Sample records for cells restrict yersinia

  1. CD8(+) T cells specific to a single Yersinia pseudotuberculosis epitope restrict bacterial replication in the liver but fail to provide sterilizing immunity.

    Shen, Haiqian; Gonzalez-Juarbe, Norberto; Blanchette, Krystle; Crimmins, Gregory; Bergman, Molly A; Isberg, Ralph R; Orihuela, Carlos J; Dube, Peter H


    CD8(+) T cells use contact-dependent cytolysis of target cells to protect the host against intracellular pathogens. We have previously shown that CD8(+) T cells and perforin are required to protect against the extracellular pathogen Yersinia pseudotuberculosis. Here we establish an experimental system where CD8(+) T cells specific to a single model antigen are the only memory response present at time of challenge. Using mice immunized with a vaccine strain of Listeria monocytogenes that expresses secreted ovalbumin (Lm-OVA), we show that OVA-specific CD8(+) T cells are generated and provide limited protection against challenge with virulent OVA(+)Y. pseudotuberculosis. Perforin expression by OVA-specific CD8(+) T cells was required, as Lm-OVA-immunized perforin-deficient mice showed higher bacterial burden as compared to Lm-OVA-immunized perforin-sufficient mice. Surprisingly, antigen-specific T cell protection waned over time, as Lm-OVA-immune mice eventually succumbed to Yersinia infection. Kinetic analysis of infection in mice with and without OVA-specific CD8(+) T cells revealed that bacterial numbers increased sharply in OVA-naïve mice until death, while OVA-immune mice held bacterial burden to a lower level throughout the duration of illness until death. Clonal analysis of bacterial populations in OVA-naïve and OVA-immune mice at distinct time points revealed equivalent and severe bottle-neck effects for bacteria in both sets of mice immediately after intravenous challenge, demonstrating a dominant role for other aspects of the immune system regardless of CD8(+) T cell status. These studies indicate that CD8(+) T cells against a single antigen can restrict Y. pseudotuberculosis colonization in a perforin-dependent manner, but ultimately are insufficient in their ability to provide sterilizing immunity and protect against death. PMID:27268148

  2. Phosphorylated CpxR restricts production of the RovA global regulator in Yersinia pseudotuberculosis.

    Junfa Liu

    Full Text Available BACKGROUND: RovA is a global transcriptional regulator of gene expression in pathogenic Yersinia. RovA levels are kept in check by a sophisticated layering of distinct transcriptional and post-transcriptional regulatory mechanisms. In the enteropathogen Y. pseudotuberculosis, we have previously reported that the extracytoplasmic stress sensing CpxA-CpxR two-component regulatory system modulates rovA expression. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we characterized CpxR phosphorylation (CpxR∼P in vitro, and determined that phosphorylation was necessary for CpxR to efficiently bind to the PCR-amplified upstream regulatory region of rovA. The precise CpxR∼P binding site was mapped by a nuclease protection assay and directed mutagenesis confirmed that in vivo binding to the rovA promoter inhibits transcription. Reduced RovA production was most pronounced following CpxR∼P accumulation in the Yersinia cytoplasm during chronic Cpx pathway activation and by the indiscriminate phosphodonor action of acetyl phosphate. CONCLUSIONS/SIGNIFICANCE: Cpx pathway activation restricts levels of the RovA global regulator. The regulatory influence of CpxR∼P must therefore extend well beyond periplasmic quality control in the Yersinia envelope, to include genes involved in environmental survival and pathogenicity.

  3. Macrophage Activation Redirects Yersinia-Infected Host Cell Death from Apoptosis to Caspase-1-Dependent Pyroptosis

    Bergsbaken, Tessa; Cookson, Brad T.


