bluuebunny/crossref_metadata_2025 huggingface dataset:
Peptidyl-Prolyl
cis/trans
Isomerase-Independent Functional NifH Mutant of
Azotobacter vinelandii
Authors: Gavini Nara , Tungtur Sudheer , Pulakat Lakshmi
Published: August 2006

Peptidyl-prolyl
cis/trans
isomerases (PPIases) play a pivotal role in catalyzing the correct folding of many prokaryotic and eukaryotic proteins that are implicated in a variety of biological functions, ranging from cell cycle regulation to bacterial infection. The
nif
accessory protein NifM, which is essential for the biogenesis of a functional NifH component of nitrogenase, is a PPIase. To understand the nature of the molecular signature that defines the NifM dependence of NifH, we screened a library of
nifH
mutants in the nitrogen-fixing bacterium
Azotobacter vinelandii
for mutants that acquired NifM independence. Here, we report that NifH can acquire NifM independence when the conserved Pro258 located in the C-terminal region of NifH, which wraps around the other subunit in the NifH dimer, is replaced by serine.
A Novel Branching Enzyme of the GH-57 Family in the Hyperthermophilic Archaeon
Thermococcus kodakaraensis
KOD1
Authors: Murakami Taira , Kanai Tamotsu , Takata Hiroki , Kuriki Takashi , Imanaka Tadayuki
Published: August 2006

Branching enzyme (BE) catalyzes formation of the branch points in glycogen and amylopectin by cleavage of the α-1,4 linkage and its subsequent transfer to the α-1,6 position. We have identified a novel BE encoded by an uncharacterized open reading frame (TK1436) of the hyperthermophilic archaeon
Thermococcus kodakaraensis
KOD1. TK1436 encodes a conserved protein showing similarity to members of glycoside hydrolase family 57 (GH-57 family). At the C terminus of the TK1436 protein, two copies of a helix-hairpin-helix (HhH) motif were found. TK1436 orthologs are distributed in archaea of the order
Thermococcales
, cyanobacteria, some actinobacteria, and a few other bacterial species. When recombinant TK1436 protein was incubated with amylose used as the substrate, a product peak was detected by high-performance anion-exchange chromatography, eluting more slowly than the substrate. Isoamylase treatment of the reaction mixture significantly increased the level of short-chain α-glucans, indicating that the reaction product contained many α-1,6 branching points. The TK1436 protein showed an optimal pH of 7.0, an optimal temperature of 70°C, and thermostability up to 90°C, as determined by the iodine-staining assay. These properties were the same when a protein devoid of HhH motifs (the TK1436ΔH protein) was used. The average molecular weight of branched glucan after reaction with the TK1436ΔH protein was over 100 times larger than that of the starting substrate. These results clearly indicate that TK1436 encodes a structurally novel BE belonging to the GH-57 family. Identification of an overlooked BE species provides new insights into glycogen biosynthesis in microorganisms.
Paired-End Sequence Mapping Detects Extensive Genomic Rearrangement and Translocation during Divergence of
Francisella tularensis
subsp.
tularensis
and
Francisella tularensis
subsp.
holarctica
Populations
Authors: Dempsey Michael P. , Nietfeldt Joseph , Ravel Jacques , Hinrichs Steven , Crawford Robert , Benson Andrew K.
Published: August 2006

Comparative genome hybridization of the
Francisella tularensis
subsp.
tularensis
and
F. tularensis
subsp.
holarctica
populations have shown that genome content is highly conserved, with relatively few genes in the
F. tularensis
subsp.
tularensis
genome being absent in other
F. tularensis
subspecies. To determine if organization of the genome differs between global populations of
F. tularensis
subsp.
tularensis
and
F. tularensis
subsp.
holarctica
, we have used paired-end sequence mapping (PESM) to identify regions of the genome where synteny is broken. The PESM approach compares the physical distances between paired-end sequencing reads of a library of a wild-type reference
F. tularensis
subsp.
holarctica
strain to the predicted lengths between the reads based on map coordinates of two different
F. tularensis
genome sequences. A total of 17 different continuous regions were identified in the
F. tularensis
subsp.
holarctica
genome (CR
holar
c
tica
) which are noncontiguous in the
F. tularensis
subsp.
tularensis
genome. Six of the 17 different CR
holarctica
are positioned as adjacent pairs in the
F. tularensis
subsp.
tularensis
genome sequence but are translocated in
F. tularensis
subsp.
holarctica
, implying that their arrangements are ancestral in
F. tularensis
subsp.
tularensis
and derived in
F. tularensis
subsp.
holarctica
. PCR analysis of the CR
holarctica
in 88 additional
F. tularensis
subsp.
tularensis
and
F. tularensis
subsp.
holarctica
isolates showed that the arrangements of the CR
holarctica
are highly conserved, particularly in
F. tularensis
subsp.
holarctica
, consistent with the hypothesis that global populations of
F. tularensis
subsp.
holarctica
have recently experienced a periodic selection event or they have emerged from a recent clonal expansion. Two unique
F. tularensis
subsp.
tularensis
-like strains were also observed which likely are derived from evolutionary intermediates and may represent a new taxonomic unit.
The Global Regulator Spx Functions in the Control of Organosulfur Metabolism inBacillus subtilis
Authors: Choi Soon-Yong , Reyes Dindo , Leelakriangsak Montira , Zuber Peter
Published: August 2006

