bluuebunny/crossref_metadata_2025 huggingface dataset:
Farnesylpyridinium, an analog of isoprenoid farnesol, induces apoptosis but suppresses apoptotic body formation in human promyelocytic leukemia cells
Authors: Hamada Masahiro , Nishio Kyo-ichi , Doe Matsumi , Usuki Yoshinosuke , Tanaka Toshio
Published: October 2002

1‐Farnesylpyridinium (FPy), an analog of isoprenoid farnesol, initially induced morphological changes similar to those of typical apoptosis in human leukemia HL‐60 cells but FPy‐treated cells were characterized by the absolute absence of final apoptotic events such as fragmentation into apoptotic bodies. FPy‐induced cell death was considered to be apoptotic on the basis of the induction of DNA fragmentation and the protection against these events by the coaddition of a pan‐caspase inhibitor. The increase in the cytoplasmic cytochrome c level supported the possibility that FPy‐treated cells should have the ability to complete the entire apoptotic process ending in cell fragmentation and apoptotic body formation. At concentrations too low to induce apoptosis, FPy could suppress the induction of apoptotic body formation in HL‐60 cells by typical inducers of apoptosis such as actinomycin D or anisomycin. FPy exhibited a cytochalasin‐like effect on spatial arrangement of actin filament independent of its apoptosis‐inducing activity.
Lipid domain formation and ligand–receptor distribution in lipid bilayer membranes investigated by atomic force microscopy
Authors: Kaasgaard Thomas , Mouritsen Ole G. , Jørgensen Kent
Published: October 2002

A novel experimental technique, based on atomic force microscopy (AFM), is proposed to visualize the lateral organization of membrane systems in the nanometer range. The technique involves the use of a ligand–receptor pair, biotin–avidin, which introduces a height variation on a solid‐supported lipid bilayer membrane. This leads to a height amplification of the lateral membrane organization that is large enough to be clearly imaged by scanning AFM. The power of the technique is demonstrated for a binary dipalmitoylphosphocholine–diarachidoylphosphocholine lipid mixture which is shown to exhibit a distinct lateral lipid domain formation. The new and simple ligand–receptor‐based AFM approach opens up new ways to investigate lipid membrane microstructure in the nanometer range as well as the lateral distribution of ligand–lipid and receptor–protein complexes in supported membrane systems.
Analysis of gene expression in rose petals using expressed sequence tags
Authors: Channelière Stéphanie , Rivière Stéphane , Scalliet Gabriel , Szecsi Judit , Jullien Frédéric , Dolle Caroline , Vergne Philippe , Dumas Christian , Bendahmane Mohammed , Hugueney Philippe , Cock J.Mark
Published: October 2002

Single‐pass sequences were obtained from the 5′‐ends of a total of 1794 rose petal cDNA clones. Cluster analysis identified 242 groups of sequences and 635 singletons indicating that the database represents a total of 877 genes. Putative functions could be assigned to 1151 of the transcripts. Expression analysis indicated that transcripts of several of the genes identified accumulated specifically in petals and stamens. The cDNA library and expressed sequence tag database described here represent a valuable resource for future research aimed at improving economically important rose characteristics such as flower form, longevity and scent.
Rat ENaC expressed in Xenopus laevis oocytes is activated by cAMP and blocked by Ni2+
Authors: Segal Andrei , Cucu Dana , Van Driessche Willy , Weber Wolf-Michael
Published: October 2002

We used oocytes of the South African clawed toad Xenopus laevis to express the three subunits of the epithelial Na+ channel from rat distal colon (rENaC). We combined conventional dual‐microelectrode voltage‐clamp with continuous capacitance (C
m) measurements and noise analysis to evaluate the effects of cAMP and Ni2+ on rENaC. Control oocytes or rENaC‐expressing oocytes exhibited no spontaneous fluctuations in current. However, in rENaC‐expressing oocytes amiloride induced a marked plateau‐shaped rise of the power density spectra. Recordings using four different concentrations of amiloride revealed that the blocker–channel interactions were of the first order. A cocktail of the membrane permeant cAMP analogue chlorophenylthio‐cAMP and IBMX (cAMP cocktail) increased amiloride‐sensitive current (I
ami) and conductance (G
ami). Furthermore, C
m was also increased following cAMP application, indicating an increase in plasma membrane surface area. Noise analysis showed that cAMP increased the number of active channels in the oocyte membrane while single‐channel current decreased. From these data we conclude that cAMP triggered exocytotic delivery of preformed rENaCs to the plasma membrane. Ni2+ (2.5 mM) inhibited about 60% of the rENaC current and conductance while C
m remained unaffected. Noise analysis revealed that this inhibition could be attributed to a decrease in the apparent channel density, while single‐channel current did not change significantly. These observations argue for direct effects of Ni2+ on channel activity rather than induction of endocytotic removal of active channels from the plasma membrane.
Docking studies reveal a selective binding of D‐penicillamine to the transactivator protein of human immunodeficiency virus type 1
Authors: Demirhan Ilhan , Kanyalkar Meena , Chandra Angelika , Doerr Hans Wilhelm , Coutinho Evans , Loewer Johannes , Saran Anil , Chandra Prakash
Published: October 2002

