bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutant huntingtin aggregates do not sensitize cells to apoptotic stressors
Authors: Chun Wanjoo , Lesort Mathieu , Lee Matthew , Johnson Gail V.W.
Published: October 2002

It has been postulated that neuronal inclusions composed of mutant huntingtin may play a causative role in the pathogenesis of Huntington's disease. To study the putative role of aggregates in modulating apoptotic vulnerability, SH‐SY5Y cell lines stably expressing truncated huntingtin with 18 (wild‐type) (N63–18Q) or 82 (mutant) (N63–82Q) glutamine repeats were established. Aggregates were observed in ∼13% of the N63–82Q cells; no aggregates were observed in the N63–18Q cells. In response to apoptotic stimuli such as staurosporine or hyperosmotic stress, caspase‐3 activity was significantly greater in the N63–82Q cells compared to the N63–18Q cells. However, double immunostaining for huntingtin and active caspase‐3 revealed that the presence of aggregates did not correlate with the presence of active caspase‐3, indicating that aggregates do not contribute to the increase in apoptosis in the N63–82Q cells.
Nitric oxide affects plant mitochondrial functionality in vivo
Authors: Zottini Michela , Formentin Elide , Scattolin Michela , Carimi Francesco , Lo Schiavo Fiorella , Terzi Mario
Published: October 2002

In this report, we show that nitric oxide affects mitochondrial functionality in plant cells and reduces total cell respiration due to strong inhibition of the cytochrome pathway. The residual respiration depends on the alternative pathway and novel synthesis of alternative oxidase occurs. These modifications are associated with depolarisation of the mitochondrial membrane potential and release of cytochrome c from mitochondria, suggesting a conserved signalling pathway in plants and animals. This signal cascade is triggered at the mitochondrial level and induces about 20% of cell death. In order to achieve a higher level of cell death, the addition of H2O2 is necessary.
Nicotine‐modulated formation of spiroiminodihydantoin nucleoside via 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine in 2′‐deoxyguanosine–hypochlorous acid reaction
Authors: Suzuki Toshinori , Ohshima Hiroshi
Published: October 2002

Hypochlorous acid (HOCl) is generated by myeloperoxidase of activated neutrophils which kill invading microorganisms, but also cause DNA damage in inflamed tissues. We report here that spiroiminodihydantoin nucleoside (dS), a further oxidized product of 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine (8‐oxo‐dG), is formed, in addition to 8‐chloro‐2′‐deoxyguanosine and 8‐oxo‐dG, by reaction of 2′‐deoxyguanosine with HOCl. Presence of low concentrations of nicotine significantly enhanced the yields of these HOCl‐modified nucleosides. Our results imply that nicotine may enhance genotoxicity and tissue damage caused by neutrophil activation. dS may also serve as a new biomarker for oxidative DNA damage induced by oxidants such as HOCl.
Crystal structure of a clavaminate synthase–Fe(II)–2‐oxoglutarate–substrate–NO complex: evidence for metal centred rearrangements
Authors: Zhang Zhihong , Ren Jing-shan , Harlos Karl , McKinnon Colin H , Clifton Ian J , Schofield Christopher J
Published: October 2002

Clavaminate synthase (CAS), a 2‐oxoglutarate (2OG) dependent dioxygenase, catalyses three steps in the biosynthesis of clavulanic acid. Crystals of CAS complexed with Fe(II), 2OG and deoxyguanidinoproclavaminate were exposed to nitric oxide (NO) acting as a dioxygen analogue. Prior to exposure with NO, the active site Fe(II) is octahedrally coordinated by a water molecule, the 2‐oxo and 1‐carboxylate groups of 2OG, and the side‐chains of an aspartyl and two histidinyl residues. NO binds to the position previously occupied by the 2OG 1‐carboxylate concomitant with rearrangement of the latter to the position previously occupied by the displaced water.
The plant Rad50–Mre11 protein complex
Authors: Daoudal-Cotterell S , Gallego M.E , White C.I
Published: October 2002

The Rad50–Mre11–Xrs2/Nbs1 protein complex plays critical roles in cellular processes involving DNA ends. This complex is implicated in DNA recombination and replication, meiosis, telomere maintenance and cellular DNA damage responses. The Rad50 and Mre11 proteins are essential for viability in animals, although not in yeast. We have prepared antibodies to the Rad50 protein of the model plant Arabidopsis thaliana which recognize a 175 kDa protein in wild‐type Arabidopsis protein extracts. Furthermore, we report here demonstration of the existence of the Rad50–Mre11 complex by co‐immunoprecipitation of the Rad50 and Mre11 proteins from the plant cell extracts.
Blue light specific and differential expression of a plastid σ factor, Sig5 in Arabidopsis thaliana
Authors: Tsunoyama Yuichi , Morikawa Kazuya , Shiina Takashi , Toyoshima Yoshinori
Published: October 2002

