bluuebunny/crossref_metadata_2025 huggingface dataset:
A Computer-Aided Design Tool in Java for Planar Gripper Design
Authors: Tao Zhang Mike , Goldberg Ken
Published: March 2004

We describe an Internet-based CAD tool that automatically designs gripper jaws that will rotate a given rigid convex polygonal part from a selected stable resting orientation to a desired final orientation to facilitate insertion or assembly. In this paper we describe the CAD algorithm, its Java implementation, and improvements that relax input specifications and improve computational efficiency. We illustrate with physical experiments using a machined aluminum part and the resulting gripper as designed by the CAD tool. We also consider cases where a solution may not exist and derive a class of triangular parts that cannot be rotated with the planar grippers. The CAD tool, implemented as a Java applet, is available online at www.ieor.berkeley.edu/∼goldberg/sa-gripper/.
Exploiting Self-Similarity in Geometry for Voxel Based Solid Modeling
Authors: Udeshi Tushar , Parker Eric
Published: March 2004

Voxel-based modeling techniques are known for their robustness and flexibility. However, they have three major shortcomings: (1) Memory intensive, since a large number of voxels are needed to represent high-resolution models (2) Computationally expensive, since a large number of voxels need to be visited (3) Computationally expensive isosurface extraction is needed to visualize the results. We describe techniques which alleviate these by taking advantage of self-similarity in the data making voxel-techniques practical and attractive. We describe algorithms for MEMS process emulation, isosurface extraction and visualization which utilize these techniques.
2nd-CAD: A Tool for Conceptual Systems Design in Electromechanical Domain
Authors: Vargas-Hernandez Noe , Shah Jami J.
Published: March 2004

This paper presents a framework and information model for the development of SECOND-CAD (Systems Engineering CONceptual Design-CAD), or 2nd-CAD, a Computer Aided Conceptual Design (CACD) tool for Electromechanical Systems. The conceptual design tasks supported include functional design, behavior modeling, and component selection from standard industrial supply catalogs for mechanical, fluid, and electric engineering domains. 2nd-CAD is composed of three entity catalogs the designer uses to create three interconnected structures for function, behavior, and component. The logical model behind 2nd-CAD is one of the major contributions of this research. It allows the user to define entities based on popular taxonomies; this eases data exchange with other tools. When constructing structures, only technically feasible relationships are permitted and if an element in a structure is modified, the change is propagated throughout the structure. It reuses the entities’ information content to create new structures and since the three structures are interconnected, changes can be traced for design validation. 2nd-CAD’s functional requirements, logical design, and physical implementation are discussed in this paper.
There's a Problem in the Furnace
Authors: Tower John
Published: March 2004

A long-sought-after mutation in the
manganese superoxide dismutase
gene
MnSOD
has been isolated in
Drosophila melanogaster
. The
MnSOD
null mutation is recessive lethal, with death occurring just after eclosion. In this Perspective, I discuss these results and related work in mice within the context of the oxidative damage theory of aging.
Riddled With Errors
Authors: Neeper Shawne
Published: March 2004

As a factory ages and parts deteriorate, new production errors can appear, resulting in damaged goods. The same might be true of men's testicles. A new study finds that old mice have more--and different--DNA damage in their sperm than do younger animals. The results might help clarify the roots of disorders that are linked to paternal age.
Hop on Board
Authors: Davenport R. John
Published: March 2004

BANFF, CANADA
--Taking the bus instead of walking encourages fat storage, and a cellular version of public transit might also cultivate blubber. A protein that carries messenger RNAs around the cell totes the instructions for a protein that plays a key role in the beginning stages of fat-cell maturation, according to new work presented here 6 March 2004 at the Molecular Control of Adipogenesis and Obesity Keystone Symposium. The work illuminates a previously uninvestigated control point in fat-cell specialization and suggests a possible new target for drugs to shrink flab.
Vintage Gerontology
Authors: Chen Ingfei
Published: March 2004

Judith Campisi meandered from college chemistry major to gas station clerk, Avon lady, and hippie folksinger before pursuing a career in cell and molecular biology. Studying cancer and the cell cycle at Boston University in the mid-'80s, she was initially skeptical that senescence, a stage in which cells stop splitting but do not die, had anything to do with whole-organism aging. But her own research into the question changed her mind. Today, based at Lawrence Berkeley National Laboratory in Berkeley, California, and the Buck Institute for Age Research in nearby Novato, she is exploring the paradoxical hypothesis that cell senescence might prevent the development of tumors early in life as well as promote the cancers of old age; she also studies the premature human aging disorder called Werner syndrome and cellular resistance to oxidative stress. Since moving to California, Campisi has taken an interest in another aging process: the kind that produces a fine Syrah. An enophile, she enjoys tasting and making wine.
Glycation Stimulates Amyloid Formation
Authors: Obrenovich Mark E. , Monnier Vincent M.
Published: March 2004

Amyloidosis comprises a group of systemic and localized diseases with varied clinical presentations. In these diseases, amyloid forms when proteins with a largely α-helical structure lose their original conformation and are converted into a predominantly β-sheet form, thereby increasing their propensity to form highly insoluble and fibrillar aggregates. Most soluble amyloid precursor proteins have substantial β-pleated sheet secondary structure, and extensive β-pleated sheet structure occurs in all of the deposited fibrils. The aberrant deposition of proteins as cellular inclusions or plaques in the form of amyloid fibrils is a characteristic hallmark of all amyloid diseases (or amyloidoses) and of the so-called conformational diseases. Environmental and genetic factors are known to be involved, but the mechanism by which this process happens still is poorly understood. Here we report a new finding from the Dutch group of Gebbink and colleagues, which points to the posttranslational process of glycation as a key mechanism in the formation of amyloid. These researchers showed that glycation causes albumin, a globular protein with a largely α-helical structure, to adopt a
β-pleated sheet structure and the quaternary structural element known as the cross-β conformation. These are features commonly shared by all amyloids. This research is the first to show glycation as a predisposing factor for amyloidosis.
Lipid-Control Allies
Authors: Neeper Shawne
Published: March 2004

