bluuebunny/crossref_metadata_2025 huggingface dataset:
Superoxide radicals can act synergistically with hypochlorite to induce damage to proteins
Authors: Hawkins Clare L. , Rees Martin D. , Davies Michael J.
Published: October 2002

Activated phagocytes generate both superoxide radicals via a respiratory burst, and HOCl via the concurrent release of the haem enzyme myeloperoxidase. Amine and amide functions on proteins and carbohydrates are major targets for HOCl, generating chloramines (RNHCl) and chloramides (RC(O)NClR′), which can accumulate to high concentrations (>100 μM). Here we show that superoxide radicals catalyse the decomposition of chloramines and chloramides to reactive nitrogen‐centred radicals, and increase the extent of protein fragmentation compared to that observed with either superoxide radicals or HOCl, alone. This synergistic action may be of significance at sites of inflammation, where both superoxide radicals and chloramines/chloramides are formed simultaneously.
Calmodulin signaling via the IQ motif
Authors: Bähler Martin , Rhoads Allen
Published: October 2002

The IQ motif is widely distributed in both myosins and non‐myosins and is quite common in the database that includes more than 900 Pfam entries. An examination of IQ motif‐containing proteins that are known to bind calmodulin (CaM) indicates a wide diversity of biological functions that parallel the Ca2+‐dependent targets. These proteins include a variety of neuronal growth proteins, myosins, voltage‐operated channels, phosphatases, Ras exchange proteins, sperm surface proteins, a Ras Gap‐like protein, spindle‐associated proteins and several proteins in plants. The IQ motif occurs in some proteins with Ca2+‐dependent CaM interaction where it may promote Ca2+‐independent retention of CaM. The action of the IQ motif may result in complex signaling as observed for myosins and the L‐type Ca2+ channels and is highly localized as required for sites of neuronal polarized growth and plasticity, fertilization, mitosis and cytoskeletal organization. The IQ motif associated with the unconventional myosins also promotes Ca2+ regulation of the vectorial movement of cellular constituents to these sites. Additional regulatory roles for this versatile motif seem likely.
Non‐antisense cellular responses to oligonucleotides
Authors: Anselmet Alain , Mayat Ebrahim , Wietek Stefan , Layer Paul G , Payrastre Bernard , Massoulié Jean
Published: October 2002

Oligonucleotides induce various cellular responses which are not due to the blockade of protein synthesis by an antisense mechanism. Oligonucleotides presenting double‐stranded or G‐quartet structures (ribo‐ or deoxyribonucleotides, phosphodiester or phosphorothioated) induce retraction of neurites and aggregation of chicken retinal cells within 10–20 h. This effect is reversible, non‐toxic; it appears to require internalization and can be mimicked by treatment of the cells with an RGDS peptide. The oligonucleotides appear to trigger a cascade of intracellular events, affecting the adhesive properties of integrins. In addition, a subset of oligonucleotides induced platelet aggregation, probably through their interaction with membrane receptors. Recognition of these effects is important for the design and interpretation of antisense experiments.
The integrin αVβ6 binds and activates latent TGFβ3
Authors: Annes Justin P. , Rifkin Daniel B. , Munger John S.
Published: October 2002

Transforming growth factors‐β (TGFβ1, 2 and 3) are secreted in a complex with their propeptides (latency‐associated peptide 1 (LAP1), 2 and 3). TGFβ signaling requires the dissociation of LAP and TGFβ, a process termed latent TGFβ activation. This process is a critical but incompletely understood step in the regulation of TGFβ function. In particular, the extent to which activation mechanisms differ among the three TGFβ isoforms is relatively unexplored. We show here that αVβ6 binds and activates latent TGFβ3.
EVH1 domains: structure, function and interactions
Authors: Ball Linda J. , Jarchau Thomas , Oschkinat Hartmut , Walter Ulrich
Published: October 2002

Drosophila enabled/vasodilator‐stimulated phosphoprotein homology 1 (EVH1) domains are 115 residue protein–protein interaction modules which provide essential links for their host proteins to various signal transduction pathways. Many EVH1‐containing proteins are associated closely with actin‐based structures and are involved in re‐organization of the actin cytoskeleton. EVH1 domains are also present in proteins enriched in neuronal tissue, thus implicating them as potential mediators of synaptic plasticity, linking them to memory formation and learning. Like Src homology 3, WW and GYF domains and profilin, EVH1 domains recognize and bind specific proline‐rich sequences (PRSs). The binding is of low affinity, but tightly regulated by the high specificity encoded into residues in the protein:peptide interface. In general, a small (3–6 residue) ‘core’ PRS in the target protein binds a ‘recognition pocket’ on the domain surface. Further affinity‐ and specificity‐increasing interactions are then formed between additional domain epitopes and peptide ‘core‐flanking’ residues. The three‐dimensional structures of EVH1:peptide complexes now reveal, in great detail, some of the most important features of these interactions and allow us to better understand the origins of specificity, ligand orientation and sequence degeneracy of target peptides, in low affinity signalling complexes.
MINT: a Molecular INTeraction database
Authors: Zanzoni Andreas , Montecchi-Palazzi Luisa , Quondam Michele , Ausiello Gabriele , Helmer-Citterich Manuela , Cesareni Gianni
Published: October 2002

