bluuebunny/crossref_metadata_2025 huggingface dataset:
MxiE Regulates Intracellular Expression of Factors Secreted by theShigella flexneri2a Type III Secretion System
Authors: Kane Colleen D. , Schuch Raymond , Day William A. , Maurelli Anthony T.
Published: October 2002

Themxi-spalocus on the virulence plasmid ofShigella flexneriencodes components of the type III secretion system.mxiE, a gene within this locus, encodes a protein that is homologous to the AraC/XylS family of transcriptional regulators, but currently its role in pathogenesis remains undefined. We characterized the virulence phenotype of a nonpolarmxiEmutant and found that this mutant retained the ability to invade mammalian cells in tissue culture and secrete Ipas (type III effectors required for host cell invasion), although it was less efficient than wild-typeShigellaat cell-to-cell spread. Despite its invasive properties in culture, themxiEmutant was completely avirulent in an animal model. Potential targets for MxiE activation were identified by using promoter-green fluorescent protein fusions, and gene expression was examined under various growth conditions. Six MxiE-regulated genes were discovered:ospB,ospC1,ospE2,ospF,virA, andipaH9.8. Notably, activation of these genes only occurred within the intracellular environment of the host and not during growth at 37°C in liquid culture. Interestingly, all of the MxiE-regulated proteins previously have been shown to be secreted through the type III secretion system and are putative virulence factors. Our findings suggest that some of these Osp proteins may be involved in postinvasion events related to virulence. Since bacterial pathogens adapt to multiple environments during the course of infecting a host, we propose thatShigellaevolved a mechanism to take advantage of a unique intracellular cue, which is mediated through MxiE, to express proteins when the organism reaches the eukaryotic cytosol.
Mutations in the
Bacillus subtilis glnRA
Operon That Cause Nitrogen Source-Dependent Defects in Regulation of TnrA Activity
Authors: Fisher Susan H. , Wray Lewis V.
Published: October 2002

The
Bacillus subtilis
nitrogen transcriptional factor TnrA is inactive in cells grown with excess nitrogen, e.g., glutamine or glutamate plus ammonium, because feedback-inhibited glutamine synthetase (product of
glnA
) binds to TnrA and blocks its DNA-binding activity. Two conditional mutations that allow TnrA-dependent gene expression in cells grown with glutamate plus ammonium, but not in glutamine-grown cells, were characterized. One mutant contained a mutation in the
glnA
ribosome binding site, while the other mutant synthesized a truncated GlnR protein that constitutively repressed
glnRA
expression. The levels of glutamine synthetase were reduced in both mutants. As a result, when these mutants are grown with excess nitrogen in the absence of glutamine, there is insufficient production of the feedback inhibitors necessary to convert glutamine synthetase into its feedback-inhibited form and TnrA-activated genes are expressed at high levels.
Development of an In Vitro Integration Assay for theBacteroidesConjugative Transposon CTnDOT
Authors: Cheng Qi , Wesslund Neil , Shoemaker Nadja B. , Salyers Abigail A. , Gardner Jeffrey F.
Published: October 2002

Integrated self-transmissible elements called conjugative transposons (CTns) are responsible for the transfer of antibiotic resistance genes in many different species of bacteria. One of the best characterized of these newly recognized elements is theBacteroidesCTn, CTnDOT. CTnDOT is thought to have a circular transfer intermediate that transfers to and integrates into the genome of the recipient cell. Previous investigations of the mechanism of CTnDOT integration have been hindered by the lack of an in vitro system for checking this model of integration and determining whether the CTnDOT integrase alone was sufficient to catalyze the integration reaction or whether host factors might be involved. We report here the development of an in vitro system in which a plasmid containing the joined ends of CTnDOT integrates into a plasmid carrying a CTnDOT target site. To develop this in vitro system, a His-tagged version of the integrase gene of CTnDOT was cloned and shown to be active in vivo. The protein produced by this construct was partially purified and then added to a reaction mixture that contained the joined ends of the circular form of CTnDOT and a plasmid carrying one of the CTnDOT target sites. Integration was demonstrated by using a fairly simple mixture of components, but integration was stimulated by aBacteroidesextract or by purifiedEscherichia coliintegration host factor. The results of this study demonstrate both that the circular form of CTnDOT is the form that integrates into the target site and that host factors are involved in the integration process.
SipY Is the
Streptomyces lividans
Type I Signal Peptidase Exerting a Major Effect on Protein Secretion
Authors: Palacín Arantxa , Parro Víctor , Geukens Nick , Anné Jozef , Mellado Rafael P.
Published: October 2002

Most bacteria contain one type I signal peptidase (SPase) for cleavage of signal peptides from secreted proteins. The developmental complex bacterium
Streptomyces lividans
has the ability to produce and secrete a significant amount of proteins and has four different type I signal peptidases genes (
sipW
,
sipX
,
sipY
, and
sipZ
) unusually clustered in its chromosome. Functional analysis of the four SPases was carried out by phenotypical and molecular characterization of the different individual
sip
mutants. None of the
sip
genes seemed to be essential for bacterial growth. Analysis of total extracellular proteins indicated that SipY is likely to be the major
S. lividans
SPase, since the
sipY
mutant strain is highly deficient in overall protein secretion and extracellular protease production, showing a delayed sporulation phenotype when cultured in solid medium.
Fe2+-Tetracycline-Mediated Cleavage of the Tn10Tetracycline Efflux Protein TetA Reveals a Substrate Binding Site near Glutamine 225 in Transmembrane Helix 7
Authors: McMurry Laura M. , Aldema-Ramos Mila L. , Levy Stuart B.
Published: October 2002