Spx is a global transcriptional regulator of the oxidative stress response inBacillus subtilis. Its target is RNA polymerase, where it contacts the α subunit C-terminal domain. Recently, evidence was presented that Spx participates in sulfate-dependent control of organosulfur utilization operons, including theytmI,yxeI,ssu, andyrrToperons. TheyrrToperon includes the genes that function in cysteine synthesis fromS-adenosylmethionine through intermediatesS-adenosylhomocysteine, ribosylhomocysteine, homocysteine, and cystathionine. These operons are also negatively controlled by CymR, the repressor of cysteine biosynthesis operons. All of the operons are repressed in media containing cysteine or sulfate but are derepressed in medium containing the alternative sulfur source, methionine. Spx was found to negatively control the expression of these operons in sulfate medium, in part, by stimulating the expression of thecymRgene. In addition, microarray analysis, monitoring ofyrrT-lacZfusion expression, and in vitro transcription studies indicate that Spx directly activatesyrrToperon expression during growth in medium containing methionine as sole sulfur source. These experiments have uncovered additional roles for Spx in the control of gene expression during unperturbed, steady-state growth.
The Nitric Oxide-Responsive Regulator NsrR Controls ResDE-Dependent Gene Expression
Authors: Nakano Michiko M. , Geng Hao , Nakano Shunji , Kobayashi Kazuo
Published: August 2006

The ResD-ResE signal transduction system is essential for aerobic and anaerobic respiration inBacillus subtilis. ResDE-dependent gene expression is induced by oxygen limitation, but full induction under anaerobic conditions requires nitrite or nitric oxide (NO). Here we report that NsrR (formerly YhdE) is responsible for the NO-dependent up-regulation of the ResDE regulon. The null mutation ofnsrRled to aerobic derepression ofhmp(flavohemoglobin gene) partly in a ResDE-independent manner. In addition to its negative role in aerobichmpexpression, NsrR plays an important role under anaerobic conditions for regulation of ResDE-controlled genes, includinghmp. ResDE-dependent gene expression was increased by thensrRmutation in the absence of NO, but the expression was decreased by the mutation when NO was present. Consequently,B. subtiliscells lacking NsrR no longer sense and respond to NO (and nitrite) to up-regulate the ResDE regulon. Exposure to NO did not significantly change the cellular concentration of NsrR, suggesting that NO likely modulates the activity of NsrR. NsrR is similar to the recently described nitrite- or NO-sensitive transcription repressors present in various bacteria. NsrR likely has an Fe-S cluster, and interaction of NO with the Fe-S center is proposed to modulate NsrR activity.
TheBordetellaBfe System: Growth and Transcriptional Response to Siderophores, Catechols, and Neuroendocrine Catecholamines
Authors: Anderson Mark T. , Armstrong Sandra K.
Published: August 2006

Ferric enterobactin utilization byBordetella bronchisepticaandBordetella pertussisrequires the BfeA outer membrane receptor. Under iron-depleted growth conditions, transcription ofbfeAis activated by the BfeR regulator by a mechanism requiring the siderophore enterobactin. In this study, enterobactin-induciblebfeAtranscription was shown to be TonB independent. To determine whether other siderophores or nonsiderophore catechols could be utilized by the Bfe system, various compounds were tested for the abilities to promote the growth of iron-starvedB. bronchisepticaand inducebfeAtranscription. The BfeA receptor transported ferric salmochelin, corynebactin, and the synthetic siderophores TRENCAM and MECAM. Salmochelin and MECAM inducedbfeAtranscription in iron-starvedBordetellacells, but induction by corynebactin and TRENCAM was minimal. The neuroendocrine catecholamines epinephrine, norepinephrine, and dopamine exhibited a remarkable capacity to induce transcription ofbfeA. Norepinephrine treatment ofB. bronchisepticaresulted in BfeR-dependentbfeAtranscription, elevated BfeA receptor production, and growth stimulation. Pyrocatechol, carbidopa, and isoproterenol were similarly strong inducers ofbfeAtranscription, whereas tyramine and 3,4-dihydroxymandelic acid demonstrated low inducing activity. The results indicate that the inducer structure requires a catechol group for function and that the ability to inducebfeAtranscription does not necessarily correlate with the ability to stimulate bacterial growth. The expanded range of catechol siderophores transported by the BfeA receptor demonstrates the potential versatility of theBordetellaBfe iron retrieval system. The finding that catecholamine neurotransmitters activatebfeAtranscription and promote growth suggests thatBordetellacells can perceive and may benefit from neuroendocrine catecholamines on the respiratory epithelium.
Relationship among Several Key Cell Cycle Events in the Developmental Cyanobacterium
Anabaena
sp. Strain PCC 7120
Authors: Sakr Samer , Thyssen Melilotus , Denis Michel , Zhang Cheng-Cai
Published: August 2006

