bluuebunny/crossref_metadata_2025 huggingface dataset:
Sampling Issues in the Administration of Justice
Authors: Aitken Colin
Published: February 2005

Colin Aitken first became interested in sampling in the context of the administration of justice when the case of US versus Shonubi was brought to his attention. Shonubi was a Nigerian working in New York. He was arrested at Kennedy Airport when seen behaving in a suspicious manner in the baggage hall, having returned on a flight from Nigeria. He was found to have 103 bags of a white substance inside him, four of which were examined. The white substance was identified as heroin. Shonubi was arrested, tried and found guilty of drug smuggling. The question then arose as to the length of sentence he should be given.
EFFECTS OF COLLAGEN ADDITION ON THE FUNCTIONALITY OF PSE‐LIKE AND NORMAL BROILER BREAST MEAT IN A CHUNKED AND FORMED DELI ROLL
Authors: SCHILLING M.W. , DAIGLE S.P. , ALVARADO C.Z. , MARRIOTT N.G. , WANG H.
Published: February 2005

A Randomized Complete Block Design with six replications was utilized to test the treatment effects of raw material and the inclusion of chicken collagen on the protein functionality of chunked and formed chicken breast manufactured from 100% pale, soft, and exudative like (PSE‐like) and 100% normal broiler breast with either 0 or 1.5% collagen. Inclusion of collagen was effective in decreasing cooking loss and increasing protein–protein bind. Differences (P < 0.05) revealed that collagen inclusion caused PSE‐like meat to have lower cooking loss. Collagen addition also increased (P < 0.05) bind so that the PSE‐like treatment was not different from the normal treatment with collagen added. However, PSE‐like with no collagen had lower (P < 0.05) protein–protein bind (kg) than normal meat with collagen. These results reveal that addition of chicken collagen has the potential to improve quality characteristics of chunked and formed deli rolls formulated from both PSE‐like and normal raw material.
How Science Survived: Medieval Manuscripts' "Demography" and Classic Texts' Extinction
Authors: Cisne John L.
Published: February 2005

Determining what fraction of texts and manuscripts have survived from Antiquity and the Middle Ages has been highly problematic. Analyzing the transmission of texts as the “paleodemography” of their manuscripts yields definite and surprisingly high estimates. Parchment copies of the foremost medieval textbooks on arithmetical and calendrical calculation closely fit age distributions expected for populations with logistic growth and manuscripts with exponential survivorship. The estimated half-lives of copies agree with Bischoff's paleographically based suggestion that roughly one in seven manuscripts survive in some form from ninth-century Carolingian workshops. On this basis, many if not most of the leading technical titles circulating in Latin probably survived, even from late Antiquity.
Dynamics of Saturn's Magnetosphere from MIMI During Cassini's Orbital Insertion
Authors: Krimigis S. M. , Mitchell D. G. , Hamilton D. C. , Krupp N. , Livi S. , Roelof E. C. , Dandouras J. , Armstrong T. P. , Mauk B. H. , Paranicas C. , Brandt P. C. , Bolton S. , Cheng A. F. , Choo T. , Gloeckler G. , Hayes J. , Hsieh K. C. , Ip W.-H. , Jaskulek S. , Keath E. P. , Kirsch E. , Kusterer M. , Lagg A. , Lanzerotti L. J. , LaVallee D. , Manweiler J. , McEntire R. W. , Rasmuss W. , Saur J. , Turner F. S. , Williams D. J. , Woch J.
Published: February 2005

The Magnetospheric Imaging Instrument (MIMI) onboard the Cassini spacecraft observed the saturnian magnetosphere from January 2004 until Saturn orbit insertion (SOI) on 1 July 2004. The MIMI sensors observed frequent energetic particle activity in interplanetary space for several months before SOI. When the imaging sensor was switched to its energetic neutral atom (ENA) operating mode on 20 February 2004, at â¼10
3
times Saturn's radius
R
S
(0.43 astronomical units), a weak but persistent signal was observed from the magnetosphere. About 10 days before SOI, the magnetosphere exhibited a day-night asymmetry that varied with an â¼11-hour periodicity. Once Cassini entered the magnetosphere, in situ measurements showed high concentrations of H
+
, H
2
+
, O
+
, OH
+
, and H
2
O
+
and low concentrations of N
+
. The radial dependence of ion intensity profiles implies neutral gas densities sufficient to produce high loss rates of trapped ions from the middle and inner magnetosphere. ENA imaging has revealed a radiation belt that resides inward of the D ring and is probably the result of double charge exchange between the main radiation belt and the upper layers of Saturn's exosphere.
Saturn's Variable Magnetosphere
Authors: Gombosi Tamas I. , Hansen Kenneth C.
Published: February 2005

Since the Cassini spacecraft reached Saturn's orbit in 2004, its instruments have been sending back a wealth of data on the planet's magnetosphere (the region dominated by the magnetic field of the planet). In this Viewpoint, we discuss some of these results, which are reported in a collection of reports in this issue. The magnetosphere is shown to be highly variable and influenced by the planet's rotation, sources of plasma within the planetary system, and the solar wind. New insights are also gained into the chemical composition of the magnetosphere, with surprising results. These early results from Cassini's first orbit around Saturn bode well for the future as the spacecraft continues to orbit the planet.
Optimization of Virulence Functions Through Glucosylation of
Shigella
LPS
Authors: West Nicholas P. , Sansonetti Philippe , Mounier Joëlle , Exley Rachel M. , Parsot Claude , Guadagnini Stéphanie , Prévost Marie-Christine , Prochnicka-Chalufour Ada , Delepierre Muriel , Tanguy Myriam , Tang Christoph M.
Published: February 2005

