bluuebunny/crossref_metadata_2025 huggingface dataset:
CD66 receptor specificity exhibited by neisserial Opa variants is controlled by protein determinants in CD66 N-domains
Authors: Bos Martine P. , Kuroki Motomu , Krop-Watorek Anna , Hogan Daniel , Belland Robert J.
Published: July 2002

Neisseria gonorrhoeae
strain MS11 is able to express 11 different opacity (Opa) proteins on its outer surface. A number of these Opa proteins have been shown to function as adhesins through binding of CD66 receptors present on human cells. CD66 antigens, or carcinoembryonic antigen family members, constitute a family of glycoproteins belonging to the immunoglobulin superfamily. Opa variants recognize this class of receptors in a differential manner such that certain Opa variants recognize up to four different CD66 receptors (CD66a, -c, -d, and -e), whereas others recognize only two (CD66a and -e) or none. We explored the basis for this receptor tropism in the present study. Our data show that glycoforms of CD66e and deglycosylated CD66e are recognized by gonococci in an Opa-specific manner. Binding by Opa variants of recombinant N-terminal domains of CD66 receptors expressed in
Escherichia coli
reflected the adherence specificities of Opa variants to HeLa cells expressing native CD66 molecules. These data indicate that recognition of CD66 receptors by Opa variants is mediated by the protein backbone of the CD66 N-domains. Furthermore, by using chimeric constructs between different CD66 N-domains we identified distinct binding regions on the CD66e N-domain for specific groups of Opa variants, suggesting that the differential recognition of CD66 receptors by Opa variants is dictated by the presence of specific binding regions on the N-domain of the receptor.
A pyrimidine-rich exonic splicing suppressor binds multiple RNA splicing factors and inhibits spliceosome assembly
Authors: Zheng Zhi Ming , Huynen Martijn , Baker Carl C.
Published: July 2002

The bovine papillomavirus type 1 (BPV-1) exonic splicing suppressor (ESS) is juxtaposed immediately downstream of BPV-1 splicing enhancer 1 and negatively modulates selection of a suboptimal 3′ splice site at nucleotide 3225. The present study demonstrates that this pyrimidine-rich ESS inhibits utilization of upstream 3′ splice sites by blocking early steps in spliceosome assembly. Analysis of the proteins that bind to the ESS showed that the U-rich 5′ region binds U2AF
65
and polypyrimidine tract binding protein, the C-rich central part binds 35- and 54–55-kDa serine/arginine-rich (SR) proteins, and the AG-rich 3′ end binds alternative splicing factor/splicing factor 2. Mutational and functional studies indicated that the most critical region of the ESS maps to the central C-rich core (GGCUCCCCC). This core sequence, along with additional nonspecific downstream nucleotides, is sufficient for partial suppression of spliceosome assembly and splicing of BPV-1 pre-mRNAs. The inhibition of splicing by the ESS can be partially relieved by excess purified HeLa SR proteins, suggesting that the ESS suppresses pre-mRNA splicing by interfering with normal bridging and recruitment activities of SR proteins.
Protein splicing in
trans
by purified N- and C-terminal fragments of the
Mycobacterium tuberculosis
RecA intein
Authors: Mills Kenneth V. , Lew Belinda M. , Jiang Shu-qin , Paulus Henry
Published: July 2002

Protein splicing involves the self-catalyzed excision of protein
splicing elements, or inteins, from flanking polypeptide sequences, or
exteins, leading to the formation of new proteins in which the exteins
are linked directly by a peptide bond. To study the enzymology of this
interesting process we have expressed and purified N- and C-terminal
segments of the
Mycobacterium tuberculosis
RecA intein,
each ≈100 amino acids long, fused to appropriate exteins. These
fragments were reconstituted into a functional protein splicing element
by renaturation from 6 M urea. When renaturation was carried out in the
absence of thiols, the reconstituted splicing element accumulated as an
inactive disulfide-linked complex of the two intein fragments, which
could be induced to undergo protein splicing by reduction of the
disulfide bond. This provided a useful tool for separately
investigating the requirements for the reconstitution of the intein
fragments to yield a functional protein splicing element and for the
protein splicing process
per se
. For example, the pH
dependence of these processes was quite different, with reconstitution
being most efficient at pH 8.5 and splicing most rapid at pH 7.0. The
availability of such an
in vitro
protein splicing system
opens the way for the exploration of intein structure and the unusual
enzymology of protein splicing. In addition, this
trans
-splicing system is a potential protein ligase that
can link any two polypeptides fused to the N- and C-terminal intein
segments.
Conversion of agonist site to metal-ion chelator site in the β
2
-adrenergic receptor
Authors: Elling Christian E. , Thirstrup Kenneth , Holst Birgitte , Schwartz Thue W.
Published: July 2002

Previously metal-ion sites have been used as structural and functional probes in seven transmembrane receptors (7TM), but as yet all the engineered sites have been inactivating. Based on presumed agonist interaction points in transmembrane III (TM-III) and -VII of the β
2
-adrenergic receptor, in this paper we construct an activating metal-ion site between the amine-binding Asp-113 in TM-III—or a His residue introduced at this position—and a Cys residue substituted for Asn-312 in TM-VII. No increase in constitutive activity was observed in the mutant receptors. Signal transduction was activated in the mutant receptors not by normal catecholamine ligands but instead either by free zinc ions or by zinc or copper ions in complex with small hydrophobic metal-ion chelators. Chelation of the metal ions by small hydrophobic chelators such as phenanthroline or bipyridine protected the cells from the toxic effect of, for example Cu
2+
, and in several cases increased the affinity of the ions for the agonistic site. Wash-out experiments and structure–activity analysis indicated, that the high-affinity chelators and the metal ions bind and activate the mutant receptor as metal ion guided ligand complexes. Because of the well-understood binding geometry of the small metal ions, an important distance constraint has here been imposed between TM-III and -VII in the active, signaling conformation of 7TM receptors. It is suggested that atoxic metal-ion chelator complexes could possibly in the future be used as generic, pharmacologic tools to switch 7TM receptors with engineered metal-ion sites on or off at will.
Excitonic couplings and electronic coherence in bridged naphthalene dimers
Authors: Tretiak Sergei , Zhang Wei Min , Chernyak Vladimir , Mukamel Shaul
Published: July 2002

