A Polymerase Chain Reaction-Based Method to Detect Cisplatin Adducts in Specific Genes

Every bulky lesion in DNA can potentially inhibit the Taq DNA polymerase and thereby decrease the amplification produced in the polymerase chain reaction. We investigated the feasibility of using this inhibition to quantify DNA lesions produced by the anticancer drug cisplatin. Products were detected by electrophoresis followed by ethidium bromide staining. Quantitation was obtained by including [32P]dCTP in the amplification reaction and subsequently assessing the incorporated radioactivity. Hamster genomic DNA was platinated in vitro to defined levels and amplified with primers that produce either a 150, 750 or 2,000 base pair fragment. The degree of inhibition of PCR agreed with the predicted level of DNA platination in each size of fragment, suggesting that the polymerase was inhibited by every cisplatin-induced lesion. This method was used to detect cisplatin-induced lesions in the adenine phosphoribosyltransferase gene of CHO cells. Cells were incubated with 0–125μM cisplatin for 2 h, the DNA was purified and subjected to PCR. A significant decrease in amplification of the 2 kbp fragment was observed in DNA from cells incubated with cisplatin at 75 μM. The degree of inhibition agreed closely with the amount of DNA damage in the overall genome as measured by atomic absorption. No change was detected in amplification of the 150 base fragment which can therefore be used to normalize data for any variations between DNA samples. This assay has the same sensitivity as other methods currently used for the analysis of gene-specific damage. The advantage of this assay is that it obviates the need for specific endonuclease complexes to recognize and cleave DNA adducts as previously required when analyzing damage in specific genomic sequences

Somatostatin subtype-2 receptor-targeted metal-based anticancer complexes

Conjugates of a dicarba analogue of octreotide, a potent somatostatin agonist whose receptors are overexpressed on tumor cells, with [PtCl 2(dap)] (dap = 1-(carboxylic acid)-1,2-diaminoethane) (3), [(η 6-bip)Os(4-CO 2-pico)Cl] (bip = biphenyl, pico = picolinate) (4), [(η 6-p-cym)RuCl(dap)] + (p-cym = p-cymene) (5), and [(η 6-p-cym)RuCl(imidazole-CO 2H)(PPh 3)] + (6), were synthesized by using a solid-phase approach. Conjugates 3-5 readily underwent hydrolysis and DNA binding, whereas conjugate 6 was inert to ligand substitution. NMR spectroscopy and molecular dynamics calculations showed that conjugate formation does not perturb the overall peptide structure. Only 6 exhibited antiproliferative activity in human tumor cells (IC 50 = 63 ± 2 μ in MCF-7 cells and IC 50 = 26 ± 3 μ in DU-145 cells) with active participation of somatostatin receptors in cellular uptake. Similar cytotoxic activity was found in a normal cell line (IC 50 = 45 ± 2.6 μ in CHO cells), which can be attributed to a similar level of expression of somatostatin subtype-2 receptor. These studies provide new insights into the effect of receptor-binding peptide conjugation on the activity of metal-based anticancer drugs, and demonstrate the potential of such hybrid compounds to target tumor cells specifically. © 2012 American Chemical Society

Designing organometallic compounds for catalysis and therapy

Bioorganometallic chemistry is a rapidly developing area of research. In recent years organometallic compounds have provided a rich platform for the design of effective catalysts, e.g. for olefin metathesis and transfer hydrogenation. Electronic and steric effects are used to control both the thermodynamics and kinetics of ligand substitution and redox reactions of metal ions, especially Ru II. Can similar features be incorporated into the design of targeted organometallic drugs? Such complexes offer potential for novel mechanisms of drug action through incorporation of outer-sphere recognition of targets and controlled activation features based on ligand substitution as well as metal- and ligand-based redox processes. We focus here on η 6-arene, η 5-cyclopentadienyl sandwich and half-sandwich complexes of Fe II, Ru II, Os II and Ir III with promising activity towards cancer, malaria, and other conditions. © 2012 The Royal Society of Chemistry

Quantification of damage in DNA recovered from highly degraded samples – a case study on DNA in faeces

BACKGROUND: Poorly preserved biological tissues have become an important source of DNA for a wide range of zoological studies. Measuring the quality of DNA obtained from these samples is often desired; however, there are no widely used techniques available for quantifying damage in highly degraded DNA samples. We present a general method that can be used to determine the frequency of polymerase blocking DNA damage in specific gene-regions in such samples. The approach uses quantitative PCR to measure the amount of DNA present at several fragment sizes within a sample. According to a model of random degradation the amount of available template will decline exponentially with increasing fragment size in damaged samples, and the frequency of DNA damage (λ) can be estimated by determining the rate of decline. RESULTS: The method is illustrated through the analysis of DNA extracted from sea lion faecal samples. Faeces contain a complex mixture of DNA from several sources and different components are expected to be differentially degraded. We estimated the frequency of DNA damage in both predator and prey DNA within individual faecal samples. The distribution of fragment lengths for each target fit well with the assumption of a random degradation process and, in keeping with our expectations, the estimated frequency of damage was always less in predator DNA than in prey DNA within the same sample (mean λ(predator )= 0.0106 per nucleotide; mean λ(prey )= 0.0176 per nucleotide). This study is the first to explicitly define the amount of template damage in any DNA extracted from faeces and the first to quantify the amount of predator and prey DNA present within individual faecal samples. CONCLUSION: We present an approach for characterizing mixed, highly degraded PCR templates such as those often encountered in ecological studies using non-invasive samples as a source of DNA, wildlife forensics investigations and ancient DNA research. This method will allow researchers to measure template quality in order to evaluate alternate sources of DNA, different methods of sample preservation and different DNA extraction protocols. The technique could also be applied to study the process of DNA decay

