Paul Teesdale-Spittle

Characteristics of a novel deep red/infrared fluorescent cell-permeant DNA probe, DRAQ5, in intact human cells analyzed by flow cytometry, confocal and multiphoton microscopy.

BACKGROUNDnThe multiparameter fluorometric analysis of intact and fixed cells often requires the use of a nuclear DNA discrimination signal with spectral separation from visible range fluorochromes. We have developed a novel deep red fluorescing bisalkylaminoanthraquinone, DRAQ5 (Ex(lambdamax) 646 nm; Em(lambdamax) 681 nm; Em(lambdarange) 665->800 nm), with high affinity for DNA and a high capacity to enter living cells. We describe here the spectral characteristics and applications of this synthetic compound, particularly in relation to cytometric analysis of the cell cycle.nnnMETHODSnCultured human tumor cells were examined for the ability to nuclear locate DRAQ5 using single and multiphoton laser scanning microscopy (LSM) and multiparameter flow cytometry.nnnRESULTSnMultiparameter flow cytometry shows that the dye can rapidly report the cellular DNA content of live and fixed cells at a resolution level adequate for cell cycle analysis and the cycle-specific expression of cellular proteins (e.g., cyclin B1). The preferential excitation of DRAQ5 by laser red lines (633/647 nm) was found to offer a means of fluorescence signal discrimination by selective excitation, with greatly reduced emission overlap with UV-excitable and visible range fluophors as compared with propidium iodide. LSM reveals nuclear architecture and clearly defines chromosomal elements in live cells. DRAQ5 was found to permit multiphoton imaging of nuclei using a 1,047-nm emitting mode-locked YLF laser. The unusual spectral properties of DRAQ5 also permit live cell DNA analysis using conventional 488 nm excitation and the single-photon imaging of nuclear fluorescence using laser excitation between 488 nm and low infrared (IR; 780 nm) wavelengths. Single and multiphoton microscopy studies revealed the ability of DRAQ5 to report three-dimensional nuclear structure and location in live cells expressing endoplasmic reticulum targeted-GFP, MitoTracker-stained mitochondria, or a vital cell probe for free zinc (Zinquin).nnnCONCLUSIONnThe fluorescence excitation and emission characteristics of DRAQ5 in living and fixed cells permit the incorporation of the measurement of cellular DNA content into a variety of multiparameter cytometric analyses.

Inhibition of glutathione S‐transferases (GSTs) from parasitic nematodes by extracts from traditional Nigerian medicinal plants

Piliostigma thonningii, Ocimum gratissimum, Nauclea latifolia and Alstonia boonei are used in Nigerian traditional medicines against gastrointestinal helminths of animals and man. Proanthocyanidins were detected in Piliostigma and Nauclea, but not Alstonia or Ocimum. Extracts of these plants killed 50% of brine shrimp nauplii at <10u2005ppm (Nauclea), 100u2005ppm (Piliostigma) and <1000u2005ppm (Ocimum and Alstonia), the Nauclea LD50 being similar to the anthelmintic drug piperazine. Extracts were also toxic to the parasitic nematode Haemonchus infective L3 stage. Nematode glutathione‐S‐transferases (GSTs) are potential drug targets. Apart from Alstonia all the medicinal plants contained heat‐stable inhibitory activities against recombinant Ascaris and Onchocerca GSTs in vitro. Piliostigma, Ocimum and Nauclea had IC50s of 2, 10 and 15u2005µg/mL respectively for Ascaris GST and 4, 8, 28u2005µg/mL respectively for Onchocerca GST. We suggest that the inhibitory properties of some of these Nigerian plant extracts against GST may contribute to the pharmacological basis of their efficacy against helminths in traditional herbal use. Copyright

A common class of nematode glutathione S-transferase (GST) revealed by the theoretical proteome of the model organism Caenorhabditis elegans

The genome verified C. elegans free-living nematode model is a new tool for investigating gene expression in human and animal nematode parasites. There is limited information on designating glutathione S-transferase (GST) to specific classes in lower invertebrates such as nematodes. Following cloning, amino acid sequence alignment, recombinant expression and Western blotting we provide evidence of a new GST class in nematodes or lower invertebrates.

β-carbonyl substituted glutathione conjugates as inhibitors of O. volvulus GST2.

A series of beta-carbonyl substituted glutathione conjugates were prepared and evaluated as inhibitors of OvGST2. Their specificity for the parasite derived protein was assessed through comparison with their inhibition of human piGST. Inhibition of OvGST2 has been demonstrated at low micromolar concentrations for these conjugates and selectivity for OvGST2 over human pi-GST of greater than 10-fold has been achieved.