DOCK and Affinity studies were carried out to study the binding of D‐ and L‐penicillamine to the transactivator protein (tat) of human immunodeficiency virus type 1 (HIV‐1). These studies reveal a selective binding of D‐penicillamine to the cysteine‐rich region covering amino acid residues 20–38 of the tat protein. A careful analysis of the components of the binding energy of the D‐ and L‐isomers reveals that the D‐isomer has a more favorable van der Waals interaction resulting from an optimal placement of the dimethylthiomethyl side chain in the binding site. This observation matches the experimental data that D‐penicillamine is a more potent inhibitor of tat‐mediated transactivation than the L‐isomer. The docking and experimental data offer an interesting approach to design structural molecules with potential application to block signal functions of the tat protein in HIV‐1 pathogenesis.
A naturally occurring NAR variable domain binds the Kgp protease from Porphyromonas gingivalis
Authors: Nuttall Stewart D , Krishnan Usha V , Doughty Larissa , Nathanielsz Anne , Ally Nafisa , Pike Robert N , Hudson Peter J , Kortt Alexander A , Irving Robert A
Published: October 2002

The new antigen receptor (NAR) from sharks consists of a single immunoglobulin variable domain attached to five constant domains, and is hypothesised to function as an antibody. Two closely related NARs with affinity for the Kgp (lysine‐specific) gingipain protease from Porphyromonas gingivalis were selected by panning an NAR variable domain library. When produced in Escherichia coli, these recombinant NARs were stable, correctly folded, and specifically bound Kgp (K
d=1.31±0.26×10−7 M). Binding localised to the Kgp adhesin domains, however without inhibiting adhesin activity. These naturally occurring proteins indicate an immune response to pathogenic bacteria and suggest that the NAR is a true antibody‐like molecule.
Conformational analysis of the GTP‐binding protein MxA using limited proteolysis
Authors: Varne Anjali , Muthukumaraswamy Karthika , Jatiani Shashidhar S , Mittal Rohit
Published: October 2002

Guanosine triphosphate (GTP)‐binding proteins are known to function as molecular switches that cycle between GTP‐bound and guanosine diphosphate (GDP)‐bound states. Switching is achieved by the fact that G‐proteins in the GTP‐bound conformation can interact with a certain set of effector molecules while they interact with a different set of partners in their GDP‐bound conformation. The antiviral properties of the interferon‐induced MxA protein are critically dependent on the ability of MxA to bind GTP. Using limited proteolysis we analyzed the conformations of the MxA protein under nucleotide‐free, GDP‐bound, and GTP‐bound conditions. We find that whereas the conformations of nucleotide‐free MxA and GDP‐bound MxA are essentially similar, GTP‐binding causes a dramatic change in the conformation of MxA.
Differential stability of tetraspanin/tetraspanin interactions: role of palmitoylation
Authors: Charrin Stéphanie , Manié Serge , Oualid Michael , Billard Martine , Boucheix Claude , Rubinstein Eric
Published: October 2002

The tetraspanins associate with various surface molecules and with each other to build a network of molecular interactions, the tetraspanin web. The interaction of tetraspanins with each other seems to be central for the assembly of the tetraspanin web. All tetraspanins studied, CD9, CD37, CD53, CD63, CD81, CD82 and CD151, were found to incorporate [3H]palmitate. By site‐directed mutagenesis, CD9 was found to be palmitoylated at any of the four internal juxtamembrane regions. The palmitoylation of CD9 did not influence the partition in detergent‐resistant membranes but contributed to the interaction with CD81 and CD53. In particular, the resistance of the CD9/CD81 interaction to EDTA, which disrupts other tetraspanin/tetraspanin interactions, was entirely dependent on palmitoylation.
Transgene‐driven protein expression specific to the intermediate pituitary melanotrope cells of Xenopus laevis
Authors: Jansen Eric J.R , Holling Tjadine M , van Herp François , Martens Gerard J.M
Published: October 2002

In the present study, we examined the amphibian Xenopus laevis as a model for stable transgenesis and in particular targeted transgene protein expression to the melanotrope cells in the intermediate pituitary. For this purpose, we have fused a Xenopus proopiomelanocortin (POMC) gene promoter fragment to the gene encoding the reporter green fluorescent protein (GFP). The transgene was integrated into the Xenopus genome as short concatemers at one to six different integration sites and at a total of one to ∼20 copies. During early development the POMC gene promoter fragment gave rise to GFP expression in the total prosencephalon, whereas during further development expression became more restricted. In free‐swimming stage 40 embryos, GFP was found to be primarily expressed in the melanotrope cells of the intermediate pituitary. Immunohistochemical analysis of cryosections of brains/pituitaries from juvenile transgenic frogs revealed the nearly exclusive expression of GFP in the intermediate pituitary. Metabolic labelling of intermediate and anterior pituitaries showed newly synthesized GFP protein to be indeed primarily expressed in the intermediate pituitary cells. Hence, stable Xenopus transgenesis with the POMC gene promoter is a powerful tool to study the physiological role of proteins in a well‐defined neuroendocrine system and close to the in vivo situation.
First purification of the antiquitin protein and demonstration of its enzymatic activity
Authors: Tang Wai-Kwan , Cheng Christopher H.K , Fong Wing-Ping
Published: October 2002