The electronic excitations of naphthalene and a family of bridged naphthalene dimers are calculated and analyzed by using the Collective Electronic Oscillator method combined with the oblique Lanczos algorithm. All experimentally observed trends in absorption profiles and radiative lifetimes are reproduced. Each electronic excitation is linked to the corresponding real-space transition density matrix, which represents the motions of electrons and holes created in the molecule by photon absorption. Two-dimensional plots of these matrices help visualize the degree of exciton localization and explain the dependence of the electronic interaction between chromophores on their separation.
Repetitive-DNA elements are similarly distributed on
Caenorhabditis elegans
autosomes
Authors: Surzycki Stefan A. , Belknap William R.
Published: July 2002

The positions of ≈4,800 individual miniature inverted-repeat transposable element (MITE)-like repeats from four families were mapped on the
Caenorhabditis
elegans
chromosomes. These families represent 1–2% of the total sequence of the organism. The four MITE families (
Cele1
,
Cele2
,
Cele14
, and
Cele42
) displayed distinct chromosomal distribution profiles. For example, the
Cele14
MITEs were observed clustering near the ends of the autosomes. In contrast, the
Cele2
MITEs displayed an even distribution through the central autosome domains, with no evidence for clustering at the ends. Both the number of elements and the distribution patterns of each family were conserved on all five
C. elegans
autosomes. The distribution profiles indicate chromosomal polarity and suggest that the current genetic and physical maps of chromosomes II, III, and X are inverted with respect to the other chromosomes. The degree of conservation of both the number and distribution of these elements on the five autosomes suggests a role in defining specific chromosomal domains.
RAD50function is essential for telomere maintenance inArabidopsis
Authors: Gallego Maria E. , White Charles I.
Published: July 2002

We have identified and characterized anArabidopsis thaliana rad50mutant plant containing a T-DNA insertion in theAtRAD50gene and showing both meiotic and DNA repair defects. We report here thatrad50/rad50mutant cells show a progressive shortening of telomeric DNA relative to heterozygousrad50/RAD50controls and that the mutant cell population rapidly enters a crisis, with the majority of the cells dying. Survivingrad50mutant cells have longer telomeres than wild-type cells, indicating the existence in plants of an alternativeRAD50-independent mechanism for telomere maintenance. These results report the role of a protein essential for double-strand break repair in telomere maintenance in higher eukaryotes.
Reduced [11C]flumazenil radioligand binding in the thalamus in alcoholics
Authors: Neiman Jack , Litton Jan‐Eric
Published: July 2002

Positron emission tomography (PET) provides in vivo quantitative measurement of radioligand binding to central neuroreceptors. In this report we present the history and PET findings of the thalamic region in two patients with diagnosis of alcohol dependence using the radioligand [11C]flumazenil (Ro 15–1788), a benzodiazepine receptor antagonist. This abnormality in the thalamus may reflect an early alcohol‐induced brain lesion or contribute to the development of alcoholism in some subjects.
Mutant Rab7 Causes the Accumulation of Cathepsin D and Cation-independent Mannose 6–Phosphate Receptor in an Early Endocytic Compartment
Authors: Press Barry , Feng Yan , Hoflack Bernard , Wandinger-Ness Angela
Published: July 2002

Stable BHK cell lines inducibly expressing wild-type or dominant negative mutant forms of the rab7 GTPase were isolated and used to analyze the role of a rab7-regulated pathway in lysosome biogenesis. Expression of mutant rab7N125I protein induced a dramatic redistribution of cation-independent mannose 6–phosphate receptor (CI-MPR) from its normal perinuclear localization to large peripheral endosomes. Under these circumstances ∼50% of the total receptor and several lysosomal hydrolases cofractionated with light membranes containing early endosome and Golgi markers. Late endosomes and lysosomes were contained exclusively in well-separated, denser gradient fractions. Newly synthesized CI-MPR and cathepsin D were shown to traverse through an early endocytic compartment, and functional rab7 was crucial for delivery to later compartments. This observation was evidenced by the fact that 2 h after synthesis, both markers were more prevalent in fractions containing light membranes. In addition, both were sensitive to HRP-DAB– mediated cross-linking of early endosomal proteins, and the late endosomal processing of cathepsin D was impaired. Using similar criteria, the lysosomal membrane glycoprotein 120 was not found accumulated in an early endocytic compartment. The data are indicative of a post-Golgi divergence in the routes followed by different lysosome-directed molecules.
Corequirement of Specific Phosphoinositides and Small GTP-binding Protein Cdc42 in Inducing Actin Assembly in Xenopus Egg Extracts
Authors: Le Ma  , Cantley Lewis C. , Janmey Paul A. , Kirschner Marc W.
Published: July 2002

