bluuebunny/crossref_metadata_2025 huggingface dataset:
Efficient Targeting of Protein Antigen to the Dendritic Cell Receptor DEC-205 in the Steady State Leads to Antigen Presentation on Major Histocompatibility Complex Class I Products and Peripheral CD8+ T Cell Tolerance
Authors: Bonifaz Laura , Bonnyay David , Mahnke Karsten , Rivera Miguel , Nussenzweig Michel C. , Steinman Ralph M.
Published: December 2002

To identify endocytic receptors that allow dendritic cells (DCs) to capture and present antigens on major histocompatibility complex (MHC) class I products in vivo, we evaluated DEC-205, which is abundant on DCs in lymphoid tissues. Ovalbumin (OVA) protein, when chemically coupled to monoclonal αDEC-205 antibody, was presented by CD11c+ lymph node DCs, but not by CD11c− cells, to OVA-specific, CD4+ and CD8+ T cells. Receptor-mediated presentation was at least 400 times more efficient than unconjugated OVA and, for MHC class I, the DCs had to express transporter of antigenic peptides (TAP) transporters. When αDEC-205:OVA was injected subcutaneously, OVA protein was identified over a 4–48 h period in DCs, primarily in the lymph nodes draining the injection site. In vivo, the OVA protein was selectively presented by DCs to TCR transgenic CD8+ cells, again at least 400 times more effectively than soluble OVA and in a TAP-dependent fashion. Targeting of αDEC-205:OVA to DCs in the steady state initially induced 4–7 cycles of T cell division, but the T cells were then deleted and the mice became specifically unresponsive to rechallenge with OVA in complete Freund's adjuvant. In contrast, simultaneous delivery of a DC maturation stimulus via CD40, together with αDEC-205:OVA, induced strong immunity. The CD8+ T cells responding in the presence of agonistic αCD40 antibody produced large amounts of interleukin 2 and interferon γ, acquired cytolytic function in vivo, emigrated in large numbers to the lung, and responded vigorously to OVA rechallenge. Therefore, DEC-205 provides an efficient receptor-based mechanism for DCs to process proteins for MHC class I presentation in vivo, leading to tolerance in the steady state and immunity after DC maturation.
Business Enterprises and Global Worlds
Authors: Jones Geoffrey
Published: December 2002

The role of business enterprise in integrating economies is one of the central historical themes of the last two centuries. Although globalization—both in its current iteration and in its nineteenth-century form—has been widely studied, the role of the firm, as opposed to macroeconomic forces, has yet to receive sufficient attention. Many research questions remain, including the role of the United States as a host country, the place of multinationals based in emerging markets, and the importance of understudied sectors such as retailing. Business historians should shift the focus of queries from “why” to “how” and go beyond the discipline's traditional organization along national lines to study the behavior of firms worldwide.
Markets in the Meadows: Department Stores and Shopping Centers in the Decentralization of Philadelphia, 1920–1980
Authors: Dyer Stephanie
Published: December 2002

Through a study of the history of department stores and shopping centers in the Philadelphia metropolitan region between 1920 and 1980, I explore the historical role of retailing in the evolution of the "sprawl" landscape that came to dominate urban form in the United States by the end of the twentieth century. Philadelphia is an important case study for this topic because of its long history of urbanization, during which it developed from a colonial port city to a nine-teenth-century industrial metropolis to a model of twentieth-century deindustrialization. Through all the phases of its growth, Philadelphia remained an important consumer marketplace serving the regional hinterland of the Delaware Valley. I sought to understand the forces that led to the city's twentieth-century decline as the primary site of consumption for the Delaware Valley, when major sites for distributing consumer goods developed in the hinterland itself.
A Nation of Realtors®: The Professionalization of Real Estate Brokerage and the Construction of a New American Middle Class
Authors: Hornstein Jeffrey M.
Published: December 2002

The rise of professional real estate brokerage is an ideal window into the internal dynamics of the cultural transformation of the American middle class in the twentieth century. Emerging as a full-time occupation in the late nineteenth century, real estate brokerage embodied a variety of early twentieth-century cultural, social, business, and economic trends, including the drive to professionalize business, the rapid expansion of white-collar labor and its feminization, the rise of independent contracting as a prominent form of labor relations, and the enormous growth of the home-building and -selling industries. As many scholars have noted, the home became a crucial site of both consumption and middle-class identification in the early twentieth century.
Projects, Management, and Protean Times: Engineering Enterprise in the United States, 1870–1960
Authors: Pinney Benjamin W.
Published: December 2002

The management of innovative work in high-technology fields has been a “hot” topic in recent years. In fields such as innovation management, product development, organization design, corporate strategy, economic sociology, organizational economics, engineering management, and knowledge management, scholars have examined subjects ranging from the sociology of creativity to the boundaries of firms in networked industries. They have remarked on the implications of contingent work, outsourcing, globalization, and the demise of the one-company career in studies concerned with issues from regional and national competitiveness to the evolution of labor markets and the social contract. Authors of articles and books published by the popular business press have trumpeted “nimble” and “virtual” organizational forms required by unprecedented conditions, and authors of scholarly essays have addressed the management of relentless change, the option-based valuation of interfirm networks, and knowledge in relation to organizational boundaries.
Consolidating Power: Technology, Ideology, and Philadelphia's Growth in the Early Republic
Authors: Schocket Andrew M.
Published: December 2002

