bluuebunny/crossref_metadata_2025 huggingface dataset:
Direct Regulation ofBacillus subtilis phoPRTranscription by Transition State Regulator ScoC
Authors: Kaushal Bindiya , Paul Salbi , Hulett F. Marion
Published: April 2010

Induction of the Pho response inBacillus subtilisoccurs when the Piconcentrations in the growth medium fall below 0.1 mM, a condition which results in slowed cellular growth followed by entry into stationary phase. ThephoPRpromoter region contains three σA-responsive promoters; only promoter PA4is PhoP autoregulated. Expression of thephoPRoperon is postexponential, suggesting the possibility of a repressor role for a transition-state-regulatory protein(s). Expression of aphoPRpromoter-lacZfusion in ascoCloss-of-function mutant strain grown in low-phosphate defined medium was significantly higher than expression in the wild-type strain during exponential growth or stationary phase. Derepression in thescoCstrain from aphoPpromoter fusion containing a mutation in the CcpA binding site (cre1) was further elevated approximately 1.4-fold, indicating that the repressor effects of ScoC and CcpA onphoPexpression were cumulative. DNase I footprinting showed protection of putative binding sites by ScoC, which included the −10 and/or −35 elements of five (PB1, PE2, PA3, PA4, and PA6) of the six promoters within thephoPRpromoter region. PA6was expressedin vivofrom thephoP cre1promoter fusion in both wild-type andscoCstrains. Evidence for ScoC repressionin vivowas shown by primer extension for PA4and PA3from the wild-type promoter and for PA4and PE2from thephoP cre1promoter. The latter may reflect ScoC repression of sporulation that indirectly affectsphoPRtranscription. ScoC was shown to repress PA6, PA4, PE2, and PB1in vitro.
A Solvent-Exposed Patch in Chaperone-Bound YopE Is Required for Translocation by the Type III Secretion System
Authors: Rodgers Loren , Mukerjea Romila , Birtalan Sara , Friedberg Devorah , Ghosh Partho
Published: April 2010

Most effector proteins of bacterial type III secretion (T3S) systems require chaperone proteins for translocation into host cells. Such effectors are bound by chaperones in a conserved and characteristic manner, with the chaperone-binding (Cb) region of the effector wound around the chaperone in a highly extended conformation. This conformation has been suggested to serve as a translocation signal in promoting the association between the chaperone-effector complex and a bacterial component required for translocation. We sought to test a prediction of this model by identifying a potential association site for the
Yersinia pseudotuberculosis
chaperone-effector pair SycE-YopE. We identified a set of residues in the YopE Cb region that are required for translocation but are dispensable for expression, SycE binding, secretion into the extrabacterial milieu, and stability in mammalian cells. These residues form a solvent-exposed patch on the surface of the chaperone-bound Cb region, and thus their effect on translocation is consistent with the structure of the chaperone-bound Cb region serving as a signal for translocation.
Loss of the WaaL O-Antigen Ligase Prevents Surface Activation of the Flagellar Gene Cascade in
Proteus mirabilis
Authors: Morgenstein Randy M. , Clemmer Katy M. , Rather Philip N.
Published: April 2010

Proteus mirabilis
is a Gram-negative bacterium that undergoes a physical and biochemical change from a vegetative swimmer cell (a typical Gram-negative rod) to an elongated swarmer cell when grown on a solid surface. In this study, we report that a transposon insertion in the
waaL
gene, encoding O-antigen ligase, blocked swarming motility on solid surfaces but had little effect on swimming motility in soft agar. The
waaL
mutant was unable to differentiate into a swarmer cell. Differentiation was also prevented by a mutation in
wzz
, encoding a chain length determinant for O antigen, but not by a mutation in
wzyE
, encoding an enzyme that polymerizes enterobacterial common antigen, a surface polysaccharide different from the lipid A::core. In wild-type
P. mirabilis
, increased expression of the
flhDC
operon occurs after growth on solid surfaces and is required for the high-level expression of flagellin that is characteristic of swarmer cells. However, in both the
waaL
and the
wzz
mutants, the
flhDC
operon was not activated during growth on agar. A loss-of-function mutation in the
rcsB
response regulator or overexpression of
flhDC
restored swarming to the
waaL
mutant, despite the absence of O antigen. Therefore, although O antigen may serve a role in swarming by promoting wettability, the loss of O antigen blocks a regulatory pathway that links surface contact with the upregulation of
flhDC
expression.
A Highly Selective Oligopeptide Binding Protein from the Archaeon
Sulfolobus Solfataricus
Authors: Gogliettino M. , Balestrieri M. , Pocsfalvi G. , Fiume I. , Natale L. , Rossi M. , Palmieri G.
Published: April 2010

SSO1273 of
Sulfolobus solfataricus
was identified as a cell surface-bound protein by a proteomics approach. Sequence inspection of the genome revealed that the open reading frame of
sso1273
is associated in an operon-like structure with genes encoding all the remaining components of a canonical protein-dependent ATP-binding cassette (ABC) transporter.
sso1273
gene expression and SSO1273 protein accumulation on the cell surface were demonstrated to be strongly induced by the addition of a peptide mixture (tryptone) to the culture medium. The native protein was obtained in multimeric form, mostly hexameric, under the purification conditions used, and it was characterized as an oligopeptide binding protein, named
S. solfataricus
OppA (OppA
Ss
). OppaA
Ss
possesses typical sequence patterns required for glycosylphosphatidylinositol lipid anchoring, resulting in an N-linked glycoprotein with carbohydrate moieties likely composed of high mannose and/or hybrid complex carbohydrates. OppA
Ss
specifically binds oligopeptides and shows a marked selectivity for the amino acid composition of substrates when assayed in complex peptide mixtures. Moreover, a truncated version of OppA
Ss
, produced in recombinant form and including the putative binding domain, showed a low but significant oligopeptide binding activity.
Roles of CcrA and CcrB in Excision and Integration of Staphylococcal Cassette Chromosome
mec
, a
Staphylococcus aureus
Genomic Island
Authors: Wang Lei , Archer Gordon L.
Published: April 2010

