bluuebunny/crossref_metadata_2025 huggingface dataset:
Placement of 19F into the center of GB1: effects on structure and stability
Authors: Campos-Olivas Ramón , Aziz Rehan , Helms Gregory L , Evans Jeremy N.S , Gronenborn Angela M
Published: October 2002

A structural and thermodynamic characterization of 5F‐Trp‐substituted immunoglobulin binding domain B1 of streptococcal protein G (GB1) was carried out by nuclear magnetic resonance and circular dichroism spectroscopy. A single fluorine reporter atom was positioned at the center of the three‐dimensional structure, uniquely poised to be exploited for studying interior properties of this protein. We demonstrate that the introduction of 5F‐Trp does not affect the global and local architecture of GB1 and has no influence on the thermodynamic stability. The favorable properties of the fluorinated GB1 render this molecule a desirable model system for the development of spectroscopic methodology and theoretical calculations.
Methionine oxidation inhibits fibrillation of human α‐synuclein in vitro
Authors: Uversky Vladimir N , Yamin Ghiam , Souillac Pierre O , Goers John , Glaser Charles B , Fink Anthony L
Published: October 2002

We examined the effect of methionine oxidation of human recombinant α‐synuclein on its structural properties and propensity to fibrillate. Both oxidized and non‐oxidized α‐synucleins were natively unfolded under conditions of neutral pH, with the oxidized protein being slightly more disordered. Both proteins adopted identical partially folded conformations under conditions of acidic pH. The fibrillation of α‐synuclein at neutral pH was completely inhibited by methionine oxidation. This inhibitory effect was eliminated at low pH. The addition of oxidized α‐synuclein to the unoxidized form led to a substantial inhibition of α‐synuclein fibrillation.
A nuclear transcription factor related to plastid ribosome biogenesis is synthesised early during germination and priming
Authors: Achard Patrick , Job Dominique , Mache Régis
Published: October 2002

Germination is a short developmental process during which many new proteins are synthesised. We have chosen the previously characterised RPL21 gene encoding plastid‐localised ribosomal proteins RPL21, to analyse activation of gene expression during germination. Transcription activation occurs at the P1 promoter during the first hours following imbibition and coincides with the appearance of a trans‐acting factor that we named AUBE1. AUBE1 binds specifically to a short DNA fragment that encompasses the P1 promoter of the RPL21 gene. The protein has a size of 28–30 kDa and is transiently expressed during the early phase of germination. Using the properties of primed seeds we show that AUBE1 is maintained after desiccation of primed seeds. We conclude that AUBE1 can be used as a marker in spinach seed priming.
A point mutation at the subunit interface of hypoxanthine–guanine–xanthine phosphoribosyltransferase impairs activity: role of oligomerization in catalysis
Authors: Subbayya I.N.Sujay , Balaram Hemalatha
Published: October 2002

Hypoxanthine–guanine–xanthine phosphoribosyltransferase (HGXPRT) from Plasmodium falciparum catalyzes the phosphoribosylation of hypoxanthine, guanine and xanthine. The functionally active form of HGXPRT is a tetramer but interface residues do not contribute to catalysis. Here we report the characterization of an interface mutant Y96C, which has a decreased k
cat, an increase in the K
m for phosphoribosyl pyrophosphate (PRPP) and no change in K
m for the purine bases when compared to the wild type enzyme. The mutant enzyme does not tetramerize in the presence of PRPP, unlike the wild type in which the tetramer is stabilized by PRPP. This is the first report of a HGXPRT mutation, at a unique interface where non‐adjacent subunits interact, that impairs catalysis.
Halocidin: a new antimicrobial peptide from hemocytes of the solitary tunicate, Halocynthia aurantium
Authors: Jang Woong Sik , Kim Kyu Nam , Lee Young Shin , Nam Myung Hee , Lee In Hee
Published: October 2002

From hemocytes of the tunicate Halocynthia aurantium we purified a new antimicrobial peptide named halocidin. The native peptide had a mass of 3443 Da and comprised two different subunits containing 18 amino acid residues (WLNALLHHGLNCAKGVLA) and 15 residues (ALLHHGLNCAKGVLA), which were linked covalently by a single cystine disulfide bond. Two different monomers were separately synthesized and used to make three additional isoforms (15 residue homodimer, 18 residue homodimer, heterodimer). In antimicrobial assays performed with synthetic peptides of halocidin, it was confirmed that congeners of the 18 residue monomer were more active than those of the 15 residue monomer against methicillin‐resistant Staphylococcus aureus and multidrug‐resistant Pseudomonas aeruginosa.
Oligomerization of DsRed is required for the generation of a functional red fluorescent chromophore
Authors: Sacchetti Andrea , Subramaniam Vinod , Jovin Thomas M , Alberti Saverio
Published: October 2002