Shigella
, the leading cause of bacillary dysentery, uses a type III secretion system (TTSS) to inject proteins into human cells, leading to bacterial invasion and a vigorous inflammatory response. The bacterium is protected against the response by the O antigen of lipopolysaccharide (LPS) on its surface. We show that bacteriophage-encoded glucosylation of
Shigella
O antigen, the basis of different serotypes, shortens the LPS molecule by around half. This enhances TTSS function without compromising the protective properties of the LPS. Thus, LPS glucosylation promotes bacterial invasion and evasion of innate immunity, which may have contributed to the emergence of serotype diversity in
Shigella
.
Susceptibility of Germfree Phagocyte Oxidase- and Nitric Oxide Synthase 2-Deficient Mice, Defective in the Production of Reactive Metabolites of Both Oxygen and Nitrogen, to Mucosal and Systemic Candidiasis of Endogenous Origin
Authors: Balish Edward , Warner Thomas F. , Nicholas Peter J. , Paulling Emily E. , Westwater Caroline , Schofield David A.
Published: February 2005

Mice deficient for phagocyte oxidase (Phox) and nitric oxide synthase 2 (NOS2) (gp91
phox−/−
/NOS2
−/−
), defective in the production of both reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI), were used to investigate the role of phagocytic cells during mucosal and systemic candidiasis of endogenous origin. The alimentary tracts of germfree mice were colonized with
Candida albicans
wild type or each of two hyphal signaling-defective mutants (
efg1/efg1
and
efg1/efg1 cph1/cph1
). All
Candida
-colonized gp91
phox−/−
/NOS2
−/−
mice were moribund within 12 to 15 days after oral inoculation.
C. albicans
wild-type and mutant strains colonized the alimentary tracts equally well and were able to translocate, most likely via Peyer's patches and mesenteric lymph nodes, to the internal organs and trigger the formation of abscesses; however, the wild-type and mutant strains did not survive in the abscessed murine tissues. Surprisingly, there was no significant difference in the ability of peritoneal exudate cells from gp91
phox−/−
/NOS2
−/−
, NOS2
−/−
, gp91
phox−/−
, or immunocompetent C57BL/6 mice to kill
C. albicans
in vitro. This suggests that anti
-Candida
factors other than ROI and RNI can control the growth of
C. albicans
and that gp91
phox−/−
/NOS2
−/−
mice die due to the inability of the host to control its inflammatory response to
Candida
. Correspondingly, reverse transcription-PCR analysis showed increased expression of the cytokines gamma interferon, tumor necrosis factor alpha, and the chemokines MIP-2 and KC at the site of infection, while interleukin-15 expression remained relatively unchanged between germfree and infected tissues. These studies indicate that defects in ROI and RNI enabled
C. albicans
to translocate and disseminate to the internal organs, resulting in an uncontrolled immune response, severe pathology, and death; however, ROI and RNI were not required for the killing of phagocytized
C. albicans
, indicating that other anti-
Candida
factors either compensate or are sufficient for the killing of phagocytized
Candida
.
Toll-Like Receptor 2 Mediates Cellular Activation by the B Subunits of Type II Heat-Labile Enterotoxins
Authors: Hajishengallis George , Tapping Richard I. , Martin Michael H. , Nawar Hesham , Lyle Elizabeth A. , Russell Michael W. , Connell Terry D.
Published: February 2005

The type II heat-labile enterotoxins (LT-IIa and LT-IIb) ofEscherichia colihave an AB5subunit structure similar to that of cholera toxin (CT) and other type I enterotoxins, despite significant differences in the amino acid sequences of their B subunits and different ganglioside receptor specificities. LT-II holotoxins and their nontoxic B subunits display unique properties as immunological adjuvants distinct from those of CT and its B subunits. In contrast to type II holotoxins, the corresponding pentameric B subunits, LT-IIaB and LT-IIbB, stimulated cytokine release in both human and mouse cells dependent upon Toll-like receptor 2 (TLR2). Induction of interleukin-1β (IL-1β), IL-6, IL-8, or tumor necrosis factor alpha in human THP-1 cells by LT-IIaB or LT-IIbB was inhibited by anti-TLR2 but not by anti-TLR4 antibody. Furthermore, transient expression of TLR1 and TLR2 in human embryonic kidney 293 cells resulted in activation of a nuclear factor-κB-dependent luciferase gene in response to LT-IIaB or LT-IIbB. Moreover, peritoneal macrophages from TLR2-deficient mice failed to respond to LT-IIaB or LT-IIbB, in contrast to wild-type or TLR4-deficient cells. These results demonstrate that besides their established binding to gangliosides, the B subunits of type II enterotoxins also interact with TLR2. Although a ganglioside-nonbinding mutant (T34I) of LT-IIaB effectively induced cytokine release, a phenotypically similar point mutation (T13I) in LT-IIbB abrogated cytokine induction, suggesting a variable requirement for gangliosides as coreceptors in TLR2 agonist activity. TLR2-dependent activation of mononuclear cells by type II enterotoxin B subunits appears to be a novel mechanism whereby these molecules may exert their immunomodulatory and adjuvant activities.
Citrobacter rodentium lifA
/
efa1
Is Essential for Colonic Colonization and Crypt Cell Hyperplasia In Vivo
Authors: Klapproth Jan-Michael A. , Sasaki Maiko , Sherman Melanie , Babbin Brian , Donnenberg Michael S. , Fernandes Paula J. , Scaletsky Isabel C. A. , Kalman Daniel , Nusrat Asma , Williams Ifor R.
Published: February 2005

