bluuebunny/crossref_metadata_2025 huggingface dataset:
Rab17 Regulates Membrane Trafficking through Apical Recycling Endosomes in Polarized Epithelial Cells
Authors: Zacchi Paola , Stenmark Harald , Parton Robert G. , Orioli Donata , Lim Filip , Giner Angelika , Mellman Ira , Zerial Marino , Murphy Carol
Published: July 2002

A key feature of polarized epithelial cells is the ability to maintain the specific biochemical composition of the apical and basolateral plasma membrane domains while selectively allowing transport of proteins and lipids from one pole to the opposite by transcytosis. The small GTPase, rab17, a member of the rab family of regulators of intracellular transport, is specifically induced during cell polarization in the developing kidney. We here examined its intracellular distribution and function in both nonpolarized and polarized cells. By confocal immunofluorescence microscopy, rab17 colocalized with internalized transferrin in the perinuclear recycling endosome of BHK-21 cells. In polarized Eph4 cells, rab17 associated with the apical recycling endosome that has been implicated in recycling and transcytosis. The localization of rab17, therefore, strengthens the proposed homology between this compartment and the recycling endosome of nonpolarized cells. Basolateral to apical transport of two membrane-bound markers, the transferrin receptor and the FcLR 5-27 chimeric receptor, was specifically increased in Eph4 cells expressing rab17 mutants defective in either GTP binding or hydrolysis. Furthermore, the mutant proteins stimulated apical recycling of FcLR 5-27. These results support a role for rab17 in regulating traffic through the apical recycling endosome, suggesting a function in polarized sorting in epithelial cells.
Lessons from Keratin 18 Knockout Mice: Formation of Novel Keratin Filaments, Secondary Loss of Keratin 7 and Accumulation of Liver-specific Keratin 8-Positive Aggregates
Authors: Magin Thomas M. , Schröder Rolf , Leitgeb Sabine , Wanninger Frederique , Zatloukal Kurt , Grund Christine , Melton David W.
Published: July 2002

Here, we report on the analysis of keratin 18 null mice. Unlike the ablation of K8, which together with K18 is expressed in embryonic and simple adult epithelia, K18 null mice are viable, fertile, and show a normal lifespan. In young K18 null mice, hepatocytes were completely devoid of keratin filaments. Nevertheless, typical desmosomes were formed and maintained. Old K18 null mice, however, developed a distinctive liver pathology with abnormal hepatocytes containing K8-positive aggregates. These stained positively for ubiquitin and MM120-1 and were identified as Mallory bodies, one hallmark of human alcoholic hepatitis. This is the first demonstration that the ablation of one keratin leads to the accumulation of its single partner. Another striking finding was the absence or drastic down regulation of K7 in several tissues despite its ongoing transcription. Moreover, K18 null mice revealed new insights in the filament-forming capacity of the tail-less K19 in vivo. Due to the unexpected secondary loss of K7, only K8/19 are expressed in the uterine epithelium of K18 null mice. Immunoelectron microscopy of this tissue demonstrated the presence of typical K8/19 IF, thus highlighting in vivo that K19 is a fully competent partner for K8.
ZYG-9, A Caenorhabditis elegans Protein Required for Microtubule Organization and Function, Is a Component of Meiotic and Mitotic Spindle Poles
Authors: Matthews Lisa R. , Carter Philip , Thierry-Mieg Danielle , Kemphues Ken
Published: July 2002

We describe the molecular characterization of zyg-9, a maternally acting gene essential for microtubule organization and function in early Caenorhabditis elegans embryos. Defects in zyg-9 mutants suggest that the zyg-9 product functions in the organization of the meiotic spindle and the formation of long microtubules. One-cell zyg-9 embryos exhibit both meiotic and mitotic spindle defects. Meiotic spindles are disorganized, pronuclear migration fails, and the mitotic apparatus forms at the posterior, orients incorrectly, and contains unusually short microtubules. We find that zyg-9 encodes a component of the meiotic and mitotic spindle poles. In addition to the strong staining of spindle poles, we consistently detect staining in the region of the kinetochore microtubules at metaphase and early anaphase in mitotic spindles. The ZYG-9 signal at the mitotic centrosomes is not reduced by nocodazole treatment, indicating that ZYG-9 localization to the mitotic centrosomes is not dependent upon long astral microtubules. Interestingly, in embryos lacking an organized meiotic spindle, produced either by nocodazole treatment or mutations in the mei-1 gene, ZYG-9 forms a halo around the meiotic chromosomes. The protein sequence shows partial similarity to a small set of proteins that also localize to spindle poles, suggesting a common activity of the proteins.
Slk19p Is a Centromere Protein That Functions to Stabilize Mitotic Spindles
Authors: Zeng Xuemei , Kahana Jason A. , Silver Pamela A. , Morphew Mary K. , McIntosh J. Richard , Fitch Ian T. , Carbon John , Saunders William S.
Published: July 2002

We have identified a novel centromere-associated gene product from Saccharomyces cerevisiae that plays a role in spindle assembly and stability. Strains with a deletion of SLK19 (synthetic lethal Kar3p gene) exhibit abnormally short mitotic spindles, increased numbers of astral microtubules, and require the presence of the kinesin motor Kar3p for viability. When cells are deprived of both Slk19p and Kar3p, rapid spindle breakdown and mitotic arrest is observed. A functional fusion of Slk19p to green fluorescent protein (GFP) localizes to kinetochores and, during anaphase, to the spindle midzone, whereas Kar3p-GFP was found at the nuclear side of the spindle pole body. Thus, these proteins seem to play overlapping roles in stabilizing spindle structure while acting from opposite ends of the microtubules.
Integrating Conflicting Chemotactic Signals
Authors: Foxman Ellen F. , Kunkel Eric J. , Butcher Eugene C.
Published: July 2002

