bluuebunny/crossref_metadata_2025 huggingface dataset:
Role for Complement in Development ofHelicobacter-Induced Gastritis in Interleukin-10-Deficient Mice
Authors: Ismail Hanan F. , Zhang Juan , Lynch Richard G. , Wang Yi , Berg Daniel J.
Published: November 2003

The mechanisms by which the immune response can eradicate gastricHelicobacterinfection are unknown. We hypothesized thatHelicobacter-induced activation of the complement system could promote both inflammation and eradication ofHelicobacterfrom the stomach. In vitro studies demonstrated thatHelicobacter felisactivates complement in normal mouse serum but not in serum from Rag2−/−mice, indicating thatH. felisactivates complement through the classical pathway. Next, we infected complement-depleted wild-type control and interleukin-10-deficient (IL-10−/−) mice withH. felis. Helicobacterinfection of wild-type mice elicited a mild, focal gastritis and did not alter serum complement levels. Infection of IL-10−/−mice withH. feliselicited severe gastritis. After the initial colonization, the IL-10−/−mice completely clearedHelicobacterfrom the stomach by day 8. In contrast to wild-type mice,H. felis-infected IL-10−/−mice had a marked increase in serum complement levels. Complement depletion of wild-type mice did not affect the intensity of gastric inflammation or the extent ofHelicobactercolonization compared to that for the wild-type control mice. In contrast, complement depletion ofHelicobacter-infected IL-10−/−mice decreased the severity of gastritis, decreased theHelicobacter-induced infiltration of neutrophils into the stomach, and delayed the clearance of bacteria. In vitro studies of stimulated splenocytes and neutrophils from IL-10−/−mice produced a twofold increase in complement production compared to that for wild-type mice. Pretreatment with IL-10 inhibited this increase. These studies identify a role for complement in the local immune response to gastricHelicobacterin IL-10−/−mice and suggest a role for IL-10 in the regulation of complement production.
Haemophilusducreyi
Requires an Intact
flp
Gene Cluster for VirulenceinHumans
Authors: Spinola Stanley M. , Fortney Kate R. , Katz Barry P. , Latimer Jo L. , Mock Jason R. , Vakevainen Merja , Hansen Eric J.
Published: November 2003

An
intact
Haemophilus ducreyi flp
operon is essential for
microcolony formation in vitro.
tadA
is the 9th of 15 genes in
the operon and has homology to NTPases of type IV secretion systems.
Fifteen human volunteers were experimentally infected with both
H.
ducreyi
35000HP and the
tadA
mutant, 35000HP.400. Papules
developed at similar rates at sites inoculated with the mutant and
parent, while pustules formed at 36.4% of parent sites and at
0% of mutant sites (
P
= 0.001). Compared to
35000HP, 35000HP.400 had only a modest but significant reduction in
lesion scores in the temperature-dependent rabbit model of chancroid.
These data suggest that proteins secreted by the
flp
locus are
required for full expression of virulence by
H. ducreyi
in
humans but have less of a role in virulence in an animal model of
infection.
Identification of B- and T-Cell Epitopes within the Fibronectin-Binding Domain of the SfbI Protein ofStreptococcus pyogenes
Authors: Schulze Kai , Medina Eva , Chhatwal Gursharan S. , Guzmán Carlos A.
Published: November 2003

The fibronectin-binding repeats of the SfbI protein ofStreptococcus pyogenesconstitute the minimal domain able to confer protection against lethal infection. We investigated the presence of B- and T-cell epitopes within this region in congenic mice. One linear B-cell epitope was recognized by BALB/b and BALB/k mice, whereas two epitopes were found in BALB/c animals. A unique T-cell epitope was recognized by all three mouse strains. All identified epitopes clustered in a 30-amino-acid fragment. These results suggest that this polypeptide may be suitable for incorporation into a polyepitope-based vaccine formulation againstS. pyogenes.
Immunogenicityof
Borrelia burgdorferi
Arthritis-RelatedProtein
Authors: Feng Sunlian , Hodzic Emir , Freet Kimberly , Barthold Stephen W.
Published: November 2003

Immunization
against arthritis-related protein (Arp) elicits antibody in mice that
resolves arthritis but is not protective against challenge with
Borrelia burgdorferi
. In mice immunized against Arp, an
unrelated 37-kDa protein (P37-42), outer surface protein A (OspA), or
glutathione
S
-transferase (GT) and then challenged by syringe
or tick, only OspA conferred protection. Passive transfer of Arp
antiserum into infected SCID mice induced arthritis resolution, but
antisera to P37-42, OspA, GT, or six overlapping Arp peptide fragments
did not. Results suggest that the arthritis-resolving immunogenicity is
specific to Arp, but the relevant epitopes may be
conformational.
Abortive Potency ofChlamydophila abortusin Pregnant Mice Is Not Directly Correlated with Placental and Fetal Colonization Levels
Authors: Bouakane Amel , Benchaîeb Ilhem , Rodolakis Annie
Published: November 2003

Abortion, placental and fetal colonization, and levels of gamma interferon were analyzed for fourChlamydophila abortusstrains presenting antigenic variations in a mouse model. Expression of virulence of these strains varied and indicated that abortion was not directly related to the number of bacteria in the placenta, and thus, other factors may have an important role in activating the abortion process.
Mycobacterium aviumsubsp. paratuberculosisInfection Causes Suppression of RANTES, Monocyte Chemoattractant Protein 1, and Tumor Necrosis Factor Alpha Expression in Peripheral Blood of Experimentally Infected Cattle
Authors: Buza Joram J. , Mori Yasuyuki , Bari Abusaleh M. , Hikono Aodon-geril Hirokazu , Hirayama Sachiyo , Shu Yujing , Momotani Eiichi
Published: November 2003

