We examined the influence of orthopaedic material and computerized high-density detection (HDD) on analysis of bone mass and soft tissue composition performed by dual-energy X-ray absorptiometry (DXA). Measurements of total and regional bone area, bone mineral content (BMC), areal bone mineral density (BMD), lean tissue mass (LTM) and fat tissue mass (FTM) were made using a Norland XR-26 DXA scanner with dynamically changing samarium filtration. Twenty-one subjects who were free of metal implants were measured without and with a Biomet femoral prosthesis (titanium) placed on the proximal part of the femoral region. Twenty-one women with an endogenous prosthesis in the proximal femur were measured once. Analyses of tissue composition were performed without and with HDD using software provided by the manufacturer. Measurements were considerably affected by exogenous metal with overestimation of LTM and underestimation of FTM and bone area. BMC and BMD were over- or underestimated depending on the anatomical region. Enabling the HDD mode, values of bone area and tissue mass came closer to the expected values (-metal/-HDD) but were in general still significantly different from these. For the total body, the following significant changes were found after application of metal (+metal/-HDD vs. +metal/+HDD, mean values): bone area -19.8% vs. -6.9%, BMC +1.1% vs. -2.1%, BMD +26.5% vs. +4.7%, LTM +12.4% vs. +3.7%, FTM -15.8% vs. -7.0%. A similar pattern of change in tissue composition and bone area was found for the subregions of the body. Changes in tissue composition after HDD were similar in subjects with exogenous and endogenous metal, indicating that the experimental model was appropriate. In conclusion, measurements of tissue composition were substantially influenced by orthopaedic metal. HDD partly corrected for the artefacts induced by the metal.

Specific cleavage of RNA is catalysed by short oligodeoxynucleotides termed DNAzymes. DNAzymes consist of two binding arms that hybridize to a predetermined RNA sequence and a catalytic core that cleaves a phosphodiester bond held between the binding arms. DNAzymes are exemplified by the well-studied 10-23 DNAzyme, which compared with protein ribonucleases is highly specific, albeit slow. Here we report a significant improvement in cleavage kinetics, while maintaining specificity, by incorporation of LNA (locked nucleic acid) and alpha-L-LNA nucleotides into the binding arms of 10-23 DNAzyme. DNAzymes modified in this way (LNAzymes) enhance cleavage of a phosphodiester bond presented in a short RNA substrate as well as in longer and highly structured substrates, and efficient cleavage is maintained from single- to multiple-turnover conditions. Analysis of the cleavage reaction indicates that substrate hybridization is boosted by the presence of the locked residues within the LNAzymes, while no apparent change occurs in the catalytic strand-scission step.

A titration study of the dimeric Asp(B9) mutant of human insulin was performed using two-dimensional NMR spectroscopy. Based on 10 NOESY spectra recorded in the pH range 1.73-3.93, the pKa values of the seven carboxyl groups in the mutant were determined, and the titration shifts of 46 pH-dependent protons in non-ionizable groups were investigated. Further, the pKa values of the two histidine imidazole rings were determined from a series of 1D spectra recorded in the pH range 6.65-10.0. The titration shifts of all pH-dependent protons were analyzed by a nonlinear least-squares fitting procedure, using an equation that describes a one-step titration. Also the pH dependence of the exchange rate of the amide proton of Phe(B24) was determined in the applied pH range. On the basis of the experimental results, it is concluded that the Asp(B9) residue forms an N-cap of the B-chain alpha-helix through an interaction between the side-chain carboxyl group of the residue and the dipole of the helix. Further, the titration data show that salt bridges are established between Glu(B13) and His(B10) and between Asn(A21) and Arg(B22) at pH values, where the interacting groups are ionized, and that a hydrogen bond exists between the amide proton of Val(A3) and the C-terminal carboxyl group of Thr(B30). Most surprisingly, the data analysis shows that the Asp(B9) insulin exists as a dimer throughout the investigated pH range, that is, also at pH values where there is a substantial negative charge repulsion in the monomer-monomer interface of the dimer.

A new 2D NMR pulse sequence for E.COSY-type measurement of J(HH) coupling constants is introduced. It exploits a heteronuclear spin, e.g., 13C, for displacement in the omega(1) frequency dimension via a large heteronuclear J coupling. The experiment is demonstrated by application to a heptapeptide at the natural abundance 13C level. It is suitable, for example, for measurement of 3J(HH) and 4J(HH) coupling constants in peptides and proteins.