The coral red fluorescent protein (DsRed) absorbs and emits light at much higher wavelengths than the structurally homologous green fluorescent protein, raising questions about the properties of its chromophore. We have analyzed the relationship between the aggregation state and fluorescence of native, 6‐histidine‐tagged, or maltose‐binding protein‐fused DsRed. In all cases, newly synthesized DsRed molecules were largely monomeric and devoid of covalently closed chromophores. Maturation in vitro induces the expression of red fluorescent chromophores but only in oligomeric forms of the protein, whereas monomers are essentially devoid of fluorescence. NaOH‐denatured samples demonstrated a generalized breakdown of the DsRed oligomers to monomers, which refolded after neutralization into weakly green fluorescent and still monomeric species. Red fluorescent chromophores were regenerated only upon oligomerization. These findings demonstrate that ‘red’ chromophores form and are functional only as oligomers, and suggest that the smallest red fluorescent functional unit is a dimer. A comparison of alkali‐, acid‐ and guanidinium‐denatured DsRed indicates that stabilization of the DsRed chromophore by concerted steps of folding and oligomerization may play a critical role in its maturation process.
Aldosterone augments endothelin‐1‐induced cardiac myocyte hypertrophy with the reinforcement of the JNK pathway
Authors: Oshima Yuichi , Fujio Yasushi , Funamoto Masanobu , Negoro Shinji , Izumi Masahiro , Nakaoka Yoshikazu , Hirota Hisao , Yamauchi-Takihara Keiko , Kawase Ichiro
Published: October 2002

Aldosterone is thought to regulate cardiac work independently of sodium retention, though the mechanisms remain to be known. In the present study, we have demonstrated that aldosterone reinforces endothelin‐mediated cardiac hypertrophy with the increase in cell surface area and upregulation of the transcripts characteristic of hypertrophy. We have also shown that aldosterone augments c‐Jun N‐terminal kinase activation induced by endothelin‐1. Taken together, it is suggested that aldosterone modulates cardiac hypertrophy, at least partially, synergistically with extracellular signals that have been shown to be involved in cardiac remodeling.
4‐Isoavenaciolide covalently binds and inhibits VHR, a dual‐specificity phosphatase
Authors: Ueda Kazunori , Usui Takeo , Nakayama Hiroshi , Ueki Masashi , Takio Koji , Ubukata Makoto , Osada Hiroyuki
Published: October 2002

A potent inhibitor of a dual‐specificity protein phosphatase, VHR (vaccinia H1 related), was isolated during a screening of microbial metabolites. This inhibitor was identified as 4‐isoavenaciolide (4‐iA), and was determined to irreversibly inhibit VHR phosphatase activity with a 50% inhibitory concentration of 1.2 μM. Detailed tandem mass spectrometry analyses of proteolysed fragments revealed that two molecules of 4‐iA bound a molecule of VHR at the two different fragments: one containing the catalytic domain and the other containing the α6 helix positioned surface domain. As 4‐iA possesses a reactive exo‐methylene moiety, it is possible that 4‐iA inhibits VHR through the direct binding to the cysteine residue in the catalytic site (Cys124). Furthermore, 4‐iA inhibited dual‐specificity protein phosphatases and tyrosine phosphatases, but did not inhibit serine/threonine phosphatases. These results suggest that 4‐iA is a cysteine‐targeting inhibitor of protein phosphatases with a common HCX5RS/T motif in the catalytic site.
Antepartum prophylactic transabdominal amnioinfusion in preterm pregnancies complicated by oligohydramnios
Authors: Turhan N.Ö , Atacan N
Published: October 2002

Objective: To assess the role of amnioinfusion in preterm pregnancies with oligohydramnios. Method: 29 women between 23 and 35 weeks’ gestation were enrolled in the study. Transabdominal amnioinfusion was performed in 15 pregnancies, 14 patients were managed expectantly. The latency period and perinatal outcome of both groups were compared using the Mann–Whitney U‐test and χ2‐test. Result: The amniotic fluid index significantly increased from a median value of 6 to 11 cm (P<0.0001) in the amnioinfusion group after amnioinfusion. The latency period of the amnioinfusion group was significantly longer (median 15 vs. 8 days P<0.05). Gestational week of the amnioinfusion group was earlier on admission (median 30.6 vs. 33.4 weeks, P=0.01) but at delivery this diversity disappeared (median 33.4 vs. 34.8 weeks, P=0.10). The perinatal outcomes of the two groups were similar. Conclusion: Transabdominal amnioinfusion prolongs the latency period and improves perinatal outcome in preterm pregnancies complicated by oligohydramnios.
Toward a theory of project interdependencies in high tech R&D environments
Authors: Verma Devesh , Sinha Kingshuk K
Published: October 2002

In today’s intensely competitive market place, high technology firms are challenged with the task of managing multiple‐concurrent research and development (R&D) projects with constrained resources. As success in the business world depends on the ability to do more with less, it is important for these high technology firms to understand why certain projects consume less monetary resources but still achieve superior product development outcomes. To address this question, we develop a theoretical framework for understanding the interdependencies between projects and their relationship to project performance in a multiple‐concurrent R&D environment. The framework is developed through multiple‐case studies of projects undertaken by a Fortune 500 high technology manufacturing firm. The seven projects that comprised the sample for the multiple‐case study were chosen from a larger portfolio of projects belonging to a technology group at the firm’s corporate R&D center. The choice of the seven projects was guided by a data envelopment analysis (DEA)‐based project performance metric, also proposed in this paper.
Mechanobiology of initial pseudarthrosis formation with oblique fractures
Authors: Loboa Elizabeth G. , Beaupré Gary S. , Carter Dennis R.
Published: October 2002