The gene encoding resistance to methicillin and other β-lactam antibiotics in staphylococci,
mecA
, is carried on a genomic island, SCC
mec
(for staphylococcal cassette chromosome
mec
). The chromosomal excision and integration of types I to IV SCC
mec
are catalyzed by the site-specific recombinases CcrA and CcrB, the genes for which are encoded on each element. We sought to identify the relative contributions of CcrA and CcrB in the excision and integration of SCC
mec
. Purified CcrB but not CcrA was shown to mediate the gel shift of chromosomal target integration sequences (
attB
) in electrophoretic mobility shift assays. However, preincubation of CcrB-DNA complexes with increasing concentrations of CcrA blocked gel shift. The interaction of CcrB and CcrA was confirmed by
Escherichia coli
two-hybrid analysis. SCC
mec
excision mediated by plasmid-encoded and inducible
ccrA
,
ccrB
, or both genes was assessed by PCR in
Staphylococcus aureus
. CcrB alone could mediate excision but excision was at an alternate
att
site (
attR2
) within the right extremity of SCC
mec
. In contrast, both CcrB and CcrA were required to mediate excision at the chromosomal
attB
site (called
attR
when SCC
mec
is integrated). Insertion of a plasmid containing the SCC
mec att
site (
attS
) into the chromosome required both CcrA and CcrB, but CcrA overexpression lowered integration frequency. Thus, while CcrB binds DNA, interaction between CcrA and CcrB, in a precise ratio, is required for
attB
site-specific excision and SCC
mec
chromosomal insertion.
Lumbar Tuberculosis Associated with Membranous Nephropathy and Interstitial Nephritis
Authors: Yuan Quan , Sun Li , Feng Jiangmin , Liu Nan , Jiang Yi , Ma Jianfei , Wang Lining
Published: April 2010

Tuberculosis is a common disease worldwide. However, it now is clear that tuberculosis can affect the kidney more insidiously. We describe a case of lumbar tuberculosis associated with simultaneous membranous nephropathy and interstitial nephritis, in which recovery of renal function occurred after treatment with steroids in addition to antituberculosis agents.
Molecular Detection and Identification of
Zygomycetes
Species from Paraffin-Embedded Tissues in a Murine Model of Disseminated Zygomycosis: a Collaborative European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Fungal Infection Study Group (EFISG) Evaluation
Authors: Dannaoui Eric , Schwarz Patrick , Slany Michal , Loeffler Juergen , Jorde Anne Tomine , Cuenca-Estrella Manuel , Hauser Philippe M. , Shrief Raghdaa , Huerre Michel , Freiberger Tomas , Gaustad Peter , Rodriguez-Tudela Juan-Luis , Bille Jacques , Denning David W. , Bretagne Stéphane , Lortholary Olivier
Published: April 2010

The present study was performed to assess the interlaboratory reproducibility of the molecular detection and identification of species of
Zygomycetes
from formalin-fixed paraffin-embedded kidney and brain tissues obtained from experimentally infected mice. Animals were infected with one of five species (
Rhizopus oryzae
,
Rhizopus microsporus
,
Lichtheimia corymbifera
,
Rhizomucor pusillus
, and
Mucor circinelloides
). Samples with 1, 10, or 30 slide cuts of the tissues were prepared from each paraffin block, the sample identities were blinded for analysis, and the samples were mailed to each of seven laboratories for the assessment of sensitivity. A protocol describing the extraction method and the PCR amplification procedure was provided. The internal transcribed spacer 1 (ITS1) region was amplified by PCR with the fungal universal primers ITS1 and ITS2 and sequenced. As negative results were obtained for 93% of the tissue specimens infected by
M. circinelloides
, the data for this species were excluded from the analysis. Positive PCR results were obtained for 93% (52/56), 89% (50/56), and 27% (15/56) of the samples with 30, 10, and 1 slide cuts, respectively. There were minor differences, depending on the organ tissue, fungal species, and laboratory. Correct species identification was possible for 100% (30 cuts), 98% (10 cuts), and 93% (1 cut) of the cases. With the protocol used in the present study, the interlaboratory reproducibility of ITS sequencing for the identification of major
Zygomycetes
species from formalin-fixed paraffin-embedded tissues can reach 100%, when enough material is available.
Cyclomodulins in Urosepsis Strains of
Escherichia coli
Authors: Dubois Damien , Delmas Julien , Cady Anne , Robin Frédéric , Sivignon Adeline , Oswald Eric , Bonnet Richard
Published: April 2010

Determinants of urosepsis in
Escherichia coli
remain incompletely defined. Cyclomodulins (CMs) are a growing functional family of toxins that hijack the eukaryotic cell cycle. Four cyclomodulin types are actually known in
E. coli
: cytotoxic necrotizing factors (CNFs), cycle-inhibiting factor (Cif), cytolethal distending toxins (CDTs), and the
pks
-encoded toxin. In the present study, the distribution of CM-encoding genes and the functionality of these toxins were investigated in 197
E. coli
strains isolated from patients with community-acquired urosepsis (
n
= 146) and from uninfected subjects (
n
= 51). This distribution was analyzed in relation to the phylogenetic background, clinical origin, and antibiotic resistance of the strains. It emerged from this study that strains harboring the
pks
island and the
cnf1
gene (i) were strongly associated with the B2 phylogroup (
P
, <0.001), (ii) frequently harbored both toxin-encoded genes in phylogroup B2 (33%), and (iii) were predictive of a urosepsis origin (
P
, <0.001 to 0.005). However, the prevalences of the
pks
island among phylogroup B2 strains, in contrast to those of the
cnf1
gene, were not significantly different between fecal and urosepsis groups, suggesting that the
pks
island is more important for the colonization process and the
cnf1
gene for virulence.
pks
-
or cnf1
-harboring strains were significantly associated with susceptibility to antibiotics (amoxicillin, cotrimoxazole, and quinolones [
P
, <0.001 to 0.043]). Otherwise, only 6% and 1% of all strains harbored the
cdtB
and
cif
genes, respectively, with no particular distribution by phylogenetic background, antimicrobial susceptibility, or clinical origin.
Use of Luminescent
Leptospira interrogans
for Enumeration in Biological Assays
Authors: Murray Gerald L. , King Amy M. , Srikram Amporn , Sermswan Rasana W. , Adler Ben
Published: April 2010

