bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of a CD36-related Thrombospondin 1–binding Domain in HIV-1 Envelope Glycoprotein gp120: Relationship to HIV-1–specific Inhibitory Factors in Human Saliva
Authors: Crombie René , Silverstein Roy L. , MacLow Clarinda , Pearce S. Frieda A. , Nachman Ralph L. , Laurence Jeffrey
Published: July 2002

Human and non–human primate salivas retard the infectivity of HIV-1 in vitro and in vivo. Because thrombospondin 1 (TSP1), a high molecular weight trimeric glycoprotein, is concentrated in saliva and can inhibit the infectivity of diverse pathogens in vitro, we sought to determine the role of TSP1 in suppression of HIV infectivity. Sequence analysis revealed a TSP1 recognition motif, previously defined for the CD36 gene family of cell adhesion receptors, in conserved regions flanking the disulfide-linked cysteine residues of the V3 loop of HIV envelope glycoprotein gp120, important for HIV binding to its high affinity cellular receptor CD4. Using solid-phase in vitro binding assays, we demonstrate direct binding of radiolabeled TSP1 to immobilized recombinant gp120. Based on peptide blocking experiments, the TSP1–gp120 interaction involves CSVTCG sequences in the type 1 properdin-like repeats of TSP1, the known binding site for CD36. TSP1 and fusion proteins derived from CD36-related TSP1-binding domains were able to compete with radiolabeled soluble CD4 binding to immobilized gp120. In parallel, purified TSP1 inhibited HIV-1 infection of peripheral blood mononuclear cells and transformed T and promonocytic cell lines. Levels of TSP1 required for both viral aggregation and direct blockade of HIV-1 infection were physiologic, and affinity depletion of salivary TSP1 abrogated >70% of the inhibitory effect of whole saliva on HIV infectivity. Characterization of TSP1–gp120 binding specificity suggests a mechanism for direct blockade of HIV infectivity that might be exploited to retard HIV transmission that occurs via mucosal routes.
Early Regeneration of Thymic Progenitors in Rhesus Macaques Infected with Simian Immunodeficiency Virus
Authors: Wykrzykowska Joanna J. , Rosenzweig Michael , Veazey Ronald S. , Simon Meredith A. , Halvorsen Katherine , Desrosiers Ronald C. , Johnson R. Paul , Lackner Andrew A.
Published: July 2002

The thymus plays a critical role in the maturation and production of T lymphocytes and is a target of infection by human immunodeficiency virus (HIV) and the related simian immunodeficiency virus (SIV). Using the SIV/macaque model of AIDS, we examined the early effects of SIV on the thymus. We found that thymic infection by SIV resulted in increased apoptosis 7–14 d after infection, followed by depletion of thymocyte progenitors by day 21. A marked rebound in thymocyte progenitors occurred by day 50 and was accompanied by increased levels of cell proliferation in the thymus. Our results demonstrate a marked increase in thymic progenitor activity very early in the course of SIV infection, long before marked declines in peripheral CD4+ T cell counts.
Analysis of Gag-specific Cytotoxic T Lymphocytes in Simian Immunodeficiency Virus–infected Rhesus Monkeys by Cell Staining with a Tetrameric Major Histocompatibility Complex Class I–Peptide Complex
Authors: Kuroda Marcelo J. , Schmitz Jörn E. , Barouch Dan H. , Craiu Abie , Allen Todd M. , Sette Alessandro , Watkins David I. , Forman Meryl A. , Letvin Norman L.
Published: July 2002

A tetrameric recombinant major histocompatibility complex (MHC) class I–peptide complex was used as a staining reagent in flow cytometric analyses to quantitate and define the phenotype of Gag-specific cytotoxic T lymphocytes (CTLs) in the peripheral blood of simian immunodeficiency virus macaque (SIVmac)-infected rhesus monkeys. The heavy chain of the rhesus monkey MHC class I molecule Mamu-A*01 and β2-microglobulin were refolded in the presence of an SIVmac Gag synthetic peptide (p11C, C–M) representing the optimal nine–amino acid peptide of Mamu-A*01–restricted predominant CTL epitope to create a tetrameric Mamu-A*01/p11C, C–M complex. Tetrameric Mamu-A*01/p11C, C–M complex bound to T cells of SIVmac-infected, Mamu-A*01+, but not uninfected, Mamu-A*01+, or infected, Mamu-A*01− rhesus monkeys. Specific staining of peripheral blood mononuclear cells (PBMC) from SIVmac-infected, Mamu-A*01+ rhesus monkeys was only found in the cluster of differentiation (CD)8α/β+ T lymphocyte subset and the percentage of CD8α/β+ T cells in the peripheral blood of four SIVmac-infected, Mamu-A*01+ rhesus monkeys staining with this complex ranged from 0.7 to 10.3%. Importantly, functional SIVmac Gag p11C-specific CTL activity was seen in sorted and expanded tetrameric Mamu-A*01/p11C, C–M complex–binding, but not nonbinding, CD8α/β+ T cells. Furthermore, the percentage of CD8α/β+ T cells binding this tetrameric Mamu-A*01/p11C, C–M complex correlated well with p11C-specific cytotoxic activity as measured in both bulk and limiting dilution effector frequency assays. Finally, phenotypic characterization of the cells binding this tetrameric complex indicated that this lymphocyte population is heterogeneous. These studies indicate the power of this approach for examining virus-specific CTLs in in vivo settings.
Yersinia-induced Apoptosis In Vivo Aids in the Establishment of a Systemic Infection of Mice
Authors: Monack Denise M. , Mecsas Joan , Bouley Donna , Falkow Stanley
Published: July 2002