The number of tools to fight heart disease could be mushrooming. Two fungal compounds slow down artery-clogging lipid buildup in mice, a new study finds, and they work through a different pathway than current treatments do. The drugs could reinforce existing therapy or take over when other medicines fail.
B6D2F1 Mouse
Authors: Harper James M.
Published: March 2004

This document contains a summary of the biological characteristics of the B6D2F1 mouse, a first-generation (F
1
) hybrid from a cross of a C57BL/6 female with a DBA/2 male. Included are longevity statistics calculated from data provided by the National Center for Toxicological Research.
Search for Methuselah Genes Heats Up
Authors: Longo Valter D.
Published: March 2004

In a recent article, Morley and Morimoto confirm previous studies suggesting that the transcriptional activator heat shock factor 1 (HSF-1) regulates stress resistance genes that extend longevity independently of DAF-16. They also show that overexpression of HSF-1 in neurons or body-wall muscle cells is sufficient to extend longevity. The role of multiple transcription factors in extending life span in yeast and worms raises the possibility that many transcriptional regulators can contribute to longevity extension.
Stop a Tumor, Save a Cell
Authors: Leslie Mitch
Published: March 2004

Tumor-suppressing proteins are supposed to be tough guys that execute faulty cells or squelch their reproduction. But according to a new study, one such protein nurtures some types of cells, fostering their recovery from starvation. The results bolster a recently discovered metabolic link between diabetes and cancer and could help researchers design new drugs to battle the diseases.
2003: Signaling Breakthroughs of the Year
Authors: Adler Elizabeth M. , Gough Nancy R. , Ray L. Bryan
Published: March 2004

This annual feature summarizes major advances in signaling research published during the past year. Notable findings in signaling research nominated by leading researchers in signal transduction and by the STKE editors are described, along with the potential new avenues of research opened by these findings.
A New Look at an Old Problem: The Survival and Organ-Specific Growth of Metastases
Authors: Vander Griend Donald J. , Rinker-Schaeffer Carrie W.
Published: March 2004

Despite improvements in cancer detection and therapy, metastatic disease is largely incurable. Recent research indicates that tumor cells disseminate widely early in the process of pathogenesis, and that the survival and proliferation of these cells--and thus the development of metastases--depend on interactions between the disseminated cells and their particular microenvironment. Proliferative signals and the inhibition of proapoptotic responses are both critically involved in the development of clinically significant metastases. Identification of the underlying signaling cascades may provide additional targets for antimetastatic therapy.
Stimulation of Cells Using EGF-Coated Magnetic Beads
Authors: Kempiak Stephan J. , Segall Jeffrey E.
Published: March 2004

The ability to determine the spatial localization of signals can assist in defining the regulation of specific signaling pathways within the cell. Localized application of a stimulus, followed by imaging of various proteins or their phosphorylated products, allows visualization of the propagation scales of responses to the stimulus. To obtain a localized stimulus of epidermal growth factor (EGF), we bound the ligand to magnetic beads, which could be rapidly pulled onto cell surfaces by a magnet. This method allows the spatial analysis of EGF-induced signaling pathways.
Local Excitation, Global Inhibition Mechanism for Gradient Sensing: An Interactive Applet
Authors: Kutscher Brett , Devreotes Peter , Iglesias Pablo A.
Published: March 2004

We have proposed a model in which cells detect gradients of chemoattractant by balancing a fast local excitation and a slower global inhibition. To illustrate this general mechanism, we have developed an interactive applet that mimics laboratory experiments in which either spatially homogeneous or heterogeneous stimuli of chemoattractant are applied.
Mechanosensitive Channels: Multiplicity of Families and Gating Paradigms
Authors: Sukharev Sergei , Corey David P.
Published: March 2004

Mechanosensitive ion channels are the primary transducers that convert mechanical force into an electrical or chemical signal in hearing, touch, and other mechanical senses. Unlike vision, olfaction, and some types of taste, which all use similar kinds of primary heterotrimeric GTP-binding protein–coupled receptors, mechanosensation relies on diverse types of transducer molecules. Unrelated types of channels can be used for the perception of various mechanical stimuli, not only in distant groups of organisms, but also in separate locations of the same organism. The extreme sensitivity of the transduction mechanism in the auditory system, which relies on an elaborate structure of rigid cilia, filamentous links, and molecular motors to focus force on transduction channels, contrasts with that of the bacterial channel MscL, which is opened by high lateral tension in the membrane and fulfills a safety-valve rather than a sensory function. The spatial scales of conformational movement and force in these two systems are described, and are shown to be consistent with a general physical description of mechanical channel gating. We outline the characteristics of several types of mechanosensitive channels and the functional contexts in which they participate in signaling and cellular regulation in sensory and nonsensory cells.
The Canonical Ethylene Signaling Pathway
Authors: Stepanova Anna N. , Alonso Jose M.
Published: March 2004