Antiquitin is an evolutionarily conserved protein believed to play a role in the regulation of cellular turgor. Based on sequence analysis, this protein is classified as a member of the aldehyde dehydrogenase superfamily. All previous studies on antiquitin have been confined to the nucleotide level, and the protein has never been purified and characterized. In the present investigation, the antiquitin protein was purified for the first time. An acetaldehyde‐oxidizing protein was isolated from the liver of black seabream (Mylio macrocephalus) by chromatographies on α‐cyanocinnamate Sepharose and Affi‐gel Blue agarose, followed by ammonium sulfate precipitation. The purified protein was identified as antiquitin by the first 18 N‐terminal amino acid residues which showed 83.3% identity with the deduced sequence of human antiquitin. Electrophoretic mobility studies indicated that black seabream antiquitin is a tetramer with a subunit molecular mass of 57.5 kDa. Kinetic analysis of the purified protein indicated that it catalyzes the oxidation of acetaldehyde with K
m and V
max values of 2.0 mM and 1.3 U/mg, respectively. The longer aliphatic propionaldehyde and the aromatic benzaldehyde are also substrates of the purified enzyme. The enzyme is highly specific towards NAD+ as the coenzyme and is totally inactive towards NADP+. Maximal enzymatic activity was found at about pH 9–10.
Corticosteroid inhibits IL‐4 signaling through down‐regulation of IL‐4 receptor and STAT6 activity
Authors: So Eui-Young , Kim Seol-Hee , Cho Byoung-Soo , Park Hyun-Hee , Lee Choong-Eun
Published: October 2002

Corticosteroids are potent anti‐inflammatory and immunosuppressive agents which down‐regulate cytokine production and action. Yet, contradictory results have been reported for their effects on the interleukin (IL)‐4‐mediated response. Using type II Fc receptor for IgE/CD23 as a target gene, here we report that corticosteroids at 10−4–10−6M inhibit the IL‐4 signaling pathway in human primary immune cells by down‐regulation of the IL‐4‐induced IL‐4 receptor expression and STAT6 activation. Although functional antagonism between steroid receptor and STAT6 for their transcriptional activity has been recently described, this is the first report that steroid inhibits the IL‐4‐induced STAT6 activity at the level of tyrosine phosphorylation and target DNA binding.
The LIM domain protein Lmo2 binds to AF6, a translocation partner of the MLL oncogene
Authors: Bégay-Müller Valérie , Ansieau Stéphane , Leutz Achim
Published: October 2002

The LIM only protein Lmo2 plays an important role in hematopoiesis and leukemogenesis. Lmo2 acts as a bridging molecule between components of hematopoietic gene regulatory protein complexes. We used the yeast two‐hybrid system to identify novel Lmo2 interacting proteins and found that the AF6 protein binds to Lmo2. AF6 is a recurrent fusion partner of MLL, the human homolog of Drosophila trithorax chromatin remodeling protein that is involved in childhood leukemia and mixed lineage leukemia. Our data support the notion that recurrent fusion partners of chimeric MLL proteins recruit hematopoietic gene regulatory complexes.
De novo expression of uncoupling protein 3 is associated to enhanced mitochondrial thioesterase‐1 expression and fatty acid metabolism in liver of fenofibrate‐treated rats
Authors: Lanni Antonia , Mancini Francesco P , Sabatino Lina , Silvestri Elena , Franco Renato , De Rosa Gaetano , Goglia Fernando , Colantuoni Vittorio
Published: October 2002

Uncoupling protein 3 (UCP3) is a member of the mitochondrial carrier superfamily, preferentially expressed in skeletal muscle. Its function is not fully understood and it is debated whether it uncouples oxidative phosphorylation as does UCP1 in brown adipose tissue. Recent evidences suggest a role for UCP3 in the flux of fatty acids in and out mitochondria and their utilization in concert with mitochondrial thioesterase‐1 (MTE‐1). In fact, mice overexpressing muscle UCP3 also show high levels of MTE‐1. Fenofibrate is a hypolipidemic drug that prevents body weight gain in diet‐induced obese rats and enhances lipid metabolism by activating peroxisome proliferator‐activated receptors (PPARs). Because fatty acids and fenofibrate stimulate PPARs and in turn UCP3, we investigated whether UCP3 expression might be induced ‘de novo’ in situations of increased hepatic mitochondrial fatty acid utilization caused by a combined effect of a high‐fat diet and fenofibrate treatment. We also investigated whether Mte‐1 expression and β‐oxidation were affected. We show here that Ucp3 is induced in liver of fenofibrate‐treated rats at the mRNA and protein level. Expression was restricted to hepatocytes and was unevenly distributed in the liver. No increase in cell proliferation, inflammatory or fibrotic responses was found. Mte‐1 expression and mitochondrial β‐oxidation were upregulated. Thus, Ucp3 can be transactivated in tissues where it is normally silent and fenofibrate can attain this effect in liver. The data demonstrate that UCP3 is involved in fatty acid utilization and support the notion that UCP3 and MTE‐1 are linked within the same metabolic pathway.
Molecular dynamics simulations revealed Ca2+‐dependent conformational change of Calmodulin
Authors: Komeiji Yuto , Ueno Yutaka , Uebayasi Masami
Published: October 2002

Molecular dynamics simulations were performed to simulate Ca2+‐dependent conformational change of calmodulin (CaM). Simulations of the fully Ca2+‐bound form of CaM (Holo‐CaM) and the Ca2+‐free form (Apo‐CaM) were performed in solution for 4 ns starting from the X‐ray crystal structure of Holo‐CaM. A striking difference was observed between the trajectories of Holo‐CaM and Apo‐CaM: the central helix remained straight in the former but became largely bent in the latter. Also, the flexibility of Apo‐CaM was higher than that of Holo‐CaM. The results indicated that the bound Ca2+ ions harden the structure of CaM.
Zinc accelerates dengue virus type 2‐induced apoptosis in Vero cells
Authors: Shafee Norazizah , AbuBakar Sazaly
Published: October 2002