Pathogenic Yersinia cause a systemic infection in mice that is dependent on the presence of a large plasmid encoding a number of secreted virulence proteins called Yops. We previously demonstrated that a plasmid-encoded Yop, YopJ, was essential for inducing apoptosis in cultured macrophages. Here we report that YopJ is a virulence factor in mice and is important for the establishment of a systemic infection. The oral LD50 for a yopJ mutant Yersinia pseudotuberculosis increases 64-fold compared with wild-type. Although the yopJ mutant strain is able to reach the spleen of infected mice, the mutant strain seldom reaches the same high bacterial load that is seen with wild-type Yersinia strain and begins to be cleared from infected spleens on day 4 after infection. Furthermore, when in competition with wild-type Yersinia in a mixed infection, the yopJ mutant strain is deficient for spread from the Peyer's patches to other lymphoid tissue. We also show that wild-type Yersinia induces apoptosis in vivo of Mac-1+ cells from infected mesenteric lymph nodes or spleens, as measured by quantitative flow cytometry of TUNEL (Tdt-mediated dUTP–biotin nick-end labeling)-positive cells. The levels of Mac-1+, TUNEL+ cells from tissue infected with the yopJ mutant strain were equivalent to the levels detected in cells from uninfected tissue. YopJ is necessary for the suppression of TNF-α production seen in macrophages infected with wild-type Yersinia, based on previous in vitro studies (Palmer, L.E., S. Hobbie, J.E. Galan, and J.B. Bliska. 1998. Mol. Microbiol. 27:953–965). We conclude here that YopJ plays a role in the establishment of a systemic infection by inducing apoptosis and that this is consistent with the ability to suppress the production of the proinflammatory cytokine tumor necrosis factor α.
A Signal Transducer and Activator of  Transcription (Stat)4-independent Pathway for the Development of  T Helper Type 1 Cells
Authors: Kaplan Mark H. , Wurster Andrea L. , Grusby Michael J.
Published: July 2002

The differentiation of T helper (Th) cells is regulated by members of the signal transducer and activator of transcription (STAT) family of signaling molecules. We have generated mice lacking both Stat4 and Stat6 to examine the ability of Th cells to develop in the absence of these two transcription factors. Stat4, Stat6−/− lymphocytes fail to differentiate into interleukin (IL)-4–secreting Th2 cells. However, in contrast to Stat4−/− lymphocytes, T cells from Stat4, Stat6−/− mice produce significant amounts of interferon (IFN)-γ when activated in vitro. Although Stat4, Stat6−/− lymphocytes produce less IFN-γ than IL-12–stimulated control lymphocytes, equivalent numbers of IFN-γ–secreting cells can be generated from cultures of Stat4, Stat6−/− lymphocytes activated under neutral conditions and control lymphocytes activated under Th1 cell–promoting conditions. Moreover, Stat4, Stat6−/− mice are able to mount an in vivo Th1 cell–mediated delayed-type hypersensitivity response. These results support a model of Th cell differentiation in which the generation of Th2 cells requires Stat6, whereas a Stat4-independent pathway exists for the development of Th1 cells.
In Vivo Survival of  Viral Antigen–specific T Cells that Induce Experimental Autoimmune Encephalomyelitis
Authors: Ufret-Vincenty Rafael L. , Quigley Laura , Tresser Nancy , Pak Seong Hee , Gado Ameer , Hausmann Stefan , Wucherpfennig Kai W. , Brocke Stefan
Published: July 2002

A peptide derived from the human papillomavirus L2 protein is recognized by a myelin basic protein (MBP)-specific T cell clone from a multiple sclerosis patient and by MBP-specific autoantibodies purified from multiple sclerosis brain tissue. We now show in mice that low doses of this papillomavirus peptide were optimal in selecting a subpopulation of papillomavirus peptide–specific T cells that cross-reacted with MBP(87–99) and with an unrelated viral peptide derived from the BSLF1 protein of Epstein-Barr virus (EBV). These low dose viral peptide– specific T cell lines were highly encephalitogenic. Splenocytes from mice transferred with viral peptide–specific T cells showed a vigorous response to both the papillomavirus and MBP peptides, indicating that viral antigen–specific T cells survived for a prolonged time in vivo. The EBV peptide, unable to prime and select an autoreactive T cell population, could still activate the low dose papillomavirus peptide–specific cells and induce central nervous system (CNS) autoimmunity. Cytokine profiles of papillomavirus peptide–specific encephalitogenic T cells and histopathology of CNS lesions resembled those induced by MBP. These results demonstrate conserved aspects in the recognition of the self-antigen and a cross-reactive viral peptide by human and murine MBP-specific T cell receptors. We demonstrate that a viral antigen, depending on its nature, dose, and number of exposures, may select autoantigen-specific T cells that survive in vivo and can trigger autoimmune disease after adoptive transfer.
Modulation of Immune Complex–induced Inflammation In Vivo by the Coordinate Expression of Activation and Inhibitory Fc Receptors
Authors: Clynes Raphael , Maizes Jay S. , Guinamard Rodolphe , Ono Masao , Takai Toshiyuki , Ravetch Jeffrey V.
Published: July 2002

