bluuebunny/crossref_metadata_2025_split huggingface dataset:
Lengthening of double‐looped tendon graft constructs in three regions after cyclic loading: A study using Roentgen stereophotogrammetric analysis
Authors: Roos P. J. , Hull M. L. , Howell S. M. 
Published: January 2004

AbstractLengthening of a double‐looped tendon graft construct used to reconstruct the anterior cruciate ligament (ACL) can result in an increase in anterior knee laxity and affect the stability of the reconstructed knee. Three possible regions where lengthening of the construct can occur are (1) the region of the tibial fixation, (2) the region of the femoral fixation, and (3) the region of the graft between the fixations. One objective of this study was to demonstrate the feasibility of using Roentgen stereophotogrammetric analysis (RSA) to determine the lengthening in each region of a double‐looped graft construct subjected to cyclic loading. A second objective was to determine which region(s) contributes most to an increase in length of this graft construct. Radio‐opaque markers were attached to ten grafts to measure the lengthening in each of the three regions. Each graft was passed through a tibial tunnel in a bovine tibia, looped around a rigid cross‐pin, and fixed to the tibia with a Washerloc fixation device. The grafts were cyclically loaded for 225,000 cycles from 20 to 170 N. Prior to and at intervals during the cyclic loading, simultaneous radiographs were taken of the tibia and graft. RSA was used to determine the 3‐dimensional coordinates of the markers from which the lengthening in each region was computed at each interval. The regions of the tibial and femoral fixations were the largest contributors to the increase in length of the graft, with maximum average values of 0.91 and 0.76 mm respectively after 225,000 cycles. The region between the fixations contributed least to lengthening of the graft, with a maximum average value of 0.23 mm. More than 90% of the lengthening in each region occurred before 100,000 cycles of loading. RSA proved to be a useful method for measuring lengthening in all three regions of the graft construct. Lengthening of the graft construct in both regions of fixation is sufficiently large that the combined contributions may cause a recurrence of instability in some knees. © 2003 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved.
Targeting of EWS/FLI‐1 by RNA interference attenuates the tumor phenotype of Ewing's sarcoma cells in vitro
Authors: Chansky Howard A. , Barahmand‐pour Fariba , Mei Qi , Kahn‐Farooqi Waqqar , Zielinska‐Kwiatkowska Anna , Blackburn Michael , Chansky Kari , Conrad Ernest U. , Bruckner James D. , Greenlee Theodore K. , Yang Liu 
Published: January 2004

AbstractThe defining cytogenetic abnormality of Ewing's sarcoma is the presence of a balanced t(11;22) translocation expressing the EWS/FLI‐1 chimeric fusion protein. The effect of EWS/FLI‐1 appears to be dominant negative since over‐expression of EWS does not overcome the sarcoma phenotype. Previous studies have shown that EWS/FLI‐1 as well as related sarcoma fusion proteins are necessary and sufficient to induce transformation both in vitro and in vivo. In this study we report that synthetic small interfering RNA (siRNA) specifically suppresses EWS/FLI‐1 fusion gene expression in SK‐ES Ewing's sarcoma cells. Knockdown of the EWS/FLI‐1 fusion protein is correlated with decreased cell proliferation and increased apoptosis. We demonstrate that Ewing's sarcoma tumors as well as Ewing's sarcoma cell lines predominantly express the CXCR4 chemokine receptor. Using an in vitro invasion assay, the SDF‐1 ligand of CXCR4 was shown to be a potent stimulus of invasion by SK‐ES cells. Knockdown of EWS/FLI‐1 by RNA interference abrogates the invasiveness of SK‐ES cells. These experiments suggest that targeted silencing of the EWS/FLI‐1 fusion gene by siRNA represents a promising strategy to study the loss of EWS/FLI‐1 protein in Ewing's sarcoma cells of otherwise identical genetic background. © 2004 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved.
Fractional cointegration and real exchange rates
Authors: Caporale Guglielmo Maria , Gil‐Alana Luis A. 
Published: January 2004

AbstractThis paper uses fractional integration and cointegration to model the DM–US dollar and the yen–US dollar real exchange rates in terms of both monetary and real factors, more specifically real interest rate and labour productivity differentials. We find that whilst the individual series may be integrated of order 1, their long‐run relationship might have a fractionally cointegrated structure. This means that mean reversion occurs, consistently with the findings of other studies. However, it also indicates, in contrast to such studies, that the cointegrating relationship possesses long memory. In other words, the error correction term responds slowly to shocks, implying that deviations from equilibrium are long‐lived. It appears that only a combination of real and monetary variables can accurately track down the movements of real exchange rates.
MinD and role of the deviant Walker A motif, dimerization and membrane binding in oscillation
Authors: Lutkenhaus Joe , Sundaramoorthy M. 
Published: January 2004

