bluuebunny/crossref_metadata_2025 huggingface dataset:
Learning to mark: a qualitative study of the experiences and concerns of medical markers
Authors: Hawthorne Kamila , Wood Fiona , Hood Kerenza , Cannings-John Rebecca , Houston Helen
Published: April 2006

Background

Although there is published research on the methods markers use in marking various types of assessment, there is relatively little information on the processes markers use in approaching a marking exercise. This qualitative paper describes the preparation and experiences of general practice (GP) teachers who undertake marking a written assessment in an undergraduate medical course.

Methods

Semi-structured interviews were conducted with seven of the 16 GP tutors on an undergraduate course. The purposive sample comprised two new markers, two who had marked for a couple of years and three experienced markers. Each respondent was interviewed twice, once following a formative assessment of a written case study, and again after a summative assessment. All interviews were audio-taped and analysed for emerging themes. A respondent validation exercise was conducted with all 16 GP tutors.

Results

Markers had internal concerns about their ability to mark fairly and made considerable efforts to calibrate their marking. They needed guidance and coaching when marking for the first time and adopted a variety of marking styles, reaching a decision through a number of routes. Dealing with pass/fail borderline scripts and the consequences of the mark on the student were particular concerns. Even experienced markers felt the need to calibrate their marks both internally and externally

Conclusion

Previous experience of marking appears to improve markers' confidence and is a factor in determining the role which markers adopt. Confidence can be improved by giving clear instructions, along with examples of marking. The authors propose that one method of providing this support and coaching could be by a process of peer review of a selection of papers prior to the main marking. New markers in particular would benefit from further guidance, however they are influenced by others early on in their marking career and course organisers should be mindful of this when arranging double marking.
CREB Activity Maintains the Survival of Cingulate Cortical Pyramidal Neurons in the Adult Mouse Brain
Authors: Ao Hushan , Ko Shanelle W , Zhuo Min
Published: April 2006

Cyclic AMP-responsive element binding protein (CREB) activity is known to contribute to important neuronal functions, such as synaptic plasticity, learning and memory. Using a microelectroporation technique to overexpress dominant negative mutant CREB (mCREB) in the adult mouse brain, we found that overexpression of mCREB in the forebrain cortex induced neuronal degeneration. Our findings suggest that constitutively active CREB phosphorylation is important for the survival of mammalian cells in the brain.
Ca2+-linked upregulation and mitochondrial production of nitric oxide in the mouse preimplantation embryo
Authors: Manser Rosemary C. , Houghton Franchesca D.
Published: April 2006

Previous studies have demonstrated a role for the signalling agent nitric oxide in regulating preimplantation embryo development. We have now investigated the biochemical mode of action of nitric oxide in mouse embryos in terms of mitochondrial function and Ca2+ signalling. DETA-NONOate, a nitric oxide donor, decreased day 4 blastocyst cell number and oxygen consumption, consistent with a role for nitric oxide in the inhibition mitochondrial cytochrome c oxidase. Using live cell imaging and the nitric-oxide-sensitive probe DAF-FM diacetate, nitric oxide was detected at all stages of preimplantation development and FRET analysis revealed a proportion of the nitric oxide to be colocalised with mitochondria. This suggests that mitochondria of preimplantation embryos produce nitric oxide to regulate their own oxygen consumption. Inhibiting or uncoupling the electron transport chain induced an increase in nitric oxide and [Ca2+]i as well as disruption of Ca2+ deposits at the plasma membrane, suggesting that mitochondrial disruption can quickly compromise cellular function through Ca2+-stimulated nitric oxide production. A link between antimycin-A-induced apoptosis and nitric oxide signalling is proposed.
Maximization of the Chemiluminescence Efficiency of 1,4,5-Triarylhydroperoxy-4H-isoimidazoles
Authors: Kimura Masaru , Nishikawa Hideki , Kura Hisanori , Lim Hyung , White Emil H
Published: April 2006

A bell shape correlation curve has been found for a plot of the Hammett σ constant and the quantum yields of the formation of excited amidines from the chemiluminescent reaction of 2-(p-dimethylaminophenyl)-4,5-di(p-X–phenyl)-4-hydroperoxy-4H-isoimidazoles (X = OCH3, H, F, Cl, CF3, and CN) with bases.
Stereoselective Synthesis of O-(2-Azido-2-deoxy-α-D-galactopyranosyl)-L-serine and -L-threonine Derivatives by Using the Catalytic Amount of Active Acidic Species
Authors: Matsubara Koki , Mukaiyama Teruaki
Published: April 2006

In the presence of a catalytic amount of SnCl3(ClO4) or SiCl3(ClO4), O-(2-azido-2-deoxy-α-D-galactopyranosyl)-L-serine and -L-threonine are stereoselectively synthesized in excellent yields from 1-O-acetyl-2-azido-2-deoxy-3,4,6-tri-O-benzyl-D-galactopyranose and trimethylsilylated L-serine or L-threonine, respectively.
Novel Tetranuclear Nickel(II) Complex with a Mixed-Spin State Generated by Pairing of Dimers
Authors: Mikuriya Masahiro , Nakadera Keiichi , Kotera Takanori
Published: April 2006

A tetranuclear nickel(II) complex with 2,6-bis(salicylideneaminomethyl)-4-methylphenol (H3L), [Ni4(L)2(pz)2(CH3OH)] (pz = pyrazolate ion) has been synthesized and characterized by electronic spectra and magnetic susceptibilites (80–300 K). The X-ray crystal structure analysis has demonstrated that it consists of tetranuclear (low-spin–high-spin–high-spin–low-spin) molecules with a zigzag structure.
Trans → cis One-way Photoisomerization of an Olefin Substituted with an Indole and a Pyridine Ring. Effect of Intramolecular Hydrogen Bond
Authors: Arai Tatsuo , Iwasaki Takeshi , Tokumaru Katsumi
Published: April 2006