When grown in the absence of a source of combined nitrogen, the filamentous cyanobacterium
Anabaena
sp. strain PCC 7120 develops, within 24 h, a differentiated cell type called a heterocyst that is specifically involved in the fixation of N
2
. Cell division is required for heterocyst development, suggesting that the cell cycle could control this developmental process. In this study, we investigated several key events of the cell cycle, such as cell growth, DNA synthesis, and cell division, and explored their relationships to heterocyst development. The results of analyses by flow cytometry indicated that the DNA content increased as the cell size expanded during cell growth. The DNA content of heterocysts corresponded to the subpopulation of vegetative cells that had a big cell size, presumably those at the late stages of cell growth. Consistent with these results, most proheterocysts exhibited two nucleoids, which were resolved into a single nucleoid in most mature heterocysts. The ring structure of FtsZ, a protein required for the initiation of bacterial cell division, was present predominantly in big cells and rarely in small cells. When cell division was inhibited and consequently cells became elongated, little change in DNA content was found by measurement using flow cytometry, suggesting that inhibition of cell division may block further synthesis of DNA. The overexpression of
minC
, which encodes an inhibitor of FtsZ polymerization, led to the inhibition of cell division, but cells expanded in spherical form to become giant cells; structures with several cells attached together in the form of a cloverleaf could be seen frequently. These results may indicate that the relative amounts of FtsZ and MinC affect not only cell division but also the placement of the cell division planes and the cell morphology. MinC overexpression blocked heterocyst differentiation, consistent with the requirement of cell division in the control of heterocyst development.
Feedback-Resistant Mutations in
Bacillus subtilis
Glutamine Synthetase Are Clustered in the Active Site
Authors: Fisher Susan H. , Wray Lewis V.
Published: August 2006

The feedback-inhibited form of
Bacillus subtilis
glutamine synthetase regulates the activity of the TnrA transcription factor through a protein-protein interaction that prevents TnrA from binding to DNA. Five mutants containing feedback-resistant glutamine synthetases (E65G, S66P, M68I, H195Y, and P318S) were isolated by screening for colonies capable of cross-feeding Gln
−
cells. In vitro enzymatic assays revealed that the mutant enzymes had increased resistance to inhibition by glutamine, AMP, and methionine sulfoximine. The mutant proteins had a variety of enzymatic alterations that included changes in the levels of enzymatic activity and in substrate

K
m

values. Constitutive expression of TnrA- and GlnR-regulated genes was seen in all five mutants. In gel mobility shift assays, the E65G and S66P enzymes were unable to inhibit TnrA DNA binding, while the other three mutant proteins (M68I, H195Y, and P318S) showed partial inhibition of TnrA DNA binding. A homology model of
B. subtilis
glutamine synthetase revealed that the five mutated amino acid residues are located in the enzyme active site. These observations are consistent with the hypothesis that glutamine and AMP bind at the active site to bring about feedback inhibition of glutamine synthetase.
Requirement for
Vibrio cholerae
Integration Host Factor in Conjugative DNA Transfer
Authors: McLeod Sarah M. , Burrus Vincent , Waldor Matthew K.
Published: August 2006

The requirement for host factors in the transmission of integrative and conjugative elements (ICEs) has not been extensively explored. Here we tested whether integration host factor (IHF) or Fis, two host-encoded nucleoid proteins, are required for transfer of SXT, a
Vibrio cholerae
-derived ICE that can be transmitted to many gram-negative species. Fis did not influence the transfer of SXT to or from
V. cholerae
. In contrast, IHF proved to be required for
V. cholerae
to act as an SXT donor. In the absence of IHF,
V. cholerae
displayed a modest defect for serving as an SXT recipient. Surprisingly, SXT integration into or excision from the
V. cholerae
chromosome, which requires an SXT-encoded integrase related to λ integrase, did not require IHF. Therefore, the defect in SXT transmission in the
V. cholerae
IHF mutant is probably not related to IHF's ability to promote DNA recombination. The
V. cholerae
IHF mutant was also highly impaired as a donor of RP4, a broad-host-range conjugative plasmid. Thus, the
V. cholerae
IHF mutant appears to have a general defect in conjugation.
Escherichia coli
IHF mutants were not impaired as donors or recipients of SXT or RP4, indicating that IHF is a
V. cholerae
-specific conjugation factor.
Mutator and Antimutator Effects of the Bacteriophage P1hotGene Product
Authors: Chikova Anna K. , Schaaper Roel M.
Published: August 2006

The Hot (homolog of theta) protein of bacteriophage P1 can substitute for theEscherichia coliDNA polymerase III θ subunit, as evidenced by its stabilizing effect on certaindnaQmutants that carry an unstable polymerase III ε proofreading subunit (antimutator effect). Here, we show that Hot can also cause an increase in the mutability of variousE. colistrains (mutator effect). Thehotmutator effect differs from the one caused by the lack of θ. Experiments using chimeric θ/Hot proteins containing various domains of Hot and θ along with a series of point mutants show that both N- and C-terminal parts of each protein are important for stabilizing the ε subunit. In contrast, the N-terminal part of Hot appears uniquely responsible for its mutator activity.
Transport of Multidrug Resistance Substrates by theStreptococcus agalactiaeHemolysin Transporter
Authors: Gottschalk Birgit , Bröker Gerd , Kuhn Melanie , Aymanns Simone , Gleich-Theurer Ute , Spellerberg Barbara
Published: August 2006

