A study of the reproducibility of a fluorescence in situ hybridization bladder cancer detection assay

OBJECTIVE: To assess the reproducibility of the UroVysion fluorescence in situ hybridization (FISH) bladder cancer detection assay. STUDY DESIGN: Thirteen specimens (2 negative, 3 low-level positive [1-10% abnormal cells], 5 mid-level positive [11-75%], and 3 high-level positive [>75%]) were analyzed by 7 cytotechnologists. Each cytotechnologist rendered an overall diagnosis of positive or negative and determined the percentage of abnormal urothelial cells for all positive specimens. RESULTS: The interobserver reproducibility of the assay was 100% for mid-level and high-level positive specimens, 93% for negative specimens, and 78% for low-level positive specimens. The range of percent abnormal determinations was highest for mid-level positive specimens, with mean SDs of 1.8%, 16.4% and 10.1% for the low-, mid-, and high-level positives, respectively. CONCLUSION: There was a high level of reproducibility among the mid- and high-level positive specimens. The reproducibility for low-level positive specimens was lowest, suggesting that such specimens should be reviewed by a second technologist to ensure an accurate diagnosis. The findings of this study are important for further elucidating the clinical value of quantitative FISH analysis in the management of patients undergoing FISH testing for bladder cancer.     

Atrial natriuretic peptide (ANP) in chronic obstructive pulmonary disease (COPD): the relationship between plasma ANP and lung function. Effects of exercise and of the calcium antagonist, isradipine, on plasma ANP. A randomised, double-blind, placebo-controlled study.

In patients with severe chronic obstructive pulmonary disease (COPD) an increased pulmonary arterial pressure (PAP), a raised plasma level of atrial natriuretic peptide (ANP) and a correlation between increasing PAP and increasing plasma ANP have been shown. Furthermore, a negative correlation between lung function and PAP has been reported, and calcium antagonists have been claimed to decrease PAP. The purpose of the present study was to investigate whether 1) a negative correlation between lung function and plasma ANP could be demonstrated, whether 2) plasma ANP would increase during exercise in patients with COPD, and whether (3), in a randomised, placebo-controlled, double-blind design, a calcium antagonist was able to decrease plasma ANP at rest and modify the expected increase in plasma ANP during exercise. Eighteen patients with severe COPD were investigated. Plasma ANP was measured at rest and during exercise before and two hours after ingestion of either a single dose of 5 mg of isradipine, or a single dose of placebo. At rest, a correlation between lung function (forced vital capacity) and plasma ANP was found (rho = -0.49, P = 0.05). During the first exercise period, before ingestion of isradipine or placebo, the median level of ANP increased from 74 pg/ml at rest to 97 pg/ml at exhaustion (P less than 0.0002) (all patients). Administration of isradipine did not alter resting levels or exercise induced increases in plasma ANP. It is concluded, that in patients with severe COPD plasma ANP tends to be higher the more severely FVC is reduced. Plasma ANP increases during exercise. The calcium antagonist, isradipine, does not alter resting levels or exercise induced levels of plasma ANP.     

Thermodynamics of metal ion binding. 2. Metal ion binding by carbonic anhydrase variants

The ability to construct molecular motifs with predictable properties in aqueous solution requires an extensive knowledge of the relationships between structure and energetics. The design of metal binding motifs is currently an area of intense interest in the bioorganic community. To date synthetic motifs designed to bind metal ions lack the remarkable affinities observed in biological systems. To better understand the structural basis of metal ion affinity, we report here the thermodynamics of binding of divalent zinc ions to wild-type and mutant carbonic anhydrases and the interpretation of these parameters in terms of structure. Mutations were made both to the direct His ligand at position 94 and to indirect, or second-shell, ligands Gln-92, Glu-117, and Thr-199. The thermodynamics of ligand binding by several mutant proteins is complicated by the development of a second zinc binding site on mutation; such effects must be considered carefully in the interpretation of thermodynamic data. In all instances modification of the protein produces a complex series of changes in both the enthalpy and entropy of ligand binding. In most cases these effects are most readily rationalized in terms of ligand and protein desolvation, rather than in terms of changes in the direct interactions of ligand and protein. Alteration of second-shell ligands, thought to function primarily by orienting the direct ligands, produces profoundly different effects on the enthalpy of binding, depending on the nature of the residue. These results suggest a range of activities for these ligands, contributing both enthalpic and entropic effects to the overall thermodynamics of binding. Together, our results demonstrate the importance of understanding relationships between structure and hydration in the construction of novel ligands and biological polymers.     

Attenuated protein expression vectors for use in siRNA rescue experiments

Transient transfection of small interfering RNA (siRNA) provides a powerful approach for studying cellular protein functions, particularly when the target protein can be re-expressed from an exogenous siRNA-resistant construct in order to rescue the knockdown phenotype, confirm siRNA target specificity, and support mutational analyses. Rescue experiments often fail, however, when siRNA-resistant constructs are expressed at suboptimal levels. Here, we describe an ensemble of mammalian protein expression vectors with CMV promoters of differing strengths. Using CHMP2A rescue of HIV-1 budding, we show that these vectors can combine high-transfection efficiencies with tunable protein expression levels to optimize the rescue of cellular phenotypes induced by siRNA transfection.