SummaryThe ATPase activity of MinD is required for it to oscillate between the ends of the cell and spatially regulate cell division in Escherichia coli. It is a member of a functionally diverse subgroup of ATPases which are involved in activities ranging from nitrogen fixation (NifH) to plasmid segregation (ParA). All members of the subgroup have a deviant Walker A motif which contains a conserved ‘signature’ lysine that characterizes this subgroup. In the NifH homodimer the signature lysines make intermonomer contact with the bound nucleotides indicating a role in ATP hydrolysis. ATP binding to NifH leads to formation of an active dimer that associates with a partner that is also a dimer. Because ATP hydrolysis is coupled to formation of the complex, the complex is only transient. In the presence of ATP MinD binds MinC and goes to the membrane, however, the ATPase is not stimulated and the complex is stable. Subsequent interaction of this complex with MinE, however, leads to ATPase stimulation and release of the Min proteins from the membrane. The sequential interaction of MinD with these two proteins, which is dictated by the membrane, is critical to the oscillatory mechanism involved in spatial regulation of division.
A Streptomyces coelicolor functional orthologue of Escherichia coli RNase E shows shuffling of catalytic and PNPase‐binding domains
Authors: Lee Kangseok , Cohen Stanley N. 
Published: January 2004

SummaryPrevious work has detected an RNase E‐like endoribonucleolytic activity in cell extracts obtained from Streptomyces. Here, we identify a Streptomyces coelicolor gene, rns, encoding a 140 kDa protein (RNase ES) that shows endoribonucleolytic cleavage specificity characteristic of RNase E, confers viability on and allows propagation of Escherichia coli cells lacking RNase E and accomplishes RNase E‐like regulation of plasmid copy number in E. coli. However, notwithstanding its complementation of rne‐deleted E. coli, RNase ES did not accurately process 9S rRNA from E. coli. Additionally, whereas RNase E is normally required for E. coli survival, rns is not an essential gene in S. coelicolor. Deletion analysis mapped the   catalytic   domain   of   RNase   ES   near   its   centre and showed that regions located near the RNase ES termini interact with an S. coelicolor homologue of polynucleotide phosphorylase (PNPase) – a major component of E. coli RNase E‐based degradosomes. The interacting arginine‐ and proline‐rich segments resemble the C‐terminally located degradosome scaffold region of E. coli RNase E. Our results indicate that RNase ES is a structurally shuffled RNase E homologue showing evolutionary conservation of functional RNase E‐like enzymatic activity, and suggest the existence of degradosome‐like complexes in Gram‐positive bacteria.
SecA2 functions in the secretion of superoxide dismutase A and in the virulence of Mycobacterium tuberculosis
Authors: Braunstein Miriam , Espinosa Benjamin J. , Chan John , Belisle John T. , R. Jacobs William 
Published: January 2004

SummaryTuberculosis remains a severe worldwide health threat. A thorough understanding of Mycobacterium tuberculosis pathogenesis will facilitate the development of new treatments for tuberculosis. Numerous bacterial pathogens possess specialized protein secretion systems that are dedicated to the export of virulence factors. Mycobacterium tuberculosis is part of a developing group of pathogenic bacteria that share the uncommon property of possessing two secA genes (secA1 and secA2). In mycobacteria, SecA1 is the essential ‘housekeeping’ SecA protein whereas SecA2 is an accessory secretion factor. Here we demonstrate that SecA2 contributes to the pathogenesis of M. tuberculosis. A deletion of the secA2 gene in M. tuberculosis attenuates the virulence of the organism in mice. By comparing the profile of proteins secreted by wild‐type M. tuberculosis and the ΔsecA2 mutant, we identified superoxide dismutase A (SodA) as a protein dependent on SecA2 for secretion. SodA lacks a classical signal sequence for protein export. Our data suggests that SecA2‐dependent export is a new type of secretion pathway that is part of a virulence mechanism of M. tuberculosis to elude the oxidative attack of macrophages.
A new Schizosaccharomyces pombe base excision repair mutant, nth1, reveals overlapping pathways for repair of DNA base damage
Authors: Osman Fekret , Bjørås Magnar , Alseth Ingrun , Morland Ingrid , McCready S. , Seeberg Erling , Tsaneva Irina 
Published: January 2004

