bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhancement of transglutaminase activity by NMR identification of its flexible residues affecting the active site
Authors: Shimba Nobuhisa , Shinohara Mina , Yokoyama Kei-ichi , Kashiwagi Tatsuki , Ishikawa Kohki , Ejima Daisuke , Suzuki Ei-ichiro
Published: October 2002

Incorporation of inter‐ or intramolecular covalent cross‐links into food proteins with microbial transglutaminase (MTG) improves the physical and textural properties of many food proteins, such as tofu, boiled fish paste, and sausage. By using nuclear magnetic resonance, we have shown that the residues exhibiting relatively high flexibility in MTG are localized in the N‐terminal region; however, the N‐terminal region influences the microenvironment of the active site. These results suggest that the N‐terminal region is not of primary importance for the global fold, but influences the substrate binding. Therefore, in order to increase the transglutaminase activity, the N‐terminal residues were chosen as candidates for site‐directed replacement and deletion. We obtained several mutants with higher activity, del1–2, del1–3, and S2R. We propose a strategy for enzyme engineering targeted toward flexible regions involved in the enzymatic activity. In addition, we also briefly describe how the number of glutamine residues in a substrate protein can be increased by mixing more than two kinds of TGases with different substrate specificities.
Alternative arrangements of catalytic residues at the active sites of restriction enzymes
Authors: Tamulaitis Gintautas , Solonin Alexander S. , Siksnys Virginijus
Published: October 2002

A catalytic sequence motif PDX10–30(E/D)XK is found in many restriction enzymes. On the basis of sequence similarities and mapping of the conserved residues to the crystal structure of NgoMIV we suggest that residues D160, K182, R186, R188 and E195 contribute to the catalytic/DNA binding site of the Ecl18kI restriction endonuclease. Mutational analysis confirms the functional significance of the conserved residues of Ecl18kI. Therefore, we conclude that the active site motif 159VDX21KX12E of Ecl18kI differs from the canonical PDX10–30(E/D)XK motif characteristic for most of the restriction enzymes. Moreover, we propose that two subfamilies of endonucleases Ecl18kI/PspGI/EcoRII and Cfr10I/Bse634I/NgoMIV, specific, respectively, for CCNGG/CCWGG and RCCGGY/GCCGGC sites, share conserved active site architecture and DNA binding elements.
Hepatic amino acid‐dependent signaling is under the control of AMP‐dependent protein kinase
Authors: Dubbelhuis Peter F , Meijer Alfred J
Published: October 2002

It has become increasingly clear in recent years that amino acids can stimulate a signal transduction pathway resulting in the phosphorylation of mammalian target of rapamycin downstream targets. We have now found that amino acid‐dependent phosphorylation of p70S6 kinase and of S6 in hepatocytes is prevented when AMP‐dependent protein kinase (AMPK) is activated by either the purine ribonucleoside analogue AICAriboside, fructose or glycerol. Insulin‐dependent phosphorylation of protein kinase B is not affected by AMPK activation. Protein synthesis is strongly inhibited when AMPK is activated. It is concluded that amino acid‐dependent signaling, a protein‐anabolic signal, can be effectively antagonized by activation of AMPK.
Earthworm species of the genusEiseniacan be phenotypically differentiated by metabolic profiling
Authors: Bundy Jacob G , Spurgeon David J , Svendsen Claus , Hankard Peter K , Osborn Daniel , Lindon John C , Nicholson Jeremy K
Published: October 2002

The universality of low molecular weight metabolites allows rapid and straightforward investigation of the biochemistry of genetically uncharacterised species. Thus ex vivo metabolic profiling in combination with multivariate data analysis (metabonomics) offers great potential in comparative biology. Here we present the first use of high resolution nuclear magnetic resonance (NMR) spectroscopy to distinguish closely related animal species via their metabolic phenotype (metabotype). We have profiled the threeEisenia(Oligochaeta, Lumbricidae) speciesEisenia fetida,Eisenia andreiandEisenia venetausing tissue extracts and coelomic fluid analysis. The low molecular weight biochemical profiles of tissue extracts were highly conserved for all three species, withE. fetidaandE. andreibeing more similar to each other than toE. veneta. However the metabolic profiles of the coelomic fluid of the different species were highly distinctive – the NMR spectra allowed unequivocal identification of species. Multivariate statistics were also used to quantify these spectral differences and to enable simplified graphical visualisation of species similarity. These results show that two morphologically undistinguishable species (E. fetidaandE. andrei) differ markedly in their biochemical profiles despite apparently occupying the same ecological niche, and indicate that metabolic phenotype profiling can be used as a powerful functional genomics tool.
Overexpression of human metallothionein‐III prevents hydrogen peroxide‐induced oxidative stress in human fibroblasts
Authors: You Ho Jin , Lee Kyung Jin , Jeong Hye Gwang
Published: October 2002