Dengue virus type 2 (DENV‐2) infection induced apoptotic cellular DNA fragmentation in Vero cells within 8 days of infection. The addition of high concentrations of extracellular Zn2+ but not Ca2+, Mg2+ or Mn2+ to the cell culture medium hastened the detection of apoptosis to within 4 h after infection. No apoptotic cellular DNA fragmentation was detected in the cell culture treated with Zn2+ alone or infected with heat‐ or ultraviolet light‐inactivated DENV‐2 in the presence of Zn2+. These results suggest that (i) apoptosis is induced in African green monkey kidney cells infected with live DENV‐2 and (ii) the addition of high extracellular Zn2+ accelerates detection of apoptosis in the DENV‐2‐infected cells.
Polyene antibiotic amphotericin B in monomolecular layers: spectrophotometric and scanning force microscopic analysis
Authors: Gruszecki Wieslaw I. , Gagos Mariusz , Kernen Peter
Published: October 2002

Monolayers of amphotericin B (AmB) and monolayers composed of AmB and dipalmitoylphosphatidylcholine (DPPC) were formed at the argon–water interface and deposited on a solid support by means of the Langmuir–Blodgett technique. The hypsochromic shift observed in the absorption spectra of monolayers is indicative of aggregated structures of AmB. The exciton splitting theory allowed us to calculate the distance between neighboring molecules in the aggregates as 7.8 Å. Scanning force microscopy of the AmB monolayers revealed the formation of a homogeneous monolayer composed of molecules separated by a distance of 6–8 Å. Microscopy also reveals the formation of cylindrical structures of AmB with a diameter close to 17 Å (internal diameter close to 6 Å) in the monolayers containing additionally 10 mol% DPPC.
Myometrial maxi‐K channel β1 subunit modulation during pregnancy and after 17β‐estradiol stimulation
Authors: Benkusky Nancy A , Korovkina Victoria P , Brainard Adam M , England Sarah K
Published: October 2002

Myometrial maxi‐K channels are modulated by β subunits. We aimed to determine whether β subunits are modulated to affect uterine excitability during gestation. RNase protection analyses revealed that mouse β1 subunit transcripts are regulated during gestation with peak expression at day 14 of pregnancy. Immunohistochemical analysis indicates an increase of this subunit during gestation. Upregulation of the β1 transcript occurs with 4‐day exposure to 17β‐estradiol but not progesterone, and acute estradiol exposure has no effect on β1 transcript expression. These findings verify that β1 subunit transcript is regulated in mouse myometrium during gestation and estrogens may contribute to this increase.
A novel p53‐inducible apoptogenic gene, PRG3, encodes a homologue of the apoptosis‐inducing factor (AIF)
Authors: Ohiro Yoichi , Garkavtsev Igor , Kobayashi Shinichiro , Sreekumar Kodangattil R. , Nantz Regan , Higashikubo Bryan T. , Duffy Shannon L. , Higashikubo Ryuji , Usheva Anny , Gius David , Kley Nikolai , Horikoshi Nobuo
Published: October 2002

The p53 tumor suppressor protein induces cell cycle arrest or apoptosis in response to cellular stresses. We have identified PRG3 (53‐esponsive ene 3), which is induced specifically under p53‐dependent apoptotic conditions in human colon cancer cells, and encodes a novel polypeptide of 373 amino acids with a predicted molecular mass of 40.5 kDa. PRG3 has significant homology to bacterial oxidoreductases and the apoptosis‐inducing factor, AIF, and the gene was assigned to chromosome 10q21.3–q22.1. Expression of PRG3 was induced by the activation of endogenous p53 and it contains a p53‐responsive element. Unlike AIF, PRG3 localizes in the cytoplasm and its ectopic expression induces apoptosis. An amino‐terminal deletion mutant of PRG3 that lacks a putative oxidoreductase activity retains its apoptotic activity, suggesting that the oxidoreductase activity is dispensable for the apoptotic function of PRG3. The PRG3 gene is thus a novel p53 target gene in a p53‐dependent apoptosis pathway.
Biochemical properties of platelet microparticle membranes formed during exocytosis resemble organelles more than plasma membrane
Authors: Olas Beata , Lundell Kerstin , Holmsen Holm , Fukami Miriam H
Published: October 2002

Studies of [3H]glycerol turnover in phosphatidylcholine (PC) in platelets revealed two metabolic pools, a ‘low turnover PC’ in collagen‐induced microparticles with specific radioactivity only 10% of that found in the ‘high turnover PC’ of bulk platelet PC. Isolated organelle fractions of [3H]glycerol‐labelled platelets contained [3H]PC with specific radioactivities about 20% of that in membrane fractions. These results together with studies on distribution of concanavalin A‐FITC and GPlb, a plasma membrane receptor, indicate that microparticles formed during exocytosis are not simple vesiculations of plasma membrane, but they seem rather to originate from a relatively metabolically static membrane pool not accessible to extracellular reagents.
Viral load of high‐risk human papillomavirus in cervical squamous intraepithelial lesions
Authors: Sun C.A. , Liu J.F. , Wu D.M. , Nieh S. , Yu C.P. , Chu T.Y.
Published: October 2002