The transcription of plastid gene psbD is under the control of the BLRP (lue‐ight‐esponsive romoter) recognized by plastid‐encoded RNA polymerase, in which nuclear‐encoded σ factors play a crucial role in the promoter recognition. We examined the effects of light on mRNA levels of six different SIG genes in Arabidopsis and found that blue light extensively induced the accumulation of SIG5 transcripts, but red light did not. The blue light specificity was not observed in the accumulations of remaining five SIG genes. The blue light dependency of the SIG5 expression well explains the light‐dependent behavior of the psbD BLRP.
The biosynthetic pathway for lipoic acid is present in plastids and mitochondria in Arabidopsis thaliana1
Authors: Yasuno Rie , Wada Hajime
Published: October 2002

In eukaryotes, the biosynthetic pathway for lipoic acid is present in mitochondria. However, it has been hypothesized that, in plants, the biosynthetic pathway is present in plastids in addition to mitochondria. In this study, Arabidopsis thaliana LIP1p cDNA for a plastidial form of lipoic acid synthase has been identified. We show that it encodes a lipoic acid synthase by demonstrating its ability to complement an Escherichia coli mutant lacking lipoic acid synthase activity. We also show that LIP1p is targeted to chloroplasts. These findings suggest that the biosynthetic pathway for lipoic acid is present not only in mitochondria but also in plastids.
A metacaspase of Trypanosoma brucei causes loss of respiration competence and clonal death in the yeast Saccharomyces cerevisiae
Authors: Szallies Alexander , Kubata Bruno K. , Duszenko Michael
Published: October 2002

Metacaspases constitute a new group of cysteine proteases homologous to caspases. Heterologous expression of Trypanosoma brucei metacaspase TbMCA4 in the budding yeast Saccharomyces cerevisiae resulted in growth inhibition, mitochondrial dysfunction and clonal death. The metacaspase orthologue of yeast, ScMCA1 (YOR197w), exhibited genetic interaction with WWM1 (YFL010c), which encodes a small WW domain protein. WWM1 overexpression resulted in growth arrest and clonal death, which was suppressed by concomitant overexpression of ScMCA1. GFP‐fusion reporters of WWM1, ScMCA1 and TbMCA4 localized to the nucleus. Taken together, we suggest that metacaspases may play a role in nuclear function controlling cellular proliferation coupled to mitochondrial biogenesis.
The dystrophin gene is alternatively spliced throughout its coding sequence
Authors: Sironi M , Cagliani R , Pozzoli U , Bardoni A , Comi G.P , Giorda R , Bresolin N
Published: October 2002

We have analysed splicing patterns in the human dystrophin gene region encoding the rod and cysteine‐rich domains in normal skeletal muscle, brain and heart tissues. Sixteen novel alternative transcripts were identified, the majority of them being present in all three tissues. Tissue‐specific variants were also identified, suggesting a functional role of transcriptional diversity. Transcript analysis in dystrophinopathic autoptic and bioptic specimens revealed that pre‐mRNAs secondary structure formation and relative strength of exon/exon association play little or no role in directing alternative splicing events. This analysis also showed that independent deletion events leading to the loss of the same exons may be associated with transcriptional variability.
Identification of casein kinase Iα interacting protein partners
Authors: Dubois Thierry , Howell Steven , Zemlickova Eva , Aitken Alastair
Published: October 2002

Casein kinase Iα (CKIα) belongs to a family of serine/threonine protein kinases involved in membrane trafficking, RNA processing, mitotic spindle formation and cell cycle progression. In this report, we identified several CKIα interacting proteins including RCC1, high mobility group proteins 1 and 2 (HMG1, HMG2), Erf, centaurin‐α1, synaptotagmin IX and CPI‐17 that were isolated from brain as CKIα co‐purifying proteins. Actin, importin‐α1, importin‐β, PP2Ac, centaurin‐α1, and HMG1 were identified by affinity chromatography using a peptide column comprising residues 214–233 of CKIα. We have previously shown that centaurin‐α1 represents a CKIα partner both in vitro and in vivo. The nuclear protein regulator of chromosome condensation 1 (RCC1) is a guanosine nucleotide exchange factor for Ran which is involved in nuclear transport and mitotic spindle formation. Here we show that CKIα and RCC1 interact in brain and in cultured cells. However, the interaction does not involve residues 217–233 of CKIα which are proposed from X‐ray structures to represent an anchoring site for CKI partners. Formation of the RCC1/CKIα complex is consistent with the association of the kinase with mitotic spindles. In conclusion, we have identified a number of novel CKIα protein partners and their relations to CKI are discussed.
The role of protein glycosylation in the control of cellular N‐sialyltransferase activity
Authors: Breen Kieran C
Published: October 2002

Protein glycosylation, which is a key post‐translational event, is catalysed by the glycosyltransferase family of enzymes. There is an increasing body of evidence to suggest that these enzymes may themselves be glycosylated, possibly as an autocatalytic event. Using a novel in vitro system, we have investigated the role of enzyme glycosylation in sialyltransferase catalytic activity. The enzyme activity is glycosylation dependent, with the penultimate galactose residue on complex N‐linked oligosaccharides playing a pivotal role. These results serve to underline the complexity of the glycosylation process.
Melanophilin directly links Rab27a and myosin Va through its distinct coiled‐coil regions
Authors: Nagashima Kazuaki , Torii Seiji , Yi Zhaohong , Igarashi Michihiro , Okamoto Koichi , Takeuchi Toshiyuki , Izumi Tetsuro
Published: October 2002