Mechanical stresses play an important role in regulating tissue differentiation in a variety of contexts during skeletal development and regeneration. It has been shown that some intermittent loading at a fracture site can accelerate secondary fracture healing. However, it has not been shown how the stress and strain histories resulting from mechanical loading of a fracture might, in some cases, inhibit normal fracture healing and induce pseudarthrosis formation. In this study, finite element analysis is used to calculate hydrostatic stress and maximum principal tensile strain patterns in regenerating tissue around the site of an oblique fracture. Using a mechanobiologic view on tissue differentiation, we compared calculated stress and strain patterns within the fracture callus to the histomorphology of a typical oblique pseudarthrosis. Tissue differentiation predictions were consistent with the characteristic histomorphology of oblique pseudarthrosis: in the interfragmentary gap, tensile strains led to “cleavage” of the callus; at the ends of both fracture fragments, hydrostatic pressure and tensile strain caused fibrocartilage formation; and, at discrete locations of the periosteum at the oblique fracture ends, mild hydrostatic tension caused bone formation. We also found that discrete regions of high hydrostatic pressure correlated with locations of periosteal bone resorption. When previous findings with distraction osteogenesis are considered with these observations, it appears that low levels of hydrostatic pressure may be conducive to periosteal cartilage formation but high hydrostatic pressure may induce periosteal bone resorption during bone healing. We concluded that tissue differentiation in pseudarthrosis formation is consistent with concepts previously presented for understanding fracture healing, distraction osteogenesis, and joint formation. © 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
The role of collagen in determining bone mechanical properties
Authors: Wang Xiaodu , Bank Ruud A. , Tekoppele Johan M. , Agrawal C. Mauli
Published: October 2002

The hypothesis of this study was that collagen denaturation would lead to a significant decrease in the toughness of bone, but has little effect on the stiffness of bone. Using a heating model, effects of collagen denaturation on the biomechanical properties of human cadaveric bone were examined. Prior to testing, bone specimens were heat treated at varied temperatures (37–200°C) to induce different degrees of collagen denaturation. Collagen denaturation and mechanical properties of bone were determined using a selective digestion technique and three‐point bending tests, respectively. The densities and weight fractions of the mineral and organic phases in bone also were determined. A repeated measures analysis of variance showed that heating had a significant effect on the biomechanical integrity of bone, corresponding to the degree of collagen denaturation. The results of this study indicate that the toughness and strength of bone decreases significantly with increasing collagen denaturation, whereas the elastic modulus of bone is almost constant irrespective of collagen denaturation. These results suggest that the collagen network plays an important role in the toughness of bone, but has little effect on the stiffness of bone, thereby supporting the hypothesis of this study. © 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Mechanisms of chondrocyte adhesion to cartilage: role of β1‐integrins, CD44, and annexin V
Authors: Kurtis Melissa S. , Tu Buu P. , Gaya Omar A. , Mollenhauer JÜRgen , Knudson Warren , Loeser Richard F. , Knudson Cheryl B. , Sah Robert L.
Published: October 2002

The initial adhesion of transplanted chondrocytes to surrounding host cartilage may be important in the repair of articular defects. Adhesion may position cells to secrete molecules that fill the defect and integrate repair tissue with host tissue. While chondrocytes are known to become increasingly adherent to cartilage with time, the molecular basis for this is unknown. The objective of this study was to investigate the role of β1‐integrin, CD44, and annexin V receptors in chondrocyte adhesion to cartilage. Chondrocytes were cultured in high density monolayer, released with trypsin, and allowed to recover in suspension for 2 h at 37°C. Under these conditions, flow cytometry analysis showed that chondrocytes expressed β1‐integrins, CD44, and annexin V. In a rapid screening assay to assess chondrocyte adhesion to cartilage, cell detachment decreased from 79% at 10 min following transplantation to 10% at 320 min. Treatment of cells with a monoclonal antibody to block β1‐integrins significantly increased chondrocyte detachment from cartilage compared to untreated controls. Similarly, results from a parallel‐plate shear flow adhesion assay showed that blocking β1‐integrins significantly increased chondrocyte detachment from cartilage compared to untreated controls at each level of applied shear (0–70 Pa). In both assays, treatment of cells with reagents that block CD44 (hyaluronan oligosaccharides or monoclonal Ab IM7) or annexin V (polyclonal Ab #8958) had no detectable effect on adhesion. With cartilage treated with chondroitinase ABC, blocking β1‐integrins also increased chondrocyte detachment, while blocking CD44 and annexin V also had no detectable effect. Under the conditions studied here, β1‐integrins appear to mediate chondrocyte adhesion to a cut cartilage surface. Delineation of the mechanisms of adhesion may have clinical implications by allowing cell manipulations or matrix treatments to enhance chondrocyte adhesion and retention at a defect site. © 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
The effect of ovariectomy and ovarian steroid treatment on growth hormone and insulin‐like growth factor‐I levels in the rat femur
Authors: Suliman Isam A. , El‐Bakri Nahid K. , Adem Abdu , Mustafa Amged , Lindgren J Urban
Published: October 2002