    Infection of macrophages by Yersinia species results in YopJ-dependent apoptosis, and naïve macrophages are highly susceptible to this form of cell death. Previous studies have demonstrated that macrophages activated with lipopolysaccharide (LPS) prior to infection are resistant to YopJ-dependent cell death; we found this simultaneously renders macrophages susceptible to killing by YopJ− Yersinia pseudotuberculosis (Yptb). YopJ− Yptb-induced macrophage death was dependent on caspase-1 activat...

  4. Enhancement of invasiveness of Yersinia enterocolitica and Escherichia coli in HEp-2 cells by centrifugation.

    Vesikari, T; Bromirska, J; Mäki, M


    Centrifugation enhanced the infectivity of invasive Escherichia coli and Yersinia enterocolitica for HEp-2 cells. Noninvasive bacteria were not endocytosed after centrifugation. The centrifugation procedure may increase the sensitivity of testing for bacterial invasiveness in cell culture without causing false-positive results.

  5. Yersinia enterocolitica YopP inhibits MAP kinase-mediated antigen uptake in dendritic cells

    Autenrieth, S. E.; Adkins, Irena; Rösemann, R.; Gunst, D.; Zahir, N.; Kracht, M.; Ruckdeschel, K.; Wagner, H.; Borgmann, S.; Autenrieth, I. B.


    Roč. 9, č. 2 (2007), s. 425-437. ISSN 1462-5814 Institutional research plan: CEZ:AV0Z50200510 Keywords : yersinia enterocolitica * dendritic cells * immunity Subject RIV: EC - Immunology Impact factor: 5.293, year: 2007

  6. Yersinia outer proteins E, H, P, and T differentially target the cytoskeleton and inhibit phagocytic capacity of dendritic cells

    Adkins, Irena; Köberle, M.; Gröbner, S.; Bohn, E.; Autenrieth, I. B.; Borgmann, S.


    Roč. 297, - (2007), s. 235-244. ISSN 1438-4221 Institutional research plan: CEZ:AV0Z50200510 Keywords : yersinia * yops * dendritic cells Subject RIV: EC - Immunology Impact factor: 2.524, year: 2007

  7. Radiometric investigation of biosynthesis of certain macromolecules in cell cultures inoculated with intracellularly multiplied Yersinia pseudotuberculosis and Listeria monocytogenes

    The study was conducted to investigate the changes in the biosynthesis of DNA, RNA and protein in epithelian cells from monkey kidney in the case of invasion by initial and passage Yersinia and Listeria strains, multiplied intracellularly. The Yersinia pseudotuberculosis strains IB 373 and IB 373-GP and Listeria monocytogenes strains 760 1,2a and 383-4b obtained by several passages through guinea pigs and their passage subculture were used. Inoculated cultures were labelled with 3H-thymidine, 3H uridine and 14C-valine. The results showed that in all cases 3H-thymidine and 3H-uridine inclusions into cell cultures, invaded with intracellularly multiplied Yersinia strains showed statistically higher values as compared to those from cell cultures invaded with the initial strain. 14C-valine inclusion in cells, invaded by passage strains, decreased reaching its lowest values in cell populations invaded by Yersiniae isolated during the 9th passage. In cell cultures, invaded by passage L. monocytogenes strains, a rise in the levels of 3H-thymidine and 3H-uridine inclusions was observed. Inclusion values of the precursors were higher in cell populations invaded by the monocytogenes 760-1,2a initial strain and by passage strains obtained from it. The 14C-valine inclusion varied in epithelial cells invaded by Listeriae of sero-type 1,2a and by those of sero type 4b. In the first group the highest inclusion values were recorded in epithelian cells invaded by Listeriae of the 1st passage and in the second in those invaded by Listerae isolated during the 3rd and 4th passages when their most active multiplication accured

  8. Involvement of M cells in the bacterial invasion of Peyer's patches: a common mechanism shared by Yersinia enterocolitica and other enteroinvasive bacteria.