Blood from cattle with subclinicalMycobacterium aviumsubsp.paratuberculosisinfection was stimulated withM. aviumsubsp.paratuberculosisantigens, and expression of interleukin-1β (IL-1β), tumor necrosis factor alpha (TNF-α), RANTES, monocyte chemoattractant protein 1 (MCP-1), and IL-8 was measured. Expression of TNF-α, RANTES, and MCP-1 was lower in infected than in uninfected cattle. The reduced response may weaken protective immunity and perpetuate infection.
Enhanced Phagocytosis ofCandidaSpecies Mediated by Opsonization with a Recombinant Human Antibody Single-Chain Variable Fragment
Authors: Wellington Melanie , Bliss Joseph M. , Haidaris Constantine G.
Published: November 2003

Specific antibody opsonization significantly enhances the level of phagocytosis ofCandidain the absence of complement. Furthermore, we have described a system using a recombinant human antibody single-chain variable fragment that allows a comparative study of phagocytosis of multipleCandidaspecies opsonized via a common antigen.
Stability and Morphology Comparisons of Self-AssembledVirus-Like Particles from Wild-Type and Mutant Human Hepatitis B VirusCapsidProteins
Authors: Newman Margaret , Suk Fat-Moon , Cajimat Maria , Chua Pong Kian , Shih Chiaho
Published: November 2003

Instead
of displaying the wild-type selective export of virions containing
mature genomes, human hepatitis B virus (HBV) mutant I97L, changing
from an isoleucine to a leucine at amino acid 97 of HBV core antigen
(HBcAg), lost the high stringency of selectivity in genome maturity
during virion export. To understand the structural basis of this
so-called “immature secretion” phenomenon, we compared
the stability and morphology of self-assembled capsid particles from
the wild-type and mutant I97L HBV, in either full-length (HBcAg1-183)
or truncated core protein contexts (HBcAg1-149 and HBcAg1-140). Using
negative staining and electron microscopy, full-length particles appear
as “thick-walled” spherical particles with little
interior space, whereas truncated particles appear as“
thin-walled” spherical particles with a much larger
inner space. We found no significant differences in capsid stability
between wild-type and mutant I97L particles under denaturing pH and
temperature in either full-length or truncated core protein contexts.
In general, HBV capsid particles (HBcAg1-183, HBcAg1-149, and
HBcAg1-140) are very robust but will dissociate at pH 2 or 14, at
temperatures higher than 75°C, or in 0.1% sodium dodecyl
sulfate (SDS). An unexpected upshift banding pattern of the SDS-treated
full-length particles during agarose gel electrophoresis is most likely
caused by disulfide bonding of the last cysteine of HBcAg. HBV capsids
are known to exist in natural infection as dimorphic T=3 or
T=4 icosahedral particles. No difference in the ratio between
T=3 (78%) and T=4 particles (20.3%) are
found between wild-type HBV and mutant I97L in the context of
HBcAg1-140. In addition, we found no difference in capsid stability
between T=3 and T=4 particles successfully separated by
using a novel agarose gel electrophoresis
procedure.
Intra- and Intermolecular Disulfide Bonds of theGP2bGlycoprotein of Equine Arteritis Virus: Relevance forVirus Assembly andInfectivity
Authors: Wieringa Roeland , de Vries Antoine A. F. , Post Sabine M. , Rottier Peter J. M.
Published: November 2003

Equine arteritis virus(EAV) is an enveloped, positive-strand RNA virus belonging to the familyArteriviridaeof the orderNidovirales. EAV virions contain six different envelope proteins. The glycoprotein GP5(previously named GL) and the unglycosylated membrane protein M are the major envelope proteins, while the glycoproteins GP2b(previously named GS), GP3, and GP4are minor structural proteins. The unglycosylated small hydrophobic envelope protein E is present in virus particles in intermediate molar amounts compared to the other transmembrane proteins. The GP5and M proteins are both essential for particle assembly. They occur as covalently linked heterodimers that constitute the basic protein matrix of the envelope. The GP2b, GP3, and GP4proteins occur as a heterotrimeric complex in which disulfide bonds play an important role. The function of this complex has not been established yet, but the available data suggest it to be involved in the viral entry process. Here we investigated the role of the four cysteine residues of the mature GP2bprotein in the assembly of the GP2b/GP3/GP4complex. Open reading frames encoding cysteine-to-serine mutants of the GP2bprotein were expressed independently or from a full-length infectious EAV cDNA clone. The results of these experiments support a model in which the cysteine residue at position 102 of GP2bforms an intermolecular cystine bridge with one of the cysteines of the GP4protein, while the cysteine residues at positions 48 and 137 of GP2bare linked by an intrachain disulfide bond. In this model, another cysteine residue in the GP4protein is responsible for the covalent association of GP3with the disulfide-linked GP2b/GP4heterodimer. In addition, our data highlight the importance of the correct association of the minor EAV envelope glycoproteins for their efficient incorporation into viral particles and for virus infectivity.
Reduction of Natural Adenovirus Tropism to Mouse Liverby Fiber-Shaft Exchange in Combination with both CAR- andαv Integrin-BindingAblation
Authors: Koizumi Naoya , Mizuguchi Hiroyuki , Sakurai Fuminori , Yamaguchi Teruhide , Watanabe Yoshiteru , Hayakawa Takao
Published: November 2003