The structural simplicity of the plant hormone ethylene contrasts with its dramatic effects in various developmental processes, as well as in the responses to a diversity of environmental signals. This broad spectrum of responses is mediated by a single well-conserved signaling cascade. Ethylene is perceived by a family of two-component histidine kinase receptors that become inactivated upon ethylene binding. In the absence of the hormone, the receptors activate CTR1, a negative regulator of ethylene responses. Sequence similarity between CTR1 and the Raf protein kinases implies involvement of a mitogen-activated protein kinase (MAPK) cascade in this signaling pathway. The first candidate MAPKs in the ethylene response pathway have been identified. EIN2 acts downstream of CTR1 and the possible kinase cascade. Although the biochemical function of EIN2 is not understood, its critical role is manifested by the complete ethylene insensitivity of the
EIN2
loss-of-function mutants. Downstream of EIN2 there is a family of plant-specific EIN3-like transcription factors that mediate all known ethylene responses. The regulation of EIN3 stability by ethylene is accomplished by F-box-containing proteins that participate in the formation of a SCF complex, which targets proteins for degradation by the proteasome. A large number of ethylene-regulated genes have been identified, among them the AP2-domain containing
ERF1
and
EDF
family of transcription factors, which suggests the participation of a transcriptional cascade in the ethylene response. The differential regulation of some components of this complex nuclear cascade by other signaling pathways provides a possible mechanism for interaction and signal integration. As new points of intersection with other pathways and additional participants in the pathway are identified, the Connections Map will be updated to include this new information.
The Role of Membrane-Bound Ankyrin-Repeat Protein ACD6 in Programmed Cell Death and Plant Defense
Authors: Dong Xinnian
Published: March 2004

Programmed cell death (PCD) is a common defense response in plants against pathogen infection. The recently cloned
ACD6
gene was identified in an
Arabidopsis
mutant, accelerated cell death 6 (
acd6
), that undergoes PCD in the absence of a pathogen challenge.
ACD6
is a founding member of a large family of genes that encode proteins with a short amino-terminal region, nine ankyrin repeats in the middle, and five putative transmembrane domains in the carboxyl-terminal region. Characterization of the original gain-of-function
acd6
mutant and a transferred-DNA knockout mutant
acd6-T
showed that ACD6 is an activator of the defense pathway against bacterial pathogens and plays a role in PCD through regulation of the defense signal salicylic acid (SA). SA mediates not only downstream pathogenesis-related (
PR
) genes, which encode proteins with antimicrobial activities, but also
ACD6
, forming a feedback signal amplification loop.
Focus Issue: Evolution of Language
Authors: Ray L. Bryan , Gough Nancy R.
Published: March 2004

This week’s issue of STKE complements a special issue of
Science
that explores the Evolution of Language. Translation of biological knowledge into standardized and machine-readable formats is needed to enable efficient exchange information and modeling of signaling mechanisms, as well as to promote integration of information on cell signaling with records in other databases of biological information. Two Perspectives describe different symbolic languages being applied to this problem. STKE is also tackling the problem of making cell signaling pathway information in the Connections Maps database accessible to novices and experts through the Web site, as well as to computers for analysis through a machine-readable file. First approaches involve creating an XML schema based on the Systems Biology Markup Language.
Molecular Interaction Maps--A Diagrammatic Graphical Language for Bioregulatory Networks
Authors: Aladjem Mirit I. , Pasa Stefania , Parodi Silvio , Weinstein John N. , Pommier Yves , Kohn Kurt W.
Published: March 2004

Molecular interaction maps (MIMs) use a clear, accurate, and versatile graphical language to depict complex biological processes. Here, we discuss the main features of the MIM language and its potential uses. MIMs can be used as database resources and simulation guides, and can serve to generate new hypotheses regarding the roles of specific molecules in the bioregulatory networks that control progression through the cell cycle, differentiation, and cell death.
Promiscuity Rules? The Dispensability of Cyclin E and Cdk2
Authors: Su Tin Tin , Stumpff Jason
Published: March 2004

The canonical view of the mammalian cell cycle arose from studies of cultured cells rather than mutant organisms. It depicts the many complexes of cyclin and Cdk (cyclin/Cdk) as fulfilling unique and essential steps that dictate the sequential order of cell cycle events. Recent analyses of knockout mice challenge this view. Cdk2 and cyclin E, long thought to be essential, are largely dispensable. Here, we discuss the phenotypes of these and other cyclin/Cdk mutants in genetically tractable metazoa (mouse, fly, and nematode) and explore possible reasons behind similarities and differences among experimental systems and cell types.
Effect ofEscherichia coliandLactobacillus rhamnosuson Macrophage Inflammatory Protein 3α, Tumor Necrosis Factor Alpha, and Transforming Growth Factor β Release by Polarized Rat Uterine Epithelial Cells in Culture
Authors: Crane-Godreau Mardi A. , Wira Charles R.
Published: March 2004