    Grützkau, A; Hanski, C; Hahn, H.; Riecken, E O


    Recent evidence indicates that ileal Peyer's patches represent the main infection route for Yersinia enterocolitica. In this study transmission and scanning electron microscopy have shown that only a small fraction of bacteria present in the lumen adhere to the follicle-associated murine epithelium with no discernible preference for either M or absorptive cells. Yersiniae attached to M cells are phagocytosed and transported from the lumen into the lamina propria. No invasion of columnar absor...

  9. Innate immune response during Yersinia infection: critical modulation of cell death mechanisms through phagocyte activation

    Bergsbaken, Tessa; Cookson, Brad T.


    Yersinia pestis, the etiological agent of plague, is one of the most deadly pathogens on our planet. This organism shares important attributes with its ancestral progenitor, Yersinia pseudotuberculosis, including a 70-kb virulence plasmid, lymphotropism during growth in the mammalian host, and killing of host macrophages. Infections with both organisms are biphasic, where bacterial replication occurs initially with little inflammation, followed by phagocyte influx, inflammatory cytokine produ...

  10. Absence of Toll-Like Receptor 4 Signaling Results in Delayed Yersinia enterocolitica YopP-Induced Cell Death of Dendritic Cells

    Gröbner, S.; Schulz, S.; Adkins, Irena; Gunst, D. S. J.; Waibel, M.; Wesselborg, S.; Borgmann, S.; Autenrieth, I. B.


    Roč. 75, č. 1 (2007), s. 512-517. ISSN 0019-9567 Institutional research plan: CEZ:AV0Z50200510 Keywords : yersinia enterocolitica * toll-like receptor 4 * dendritic cells Subject RIV: EC - Immunology Impact factor: 3.996, year: 2007

  11. Rare Infections: Yersinia Enterocolitica and Yersinia Pseudotuberculosis

    ... Health Issues Listen Text Size Email Print Share Rare Infections: Yersinia Enterocolitica and Yersinia Pseudotuberculosis Page Content ... infected animal, drinking contaminated well water, or on rare occasions, from contaminated transfusions. The infections are increasing ...

  12. Yersinia pseudotuberculosis Spatially Controls Activation and Misregulation of Host Cell Rac1.


    Full Text Available Yersinia pseudotuberculosis binds host cells and modulates the mammalian Rac1 guanosine triphosphatase (GTPase at two levels. Activation of Rac1 results from integrin receptor engagement, while misregulation is promoted by translocation of YopE and YopT proteins into target cells. Little is known regarding how these various factors interplay to control Rac1 dynamics. To investigate these competing processes, the localization of Rac1 activation was imaged microscopically using fluorescence resonance energy transfer. In the absence of translocated effectors, bacteria induced activation of the GTPase at the site of bacterial binding. In contrast, the entire cellular pool of Rac1 was inactivated shortly after translocation of YopE RhoGAP. Inactivation required membrane localization of Rac1. The translocated protease YopT had very different effects on Rac1. This protein, which removes the membrane localization site of Rac1, did not inactivate Rac1, but promoted entry of cleaved activated Rac1 molecules into the host cell nucleus, allowing Rac1 to localize with nuclear guanosine nucleotide exchange factors. As was true for YopE, membrane-associated Rac1 was the target for YopT, indicating that the two translocated effectors may compete for the same pool of target protein. Consistent with the observation that YopE inactivation requires membrane localization of Rac1, the presence of YopT in the cell interfered with the action of the YopE RhoGAP. As a result, interaction of target cells with a strain that produces both YopT and YopE resulted in two spatially distinct pools of Rac1: an inactive cytoplasmic pool and an activated nuclear pool. These studies demonstrate that competition between bacterial virulence factors for access to host substrates is controlled by the spatial arrangement of a target protein. In turn, the combined effects of translocated bacterial proteins are to generate pools of a single signaling molecule with distinct localization and

  13. Yersinia enterocolitica targets cells of the innate and adaptive immune system by injection of Yops in a mouse infection model.