On direct irradiation in benzene 2-[2-(2-pyridyl)ethenyl]indole undegoes trans-to-cis one-way isomerization in the excited singlet state.
One-Way Photoisomerization of Dienes Substituted by an Anthracene Nucleus
Authors: Gong Yuefa , Arai Tatsuo , Tokumaru Katsumi
Published: April 2006

On benzil sensitization, 1-(2-anthryl)-1-cis,3-trans-pentadiene and cis-1-(2-anthryl)-4-methyl-1,3-pentadiene underwent one-way cis → trans isomerization with high quantum efficiencies in an adiabatic process at the excited triplet state.
Steady-State Flow Analysis. A New Approach to Rapid and Sensitive Analysis for Long-Period Reactions in Continuous-Flow System
Authors: Zhang Chongliang , Narusawa Yoshio , Kitahama Shizuo
Published: April 2006

Air-segmented flow analysis, flow-injection analysis and sequential-injection analysis which characterized with sample zone dispersion and/or sequentially, do not suit to an analysis for long-period reactions because of their sharp decreases in analytical speed and sensitivity. A new approach, based on the combined techniques of a parallel double-line FIA, a stopped-flow FIA and a steady-state determination model, is proposed to develop a rapid and sensitive method for filling a gap of the application to long-period reactions in the present continuous-flow analysis.
Preparation of Partially Protected Peptide Thioesters Containing a Cysteine Residue and Their Segment Condensation
Authors: Kwon Yeondae , Zhang Ruoheng , Bemquerer Marcelo P , Tominaga Mineko , Hojo Hironobu , Aimoto Saburo
Published: April 2006

Methods for the preparation of partially-protected cysteine-containing peptide segments were developed. The peptide thioester, obtained via a solid-phase method of Boc chemistry, was used for segment condensation with an amino component peptide in the presence of silver ions and N-hydroxysuccinimide to give a desired product in good yield.
One-pot Preparation of 2-Chloromethyldioxolanes and 2-Aminothiazoles from Chloromethyltrioxanes
Authors: Wakasugi Takashi , Miyakawa Tadashi , Suzuki Fukuichi , Itsuno Shinichi , Ito Koichi
Published: April 2006

Thermal degradation of chloromethyltrioxanes in the presence of catalytic amount of montmorillonite clay generated α-chloroaldehydes with high purity, which were treated in situ with ethylene glycol or thiourea to afford 2-chloromethyldioxolanes and 2-aminothiazoles, respectively. The clay catalysts used were removed by filtration.
Synthesis and Structure of a Pyridine Complex of Trinuclear Molybdenum Cluster [Mo3S4Cl4(py)5]
Authors: Mizutani Jun , Imoto Hideo , Saito Taro
Published: April 2006

A pyridine complex of trinuclear molybdenum chloro sulfido cluster [Mo3S4Cl4(py)5] has been synthesized by the excision reaction of a solid state cluster compound Mo3S7Cl4 with pyridine and triphenylphosphine. The structure has been determined by the single crystal X-ray structure analysis.
Poly(phenylenevinylene) Bearing Built-in tert-Butylnitroxide. A Polyradical Ferromagnetically Coupled in the Intrachain
Authors: Kaneko Takashi , Toriu Shuichi , Kuzumaki Yoshihiro , Nishide Hiroyuki , Tsuchida Eishun
Published: April 2006

Poly(1,4- or 1,2-phenylenevinylene)s bearing a 2- or 4-substituted nitroxide radical were synthesized by polymerizing [bromo[N-tert-butyl-N-(trialkylsiloxy)amino]]styrenes with a palladium catalyst. The polyradicals were chemically stable, and their ESR and SQUID measurements indicated an intramolecular and through-bond ferromagnetic coupling.
High Resolution Solid State NMR in Liquids 3. 1H NMR Study of Organic Nano-Particles
Authors: Satoh Naoki , Kimura Keisaku
Published: April 2006

High resolution 1H NMR spectra of terephthalic acid (TPA) ultrafine particles (UFP’s) were obtained by means of motional narrowing caused by Brownian motion of UFP’s in a liquid phase of CCl4. In the case of dispersing condition of UFP’s, highly resolved signals from benzoic protons were obtained at 6.85, 6.95, 7.08, 7.25, 7.50 and 7.65 ppm with respect to tetramethylsilane (TMS), even though only a signal at 8.15 ppm was obtained from moleculary dispersed TPA in acetone. Particle size was obtained to be ca. 15 nm by a dynamic laser scattering method.
Reaction of Lanthanum(III) and Gadolinium(III) Complexes Coordinated by Pendant-armed Macrocyclic Ligand with Salicylaldehyde
Authors: Kanesato Masatoshi , Yokoyama Toshiro , Itabashi Osamu
Published: April 2006

In the reaction of Lanthanum(III) and Gadolinium(III) complexes coordinated by the ligand 1,9-bis(2-aminoethyl)-1,4,6,9,12,14-hexaazacyclohexadecane with salicylaldehyde, only Gd3+ was led to an unexpected complex with the ligand tris{2-(salicylideneamino)ethyl}amine. The difference of the reactivity was applied to the separation of La3+ and Gd3+.
Reactions of Alkyl malonates with Molybdenum and Tungsten Complexes. Syntheses and Structure of Hydrido-Malonato Complexes
Authors: Minato Makoto , Kurishima Susumu , Nagai Ko-ichiro , Yamasaki Mikio , Ito Takashi
Published: April 2006