Autoantibodies and immune complexes are major pathogenic factors in autoimmune injury, responsible for initiation of the inflammatory cascade and its resulting tissue damage. This activation results from the interaction of immunoglobulin (Ig)G Fc receptors containing an activation motif (ITAM) with immune complexes (ICs) and cytotoxic autoantibodies which initiates and propagates an inflammatory response. In vitro, this pathway can be interrupted by coligation to FcγRIIB, an IgG Fc receptor containing an inhibitory motif (ITIM). In this report, we describe the in vivo consequences of FcγRII deficiency in the inflammatory response using a mouse model of IC alveolitis. At subthreshold concentrations of ICs that fail to elicit inflammatory responses in wild-type mice, FcγRII-deficient mice developed robust inflammatory responses characterized by increased hemorrhage, edema, and neutrophil infiltration. Bronchoalveolar fluids from FcγRII−/− stimulated mice contain higher levels of tumor necrosis factor and chemotactic activity, suggesting that FcγRII deficiency lowers the threshold of IC stimulation of resident cells such as the alveolar macrophage. In contrast, complement- and complement receptor–deficient mice develop normal inflammatory responses to suprathreshold levels of ICs, while FcRγ−/− mice are completely protected from inflammatory injury. An inhibitory role for FcγRII on macrophages is demonstrated by analysis of FcγRII−/− macrophages which show greater phagocytic and calcium flux responses upon FcγRIII engagement. These data reveal contrasting roles for the cellular receptors for IgG on inflammatory cells, providing a regulatory mechanism for setting thresholds for IC sensitivity based on the ratio of ITIM to ITAM FcγR expression. Exploiting the FcγRII inhibitory pathway could thus provide a new therapeutic approach for modulating antibody-triggered inflammation.
Coupling and Uncoupling of Tumor Immunity and Autoimmunity
Authors: Bowne Wilbur B. , Srinivasan Roopa , Wolchok Jedd D. , Hawkins William G. , Blachere Nathalie E. , Dyall Ruben , Lewis Jonathan J. , Houghton Alan N.
Published: July 2002

Self-antigens, in the form of differentiation antigens, are commonly recognized by the immune system on melanoma and other cancers. We have shown previously that active immunization of mice against the melanocyte differentiation antigen, a tyrosinase-related protein (TRP) gp75TRP-1 (the brown locus protein) expressed by melanomas, could induce tumor immunity and autoimmunity manifested as depigmentation. In this system, tumor immunity and autoimmunity were mediated by autoantibodies. Here, we characterize immunity against another tyrosinase family glycoprotein TRP-2 (the slaty locus protein), using the same mouse model and method of immunization. As observed previously for gp75TRP-1, immunity was induced by DNA immunization against a xenogeneic form of TRP-2, but not against the syngeneic gene, and depended on CD4+ cells. Immunization against TRP-2 induced autoantibodies and autoreactive cytotoxic T cells. In contrast to immunization against gp75TRP-1, both tumor immunity and autoimmunity required CD8+ T cells, but not antibodies. Only autoimmunity required perforin, whereas tumor immunity proceeded in the absence of perforin. Thus, immunity induced against two closely related autoantigens that are highly conserved throughout vertebrate evolution involved qualitatively different mechanisms, i.e., antibody versus CD8+ T cell. However, both pathways led to tumor immunity and identical phenotypic manifestations of autoimmunity.
Assessment of Caspase Activities in Intact Apoptotic Thymocytes Using Cell-Permeable Fluorogenic Caspase Substrates
Authors: Komoriya Akira , Packard Beverly Z. , Brown Martin J. , Wu Ming-Lei , Henkart Pierre A.
Published: July 2002

To detect caspase activities in intact apoptotic cells at the single cell level, cell-permeable fluorogenic caspase substrates were synthesized incorporating the optimal peptide recognition motifs for caspases 1, 3/7, 6, 8, and 9. Caspase activities were then assessed at various times after in vitro treatment of mouse thymocytes with dexamethasone or anti-Fas antibody. Dexamethasone induced the following order of appearance of caspase activities as judged by flow cytometry: LEHDase, WEHDase, VEIDase, IETDase, and DEVDase. Since the relative order of caspases 3 (DEVDase) and 6 (VEIDase) in the cascade has been controversial, this caspase activation order was reexamined using confocal microscopy. The VEIDase activity appeared before DEVDase in every apoptotic cell treated with dexamethasone. In contrast, anti-Fas stimulation altered this sequence: IETDase was the first measurable caspase activity and DEVDase preceded VEIDase. In an attempt to determine the intracellular target of the potent antiapoptotic agent carbobenzoxy-valyl-alanyl-aspartyl(β-methyl ester)-fluoromethyl ketone (Z-VAD[OMe]-FMK), we examined its ability to inhibit previously activated intracellular caspases. However, no significant reductions of these activities were observed. These fluorogenic caspase substrates allow direct observation of the caspase cascade in intact apoptotic cells, showing that the order of downstream caspase activation is dependent on the apoptotic stimulus.
Role for Cathepsin F in Invariant Chain Processing and Major Histocompatibility Complex Class II Peptide Loading by Macrophages
Authors: Shi Guo-Ping , Bryant Rebecca A.R. , Riese Richard , Verhelst Steven , Driessen Christoph , Li Zhenqiang , Bromme Dieter , Ploegh Hidde L. , Chapman Harold A.
Published: July 2002

The major histocompatibility complex (MHC) class II–associated invariant chain (Ii) regulates intracellular trafficking and peptide loading of MHC class II molecules. Such loading occurs after endosomal degradation of the invariant chain to a ∼3-kD peptide termed CLIP (class II–associated invariant chain peptide). Cathepsins L and S have both been implicated in degradation of Ii to CLIP in thymus and peripheral lymphoid organs, respectively. However, macrophages from mice deficient in both cathepsins S and L can process Ii and load peptides onto MHC class II dimers normally. Both processes are blocked by a cysteine protease inhibitor, indicating the involvement of an additional Ii-processing enzyme(s). Comparison of cysteine proteases expressed by macrophages with those found in splenocytes and dendritic cells revealed two enzymes expressed exclusively in macrophages, cathepsins Z and F. Recombinant cathepsin Z did not generate CLIP from Ii–MHC class II complexes, whereas cathepsin F was as efficient as cathepsin S in CLIP generation. Inhibition of cathepsin F activity and MHC class II peptide loading by macrophages exhibited similar specificity and activity profiles. These experiments show that cathepsin F, in a subset of antigen presenting cells (APCs), can efficiently degrade Ii. Different APCs can thus use distinct proteases to mediate MHC class II maturation and peptide loading.
Visualization of Myelin Basic Protein (Mbp) T Cell Epitopes in Multiple Sclerosis Lesions Using a Monoclonal Antibody Specific for the Human Histocompatibility Leukocyte Antigen (Hla)-Dr2–Mbp 85–99 Complex
Authors: Krogsgaard Michelle , Wucherpfennig Kai W. , Canella Barbara , Hansen Bjarke E. , Svejgaard Arne , Pyrdol Jason , Ditzel Henrik , Raine Cedric , Engberg Jan , Fugger Lars
Published: July 2002