Streptococcus agalactiae(group B streptococcus [GBS]) causes neonatal sepsis, pneumonia, and meningitis, as well as infections of the bovine udder. TheS. agalactiaehemolysin is regarded as an important virulence factor, and hemolysin expression is dependent on thecylgene cluster.cylAandcylBencode the ATP binding and transmembrane domains of a typical ATP binding cassette (ABC) transporter. The deduced proteins contain the signature sequence of a multidrug resistance (MDR) transporter, and mutation of the genes results in a nonhemolytic and nonpigmented phenotype. To further elucidate the function of the putative transporter, nonpolar deletion mutants ofcylAwere constructed. These mutants are nonhemolytic and can be complemented by the transporter genes. Wild-type strain and nonhemolyticcylAandcylKdeletion mutants were exposed to known substrates of MDR transporters. Mutation ofcylAsignificantly impaired growth in the presence of daunorubicin, doxorubicin, and rhodamine 6G and resulted in a decreased export of doxorubicin from the cells. The mutation ofcylK, a gene of unknown function located downstream fromcylA, caused a loss of hemolysis but had no effect on the transport of MDR substrates. Furthermore, the hemolytic activity of the wild-type strain was inhibited by reserpine in a dose-dependent manner. We conclude that CylAB closely resembles an ABC-type MDR transporter and propose that the GBS hemolysin molecule represents a natural substrate of the transporter.
Comparison of the Genome Sequence of the Poultry PathogenBordetella aviumwith Those ofB. bronchiseptica,B. pertussis, andB. parapertussisReveals Extensive Diversity in Surface Structures Associated with Host Interaction
Authors: Sebaihia Mohammed , Preston Andrew , Maskell Duncan J. , Kuzmiak Holly , Connell Terry D. , King Natalie D. , Orndorff Paul E. , Miyamoto David M. , Thomson Nicholas R. , Harris David , Goble Arlette , Lord Angela , Murphy Lee , Quail Michael A. , Rutter Simon , Squares Robert , Squares Steven , Woodward John , Parkhill Julian , Temple Louise M.
Published: August 2006

Bordetella aviumis a pathogen of poultry and is phylogenetically distinct fromBordetella bronchiseptica,Bordetella pertussis, andBordetella parapertussis, which are other species in theBordetellagenus that infect mammals. In order to understand the evolutionary relatedness ofBordetellaspecies and further the understanding of pathogenesis, we obtained the complete genome sequence ofB. aviumstrain 197N, a pathogenic strain that has been extensively studied. With 3,732,255 base pairs of DNA and 3,417 predicted coding sequences, it has the smallest genome and gene complement of the sequenced bordetellae. In this study, the presence or absence of previously reported virulence factors fromB. aviumwas confirmed, and the genetic bases for growth characteristics were elucidated. Over 1,100 genes present inB. aviumbut not inB. bronchisepticawere identified, and most were predicted to encode surface or secreted proteins that are likely to define an organism adapted to the avian rather than the mammalian respiratory tracts. These include genes coding for the synthesis of a polysaccharide capsule, hemagglutinins, a type I secretion system adjacent to two very large genes for secreted proteins, and unique genes for both lipopolysaccharide and fimbrial biogenesis. Three apparently complete prophages are also present. The BvgAS virulence regulatory system appears to have polymorphisms at a poly(C) tract that is involved in phase variation in other bordetellae. A number of putative iron-regulated outer membrane proteins were predicted from the sequence, and this regulation was confirmed experimentally for five of these.
Class 1 Integrons Potentially Predating the Association with Tn402-Like Transposition Genes Are Present in a Sediment Microbial Community
Authors: Stokes H. W. , Nesbø Camilla L. , Holley Marita , Bahl Martin I. , Gillings Michael R. , Boucher Yan
Published: August 2006

Integrons are genetic elements that contribute to lateral gene transfer in bacteria as a consequence of possessing a site-specific recombination system. This system facilitates the spread of genes when they are part of mobile cassettes. Most integrons are contained within chromosomes and are confined to specific bacterial lineages. However, this is not the case for class 1 integrons, which were the first to be identified and are one of the single biggest contributors to multidrug-resistant nosocomial infections, carrying resistance to many antibiotics in diverse pathogens on a global scale. The rapid spread of class 1 integrons in the last 60 years is partly a result of their association with a specific suite of transposition functions, which has facilitated their recruitment by plasmids and other transposons. The widespread use of antibiotics has acted as a positive selection pressure for bacteria, especially pathogens, which harbor class 1 integrons and their associated antibiotic resistance genes. Here, we have isolated bacteria from soil and sediment in the absence of antibiotic selection. Class 1 integrons were recovered from four different bacterial species not known to be human pathogens or commensals. All four integrons lacked the transposition genes previously considered to be a characteristic of this class. At least two of these integrons were located on a chromosome, and none of them possessed antibiotic resistance genes. We conclude that novel class 1 integrons are present in a sediment environment in various bacteria of the β-proteobacterial class. These data suggest that the dispersal of this class may have begun before the “antibiotic era.”
Salicylic Acid Activates Sigma Factor B by
rsbU
-Dependent and -Independent Mechanisms
Authors: Palma Marco , Bayer Arnold , Kupferwasser Leon I. , Joska Tammy , Yeaman Michael R. , Cheung Ambrose
Published: August 2006