The
primary receptor, the coxsackievirus and adenovirus
receptor (CAR), and the secondary receptor, αv
integrins, are the tropism determinants of adenovirus (Ad) type 5.
Inhibition of the interaction of both the fiber with CAR and the penton
base with the αv integrin appears to be crucial to the
development of targeted Ad vectors, which specifically transduce a
given cell population. In this study, we developed Ad vectors with
ablation of both CAR and αv integrin binding by
mutating the fiber knob and the RGD motif of the penton base. We also
replaced the fiber shaft domain with that derived from Ad type 35. High
transduction efficiency in the mouse liver was suppressed approximately
130- to 270-fold by intravenous administration of the double-mutant Ad
vectors, which mutated two domains each of the fiber knob and shaft and
the RGD motif of the penton base compared with those of conventional Ad
vectors (type 5). Most significantly, the triple-mutant Ad vector
containing the fiber knob with ablation of CAR binding ability, the
fiber shaft of Ad type 35, and the penton base with a deletion of the
RGD motif mediated a >30,000-fold lower level of mouse liver
transduction than the conventional Ad vectors. This triple-mutant Ad
vector also mediated reduced transduction in other organs (the spleen,
kidney, heart, and lung). Viral DNA analysis showed that systemically
delivered triple-mutant Ad vector was primarily taken up by liver
nonparenchymal cells and that most viral DNAs were easily degraded,
resulting in little gene expression in the liver. These results suggest
that the fiber knob, fiber shaft, and RGD motif of the penton base each
plays an important role in Ad vector-mediated transduction to the mouse
liver and that the triple-mutant Ad vector exhibits little tropism to
any organs and appears to be a fundamental vector for targeted Ad
vectors.
Barrier-to-Autointegration Factor BAF Binds p55 Gagand Matrix and Is a Host Component of Human ImmunodeficiencyVirus Type 1Virions
Authors: Mansharamani Malini , Graham David R. M. , Monie Daphne , Lee Kenneth K. , Hildreth James E. K. , Siliciano Robert F. , Wilson Katherine L.
Published: November 2003

Barrier-to-autointegration
factor (BAF) is a conserved human chromatin protein exploited by
retroviruses. Previous investigators showed that BAF binds
double-stranded DNA nonspecifically and is a host component of
preintegration complexes (PICs) isolated from cells infected with human
immunodeficiency virus type 1 (HIV-1) or Moloney murine leukemia virus.
BAF protects PIC structure and stimulates the integration of
salt-stripped PICs into target DNA in vitro. PICs are thought to
acquire BAF from the cytoplasm during infection. However, we identified
two human tissues (of 16 tested) in which BAF mRNA was not detected:
thymus and peripheral blood leukocytes, which are enriched in
CD4
+
T lymphocytes and macrophage precursors,
respectively. BAF protein was detected in activated but not resting
CD4
+
T lymphocytes; thus, if BAF were essential for
PIC function, we hypothesized that virions might “bring their
own BAF.” Supporting this model, BAF copurified with HIV-1
virions that were digested with subtilisin to remove microvesicle
contaminants, and BAF was present in approximately zero to three copies
per virion. In three independent assays, BAF bound directly to both p55
Gag (the structural precursor of HIV-1 virions) and its cleaved
product, matrix. Using lysates from cells overexpressing Gag,
endogenous BAF and Gag were coimmunoprecipitated by antibodies against
Gag. Purified recombinant BAF had low micromolar affinities (1.1 to 1.4μ
M) for recombinant Gag and matrix. We conclude that BAF is
present at low levels in incoming virions, in addition to being
acquired from the cytoplasm of newly infected cells. We further
conclude that BAF might contribute to the assembly or activity of HIV-1
PICs through direct binding to matrix, as well as
DNA.
An InternalRibosome Entry Site Directs Translation of the Murine Gammaherpesvirus68 MK3 Open ReadingFrame
Authors: Coleman Heather M. , Brierley Ian , Stevenson Philip G.
Published: November 2003

The
gammaherpesviruses characteristically drive the proliferation of
latently infected lymphocytes. The murine gammaherpesvirus 68 (MHV-68)
MK3 protein contributes to this process in vivo by evading
CD8
+
-T-cell recognition during latency, as well as
during lytic infection. We analyzed some of the molecular mechanisms
that control MK3 expression. No dedicated MK3 mRNA was detected.
Instead, the MK3 open reading frame (ORF) was transcribed as part of a
bicistronic mRNA, downstream of a previously unidentified ORF, 13M. The
13M/MK3 promoter appeared to extend approximately 1 kb 5′ of
the transcription start site and included elements both dependent on
and independent of the ORF50 lytic transactivator. MK3 was translated
from the bicistronic transcript by virtue of an internal ribosome entry
site (IRES) element. RNA structure mapping identified two stem-loops
between 13M and MK3 that were sufficient for IRES activity in a
bicistronic reporter plasmid and a third stem-loop just within the MK3
coding sequence, with a subtler, perhaps regulatory role. Overall,
translation of the MHV-68 MK3 bore a striking resemblance to that of
the Kaposi's sarcoma-associated herpesvirus vFLIP, suggesting that
IRES elements are a common theme of latent gammaherpesvirus immune
evasion in proliferating
cells.
Chromosomal Integration and Homologous Gene Targetingby Replication-Incompetent Vectors Based on the Autonomous ParvovirusMinute Virus ofMice
Authors: Hendrie Paul C. , Hirata Roli K. , Russell David W.
Published: November 2003

The
molecular mechanisms responsible for random integration and gene
targeting by recombinant adeno-associated virus (AAV) vectors are
largely unknown, and whether vectors derived from autonomous
parvoviruses transduce cells by similar pathways has not been
investigated. In this report, we constructed vectors based on the
autonomous parvovirus minute virus of mice (MVM) that were designed to
introduce a neomycin resistance expression cassette (
neo
) into
the X-linked human hypoxanthine phosphoribosyl transferase
(
HPRT
) locus. High-titer, replication-incompetent MVM vector
stocks were generated with a two-plasmid transfection system that
preserved the wild-type characteristic of packaging only one DNA
strand. Vectors with inserts in the forward or reverse orientations
packaged noncoding or coding strands, respectively. In human HT-1080
cells, MVM vector random integration frequencies
(

neo
+

colonies) were comparable to those
obtained with AAV vectors, and no difference was observed for noncoding
and coding strands.
HPRT
gene-targeting frequencies
(
HPRT
mutant colonies) were lower with MVM
vectors, and the noncoding strand frequency was threefold greater than
that of the coding strand. Random integration and gene-targeting events
were confirmed by Southern blot analysis of G418- and 6-thioguanine
(6TG)-resistant clones. In separate experiments, correction of an
alkaline phosphatase (AP) gene by gene targeting was nine times more
effective with a coding strand vector. The data suggest that
single-stranded parvoviral vector genomes are substrates for gene
targeting and possibly for random integration as
well.
Protection against Murine Leukemia Virus-InducedSpongiform Myeloencephalopathy in Mice Overexpressing Bcl-2 butNot in Mice Deficient for Interleukin-6, Inducible NitricOxide Synthetase, ICE, Fas, Fas Ligand, or TNF-R1Genes
Authors: Jolicoeur Paul , Hu Chunyan , Mak Tak W. , Martinou Jean-Claude , Kay Denis G.
Published: November 2003