TetA specified by Tn10is a class B member of a group of related bacterial transport proteins of 12 transmembrane alpha helices that mediate resistance to the antibiotic tetracycline. A tetracycline-divalent metal cation complex is expelled from the cell in exchange for a entering proton. The site(s) where tetracycline binds to this export pump is not known. We found that, when chelated to tetracycline, Fe2+cleaved the backbone of TetA predominantly at a single position, glutamine 225 in transmembrane helix 7. The related class D TetA protein from plasmid RA1 was cut at exactly the same position. There was no cleavage with glycylcycline, an analog of tetracycline that does not bind to TetA. The Fe2+-tetracycline complex was not detectably transported by TetA. However, cleavage products of the same size as with Fe2+occurred with Co2+, known to be cotransported with tetracycline. The known substrate Mg2+-tetracycline interfered with cleavage by Fe2+. These findings suggest that cleavage results from binding at a substrate-specific site. Fe2+is known to be able to cleave amide bonds in proteins at distances up to approximately 12 Å. We conclude that the α carbon of glutamine 225 is probably within 12 Å of the position of the Fe2+ion in the Fe2+-tetracycline complex bound to the protein.
Role for Phosphoglucomutase in
Vibrio fischeri-Euprymna scolopes
Symbiosis
Authors: DeLoney Cindy R. , Bartley Therese M. , Visick Karen L.
Published: October 2002

Vibrio fischeri
, a luminescent marine bacterium, specifically colonizes the light organ of its symbiotic partner, the Hawaiian squid
Euprymna scolopes.
In a screen for
V. fischeri
colonization mutants, we identified a strain that exhibited on average a 10-fold decrease in colonization levels relative to that achieved by wild-type
V. fischeri
. Further characterization revealed that this defect did not result from reduced luminescence or motility, two processes required for normal colonization. We determined that the transposon in this mutant disrupted a gene with high sequence identity to the
pgm
(phosphoglucomutase) gene of
Escherichia coli
, which encodes an enzyme that functions in both galactose metabolism and the synthesis of UDP-glucose. The
V. fischeri
mutant grew poorly with galactose as a sole carbon source and was defective for phosphoglucomutase activity, suggesting functional identity between
E. coli
Pgm and the product of the
V. fischeri
gene, which was therefore designated
pgm
. In addition, lipopolysaccharide profiles of the mutant were distinct from that of the parent strain and the mutant exhibited increased sensitivity to various cationic agents and detergents. Chromosomal complementation with the wild-type
pgm
allele restored the colonization ability to the mutant and also complemented the other noted defects. Unlike the
pgm
mutant, a galactose-utilization mutant (
galK
) of
V. fischeri
colonized juvenile squid to wild-type levels, indicating that the symbiotic defect of the
pgm
mutant is not due to an inability to catabolize galactose. Thus,
pgm
represents a new gene required for promoting colonization of
E. scolopes
by
V. fischeri
.
Staphylococcus aureusContains Two Low-Molecular-Mass Phosphotyrosine Protein Phosphatases
Authors: Soulat Didier , Vaganay Elisabeth , Duclos Bertrand , Genestier Anne-Laure , Etienne Jérôme , Cozzone Alain J.
Published: October 2002

The analysis of the different amino acid sequences deduced from the complete genome sequence of the gram-positive bacteriumStaphylococcus aureussuggested the presence of two eukaryotic-protein-like low-molecular-mass phosphotyrosine protein phosphatases, which are usually found in gram-negative bacteria. To check this prediction, the corresponding genes were cloned and overexpressed in anEscherichia colisystem. Two distinct proteins with an apparent molecular mass of 23 kDa each, PtpA and PtpB, were produced and then purified by affinity chromatography and assayed for enzymatic properties. As expected, they both exhibited phosphatase activity in vitro, with a maximum value at a pH of around 6.2 and at a temperature of 40°C. In addition, their kinetic constants, their specificity for phosphotyrosine residues, and their sensitivity to two phosphatase inhibitors,N-ethylmaleimide and orthovanadate, matched those of acid low-molecular-mass phosphotyrosine protein phosphatases.
PCR-Based Method for Detecting Viral Penetration of Medical Exam Gloves
Authors: Broyles John M. , O'Connell Kevin P. , Korniewicz Denise M.
Published: October 2002

The test approved by the U.S. Food and Drug Administration for assessment of the barrier quality of medical exam gloves includes visual inspection and a water leak test. Neither method tests directly the ability of gloves to prevent penetration by microorganisms. Methods that use microorganisms (viruses and bacteria) to test gloves have been developed but require classical culturing of the organism to detect it. We have developed a PCR assay for bacteriophage φX174 that allows the rapid detection of penetration of gloves by this virus. The method is suitable for use with both latex and synthetic gloves. The presence of glove powder on either latex or synthetic gloves had no effect on the ability of the PCR assay to detect bacteriophage DNA. The assay is rapid, sensitive, and inexpensive; requires only small sample volumes; and can be automated.
Evaluation of Performance of Four Genotypic Methods for Studying the Genetic Epidemiology of
Aspergillus fumigatus
Isolates
Authors: Lasker Brent A.
Published: October 2002