Both phosphoinositides and small GTP-binding proteins of the Rho family have been postulated to regulate actin assembly in cells. We have reconstituted actin assembly in response to these signals in Xenopus extracts and examined the relationship of these pathways. We have found that GTPγS stimulates actin assembly in the presence of endogenous membrane vesicles in low speed extracts. These membrane vesicles are required, but can be replaced by lipid vesicles prepared from purified phospholipids containing phosphoinositides. Vesicles containing phosphatidylinositol (4,5) bisphosphate or phosphatidylinositol (3,4,5) trisphosphate can induce actin assembly even in the absence of GTPγS. RhoGDI, a guanine-nucleotide dissociation inhibitor for the Rho family, inhibits phosphoinositide-induced actin assembly, suggesting the involvement of the Rho family small G proteins. Using various dominant mutants of these G proteins, we demonstrate the requirement of Cdc42 for phosphoinositide-induced actin assembly. Our results suggest that phosphoinositides may act to facilitate GTP exchange on Cdc42, as well as to anchor Cdc42 and actin nucleation activities. Hence, both phosphoinositides and Cdc42 are required to induce actin assembly in this cell-free system.
Rho Guanosine Triphosphatase Mediates the Selective Stabilization of Microtubules Induced by Lysophosphatidic Acid
Authors: Cook Tiffani A. , Nagasaki Takayuki , Gundersen Gregg G.
Published: July 2002

The asymmetric distribution of stable, posttranslationally modified microtubules (MTs) contributes to the polarization of many cell types, yet the factors controlling the formation of these MTs are not known. We have found that lysophosphatidic acid (LPA) is a major serum factor responsible for rapidly generating stable, detyrosinated (Glu) MTs in serum-starved 3T3 cells. Using C3 toxin and val14 rho we showed that rho was both necessary and sufficient for the induction of Glu MTs by LPA and serum. Unlike previously described factors that induce MT stability, rho induced the stabilization of only a subset of the MTs and, in wound-edge cells, these stable MTs were appropriately oriented toward the leading edge of the cell. LPA had little effect on individual parameters of MT dynamics, but did induce long states of pause in a subset of MTs near the edge of the cell. Rho stimulation of MT stability was independent of actin stress fiber formation. These results identify rho as a novel regulator of the MT cytoskeleton that selectively stabilizes MTs during cell polarization by acting as a switch between dynamic and stable states of MTs rather than as a modulator of MT assembly and disassembly.
Growth Factor–induced p42/p44 MAPK Nuclear Translocation and Retention Requires Both MAPK Activation and Neosynthesis of Nuclear Anchoring Proteins
Authors: Lenormand Philippe , Brondello Jean-Marc , Brunet Anne , Pouysségur Jacques
Published: July 2002

Mitogen-activated protein kinases (p42/p44 MAPK, also called Erk2 and Erk1) are key mediators of signal transduction from the cell surface to the nucleus. We have previously shown that the activation of p42/p44 MAPK required for transduction of mitogenic signaling is associated with a rapid nuclear translocation of these kinases. However, the means by which p42 and p44 MAPK translocate into the nucleus after cytoplasmic activation is still not understood and cannot simply be deduced from their protein sequences. In this study, we have demonstrated that activation of the p42/ p44 MAPK pathway was necessary and sufficient for triggering nuclear translocation of p42 and p44 MAPK. First, addition of the MEK inhibitor PD 98059, which blocks activation of the p42/p44 MAPK pathway, impedes the nuclear accumulation, whereas direct activation of the p42/p44 MAPK pathway by the chimera ΔRaf-1:ER is sufficient to promote nuclear accumulation of p42/p44 MAPK. In addition, we have shown that this nuclear accumulation of p42/p44 MAPK required the neosynthesis of short-lived proteins. Indeed, inhibitors of protein synthesis abrogate nuclear accumulation in response to serum and accelerate p42/p44 MAPK nuclear efflux under conditions of persistent p42/p44 MAPK activation. In contrast, inhibition of targeted proteolysis by the proteasome synergistically potentiated p42/p44 MAPK nuclear localization by nonmitogenic agonists and markedly prolonged nuclear localization of p42/p44 MAPK after mitogenic stimulation. We therefore conclude that the MAPK nuclear translocation requires both activation of the p42/p44 MAPK module and neosynthesis of short-lived proteins that we postulate to be nuclear anchors.
Hydrogen Peroxide Inhibition of Nuclear Protein Import Is Mediated by the Mitogen-Activated Protein Kinase, Erk2
Authors: Czubryt Michael P. , Austria J. Alejandro , Pierce Grant N.
Published: July 2002

H2O2 alters gene expression in many cell types. Alterations in nuclear import of transcription factors or similar key proteins may be responsible for these changes. To investigate this possibility, a cytosolic nuclear import cocktail was treated with varying [H2O2] and used in import assays. H2O2 caused a dose- and time-dependent inhibition of import at concentrations as low as 100 μM. Catalase reversed this effect. H2O2 treatment of permeablized cells did not affect import, suggesting that H2O2 was acting on a cytosolic factor. Treatment of import cocktail with two different free radical generating systems had no effect, but treatment of permeablized cells inhibited import, suggesting H2O2 works via a distinct process from hydroxyl or superoxide radicals. Pretreatment of import cocktail with genistein reversed the effect of H2O2 on import. Western blotting revealed that H2O2 activated ERK2. The specific MEK1/2 inhibitor, PD98059, completely blocked the effects of H2O2 on import. Activated ERK2 mimicked H2O2's effect on import. Immunocytochemistry revealed that H2O2 treatment of whole cells increased cytosolic Ran/TC4 levels, an effect reversible by catalase or PD98059. These data demonstrate that H2O2 inhibits nuclear protein import and that this effect is mediated by mitogen-activated protein (MAP) kinase activation, possibly by altering Ran/TC4 function.
Reverse Transcriptase Activity in Mature Spermatozoa of Mouse
Authors: Giordano Roberto , Magnano Anna Rosa , Zaccagnini Germana , Pittoggi Carmine , Moscufo Nicola , Lorenzini Rodolfo , Spadafora Corrado
Published: July 2002