Protein interaction databases represent unique tools to store, in a computer readable form, the protein interaction information disseminated in the scientific literature. Well organized and easily accessible databases permit the easy retrieval and analysis of large interaction data sets. Here we present MINT, a database (http://cbm.bio.uniroma2.it/mint/index.html) designed to store data on functional interactions between proteins. Beyond cataloguing binary complexes, MINT was conceived to store other types of functional interactions, including enzymatic modifications of one of the partners. Release 1.0 of MINT focuses on experimentally verified protein–protein interactions. Both direct and indirect relationships are considered. Furthermore, MINT aims at being exhaustive in the description of the interaction and, whenever available, information about kinetic and binding constants and about the domains participating in the interaction is included in the entry. MINT consists of entries extracted from the scientific literature by expert curators assisted by ‘MINT Assistant’, a software that targets abstracts containing interaction information and presents them to the curator in a user‐friendly format. The interaction data can be easily extracted and viewed graphically through ‘MINT Viewer’. Presently MINT contains 4568 interactions, 782 of which are indirect or genetic interactions.
PTB or not PTB – that is the question
Authors: Yan Kelley S , Kuti Miklos , Zhou Ming-Ming
Published: October 2002

Phosphotyrosine binding (PTB) domains are structurally conserved modules found in proteins involved in numerous biological processes including signaling through cell‐surface receptors and protein trafficking. While their original discovery is attributed to the recognition of phosphotyrosine in the context of NPXpY sequences – a function distinct from that of the classical src homology 2 (SH2) domain – recent studies show that these protein modules have much broader ligand binding specificities. These studies highlight the functional diversity of the PTB domain family as generalized protein interaction domains, and reinforce the concept that evolutionary changes of structural elements around the ligand binding site on a conserved structural core may endow these protein modules with the structural plasticity necessary for functional versatility.
Light control over the size of an antenna unit building block as an efficient strategy for light harvesting in photosynthesis
Authors: Yakovlev A.G , Taisova A.S , Fetisova Z.G
Published: October 2002

It was shown that an increase in the bacteriochlorophyll (BChl) c antenna size observed upon lowering growth light intensities led to enhancement of the hyperchromism of the BChl c Qy
absorption band of the green photosynthetic bacterium Chloroflexus aurantiacus. With femtosecond difference absorption spectroscopy, it was shown that the amplitude of bleaching of the oligomeric BChl c Qy
band (as compared to that for monomeric BChl a) increased with increasing BChl c content in chlorosomes. This BChl c bleaching amplitude was about doubled as the chlorosomal antenna size was about trebled. Both sets of findings clearly show that a unit BChl c aggregate in the chlorosomal antenna is variable in size and governed by the grow light intensity, thus ensuring the high efficiency of energy transfer within the BChl c antenna regardless of its size.
On the pro‐oxidant effects of haemozoin
Authors: Oliveira Marcus F , Timm Bruno L , Machado Ednildo A , Miranda Kildare , Attias Márcia , Silva José R , Dansa-Petretski Marı́lvia , de Oliveira Marco A , de Souza Wanderley , Pinhal Nelson M , Sousa João J.F , Vugman Ney V , Oliveira Pedro L
Published: October 2002

Haemozoin (Hz) is a haem aggregate produced in some blood‐feeding organisms. There is a general belief that Hz formation would be a protective mechanism against haem toxicity. Here we show that when aggregated into Hz, haem is less deleterious than its free form. When haem was added to phosphatidylcholine (PC) liposomes, there was an intense stimulation of oxygen consumption, which did not occur when Hz was incubated with the same preparation. Evaluation of oxygen radical attack to lipids, by measurement of thiobarbituric acid reactive substances (TBARS), showed significantly lower levels of lipid peroxidation in samples containing PC liposomes incubated with Hz than with haem. However, TBARS production induced by Hz was much higher when using 2‐deoxyribose (2‐DR) as substrate, than with PC liposomes. Spin‐trapping analysis by electron paramagnetic resonance (EPR) of Hz and tert‐butylhydroperoxide (tert‐BuOOH) showed that production of methoxyl and tert‐butoxyl radicals was only slightly reduced compared to what was observed with haem. Interestingly, when large Hz crystals were used in 2‐DR TBARS assays and tert‐BuOOH EPR experiments, the pro‐oxidant effects of Hz were strongly reduced. Moreover, increasing concentrations of Hz did not induce erythrocyte lysis, as occurred with haem. Thus, the reduced capacity of Hz to impose radical damage seems to result from steric hindrance of substrates to access the aggregated haem, that becomes less available to participate in redox reactions.
Cooperative activity of phospholipid‐N‐methyltransferases localized in different membranes
Authors: Janssen Marjolein J.F.W , de Jong H.Mark , de Kruijff Ben , de Kroon Anton I.P.M
Published: October 2002

The possibility that the phospholipid‐N‐methyltransferases from yeast are capable of acting upon a phospholipid substrate, localized in a different membrane than in which the enzymes reside (‘trans‐catalysis’ hypothesis), was investigated using cho2 and opi3 gene disruptant strains, which are defective in phosphatidylethanolamine transferase (PEMT) and phospholipid methyltransferase (PLMT), respectively. When cell homogenates or microsomes of the two disruptant strains are mixed, the combined methyltransferase activity, measured as the incorporation of [3H]methyl label from S‐adenosyl methionine, exceeds that expected based on the separate activities of PEMT and PLMT. The increased incorporation implies that monomethylphosphatidylethanolamine generated by PEMT becomes available for PLMT, as evidenced by increased synthesis of dimethylphosphatidylethanolamine and phosphatidylcholine. The kinetics of the cooperativity suggest a collision‐based process, enabling either transport of substrate or ‘trans‐catalysis’.
Tandem termination signals: myth or reality?
Authors: Major Louise L , Edgar Tina D , Yee Yip Po , Isaksson Leif A , Tate Warren P
Published: October 2002