Few contemporary American issues are more controversial than the powerful role of corporations (both business and municipal) in society, politics, and the economy. Yet strikingly, few scholars have investigated the deepest historical roots of American corporate power. Most historians who have considered this issue have focused their research on the Gilded Age and beyond because of the post-Civil War rise of industrial capitalism, the increased prominence of corporations on the national scene, and the dramatic growth of city governments in the context of late nineteenth-century large-scale immigration and the provision of citywide service and transportation infrastructure. Consequently, they have minimized the origins of American corporate power in the first decades of the republic, a crucial issue in the development of American business, American cities, and the nation.
Women Entrepreneurs and Italian Industrialization: Conjectures and Avenues for Research
Authors: Curli Barbara
Published: December 2002

This article examines the “state of the art” and the current debates on the subject of women entrepreneurs, presenting some preliminary observations and hypotheses regarding the role of business-women in Italian economic development. Reasons for the new historiographic interest in female entrepreneurship are identified, and the primary methodological difficulties encountered in its historical study—starting with establishing the definition and the statistical parameters of the woman entrepreneur and discussing her social and juridical “invisibility”—are summarized. Finally, suggestions are made about possible directions for research on key historical trends important in shaping female entrepreneurial abilities in the Italian context.
Competition and Cooperation: A Comparative Analysis of SEMATECH and the VLSI Research Project
Authors: Thornberry Jon B.
Published: December 2002

In this article I present a comparative analysis of two government-funded research and development consortia, Japan's Very Large Scale Integration Research Project (VLSI) and the American consortium Semiconductor Manufacturing Technology (SEMATECH), designed with the VLSI project as a model. An examination of the semiconductor industries in Japan and the United States reveals some important factors influencing the interaction of the consortia's member companies. Three main elements affected interfirm cooperation within the two organizations: first, past government policies influenced industry structure, firm capabilities, and firm behavior; second, the way firms competed had a material impact on how well they were able to cooperate; and third, the social structure and traditions of the semiconductor industries in the two countries mediated the transition from cooperation to competition.
Photosynthetic Light Harvesting by Carotenoids: Detection of an Intermediate Excited State
Authors: Cerullo G. , Polli D. , Lanzani G. , De Silvestri S. , Hashimoto H. , Cogdell R. J.
Published: December 2002

We present the first direct evidence of the presence of an intermediate singlet excited state (S
x
) mediating the internal conversion from S
2
to S
1
in carotenoids. The S
2
to S
x
transition is extremely fast and is completed within approximately 50 femtoseconds. These results require a reassessment of the energy transfer pathways from carotenoids to chlorophylls in the primary step of photosynthesis.
Interannual Variability in the North Atlantic Ocean Carbon Sink
Authors: Gruber Nicolas , Keeling Charles D. , Bates Nicholas R.
Published: December 2002

The North Atlantic is believed to represent the largest ocean sink for atmospheric carbon dioxide in the Northern Hemisphere, yet little is known about its temporal variability. We report an 18-year time series of upper-ocean inorganic carbon observations from the northwestern subtropical North Atlantic near Bermuda that indicates substantial variability in this sink. We deduce that the carbon variability at this site is largely driven by variations in winter mixed-layer depths and by sea surface temperature anomalies. Because these variations tend to occur in a basinwide coordinated pattern associated with the North Atlantic Oscillation, it is plausible that the entire North Atlantic Ocean may vary in concert, resulting in a variability of the strength of the North Atlantic carbon sink of about ±0.3 petagrams of carbon per year (1 petagram = 10
15
grams) or nearly ±50%. This extrapolation is supported by basin-wide estimates from atmospheric carbon dioxide inversions.
CREM-Dependent Transcription in Male Germ Cells Controlled by a Kinesin
Authors: Macho Betina , Brancorsini Stefano , Fimia Gian Maria , Setou Mitsutoshi , Hirokawa Nobutaka , Sassone-Corsi Paolo
Published: December 2002

ACT is a LIM-only protein expressed exclusively in round spermatids, where it cooperates with transcriptional activator CREM in regulating various postmeiotic genes. Targeted inactivation of CREM leads to a complete block of mouse spermiogenesis. We sought to identify the regulatory steps controlling the functional interplay between CREM and ACT. We found that ACT selectively associates with KIF17b, a kinesin highly expressed in male germ cells. The ACT-KIF17b interaction is restricted to specific stages of spermatogenesis and directly determines the intracellular localization of ACT. Sensitivity to leptomycin B indicates that KIF17b can be actively exported from the nucleus through the Crm1 receptor. Thus, a kinesin directly controls the activity of a transcriptional coactivator by a tight regulation of its intracellular localization.
Structure of the LDL Receptor Extracellular Domain at Endosomal pH
Authors: Rudenko Gabby , Henry Lisa , Henderson Keith , Ichtchenko Konstantin , Brown Michael S. , Goldstein Joseph L. , Deisenhofer Johann
Published: December 2002

The low-density lipoprotein receptor mediates cholesterol homeostasis through endocytosis of lipoproteins. It discharges its ligand in the endosome at pH < 6. In the crystal structure at pH = 5.3, the ligand-binding domain (modules R2 to R7) folds back as an arc over the epidermal growth factor precursor homology domain (the modules A, B, β propeller, and C). The modules R4 and R5, which are critical for lipoprotein binding, associate with the β propeller via their calcium-binding loop. We propose a mechanism for lipoprotein release in the endosome whereby the β propeller functions as an alternate substrate for the ligand-binding domain, binding in a calcium-dependent way and promoting lipoprotein release.
Calibration of Sulfate Levels in the Archean Ocean
Authors: Habicht Kirsten S. , Gade Michael , Thamdrup Bo , Berg Peter , Canfield Donald E.
Published: December 2002