In the present investigation, 49
Aspergillus fumigatus
isolates obtained from four nosocomial outbreaks were typed by
Afut1
restriction fragment length polymorphism (RFLP) analysis and three PCR-based molecular typing methods: random amplified polymorphic DNA (RAPD) analysis, sequence-specific DNA primer (SSDP) analysis, and polymorphic microsatellite markers (PMM) analysis. The typing methods were evaluated with respect to discriminatory power (
D
), reproducibility, typeability, ease of use, and ease of interpretation to determine their performance and utility for outbreak and surveillance investigations.
Afut1
RFLP analysis detected 40 types. Thirty types were observed by RAPD analysis. PMM analysis detected 39 allelic types, but SSDP analysis detected only 14 types. All four methods demonstrated 100% typeability. PMM and RFLP analyses had comparable high degrees of discriminatory power (
D
= 0.989 and 0.988, respectively). The discriminatory power of RAPD analysis was slightly lower (
D
= 0.971), whereas SSDP analysis had the lowest discriminatory power (
D
= 0.889). Overall, SSDP analysis was the easiest method to interpret and perform. The profiles obtained by PMM analysis were easier to interpret than those obtained by RFLP or RAPD analysis. Bands that differed in staining intensity or that were of low intensity were observed by RAPD analysis, making interpretation more difficult. The reproducibilities with repeated runs of the same DNA preparation or with different DNA preparations of the same strain were high for all the methods. A high degree of genetic variation was observed in the test population, but isolates were not always similarly divided by each method. Interpretation of band profiles requires understanding of the molecular mechanisms responsible for genetic alternations. PMM analysis and
Afut1
RFLP analysis, or their combination, appear to provide the best overall discriminatory power, reproducibility, ease of interpretation, and ease of use. This investigation will aid in planning epidemiologic and surveillance studies of
A. fumigatus
.
Phenotypic and Genetic Diversity of Invasive Pneumococcal Isolates Recovered from French Children
Authors: Doit Catherine , Loukil Chawki , Geslin Pierre , Bingen Edouard
Published: October 2002

We investigated the phenotypic and genetic diversity (by ribotyping) of
Streptococcus pneumoniae
isolates recovered from French children, by blood culture from meningitis-free patients (
n
= 244) and by cerebrospinal fluid culture from patients with meningitis (
n
= 154). Isolates belonging to serotypes associated with carriage and penicillin-resistant isolates were significantly more frequent in children under 2 years of age than in older children. The seven-valent pneumococcal conjugate vaccine covered 68% of strains associated with bacteremia and 61% of strains associated with meningitis in children under 2 years. Although some serotypes were recovered more frequently from children with bacteremia than from those with meningitis, no difference in the genetic backgrounds of the two groups of strains was found.
DNA Fingerprinting of
Pasteurella multocida
Recovered from Avian Sources
Authors: Amonsin Alongkorn , Wellehan James F. X. , Li Ling-Ling , Laber Judy , Kapur Vivek
Published: October 2002

Repetitive sequence-based PCR (rep-PCR) and amplified fragment length polymorphism (AFLP) were used to characterize a sample of 43 field isolates and 4 attenuated vaccine strains of
Pasteurella multocida
recovered from multiple avian species. Both rep-PCR and AFLP assays were rapid and reproducible, with high indices of discrimination. Concordance analyses of rep-PCR and AFLP with somatic serotyping indicate that, in general, somatic serotyping is a poor indicator of genetic relatedness among isolates of
P. multocida
. In addition, the data provide evidence of host specificity of
P. multocida
clones. Overall, the results of our study indicate that the rep-PCR and AFLP techniques enable rapid fingerprinting of
P. multocida
isolates from multiple avian species and enhance the investigation of fowl cholera outbreaks.
Cross-Infection Due to Imipenem-Resistant
Bacteroides fragilis
Associated with a Totally Implantable Venous Port
Authors: Arpin Corinne , Dubois Véronique , Rogues Anne-Marie , Menard Fanny , Gavinet Anne-Marie , Maire Jean-Pierre , Gachie Jean-Pierre , Bezian Marie-Christine , Quentin Claudine
Published: October 2002

Four patients in an oncology ward developed
Bacteroides fragilis
bacteremia over a 12-day period. Cross-infection between two of them, due to an imipenem-resistant strain, was demonstrated by epidemiological investigation and genotypic typing methods (arbitrarily primed PCR fingerprinting and nucleotide sequencing of the
cfiA
genes and upstream IS
1186/
IS
1168
elements).
Detection of Enteroaggregative
Escherichia coli
with Formalin-Preserved HEp-2 Cells
Authors: Miqdady Mohamad S. , Jiang Zhi-Dong , Nataro James P. , DuPont Herbert L.
Published: October 2002

Formalin-stored HEp-2 cells were used to assay
Escherichia coli
for adherence. Cells refrigerated in formalin for up to 28 days and used in a wet assay format demonstrated an assay sensitivity ranging from 94 to 98% to detect enteroaggregative
E. coli
(EAEC). HEp-2 cells first fixed and stored with formalin and then stored dry in ambient conditions for 6 weeks demonstrated an assay sensitivity of 92% to detect EAEC. Using formalin-fixed HEp-2 cells will improve the efficiency of EAEC identification.
GenoType Mycobacterium Assay for Identification of Mycobacterial Species Isolated from Human Clinical Samples by Using Liquid Medium
Authors: Ruiz P. , Gutierrez J. , Zerolo F. J. , Casal M.
Published: October 2002

