Refill adherence to repeat prescriptions of cancer drugs to ambulatory patients.

The objective was to study the refill adherence among ambulatory patients with prescribed cancer drugs. The study was based on copies of repeat prescriptions, which were collected at three large Swedish pharmacies during the last 3 months of 2004. Copies of 141 repeat prescriptions were analysed. There was no statistical significant difference between the number of patients with undersupply of cancer drugs (i.e. <80% of prescribed cancer drugs) and that of patients with undersupply of all other drugs, or between the number of patients with oversupply of cancer drugs (>120% of prescribed cancer drugs) and that of patients with oversupply of all other drugs. Undersupply of drugs was found among 14% of the patients. The median treatment gap for these patients was 39 (range 29-49 days) per 98-100 days of prescribed treatment time, meaning that the undersupply leads to treatment gaps that may jeopardize their therapeutic outcome. It is reasonable to expect that more seriously ill patients would be adherent to prescribed medication, and consequently that cancer patients would have high adherence. However, our data show that cancer patients on oral long-term treatment have a non-adherence similar to that of patients in general.

Surface interactions in the complex between cytochrome f and the E43Q/D44N and E59K/E60Q plastocyanin double mutants as determined by (1)H-NMR chemical shift analysis.

A combination of site-directed mutagenesis and NMR chemical shift perturbation analysis of backbone and side-chain protons has been used to characterize the transient complex of the photosynthetic redox proteins plastocyanin and cytochrome f. To elucidate the importance of charged residues on complex formation, the complex of cytochrome f and E43Q/D44N or E59K/E60Q spinach plastocyanin double mutants was studied by full analysis of the (1)H chemical shifts by use of two-dimensional homonuclear NMR spectra. Both mutants show a significant overall decrease in chemical shift perturbations compared with wild-type plastocyanin, in agreement with a large decrease in binding affinity. Qualitatively, the E43Q/D44N mutant showed a similar interaction surface as wild-type plastocyanin. The interaction surface in the E59K/E60Q mutant was distinctly different from wild type. It is concluded that all four charged residues contribute to the affinity and that residues E59 and E60 have an additional role in fine tuning the orientation of the proteins in the complex.

Interactions of the NADP(H)-binding domain III of proton-translocating transhydrogenase from escherichia coli with NADP(H) and the NAD(H)-binding domain I studied by NMR and site-directed mutagenesis.

Using the purified NADP(H)-binding domain of proton-translocating Escherichia coli transhydrogenase (ecIII) overexpressed in (15)N- and (2)H-labeled medium, together with the purified NAD(H)-binding domain from E. coli (ecI), the interface between ecIII and ecI, the NADP(H)-binding site and the influence on the interface by NAD(P)(H) was investigated in solution by NMR chemical shift mapping. Mapping of the NADP(H)-binding site showed that the NADP(H) substrate is bound to ecIII in an extended conformation at the C-terminal end of the parallel beta-sheet. The distribution of chemical shift perturbations in the NADP(H)-binding site, and the nature of the interaction between ecI and ecIII, indicated that the nicotinamide moiety of NADP(H) is located near the loop comprising residues P346-G353, in agreement with the recently determined crystal structures of bovine [Prasad, G. S., et al. (1999) Nat. Struct. Biol. 6, 1126-1131] and human heart [White, A. W., et al. (2000) Structure 8, 1-12] transhydrogenases. Further chemical shift perturbation analysis also identified regions comprising residues G389-I406 and G430-V434 at the C-terminal end of ecIII's beta-sheet as part of the ecI-ecIII interface, which were regulated by the redox state of the NAD(P)(H) substrates. To investigate the role of these loop regions in the interaction with domain I, the single cysteine mutants T393C, R425C, G430C, and A432C were generated in ecIII and the transhydrogenase activities of the resulting mutant proteins characterized using the NAD(H)-binding domain I from Rhodospirillum rubrum (rrI). All mutants except R425C showed altered NADP(H) binding and domain interaction properties. In contrast, the R425C mutant showed almost exclusively changes in the NADP(H)-binding properties, without changing the affinity for rrI. Finally, by combining the above conclusions with information obtained by a further characterization of previously constructed mutants, the implications of the findings were considered in a mechanistic context.