In two Escherichia coli genomes, laboratory strain K‐12 and pathological strain O157:H7, tandem termination codons as a group are slightly over‐represented as termination signals. Individually however, they span the range of representations, over, as expected, or under, in one or both of the strains. In vivo, tandem termination codons do not make more efficient signals. The second codon can act as a backstop where readthrough of the first has occurred, but not at the expected efficiency. UGAUGA remains an enigma, highly over‐represented, but with the second UGA a relatively inefficient back up stop codon.
Transforming growth factor‐β1 induces collagen synthesis and accumulation via p38 mitogen‐activated protein kinase (MAPK) pathway in cultured L6E9 myoblasts
Authors: Rodrı́guez-Barbero A , Obreo J , Yuste L , Montero J.C , Rodrı́guez-Peña A , Pandiella A , Bernabéu C , López-Novoa J.M
Published: October 2002

Transforming growth factor‐β (TGF‐β) plays a pivotal role in the extracellular matrix accumulation observed in chronic progressive tissue fibrosis, but the intracellular signaling mechanism by which TGF‐β stimulates this process remains poorly understood. We examined whether mitogen‐activated protein kinase (MAPK) routes were involved in TGF‐β1‐induced collagen expression in L6E9 myoblasts. TGF‐β1 induced p38 and extracellular signal‐regulated kinase (ERK) 1/2 phosphorylation whereas no effect on Jun N‐terminal kinase phosphorylation was observed. Biochemical blockade of p38 but not of the ERK MAPK pathway abolished TGF‐β1‐induced α2(I) collagen mRNA expression and accumulation. These data indicate that TGF‐β1‐induced p38 activation is involved in TGF‐β1‐stimulated collagen synthesis.
Site‐directed mutagenesis of the type II TGF‐β receptor indicates a ligand‐binding site distinct from that of the type II activin receptor
Authors: Guimond Alain , Sulea Traian , Pen Ally , Ear Pohien , O'Connor-McCourt Maureen D
Published: October 2002

Site‐directed mutagenesis was used to map the ligand‐binding surface of the type II transforming growth factor‐β receptor extracellular domain (TβRII‐ECD). Two putative ligand‐binding sites were probed, the first being a predicted hydrophobic patch, the second being the finger 1 surface loop. Nine residues were mutated in the context of full‐length TβRII and the effect of these mutations on ligand‐binding and receptor signaling was analyzed. Complementary information was obtained by examining ‘natural’ evolutionary TβRII mutations. Together, the results indicate that residues within the finger 1 region, but not the hydrophobic patch, of the TβRII‐ECD are required for productive ligand‐binding. We conclude that, surprisingly, the ECDs of TβRII and type II activin receptor utilize distinct interacting surfaces for binding their respective ligands.
Arabidopsis glutathione‐dependent formaldehyde dehydrogenase is an S‐nitrosoglutathione reductase
Authors: Sakamoto Atsushi , Ueda Manami , Morikawa Hiromichi
Published: October 2002

S–Nitrosoglutathione (GSNO), an adduct of nitric oxide (NO) with glutathione, is known as a biological NO reservoir. Heterologous expression in Escherichia coli of a cDNA encoding a glutathione‐dependent formaldehyde dehydrogenase of Arabidopsis thaliana showed that the recombinant protein reduces GSNO. The identity of the cDNA was further confirmed by functional complementation of the hypersensitivity to GSNO of a yeast mutant with impaired GSNO metabolism. This is the first demonstration of a plant GSNO reductase, suggesting that plants possess the enzymatic pathway that modulates the bioactivity and toxicity of NO.
MAP kinase mediates growth factor‐induced nuclear translocation of estrogen receptor α
Authors: Lu Qing , Ebling Heather , Mittler Jens , Baur Wendy E , Karas Richard H
Published: October 2002

In addition to mediating the classical transcriptional effects of estrogen, estrogen receptors (ERs) are now known to regulate gene expression in the absence of estrogen by ligand‐independent activation pathways, and to mediate the rapid, non‐genomic effects of estrogen as well. ERs have been shown to associate with the cell membrane, and recent studies demonstrate that this subpopulation of membrane‐associated ER mediates the rapid effects of estrogen. To date, however, little is known regarding the pathways that regulate the distribution of the ER between the nuclear and membrane fractions. In the current study, we demonstrate membrane localization of transiently transfected ERα in human vascular smooth muscle cells, and translocation of ERα from the membrane to the nucleus in response to both estrogen‐dependent and estrogen‐independent stimulation. Mutational analyses identified serine 118 as the critical residue regulating nuclear localization following estrogen‐independent stimulation, but not following estrogen stimulation. Induction of nuclear localization of ERα by estrogen‐independent, but not estrogen‐dependent stimulation was blocked by both pharmacologic and genetic inhibition of mitogen‐activated protein (MAP) kinase activation. Furthermore, constitutive activation of MAP kinase resulted in nuclear translocation of ERα. These overexpression studies support that MAP kinase‐mediated phosphorylation of ERα induces nuclear localization of the ER in response to estrogen‐independent, but not estrogen‐dependent stimulation, demonstrating stimulus‐specific molecular pathways regulate the nuclear localization of the ER. These findings identify a previously unrecognized pathway that regulates the intracellular localization of the ER, and represent the first demonstration that the distribution of the ER between membrane and nuclear compartments is regulated by physiologic stimuli.
Caspase‐3 is not essential for DNA fragmentation in MCF‐7 cells during apoptosis induced by the pyrrolo‐1,5‐benzoxazepine, PBOX‐6
Authors: Mc Gee Margaret M , Hyland Edel , Campiani Giuseppe , Ramunno Anna , Nacci Vito , Zisterer Daniela M
Published: October 2002