The GenoType Mycobacterium assay was used to identify 98 mycobacteria isolates by using liquid cultures from positive BACTEC, MGIT, and ESP bottles. This system identifies 16 mycobacteria. There was complete agreement between the GenoType results and the laboratory identifications for
Mycobacterium tuberculosis
complex and other
Mycobacterium
spp. GenoType also identified mixed mycobacterial infections.
Serologic and Molecular Detection of
Ehrlichia chaffeensis
and
Anaplasma phagocytophila
(Human Granulocytic Ehrlichiosis Agent) in Korean Patients
Authors: Heo Eun-jeong , Park Jin-ho , Koo Ja-ryong , Park Man-suk , Park Mi-yeoun , Dumler J. Stephen , Chae Joon-seok
Published: October 2002

Sera from 491 Korean patients with acute febrile diseases were tested for
Ehrlichia chaffeensis
and
Anaplasma phagocytophila
antibodies by indirect immunofluorescence assay (IFA), Western blotting, and TaqMan real-time PCR. Overall, 0.4% of sera reacted with
E. chaffeensis
, and 1.8% reacted with
A. phagocytophila
in IFAs. This is the first report of detection of antibodies to
A. phagocytophila
and
E. chaffeensis
in Korea and suggests the presence of
A. phagocytophila
and
E. chaffeensis
or antigenically similar species.
Alphavirus Minus-Strand RNA Synthesis: Identification of a Role for Arg183 of the nsP4 Polymerase
Authors: Fata Cori L. , Sawicki Stanley G. , Sawicki Dorothea L.
Published: October 2002

A partially conserved region spanning amino acids 142 to 191 of the Sindbis virus (SIN) nsP4 core polymerase is implicated in host restriction, elongation, and promoter recognition. We extended the analysis of this region by substituting Ser, Ala, or Lys for a highly conserved Arg183 residue immediately preceding its absolutely conserved Ser184-Ala-Val-Pro-Ser188 sequence. In chicken cells, the nsP4 Arg183 mutants had a nonconditionally lethal, temperature-sensitive (
ts
) growth phenotype caused by a
ts
defect in minus-strand synthesis whose extent varied with the particular amino acid substituted (Ser>Ala>Lys). Plus-strand synthesis by nsP4 Arg183 mutant polymerases was unaffected when corrected for minus-strand numbers, although 26S mRNA synthesis was enhanced at the elevated temperature compared to wild type. The
ts
defect was not due to a failure to form or accumulate nsP4 at 40°C. In contrast to their growth in chicken cells, the nsP4 Arg183 mutants replicated equally poorly, if at all, in mosquito cells. We conclude that Arg183 within the Pro180-Asn-Ile-Arg-Ser184 sequence of the SIN nsP4 polymerase contributes to the efficient initiation of minus strands or the formation of its replicase and that a host factor(s) participates in this event.
The Metastatic Suppressor Nm23-H1 Interacts with EBNA3C at Sequences Located between the Glutamine- and Proline-Rich Domains and Can Cooperate in Activation of Transcription
Authors: Subramanian Chitra , Robertson Erle S.
Published: October 2002

Epstein-Barr virus (EBV) is a lymphotrophic herpesvirus infecting most of the world's population. It is associated with a number of human lymphoid and epithelial tumors and lymphoproliferative diseases in immunocompromised patients. Recent studies have shown an in vitro and in vivo interaction between the EBV nuclear antigen 3C (EBNA3C) and the metastatic suppressor Nm23-H1, known to be downregulated in human invasive breast carcinoma. In this study, we have identified the domain of EBNA3C that specifically binds to Nm23-H1. This domain lies within the region comprising amino acids 637 to 675 of EBNA3C flanked by the proline- and glutamine-rich domains. Furthermore, we show that Nm23-H1 activates transcription when fused to the Gal4 DNA-binding domain and is coexpressed with a luciferase reporter construct containing the Gal4 binding sites upstream of a basal promoter. Gal4-Nm23-H1, when tethered to the promoter by binding to the Gal4 DNA binding sequences, consistently activated transcription. The level of activation increased when increasing amounts of Gal4-Nm23-H1 were introduced into the system. Moreover, EBNA3C when cotransfected with Gal4-Nm23-H1 enhanced the transcriptional activity. These results suggest that Nm23-H1 may have intrinsic transcription activities in EBV-infected cells and that this activity can be modulated in the presence of the essential latent antigen EBNA3C.
Down Regulation of the Interleukin-8 Promoter by Human Papillomavirus Type 16 E6 and E7 through Effects on CREB Binding Protein/p300 and P/CAF
Authors: Huang Shih-Min , McCance D. J.
Published: October 2002