We show here that a reverse transcriptase (RT) activity is present in murine epididymal spermatozoa. Sperm cells incubated with human poliovirus RNA can take up exogenous RNA molecules and internalize them in nuclei. Direct PCR amplification of DNA extracted from RNA-incubated spermatozoa indicate that poliovirus RNA is reverse-transcribed in cDNA fragments. PCR analysis of two-cell embryos shows that poliovirus RNA-challenged spermatozoa transfer retrotranscribed cDNA molecules into eggs during in vitro fertilization. Finally, RT molecules can be visualized on sperm nuclear scaffolds by immunogold electron microscopy. These results, therefore, reveal a novel metabolic function in spermatozoa, which may play a role during early embryonic development.
α4-Integrin Mediates Neutrophil-Induced Free Radical Injury to Cardiac Myocytes
Authors: Poon Betty Y. , Ward Christopher A. , Cooper Conan B. , Giles Wayne R. , Burns Alan R. , Kubes Paul
Published: July 2002

Previous work has demonstrated that circulating neutrophils (polymorphonuclear leukocytes [PMNs]) adhere to cardiac myocytes via β2-integrins and cause cellular injury via the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme system. Since PMNs induced to leave the vasculature (emigrated PMNs) express the α4-integrin, we asked whether (a) these PMNs also induce myocyte injury via NADPH oxidase; (b) β2-integrins (CD18) still signal oxidant production, or if this process is now coupled to the α4-integrin; and (c) dysfunction is superoxide dependent within the myocyte or at the myocyte–PMN interface. Emigrated PMNs exposed to cardiac myocytes quickly induced significant changes in myocyte function. Myocyte shortening was decreased by 30–50% and rates of contraction and relaxation were reduced by 30% within the first 10 min. Both α4-integrin antibody (Ab)-treated PMNs and NADPH oxidase–deficient PMNs were unable to reduce myocyte shortening. An increased level of oxidative stress was detected in myocytes within 5 min of PMN adhesion. Addition of an anti–α4-integrin Ab, but not an anti-CD18 Ab, prevented oxidant production, suggesting that in emigrated PMNs the NADPH oxidase system is uncoupled from CD18 and can be activated via the α4-integrin. Addition of exogenous superoxide dismutase (SOD) inhibited all parameters of dysfunction measured, whereas overexpression of intracellular SOD within the myocytes did not inhibit the oxidative stress or the myocyte dysfunction caused by the emigrated PMNs. These findings demonstrate that profound molecular changes occur within PMNs as they emigrate, such that CD18 and associated intracellular signaling pathways leading to oxidant production are uncoupled and newly expressed α4-integrin functions as the ligand that signals oxidant production. The results also provide pathological relevance as the emigrated PMNs have the capacity to injure cardiac myocytes through the α4-integrin–coupled NADPH oxidase pathway that can be inhibited by extracellular, but not intracellular SOD.
Disruption of cytoskeletal integrity impairs Gi-mediated signaling due to displacement of Gi proteins
Authors: Bloch Wilhelm , Fan Yun , Han Ji , Xue Sheng , Schöneberg Torsten , Ji Guanju , Lu Zhong J. , Walther Michael , Fässler Reinhard , Hescheler Jürgen , Addicks Klaus , Fleischmann Bernd K.
Published: July 2002

β1 integrins play a crucial role as cytoskeletal anchorage proteins. In this study, the coupling of the cytoskeleton and intracellular signaling pathways was investigated in β1 integrin deficient (−/−) embryonic stem cells. Muscarinic inhibition of the L-type Ca2+ current (ICa) and activation of the acetylcholine-activated K+ current (IK,ACh) was found to be absent in β1 integrin−/− cardiomyocytes. Conversely, β adrenoceptor-mediated modulation of ICa was unaffected by the absence of β1 integrins. This defect in muscarinic signaling was due to defective G protein coupling. This was supported by deconvolution microscopy, which demonstrated that Gi exhibited an atypical subcellular distribution in the β1 integrin−/− cardiomyocytes. A critical role of the cytoskeleton was further demonstrated using cytochalasin D, which displaced Gi and impaired muscarinic signaling. We conclude that cytoskeletal integrity is required for correct localization and function of Gi-associated signaling microdomains.
Maximal Proliferation of Cytotoxic T Lymphocytes Requires Reverse Signaling through Fas Ligand
Authors: Suzuki Ivy , Fink Pamela J.
Published: July 2002

