Standardisation of metalloimmunoassay protocols for assessment of silver nanoparticle antibody conjugates.

The application of silver nanoparticles to electrochemical metalloimmunoassays has previously been reported (Szymanski et al., 2009) and is now used in the electrochemical Argento immunoassay platform via UK Patent No. 2458420. The development of an immunoassay in this format requires the optimisation of the antibody to silver nanoparticle conjugation process. Issues such as pH, antibody concentration and other factors can affect the assay performance. In order to determine the effect of these variables it is necessary to understand and control the effects of other factors that may affect the assay signal. In this study a number of conditions which affect the assay signal were identified and methods developed to minimise variability in the assay signal, resulting in a standardised method allowing easy comparisons of silver nanoparticle conjugates prepared and assayed at different times.

A novel optical biosensor format for the detection of clinically relevant TP53 mutations.

The TP53 gene has been the subject of intense research since the realisation that inactivation of this gene is common to most cancer types. Numerous publications have linked TP53 mutations in general or at specific locations to patient prognosis and therapy response. The findings of many studies using general approaches such as immunohistochemistry or sequencing are contradictory. However, the detection of specific mutations, especially those occurring in the structurally important L2 and L3 zinc binding domains, which are the most common sites of TP53 mutations, have been linked to patient prognosis and more strongly to radiotherapy and chemotherapy resistance in several major cancers. In this study, the TI-SPR-1 surface plasmon resonance system and Texas Instruments Spreeta chips were used to develop a DNA biosensor based on thiolated probes complementary to these domains. The sensors were able to detect these mutations in both oligonucleotides and PCR products with normal and mutant TP53 DNA, but the difference in hybridisation signal was small. Preliminary experiments to enhance the signal using Escherichia coli mismatch repair proteins, MutS and single strand binding protein were carried out. It was found that MutS was unable to bind to mismatch oligonucleotides, but single strand binding protein was able to bind to single stranded probes, which had not hybridised to the target, resulting in a three-fold increase in the sensitivity of the biosensor. While further work needs to be carried out to optimise the system, these preliminary experiments indicate that the TI-SPR-1 can be used for the detection of clinically relevant mutations in the TP53 gene and that the sensitivity can be increased significantly using single strand binding protein. This system has a number of advantages over current mutation detection technologies, including lower cost, ease of sensor preparation and measurement procedures, technical simplicity and increased speed due to the lack of need for gel electrophoresis.

Comparative effects of cladribine, fludarabine and pentostatin on nucleotide metabolism in T- and B-cell lines.

BACKGROUND AND AIMS: the purine nucleoside analogues cladribine (CdA), fludarabine (F-Ara-AMP) and pentostatin (dCf), are effective therapy for a range of T- and B-cell lymphoid malignancies. The effects upon nucleotide metabolism in human CCRF-CEM T-cell leukaemia and Raji B-cell lymphoma cell lines of these drugs have been compared to assess possible mechanisms of cytotoxicity. METHODS: Leukaemia cells were exposed to a purine nucleoside analogue and perchloric acid extracts were analysed by HPLC for 2'-deoxynucleoside-5'-triphosphates (dNTPs), nucleoside-5'-triphosphates (NTPs) and drug metabolites. RESULTS: After addition of a purine nucleoside analogue, CdA-TP and F-Ara-ATP accumulate in cells while the levels of dCf-TP formed were not detectable by ultra-violet absorbance. In response to accumulating concentrations of drug triphosphate, the cellular levels of dNTPs initially decrease (0-4 h), then accumulate above their initial levels (4-10 h) before slowly declining beyond 10 h. NTPs also accumulate during the period 4-10 h before declining at later times. CONCLUSION: The temporal effects on the levels of dNTPs and NTPs of the 3 purine nucleoside analogues are similar against CCRF-CEM and Raji cells. However, CdA induces major depletions of dTTP, dGTP and dATP in CCRF-CEM cells and F-Ara-A induces a major accumulation of dATP in Raji cells.

Inosine-5'-monophosphate analogues as inhibitors of human IMP cyclohydrolase and cellular growth.

The catalytic mechanism for the enzyme, IMP cyclohydrolase, may involve a reaction intermediate with negative charge in the 2-position of the purine ring (Szabados, E., Hindmarsh, E., Phillips, L., Duggleby, R.G. & Christopherson, R.I. (1994) Biochemistry 33, 14237-14245). Three analogues of IMP have been synthesised where fluorine, chlorine or bromine has been substituted in the 2-position on the purine ring. These analogues with an electronegative substituent may resemble a reaction intermediate for IMP cyclohydrolase; 2-fluoro IMP is a potent inhibitor of the enzyme with a Ki value of 0.19 microM, while 2-chloro IMP has a Ki of 1.9 microM and 2-bromo IMP is not inhibitory. However, IMP cyclohydrolase is not inhibited in human CCRF-CEM leukaemia cells exposed to 2-fluoro inosine although it is toxic to these cells with an IC50 value of 4.9 microM.

Reorganization of terminator DNA upon binding replication terminator protein: implications for the functional replication fork arrest complex.