Previously, we have identified a large gene (
lifA
, for lymphocyte inhibitory factor A) in enteropathogenic
Escherichia coli
(EPEC) encoding a protein termed lymphostatin that suppresses cytokine expression in vitro. This protein also functions as an adhesion factor for enterohemorrhagic
E. coli
(EHEC) and Shiga toxin-producing
E. coli
and is alternatively known as
efa1
(EHEC factor for adherence 1). The
lifA
/
efa1
gene is also present in
Citrobacter rodentium
, an enteric pathogen that causes a disease termed transmissible murine colonic hyperplasia (TMCH), which induces colitis and massive crypt cell proliferation, in mice. To determine if
lifA
/
efa1
is required for
C. rodentium
-induced colonic pathology in vivo, three in-frame mutations were generated, disrupting the glycosyltransferase (GlM12) and protease (PrMC31) motifs and a domain in between that does not encode any known activity (EID3). In contrast to infection with wild-type
C. rodentium
, that with any of the
lifA
/
efa1
mutant strains did not induce weight loss or TMCH. Enteric infection with motif mutants GlM12 and PrM31 resulted in significantly reduced colonization counts during the entire 20-day course of infection. In contrast, EID3 was indistinguishable from the wild type during the initial colonic colonization, but cleared rapidly after day 8 of the infection. The colonic epithelium of all infected mice displayed increased epithelial regeneration. However, significantly increased regeneration was observed by day 20 only in mice infected with the wild-type in comparison to those infected with
lifA
/
efa1
mutant EID3. In summary,
lifA
/
efa1
is a critical gene outside the locus for enterocyte effacement that regulates bacterial colonization, crypt cell proliferation, and epithelial cell regeneration.
Natural Killer Cells andHelicobacter pyloriInfection: Bacterial Antigens and Interleukin-12 Act Synergistically To Induce Gamma Interferon Production
Authors: Yun Cheol H. , Lundgren Anna , Azem Josef , Sjöling Åsa , Holmgren Jan , Svennerholm Ann-Mari , Lundin B. Samuel
Published: February 2005

Helicobacter pyloriis known to induce a local immune response, which is characterized by activation of lymphocytes and the production of IFN-γ in the stomach mucosa. Since not only T cells, but also natural killer (NK) cells, are potent producers of gamma interferon (IFN-γ), we investigated whether NK cells play a role in the immune response toH. pyloriinfection. Our results showed that NK cells were present in both the gastric and duodenal mucosae but thatH. pyloriinfection did not affect the infiltration of NK cells into the gastrointestinal area. Furthermore, we could show that NK cells could be activated directly byH. pyloriantigens, asH. pyloribacteria, as well as lysate fromH. pylori, induced the secretion of IFN-γ by NK cells. NK cells were also activated without direct contact when separated from the bacteria by an epithelial cell layer, indicating that the activation of NK cells byH. pylorican also occur in vivo, in the infected stomach mucosa. Moreover, the production of IFN-γ by NK cells was greatly enhanced when a small amount of interleukin-12 (IL-12) was added, and this synergistic effect was associated with increased expression of the IL-12 receptor β2. It was further evident that bacterial lysate alone was sufficient to induce the activation of cytotoxicity-related molecules. In conclusion, we demonstrated that NK cells are present in the gastroduodenal mucosa of humans and that NK cells produce high levels of IFN-γ when stimulated with a combination ofH. pyloriantigen and IL-12. We propose that NK cells play an active role in the local immune response toH. pyloriinfection.
Innate Immunity to the Pathogenic Fungus
Coccidioides posadasii
Is Dependent on Toll-Like Receptor 2 and Dectin-1
Authors: Viriyakosol Suganya , Fierer Joshua , Brown Gordon D. , Kirkland Theo N.
Published: February 2005

Coccidioides posadasii
is a pathogenic fungus that causes endemic and epidemic coccidioidomycosis in the deserts of North, Central, and South America. How the innate immune system responds to the organism is not well understood. Here we show that elicited mouse peritoneal macrophages respond to spherules (the tissue form of the fungus) by producing proinflammatory cytokines as measured by quantitative PCR of cellular transcripts and by enzyme-linked immunosorbent assay (ELISA) assays for secreted protein. We examined the contribution of Toll-like receptors (TLR) and MyD88 in macrophage responses to formalin-killed spherules (FKS) by comparing cytokine responses of elicited macrophages from different knockout mice. FKS were added to elicited mouse peritoneal macrophages from wild-type, TLR2
−/−
, and MyD88
−/−
cells, and wild-type cells made more tumor necrosis factor alpha, MIP-2, and interleukin 6 than did the mutant macrophages. In contrast, the C3H/HeJ mice, which have a point mutation in TLR4, and TLR4
−/−
B6 mice exhibited no defect in cytokine production compared to the control mice. We also investigated the role of the macrophage β-glucan receptor, Dectin-1. RAW 264.7 macrophages overexpressing Dectin-1 produced more cytokines in respond to FKS, live spherules, and purified β-glucan than did control RAW cells. Blockage of Dectin-1 with antibodies inhibited cytokine production in elicited mouse peritoneal macrophages. Taken together, these results show that cytokine responses in mouse peritoneal macrophages to
C. posadasii
spherules are dependent on TLR2, MyD88, and Dectin-1.
Identification and Characterization of an Immunodominant 28-Kilodalton
Coxiella burnetii
Outer Membrane Protein Specific to Isolates Associated with Acute Disease
Authors: Zhang Guoquan , To Ho , Russell Kasi E. , Hendrix Laura R. , Yamaguchi Tsuyoshi , Fukushi Hideto , Hirai Katsuya , Samuel James E.
Published: February 2005