Rab GTPases regulate the membrane transport pathways by recruiting their specific effector proteins. Melanophilin, a putative Rab effector, has recently been identified as a gene that is mutated in leaden mice, in which peripheral localization of melanosomes is impaired in melanocytes. Genetic studies suggest that three coat‐color mutation genes, dilute (MyoVad
), ashen (Rab27aash
), and leaden (Mlphln
), act in the same or overlapping pathways. Here we have cloned and characterized a human melanophilin homolog, which belongs to the rabphilin3/granuphilin‐like Rab effector family. Cosedimentation assays using recombinant proteins reveal that melanophilin directly binds to Rab27a and myosin Va through its N‐terminal and its first C‐terminal coiled‐coil region, respectively. Moreover, we show that Rab27a, melanophilin, and myosin Va form a ternary complex in the human melanocyte cell line HMV‐II. These findings suggest that melanophilin has a role in bridging Rab27a on melanosomes and myosin Va on actin filaments during melanosome transport. We also propose that the Rab‐binding region conserved in a novel rabphilin3/granuphilin‐like Rab effector family constitutes an α‐helix‐based coiled‐coil structure.
ARF6: a newly appreciated player in G protein‐coupled receptor desensitization
Authors: Hunzicker-Dunn Mary , Gurevich Vsevolod V. , Casanova James E. , Mukherjee Sutapa
Published: October 2002

The luteinizing hormone/choriogonadotropin hormone receptor (LH/CG R) signals to regulate ovulation, corpus luteum formation, and fetal survival during pregnancy. Agonist binding to the LH/CG R is poorly reversible, emphasizing the importance of a cellular mechanism to temper signaling by a potentially persistently active receptor. Like other G protein‐coupled receptors (GPCRs), signaling by this receptor is modulated by its binding of an arrestin. We have identified ADP ribosylation factor 6 (ARF6) as a protein whose activation state is regulated by the LH/CG R and which functions to regulate the availability of plasma membrane‐docked arrestin 2 to this receptor. We hypothesize that ARF6 might also serve GPCRs other than the LH/CG R to regulate the availability of arrestin 2 for receptor desensitization.
Palindromic repetitive elements in the mitochondrial genome of Volvox1
Authors: Aono Naoki , Shimizu Toshinobu , Inoue Tan , Shiraishi Hideaki
Published: October 2002

Group I introns were found in the cob and cox I genes of Volvox carteri. These introns contain tandem arrays of short palindromic sequences that are related to each other. Inspection of other regions in the mtDNA revealed that similar palindromic repetitive sequences are dispersed in the non‐protein coding regions of the mitochondrial genome. Analysis of the group I intron in the cob gene of another member of Volvocaceae, Volvox aureus, has shown that its sequence is highly homologous to its counterpart in V. carteri with the exception of a cluster of palindromic sequences not found in V. carteri. This indicates that the palindromic clusters were inserted into the introns after divergence of the two species, presumably due to frequent insertions of the palindromic elements during evolution of the Volvocaceae. Possible involvement of the palindromic repetitive elements in the molecular evolution of functional RNAs is discussed.
RNA interference (RNAi) induction with various types of synthetic oligonucleotide duplexes in cultured human cells
Authors: Hohjoh Hirohiko
Published: October 2002

Various types of synthetic oligonucleotide duplexes against the Photinus luciferase gene were tested on their induction of the sequence‐specific RNA interference (RNAi) activity in transfected human cells. Results indicate that RNA duplexes with ribonucleotide 3′ overhangs rather than those with deoxyribonucleotide 3′ overhangs induce more efficient RNAi activity, and that sense‐stranded DNA/antisense‐stranded RNA hybrids induce a moderate RNAi activity. These results suggest that there is a difference in the potential of oligonucleotide duplexes to be RNAi mediators, i.e. short interfering RNAs (siRNAs), between human RNAi and invertebrate RNAi. The data further show that different siRNAs induce different levels of RNAi.
Serine 774 and amino acids 296 to 437 comprise the major C4 determinants of the C4 phosphoenolpyruvate carboxylase of Flaveria trinervia
Authors: Engelmann Sascha , Bläsing Oliver E , Westhoff Peter , Svensson Per
Published: October 2002

C4 phosphoenolpyruvate carboxylases have evolved several times independently from non‐photosynthetic C3 ancestral enzymes. To identify C4‐specific determinants at the amino acid level the two orthologous ppcA PEPCases of Flaveria trinervia (C4) and Flaveria pringlei (C3) were used as a model system. In a previous publication [Bläsing et al., J. Biol. Chem. 275 (2000) 27917–27923] it was reported that the serine at position 774 is an invariant residue in all C4 phosphoenolpyruvate carboxylases. Here we show by swapping experiments and site‐directed mutagenesis that the serine 774 and amino acids 296–437 explain two thirds of the C4 characteristic phosphoenolpyruvate saturation kinetics when investigated in the C3 background. In addition, the results indicate that the determinants functionally interact with each other.
Processing of proendothelin‐1 by members of the subtilisin‐like pro‐protein convertase family
Authors: Blais Véronique , Fugère Martin , Denault Jean-Bernard , Klarskov Klaus , Day Robert , Leduc Richard
Published: October 2002