    Martin Köberle


    Full Text Available Yersinia enterocolitica (Ye evades the immune system of the host by injection of Yersinia outer proteins (Yops via a type three secretion system into host cells. In this study, a reporter system comprising a YopE-beta-lactamase hybrid protein and a fluorescent staining sensitive to beta-lactamase cleavage was used to track Yop injection in cell culture and in an experimental Ye mouse infection model. Experiments with GD25, GD25-beta1A, and HeLa cells demonstrated that beta1-integrins and RhoGTPases play a role for Yop injection. As demonstrated by infection of splenocyte suspensions in vitro, injection of Yops appears to occur randomly into all types of leukocytes. In contrast, upon infection of mice, Yop injection was detected in 13% of F4/80(+, 11% of CD11c(+, 7% of CD49b(+, 5% of Gr1(+ cells, 2.3% of CD19(+, and 2.6% of CD3(+ cells. Taking the different abundance of these cell types in the spleen into account, the highest total number of Yop-injected cells represents B cells, particularly CD19(+CD21(+CD23(+ follicular B cells, followed by neutrophils, dendritic cells, and macrophages, suggesting a distinct cellular tropism of Ye. Yop-injected B cells displayed a significantly increased expression of CD69 compared to non-Yop-injected B cells, indicating activation of these cells by Ye. Infection of IFN-gammaR (receptor- and TNFRp55-deficient mice resulted in increased numbers of Yop-injected spleen cells for yet unknown reasons. The YopE-beta-lactamase hybrid protein reporter system provides new insights into the modulation of host cell and immune responses by Ye Yops.

  14. Cytotoxic T-Cell-Mediated Response against Yersinia pseudotuberculosis in HLA-B27 Transgenic Rat

    Falgarone, Géraldine; Blanchard, Hervé S.; Riot, Bertrand; Simonet, Michel; Breban, Maxime


    Yersinia-induced reactive arthritis is highly associated with HLA-B27, the role of which in defense against the triggering bacteria remains unclear. The aim of this study was to examine the capacity of rats transgenic for HLA-B27 to mount a cytotoxic T-lymphocyte (CTL) response against Y. pseudotuberculosis and to determine the influence of the HLA-B27 transgene on this response. Rats transgenic for HLA-B*2705 and human β2-microglobulin of the 21-4L line, which do not spontaneously develop di...

  15. The "occlusis" model of cell fate restriction.

    Lahn, Bruce T


    A simple model, termed "occlusis", is presented here to account for both cell fate restriction during somatic development and reestablishment of pluripotency during reproduction. The model makes three assertions: (1) A gene's transcriptional potential can assume one of two states: the "competent" state, wherein the gene is responsive to, and can be activated by, trans-acting factors in the cellular milieu, and the "occluded" state, wherein the gene is blocked by cis-acting, chromatin-based mechanisms from responding to trans-acting factors such that it remains silent irrespective of whether transcriptional activators are present in the milieu. (2) As differentiation proceeds in somatic lineages, lineage-inappropriate genes shift progressively and irreversibly from competent to occluded state, thereby leading to the restriction of cell fate. (3) During reproduction, global deocclusion takes place in the germline and/or early zygotic cells to reset the genome to the competent state in order to facilitate a new round of organismal development. PMID:20954221

  16. Small molecule inhibitors of the Yersinia type III secretion system impair the development of Chlamydia after entry into host cells

    Henriques-Normark Birgitta


    Full Text Available Abstract Background Chlamydiae are obligate intracellular pathogens that possess a type III secretion system to deliver proteins into the host cell during infection. Small molecule inhibitors of type III secretion in Yersinia, termed INPs (Innate Pharmaceuticals AB were reported to strongly inhibit Chlamydia growth in epithelial cells. In this study we have analyzed the effect of these drugs on bacterial invasiveness. Results We demonstrate that INPs affect Chlamydia growth in a dose dependent manner after bacterial invasion. The efficiency of C. trachomatis L2 and C. caviae GPIC entry into host cells was not altered in the presence of INPs. In C. caviae, entry appears to proceed normally with recruitment of actin and the small GTPases Rac, Cdc42 and Arf6 to the site of bacterial entry. Conclusion INPs have a strong inhibitory effect on Chlamydia growth. However, bacterial invasion is not altered in the presence of these drugs. In the light of these results, we discuss several hypotheses regarding the mode of action of INPs on type III secretion during the Chlamydia infectious cycle.