Coxiella burnetii
causes acute Q fever in humans and occasional chronic infections that typically manifest as endocarditis or hepatitis. Isolates associated with acute disease were found to be distinct from a group of chronic disease isolates by a variety of biochemical parameters and in a guinea pig fever model of acute disease, suggesting a difference in virulence potential. We compared antigenic polypeptides among
C. burnetii
isolates and found an immunodominant 28-kDa protein in acute group isolates but not in chronic group isolates (T. Ho, A. Hotta, G. Q. Zhang, S. V. Nguyen, M. Ogawa, T. Yamaguchi, H. Fukushi, and K. Hirai, Microbiol. Immunol.
42:
81-85, 1998). In order to clone the
adaA
gene, the N-terminal amino acid sequence of
adaA
was determined and a 59-bp fragment was amplified from Nine Mile phase I DNA by PCR. The putative gene fragment was used to screen a lambda ZAP II genomic DNA library, and an open reading frame expressing a 28-kDa immunoreactive protein was identified. Sequence analysis predicted a gene encoding an ∼28-kDa mature protein with a typical signal sequence. The
adaA
(acute disease antigen A) gene was detected in acute group
C. burnetii
isolates but not identified in chronic group isolates by PCR and Southern blotting. A typical signal peptide was predicted in
adaA
, and specific antibody to
adaA
reacted with the purified membrane fraction of acute group isolates by Western blotting, suggesting that
adaA
is exposed on the outer surface of
C. burnetii. adaA
was overexpressed in pET23a as a fusion protein in
Escherichia coli
to develop anti-recombinant
adaA
(anti-r
adaA
) specific antibody, which recognized a ∼28-kDa band in acute group isolates but not in chronic group isolates. In addition, immunoblotting indicates that r
adaA
reacted with sera derived from animals infected with acute group isolates but did not react with sera from animals infected with chronic group isolates. These results support the idea that an
adaA
gene-targeted PCR assay and an r
adaA
antigen-based serodiagnostic test may be useful for differential diagnosis of acute and chronic Q fever.
Rapid Loss of Motility of
Helicobacter pylori
in the Gastric Lumen In Vivo
Authors: Schreiber Sören , Bücker Roland , Groll Claudia , Azevedo-Vethacke Marina , Garten Désirée , Scheid Peter , Friedrich Susanne , Gatermann Sören , Josenhans Christine , Suerbaum Sebastian
Published: February 2005

The human pathogen
Helicobacter pylori
has infected more than half of the world's population. Nevertheless, the first step of infection, the acute colonization of the gastric mucus, is poorly understood. For successful colonization,
H. pylori
must retain active motility in the gastric lumen until it reaches the safety of the mucus layer. To identify the factors determining the acute colonization, we inserted bacteria into the stomach of anesthetized Mongolian gerbils. We adjusted the gastric juice to defined pH values of between 2.0 and 6.0 by using an autotitrator. Despite the fact that
Helicobacter
spp. are known to survive low pH values for a certain time in vitro, the length of time that
H. pylori
persisted under the assay conditions within the gastric juice in vivo was remarkably shorter. In the anesthetized animal we found
H. pylori
to be irreversibly immotile in less than 1 min at lumen pH values of 2 and 3. At pH 4 motility was lost after 2 min. However, the period of motility increased to more than 15 min at pH 6. Blocking pepsins in the gastric lumen in vivo by using pepstatin significantly increased the period of motility. It was possible to simulate the rapid in vivo immotilization in vitro by adding pepsins. We conclude that pepsin limits the persistence of
H. pylori
in the gastric chymus to only a few minutes by rapidly inhibiting active motility. It is therefore likely that this short period of resistance in the gastric lumen is one of the most critical phases of
Helicobacter
infection.
Monocytes Are Highly Sensitive toClostridium difficileToxin A-Induced Apoptotic and Nonapoptotic Cell Death
Authors: Solomon K. , Webb J. , Ali N. , Robins R. A. , Mahida Y. R.
Published: February 2005

In this study we investigated the in vitro responses of peripheral blood mononuclear preparations and purified monocytes toClostridium difficiletoxin A. In contrast to the responses of T and B cells, exposure to toxin A led to a rapid loss of monocytes in a time- and dose-dependent fashion (the majority of cells were lost within 24 h of exposure to >100 ng of toxin per ml). Transmission electron microscopy, flow cytometry, and fluorescence microscopy after propidium iodide and Hoechst staining showed that cell death in purified preparations of monocytes following exposure to 100 and 1,000 ng of toxin A per ml occurred by apoptosis. Further studies showed that 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazole-carbocyanine iodide aggregates were retained within toxin A-exposed monocyte mitochondria, but cytochromecwas released, suggesting that the apoptotic cascade was triggered in the absence of mitochondrial permeability transition. There was also an increase in caspase-3 activity in toxin A-stimulated monocytes. Following exposure to very high concentrations of toxin A (30 μg/ml), monocyte cell death was predominantly of the necrotic type, with rapid extracellular release of lactate dehydrogenase. These studies demonstrated thatC. difficiletoxin A has a cell-specific effect, in which monocytes exhibit greater susceptibility than lymphocytes and their death is induced in a concentration-dependent manner.
Identification of Enterohemorrhagic
Escherichia coli
O26:H
−
Genes Required for Intestinal Colonization in Calves
Authors: van Diemen Pauline M. , Dziva Francis , Stevens Mark P. , Wallis Timothy S.
Published: February 2005

Enterohemorrhagic
Escherichia coli
(EHEC) infections in humans are an important public health problem and are commonly acquired via contact with ruminant feces. The serogroups that are predominantly associated with human infection in the United States and Europe are O157 and O26. Serotypes O157:H7 and O26:H
−
differ in their virulence and tissue tropism in calves and therefore may colonize calves by distinct mechanisms. The mechanisms underlying EHEC intestinal colonization and pathogenesis are poorly understood. Signature-tagged mutagenesis was used to identify 59 genes of EHEC O26:H
−
that are required for the intestinal colonization of calves. Our results indicate important roles for locus of enterocyte effacement (LEE)-encoded type III secreted proteins in intestinal colonization. In addition, colonization is facilitated by cytotoxins, putative type III secreted proteins unlinked to the LEE, a putative fimbrial operon, and numerous genes involved in central metabolism and transport and genes of unknown function. Our data also imply that the elaboration of type I fimbriae by EHEC O26:H
−
is disadvantageous for persistence within the bovine intestines. These observations have important implications for the design of vaccines to control these important zoonotic pathogens.
Distinct Roles of Pattern Recognition Receptors CD14 and Toll-Like Receptor 4 in Acute Lung Injury
Authors: Jeyaseelan Samithamby , Chu Hong Wei , Young Scott K. , Freeman Mason W. , Worthen G. Scott
Published: February 2005