Leukocytes navigate through complex chemoattractant arrays, and in so doing, they must migrate from one chemoattractant source to another. By evaluating directional persistence and chemotaxis during neutrophil migration under agarose, we show that cells migrating away from a local chemoattractant, against a gradient, display true chemotaxis to distant agonists, often behaving as if the local gradient were without effect. We describe two interrelated properties of migrating cells that allow this to occur. First, migrating leukocytes can integrate competing chemoattractant signals, responding as if to the vector sum of the orienting signals present. Second, migrating cells display memory of their recent environment: cells' perception of the relative strength of orienting signals is influenced by their history, so that cells prioritize newly arising or newly encountered attractants. We propose that this cellular memory, by promoting sequential chemotaxis to one attractant after another, is in fact responsible for the integration of competitive orienting signals over time, and allows combinations of chemoattractants to guide leukocytes in a step-by-step fashion to their destinations within tissues.
Cdc42 and Rac Stimulate Exocytosis of Secretory Granules by Activating the Ip3/Calcium Pathway in Rbl-2h3 Mast Cells
Authors: Hong-Geller Elizabeth , Cerione Richard A.
Published: July 2002

We have expressed dominant-active and dominant-negative forms of the Rho GTPases, Cdc42 and Rac, using vaccinia virus to evaluate the effects of these mutants on the signaling pathway leading to the degranulation of secretory granules in RBL-2H3 cells. Dominant-active Cdc42 and Rac enhance antigen-stimulated secretion by about twofold, whereas the dominant-negative mutants significantly inhibit secretion. Interestingly, treatment with the calcium ionophore, A23187, and the PKC activator, PMA, rescues the inhibited levels of secretion in cells expressing the dominant-negative mutants, implying that Cdc42 and Rac act upstream of the calcium influx pathway. Furthermore, cells expressing the dominant-active mutants exhibit elevated levels of antigen-stimulated IP3 production, an amplified antigen-stimulated calcium response consisting of both calcium release from internal stores and influx from the extracellular medium, and an increase in aggregate formation of the IP3 receptor. In contrast, cells expressing the dominant-negative mutants display the opposite phenotypes. Finally, we are able to detect an in vitro interaction between Cdc42 and PLCγ1, the enzyme immediately upstream of IP3 formation. Taken together, these findings implicate Cdc42 and Rac in regulating the exocytosis of secretory granules by stimulation of IP3 formation and calcium mobilization upon antigen stimulation.
Fusion of Constitutive Membrane Traffic with the Cell Surface
Observed by Evanescent Wave Microscopy
Authors: Toomre Derek , Steyer Jürgen A. , Keller Patrick , Almers Wolfhard , Simons Kai
Published: July 2002

Monitoring the fusion of constitutive traffic with the plasma membrane has remained largely elusive. Ideally, fusion would be monitored with high spatial and temporal resolution. Recently, total internal reflection (TIR) microscopy was used to study regulated exocytosis of fluorescently labeled chromaffin granules. In this technique, only the bottom cellular surface is illuminated by an exponentially decaying evanescent wave of light. We have used a prism type TIR setup with a penetration depth of ∼50 nm to monitor constitutive fusion of vesicular stomatitis virus glycoprotein tagged with the yellow fluorescent protein. Fusion of single transport containers (TCs) was clearly observed and gave a distinct analytical signature. TCs approached the membrane, appeared to dock, and later rapidly fuse, releasing a bright fluorescent cloud into the membrane. Observation and analysis provided insight about their dynamics, kinetics, and position before and during fusion. Combining TIR and wide-field microscopy allowed us to follow constitutive cargo from the Golgi complex to the cell surface. Our observations include the following: (1) local restrained movement of TCs near the membrane before fusion; (2) apparent anchoring near the cell surface; (3) heterogeneously sized TCs fused either completely; or (4) occasionally larger tubular-vesicular TCs partially fused at their tips.
Determinants of the Nuclear Localization of the Heterodimeric DNA Fragmentation Factor (Icad/Cad)
Authors: Lechardeur Delphine , Drzymala Luke , Sharma Manu , Zylka Danuta , Kinach Robert , Pacia Joanna , Hicks Christopher , Usmani Nawaid , Rommens Johanna M. , Lukacs Gergely L.
Published: July 2002

Programmed cell death or apoptosis leads to the activation of the caspase-activated DNase (CAD), which degrades chromosomal DNA into nucleosomal fragments. Biochemical studies revealed that CAD forms an inactive heterodimer with the inhibitor of caspase-activated DNase (ICAD), or its alternatively spliced variant, ICAD-S, in the cytoplasm. It was initially proposed that proteolytic cleavage of ICAD by activated caspases causes the dissociation of the ICAD/CAD heterodimer and the translocation of active CAD into the nucleus in apoptotic cells. Here, we show that endogenous and heterologously expressed ICAD and CAD reside predominantly in the nucleus in nonapoptotic cells. Deletional mutagenesis and GFP fusion proteins identified a bipartite nuclear localization signal (NLS) in ICAD and verified the function of the NLS in CAD. The two NLSs have an additive effect on the nuclear targeting of the CAD–ICAD complex, whereas ICAD-S, lacking its NLS, appears to have a modulatory role in the nuclear localization of CAD. Staurosporine-induced apoptosis evoked the proteolysis and disappearance of endogenous and exogenous ICAD from the nuclei of HeLa cells, as monitored by immunoblotting and immunofluorescence microscopy. Similar phenomenon was observed in the caspase-3–deficient MCF7 cells upon expressing procaspase-3 transiently. We conclude that a complex mechanism, involving the recognition of the NLSs of both ICAD and CAD, accounts for the constitutive accumulation of CAD/ICAD in the nucleus, where caspase-3–dependent regulation of CAD activity takes place.
Numb Is an Endocytic Protein
Authors: Santolini Elisa , Puri Claudia , Salcini Anna Elisabetta , Gagliani Maria Cristina , Pelicci Pier Giuseppe , Tacchetti Carlo , Di Fiore Pier Paolo
Published: July 2002