  17. Restriction of porcine parvovirus replication in nonpermissive cells.

    Oraveerakul, K; Choi, C S; Molitor, T W


    Swine testicle (ST) cells and Madin-Darby canine kidney (MDCK) cells differ in their ability to support replication of porcine parvovirus (PPV). Viral replication events in ST cells, a permissive cell type, and MDCK cells, a nonpermissive cell type, were compared in an attempt to elucidate putative mechanisms of restrictive virus replication. Radiolabeled PPV bound to the cell surface of both cell types equally well and the binding was shown to be PPV specific, indicating that the restriction...

  18. Tetherin restricts productive HIV-1 cell-to-cell transmission.

    Nicoletta Casartelli

    Full Text Available The IFN-inducible antiviral protein tetherin (or BST-2/CD317/HM1.24 impairs release of mature HIV-1 particles from infected cells. HIV-1 Vpu antagonizes the effect of tetherin. The fate of virions trapped at the cell surface remains poorly understood. Here, we asked whether tetherin impairs HIV cell-to-cell transmission, a major means of viral spread. Tetherin-positive or -negative cells, infected with wild-type or DeltaVpu HIV, were used as donor cells and cocultivated with target lymphocytes. We show that tetherin inhibits productive cell-to-cell transmission of DeltaVpu to targets and impairs that of WT HIV. Tetherin accumulates with Gag at the contact zone between infected and target cells, but does not prevent the formation of virological synapses. In the presence of tetherin, viruses are then mostly transferred to targets as abnormally large patches. These viral aggregates do not efficiently promote infection after transfer, because they accumulate at the surface of target cells and are impaired in their fusion capacities. Tetherin, by imprinting virions in donor cells, is the first example of a surface restriction factor limiting viral cell-to-cell spread.

  19. Yersinia enterocolitica Monographic Study

    Emil Tirziu; Ciceronis Cumpanasoiu; Radu Valentin Gros; Monica Seres


    Germs from Yersinia genus have a vast ecologic niche, being met at different domestic and wild animal species, but also in food, water and soil. The majority of yersinis live in the digestive tract of human and numerous animal species, especially rodents, but also in soil, plant debris, waters etc. Numerous species of Yersinia genus could produce characteristic infections in human, the main source of infections is represented by rodents and hematophagous insects or, more frequently, by water ...

  20. Yersinia enterocolitica Monographic Study

    Emil Tirziu


    Full Text Available Germs from Yersinia genus have a vast ecologic niche, being met at different domestic and wild animal species, but also in food, water and soil. The majority of yersinis live in the digestive tract of human and numerous animal species, especially rodents, but also in soil, plant debris, waters etc. Numerous species of Yersinia genus could produce characteristic infections in human, the main source of infections is represented by rodents and hematophagous insects or, more frequently, by water or contaminated food. In a 1999 study, Mead and coauthors established that the Yersinia enterocolitica prevalence in food, in USA, is around 90%. Foods of animal origin more frequently contaminated with Yersinia enterocolitica are: pork, poultry, beef and lamb meat, milk, ice-cream, sea fruits etc., among them pork meat and milk represents the sources of the most numerous toxi-infection outbreaks in human, in different world regions. Bacteria determine infections which interest the digestive tract in numerous animal species and human, with diarrhea, lymphadenitis, pneumonia and abortion are the most important symptoms. Yersinia enterocolitica enter the human body regularly by oral ingestion, and localize itself with predilection in the distal portion of the ileum and at the ileocaecal appendix and proximal colon level, were determine a terminal ileitis with lymphadenitis, acute enterocolitis, and secondary accompanied with nodosum erythema, poliartritis that could be complicated with septicemia, sometimes leading to death.