Molybdenum and tungsten hydrido-malonato complexes have been prepared by the photochemical or thermal reactions of [MoH4(dppe)2] or trans-[W(N2)2(dppe)2] with an excess of alkyl malonates. The structure of the complex is reported, and a possible mechanism is proposed for the reaction.
Separation of Lead(II) by Solvent Extraction and Membrane Transport with a Dibenzopolyether Dicarboxylic Acid Ionophore
Authors: Hayashita Takashi , Fujimoto Takashi , Morita Yuki , Bartsch Richard A
Published: April 2006

The lipophilic di-ionizable ionophore 1,5-Bis[2-(2′-carboxyheptyloxy)phenoxy]-3-oxapentane (1) provides a selective solvent extraction of Pb(II) from Cu(II). The stoicheometry of the extracted species is determined to be 1:1 by equilibrium analysis. Ionophore 1 also functions as a carrier in polymeric plasticizer membrane for separation of Pb(II) from Cu(II).
JunB as a Downstream Mediator of PTHrP Actions in Cementoblasts
Authors: Berry Janice E , Ealba Erin L , Pettway Glenda J , Datta Nabanita S , Swanson Erica C , Somerman Martha J , McCauley Laurie K
Published: April 2006

The role of AP-1 family members in the action of PTHrP was examined in cementoblasts. PTHrP increased mRNA and protein levels of all Fos members, but only one Jun member (JunB) was increased. Overexpression of JunB in cementoblasts mimicked actions of PTHrP to support osteoclastogenesis and inhibit cementoblast differentiation, suggesting that the actions of PTHrP on mesenchymal cells operate through JunB.

Introduction: Cementoblasts are mesenchymal cells that share phenotypic features with osteoblasts in vitro; however, unlike osteoblasts, cementoblasts rarely support osteoclastogenesis in vivo. The osteoblast-mediated support of osteoclastogenesis involves PTH-induced reduction in osteoprotegerin (OPG) expression. PTH acts on osteoblastic cells through specific signaling pathways and transcription factors such as activator protein 1 (AP-1). The purpose of this study was to determine the impact of PTH-related protein (PTHrP) on AP-1 transcription factors in cementoblasts and the role of JunB in the actions of PTHrP.

Materials and Methods: Cementoblastic cells were treated with PTHrP and evaluated for mRNA and protein levels of AP-1 family members. Stable transfectants of OCCM cells overexpressing JunB were evaluated for OPG production, ability to support osteoclastogenesis, and measures of proliferation and differentiation.

Results: PTHrP treatment in vitro resulted in a time-dependent upregulation of mRNA and proteins for the Fos family members, but only JunB of the Jun family. OPG mRNA and protein levels were reduced by PTHrP in OCCM and were lower in JunB overexpressing cells than controls. In co-culture experiments, TRACP+ cells were increased with RANKL treatment in JunB overexpressing cells compared with controls. Cementoblast differentiation was reduced with overexpression of JunB as measured by a decrease in mineralized nodule formation and gene expression for bone sialoprotein and osterix. Measures of proliferation including cell number and cyclin D1 levels were increased in JunB overexpressing clones. In vivo, cementoblast implants exhibited a cementoblastoid nature with copious mineral-like matrix, whereas JunB-overexpressing implants were densely cellular with little mineralized matrix.

Conclusions: JunB was the only Jun family member increased by PTHrP, and its overexpression showed similar patterns of gene expression and OPG production as PTHrP treatment of controls. These data suggest that JunB may be a key mediator of PTHrP actions in cementoblasts.
Suppression of Adjuvant-Induced Arthritic Bone Destruction by Cyclooxygenase-2 Selective Agents With and Without Inhibitory Potency Against Carbonic Anhydrase II
Authors: Katagiri Mika , Ogasawara Toru , Hoshi Kazuto , Chikazu Daichi , Kimoto Aishi , Noguchi Masahiro , Sasamata Masao , Harada Shun-ichi , Akama Hideto , Tazaki Hatsue , Chung Ung-il , Takato Tsuyoshi , Nakamura Kozo , Kawaguchi Hiroshi
Published: April 2006

In vitro assays revealed that COX-2 inhibitors with CA II inhibitory potency suppressed both differentiation and activity of osteoclasts, whereas that without the potency reduced only osteoclast differentiation. However, all COX-2 inhibitors similarly suppressed bone destruction in adjuvant-induced arthritic rats, indicating that suppression of osteoclast differentiation is more effective than that of osteoclast activity for the treatment.

Introduction: Cyclooxygenase (COX)-2 and carbonic anhydrase II (CA II) are known to play important roles in the differentiation of osteoclasts and the activity of mature osteoclasts, respectively. Because several COX-2 selective agents were recently found to possess an inhibitory potency against CA II, this study compared the bone sparing effects of COX-2 selective agents with and without the CA II inhibitory potency.

Materials and Methods: Osteoclast differentiation was determined by the mouse co-culture system of osteoblasts and bone marrow cells, and mature osteoclast activity was measured by the pit area on a dentine slice resorbed by osteoclasts generated and isolated from bone marrow cells. In vivo effects on arthritic bone destruction were determined by radiological and histological analyses of hind-paws of adjuvant-induced arthritic (AIA) rats.

Results: CA II was expressed predominantly in mature osteoclasts, but not in the precursors. CA II activity was inhibited by sulfonamide-type COX-2 selective agents celecoxib and JTE-522 similarly to a CA II inhibitor acetazolamide, but not by a methylsulfone-type COX-2 inhibitor rofecoxib. In vitro assays clearly revealed that celecoxib and JTE-522 suppressed both differentiation and activity of osteoclasts, whereas rofecoxib and acetazolamide suppressed only osteoclast differentiation and activation, respectively. However, bone destruction in AIA rats was potently and similarly suppressed by all COX-2 selective agents whether with or without CA II inhibitory potency, although only moderately by acetazolamide.