SummaryEndonuclease III (Nth) enzyme from Escherichia coli is involved in base excision repair of oxidised pyrimidine residues in DNA. The Schizosaccharomyces pombe Nth1 protein is a sequence and functional homologue of E. coli Nth, possessing both DNA glycosylase and apurinic/apyrimidinic (AP) lyase activity. Here, we report the construction and characterization of the S. pombe nth1 mutant. The nth1 mutant exhibited no enhanced sensitivity to oxidising agents, UV or γ‐irradiation, but was hypersensitive to the alkylating agent methyl methanesulphonate (MMS). Analysis of base excision from DNA exposed to [3H]methyl‐N‐nitrosourea showed that the purified Nth1 enzyme did not remove alkylated bases such as 3‐methyladenine and 7‐methylguanine whereas methyl‐formamidopyrimidine was excised efficiently. The repair of AP sites in S. pombe has previously been shown to be independent of Apn1‐like AP endonuclease activity, and the main reason for the MMS sensitivity of nth1 cells appears   to   be   their   lack   of   AP   lyase   activity.   The nth1 mutant   also   exhibited   elevated   frequencies   of spontaneous mitotic intrachromosomal recombination,   which   is   a   phenotype   shared   by   the   MMS‐hypersensitive DNA repair mutants rad2, rhp55 and NER repair mutants rad16, rhp14, rad13 and swi10. Epistasis analyses of nth1 and these DNA repair mutants suggest that several DNA damage repair/tolerance pathways participate in the processing of alkylation and spontaneous DNA damage in S. pombe.
Genetic and biochemical characterization of MbeA, the relaxase involved in plasmid ColE1 conjugative mobilization
Authors: Varsaki Athanasia , Lucas María , Afendra Amalia S. , Drainas Constantin , De La Cruz Fernando 
Published: January 2004

SummaryMbeA is a 60 kDa protein encoded by plasmid ColE1. It plays a key role in conjugative mobilization. MbeA*, a slightly truncated version of MbeA, was purified for in vitro analysis. MbeA* catalysed DNA cleavage and strand‐transfer reactions using oligonucleotides embracing the ColE1 nic site, which was mapped to 5′‐(1469)CTGG/CTTA(1462)‐3′. Thus MbeA is the relaxase for ColE1 conjugal mobilization, in spite of the fact that it lacks a three histidine motif considered the invariant signature of conjugative relaxases. Amino acid sequence comparisons suggest MbeA is nevertheless related to the common relaxase protein family. For instance, MbeA residue Y19 could correspond to the invariant tyrosine in Motif I, whereas H97, E104 and N106 may constitute the equivalent residues to the histidine triad in Motif III. This hypothesis was tested by site‐directed mutagenesis. MbeA amino acid residues Y19, H97, E104 and N106 were changed to alanine. MbeA mutant N106A showed reduced oligonucleotide cleavage and strand‐transfer activities, whereas mutation in the other three residues resulted in proteins without detectable activity, suggesting they  are  directly  implicated  in  catalysis  of  DNA‐cleavage and strand‐transfer reactions. A double substitution of E104 and N106 by histidines, therefore reconstituting the canonical histidine triad, restored relaxase activities to 1% of wild type. Thus, MbeA is a    variant    of    the    common    relaxase    theme    with    a HEN signature motif, which has to be added to the canonical three histidine motif of previously reported relaxases.
An archaeal XPF repair endonuclease dependent on a heterotrimeric PCNA
Authors: Roberts J. A. , Bell S. D. , White M. F. 
Published: January 2004

SummaryArchaea share many similarities with eukarya in their information processing pathways and have proven to be a useful model for studies of DNA replication and transcription, but DNA repair pathways are not well understood in archaea. Nucleotide Excision Repair (NER) deals with many bulky DNA lesions and involves over 30 proteins in eukarya. Archaeal NER has not been characterized biochemically, but homologues of the human repair nucleases XPF and XPG have been identified by homology searches. Crenarchaeal XPF proteins have a simplified domain structure, consisting of the C‐terminal nuclease domain conserved   in   XPF   and   Mus81   but   lacking   the   N‐terminal ‘helicase’ domain that is found in eukaryal and euryarchaeal sequences. Unexpectedly, Sulfolobus XPF is only active in the presence of the sliding clamp PCNA, which is a heterotrimer in this organism. Interactions with two of the three subunits of PCNA are mediated via a C‐terminal interaction motif. The PCNA‐XPF complex acts as a structure‐specific nuclease on a similar range of DNA flap, bubble and junction substrates as the human protein, suggesting a fundamental conservation through billions of years of evolution.
Origins of metal ion selectivity in the DtxR/MntR family of metalloregulators
Authors: Guedon Emmanuel , Helmann John D. 
Published: January 2004