The size of the marine sulfate reservoir has grown through Earth's history, reflecting the accumulation of oxygen into the atmosphere. Sulfur isotope fractionation experiments on marine and freshwater sulfate reducers, together with the isotope record, imply that oceanic Archean sulfate concentrations were <200 μM, which is less than one-hundredth of present marine sulfate levels and one-fifth of what was previously thought. Such low sulfate concentrations were maintained by volcanic outgassing of SO
2
gas, and severely suppressed sulfate reduction rates allowed for a carbon cycle dominated by methanogenesis.
Coordinated Nonvectorial Folding in a Newly Synthesized Multidomain Protein
Authors: Jansens Annemieke , van Duijn Esther , Braakman Ineke
Published: December 2002

The low-density lipoprotein receptor (LDL-R) is a typical example of a multidomain protein, for which in vivo folding is assumed to occur vectorially from the amino terminus to the carboxyl terminus. Using a pulse-chase approach in intact cells, we found instead that newly synthesized LDL-R molecules folded by way of “collapsed” intermediates that contained non-native disulfide bonds between distant cysteines. The most amino-terminal domain acquired its native conformation late in folding instead of during synthesis. Thus, productive LDL-R folding in a cell is not vectorial but is mostly posttranslational, and involves transient long-range non-native disulfide bonds that are isomerized into native short-range cysteine pairs.
Mass-Independent Sulfur of Inclusions in Diamond and Sulfur Recycling on Early Earth
Authors: Farquhar J. , Wing B. A. , McKeegan K. D. , Harris J. W. , Cartigny P. , Thiemens M. H.
Published: December 2002

Populations of sulfide inclusions in diamonds from the Orapa kimberlite pipe in the Kaapvaal-Zimbabwe craton, Botswana, preserve mass-independent sulfur isotope fractionations. The data indicate that material was transferred from the atmosphere to the mantle in the Archean. The data also imply that sulfur is not well mixed in the diamond source regions, allowing for reconstruction of the Archean sulfur cycle and possibly offering insight into the nature of mantle convection through time.
Microporous and Photoluminescent Chalcogenide Zeolite Analogs
Authors: Zheng Nanfeng , Bu Xianhui , Wang Bing , Feng Pingyun
Published: December 2002

Crystalline semiconducting sulfide and selenide zeolite analogs were synthesized that possess four-connected, three-dimensional tetrahedral networks built from tetravalent (M
4+
= Ge
4+
or Sn
4+
, where M = meta) and trivalent (M
3+
= Ga
3+
or In
3+
) cations. Microporous materials were obtained in all four combinations of M
4+
and M
3+
, and some of them were thermally stable up to at least 380°C. These materials exhibit framework topologies with pore size ranging from 12 to 24 tetrahedral atoms, high surface area, high framework charge density and ion exchange capacity, and tunable electronic and optical properties.
Structure-Assigned Optical Spectra of Single-Walled Carbon Nanotubes
Authors: Bachilo Sergei M. , Strano Michael S. , Kittrell Carter , Hauge Robert H. , Smalley Richard E. , Weisman R. Bruce
Published: December 2002

Spectrofluorimetric measurements on single-walled carbon nanotubes (SWNTs) isolated in aqueous surfactant suspensions have revealed distinct electronic absorption and emission transitions for more than 30 different semiconducting nanotube species. By combining these fluorimetric results with resonance Raman data, each optical transition has been mapped to a specific (
n
,
m
) nanotube structure. Optical spectroscopy can thereby be used to rapidly determine the detailed composition of bulk SWNT samples, providing distributions in both tube diameter and chiral angle. The measured transition frequencies differ substantially from simple theoretical predictions. These deviations may reflect combinations of trigonal warping and excitonic effects.
Reason and Passion in Bioethics
Authors: O'Neill Onora
Published: December 2002

Life, Liberty and the Defense of Dignity
The Challenge for Bioethics. Leon R. Kass. Encounter Books, San Francisco, 2002. 319 pp. $26.95. ISBN 1-893554-55-4.

In this "series of mediations on cloning, embryo research, the Human Genome Project, the sale of organs, and the assault on mortality itself," the chair of Bush's Council on Bioethics argues against the "runaway scientism" he fears will determine the course of biomedical research.
Swept Into the Modern Along with Science
Authors: Shahn Ezra
Published: December 2002

Exploring the Invisible
Art, Science, and the Spiritual. Lynn Gamwell. Princeton University Press, Princeton, NJ, 2002. 344 pp. $49.95, £35. ISBN 0-691-08972-8.

In this exploration of the impact of modern science on the visual arts, Gamwell argues that the shift to a scientific worldview led to the emergence of abstract art.
Generation of
Helicobacter pylori
Ghosts by PhiX Protein E-Mediated Inactivation and Their Evaluation as Vaccine Candidates
Authors: Panthel Klaus , Jechlinger Wolfgang , Matis Alexander , Rohde Manfred , Szostak Michael , Lubitz Werner , Haas Rainer
Published: December 2002

Bacterial ghosts are empty cell envelopes, which may be generated by the controlled expression of the PhiX174 lysis gene
E
in gram-negative bacteria to obtain vaccine candidates. We describe here the application of this technology to
Helicobacter pylori
. The lysis gene cassette was cloned into an
Escherichia coli-Helicobacter pylori
shuttle vector and introduced into an
H. pylori
recipient strain by bacterial conjugation. Temperature induction of the lysis gene cassette revealed a quantitative killing of the
H. pylori
culture without induction of lysis-resistant bacteria. Biochemical and transmission electron microscopic studies identified structurally intact
H. pylori.
Prophylactic oral vaccination experiments using these
H. pylori
ghosts in the BALB/c mouse model showed a significant reduction of the bacterial load in the ghost group, as measured by a quantitative bacterial reisolation procedure. Ten of 10 and 5 of 10 mice were protected, respectively, without the use of a mucosal adjuvant. Coadministration of ghosts with cholera toxin as mucosal adjuvant resulted in a complete protection of 10 of 10 and 8 of 8 mice against
H. pylori
challenge, with three animals showing a sterile immunity.
Multigenic Control of Disease Severity after VirulentMycobacterium tuberculosisInfection in Mice
Authors: Sánchez Fabio , Radaeva Tatiana V. , Nikonenko Boris V. , Persson Ann-Sophie , Sengul Selim , Schalling Martin , Schurr Erwin , Apt Alexander S. , Lavebratt Catharina
Published: December 2002