Previously, we reported that human papillomavirus (HPV) type 16 E6 binds to C/H1, C/H3, and the C-terminal domains of coactivators p300 and CBP, causing the modulation of the transcription of certain genes controlled by NF-κB (p65 orrelA) and p53. To establish the biological significance of these observations, we have focused on the transcriptional regulation of interleukin-8 (IL-8), a potent chemoattractant for T lymphocytes and neutrophils, which is also essential for the initiation of the local immune response. The IL-8 promoter is regulated by NF-κB/p65 in response to tumor necrosis factor alpha and requires the cooperation of the coactivators CBP/p300 and steroid receptor coactivator 1 (SRC-1) and the p300/CBP-associated factor (P/CAF) for optimal activation. Here we report that, in the presence of HPV-16 E6, the promoter activity of IL-8 was repressed. Moreover, from the mutational analysis of the IL-8 promoter, we found that E6 down-regulates the IL-8 promoter activity through the NF-κB/p65 binding site. This inhibition appears to result from the ability of HPV-16 E6 to compete with NF-κB/p65 and SRC-1 for binding to the N terminus and C terminus of CBP, respectively. Reporter data also showed that E7 represses IL-8 promoter activity, though to a lesser extent than E6 but, like E6, the repression by E7 is through the NF-κB/p65 binding site. E7 was shown for the first time to bind to P/CAF, and the binding was necessary for the down regulation of the IL-8 promoter. E6 and E7 together inhibited transcription of the IL-8 promoter to a greater extent than either alone. Finally, by RNase protection assay, we showed that the synthesis of endogenous IL-8 mRNA was repressed in keratinocytes stably expressing E6 and E7. Taken together, the results provide evidence that E6 and E7 can cooperatively disrupt IL-8 transcription through disruption of transcriptional active complexes, and this may have important consequences for immune responses in infected hosts.
Release of Macrophage Migration Inhibitory Factor and CXCL8/Interleukin-8 from Lung Epithelial Cells Rendered Necrotic by Influenza A Virus Infection
Authors: Arndt Ute , Wennemuth Gunther , Barth Peter , Nain Marianne , Al-Abed Yousef , Meinhardt Andreas , Gemsa Diethard , Bacher Michael
Published: October 2002

Bronchiolar epithelial cells are the prime targets for influenza A virus infection. It still remains to be clarified which signals are generated from these cells to initiate an immune response. Among chemokines, viral infection of primary lung epithelial cells triggered exclusively the release of CXCL8/interleukin-8 (IL-8), which contrasts with our previous observation that influenza A virus induced in monocytes the expression of mononuclear-leukocyte-attracting chemokines and even suppressed the production of neutrophil-attracting chemokines. Therefore, we speculated that it may be advantageous for respiratory epithelial cells to release primarily neutrophil-attracting CXCL8/IL-8 since neutrophils rapidly remove necrotic debris and are the first line of defense against bacterial superinfections. This concept has also been supported by our finding that influenza A virus infection led to necrosis of lung epithelial cells. This is in striking contrast to previous studies where influenza A virus infection induced apoptosis in monocytes and epithelial cells from origins other than the lung. Thus, the cell type instead of the virus determines which death pathway will be followed. In addition to the release of CXCL8/IL-8, we obtained a massive release of macrophage migration inhibitory factor (MIF) from virus-infected lung cells. However, whereas the CXCL8/IL-8 secretion was accompanied by induced gene activation, the transcription rate of MIF remained unchanged during the infection course and the virus-induced MIF release was predominantly a discharge from intracellular stores, suggesting that MIF is passively released upon cell death. Despite virus induced necrosis, the passively liberated MIF remained bioactive. Considering the well-established immunostimulatory effects of MIF on different leukocyte subsets, is its very likely that enhanced levels of MIF may contribute to the host immune response during the acute phase of influenza A virus infection in humans.
Interaction of the Vp3 Nuclear Localization Signal with the Importin α2/β Heterodimer Directs Nuclear Entry of Infecting Simian Virus 40
Authors: Nakanishi Akira , Shum Dorothy , Morioka Hiroshi , Otsuka Eiko , Kasamatsu Harumi
Published: October 2002

For nuclear entry of large nucleoprotein complexes, it is thought that one key nuclear localization signal (NLS) of a protein component becomes exposed to mediate importin recognition. We show that the nuclear entry of simian virus 40 involves a dynamic interplay between two distinct interiorly situated capsid NLSs, the Vp1 NLS and the Vp3 NLS, and the selective exposure and importin recognition of the Vp3 NLS. The Vp3 NLS-null mutants assembled normally into virion-like particles (VLP) in mutant DNA-transfected cells. When used to infect a new host, the null VLP entered the cell normally but was impaired in viral DNA nuclear entry due to a lack of recognition by the importin α2/β heterodimer, leading to reduced viability. Both Vp3 and Vp1 NLSs directed importin interaction in vitro, but the Vp1 NLS, which overlaps the Vp1 DNA binding domain, did not bind importins in the presence of DNA. The results suggest that certain canonical NLSs within a nucleoprotein complex, such as the Vp1 NLS, can be masked from functioning by binding to the nucleic acid component and that the availability of an NLS that is not masked and can become exposed for importin binding, such as the Vp3 NLS, is a general feature of the nuclear entry of the nucleoprotein complexes, including those of other animal viruses.
Synthesis, Characterization, and Chemistry of Dimethyl Sulfide Derivatives of closo-B10H102-
Authors: Hall Heather D. , Ulrich Bradley D. , Kultyshev Roman G. , Liu Jianping , Liu Shengming , Meyers Edward A. , Gréau Sandra , Shore Sheldon G.
Published: October 2002