Effector caspases‐3, ‐6 and ‐7 are responsible for producing the morphological features associated with apoptosis, such as DNA fragmentation. The present study demonstrates that a member of a novel series of pyrrolo‐1,5‐benzoxazepines, PBOX‐6, induces apoptosis in MCF‐7 cells, which lack caspase‐3. Apoptosis was accompanied by DNA fragmentation and the activation of caspase‐7, but not caspases‐3 and ‐6. Inhibition of caspase‐7 activity reduced the extent of apoptosis induced, indicating that activation of caspase‐7 is involved in the mechanism by which PBOX‐6 induces apoptosis in MCF‐7 cells. This study suggests that caspase‐3 is not necessarily essential for DNA fragmentation and the morphological changes associated with apoptosis.
Yeast Npi3/Bro1 is involved in ubiquitin‐dependent control of permease trafficking
Authors: Springael Jean-Yves , Nikko Elina , André Bruno , Marini Anne-Marie
Published: October 2002

The membrane traffic and stability of the general amino acid permease Gap1 of Saccharomyces cerevisiae are under nitrogen control. Addition of a preferential nitrogen source such as ammonium to cells growing on a poor nitrogen source induces internalization of the permease and its subsequent degradation in the vacuole. This down‐regulation requires ubiquitination of Gap1 through a process involving ubiquitin ligase Npi1/Rsp5, ubiquitin hydrolase Npi2/Doa4, and Bul1/2, two Npi1/Rsp5 interacting proteins. Here we report that yet another protein, Npi3, is involved in the regulation of Gap1 trafficking. We show that Npi3 is required for NH4
+‐induced down‐regulation of Gap1, and particularly for efficient ubiquitination of the permease. Npi3 plays a pleiotropic role in permease down‐regulation, since it is also involved in ubiquitination and stress‐induced down‐regulation of the uracil permease Fur4 and in glucose‐induced degradation of hexose transporters Hxt6/7. We further provide evidence that Npi3 is required for direct vacuolar sorting of neosynthesized Gap1 permease as it occurs in npr1 mutant cells. NPI3 is identical to BRO1, a gene encoding a protein of unknown biochemical function and recently proposed to be involved in protein turnover. Npi3/Bro1 homologues include fungal proteins required for proteolytic cleavage of zinc finger proteins and the mouse Aip1 protein involved in apoptosis. We propose that proteins of the Npi3/Bro1 family, including homologues from higher species, may play a conserved role in ubiquitin‐dependent control of membrane protein trafficking.
Nuclear Apaf‐1 and cytochrome c redistribution following stress‐induced apoptosis
Authors: Ruiz-Vela Antonio , González de Buitrago Gonzalo , Martı́nez-A Carlos
Published: October 2002

Apoptotic protease activating factor‐1 (Apaf‐1) and cytochrome c are cofactors critical for inducing caspase‐9 activation following stress‐induced apoptosis. One consequence of caspase‐9 activation is nuclear–cytoplasmic barrier disassembly, which is required for nuclear caspase‐3 translocation. In the nucleus, caspase‐3 triggers proteolysis of the caspase‐activated DNA nuclease (CAD) inhibitor, causing CAD induction and subsequent DNA degradation. Here we demonstrate that apoptotic cells show perinuclear cytochrome c aggregation, which may be critical for nuclear redistribution of cytochrome c and Apaf‐1. We thus indicate that the nuclear redistribution of these cofactors concurs with the previously reported caspase‐9‐induced nuclear disassembly, and may represent an early apoptotic hallmark.
Circular dichroism spectroscopy of fluorescent proteins
Authors: Visser Nina V , Hink Mark A , Borst Jan Willem , van der Krogt Gerard N.M , Visser Antonie J.W.G
Published: October 2002

Circular dichroism (CD) spectra have been obtained from several variants of green fluorescent protein: blue fluorescent protein (BFP), enhanced cyan fluorescent protein (CFP), enhanced green fluorescent protein (GFP), enhanced yellow fluorescent protein (YFP), all from Aequorea victoria, and the red fluorescent protein from the coral species Discosoma (DsRed). We demonstrate that CD spectra in the spectral fingerprint region of the chromophore yield spectra that after normalization are not coincident with the normalized absorbance spectra of GFP, YFP and DsRed. On the other hand, the CD spectra of BFP and CFP coincide with the absorbance spectra. The resolution of absorption and CD spectra into Gaussian bands confirmed the location of the different electronic band positions of GFP and YFP as reported in the literature using other techniques. In the case of BFP and CFP the location of Gaussian bands provided information of the vibrational progression of the electronic absorption bands. The CD spectrum of DsRed is anomalous in the sense that the major CD band has a clear excitonic character. Far‐UV CD spectra of GFP confirmed the presence of the high β‐sheet content of the polypeptide chain in the three‐dimensional structure.
Optimization of GFP levels for analyzing Salmonella gene expression during an infection
Authors: Wendland Meike , Bumann Dirk
Published: October 2002