Metallothioneins (MT) are ubiquitous low molecular weight metal binding proteins that may act as antioxidants. In the present study, the cloned human MT‐III coding region was permanently transfected into GM00637 cells in order to investigate the antioxidative effects of this brain‐specific MT isoform. GM00637/MT‐III cells overexpressed MT‐III mRNA versus pcDNA3 plasmid‐transfected control cells (GM00637). When challenged with H2O2, the GM00637/MT‐III cells displayed significantly more resistance than the GM00637 cells, as determined by cell cytotoxicity, lactate dehydrogenase leakage, and lipid peroxidation. In addition, the GM00637/MT‐III cells were highly protected from the H2O2‐induced production of reactive oxygen species and DNA damage. These results directly support the antioxidative role of MT‐III, and demonstrate MT‐III can scavenge free oxygen radicals and protect cells from oxidative stress.
The proline rich region of the Tec homology domain of ITK regulates its activity
Authors: Hao Shengli , August Avery
Published: October 2002

Inducible T‐cell kinase (ITK) is a member of the Tec family of tyrosine kinases that are involved in signals emanating from cytokine receptors, antigen receptors and other lymphoid cell surface receptors. Stimulation of tyrosine phosphorylation and activation of ITK by the T‐cell antigen receptor, CD28 and CD2 requires the presence of the Src family kinase Lck in T‐cells. We have previously demonstrated that the activation of ITK by Src family kinases uses a phosphatidylinositol 3‐kinase pathway, which recruits ITK to the membrane via its pleckstrin homology (PH) domain where it is acted upon by Src. We have further explored the mechanism of this requirement for Src family kinases in the activation of ITK. We found that deletion of the proline rich sequence found in the Tec homology domain of ITK results in reduced basal activity of ITK approximately 50%. These differences in the basal activity of ITK were observed when the PH domain was deleted or when the kinase was membrane targeted. Furthermore, this deletion reduces the ability of the Src family kinase Lck to activate ITK, as well as to induce the ITK mediated tyrosine phosphorylation of its substrate PLCγ1. By contrast, deletion of the SH3 domain of ITK resulted in a two‐fold increase in the basal activity of ITK, and allowed this mutant to have an enhanced response to Lck. These results suggest that the proline rich region within the Tec homology domain of ITK regulates its basal activity and its response to Src family kinase signals.
Modulation of the human polycystin‐L channel by voltage and divalent cations
Authors: Liu Yan , Li Qiang , Tan Miao , Zhang Yu-Yang , Karpinski Edward , Zhou Jing , Chen Xing-Zhen
Published: October 2002

Polycystin‐L (PCL) is highly homologous in sequence and membrane topology to polycystin‐2, the product of the second gene responsible for autosomal dominant polycystic kidney disease (ADPKD). PCL and polycystin‐2 were recently shown to be Ca2+‐permeable, Ca2+‐activated cation channels. Further characterization of polycystins will help in the understanding of cystogenesis and pathogenesis of ADPKD. In the present study, we expressed human PCL in Xenopus oocytes and studied its function utilizing patch‐clamp and two‐electrode voltage clamp techniques. In addition to its permeability to Ca2+, K+ and Na+, PCL was highly permeable to NH4
+ and Cs+ with a permeability ratio NH4
+:Cs+:Na+ of 2.2:1.02:1. Voltage modulation of channel properties was studied using cell‐attached (C‐A) and excised inside‐out (I‐O) patches. In the C‐A mode, the open probability (NPo) of PCL at negative potentials (NPo=0.22) was higher than at positive potentials (NPo=0.05). The mean open time averaged 31.6 ms at negative potentials, and 6.2 ms at positive potentials; single‐channel activity exhibited bursts with a mean interburst time of 178 ms. Using I‐O patches under symmetrical ionic conditions, single‐channel inward conductance was significantly larger than outward conductance, indicating a slight inward rectification. External Mg2+ inhibited the PCL channel currents. The inhibitory effect was voltage‐dependent and substantially reduced by depolarization. The time course of inactivation depended on external calcium concentration but was independent of voltage and peak current. This study shows that although PCL is not a voltage‐gated channel, its channel activity and inhibition by Mg2+ are modulated by membrane potential.
Chemosensitivity of human prostate cancer cells PC3 and LNCaP to genistein isoflavone and β‐lapachone
Authors: Kumi‐Diaka J.
Published: October 2002

A wide spectrum of anti‐cancer activity of genistein and β‐lapachone in various tumors has been reported in single treatments. In this study the combined effects of genistein and β‐lapachone on the chemosensitivity of LNCaP and PC3 human prostate cancer cells was determined in vitro, using 3‐〚4,5‐dimethylthiazol‐2‐yl〛‐2‐,5‐diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) to study treatment‐induced growth inhibition and cytotoxicity and, annexin V‐fluoresceine (FI) and terminal deoxyribonucleotidyl transferase‐mediated dUTP nick‐end labeling (TUNEL)–propidium iodide (PI) assays to determine potential treatment‐induced apoptosis and/or necrosis. The results showed: i) that both PC3 and LNCaP are sensitive to single and combination treatments regardless of hormone sensitivity status, ii) that treatment induced dual death pathways (apoptosis and necrosis) in both cell types, iii) that growth inhibition in both cell types correlated positively with cell death via apoptosis at lower drug concentrations and necrosis at higher concentrations, iv) that combination of genistein and β‐lapachone had synergistic inhibitory effects on growth and proliferation in both cell types. The synergistic inhibitory effect was correlated positively with treatment‐induced cell death via apoptosis and necrosis. The overall results indicate that combination treatments with β‐lapachone and genistein are more potent in killing both PC3 and LNCaP cancer cells than treatment with either genistein or β‐lapachone alone. β‐lapachone acts at the G1 and S phase checkpoints in the cell cycle, while genistein induces cell cycle arrest at the G2–M stage. The current results are therefore in agreement with the hypothesis that drug combinations that target cell cycles at different critical checkpoints would be more effective in causing cell death. This result provides a rationale for in vivo studies to determine whether β‐lapachone—genistein combination will provide effective chemotherapy for prostate cancer, regardless of the tumor sensitivity to hormone.
New magnet‐sensitive structures in bacterial and archaeal cells
Authors: Vainshtein M. , Suzina N. , Kudryashova E. , Ariskina E.
Published: October 2002