Conclusions: Suppression of osteoclast differentiation by COX-2 inhibition is more effective than suppression of mature osteoclast activity by CA II inhibition for the treatment of arthritic bone destruction.
The 25-Hydroxyvitamin D 1-Alpha-Hydroxylase Gene Maps to the Pseudovitamin D-Deficiency Rickets (PDDR) Disease Locus
Authors: St-Arnaud René , Messerlian Serge , Moir Janet M. , Omdahl John L. , Glorieux Francis H.
Published: April 2006

Pseudovitamin D-deficiency rickets (PDDR) is an autosomal recessive disorder that may be due to impaired activity of 25-hydroxyvitamin D-1α-hydroxylase, a renal cytochrome P450 enzyme (P4501α) of the vitamin D pathway. The disease locus for PDDR has been mapped by linkage analysis to 12q13-q14, but the molecular defect underlying the enzyme dysfunction has remained elusive due to the lack of sequence information for the P4501α gene (hereafter referred to as 1α-OHase). We have used a probe derived from the rat 25-hydroxyvitamin D-24-hydroxylase (CYP24; 24-OHase) sequence to identify and clone the 1α-OHase cDNA. The full-length 1α-OHase clone of 2.4 kb codes for a protein of predicted Mr 55 kDa. Functional activity of the cloned sequence was assessed using transient transfection, and the production of authentic 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] was confirmed using high performance liquid chromatography fractionation and time-of-flight mass spectrometry. The expression of the gene was analyzed in vitamin D–replete animals; treatment with 1α,25(OH)2D3 reduced 1α-OHase transcript levels by 70%, while administration of parathyroid hormone led to a 2-fold increase in the expression of the gene, thus confirming the hormonal regulation previously described using biochemical methods. The rat cDNA was used to obtain a human genomic clone. Interestingly, the human 1α-OHase gene mapped to 12q13.1-q13.3, providing strong evidence that a mutation in the 1α-OHase gene is responsible for the PDDR phenotype. The availability of a cloned sequence for 1α-OHase generates novel tools for the study of the molecular etiology of PDDR, and will allow the investigation of other disturbances of vitamin D metabolism.
Different Morphometric and Densitometric Parameters Predict Cervical and Trochanteric Hip Fracture: The EPIDOS Study
Authors: Duboeuf F. , Hans D. , Schott A. M. , Kotzki P. O. , Favier F. , Marcelli C. , Meunier P. J. , Delmas P. D.
Published: April 2006

We used an experimental software measuring the hip axis length (HAL) and bone mineral density (BMD) in specific regions of the lower and upper part of the femoral neck on dual-energy X-ray absorptiometry scans. To determine whether these parameters were significant predictors of the type of hip fracture, we measured 167 healthy women (controls), 24 women with trochanteric, and 42 women with cervical hip fractures within the EPIDOS prospective cohort. EPIDOS is a multicenter prospective study on risk factors for hip fracture performed in 7575 elderly women living at home, aged 75–95 and conducted in five French centers (Amiens, Lyon, Montpellier, Paris, Toulouse). Measurements were performed on data acquired at baseline before the occurrence of fracture. In the cervical fracture group, HAL was significantly longer than in controls (94.2 vs. 92.3, p = 0.03), and the associated odds ratio (OR) adjusted for age, weight, and total femoral neck BMD was significant (OR = 1.64, 95% confidence interval [CI] 1.06–2.55). In contrast, HAL was not significantly different from controls in the trochanteric fracture group. Femoral neck diameter was not a predictor of fracture. The upper and lower femoral neck BMD was lower in the trochanteric fracture group than in controls, and both measurements predicted trochanteric femoral neck fracture. In contrast, the prediction of cervical femoral neck fracture was enhanced by measuring only the upper part of the femoral neck (OR = 2.79 vs. 1.97 for the total femoral neck) while BMD of the lower part was not different from controls. Hip axis length is a predictor of femoral neck fracture. Femoral neck BMD distribution is different between cervical and trochanteric fractures. These results support the hypothesis of a different pathophysiological mechanism between the two types of hip fractures.
Influence of the Vitamin D Receptor Gene Alleles on Bone Mineral Density in Postmenopausal and Osteoporotic Women
Authors: Dr. Vandevyver Caroline , Wylin Tine , Cassiman Jean-Jacques , Raus Jef , Geusens Piet
Published: April 2006

It is well established that genetic factors contribute to bone turnover and bone density. Evidence exists suggesting that a major part of this genetic influence may be due to polymorphisms in the vitamin D receptor (VDR) gene. However, it is not clear whether the VDR genotype effect persists in elderly women. In the present study, the relationship between the BsmI, ApaI, and TaqI polymorphisms in the VDR gene, and the bone mineral density (BMD) at the lumbar spine, the femoral neck (FN), and the proximal radius was investigated in a large group of elderly women (75.5 ± 5.0 years) of Caucasian origin and in 84 Type I osteoporotic women (66.6 ± 8.4 years). We did not find a correlation between the VDR genotypes and BMD in elderly women. However, a significantly higher FN-BMD was observed in obese (body mass index [BMI] > 30 kg/m2) versus nonobese (BMI < 30 kg/m2) women (p < 0.01). This relationship was observed for all BsmI genotypes. Furthermore, the FN-BMD of nonobese women with bb BsmI genotype was 5% higher than that of women with the BB genotype (p = 0.04). We conclude that the VDR gene polymorphisms influence the FN-BMD in nonobese postmenopausal women. In a second part of the study, possible correlations between the VDR gene polymorphisms and osteoporosis Type I were analyzed. Our data could not reveal any association between these parameters.
Three-Fold Induction of Renal 25-Hydroxyvitamin D3-24-Hydroxylase Activity and Increased Serum 24,25-Dihydroxyvitamin D3 Levels Are Correlated with the Healing Process After Chick Tibial Fracture
Authors: Seo Eun-Gyoung , Prof. Norman Anthony W.
Published: April 2006

