Transmenopausal changes in cortical bone quality.

Bone's resistance to fracture depends on its amount and quality, the latter including its structural and material/compositional properties. Bone material properties are dependent on bone turnover rates, which are significantly elevated immediately following menopause. Previously published data reported that following menopause, the amount of organic matrix synthesized at actively forming surfaces is significantly decreased, while glycosaminoglycan content was also modulated at resorbing surfaces, in the cancellous compartment. In the present study, we used Raman microspectroscopic analysis of paired iliac crest biopsies obtained before and shortly after menopause (1 year after cessation of menses) in healthy females to investigate changes in material/compositional properties due to menopause, in the cortical compartment. Specifically, the mineral/matrix ratio, the relative proteoglycan content, the mineral maturity/crystallinity, and the relative pyridinoline collagen cross-link content were determined at actively forming intracortical surfaces (osteons) as a function of tissue age, as well as in interstitial bone. Results indicated that it is the freshly synthesized organic matrix content that significantly declines following menopause, in agreement with what was previously reported for the cancellous compartment. This decline was not evident in the freshly deposited mineral content. None of the compositional/quality properties were altered following menopause either. Finally, no differences in any of the monitored parameters were evident in cortical interstitial bone.

Bone remodeling and bone matrix quality before and after menopause in healthy women.

Acceleration of remodeling activity after menopause leads to bone loss and fragility, however, whether this is associated with modifications of bone matrix quality has been less studied. The impact of variation in bone remodeling rate on bone matrix has been studied mainly in pathologies or anti-osteoporotic treatments. However, in healthy women this has been less studied. We analyzed, at the global level, bone matrix quality in bone biopsies from 3 groups of healthy women (20 per group): 1) before menopause (PreM), 2) 1 year after menopause (PostM, paired biopsies with preM), and 3) 14 (±9) years after menopause (LT-PostM). The mean degree of mineralization (DMB) and heterogeneity index (HI) of mineralization were assessed by X-ray microradiography on whole bone matrix; intrinsic properties (mineral/organic ratio, mineral maturity, mineral crystallinity, collagen maturity) were assessed by Fourier Transform Infrared microspectroscopy, microhardness by microindentation, both at a global level and calculated by mean of several measurements over the whole tissue area. In PostM compared to PreM (bone remodeling rate had doubled), mean DMB measured on the entire bone plane (whole bone matrix) of the sample was not different. HI was increased in trabecular bone indicating a higher heterogeneity of mineralization. However, in PostM, mineral/organic ratio (trabecular) and microhardness (cortical and trabecular) were decreased, whereas mineral/collagen maturation or crystal size/perfection were unchanged. Thus, in PostM, the local mineral content and microhardness were first affected. In LT-PostM (bone remodeling rate was 3 times higher), the mean DMB was still not different. However, the mineral/organic ratio, microhardness, mineral maturity, crystallinity all were lower compared to PreM and PostM, in both cortical and trabecular bone. Bone remodeling rate was negatively correlated with microhardness, DMB, mineral/organic and crystallinity. This suggests that increases in bone remodeling rates after menopause have a direct impact on bone quality by inducing the formation of more extensive "immature" bone areas, but the amount of immature bone does not cause modification of the global DMB.

Simultaneous XAFS measurements of multiple samples.

A four-channel ionization chamber has been designed, constructed and tested. This ionization chamber allows X-ray absorption spectra to be collected in transmission from up to four samples simultaneously. This results in spectra that are free of systematic uncertainty in relative energy alignment introduced by scan-to-scan stability of the monochromator or of numerical uncertainty associated with a post-processing alignment algorithm, allowing, in a single shot, an absolute measure of edge shift between four samples of different valence. As four samples can be measured in parallel, the time expended over the course of an experiment to cycle the measurement environment between its rest state and the measurement condition is substantially reduced. The ionization chamber is simple in design and could be implemented at virtually any XAFS beamline with a horizontal fan of radiation such as that provided by a bend magnet or wiggler.

Cancellous and cortical bone architecture and turnover at the iliac crest of postmenopausal osteoporotic women treated with parathyroid hormone 1-84.