Acute lung injury (ALI) induced by lipopolysaccharide (LPS) is a major cause of mortality among humans. ALI is characterized by microvascular protein leakage, neutrophil influx, and expression of proinflammatory mediators, followed by severe lung damage. LPS binding to its receptors is the crucial step in the causation of these multistep events. LPS binding and signaling involves CD14 and Toll-like receptor 4 (TLR4). However, the relative contributions of CD14 and TLR4 in the induction of ALI and their therapeutic potentials are not clear in vivo. Therefore, the aim of the present study was to compare the roles of CD14 and TLR4 in LPS-induced ALI to determine which of these molecules is the more critical target for attenuating ALI in a mouse model. Our results show that CD14 and TLR4 are necessary for low-dose (300-μg/ml) LPS-induced microvascular leakage, NF-κB activation, neutrophil influx, cytokine and chemokine (KC, macrophage inflammatory protein 2, tumor necrosis factor alpha, interleukin-6) expression, and subsequent lung damage. On the other hand, when a 10-fold-higher dose of LPS (3 mg/ml) was used, these responses were only partially dependent on CD14 and they were totally dependent on TLR4. The CD14-independent LPS response was dependent on CD11b. A TLR4 blocking antibody abolished microvascular leakage, neutrophil accumulation, cytokine responses, and lung pathology with a low dose of LPS but only attenuated the responses with a high dose of LPS. These data are the first to demonstrate that LPS-induced CD14-depdendent and -independent (CD11b-dependent) signaling pathways in the lung are entirely dependent on TLR4 and that blocking TLR4 might be beneficial in lung diseases caused by LPS from gram-negative pathogens.
Role for Flagella but Not Intimin in the Persistent Infection of the Gastrointestinal Tissues of Specific-Pathogen-Free Chicks by Shiga Toxin-Negative
Escherichia coli
O157:H7
Authors: Best Angus , La Ragione Roberto M. , Sayers A. Robin , Woodward Martin J.
Published: February 2005

Shiga toxin (Stx)-positive
Escherichia coli
O157:H7 readily colonize and persist in specific-pathogen-free (SPF) chicks, and we have shown that an Stx-negative
E. coli
O157:H7 isolate (NCTC12900) readily colonizes SPF chicks for up to 169 days after oral inoculation at 1 day of age. However, the role of intimin in the persistent colonization of poultry remains unclear. Thus, to investigate the role of intimin and flagella, which is a known factor in the persistence of non-O157
E. coli
in poultry, isogenic single- and double-intimin and aflagellar mutants were constructed in
E. coli
O157:H7 isolate NCTC12900. These mutants were used to inoculate (10
5
CFU) 1-day-old SPF chicks. In general, significant attenuation of the aflagellate and intimin-aflagellate mutants, but not the intimin mutant, was noted at similar time points between 22 and 92 days after inoculation. The intimin-deficient mutant was still being shed at the end of the experiment, which was 211 days after inoculation, 84 days more than the wild type. Shedding of the aflagellar and intimin-aflagellar mutants ceased 99 and 113 days after inoculation, respectively. Histological analysis of gastrointestinal tissues from inoculated birds gave no evidence for true microcolony formation by NCTC12900 or intimin and aflagellar mutants to epithelial cells. However, NCTC12900 mutant derivatives associated with the mucosa were observed as individual cells and/or as large aggregates. Association with luminal contents was also noted. These data suggest that O157 organisms do not require intimin for the persistent colonization of chickens, whereas flagella do play a role in this process.
Bacterial and Host Factors Implicated in Nasal Carriage of Methicillin-Resistant
Staphylococcus aureus
in Mice
Authors: González-Zorn Bruno , Senna Jose P. M. , Fiette Laurence , Shorte Spencer , Testard Aurélie , Chignard Michel , Courvalin Patrice , Grillot-Courvalin Catherine
Published: February 2005

Nasal carriage is a major risk factor for
Staphylococcus aureus
infection, especially for methicillin-resistant strains (MRSA). Using a mouse model of nasal carriage, we have compared several
S. aureus
strains and demonstrated increased colonization levels by MRSA in cystic fibrosis transmembrane conductance regulator-deficient mice and Toll-like receptor 2 (TLR2)-deficient mice but not TLR4-deficient mice.
Degradation of the Proapoptotic Proteins Bik, Puma, and Bim with Bcl-2 Domain 3 Homology in
Chlamydia trachomatis
-Infected Cells
Authors: Dong Feng , Pirbhai Mustak , Xiao Yangming , Zhong Youmin , Wu Yimou , Zhong Guangming
Published: February 2005

We have previously correlated
Chlamydia trachomatis
antiapoptotic activity with the blockade of mitochondrial cytochrome
c
release and the inhibition of Bax and Bak activation. We now report that
C. trachomatis
infection leads to degradation of Bik, Puma, and Bim, three upstream proapoptotic BH3-only proteins of the Bcl-2 family that can transmit death signals to mitochondria by inhibiting the Bcl-2 antiapoptotic proteins and/or activating the Bcl-2 proapoptotic members, such as Bax and Bak. This observation has provided new information on the chlamydial antiapoptosis mechanisms.
Deletion of Highly Conserved Arginine-Rich RNA Binding Motif in Cowpea Chlorotic Mottle Virus Capsid Protein Results in Virion Structural Alterations and RNA Packaging Constraints
Authors: Annamalai Padmanaban , Apte Swapna , Wilkens Stephan , Rao A. L. N.
Published: February 2005