Following challenge with virulentMycobacterium tuberculosis, mice of the I/St inbred strain exhibit shorter survival time, more rapid body weight loss, higher mycobacterial loads in organs, and more severe lung histopathology than mice of the A/Sn strain. We previously performed a genome-wide scan for quantitative trait loci (QTLs) that control the severity ofM. tuberculosis-triggered disease in [(A/Sn × I/St) F1 × I/St] backcross-1 (BC1) mice and described several QTLs that are significantly or suggestively linked to body weight loss. In the present study we expanded our analysis by including the survival time phenotype and by genotyping 406 (A/Sn × I/St) F2 mice for the previously identified chromosomal regions of interest. The previously identified 12-cM-wide QTL on distal mouse chromosome 3 was designatedtbs1(tuberculosisseverity1); the location of the QTL on proximal chromosome 9 was narrowed to a 9-cM interval, and this QTL was designatedtbs2. Allelic variants of thetbs2locus appeared to be involved in control of both body weight loss and survival time. Also, the data strongly suggested that a QTL located in the vicinity of theH-2complex on chromosome 17 is involved in control of tuberculosis in mice of both genders, whereas thetbs1locus seemed to have an effect on postinfection body weight loss in female mice. Interestingly, these loci appeared to interact with each other, which suggests that there might be a basic genetic network for the control of intracellular parasites. Overall, linkage data reported here for F2 mice are in agreement with, and add to, our previous findings concerning the control ofM. tuberculosis-triggered disease in the BC1 segregation.
Fibronectin Bound to the Surface of
Staphylococcus aureus
Induces Association of Very Late Antigen 5 and Intracellular Signaling Factors with Macrophage Cytoskeleton
Authors: Shinji Hitomi , Seki Keiko , Tajima Akiko , Uchida Atsuko , Masuda Shogo
Published: December 2002

Staphylococcus aureus
Cowan I and a clinically isolated coagulase-negative
Staphylococcus
strain,
S. saprophyticus
10312, were found to have two fibronectin binding proteins, FnBPA and FnBPB. While both staphylococci bound to serum fibronectin to a similar extent, fibronectin binding significantly increased the phagocytic activity of macrophages against
S. aureus
(by ca. 150%) but not against
S. saprophyticus
. This enhancing effect of fibronectin was inhibited by an RGD sequence-containing peptide and also by anti-very late antigen 5 antibody. This suggests that the effect is mediated by very late antigen 5 expressed on macrophages. In macrophages ingesting fibronectin-bound Cowan I, α
5
and β
1
chains were associated with the cytoskeleton. Cytosolic signaling factors such as paxillin, c-Src, and c-Csk were also associated with the cytoskeleton. On the contrary, β
3
integrin transiently disappeared from the cytoskeleton when macrophages ingested the fibronectin-treated
S. aureus
Cowan I. Furthermore, the Src kinase family tyrosine kinase Lyn dissociated from the cytoskeleton. These cellular components did not respond in a fibronectin-dependent manner when macrophages phagocytosed
S. saprophyticus
. This means that only fibronectin-treated
S. aureus
Cowan I induces the accumulation of very late antigen 5, which in turn induces the association of paxillin and tyrosine kinases. It is thought that the phagocytic activity of macrophages against fibronectin-treated
S. aureus
was increased by signaling via the activation of very late antigen 5.
Lipooligosaccharide-DeficientNeisseria meningitidisShows Altered Pilus-Associated Characteristics
Authors: Albiger Barbara , Johansson Linda , Jonsson Ann-Beth
Published: December 2002

Molecular interaction between host mucosal surfaces and outer membrane components of microbes is crucial in the infection process. The outer membrane of pathogenicNeisseriacontains surface molecules such as pili, PilC, and Opa and a monolayer of lipooligosaccharide (LOS), all of which are involved in the interaction with host cells. Pili mediate the initial attachment to human epithelial cells, which is followed by tight contact between bacteria and the eucaryotic cells, leading to bacterial invasion. To further examine the basis for bacterium-host cell contact, we constructed an LOS-deficientNeisseria meningitidisserogroup C mutant. LOS deficiency was without exception accompanied by altered colony opacity and morphology, which most likely represented an “on” switch for Opa540 expression, and by reduced levels of the iron-regulated proteins FetA and FbpA. We show here that LOS is essential for pilus-associated adherence but dispensable for fiber formation and twitching motility. The absence of attachment to epithelial cells could not be attributed to altered levels of piliation or defects in the pilus adhesion phenotype. Further, LOS mutants do not invade host cells and have lost the natural competence for genetic transformation.
Superoxide Dismutase Influences the Virulence ofCryptococcus neoformansby Affecting Growth within Macrophages
Authors: Cox Gary M. , Harrison Thomas S. , McDade Henry C. , Taborda Carlos P. , Heinrich Garrett , Casadevall Arturo , Perfect John R.
Published: December 2002