Rapid and reliable
in vitro
methods for the detection of pathogenic leptospires, such as
Leptospira interrogans
, are lacking. The present study investigated the use of luminescence to replace the existing enumeration techniques. Transposon Tn
SC189
was modified to incorporate the
luxCDABE
cassette from
Photorhabdus luminescens
and was used to construct luminescent
Leptospira
spp. There was a linear relationship between luminescence and cell number, with the theoretical detection limit being less than 10
4
leptospires. A comparison of enumeration by a standard method (counting by dark-field microscopy) and enumeration by luminescence was conducted with luminescent
L. interrogans
. There was a good correlation between the two methods of enumeration (
R
2
= 0.766), although variation in the luminescence early and late in growth phase reduced the degree of correlation. To demonstrate the utility of luminescence as a viability and cell number reporter,
in vitro
assays, including MIC determination, an extracellular matrix binding experiment, and a complement killing experiment, were conducted. In each case, the results obtained by luminescence matched those obtained by traditional means with high correlations (binding assay
R
2
= 0.916, complement killing assay
R
2
= 0.988). A strain expressing the
luxCDABE
transposon retained virulence in the hamster model of infection. Despite some variation in luminescence as a result of the growth phase or the particular assay conditions, enumeration by luminescence was found to be a quick, reliable, and highly sensitive method for the
in vitro
detection of leptospires that has the potential to replace more time-consuming methods of enumeration.
Identification and Characterization of the 480-Kilodalton Template-Specific RNA-Dependent RNA Polymerase Complex of
Red Clover Necrotic Mosaic Virus
Authors: Mine Akira , Takeda Atsushi , Taniguchi Takako , Taniguchi Hisaaki , Kaido Masanori , Mise Kazuyuki , Okuno Tetsuro
Published: April 2010

Replication of positive-strand RNA viruses occurs through the assembly of membrane-associated viral RNA replication complexes that include viral replicase proteins, viral RNA templates, and host proteins.
Red clover necrotic mosaic virus
(RCNMV) is a positive-strand RNA plant virus with a genome consisting of RNA1 and RNA2. The two proteins encoded by RNA1, a 27-kDa protein (p27) and an 88-kDa protein containing an RNA-dependent RNA polymerase (RdRP) motif (p88), are essential for RCNMV RNA replication. To analyze RCNMV RNA replication complexes, we used blue-native polyacrylamide gel electrophoresis (BN/PAGE), which enabled us to analyze detergent-solubilized large membrane protein complexes. p27 and p88 formed a complex of 480 kDa in RCNMV-infected plants. As a result of sucrose gradient sedimentation, the 480-kDa complex cofractionated with both endogenous template-bound and exogenous template-dependent RdRP activities. The amount of the 480-kDa complex corresponded to the activity of exogenous template-dependent RdRP, which produced RNA fragments by specifically recognizing the 3′-terminal core promoter sequences of RCNMV RNAs, but did not correspond to the activity of endogenous template-bound RdRP, which produced genome-sized RNAs without the addition of RNA templates. These results suggest that the 480-kDa complex contributes to template-dependent RdRP activities. We subjected those RdRP complexes to affinity purification and analyzed their components using two-dimensional BN/sodium dodecyl sulfate-PAGE (BN/SDS-PAGE) and mass spectrometry. The 480-kDa complex contained p27, p88, and possible host proteins, and the original affinity-purified RdRP preparation contained HSP70, HSP90, and several ribosomal proteins that were not detected in the 480-kDa complex. A model for the formation of RCNMV RNA replication complexes is proposed.
Nonpathogenic Simian Immunodeficiency Virus Infection of Sooty Mangabeys Is Not Associated with High Levels of Autologous Neutralizing Antibodies
Authors: Li Bing , Stefano-Cole Kelly , Kuhrt David M. , Gordon Shari N. , Else James G. , Mulenga Joseph , Allen Susan , Sodora Donald L. , Silvestri Guido , Derdeyn Cynthia A.
Published: April 2010

Simian immunodeficiency virus (SIV) infection of natural-host species, such as sooty mangabeys (SMs), is characterized by a high level of viral replication and a low level of generalized immune activation, despite evidence of an adaptive immune response. Here the ability of SIV-infected SMs to mount neutralizing antibodies (Nab) against autologous virus was compared to that of human immunodeficiency virus type 1 (HIV-1) subtype C-infected subjects. While high levels of Nab were observed in HIV-1 infection, samples obtained at comparable time points from SM exhibited relatively low titers of autologous Nab. Nevertheless, SM plasma with higher Nab titers also contained elevated peripheral CD4
+
T-cell levels, suggesting a potential immunologic benefit for SMs. These data indicate that AIDS resistance in these primates is not due to high Nab titers and raise the possibility that low levels of Nab might be an inherent feature of natural-host SIV infections.
The Crystal Structure of D212 from
Sulfolobus
Spindle-Shaped Virus Ragged Hills Reveals a New Member of the PD-(D/E)XK Nuclease Superfamily
Authors: Menon Smita K. , Eilers Brian J. , Young Mark J. , Lawrence C. Martin
Published: April 2010