Side-chain interactions in the plastocyanin-cytochrome f complex.

Cytochrome f and plastocyanin are redox partners in the photosynthetic electron-transfer chain. Electron transfer from cytochrome f to plastocyanin occurs in a specific short-lived complex. To obtain detailed information about the binding interface in this transient complex, the effects of binding on the backbone and side-chain protons of plastocyanin have been analyzed by mapping NMR chemical-shift changes. Cytochrome f was added to plastocyanin up to 0.3 M equiv, and the plastocyanin proton chemical shifts were measured. Out of approximately 500 proton resonances, 86% could be observed with this method. Nineteen percent demonstrate significant chemical-shift changes and these protons are located in the hydrophobic patch (including the copper ligands) and the acidic patches of plastocyanin, demonstrating that both areas are part of the interface in the complex. This is consistent with the recently determined structure of the complex [Ubbink, M., Ejdebäck, M., Karlsson, B. G., and Bendall, D. S. (1998) Structure 6, 323-335]. The largest chemical-shift changes are found around His87 in the hydrophobic patch, which indicates tight contacts and possibly water exclusion from this part of the protein interface. These results support the idea that electron transfer occurs via His87 to the copper in plastocyanin and suggest that the hydrophobic patch determines the specificity of the binding. The chemical-shift changes in the acidic patches are significant but small, suggesting that the acidic groups are involved in electrostatic interactions but remain solvent exposed. The existence of small differences between the present data and those used for the structure may imply that the redox state of the metals in both proteins slightly affects the structure of the complex. The chemical-shift mapping is performed on unlabeled proteins, making it an efficient way to analyze effects of mutations on the structure of the complex.

Proton translocating nicotinamide nucleotide transhydrogenase from E. coli. Mechanism of action deduced from its structural and catalytic properties.

Transhydrogenase couples the stereospecific and reversible transfer of hydride equivalents from NADH to NADP(+) to the translocation of proton across the inner membrane in mitochondria and the cytoplasmic membrane in bacteria. Like all transhydrogenases, the Escherichia coli enzyme is composed of three domains. Domains I and III protrude from the membrane and contain the binding site for NAD(H) and NADP(H), respectively. Domain II spans the membrane and constitutes at least partly the proton translocating pathway. Three-dimensional models of the hydrophilic domains I and III deduced from crystallographic and NMR data and a new topology of domain II are presented. The new information obtained from the structures and the numerous mutation studies strengthen the proposition of a binding change mechanism, as a way to couple the reduction of NADP(+) by NADH to proton translocation and occurring mainly at the level of the NADP(H) binding site.

NMR characterization of the NADP(H)-binding domain of Escherichia coli transhydrogenase: sequential assignment and global fold.

The soluble NADP(H)-binding domain of Escherichia coli transhydrogenase (186 amino acids, 20.4 kDa, rotational correlation time 14 ns) was characterized using NMR techniques. The global fold is similar to that of a classical dinucleotide-binding fold with six parallel beta-strands in a central sheet surrounded by helices and irregular structures, but is lacking both alphaD and alphaE. The substrate is bound in an extended conformation at the C-terminal end of the parallel beta-sheet and our data support the notion of a redox dependent structural rearrangement.

Macroscopic and radiographic examination of proximal root surface caries.

The purpose of the study was to compare macroscopic and radiographic examination of proximal root surface caries of extracted teeth from patients aged 65-95 years. Although the study conditions for macroscopic and radiographic diagnosis favored more sensitive evaluations than routine clinical conditions, there was a 24% disagreement in diagnosis. This finding indicates that under routine clinical conditions it is difficult to register with certainty all superficial root carious lesions. Even in the absence of clinically detectable root surface caries, preventive measures should be considered for elderly people with exposed root surfaces.