Entry of bacteria from the vagina into the uterus raises the question of uterine epithelial cell (UEC) signaling in response to the presence of bacteria. Our model system helps to define microbially elicited UEC basolateral cytokine release, important in regulating underlying stromal immune cell protection. UECs from adult rats were grown in cell culture inserts to establish a confluent polarized monolayer as was determined by transepithelial resistance (TER). Polarized epithelial cell cultures were treated apically with live or heat-killedEscherichia coliorLactobacillus rhamnosusprior to collection of basolateral media after 24 h of incubation. Coculture of polarized UECs with liveE. colihad no effect on epithelial cell TER. In response to exposure to liveE. coli, epithelial cell basolateral release of macrophage inflammatory protein 3α (MIP3α) and tumor necrosis factor alpha (TNF-α) increased at a time when basolateral release of biologically active transforming growth factor β (TGF-β) decreased. Incubation of UECs with heat-killedE. coliresulted in an increased basolateral release of MIP3α and TNF-α, without affecting TER or TGF-β. In contrast toE. coli, live or heat-killedL. rhamnosushad no effect on TER or cytokine release. These studies indicate that polarized rat UECs respond to gram-negativeE. coliby releasing the cytokines MIP3α and TNF-α, signals important to both the innate and adaptive immune systems. These findings suggest that UEC responses to bacteria are selective and important in initiating and regulating immune protection in the female reproductive tract.
Toll-Like Receptor 4 Contributes to Efficient Control of Infection with the Protozoan ParasiteLeishmania major
Authors: Kropf Pascale , Freudenberg Marina A. , Modolell Manuel , Price Helen P. , Herath Shanti , Antoniazi Simone , Galanos Chris , Smith Deborah F. , Müller Ingrid
Published: March 2004

The essential role of Toll-like receptors (TLR) in innate immune responses to bacterial pathogens is increasingly recognized, but very little is known about the role of TLRs in host defense against infections with eukaryotic pathogens. For the present study, we investigated whether TLRs contribute to the innate and acquired immune response to infection with the intracellular protozoan parasiteLeishmania major. Our results show that TLR4 contributes to the control of parasite growth in both phases of the immune response. We also addressed the mechanism that results in killing or growth of the intracellular parasites. Control of parasite replication correlates with the early induction of inducible nitric oxide synthase in TLR4-competent mice, whereas increased parasite survival in host cells from TLR4-deficient mice correlates with a higher activity of arginase, an enzyme known to promote parasite growth. This is the first study showing that TLR4 contributes to the effective control ofLeishmaniainfection in vivo.
Immunoglobulin-Mediated Agglutination of and Biofilm Formation by
Escherichia coli
K-12 Require the Type 1 Pilus Fiber
Authors: Orndorff Paul E. , Devapali Aditya , Palestrant Sarah , Wyse Aaron , Everett Mary Lou , Bollinger R. Randal , Parker William
Published: March 2004

The binding of human secretory immunoglobulin A (SIgA), the primary immunoglobulin in the gut, to
Escherichia coli
is thought to be dependent on type 1 pili. Type 1 pili are filamentous bacterial surface attachment organelles comprised principally of a single protein, the product of the
fimA
gene. A minor component of the pilus fiber (the product of the
fimH
gene, termed the adhesin) mediates attachment to a variety of host cell molecules in a mannose inhibitable interaction that has been extensively described. We found that the aggregation of
E. coli
K-12 by human secretory IgA (SIgA) was dependent on the presence of the pilus fiber, even in the absence of the mannose specific adhesin or in the presence of 25 mM α-CH
3
Man. The presence of pilus without adhesin also facilitated SIgA-mediated biofilm formation on polystyrene, although biofilm formation was stronger in the presence of the adhesin. IgM also mediated aggregation and biofilm formation in a manner dependent on pili with or without adhesin. These findings indicate that the pilus fiber, even in the absence of the adhesin, may play a role in biologically important processes. Under conditions in which
E. coli
was agglutinated by SIgA, the binding of SIgA to
E. coli
was not increased by the presence of the pili, with or without adhesin. This observation suggests that the pili, with or without adhesin, affect factors such as cell surface rigidity or electrostatic repulsion, which can affect agglutination but which do not necessarily determine the level of bound immunoglobulin.
Lon Protease Activity Causes Down-Regulation of
Salmonella
Pathogenicity Island 1 Invasion Gene Expression after Infection of Epithelial Cells
Authors: Boddicker Jennifer D. , Jones Bradley D.
Published: March 2004

Salmonella enterica
serovar Typhimurium causes self-limiting gastroenteritis in humans and a typhoid-like disease in mice that serves as a model for typhoid infections in humans. A critical step in
Salmonella
pathogenesis is the invasion of enterocytes and M cells of the small intestine via expression of a type III secretion system, encoded on
Salmonella
pathogenicity island 1 (SPI-1), that secretes effector proteins into host cells, leading to engulfment of the bacteria within large membrane ruffles. The in vitro regulation of invasion genes has been the subject of much scientific investigation. Transcription of the
hilA
gene, which encodes an OmpR/ToxR-type transcriptional activator of downstream invasion genes, is increased during growth under high-osmolarity and low-oxygen conditions, which presumably mimic the environment found within the small intestine. Several negative regulators of invasion gene expression have been identified, including HilE, Hha, and Lon protease. Mutations within the respective genes increase the expression of
hilA
when the bacteria are grown under environmental conditions that are not favorable for
hilA
expression and invasion. In this study, the intracellular expression of invasion genes was examined, after bacterial invasion of HEp-2 epithelial cells, using
Salmonella
strains containing plasmid-encoded short-half-life green fluorescent protein reporters of
hilA
,
hilD
,
hilC
, or
sicA
expression. Interestingly, the expression of SPI-1 genes was down-regulated after invasion, and this was important for the intracellular survival of the bacteria. In addition, the effects of mutations in genes encoding negative regulators of invasion on intracellular
hilA
expression were examined. Our results indicate that Lon protease is important for down-regulation of
hilA
expression and intracellular survival after the invasion of epithelial cells.
Analysis of Relative Levels of Production of Pertussis Toxin Subunits and Ptl Proteins in
Bordetella pertussis
Authors: Cheung Anissa M. , Farizo Karen M. , Burns Drusilla L.
Published: March 2004