Susceptibility to multiple sclerosis (MS) is associated with the human histocompatibility leukocyte antigen (HLA)-DR2 haplotype, suggesting that major histocompatibility complex class II–restricted presentation of central nervous system–derived antigens is important in the disease process. Antibodies specific for defined HLA-DR2–peptide complexes may therefore be valuable tools for studying antigen presentation in MS. We have used phage display technology to select HLA-DR2–peptide-specific antibodies from HLA-DR2–transgenic mice immunized with HLA-DR2 molecules complexed with an immunodominant myelin basic protein (MBP) peptide (residues 85–99). Detailed characterization of one clone (MK16) demonstrated that both DR2 and the MBP peptide were required for recognition. Furthermore, MK16 labeled intra- and extracellular HLA-DR2–MBP peptide complexes when antigen-presenting cells (APCs) were pulsed with recombinant MBP. In addition, MK16 inhibited interleukin 2 secretion by two transfectants that expressed human MBP–specific T cell receptors. Analysis of the structural requirement for MK16 binding demonstrated that the two major HLA-DR2 anchor residues of MBP 85–99 and the COOH-terminal part of the peptide, in particular residues Val-96, Pro-98, and Arg-99, were important for binding. Based on these results, the antibody was used to determine if the HLA-DR2–MBP peptide complex is presented in MS lesions. The antibody stained APCs in MS lesions, in particular microglia/macrophages but also in some cases hypertrophic astrocytes. Staining of APCs was only observed in MS cases with the HLA-DR2 haplotype but not in cases that carried other haplotypes. These results demonstrate that HLA-DR2 molecules in MS lesions present a myelin-derived self-peptide and suggest that microglia/macrophages rather than astrocytes are the predominant APCs in these lesions.
Interleukin 8 Receptor Deficiency Confers Susceptibility to Acute Experimental Pyelonephritis and May Have a Human Counterpart
Authors: Frendéus Björn , Godaly Gabriela , Hang Long , Karpman Diana , Lundstedt Ann-Charlotte , Svanborg Catharina
Published: July 2002

Neutrophils migrate to infected mucosal sites that they protect against invading pathogens. Their interaction with the epithelial barrier is controlled by CXC chemokines and by their receptors. This study examined the change in susceptibility to urinary tract infection (UTI) after deletion of the murine interleukin 8 receptor homologue (mIL-8Rh). Experimental UTIs in control mice stimulated an epithelial chemokine response and increased chemokine receptor expression. Neutrophils migrated through the tissues to the epithelial barrier that they crossed into the lumen, and the mice developed pyuria. In mIL-8Rh knockout (KO) mice, the chemokine response was intact, but the epithelial cells failed to express IL-8R, and neutrophils accumulated in the tissues. The KO mice were unable to clear bacteria from kidneys and bladders and developed bacteremia and symptoms of systemic disease, but control mice were fully resistant to infection. The experimental UTI model demonstrated that IL-8R–dependent mechanisms control the urinary tract defense, and that neutrophils are essential host effector cells. Patients prone to acute pyelonephritis also showed low CXC chemokine receptor 1 expression compared with age-matched controls, suggesting that chemokine receptor expression may also influence the susceptibility to UTIs in humans. The results provide a first molecular clue to disease susceptibility of patients prone to acute pyelonephritis.
Role of the Parasite-Derived Prostaglandin D2 in the Inhibition of Epidermal Langerhans Cell Migration during Schistosomiasis Infection
Authors: Angeli Véronique , Faveeuw Christelle , Roye Olivier , Fontaine Josette , Teissier Elisabeth , Capron André , Wolowczuk Isabelle , Capron Monique , Trottein François
Published: July 2002

Epidermal Langerhans cells (LCs) play a key role in immune defense mechanisms and in numerous immunological disorders. In this report, we show that percutaneous infection of C57BL/6 mice with the helminth parasite Schistosoma mansoni leads to the activation of LCs but, surprisingly, to their retention in the epidermis. Moreover, using an experimental model of LC migration induced by tumor necrosis factor (TNF)-α, we show that parasites transiently impair the departure of LCs from the epidermis and their subsequent accumulation as dendritic cells in the draining lymph nodes. The inhibitory effect is mediated by soluble lipophilic factors released by the parasites and not by host-derived antiinflammatory cytokines, such as interleukin-10. We find that prostaglandin (PG)D2, but not the other major eicosanoids produced by the parasites, specifically impedes the TNF-α–triggered migration of LCs through the adenylate cyclase–coupled PGD2 receptor (DP receptor). Moreover, the potent DP receptor antagonist BW A868C restores LC migration in infected mice. Finally, in a model of contact allergen-induced LC migration, we show that activation of the DP receptor not only inhibits LC emigration but also dramatically reduces the contact hypersensitivity responses after challenge. Taken together, we propose that the inhibition of LC migration could represent an additional stratagem for the schistosomes to escape the host immune system and that PGD2 may play a key role in the control of cutaneous immune responses.
Shear-dependent Eosinophil Transmigration on Interleukin 4–stimulated Endothelial Cells
Authors: Cuvelier Susan L. , Patel Kamala D.
Published: July 2002