Fas ligand (FasL/CD95L) is best known for its role in delivering apoptotic signals through its receptor, Fas (APO-1/CD95). In this study, we present evidence that FasL has a second role as a signaling receptor. Alloantigen-specific proliferation by multiple FasL− murine CTL lines is depressed compared to that of FasL+ CTL lines. FasL− CTLs kill efficiently on a per recovered cell basis and can achieve wild-type levels of proliferation upon stimulation by optimal doses of anti-CD3, suggesting the lack of a costimulatory signal during antigen stimulation. To test this hypothesis directly, soluble FasIgG, a fusion protein of murine Fas and human IgG1, was added to FasL+ CTLs to demonstrate that blocking cell surface Fas–FasL interactions mimics the depression observed for FasL− CTLs. In addition, plate-bound FasIgG in conjunction with suboptimal anti-CD3 stimulation augments proliferative signals in FasL+ but not FasL− CTLs. In contrast to these results with CD8+ T cells, alloantigen-stimulated FasL− CD4+ T cells proliferate vigorously compared to FasL+ cells. These data demonstrate that reverse signaling through FasL is required for CTLs to achieve maximal proliferation and may provide clues to differences in the homeostatic regulation of activated CD4+ and CD8+ T cells during an immune response.
The Phosphotyrosine Phosphatase SHP-2 Participates in a Multimeric Signaling Complex and Regulates T Cell Receptor (TCR) coupling to the Ras/Mitogen-activated Protein Kinase (MAPK) Pathway in Jurkat T Cells
Authors: Frearson Julie A. , Alexander Denis R.
Published: July 2002

Src homology 2 (SH2) domain–containing phosphotyrosine phosphatases (SHPs) are increasingly being shown to play critical roles in protein tyrosine kinase–mediated signaling pathways. The role of SHP-1 as a negative regulator of T cell receptor (TCR) signaling has been established. To further explore the function of the other member of this family, SHP-2, in TCR-mediated events, a catalytically inactive mutant SHP-2 was expressed under an inducible promoter in Jurkat T cells. Expression of the mutant phosphatase significantly inhibited TCR-induced activation of the extracellular-regulated kinase (ERK)-2 member of the mitogen-activated protein kinase (MAPK) family, but had no effect on TCR-ζ chain tyrosine phosphorylation or TCR-elicited Ca2+ transients. Inactive SHP-2 was targeted to membranes resulting in the selective increase in tyrosine phosphorylation of three membrane-associated candidate SHP-2 substrates of 110 kD, 55-60 kD, and 36 kD, respectively. Analysis of immunoprecipitates containing inactive SHP-2 also indicated that the 110-kD and 36-kD Grb-2–associated proteins were putative substrates for SHP-2. TCR-stimulation of Jurkat T cells expressing wild-type SHP-2 resulted in the formation of a multimeric cytosolic complex composed of SHP-2, Grb-2, phosphatidylinositol (PI) 3′-kinase, and p110. A significant proportion of this complex was shown to be membrane associated, presumably as a result of translocation from the cytosol. Catalytically inactive SHP-2, rather than the wild-type PTPase, was preferentially localized in complex with Grb-2 and the p85 subunit of PI 3′-kinase, suggesting that the dephosphorylating actions of SHP-2 may regulate the association of these signaling molecules to the p110 complex. Our results show that SHP-2 plays a critical role in linking the TCR to the Ras/MAPK pathway in Jurkat T cells, and also provide some insight into the molecular interactions of SHP-2 that form the basis of this signal transduction process.
Two Enteropathogenic Escherichia coli Type III Secreted Proteins, EspA and EspB, Are Virulence Factors
Authors: Abe Akio , Heczko Ursula , Hegele Richard G. , Brett Finlay B.
Published: July 2002

Enteropathogenic Escherichia coli (EPEC) belongs to a family of related bacterial pathogens, including enterohemorrhagic Escherichia coli (EHEC) O157:H7 and other human and animal diarrheagenic pathogens that form attaching and effacing (A/E) lesions on host epithelial surfaces. Bacterial secreted Esp proteins and a type III secretion system are conserved among these pathogens and trigger host cell signal transduction pathways and cytoskeletal rearrangements, and mediate intimate bacterial adherence to epithelial cell surfaces in vitro. However, their role in pathogenesis is still unclear. To investigate the role of Esp proteins in disease, mutations in espA and espB were constructed in rabbit EPEC serotype O103 and infection characteristics were compared to that of the wild-type strain using histology, scanning and transmission electron microscopy, and confocal laser scanning microscopy in a weaned rabbit infection model. The virulence of EspA and EspB mutant strains was severely attenuated. Additionally, neither mutant strain formed A/E lesions, nor did either one cause cytoskeletal actin rearrangements beneath the attached bacteria in the rabbit intestine. Collectively, this study shows for the first time that the type III secreted proteins EspA and EspB are needed to form A/E lesions in vivo and are indeed virulence factors. It also confirms the role of A/E lesions in disease processes.
Humoral and Cell-mediated Autoimmune Reactions to Human Acidic Ribosomal P2 Protein in Individuals Sensitized to Aspergillus fumigatus P2 Protein
Authors: Mayer Christina , Appenzeller Ulrich , Seelbach Heike , Achatz Gernot , Oberkofler Hannes , Breitenbach Michael , Blaser Kurt , Crameri Reto
Published: July 2002

A panel of cDNAs encoding allergenic proteins was isolated from an Aspergillus fumigatus cDNA library displayed on the surface of filamentous phage. Solid phase–immobilized serum immunoglobulin E (IgE) from A. fumigatus–allergic individuals was used to enrich phage displaying IgE-binding molecules. One of the cDNAs encoded a 11.1-kD protein that was identified as acidic ribosomal phosphoprotein type 2 (P2 protein). The allergen, formally termed rAsp f 8, shares >62% sequence identity and >84% sequence homology to corresponding eukaryotic P2 proteins, including human P2 protein. The sequences encoding human and fungal P2 protein were subcloned, expressed in Escherichia coli as His6-tagged fusion proteins, and purified by Ni2+–chelate affinity chromatography. Both recombinant P2 proteins were recognized by IgE antibodies from allergic individuals sensitized to the A. fumigatus P2 protein and elicited strong type 1–specific skin reactions in these individuals. Moreover, human and fungal P2 proteins induced proliferative responses in peripheral blood mononuclear cells of A. fumigatus– allergic subjects sensitized to the fungal P2 protein. These data provide strong evidence for in vitro and in vivo humoral and cell-mediated autoreactivity to human P2 protein in patients suffering from chronic A. fumigatus allergy.
Interleukin 15 Induces Endothelial Hyaluronan Expression in Vitro and Promotes Activated T Cell Extravasation through a Cd44-Dependent Pathway in Vivo
Authors: Estess Pila , Nandi Animesh , Mohamadzadeh Mansour , Siegelman Mark H.
Published: July 2002