Pertussis toxin is transported across the outer membrane of
Bordetella pertussis
by the type IV secretion system known as the Ptl transporter, which is composed of nine different proteins. In order to determine the relative levels of production of pertussis toxin subunits and Ptl proteins in
B. pertussis
, we constructed translational fusions of the gene for alkaline phosphatase,
phoA
, with various
ptx
and
ptl
genes. Comparison of the alkaline phosphatase activity of strains containing
ptx
′- or
ptl
′-
phoA
fusions indicated that pertussis toxin subunits are produced at higher levels than Ptl proteins, which are encoded by genes located toward the 3′ end of the
ptx-ptl
operon. We also engineered strains of
B. pertussis
by introducing multiple copies of the
ptl
genes or subsets of these genes and then examined the ability of each of these strains to secrete pertussis toxin. From these studies, we determined that certain Ptl proteins appear to be limiting in the secretion of pertussis toxin from the bacteria. These results represent an important first step in assessing the stoichiometric relationship of pertussis toxin and its transporter within the bacterial cell.
Phagocytosis of Apoptotic Cells Increases the Susceptibility of Macrophages to Infection withCoxiella burnetiiPhase II through Down-Modulation of Nitric Oxide Production
Authors: Zamboni Dario S. , Rabinovitch Michel
Published: March 2004

Coxiella burnetii, the agent of Q fever in humans and coxiellosis in other mammals, is an obligate intracellular bacterium which is sheltered and multiplies within typically large phagolysosome-like replicative vacuoles (LRVs). We have previously shown that, compared with fibroblasts, mouse resident peritoneal macrophages control the development of LRVs and bacterial multiplication within these vacuoles. Earlier experiments with the nitric oxide (NO) synthase inhibitor aminoguanidine (AG) revealed that the control is exerted by NO induced by the bacteria. We report here that phagocytosis of apoptotic-like, but not of aldehyde-killed, lymphocytes by the macrophages reduced the production of NO induced by the bacteria and increased the development of LRVs and, therefore, the total bacterial load in the cultures. Experiments with macrophages from mice deficient for inducible NO synthase (iNOS−/−) confirmed the involvement of NO in the control of infection, since neither apoptotic lymphocytes nor AG affected the development of LRVs in these phagocytes. Since macrophages are important for the clearance of apoptotic bodies andC. burnetiiis able to induce apoptosis in human monocytes, the phenomenon shown here may be biologically relevant to the development of Q fever and coxiellosis.
Vaccine Potential of theNeisseria meningitidis2086 Lipoprotein
Authors: Fletcher Leah D. , Bernfield Liesel , Barniak Vicki , Farley John E. , Howell Alan , Knauf Melissa , Ooi Peggy , Smith Robert P. , Weise Paige , Wetherell Mike , Xie Xiaoling , Zagursky Robert , Zhang Ying , Zlotnick Gary W.
Published: March 2004

A novel antigen that induces cross-reactive bactericidal antibodies against a number ofNeisseria meningitidisstrains is described. This antigen, a ∼28-kDa lipoprotein called LP2086, was first observed within a complex mixture of soluble outer membrane proteins (sOMPs) following a series of fractionation, protein purification, and proteomics steps. Approximately 95 different neisserial isolates tested positive by Western blotting and PCR screening methods for the presence of the protein and the gene encoding LP2086. The strains tested included isolates ofN. meningitidisserogroups A, B, C, W135, and Y,Neisseria gonorrhoeae, andNeisseria lactamica. To better understand the microheterogeneity of this protein, the 2086 genes from 63 neisserial isolates were sequenced. Two different subfamilies of LP2086 were identified based on deduced amino acid sequence homology. A high degree of amino acid sequence similarity exists within each 2086 subfamily. The highest degree of genetic diversity was seen between the two subfamilies which share approximately 60 to 75% homology at the nucleic acid level. Flow cytometry (fluorescence-activated cell sorting) analyses and electron microscopy indicated that the LP2086 is localized on the outer surface ofN. meningitidis. Antiserum produced against a single protein variant was capable of eliciting bactericidal activity against strains expressing different serosubtype antigens. Combining one recombinant lipidated 2086 (rLP2086) variant from each subfamily with two rPorA variants elicited bactericidal activity against all strains tested. The rLP2086 family of antigens are candidates worthy of further vaccine development.
Toll-Like Receptor 2 Is Required for Optimal Control ofListeria monocytogenesInfection
Authors: Torres David , Barrier Mathieu , Bihl Franck , Quesniaux Valerie J. F. , Maillet Isabelle , Akira Shizuo , Ryffel Bernhard , Erard François
Published: March 2004