Novel Sources of Mammalian C-S Lyase Activity

The C‐S lysis of L‐cysteine conjugates is one biotransformation pathway which is responsible for the generation of mutagenic and cytotoxic metabolic species. Thirteen cysteine S‐conjugates were synthesized in our laboratories and incubated with aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT) enzymes from porcine heart tissue. The C‐S lyase (CSL) activity for each enzyme‐substrate combination was determined.

The C-S lysis of L-cysteine conjugates by aspartate and alanine aminotransferase enzymes

One biotransformation pathway which is responsible for the generation of mutagenic and cytotoxic metabolites is that of the C-S lysis (CSL) of L-cysteine conjugates. Thirteen cysteine S-conjugates, synthesised in our labora tories, were incubated with porcine heart aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT), and the C-S lyase activity for each enzyme-sub strate combination was determined. ASAT and ALAT were shown to exhibit CSL activity. It was also demonstrated that this activity was inhibited in the presence of the pyri doxal phosphate (PLP)-dependent enzyme inhibitor amino(oxyacetic acid) (AOAA) confirming the pyridoxal phosphate dependent mechanism by which C-S lysis is known to take place. Since the activities of these enzymes are used as biomarkers for the assessment of organ dam age, the potential interaction of L-cysteine conjugates with them may suppress their activity through direct inhibition.

Cysteine conjugate β-lyase activity in three species of parasitic helminth

Abstract Living organisms employ a variety of metabolic pathways when detoxifying xenobiotic compounds, including the formation of cysteine S- conjugates via glutathione conjugation. However, cysteine conjugate β-lyase (CCBL) catalysed β-cleavage, of certain cysteine conjugates, is known to cause cytotoxicity. This study represents the first investigation into the expression of CCBL and other associated enzymes in helminth species. A survey of the three major groups of parasitic helminths [cestodes ( Moniezia expansa ), digeneans ( Fasciola hepatica ) and nematodes ( Necator americanus , Heligmosomoides polygyrus )] has been made. The presence of CCBL enzymes within Moniezia expansa , Necator americanus and Heligmosomoides polygyrus has been established. Each species was screened for γ-glutamyl transpeptidase activity and transaminase activity towards l -aspartate, l -alanine, l -albizziin and l -phenylalanine. Aspartate and alanine aminotransferase activity were detected in all four species tested. γ-Glutamyl transpeptidase activity was only detected in Moniezia expansa and Necator americanus .

Anthraquinone-peptides as inhibitors of AP-1 transcription factor.

Peptide-1-[N-[2-succinamidylethyl]amino]anthraquinones containing five seven amino acid residues including the KCR motif important in AP-1 protein binding to DNA have been synthesised as potential transcription factor inhibitors. These anthraquinone-peptides showed DNA intercalative binding and inhibition of AP-1 protein binding to its DNA consensus sequence.

Purification and characterisation of a novel cysteine conjugate β-lyase from the tapeworm Moniezia expansa.

The paper presents the first report of the purification of an invertebrate cysteine conjugate beta-lyase (CCBL). CCBL activity was shown to predominate within the cytosolic fraction of tissue from the tapeworm Moniezia expansa. The monomeric cytosolic enzyme was isolated with a M(r) of 72 kDa and co-purified with transaminase activity towards L-aspartate. The substrate profile for M. expansa CCBL is different from that of mammalian CCBLs. Exploiting the differences in mammalian and parasite substrate profiles will facilitate the development of helminth targeted conjugates which will not be activated by host (mammalian) CCBLs.

Characterisation of fatty acid multilayers using a TSM biosensor.

Thickness shear mode (TSM) biosensors have many potential applications within the pharmaceutical sciences as a means of measuring mass changes in the nanogram range, film thickness, viscosity and shear moduli. This study addresses the possible use of the TSM sensor as a biosensor for measuring drug partition coefficients. In order to realise this potential, some fundamental understanding is required of the behaviour of lipid films on the sensor. The present study characterises the behaviour of fatty acid multilayers as a suitable model chemical system. Frequency shifts and impedance spectra are presented for multilayers of three fatty acid films coated on to the sensor using a Langmuir-Blodgett trough. The results indicate that the frequency shift is non-linear at lower numbers of fatty acid layers but the response is Sauerbrey-like at higher numbers of layers. Also at high numbers of layers, changes in the impedance spectra indicate viscoelastic behaviour in thicker membranes. An inverse relationship is observed between chain length and frequency shift, which is attributed to variations in the topography of the sensor surface. This work demonstrates the importance of fully characterising the physical behaviour of the lipid multilayers prior to using these systems for the measurement of drug partition coefficients.