Termination of DNA replication in Bacillus subtilis involves the polar arrest of replication forks by a specific complex formed between the replication terminator protein (RTP) and DNA terminator sites. While determination of the crystal structure of RTP has facilitated our understanding of how a single RTP dimer interacts with terminator DNA, additional information is required in order to understand the assembly of a functional fork arrest complex, which requires an interaction between two RTP dimers and the terminator site. In this study, we show that the conformation of the major B.subtilis DNA terminator,TerI, becomes considerably distorted upon binding RTP. Binding of the first dimer of RTP to the B site of TerI causes the DNA to become slightly unwound and bent by approximately 40 degrees. Binding of a second dimer of RTP to the A site causes the bend angle to increase to approximately 60 degrees . We have used this new data to construct two plausible models that might explain how the ternary terminator complex can block DNA replication in a polar manner. In the first model, polarity of action is a consequence of the two RTP-DNA half-sites having different conformations. These different conformations result from different RTP-DNA contacts at each half-site (due to the intrinsic asymmetry of the terminator DNA), as well as interactions (direct or indirect) between the RTP dimers on the DNA. In the second model, polar fork arrest activity is a consequence of the different affinities of RTP for the A and B sites of the terminator DNA, modulated significantly by direct or indirect interactions between the RTP dimers.

An alternative mechanism for the inhibition of cholesterol biosynthesis in HepG2 cells by N-[(1,5,9)-trimethyldecyl]-4 alpha,10-dimethyl-8-aza-trans-decal-3 beta-ol (MDL 28,815).

Compounds that block hepatic cholesterol biosynthesis and secretion may be useful hypocholesterolemic agents. N-[(1,5,9)-trimethyldecyl]-4 alpha,10-dimethyl-8-aza-trans-decal-3 beta-ol (MDL 28,815) has been shown to block cholesterol biosynthesis in 3T3 fibroblasts and it causes cellular accumulation of squalene 2,3-epoxide and squalene 2,3:23,24-diepoxide (squalene epoxides), which suggests that it inhibits 2,3-oxidosqualene cyclase. The purpose of the present report was to determine whether MDL 28,815 acts only at the level of 2,3-oxidosqualene cyclase or whether other enzymes in the cholesterol biosynthetic pathway are affected. HepG2 cells, grown in lipoprotein-deficient serum, were incubated with MDL 28,815 and 14C-acetate to radiolabel cholesterol and the intermediates in the cholesterol biosynthetic pathway. Blockade of cholesterol biosynthesis by MDL 28,815 in these cells was associated with the accumulation of two metabolites, one of which was 5 alpha-cholest-8-en-3 beta-ol. The other metabolite was identified by a combination of ultraviolet spectrometry, gas chromatography, mass spectroscopy and analytical high-performance liquid chromatography as 5 alpha-cholest-8,14-dien-3 beta-ol. Maximal blockade of cholesterol biosynthesis was associated with the accumulation of these two metabolites and, in particular, 5 alpha-cholest-8,14-dien-3 beta-ol, rather than with squalene epoxides. These results suggest that MDL 28,815 blocks cholesterol biosynthesis primarily by the inhibition of sterol-delta 14-reductase, and possibly sterol-delta 8-ene isomerase, rather than 2,3-oxidosqualene cyclase.

Cardiac Rehabilitation: Then and Now.

In brief: Cardiac rehabilitation, born of cardiology in order to care for patients with coronary heart disease, has evolved into an accepted area of clinical practice. As more and more patients survive a coronary event, the need for state-of-the-art methods of rehabilitation will be increasingly needed. The author reviews the history of cardiac rehabilitation, discusses its current status, and predicts What courses of action will be needed as the field continues to evolve.

Isolated perfused lung histamine release, lipid peroxidation, and tissue superoxide dismutase from rats exposed to normobaric hyperoxia.

Female Sprague-Dawley inbred rats were exposed to either 1 atm of 100% O2 for 24 h, or 65% O2 for 5 days, with or without pretreatment with disulfiram, an inhibitor of lung CuZn-SOD. After O2 exposure, the rats were killed, the lungs removed, and isolated perfused lungs (IPLs) prepared. The IPLs were perfused with modified Krebs-Henseleit buffer, and perfusate histamine, malondialdehyde (MDA), and lung tissue CuZn-SOD activity examined. Disulfiram administration decreased the LT50 of O2-exposed rats from 65 to 36 h. Histamine and MDA in the perfusate from the IPL prepared from rats exposed to 100% O2 for 24 h were markedly increased. When rats were pretreated with disulfiram and exposed to 100% O2 for 24 h, histamine and MDA were increased an additional 77% and 45%, respectively. In separate experiments, 100% O2 exposure significantly decreased lung CuZn-SOD activity by 40% while IPL histamine and MDA were significantly increased. However, exposure of rats to 65% O2 for 5 days decreased lung CuZn-SOD by 69% but did not affect IPL histamine release or perfusate MDA. These studies suggest that IPL histamine release and/or MDA may be an early biochemical marker for pulmonary O2 toxicity, that lung CuZn-SOD activity may not be the only determinant in O2 toxicity, and other defense mechanisms may play a vital protective role during sublethal O2 exposures.

Lead Selection and Time of Monitoring in Diagnostic Exercise Testing.

In brief: The authors examined the effects of variations in lead selection and time of monitoring during recovery on the predictive value and sensitivity of exercise testing. Eighty-six patients received a standard Bruce protocol exercise test and follow-up angiography. The results showed that 12-lead monitoring for at least six minutes of recovery had more sensitivity for diagnosing coronary artery disease, and the greater the ST-segment depression, the greater the predictive value of the test.

Effect of a conditioning program in patients taking propranolol for angina pectoris.

17 patients with stable angina pectoris due to coronary occlusive disease had serial graded exercise tests performed while on no medication, after treatment with propranolol and after an 8-week exercise reconditioning regimen while taking propranolol. The patient whose angina-free exercise capacity on beta-blockade is 2.5 METs or greater is likely to benefit from an exercise program.