Structural studies have made significant contributions to our understanding of
Sulfolobus
spindle-shaped viruses (
Fuselloviridae
), an important model system for archaeal viruses. Continuing these efforts, we report the structure of D212 from
Sulfolobus
spindle-shaped virus Ragged Hills. The overall fold and conservation of active site residues place D212 in the PD-(D/E)XK nuclease superfamily. The greatest structural similarity is found to the archaeal Holliday junction cleavage enzymes, strongly suggesting a role in DNA replication, repair, or recombination. Other roles associated with nuclease activity are also considered.
Through Its Nonstructural Protein NS1, Parvovirus H-1 Induces Apoptosis via Accumulation of Reactive Oxygen Species
Authors: Hristov Georgi , Krämer Melanie , Li Junwei , El-Andaloussi Nazim , Mora Rodrigo , Daeffler Laurent , Zentgraf Hanswalter , Rommelaere Jean , Marchini Antonio
Published: April 2010

The rat parvovirus H-1 (H-1PV) attracts high attention as an anticancer agent, because it is not pathogenic for humans and has oncotropic and oncosuppressive properties. The viral nonstructural NS1 protein is thought to mediate H-1PV cytotoxicity, but its exact contribution to this process remains undefined. In this study, we analyzed the effects of the H-1PV NS1 protein on human cell proliferation and cell viability. We show that NS1 expression is sufficient to induce the accumulation of cells in G
2
phase, apoptosis via caspase 9 and 3 activation, and cell lysis. Similarly, cells infected with wild-type H-1PV arrest in G
2
phase and undergo apoptosis. Furthermore, we also show that both expression of NS1 and H-1PV infection lead to higher levels of intracellular reactive oxygen species (ROS), associated with DNA double-strand breaks. Antioxidant treatment reduces ROS levels and strongly decreases NS1- and virus-induced DNA damage, cell cycle arrest, and apoptosis, indicating that NS1-induced ROS are important mediators of H-1PV cytotoxicity.
Long-Term Evolution of theLuteoviridae: Time Scale and Mode of Virus Speciation
Authors: Pagán Israel , Holmes Edward C.
Published: April 2010

Despite their importance as agents of emerging disease, the time scale and evolutionary processes that shape the appearance of new viral species are largely unknown. To address these issues, we analyzed intra- and interspecific evolutionary processes in theLuteoviridaefamily of plant RNA viruses. Using the coat protein gene of 12 members of the family, we determined their phylogenetic relationships, rates of nucleotide substitution, times to common ancestry, and patterns of speciation. An associated multigene analysis enabled us to infer the nature of selection pressures and the genomic distribution of recombination events. Although rates of evolutionary change and selection pressures varied among genes and species and were lower in some overlapping gene regions, all fell within the range of those seen in animal RNA viruses. Recombination breakpoints were commonly observed at gene boundaries but less so within genes. Our molecular clock analysis suggested that the origin of the currently circulatingLuteoviridaespecies occurred within the last 4 millennia, with intraspecific genetic diversity arising within the last few hundred years. Speciation within theLuteoviridaemay therefore be associated with the expansion of agricultural systems. Finally, our phylogenetic analysis suggested that viral speciation events tended to occur within the same plant host species and country of origin, as expected if speciation is largely sympatric, rather than allopatric, in nature.
Bacteriophage Lambda: a Paradigm Revisited
Authors: Fogg Paul C. M. , Allison Heather E. , Saunders Jon R. , McCarthy Alan J.
Published: April 2010

Bacteriophage lambda has an archetypal immunity system, which prevents the superinfection of its
Escherichia coli
lysogens. It is now known that superinfection can occur with toxigenic lambda-like phages at a high frequency, and here we demonstrate that the superinfection of a lambda lysogen can lead to the acquisition of additional lambda genomes, which was confirmed by Southern hybridization and quantitative PCR. As many as eight integration events were observed but at a very low frequency (6.4 × 10
−4
) and always as multiple insertions at the established primary integration site in
E. coli
. Sequence analysis of the complete immunity region demonstrated that these multiply infected lysogens were not immunity mutants. In conclusion, although lambda superinfection immunity can be confounded, it is a rare event.
Hepatitis C Virus Transmission Bottlenecks Analyzed by Deep Sequencing
Authors: Wang Gary P. , Sherrill-Mix Scott A. , Chang Kyong-Mi , Quince Chris , Bushman Frederic D.
Published: April 2010

Hepatitis C virus (HCV) replication in infected patients produces large and diverse viral populations, which give rise to drug-resistant and immune escape variants. Here, we analyzed HCV populations during transmission and diversification in longitudinal and cross-sectional samples using 454/Roche pyrosequencing, in total analyzing 174,185 sequence reads. To sample diversity, four locations in the HCV genome were analyzed, ranging from high diversity (the envelope hypervariable region 1 [HVR1]) to almost no diversity (the 5′ untranslated region [UTR]). For three longitudinal samples for which early time points were available, we found that only 1 to 4 viral variants were present, suggesting that productive infection was initiated by a very small number of HCV particles. Sequence diversity accumulated subsequently, with the 5′ UTR showing almost no diversification while the envelope HVR1 showed >100 variants in some subjects. Calculation of the transmission probability for only a single variant, taking into account the measured population structure within patients, confirmed initial infection by one or a few viral particles. These findings provide the most detailed sequence-based analysis of HCV transmission bottlenecks to date. The analytical methods described here are broadly applicable to studies of viral diversity using deep sequencing.
Cell Entry of the Aphthovirus Equine Rhinitis A Virus Is Dependent on Endosome Acidification
Authors: Groppelli Elisabetta , Tuthill Tobias J. , Rowlands David J.
Published: April 2010