Leukocyte infiltration into inflammatory sites is regulated by the expression of adhesion and activation proteins, yet the role of these proteins in shear-dependent transmigration is poorly understood. We examined eosinophil recruitment on cytokine-stimulated human umbilical vein endothelial cells (HUVECs) under laminar flow conditions. Eosinophils rapidly transmigrated on interleukin (IL)-4–, but not TNF-stimulated HUVECs. Transmigration was shear dependent, with up to 90% of eosinophils transmigrating in the presence of shear and less than 25% of cells transmigrating under static conditions. Eosinophils express CC chemokine receptor CCR3 and are responsive to various CC chemokines. The effects of chemokines are mediated primarily through Gαi, which is pertussis toxin sensitive. Greater than 65% of shear-dependent eosinophil transmigration on IL-4–stimulated HUVECs was blocked by either pertussis toxin or by an anti-CCR3 monoclonal antibody. Using reverse transcription polymerase chain reaction (RT-PCR) and Western blots, we found that IL-4–stimulated HUVECs produce both mRNA and protein for eotaxin-3. Eotaxin-3 was both released by HUVECs and expressed on the endothelial cell surface. Pretreatment of HUVECs with an anti–eotaxin-3 antibody blocked eosinophil transmigration to the same extent as an anti-CCR3 antibody. These results indicate that IL-4–stimulated HUVECs support shear-dependent eosinophil transmigration by upregulating eotaxin-3, and that surface association is critical for the role of eotaxin-3 in transmigration.
αβ T Cell Receptor-positive Cells and Interferon-γ, but not Inducible Nitric Oxide Synthase, Are Critical for Granuloma Necrosis in a Mouse Model of Mycobacteria-induced Pulmonary Immunopathology
Authors: Ehlers Stefan , Benini Jochen , Held Heinz-Dieter , Roeck Christiane , Alber Gottfried , Uhlig Stefan
Published: July 2002

The immunological basis of tuberculin-induced necrosis, known for more than a century as “Koch's phenomenon,” remains poorly understood. Aerosol infection in mice with the highly virulent Mycobacterium avium strain TMC724 causes progressive pulmonary pathology strongly resembling caseating necrosis in human patients with tuberculosis. To identify the cellular and molecular mediators causing this pathology, we infected C57BL/6 mice and mice selectively deficient in recombinase activating gene (RAG)-1, αβ T cell receptor (TCR), γδ TCR, CD4, CD8, β2-microglobulin, interferon (IFN)-γ, interleukin (IL)-10, IL-12p35, IL-12p35/p40, or iNOS with M. avium by aerosol and compared bacterial multiplication, histopathology, and respiratory physiology in these mice. The bacterial load in the lung was similarly high in all mouse groups. Pulmonary compliance, as a surrogate marker for granulomatous infiltrations in the lung, deteriorated to a similar extent in all groups of mice, except in αβ TCR-knockout (KO) and IL-12–KO mice in which compliance was higher, and in IFN-γ and inducible nitric oxide synthase–KO mice in which compliance was reduced faster. Progressive caseation of pulmonary granulomas never occurred in αβ TCR-KO, IL-12–KO, and IFN-γ–KO mice and was reduced in CD4-KO mice. In summary, αβ TCR+ cells and IFN-γ are essential for the development of mycobacteria-induced pulmonary caseous necrosis. In contrast, high mycobacterial load and extensive granulomatous infiltration per se are not sufficient to cause caseation, nor is granuloma necrosis linked to the induction of nitric oxide.
HERG-like K+ Channels in Microglia
Authors: Zhou Wei , Cayabyab Francisco S. , Pennefather Peter S. , Schlichter Lyanne C. , DeCoursey Thomas E.
Published: July 2002

A voltage-gated K+ conductance resembling that of the human ether-à-go-go-related gene product (HERG) was studied using whole-cell voltage-clamp recording, and found to be the predominant conductance at hyperpolarized potentials in a cell line (MLS-9) derived from primary cultures of rat microglia. Its behavior differed markedly from the classical inward rectifier K+ currents described previously in microglia, but closely resembled HERG currents in cardiac muscle and neuronal tissue. The HERG-like channels opened rapidly on hyperpolarization from 0 mV, and then decayed slowly into an absorbing closed state. The peak K+ conductance–voltage relation was half maximal at −59 mV with a slope factor of 18.6 mV. Availability, assessed by a hyperpolarizing test pulse from different holding potentials, was more steeply voltage dependent, and the midpoint was more positive (−14 vs. −39 mV) when determined by making the holding potential progressively more positive than more negative. The origin of this hysteresis is explored in a companion paper (Pennefather, P.S., W. Zhou, and T.E. DeCoursey. 1998. J. Gen. Physiol. 111:795–805). The pharmacological profile of the current differed from classical inward rectifier but closely resembled HERG. Block by Cs+ or Ba2+ occurred only at millimolar concentrations, La3+ blocked with Ki = ∼40 μM, and the HERG-selective blocker, E-4031, blocked with Ki = 37 nM. Implications of the presence of HERG-like K+ channels for the ontogeny of microglia are discussed.
The β Subunit Increases the Ca2+ Sensitivity of Large Conductance Ca2+-activated Potassium Channels by Retaining the Gating in the Bursting States
Authors: Nimigean Crina M. , Magleby Karl L.
Published: July 2002