Treatment with parathyroid hormone [PTH(1-84)] increases lumbar spine bone mineral density and decreases vertebral fractures, but its effects on bone microarchitecture are unknown. We obtained iliac crest biopsies from postmenopausal osteoporotic women given placebo (n=8) or 100 microg PTH(1-84) for 18 (n=8) or 24 (n=7) months to assess cancellous and cortical bone formation and structure. At 18 months, cancellous bone volume (BV/TV) measured by microcomputed tomography and histomorphometry was 45-48% higher in subjects treated with PTH(1-84) versus placebo, a result of higher trabecular number (Tb.N) and thickness. The higher Tb.N appeared to result from intratrabecular tunneling. Connectivity density was higher and structure model index was lower, indicating a better connected and more plate-like trabecular architecture. Cancellous bone formation rate (BFR) was 2-fold higher in PTH(1-84)-treated subjects, primarily because of greater mineralizing surface. Osteoblast and osteoid surfaces were a nonsignificant 58% and 35%, respectively, higher with PTH(1-84) treatment. Osteoclast and eroded surface were unaffected by PTH(1-84). There were no effects of PTH(1-84) treatment on cortical thickness, or endocortical or periosteal BFR, but cortical porosity tended to be higher. Although cancellous BFR was lower at 24 than at 18 months, measures of cancellous and cortical bone structure were similar at both timepoints. The bone produced by PTH(1-84) had normal lamellar structure and mineralization with no abnormal histology. In conclusion, when compared with placebo, treatment of osteoporotic women with PTH(1-84) was associated with higher BV/TV and trabecular connectivity, with a more plate-like architecture, all consistent with the lower vertebral fracture incidence.

Intratrabecular tunneling increases trabecular number throughout the skeleton of ovariectomized rhesus monkeys treated with parathyroid hormone 1-84.

Daily treatment of ovariectomized (OVX) adult rhesus monkeys with human parathyroid hormone (PTH) 1-84 for 16 months increases trabecular bone volume (BV/TV), number (Tb.N) and connectivity at lumbar vertebra-3 (L3) and thoracic vertebra-10. We proposed that the increased Tb.N and connectivity was achieved by stimulation of intratrabecular tunneling. Using histomorphometry to determine frequency of events, we have now quantified intratrabecular tunneling at L3 and extended it to investigate the effects of PTH(1-84) treatment on trabecular bone at the proximal femur, distal radius and iliac crest of these animals. At L3, tunneling frequency was low in control sham and OVX animals ( approximately 0.05/mm(2)) but increased significantly in PTH(1-84)-treated animals (0.27, 0.49 and 0.95/mm(2) with the 5, 10 and 25 microg/kg doses, respectively). Very similar tunneling frequencies were observed at all skeletal sites in all groups. Iliac crest biopsies were also collected at baseline and after 6 months of treatment and showed significant time- and dose-related increases in tunnels. Although the pattern and magnitude of response varied slightly from site to site, PTH(1-84) treatment significantly increased Tb.N, as well as BV/TV and bone formation rate at all skeletal sites. A modest but statistically significant increase in trabecular thickness occurred only at the iliac crest. In summary, intratrabecular tunneling is rare in control monkeys, but increased substantially with PTH(1-84) treatment. This phenomenon provides a plausible explanation for the PTH(1-84)-induced increase in Tb.N observed in OVX monkeys. Moreover, these analyses allowed a comparison of the effects PTH(1-84) treatment on trabecular bone at multiple locations.

Characterization of the flow of anisotropic colloidal particles using energy-dispersive X-ray diffraction.

The technique of energy-dispersive X-ray diffraction to study the orientation of microscopic crystalline particles dispersed in a liquid has been described recently. This complements previous neutron diffraction studies by permitting measurements at higher spatial resolution. Work with synchrotron radiation and high-energy X-rays has allowed studies on liquid dispersions flowing in pipes with a thickness of about 1 cm and a spatial resolution of 100 mum. Kaolinite is often found as a dispersion of monocrystalline, microscopic plates. The crystallographic layer structure is commensurate with the particle shape: the 00l direction is normal to the plane of the plates. Measurements of diffraction of the flowing liquid dispersion in a pipe oriented in various directions to the incident beam can be used to deduce the average orientation and order parameters of the particles. The competing effects of alignment with walls and in flow fields were observed. Further work has measured the orientation near a bend in a pipe.

Pt L-edge XANES as a probe of Pt clusters.

The sensitivity of Pt L-edge XANES to local geometric and electronic structure in various Pt(n) clusters is investigated using the ab initio self-consistent FEFF8 code. Calculations based on FEFF8 are found to be in good agreement with experiment. For pure Pt clusters the XANES can distinguish between 2- and 3-dimensional clusters. Self-consistency is important in determining the variation of XANES with cluster size. The effect of a support is also studied. In Pt-Cl(x) clusters the presence of a Cl-Pt bond leads to a "hybridization peak," i.e., a peak in the Cl d-density of states (d-DOS) mixed with Pt d-states. For Pt-H clusters hydrogen addition is well correlated with the growth of a broad shoulder on the white line. This change is attributed largely to AXAFS, i.e., to a corresponding change in the atomic background absorption.

Effect of hydrogen adsorption on the x-ray absorption spectra of small Pt clusters.

Hydrogen adsorption on Pt(6)H(n) clusters leads to striking changes in the Pt L(2,3) x-ray absorption spectra. These effects are interpreted using a self-consistent real space Green's function approach. Calculations show that they are due largely to changes in the atomic background contribution to x-ray absorption (i.e., atomic x-ray absorption fine structure) and to reduced Pt-Pt scattering at the edge, while Pt-H multiple scattering is relatively weak. The origin of both effects is traced to the change in the local Pt potential due to Pt-H bonding.