The control ofListeria monocytogenesinfection depends on the rapid activation of the innate immune system, likely through Toll-like receptors (TLR), since mice deficient for the common adapter protein of TLR signaling, myeloid differentiation factor 88 (MyD88), succumb toListeriainfection. In order to test whether TLR2 is involved in the control of infections, we compared the host response in TLR2-deficient mice with that in wild-type mice. Here we show that TLR2-deficient mice are more susceptible to systemic infection byListeriathan are wild-type mice, with a reduced survival rate, increased bacterial burden in the liver, and abundant and larger hepatic microabscesses containing increased numbers of neutrophils. The production of tumor necrosis factor, interleukin-12, and nitric oxide and the expression of the costimulatory molecules CD40 and CD86, which are necessary for the control of infection, were reduced in TLR2-deficient macrophages and dendritic cells stimulated byListeriaand were almost abolished in the absence of MyD88, coincident with the high susceptibility of MyD88-deficient mice to in vivo infection. Therefore, the present data demonstrate a role for TLR2 in the control ofListeriainfection, but other MyD88-dependent signals may contribute to host resistance.
Effect of Bronchoalveolar Lavage Fluid fromPneumocystis carinii- Infected Hosts on Phagocytic Activity of Alveolar Macrophages
Authors: Lasbury Mark E. , Lin Peimao , Tschang Dennis , Durant Pamela J. , Lee Chao-Hung
Published: March 2004

Alveolar macrophages fromPneumocystis carinii-infected rats are defective in phagocytosis. To investigate whether this defect is due to a certain factor present inP. carinii-infected lungs, alveolar macrophages from uninfected rats were incubated with bronchoalveolar lavage (BAL) fluid samples fromP. carinii-infected rats. Alveolar macrophages treated with these BAL fluid samples became defective in phagocytosis but remained normal when treated with BAL fluid samples from noninfected orToxoplasma gondii-infected rats. The suppressive activity of the BAL fluid samples fromP. carinii-infected rats on phagocytosis was retained when the BAL fluid samples were passed through a filter with a pore size of 0.45 μm but was lost when the BAL fluid samples were digested with proteases such as trypsin, pepsin, papain, or endopeptidase Gly-C. Lipid fractions of these BAL fluid samples had no suppressive activity on phagocytosis. The suppressive activity of these BAL fluid samples was also lost when they were incubated with concanavalin A-agarose beads, suggesting that the inhibitor is a glycoprotein. The inhibitor was estimated to be larger than 100,000 Da by exclusion filtration. After binding to the concanavalin A-agarose beads, the inhibitor in BAL fluid samples andP. cariniilysate could be eluted with 200 mM methylmannose. Treatment of both the crude BAL fluid samples andP. cariniilysate and the 200 mM methylmannose eluate with antibody against the major surface glycoprotein ofP. cariniieliminated their suppressive activity. These results suggest that the factor capable of suppressing the phagocytic activity of alveolar macrophages isP. cariniimajor surface glycoprotein or one or more of its derivatives.
Normally Occurring Human Anti-GM1Immunoglobulin M Antibodies and the Immune Response to Bacteria
Authors: Alaniz María E. , Lardone Ricardo D. , Yudowski Silvia L. , Farace María I. , Nores Gustavo A.
Published: March 2004

Anti-GM1antibodies of the immunoglobulin M (IgM) isotype are normal components of the antibody repertoire of adult human serum. Using a sensitive high-performance thin-layer chromatography (HPTLC) immunostaining assay, we found that these antibodies were absent in the umbilical vein and children <1 month of age but could be detected after 1 month of age. Although most of the children older than 6 months of age were positive, there were still a few negative children. The appearance of anti-GM1IgM antibodies showed a perfect concordance with two well-characterized antibacterial antibodies, anti-Forssman and anti-blood group A, which indicates a similar origin. We also studied IgM reactivity with lipopolysaccharides (LPSs) from gram-negative bacteria isolated from stool samples from healthy babies and fromEscherichia coliHB101 in serum from individuals of different ages. We found a positive reaction with both LPSs in all the children more than 1 month of age analyzed, even in those that were negative for anti-GM1antibodies. Anti-GM1IgM antibodies were purified from adult serum by affinity chromatography and tested for the ability to bind LPSs from different bacteria. This highly specific preparation showed reactivity only with LPS from a strain ofCampylobacter jejuniisolated from a patient with diarrhea. We conclude that normally occurring IgM antibodies are generated after birth, probably during the immune defense against specific bacterial strains.
Detergent-Resistant Membrane Microdomains Facilitate Ib Oligomer Formation and Biological Activity of
Clostridium perfringens
Iota-Toxin
Authors: Hale Martha L. , Marvaud Jean-Christophe , Popoff Michel R. , Stiles Bradley G.
Published: March 2004

Clostridium perfringens
iota-toxin consists of two separate proteins identified as a cell binding protein, iota b (Ib), which forms high-molecular-weight complexes on cells generating Na
+
/K
+
-permeable pores through which iota a (Ia), an ADP-ribosyltransferase, presumably enters the cytosol. Identity of the cell receptor and membrane domains involved in Ib binding, oligomer formation, and internalization is currently unknown. In this study, Vero (toxin-sensitive) and MRC-5 (toxin-resistant) cells were incubated with Ib, after which detergent-resistant membrane microdomains (DRMs) were extracted with cold Triton X-100. Western blotting revealed that Ib oligomers localized in DRMs extracted from Vero, but not MRC-5, cells while monomeric Ib was detected in the detergent-soluble fractions of both cell types. The Ib protoxin, previously shown to bind Vero cells but not form oligomers or induce cytotoxicity, was detected only in the soluble fractions. Vero cells pretreated with phosphatidylinositol-specific phospholipase C before addition of Ib indicated that glycosylphosphatidyl inositol-anchored proteins were minimally involved in Ib binding or oligomer formation. While pretreatment of Vero cells with filipin (which sequesters cholesterol) had no effect, methyl-β-cyclodextrin (which extracts cholesterol) reduced Ib binding and oligomer formation and delayed iota-toxin cytotoxicity. These studies showed that iota-toxin exploits DRMs for oligomer formation to intoxicate cells.
Identification and Molecular Characterization of a Gene Encoding a ProtectiveLeishmania amazonensisTrp-Asp (WD) Protein
Authors: Campbell Kimberly , Popov Vsevolod , Soong Lynn
Published: March 2004