Growth hormone (GH) and insulin‐like growth factor‐I (IGF‐I) are known to play an important role in bone metabolism. The regulation of plasma levels of GH and IGF‐I by ovarian steroids is well known, however, their effect on local GH and IGF‐I is still unclear. In this study, we investigated the effect of ovariectomy and ovarian steroid treatment on the femur GH and IGF‐I levels as well as on bone density in the rat. Nine month‐old rats were ovariectomized (OVX) or sham‐operated (SHAM) and 9 weeks after the surgery they were treated with daily s.c. injections of either 17β‐estradiol (OVX + E), progesterone (OVX + P), or vehicle (OVX + V) for another 10 weeks. GH and IGF‐I levels in the femur extracts were measured by specific radioimmunoassay (RIA). Ovariectomy decreased GH and had no effect on IGF‐I levels. Estradiol treatment increased femur GH and IGF‐I levels compared to SHAM rats. Progesterone restored GH and increased IGF‐I levels. Ovariectomy decreased, estrogen restored and progesterone partially restored femur bone density. Our results demonstrate that ovariectomy and ovarian steroids modulate the levels of GH and IGF‐I in the bone of aged OVX rats. However, these effects appear to be limited to supraphysiological concentrations of estradiol and progesterone. © 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
EMG activity of trunk muscles and torque output during isometric axial rotation exertion: a comparison between back pain patients and matched controls
Authors: Ng Joseph K.‐F. , Richardson Carolyn A. , Parnianpour Mohamad , Kippers Vaughan
Published: October 2002

Abnormal patterns of trunk muscle activity could affect the biomechanics of spinal movements and result in back pain. The present study aimed to examine electromyographic (EMG) activity of abdominal and back muscles as well as triaxial torque output during isometric axial rotation at different exertion levels in back pain patients and matched controls. Twelve back pain patients and 12 matched controls performed isometric right and left axial rotation at 100%, 70%, 50% and 30% maximum voluntary contractions in a standing position. Surface EMG activity of rectus abdominis, external oblique, internal oblique, latissimus dorsi, iliocostalis lumborum and multifidus were recorded bilaterally. The primary torque in the transverse plane and the coupling torques in sagittal and coronal planes were measured. Results showed that there was a trend (P = 0.08) of higher flexion coupling torque during left axial rotation exertion in back pain patients. Higher activity for external oblique and lower activity for multifidus was shown during left axial rotation exertion in back pain group when compared to the control group. In right axial rotation, back pain patients exhibited lesser activity of rectus abdominis at higher levels of exertion when compared with matched controls. These findings demonstrated that decreased activation of one muscle may be compensated by overactivity in other muscles. The reduced levels of activity of the multifidus muscle during axial rotation exertion in back pain patients may indicate that spinal stability could be compromised. Future studies should consider these alternations in recruitment patterns in terms of spinal stability and internal loading. The findings also indicate the importance of training for coordination besides the strengthening of trunk muscles during rehabilitation process. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Stretch and interleukin‐1β induce matrix metalloproteinases in rabbit tendon cells in vitro
Authors: Archambault Joanne , Tsuzaki Mari , Herzog Walter , Banes Albert J.
Published: October 2002

Little is known about the factors that initiate and propagate tendon overuse injuries, but chronic inflammation and matrix destruction have been implicated. The purpose of this study was to evaluate the production of cyclooxygenase II (COX‐2) and matrix metalloproteinases (MMPs) by tendon cells exposed to cyclic strain and inflammatory cytokines in vitro. Rabbit Achilles tendon cells were subjected to a stretching protocol with 5% elongation at 0.33 Hz for 6 h, or treated with 1000 pM interleukin‐1β (IL‐1β), or exposed to IL‐1β and stretching together. Gene expression was evaluated by RT‐PCR and production of stromelysin was quantified with an ELISA. IL‐1β induced the expression of the collagenase‐1 and stromelysin‐1 genes. Production of stromelysin proenzyme by cells stimulated with IL‐1β was 17 times higher than production by control cells. Cells exposed to IL‐1β and stretching produced 20 times more stromelysin than control cells. Cells subjected to stretching alone did not produce more stromelysin than control cells. The synergistic effect of IL‐1β and stretching was observed at doses of IL‐1β ranging from 10 to 1000 pM. These data suggest that mechanical load and inflammatory cytokines can initiate a matrix destructive pathway in tendon that is more pronounced than with mechanical loading or inflammation alone. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Altered swelling behavior of femoral cartilage following joint immobilization in a canine model
Authors: Narmoneva Daria A. , Cheung Herman S. , Wang Jean Y. , Howell David S. , Setton Lori A.
Published: October 2002

Periods of reduced joint loading have been shown to induce changes in the biochemical composition, metabolism and mechanics of articular cartilage. In this study, changes in cartilage swelling behavior were studied following a 4‐week period of joint immobilization, using a recently developed osmotic loading technique [J. Biomech. 32 (1999) 401–408]. The magnitude and distribution of swelling strains were measured in cartilage–bone samples equilibrated in physiological and hypotonic saline, relative to a hypertonic reference NaCl solution. Physicochemical parameters (glycosaminoglycan fixed charge density and water volume fraction) were determined in site‐matched cartilage samples. The experimental data for swelling strains, fixed charge density and water volume fraction were used with a triphasic mechano‐chemical theory [J. Biomech. Eng. 113 (1991) 245–258] to determine the effect of joint immobilization on the tensile modulus of the cartilage solid matrix. Four weeks of immobilization resulted in a significant increase in the magnitude of swelling‐induced strains, and a significant decrease in fixed charge density in cartilage, as compared with the contralateral controls. Joint immobilization also resulted in decreases in values for the modulus of cartilage, as compared with the contralateral controls. Our results suggest that 4 weeks of joint immobilization had a significant effect on cartilage mechanical function that may be linked to collagen changes in the cartilage extracellular matrix. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Differential apoptotic response of J774 macrophages to alumina and ultra‐high‐molecular‐weight polyethylene particles
Authors: Petit Alain , Catelas Isabelle , Antoniou John , Zukor David J. , Huk Olga L.
Published: October 2002