Green fluorescent protein (GFP) is an attractive reporter for Salmonella gene expression analysis but might interfere with virulence when expressed at high levels. To identify suitable GFP levels, we constructed a series of Salmonella strains expressing different amounts of GFP and measured their fluorescence and colonization levels in infected mice. The results show that GFP concentrations in the range of 7000–200 000 molecules per Salmonella cell are detectable in ex vivo samples using flow cytometry, and cause no major Salmonella virulence defect. Appropriate GFP levels can be obtained with weak promoters and stable GFP, or strong promoters and destabilized GFP.
Activation of Pyk2/RAFTK induces tyrosine phosphorylation of α‐synuclein via Src‐family kinases
Authors: Nakamura Takeshi , Yamashita Hiroshi , Nagano Yoshito , Takahashi Tetsuya , Avraham Shalom , Avraham Hava , Matsumoto Masayasu , Nakamura Shigenobu
Published: October 2002

α‐Synuclein (αS) is a neuronal protein that has been implicated in the pathogenesis of Parkinson's disease. The present report demonstrates that the protein tyrosine kinase Pyk2/RAFTK is involved in cell stress‐induced tyrosine phosphorylation of αS. Hyperosmotic stress induced tyrosine phosphorylation of αS via Pyk2/RAFTK at tyrosine residue 125. Pyk2/RAFTK‐mediated phosphorylation of αS was primarily achieved with Src‐family kinases. In addition, osmotic stress‐induced phosphorylation of αS was dependent on Pyk2/RAFTK activation. Accordingly, such results indicate that Pyk2/RAFTK lies upstream of Src‐family kinases in the signaling cascade by which osmotic stress induces tyrosine phosphorylation of αS.
The centrosome cycle
Authors: Meraldi P , Nigg E.A
Published: October 2002

The centrosome is the major microtubule‐organizing center of animal cells. It influences cell shape and polarity and directs the formation of the bipolar mitotic spindle. Numerical and structural centrosome aberrations have been implicated in disease, notably cancer. In dividing cells, centrosomes need to be duplicated and segregated in synchrony with chromosomes. This centrosome cycle requires a series of structural and functional transitions that are regulated by both phosphorylation and proteolysis. Here we summarize recent information on the regulation of the centrosome cycle and its coordination with the chromosomal cell cycle.
Lentiviral vector transduction of male germ line stem cells in mice
Authors: Nagano Makoto , Watson Deborah J , Ryu Buom-Yong , Wolfe John H , Brinster Ralph L
Published: October 2002

Transgenesis can be achieved in mice by retroviral transduction of male germ line stem cells (GSCs). However, the transduction efficiency by a Moloney murine leukemia virus (MMLV)‐based vector is low, probably due to the characteristically slow cell cycle of stem cells. Since lentiviral vectors can transduce non‐dividing cells, they have the potential to efficiently transduce GSCs. Here we report that male GSCs of mice can be transduced in vitro by a lentiviral vector and generate complete spermatogenesis when transplanted into infertile host testes. Transduction efficiencies were comparable to those for MMLV transduction using similar experimental conditions. The results suggest that both lentiviral and MMLV vectors could be effective in transducing GSCs of other species. In addition, these and previous studies suggest that transduction of immature donor stem cells transplanted into immature recipient testes will provide the most efficient system for male germ line modification.
Keratinocytes enriched for stem cells are protected from anoikis via an integrin signaling pathway in a Bcl‐2 dependent manner
Authors: Tiberio Rossana , Marconi Alessandra , Fila Chiara , Fumelli Cristiana , Pignatti Marco , Krajewski Stan , Giannetti Alberto , Reed John C , Pincelli Carlo
Published: October 2002

Because inhibition of integrin signaling induces apoptosis, we investigated whether keratinocytes expressing β1 and α6β4 integrins (enriched for stem cells) are protected from cell death. Keratinocytes rapidly adhering to type IV collagen expressed highest levels of β1 and α6β4 and of the anti‐apoptotic stem cell marker p63. Apoptotic cells were significantly higher in slowly adhering than in rapidly adhering keratinocytes. Anti‐β1 integrin caused a significant increase in apoptotic cells, while it decreased Bcl‐2 levels in stem keratinocytes. Bax and Bad proteins were higher in slowly adhering than in rapidly adhering cells. By contrast, Bcl‐2, Bcl‐x and Mcl‐1 proteins were highest in rapidly adhering keratinocytes and nearly absent in slowly adhering cells. After addition of anti‐β1 integrin, the apoptotic rate was significantly higher in HaCaT cells not expressing Bcl‐2 than in controls. These results indicate that keratinocytes enriched for stem cells are protected from apoptosis via β1 integrin, in a Bcl‐2 dependent manner.
The enzymatic activity of ADAM8 and ADAM9 is not regulated by TIMPs
Authors: Amour Augustin , Knight C.Graham , English William R , Webster Ailsa , Slocombe Patrick M , Knäuper Vera , Docherty Andrew J.P , Becherer J.David , Blobel Carl P , Murphy Gillian
Published: October 2002