The objects of the investigation were: distribution of intracellular magnet‐sensitive structures among different taxonomic groups of prokaryotes, localisation and organisation of the magnet‐sensitive inclusions (MsI) in cells. The MsI were discovered in representatives of both prokaryotic domains (Bacteria and Archaea), 2 kingdoms and 7 orders of bacteria. They were some amorphous or non‐crystalline globules with the electron‐transparent centre surrounded with an electron‐dense homogenous matrix. The magnetic nature of the structures was shown by attraction with an applied magnet both for the cell suspensions and for the MsI isolated and separated from the destroyed cells. The MsI were studied with transparent electron microscopy and with X‐ray analyses. When the cells were grown in the iron‐containing nutrient medium, the matrix was enriched with iron. It was shown also that some bacteria grown with cobalt or with chromium contained the cobalt‐ or chromium‐enriched magnetic inclusions.
Miranda, a protein involved in neuroblast asymmetric division, is associated with embryonic centrosomes of Drosophila melanogaster
Authors: Mollinari Cristiana , Lange Bodo , González Cayetano
Published: October 2002

Miranda, a highly coiled‐coil protein, has been shown to be one of the key components necessary for the differential segregation of fate‐determining factors during neurogenesis in Drosophila embryos. The multidomain protein Miranda, by segregating the transcription factor Prospero, guarantees the generation of cell diversity during the formation of the embryonic nervous system. While looking for new molecular components of the centrosome in Drosophila embryos, we have isolated a short isoform of Miranda. An antibody was raised directed against the central coiled‐coil region of Miranda, and the pattern of expression of the protein was studied in details throughout Drosophila development. Here, we show that Miranda has a broad pattern of expression and is a rather ubiquitous molecule. Immunofluorescence on syncytial Drosophila embryos shows that Miranda has a dynamic redistribution and is associated with centrosomes. Electron microscopy on purified syncytial centrosomes shows that Miranda is located on the pericentriolar material along with γ‐tubulin. Taken all together, our data indicate for the first time that Miranda belongs to a growing class of proteins that concentrate at the centrosome in a cell‐cycle and stage‐specific manner. The observations that Miranda has a broad range of expression, as well as a dynamic and cell‐cycle dependent subcellular localization, suggest that it may have alternative functions outside the embryonic nervous system. These potential new functions are discussed in this paper.
The impact of strategic operations management decisions on community hospital performance
Authors: Li Ling X. , Benton W.C. , Leong G.Keong
Published: October 2002

Over the past decade, 10% of community hospitals have closed. In this challenging time, our study presents hospital administrators with some valuable information that can help improve community hospitals’ performance. The purpose of this paper is to develop a strategic operations management model that links long‐term service choices, intermediate operations decisions, and hospital performance given the structural constraints of location, size, and medical teaching status. Data collected from 151 community hospitals are used to test the model. The research identifies strategic operations management decisions in the US community hospitals, shows their causal relationships, and identifies their effects on hospital performance. Specifically, we find that intermediate infrastructural operations decisions affect a community hospital’s cost, quality, and financial performance after the structural decisions of location and size have set the stage. Our study also reveals that community hospitals have adopted new staff and demand management decisions in response to the market needs.
Weight‐bearing alters the expression of collagen types I and II, BMP 2/4 and osteocalcin in the early stages ofdistraction osteogenesis
Authors: Radomisli Timothy E. , Moore Douglas C. , Barrach Hans J. , Keeping Hugh S. , Ehrlich Michael G.
Published: October 2002

This study was performed to investigate the effect of loading on the biology of newly forming bone during limb lengthening. Unilateral 2.0 mm femoral lengthenings were performed in 20 male Sprague Dawley rats. Half (n = 10) of the animals were allowed to bear weight freely, while the other half were prevented from weight‐bearing via an ipsilateral through‐knee amputation. The animals in each group were sacrificed after one (n = 5) or four (n = 5) days of consolidation (post‐operative days seven and 10, respectively). In situ hybridization for osteocalcin and collagen I, and antibody staining for collagen II and BMP 2/4 were used to evaluate the molecular influence of loading.