Some murine leukemia viruses (MuLVs), among them Cas-Br-E andts-1 MuLVs, are neurovirulent, inducing spongiform myeloencephalopathy and hind limb paralysis in susceptible mice. It has been shown that theenvgene of these viruses harbors the determinant of neurovirulence. It appears that neuronal loss occurs by an indirect mechanism, since the target motor neurons have not been found to be infected. However, the pathogenesis of the disease remains unclear. Several lymphokines, cytokines, and other cellular effectors have been found to be aberrantly expressed in the brains of infected mice, but whether these are required for the development of the neurodegenerative lesions is not known. In an effort to identify the specific effectors which are indeed required for the initiation and/or development of spongiform myeloencephalopathy, we inoculated gene-deficient (knockout [KO]) mice withts-1 MuLV. We show here that interleukin-6 (IL-6), inducible nitric oxide synthetase (iNOS), ICE, Fas, Fas ligand (FasL), and TNF-R1 KO mice still develop signs of disease. However, transgenic mice overexpressing Bcl-2 in neurons (NSE/Bcl-2) were largely protected from hind limb paralysis and had less-severe spongiform lesions. These results indicate that motor neuron death occurs in this disease at least in part by a Bcl-2-inhibitable pathway not requiring the ICE, iNOS, Fas/FasL, TNF-R1, and IL-6 gene products.
Three-Dimensional Structures of the A, B, and CCapsids of Rhesus Monkey Rhadinovirus: Insights into GammaherpesvirusCapsid Assembly, Maturation, and DNAPackaging
Authors: Yu Xue-Kui , O'Connor Christine M. , Atanasov Ivo , Damania Blossom , Kedes Dean H. , Zhou Z. Hong
Published: November 2003

Rhesus
monkey rhadinovirus (RRV) exhibits high levels of sequence homology to
human gammaherpesviruses, such as Kaposi's sarcoma-associated
herpesvirus, and grows to high titers in cell cultures, making it a
good model system for studying gammaherpesvirus capsid structure and
assembly. We have purified RRV A, B, and C capsids, thus for the first
time allowing direct structure comparisons by electron cryomicroscopy
and three-dimensional reconstruction. The results show that the shells
of these capsids are identical and are each composed of 12 pentons, 150
hexons, and 320 triplexes. Structural differences were apparent inside
the shells and through the penton channels. The A capsid is empty, and
its penton channels are open. The B capsid contains a scaffolding core,
and its penton channels are closed. The C capsid contains a DNA genome,
which is closely packaged into regularly spaced density shells (25Å
apart), and its penton channels are open. The different
statuses of the penton channels suggest a functional role of the
channels during capsid maturation, and the overall structural
similarities of RRV capsids to alphaherpesvirus capsids suggest a
common assembly and maturation pathway. The RRV A capsid reconstruction
at a 15-Å resolution, the best achieved for gammaherpesvirus
particles, reveals overall structural similarities to alpha- and
betaherpesvirus capsids. However, the outer regions of the capsid,
including densities attributed to the Ta triplex and the small
capsomer-interacting protein (SCIP or ORF65), exhibit prominent
differences from their structural counterparts in alphaherpesviruses.
This structural disparity suggests that SCIP and the triplex, together
with tegument and envelope proteins, confer structural and potentially
functional specificities to alpha-, beta-, and
gammaherpesviruses.
Mechanismof Action and Antiviral Activity of Benzimidazole-Based AllostericInhibitors of the Hepatitis C Virus RNA-Dependent RNAPolymerase
Authors: Tomei Licia , Altamura Sergio , Bartholomew Linda , Biroccio Antonino , Ceccacci Alessandra , Pacini Laura , Narjes Frank , Gennari Nadia , Bisbocci Monica , Incitti Ilario , Orsatti Laura , Harper Steven , Stansfield Ian , Rowley Michael , De Francesco Raffaele , Migliaccio Giovanni
Published: November 2003

The RNA-dependent RNA polymerase of hepatitis C virus (HCV) is the catalytic subunit of the viral RNA amplification machinery and is an appealing target for the development of new therapeutic agents against HCV infection. Nonnucleoside inhibitors based on a benzimidazole scaffold have been recently reported. Compounds of this class are efficient inhibitors of HCV RNA replication in cell culture, thus providing attractive candidates for further development. Here we report the detailed analysis of the mechanism of action of selected benzimidazole inhibitors. Kinetic data and binding experiments indicated that these compounds act as allosteric inhibitors that block the activity of the polymerase prior to the elongation step. Escape mutations that confer resistance to these compounds map to proline 495, a residue located on the surface of the polymerase thumb domain and away from the active site. Substitution of this residue is sufficient to make the HCV enzyme and replicons resistant to the inhibitors. Interestingly, proline 495 lies in a recently identified noncatalytic GTP-binding site, thus validating it as a potential allosteric site that can be targeted by small-molecule inhibitors of HCV polymerase.
SignalsThat Dictate Nuclear Localization of Human Papillomavirus Type 16Oncoprotein E6 in LivingCells
Authors: Tao Mingfang , Kruhlak Michael , Xia Shuhua , Androphy Elliot , Zheng Zhi-Ming
Published: November 2003