The ADAM family of proteases are type I transmembrane proteins with both metalloproteinase and disintegrin containing extracellular domains. ADAMs are implicated in the proteolytic processing of membrane‐bound precursors and involved in modulating cell–cell and cell–matrix interactions. ADAM8 (MS2, CD156) has been identified in myeloid and B cells. In this report we demonstrate that soluble ADAM8 is an active metalloprotease in vitro and is able to hydrolyse myelin basic protein and a variety of peptide substrates based on the cleavage sites of membrane‐bound cytokines, growth factors and receptors which are known to be processed by metalloproteinases. Interestingly, although ADAM8 was inhibited by a number of peptide analogue hydroxamate inhibitors, it was not inhibited by the tissue inhibitors of metalloproteinases (TIMPs). We also demonstrate that the activity of recombinant soluble ADAM9 (meltrin‐γ, MDC9) lacks inhibition by the TIMPs, but can be inhibited by hydroxamate inhibitors. The lack of TIMP inhibition of ADAM8 and 9 contrasts with other membrane‐associated metalloproteinases characterised to date in this respect (ADAM10, 12, 17, and the membrane‐type metalloproteinases) which have been implicated in protein processing at the cell surface.
Species specificity of the cytokine function of phosphoglucose isomerase
Authors: Amraei Mohammad , Nabi Ivan R
Published: October 2002

Phosphoglucose isomerase (PGI) is a cytosolic glycolytic enzyme that also functions as an extracellular cytokine (neuroleukin/autocrine motility factor (AMF)/maturation factor). Contrary to mammalian PGI, bacterial PGI was not internalized by the PGI/AMF receptor (gp78/AMF‐R) and neither bacterial nor yeast PGI competed with mammalian PGI for receptor binding and internalization. Furthermore, while the bacterial, yeast and mammalian preparations all exhibited isomerase activity, only mammalian PGI stimulated the motility of NIH‐3T3 fibroblasts. The conserved active site of PGI is therefore not sufficient for receptor binding and cytokine activity of PGI. However, synthetic peptides corresponding to distinct peripheral mammalian PGI sequences did not inhibit internalization of mammalian PGI. Our data therefore argue that the cytokine activity of PGI is specific to mammalian PGI but cannot exclude the possibility that the receptor binding motif of PGI is complex and includes elements within and without the active site.
Residual and recurrent lesions after conization for cervical intraepithelial neoplasia grade 3
Authors: Milojkovic M
Published: October 2002

Objectives: To evaluate the importance of regular and long‐term follow‐up of patients who had conization for cervical intraepithelial neoplasia grade 3 (CIN 3); and to assess the risk of residual/recurrent lesions of the cervix uteri. Methods: Retrospective analysis of 934 patients with CIN 3 who underwent conization over the period 1 January 1985–31 December 1997. The analysis included 38 (4.1%) patients who had involved resection margins and 896 (95.9%) patients who had free resection margins. Results: Twenty‐three out of 934 (2.5%) patients had another operation (reconization in 17 and hysterectomy in six) whereas one patient had a biopsy. Residual or recurrent lesion after conization was found in 23 patients out of 934 (2.5%). Three patients had invasive cervical cancer (0.3%), 19 had CIN 3 (2.0%) and one patient had CIN 1 (0.1%). One of the 24 patients (4.2%) was diagnosed with no lesion after repeated operation. Eleven patients out of 38 (28.9%) with involved resection margins during the first conization had residual/recurrent disease, compared with eight out of 896 patients (0.9%) who had free resection margins. Conclusion: Another operation is recommended only in cases when, on the basis of cytological, colposcopical and histological findings, the gynecologist suspect the residual/recurrent lesions.
Minilaparotomy‐assisted vaginal hysterectomy
Authors: Ahmed A.A. , Stachurski J. , Abdel Aziz E. , Bone C.
Published: October 2002

Objectives: To determine the feasibility and acceptability of minilaparotomy‐assisted vaginal hysterectomy. Methods: A prospective pilot study in a general hospital was conducted. Twenty patients who were on the waiting list for abdominal hysterectomy were included in the study. All these patients had one or more relative contraindications to vaginal hysterectomy. The hysterectomy procedure was started vaginally in all cases. A minilaparotomy incision was performed to complete the procedure if vaginal hysterectomy was not feasible. Results were analyzed on the intention to treat basis. Results: The procedure was successfully completed as intended in 19/20 patients (95%). Six patients had the procedure completed vaginally (30%). Thirteen patients had the procedure completed with minilaparotomy assistance (65%). The mean operative time was 63±24.8 min (±S.D.). The median estimated blood loss was 155 ml (range: 20–800). One bladder injury occurred. The overall post‐operative complication rate was 35% (7/20). This included urinary retention necessitating catheterization for 24 h (n=3), urinary infection (n=2), vaginal infection (n=1) and wound hematoma (n=1). The mean post‐operative pain score on a scale from 1 to 10 was 3.1. The overall patient satisfaction based on a scale from 1 to 10 was 9.23 (range: 8–10). Conclusions: Minilaparotomy‐assisted vaginal hysterectomy is a feasible and safe procedure. Our results suggest that this approach is potentially useful in increasing the proportion of hysterectomies performed vaginally.
Inducible displacements of cemented tibial components during weight‐bearing and knee extension. Observations during dynamic radiostereometry related to joint positions and 2 years history of migration in 16 TKR
Authors: Uvehammer J. , Kärrholm J.
Published: October 2002