Numb is a protein that in Drosophila determines cell fate as a result of its asymmetric partitioning at mitosis. The function of Numb has been linked to its ability to bind and to biologically antagonize Notch, a membrane receptor that also specifies cell fate. The biochemical mechanisms underlying the action of Numb, however, are still largely unknown. The wide pattern of expression of Numb suggests a general function in cellular homeostasis that could be additional to, or part of, its action in fate determination. Such a function could be endocytosis, as suggested by the interaction of Numb with Eps15, a component of the endocytic machinery. Here, we demonstrate that Numb is an endocytic protein. We found that Numb localizes to endocytic organelles and is cotrafficked with internalizing receptors. Moreover, it associates with the appendage domain of α adaptin, a subunit of AP2, a major component of clathrin-coated pits. Finally, fragments of Numb act as dominant negatives on both constitutive and ligand-regulated receptor-mediated internalization, suggesting a general role for Numb in the endocytic process.
The High Mobility Group (Hmg) Boxes of the Nuclear Protein Hmg1 Induce Chemotaxis and Cytoskeleton Reorganization in Rat Smooth Muscle Cells
Authors: Degryse Bernard , Bonaldi Tiziana , Scaffidi Paola , Müller Susanne , Resnati Massimo , Sanvito Francesca , Arrigoni Gianluigi , Bianchi Marco E.
Published: July 2002

HMG1 (high mobility group 1) is a ubiquitous and abundant chromatin component. However, HMG1 can be secreted by activated macrophages and monocytes, and can act as a mediator of inflammation and endotoxic lethality. Here we document a role of extracellular HMG1 in cell migration. HMG1 (and its individual DNA-binding domains) stimulated migration of rat smooth muscle cells in chemotaxis, chemokinesis, and wound healing assays. HMG1 induced rapid and transient changes of cell shape, and actin cytoskeleton reorganization leading to an elongated polarized morphology typical of motile cells. These effects were inhibited by antibodies directed against the receptor of advanced glycation endproducts, indicating that the receptor of advanced glycation endproducts is the receptor mediating the HMG1-dependent migratory responses. Pertussis toxin and the mitogen-activated protein kinase kinase inhibitor PD98059 also blocked HMG1-induced rat smooth muscle cell migration, suggesting that a Gi/o protein and mitogen-activated protein kinases are required for the HMG1 signaling pathway. We also show that HMG1 can be released by damage or necrosis of a variety of cell types, including endothelial cells. Thus, HMG1 has all the hallmarks of a molecule that can promote atherosclerosis and restenosis after vascular damage.
Cooh-Terminal Truncations Promote Proteasome-Dependent Degradation of Mature Cystic Fibrosis Transmembrane Conductance Regulator from Post-Golgi Compartments
Authors: Benharouga Mohamed , Haardt Martin , Kartner Norbert , Lukacs Gergely L.
Published: July 2002

Impaired biosynthetic processing of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel, constitutes the most common cause of CF. Recently, we have identified a distinct category of mutation, caused by premature stop codons and frameshift mutations, which manifests in diminished expression of COOH-terminally truncated CFTR at the cell surface. Although the biosynthetic processing and plasma membrane targeting of truncated CFTRs are preserved, the turnover of the complex-glycosylated mutant is sixfold faster than its wild-type (wt) counterpart. Destabilization of the truncated CFTR coincides with its enhanced susceptibility to proteasome-dependent degradation from post-Golgi compartments globally, and the plasma membrane specifically, determined by pulse–chase analysis in conjunction with cell surface biotinylation. Proteolytic cleavage of the full-length complex-glycosylated wt and degradation intermediates derived from both T70 and wt CFTR requires endolysosomal proteases. The enhanced protease sensitivity in vitro and the decreased thermostability of the complex-glycosylated T70 CFTR in vivo suggest that structural destabilization may account for the increased proteasome susceptibility and the short residence time at the cell surface. These in turn are responsible, at least in part, for the phenotypic manifestation of CF. We propose that the proteasome-ubiquitin pathway may be involved in the peripheral quality control of other, partially unfolded membrane proteins as well.
Maintenance of Golgi structure and function depends on the integrity of ER export
Authors: Ward Theresa H. , Polishchuk Roman S. , Caplan Steve , Hirschberg Koret , Lippincott-Schwartz Jennifer
Published: July 2002

The Golgi apparatus comprises an enormous array of components that generate its unique architecture and function within cells. Here, we use quantitative fluorescence imaging techniques and ultrastructural analysis to address whether the Golgi apparatus is a steady-state or a stable organelle. We found that all classes of Golgi components are dynamically associated with this organelle, contrary to the prediction of the stable organelle model. Enzymes and recycling components are continuously exiting and reentering the Golgi apparatus by membrane trafficking pathways to and from the ER, whereas Golgi matrix proteins and coatomer undergo constant, rapid exchange between membrane and cytoplasm. When ER to Golgi transport is inhibited without disrupting COPII-dependent ER export machinery (by brefeldin A treatment or expression of Arf1[T31N]), the Golgi structure disassembles, leaving no residual Golgi membranes. Rather, all Golgi components redistribute into the ER, the cytoplasm, or to ER exit sites still active for recruitment of selective membrane-bound and peripherally associated cargos. A similar phenomenon is induced by the constitutively active Sar1[H79G] mutant, which has the additional effect of causing COPII-associated membranes to cluster to a juxtanuclear region. In cells expressing Sar1[T39N], a constitutively inactive form of Sar1 that completely disrupts ER exit sites, Golgi glycosylation enzymes, matrix, and itinerant proteins all redistribute to the ER. These results argue against the hypothesis that the Golgi apparatus contains stable components that can serve as a template for its biogenesis. Instead, they suggest that the Golgi complex is a dynamic, steady-state system, whose membranes can be nucleated and are maintained by the activities of the Sar1–COPII and Arf1–coatomer systems.
Purification of pseudopodia from polarized cells reveals redistribution and activation of Rac through assembly of a CAS/Crk scaffold
Authors: Cho Samuel Y. , Klemke Richard L.
Published: July 2002