Salicylic acid (SAL) may impact
Staphylococcus aureus
virulence by activating the
sigB
operon (
rsbU-V-W-sigB
), thus leading to reductions in alpha-toxin production and decreased fibronectin binding (L. I. Kupferwasser et al., J. Clin. Investig. 112:222-233, 2003). As these prior studies were performed in strain RN6390 (an
rsbU
mutant) and its
rsbU
-repaired variant, SH1000, the current investigation was designed to determine if the SAL effect occurs via
rsbU-
and/or
rsbV-
dependent pathways in an
rsbU-
intact
S. aureus
strain (FDA486). We thus quantified the transcription from two
sigB
-dependent promoters (
asp
23 and
sarA
P3) in FDA486 in response to SAL exposure in vitro, using isogenic single-knockout constructs of
rsbU
,
rsbV
, or
rsbW
and a green fluorescent protein reporter system. SAL induced
sarA
P3 and
asp23
promoter activities in a dose-dependent manner in the parental strain. In contrast,
sigB
activation by SAL was progressively more mitigated in the
rsbU
and
rsbV
mutants. As predicted, SAL caused significant reductions in both alpha-toxin production and fibrinogen and fibronectin binding in the parental strain. The extent of these reductions, compared with the parent, was reduced in the
rsb
mutants (
rsbV
>
rsbU
), especially at low SAL concentrations. Since generation of the free SigB protein usually requires a sequential
rsbU-V-W-sigB
activation cascade, the present phenotypic and genotypic data suggest key roles for both
rsbU
and
rsbV
in SAL-mediated activation of
sigB
in strains with a fully intact
sigB
operon.
International survey of seat belt use exemptions
Authors: Weiss H , Sirin H , Levine J A , Sauber E
Published: August 2006

Background:Substantial evidence of seatbelt efficacy has been shown by several studies, and it is widely recommended that motor vehicle occupants use properly fitted seat belts. However, some (but a heretofore unknown number of) countries with national seat belt laws permit various exemptions which may lower use rates. The aim of this study was to survey the variety of exemptions to national seat belt laws.

Methods:This investigation relied on identifying respondents from national traffic safety agencies, other governmental and non-governmental organizations, Internet searches, personal contacts, and other sources. Questionnaires were deployed through a web based survey supplemented by email and postal versions.

Results:Responses were received from 30 countries of which 28 (93.7%) had a national seat belt law. About two thirds (63.7%) of the 28 national laws applied to both front and back seat passengers. The leading exemption types included vehicles made before a certain year (n = 13), antique vehicles (n = 12), military vehicles (n = 11), buses (n = 9), and emergency vehicles (n = 8). Most responding countries reported one or more specific categories of individuals as exempt including those with medical exemptions (n = 20), taxi drivers (n = 11), police (n = 9), emergency medical personnel (n = 8), physically disabled people (n = 6), and pregnant women (n = 6). Out of 26 responses to the question regarding current level of enforcement, 42.3% felt enforcement was “very good or good” and 57.7% characterized it as “fair or poor”.

Conclusions:This study represents one of the largest international traffic law surveys reported. Most national seatbelt laws offer perilous exemptions to a broad array of vehicle types and road user groups. These findings, coupled with concern over the level of enforcement in the majority of countries surveyed, suggest that international road safety efforts have a long way to go to improve coverage and enforcement of national seat belt laws.
Economic disparity in bicycle helmet use by children six years after the introduction of legislation
Authors: Macpherson A K , Macarthur C , To T M , Chipman M L , Wright J G , Parkin P C
Published: August 2006

Background:Studies evaluating the effectiveness of bicycle helmet legislation often focus on short term outcomes. The long term effect of helmet legislation on bicycle helmet use is unknown.

Objective:To examine bicycle helmet use by children six years after the introduction of the law, and the influence of area level family income on helmet use.

Methods:The East York (Toronto) health district (population 107 822) was divided into income areas (designated as low, mid, and high) based on census tract data from Statistics Canada. Child cyclists were observed at 111 preselected sites (schools, parks, residential streets, and major intersections) from April to October in the years 1995–1997, 1999, and 2001. The frequency of helmet use was determined by year, income area, location, and sex. Stratified analysis was used to quantify the relation between income area and helmet use, after controlling for sex and bicycling location.

Results:Bicycle helmet use in the study population increased from a pre-legislation level of 45% in 1995 to 68% in 1997, then decreased to 46% by 2001. Helmet use increased in all three income areas from 1995 to 1997, and remained above pre-legislation rates in high income areas (85% in 2001). In 2001, six years post-legislation, the proportion of helmeted cyclists in mid and low income areas had returned to pre-legislation levels (50% and 33%, respectively). After adjusting for sex and location, children riding in high income areas were significantly more likely to ride helmeted than children in low income areas across all years (relative risk = 3.4 (95% confidence interval, 2.7 to 4.3)).

Conclusion:Over the long term, the effectiveness of bicycle helmet legislation varies by income area. Alternative, concurrent, or ongoing strategies may be necessary to sustain bicycle helmet use among children in mid and low income areas following legislation.
The properties of the International Classification of the External Cause of Injury when used as an instrument for injury prevention research
Authors: Scott D , Harrison J , Purdie D , Bain C , Najman J , Nixon J , Spinks A B , McClure R J
Published: August 2006

Objective:To demonstrate properties of the International Classification of the External Cause of Injury (ICECI) as a tool for use in injury prevention research.

Methods:The Childhood Injury Prevention Study (CHIPS) is a prospective longitudinal follow up study of a cohort of 871 children 5–12 years of age, with a nested case crossover component. The ICECI is the latest tool in the International Classification of Diseases (ICD) family and has been designed to improve the precision of coding injury events. The details of all injury events recorded in the study, as well as all measured injury related exposures, were coded using the ICECI. This paper reports a substudy on the utility and practicability of using the ICECI in the CHIPS to record exposures. Interrater reliability was quantified for a sample of injured participants using the Kappa statistic to measure concordance between codes independently coded by two research staff.