The inducible displacements of the tibial component caused by active extension were studied in 16 knees 1 yr after an AMK total knee arthroplasty with either flat, concave or posterior‐stabilised (PS) designs of the joint area. Continuous change of the position of the tibial component occurred with proceeding extension. Rocking, subsidence and lift‐off at different localisations were observed. In 3 of 4 knees with flat inserts the tibial component tilted anteriorly from 45° to 35° of flexion. A similar anterior tilt was seen in 2 of 6 with concave inserts and 5 of 6 with the PS design, but the tilting started later, when the knee had 5–20° more extension. From 45–15° of flexion most components tilted into valgus. Three knees (1 concave with, 1 concave without PCL and 1 PS) showed a sudden tilt into varus direction followed by a rocking motion in the opposite direction. The other types of displacements studied showed a more uniform pattern. The inducible maximum translation (MTPM) at 20° of extension tended to be associated with increased migration between 0 and 2 yr when measured with the same parameter (Spearman's rho = 0.54, P = 0.03). Increased medial displacement of the center of the proximal tibia at 25° was associated with increased anterior tilt. This type of motion was most commonly seen with the concave design. Our observations demonstrate that the forces acting on the tibial component vary during active extension, which results in rocking movements. This will influence the migration and the patttern of wear, factors of importance for the clinical longevity of a total knee replacement (TKR). © 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Low molecular weight heparin aggravates infectious arthritis triggered by Staphylococcus aureus
Authors: Sakiniene Egidija , Tarkowski Andrzej
Published: October 2002

Purpose. Staphylococcus aureus is responsible for the majority of wound infections in clean surgical procedures that involve implantation of foreign material, grafts or prosthetic devices. The aim of the study was to assess the effect of low molecular weight heparin on the development and progression of S. aureus arthritis.

Materials and methods. The murine model of hematogenously acquired septic arthritis was used injecting intravenously toxic shock syndrome toxin‐1 (TSST‐1) producing S. aureus of LS‐1 strain. Mice lacking prosthetic implants were treated with intraperitoneal injections of low molecular weight heparin, used routinely as anti‐thrombotic prophylaxis following joint prosthetic surgery. Evaluation of arthritis was performed clinically and histopathologically. In addition, the effect of low molecular weight heparin on T cell dependent and independent inflammation was assessed.

Results. Seven days after inoculation with bacteria 18 out of 19 low molecular weight heparin treated mice displayed clinical symptoms of arthritis as compared to 9 out of 23 control animals (p < 0.05), and the severity of arthritis, expressed as arthritic index, was 2.6 ± 0.5 versus 1.6 ± 0.5 (p = 0.05). The histopathological examination confirmed the clinical findings showing that both inflammation and joint destruction were more substantial in heparin treated animals.

Conclusion. Our findings indicate that the routine anti‐coagulation treatment with heparin contributes to more severe course of joint infection.S © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Abnormal knee joint position sense in individuals with patellofemoral pain syndrome
Authors: Baker Vanessa , Bennell Kim , Stillman Barry , Cowan Sallie , Crossley Kay
Published: October 2002

The purpose of this cross‐sectional study was to compare knee joint position sense (JPS) in 20 individuals with and 20 without patellofemoral pain syndrome (PFPS). Five active tests with ipsilateral limb matching responses were performed at 20° and 60° flexion under non‐weightbearing conditions, and at 40° flexion under uni‐ and bi‐lateral weightbearing conditions. The response errors were calculated as the difference between each target and response position (accuracy) and the standard deviation of these differences (reliability).

JPS was: (i) significantly less accurate and less consistent in the knees with PFPS during both the non‐weightbearing and weightbearing tests when compared to the control subject knees; (ii) less accurate when the symptomatic and asymptomatic knees of the 12 uni‐lateral PFPS subjects were compared and (iii) less accurate in the asymptomatic knees of the uni‐lateral PFPS subjects and knees of the control subjects. The maximum intensity of pain experienced during each knee JPS test was not correlated to any of the JPS test results.

The results confirm abnormal knee joint proprioception in individuals with PFPS. Although it cannot be determined whether the abnormality precedes or follows the development of PFPS, the results support including proprioceptive reeducation in management of PFPS. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
α‐Smooth muscle actin is expressed by fibroblastic cells of the ovine anterior cruciate ligament and its free tendon graft during remodeling
Authors: Weiler Andreas , Unterhauser Frank N. , Bail Hermann‐J. , Hüning Martin , Haas Norbert P.
Published: October 2002

Contractile fibroblastic cells expressing the α‐smooth muscle actin isoform, so‐called myofibroblasts, have been identified to play a possible role during the healing of the medial collateral ligament by means of restoring the tissues in situ strain via extracellular matrix contraction. Recently, these cells have also been identified to be a normal part of the human anterior cruciate ligament. It has been hypothesized that myofibroblasts play a role in the wrinkling of the extracellular matrix. The goal of the present study was to identify myofibroblasts in the intact ovine anterior cruciate ligament and a free autologous tendon graft during remodeling after anterior cruciate ligament reconstruction. In 36 mature merino sheep the anterior cruciate ligament was replaced with an ipsilateral Achilles tendon split graft. Midsubstance tissue samples were immunostained for α‐smooth muscle actin at 6, 9, 12, 24, 52, and 104 weeks. Myofibroblasts were identified in the intact ovine anterior cruciate ligament as well as in the Achilles tendon graft prior to implantation. During remodeling the first myofibroblasts were found at six weeks within newly formed fiber bundles. At 24, 52, and 104 weeks myofibroblast distribution and cell density were similar to those of the intact ovine anterior cruciate ligament. These findings indicate that α‐smooth muscle actin containing fibroblastic cells are a regular part of the intact as well as the remodeled anterior cruciate ligament. There is evidence that myofibroblasts may be involved in maintaining tissue homeostasis in the mature ligament e.g., by means of crimp formation. The presence of these cells during the early remodeling may further indicate that α‐smooth muscle actin containing fibroblastic cells are involved in the earliest stages of fiber bundle formation. The role and function of this special cell type for the anterior cruciate ligament needs to be further clarified. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Human atrophic fracture non‐unions are not avascular
Authors: Reed A. A. C. , Joyner C. J. , Brownlow H. C. , Simpson A. H. R. W.
Published: October 2002