To investigate the possible biological actions of 24,25-dihydroxyvitamin D3 (24,25(OH)2D3), a tibial fracture-healing model was established in White Leghorn chicks. Three-week-old White Leghorn chicks fed a vitamin D3–replete diet were divided into four groups (control, anesthetized, sham, and fractured). On varying days after tibial fracture (F) or sham manipulation (S), renal 25(OH)D3-1α-hydroxylase and 25(OH)D3-24-hydroxylase (24-hydroxylase) activities and serum Ca2+ concentrations were measured. Metofane anesthesia was found to have no effect on the activity of either of the hydroxylases; the activities of the hydroxylases in the control, anesthetized, and sham-operated birds were similar. By 10 days after tibial fracture, the renal 24-hydroxylase activity increased more than 3-fold in F (1.33 ± 0.07 pmol/mg of protein) as compared with S (0.42 ± 0.03 pmol/mg of protein) (p < 0.0001). A time-dependent study of the renal 24-hydroxylase activity during the fracture repair process revealed a slow increase from the first day after fracture, a higher activity at 8 days, which peaked at 10–11 days, which is consistent with the formation of the callus. The 24-hydroxylase activity then returned to the same level as the sham group 14 days after fracture. There was no significant difference in serum Ca2+ levels between the F and S groups over the 3-week postfracture period. Serum levels of vitamin D3 metabolites were also measured during the fracture healing process: a 3.4× increase of the 24,25(OH)2D3 level in the fractured group (3.64 ± 1.16 nM) was observed as compared with the control groups (1.08 ± 0.49 nM) at 10 days after fracture (p = 0.068). No significant differences were observed in the plasma levels of 25(OH)D3 or 1α,25(OH)2D3 between the group with a fracture and the controls. Exposure of primary chick kidney cells in culture to serum obtained from chicks with a tibial fracture for 20 h resulted in an ∼40% increase in the activity of the 24-hydroxylase as compared with cells exposed to serum from control birds. These results suggest that 24,25(OH)2D3 is involved in the early process of fracture repair and that there is some form of physiological communication between the fractured bone and the kidney so as to increase the renal 24-hydroxylase and the circulating concentration of this metabolite.
Quantitative Ultrasound of the Calcaneus Reflects the Mechanical Properties of Calcaneal Trabecular Bone
Authors: Bouxsein M. L. , Radloff S. E.
Published: April 2006

To provide new information about the potential role of quantitative ultrasound (QUS) in fracture risk prediction, we assessed whether QUS and densitometric variables derived from measurements of intact cadaveric feet were associated with the mechanical properties of calcaneal trabecular bone. We obtained 31 intact cadaveric feet from the local anatomic gifts program, including 13 men and 18 women, with a mean age of 77 years (range 50–91 years). Broadband ultrasound attenuation (BUA) and ultrasound transit velocity (SOS) were assessed in the intact cadaveric feet using a water-based ultrasound system. In addition, we measured the bone mineral density (BMD) of the posterior calcaneus using dual-energy X-ray absorptiometry. Cubes of trabecular bone were then removed from the calcaneus at approximately the same location as the QUS measurements were obtained, and the elastic modulus and ultimate strength of the trabecular bone specimens were measured by compressing them in the mediolateral direction. We found that QUS variables were moderately to strongly correlated with the mechanical properties of calcaneal trabecular bone (r2 = 0.48–0.63, p < 0.001 for all). However, the strongest associations with the mechanical properties trabecular bone were provided by calcaneus BMD and trabecular bone apparent density (r2 = 0.66–0.88). BUA and densitometric measurements were independently associated with elastic modulus, but not with ultimate strength. Our results indicate that QUS measurements of the intact heel are associated with the mechanical properties of calcaneal trabecular bone and, in some cases, provide information in addition to that provided by BMD or density measurements.
Characterization of Native and Recombinant Bone Sialoprotein: Delineation of the Mineral-Binding and Cell Adhesion Domains and Structural Analysis of the RGD Domain
Authors: Stubbs John T. , Mintz Keith P. , Eanes Edward D. , Torchia Dennis A. , Fisher Larry W.
Published: April 2006

Bone sialoprotein is a small, sulfated, and phosphorylated integrin-binding glycoprotein apparently found only in tissues that eventually mineralize. Nondenatured bone sialoprotein (BSP) purified from rat osteosarcoma cell line (UMR 106–01 BSP) culture media is shown to have a hydroxyapatite Kd ≈ 2.6 × 10−9 M, perhaps the strongest affinity for this mineral of any of the matrix proteins. Both native BSP and a 47 kD fragment of UMR-BSP (Fragment 1 ∼133A–∼265Y) are more potent inhibitors of seeded hydroxyapatite crystal growth than recombinant human BSP fragments lacking post-translational modifications. The recombinant proteins, however, do show reproducible inhibitory activity, suggesting that at least some of the strong mineral-binding properties are encoded directly within the protein sequence itself. BSP facilitates the adhesion of several cell types through its integrin binding (RGD) tripeptide sequence. Nuclear magnetic resonance (NMR) analysis of a15N-enriched 59 amino acid recombinant domain containing the RGD tripeptide shows that the structure of this isolated domain is highly flexible with or without 5 mM calcium. Previous work has also shown that an endogenous fragment of UMR-BSP (Fragment 1) supports cell adhesion in the absence of the RGD sequence. In this report, non-RGD cell adhesion sites are localized within conserved amino- and carboxy-terminal tyrosine-rich domains of recombinant human BSP. Given the proximity of the latter non-RGD cell adhesion site to the RGD tripeptide, a model of BSP-receptor interactions is presented.
Clodronate and Liposome-Encapsulated Clodronate Are Metabolized to a Toxic ATP Analog, Adenosine 5′-(β,γ-Dichloromethylene) Triphosphate, by Mammalian Cells In Vitro
Authors: Frith Julie C. , Mönkkönen Jukka , Blackburn G. Michael , Russell R. Graham G. , Rogers Michael J.
Published: April 2006