Initiation of cell migration requires morphological polarization with formation of a dominant leading pseudopodium and rear compartment. A molecular understanding of this process has been limited, due to the inability to biochemically separate the leading pseudopodium from the rear of the cell. Here we examine the spatio-temporal localization and activation of cytoskeletal-associated signals in purified pseudopodia directed to undergo growth or retraction. Pseudopodia growth requires assembly of a p130Crk-associated substrate (CAS)/c-CrkII (Crk) scaffold, which facilitates translocation and activation of Rac1. Interestingly, Rac1 activation then serves as a positive-feedback loop to maintain CAS/Crk coupling and pseudopodia extension. Conversely, disassembly of this molecular scaffold is critical for export and down regulation of Rac1 activity and induction of pseudopodia retraction. Surprisingly, the uncoupling of Crk from CAS during pseudopodium retraction is independent of changes in focal adhesion kinase activity and CAS tyrosine phosphorylation. These findings establish CAS/Crk as an essential scaffold for Rac1-mediated pseudopodia growth and retraction, and illustrate spatio-temporal segregation of cytoskeletal signals during cell polarization.
A requirement for cytoplasmic dynein and dynactin in intermediate filament network assembly and organization
Authors: Helfand Brian T. , Mikami Atsushi , Vallee Richard B. , Goldman Robert D.
Published: July 2002

We present evidence that vimentin intermediate filament (IF) motility in vivo is associated with cytoplasmic dynein. Immunofluorescence reveals that subunits of dynein and dynactin are associated with all structural forms of vimentin in baby hamster kidney-21 cells. This relationship is also supported by the presence of numerous components of dynein and dynactin in IF-enriched cytoskeletal preparations. Overexpression of dynamitin biases IF motility toward the cell surface, leading to a perinuclear clearance of IFs and their redistribution to the cell surface. IF-enriched cytoskeletal preparations from dynamitin-overexpressing cells contain decreased amounts of dynein, actin-related protein-1, and p150Glued relative to controls. In contrast, the amount of dynamitin is unaltered in these preparations, indicating that it is involved in linking vimentin cargo to dynactin. The results demonstrate that dynein and dynactin are required for the normal organization of vimentin IF networks in vivo. These results together with those of previous studies also suggest that a balance among the microtubule (MT) minus and plus end–directed motors, cytoplasmic dynein, and kinesin are required for the assembly and maintenance of type III IF networks in interphase cells. Furthermore, these motors are to a large extent responsible for the long recognized relationships between vimentin IFs and MTs.
Turnover of CD4+ and CD8+ T Lymphocytes in HIV-1 Infection as Measured by Ki-67 Antigen
Authors: Sachsenberg Nicolas , Perelson Alan S. , Yerly Sabine , Schockmel Gérard A. , Leduc Dominique , Hirschel Bernard , Perrin Luc
Published: July 2002

We investigated CD4+ and CD8+ T cell turnover in both healthy and HIV-1–infected adults by measuring the nuclear antigen Ki-67 specific for cell proliferation. The mean growth fraction, corresponding to the expression of Ki-67, was 1.1% for CD4+ T cells and 1.0% in CD8+ T cells in healthy adults, and 6.5 and 4.3% in HIV-1–infected individuals, respectively. Analysis of CD45RA+ and CD45RO+ T cell subsets revealed a selective expansion of the CD8+ CD45RO+ subset in HIV-1–positive individuals. On the basis of the growth fraction, we derived the potential doubling time and the daily turnover of CD4+ and CD8+ T cells. In HIV-1–infected individuals, the mean potential doubling time of T cells was five times shorter than that of healthy adults. The mean daily turnover of CD4+ and CD8+ T cells in HIV-1–infected individuals was increased 2- and 6-fold, respectively, with more than 40-fold interindividual variation. In patients with <200 CD4+ counts, CD4+ turnover dropped markedly, whereas CD8+ turnover remained elevated. The large variations in CD4+ T cell turnover might be relevant to individual differences in disease progression.
The Specificity of Peptides Bound to Human Histocompatibility Leukocyte Antigen (HLA)-B27 Influences the Prevalence of Arthritis in HLA-B27 Transgenic Rats
Authors: Zhou Ming , Sayad Alain , Simmons William A. , Jones Richard C. , Maika Shanna D. , Satumtira Nimman , Dorris Martha L. , Gaskell Simon J. , Bordoli Robert S. , Balfour Sartor R. , Slaughter Clive A. , Richardson James A. , Hammer Robert E. , Taurog Joel D.
Published: July 2002

Human histocompatibility leukocyte antigen B27 is highly associated with the rheumatic diseases termed spondyloarthropathies, but the mechanism is not known. B27 transgenic rats develop a spontaneous disease resembling the human spondyloarthropathies that includes arthritis and colitis. To investigate whether this disease requires the binding of specific peptides to B27, we made a minigene construct in which a peptide from influenza nucleoprotein, NP383-391 (SRYWAIRTR), which binds B27 with high affinity, is targeted directly to the ER by the signal peptide of the adenovirus E3/gp19 protein. Rats transgenic for this minigene, NP1, were made and bred with B27 rats. The production of the NP383-391 peptide in B27+NP1+ rats was confirmed immunologically and by mass spectrometry. The NP1 product displaced ∼90% of the 3H-Arg-labeled endogenous peptide fraction in B27+NP1+ spleen cells. Male B27+NP1+ rats had a significantly reduced prevalence of arthritis, compared with B27+NP− males or B27+ males with a control construct, NP2, whereas colitis was not significantly affected by the NP1 transgene. These findings support the hypothesis that B27-related arthritis requires binding of a specific peptide or set of peptides to B27, and they demonstrate a method for efficient transgenic targeting of peptides to the ER.
Protein Kinase B Regulates T Lymphocyte Survival, Nuclear Factor κb Activation, and Bcl-XL Levels in Vivo
Authors: Jones Russell G. , Parsons Michael , Bonnard Madeleine , Chan Vera S.F. , Yeh Wen-Chen , Woodgett James R. , Ohashi Pamela S.
Published: July 2002

