory species are connected by substantial gene flow. However, the present study reveals the presence of two genetically divergent lineages of this taxonin Australia. One shows close genetic and morphological similarity with the nearly cosmopolitan Plutella xylostella. The second lineage possesses a similar external morphology, but marked sequence divergence in the barcode region of the cytochrome c oxidase I gene, coupled with clear differences in genitalia. As a consequence, members of this lineage are described as a new species, Plutella australiana Landry & Hebert, which is broadly distributed in the eastern half of Australia.