Clodronate, alendronate, and other bisphosphonates are widely used in the treatment of bone diseases characterized by excessive osteoclastic bone resorption. The exact mechanisms of action of bisphosphonates have not been identified but may involve a toxic effect on mature osteoclasts due to the induction of apoptosis. Clodronate encapsulated in liposomes is also toxic to macrophages in vivo and may therefore be of use in the treatment of inflammatory diseases. It is generally believed that bisphosphonates are not metabolized. However, we have found that mammalian cells in vitro (murine J774 macrophage-like cells and human MG63 osteosarcoma cells) can metabolize clodronate (dichloromethylenebisphosphonate) to a nonhydrolyzable adenosine triphosphate (ATP) analog, adenosine 5′-(β,γ-dichloromethylene) triphosphate, which could be detected in cell extracts by using fast protein liquid chromatography. J774 cells could also metabolize liposome-encapsulated clodronate to the same ATP analog. Liposome-encapsulated adenosine 5′-(β,γ-dichloromethylene) triphosphate was more potent than liposome-encapsulated clodronate at reducing the viability of cultures of J774 cells and caused both necrotic and apoptotic cell death. Neither alendronate nor liposome-encapsulated alendronate were metabolized. These results demonstrate that the toxic effect of clodronate on J774 macrophages, and probably on osteoclasts, is due to the metabolism of clodronate to a nonhydrolyzable ATP analog. Alendronate appears to act by a different mechanism.
Articular Chondrocytes Produce Factors That Inhibit Maturation of Sternal Chondrocytes in Serum-Free Agarose Cultures: A TGF-β Independent Process
Authors: D'Angelo Marina , Pacifici Maurizio
Published: April 2006

Under normal conditions, articular chondrocytes persist throughout postnatal life, whereas “transient” chondrocytes, which constitute the bulk of prenatal and early postnatal cartilaginous skeleton, undergo maturation, hypertrophy, and replacement by bone cells. The mechanisms regulating the markedly different behavior and fate of articular and transient chondrocytes are largely unclear. In the present study, we asked whether articular chondrocytes possess dominant antimaturation properties which may subtend their ability to persist throughout life. Adult chicken articular chondrocytes and transient maturing chondrocytes from the core region of day 17 chick embryo cephalic sternum were cultured or cocultured in serum-free agarose conditions. When the sternal cells were grown by themselves, they quickly developed into hypertrophic type X collagen-synthesizing cells; however, when they were cocultured with as few as 10% articular chondrocytes or fed with articular chondrocyte-conditioned medium, their maturation was markedly impaired, as revealed by a sharp drop in type X collagen synthesis. A similar, albeit less potent, antimaturation activity characterized resting and proliferating immature chondrocytes isolated from other regions of embryonic sternum. Transforming growth factor-β2 (TGF-β2) was previously suggested to be an inhibitor of chondrocyte maturation. We found, however, that treatment with a neutralizing antiserum to TGF-β did not counteract the inhibition of maturation in cocultures of articular and maturing core sternal chondrocytes. Indeed, articular chondrocytes produced and accumulated relatively low levels of TGF-β in their culture medium, about 15 ng/ml/48 h, of which over 90% was latent; surprisingly, maturing sternal core chondrocytes accumulated over 10-fold more TGF-β in the medium, about 150 ng/ml/48 h, of which over 20% was endogenously active. These results indicate that articular chondrocytes do possess dominant antimaturation properties which appear to be TGF-β independent. The TGF-βs may thus have a more prominent role in the terminal phases of chondrocyte maturation, as indicated by their abundance and greater activity in hypertrophic chondrocytes.
Association of Bone Mineral Density with Apolipoprotein E Phenotype
Authors: Shiraki Masataka , Shiraki Yumiko , Aoki Choju , Hosoi Takayuki , Inoue Satoshi , Kaneki Masao , Ouchi Yasuyoshi
Published: April 2006

The phenotypes of apolipoprotein E (Apo E) and their relationship with the bone mineral density (BMD) were examined in 284 unrelated postmenopausal Japanese women aged 47–82 years (64.0 ± 1.0 years, mean ± SE). The Apo E phenotype was analyzed by the isoelectric focusing method, followed by immunoblotting. The relationship between the Apo E phenotype and the vitamin D receptor (VDR) gene or estrogen receptor (ER) gene genotypes was also studied in the same population. The Apo E phenotypic frequencies in our population were 9.9% for E3/2, 66.5% for E3/3, 1.8% for E4/2, 19.7% for E4/3, and 2.1% for E4/4. We classified these phenotypes into three categories: Apo E4−/− (E3/2 and E3/3, n = 217), Apo E4+/− (E4/3 and E4/2, n = 61), and Apo E4+/+ (E4/4, n = 6). The age, body weight, body height, and years since menopause were not significantly different among these three categories. The lumbar BMD values in these three groups were significantly different in the order of E4−/− (0.91 ± 0.01 g/cm2), E4+/− (0.85 ± 0.02 g/cm2), and E4+/+ (0.83 ± 0.06 g/cm2) (p = 0.031). The same trend was also observed for the Z score of the total BMD (p = 0.022). The serum level of intact osteocalcin in E4+/+ (15.2 ± 5.7 ng/ml) was higher than in E4−/− (7.7 ± 0.3 ng/ml) or E4+/− (7.7 ± 0.7 ng/ml) (p = 0.004 by analysis of variance). However, there were no other significant differences in the serum or urinary levels of bone turnover markers. Serum cholesterol in the E4+/+ group tended to be higher than in the other two groups (p = 0.05). There were no significant associations of the VDR and ER genotypes with the Apo E4 phenotype. A multivariate linear regression analysis revealed Apo E4 to be a significant, independent predictor of the Z score of the lumbar BMD. The effect of the Apo E4 allele on the Z score of the lumbar BMD (−0.493 ± 0.152) was not significantly different from that in the AAB of VDR (−0.616 ± 0.225) or PPxx of ER (−0.785 ± 0.314). In conclusion, the Apo E4 allele is associated with a low bone mass in postmenopausal Japanese.
Vitamin D Receptor Gene Polymorphisms, Bone Turnover, and Rates of Bone Loss in Older African-American Women
Authors: Zmuda Joseph M. , Cauley Jane A. , Danielson Michelle E. , Wolf Randi L. , Ferrell Robert E.
Published: April 2006