Human
papillomavirus (HPV) type 16 E6 (16E6) is an oncogenic, multifunctional
nuclear protein that induces p53 degradation and perturbs normal cell
cycle control, leading to immortalization and transformation of
infected keratinocytes and epithelial cells. Although it is unclear how
16E6 disrupts the epigenetic profile of host genes, its presence in the
nucleus is a key feature. The present report describes intrinsic
properties of 16E6 that influence its nuclear import in living cells.
When the coding region of full-length 16E6 was inserted in frame into
the C terminus of green fluorescent protein (GFP), it effectively
prevented the 16E6 pre-mRNA from being spliced and led to the
expression of a GFP-E6 fusion which localized predominantly
to the nucleus. Further studies identified three novel nuclear
localization signals (NLSs) in 16E6 that drive the protein to
accumulate in the nucleus. We found that all three NLS sequences are
rich in positively charged basic residues and that point mutations in
these key residues could abolish the retention of 16E6 in the nucleus
as well as the p53 degradation and cell immortalization activities of
the protein. When inserted into corresponding regions of low-risk HPV
type 6 E6, the three NLS sequences described for 16E6 functioned
actively in converting the normally cytoplasmic HPV type 6 E6 into a
nuclear protein. The separate NLS sequences, however, appear to play
different roles in nuclear import and retention of HPV E6. The
discovery of three unique NLS sequences in 16E6 provides new insights
into the nuclear association of 16E6 which may reveal other novel
activities of this important oncogenic
protein.
Association of Adenovirus with the MicrotubuleOrganizingCenter
Authors: Bailey Christopher J. , Crystal Ronald G. , Leopold Philip L.
Published: November 2003

Adenoviruses
(Ad) must deliver their genomes to the nucleus of the target cell to
initiate an infection. Following entry into the cell and escape from
the endosome, Ad traffics along the microtubule cytoskeleton toward the
nucleus. In the final step in Ad trafficking, Ad must leave the
microtubule and establish an association with the nuclear envelope. We
hypothesized that in cells lacking a nucleus, the capsid moves to and
associates with the microtubule organizing center (MTOC). To test this
hypothesis, we established an experimental system to examine Ad
trafficking in enucleated cells compared to Ad trafficking in intact,
mock-enucleated cells. Enucleation of a monolayer of A549 human lung
epithelial cells was accomplished by depolymerization of the actin
cytoskeleton followed by centrifugation. Upon infection of enucleated
cells with Cy3-labeled Ad, the majority of Ad capsid trafficked to a
discrete, centrally located site which colocalized with pericentrin, a
component of the MTOC. MTOC-associated Ad had escaped from endosomes
and thus had direct access to MTOC components. Ad localization at this
site was sensitive to the microtubule-depolymerizing agent nocodazole,
but not to the microfilament-depolymerizing agent cytochalasin B,
indicating that intact microtubules were required to maintain the
localization with the MTOC. Ad localization to the MTOC in the
enucleated cells was stable, as demonstrated by continuing Ad
localization with pericentrin for more than 5 h after
infection, a strong preference for Ad arrival at rather than Ad
departure from the MTOC, and minimal redistribution of Ad between MTOCs
within a single cell. In summary, the data demonstrate that the Ad
capsid establishes a stable interaction with the MTOC when a nucleus is
not present, suggesting that dissociation of Ad from microtubules
likely requires nuclear
factors.
Novel System for the Simultaneous Analysis ofGeminivirus DNA Replication and Plant Interactions inNicotianabenthamiana
Authors: Hong Yiguo , Stanley John , van Wezel Rene
Published: November 2003

The origin of replication of African cassava mosaic virus (ACMV) and a gene expression vector based onPotato virus Xwere exploited to devise an in planta system for functional analysis of the geminivirus replication-associated protein (Rep) in transgenicNicotiana benthamianaline pOri-2. This line contains an integrated copy of a tandem repeat of the ACMV origin of replication flanking nonviral sequences that can be mobilized and replicated by Rep as an episomal replicon. A Rep-GFP fusion protein can also mobilize and amplify the replicon, facilitating Rep detection in planta. The activity of Rep and its mutants, Rep-mediated host response, and the correlation between Rep intracellular localization and biological functions could be effectively assessed by using this in planta system. Our results indicate that modification of amino acid residues R2, R5, R7and K11or H56, L57and H58prevent Rep function in replication. This defect correlates with possible loss of Rep nuclear localization and inability to trigger the host defense mechanism resembling a hypersensitive response.
Characterizationof Murine Leukemia Virus Restriction inMammals
Authors: Besnier Caroline , Ylinen Laura , Strange Benjamin , Lister Adrian , Takeuchi Yasuhiro , Goff Stephen P. , Towers Greg J.
Published: November 2003

Restriction
of murine leukemia virus (MLV) was examined in cells from a range of
mammals. All nonmurine restrictions were saturable blocks to N-tropic
MLV infection, and several were prior to reverse transcription. We
demonstrate restriction in cells from bat and show that if we express
the murine restriction factor Fv1 in human cells, then Fv1, not the
human host, defines the stage at which infection is
blocked.
HumanImmunodeficiency Virus Type 1 DNA Nuclear Import and Integration AreMitosis Independent in CyclingCells
Authors: Katz Richard A. , Greger James G. , Boimel Pamela , Skalka Anna Marie
Published: November 2003

An
essential step in human immunodeficiency virus type 1 (HIV-1)
replication is the movement of the viral preintegration complex from
the cytoplasm into the nucleus. The pathway(s) and timing for HIV-1 DNA
nuclear entry in cycling cells have not been established. Here, we show
that if cycling cells are infected before S phase, viral DNA can be
integrated prior to passage of the host DNA replication fork through
the integration site, as indicated by stable inheritance in both
daughter cells. We conclude that efficient nuclear entry can occur
independently of mitotic nuclear disassembly in cycling
cells.
FolateReceptor Alpha and Caveolae Are Not Required for Ebola VirusGlycoprotein-Mediated ViralInfection
Authors: Simmons Graham , Rennekamp Andrew J. , Chai Ning , Vandenberghe Luk H. , Riley James L. , Bates Paul
Published: November 2003