Equine rhinitis A virus (ERAV) is genetically closely related to foot-and-mouth disease virus (FMDV), and both are now classified within the genus
Aphthovirus
of the family
Picornaviridae
. For disease security reasons, FMDV can be handled only in high-containment facilities, but these constraints do not apply to ERAV, making it an attractive alternative for the study of aphthovirus biology. Here, we show, using immunofluorescence, pharmacological agents, and dominant negative inhibitors, that ERAV entry occurs (as for FMDV) via clathrin-mediated endocytosis and acidification of early endosomes. This validates the use of ERAV as a model system to study the mechanism of cell entry by FMDV.
A Single Coxsackievirus B2 Capsid Residue Controls Cytolysis and Apoptosis in Rhabdomyosarcoma Cells
Authors: Gullberg Maria , Tolf Conny , Jonsson Nina , Polacek Charlotta , Precechtelova Jana , Badurova Miriam , Sojka Martin , Mohlin Camilla , Israelsson Stina , Johansson Kjell , Bopegamage Shubhada , Hafenstein Susan , Lindberg A. Michael
Published: April 2010

Coxsackievirus B2 (CVB2), one of six human pathogens of the group B coxsackieviruses within the enterovirus genus of
Picornaviridae
, causes a wide spectrum of human diseases ranging from mild upper respiratory illnesses to myocarditis and meningitis. The CVB2 prototype strain Ohio-1 (CVB2O) was originally isolated from a patient with summer grippe in the 1950s. Later on, CVB2O was adapted to cytolytic replication in rhabdomyosarcoma (RD) cells. Here, we present analyses of the correlation between the adaptive mutations of this RD variant and the cytolytic infection in RD cells. Using reverse genetics, we identified a single amino acid change within the exposed region of the VP1 protein (glutamine to lysine at position 164) as the determinant for the acquired cytolytic trait. Moreover, this cytolytic virus induced apoptosis, including caspase activation and DNA degradation, in RD cells. These findings contribute to our understanding of the host cell adaptation process of CVB2O and provide a valuable tool for further studies of virus-host interactions.
Regulation of
De Novo
-Initiated RNA Synthesis in Hepatitis C Virus RNA-Dependent RNA Polymerase by Intermolecular Interactions
Authors: Chinnaswamy S. , Murali A. , Li P. , Fujisaki K. , Kao C. C.
Published: April 2010

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) has been proposed to change conformations in association with RNA synthesis and to interact with cellular proteins.
In vitro
, the RdRp can initiate
de novo
from the ends of single-stranded RNA or extend a primed RNA template. The interactions between the Δ1 loop and thumb domain in NS5B are required for
de novo
initiation, although it is unclear whether these interactions are within an NS5B monomer or are part of a higher-order NS5B oligomeric complex. This work seeks to address how polymerase conformation and/or oligomerization affects
de novo
initiation. We have shown that an increasing enzyme concentration increases
de novo
initiation by the genotype 1b and 2a RdRps while primer extension reactions are not affected or inhibited under similar conditions. Initiation-defective mutants of the HCV polymerase can increase
de novo
initiation by the wild-type (WT) polymerase. GTP was also found to stimulate
de novo
initiation. Our results support a model in which the
de novo
initiation-competent conformation of the RdRp is stimulated by oligomeric contacts between individual subunits. Using electron microscopy and single-molecule reconstruction, we attempted to visualize the low-resolution conformations of a dimer of a
de novo
initiation-competent HCV RdRp.
Effect of Mutations in VP5* Hydrophobic Loops on Rotavirus Cell Entry
Authors: Kim Irene S. , Trask Shane D. , Babyonyshev Marina , Dormitzer Philip R. , Harrison Stephen C.
Published: April 2010

Experiments in cell-free systems have demonstrated that the VP5* cleavage fragment of the rotavirus spike protein, VP4, undergoes a foldback rearrangement that translocates three clustered hydrophobic loops from one end of the molecule to the other. This conformational change resembles the foldback rearrangements of enveloped virus fusion proteins. By recoating rotavirus subviral particles with recombinant VP4 and VP7, we tested the effects on cell entry of substituting hydrophilic for hydrophobic residues in the clustered VP5* loops. Several of these mutations decreased the infectivity of recoated particles without preventing either recoating or folding back. In particular, the V391D mutant had a diminished capacity to interact with liposomes when triggered to fold back by serial protease digestion in solution, and particles recoated with this mutant VP4 were 10,000-fold less infectious than particles recoated with wild-type VP4. Particles with V391D mutant VP4 attached normally to cells and internalized efficiently, but they failed in the permeabilization step that allows coentry of the toxin α-sarcin. These findings indicate that the hydrophobicity of the VP5* apex is required for membrane disruption during rotavirus cell entry.
Either ZEB1 or ZEB2/SIP1 Can Play a Central Role in Regulating the Epstein-Barr Virus Latent-Lytic Switch in a Cell-Type-Specific Manner
Authors: Ellis Amy L. , Wang Zhenxun , Yu Xianming , Mertz Janet E.
Published: April 2010