Objectives: This case‐control study was conducted to investigate the role of viral load of high‐risk human papillomaviruses (HPVs) in the development of cervical squamous intraepithelial lesions (SILs) and invasive cancers. Methods: A total of 30 female cases who had histological evidence of low‐grade SIL (n=10) or high‐grade SIL and above (n=20) were identified as the case group at the Tri‐Service General Hospital, Taipei between September 1998 and March 1999. In addition, 80 female controls who had normal cervical cytology were enrolled and individually matched on age (±5 years) and date of recruitment to each case. Cervical swabs collected from study subjects were tested for the positivity and viral load of high‐risk HPVs by Hybrid Capture II assay. Additionally, subjects completed a risk factor questionnaire. Results: Among sex behavioral factors studied, younger age at first intercourse was associated with a significantly elevated risk of cervical SIL and invasive cancers. With respect to HPV infection, high‐risk HPV DNA was present in 70% (21/30) of case and 21% (17/80) of control subjects, resulting in an odds ratio (OR) of 6.6 [95% confidence interval (C.I.)=2.6–17.0]. Moreover, women who had a high viral load were at significantly greater risk for cervical SIL and invasive cancers than those who were infected with a low viral load (OR=18.0, 95% C.I.=3.0–108.5). Conclusions: Among the variables tested, infection with a high viral load of high‐risk HPVs is the strongest determinant for cervical SIL and cervical cancers in Taiwan.
Modularity, product variety, production volume, and component sourcing: theorizing beyond generic prescriptions
Authors: Salvador F , Forza C , Rungtusanatham M
Published: October 2002

Research in operations management suggests that firms can mitigate the negative impact of product variety on operational performance by deliberately pursuing modularity in the design of product family architectures. However, modularity is not a dichotomous property of a product, as different types of modularity can be embedded into a product family architecture. The present paper explores how manufacturing characteristics affect the appropriate type of modularity to be embedded into the product family architecture, and how the types of modularity relate to component sourcing. The study is based on a qualitative research design involving a multiple case study methodology to examine six product families belonging to six European companies. The themes derived through case analyses are synthesized in the form of empirical generalizations. Insights from these empirical generalizations are subsequently developed into two propositions explaining why and under what conditions these empirical generalizations might hold for a product family outside of the original sample. The theoretical results formalize, first of all, a type of modularity (i.e. combinatorial modularity) not currently described in literature. Second, the theoretical propositions suggest that when the desired level of product variety is low (high) relative to total production volume, component swapping modularity (combinatorial modularity) helps to maximize operational performance. Finally, the complexity of component families outsourced to suppliers and the geographical proximity of component family suppliers affect the extent to which the product variety–operational performance trade‐off can be mitigated through modularity.
Tensile properties of the physis vary with anatomic location, thickness, strain rate and age
Authors: Williams John L. , Do Pat D. , Eick J. David , Schmidt Thomas L.
Published: October 2002

The variable outcome of physeal distraction has raised questions as to the mechanism by which bone lengthening is achieved. Is it by stretching of the matrix or does it stimulate growth? In order to explore the contribution of matrix stretching, we sought to answer the following questions in an animal model: (a) Are the tensile properties of the lateral side of the proximal tibial physis different from the medial? (b) Are the tensile properties strain‐rate dependent? (c) Does the growth plate fracture through any preferred zone in tension? (d) Are the tensile properties of the bovine growth plate a function of age? (e) Are thicker growth plates weaker in tension? (f) Are the tensile properties of the bovine growth plate comparable to those of a child's? We compared bone—cartilage—bone specimens (0.5 ± 2.5 mm2 in cross‐section) from the lateral, central and medial regions of the proximal tibial growth plates of 12‐ to 18‐month heifers. 70 specimens were tested to failure in tension at 0.0004, 0.004 and 0.04 mm/s. Tensile strength and tangent modulus were 33% and 25% greater, respectively, on the lateral side compared with the medial, and both were increased at the higher strain rates. We found no difference in the ultimate strains by region or strain rate. Thicker growth plates were weaker. Scanning electron microscopy revealed a three‐dimensional fracture pattern extending from the upper columnar into the reserve zone. Bundles of intact chondrons remained intact, but only on the metaphyseal side, having been torn from an interterritorial matrix which remained mostly on the epiphyseal side of the fracture. We compared 21 specimens of 12‐ to 18‐month and 19 specimens of 5‐month calves from similar regions of the proximal tibia. These were tested to failure in tension at 0.004 mm/s. The older bovine growth plate was 25% thinner, 34% stronger and failed at 65% greater strain. For comparison, we tested eight samples from the femoral capital growth plate of two cerebral palsy patients. These were twice as thick as our bovine samples and about half as strong, but with similar ultimate strain values. © 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
The angiogenic response to skeletal injury is preserved in the elderly
Authors: Street J. T. , Wang J. H. , Wu Q. D. , Wakai A. , McGuinness A. , Redmond H. P.
Published: October 2002