Endothelial cells (ECs) secrete numerous bioactive peptides that are initially synthesized as inactive precursor proteins. One of these, proendothelin‐1 (proET‐1), undergoes proteolysis at specific pairs of basic amino acids. Here, we wished to examine the role of mammalian convertases in this event. Northern blot analysis shows that only furin and PC7 are expressed in ECs. In vitro cleavage of proET‐1 by furin or PC7 demonstrated that both enzymes efficiently and specifically process proET‐1. These data reveal that furin and PC7 have similar specificities towards proET‐1 and suggest that both enzymes may participate in the maturation of proET‐1 in ECs.
A caged sperm‐activating peptide that has a photocleavable protecting group on the backbone amide
Authors: Tatsu Yoshiro , Nishigaki Takuya , Darszon Alberto , Yumoto Noboru
Published: October 2002

A backbone‐caged sperm‐activating peptide (caged speract) that has a 2‐nitrobenzyl group at a backbone amide and a vastly reduced affinity for its receptor (IC50=950 nM) was synthesized. UV irradiation of caged speract photocleaves the 2‐nitrobenzyl group (τ
1/2=26 μs), restoring its affinity (IC50=0.67 nM) and ability to increase sperm intracellular pH and Ca2+, as intact speract. Backbone caging of the biological activity was more efficient than side chain caging, which adds a nitrobenzyl group on the peptide side chain. The backbone caging strategy described can be used as a general procedure to cage biologically active peptides, which have no side chain for introduction of a caging group.
E‐selectin and ICAM‐1 are incorporated into detergent‐insoluble membrane domains following clustering in endothelial cells
Authors: Tilghman Robert W , Hoover Richard L
Published: October 2002

Here we present data supporting the role of lipid rafts in endothelial cells during leukocyte adhesion. Following adhesion of THP‐1 cells or antibody‐mediated clustering, both E‐selectin and intercellular adhesion molecule‐1 (ICAM‐1) partitioned into the detergent‐insoluble portion of the endothelial cellular lysate. Sucrose gradient centrifugation revealed the partitioning of clustered E‐selectin and ICAM‐1 with the low‐density fraction where they co‐fractionated with src family kinases, markers of lipid rafts. Depleting the plasma membrane of cholesterol inhibited clustering of adhesion molecules following their antibody‐induced crosslinking and inhibited their association with src kinases. Thus, our data suggest that E‐selectin and ICAM‐1 associate with lipid rafts in human endothelial cells following leukocyte adhesion.
Differential regulation of transcription and induction of programmed cell death by human p53‐family members p63 and p73
Authors: Dietz Sebastian , Rother Karen , Bamberger Casimir , Schmale Hartwig , Mössner Joachim , Engeland Kurt
Published: October 2002

The p53 tumor suppressor acts as a transcription factor and has a central function in controlling apoptosis. With p63 and p73 two genes coding for proteins homologous to p53 have been identified. We describe the properties of seven human p63 and p73 proteins as transcriptional activators of p21WAF1/CIP1 expression and apoptotic inducers in direct comparison to p53 in the same assay systems employing DLD‐1‐tet‐off colon cells. Programmed cell death is detected in cells expressing high levels of p53 and p73α. Cells overexpressing TAp63α, TAp63γ, TA*p63α, TA*p63γ, ΔNp63α, and ΔNp63γ display low or no detectable apoptosis.
Misoprostol use in obstetrics and gynecology in Brazil, Jamaica, and the United States
Authors: Clark S. , Blum J. , Blanchard K. , Galvão L. , Fletcher H. , Winikoff B.
Published: October 2002

Objectives: To investigate current clinical use of misoprostol for the treatment of a range of reproductive health indications by providers in Brazil, Jamaica, and the United States. Methods: Using a ‘snowball’ sampling technique, we surveyed 228 gynecologists and obstetricians in Brazil (n=123), Jamaica (n=52), and the United States (n=53). Results: Providers use misoprostol for labor induction (46%), postpartum hemorrhage (8%), intra‐uterine fetal death (61%), cervical priming (21%), missed abortion (57%), and incomplete abortion (16%) as well as first and second trimester abortion induction (27% and 13%, respectively). Conclusions: There is considerable variation in the regimens used; moreover, the regimens commonly used in clinical practice often differ from those recommended in the medical literature. While misoprostol is an appealing alternative for many reproductive health indications in developing countries, the varied regimens and lack of registration raise critical medical and policy questions.
Transplantation of the human uterus
Authors: Fageeh W , Raffa H , Jabbad H , Marzouki A
Published: October 2002