There was more new bone in the distraction gap of the weight‐bearing animals than there was in the non‐weight‐bearing animals. BMP 2/4 expression, and the messages for collagen I and osteocalcin, were more abundant in tissue from the weight‐bearing animals; collagen II was higher in the non‐weight‐bearing animals. This suggests that early regenerate tissue is capable of responding to loading, and that weight‐bearing appears to stimulate intramembranous ossification. These findings support the concept of early weight‐bearing after limb lengthening.
Relative roles of microdamage and microfracture in the mechanical behavior of trabecular bone
Authors: Yeh Oscar C. , Keaveny Tony M.
Published: October 2002

Compared to trabecular microfracture, the biomechanical consequences of the morphologically more subtle trabecular micro‐damage are unclear but potentially important because of its higher incidence. A generic three‐dimensional finite element model of the trabecular bone microstructure was used to investigate the relative biomechanical roles of these damage categories on reloading elastic modulus after simulated overloads to various strain levels. Microfractures of individual trabeculae were modeled using a maximum fracture strain criterion, for three values of fracture strain (2%, 8%, and 35%). Microdamage within the trabeculae was modeled using a strain‐based modulus reduction rule based on cortical bone behavior. When combining the effects of both microdamage and microfracture, the model predicted reductions in apparent modulus upon reloading of over 60% at an applied apparent strain of 2%, in excellent agreement with previously reported experimental data. According to the model, up to 80% of the trabeculae developed microdamage at 2% apparent strain, and between 2% and 10% of the trabeculae were fractured, depending on which fracture strain was assumed. If microdamage could not occur but microfracture could, good agreement with the experimental data only resulted if the trabecular hard tissue had a fracture strain of 2%. However, a high number of fractures (10% of the trabeculae) would need to occur for this case, and this has not been observed in published damage morphology studies. We conclude therefore that if the damage behavior of trabecular hard tissue is similar to that of cortical bone, then extensive microdamage is primarily responsible for the large loss in apparent mechanical properties that can occur with overloading of trabecular bone. © 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Osteogenic protein‐1 induced bone formation in an infected segmental defect in the rat femur
Authors: Chen Xinqian , Kidder Louis S. , Lew William D.
Published: October 2002

The goal of this study was to use a segmental defect model in the rat femur to determine if osteogenic protein‐1 (OP‐1) is capable of inducing bone formation in the presence of bacterial contamination. A 6 mm segmental defect was surgically created and stabilized with a polyacetyl plate and Kirschner wires in one femur in each of 126 Sprague–Dawley rats. The animals were divided into eight groups in which the defect was either left untreated, or subjected to various combinations of OP‐1 (11 or 50 μg), lyophilized bovine type I collagen (carrier for the OP‐1), and 105 colony‐forming units of Staphylococcus aureus. The animals were euthanized at either 2, 4, or 9 weeks. Quantitative radiographic and histologic analyses were performed on the harvested tissue. The initial contamination progressed to infection in all animals receiving bacteria, as determined by qualitative bacteriology. There was very little, if any, bone formation in the untreated defects, and in the contaminated defects with or without collagen carrier. Bone formation was significantly greater in contaminated defects with either dose of OP‐1, compared with contaminated defects without OP‐1. The 50 μg dose of OP‐1 induced significantly more bone formation than the 11 μg dose, both with and without bacteria. This investigation has demonstrated that OP‐1 maintains its osteoinductive capability in a contaminated segmental defect. OP‐1 may potentially be used in the clinical management of contaminated fractures. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Integrin expression by human osteoblasts cultured on degradable polymeric materials applicable for tissue engineered bone
Authors: El‐Amin Saadiq F. , Attawia Mohamed , Lu Helen H. , Shah Asist K. , Chang Richard , Hickok Noreen J. , Tuan Rocky S. , Laurencin Cato T.
Published: October 2002

The use of biodegradable polymers in the field of orthopaedic surgery has gained increased popularity, as surgical pins and screws, and as potential biological scaffolds for repairing cartilage and bone defects. One such group of polymers that has gained considerable attention are the polyesters, poly(lactide‐co‐glycolide) (PLAGA) and polylactic acid (PLA), because of their minimal tissue inflammatory response, favorable biocompatibility and degradation characteristics. The objective of this study was to evaluate human osteoblastic cell adherence and growth on PLAGA and PLA scaffolds by examining integrin receptor (α2, α3, α4, α5, α6 and β1) expression. Primary human osteoblastic cells isolated from trabecular bone adhered efficiently to both PLAGA and PLA, with the rate of adherence on PLAGA comparable to that of control tissue culture polystyrene (TCPS), and significantly higher than on PLA polymers at 3, 6 and 12 h. Human osteoblastic phenotypic expression, alkaline phosphatase (ALP) activity was positive on both degradable matrices, whereas osteocalcin levels were significantly higher on cells grown on PLAGA than on PLA composites. Interestingly, the integrin subunits, α2, α3, α4, α5, α6 and β1 were all expressed at higher levels by osteoblasts cultured on PLAGA than those on PLA as analyzed by westerns blots and by flow cytometry. Among the integrins, α2, α5 and β1 showed the greatest difference in levels between the two surfaces. Thus, both PLA and PLAGA support osteoblastic adhesion and its accompanying engagement of integrin receptor and expression of osteocalcin and ALP. However PLAGA consistently appeared to be a better substrate for osteoblastic cells based on these parameters. This study is one of the first to investigate the ability of primary human osteoblastic cells isolated from trabecular bone to adhere to the biodegradable polymers PLAGA and PLA, and to examine the expression of their key adhesion receptors (integrins) on these substrates. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Tissue transglutaminase localization and activity regulation in the extracellular matrix of articular cartilage
Authors: Summey Brett T. , Graff Ronald D. , Lai Thung‐Sheng , Greenberg Charles S. , Lee Greta M.
Published: October 2002