A small proportion of fractures progress to non‐union. Non‐unions are routinely classified into two groups either hypertrophic or atrophic according to their radiological appearance. It is a common preconception that non‐unions with a hypertrophic appearance on X‐ray are biologically active and vascular with potential to heal given the correct stable environment. Atrophic non‐unions are considered to be avascular and inert and will not heal even under the correct stable environment. Non‐unions are either infected or aseptic. In the present study, we tested the hypothesis that aseptic atrophic non‐unions are less vascular than aseptic hypertrophic non‐unions and healing fractures. Biopsies were taken from the fracture gap of patients with healing fractures, hypertrophic non‐unions and atrophic non‐unions. A dual labelling technique was used with antibodies against CD31 (JC70) and Collagen IV. Blood vessels were quantified using a Chalkley point eyepiece graticule. There was no statistically significant difference in the median vessel count between the three fracture groups. These findings do not support the hypothesis that established atrophic fracture non‐union are less vascular than hypertrophic non‐unions or healing fractures. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Titanium particles induce the immediate early stress responsive chemokines IL‐8 and MCP‐1 in osteoblasts
Authors: Fritz Elizabeth A. , Glant Tibor T. , Vermes Csaba , Jacobs Joshua J. , Roebuck Kenneth A.
Published: October 2002

Exposure of human osteoblasts to ultrafine titanium (Ti) particles has been shown to alter osteoblast gene expression. We previously reported that Ti particles can increase IL‐6 release and suppress the gene expression of procollagens α1[I] and α1[III] in human osteoblasts. In this study, we now demonstrate that Ti particles can rapidly induce the chemotactic cytokines interleukin‐8 (IL‐8) and monocyte chemoattractant protein‐1 (MCP‐1), two immediate early stress responsive chemokines important for the activation and chemotaxis of neutrophils and macrophages, respectively. In MG‐63 osteosarcoma cells and bone marrow derived primary osteoblasts Ti particles selectively increased the steady state levels of IL‐8 and MCP‐1 mRNA in a time and concentration dependent manner. The increased chemokine mRNA correlated with increased secretion of IL‐8 and MCP‐1 protein. Actinomycin D, a potent RNA polymerase II inhibitor, blocked the Ti particle induction of IL‐8 and MCP‐1 mRNA expression, whereas cycloheximide, which inhibits protein synthesis, failed to inhibit chemokine gene expression suggesting Ti particles directly target activation of chemokine gene transcription. Consistent with a transcriptional mechanism not involving new protein synthesis, we demonstrate that Ti particles induce the binding of the p65 and p50 subunits of the latent transcription factor NF‐κB to the IL‐8 gene promoter. Taken together, these data demonstrate that Ti particles can activate transcription of the stress responsive chemokine genes IL‐8 and MCP‐1 in human osteoblasts. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Cell outgrowth from the human ACL in vitro: regional variation and response to TGF‐β1
Authors: Murray Martha Meaney , Bennett Rebecca , Zhang Xiuying , Spector Myron
Published: October 2002

One of the new methods being developed to stimulate healing of the human anterior cruciate ligament (ACL) after rupture is the implantation of a biodegradable scaffold which the host cells invade, populate and remodel. One of the cellular behaviors critical to the success of this method is cell outgrowth from the ligament remnants onto an adjacent scaffold. As morphological differences have been previously reported in the proximal and distal human ACL, the primary aim of this study was to determine if the cells from the proximal and distal ACL had different outgrowth behaviors as well. A second aim was to determine whether TGF‐β1, reported to be a mitogen for fibroblasts, would affect the outgrowth behaviors from the proximal and distal ACL. To achieve these aims, explants from both the proximal and distal human ACL were placed into culture under various conditions and outgrowth measured for 42 days. In explants cultured with 10% fetal bovine serum (high serum group) the explants from the proximal ACL had an earlier start of outgrowth than the distal explants (p < 0.015), and outgrowth rates were similar in the two groups. In explants cultured with 2% fetal bovine serum (low serum group), the explants from the proximal ACL had an earlier start to outgrowth (p < 0.003) as well as a faster rate of outgrowth (p < 0.004) than the distal explants. The addition of TGF‐β1 to the low serum cultures significantly slowed the rate of outgrowth from both groups of ACL explants (p < 0.025). These results suggest that outgrowth behaviors are different in the proximal and distal human ACL, and that TGB‐β1 has an inhibitory effect on cell outgrowth from ACL explants. However, this study is only a first step, and additional experiments are needed to further optimize tissue engineering parameters for enhancement of the repair of the ACL. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.