Bone mineral density (BMD) is under genetic control. Some studies in Caucasian and Asian women suggest that polymorphisms in the vitamin D receptor (VDR) gene are associated with BMD and the rate of postmenopausal bone loss. We determined if similar associations exist in 101 African-American women aged 65 years and older (71 ± 5 years, mean ± SD). We also examined the relation between VDR genotype and fractional45Ca absorption and markers of bone formation (osteocalcin) and resorption (N-telopeptides) in these women. BMD was measured at the proximal femur and whole body at baseline and after 1.9 ± 0.4 years (femur only) on a Hologic QDR-2000 densitometer using dual-energy X-ray absorptiometry. Calcaneal BMD was measured with single x-ray absorptiometry. VDR gene polymorphisms were defined by the endonucleases BsmI, ApaI, and TaqI. These polymorphisms were not associated with BMD at any skeletal site or with markers of bone turnover. There was a significant interaction between age and VDR genotype where the oldest women (>70 years) with the tt genotype experienced greater hip bone loss than women with the TT genotype (−2.1%/year vs. −0.4%/year, respectively), whereas heterozygous women experienced an intermediate rate of bone loss (−1.3%/year). Women homozygous for the B allele had 14% lower fractional45Ca absorption compared with women homozygous for the b allele, although this difference was not statistically significant (p = 0.08). We conclude that VDR gene polymorphisms are not associated with BMD or indices of bone turnover in this population of older African-American women. However, DNA sequence variation in the VDR gene or a nearby locus may influence intestinal calcium transport and the rate of postmenopausal bone loss in African-American women.
Universal Standardization of Bone Density Measurements: A Method with Optimal Properties for Calibration Among Several Instruments
Authors: Hui Siu L. , Gao Sujuan , Zhou Xiao-Hua , Johnston C. Conrad , Lu Ying , Glüer Claus C. , Grampp Stephen , Genant Harry
Published: April 2006

The International Dual-Photon X-Ray Absorptiometry (DXA) Standardization Committee (IDSC) conducted a cross-calibration study among three models of DXA machines from three different manufacturers. In that study, 100 subjects were scanned on all three machines. A set of equations were derived to convert bone mineral density (BMD) on each machine to a “standardized BMD” (sBMD) such that sBMD from the same subject derived from different machines would be approximately the same. In a reanalysis of the cross-calibration data, we showed that the conversion method used in the IDSC study did not achieve several optimal properties desirable in such conversions. We derived new conversion equations to sBMD based on minimizing differences among sBMD from the three machines. More important is that the new conversions have no residual bias that was present in the IDSC conversions. The performance of the methods were compared on the cross-calibration data as well as an external data set. We conclude that the IDSC conversions are adequate for clinical use on other machines worldwide, but that researchers should standardize their own machines in a laboratory using the new method.
Methionine-Enkephalin in Bone and Joint Tissues
Authors: Elhassan Adlan M. , Lindgren J. U. , Hultenby K. , Bergstrom J. , Adem Abdu
Published: April 2006

Methionine-enkephalin (met-enk), an endogenous opiate, mimics many of the effects of morphine by binding to opiate receptors, thereby eliciting similar cellular and behavioral effects. Using biochemical and immunohistochemical techniques, several peptides have been identified in bone and joint tissues. Here we report, for the first time, the presence as well as concentration of met-enk in bone and joint tissues. Immunohistochemistry using electron and immunoflourescence microscopy showed cellular and neuronal distribution of met-enk in bone and joint tissues. The concentration of met-enk analyzed by high performance liquid chromatography electrochemical detection or radioimmunoassay was high in bone marrow, periosteum, ankle joint tissue, and cortical bone. Analysis by fast atom bombardment mass spectrometry suggested that the recovered fragment was met-enk. Administration of met-enk inhibits osteoblast cell growth in culture, which is reversible by naltrexone. In arthritic rats, the concentration of met-enk was significantly decreased in ankle joints compared with controls, suggesting a role for met-enk in the pathophysiology of adjuvant arthritis.
Nonsteroidal Anti-Inflammatory Drugs and Bone Mineral Density in Older Women: The Rancho Bernardo Study
Authors: Morton Deborah J. , Dr. Barrett-Connor Elizabeth L. , Schneider Diane L.
Published: April 2006