The reaction of Cs2[B10H10] with DMSO in acid produces the disubstituted 1,10-, 1,6-, and 2,7(8)-isomers of (Me2S)2B10H8 as well as the monosubstituted [(Me2S)B10H9]- anion. Through a modified procedure, the trisubstituted compound 1,10-(Me2S)2-2-(MeS)B10H7 was prepared and characterized. The 1,10-(Me2S)2B10H8 isomer was converted to the enantiomers 2,7-(Me2S)2B10H8 and 2,8-(Me2S)2B10H8 which were separated on a chiral column. The 1,6-(Me2S)2B10H8 isomer was converted to a mixture of 1,10-(Me2S)2B10H8 and 2,3-(Me2S)2B10H8. These polyhedral rearrangements are believed to occur through the diamond-square-diamond mechanism. The 1,6- and 1,10-(Me2S)2B10H8 isomers were reduced with alkali metal in liquid ammonia to produce the dianions [1,6-(MeS)2B10H8]2- and [1,10-(MeS)2B10H8]2-, respectively. Sodium ethanethiolate was used for the reduction of [1-(Me2S)B10H9]- and 1,10-(Me2S)2B10H8 to form [1-(MeS)B10H9]2- and [1-(MeS)-10-(Me2S)B10H8]-, respectively. The structures of 1,10-(Me2S)2B10H8, 1,6-(Me2S)2B10H8, 2,8-(Me2S)2B10H8, 2,3-(Me2S)2B10H8, 1,10-(Me2S)2-2-(MeS)B10H7, [1-(MeS)-10-(Me2S)B10H8]-, and [1,6-(MeS)2B10H8]2- were determined by single-crystal X-ray diffraction analysis.
A Novel Approach to the Syntheses of Functionalized, Water-Soluble Icosahedral Carboranyl Anions. Crystal Structure of Methyl N-[(Trimethylamineboryl)carbonyl]-L-tyrosinate: A Synthon for Novel Carboranylpeptides
Authors: Spielvogel Bernard F. , Rana Geeta , Vyakaranam Kamesh , Grelck Kurt , Dicke Kari E. , Dolash Bridget D. , Li Shou-Jian , Zheng Chong , Maguire John A. , Takagaki Masao , Hosmane Narayan S.
Published: October 2002

New functionalized water-soluble carboranyl anions have been prepared from ortho-carborane through lithiation and subsequent derivatization. The reaction of Li2[1,2-C2B10H10] with Me3NBH2X produced the carboranylborane dianions, [1,2-(BH2X)-1,2-C2B10H10]2- (X = H (2a), CN (2b), COOMe (2c), COOH (2d)), while the reaction of [1-R-2-Li-C2B10H10]- with Me3NBH2X produced the monoanions [1-R-2-BH2X-C2B10H10]- (R = C6H5, X = H (2e), CN (2f), COOH (2h), COOCH3 (2g), CONHCH(CH2OH)COOMe (2i), CONHCH(CHMe2)COOMe (2j), CONHCH(4-CH2C6H4OH)- COOMe (2k); R = Me, X = H (2l), COOH (2m)). These water-soluble carboranylboranes have the potential of being effective hydrophilic boron neutron capture therapy (BNCT) agents. The crystal structure of the peptide precursor Me3NBH2CONHCH(4-CH2C6H4OH)COOMe (3b) which showed a special promise as a BNCT agent is also described.
Biochemical changes as a result of prolonged strenuous exercise
Authors: Warburton D E R , Welsh R C , Haykowsky M J , Taylor D A , Humen D P
Published: October 2002

Objective: To briefly review biochemical changes that may result from prolonged strenuous exercise and to relate these changes to health risk.

Methods: Medline and Sports Discus databases were searched for relevant articles. Additional articles were found using cross referencing and the authors' knowledge of the subject area.

Results: Prolonged strenuous exercise may result in a series of biochemical changes that are of concern from a health point of view. Generally, these changes are benign, but some, especially hyponatraemia, are potentially life threatening occurrences.

Conclusion: Doctors and athletes should be aware of the potentially adverse biochemical changes, especially hyponatraemia, that may result from prolonged strenuous exercise.
Increasing age and experience: are both protective against motorcycle injury? A case-control study
Authors: Mullin Bernadette , Jackson Rodney , Langley John , Norton Robyn
Published: October 2002

Objectives—To assess the associations between age, experience, and motorcycle injury.

Setting—Motorcycle riding on non-residential roads between 6 am and midnight over a three year period from February 1993 in Auckland, New Zealand.

Methods—A population based case-control study was conducted. Cases were 490 motorcycle drivers involved in a crash and controls were 1518 drivers identified at random roadside surveys. Crash involvement was defined in terms of a motorcycle crash resulting in either a driver or pillion passenger being killed, hospitalised, or presenting to a public hospital emergency department with an injury severity score ≥5.

Results—There was a strong and consistent relationship between increasing driver age and decreasing risk of moderate to fatal injury. In multivariate analyses, drivers older than 25 years had more than 50% lower risk than those aged from 15–19 years (odds ratio (OR) 0.46; 95% confidence interval (CI) 0.26 to 0.81). In univariate analyses, a protective effect from riding more than five years compared with less than two years was observed. However, this protection was not sustained when driver age and other potential confounding variables were included in the analyses. Familiarity with the specific motorcycle was the only experience measure associated with a strong protective effect (OR (≥10 000 km experience) 0.52; 95% CI 0.35 to 0.79) in multivariate analyses.

Conclusions—Current licensing regulations should continue to emphasise the importance of increased age and might consider restrictions that favour experience with a specific motorcycle.
Host and environment factors for exposure to poisons: a case-control study of preschool children in Thailand
Authors: Chatsantiprapa K , Chokkanapitak J , Pinpradit N
Published: October 2002

Objective—To identify host and environment characteristics associated with poison exposure among preschool children in Thailand.

Setting—A matched case-control study in 20 public hospitals in Khon Kaen province.

Methods—Cases comprised 100 consecutive preschool children attending hospital between September 1997 and February 1999 because of reported exposure to a poison. Controls were three children matched by age, sex and area of residence to each case, who had never attended a hospital for this reason. Risk factors were elicited by interviewing the children's caregivers at their homes using a structured questionnaire and analyzed using conditional logistic regression.