The N-proximal region of cowpea chlorotic mottle virus (CCMV) capsid protein (CP) contains an arginine-rich RNA binding motif (ARM) that is also found in the CPs of other members of
Bromoviridae
and in other RNA binding proteins such as the Tat and Rev proteins of human immunodeficiency virus. To assess the critical role played by this motif during encapsidation, a variant of CCMV RNA3 (C3) precisely lacking the ARM region (C3/Δ919) of its CP gene was constructed. The biology and the competence of the matured CP derived in vivo from C3/Δ919 to assemble and package progeny RNA was examined in whole plants. Image analysis and computer-assisted three-dimensional reconstruction of wild-type and mutant virions revealed that the CP subunits bearing the engineered deletion assembled into polymorphic virions with altered surface topology. Northern blot analysis of virion RNA from mutant progeny demonstrated that the engineered mutation down-regulated packaging of all four viral RNAs; however, the packaging effect was more pronounced on genomic RNA1 and RNA2 than genomic RNA3 and its CP mRNA. In vitro assembly assays with mutant CP subunits and RNA transcripts demonstrated that the mutant CP is inherently not defective in packaging genomic RNA1 (53%) and RNA2 (54%), but their incorporation into virions was competitively inhibited by the presence of other viral RNAs. Northern blot analysis of RNA encapsidation in vivo of two distinct bromovirus RNA3 chimeras, constructed by exchanging CPs having the Δ919 deletion, demonstrated that the role of the conserved N-terminal ARM in recognizing and packaging specific RNA is distinct for each virus.
Genetic Analysis of the Human Papillomavirus Type 31 Differentiation-Dependent Late Promoter
Authors: Bodily Jason M. , Meyers Craig
Published: February 2005

Human papillomaviruses infect stratifying squamous epithelia, causing benign and malignant lesions. Upon differentiation of the host keratinocyte, the virus undergoes a dramatic increase in both DNA replication and transcription from the late promoter, leading to expression of late genes and virion morphogenesis. In human papillomavirus type 31 (HPV31), the late promoter is designated p742 and includes multiple start sites embedded within the E7 gene. In this report, we mapped viral DNA elements that control transcriptional activity from p742. Enhancer elements in the viral upstream regulatory region positively regulate this promoter. The region containing the transcriptional start sites is dispensable for activity, and at least two separate elements in the E6/E7 region are capable of supporting transcription. Of these, we mapped one to a 150-bp region of the E7 open reading frame and designate it the core p742 promoter. Using GF109203X, an inhibitor of protein kinase C signaling, we show that p742 activation is independent of viral genome amplification. Finally, we mapped elements in the region of p742 that confer responsiveness to differentiation and show that the upstream regulatory region does not contribute to the differentiation response of p742. These studies are an important step toward understanding the functioning and regulation of this multiple-start promoter.
Identification of Sites in Adenovirus Hexon for Foreign Peptide Incorporation
Authors: Wu Hongju , Han Tie , Belousova Natalya , Krasnykh Victor , Kashentseva Elena , Dmitriev Igor , Kataram Manjula , Mahasreshti Parameshwar J. , Curiel David T.
Published: February 2005

Adenovirus type 5 (Ad5) is one of the most promising vectors for gene therapy applications. Genetic engineering of Ad5 capsid proteins has been employed to redirect vector tropism, to enhance infectivity, or to circumvent preexisting host immunity. As the most abundant capsid protein, hexon modification is particularly attractive. However, genetic modification of hexon often results in failure of rescuing viable viruses. Since hypervariable regions (HVRs) are nonconserved among hexons of different serotypes, we investigated whether the HVRs could be used for genetic modification of hexon by incorporating oligonucleotides encoding six histidine residues (His
6
) into different HVRs in the Ad5 genome. The modified viruses were successfully rescued, and the yields of viral production were similar to that of unmodified Ad5. A thermostability assay suggested the modified viruses were stable. The His
6
epitopes were expressed in all modified hexon proteins as assessed by Western blotting assay, although the intensity of the reactive bands varied. In addition, we examined the binding activity of anti-His tag antibody to the intact virions with the enzyme-linked immunosorbent assay and found the His
6
epitopes incorporated in HVR2 and HVR5 could bind to anti-His tag antibody. This suggested the His
6
epitopes in HVR2 and HVR5 were exposed on virion surfaces. Finally, we examined the infectivities of the modified Ad vectors. The His
6
epitopes did not affect the native infectivity of Ad5 vectors. In addition, the His
6
epitopes did not appear to mediate His
6
-dependent viral infection, as assessed in two His
6
artificial receptor systems. Our study provided valuable information for studies involving hexon modification.
B1 Lymphocytes and Myeloid Dendritic Cells in Lymphoid Organs Are Preferential Extratumoral Sites of Parvovirus Minute Virus of Mice Prototype Strain Expression
Authors: Raykov Zahari , Savelyeva Larissa , Balboni Ginette , Giese Thomas , Rommelaere Jean , Giese Nathalia A.
Published: February 2005