SeveralLeishmaniaproteins have been identified and characterized in pursuit of understanding pathogenesis and protection in cutaneous leishmaniasis. In the present study, we utilized sera from infected BALB/c mice to screen aLeishmania amazonensisamastigote cDNA expression library and obtained the full-length gene that encodes a novel Trp-Asp (WD) protein designated LAWD (forLeishmaniaantigenic WD protein). The WD family of proteins mediates protein-protein interactions and coordinates the formation of protein complexes. The single-copy LAWD gene is transcribed as a ∼3.1-kb mRNA in both promastigotes and amastigotes, with homologues being detected in several otherLeishmaniaspecies. Immunoelectron microscopy revealed a predominant localization of the LAWD protein in the flagellar pocket. Analyses of sera from human patients with cutaneous and mucocutaneous leishmaniasis indicated that these individuals mounted significant humoral responses against LAWD. Given that recombinant LAWD protein elicited the production of high levels of gamma interferon, but no detectable levels of interleukin-10 (IL-10), in CD4+cells ofL. amazonensis-infected mice, we further examined whether LAWD could elicit protective immunity. DNA vaccination with the LAWD and IL-12 genes significantly delayed lesion development, which correlated with a dramatic reduction in parasite burdens. Thus, we have successfully identified a promising vaccine candidate and antigenic vehicle to aid in the dissection of the complicated pathogenic immune response ofL. amazonensis.
Growth Control of Small-Colony Variants by Genetic Regulation of the Hemin Uptake System
Authors: Roggenkamp Andreas , Hoffmann Harald , Hornef Mathias W.
Published: March 2004

Small-colony variants (SCVs) are slow-growing variants of human bacterial pathogens. They are associated with chronic persistent infections, and their biochemical identification and antimicrobial treatment are impaired by altered metabolic properties. To contribute to the understanding of SCV-mediated infections, we analyzed a clinical SCV isolate derived from a chronic prosthetic hip infection. A sequence analysis of housekeeping genes identified an
Enterobacter hormaechei
-like organism. The SCV phenotype, with growth as microcolonies, was caused by a block within the heme biosynthesis pathway through deletion of the
hemB
locus, as shown by hybridization and complementation experiments. At a low frequency, large-colony variants (LCVs) arose that were dependent on exogenous hemin. To investigate this phenomenon, we cloned and sequenced the 5.8-kb hemin uptake system, denoted
ehu
. Gene expression analysis indicated regulation of this locus in wild-type bacteria by the global iron regulator Fur. Inactivation of Fur in LCVs caused the derepression of
ehu
expression and facilitated bacterial growth. Genetic alterations of the
fur
locus in LCVs were identified as insertions of IS
1A
elements and point mutations. In contrast, SCVs could utilize exogenous hemin only in the absence of iron. Thus, we provide the first molecular characterization of the growth properties of a clinical SCV isolate, which may help to improve the diagnostic and therapeutic management of patients with chronic persistent infections.
Molecular Cloning and Characterization of
cgt
, the
Brucella abortus
Cyclic β-1,2-Glucan Transporter Gene, and Its Role in Virulence
Authors: Roset Mara S. , Ciocchini Andrés E. , Ugalde Rodolfo A. , Iñón de Iannino Nora
Published: March 2004

The animal pathogen
Brucella abortus
contains a gene
cgt
, which complemented
Sinorhizobium meliloti
nodule development (
ndvA
) and
Agrobacterium tumefaciens
chromosomal virulence (
chvA
) mutants. Complemented strains recovered the presence of anionic cyclic β-1,2-glucan, motility, tumor induction in
A. tumefaciens
, and nodule occupancy in
S. meliloti
, all traits strictly associated with the presence of cyclic β-1,2-glucan in the periplasm. Nucleotide sequencing revealed that
B. abortus cgt
contains a 1,797-bp open reading frame coding for a predicted membrane protein of 599 amino acids (65.9 kDa) that is 58.5 and 59.9% identical to
S. meliloti
NdvA and
A. tumefaciens
ChvA, respectively. Additionally,
B. abortus cgt
, like
S. meliloti ndvA
and
A. tumefaciens chvA
possesses ATP-binding motifs and the ABC signature domain features of a typical ABC transporter. Characterization of Cgt was carried out by the construction of null mutants in
B. abortus
2308 and S19 backgrounds. Both mutants do not transport cyclic β-1,2-glucan to the periplasm, as shown by the absence of anionic cyclic glucan, and they display reduced virulence in mice and defective intracellular multiplication in HeLa cells. These results suggest that cyclic β-1,2-glucan must be transported into the periplasmatic space to exert its action as a virulence factor.
Fusobacterium nucleatum
Induces Premature and Term Stillbirths in Pregnant Mice: Implication of Oral Bacteria in Preterm Birth
Authors: Han Yiping W. , Redline Raymond W. , Li Mei , Yin Lihong , Hill Gale B. , McCormick Thomas S.
Published: March 2004