Coexpression of the β subunit (KV,Caβ) with the α subunit of mammalian large conductance Ca2+- activated K+ (BK) channels greatly increases the apparent Ca2+ sensitivity of the channel. Using single-channel analysis to investigate the mechanism for this increase, we found that the β subunit increased open probability (Po) by increasing burst duration 20–100-fold, while having little effect on the durations of the gaps (closed intervals) between bursts or on the numbers of detected open and closed states entered during gating. The effect of the β subunit was not equivalent to raising intracellular Ca2+ in the absence of the beta subunit, suggesting that the β subunit does not act by increasing all the Ca2+ binding rates proportionally. The β subunit also inhibited transitions to subconductance levels. It is the retention of the BK channel in the bursting states by the β subunit that increases the apparent Ca2+ sensitivity of the channel. In the presence of the β subunit, each burst of openings is greatly amplified in duration through increases in both the numbers of openings per burst and in the mean open times. Native BK channels from cultured rat skeletal muscle were found to have bursting kinetics similar to channels expressed from alpha subunits alone.
Insulin Stimulates Transepithelial Sodium Transport by Activation of a Protein Phosphatase That Increases Na-K Atpase Activity in Endometrial Epithelial Cells
Authors: Deachapunya Chatsri , Palmer-Densmore Melissa , O'Grady Scott M.
Published: July 2002

The objective of this study was to investigate the effects of insulin and insulin-like growth factor I on transepithelial Na+ transport across porcine glandular endometrial epithelial cells grown in primary culture. Insulin and insulin-like growth factor I acutely stimulated Na+ transport two- to threefold by increasing Na+-K+ ATPase transport activity and basolateral membrane K+ conductance without increasing the apical membrane amiloride-sensitive Na+ conductance. Long-term exposure to insulin for 4 d resulted in enhanced Na+ absorption with a further increase in Na+-K+ ATPase transport activity and an increase in apical membrane amiloride-sensitive Na+ conductance. The effect of insulin on the Na+-K+ ATPase was the result of an increase in Vmax for extracellular K+ and intracellular Na+, and an increase in affinity of the pump for Na+. Immunohistochemical localization along with Western blot analysis of cultured porcine endometrial epithelial cells revealed the presence of α-1 and α-2 isoforms, but not the α-3 isoform of Na+-K+ ATPase, which did not change in the presence of insulin. Insulin-stimulated Na+ transport was inhibited by hydroxy-2-naphthalenylmethylphosphonic acid tris-acetoxymethyl ester [HNMPA-(AM)3], a specific inhibitor of insulin receptor tyrosine kinase activity, suggesting that the regulation of Na+ transport by insulin involves receptor autophosphorylation. Pretreatment with wortmannin, a specific inhibitor of phosphatidylinositol 3–kinase as well as okadaic acid and calyculin A, inhibitors of protein phosphatase activity, also blocked the insulin-stimulated increase in short circuit and pump currents, suggesting that activation of phosphatidylinositol 3–kinase and subsequent stimulation of a protein phosphatase mediates the action of insulin on Na+-K+ ATPase activation.
Ion–Ion Interactions at the Selectivity Filter
Authors: Immke David , Korn Stephen J.
Published: July 2002

In the Kv2.1 potassium channel, binding of K+ to a high-affinity site associated with the selectivity filter modulates channel sensitivity to external TEA. In channels carrying Na+ current, K+ interacts with the TEA modulation site at concentrations ≤30 μM. In this paper, we further characterized the TEA modulation site and examined how varying K+ occupancy of the pore influenced the interaction of K+ with this site. In the presence of high internal and external [K+], TEA blocked 100% of current with an IC50 of 1.9 ± 0.2 mM. In the absence of a substitute permeating ion, such as Na+, reducing access of K+ to the pore resulted in a reduction of TEA efficacy, but produced little or no change in TEA potency (under conditions in which maximal block by TEA was just 32%, the IC50 for block was 2.0 ± 0.6 mM). The all-or-none nature of TEA block (channels were either completely sensitive or completely insensitive), indicated that one selectivity filter binding site must be occupied for TEA sensitivity, and that one selectivity filter binding site is not involved in modulating TEA sensitivity. At three different levels of K+ occupancy, achieved by manipulating access of internal K+ to the pore, elevation of external [K+] shifted channels from a TEA-insensitive to -sensitive state with an EC50 of ∼10 mM. Combined with previous results, these data demonstrate that the TEA modulation site has a high affinity for K+ when only one K+ is in the pore and a low affinity for K+ when the pore is already occupied by K+. These results indicate that ion–ion interactions occur at the selectivity filter. These results also suggest that the selectivity filter is the site of at least one low affinity modulatory effect of external K+, and that the selectivity filter K+ binding sites are not functionally interchangeable.
Kinetic, Mechanistic, and Structural Aspects of Unliganded Gating of Acetylcholine Receptor Channels
Authors: Grosman Claudio , Auerbach Anthony
Published: July 2002

The spontaneous activity of adult mouse muscle acetylcholine receptor channels, transiently expressed in HEK-293 cells, was studied with the patch-clamp technique. To increase the frequency of unliganded openings, mutations at the 12′ position of the second transmembrane segment were engineered. Our results indicate that: (a) in both wild type and mutants, a C ↔ O kinetic scheme provides a good description of spontaneous gating. In the case of some mutant constructs, however, additional states were needed to improve the fit to the data. Similar additional states were also needed in one of six patches containing wild-type acetylcholine receptor channels; (b) the δ12′ residue makes a more pronounced contribution to unliganded gating than the homologous residues of the α, β, and ε subunits; (c) combinations of second transmembrane segment 12′ mutations in the four different subunits appear to have cumulative effects; (d) the volume of the side chain at δ12′ is relevant because residues larger than the wild-type Ser increase spontaneous gating; (e) the voltage dependence of the unliganded gating equilibrium constant is the same as that of diliganded gating, but the voltage dependences of the opening and closing rate constants are opposite (this indicates that the reaction pathway connecting the closed and open states of the receptor changes upon ligation); (f) engineering binding-site mutations that decrease diliganded gating (αY93F, αY190W, and αD200N) reduces spontaneous activity as well (this suggests that even in the absence of ligand the opening of the channel is accompanied by a conformational change at the binding sites); and (g) the diliganded gating equilibrium constant is also increased by the 12′ mutations. Such increase is independent of the particular ligand used as the agonist, which suggests that these mutations affect mostly the isomerization step, having little, if any, effect on the ligand-affinity ratio.
Structural Determinants of Pip2 Regulation of Inward Rectifier KATP Channels
Authors: Shyng Show-Ling , Cukras Catherine A. , Harwood Jane , Nichols Colin G.
Published: July 2002