The serine/threonine kinase protein kinase B (PKB)/Akt mediates cell survival in a variety of systems. We have generated transgenic mice expressing a constitutively active form of PKB (gag-PKB) to examine the effects of PKB activity on T lymphocyte survival. Thymocytes and mature T cells overexpressing gag-PKB displayed increased active PKB, enhanced viability in culture, and resistance to a variety of apoptotic stimuli. PKB activity prolonged the survival of CD4+CD8+ double positive (DP) thymocytes in fetal thymic organ culture, but was unable to prevent antigen-induced clonal deletion of thymocytes expressing the major histocompatibility complex class I–restricted P14 T cell receptor (TCR). In mature T lymphocytes, PKB can be activated in response to TCR stimulation, and peptide-antigen–specific proliferation is enhanced in T cells expressing the gag-PKB transgene. Both thymocytes and T cells overexpressing gag-PKB displayed elevated levels of the antiapoptotic molecule Bcl-XL. In addition, the activation of peripheral T cells led to enhanced nuclear factor (NF)-κB activation via accelerated degradation of the NF-κB inhibitory protein IκBα. Our data highlight a physiological role for PKB in promoting survival of DP thymocytes and mature T cells, and provide evidence for the direct association of three major survival molecules (PKB, Bcl-XL, and NF-κB) in vivo in T lymphocytes.
The Streptococcal Superantigen Smez Exhibits Wide Allelic Variation, Mosaic Structure, and Significant Antigenic Variation
Authors: Proft Thomas , Moffatt S. Louise , Weller Kylie D. , Paterson A. , Martin Diana , Fraser John D.
Published: July 2002

The frequencies of the newly identified streptococcal superantigen genes smez, spe-g, and spe-h were determined in a panel of 103 clinical isolates collected between 1976 and 1998 at various locations throughout New Zealand. smez and spe-g were found in every group A Streptococcus (GAS) isolate, suggesting a chromosomal location. The spe-h gene was found in only 24% of the GAS isolates and is probably located on a mobile DNA element. The smez gene displays extensive allelic variation and appears to be in linkage equilibrium with the M/emm type. 22 novel smez alleles were identified from 21 different M/emm types in addition to the already reported alleles smez and smez-2 with sequence identities between 94.5 and 99.9%. Three alleles are nonfunctional due to a single base pair deletion. The remaining 21 alleles encode distinct SMEZ variants. The mosaic structure of the smez gene suggests that this polymorphism has arisen from homologous recombination events rather than random point mutation. The recently resolved SMEZ-2 crystal structure shows that the polymorphic residues are mainly surface exposed and scattered over the entire protein. The allelic variation did not affect either Vβ specificity or potency, but did result in significant antigenic differences. Neutralizing antibody responses of individual human sera against different SMEZ variants varied significantly. 98% of sera completely neutralized SMEZ-1, but only 85% neutralized SMEZ-2, a very potent variant that has not yet been found in any New Zealand isolate. SMEZ-specific Vβ8 activity was found in culture supernatants of 66% of the GAS isolates, indicating a potential base for the development of a SMEZ targeting vaccine.
Mast Cells Control Neutrophil Recruitment during T Cell–Mediated Delayed-Type Hypersensitivity Reactions through Tumor Necrosis Factor and Macrophage Inflammatory Protein 2
Authors: Biedermann Tilo , Kneilling Manfred , Mailhammer Reinhard , Maier Konrad , Sander Christian A. , Kollias George , Kunkel Steven L. , Hültner Lothar , Röcken Martin
Published: July 2002

Polymorphonuclear leukocytes (PMNs) characterize the pathology of T cell–mediated autoimmune diseases and delayed-type hypersensitivity reactions (DTHRs) in the skin, joints, and gut, but are absent in T cell–mediated autoimmune diseases of the brain or pancreas. All of these reactions are mediated by interferon γ–producing type 1 T cells and produce a similar pattern of cytokines. Thus, the cells and mediators responsible for the PMN recruitment into skin, joints, or gut during DTHRs remain unknown. Analyzing hapten-induced DTHRs of the skin, we found that mast cells determine the T cell–dependent PMN recruitment through two mediators, tumor necrosis factor (TNF) and the CXC chemokine macrophage inflammatory protein 2 (MIP-2), the functional analogue of human interleukin 8. Extractable MIP-2 protein was abundant during DTHRs in and around mast cells of wild-type (WT) mice but absent in mast cell–deficient WBB6F1-KitW/KitW-v (KitW/KitW-v) mice. T cell–dependent PMN recruitment was reduced >60% by anti–MIP-2 antibodies and >80% in mast cell–deficient KitW/KitW-v mice. Mast cells from WT mice efficiently restored DTHRs and MIP-2–dependent PMN recruitment in KitW/KitW-v mice, whereas mast cells from TNF−/− mice did not. Thus, mast cell–derived TNF and MIP-2 ultimately determine the pattern of infiltrating cells during T cell–mediated DTHRs.
Salmonella Exploits Caspase-1 to Colonize Peyer's Patches in a Murine Typhoid Model
Authors: Monack Denise M. , Hersh David , Ghori Nafisa , Bouley Donna , Zychlinsky Arturo , Falkow Stanley
Published: July 2002