We recently identified apoptosis in in vitro wear particle‐stimulated macrophages. The recent explosion of interest in apoptosis lies in the fact that it is under positive and negative regulation through evolutionary conserved biochemical pathways. It may also be possible to modulate macrophage apoptosis in the treatment of periprosthetic osteolysis. The purpose of this study was to compare the macrophage response to identically sized particles of alumina ceramic (Al2O3) and ultra‐high‐molecular‐weight polyethylene (UHMWPE) in terms of TNF‐α release and induction of apoptosis. J774 mouse macrophages were incubated for 0–24 h in the presence of Al2O3 and UHMWPE particles. TNF‐α release was measured by ELISA; Poly(ADP‐ribose)polymerase (PARP) and caspase‐3 expression was analyzed by Western blot; DNA fragmentation (DNA laddering) was visualized on agarose gel containing ethidium bromide. Al2O3 particles induced TNF‐α release after 4 h incubation with concentrations reaching 483 and 800 pg/ml after 24 h with 125 and 250 particles/macrophage, respectively (control = 161 pg/ml) (P < 0.05 vs. control). The same concentrations of UHMWPE particles induced a much larger and significant TNF‐α release after only 1 h incubation, increasing up to 6250 pg/ml after 24 h (P < 0.05 vs. control). Western blot analysis demonstrated that the active caspase‐3 fragment (17 kDa) and the proteolytic PARP fragment (85 kDa) were expressed after 2 h incubation with 125 and 250 Al2O3 particles/macrophage. The active caspase‐3 and the PARP fragment had lower expression and appeared after a longer incubation time (8 h) with 125 and 250 UHMWPE particles/macrophage. Finally, DNA fragmentation (DNA laddering) was observed after 16 h with 125 and 250 particles of Al2O3 per macrophage whereas no laddering was induced by UHMWPE particles even after 24 h incubation. This study shows that although both Al2O3 and UHMWPE particles induce TNF‐α release, this stimulation was much greater (8–10 times higher) with UHMWPE than A12O3 (P < 0.05 vs. control). As well, the induction of apoptosis, as measured by activation of caspase‐3, PARP cleavage and DNA laddering, is different for these two particles, being faster and more important with Al2O3 than UHMWPE. We hypothesize that the ability of Al2O3 to induce macrophage apoptosis may explain the lower TNF‐α release observed with these particles and explain the differences seen in osteolysis patterns of ceramic–ceramic (CC) vs. metal–polyethylene (Mpe) articulations. In conclusion, apoptosis may be a major internal mechanism to decrease macrophage activity and may be a desired therapeutic endpoint. The identification of an apoptosis‐related pathway in the macrophage response to ceramic particles provides crucial data for a rational approach in the treatment and/or prevention of periprosthetic osteolysis. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Temporal expression patterns of BMP receptors and collagen II (B) during periosteal chondrogenesis
Authors: Sanyal Arunik , Oursler Merry Jo , Clemens Victoria R. , Fukumoto Takumi , Fitzsimmons James S. , O'Driscoll Shawn W.
Published: October 2002

Articular cartilage has a limited ability to repair itself. Periosteal grafts have chondrogenic potential and are used clinically to repair defects in articular cartilage. An organ culture model system for in vitro rabbit periosteal chondrogenesis has been established to study the molecular events of periosteal chondrogenesis in vitro. In this model, bone morphogenetic protein‐2 (BMP2) mRNA expression was found to be upregulated in the first 12 h. BMPs usually transduce their signals through a receptor complex that includes type II and either type IA or type IB BMP receptors. Receptors IA and IB play distinct roles during limb development. We have examined the temporal expression patterns for the mRNAs of these receptors using our experimental model. The mRNA expression patterns of these three BMP receptors differed from one another in periosteal explants during chondrogenesis. When these explants were cultured under chondrogenic conditions (agarose suspension with TGF‐β1 added to the media for the first 2 days), the expression of BMPRII mRNA and that of BMPRIA mRNA varied only slightly and persisted over a long time. In contrast, the expression of BMPRIB mRNA was upregulated within 12 h, peaked at day 5, and fell to a level that was barely detected beyond day 21. Moreover, the expression of BMPRIB mRNA preceded that of collagen type IIB mRNAs, a marker for matrix‐depositing chondrocytes. These data support a role for coordinate expression of BMP2 and its receptors early during periosteal chondrogenesis. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Upregulation of basal TGFβ1 levels by EMF coincident with chondrogenesis – implications for skeletal repair and tissue engineering
Authors: Aaron Roy K. , Wang Shuo , Ciombor Deborah McK.
Published: October 2002

Members of the TGFβ/BMP gene family regulate cartilage and bone development. These genes are re‐expressed in bone repair and are thought to mediate chondro‐ and osteoprogenitor cell differentiation. These observations have led to a therapeutic strategy of introducing these growth factors into experimental cartilage and bone defects. Therapeutic efficacy, however, has been limited by diffusion or inactivation of these growth factors from the desired site and by the inability to deliver sustained concentrations of growth factors. This study demonstrates an increase in basal TGFβ mRNA and protein levels in association with chondrogenic differentiation in endochondral ossification. mRNA is increased by 158%; protein by 23%, and cells immunopositive for TGFβ by 343% at maximal TGFβ expression. Importantly, the pattern of TGFβ expression is preserved throughout the developmental sequence. Our data suggest that the exposure to a specific electromagnetic field (EMF) enhances, but does not disorganize, chondrogenesis and endochondral calcification as well as the normal physiologic expression of TGFβ. The ability to increase TGFβ at a moderately low dose for sustained periods of time without disorganizing its physiology suggests the ability to establish temporal concentration gradients of growth factors for the purpose of stimulating skeletal repair. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Serum cartilage‐derived retinoic acid‐sensitive protein (CD‐RAP) levels in Swarm rat chondrosarcoma
Authors: Yonekawa Masahiro , Kondo Seiji , Sugiura Hideshi , Kobayashi Kenji , Watanabe Tsuyoshi , Kimata Koji , Sandell L. J. , Iwata Hisashi
Published: October 2002