Human uterine transplantation was performed on 6 April 2000 on a 26‐year‐old female who lost her uterus 6 years earlier due to post‐partum hemorrhage. The donor, a 46‐year‐old patient with multiloculated ovarian cysts, underwent a hysterectomy modified to preserve tissue and vascular integrity. The donor uterus was connected in the orthotopic position to the recipient's vaginal vault and additional fixation was achieved by shortening the uterosacral ligament. The uterine arteries and veins were extended using reversed segments of the great saphenous vein, then connected to the external iliac arteries and veins, respectively. Immunosuppression was maintained by oral cyclosporine A (4 mg/kg/body wt.), azathioprine (1 mg/kg/body wt.) and prednisolone (0.2 mg/kg/body wt.). Allograft rejection was monitored by Echo–Doppler studies, magnetic resonance imaging (MRI), and measurement of the CD4/CD8 ratio in peripheral blood by fluorescence activated cell sorter (FACS scan). An episode of acute rejection was treated and controlled on the ninth day with anti‐thymocytic globulin (ATG). The transplanted uterus responded well to combined estrogen–progesterone therapy, with endometrial proliferation up to 18 mm. The patient had two episodes of withdrawal bleeding upon cessation of the hormonal therapy. Unfortunately, she developed acute vascular thrombosis 99 days after transplantation, and hysterectomy was necessary. Macro‐ and microscopic histopathological examination revealed acute thrombosis in the vessels of the uterine body, with resulting infarction. Both fallopian tubes remained viable, however, with no evidence of rejection. The acute vascular occlusion appeared to be caused by inadequate uterine structure support, which led to probable tension, torsion, or kinking of the connected vascular uterine grafts.
Print versus electronic surveys: a comparison of two data collection methodologies
Authors: Boyer Kenneth K. , Olson John R. , Calantone Roger J. , Jackson Eric C.
Published: October 2002

This paper compares the responses of consumers who submitted answers to a survey instrument focusing on Internet purchasing patterns both electronically and using traditional paper response methods. We present the results of a controlled experiment within a larger data collection effort. The same survey instrument was completed by 416 Internet customers of a major office supplies company, with approximately 60% receiving the survey in paper form and 40% receiving the electronic version. In order to evaluate the efficacy of electronic surveys relative to traditional, printed surveys we conduct two levels of analysis. On a macro‐level, we compare the two groups for similarity in terms of fairly aggregate, coarse data characteristics such as response rates, proportion of missing data, scale means and inter‐item reliability. On a more fine‐grained, micro‐level, we compare the two groups for aspects of data integrity such as the presence of data runs and measurement errors. This deeper, finer‐grained analysis allows an examination of the potential benefits and flaws of electronic data collection.

Our findings suggest that electronic surveys are generally comparable to print surveys in most respects, but that there are a few key advantages and challenges that researchers should evaluate. Notably, our sample indicates that electronic surveys have fewer missing responses and can be coded/presented in a more flexible manner (namely, contingent coding with different respondents receiving different questions depending on the response to earlier questions) that offers researchers new capabilities.
Unveiling the structure of supply networks: case studies in Honda, Acura, and DaimlerChrysler
Authors: Choi Thomas Y , Hong Yunsook
Published: October 2002

Three complete supply networks have been mapped in this study. These supply networks pertain to the center console assembly and come from three different product lines—Honda Accord, Acura CL/TL, and DaimlerChrysler (DCX) Grand Cherokee. Based on these three cases of supply networks, propositions are built concerning how the structure of supply networks operates. Based on the extant literature, we frame structure in three dimensions—formalization, centralization, and complexity. As an underlying methodology, we first conduct the within‐case analysis and then expand the analysis to cross‐case context. The three structural dimensions affect one another progressively, and the cost consideration appears to be the overarching force that shapes the supply‐network structure.
Grappling with a gusher! Manufacturing’s response to business success in small and medium enterprises
Authors: Corbett L.M , Campbell‐Hunt C
Published: October 2002

This study examined how the operations in six New Zealand manufacturers responded to the turbulence in their business environment brought on by the success of their products. It was carried out in response to various calls in the business and operations strategies literature for more studies on how companies create configurations of capabilities that lead to competitive advantage, how these literature can be better integrated, and on internal fit within manufacturing. Case research and historiographic methods were used to record the competitive evolution of these firms. The study used semi‐structured interviews with Chief Executive Officers (CEOs) and operations managers, analysis of company and public records and a manufacturing practices survey instrument. We suggest that when small and medium‐sized enterprises (SMEs) are faced with sudden and dramatic success from an innovation, they need to focus the company’s energy and resources on that product and its niche. Then, in terms of manufacturing strategy, the key attribute to develop is an operation that can produce short runs at little or no cost penalty, achieve requisite product variety, provide short manufacturing throughput times, and operate at high levels of quality and delivery dependability. We find that an approach that develops manufacturing capabilities that are related to market requirements is preferred to panacea‐based approaches. Also, we see no common pattern in manufacturing capability development.
Integration of engineered cartilage
Authors: Obradovic B. , Martin I. , Padera R. F. , Treppo S. , Freed L. E. , Vunjak‐Navakovic G.
Published: October 2002

The structure and function of cartilaginous constructs, engineered in vitro using bovine articular chondrocytes, biodegradable scaffolds and bioreactors, can be modulated by the conditions and duration of tissue cultivation. We hypothesized that the integrative properties of engineered cartilage depend on developmental stage of the construct and the extracellular matrix content of adjacent cartilage, and that some aspects of integration can be studied under controlled in vitro conditions. Disc‐shaped constructs (cultured for 5±1 days or 5±1 weeks) or explants (untreated or trypsin treated cartilage) were sutured into ring‐shaped explants (untreated or trypsin treated cartilage) to form composites that were cultured for an additional 1‐8 weeks in bioreactors and evaluated biochemically, histologically and mechanically (compressive stiffness of the central disk, adhesive strength of the integration interface). Immature constructs had poorer mechanical properties but integrated better than either more mature constructs or cartilage explants. Integration of immature constructs involved cell proliferation and the progressive formation of cartilaginous tissue, in contrast to the integration of more mature constructs or native cartilage which involved only the secretion of extracellular matrix components. Integration patterns correlated with the adhesive strength of the disc‐ring interface, which was markedly higher for immature constructs than for either more mature constructs or cartilage explants. Trypsin treatment of the adjacent cartilage further enhanced the integration of immature constructs. © 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Shape and size of isolated bone mineralites measured using atomic force microscopy
Authors: Eppell Steven J. , Tong Weidong , Katz J. Lawrence , Kuhn Liisa , Glimcher Melvin J.
Published: October 2002