Results:There were 767 diaries collected at baseline and event details from 563 injuries and exposure details from injury crossover periods. There were no event, location, or activity details which could not be coded using the ICECI. Kappa statistics for concordance between raters within each of the dimensions ranged from 0.31 to 0.93 for the injury events and 0.94 and 0.97 for activity and location in the control periods.

Discussion:This study represents the first detailed account of the properties of the ICECI revealed by its use in a primary analytic epidemiological study of injury prevention. The results of this study provide considerable support for the ICECI and its further use.
Clean cut cigarettes: fictitious launch foreshadows actual tobacco industry strategy
Authors: Henningfield Jack E , Connolly Gregory N , Kemper Katherine E , Gitchell Joseph G , Pinney John M
Published: August 2006

A fictitious cigarette brand, “launched” at the 11th World Conference on Tobacco or Health, appears to have been adopted by Philip Morris
Parental smoking and children’s respiratory health: independent effects of prenatal and postnatal exposure
Authors: Pattenden Sam , Antova Temenuga , Neuberger Manfred , Nikiforov Bojidar , De Sario Manuela , Grize Leticia , Heinrich Joachim , Hruba Frantiska , Janssen Nicole , Luttmann-Gibson Heike , Privalova Larissa , Rudnai Peter , Splichalova Anna , Zlotkowska Renata , Fletcher Tony
Published: August 2006

Objectives:Adverse effects have been reported of prenatal and/or postnatal passive exposure to smoking on children’s health. Uncertainties remain about the relative importance of smoking at different periods in the child’s life. We investigate this in a pooled analysis, on 53 879 children from 12 cross-sectional studies—components of the PATY study (Pollution And The Young).

Methods:Effects were estimated, within each study, of three exposures: mother smoked during pregnancy, parental smoking in the first two years, current parental smoking. Outcomes were: wheeze, asthma, “woken by wheeze”, bronchitis, nocturnal cough, morning cough, “sensitivity to inhaled allergens” and hay fever. Logistic regressions were used, controlling for individual risk factors and study area. Heterogeneity between study-specific results, and mean effects (allowing for heterogeneity) were estimated using meta-analytical tools.

Results:There was strong evidence linking parental smoking to wheeze, asthma, bronchitis and nocturnal cough, with mean odds ratios all around 1.15, with independent effects of prenatal and postnatal exposures for most associations.

Conclusions:Adverse effects of both pre- and postnatal parental smoking on children’s respiratory health were confirmed. Asthma was most strongly associated with maternal smoking during pregnancy, but postnatal exposure showed independent associations with a range of other respiratory symptoms. All tobacco smoke exposure has serious consequences for children’s respiratory health and needs to be reduced urgently.
Tobacco interests or the public interest: 20 years of industry strategies to undermine airline smoking restrictions: Table 1
Authors: Lopipero Peggy , Bero Lisa A
Published: August 2006

Objectives:To understand the evolution of 20 years of tobacco industry strategies to undermine federal restrictions of smoking on aircraft in the United States.

Design:We searched and analysed internal tobacco industry records, public documents, and other related research.

Results:The industry viewed these restrictions as a serious threat to the social acceptability of smoking. Its initial efforts included covert letter-writing campaigns and lobbying of the airline industry, but with the emergence of proposals to ban smoking, the tobacco companies engaged in ever increasing efforts to forestall further restrictions. Tactics to dominate the public record became especially rigorous. The industry launched an aggressive public relations campaign that began with the promotion of industry sponsored petition drives and public opinion surveys. Results from polling research that produced findings contrary to the industry’s position were suppressed. In order to demonstrate smoker outrage against a ban, later efforts included the sponsorship of smokers’ rights and other front groups. Congressional allies and industry consultants sought to discredit the science underlying proposals to ban smoking and individual tobacco companies conducted their own cabin air quality research. Faced with the potential of a ban on all domestic flights, the industry sought to intimidate an air carrier and a prominent policymaker. Despite the intensification of tactics over time, including mobilisation of an army of lobbyists and Congressional allies, the tobacco industry was ultimately defeated.

Conclusions:Our longitudinal analysis provides insights into how and when the industry changed its plans and provides public health advocates with potential counterstrategies.
Why not seek clever regulation? A reply to Liberman
Authors: Borland Ron
Published: August 2006

Ron Borland replies to Jonathan Liberman
Decondensation ofXenopussperm chromatin usingSaccharomyces cerevisiaewhole-cell extractsThis paper is one of a selection of papers published in this Special Issue, entitled The Nucleus: A Cell Within A Cell.
Authors: Harkness Troy A.A.
Published: August 2006