Results—Number of siblings, whether the child was “medicine eating” and “rushing to explore new objects”, the child's activities while the caregiver was working during the daytime, the child's distance from the caregiver while the caregiver was working, whether the child was taken to a non-agricultural worksite, and the number of used containers around the residence, were all statistically significant in univariate analyses. However, when all significant variables were included in a multivariate model, only “medicine eating” and the number of used containers around the residence remained statistically significant.

Conclusion—Attention to “medicine eating” and used containers through increased supervision could be a protective factor against poison exposure for these children. To reduce risk, caregivers should not refer to medicines as foods and used containers should be systematically collected for disposal or recycling.
Risk factors for burns in children: crowding, poverty, and poor maternal education
Authors: Delgado J , Ramírez-Cardich M E , Gilman R H , Lavarello R , Dahodwala N , Bazán A , Rodríguez V , Cama R I , Tovar M , Lescano A
Published: October 2002

Objective: To characterize the presentation of burns in children and risk factors associated with their occurrence in a developing country as a basis for future prevention programs.

Design: Case-control study.

Setting: Burn unit of the National Institute of Child Health (Instituto Nacional de Salud del Niño) in Lima, Peru.

Methods: A questionnaire was administered to all consenting guardians of children admitted to the burns (cases) and general medicine (controls) units during a period of 14 months. Guardians of patients were questioned regarding etiology of the injury, demographic and socioeconomic data.

Results: 740 cases and controls were enrolled. Altogether 77.5% of the cases burns occurred in the patient's home, with 67.8% in the kitchen; 74% were due to scalding. Most involved children younger than 5 years. Lack of water supply (odds ratio (OR) 5.2, 95% confidence interval (CI) 2.1 to 12.3), low income (OR 2.8, 95% CI 2.0 to 3.9), and crowding (OR 2.5, 95%CI 1.7 to 3.6) were associated with an increased risk. The presence of a living room (OR 0.6, 95% CI 0.4 to 0.8) and better maternal education (OR 0.6, 95% CI 0.5 to 0.9) were protective factors.

Conclusions: To prevent burns interventions should be directed to low socioeconomic status groups; these interventions should be designed accordingly to local risk factors.
Religious meddling: a comment on Skene and Parker
Authors: Coady C A J
Published: October 2002

The question of churches resorting to the courts to influence public policy is one that concerns the appropriate role of the courts and the appropriate conduct of religious authorities. I agree with Skene and Parker that there is no principled legal reason to exclude such interventions out of hand; but my comments are principally addressed to the political and religious reasons for being rightly concerned about such activity. These advert both to the nature of the liberal democratic compromise and to the nature and scope of religious authority (at least within Christianity).
Response to Kuhse
Authors: Perkin R M , Resnik D B
Published: October 2002

In this short paper, we respond to critics of our original paper, The agony of agonal respiration: is the last gasp necessary?. A common thread in both Hawryluck’s and Kuhse’s responses is the difficulties encountered when using the agent’s intentions to make moral distinctions between using neuromuscular blocking drugs to palliate versus using neuromuscular blocking drugs to kill. Although this difficulty does exist we maintain that the intentions of the physician must matter when providing end-of-life care.
Surveys on mammography frequency in Geneva
Authors: Lutz J-M. , Reith-Chaton J. , Fioretta G. , Cerny V. , Bouchardy C.
Published: October 2002

Two surveys were conducted in Geneva, in 1991 and 1995, to assess the coverage of mammography before the introduction of a breast cancer screening programme. Women who attended for mammographies did so at their own request, or were referred by doctors (more by gynaecologists than general practitioners). In 1995, the total female population was around 200 000, of which the target population for screening (age group 50 to 69) was 46 000 persons. The total number of mammographies observed increased by 23%, with a higher increase for “routine” mammographies (+35%). However, these figures hide certain trends, with a high proportion of educated women performing regular breast investigations. The two year coverage rate for mammography among the 50 to 69 age group is estimated at 38%, which means that about 20 000 women within this target population in Geneva still escape screening.
Primary care perspective of prostate cancer screening after national guidance: a questionnaire survey
Authors: Kalsi G.S. , Rajaratnam G. , Bridgman S.A.
Published: October 2002

National guidance (executive letter) EL(97)12 stated that population screening should not be provided by the NHS, or be offered to the public until there is effective screening technology for prostate cancer. The study set out to determine the views of general practitioners and, indirectly, their practice staff on prostate cancer screening in primary care upon receiving EL(97)12. This postal questionnaire survey reveals that 81% (95% CI 75% to 87%) of responding general practitioners in North Staffordshire agreed with EL(97)12 and one in ten said that the executive letter changed their views, suggesting that such national guidance has an effect.
The Italian experience of a Pap test and speculoscopy based screening programme
Authors: Boselli F. , De Martis S. , Rivasi F. , Toni A. , Abbiati R. , Chiossi G.
Published: October 2002

Objectives The main objective of the study was to evaluate if speculoscopy, a magnified chemiluminescent examination, combined with a Pap smear, could improve the detection of early cervical lesions compared with the Pap smear alone.

Setting Pap tests and speculoscopies were performed in two family planning centres located in the surrounding areas of Modena. Colposcopic investigations and biopsies of the uterine cervix were performed in a second level centre (Gynaecological Prevention Centre of Modena Policlinico). Histological specimens were analysed at the Section of Pathological Anatomy of Modena Policlinico.