The inorganic phase of bone is comprised primarily of very small mineralites. The size and shape of these mineralites play fundamental roles in maintaining ionic homeostasis and in the biomechanical function of bone. Using atomic force microscopy, we have obtained direct three‐dimensional visual evidence of the size and shape of native protein‐free mineralites isolated from mature bovine bone. Approximately 98% of the mineralites are less than 2 nm thick displaying a plate‐like habit. Distributions of both thickness and width show single peaks. The distribution of lengths may be multimodal with distinct peaks separated by ∼6 nm. Application of our results is expected to be of use in the design of novel orthopaedic biomaterials. In addition, they provide more accurate inputs to molecular‐scale models aimed at predicting the physiological and mechanical behavior of bone. © 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Three‐dimensional analysis of shoulder girdle and supraspinatus motion patterns in patients with impingement syndrome
Authors: Graichen H. , Stammberger T. , Bonél H. , Wiedemann E. , Englmeier K‐H. , Reiser M. , Eckstein F.
Published: October 2002

Alterations of the shoulder girdle motion have been suggested to be associated with shoulder disorders. The objective of this study was to perform a three‐dimensional (3D) motion analysis of the supraspinatus muscle and shoulder girdle in patients with different stages of impingement syndrome. 20 patients with unilateral impingement and 14 normal controls were investigated at 30°, 90°, and 120° of abduction with and without abducting muscle activity. The spatial relationship between the shoulder girdle elements and the supraspinatus was quantified from open MRI data. No significant alterations in glenoid rotation were observed between the patients and asymptomatic volunteers. However, while in the healthy volunteers the values showed a normal distribution (28.5 ± 3.6° at 90° abduction with muscle activity), the patients (30.5° ± 9.7°) contained a subset of five individuals with an obvious increase in glenoid rotation angle (>40°) compared with controls (>2.5 standard deviations higher than the mean) and with the healthy contralateral side. These five patients also displayed alterations in the scapulo‐humeral rhythm and supraspinatus motion, but not in clavicular position. The study shows that only a specific subset of patients with impingement syndrome demonstrates complex changes in shoulder girdle and supraspinatus motion patterns, suggesting that this subset may benefit from an alternative type of treatment. © 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Early cell adhesion events differ between osteoporotic and non‐osteoporotic osteoblasts
Authors: Perinpanayagam H. , Zaharias R. , Stanford C. , Brand R. , Keller J. , Schneider G.
Published: October 2002

In osteoporosis, the regenerative capacity of bone is compromised, which may involve altered osteoblast (OB) activity. This could be attributed to an inappropriate synthesis and assembly of an extracellular matrix (ECM), altered cell adhesion to the ECM, or be due to inappropriate downstream activation of adhesion‐mediated signaling cascades through proteins such as focal adhesion kinase (FAK). The purpose of our study was to compare early adhesion‐mediated events using previously described and characterized clinically derived OBs obtained from human patients undergoing major joint arthroplasty for osteoporosis or osteoarthritis. The presence or absence of osteoporosis was established with a radiographic index. Using light microscopy and crystal violet staining, we show that OB cells derived from sites of osteoporosis do not attach and spread as well as non‐osteoporotic (OP) OB cells. OP cells initially have a more rounded morphology, and show significantly less (P < 0.001) attachment to serum‐coated tissue culture plastic over a 24 h time period. Immunofluorescent labeling after 24 h of attachment showed that OP OB focal adhesions (FAs) and stress fibers were less defined, and that the OP cells were smaller and had a more motile phenotype. When normalized protein lysates were Western blotted for phosphotyrosine (PY) a band corresponding to pp125FAK was identified. FAK tyrosine phosphorylation was evident at 6 h in both the OP and non‐OP OBs, but decreased or was absent through 24 h in OP OBs. These results suggest early adhesion‐mediated events, such as cell adhesion, attachment, and FAK signaling via PY may be altered in OP OBs. © 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
The gastrocnemius muscle is an antagonist of the anterior cruciate ligament
Authors: Fleming Braden C. , Renstrom Per A. , Ohlen Goran , Johnson Robert J. , Peura Glenn D. , Beynnon Bruce D. , Badger Gary J.
Published: October 2002