Development of selective, active platinum complexes

The development of Pt-complexes of the type 1,2-bis(4-hydroxyphenyl)ethylenediamines as ligands for estrogen receptors of receptor-pos. and -neg. mammary cancer cells is discussed. The degree of receptor affinity of the ligand was dependent on its configuration, i.e. the R,R- and the S,S-conformations, in contrast to the R,S conformation, were more effective for inhibition of estradiol-receptor interactions. An ethylenediamine structure was essential for antitumor activity of these compds. Exptl. results with various tumor systems demonstrated that R,R-dichloro-1,2-bis(4-hydroxyphenyl)ethylenediamine-Pt(II)-complex (I) [91326-63-5] was the most active compd. I as an antitumor drug seems promising since it has only slight nephrotoxic effects and no myelotoxic effects

Evaluation of reversed phase versus normal phase column combination for the quantitative analysis of common commercial available middle distillates using GC x GC-TOFMS and Visual Basic Script.

Normal and reversed phase column combinations for comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry were evaluated concerning their suitability for the analysis of common commercial available middle distillates. Compound classes were identified and quantified by applying a previously developed data evaluation method (Jennerwein et al., 2014) [1] for middle distillates based on GC x GC-TOFMS using "normal phase" column combination and Visual Basic Scripting (VBS). The GC x GC-TOFMS methodology was transferred to a "reversed phase" column combination and it could be found, that this kind of column combination provides advantages for the quantification of petrochemical samples in terms of precision of the results. Special improvements were observed for the quantification of aromatics and paraffins

Complete group-type quantification of petroleum middle distillates based on comprehensive two-dimensional Gas Chromatography Time-of-Flight Mass Spectrometry (GCxGC-TOFMS) and visual basic scripting.

The subject of the presented work was the development of a two-dimensional GCxGC time-of-flight mass spectrometric method (GCxGC-TOFMS) for the complete group-type quantification of petroleum middle distillates. The development of this method was possible due to the inherent features of GCxGC-TOFMS, namely the structured arrangement of compound groups and the mass fragmentation pattern, which provide the possibility of using Visual Basic Scripts as an analytical tool and thereby the classification of several thousand different compounds. The analysis method was focused on common petroleum based fuels from light to heavier middle distillation fractions. For the implementation of an absolute quantification method, a set of standard substances representing the main substance classes and carbon numbers within middle distillates and well-separated and recognizable internal standards were thoroughly chosen in order to obtain individual response factors and group specific response curves. The results of the qualitative and quantitative analysis were compared to well-established standard methods used in the petrochemical industry. The quantification of aromatic hydrocarbons was compared to EN 12916, a high performance liquid chromatography (HPLC) method that provides a rough separation between mono-, di-, and higher aromatics. Further, the quantification of the fatty acid methyl ester (FAME) content in diesel fuel was compared to EN 14078 and the distribution of single FAMEs was compared to EN 14103. It could be stated that an absolute quantification as the here presented method has not been reported before and the results were in good agreement with the reference methods. Furthermore, the here presented GCxGC-TOFMS quantification method is able to itemize according substance classes and carbon number. The detection limit of the method allows accurate and sensitive quantification for different limiting values of middle distillates with a single method

Proof of concept of high-temperature comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry for two-dimensional simulated distillation of crude oils.

In this work, a reversed-phase high-temperature comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GC × GC-TOFMS) approach for the qualitative and quantitative analyses of crude oils will be presented. The proposed setup provides the best utilization of the two-dimensional separation space for carbon numbers between C 10 and C 60 . Visual Basic Script (VBS) was successfully applied for data processing to achieve comprehensive classification of the main compound classes. On this basis, crude oils from different origins could be compared by their composition. Real distillation cuts following ASTM D2892 and ASTM D5236 were applied for the development of area-based templates representing virtual boiling point cuts. By this approach, a quantification of an artificial crude oil sample with a defined initial boiling point was evaluated versus the quantitative result according to ASTM D7169 (one-dimensional simulated distillation for high boiling samples, hereinafter 1D-SimDist), and by this, a two-dimensional simulated distillation (2D-SimDist) was successfully developed