Cartilage‐derived retinoic acid‐sensitive protein (CD‐RAP) is a new protein that was isolated from bovine articular chondrocytes and human melanoma cell lines (melanoma inhibitory protein or MIA). In normal tissue its expression is limited to cartilage, and in morbid tissue to melanoma, chondrosarcoma, and breast cancer. Serum levels of CD‐RAP/MIA correlate with the progression of malignant melanoma, but there have been no reports on chondrosarcoma. Here, it was first demonstrated by RT‐PCR and immuno‐histological methods that CD‐RAP was expressed in tissue from a Swarm rat chondrosarcoma that was used as an experimental model. The course following tumor transplantation and changes in serum CD‐RAP after tumor excision were then observed to investigate whether serum CD‐RAP could be used as a marker of tumor activity. Consequently, serum CD‐RAP in control rats tended to decrease as the animal grew, whereas it rose in proportion to tumor proliferation in rats that had received a tumor graft. Serum CD‐RAP levels dropped rapidly following excision of the tumor in a group of tumor‐excised rats. In those rats which had a recurrence following excision of the tumor, serum CD‐RAP rose prior to the appearance of the tumor. Serum CD‐RAP thus sensitively reflected tumor onset and proliferation, so that it appeared to be an effective marker of tumor activity for Swarm rat chondrosarcoma. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Delayed tendon reattachment does not reverse atrophy and fat accumulation of the supraspinatus — an experimental study in rabbits
Authors: Matsumoto Fujio , Uhthoff Hans K. , Trudel Guy , Loehr Joachim F.
Published: October 2002

Muscle atrophy and fat accumulation occur after rotator cuff tearing. Whether these changes are reversible after a successful repair is still unknown. Imaging allows only a semi‐quantitative assessment of muscle atrophy and fat deposition. As only experimental studies permit a quantification of both, a unilateral detachment and wrapping in a polyvinylidine fluoride membrane of the supraspinatus was done in 22 rabbits. After 12 weeks, the polyvinylidine fluoride membrane was removed in 10 rabbits and the tendon inserted into a bony trough. The opposite shoulder served as control. All rabbits were killed at 24 weeks. Accumulation of fat on the muscle surface as well as between the muscle fascicles and occasionally in the sarcoplasm was increased in both experimental groups when compared to controls (non‐reattached 12.8%, reattached 23.5% vs controls 5.4%; all p < 0.005). The muscle atrophy was significant (non‐reattached 9.2 ml, reattached 9.1 ml vs controls 10.7 ml; both p < 0.005). Neither atrophy nor fat accumulation were reversed by surgical reattachment of the supraspinatus tendon. On the contrary, the fat accumulation was higher in the reattached group (p < 0.005). The trauma of reattachment, the delay between detachment and reattachment or an insufficient duration of recovery may have contributed to both, the lack of reversibility of supraspinatus atrophy and fat accumulation. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Effects of different exposures of hyperbaric oxygen on ligament healing in rats
Authors: Ishii Yoshimasa , Ushida Takashi , Tateishi Tetsuya , Shimojo Hitoshi , Miyanaga Yutaka
Published: October 2002

Hyperbaric oxygen (HBO) is a method of augmenting, intermittently, oxygen availability to tissues. We examined the effect of three different HBO exposures on the healing of experimentally induced ligament lacerations in the right hind limb of 44 male Wistar rats. Animals were divided into four groups after ligament injury: (a) control group, animals breathed room air at 1 ATA (atmosphere absolute) in a hyperbaric chamber for 60 min; (b) HBO treatment at 1.5 ATA for 30 min once a day, (c) HBO treatment at 2 ATA for 30 min once a day, (d) 2 ATA for 60 min once a day. At 14 days post‐ligament injury, we compared the ligaments of the four treatment groups for gross appearance, histology and expression of pro‐α(I) mRNA by northern hybridization. Our results indicate that HBO was effective in promoting ligament healing compared to control (p < 0.01). Of these three exposures, HBO at 2 ATA for 60 min was the most effective, resulting in enhanced extra‐cellular matrix deposition as measured by collagen synthesis. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Assessment of specific mRNA levels in cartilage regions in a lapine model of osteoarthritis
Authors: Le Graverand Marie‐Pierre Hellio , Eggerer Jonna , Vignon Eric , Otterness Ivan G. , Barclay Leona , Hart David A.
Published: October 2002