Since the proximal tendon of the gastrocnemius muscle wraps around the posterior aspect of the tibia, its contraction could potentially strain the anterior cruciate ligament (ACL) by pushing the tibia anteriorly. However, the relationship between contraction of the gastrocnemius muscle and ACL strain has not been studied in vivo. The objectives of this study were to evaluate the ACL strain response due to isolated contractions of the gastrocnemius muscle and to determine how these strains are affected by co‐contraction with the hamstrings and quadriceps muscles. Six subjects with normal ACLs participated in the study: they underwent spinal anesthesia to ensure that their leg musculature was relaxed. Transcutaneous electrical muscle stimulation (TEMS) was used to induce contractions of the gastrocnemius, quadriceps and hamstrings muscles while the strains in the anteromedial bundle of the ACL were measured using a differential variable reluctance transducer. The ACL strain values produced by contraction of the gastrocnemius muscle were dependent on the magnitude of the ankle torque and knee flexion angle. Strains of 2.8% and 3.5% were produced at 5° and 15° of knee flexion, respectively. The ACL was not strained at 30° and 45°. Changes in ankle angle did not significantly affect these strain values. Co‐contraction of the gastrocnemius and quadriceps muscles produced ACL strain values that were greater than those produced by isolated activation of either muscle group when the knee was at 15° and 30°. Co‐contraction of the gastrocnemius and hamstrings muscles produced strains that were higher than those produced by the isolated contraction of the hamstrings muscles. At 15° and 30° of knee flexion, the co‐contraction strain values were less than those produced by stimulation of the gastrocnemius muscle alone. This study verified that the gastrocnemius muscle is an antagonist of the ACL. Since the gastrocnemius is a flexor of the knee, this finding may have important clinical ramifications in ACL rehabilitation since flexor torques are generally thought to be protective of a healing ACL graft. © 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Evaluation of quadriceps strength and voluntary activation after unicompartmental arthroplasty for medial osteoarthritis of the knee
Authors: Machner Andreas , Pap Géza , Awiszus Friedemann
Published: October 2002

Introduction: In early and moderate stages of osteoarthritis (OA) of the knee, arthrogenous muscle inhibition (AMI) is an important factor for the initiation and the progression of the disease. Although AMI has been shown to be reduced after physiotherapeutical exercises resulting in significant improvements in disability, implantation of unicondylar knee arthroplasties is much provided in these stages of OA. Therefore, in the present study we investigate changes in quadriceps muscle after implantation of such prostheses as compared to physiotherapeutical treatment, alone.

Methods: In eighteen patients with bilateral moderate knee OA, who were treated with unicondylar knee arthroplasty we investigated voluntary activation (VA) and maximum voluntary contraction (MVC) of the quadriceps femoris muscle. There were 7 males and 11 females, the mean age at time of operation was 67 years (range 58–76 years). Measurements on both sides were performed preoperatively and 18 months postoperatively using the twitch‐interpolation technique.

Results: Follow‐up assessment revealed a significant VA and MVC increase in both the surgically treated knees and in the contralateral knees treated by physiotherapy alone. However, VA and MVC improvements were significantly higher in the operated on knees than in those treated by physiotherapy alone.

Discussion: Both physiotherapeutical exercise and unicondylar knee replacements lead to an improvement of quadriceps motor function in knee OA. The greater improvement in knees with both knee replacement and physiotherapy might be related to the intraoperative removal of arthritic tissue in these knees. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Androgen receptors in Dupuytren's contracture
Authors: Pagnotta A. , Specchia N. , Greco F.
Published: October 2002

Palmar fascia tissue and cultured cells from patients with Dupuytren's contracture and from normal subjects were characterized and analyzed for androgen receptor expression. Androgen receptors have never been studied in Dupuytren's myofibroblasts and may have a role in its high male predominance. Surgical samples were collected from eight patients undergoing surgery for Dupuytren's contracture and from four patients with carpal tunnel syndrome, used as control tissue. Immunohistochemical analysis was performed on tissue samples and on cell cultures with anti‐androgen receptor, anti‐α‐smooth muscle actin, anti‐fibronectin, and anti‐type I and III collagen antibodies using the biotin–avidin peroxidase method as revelatory system. Immunostaining for androgen receptors in tissue samples and cultured cells revealed nuclear reaction in many Dupuytren's myofibroblasts, but in few fibroblasts of the normal palmar fascia. In a double‐labeling study, androgen receptors were seen to co‐localize with α‐actin in both cell cultures and tissue samples.

We present the first evidence that the palmar fascia is a target tissue for androgen action and that the expression of androgen receptors in Dupuytren's contracture is considerably higher than in the normal palmar fascia. Further studies will need to evaluate whether the androgen‐responsive state of the tissue is related to the high incidence of Dupuytren's contracture in the male sex. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
The involvement of β1 integrin in the modulation by collagen of chondrocyte‐response to transforming growth factor‐β1
Authors: Lee Jin Woo , Qi Wen Ning , Scully Sean P.
Published: October 2002

The physiologic response of chondrocytes to maintenance of the matrix and response to injury likely involves signaling from multiple sources including soluble cytokines, mechanical stimulation, and signaling from the extracellular matrix. The signaling from the extracellular matrix may serve to effect cell differentiation and to modulate the response to cytokines. We have previously reported that type II collagen modulates the response of bovine articular chondrocytes to TGF‐β1. The molecular nature of the signaling mechanism has not been elucidated but presumably involves a similar mechanism by which the cell attaches to the surrounding matrix.