Summary Corynebacterium diphtheriae
DtxR is an iron‐specific repressor of diphtheria toxin expression and iron homeostasis functions. A homologue, MntR, serves as a manganese‐specific repressor of Mn(II) uptake in
Bacillus subtilis
. When expressed in
B. subtilis
, DtxR regulates gene expression in response to either iron or manganese with comparable sensitivity. Replacement of two amino acids in the metal‐sensing site with the corresponding residues from MntR results in a DtxR mutant that is highly selective for Mn(II). However, iron responsiveness can be partially restored in a
fur
mutant in which iron uptake is derepressed and intracellular iron pools elevated. Conversely, if the putative metal‐binding residues in MntR are altered to those in DtxR, the resulting protein responds to both iron and manganese. These results suggest that the composition and geometry of the metal‐binding site plays a major role in defining the metal‐selectivity in this protein family. However, the broadened selectivity of DtxR when expressed in
B. subtilis
, and the effects of a
fur mutation, demonstrate that cellular milieu also influences metal responsiveness.
LuxO controls luxR expression in Vibrio harveyi: evidence for a common regulatory mechanism in Vibrio
Authors: Miyamoto Carol M. , Dunlap Paul V. , Ruby Edward G. , Meighen Edward A. 
Published: January 2004

SummaryQuorum‐sensing control of luminescence in Vibrio harveyi, which involves an indirect autoinducer‐mediated phosphorelay signal transduction system, contrasts with the prototypical quorum‐sensing system of Vibrio fischeri, in which the autoinducer and the transcriptional activator LuxR directly activate lux operon expression. In V. harveyi, a regulator not homologous to V. fischeri LuxR and also designated LuxR (LuxRvh), binds specifically to the lux operon promoter region and activates the expression of luminescence. A direct connection has not been identified previously between V. harveyi LuxRvh and the autoinducer‐mediated phosphorelay system. Here, we demonstrate by mobility shift assays and measurement of luxRvh mRNA levels with luxO+ and luxO– cells that the central response regulator of the V. harveyi phosphorelay system (LuxO) represses the level of LuxRvh. Expression of a luxRvh‐bearing plasmid strongly stimulated luminescence of a luxO– mutant but had no effect on luminescence of wild‐type luxO+ cells, indicating tight regulation of luxRvh by LuxO. Furthermore, luxO null mutants of V. fischeri MJ‐1 and two autoinducer mutants, MJ‐211 (luxI–) and MJ‐215 (luxI–ainS–), emitted more light and exhibited more elevated levels of litR, a newly identified V. harveyi luxRvh homologue, than their luxO+ counterparts. These results suggest that activity of the autoinducer‐mediated phosphorelay system is coupled to LuxRvh/LitR control of luminescence through LuxO in V. harveyi and V. fischeri. The presence of homologues of V. harveyi LuxRvh, LuxO and other phosphorelay system proteins in various Vibrio species and the control of LuxRvh and its homologues by LuxO identified here in V. harveyi and V. fischeri and recently in Vibrio cholerae suggest that the luxO–luxRvh couple is a central feature of this quorum‐sensing system in members of the genus Vibrio.
virK,somAandrcsCare important for systemicSalmonella entericaserovar Typhimurium infection and cationic peptide resistance
Authors: Detweiler Corrella S. , Monack Denise M. , Brodsky Igor E. , Mathew Hanza , Falkow Stanley 
Published: January 2004

SummarySalmonellamust express and deploy a type III secretion system located inSalmonellapathogenicity island 2 (SPI‐2) in order to survive in host phagocytic vacuoles and to cause systemic infection in mouse models of typhoid fever. A genome‐wide approach to screening forSalmonellagenes that are transcriptionally co‐regulatedin vitrowith SPI‐2 genes was used to identify bacterial loci that might function in a mouse model of systemic disease. Strains with mutations in three SPI‐2 co‐expressed genes were constructed and tested for their ability to cause disease in mice. We found thatvirK, a homologue of aShigellavirulence determinant, andrcsC, a sensor kinase, are important at late stages of infection. A secondSalmonellagene that has VirK homology,somA, is also important for systemic infection in mice. We have shown that expression of bothvirKandsomArequires the transcription factor PhoP, whereasrcsCdoes not. Additionally,rcsCexpression does not require the transcription factor OmpR, but expression of one of the known targets of RcsC, theyojN rcsBputative operon, does require OmpR.virK,somAandrcsCare expressed in tissue culture macrophages and conferSalmonellaresistance to the cationic peptide polymyxin B. We conclude thatvirK,somAandrcsCare important for late stages ofSalmonellaenteric fever, and that they probably contribute to the remodelling of the bacterial outer membrane in response to the host environment.
The role of DNA base excision repair in the pathogenesis of Salmonella enterica serovar Typhimurium
Authors: Suvarnapunya Akamol E. , Lagassé H. A. Daniel , Stein Murry A. 
Published: January 2004