Folate
receptor alpha (FRα) has been described as a factor involved in
mediating Ebola virus entry into cells
(6). Furthermore, it was
suggested that interaction with FRα results in internalization
and subsequent viral ingress into the cytoplasm via caveolae
(9). Descriptions of
cellular receptors for Ebola virus and its entry mechanisms are of
fundamental importance, particularly with the advent of vectors bearing
Ebola virus glycoprotein (GP) being utilized for gene transfer into
cell types such as airway epithelial cells. Thus, the ability of
FRα to mediate efficient entry of viral pseudotypes carrying GP
was investigated. We identified cell lines and primary cell types such
as macrophages that were readily infected by GP pseudotypes despite
lacking detectable surface FRα, indicating that this receptor
is not essential for Ebola virus infection. Furthermore, we find that
T-cell lines stably expressing FRα are not infectible,
suggesting that FRα is also not sufficient to mediate entry.
T-cell lines lack caveolae, the predominant route of
FRα-mediated folate metabolism. However, the coexpression of
FRα with caveolin-1, the major structural protein of caveolae,
was not able to rescue infectivity in a T-cell line. In addition, other
cell types lacking caveolae are fully infectible by GP pseudotypes.
Finally, a panel of ligands to and soluble analogues of FRα
were unable to inhibit infection on a range of cell lines, questioning
the role of FRα as an important factor for Ebola virus
entry.
Integrinα3β1 Is an Alternative Cellular Receptor for AdenovirusSerotype5
Authors: Salone Barbara , Martina Yuri , Piersanti Stefania , Cundari Enrico , Cherubini Gioia , Franqueville Laure , Failla Cristina M. , Boulanger Pierre , Saggio Isabella
Published: November 2003

Many
adenovirus serotypes enter cells by high-affinity binding to the
coxsackievirus-adenovirus receptor (CAR) and integrin-mediated
internalization. In the present study, we analyzed the possible
receptor function of α3β1 for adenovirus serotype 5
(Ad5). We found that penton base and integrin α3β1
could interact in vitro. In vivo, both Ad5-cell binding and
virus-mediated transduction were inhibited in the presence of
anti-α3 and anti-β1 function-blocking antibodies, and
this occurred in both CAR-positive and CAR-negative cell lines. Peptide
library screenings and data from binding experiments with wild-type and
mutant penton base proteins suggest that the Arg-Gly-Asp (RGD) in the
penton base protein, the best known integrin binding motif, is only
part of the binding interface with α3β1, which involved
multiple additional contact
sites.
Identification of α-Chloro Fatty Aldehydes and Unsaturated Lysophosphatidylcholine Molecular Species in Human Atherosclerotic Lesions
Authors: Thukkani Arun K. , McHowat Jane , Hsu Fong-Fu , Brennan Marie-Luise , Hazen Stanley L. , Ford David A.
Published: November 2003

Background—

A role for myeloperoxidase (MPO) as a mediator of coronary artery disease and acute coronary syndromes has recently received considerable attention. Although active MPO and hypochlorite-modified proteins and peptides have been detected in human atherosclerotic lesions, detection of novel chlorinated oxidized lipid species with proatherogenic properties in vivo has not yet been reported. In this study we show that MPO-generated reactive chlorinating species promote selective oxidative cleavage of plasmalogens, liberating α-chloro fatty aldehydes and unsaturated lysophosphatidylcholine in human atherosclerotic lesions.

Methods and Results—

Stable isotope dilution gas chromatography–mass spectrometry methods were used to identify and quantitate the α-chloro fatty aldehyde, 2-chlorohexadecanal, in atherosclerotic versus normal human aorta. Compared with normal aorta, 2-chlorohexadecanal levels were elevated more than 1400-fold in atherosclerotic tissues. Parallel electrospray ionization mass spectrometry studies confirmed 34- and 20-fold increases in the plasmalogen cooxidation products, unsaturated lysophosphatidylcholine molecular species containing linoleic and arachidonic acid, respectively, within atherosclerotic compared with normal aorta. Unsaturated lysophosphatidylcholine containing docosahexaenoic acid was also detected in atherosclerotic but not in normal aorta. Exposure of primary human coronary artery endothelial cells to plasmalogen-derived lysophosphatidylcholine molecular species produced marked increases in P-selectin surface expression.

Conclusions—

The present studies demonstrate that plasmalogens are attacked by MPO-derived reactive chlorinating species within human atheroma. The resultant species formed, α-chloro fatty aldehydes and unsaturated lysophospholipids, possess proatherogenic properties, as shown by induction of P-selectin surface expression in primary human coronary artery endothelial cells.
Blood Pressure, C-Reactive Protein, and Risk of Future Cardiovascular Events
Authors: Blake Gavin J. , Rifai Nader , Buring Julie E. , Ridker Paul M
Published: November 2003

Background—

Accumulating data suggest a link between blood pressure and vascular inflammation.