Salmonella typhimurium invades host macrophages and induces apoptosis and the release of mature proinflammatory cytokines. SipB, a protein translocated by Salmonella into the cytoplasm of macrophages, is required for activation of Caspase-1 (Casp-1, an interleukin [IL]-1β–converting enzyme), which is a member of a family of cysteine proteases that induce apoptosis in mammalian cells. Casp-1 is unique among caspases because it also directly cleaves the proinflammatory cytokines IL-1β and IL-18 to produce bioactive cytokines. We show here that mice lacking Casp-1 (casp-1−/− mice) had an oral S. typhimurium 50% lethal dose (LD50) that was 1,000-fold higher than that of wild-type mice. Salmonella breached the M cell barrier of casp-1−/− mice efficiently; however, there was a decrease in the number of apoptotic cells, intracellular bacteria, and the recruitment of polymorphonuclear lymphocytes in the Peyer's patches (PP) as compared with wild-type mice. Furthermore, Salmonella did not disseminate systemically in the majority of casp-1−/− mice, as demonstrated by significantly less colonization in the PP, mesenteric lymph nodes, and spleens of casp-1−/− mice after an oral dose of S. typhimurium that was 100-fold higher than the LD50. The increased resistance in casp-1−/− animals appears specific for Salmonella infection since these mice were susceptible to colonization by another enteric pathogen, Yersinia pseudotuberculosis, which normally invades the PP. These results show that Casp-1, which is both proapoptotic and proinflammatory, is essential for S. typhimurium to efficiently colonize the cecum and PP and subsequently cause systemic typhoid-like disease in mice.
Signal Transduction by Cxc Chemokine Receptor 4
Authors: Tilton Bettina , Ho Liza , Oberlin Estelle , Loetscher Pius , Baleux Françoise , Clark-Lewis Ian , Thelen Marcus
Published: July 2002

We report that stromal cell–derived factor (SDF)-1 has the remarkable capacity to induce sustained signaling through CXC chemokine receptor 4 (CXCR4). In contrast to other chemokines, such as monocyte chemotactic protein 1 (CC chemokine receptor 2 [CCR2]), macrophage inflammatory protein 1β (CCR5), liver and activation-regulated chemokine (LARC [CCR6]), Epstein-Barr virus–induced molecule 1 ligand chemokine (ELC [CCR7]), and IP10 (CXCR3), SDF-1 stimulates the prolonged activation of protein kinase B and extracellular signal–regulated kinase (ERK)-2. Activation of protein kinase B is reversed by displacement of SDF-1 from CXCR4 or inhibition of phosphatidylinositol 3-kinase. Although increasing concentrations of SDF-1 enhance CXCR4 internalization, kinase activation is prolonged. In addition, restimulation yields >60% of initial protein kinase B activity, indicating that the remaining receptors are not desensitized. Furthermore, activation is prolonged by inhibiting SDF-1 degradation. The sustained activation of cell survival and mitogenic pathways may account for the unique role of SDF-1 and CXCR4 in embryogenesis and lymphopoiesis.
Chemoattractants Induce a Rapid and Transient Upregulation of Monocyte α4 Integrin Affinity for Vascular Cell Adhesion Molecule 1 Which Mediates Arrest
Authors: Chan Jason R. , Hyduk Sharon J. , Cybulsky Myron I.
Published: July 2002

Chemoattractants and chemokines induce arrest of rolling monocytes during emigration from blood into tissues. In this study, we demonstrated that α4 integrin affinity for vascular cell adhesion molecule (VCAM)-1 was upregulated rapidly and transiently by chemoattractants and stromal cell–derived factor (SDF)-1α and mediated monocyte arrest. α4 integrin affinity changes were detected and blocked using soluble VCAM-1/Fc (sVCAM-1/Fc). In a flow cytometry assay, markedly increased sVCAM-1/Fc binding to human blood monocytes or U937 cells transfected with formyl peptide (FP) receptor was detected 30 s after FP or SDF-1α treatment and declined after 2 min. In a parallel plate flow chamber assay, FP, C5a, platelet-activating factor, or SDF-1α coimmobilized with VCAM-1 induced leukocyte arrest, which was blocked by inclusion of sVCAM-1/Fc but not soluble nonimmune immunoglobulin G in the assay buffer.
Glucocorticoids Attenuate T Cell Receptor Signaling
Authors: Van Laethem François , Baus Erika , Smyth Lesley A. , Andris Fabienne , Bex Françoise , Urbain Jacques , Kioussis Dimitris , Leo Oberdan
Published: July 2002

Glucocorticoids (GCs) affect peripheral immune responses by inhibiting T cell immunity at several stages of the activation cascade, causing impaired cytokine production and effector function. The recent demonstration that the thymic epithelium and possibly thymocytes themselves produce steroids suggests that endogenous GCs also play a role in the control of T cell development. As both peripheral responsiveness and thymic differentiation appear to be regulated by the quantity and quality of intracellular signals issued by antigen–major histocompatibility complex-engaged T cell receptor (TCR) complexes, we investigated the effects of GCs on the signaling properties of T cells stimulated by anti-CD3 monoclonal antibodies or agonist peptides. We demonstrate in this work that dexamethasone, a synthetic GC, inhibits the early signaling events initiated upon TCR ligation, such as tyrosine phosphorylation of several TCR-associated substrates including the ζ chain, the ZAP70 kinase, and the transmembrane adapter molecule linker for activation of T cells. Hypophosphorylation was not a consequence of reduced kinase activity of src protein tyrosine kinases, but was correlated with an altered- membrane compartmentalization of these molecules. These observations indicate that in addition to their well-described ability to interfere with the transcription of molecules involved in peripheral responses, GCs inhibit T cell activation by affecting the early phosphorylating events induced after TCR ligation.
The Adenylate Cyclase Toxin of Bordetella pertussis Binds to Target Cells via the αMβ2 Integrin (Cd11b/Cd18)
Authors: Guermonprez Pierre , Khelef Nadia , Blouin Eric , Rieu Philippe , Ricciardi-Castagnoli Paola , Guiso Nicole , Ladant Daniel , Leclerc Claude
Published: July 2002