Tissue transglutaminase (tTG) catalyzes a Ca2+‐dependent transglutaminase (TGase) activity which cross‐links proteins and stabilizes many tissues [C.S. Greenberg et al. FASEB J. 5 (1991) 3071]. Because cartilage is subjected to great stress in vivo, an enzyme that strengthens and stabilizes tissue could play an integral role in maintaining cartilage integrity. The purpose of this study was to determine if active tTG is present in the extracellular matrix (ECM) of adult human osteoarthritic articular cartilage. Using a TGase activity assay along with immunolabeling for tTG of cartilage sections, TGase activity and tTG immunoreactivity were localized in the ECM in cartilage sections, predominantly in the superficial layer. Previous in vitro studies have demonstrated that the Mg‐GTP complex inhibits the TGase activity of tTG [T.S. Lai et al. J. Biol. Chem. 273 (1998) 1776]. To investigate the in situ regulation of the TGase activity of tTG, a TGase activity assay was done with a dose response of GTP, measuring incorporation of fluorescein cadaverine. TGase activity was inhibited by GTP in a similar manner as in vitro. These results not only confirm tTG presence in the ECM, but also indicate tTG as the major TGase activity of the ECM. Secondly, the study provides a possible mechanism by which extracellular tTG is regulated in vivo. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Estrogen regulation of growth and alkaline phosphatase expression by cultured human bone marrow stromal cells
Authors: Holzer Gerold , Einhorn Thomas A. , Majeska Robert J.
Published: October 2002

Estrogen has been reported to regulate the growth and differentiation of cultured murine osteoprogenitor cells in bone marrow stroma. This study tested the ability of 17β‐estradiol (E2) to regulate growth and expression of alkaline phosphatase (ALP), an osteoblastic differentiation marker, in strains of normal human bone marrow stromal cells derived from different donors. In eight strains examined, E2 at 1 and 10 nM produced at most modest effectxs on growth and ALP activity. Growth inhibition, seen in 4 of the 8 strains, was more common than stimulation (2 of the 8 strains); the greatest observed E2 effect was an inhibition of ca. 50%. E2 altered ALP activity less dramatically than cell growth. Differences from control in total ALP per culture were seen in only two strains; one was a reduction, one an increase. Colony forming assays were used to determine if E2 changed the proportion of ALP‐expressing cells in marrow stromal cell cultures. In contrast to growth experiments, ALP expression under colony forming conditions (200 cells per 35 mm‐diameter well) was dependent on the type of serum supplementation used. Under permissive conditions using medium supplemented with 10% charcoal‐treated fetal bovine serum, 10 nM E2 increased the number of ALP‐positive colonies (cfu‐ap) but not the total number of colonies formed (cfu‐f). When cells cultured in the presence or absence of 10 nM E2 were replated at colony forming densities, significantly higher proportions of cfu‐ap were found in 2 of 6 strains examined, while pretreatment with E2 affected the number of cfu‐f in only 1 of the 6 strains. Similar results were obtained when colony formation was carried out in the presence of dexamethasone and ascorbate, although these agents themselves increased the formation of both cfu‐f and cfu‐ap. These results show that the direct effects of E2 on human marrow stromal cells are small and vary depending on the cell strain and on the experimental conditions; however, the E2 actions observed in this study were consistent with reports that E2 exerts direct actions on osteoblasts and osteoblast progenitor cells that favor rather than suppress their phenotypic expression. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Quantitative cartilage imaging of the human hind foot: Precision and inter‐subject variability
Authors: Al‐Ali Dina , Graichen Heiko , Faber Sonja , Englmeier Karl‐Hans , Reiser Maximilian , Eckstein Felix
Published: October 2002

Alterations of ankle cartilage are observed in degenerative and inflammatory joint disease, but cartilage cannot be directly visualized by radiography. The purpose of this study was therefore to analyze the feasibility and precision of quantitative cartilage imaging in the human hind foot (talocrural, talotarsal, and intertarsal joints), and to report the inter‐subject variability for cartilage volume, thickness and surface areas. The feet of 16 healthy volunteers were imaged using a 3D gradient‐echo magnetic resonance imaging sequence with water‐excitation. After interpolation to a resolution of 1 ± 0.125 ± 0.125 mm3 the cartilage plates were segmented, and the cartilage volume, thickness, and surface areas determined. The precision (four repeated measurements) was examined in eight volunteers, the RMS average CV% being 2.1% to 10.9% in single joint surfaces, and ⩽ 3% for the cumulative values of all joints. The mean cartilage thickness ranged from 0.57 ± 0.08 (navicular surface) to 0.89 ± 0.19 mm (trochlear surface for tibia). In conclusion this study shows that it is feasible to quantify thin cartilage layers in the hind foot under in vivo imaging conditions, and that the precision errors are substantially smaller than the inter‐subject variability in healthy subjects. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Identification of the metalloproteinase stromelysin in the physis
Authors: Armstrong Ann L. , Barrach Hans J. , Ehrlich Michael G.
Published: October 2002

For long bone growth to occur, calcification of the matrix must commence in the lower hypertrophic zone of the growth plate. It is generally accepted that physeal proteoglycans help regulate mineralization, and that at least in vitro, intact proteoglycans can inhibit mineralization. Thus degradation of proteoglycan may be a necessary step prior to calcification. Previous work in our laboratory has demonstrated the presence of neutral metallo‐proteases in the growth plate with highest levels in the hypertrophic zone, where calcification occurs.