Osteoarthritis (OA) is the most common form of arthritis and patients with meniscal and ligament injuries of the knee are at high risk to develop the disease. The purpose of this study was to evaluate molecular and structural changes occurring in four articular cartilage (AC) regions from the knees of anterior cruciate ligament (ACL)‐transected rabbits at 3 and 8 weeks post‐surgery. Rabbit AC from the lateral and medial femoral condyles (LFC and MFC) as well as from the medial and lateral tibial plateau (MTP and LTP) were processed for histology and for semi‐quantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis for a subset of relevant molecules (collagen II, aggrecan, biglycan, decorin, fibromodulin, MMP‐1,‐3,‐13, and TIMP‐1). While the most severe histological changes were observed in the MTP starting as early as 3 weeks post‐ACL transection based on Mankin scores, histological examination demonstrated a progression of osteoarthritic changes in the MFC from 3 to 8 weeks post‐surgery. In contrast, very few changes were observed within both the LFC and LTP, and these changes did not worsen with increasing time after surgery. The water content increased significantly in the MFC at 8 weeks post‐ACL transection and at both 3 and 8 weeks post‐ACL transection in the MTP. Significant decreases in DNA content were observed for the MFC, LTP and MTP at 8 weeks post‐ACL transection. Total RNA yields from the MFC and MTP were significantly elevated at 8 weeks post‐ACL transection, while in the lateral compartment total RNA was unchanged following ACL transection. Analysis of mRNA levels for a subset of matrix molecules, proteinases and proteinase inhibitors, by RT‐PCR demonstrated significant region‐specific changes at the mRNA level following ACL transection. These results show that following ACL transection, complex molecular, as well as structural changes occur early in cartilage and that the observed changes are both region‐specific and time‐dependent. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Coordinated expression of scleraxis and Sox9 genes during embryonic development of tendons and cartilage
Authors: Asou Yoshinori , Nifuji Akira , Tsuji Kunikazu , Shinomiya Kenichi , Olson Eric N. , Koopman Peter , Noda Masaki
Published: October 2002

Embryonic development of tendons is in close association with that of cartilage and bone. Although these tissues are derived from mesenchymal progenitor cells which also give rise to muscle and fat, their fates clearly diverse in early embryonic stages. Transcription factors may play pivotal roles in the process of determination and differentiation of tendon cells as well as other cells in the skeletal system. Scleraxis, a basic helix‐loop‐helix (bHLH) type transcription factor, is expressed in mesenchymal progenitors that later form connective tissues including tendons. Sox9 is an HMG‐box containing transcription factor, which is expressed at high levels in chondrocytes. We hypothesized that the two transcription factors regulate the fate of cells that interact with each other at the interface between the two tissues during divergence of their differentiation pathways. To address this point, we investigated scleraxis and Sox9 mRNA expression during mouse embyogenesis focusing on the coordinated development of tendons and skeletons. In the early stage of mesenchymal tissue development at 10.5 d.p.c., scleraxis and Sox9 transcripts were expressed in the mesenchymal progenitor cells in the appendicular and axial mesenchyme. At 11.5 d.p.c., scleraxis transcripts were observed in the mesenchymal tissue surrounding skeletal primordia which express Sox9. From this stage, scleraxis expression was closely associated with, but distinct from, formation of skeletal primordia. At 13.5 d.p.c., scleraxis was expressed broadly in the interface between muscle and skeletal primordia while Sox9 expression is confined within the early skeletal primordia. Then, at 15.5 d. p.c., scleraxis transcripts were more restricted to tendons. These observations revealed the presence of temporal and spatial association of scleraxis expression during embryonic development of tendon precursor cells in close association with that of Sox9 expression in chondrogenic cells in skeletal tissues. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Quantitative computed tomography estimates of the mechanical properties of human vertebral trabecular bone
Authors: Kopperdahl David L. , Morgan Elise F. , Keaveny Tony M.
Published: October 2002

The objective of this study was to report our quantitative computed tomography (QCT) density‐mechanical property regressions for trabecular bone for use in biomechanical modelling of the human spine. Cylindrical specimens of human vertebral trabecular bone (from T10 to L4) were cored from 32 cadavers (mean ± SD age = 70.1 ± 16.8; 13 females, 19 males) and scanned using QCT. Mechanical tests were conducted using a protocol that minimized end‐artifacts over the apparent density range tested (0.09–0.38 g/ cm3). To account for the presence of multiple specimens per donor in this data set, donor was treated as a random effect in the regression model. Mean modulus (319 ± 189 MPa) was higher and mean yield strain (0.78 ± 0.06%) was lower than typical values reported previously due to minimization of the end‐artifact errors. QCT density showed a strong positive correlation with modulus (n = 76) and yield stress (r2 = 0.90–0.95, n = 53, p < 0.001). There was a weak positive linear correlation with yield strain (r2 = 0.58, n = 53, p = 0.07). Prediction errors, incurred when estimating modulus or strength for specimens from a new donor, were 30–36% of the mean values of these properties. Direct QCT density‐mechanical property regressions gave more precise predictions of mechanical properties than if physically measured wet apparent density was used as an intermediate variable to predict mechanical properties from QCT density. Use of these QCT density‐mechanical property regressions should improve the fidelity of QCT‐based biomechanical models of the human spine for whole bone and bone‐implant analyses. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Dobutamine echocardiography in idiopathic dilated cardiomyopathy: clinical and prognostic implications
Authors: Pinamonti Bruno , Perkan Andrea , Di Lenarda Andrea , Gregori Dario , Sinagra Gianfranco
Published: October 2002