  1. The YopB protein of Yersinia pseudotuberculosis is essential for the translocation of Yop effector proteins across the target cell plasma membrane and displays a contact-dependent membrane disrupting activity.

    Håkansson, S; Schesser, K; Persson, C; Galyov, E E; Rosqvist, R; Homblé, F; Wolf-Watz, H


    During infection of cultured epithelial cells, surface-located Yersinia pseudotuberculosis deliver Yop (Yersinia outer protein) virulence factors into the cytoplasm of the target cell. A non-polar yopB mutant strain displays a wild-type phenotype with respect to in vitro Yop regulation and secretion but fails to elicit a cytotoxic response in cultured HeLa cells and is unable to inhibit phagocytosis by macrophage-like J774 cells. Additionally, the yopB mutant strain was avirulent in the mouse model. No YopE or YopH protein were observed within HeLa cells infected with the yopB mutant strain, suggesting that the loss of virulence of the mutant strain was due to its inability to translocate Yop effector proteins through the target cell plasma membrane. Expression of YopB is necessary for Yersinia-induced lysis of sheep erythrocytes. Purified YopB was shown to have membrane disruptive activity in vitro. YopB-dependent haemolytic activity required cell contact between the bacteria and the erythrocytes and could be inhibited by high, but not low, molecular weight carbohydrates. Similarly, expression of YopE reduced haemolytic activity. Therefore, we propose that YopB is essential for the formation of a pore in the target cell membrane that is required for the cell-to-cell transfer of Yop effector proteins. PMID:8918459

  2. Current activities of the Yersinia effector protein YopM.

    Höfling, Sabrina; Grabowski, Benjamin; Norkowski, Stefanie; Schmidt, M Alexander; Rüter, Christian


    Yersinia outer protein M (YopM) belongs to the group of Yop effector proteins, which are highly conserved among pathogenic Yersinia species. During infection, the effectors are delivered into the host cell cytoplasm via the type 3 secretion system to subvert the host immune response and support the survival of Yersinia. In contrast to the other Yop effectors, YopM does not possess a known enzymatic activity and its molecular mechanism(s) of action remain(s) poorly understood. However, YopM was shown to promote colonization and dissemination of Yersinia, thus being crucial for the pathogen's virulence in vivo. Moreover, YopM interacts with several host cell proteins and might utilize them to execute its anti-inflammatory activities. The results obtained so far indicate that YopM is a multifunctional protein that counteracts the host immune defense by multiple activities, which are at least partially independent of each other. Finally, its functions seem to be also influenced by differences between the specific YopM isoforms expressed by Yersinia subspecies. In this review, we focus on the global as well as more specific contribution of YopM to virulence of Yersinia during infection and point out the various extra- and intracellular molecular functions of YopM. In addition, the novel cell-penetrating ability of recombinant YopM and its potential applications as a self-delivering immunomodulatory therapeutic will be discussed. PMID:25865799

  3. Yops of Yersinia enterocolitica Inhibit Receptor-Dependent Superoxide Anion Production by Human Granulocytes

    Visser, L.G.; Seijmonsbergen, E.; Nibbering, P H; van den Broek, P J; van Furth, R


    The virulence plasmid-borne genes encoding Yersinia adhesin A (YadA) and several Yersinia secreted proteins (Yops) are involved in the inhibition of phagocytosis and killing of Yersinia enterocolitica by human granulocytes. One of these Yops, YopH, dephosphorylates multiple tyrosine-phosphorylated proteins in eukaryotic cells and is involved in the inhibition of phagocytosis of Y. enterocolitica by human granulocytes. We investigated whether antibody- and complement-opsonized plasmid-bearing ...