Stromelysin (MMP‐3) is a connective tissue matrix‐degrading enzyme. It was formerly known as proteoglycanase and is generally considered to be one of the major proteoglycan degrading enzymes in cartilage. Stromelysin is implicated in cartilage destruction in osteoarthritis and may also be involved in tissue remodeling in the physis. Our goal was to determine if the neutral protease previously reported by the authors in the physis was stromelysin.

In this study we used Western blots and antibodies to stromelysin and to the stromelysin cleavage site in aggrecan, the most common form of proteoglycan, to demonstrate the presence of stromelysin in the bovine physis. When an antibody raised against the stromelysin cleavage site of aggrecan (FVDIPEN) was incubated with a Western blot, which had been run with aggrecan extracted from bovine physes, a positive reaction resulted. This suggests that there is stromelysin degradation in vivo in the physis. Two different polyclonal antibodies to stromelysin gave positive results on Western blots of purified media from growth plate cultures indicating that stromelysin is produced in vitro in culture. These antibodies also reacted with active stromelysin.

The presence of stromelysin in the physis implicates it in physeal physiology. The concentration of its activity in the lower hypertrophic zone and zone of provisional calcification suggests that it may be particularly important in mineralization. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Chronic changes in rabbit retro‐patellar cartilage and subchondral bone after blunt impact loading of the patellofemoral joint
Authors: Ewers Benjamin J. , Weaver Brian T. , Sevensma Eric T. , Haut Roger C.
Published: October 2002

Animal models of acute joint injury are useful for study of changes in joint tissues that may eventually lead to degradative disease. Our laboratory has developed a joint trauma model using a single blunt impact to the patellofemoral joint of rabbits and documented softening of retro‐patellar cartilage and thickening of its underlying bone out to 12 months post‐trauma. In the present study, we examined changes in these joint tissues out to 36 months post‐impact. Forty‐nine Flemish giant rabbits were impacted on the right patellofemoral joint and sacrificed at one of six times: 0, 4.5, 7.5, 12, 24, and 36 months post‐impact. A 30% reduction in the compressive modulus of traumatized retro‐patellar cartilage occurred at 4.5 months versus the contralateral, non‐impacted limb and remained at this reduced level out to 36 months. The fluid permeability of traumatized cartilage also increased over time from baseline and versus the non‐impacted limb. Tissue thickness increased slightly at 4.5 months and then decreased over time to a 45% difference from baseline at 36 months post‐trauma. While impacted cartilage revealed a significantly greater length of surface fissuring than contralateral, non‐impacted cartilage, no time‐dependent changes were evident in this study. Moreover, the number and depth of these impact surface lesions did not change as a function of time. Finally, the histological analyses indicated that the thickness of underlying subchondral bone increased over time from baseline and versus that in the non‐impacted limb. This long‐term study suggested an association between a decrease in the characteristic time constant of traumatized cartilage and thickening of the underlying subchondral bone. Any potential cause and effect relationship, however, must be investigated in future studies. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Reliability of detecting prosthesis/cement interface radiolucencies in total hip arthroplasty
Authors: Zhang Yongde , Putnam Aaron W. , Heiner Anneliese D. , Callaghan John J. , Brown Thomas D.
Published: October 2002

A laboratory study assessed the reliability of detecting radiolucencies at the prosthesis/cement interface in femoral components for total hip replacement. Radiolucencies (thicknesses = 0.1, 0.3, 0.5 and 0.7 mm) were created by randomly masking non‐tip Gruen zones (six per stem) in a group of 72 matte‐finish femoral components. Only half of all Gruen zones were masked. The femoral components were reproducibly implanted in composite fiberglass replicate femurs. Anteroposterior and lateral radiographs taken using conventional techniques were then presented to 10 experienced readers, who scored each of 432 non‐tip Gruen zones for radiolucency presence or absence. The series‐average radiolucency detection rate was 47.1%, with a breakdown of 9.8%, 20.7%, 69.8% and 88.0% for radiolucency thicknesses of 0.1, 0.3, 0.5 and 0.7 mm, respectively. The false positive rate was 7.5%. The findings argue strongly that in many or most instances, initial cement‐prosthesis debonding is not reliably detected from conventional plain film radiographs. Radiolucencies of at least 0.7 mm width are necessary to be accurately detectable by most viewers. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
The national identity of post-communist small states in the process of accession to the European Union: the case of Slovenia
Authors: Šabič Z. , Brglez M.
Published: October 2002

The article discusses, through conceptual elaboration and empirical investigation, the interplay between the perception of smallness and the concept of national identity in the case of Slovenia’s integration into the European Union. Unlike the prevailing literature on Slovenia, the authors do not take Slovenia’s smallness for granted. They argue that, in the particular case of Slovenian identity-building, smallness as an independent variable does not feature frequently at all. The research is based on methodological pluralism, with a special emphasis on political discourse, the mass media and public opinion in Slovenia
Clinical trials update: The Heart Protection Study, IONA, CARISA, ENRICHD, ACUTE, ALIVE, MADIT II and REMATCH
Authors: Louis Amala A. , Manousos I. Renata , Coletta Alison P. , Clark Andrew L. , Cleland John G.F.
Published: October 2002