The dobutamine echocardiographic test (DET) is frequently used in coronary artery disease to detect viable myocardium, but few data are available about its role in idiopathic dilated cardiomyopathy (IDCM). The aims of this study were to evaluate the clinical role of DET and the prognostic implications of the ‘contractile reserve’ in patients with IDCM treated with optimal medical therapy, including β‐blockade (BB). A total of 51 patients with IDCM underwent DET at diagnosis. A positive response to DET (DET+) was judged to be a significant increase (≥10 points) in left ventricular ejection fraction (LVEF) with a peak value ≥40%, and a reversed restrictive left ventricular filling pattern (RFP) if present at baseline study. Improvement at follow‐up was defined according to combined clinical and echo‐Doppler criteria. In all, 22 patients (43%) were classified as DET+. DET+ patients were less symptomatic (P<0.001), with lower heart rate (P<0.01), less enlarged left and right ventricles (P<0.0001 and P<0.05), higher LVEF (P‐0.0001), less frequent RFP (P‐0.01), and lower pulmonary pressure (P<0.01). At follow‐up (34±16 months), 21 patients had improved, while four had died and seven had received a transplant. Among clinical data, NYHA classes I–II (OR‐0.25, P‐0.07) and BB dosage (OR‐0.97, P<0.005) were significantly associated with higher transplant‐free survival at multivariate analysis. The addition of DET+ (OR‐0.34, P<0.05) showed a moderate but significant improvement of sensitivity, but the predictive power of the model remained low (sensitivity, 0.67; specificity, 0.55). Absence of left bundle branch block (OR‐0.27, P<0.01) and BB dosage (OR‐1.03, P<0.005), but not DET+, were predictive of improvement. In patients with IDCM, DET response is associated with a more favourable outcome, since it suggests an earlier stage of the disease. However, in the light of our data, the incremental prognostic power of DET response compared to clinical evaluation at enrolment, despite being significant, seems to be of limited clinical value. Further studies should be carried out in order to clarify the prognostic value of DET in IDCM patients.
Increased myocardial expression of osteopontin in patients with advanced heart failure
Authors: Stawowy Philipp , Blaschke Florian , Pfautsch Peter , Goetze Stephan , Lippek Frank , Wollert‐Wulf Brigitte , Fleck Eckart , Graf Kristof
Published: October 2002

The expression of the adhesion protein osteopontin (OP) is associated with cardiac hypertrophy and is significantly increased after transition to heart failure in experimental animal models. We, therefore, hypothesized that OP could be upregulated in heart failure in humans. In the present study, we investigated the expression of OP in myocardial biopsies obtained from patients with heart failure due to dilated cardiomyopathy (mean LVEF ‐ 30.3±4.4%, mean±S.D., n ‐ 10, group A) compared to patients with a normal left‐ventricular ejection fraction (mean LVEF ‐ 61±11.2%, n ‐ 9; group B). Myocardial immunoreactivity for OP was examined using two different antibodies against OP. The expression of cardiac myocyte OP was significantly upregulated in group A in comparison to group B (P<0.0001). Both groups also displayed OP immunoreactivity in non‐myocytes, including vascular smooth muscle cells and cardiac fibroblasts (P ‐ not significant). Statistical analysis revealed a significant correlation of increased OP immunoreactivity in cardiac myocytes of patients with impaired left ventricular function, assessed by hemodynamic data (LVEF, RVEF, LVESVI, LVEDVI and LVEDP, R ‐ −0.828, −0.671, 0.751, 0.685 and 0.461, respectively; all P<0.05). Furthermore, OP expression correlated with cardiac myocyte hypertrophy (mean diameter 21.0±1.8 μm in group A and 16.6±2.1 μm in group B; P<0.0001). In conclusion, the present study indicates, that factors and/or mechanisms involved in heart failure in patients with dilated cardiomyopathy, lead to induction of OP expression in humans.
Prevalence of chronic heart failure in Southwestern Europe: the EPICA study
Authors: Ceia Fátima , Fonseca Cândida , Mota Teresa , Morais Humberto , Matias Fernando , de Sousa António , Oliveira António Gouveia ,   on behalf of the EPICA Investigators
Published: October 2002

Aim

To estimate the prevalence of chronic heart failure (CHF) in mainland Portugal in 1998.

Methods and population

A community‐based epidemiological survey involving subjects attending primary care centres selected by a combined two‐stage sampling and stratified procedure. General practitioners (GPs) randomly selected in proportion to the population of the District, evaluated subjects attending primary care centres aged over 25 years, recruited consecutively and stratified by age. CHF cases were identified according to the Guidelines of the European Society of Cardiology for CHF diagnosis.

Results

5434 eligible subjects were evaluated by 365 GPs; 551 patients with CHF were identified. The overall prevalence and 95% CI of CHF in mainland Portugal is 4.36% (3.69–5.02%), 4.33% in males (3.19–5.46%), and 4.38% in females (3.64–5.13%). Age‐specific CHF prevalence was as follows: 1.36% in the 25–49 years‐old group (0.39–2.33%), 2.93% in the 50–59 years‐old group (5.58–9.37%), 7.63% in the 60–69 years‐old group (5.58–9.37%), 12.67% in the 70–79 years‐old group (10.73–14.6%), and 16.14% in group over 80 years old (13.81–18.47%). The prevalence of CHF due to systolic dysfunction was 1.3% and the prevalence of CHF with normal systolic function was 1.7%.

Conclusions

The overall prevalence of CHF in Portugal was slightly higher than that of other European studies and increases sharply with age. The prevalence of CHF due to systolic dysfunction is very similar to that reported by other recent European studies. The differences found may correspond to differences in methodology rather than actual differences in the population.