Richard Cosstick

A second catalytic metal ion in a group I ribozyme

Although only a subset of protein enzymes depend on the presence of a metal ion for their catalytic function, all naturally occurring RNA enzymes require metal ions to stabilize theirstructure and for catalytic competence. In the self-splicing group I intron from Tetrahymena thermophila, several divalent metals can serve structural roles, but only Mg2+ and Mn2+ promote splice-site cleavage and exon ligation,. A study of a ribozyme reaction analogous to 5′-splice-site cleavage by guanosine uncovered the first metal ion with a definitive role in catalysis. Substitution of the 3′-oxygen of the leaving group with sulphur resulted in a metal-specificity switch, indicating an interaction between the leaving group and the metal ion. Here we use 3′-(thioinosylyl)-(3′ → 5′)-uridine, IspU, as a substrate in a reaction that emulates exon ligation. Activity requires the addition of a thiophilic metal ion (Cd2+ or Mn2+), providing evidence for stabilization of the leaving group by a metal ion in that step of splicing. Based on the principle of microscopic reversibility, this metal ion activates the nucleophilic 3′-hydroxyl of guanosine in the first step of splicing, supporting the model of a two-metal-ion active site.

The RuvC protein dimer resolves Holliday junctions by a dual incision mechanism that involves base-specific contacts

The Escherichia coli RuvC protein resolves DNA intermediates produced during genetic recombination. In vitro, RuvC binds specifically to Holliday junctions and resolves them by the introduction of nicks into two strands of like polarity. In contrast to junction recognition, which occurs without regard for DNA sequence, resolution occurs preferentially at sequences that exhibit the consensus 5′‐A/TTT↓G/C‐3′ (where ↓ indicates the site of incision). Synthetic Holliday junctions containing modified cleavage sequences have been used to investigate the mechanism of cleavage. The results indicate that specific DNA sequences are required for the correct docking of DNA into the two active sites of the RuvC dimer. In addition, using chemically modified oligonucleotides to introduce a hydrolysis‐resistant 3′‐S‐phosphorothiolate linkage at the cleavage site, it was found that, as long as the sequence requirements are fulfilled, the two incisions could be uncoupled from each other. These results indicate that RuvC protein resolves Holliday junctions by a mechanism similar to that exhibited by certain restriction enzymes.

Synthesis and biological evaluation of extraordinarily potent C-10 carba artemisinin dimers against P. falciparum malaria parasites and HL-60 cancer cells

A series of artemisinin dimers incorporating a metabolically stable C-10 carba-linkage have been prepared, several of which show remarkable in vitro antimalarial activity (as low as 30 pM) versus Plasmodium falciparum and in vitro anticancer activity in the micromolar to nanomolar range versus HL-60 cell lines.

Solid phase synthesis of oligonucleotides containing 3'-thiothymidine

Abstract A 5′- O -monomethoxytritylthymidine-3′- S -thiophosphoramidite (3) has been used to prepare oligodeoxynucleotides containing 3′-thiothymidine on a solid phase support. The intermediate thiophosphites are most efficiently oxidised using tetrabutylammonium periodate.

Enzymatic DNA processing on gold nanoparticles

Double stranded DNA immobilised on gold nanoparticles is amenable to enzymatic cleavage by a range of restriction endonucleases and the efficiency of the cleavage can be quantified using fluorescence spectroscopy.

Aplastic anaemia associated with organochlorine pesticide: case reports and review of evidence.

Three patients with aplastic anaemia had a history of substantial previous exposure to organochlorine pesticides. The temporal association between chemical exposure and the onset of first symptoms of anaemia was strongly supportive. Organochlorines have the property of lipid affinity and accumulation in adipose tissue. Objective evidence of clinically important concentrations of tissue pesticide residues may be a useful confirmation of previous exposure. In the patients studied the presence of Lindane (gamma hexachlorocyclohexane) was shown using gas chromatography/mass spectrometry with selective ion monitoring of fragments obtained from one heavily exposed patient, with concentrations about five times greater than a matched control. The presence of clinically important tissue concentrations of pentachlorophenol was also confirmed in a second patient exposed to this agent. The long term safety of organochlorine pesticides remains doubtful as they were introduced before adequate toxicological screening tests had been developed. The central registration of possible haematological adverse reactions, however, forms an important epidemiological method in the study of environmental chemical hazards and should be complied with whenever possible.

Antitumour and antimalarial activity of artemisinin-acridine hybrids

Artemisinin-acridine hybrids were prepared and evaluated for their in vitro activity against tumour cell lines and a chloroquine sensitive strain of Plasmodium falciparum. They showed a 2-4-fold increase in activity against HL60, MDA-MB-231 and MCF-7 cells in comparison with dihydroartemisinin (DHA) and moderate antimalarial activity. Strong evidence that the compounds induce apoptosis in HL60 cells was obtained by flow cytometry, which indicated accumulation of cells in the G1 phase of the cell cycle.

A combined top-down bottom-up approach for introducing nanoparticle networks into nanoelectrode gaps

We report here on the fabrication of a three-dimensional array of nanoparticles which bridges the gap between lithographically defined gold electrode contacts separated by 20 nm. The nanoparticle assemblies are formed from about 5 nm gold nanoparticles and benzenedimethanethiol (BDMT) bridging ligands. These assemblies are introduced between the contacts using a layer-by-layer protocol with successive BDMT self-assembly being followed by nanoparticle adsorption until the gap is bridged. The temperature dependent electrical properties of these devices are analysed to establish whether they are consistent with the notion that the networks are built up from molecularly interlinked discrete gold nanoparticles. To aid this analysis the molecular conductance of single bridging molecules is also characterized at room temperature using a recently introduced method based on the scanning tunnelling microscope (STM). From these measurements it is concluded that the room temperature electrical properties of the nanostructured networks are limited by the small interparticle connectivity and the inherent resistance of the linker molecules.

New methods for the synthesis of 3′-S-phosphorothiolate internucleoside linkages

Abstract Efficient procedures are described for the synthesis of dinucleoside phosphorothiolates using either a Michaelis-Arbusov-type reaction or a phosphotriester approach.

A novel mechanism for the scission of double-stranded DNA: BfiI cuts both 3′–5′ and 5′–3′ strands by rotating a single active site

Metal-dependent nucleases that generate double-strand breaks in DNA often possess two symmetrically-equivalent subunits, arranged so that the active sites from each subunit act on opposite DNA strands. Restriction endonuclease BfiI belongs to the phospholipase D (PLD) superfamily and does not require metal ions for DNA cleavage. It exists as a dimer but has at its subunit interface a single active site that acts sequentially on both DNA strands. The active site contains two identical histidines related by 2-fold symmetry, one from each subunit. This symmetrical arrangement raises two questions: first, what is the role and the contribution to catalysis of each His residue; secondly, how does a nuclease with a single active site cut two DNA strands of opposite polarities to generate a double-strand break. In this study, the roles of active-site histidines in catalysis were dissected by analysing heterodimeric variants of BfiI lacking the histidine in one subunit. These variants revealed a novel mechanism for the scission of double-stranded DNA, one that requires a single active site to not only switch between strands but also to switch its orientation on the DNA.