This article continues a series of reports summarising recent research developments of particular interest to those involved in the management of patients with heart failure. A number of relevant presentations were made at the American Heart Association Scientific Sessions in Anaheim, California, between 11–14 November 2001. Summaries of the following Clinical Trials are included: The Heart Protection Study, IONA, CARISA, ENRICHD, ACUTE, ALIVE, MADIT II and REMATCH.
Short‐term effects of sinus rhythm restoration in patients with lone atrial fibrillation: a hormonal study
Authors: Jourdain P. , Bellorini M. , Funck F. , Fulla Y. , Guillard N. , Loiret J. , Thebault B. , Sadeg N. , Desnos M.
Published: October 2002

It is well known that atrial fibrillation can lead to heart failure, and is attributed to rapid ventricular rate (tachycardia‐induced cardiomyopathy). Some recent studies suggest the possible existence of an intrinsic left‐ventricular factor related to atrial fibrillation, irrespective of other elements. In order to demonstrate the implication of this factor, we measured B‐type Natriuretic Peptide, known as a functional marker of left‐ventricular dysfunction, in 40 consecutive patients with chronic non‐valvular atrial fibrillation, with low ventricular rate and absence of clinical heart failure or echocardiographic left‐ventricular dysfunction. In all patients, Brain Natriuretic Peptide (BNP) plasma level was high and dramatically decreased 24 h after external electrical cardioversion (61.4 pg/ml before cardioversion, 23.5 pg/ml 1 day after cardioversion, P<0.002). Our study demonstrates that atrial fibrillation, in absence of high ventricular rate, induces an asymptomatic cardiac alteration that is not detectable by echocardiography.
Self‐rating of quality of life provides additional prognostic information in heart failure. Insights into the EPICAL study
Authors: Alla François , Briançon Serge , Guillemin Francis , Juillière Yves , Mertès Paul‐Michel , Villemot Jean‐Pierre , Zannad Faiez ,   for the EPICAL Investigators
Published: October 2002

Background:

The relationship between quality of life (QoL) and survival have been poorly investigated. The aim of this study was to determine the value of QoL score as a prognostic factor in a prospective cohort of patients with advanced chronic heart failure (CHF).

Methods:

QoL assessment was performed with a generic questionnaire: the Duke Health Profile (DHP) and a disease‐specific instrument: the Minnesota Living With Heart Failure Questionnaire (LIhFE), in a sample of 108 patients registered in the EPICAL program (hospitalised patients with severe CHF defined by a NYHA grade III/IV, oedema or hypotension, and LVEF <30%). Prognostic value of general, physical, mental and social dimensions on survival and hospital‐free survival were tested in a Cox model.

Results:

One‐year survival rate was 76%, 1‐year hospital‐free survival 38%. QoL was significantly associated with outcomes: for both questionnaires, a 10‐point decrement in baseline score was associated with a 23–36% increase in the risk of death or hospitalisation for heart failure. For hospital‐free survival, this relationship remained significant after adjustment for others prognostic factors.

Conclusion:

QoL score is a predictive factor of survival and an independent predictive factor of hospital‐free survival in patients with advanced CHF. This assessment may provide additional information for clinical management and therapeutic decisions.
Hypertrophic responsiveness of cardiomyocytes to α‐ or β‐adrenoceptor stimulation requires sodium‐proton‐exchanger‐1 (NHE‐1) activation but not cellular alkalization
Authors: Schäfer Matthias , Schäfer Claudia , Piper Hans Michael , Schlüter Klaus‐Dieter
Published: October 2002

The influence of the sodium‐proton‐exchanger‐1 (NHE‐1) inhibitor HOE694 on α‐ or β‐adrenoceptor mediated stimulation of protein synthesis was investigated in cultured ventricular cardiomyocytes from adult rat pre‐treated with fetal calf serum to induce hypertrophic responsiveness to β‐adrenoceptor stimulation. Stimulation of α‐adrenoceptors with phenylephrine (10 μM) in bicarbonate‐free medium caused cellular alkalization (ΔpHi: +0.17±0.02, n‐5, P<0.05). HOE694, an NHE‐1 inhibitor, completely abolished this effect. [14C]phenylalanine incorporation into cellular protein mass increased in the presence of phenylephrine by 23±8%, and this effect was also abolished in the presence of HOE694. HOE694 (1 μM) neither influenced basal protein synthesis nor interfered with α‐adrenoceptor mediated activation of ERK2. Phorbol myristate acetate, a direct stimulator of protein kinase C, mimicked the effect of α‐adrenoceptor stimulation in regard to protein synthesis, but did not lead to cellular alkalization. Protein synthesis increased in the presence of isoprenaline, a β‐adrenoceptor agonist also. Again, HOE694 attenuated the stimulation of protein synthesis although isoprenaline did not cause cellular alkalization. In conclusion, the growth response to different hypertrophic stimuli, namely α‐ or β‐adrenoceptor stimulation, is attenuated in the presence of the NHE‐1 inhibitor HOE694 and this inhibition is independent from cellular alkalization.
Retail Structure of Beijing
Authors: Wang Shuguang , Jones Ken
Published: October 2002