Fusobacterium nucleatum
is a gram-negative anaerobe ubiquitous to the oral cavity. It is associated with periodontal disease. It is also associated with preterm birth and has been isolated from the amniotic fluid, placenta, and chorioamnionic membranes of women delivering prematurely. Periodontal disease is a newly recognized risk factor for preterm birth. This study examined the possible mechanism underlying the link between these two diseases.
F. nucleatum
strains isolated from amniotic fluids and placentas along with those isolated from orally related sources invaded both epithelial and endothelial cells. The invasive ability may enable
F. nucleatum
to colonize and infect the pregnant uterus. Transient bacteremia caused by periodontal infection may facilitate bacterial transmission from the oral cavity to the uterus. To test this hypothesis, we intravenously injected
F. nucleatum
into pregnant CF-1 mice. The injection resulted in premature delivery, stillbirths, and nonsustained live births. The bacterial infection was restricted inside the uterus, without spreading systemically.
F. nucleatum
was first detected in the blood vessels in murine placentas. Invasion of the endothelial cells lining the blood vessels was observed. The bacteria then crossed the endothelium, proliferated in surrounding tissues, and finally spread to the amniotic fluid. The pattern of infection paralleled that in humans. This study represents the first evidence that
F. nucleatum
may be transmitted hematogenously to the placenta and cause adverse pregnancy outcomes. The results strengthen the link between periodontal disease and preterm birth. Our study also indicates that invasion may be an important virulence mechanism for
F. nucleatum
to infect the placenta.
Absence of Interleukin-4 Determines Less Severe Pulmonary Paracoccidioidomycosis Associated with Impaired Th2 Response
Authors: Pina Adriana , Valente-Ferreira Rita C. , Molinari-Madlum Eugênia E. W. , Vaz Celidéia A. C. , Keller Alexandre C. , Calich Vera L. G.
Published: March 2004

Host resistance to paracoccidiodomycosis, the main deep mycosis in Latin America, is mainly due to cellular immunity and gamma interferon (IFN-γ) production. To assess the role of interleukin-4 (IL-4), a Th2-inducing cytokine, pulmonary paracoccidioidomycosis was studied in IL-4-deficient (IL-4−/−) and wild-type (WT) C57BL/6 mice at the innate and acquired phases of immune response. Forty-eight hours after infection, equivalent numbers of viableParacoccidioides brasiliensisyeast cells were recovered from the lungs of IL-4−/−and WT mice intratracheally infected with one million fungal cells. Alveolar macrophages from infected IL-4−/−mice controlled in vitro fungal growth more efficiently than macrophages from WT mice and secreted higher levels of nitric oxide. Compared with WT mice, IL-4−/−animals presented increased levels of pulmonary IFN-γ and augmented polymorphonuclear leukocyte influx to the lungs. Decreased pulmonary fungal loads were characterized in deficient mice at week 2 postinfection, concomitant with diminished presence of IL-10. At week 8, lower numbers of yeasts were recovered from lungs and liver of IL-4−/−mice associated with increased production of IFN-γ but impaired synthesis of IL-5 and IL-10. However, a clear shift to a Th1 pattern was not characterized, since IL-4−/−mice did not alter delayed-type hypersensitivity anergy or IL-2 levels. In addition, IL-4 deficiency resulted in significantly reduced levels of pulmonary IL-12, granulocyte-macrophage colony-stimulating factor, IL-3, monocyte chemotactic protein 1, and specific antibody isotypes. In IL-4−/−mice, well-organized granulomas restraining fungal cells replaced the more extensive lesions containing high numbers of fungi and inflammatory leukocytes developed by IL-4-sufficient mice. These results clearly showed that genetically determined deficiency of IL-4 can exert a protective role in pulmonary paracoccidioidomycosis.
MyD88-Deficient Mice Display a Profound Loss in Resistance toMycobacterium tuberculosisAssociated with Partially Impaired Th1 Cytokine and Nitric Oxide Synthase 2 Expression
Authors: Scanga Charles A. , Bafica Andre , Feng Carl G. , Cheever Allen W. , Hieny Sara , Sher Alan
Published: March 2004

Mycobacterium tuberculosispossesses agonists for several Toll-like receptors (TLRs), yet mice with single TLR deletions are resistant to acute tuberculosis. MyD88−/−mice were used to examine whether TLRs play any role in protection against aerogenicM. tuberculosisH37Rv infection. MyD88−/−mice failed to control mycobacterial replication and rapidly succumbed. Moreover, expressions of interleukin 12, tumor necrosis factor alpha, gamma interferon, and nitric oxide synthase 2 were markedly decreased in the knockout animals. These results argue that resistance toM. tuberculosismust depend on MyD88-dependent signals mediated by an as-yet-undetermined TLR or a combination of TLRs.
Spatiotemporal Analysis of Acid Adaptation-Mediated
Vibrio cholerae
Hyperinfectivity
Authors: Angelichio Michael J. , Merrell D. Scott , Camilli Andrew
Published: March 2004

Acid adaptation has previously been shown to increase the infectivity of
Vibrio cholerae
in the infant mouse model. To better understand this phenomenon, we monitored the spatial distribution and temporal changes in the ratios of acid-adapted cells to unadapted
V. cholerae
cells in the small intestine, as well as the timing of virulence factor expression. We found that the competitive advantage afforded by acid adaptation does not become manifest until greater than 3 h postinfection; thus, acid adaptation does not increase
V. cholerae
passage through the gastric acid barrier. Additionally, acid-adapted and unadapted
V. cholerae
cells colonize the same sections of the small intestine and show similar kinetics of transcriptional induction of the virulence genes
tcpA
and
ctxA.
These studies suggest that the increased infectivity of acid-adapted
V. cholerae
is due to a more rapid onset of multiplication and/or to an increased multiplication rate within the infant mouse intestine.