Phosphatidylinositol 4,5-bisphosphate (PIP2) activates KATP and other inward rectifier (Kir) channels. To determine residues important for PIP2 regulation, we have systematically mutated each positive charge in the COOH terminus of Kir6.2 to alanine. The effects of these mutations on channel function were examined using 86Rb efflux assays on intact cells and inside-out patch-clamp methods. Both methods identify essentially the same basic residues in two narrow regions (176–222 and 301–314) in the COOH terminus that are important for the maintenance of channel function and interaction with PIP2. Only one residue (R201A) simultaneously affected ATP and PIP2 sensitivity, which is consistent with the notion that these ligands, while functionally competitive, are unlikely to bind to identical sites. Strikingly, none of 13 basic residues in the terminal portion (residues 315–390) of the COOH terminus affected channel function when neutralized. The data help to define the structural requirements for PIP2 sensitivity of KATP channels. Moreover, the regions and residues defined in this study parallel those uncovered in recent studies of PIP2 sensitivity in other inward rectifier channels, indicating a common structural basis for PIP2 regulation.
Extinction of species in nonautonomous Lotka-Volterra systems
Authors: Ahmad Shair
Published: July 2002

A nonautonomous

n
n

th order Lotka-Volterra system of differential equations is considered. It is shown that if the coefficients satisfy certain inequalities, then any solution with positive components at some point will have all of its last

n
−
1

n-1

components tend to zero, while the first one will stabilize at a certain solution of a logistic equation.
Semi-classical limit of wave functions
Authors: Truman A. , Zhao H.
Published: July 2002

We study in one dimension the semi-classical limit of the exact eigenfunctionΨE(N,h)h\Psi _{E(N,h)}^{h}of the HamiltonianH=−12h2Δ+V(x)H=-\frac {1}{2} h^{2} \Delta +V(x), for a potentialVVbeing analytic, bounded below andlim|x|→∞V(x)=+∞\lim _{|x|\to \infty }V(x)=+\infty. The main result of this paper is that, for any givenE>minx∈R1V(x)E>\min _{x\in R^{1}} V(x)with two turning points, the exactL2L^{2}normalized eigenfunction|ΨE(N,h)h(q)|2|\Psi ^{h}_{E(N,h)}(q)|^{2}converges to the classical probability density, and the momentum distribution|Ψ^E(N,h)h(p)|2|\hat \Psi ^{h}_{E(N,h)}(p)|^{2}converges to the classical momentum density in the sense of distribution, ash→0h\to 0andN→∞N\to \inftywith<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" alttext="left-parenthesis upper N plus one half right-parenthesis h equals StartFraction 1 Over pi EndFraction integral Underscript upper V left-parenthesis x right-parenthesis greater-than upper E Endscripts StartRoot 2 left-parenthesis upper E minus upper V left-parenthesis x right-parenthesis right-parenthesis EndRoot d x">(N+12)h=1π∫V(x)>E2(E−V(x))dx(N+\frac {1}{2} )h =\frac {1}{\pi } \int _{V(x)>E} \sqrt {2(E-V(x))}dxfixed. In this paper we only consider the harmonic oscillator Hamiltonian. By studying the semi-classical limit of the Wigner’s quasi-probability density and using the generating function of the Laguerre polynomials, we give a complete mathematical proof of the Correspondence Principle.
On integers not of the form ±𝑝^{𝑎}±𝑞^{𝑏}
Authors: Sun Zhi-Wei
Published: July 2002

In 1975 F. Cohen and J.L. Selfridge found a 94-digit positive integer which cannot be written as the sum or difference of two prime powers. Following their basic construction and introducing a new method to avoid a bunch of extra congruences, we are able to prove that if

x
≡
47867742232066880047611079
(
mod
⁡
66483034025018711639862527490
)
,

\begin{equation*} \hspace {-1.5pc} x\equiv 47867742232066880047611079 (\operatorname {mod} 66483034025018711639862527490), \hspace {-1.5pc} \end{equation*}

then

x
x

is not of the form

±

p

a

±

q

b

\pm p^{a}\pm q^{b}

where

p
,
q

p,q

are primes and

a
,
b

a,b

are nonnegative integers.
Characterizations of generalized Hermite and sieved ultraspherical polynomials
Authors: Dette Holger
Published: July 2002

A new characterization of the generalized Hermite polyno- mials and of the orthogonal polynomials with respect to the measure

|

x

|

γ

(
1
−

x
2

)

1

/

2

d
x

|x|^\gamma (1-x^2)^{1/2}dx

is derived which is based on a “reversing property" of the coefficients in the corresponding recurrence formulas and does not use the representation in terms of Laguerre and Jacobi polynomials. A similar characterization can be obtained for a generalization of the sieved ultraspherical polynomials of the first and second kind. These results are applied in order to determine the asymptotic limit distribution for the zeros when the degree and the parameters tend to infinity with the same order.
The flow completion of a manifold with vector field
Authors: Kamber Franz , Michor Peter
Published: July 2002

For a vector field

X
X

on a smooth manifold

M
M

there exists a smooth but not necessarily Hausdorff manifold

M

R

M_{\mathbb {R}}

and a complete vector field

X

R

X_{\mathbb {R}}

on it which is the universal completion of

(
M
,
X
)

(M,X)