Superoxide dismutase (SOD) is an enzyme that converts superoxide radicals into hydrogen peroxide and molecular oxygen and has been shown to contribute to the virulence of many human-pathogenic bacteria through its ability to neutralize toxic levels of reactive oxygen species generated by the host. SOD has also been speculated to be important in the pathogenesis of fungal infections, but the role of this enzyme has not been rigorously investigated. To examine the contribution of SOD to the pathogenesis of fungal infections, we cloned the Cu,Zn SOD-encoding gene (SOD1) from the human-pathogenic yeastCryptococcus neoformansand made mutants via targeted disruption. Thesod1mutant strains had marked decreases in SOD activity and were strikingly more susceptible to reactive oxygen species in vitro. Asod1mutant was significantly less virulent than the wild-type strain and two independent reconstituted strains, as measured by cumulative survival in the mouse inhalational model. In vitro studies established that thesod1strain had attenuated growth compared to the growth of the wild type and a reconstituted strain inside macrophages producing reduced amounts of nitric oxide. These findings demonstrate that (i) the Cu,Zn SOD contributes to virulence but is not required for pathogenicity inC. neoformans; (ii) the decreased virulence of thesod1strain may be due to increased susceptibility to oxygen radicals within macrophages; and (iii) other antioxidant defense systems inC. neoformanscan compensate for the loss of the Cu,Zn SOD in vivo.
Purification and Characterization of a UDP-Glucosyltransferase Produced by
Legionella pneumophila
Authors: Belyi Iouri , Popoff Michel R. , Cianciotto Nicholas P.
Published: December 2002

Legionella pneumophila
is the agent of Legionnaires' disease. It invades and replicates within eukaryotic cells, including aquatic protozoans, mammalian macrophages, and epithelial cells. The molecular mechanisms of the
Legionella
interaction with target cells are not fully defined. In an attempt to discover novel virulence factors of
L. pneumophila
, we searched for bacterial enzymes with transferase activity. Upon screening ultrasonic extracts of virulent legionellae, we identified a uridine diphospho (UDP)-glucosyltransferase activity, which was capable of modifying a 45-kDa substrate in host cells. An approximately 60-kDa UDP-glucosyltransferase was purified from
L. pneumophila
and subjected to microsequencing. An N-terminal amino acid sequence, as well as the sequence of an internal peptide, allowed us to identify the gene for the enzyme within the unfinished
L. pneumophila
genome database. The intact gene was cloned and expressed in
Escherichia coli
, and the recombinant protein was purified and confirmed to possess an enzymatic activity similar to that of the native UDP-glucosyltransferase. We designated this gene
ugt
(
U
DP-glucosyl
t
ransferase). The
Legionella
enzyme did not exhibit significant homology with any known protein, suggesting that it is novel in structure and, perhaps, in function. Based on PCR data, an enzyme assay, and an immunoblot analysis, the glucosyltransferase appeared to be conserved in
L. pneumophila
strains but was absent from the other
Legionella
species. This study represents the first identification of a UDP-glucosyltransferase in an intracellular parasite, and therefore modification of a eukaryotic target(s) by this enzyme may influence host cell function and promote
L. pneumophila
proliferation.
Molecular Heterogeneity of
Ehrlichia chaffeensis
Isolates Determined by Sequence Analysis of the 28-Kilodalton Outer Membrane Protein Genes and Other Regions of the Genome
Authors: Cheng Chuanmin , Paddock Christopher D. , Reddy Ganta Roman
Published: December 2002

Ehrlichia chaffeensis
, a tick-transmitted rickettsial agent, is responsible for human monocytic ehrlichiosis (HME). In this study, we genetically mapped 10 isolates obtained from HME patients. Sequence analysis of the 28-kDa outer membrane protein (OMP) multigene locus spanning 6 of the 22 tandemly arranged genes identified three distinct genetic groups with shared homology among isolates within each group. Isolates in Groups I and III contained six genes each, while Group II isolates had a gene deletion. There were two regions on the locus where novel gene deletion or insertion mutations occurred, resulting in the net loss of one gene in Group II isolates. Numerous nucleotide differences among genes in isolates of each group also were detected. The shared homology among isolates in each group for the 28-kDa OMP locus suggests the derivation of clonal lineages. Transcription and translation analysis of the locus revealed differences in the expressed genes of different group isolates. Analysis of the 120-kDa OMP gene and variable-length PCR target gene showed size variations resulting from loss or gain of long, direct repeats within the protein coding sequences. To our knowledge this is the first study that looked at several regions of the genome simultaneously, and we provide the first evidence of heterogeneity resulting from gene deletion and insertion mutations in the
E. chaffeensis
genome. Diversity in different genomic regions could be the result of a selection process or of independently evolved genes.
Phagocyte NADPH Oxidase, but Not Inducible Nitric Oxide Synthase, Is Essential for Early Control of
Burkholderia cepacia
and
Chromobacterium violaceum
Infection in Mice
Authors: Segal Brahm H. , Ding Li , Holland Steven M.
Published: December 2002

Reactive oxygen and nitrogen intermediates have critical, partially overlapping roles in host defense against a variety of pathogens. Using mice deficient in generating phagocyte superoxide (p47

phox

−/−
) and mice deficient in generating inducible nitric oxide synthase (iNOS
−/−
), we examined the roles of these reactive species in host defense against
Burkholderia cepacia
and
Chromobacterium violaceum
, organisms known to have unusual virulence in chronic granulomatous disease. Intraperitoneal
B. cepacia
challenge (4.0 × 10
3
to 4.0 × 10
5
organisms/mouse) resulted in mortality in all p47

phox

−/−
mice, with the survival interval being inversely proportionate to the amount of inoculum. Pretreatment with gamma interferon did not affect survival.
C. violaceum
was strikingly virulent in p47

phox

−/−
mice (the 50% lethal dose [LD
50
] was <13 organisms). iNOS
−/−
and wild-type mice were resistant to
B. cepacia
challenges of at least 10
6
organisms per mouse, and the LD
50
of
C. violaceum
was between 10
6
and 10
7
organisms per mouse. Consistent with the survival data, numbers of organisms in cultures of
B. cepacia
from multiple sites were higher for p47