Due to their oncolytic properties and apathogenicity, autonomous parvoviruses have attracted significant interest as possible anticancer agents. Recent preclinical studies provided evidence of the therapeutic potential of minute virus of mice prototype strain (MVMp) and its recombinant derivatives. In a murine model of hemangiosarcoma, positive therapeutic outcome correlated with high intratumoral expression of MVMp-encoded genes in tumors and lymphoid organs, especially in tumor-draining lymph nodes. The source and relevance of this extratumoral expression, which came as a surprise because of the known fibrotropism of MVMp, remained unclear. In the present study, we investigated (i) whether the observed expression pattern occurs in different tumor models, (ii) which cell population is targeted by the virus, and (iii) the immunological consequences of this infection. Significant MVMp gene expression was detected in lymphoid tissues from infected tumor-free as well as melanoma-, lymphoma-, and hemangiosarcoma-bearing mice. This expression was especially marked in lymph nodes draining virus-injected tumors. Fluorescent in situ hybridization analysis, multicolor fluorescence-activated cell sorting, and quantitative reverse transcription-PCR revealed that MVMp was expressed in rare subpopulations of CD11b (Mac1)-positive cells displaying CD11c+(myeloid dendritic cells [MDC]) or CD45B (B220+[B1 lymphocytes]) markers. Apart from the late deletion of cytotoxic memory cells (CD8+CD44+CD62L−), this infection did not lead to significant alteration of the immunological profile of cells populating lymphoid organs. However, subtle changes were detected in the production of specific proinflammatory cytokines in lymph nodes from virus-treated animals. Considering the role of B1 lymphocytes and MDC in cancer and immunological surveillance, the specific ability of these cell types to sustain parvovirus-driven gene expression may be exploited in gene therapy protocols.
3′-Terminal Sequence in Poliovirus Negative-Strand Templates Is the Primary
cis
-Acting Element Required for VPgpUpU-Primed Positive-Strand Initiation
Authors: Sharma Nidhi , O'Donnell Brian J. , Flanegan James B.
Published: February 2005

The 5′ cloverleaf in poliovirus RNA has a direct role in regulating the stability, translation, and replication of viral RNA. In this study, we investigated the role of stem a in the 5′ cloverleaf in regulating the stability and replication of poliovirus RNA in HeLa S10 translation-replication reactions. Our results showed that disrupting the duplex structure of stem a destabilized viral RNA and inhibited efficient negative-strand synthesis. Surprisingly, the duplex structure of stem a was not required for positive-strand synthesis. In contrast, altering the primary sequence at the 5′-terminal end of stem a had little or no effect on negative-strand synthesis but dramatically reduced positive-strand initiation and the formation of infectious virus. The inhibition of positive-strand synthesis observed in these reactions was most likely a consequence of nucleotide alterations in the conserved sequence at the 3′ ends of negative-strand RNA templates. Previous studies suggested that VPgpUpU synthesized on the
cre
(2C) hairpin was required for positive-strand synthesis. Therefore, these results are consistent with a model in which preformed VPgpUpU serves as the primer for positive-strand initiation on the 3′AAUUUUGUC5′ sequence at the 3′ ends of negative-strand templates. Our results suggest that this sequence is the primary
cis
-acting element that is required for efficient VPgpUpU-primed positive-strand initiation.
Functional Domains within the Human Immunodeficiency Virus Type 2 Envelope Protein Required To Enhance Virus Production
Authors: Abada Paolo , Noble Beth , Cannon Paula M.
Published: February 2005

Primate lentiviruses code for a protein that stimulates virus production. In human immunodeficiency virus type 1 (HIV-1), the activity is provided by the accessory protein, Vpu, while in HIV-2 and simian immunodeficiency virus it is a property of the envelope (Env) glycoprotein. Using a group of diverse retroviruses and cell types, we have confirmed the functional equivalence of the two proteins. However, despite these similarities, the two proteins have markedly different functional domains. While the Vpu activity is associated primarily with its membrane-spanning region, we have determined that the HIV-2 Env activity requires both the cytoplasmic tail and ectodomain of the protein, with the membrane-spanning domain being less important. Within the Env cytoplasmic tail, we further defined the necessary sequence as a membrane-proximal tyrosine-based motif. Providing the two Env regions separately as distinct CD8 chimeric proteins did not increase virus release. This suggests that the two domains must be either contained within a single protein or closely associated within a multiprotein oligomer, such as the Env trimer, in order to function. Finally, we observed that wild-type levels of incorporation of the HIV-2 Env into budding viruses were not required for this activity.
Ligand-Directed Gene Targeting to Mammalian Cells by Pseudotype Baculoviruses
Authors: Kitagawa Yoshinori , Tani Hideki , Limn Chang Kwang , Matsunaga Tomoko M. , Moriishi Kohji , Matsuura Yoshiharu
Published: February 2005

The baculovirusAutographa californicamultiple nucleopolyhedrovirus (AcMNPV) can infect a variety of mammalian cells, as well as insect cells, facilitating its use as a viral vector for gene delivery into mammalian cells. Glycoprotein gp64, a major component of the budded AcMNPV envelope, is involved in viral entry into cells by receptor-mediated endocytosis and subsequent membrane fusion. We examined the potential production of pseudotype baculovirus particles transiently carrying ligands of interest in place of gp64 as a method of ligand-directed gene delivery into target cells. During amplification of a gp64-null pseudotype baculovirus carrying a green fluorescent protein gene in gp64-expressing insect cells, however, we observed the high-frequency appearance of a replication-competent virus incorporating the gp64 gene into the viral genome. To avoid generation of replication-competent revertants, we prepared pseudotype baculoviruses by transfection with recombinant bacmids without further amplification in the gp64-expressing cells. We constructed gp64-null recombinant bacmids carrying cDNAs encoding either vesicular stomatitis virus G protein (VSVG) or measles virus receptors (CD46 or SLAM). The VSVG pseudotype baculovirus efficiently transduced a reporter gene into a variety of mammalian cell lines, while CD46 and SLAM pseudotype baculoviruses allowed ligand-receptor-directed reporter gene transduction into target cells expressing measles virus envelope glycoproteins. Gene transduction mediated by the pseudotype baculoviruses could be inhibited by pretreatment with specific antibodies. These results indicate the possible application of pseudotype baculoviruses in ligand-directed gene delivery into target cells.
Immunologic Pressure within Class I-Restricted Cognate Human Immunodeficiency Virus Epitopes during Highly Active Antiretroviral Therapy
Authors: Casazza Joseph P. , Betts Michael R. , Hill Brenna J. , Brenchley Jason M. , Price David A. , Douek Daniel C. , Koup Richard A.
Published: February 2005