One dynamic and central element in China's urban landscape is its retail structure. Yet, little attention has been given to the burgeoning retail sector in the vast literature that relates to China's economic reforms. Using the latest census of commercial activity in Beijing and recent empirical work undertaken in the city, we analyze Beijing's postreform retail structure and economy. Beijing's retail sector has been greatly diversified in both ownership and format. In addition to the emergence of the numerous family-based retailers, private and joint-stock companies now represent a significant part of the retail enterprises—a strong trend towards a market-oriented economy. The presence of many overseas-invested enterprises shows that the Beijing consumer market has been effectively opened to foreign competition and that the Beijing government has a strong desire to continue this trend. The high concentration of retail facilities in Beijing proper is closely related to the urban form. The relative concentration of full-line department stores within the third ring road suggests that new entrants will be better off if they stay away from the inner city and take peripheral locations outside the third, or even the fourth, ring road, in conjunction with development of the newly planned suburban communities.
Profiting from Creativity? The Music Industry in Stockholm, Sweden and Kingston, Jamaica
Authors: Power Dominic , Hallencreutz Daniel
Published: October 2002

In this paper we present the results of detailed case-study work on two important music production centres: Stockholm (Sweden) and Kingston (Jamaica). It is shown that both are dynamic innovation and production centres for populate music. In both cases local characteristics of the organisational and firm structure in the industry are claimed to be crucial for understanding innovation processes and the competitiveness of the resulting products. However, it is shown that considerable differences exist between the two centres in terms of the profits and returns (both monetary and otherwise) on global competitiveness and success made both by local actors and by the local production system. It is suggested that two crucial areas explain these unequal returns to commercially successful creativity: interfirm and intrafirm links between the local and global; and the effectiveness of intellectual property regimes. It is suggested that work on cultural-products industries must attempt to understand not only the processes from which commercial innovation arises and in which it is embedded, but also the processes and mechanisms by which appropriate levels of returns on creativity can be secured by the local production centre.
Beyond the Sunan Model: Trajectory and Underlying Factors of Development in Kunshan, China
Authors: Dennis Wei Yehua
Published: October 2002

Since the launch of economic reforms in the late 1970s, some cities and regions in China have been growing much faster than others, stimulating scholarly research on the forces underlying uneven development. This paper, through a case study of the emergence of Kunshan in southern Jiangsu (Sunan), shows that the orthodox Sunan model centered on the development of township and village enterprises (TVEs) has become inadequate to account for the recent development and restructuring in Sunan. I argue that spatial development in China is a complicated process incorporating the role of the state, local development conditions, and foreign investment. Moreover, there are strong geographical foundations for the functioning of local states. The arguments are particularly relevant to the transitional nature of provincial China, where the state still plays a significant role in development while local and global forces have emerged as equally important forces.
Collective Technology Learning Between Transnational Corporations and Local Business Partners: The Case of West Sweden
Authors: Ivarsson Inge
Published: October 2002

Unique firm-level data from 287 majority-owned foreign affiliates (MOFAs) located in West Sweden are used to analyse the extent in which transnational corporations (TNCs) in a developed host country have established technological linkages, leading to collective technology learning where both TNCs and their local business partners benefit. The findings indicate that local business partners have established substantial levels of organised technological cooperation, not only with manufacturing MOFAs but also with wholesale MOFAs supplying industrial products. This seems to result in collective technology learning where both MOFAs and business partners in Sweden, especially customers, earn advantages. In the case of manufacturing MOFAs, local business partners in West Sweden seem to be important as cooperative partners. By using a multiple logistic regression to analyse key determinants, it was found that the size of MOFAs positively affected the establishment of technological linkages to business partners in Sweden, among both manufacturing MOFAs and sales MOFAs. A positive correlation with linkage formation was also found among manufacturing MOFAs regarding both increasing age and the extent in which they operate within competitive Swedish industry clusters.
TGFβ induces GDNF responsiveness in neurons by recruitment of GFRα1 to the plasma membrane
Authors: Peterziel H. , Unsicker K. , Krieglstein K.
Published: October 2002

We have previously shown that the neurotrophic effect of glial cell line–derived neurotrophic factor (GDNF) in vitro and in vivo requires the presence of transforming growth factor (TGF)β. Using primary neurons (chick E8 ciliary) we show that the combination of GDNF plus TGFβ promotes survival, whereas the single factors do not. This cooperative effect is inhibited by blocking the extracellular signal-regulated kinase (ERK)/MAPK pathway, but not by interfering with the PI3 kinase signaling cascade. Although there is no functional GDNF signaling in the absence of TGFβ, pretreatment with TGFβ confers GDNF responsiveness to the cells. This is not due to upregulation of GDNF receptors mRNA and protein, but to TGFβ-induced recruitment of the glycosyl-phosphatidylinositol-anchored GDNF receptor (GFR)α1 to the plasma membrane. This is supported by the fact that GDNF in the presence of a soluble GFRα1 can promote survival in the absence of TGFβ. Our data suggest that TGFβ is involved in GFRα1 membrane translocation, thereby permitting GDNF signaling and neurotrophic effects.