  4. Yersinia type III effectors perturb host innate immune responses.

    Pha, Khavong; Navarro, Lorena


    The innate immune system is the first line of defense against invading pathogens. Innate immune cells recognize molecular patterns from the pathogen and mount a response to resolve the infection. The production of proinflammatory cytokines and reactive oxygen species, phagocytosis, and induced programmed cell death are processes initiated by innate immune cells in order to combat invading pathogens. However, pathogens have evolved various virulence mechanisms to subvert these responses. One strategy utilized by Gram-negative bacterial pathogens is the deployment of a complex machine termed the type III secretion system (T3SS). The T3SS is composed of a syringe-like needle structure and the effector proteins that are injected directly into a target host cell to disrupt a cellular response. The three human pathogenic Yersinia spp. (Y. pestis, Y. enterocolitica, and Y. pseudotuberculosis) are Gram-negative bacteria that share in common a 70 kb virulence plasmid which encodes the T3SS. Translocation of the Yersinia effector proteins (YopE, YopH, YopT, YopM, YpkA/YopO, and YopP/J) into the target host cell results in disruption of the actin cytoskeleton to inhibit phagocytosis, downregulation of proinflammatory cytokine/chemokine production, and induction of cellular apoptosis of the target cell. Over the past 25 years, studies on the Yersinia effector proteins have unveiled tremendous knowledge of how the effectors enhance Yersinia virulence. Recently, the long awaited crystal structure of YpkA has been solved providing further insights into the activation of the YpkA kinase domain. Multisite autophosphorylation by YpkA to activate its kinase domain was also shown and postulated to serve as a mechanism to bypass regulation by host phosphatases. In addition, novel Yersinia effector protein targets, such as caspase-1, and signaling pathways including activation of the inflammasome were identified. In this review, we summarize the recent discoveries made on Yersinia

  5. Dietary restriction, cell proliferation and carcinogenesis: a preliminary study

    Four groups of female Swiss Webster mice were given either laboratory chow or a purified (semi-synthetic) diet (AIN-76A) either ad libitum or at 75% of the ad libitum rate for about 30 days. Three tissues, the crypt cells of the jejunum, the dermis and the basal epithelial cells of the esophagus were investigated using [3H]thymidine labelling and by counting mitoses; four other tissues, the alveolar cells of the mammary gland, the crypt cells of the duodenum and colo-rectum, and the transitional cells of the urinary bladder were examined using [3H]thymidine labelling only. In each case dietary restriction led to a reduction of cellular proliferation assessed by these indices. The potential of the approach for the study of the effects of dietary modification on the introduction of cancer is discussed. (author). 22 refs

  6. [Yersinia pestis. Bacteriology].

    Pattyn, S R


    The author discusses the evolution in the classification of the bacterium, responsible for plague: first a classification based on phenotypic characteristics, later based on genotypic characteristics, to finally arrive at an evolutionist classification. He treats the seven species of the genus Yersinia that can be distinguished by DNA hybridization. He examines the issue of sequencing and decoding the chromosome and mentions research regarding the phenomenon that the metabolism of the organisms modifies as a reaction to signals of their changing environment. Furthermore the author discusses the efforts to characterize the strains of Y. pestis (antiqua, medievalis and orientalis). Finally he comments on the discovery of a multiresistant strain, isolated in 1995 in Madagascar. PMID:10379199

  7. Catalytically active Yersinia outer protein P induces cleavage of RIP and caspase-8 at the level of the DISC independently of death receptors in dendritic cells

    Gröbner, S.; Adkins, Irena; Schulz, S.; Richter, K.; Borgmann, S.; Wesselborg, S.; Ruckdeschel, K.; Micheau, O.; Autenrieth, I. B.


    Roč. 10, č. 12 (2007), s. 1813-1825. ISSN 1360-8185 Institutional research plan: CEZ:AV0Z50200510 Keywords : yersinia enterocolitica * yopp * death receptors Subject RIV: EC - Immunology Impact factor: 3.043, year: 2007

  8. Plague virulence antigens from Yersinia enterocolitica.

    Carter, P B; Zahorchak, R J; Brubaker, R R


    The virulence of Yersinia enterocolitica, biotype 2, serotype O:8, in mice is related to its ability to produce plague V and W antigens. V and W antigens in Y. enterocolitica are shown to be immunologically identical to the previously described V and W antigens of Yersinia pestis and Yersinia pseudotuberculosis.