Cytotoxic T lymphocytes (CTL) and highly active antiretroviral therapy (HAART) are known to exert strong evolutionary pressures on the virus population during human immunodeficiency virus (HIV) infection. However, it is not known whether CTL responses continue to substantially affect viral evolution during treatment. To study the effect of immunologic pressure on viral sequences during HAART, we identified 10 targeted HIV-specific CD8
+
-T-cell epitopes in five treatment-naïve patients, sequenced each epitope in plasma-derived viruses, and then identified evidence of immunologic pressure at these epitopes by comparing the frequency of viral variants in plasma to the frequency of the CD8
+
-T-cell response for each variant identified. For one of the five patients, evidence of viral evolution was found during therapy. The sequence of the CTL-targeted epitope changed from an apparent escape variant prior to the initiation of therapy, to the sequence that is best recognized by the CTL response after the initiation of therapy, and then finally to a new escape variant during continued therapy. These data show that CTL-mediated pressure can continue to affect viral evolution after the initiation of HAART, even when treatment drives the viral load below detectable levels, and suggest that antiretroviral therapy may preferentially inhibit those virus variants that escape the CTL response.
Inhibition of Viral Replication by Ribozyme: Mutational Analysis of the Site and Mechanism of Antiviral Activity
Authors: Zhang Zhenxi , Burke John M.
Published: February 2005

A controlled mutational study was used to determine the site and mechanism of the antiviral action of ribozymes that inhibit Sindbis virus replication. A hairpin ribozyme targeting G575 of the Sindbis virus genomic RNA was designed and cloned into a minimized alphavirus amplicon vector. Cells that were stably transfected with this construct expressed low levels of a constitutive transcript containing the ribozyme plus recognition sequences for Sindbis RNA replicase. Upon infection, the ribozyme transcript was amplified to high levels by the viral replicase, resulting in decreased viral production from infected ribozyme-expressing cells. Mutations were then introduced into the viral RNA target sequence to interfere with ribozyme binding, and compensatory changes were generated in the ribozyme recognition sequence. Single mutations in the virus or ribozyme decreased the efficacy of the ribozyme's inhibition of viral replication, and compensatory mutations restored it. To confirm that ribozyme-catalyzed RNA cleavage was actually needed for inhibition, we performed tests with a cell line expressing an inactivated ribozyme and with a virus containing a single nucleotide target mutation that allowed the ribozyme to bind but blocked cleavage at the recognition site. The results show that most of the antiviral activity of ribozymes is due to ribozyme-catalyzed cleavage at the targeted RNA sequence, but some additional inhibition seems to occur through an antisense mechanism.
Size Heterogeneity in the 3′ Noncoding Region of South American Isolates of Yellow Fever Virus
Authors: Bryant Juliet E. , Vasconcelos Pedro F. C. , Rijnbrand Rene C. A. , Mutebi J. P. , Higgs Stephen , Barrett Alan D. T.
Published: February 2005

The 3′ noncoding region (3′ NCR) of flaviviruses contains secondary and tertiary structures essential for virus replication. Previous studies of yellow fever virus (YFV) and dengue virus have found that modifications to the 3′ NCR are sometimes associated with attenuation in vertebrate and/or mosquito hosts. The 3′ NCRs of 117 isolates of South American YFV have been examined, and major deletions and/or duplications of conserved RNA structures have been identified in several wild-type isolates. Nineteen isolates (designated YF-XL isolates) from Brazil, Trinidad, and Venezuela, dating from 1973 to 2001, exhibited a 216-nucleotide (nt) duplication, yielding a tandem repeat of conserved hairpin, stem-loop, dumbbell, and pseudoknot structures. YF-XL isolates were found exclusively within one subclade of South American genotype I YFV. One Brazilian isolate exhibited, in addition to the 216-nt duplication, a deletion of a 40-nt repeated hairpin (RYF) motif (YF-XL-ΔRYF). To investigate the biological significance of these 3′ NCR rearrangements, YF-XL-ΔRYF and YF-XL isolates, as well as other South American YFV isolates, were evaluated for three phenotypes: growth kinetics in cell culture, neuroinvasiveness in suckling mice, and ability to replicate and produce disseminated infections in
Aedes aegypti
mosquitoes. YF-XL-ΔRYF and YF-XL isolates showed growth kinetics and neuroinvasive characteristics comparable to those of typical South American YFV isolates, and mosquito infectivity trials demonstrated that both types of 3′ NCR variants were capable of replication and dissemination in a laboratory-adapted colony of
A. aegypti
.
Improving the Safety of a Conditional-Live Human Immunodeficiency Virus Type 1 Vaccine by Controlling both Gene Expression and Cell Entry
Authors: Das Atze T. , Baldwin Chris E. , Vink Monique , Berkhout Ben
Published: February 2005

Live attenuated human immunodeficiency virus type 1 (HIV-1) vaccines are considered unsafe because faster-replicating pathogenic virus variants may evolve after vaccination. We previously presented a conditional-live HIV-1 variant of which replication can be switched off as an alternative vaccination strategy. To improve the safety of such a vaccine, we constructed a new HIV-1 variant that depends not only on doxycycline for gene expression but also on the T20 peptide for cell entry. Replication of this virus can be limited to the level required to induce the immune system by transient administration of doxycycline and T20. Subsequent withdrawal of these inducers efficiently blocks viral replication and evolution.