We previously reported that the cellular protein ZEB1 can repress expression of the Epstein-Barr virus (EBV)
BZLF1
gene in transient transfection assays by directly binding its promoter, Zp. We also reported that EBV containing a 2-bp substitution mutation in the ZEB-binding ZV element of Zp spontaneously reactivated out of latency into lytic replication at a higher frequency than did wild-type EBV. Here, using small interfering RNA (siRNA) and short hairpin RNA (shRNA) technologies, we definitively show that ZEB1 is, indeed, a key player in maintaining EBV latency in some epithelial and B-lymphocytic cell lines. However, in other EBV-positive epithelial and B-cell lines, another zinc finger E-box-binding protein, ZEB2/SIP1, is the key player. Both ZEB1 and ZEB2 can bind Zp via the ZV element. In EBV-positive cells containing only ZEB1, knockdown of ZEB1 led to viral reactivation out of latency, with synthesis of EBV immediate-early and early lytic gene products. However, in EBV-positive cells containing both ZEBs, ZEB2, not ZEB1, was the primary ZEB family member bound to Zp. Knockdown of ZEB2, but not ZEB1, led to EBV lytic reactivation. Thus, we conclude that either ZEB1 or ZEB2 can play a central role in the maintenance of EBV latency, doing so in a cell-type-dependent manner.
Effect of an electronic nicotine delivery device (e cigarette) on desire to smoke and withdrawal, user preferences and nicotine delivery: randomised cross-over trial
Authors: Bullen C , McRobbie H , Thornley S , Glover M , Lin R , Laugesen M
Published: April 2010

Objectives

To measure the short-term effects of an electronic nicotine delivery device (“e cigarette”, ENDD) on desire to smoke, withdrawal symptoms, acceptability, pharmacokinetic properties and adverse effects.

Design

Single blind randomised repeated measures cross-over trial of the Ruyan V8 ENDD.

Setting

University research centre in Auckland, New Zealand.

Participants

40 adult dependent smokers of 10 or more cigarettes per day.

Interventions

Participants were randomised to use ENDDs containing 16 mg nicotine or 0 mg capsules, Nicorette nicotine inhalator or their usual cigarette on each of four study days 3 days apart, with overnight smoking abstinence before use of each product.

Main outcome measures

The primary outcome was change in desire to smoke, measured as “area under the curve” on an 11-point visual analogue scale before and at intervals over 1 h of use. Secondary outcomes included withdrawal symptoms, acceptability and adverse events. In nine participants, serum nicotine levels were also measured.

Results

Over 60 min, participants using 16 mg ENDD recorded 0.82 units less desire to smoke than the placebo ENDD (p=0.006). No difference in desire to smoke was found between 16 mg ENDD and inhalator. ENDDs were more pleasant to use than inhalator (p=0.016) and produced less irritation of mouth and throat (p<0.001). On average, the ENDD increased serum nicotine to a peak of 1.3 mg/ml in 19.6 min, the inhalator to 2.1 ng/ml in 32 min and cigarettes to 13.4 ng/ml in 14.3 min.

Conclusions

The 16 mg Ruyan V8 ENDD alleviated desire to smoke after overnight abstinence, was well tolerated and had a pharmacokinetic profile more like the Nicorette inhalator than a tobacco cigarette. Evaluation of the ENDD for longer-term safety, potential for long-term use and efficacy as a cessation aid is needed.

Trial registration

No.12607000587404, Australia and New Zealand Clinical Trials Register
Human electroencephalography and the tobacco industry: a review of internal documents
Authors: Panzano Vincent C , Wayne Geoffrey Ferris , Pickworth Wallace B , Connolly Gregory N
Published: April 2010

Objective

To determine the extent and implications of internal human electroencephalography (EEG) research conducted by the tobacco industry.

Methods

This study analysed internal documents that describe the results of human EEG studies conducted by tobacco manufacturers. Emphasis was placed on documents that pertain to the application of EEG to product evaluation efforts.

Results

Internal EEG research was used to determine dose-response relations and effective threshold levels for nicotine, emphasising the importance of form and mechanism of nicotine delivery for initiating robust central nervous system (CNS) effects. Internal studies also highlight the importance of human behaviour during naturalistic smoking, revealing neurophysiological markers of compensation during smoking of reduced nicotine cigarettes. Finally, internal research demonstrates the effectiveness of EEG for the evaluation of non-nicotine phenomena including smoke-component discrimination by smokers, classification of sensory characteristics and measurement of hedonics and other subjective effects.

Conclusions

Tobacco manufacturers successfully developed objective, EEG-based techniques to evaluate the influence of product characteristics on acceptance and use. Internal results suggest that complex interactions between pharmacological, sensory and behavioural factors mediate the brain changes that occur with smoking. These findings have implications for current proposals regarding the regulation of tobacco products and argue for the incorporation of objective measures of product effects when evaluating the health risks of new and existing tobacco products.
Public support in England for a total ban on the sale of tobacco products
Authors: Shahab Lion , West Robert
Published: April 2010

Background

This study aimed to determine the level of support for a sales ban on tobacco in England to provide a benchmark against which any changes over time can be assessed.

Methods

8735 people from England who participated in one of five monthly cross-sectional household surveys in 2008 were asked to indicate whether they would support the statement that ‘the government should work towards banning the sale of tobacco completely within the next 10 years’. In addition, sociodemographic and smoking characteristics were assessed.

Results

A substantial proportion of the total sample (44.5%; 95% CI 43.5% to 45.6%) would support a move towards a complete ban. While never smokers (OR 2.02; 95% CI 1.82 to 2.25) and ex-smokers (OR 1.41; 95% CI 1.21 to 1.65) were more likely to support this idea, even among current smokers, a third would favour moving towards a sales ban of tobacco products. Adjusting for other background characteristics, younger, female participants, those living in London and those from lower socioeconomic groups were most likely to support a ban. Among smokers, a higher cigarette consumption, smoking enjoyment and contentment with being a smoker were associated with opposition to a ban, while feeling uncomfortable being a smoker, wanting to be a non-smoker and being worried about future health consequences of smoking were associated with support for a ban.