Subjects The study population comprised 1000 women aged 25–64 years, invited to undergo a Pap smear every three years in accordance with the screening programme for cervical cancer started in Modena in 1997.

Methods Midwives performed the Pap smear and speculoscopy in succession. Women with a positive Pap test and/or positive speculoscopy underwent colposcopy and, if colposcopic findings were positive, targeted punch biopsies were performed.

Results A total of 1000 patients were subjected to cytology and speculoscopy examinations. Among these women, 10 had abnormal Pap smear findings whereas 144 had an abnormal speculoscopic pattern. Only three of 59 patients with a histological diagnosis of cervical intraepithelial neoplasia grade I (CIN I)/human papillomavirus and only three of seven patients with CIN II/CIN III had a positive Pap test.

Conclusions Speculoscopy combined with a Pap test can significantly increase the detection of cervical lesions when included in a screening programme.
Radiological review of interval cancers in an Australian mammographic screening programme
Authors: Amos A.F. , Kavanagh A.M. , Cawson J. ,   Radiology Quality Assurance Group of BreastScreen Victoria
Published: October 2002

Objectives To determine the proportion and features of invasive interval cancers that could be considered detectable at the time of the previous screen and the proportion of cases that could be classified as true intervals, false negatives, minimal signs, or radiographically occult lesions.

Setting BreastScreen Victoria, the Victorian component of the BreastScreen Australia mammography screening programme.

Methods Two separate review methodologies were adopted. Firstly a blinded review of interval, screen detected, and normal cases was undertaken, followed by a confirmation exercise to determine the proportion of invasive interval cancers that could be considered detectable at the time of the previous screen. Secondly, an unblinded review was performed to classify interval cases as true interval, false negative, minimal signs, or radiographically occult.

Results From the blinded review, it was estimated that 38% of interval cases may be considered “potentially detectable” at the time of screening. Comparison of the characteristics of interval and screen detected cases indicates that interval cases are more likely to be smaller, equivocal, ill defined masses. In the unblinded exercise, 41% of interval cases were classified as false negatives and a further 16% as minimal signs, 33% true intervals, and 10% radiographically occult. Of the interval cancers considered potentially detectable at screening, 97% were classified as false negatives in the unblinded review.

Conclusions This study highlights the importance of adopting staged review methods with both blinded and unblinded components. The blinded review and confirmation exercise allows the determination of the proportion of interval cases that could be considered potentially detectable at screening. The unblinded review provides an active important opportunity for professional development and review and a mechanism to link into the blinded review through further classification of interval cases.
Predictors of non-attendance for second round mammography in an Australian mammographic screening programme
Authors: O'Byrne A-M. , Kavanagh A.M. , Ugoni A. , Diver F.
Published: October 2002

Objective To determine the socioeconomic, cultural, and clinical predictors of non-attendance for second round mammography.

Design/participantsRetrospective cohort study of 121 889 women aged 50–69 years who attended for first mammography screening in the BreastScreen Victoria programme in 1995/1996 and who were recommended to be invited for routine biennial mammography. Women were considered to be non-attenders if they had not attended for rescreening within 27 months of their initial screening. Relative risk (RR) was used to compare categories for non-attendance for second screening, and a multivariate model was fitted to adjust for possible confounding.

Setting BreastScreen Victoria, a population based mammographic screening programme, which offers free biennial mammography to all women 40 years and older. The programme specifically targets women aged 50–69 years.

Results In the multivariate analysis, women from non-English speaking backgrounds were more likely not to attend for second round screening (RR ranged from 1.18 to 1.77). Indigenous women (RR 2.02, 95% confidence interval (CI) 1.61 to 2.54) and women who reported either significant symptoms (RR 1.90, 95% CI 1.76 to 2.05) or other breast symptoms (RR 2.25, 95% CI 2.15 to 2.36) at the time of first round screening were also more likely not to attend for second round screening.

Conclusions Women from non-English speaking backgrounds, indigenous women, and women who report symptoms at the time of first screening are more likely to not attend for second round screening. It is important to investigate why these women do not attend for second round screening so that services can be more appropriately tailored to their needs.
Results from the UK NHS breast screening programme 1994–1999
Authors: Blanks R.G. , Moss S.M. , Patnick J.
Published: October 2002

Objective To present results from the NHS breast screening programme (NHSBSP) from 1994 through to 1999 and to examine the extent to which interim targets are being achieved.

Methods Data were collated from all screening programmes in the UK on standard statistical returns. Performance of the screening programme was evaluated using calculated targets based on comparison with the Swedish two counties (STC) randomised controlled trial.

Results In the early years of screening (1988–1993), the NHS programme was detecting more DCIS but considerably less invasive cancers than expected, based on the STC trial. Since the screening year 1993/94 (1 April to 31 March) the standardised detection ratio (SDR) measure of the detection of invasive cancers has increased by 36% from 0.83 to 1.13. The SDR has been greater than 1 since 1996/97 suggesting that the NHSBSP has only been screening as efficiently as the STC trial since this date. Uptake for screening has been relatively stable over time at approximately 75%.

Conclusions The NHSBSP has made considerable advances in performance since its inception in 1988 and screening uptake and age standardised invasive cancer detection rates are (by 1999) well in excess of targets. The early years of screening were characterised by good uptake but a low sensitivity for the detection of invasive cancers. It is anticipated that the screening programme will have an increasing impact on breast cancer mortality, particularly in the 55–69 year age group over the coming years.