T cell recruitment to extralymphoid tissues is fundamental to the initiation and perpetuation of the inflammatory state during immune and autoimmune responses. Interleukin (IL)-15 is a proinflammatory cytokine whose described functions largely overlap with those of IL-2. The latter is attributable in large part to its binding of the heterotrimeric receptor that contains the β and γ chains of the IL-2R in combination with an unique IL-15Rα chain. However, unlike IL-2, IL-15 and its receptor have a wide tissue and cell type distribution, including endothelial cells. Here, we examine the effect of IL-15 on hyaluronan expression by endothelial cells, and investigate its role in vivo in promoting the extravasation of antigen-activated T cells through a CD44-dependent pathway. The expression of hyaluronan on primary endothelial cells and microvascular endothelial cell lines is induced by IL-15, whereas IL-2 has no such activity. Moreover, intraperitoneal administration of IL-15 or TNF-α in the absence of other exogenous proinflammatory stimuli allows the extravasation of superantigen-stimulated T cells into this site in vivo in a CD44-dependent manner. T cell recruitment induced by IL-15 requires expression of an intact IL-2Rβ chain, indicating that IL-15 operates in this context through the traditional IL-15R. The results suggest that IL-15 can regulate endothelial cell function and thereby enables a CD44-initiated adhesion pathway that facilitates entry of activated T lymphocytes into inflammatory sites. They further demonstrate a novel role for IL-15 (distinct from any of IL-2) in regulating microvascular endothelial cell adhesive function, help to understand the role of IL-15R expression on endothelium, and further support a central position for this cytokine in orchestrating multiple sequential aspects of T cell effector function and therefore chronic inflammatory processes.
Neonatally Induced Inactivation of the Vascular Cell Adhesion Molecule 1 Gene Impairs B Cell Localization and T Cell–Dependent Humoral Immune Response
Authors: Leuker Christoph E. , Labow Mark , Müller Werner , Wagner Norbert
Published: July 2002

Vascular cellular adhesion molecule (VCAM)-1 is a membrane-bound cellular adhesion molecule that mediates adhesive interactions between hematopoietic progenitor cells and stromal cells in the bone marrow (BM) and between leukocytes and endothelial as well as dendritic cells. Since VCAM-1–deficient mice die embryonically, conditional VCAM-1 mutant mice were generated to analyze the in vivo function of this adhesion molecule. Here we show that interferon-induced Cre-loxP–mediated deletion of the VCAM-1 gene after birth efficiently ablates expression of VCAM-1 in most tissues like, for example, BM, lymphoid organs, and lung, but not in brain. Induced VCAM-1 deficiency leads to a reduction of immature B cells in the BM and to an increase of these cells in peripheral blood but not in lymphoid organs. Mature recirculating B cells are reduced in the BM. In a migration assay, the number of mature B cells that appears in the BM after intravenous injection is decreased. In addition, the humoral immune response to a T cell–dependent antigen is impaired. VCAM-1 serves an important role for B cell localization and the T cell–dependent humoral immune response.
Human Immunodeficiency Virus 1 Envelope Glycoprotein Complex-Induced Apoptosis Involves Mammalian Target of Rapamycin/Fkbp12-Rapamycin–Associated Protein–Mediated P53 Phosphorylation
Authors: Castedo Maria , Ferri Karine F. , Blanco Julià , Roumier Thomas , Larochette Nathanael , Barretina Jordi , Amendola Alessandra , Nardacci Roberta , Métivier Didier , Este José A. , Piacentini Mauro , Kroemer Guido
Published: July 2002

Syncytia arising from the fusion of cells expressing a lymphotropic human immunodeficiency virus (HIV)-1–encoded envelope glycoprotein complex (Env) gene with cells expressing the CD4/CXCR4 complex undergo apoptosis through a mitochondrion-controlled pathway initiated by the upregulation of Bax. In syncytial apoptosis, phosphorylation of p53 on serine 15 (p53S15) precedes Bax upregulation, the apoptosis-linked conformational change of Bax, the insertion of Bax in mitochondrial membranes, subsequent release of cytochrome c, caspase activation, and apoptosis. p53S15 phosphorylation also occurs in vivo, in HIV-1+ donors, where it can be detected in preapoptotic and apoptotic syncytia in lymph nodes, as well as in peripheral blood mononuclear cells, correlating with viral load. Syncytium-induced p53S15 phosphorylation is mediated by the upregulation/activation of mammalian target of rapamycin (mTOR), also called FKBP12-rapamycin-associated protein (FRAP), which coimmunoprecipitates with p53. Inhibition of mTOR/FRAP by rapamycin reduces apoptosis in several paradigms of syncytium-dependent death, including in primary CD4+ lymphoblasts infected by HIV-1. Concomitantly, rapamycin inhibits p53S15 phosphorylation, mitochondrial translocation of Bax, loss of the mitochondrial transmembrane potential, mitochondrial release of cytochrome c, and nuclear chromatin condensation. Transfection with dominant negative p53 has a similar antiapoptotic action as rapamycin, upstream of the Bax upregulation/translocation. In summary, we demonstrate that phosphorylation of p53S15 by mTOR/FRAP plays a critical role in syncytial apoptosis driven by HIV-1 Env.
Effect of Bay K 8644 (−) and the β2a Subunit on Ca2+-dependent Inactivation in α1C Ca2+ Channels
Authors: Noceti Francesca , Olcese Riccardo , Qin Ning , Zhou Jianming , Stefani Enrico
Published: July 2002