Conclusion

Support for movement towards a ban on the sale of tobacco is higher than might be imagined. It is conceivable that as smoking prevalence falls further and smoking becomes more socially unacceptable, support might grow to a point where such a policy could become feasible.
The Effects of Tai Chi on Body Balance in Elderly People — A Review of Studies from the Early 21st Century
Authors: Maciaszek Janusz , Osiński W.
Published: April 2010

We performed a systematic review of studies regarding the effects of Tai Chi on the body balance of people older than 60 years. The Medline, SPORTDiscus and Academic Search Complete databases were searched for relevant studies published after the year 2000. Original randomized controlled trials, non-randomized controlled studies and observational studies were included in this review if they evaluated Tai Chi for the treatment of body balance disorder or fall prevention, and if they contained at least one of the following key words: Tai Chi, body balance, stability, elderly, old, training or posturography. These studies suggest that participation in Tai Chi may improve the body balance of elderly people; however, more information is needed to determine the extent to which Tai Chi is more effective than other methods, especially different types of physical exercise. In addition, subgroup analyses should be performed to determine the effects of Tai Chi in people with different physical characteristics. Directions for future research are discussed.
Effect of Electroacupuncture on St. 36 (Zusanli) and LI. 10 (Shousanli) Acupuncture Points on Heart Rate Variability
Authors: Chang Chi-Sen , Ko Chung-Wang , Lien Han-Chung , Chou Ming-Chih
Published: April 2010

Electrical stimulation of St. 36 (Zusanli) has been shown to enhance the regularity of gastric myoelectrical activity in healthy subjects. However, the underlying mechanism by which acupuncture alters gastric myoelectrical activity is still not clear. To elucidate the possible role of the autonomic nervous system in mediating the effect of acupuncture, we monitored heart rate variability (HRV), a widely used index of vagal discharge at the sinoatrial node of the heart before, during and after electroacupuncture. In this study, we applied two different frequencies (2 Hz and 100 Hz) of electrical stimulation at St. 36 (Zusanli) and LI. 10 (Shousanli) in 15 healthy volunteers. Low frequency (LF, sympathetic activity), high frequency (HF, vagal activity) and LF/HF ratio (sympathovagal balance) were analyzed and compared at the two different frequencies. The results showed an increase in the LF/HF ratio (indicating greater sympathetic activity) during the post-acupuncture period using 2 Hz of electrical stimulation at St. 36 (Zusanli). However, the overall change was not statistically significant. In addition, the power of LF and HF did not change significantly with electroacupuncture at St. 36 (Zusanli) and LI. 10 (Shousanli). In conclusion, applying 2 Hz or 100 Hz electroacupuncture at St. 36 (Zusanli) or LI. 10 (Shousanli) did not affect cardiovagal activity in normal volunteers. This phenomenon might be due to a difference in presentation in the autonomic nervous system between cardiac and abdominal vagal activity.
Protective Effect of Puerarin on Acute Alcoholic Liver Injury
Authors: Zhao Min , Du Yan-Qiu , Yuan Li , Wang Na-Na
Published: April 2010

To provide experimental and theoretical basis for the clinical application of Puerarin in acute alcohol poisoning, 30 Wistar rats were randomized into 3 groups as follows: (1) Group A (control) underwent normal sodium (N.S.) peritoneal injection (i.p.) and intragastric administration (i.g.); (2) Group B (alcohol) underwent an equivalent dosage of N.S. i.p. and 40% ethanol (8000 mg/kg. d).ig for 5 days; (3) Group C (Puerarin) underwent Puerarin 200 mg/kg. d. ip, and an equivalent dosage of ethanol for 5 days. The left lobes of livers were sampled, and the levels of MDA, SOD and GPX in plasma and liver homogenate were detected. The level of MDA in plasma and liver homogenate in the alcohol group was obviously higher than that in the control group (p < 0.05, respectively), while that in the Puerarin group was significantly lower than in the alcohol group (p < 0.05, respectively). The levels of SOD and GPX were opposite to that of MDA. Under a light microscope, the livers of the rats in the alcohol group showed unclear structure of hepatic lobules, stiffness of hepatic sinusoids, diffused lipid degeneration of hepatic cells, cellular swelling, and focal necrosis, while the structure remained clear in the Puerarin group. Under the electron microscope, lipid degeneration, cell organ decrease, enlargement of endoplasmic reticulum, reduced quantity of hepatins and swelling of mitochondria were observed in cells of the model group. However, the pathologistic changes were slight in the Puerarin group. In conclusion, Puerarin may have the function of inhibiting the oxidative stress induced by acute alcoholism.
Total Alkaloids of Sophora alopecuroides Increases the Expression of CD4+ CD25+ Tregs and IL-10 in Rats with Experimental Colitis
Authors: Zhou Yi , Wang He , Liang Lei , Zhao Wen-Chan , Chen Yan , Deng Hong-Zhu
Published: April 2010