Angiogenesis is essential for normal bone formation and repair. Avascularity characterizes aberrant fracture union in the elderly, while angiogenic mechanisms during cutaneous wound repair are attenuated in aged humans. We hypothesized that skeletal injury results in local (circulating) and systemic (fracture site) ‘angiogenic’ responses and that these reparative mechanisms are attenuated with advanced patient age. This prospective study examined peripheral blood and fracture hematoma from 32 patients, 16 under 40 years and 16 over the age of 75, undergoing emergent surgery for isolated fracture. The angiogenic cytokines vascular endothelial growth factor (VEGF) and platelet‐derived growth factor (PDGF) were assayed. Endothelial cell cultures were supplemented with patient plasma and fracture hematoma and angiogenesis determined in vitro by measuring cell proliferation and blood vessel tube formation. Angiogenesis was determined in vivo using a murine dorsal wound pocket model and quantification of new blood vessel formation after 7 days. We found that all injured patients, irrespective of age, have elevated plasma and fracture hematoma levels of VEGF and PDGF. These elevated cytokine concentrations translate into biologically significant angiogenic effects, in vitro and in vivo. These effects are primarily VEGF mediated and are not dependent on patient age. The biological activity of these growth factors does not diminish with advanced age. Thus skeletal injury does result in local and systemic angiogenic responses whereby angiogenic cytokine availability and activity is preserved in the aged suggesting alternative mechanisms for the development of avascularity in delayed and fracture non‐union in the elderly. © 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Hydroxyapatite crystals as a local delivery system for cisplatin: adsorption and release of cisplatin in vitro
Authors: Barroug Allal , Glimcher Melvin J.
Published: October 2002

This study describes the characteristics of the in vitro binding and release of the anti‐tumor drug cisplatin by slurries of synthetic hydroxyapatite crystals carried out in aqueous media. The adsorption of cisplatin by slurries of hydroxyapatite and its release were found to depend significantly on the ionic composition of the aqueous media used. At a constant pH of 7.4, significantly more cisplatin is adsorbed by the hydroxyapatite crystals in the slurry from a chloride‐free phosphate buffered solution or a Tris buffered solution than from a buffered phosphate solution containing chloride ions. The amount of hydroxyapatite‐bound cisplatin desorbed into solution was also progressively increased as a function of the increasing concentration of chloride in the equilibrating solution. Very little hydroxyapatite‐bound cisplatin was released from the crystals in either a Tris or phosphate buffer. These results suggest that it is the hydrated derivatives of cisplatin which are involved in the adsorption of cisplatin by hydroxyapatite crystals. The adsorption data can be expressed as a Freundlich isotherm from which the association constant can be calculated. The rate of release of cisplatin bound to crystals of hydroxyapatite is relatively slow even at the maximum concentration of chloride ions in the phosphate buffer. Approximately 33% of the total cisplatin bound to the crystals of hydroxyapatite was released after 4.25 days. An additional 15% of the remaining cisplatin bound to the hydroxyapatite cyrstals was released after an additional equilibration with fresh buffer for two weeks (58% of the total cisplatin originally bound). These findings suggest that cisplatin bound to slurries of hydroxyapatite crystals may be useful in the local treatment of malignant tumors. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Clonality studies in giant cell tumor of bone
Authors: Schwartz H. S. , Eskew J. D. , Butler M. G.
Published: October 2002

Genetic studies including chromosome analysis, telomere reduction and telomere activity, DNA microsatellites and loss of heterozygosity (LOH) studies have been performed on giant cell tumor (GCT) of bone however whether this primary skeletal neoplasm represents a monoclonal or polyclonal proliferation is unknown. Utilizing a new assay to study the polymorphic human androgen receptor locus (HUMARA), the ratio of maternal inactive X‐chromosome to the paternal inactive X (Lyon hypothesis) is determined via a methylation – specific polymerase chain reaction (PCR) technique to detect X‐chromosome polymorphisms. Characterization of the genetic tumorigenesis of this unpredictable neoplasm may lend insight into its biological behavior and offer improvements in therapeutic intervention, as new information emerges regarding osteoclastic bone resorption. Seventeen female patients with giant cell tumor of bone had their DNA harvested and their X‐chromosome inactivation pattern and polymorphisms determined and compared to control. A polyclonal proliferation pattern was identified in all informative samples studied. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Subchondral defects in caprine femora augmented with in situ setting hydroxyapatite cement, polymethylmethacrylate, or autogenous bone graft: biomechanical and histomorphological analysis after two‐years
Authors: Welch Robert D. , Berry B. Hudson , Crawford Kevin , Zhang Hong , Zobitz Mark , Bronson Dwight , Krishnan Sumant
Published: October 2002

Juxta‐articular defects pose significant challenges due to the high risk of fracture of the subchondral plate and articular cartilage. We evaluated the mechanical and histomorphological repair process of caprine subchondral femoral defects augmented with either a bioresorbable in situ setting hydroxyapatite cement (HAC), polymethylmethacrylate (PMMA), autogenous bone graft (AG), or left empty. Twelve‐mm subchondral defects were made bilaterally in the medial femoral condyles of skeletally mature goats and augmented with a test material or left empty. Femurs were harvested at varying time periods out to 2 years and evaluated for subchondral stiffness and histomorphological indices. Several defects augmented using autograft or left empty sustained focal fracture of the subchondral plate. No HAC or PMMA augmented defects showed evidence of subchondral fracture. The HAC and PMMA augmented defects showed comparable stiffness at all time points. The mean volume fraction of HAC remaining within the defects progressively decreased from 96% at 24 h to 38% at 2 years. The new bone replacing the HAC appeared to have normal physiological architecture and orientation. In situ setting hydroxyapatite cement may be a viable alternative for the repair of subchondral defects with an important advantage that while undergoing gradual resorption and replacement with host bone, mechanical integrity of the skeletal defect is maintained. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Low‐intensity pulsed ultrasound increases the fracture callus strength in diabetic BB Wistar rats but does not affect cellular proliferation
Authors: Gebauer Gregory P. , Lin Sheldon S. , Beam Heather A. , Vieira Pedro , Parsons J. Russell
Published: October 2002