Ca2+ currents recorded from Xenopus oocytes expressing only the α1C pore-forming subunit of the cardiac Ca2+ channel show Ca2+-dependent inactivation with a single exponential decay. This current-dependent inactivation is not detected for inward Ba2+ currents in external Ba2+. Facilitation of pore opening speeds up the Ca2+-dependent inactivation process and makes evident an initial fast rate of decay. Facilitation can be achieved by (a) coexpression of the β2a subunit with the α1C subunit, or (b) addition of saturating Bay K 8644 (−) concentration to α1C channels. The addition of Bay K 8644 (−) to α1Cβ2a channels makes both rates of inactivation faster. All these maneuvers do not induce inactivation in Ba2+ currents in our expression system. These results support the hypothesis of a mechanism for the Ca2+-dependent inactivation process that is sensitive to both Ca2+ flux (single channel amplitude) and open probability. We conclude that the Ca2+ site for inactivation is in the α1C pore-forming subunit and we propose a kinetic model to account for the main features of α1Cβ2a Ca2+ currents.
A posteriori error control in low-order finite element discretisations of incompressible stationary flow problems
Authors: Carstensen Carsten , Funken Stefan
Published: July 2002

Computable a posteriori error bounds and related adaptive mesh-refining algorithms are provided for the numerical treatment of monotone stationary flow problems with a quite general class of conforming and nonconforming finite element methods. A refined residual-based error estimate generalises the works of Verfürth; Dari, Duran and Padra; Bao and Barrett. As a consequence, reliable and efficient averaging estimates can be established on unstructured grids. The symmetric formulation of the incompressible flow problem models certain nonNewtonian flow problems and the Stokes problem with mixed boundary conditions. A Helmholtz decomposition avoids any regularity or saturation assumption in the mathematical error analysis. Numerical experiments for the partly nonconforming method analysed by Kouhia and Stenberg indicate efficiency of related adaptive mesh-refining algorithms.
Maximally equidistributed combined Tausworthe generators
Authors: L’Ecuyer Pierre
Published: July 2002

Tausworthe random number generators based on a primitive trinomial allow an easy and fast implementation when their parameters obey certain restrictions. However, such generators, with those restrictions, have bad statistical properties unless we combine them. A generator is called maximally equidistributed if its vectors of successive values have the best possible equidistribution in all dimensions. This paper shows how to find maximally equidistributed combinations in an efficient manner, and gives a list of generators with that property. Such generators have a strong theoretical support and lend themselves to very fast software implementations.
On the beta expansion for Salem numbers of degree 6
Authors: Boyd David
Published: July 2002

For a given

β
>
1

\beta > 1

, the beta transformation

T
=

T

β

T = T_{\beta }

is defined for

x
∈
[
0
,
1
]

x \in [0,1]

by

T
x
:=
β
x

Tx := \beta x

(mod

1
1

). The number

β
\beta

is said to be a beta number if the orbit

{

T

n

(
1
)

}

n
≥
1

\{T^{n}(1)\}_{n \ge 1}

is finite, hence eventually periodic. It is known that all Pisot numbers are beta numbers, and it is conjectured that this is true for Salem numbers, but this is known only for Salem numbers of degree

4
4

. Here we consider some computational and heuristic evidence for the conjecture in the case of Salem numbers of degree

6
6

, by considering the set of

11836
11836

such numbers of trace at most

15
15

. Although the orbit is small for the majority of these numbers, there are some examples for which the orbit size is shown to exceed

10

9

10^{9}

and for which the possibility remains that the orbit is infinite. There are also some very large orbits which have been shown to be finite: an example is given for which the preperiod length is

39420662
39420662

and the period length is

93218808
93218808

. This is in contrast to Salem numbers of degree

4
4

where the orbit size is bounded by

2
β
+
3

2\beta + 3

. An heuristic probabilistic model is proposed which explains the difference between the degree-

4
4

and degree-

6
6

cases. The model predicts that all Salem numbers of degree

4
4

and

6
6

should be beta numbers but that degree-

6
6

Salem numbers can have orbits which are arbitrarily large relative to the size of

β
\beta

. Furthermore, the model predicts that a positive proportion of Salem numbers of any fixed degree

≥
8

\ge 8

will not be beta numbers. This latter prediction is not tested here.
The practical computation of areas associated with binary quartic forms
Authors: Bean Michael
Published: July 2002

We derive formulas for practically computing the area of the region

|

F
(
x
,
y
)

|

≤
1

|F(x,y)| \leq 1

defined by a binary quartic form

F
(
X
,
Y
)
∈

R

[
X
,
Y
]

F(X,Y) \in \mathbb R [X,Y]

. These formulas, which involve a particular hypergeometric function, are useful when estimating the number of lattice points in certain regions of the type

|

F
(
x
,
y
)

|

≤
h

|F(x,y)| \leq h

and will likely find application in many contexts. We also show that for forms

F
F

of arbitrary degree, the maximal size of the area of the region

|

F
(
x
,
y
)

|

≤
1

|F(x,y)| \leq 1

, normalized with respect to the discriminant of

F
F

and taken with respect to the number of conjugate pairs of

F
(
x
,
1
)