phox

−/−
mice than for iNOS
−/−
and wild-type mice at day 4 after challenge, but numbers of organisms for different
B. cepacia
strains varied. The recovery of
C. violaceum
was strikingly greater at 18 h after challenge for p47

phox

−/−
mice than for iNOS
−/−
and wild-type mice, in which the organism burdens were virtually nil. In vitro, both
B. cepacia
and
C. violaceum
were sensitive to H
2
O
2
and to reactive nitrogen intermediates but the sensitivities of different strains varied significantly. Host defense against
B. cepacia
and
C. violaceum
is critically dependent in vivo on reactive oxygen intermediates, and these species are model organisms to further dissect host and pathogen interactions related to the generation and scavenging of microbicidal reactive intermediates.
Identification of Substrates and Chaperone from the
Yersinia enterocolitica
1B Ysa Type III Secretion System
Authors: Foultier Boris , Troisfontaines Paul , Vertommen Didier , Marenne Marie-Noëlle , Rider Mark , Parsot Claude , Cornelis Guy R.
Published: December 2002

All pathogenic
Yersinia enterocolitica
strains carry the pYV plasmid encoding the Ysc-Yop type III secretion (TTS) system, which operates at 37°C. In addition, biovar 1B
Y. enterocolitica
strains possess a second, chromosomally encoded, TTS system called Ysa, which operates, at least in vitro, under low-temperature and high-salt (LTHS) conditions. Six open reading frames,
sycB
,
yspB
,
yspC
,
yspD
,
yspA
, and
acpY
, neighbor the
ysa
genes encoding the Ysa TTS apparatus. Here we show that YspA, YspB, YspC, and YspD are secreted by the Ysa TTS system under LTHS conditions. SycB is a chaperone for YspB and YspC and stabilizes YspB. YspB, YspC, and SycB share some similarity with TTS substrates and the chaperone encoded by the Mxi-Spa locus of
Shigella flexneri
and SPI-1 of
Salmonella enterica
. In addition, Ysa also secretes the pYV-encoded YopE under LTHS conditions, indicating that YopE is a potential effector of both
Y. enterocolitica
TTS systems. YspC could also be secreted by
S. flexneri
, but no functional complementation of
ipaC
was observed, which indicates that despite their similarity the Ysa and the Mxi-Spa systems are not interchangeable. When expressed from the
yopE
promoter, YspB and YspC could also be secreted via the Ysc injectisome. However, they could not form detectable pores in eukaryotic target cells and could not substitute for YopB and YopD for translocation of Yop effectors.
THP-1 Cell Apoptosis in Response to Mycobacterial Infection
Authors: Riendeau Carrie J. , Kornfeld Hardy
Published: December 2002

We previously reported thatMycobacterium tuberculosisinfection primes human alveolar macrophages (HAM) for tumor necrosis factor alpha (TNF-α)-mediated apoptosis and that macrophage apoptosis is associated with killing internalized bacilli. Virulent mycobacterial strains elicit much less apoptosis than attenuated strains, implying that apoptosis is a defense against intracellular infection. The present study evaluated the potential for phorbol myristate acetate-differentiated THP-1 cells to mimic this response of primary macrophages. Consistent with the behavior of alveolar macrophages, attenuatedM. tuberculosisH37Ra andMycobacterium bovisBCG strongly induce THP-1 apoptosis, which requires endogenous TNF. THP-1 apoptosis is associated with reduced viability of infecting BCG. In contrast, virulent wild-typeM. tuberculosisH37Rv andM. bovisdo not increase THP-1 apoptosis over baseline. BCG induced early activation of caspase 10 and 9, followed by caspase 3. In contrast, wild-typeM. bovisinfection failed to activate any caspases in THP-1 cells. BCG-induced THP-1 apoptosis is blocked by retroviral transduction with vectors expressingcrmAbut notbcl-2. We conclude that differentiated THP-1 cells faithfully model the apoptosis response of HAM. Analysis of the THP-1 cell response to infection with virulent mycobacteria suggests that TNF death signals are blocked proximal to initiator caspase activation, at the level of TNF receptor 1 or its associated intracytoplasmic adaptor complex. Interference with TNF death signaling may be a virulence mechanism that allowsM. tuberculosisto circumvent innate defenses leading to apoptosis of infected host cells.
Entry of the Lymphogranuloma Venereum Strain ofChlamydia trachomatisinto Host Cells Involves Cholesterol-Rich Membrane Domains
Authors: Jutras Isabelle , Abrami Laurence , Dautry-Varsat Alice
Published: December 2002

Chlamydiae are bacterial pathogens which develop strictly inside the epithelial cells of their hosts. The mechanism used by chlamydiae to enter cells is not well characterized; however, it is thought to consist of a receptor-mediated process. In addition, the formation of clathrin-coated pits appears to be dispensable for chlamydiae to be internalized by host cells. Clathrin-independent endocytosis has recently been shown to occur through cholesterol-rich lipid microdomains, which are characterized by detergent insolubility. In the present study, we investigated whether these lipid domains play a role inChlamydia trachomatisserovar L2 internalization by host cells. Our results show that after binding to HeLa cells, chlamydiae are associated with detergent-resistant lipid microdomains (DRMs), which can be isolated by fractionation of infected HeLa cells and flotation on a sucrose gradient. After internalization by HeLa cells, chlamydiae were still found in DRMs. In addition, extraction of plasma membrane cholesterol inhibited infection of HeLa cells byC. trachomatis. Many of the proteins associated with DRMs are glycosylphosphatidylinositol (GPI)-anchored proteins; however, our results could not identify a role for GPI-anchored proteins in the entry process. The same results were obtained forChlamydia psittacistrain GPIC. We propose that cholesterol-rich domains participate in the entry of chlamydiae into host cells.Chlamydiabinding to cholesterol-rich domains may lead to coalescence of the bacterial cells, which could trigger internalization by host cells.
Interleukin-1 Receptor Antagonist Gene Polymorphism, Vaginal Interleukin-1 Receptor Antagonist Concentrations, and VaginalUreaplasma urealyticumColonization in Pregnant Women
Authors: Barton Parrin T. , Gerber Stefan , Skupski Daniel W. , Witkin Steven S.
Published: December 2002