Type I diabetes mellitus (DM) is associated with impaired fracture healing. Specifically, DM affects multiple phases of fracture healing including early cellular proliferation and late phases resulting in inferior biomechanical properties. Recent studies demonstrated the utility of pulsed low‐intensity ultrasound (US) to facilitate fracture healing. The current study evaluated the effects of daily application of US on mid‐diaphyseal femoral fractures in DM and non‐DM BB Wistar rats. Immunohistochemical staining for PCNA was used to evaluate cellular proliferation at 2, 4, and 7 days post‐fracture. In concordance with previous findings, DM fracture callus demonstrated decreased cellular proliferation. Importantly, the application of US did not significantly alter the proliferation in either DM or control groups. However, mechanical testing revealed significantly greater torque to failure and stiffness in US‐treated DM versus non‐US‐treated DM groups at six weeks post‐fracture. Despite the inability of US to affect the early proliferative phase of fracture healing, its application clearly results in improved mechanical properties during the late phases of healing. These findings suggest a potential role of US as an adjunct for DM fracture treatment. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Expression of smooth muscle actin in osteoblasts in human bone
Authors: Kinner B. , Spector M.
Published: October 2002

It is well known that certain connective tissue cells (viz., dermal fibroblasts) can express the gene for a muscle actin – α‐smooth muscle actin – and can contract. This process contributes to skin wound closure and is responsible for Dupuytren's contracture. The objective of this study was to determine if human osteoblasts can also express the gene for α‐smooth muscle actin. Immunohistochemistry using a monoclonal antibody for α‐smooth muscle actin was performed on human cancellous bone samples obtained from 20 individuals at the time of total joint arthroplasty. The percentages of resting and active osteoblasts on the bone surfaces containing this muscle actin isoform were evaluated. Explants of human bone were also studied for the expression of α‐smooth muscle actin in the tissue and in the outgrowing cells with time in culture. Western blot analysis was performed to quantify the α‐smooth muscle actin content of the outgrowing cells relative to smooth muscle cell controls.

Nine ± 2% (mean ± SEM; n = 20) of the cells classified as inactive osteoblasts and 69 ± 3% (n = 19) of the cells identified as active osteoblasts on the bone surface contained α‐smooth muscle actin. This difference was highly statistically significant (Student's t test, p < 0.0001). Similar profiles of α‐smooth muscle actin‐expressing cells were found in explants cultured for up to 12 weeks. Cells forming a layer on the surface of the explants and growing out from them in monolayer also contained α‐smooth muscle actin by immunohistochemistry and Western blot analysis.

Human osteoblasts can express the gene for α‐smooth muscle actin. This expression should be considered a phenotypic characteristic of this cell type, conferred by its progenitor cells: bone marrow stromal‐derived stem cells, and perhaps pericytes and smooth muscle cells. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Vascular endothelial growth factor (VEGF)‐induced angiogenesis in herniated disc resorption
Authors: Haro Hirotaka , Kato Tsuyoshi , Komori Hiromichi , Osada Motonobu , Shinomiya Kenichi
Published: October 2002

Intervertebral disc herniation is a major cause of low back pain and sciatica. Spontaneous resorption of herniated disc (HD) is frequently detected by magnetic resonance imaging (MRI). Marked infiltration by macrophages and neo‐vascularization are observed upon histogical examination of HD. In addition, enhanced MRI studies suggest that HD resorption occurs more frequently in those completely exposed to the epidural space and that this correlates with their degree of vascularization. We have postulated that the angiogenic factor, vascular endothelial growth factor (VEGF), may be implicated in the neo‐vascularization of HD tissues. Here we demonstrate that VEGF and its receptors VEGFR‐1 and VEGFR‐2 are expressed in human surgical samples of HD. Using a co‐culture system comprised of murine peritoneal macrophages and intervertebral disc tissue as a model of the acute phase of HD developed previously, an increase in macrophage VEGF protein and mRNA expression was observed upon exposure to disc tissue. Tumor necrosis factor alpha (TNF‐α) was required for this induction of VEGF. Use of a novel angiogenesis assay revealed that addition of the conditioned media from the co‐culture system resulted in an increase of vascular tubule formation. This effect was strongly inhibited by anti‐VEGF antibody, but augmented by recombinant VEGF. We conclude that VEGF induction, under the co‐culture conditions tested can result in neo‐vascularization of intervertebral disc tissue and may thus play a role in the resorption of HD. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Predictors of wound infection in hip and knee joint replacement: results from a 20 year surveillance program
Authors: Saleh Khaled , Olson Mary , Resig Scott , Bershadsky Boris , Kuskowski Mike , Gioe Terence , Robinson Harry , Schmidt Richard , McElfresh Edward
Published: October 2002

Background. Deep wound infection (DWI) in total knee (TKA) and total hip (THA) arthroplasty has been shown to highly correlate with superficial surgical site infection (SSSI). Although several studies have reported hospital factors that predispose to SSSI, patient factors have not been clearly elucidated.