Biochemical studies using highly condensed Xenopus sperm chromatin and protein extracts prepared from multiple systems have lead to the identification of conserved proteins involved in chromosome decondensation. However, mutations to these proteins are unavailable as the systems used are not amenable to genetic studies. We took a genetic approach to isolating chromosome decondensation mutants by incubating Xenopus sperm chromatin with whole-cell extracts prepared from the Hartwell library of random temperature sensitive (ts) yeast cells. We show that decondensation of Xenopus sperm chromatin using wild type yeast extracts was rapid, ATP- and extract-dependent, and resistant to heat, N-ethylmaleimide, protease K, RNase A, and micrococcal nuclease. From 100 mutant extracts screened, we obtained one strain, referred to as rmc4, that was chromosome decondensation defective. The mutant was slow growing and exhibited germination defects. Low concentrations of rmc4 extract would eventually decondense sperm heads, and fractionation of the mutant extract produced a decondensation competent fraction, suggesting the presence of an overactive inhibitor in rmc4 cells. We performed a multicopy suppressor screen that identified PDE2, a gene encoding a protein that inhibits protein kinase A (PKA) activity. As PKA was previously shown in human cells to maintain condensed chromatin, our results suggest that PKA activity is elevated in rmc4 cells, causing a decondensation defect. Thus, our experiments reveal that yeast encodes an evolutionarily conserved chromosome decondensation activity that can be genetically manipulated.
Somatic DNA recombination in a mouse genomic region, BC-1, in brain and non-brain tissueThis paper is one of a selection of papers published in this Special Issue, entitled The Nucleus: A Cell Within A Cell.
Authors: Maeda Toyoki , Mizuno Ryuzo , Sugano Masahiro , Satoh Shinji , Oyama Junichi , Sakoda Saburo , Suzuki Tomokazu , Makino Naoki
Published: August 2006

The genomic region BC-1 (GenBank acc. No. AB075899 ) on mouse chromosome 16 has been reported as a genomic region undergoing somatic DNA recombination producing circular DNA and genomic deletion in brain during late embryogenesis. The present study shows that the BC-1 circular DNA production had already started on the 13th day of embryonic age, earlier than the previous observation that the circular DNA production started on the 15th through 17th embryonic day. The BC-1 deletion was also observed in the spleen and ocular lens. In situ hybridization analysis indicated that a human-homologous region in the BC-1 sequence was expressed in the lens at a perinatal period. These data suggest that the somatic DNA recombination in the BC-1 region is not restricted to brain tissue, and that the BC-1 DNA recombination relates to lens development.
Somatic DNA recombination in the brainThis paper is one of a selection of papers published in this Special Issue, entitled The Nucleus: A Cell Within A Cell.
Authors: Maeda Toyoki , Sakoda Saburo , Suzuki Tomokazu , Makino Naoki
Published: August 2006

Possible somatic DNA recombination in the brain has been investigated by attempting to capture direct or indirect evidence of it. Until recently, the biological significance of the DNA event, the genes is involved in the recombination, or even whether the event actually occurs in the brain has remained unclear. The DNA-rearranged locus-oriented approach and the recombination activity-oriented approach have mutually contributed to the elucidation of the biological features of extra-immune system somatic DNA recombination. There have been only 2 loci proposed for the candidate, one is a repetitive sequence and the other DNA recombination is nonrepetitive locus. This review states conventional concepts and discussions chronologically and finally to the newest aspects of DNA rearrangement in the brain.
Nuclear import of spliceosomal snRNPsThis paper is one of a selection of papers published in this Special Issue, entitled The Nucleus: A Cell Within A Cell.
Authors: Rollenhagen Christiane , Panté Nelly
Published: August 2006

Uridine-rich small nuclear ribonucleoproteins (U snRNPs) are the building units of the spliceosome. These RNA and protein complexes assemble in the cytoplasm. After proper assembly and RNA maturation, mature U snRNPs are imported into the cell nucleus, where they take part in the splicing process. In this paper we review the current knowledge of how U snRNPs enter the nucleus.
Will you let me use your nucleus? How Agrobacterium gets its T-DNA expressed in the host plant cellThis paper is one of a selection of papers published in this Special Issue, entitled The Nucleus: A Cell Within A Cell.
Authors: Lacroix Benoît , Li Jianxiong , Tzfira Tzvi , Citovsky Vitaly
Published: August 2006

Agrobacterium is the only known bacterium capable of natural DNA transfer into a eukaryotic host. The genes transferred to host plants are contained on a T-DNA (transferred DNA) molecule, the transfer of which begins with its translocation, along with several effector proteins, from the bacterial cell to the host-cell cytoplasm. In the host cytoplasm, the T-complex is formed from a single-stranded copy of the T-DNA (T-strand) associated with several bacterial and host proteins and it is imported into the host nucleus via interactions with the host nuclear import machinery. Once inside the nucleus, the T-complex is most likely directed to the host genome by associating with histones. Finally, the chromatin-associated T-complex is uncoated from its escorting proteins prior to the conversion of the T-strand to a double-stranded form and its integration into the host genome.
The role of nuclear envelope calcium in modifying nuclear pore complex structureThis paper is one of a selection of papers published in this Special Issue, entitled The Nucleus: A Cell Within A Cell.
Authors: Erickson Elizabeth S. , Mooren Olivia L. , Moore David , Krogmeier Jeffrey R. , Dunn Robert C.
Published: August 2006