Ureaplasma urealyticumis the microorganism most frequently isolated from amniotic fluids of women in preterm labor. The relationship between vaginal colonization withU. urealyticum,vaginal interleukin-1 receptor antagonist (IL-1ra) levels, and the IL-1ra genotype in pregnant women was examined. Vaginal specimens, obtained with a cotton swab from 207 women in their first trimester of pregnancy, were tested for IL-1ra concentrations by enzyme-linked immunosorbent assay and forU. urealyticumand IL-1ra genotypes by PCR.U. urealyticumwas detected in 85 (41.1%) women. The median IL-1ra level was 450 ng/ml in women positive forU. urealyticum, as opposed to 225 ng/ml in women negative for this microorganism (P< 0.0001). Sixty-two percent of the 16 women who were homozygous for allele 2 of the IL-1ra gene (IL-1RN*2) were colonized withU. urealyticum, as opposed to 47% of the 49 women who wereIL-1RN*1/IL-1RN*2heterozygotes and 34% of the 133 women who wereIL-1RN*1homozygotes (P< 0.05). Median IL-1ra levels were 750 ng/ml inIL-1RN*2homozygotes, 300 ng/ml inIL-1RN*1/IL-1RN*2heterozygotes, and 250 ng/ml inIL-1RN*1homozygotes (P= 0.02). The vast majority of subjects had an uneventful pregnancy and delivered a healthy infant at term. The IL-1ra genotype orU. urealyticumcolonization was unrelated to birth weight. Pregnant women who are colonized withU. urealyticumduring the first trimester have elevated vaginal IL-1ra concentrations and a higher prevalence of theIL-1RN*2homozygote genotype than do noncolonized women.
Oral Immunization with ATP-Dependent Protease-Deficient Mutants Protects Mice against Subsequent Oral Challenge with VirulentSalmonella entericaSerovar Typhimurium
Authors: Matsui Hidenori , Suzuki Masato , Isshiki Yasunori , Kodama Chie , Eguchi Masahiro , Kikuchi Yuji , Motokawa Kenji , Takaya Akiko , Tomoyasu Toshifumi , Yamamoto Tomoko
Published: December 2002

We evaluated the efficacy of mutants with a deletion of the stress response protease gene as candidates for live oral vaccine strains againstSalmonellainfection through infection studies with mice by using aSalmonella entericaserovar Typhimurium mutant with a disruption of the ClpXP or Lon protease. In vitro, the ClpXP protease regulates flagellum synthesis and the ClpXP-deficient mutant strain exhibits hyperflagellated bacterial cells (T. Tomoyasu et al., J. Bacteriol.184:645-653, 2002). On the other hand, the Lon protease negatively regulates the efficacy of invading epithelial cells and the expression of invasion genes (A. Takaya et al., J. Bacteriol.184:224-232, 2002). When 5-week-old BALB/c mice were orally administered 5 × 108CFU of the ClpXP- or Lon-deficient strain, bacteria were detected with 103to 104CFU in the spleen, mesenteric lymph nodes, Peyer's patches, and cecum 1 week after inoculation and the bacteria then decreased gradually in each tissue. Significant increases of lipopolysaccharide-specific immunoglobulin G (IgG) and secretory IgA were detected at week 4 and maintained until at least week 12 after inoculation in serum and bile, respectively. Immunization with the ClpXP- or Lon-deficient strain protected mice against oral challenge with the serovar Typhimurium virulent strain. Both the challenged virulent and immunized avirulent salmonellae were completely cleared from the spleen, mesenteric lymph nodes, Peyer's patches, and even cecum 5 days after the challenge. These data indicate thatSalmonellawith a disruption of the ATP-dependent protease ClpXP or Lon can be useful in developing a live vaccine strain.
A GRA1 DNA Vaccine Primes Cytolytic CD8+T Cells To Control AcuteToxoplasma gondiiInfection
Authors: Scorza T. , D'Souza S. , Laloup M. , Dewit J. , De Braekeleer J. , Verschueren H. , Vercammen M. , Huygen K. , Jongert E.
Published: December 2002

Protective immunity againstToxoplasma gondiiis known to be mediated mainly by T lymphocytes and gamma interferon (IFN-γ). The contribution of CD4+and CD8+T-lymphocyte subsets to protective immune responses againstT. gondiiinfection, triggered by a GRA1 (p24) DNA vaccine, was assessed in this study. In vitro T-cell depletion experiments indicated that both CD4+and CD8+T-cell subsets produced IFN-γ upon restimulation with aT. gondiilysate. In addition, the GRA1 DNA vaccine elicited CD8+T cells that were shown to have cytolytic activity against parasite-infected target cells and a GRA1-transfected cell line. C3H mice immunized with the GRA1 DNA vaccine showed 75 to 100% protection, while 0 to 25% of the mice immunized with the empty control vector survived challenge withT. gondiicysts. In vivo T-cell depletion experiments indicated that CD8+T cells were essential for the survival of GRA1-vaccinated C3H mice during the acute phase ofT. gondiiinfection, while depletion of CD4+T cells led to an increase in brain cyst burden during the chronic phase of infection.
Role for Recombinant γ-Glutamyltransferase from
Treponema denticola
in Glutathione Metabolism
Authors: Chu Lianrui , Xu Xiaoping , Dong Zheng , Cappelli David , Ebersole Jefferey L.
Published: December 2002