Methods. All patients undergoing TKA (n = 1181) and THA (n = 1124) surgery during the period 1977–1995 at our institution were observed at the end of a 30‐day post‐operative period. Thirty‐three patients that developed SSSI during this period constituted the study group. The control group was composed of 64 matched subjects that did not develop SSSI. A chart review was applied to abstract DWI cases during the first 18 post‐operative months for the study group and for an average of 6.7 years for the control group (range 5–18.2 years). Potential risk factors for SSSI were used as predictors of SSSI in a logistic regression analysis.

Results. During the 18‐month observation period 19 out of the 33 study subjects (58%) developed DWI. No DWI was registered in the control group (the difference was significant, p < 0.0001). Of the nine pre‐operative, five intra‐operative, and five post‐operative factors examined, only hematoma formation (odds ratio = 11.8; p = 0.001) and days of post‐operative drainage (odds ratio = 1.32; p = 0.01) were significant predictors of SSSI. The cases consumed more health care resources at all stages of the medical process.

Conclusions. Our results (1) confirm the strong correlation between the probability of developing DWI and SSSI: (2) indicate that hematoma formation and persistent post‐operative drainage increase the risk of SSSI. We hypothesize that post‐operative monitoring of patients for hematoma and persistent drainage enables earlier intervention that may lower the risk of developing SSSI and subsequent DWI. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Recombinant human bone morphogenetic protein‐4 (rhBMP‐4) enhanced posterior spinal fusion without decortication
Authors: Guo Xia , Lee Kwan Man , Law Lai Pang , Chow Hung Kay Daniel , Rosier Randy , Cheng Chung Yiu Jack
Published: October 2002

In posterior spinal fusion, insufficient decortication may decrease the number of bone marrow derived ostoprogenitor stem cells and affect the success of bony fusion. The finding of bone formation through interaction between rhBMP‐4 and non‐marrow derived mesenchymal cells constituted the basis of the current study. The aim is to investigate the possibility of molecular enhancement of posterior spinal fusion by site‐specific application of rhBMP‐4 with or without surgical decortication. Eighteen adult rabbits underwent single level bilateral posterior intertransverse process spinal fusion at L5‐L6, one side with decortication, and the other side without decortication. Two animals underwent sham operation without bone grafts, the other 16 animals were randomly allocated into three groups, using hydroxyapatite‐tricalcium phosphate (HA‐TCP) ceramic blocks augmented with 0, 0.125 and 0.5 mg of rhBMP‐4 respectively. Spinal fusion morphology was evaluated with sequential X‐ray, microradiography and histomorphology. At week 7, complete bony fusion was achieved in none of the groups without rhBMP‐4 irrespective of whether the bony contact surface was decorticated or not. In the groups with low dose rhBMP‐4, complete fusion occurred in two of six un‐decorticated sites (33%) and in three of six (50%) decorticated sites. 100% complete fusion was found in the high dose rhBMP‐4 group independent of surgical decortication. The dorsal cortices of the un‐decorticated transverse processes were replaced by newly formed trabecular bone through biological remodeling. This study suggested that rhBMP‐4 can induce non‐marrow derived mesenchymal cells to differentiate into osteogenic cells and thus enhance the high success rate of posterior spinal fusion in both the decorticated and un‐decorticated model. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
The effect of long‐term methylprednisolone treatment on the femoral head in growing pigs
Authors: Drescher Wolf , Li Haisheng , Jensen Søren D. , Ingerslev Jørgen , Hansen Ebbe S. , Hauge Ellen M. , Bünger Cody
Published: October 2002

The effect of long term steroid treatment on coagulation, intraosseous pressure (IOP), femoral head (FH) blood flow, and histology in the normal organism was investigated in this study in growing pigs. From 24 growing female Danish Landrace pigs from 12 litters, 12 animals daily received 100 mg methylprednisolone orally for three months. Their 12 sister pigs served as controls without steroid treatment. Prothrombin time, activated partial thromboplastin time (aPTT), fibrinogen, and antithrombin III levels were recorded in jugular venous blood. Blood flow of the hip regions was measured by means of the radioactive microsphere technique. Metaphyseal and epiphyseal IOP of the left or right proximal femur were measured. Histomorphometry of the left or right FH epiphysis was performed. Major growth inhibition was found in the corticosteroid (CS) treated group. In CS pigs, aPTT was shortened to 50% compared to control pigs. Plasma fibrinogen was higher in the CS animals. Total FH blood flow was not different while regional blood flow in the cranial subregion of the FH epiphysis was higher in the CS group. Metaphyseal and epiphyseal IOP of the proximal femur were not different in the CS animals. Histomorphometrically, cancellous bone volume (23 ± 1% vs. 34 ± 2%; p ≤ 0.001) and bone turnover (10 ± 2% vs. 55 ± 8%; p ≤ 0.001) of the FH epiphysis was lower in the CS than in the control group. The FH epiphysis of the CS animals invariably showed an irregular cartilage‐bone interface with cartilaginous projections into the subchondral bone mainly in its cranial part. In normal growing pigs, long term high dose CS treatment has induced a hypercoagulable state of plasma via the intrinsic pathway of coagulation, cartilaginous projections into FH subchondral bone, FH osteopenia, and reduced bone turnover. Long‐term steroid treatment did not produce FH necrosis or FH IOP alterations in the immature pig model. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.