An alginate bead culture system is utilized to which exogenous type II collagen is added. The inclusion of type II collagen results in an alteration of integrin expression with a down regulation of α2. The response of the chondrocyte to TGF‐β1 can be modulated by the inclusion of exogenous type II collagen. The modulation of DNA and proteoglycan synthesis was blocked by the treatment of anti‐β1 integrin antibody (4B4) or by cyclic RGD containing peptides. These events occur at concentrations that block cell adhesion to type II collagen. Linear RGD containing peptides and anti‐anchorin antibodies had no effect on the modulation by type II collagen.

These results suggest that type II collagen binding by chondrocytes at least in part occurs through the β1 integrin. This binding results in modulation of the cell response to TGF‐β1. This modulation may serve to provide physiologic specificity to the cytokine‐signaling cascade. An understanding of the regulatory milieu of the chondrocyte may permit the stimulation of an intrinsic repair of articular cartilage in the future. A near term application of this understanding can be made to tissue engineering attempts at articular cartilage repair. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Changes in human knee ligament stiffness secondary to osteoarthritis
Authors: Fishkin Zair , Miller David , Ritter Christopher , Ziv Israel
Published: October 2002

Stiffness of the medial (MCL) and lateral (LCL) collateral ligaments was compared between a group of 10 patients undergoing total knee arthroplasty for varus degenerative osteoarthritis (OAP), a group of 10 osteoarthritic cadaveric knees (OAC), and a group of 10 non‐osteoarthritic cadaveric knees (NOA). A load‐elongation curve was obtained for the medial and lateral compartments of each knee using an instrumented Moreland spreader. A strain gage (SG) was attached to the spreader handle and strain was calibrated to load applied against the spread distance. In extension, medial compartment stiffness of the OAP, OAC, and NOA groups was 60.7 ± 16, 52.8 ± 9.3 and 21.4 ± 5.0 N/mm, respectively. Lateral compartment stiffness in extension was 29.2 ± 9.2, 33.3 ± 10.3 and 19.5 ± 5.3 N/mm, for OAP, OAC, and NOA, respectively. Differences in stiffness between the OAP and OAC groups were not statistically significant (p > 0.05). However, the osteoarthritic groups (OAP and OAC) demonstrated a statistically significantly (p < 0.05) increase in ligament stiffness when compared to the NOA group. Following knee arthroplasty, stiffer medial structures in extension may lead to flexion contracture and accelerated polyethylene wear. Adequate bone resection, in conjunction with soft tissue release may alleviate the threefold increase in stiffness observed in the medial compartment secondary to OA without jeopardizing joint stability. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Outcomes of surgical management of total HIP replacement in patients aged 65 years and older: cemented versus cementless femoral components and lateral or anterolateral versus posterior anatomical approach
Authors: Zimmerman Sheryl , Hawkes William G. , Hudson James I. , Magaziner Jay , Hebel J. Richard , Towheed Tanveer , Gardner James , Provenzano George , Kenzora John E.
Published: October 2002

This observational study compared the outcomes of 271 cases of hip osteoarthritis receiving primary total hip replacement (patients 65 years of age and older) from numerous surgeons in 12 Baltimore region hospitals from 1991–1993. The independent variables studied were: (a) totally non‐cemented prostheses (non‐cemented femoral component, non‐cemented acetabular component) versus hybrid prostheses (cemented femoral component, non‐cemented acetabular component), and (b) lateral or anterolateral surgical approach versus posterior surgical approach. Outcomes included complications during the initial hospitalization, hospital length of stay, hospital cost, readmission, and reported and/or observed physical, instrumental, neuromuscular and affective functioning and pain at 2, 6, and 12 months post surgery.

Results indicated that, while the totally non‐cemented prosthesis was more costly, there were no statistically significant differences in clinical or functional outcomes between the non‐cemented and the hybrid prostheses up to 12 months post surgery. Also, while the posterior surgical approach was associated with a non‐statistically significant higher rate of dislocation, overall, there was improved function and reduced pain in the first 12 months post‐surgery associated with this approach. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Differential expression of neural cell adhesion molecule (NCAM) after tenotomy in rabbit skeletal muscle
Authors: Jamali Amir A. , Afshar Pouya , Abrams Reid A. , Lieber Richard L.
Published: October 2002

Tenotomy is a commonly encountered event in orthopaedic surgery. In 23 rabbit extensor digitorum longus (EDL) muscles, within 24 h after tenotomy, a marked drop in maximum force production occurred. This was not explainable based on architectural changes and histological examination using standard markers for muscle injury, i.e., haematoxylin and eosin morphology, developmental myosin heavy chain (MHC) immunolabeling, and quantitation of muscle fiber type percentage, area and distribution. The expression of neural cell adhesion molecule (NCAM), a glycoprotein expressed during muscle development was measured as a function of time in these muscles. NCAM expression was increased as early as one day after tenotomy with 2.2 ± 1.2% of the fibers showing positive expression. This expression level increased significantly to 15.4 ± 15.2% after 7 days and then subsided to 13.2 ± 10.6% 21 days after tenotomy. Two‐way analysis of variance demonstrated a significant effect of time and a significant time x tenotomy method interaction. These results suggest that tenotomy leads to possible changes in muscle‐nerve connections and/or excitation–contraction (EC) coupling. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.