Factor V Leiden as a risk factor for miscarriage and reduced fertility.

The Leiden mutation is a recent discovery. It is the main cause of inherited thrombophilia and has been found in 20-60% of deep vein thrombosis cases. More recently it has been found in a significant number of cases of obstetric complications attributable to placental thrombosis. Current patient management practice for dealing with the Leiden mutation is based mainly on information about deep vein thrombosis because there is little information on pregnancy complications. There are no prospective studies examining the risk of developing pregnancy complications for Leiden mutation carriers. The aim of this study is to do that by comparing the frequency of unfavourable pregnancy outcomes among carriers with those among controls. The number of women developing miscarriages, intrauterine deaths, or infertility problems among 128 Leiden mutation carriers was compared with the number among 461 controls. The risk of having at least one miscarriage or infertility problems was 1.5 times greater for Leiden mutation carriers than controls. This result was statistically significant (95% CI 1.2, 2.7). The risk of having at least two miscarriages or infertility problems was 2.5 times greater for Leiden mutation carriers than controls. This was also statistically significant (95% CI 1.2, 5.13).

Conformations in solution of the fuscopeptins. Phytotoxic metabolites of Pseudomonas fuscovaginae.

Fuscopeptins are phytotoxic amphiphilic lipodepsipeptides containing 19 amino acid residues. They are produced by the plant pathogenic bacterium Pseudomonas fuscovaginae in two forms, A and B, which differ only in the number of methylene groups in the fatty acid chain. Their covalent structure and biological properties have been reported previously. CD and NMR spectroscopy investigations in solution revealed the absence of identifiable elements of secondary and tertiary structure for these molecules. Fuscopeptin B appears to be completely unstructured in aqueous solution, and has a large molecular flexibility. A dramatic conformational change was observed upon addition of trifluoroethanol. This study reports the complete interpretation of the two-dimensional NMR spectra and the NOE results obtained for fuscopeptin B in water/trifluoroethanol solutions; the signals relative to the peptidic moiety are identical to those observed for fuscopeptin A. The results of this investigation were used to determine the solution structure of fuscopeptin B by computer simulations applying distance geometry and simulated annealing procedures. In water/trifluoroethanol solutions the peptidic region appears to have a partly helical structure. The lactonic ring assumes defined conformations very similar to those already reported for other lipodepsipeptides. The structure for fuscopeptin B in solution is also valid for fuscopeptin A because of the negligible structural difference between the two metabolites.

NMR structure of an antisense DNA.RNA hybrid duplex containing a 3'-CH(2)N(CH(3))-O-5' or an MMI backbone linker.

The solution structure of an antisense DNA.RNA hybrid duplex, d(CGCGTT-MMI-TTGCGC).r(GCGCAAAACGCG) (designated R4), containing an MMI backbone linker [3'-CH(2)N(CH(3))-O5'], is elucidated. The structural details of the MMI linker, its structural effects on the neighboring residues, and the molecular basis of the MMI effects are examined. The lipophilic N-methyl group of MMI is peripheral to the helix, assuming a conformation that is most stable with regard to the N-O torsion angle. The MMI linker promotes a 3'-endo conformation for the sugar moieties at both 3'- and 5'-adjacent positions and a backbone kink involving distant residues along the 3'-direction. Comparison of R4 with other analogous hybrid duplexes previously studied in this laboratory reveals a new family of low-energy helical conformations that can be accommodated in stable duplexes and a common feature of C3'-modified sugars for adopting a C3'-endo pucker. The results of these studies emphasize the interplay of several factors that govern the formation of stable hybrid duplexes and provide a basis for the understanding of the biological role of the MMI modifications, which are important building blocks for a family of promising chimeric antisense oligonucleotides.

An experimental design approach to the optimisation of a flow injection analysis method for glycine.

A flow injection analysis method for the determination of glycine, based on the reaction with ortho-phtalaldehyde and N-acetylcysteine in a basic buffer, was optimised. In the first step screening of the variables, to select the most important ones, was performed using: (i) a half-fraction factorial design and (ii) a quarter-fraction factorial design, for five factors at two levels. The effects of the factors on the peak height were calculated from both screening designs and compared. For the half-fraction factorial design (resolution IV), the significance of the factor effects on the peak height was checked by: (i) comparing them with a critical effect, calculated from two-factor interactions and based on a t-test, (ii) using a non parametric approach and (iii) drawing a normal probability plot. For the quarter-fraction factorial design (resolution III) the significance of the effects of the factors on the peak height was checked using: (i) a randomization test method, (ii) the non parametric method and (iii) a normal probability plot. In the second step, the factor found to be of importance was optimised using the uniplex method.