The adenylate cyclase toxin (CyaA) of Bordetella pertussis is a major virulence factor required for the early phases of lung colonization. It can invade eukaryotic cells where, upon activation by endogenous calmodulin, it catalyzes the formation of unregulated cAMP levels. CyaA intoxication leads to evident toxic effects on macrophages and neutrophils. Here, we demonstrate that CyaA uses the αMβ2 integrin (CD11b/CD18) as a cell receptor. Indeed, the saturable binding of CyaA to the surface of various hematopoietic cell lines correlated with the presence of the αMβ2 integrin on these cells. Moreover, binding of CyaA to various murine cell lines and human neutrophils was specifically blocked by anti-CD11b monoclonal antibodies. The increase of intracellular cAMP level and cell death triggered by CyaA intoxication was also specifically blocked by anti-CD11b monoclonal antibodies. In addition, CyaA bound efficiently and triggered intracellular cAMP increase and cell death in Chinese hamster ovary cells transfected with αMβ2 (CD11b/CD18) but not in cells transfected with the vector alone or with the αXβ2 (CD11c/CD18) integrin. Thus, the cellular distribution of CD11b, mostly on neutrophils, macrophages, and dendritic and natural killer cells, supports a role for CyaA in disrupting the early, innate antibacterial immune response.
Phenotypic and Functional Analysis of Cd8+ T Cells Undergoing Peripheral Deletion in Response to Cross-Presentation of Self-Antigen
Authors: Hernandez Javier , Aung Sandra , Redmond William L. , Sherman Linda A.
Published: July 2002

Not all T cells specific for autoantigens are eliminated in the thymus, and therefore alternate mechanisms are required to prevent potentially autoreactive T cells from developing into effectors. Adoptive transfer of CD8+ T cells from influenza hemagglutinin-specific Clone 4 TCR transgenic mice into mice that express hemagluttinin in the pancreatic islets results in tolerance. This is preceded by activation of Clone 4 T cells that encounter antigen cross-presented in the draining lymph nodes of the pancreas. In this report we compare the phenotype, function, and costimulatory requirements of Clone 4 T cells activated by endogenous self-antigen, with Clone 4 T cells stimulated by influenza virus. The cells undergoing tolerance upregulate both CD69 and CD44, yet only partially downregulate CD62L, and do not express CD49d or CD25. Most importantly, they lack the ability to produce interferon-γ in response to antigen and show no cytolytic activity. Clone 4 T cells disappear after several cycles of division, apparently without leaving the site of initial activation. Surprisingly, despite the fact that such stimulation occurs through recognition of antigen that is cross-presented by a professional antigen-presenting cell, we find this activation is not dependent on costimulation through CD28. These data demonstrate that the recognition by naive CD8+ T cells of cross-presented self-antigen results in localized proliferation and deletion, without the production of effector cells.
Voltage- and Calcium-Dependent Inactivation of Calcium Channels in Lymnaea Neurons
Authors: Gera Shalini , Byerly Lou
Published: July 2002

Ca2+ channel inactivation in the neurons of the freshwater snail, Lymnaea stagnalis, was studied using patch-clamp techniques. In the presence of a high concentration of intracellular Ca2+ buffer (5 mM EGTA), the inactivation of these Ca2+ channels is entirely voltage dependent; it is not influenced by the identity of the permeant divalent ions or the amount of extracellular Ca2+ influx, or reduced by higher levels of intracellular Ca2+ buffering. Inactivation measured under these conditions, despite being independent of Ca2+ influx, has a bell-shaped voltage dependence, which has often been considered a hallmark of Ca2+-dependent inactivation. Ca2+-dependent inactivation does occur in Lymnaea neurons, when the concentration of the intracellular Ca2+ buffer is lowered to 0.1 mM EGTA. However, the magnitude of Ca2+-dependent inactivation does not increase linearly with Ca2+ influx, but saturates for relatively small amounts of Ca2+ influx. Recovery from inactivation at negative potentials is biexponential and has the same time constants in the presence of different intracellular concentrations of EGTA. However, the amplitude of the slow component is selectively enhanced by a decrease in intracellular EGTA, thus slowing the overall rate of recovery. The ability of 5 mM EGTA to completely suppress Ca2+-dependent inactivation suggests that the Ca2+ binding site is at some distance from the channel protein itself. No evidence was found of a role for serine/threonine phosphorylation in Ca2+ channel inactivation. Cytochalasin B, a microfilament disrupter, was found to greatly enhance the amount of Ca2+ channel inactivation, but the involvement of actin filaments in this effect of cytochalasin B on Ca2+ channel inactivation could not be verified using other pharmacological compounds. Thus, the mechanism of Ca2+-dependent inactivation in these neurons remains unknown, but appears to differ from those proposed for mammalian L-type Ca2+ channels.
Geodesic nets on the 2-sphere
Authors: Hass Joel , Morgan Frank
Published: July 2002

In this paper we introduce the concept of a geodesic net, an idea which plays the role among graphs that geodesics play among simple closed curves. We establish the existence of specific geodesic nets on the 2-sphere in certain cases.
Elements of provable high orders in finite fields
Authors: Gao Shuhong
Published: July 2002