SummaryThe intracellular pathogen, Salmonella enterica serovar Typhimurium, is able to proliferate in phagocytes, although reactive oxygen and nitrogen intermediates are lethal to most phagocytosed bacteria. To determine whether repair of oxidatively damaged DNA is involved in S. typhimurium intramacrophage proliferation, null mutants of the DNA base excision repair (BER) system were generated. These mutants were deficient in discrete enzymes (Δnth, Δnei, Δxth, Δnfo) or in the defined glycosylase (Δnth/nei) and endonuclease (Δxth/nfo) steps. In this study, S. typhimurium BER mutants are characterized for the first time. In vitro characterization of the Salmonella BER mutants revealed phenotypes that are mostly consistent with characterized Escherichia coli BER mutants. These strains were used to evaluate the role of BER in the context of Salmonella virulence. S. typhimurium Δxth and Δxth/nfo were significantly impaired for survival in both cultured and primary macrophages activated with interferon (IFN)‐γ. Survival of Δxth and Δxth/nfo was improved nearly to wild‐type levels in activated primary macrophages lacking both phagocyte oxidase and inducible nitric oxide synthase. In the murine typhoid fever model, Δnth/nei was fivefold attenuated and Δxth/nfo was 12‐fold attenuated compared with wild type. These data indicate that DNA oxidation is a mechanism that macrophages use to damage intracellular Salmonella, and suggest that BER‐mediated repair of this damage may be important in the establishment of Salmonella infection. We speculate that adaptation to a pathogenic lifestyle may influence the acquisition and retention of redundant BER enzymes.
DNA supercoiling contributes to disconnect σS accumulation from σS‐dependent transcription in Escherichia coli
Authors: Bordes Patricia , Conter Annie , Morales Violette , Bouvier Jean , Kolb Annie , Gutierrez Claude 
Published: January 2004

SummaryThe σS subunit of RNA polymerase is a key regulator of Escherichia coli transcription in stress conditions. σS accumulates in cells subjected to stresses such as an osmotic upshift or the entry into stationary phase. We show here that, at elevated osmolarity, σS accumulates long before the beginning of the σS‐dependent induction of osmEp, one of its target promoters. A combination of in vivo and in vitro evidence indicates that a high level of DNA negative supercoiling inhibits transcription by EσS. The variations in superhelical densities occurring as a function of growth conditions can modulate transcription of a subset of σS targets and thereby contribute to the temporal disconnection between the accumulation of σS and σS‐driven transcription. We propose that, in stress conditions leading to the accumulation of σS without lowering the growth rate, the level of DNA supercoiling acts as a checkpoint that delays the shift from the major (Eσ70) to the general stress (EσS) transcriptional machinery, retarding the induction of a subset of the σS regulon until the conditions become unfavourable enough to cause entry into stationary phase.
Multiplication of a restriction–modification gene complex
Authors: Sadykov Marat , Asami Yasuo , Niki Hironori , Handa Naofumi , Itaya Mitsuhiro , Tanokura Masaru , Kobayashi Ichizo 
Published: January 2004

SummaryPrevious works have suggested that some gene complexes encoding a restriction (R) enzyme and a cognate modification (M) enzyme may behave as selfish mobile genetic elements. RM gene complexes, which destroy ‘non‐self’ elements marked by the absence of proper methylation, are often associated with mobile genetic elements and are involved in various genome rearrangements. Here, we found amplification of a restriction–modification gene complex. BamHI gene complex inserted into the Bacillus chromosome showed resistance to replacement by a homologous stretch of DNA. Some cells became transformed with the donor without losing BamHI. In most of these transformants, multiple copies of BamHI and the donor allele were arranged as tandem repeats. When a clone carrying one copy of each allele was propagated, extensive amplification of BamHI and the donor unit was observed in a manner dependent on restriction enzyme gene. This suggests that restriction cutting of the genome participates in the am­plification. Visualization by fluorescent in situ hybridization revealed that the amplification occurred in single cells in a burst‐like fashion that is reminiscent of induction of provirus replication. The multiplication ability in a bacterium with natural capacity for DNA release, uptake and transformation will be discussed in relation to spreading of RM gene ­complexes.