Methods and Results—

We examined the relationship between blood pressure, C-reactive protein (CRP), and incident first cardiovascular events among 15 215 women followed prospectively over a median of 8.1 years. In cross-sectional analyses at baseline, median levels of CRP for women with blood pressure <120/75, 120 to 129/75 to 84, 130 to 139/85 to 89, 140 to 159/90 to 94, and ≥160/95 mm Hg were 0.96, 1.42, 2.20, 2.82, and 3.34 mg/L, respectively (
P
for trend <0.0001). Increasing categories of blood pressure were significant predictors of CRP levels at baseline. In prospective analyses, both elevated CRP levels (≥3 mg/L) and increasing categories of blood pressure were independent determinants of future cardiovascular events, and CRP had incremental prognostic value at all levels of blood pressure. The adjusted hazard ratio for women with blood pressure ≥160/95 mm Hg and CRP levels ≥3 mg/L was 8.31 (95% CI, 4.44 to 15.55,
P
<0.0001) compared with those with blood pressure <120/75 and CRP levels <3 mg/L. After participants had been divided into 4 groups on the basis of CRP levels (<3 or ≥3 mg/L) and blood pressure levels (<130/85 or ≥130/85), the risk factor–adjusted hazard ratios were as follows: low CRP/low blood pressure, 1.0; high CRP/low blood pressure, 1.87 (
P
=0.002); low CRP/high blood pressure, 2.54 (
P
<0.0001); and high CRP/high blood pressure, 3.27 (
P
<0.0001).

Conclusions—

CRP and blood pressure are independent determinants of cardiovascular risk, and their predictive value is additive.
Low [Mg
2+
]
e
Enhances Arterial Spontaneous Tone via Phosphatidylinositol 3-Kinase in DOCA-Salt Hypertension
Authors: Northcott Carrie A. , Watts Stephanie W.
Published: November 2003

Phosphatidylinositol 3-kinase (PI3K) has been implicated in low extracellular Mg
2+
concentration ( [Mg
2+
]
e
)–induced aortic contraction, and Mg
2+
deficiency has been associated with hypertension. Moreover, arterial PI3K activity is increased in hypertensive deoxycorticosterone (DOCA)-salt rats. We hypothesized that low [Mg
2+
]
e
activates PI3K, eliciting enhanced vascular contraction, PI3K activity, and norepinephrine (NE)-induced contraction. Spontaneous tone was monitored in endothelium-denuded aortic strips from sham and DOCA-salt rats exposed to low Mg
2+
(0.15 mmol/L), high Mg
2+
(4.8 mmol/L), or normal (1.17 mmol/L) physiologic salt solution (PSS) in isolated tissue baths. LY294002 (20 μmol/L), a PI3K inhibitor, or vehicle was added (30 minutes), followed by NE (10
−9
to 3 x10
−-5
mol/L). Low [Mg
2+
]
e
significantly enhanced tone in aortas from DOCA-salt and sham rats compared with normal PSS (DOCA-salt low [Mg
2+
]
e
, +51.5 +7.0 vs DOCA-salt normal PSS, +7.1 +1.4 % of initial phenylephrine [PE] contraction). LY294002 and incubation with high Mg
2+
PSS decreased tone in aortas from DOCA-salt rats (low [Mg
2+
]
e
LY294002, −-87.5 +8.8; normal PSS LY294002, −81.7 +13.7; and high [Mg
2+
]
e
, −31.2 +10.8 % of initial PE contraction). Low [Mg
2+
]
e
leftward-shifted NE-induced aortic contractions in sham and thus matched the shift observed with DOCA (−log EC
50
mol/L: sham PSS, −7.7 +0.1; DOCA-salt PSS, −8.2 +0.1; sham low [Mg
2+
]
e
, −8.2 +0.1; and DOCA-salt low [Mg
2+
]
e
, −8.1 +0.1). Moreover, this shift was inhibited by LY294002. In conclusion, low [Mg
2+
]
e
might activate PI3K, leading to enhanced tone and agonist-induced contraction observed in aortas from DOCA-salt hypertensive rats.
Role of Membrane Potential and Expression of Endothelial Factors in Restenosis After Angioplasty in SHR
Authors: Dina Janaina P. , Feres Teresa , Paiva Antonio C.M. , Paiva Therezinha B.
Published: November 2003

We examined the roles played by impaired K
+
channels, diminished nitric oxide (NO) production, endothelin release, and smooth muscle membrane potential in the increased restenosis observed in spontaneously hypertensive rat (SHR) carotid arteries after angioplasty. The SHR carotid was found to be less polarized than that of normotensive Wistar rats (NWR), and it was further depolarized by the α
2
agonist UK 14,304. This response was blocked by iberiotoxin, indicating that calcium-dependent K
+
channels operate normally in the SHR carotid. Acetylcholine caused a hyperpolarization that was significantly smaller in SHR than in NWR carotids, indicating a deficient release of NO in the SHR. After angioplasty, SHR and NWR vessels were depolarized, returning to baseline after 10 days. In the SHR but not in the NWR the contralateral carotid was also depolarized, and this was prevented by the endothelin A/B receptor antagonist bosentan. After angioplasty, endothelin-1 plasma levels increased in both SHR and NWR, but the increase was significantly more prolonged in SHR. We found that the more pronounced restenosis observed in the SHR carotid after angioplasty is not due to impairment of calcium-dependent K
+
channels but is related to the relatively depolarized vascular smooth muscles, involving endothelin release caused by reduced NO levels in that strain.
How Effective Are “Community” Stroke Screening Programs at Improving Stroke Knowledge and Prevention Practices?
Authors: DeLemos Christi Diane , Atkinson Richard Presley , Croopnick Susan Louise , Wentworth Deidre Alice , Akins Paul Taylor
Published: November 2003

Background and Purpose—

Community stroke screening is a commonly used prevention strategy to identify and educate those at risk. Although the goal of this approach is to reduce the overall occurrence of stroke, its long-term benefit remains unknown. The purpose of this study was to determine whether attendance of a stroke screening changes knowledge or prevention practices in persons at risk for stroke 3 months later.

Methods—

A stroke screening event was held following the National Stroke Association guidelines, with health screening, counseling, and education. Knowledge about stroke was measured by a questionnaire before and after the event. At 3 months, attendees identified at risk for stroke were contacted by telephone to determine their retained knowledge and any specific actions taken as a result of the health counseling.

Results—

At 3 months, 78 persons were contacted. Knowledge of stroke warning signs increased from 59% to 94% after screening but decreased to 77% at 3 months. At 3 months, 73% had done nothing to change their health practices.