F(x,1)

, increases as the number of conjugate pairs decreases; and we give explicit numerical values for these normalized maxima when

F
F

is a quartic form.
Implicit-explicit multistep finite element methods for nonlinear parabolic problems
Authors: Akrivis Georgios , Crouzeix Michel , Makridakis Charalambos
Published: July 2002

We approximate the solution of initial boundary value problems for nonlinear parabolic equations. In space we discretize by finite element methods. The discretization in time is based on linear multistep schemes. One part of the equation is discretized implicitly and the other explicitly. The resulting schemes are stable, consistent and very efficient, since their implementation requires at each time step the solution of a linear system with the same matrix for all time levels. We derive optimal order error estimates. The abstract results are applied to the Kuramoto-Sivashinsky and the Cahn-Hilliard equations in one dimension, as well as to a class of reaction diffusion equations in

R

ν

,

{\mathbb {R}} ^{\nu },

ν
=
2
,
3.

\nu = 2, 3.
Mapplet: a CORBA-based genome map viewer.
Authors: Jungfer K , Rodriguez-Tom√© P
Published: July 2002

MOTIVATION: There are a large number of genetic and physical maps, distributed at many sites. Each site offers different kinds of access methods and viewers. CORBA, the de facto standard for distributed object-oriented computing, offers new opportunities to unify the view on these maps through standard interfaces. A collaboration of Infobiogen and the EBI proposes a common IDL for maps. RESULTS: A CORBA map viewer is presented which serves as a proof of concept for the proposed IDL. It demonstrates its usefulness in the context of map viewing and its ability to handle large maps with <1000 markers. The viewer gives access to the maps of the Radiation Hybrid Database at EBI. It gives a quick overview of several large maps side by side. The marker density at each map position is displayed and different marker types can be highlighted. AVAILABILITY: Demonstration and source code at: http://sunny.ebi.ac.uk/ approximately jungfer/Mapplet CONTACT: jungfer@ebi.ac.uk
MONEY, WHO NEEDS IT? NATURAL RESOURCE DAMAGE ASSESSMENT
Authors: Flores Nicholas E. , Thacher Jennifer
Published: July 2002

A new natural resource damage assessment paradigm has been suggested that emphasizes direct analysis of compensatory restoration rather than analysis of compensating variation for damages. This article considers whether money can be avoided in damage assessment. The analysis of compensatory restoration leads to the conclusion that money should be considered when measuring preferences. Failure to consider money leaves trustees unable to judge the adequacy of compensatory restoration. The problem stems from heterogeneity over restoration scale.
Allosteric regulation of the light-harvesting system of photosystem II
Authors: Horton Peter , Ruban Alexander V. , Wentworth Mark
Published: July 2002

Non–photochemical quenching of chlorophyll fluorescence (NPQ) is symptomatic of the regulation of energy dissipation by the light–harvesting antenna of photosystem II (PS II). The kinetics of NPQ in both leaves and isolated chloroplasts are determined by the transthylakoid ΔpH and the de–epoxidation state of the xanthophyll cycle. In order to understand the mechanism and regulation of NPQ we have adopted the approaches commonly used in the study of enzyme–catalysed reactions. Steady–state measurements suggest allosteric regulation of NPQ, involving control by the xanthophyll cycle carotenoids of a protonationdependent conformational change that transforms the PS II antenna from an unquenched to a quenched state. The features of this model were confirmed using isolated light–harvesting proteins. Analysis of the rate of induction of quenching both in vitro and in vivo indicated a bimolecular second–order reaction; it is suggested that quenching arises from the reaction between two fluorescent domains, possibly within a single protein subunit. A universal model for this transition is presented based on simple thermodynamic principles governing reaction kinetics.
Crystal structure of an anti‐interleukin‐2 monoclonal antibody Fab complexed with an antigenic nonapeptide
Authors: Afonin Pavel V. , Fokin Andrey V. , Tsygannik Igor N. , Mikhailova Irina YU. , Onoprienko Lyudmila V. , Mikhaleva Inna I. , Ivanov Vadim T. , Mareeva Tat'yana YU. , Nesmeyanov Vladimir A. , Li Naiyin , Pangborn Walter A. , Duax William L. , Pletnev Vladimir Z.
Published: July 2002

The three‐dimensional structure of the Fab fragment of a monoclonal antibody (LNKB‐2) to human interleukin‐2 (IL‐2) complexed with a synthetic antigenic nonapeptide, Ac‐Lys‐Pro‐Leu‐Glu‐Glu‐Val‐Leu‐Asn‐Leu‐OMe, has been determined at 3.0 Å resolution. In the structure, four out of the six hypervariable loops of the Fab (complementarity determining regions [CDRs] L1, H1, H2, and H3) are involved in peptide association through hydrogen bonding, salt bridge formation, and hydrophobic interactions. The Tyr residues in the Fab antigen binding site play a major role in antigen–antibody recognition. The structures of the complexed and uncomplexed Fab were compared. In the antigen binding site the CDR‐L1 loop of the antibody shows the largest structural changes upon peptide binding. The peptide adopts a mostly α‐helical conformation similar to that in the epitope fragment 64–72 of the IL‐2 antigen. The side chains of residues Leu 66, Val 69, and Leu 70, which are shielded internally in the IL‐2 structure, are involved in interactions with the Fab in the complex studied. This indicates that antibody–antigen complexation involves a significant rearrangement of the epitope‐containing region of the IL‐2 with retention of the α‐helical character of the epitope fragment.