Some of the most important trafficking processes in cells involve transport across the nuclear envelope. Whether it is the import of transcription factors or the export of RNA, the only known portal across the double lipid bilayer that forms the nuclear envelope are the macromolecular pores known as nuclear pore complexes (NPCs). Understanding how signals influence the conformation of the NPC is important for testing models of, and perhaps modifying, transport across the nuclear envelope. Here we summarize high-resolution atomic force microscopy studies of NPC structure following manipulation of nuclear envelope calcium stores of nuclei from Xenopus laevis oocytes. The results show that the release of calcium from these stores through the specific activation of inositol 1,4,5-trisphosphate receptors leads to changes in NPC structure observable from both sides of the nuclear envelope. The diameter of the NPC is also sensitive to these calcium stores and increases upon calcium release. Western blot analysis reveals the presence of ryanodine receptors in the nuclear envelope of X. laevis oocytes, although in low abundance. Activation of these calcium channels also leads to the displacement of the central mass and changes in NPC diameter. This change in structure may involve a displacement of the cytoplasmic and nuclear rings of the NPC towards each other, leading to the apparent emergence of the central mass from both sides of the NPC. The changes in conformation and diameter of the NPC may alter cargo access and binding to phenylalanine-glycine repeats lining the pore, thus altering transport.
The nuclear tubular invaginations are dynamic structures inside the nucleus of HeLa cellsThis paper is one of a selection of papers published in this Special Issue, entitled The Nucleus: A Cell Within A Cell.
Authors: Lee Rebecca K.Y. , Lui Pauline P.Y. , Ngan Erika K.S. , Lui Julian C.K. , Suen Y.K. , Chan Franky , Kong S.K.
Published: August 2006

Nuclear tubules (NTs) were found in the nucleus of HeLa cells. Although no function has been ascribed to these structures, our previous data has shown that they are the sites of Ca2+release with mitochondria shuttled around. In the present study, we further characterized these NTs through different fluorescent dye-labeling and red fluorescent protein transfection experiments. We found that doxorubicin (Dox) is a good indicator to demonstrate the NTs since Dox is fluorescent and DNA is able to quench its fluorescence. By using confocal and electron microscopy, we show that the number and nature of the NTs in HeLa vary from cell to cell, ranging from tubular to intricately branched structures. Additionally, these NTs are double-membrane invaginations of the nuclear envelope and usually lie close to nucleolus. At rest, NTs appeared to be stable and their mouths are always closed. Upon Ca2+ionomycin stimulation, various forms of dynamism, including membrane protrusion to the nucleus, enlargement and shrinkage of the NTs, and distortion of the nuclear envelope and NTs were observed over a time scale of minutes. These observations suggest that the NT represents a specialized and dynamic compartment inside the nucleus under the control of Ca2+.
Mechanism regulating nuclear calcium signalingThis paper is one of a selection of papers published in this Special Issue, entitled The Nucleus: A Cell Within A Cell.
Authors: Malviya Anant N. , Klein Christian
Published: August 2006

Although the outer nuclear membrane is continuous with the endoplasmic reticulum, it is possible to isolate nuclei both intact and free from endoplasmic reticulum contaminants. The outer and the inner nuclear membranes can be purified free from cross-contamination. Evidence in support of autonomous regulation of nuclear calcium signaling relies upon the investigations with isolated nuclei. Mechanisms for generating calcium signaling in the nucleus have been identified. Two calcium transporting systems, an ATP-dependant nuclear Ca2+-ATPase and an IP4-mediated inositol 1,3,4,5-tetrakisphosphate receptor, are located on the outer nuclear membrane. Thus, ATP and IP4, depending on external free calcium concentrations, are responsible for filling the nuclear envelope calcium pool. The inositol 1,4,5-trisphosphate receptor is located on the inner nuclear membrane with its ligand binding domain facing toward the nucleoplasm. Likewise, the ryanodine receptor is located on the inner nuclear membrane and its ligand cADP-ribose is generated within the nucleus. A 120 kDa protein fragment of nuclear PLC-γ1 is stimulated in vivo by epidermal growth factor nuclear signaling coincident with the time course of nuclear membrane epidermal growth factor receptor activation. Stimulated 120 kDa protein fragment interacts with PIKE, a nuclear GTPase, and together they form a complex with PI[3]kinase serving as a module for nuclear PI[3]K stimulation. Thus, the nucleus has its own IP3generating system.
Effects of mitogen-activated protein kinases on nuclear protein importThis paper is one of a selection of papers published in this Special Issue, entitled The Nucleus: A Cell Within A Cell.
Authors: Faustino Randolph S. , Rousseau Delphine C. , Landry Melanie N. , Kostenuk Annette L. , Pierce Grant N.
Published: August 2006

ERK-2 MAP kinase activation induces inhibitory effects on nuclear protein import in vascular smooth muscle cells. The mechanism and characteristics of this effect of ERK-2 were investigated. An unusual dose-dependent effect of ERK-2 on nuclear protein import was identified. At higher concentrations (1 μg/mL) of ERK-2, nuclear protein import was stimulated, whereas lower concentrations (0.04 μg/mL) inhibited import. Intermediate concentrations exerted intermediate effects. The stimulatory and inhibitory effects at the 2 different ERK-2 concentrations were observed in both conventional, permeabilized cell assays of nuclear protein import and with in situ microinjection of smooth muscle cells. The biphasic effects of ERK-2 on import were also found for the other 2 members of the MAPK family, p38 and JNK. RanGAP was identified by structural analysis as a candidate target protein responsible for mediating the effects of ERK-2. After pretreatment with high concentrations of ERK-2, RanGAP activity was significantly increased by ~50%. In contrast, low concentrations of ERK-2 significantly attenuated RanGAP activity. These results demonstrate that all 3 members of the MAPK family can alter nuclear protein import in opposite directions depending upon the concentration of ERK-2 used. RanGAP represents the MAP kinase target whereby nuclear transport can be stimulated or inhibited.