.
Combinatorial pattern discovery in biological sequences: The TEIRESIAS algorithm.
Authors: Rigoutsos I , Floratos A
Published: July 2002

MOTIVATION: The discovery of motifs in biological sequences is an important problem. RESULTS: This paper presents a new algorithm for the discovery of rigid patterns (motifs) in biological sequences. Our method is combinatorial in nature and able to produce all patterns that appear in at least a (user-defined) minimum number of sequences, yet it manages to be very efficient by avoiding the enumeration of the entire pattern space. Furthermore, the reported patterns are maximal: any reported pattern cannot be made more specific and still keep on appearing at the exact same positions within the input sequences. The effectiveness of the proposed approach is showcased on a number of test cases which aim to: (i) validate the approach through the discovery of previously reported patterns; (ii) demonstrate the capability to identify automatically highly selective patterns particular to the sequences under consideration. Finally, experimental analysis indicates that the algorithm is output sensitive, i.e. its running time is quasi-linear to the size of the generated output.
Incremental threading optimization (TITO) to help alignment and modelling of remote homologues.
Authors: Labesse G , Mornon J
Published: July 2002

MOTIVATION: Protein sequence comparison has become a major tool for biologists. Various methods have been designed in order to reveal even remote homologies among proteins. Sequence analyses are powerful, but the lower the identity score, the more skill and time are required to perform them accurately. While necessary to confirm the sequence comparison through the potential of mean force, sequence alignment and molecular modelling are still tedious and time-consuming tasks. RESULTS: In order to help sequence threading onto known three-dimensional (3D) structures, we have developed a new program, named TITO (Tool for Incremental Threading Optimization), which uses a multiple alignment to validate and help the refinement of a sequence/structure comparison. It measures the compatibility of a family of related sequences with a known 3D structure. The coordinates of the common core are extracted, allowing further molecular modelling and 3D survey. AVAILABILITY: TITO is available by anonymous ftp at: ftp://lmcp.jussieu. fr/pub/tito CONTACT: labesse@lmcp.jussieu.fr Supplementary information: http://www.lmcp.jussieu.fr/ approximately labesse/TITO/TITO.html
GeneNet: a gene network database and its automated visualization.
Authors: Kolpakov F A , Ananko E A , Kolesov G B , Kolchanov N A
Published: July 2002

MOTIVATION: Gene networks that provide the regulation of physiological processes are the basic feature of organisms. Information regarding the regulation of gene expression and signal transduction pathways is increasing rapidly. However, the information is hard to formalize and systematize. Ways and means for automated visualization of the gene networks based on their formalized description are needed. RESULTS: The object-oriented database GeneNet and the software for its automated visualization have been developed. The main principles of a formalized description of the gene network have been worked out. Antiviral response and erythropoiesis are provided as examples to show how this is achieved. The GeneNet graphical user interface written in Java provides automated generation of the gene network diagrams and allows visualization and exploration of the GeneNet database through the Internet. A system of filters allows the selection of particular components of the network for visualization. AVAILABILITY: The GeneNet database and its graphical user interface are available at http://wwwmgs.bionet.nsc.ru/systems/MGL/GeneN et/ CONTACT: fedor@bionet.nsc.ru
Sausage: protein threading with flexible force fields
Authors: Huber Thomas , J.Russell Anthony , Ayers Daniel , E.Torda Andrew
Published: July 2002

Summary: Sausage is a protein sequence threading program, but with remarkable run-time flexibility. Using different scripts, it can calculate protein sequence-structure alignments, search structure libraries, swap force fields, create models from alignments, convert file formats and analyse results. There are several different force fields which might be classed as knowledge-based, although they do not rely on Boltzmann statistics. Different force fields are used for alignment calculations and subsequent ranking of calculated models.

Availability: Freely available to academics at ftp://ftp.rsc.anu.edu.au/pub/torda/sausage/README

Contact: Andrew.Torda@anu.edu.au
MASIA: recognition of common patterns and properties in
multiple aligned protein sequences
Authors: Zhu H. , Schein C. H. , Braun W.
Published: July 2002

Summary: MASIA is a software tool for pattern recognition in multiple aligned protein sequences. MASIA converts a sequence to a properties matrix that can be scanned in both vertical and horizontal steps. Consistent patterns are recognized based on the statistical significance of their occurrence. Preset macros can be altered on-line to seek any combination of amino acid properties or sequence characteristics. MASIA output can be used directly by our programs to predict the 3D structure of proteins.

Availability: Access MASIA at http://www.scsb.utmb.edu/masia/masia.html.

Contact: werner@newton.utmb.edu

To whom correspondence should be addressed.
Biochemical systems analysis of genome-wide expression data
Authors: Voit Eberhard O. , Radivoyevitch Tomas
Published: July 2002

Motivation: Modern methods of genomics have produced an unprecedented amount of raw data. The interpretation and explanation of these data constitute a major, well-recognized challenge.

Results: Biochemical Systems Theory (BST) is the mathematical basis of a well-established methodological framework for analyzing networks of biochemical reactions. An existing BST model of yeast glycolysis is used here to explain and interpret the glycolytic gene expression pattern of heat shocked yeast. Our analysis demonstrates that the observed gene expression profile satisfies the primary goals of increased ATP, trehalose, and NADPH production, while maintaining intermediate metabolites at reasonable levels. Based on a systematic exploration of alternative, hypothetical expression profiles, we show that the observed profile outperforms other profiles.

Conclusion: BST is a useful framework for combining DNA microarray data with enzymatic process information to yield new insights into metabolic pathway regulation.

Availability: All analyses were executed with the software PLAS©, which is freely available at http://correio.cc.fc.ul.pt/~aenf/plas.html for academic use.

Contact: VoitEO@MUSC.edu

To whom correspondence should be addressed.