Conclusions—

Community stroke screening has modest effects on health behavior, knowledge of stroke risk factors, and warning signs.
Extracellular Concentrations of Non–Transmitter Amino Acids in Peri-Infarct Tissue of Patients Predict Malignant Middle Cerebral Artery Infarction
Authors: Bosche Bert , Dohmen Christian , Graf Rudolf , Neveling Michael , Staub Frank , Kracht Lutz , Sobesky Jan , Lehnhardt Fritz-Georg , Heiss Wolf-Dieter
Published: November 2003

Background and Purpose—

Space-occupying brain edema is a life-threatening complication in patients with large middle cerebral artery (MCA) infarction. To determine predictors of this detrimental process, we investigated alterations of extracellular non–transmitter amino acid concentrations in peri-infarct tissue.

Methods—

Thirty-one patients with infarctions covering >50% of the MCA territory in early cranial CT scans were included in the study. Probes for microdialysis, intracranial pressure, and tissue oxygen pressure were placed into the noninfarcted ipsilateral frontal lobe. Positron emission tomography imaging was performed in 16 of these patients to measure cerebral blood flow in the tissue around the neuromonitoring probes.

Results—

Fourteen of the 31 patients developed a malignant MCA infarction, and 17 did not. The patients in the malignant group had significantly lower extracellular concentrations of non–transmitter amino acids than those in the benign group in the first 12 hours of neuromonitoring. At this time, CBF values determined in regions of interest around the probes by positron emission tomography and tissue oxygen pressure showed that the monitored tissues were not yet infarcted, and no differences in transmitter amino acids concentrations were found between the 2 groups. Furthermore, extracellular concentrations of non–transmitter amino acids were negatively correlated with size of infarction.

Conclusions—

We assume that reduction of non–transmitter amino acid concentrations reflects an expansion of the extracellular space by vasogenic edema formation in peri-infarct tissue of patients with malignant MCA infarction. Our findings facilitate early prediction of malignant edema formation and may help to increase knowledge of the pathophysiology of the peri-infarct zone of large MCA infarction.
In Words and Pictures: the Sun in 2 Samuel 12:7-12
Authors: van Wolde Ellen
Published: November 2003

A cognitive web with different layers of emotion spreads over the text of 2 Samuel 12, in which the sun plays an important role. The unique linguistic combinations in vv. 11-12 show the sun's personified role. Iconographic study clarifies the sun as an image loaded with strong emotional feelings. Thus, it becomes understandable why the sun could function for the contemporaneous reader as a metonym which activates the contiguity between the character of David and a material object, possibly the sun emblem on the royal throne. It is before the vigilant eye of this sun, as the all-seeing judge, that Yhwh 's verdict will be executed. Every reader since, all those who know of the sun's cultural and religious power as well as of Yhwh 's, all those who read in the textual context about desire, lust and murder and about the king's devotion to the royal throne, can experience the sparks of thrill and fear in this text.
Gazing Back At the Shulammite, Yet Again
Authors: Brenner Athalya
Published: November 2003

In this short note a rereading of SoS 7:1-7 is coupled with a reading of an episode from 'Hassan of Basra', of the Thousand and One Nights collection. The comparison between the two so-called 'poems of physical description', of the Shulammite and the Bird Woman respectively, raises once again issues of visual as against emotive content, gender provenance and gender performance for the SoS text.
Proving Yahweh Killed His Wife (Zechariah 5:5-11)
Authors: Edelman Diana
Published: November 2003

Using an alternative form of scholarship, issues concerning how meaning is determined when reading an ancient text, the development of monotheism with the resulting need to reinterpret older Yahwistic texts, and how to understand divine motivations are explored. The piece is cast as a class action suit brought by modern humans against Yahweh in the heavenly court for murdering his wife, Asherah, citing Zech. 5:5-11 as evidence to support the accusation. Yahweh is defendant, self-appointed counsel and judge, whose cross-examination highlights all three issues. The case remains unresolved, as do answers to the issues.
Rhetorische Fragen!? Eine Aufkündigung Des Konsenses Über Psalm 88:11-13 Und Seine Bedeutung Für Das Alttestamentliche Reden Von Gott Und Tod
Authors: Crüsemann Frank
Published: November 2003

“Wirst du an den Toten ein Wunder tun?" Es besteht ein breiter Konsens in der Bibelwissenschaft, dass die Fragen in Ps. 88:11ff rein rhetorisch sind und sich in ihnen eine grundsätzliche Distanz Gottes zu den Toten ausdrückt, ein “theologisches Vakuum," das nur an den Rändern des Kanons, in den apokalyptischen Vorstellungen der Spätzeit überwunden wird. Der Aufsatz will diesen Konsens in Frage stellen. Ausgangspunkt sind hymnische und prophetische Aussagen über Jhwhs Macht über die Scheol (Am 9:2; 1 Sam. 2:6), aber auch Psalm 88 selbst, wo die Distanz zu Gott durch Gott selbst bewirkt ist (v.7 u.a.), also auch durch ihn rückgängig gemacht werden kann. Hinter dem Konsens steht wohl ein neuzeitlich-naturwissenschaftliches Verständnis des physischen Todes, durch das die Macht des Todes in Erfahrungen wie Krankheit und Not vom “realen" Tod entgegen Sprache und Vorstellung der Bibel getrennt wird. Religionsgeschichtlich spricht alles dafür, dass mit der Entstehung eines radikalen Alleinverehrungsanspruchs des israelitischen Gottes, also wahrscheinlich seit der Zeit Elias, dieser Gott auch dem Totengott überlegen ist und also Macht über Tod und Totenreich hat. Dafür sprechen u.a. Erzählungen über Totenerweckungen und Entrückungen, aber auch israelitische Grabinschriften schon der Königszeit. Die Fragen in Psalm 88 sind also echte Fragen an Gott und enthalten in nuce die spätere Sprache der Auferstehung.