A method is given for constructing elements in

F

q
n

\mathbb {F}_{q^n}

whose orders are larger than any polynomial in

n
n

when

n
n

becomes large. As a by-product a theorem on multiplicative independence of compositions of polynomials is proved.
Bilinear operators on Herz-type Hardy spaces
Authors: Grafakos Loukas , Li Xinwei , Yang Dachun
Published: July 2002

The authors prove that bilinear operators given by finite sums of products of Calderón-Zygmund operators on

R

n

\mathbb {R}^{n}

are bounded from

H

K
˙

q

1

α

1

,

p

1

×
H

K
˙

q

2

α

2

,

p

2

H\dot K_{q_{1}}^{\alpha _{1},p_{1}}\times H\dot K_{q_{2}}^{\alpha _{2},p_{2}}

into

H

K
˙

q

α
,
p

H\dot K_{q}^{\alpha ,p}

if and only if they have vanishing moments up to a certain order dictated by the target space. Here

H

K
˙

q

α
,
p

H\dot K_{q}^{\alpha ,p}

are homogeneous Herz-type Hardy spaces with

1

/

p
=
1

/

p

1

+
1

/

p

2

,

1/p=1/p_{1}+1/p_{2},

0
>

p

i

≤
∞
,

0>p_{i}\le \infty ,

1

/

q
=
1

/

q

1

+
1

/

q

2

,

1/q=1/q_{1}+1/q_{2},

1
>

q

1

,

q

2

>
∞
,

1>q_{1},q_{2}>\infty ,

1
≤
q
>
∞
,

1\le q>\infty ,

α
=

α

1

+

α

2

\alpha =\alpha _{1}+\alpha _{2}

and

−
n

/

q

i

>

α

i

>
∞

-n/q_{i}>\alpha _{i}>\infty

. As an application they obtain that the commutator of a Calderón-Zygmund operator with a BMO function maps a Herz space into itself.
Power integral bases in a parametric family of totally real cyclic quintics
Authors: Gaál István , Pohst Michael
Published: July 2002

We consider the totally real cyclic quintic fields

K

n

=

Q

(

ϑ

n

)

K_{n}=\mathbb {Q}(\vartheta _{n})

, generated by a root

ϑ

n

\vartheta _{n}

of the polynomial
<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" alttext="StartLayout 1st Row f Subscript n Baseline left-parenthesis x right-parenthesis equals x Superscript 5 Baseline plus n squared x Superscript 4 Baseline minus left-parenthesis 2 n cubed plus 6 n squared plus 10 n plus 10 right-parenthesis x cubed plus left-parenthesis n Superscript 4 Baseline plus 5 n cubed plus 11 n squared plus 15 n plus 5 right-parenthesis x squared plus left-parenthesis n cubed plus 4 n squared plus 10 n plus 10 right-parenthesis x plus 1 period EndLayout">

f

n

(
x
)
=

x

5

+

n

2

x

4

−
(
2

n

3

+
6

n

2

+
10
n
+
10
)

x

3

 
+
(

n

4

+
5

n

3

+
11

n

2

+
15
n
+
5
)

x

2

+
(

n

3

+
4

n

2

+
10
n
+
10
)
x
+
1.

\begin{multline*} f_{n}(x)=x^{5}+n^{2}x^{4}-(2n^{3}+6n^{2}+10n+10)x^{3}\ +(n^{4}+5n^{3}+11n^{2}+15n+5)x^{2}+(n^{3}+4n^{2}+10n+10)x+1. \end{multline*}

Assuming that

m
=

n

4

+
5

n

3

+
15

n

2

+
25
n
+
25

m=n^{4}+5n^{3}+15n^{2}+25n+25

is square free, we compute explicitly an integral basis and a set of fundamental units of

K

n

K_{n}

and prove that

K

n

K_{n}

has a power integral basis only for

n
=
−
1
,
−
2

n=-1,-2

. For

n
=
−
1
,
−
2

n=-1,-2

(both values presenting the same field) all generators of power integral bases are computed.
Analysis of non-overlapping domain decomposition algorithms with inexact solves
Authors: Bramble James , Pasciak Joseph , Vassilev Apostol
Published: July 2002

In this paper we construct and analyze new non-overlapping domain decomposition preconditioners for the solution of second-order elliptic and parabolic boundary value problems. The preconditioners are developed using uniform preconditioners on the subdomains instead of exact solves. They exhibit the same asymptotic condition number growth as the corresponding preconditioners with exact subdomain solves and are much more efficient computationally. Moreover, this asymptotic condition number growth is bounded independently of jumps in the operator coefficients across subdomain boundaries. We also show that our preconditioners fit into the additive Schwarz framework with appropriately chosen subspace decompositions. Condition numbers associated with the new algorithms are computed numerically in several cases and compared with those of the corresponding algorithms in which exact subdomain solves are used.
Error estimation of Hermite spectral method for nonlinear partial differential equations
Authors: Guo Ben-yu
Published: July 2002

Hermite approximation is investigated. Some inverse inequalities, imbedding inequalities and approximation results are obtained. A Hermite spectral scheme is constructed for Burgers equation. The stability and convergence of the proposed scheme are proved strictly. The techniques used in this paper are also applicable to other nonlinear problems in unbounded domains.
Gromov invariants for holomorphic maps from Riemann surfaces to Grassmannians
Authors: Bertram Aaron , Daskalopoulos Georgios , Wentworth Richard
Published: July 2002

Two compactifications of the space of holomorphic maps of fixed degree from a compact Riemann surface to a Grassmannian are studied. It is shown that the Uhlenbeck compactification has the structure of a projective variety and is dominated by the algebraic compactification coming from the Grothendieck Quot Scheme. The latter may be embedded into the moduli space of solutions to a generalized version of the vortex equations studied by Bradlow. This gives an effective way of computing certain intersection numbers (known as “Gromov invariants”) on the space of holomorphic maps into Grassmannians. We carry out these computations in the case where the Riemann surface has genus one.