Previous studies have demonstrated that the total alkaloids of Sophora alopecuroides (TASA), which contains many different ingredients like sophocarpine, matrine, oxymatrine, sophoridine, sophoramine, aloperine and cytosine, were able to protect colon against ulcers caused by 2,4,6-trinitrobenze sulphonic acid (TNBS)/ethanol treated models. In order to elucidate the mechanisms by which TASA exerts its effect of anti-inflammation and immunoregulation on rats with colitis, DAI (disease activity index) and histological grading of colitis were evaluated in the animal model. Moreover, the expression of CD4+CD25+ regulatory T cells (Tregs) and IL-10 in rats with experimental colitis were observed by FCM, ELISA and RT-PCR in this study. Results showed that TASA (15, 30 or 60 mg/kg/day) significantly up-regulated CD4+CD25+Tregs (P = 0.02, P = 0.02, P = 0.03) and IL-10 levels (ELISA: P = 0.03, P = 0.02, P = 0.00; RT-PCR: P = 0.04, P = 0.02, P = 0.01) respectively and decreased the DAI and histological grading of colitis in the peripheral blood (PB) and colon of rat colitis models (3.44 ± 1.53, 4.25 ± 1.27, 4.42 ± 1.24 and 3.50 ± 1.42, 4.05 ± 1.32, 4.51 ± 1.55 vs. 7.18 ± 1.32 and 7.38 ± 1.52, P < 0.05, P < 0.01, respectively). Most interestingly, a negative correlation was demonstrated between the expression of CD4+CD25+ Tregs and DAI (Pearson rPB = -0.677, P < 0.01; Pearson rCOLON = -0.663, P < 0.01, n = 60), or histological grading of colitis (Pearson rPB = -0.725, P < 0.01; Pearson rCOLON = -0.623, P < 0.01, n = 60). Simultaneously, a positive correlation existed between CD4+CD25+ Tregs and IL-10 cytokine (IL-10 mRNA) in the colon and PB of rats (Pearson rPB = 0.789, P < 0.01, n = 60; Pearson rCOLON = 0.678, P < 0.01, n = 60). These results may explain to some extent the mechanisms of TASA on treating rats with experimental colitis.
Effects of Toosendanin on Pregnancy and Uterine Immunity Alterations in Mice
Authors: Zhang Jian-Lou , Shi Wang-Yu , Zhong Wei , Ma Ai-Tuan , Wang Xiao-Dan , Zhao Yan-Tao , Wang Meng , Zhong Xiu-Hui
Published: April 2010

This study was conducted to explore the abortifacient effect and the mechanisms of the Chinese herbal medicine component toosendanin, and to elucidate the significance of the Th1 cytokines IFN-γ and TNF-α, CD4+ and CD8+ T lymphocytes in the occurrence of abortion. Graded doses of toosendanin were given by intraperitoneal injection (i.p.) to mice at day 5, 6, 7 of gestation. The levels of Th1 cytokines (IFN-γ, TNF-α) in serum and uterine tissues from mice sacrificed at day 8 were analyzed by enzyme linked immunosorbent assay (ELISA). Presence of T lymphocytes in endometrium was detected by immunohistochemistry. The results revealed that injection of toosendanin could produce a dose-dependent toxicity. The IFN-γ, TNF-α content in serum and uterine tissues were increased significantly. The CD4+ and CD8+ T lymphocytes were also increased in the endometrium of toosendanin treated groups. In conclusion, toosendanin is pregnancy-toxic to animals and it is relevant to the increased contents of IFN-γ, TNF-α and CD4+, CD8+ T lymphocytes.
Therapeutic Efficacy Evaluation of Curcumin on Human Oral Squamous Cell Carcinoma Xenograft Using Multimodalities of Molecular Imaging
Authors: Lin Yu-Chuan , Chen Hong-Wen , Kuo Yu-Cheng , Chang Ya-Fang , Lee Yi-Jang , Hwang Jeng-Jong
Published: April 2010

Oral squamous cell carcinoma (OSCC) is the most common malignancy of the oral cavity; however the treatment approaches are still unsatisfactory. We used a luciferase-transfected animal model to evaluate the therapeutic effects of curcumin.

Human oral squamous cell carcinoma SAS cell line was stably transfected with luc gene, named SAS/luc cells. For the in vivo study, they were inoculated subcutaneously to 6-week-old male NOD/SCID mice which were separated into four groups for intraperitoneal injection (i.p.) of curcumin: control, daily with 35 mg/kg, 70 mg/kg every 2 days, and 100 mg/kg every 3 days. We applied SAS/luc bearing animal model and bioluminescent imaging (BLI) to study the inhibition effect of curcumin on tumor growth.

The cytotoxic effect of curcumin on SAS/luc cells was mainly at G2/M phase and a significant dose dependent increase of the apoptotic SAS/luc cells as represented by sub-G1 phase was shown. Therapeutic efficacy evaluated by both caliper assay and BLI showed a significant difference between curcumin-treated mice and the controls (p < 0.01). The significant inhibition effects of curcumin on the proliferation and the growth of human OSCC are observed both in vitro and in vivo. No significant body weight change (i.e. within 20%) was observed in all SAS/luc-bearing mice with or without curcumin treatment.

This SAS/luc human OSCC bearing animal model combined with multimodalities of molecular imaging permits a sensitive and non-invasive approach to evaluate the therapeutic efficacy in vivo.
Effect ofDuabanga grandiflorafor Human Skin Cells
Authors: Tsukiyama Muneo , Sugita Takio , Kikuchi Hiroko , Yasuda Yoichi , Arashima Masaki , Okumura Hidenobu , Lhieochaiphant Sorasak , Shoyama Yukihiro
Published: April 2010

The purpose of this investigation was to evaluate the effects of Duabanga grandiflora (Sonneratiaceae), which has been used as a traditional Thai medicine on human skin cells. The leaf extract of D. grandiflora actively affected several human skin cells such as skin whitening, anti-aging and anti-inflammation. It became evident that the extract stimulated the production of type III collagen. The crude extract was fractionated and analyzed for stimulation of type III collagen production, and finally by HPLC to isolate an active compound which was determined to be eugeniin by EI-mass,13C NMR,1H NMR and acidic hydrolysis. Eugeniin has strong dose dependent activity for type III collagen production, with this being the first example of stimulation activity for type III collagen production.
CONSTRUCTING SEIFERT SURFACES FROM n-BRIDGE LINK PROJECTIONS
Authors: LICATA JOAN E.
Published: April 2010

This paper presents a new algorithm for constructing Seifert surfaces from n-bridge projections of links. The algorithm, 𝔄, produces minimal complexity surfaces for large classes of braids and alternating links. In addition, we consider a family of knots for which canonical genus is strictly greater than genus, (gc(K) > g(K)), and show that 𝔄 builds surfaces realizing the knot genus g(K). We also present a generalization of Seifert's algorithm which constructs surfaces representing arbitrary relative second homology classes in a link complement.