Nonsteroidal anti-inflammatory drugs (NSAIDs) are known to inhibit synthesis of prostaglandins and may help prevent bone loss, but no study has shown the differential association of type or dose of NSAID compound with bone mineral density (BMD). The purpose of this study was to determine the relation of NSAIDs by type and dose to BMD. Participants were 932 Caucasian, community-dwelling women aged 44–98 years from southern California. Data were collected from 1988 to 1991 through the use of standardized medical questionnaires. Medication use was validated by a nurse. BMD at the ultradistal and midshaft radii were measured using single-photon absorptiometry, and at the hip and lumbar spine using dual-energy X-ray absorptiometry. Women (mean age, 72 years) were classified into 818 nonusers and 114 regular daily users of NSAIDs, of which 84 used propionic acid NSAIDs and the remainder used acetic acid NSAIDs. Occasional NSAID users were excluded. Women who used propionic acid NSAIDs, but not acetic acid NSAIDs, had higher BMD at all five sites and significantly higher BMD at the midshaft radius and lumbar spine. These differences remained after controlling for known covariates of osteoporosis. When women with self-reported osteoarthritis were excluded from the model, significantly higher BMD in propionic acid NSAID users was also observed at the femoral neck and total hip. Those who concurrently used estrogen and propionic acid NSAIDs had the highest BMD at all sites, suggesting an additive effect. We conclude that regular daily use of propionic acid NSAIDs, with or without simultaneous use of estrogen, may be helpful in preventing bone loss in older women. However, further research is needed to confirm these results before any clinical practice guidelines can be recommended due to the increased risk of serious complications associated with NSAID use.
Femoral Neck Length and Hip Fracture Risk
Authors: Michelotti John , Clark John
Published: April 2006

To determine whether there are differences in femoral skeletal geometry in fracture-prone subjects when size, positioning diagnosis, and age are controlled, we compared femoral measurements made from the uninvolved hip on 119 plane anteroposterior pelvis radiographs of women without fracture to those of the contralateral hip in a group of 43 female patients with hip fractures (neck, 23; intertrochanteric, 20). The hip was imaged in a standardized position of rotation and adduction. Race, age, and musculoskeletal diagnosis were known. Subjects were grouped by diagnosis of the opposite hip condition (rheumatoid arthritis, osteoarthritis, and normal) and compared. Measurements were also analyzed as ratios to head diameter (HD), neck diameter (ND), and pelvic width. Femoral neck length (NL) was measured from skeletal preparations and imaged in controlled positions of abduction and external rotation. No differences were found between the neck and intertrochanteric fracture groups. The differences between the fracture group and the controls were a thinner femoral cortex (measured at a point one head radius below the lesser trochanter) a larger femoral head, and a larger femoral ND in the fracture group (p < 0.025). The difference in cortical thickness was still significant when scaled by size, but the ratio of HD to ND was equivalent in fractures and controls. No difference in femoral NL could be demonstrated. The experimental measurements showed that apparent NL is significantly position sensitive and this may explain previously reported differences in fracture-prone groups.
Extracellular Ca2+ Increases Cytosolic Free Ca2+ in Freshly Isolated Rat Odontoblasts
Authors: Guo Liang , Dr. Davidson Robert M.
Published: April 2006

Recent evidence suggests that extracellular Ca2+ may modulate cell function in mineralized tissue. To determine whether dentinogenic cells, in particular, are sensitive to extracellular Ca2+, fura-2 microfluorometry was used to monitor intracellular calcium levels in odontoblasts freshly isolated from rat incisor. In response to applications of 0.5–4.0 mM extracellular calcium (CaCl2), most odontoblasts (84%; 107/128) showed an increase in intracellular calcium. For the majority of these cells (70%; 75/107), the typical response was biphasic; there was an initial, transient increase in intracellular calcium which reached peak levels within 30–50 s and decayed rapidly, followed by a slower (> 300 s) recovery toward basal levels. In general, the response of these cells to calcium was repeatable and the mean calcium concentration for the half-maximal response was ∼1.3 mM. This effect could be partially blocked by either 200 μM lanthanum, a nonspecific blocker of Ca2+ channels, or 20 μM dantrolene, a potent inhibitor of Ca2+ release from internal stores. Used in combination, lanthanum, and dantrolene nearly abolished the calcium response completely. In addition, this response was sensitive to the dihydropyridine-sensitive calcium channel blocking agent nicardipine (60 μM), indicating a role for voltage-gated calcium channels during these events. These results show that odontoblasts respond to external calcium through mechanisms involving both influx of external calcium as well as release of calcium from internal stores and suggest a role for extracellular calcium in regulating the function of these cells.
Tartrate-Resistant Acid Phosphatase 5b: A Novel Serum Marker of Bone Resorption
Authors: Halleen Jussi M. , Alatalo Sari L. , Suominen Harri , Cheng Sulin , Janckila Anthony J. , Väänänen H. Kalervo
Published: April 2006

Human serum contains two forms of tartrate-resistant acid phosphatase (TRAP), 5a and 5b. Of these, 5a contains sialic acid and 5b does not. We show here that antigenic properties and pH optimum of TRAP purified from human osteoclasts are identical to those of serum TRAP 5b and completely different from those of serum TRAP 5a, suggesting that 5b would be derived from osteoclasts and 5a from some other source. We developed a novel immunoassay specific for 5b using a monoclonal antibody O1A as capture antibody. O1A did not bind acid phosphatase derived from platelets and erythrocytes. Western analysis showed that O1A was specific for TRAP in both human bone and serum. We measured bound TRAP activity at pH 6.1, where 5b is highly active and 5a almost completely inactive. The immunoassay detected more than 90% of the initial TRAP 5b activity after 8-h incubation of serum samples at 25°C and after 3 days incubation at 4°C. Serum TRAP 5b activity decreased significantly after 6 months of hormone replacement therapy (HRT) of postmenopausal women compared with the change observed in postmenopausal women receiving placebo (p < 0.0001). Instead, no significant differences were observed between the changes in the placebo and HRT groups in total serum TRAP amount. These results show that serum TRAP 5b is a specific and sensitive marker for monitoring antiresorptive treatment. Instead, total serum TRAP cannot be used for that purpose. These findings may turn out to be a significant improvement in using serum TRAP as a resorption marker.