Volatile sulfur compounds, including hydrogen sulfide (H
2
S), have been implicated in the development of periodontal disease. Glutathione is an important thiol source for H
2
S production in periodontal pockets. Our recent studies have delineated a pathway of glutathione metabolism in
Treponema denticola
that releases H
2
S. In this pathway, γ-glutamyltransferase (GGT) has been proposed to catalyze the first step of glutathione degradation. We have cloned the gene of GGT from
T. denticola
, which contains an open reading frame of 726 bp encoding a protein of 241 amino acids. Transformation of this gene into
Escherichia coli
led to the expression of a recombinant protein. After purification by chromatography, the recombinant protein showed enzymatic activity typical of GGT, catalyzing the degradation of Na-γ-glutamyl-4-nitroaniline (GNA) and the hydrolysis of glutathione, releasing glutamic acid or glutamine and cysteinylglycine.
l
-Cysteine is not a substrate of GGT. Importantly, GNA, when added to
T. denticola
, was able to compete with glutathione and inhibit the production of H
2
S, ammonia, and pyruvate. This was accompanied by the suppression of hemoxidative and hemolytic activities of the bacteria. Purified GGT was inactivated by TLCK (
N
α-
p
-tosyl-
l
-lysine chloromethyl ketone) and proteinase K treatment. However, higher enzymatic activity was demonstrated in the presence of 2-mercaptoethanol and dithiothreitol. Our further experiments showed that the addition of recombinant GGT to
Porphyromonas gingivalis
, a bacterium without significant glutathione-metabolizing capacity, drastically increased the utilization of glutathione by the bacterium, producing H
2
S, ammonia, and pyruvate. This was again accompanied by enhanced bacterial hemoxidative and hemolytic activities. Together, the results suggest an important role for GGT in glutathione metabolism in oral bacteria.
sarU
, a
sarA
Homolog, Is Repressed by SarT and Regulates Virulence Genes in
Staphylococcus aureus
Authors: Manna Adhar C. , Cheung Ambrose L.
Published: December 2002

In searching the
Staphylococcus aureus
genome, we previously identified
sarT
, a homolog of
sarA,
which encodes a repressor for α-hemolysin synthesis. Adjacent but transcribed divergently to
sarT
is
sarU,
which encodes a 247-residue polypeptide, almost twice the length of SarA. Sequence alignment disclosed that SarU, like SarS, which is another SarA homolog, could be envisioned as a molecule with two halves, with each half being homologous to SarA. SarU, as a member of the SarA family proteins, disclosed conservation of basic residues within the helix-turn-helix motif and within the beta hairpin loop, two putative DNA binding domains within this protein family. The transcription of
sarU
is increased in a
sarT
mutant. Gel shift and transcriptional fusion studies revealed that SarT can bind to the
sarU
promoter region, probably acting as a repressor for
sarU
transcription. The expression of RNAII and RNAIII of
agr
is decreased in a
sarU
mutant. As RNAIII expression is up-regulated in a
sarT
mutant, we hypothesize that
sarT
may down regulate
agr
RNAIII expression by repressing
sarU
, a positive activator of
agr
expression. We propose that, in addition to the quorum sensing effect of the autoinducing peptide of
agr
, the
sarT
-
sarU
pathway may represent a secondary amplification loop whereby the expression of
agr
(e.g., those found in vivo) might repress
sarT
, leading to increased expression of
sarU
. Elevated
sarU
expression would result in additional amplification of the original
agr
signal.
Differential Expression of Gamma Interferon mRNA Induced by Attenuated and VirulentMycobacterium tuberculosisin Guinea Pig Cells afterMycobacterium bovisBCG Vaccination
Authors: Jeevan Amminikutty , Yoshimura Teizo , Lee Kyeong Eun , McMurray David N.
Published: December 2002

To determine whetherMycobacterium bovisBCG vaccination would alter gamma interferon (IFN-γ) mRNA expression in guinea pig cells exposed toMycobacterium tuberculosis, we cloned a cDNA encoding guinea pig IFN-γ from a spleen cell cDNA library. The cDNA is composed of 1,110 bp, with an open reading frame encoding a 166-amino-acid protein which shows 56 and 41% amino acid sequence homology to human and mouse IFN-γ, respectively. Spleen or lymph node cells from naïve and BCG-vaccinated guinea pigs were stimulated with purified protein derivative (PPD) orM. tuberculosisH37Ra or H37Rv, and the total RNA was subjected to Northern blot analysis with a32P-labeled probe derived from the cDNA clone. Compared to the IFN-γ mRNA expression in cells of naïve animals, that in spleen and lymph node cells exposed to various stimuli was enhanced after BCG vaccination. However, there was a significant reduction in IFN-γ mRNA levels when cells were stimulated with a multiplicity of infection of greater than 1 virulentM. tuberculosisbacterium per 10 cells. The enhanced IFN-γ mRNA response in BCG-vaccinated animals was associated with an increase in the proportions of CD4+T cells in the spleens, as determined by fluorescence-activated cell sorter analysis. Furthermore, the nonadherent population in the spleens enriched either by panning with anti-guinea pig immunoglobulin G-coated plates or by purification on nylon wool columns produced more IFN-γ mRNA than whole spleen cells following stimulation with concanavalin A or PPD. This indicates that T cells are principally responsible for the upregulation of IFN-γ mRNA expression following BCG vaccination. The mechanism by which virulent mycobacteria suppress IFN-γ mRNA accumulation is currently under investigation.