  9. Epitopes associated with MHC restriction site of T cells. III. I-J epitope on MHC-restricted T helper cells

    I-J epitopes were found to be associated with the functional site of the class II MHC-restricted helper T (Th) cells: Virtually all of the H-2k-restricted Th cell function of H-2kxbF1 T cells was inhibited by the anti-I-Jk mAb, leaving the H-2b-restricted function unaffected. The I-Jk epitope was inducible in Th cells of different genotype origin according to the environmental class II antigens present in the early ontogeny of T cells. Although above results suggested that I-J is the structure reflecting the inducible MHC restriction specificity, further studies revealed some interesting controversies: First, the I-J phenotype did not always correlate with the class II restriction specificity, e.g., I-Ab-restricted Th from 5R was I-Jk-positive, whereas I-Ak-restricted Th of 4R was not. Second, there was no trans expression of parental I-J phenotypes and restriction specificities in F1 Th, e.g., the I-J phenotype was detected only on I-Ab-restricted Th of (4R X 5R)F1, whereas it was absent on I-Ak-restricted Th. This strict linkage between the restriction specificity and I-J phenotype was also found on Th cells developed in bone marrow chimera constructed with intra-H-2-recombinant mice. The expression of I-Jk was always associated with the restriction specificity of the relevant host. Thus, the restriction specificity of Th cells followed the host type, and the I-J expression on Th was exactly the same as that expressed by the host haplotype. These results indicate that I-J is an isomorphic structure adaptively expressed on Th cells that is involved in the unidirectional regulatory cell interactions, and that the polymorphism cannot be explained merely by the restriction specificity of the conventional T cell receptor heterodimer

  10. Behavior of Yersinia enterocolitica in Foods.

    Bari, Md Latiful; Hossain, M Anwar; Isshiki, Kenji; Ukuku, Dike


    Yersinia enterocolitica are ubiquitous, being isolated frequently from soil, water, animals, and a variety of foods. They comprise a biochemically heterogeneous group that can survive and grow at refrigeration temperatures. The ability to propagate at refrigeration temperatures is of considerable significance in food hygiene. Virulent strains of Yersinia invade mammalian cells such as HeLa cells in tissue culture. Two chromosomal genes, inv and ail, were identified for cell invasion of mammalian. The pathogen can cause diarrhoea, appendicitis and post-infection arthritis may occur in a small proportion of cases. The most common transmission route of pathogenic Y. enterocolitica is thought to be fecal-oral via contaminated food. Direct person-to-person contact is rare. Occasionally, pathogenic Y. enterocolitica has been detected in vegetables and environmental water; thus, vegetables and untreated water are also potential sources of human yersiniosis. However, the isolation rates of pathogenic Y. enterocolitica have been low, which may be due to the limited sensitivity of the detection methods. To identify other possible transmission vehicles, different food items should be studied more extensively. Many factors related to the epidemiology of Y. enterocolitica, such as sources, transmission routes, and predominating genotypes remain obscure because of the low sensitivity of detection methods. PMID:22567332

  11. Virulence and viability of Yersinia pestis 25 years after lyophilization.

    Heckly, R J; Blank, H.


    When equal volumes of 6% lactose and a broth culture of Yersinia pestis were mixed before freezing, approximately 50% of the cells survived lyophilization and reconstitution on the following day. Concomitantly, the number of viable cells per 50% lethal dose increased from about 16 to 125 organisms. On subsequent storage of the lyophilized cells under vacuum in glass ampoules at 4 degrees C for 25 years, more than 25% of the cells remained viable. When stored cultures were assayed immediately ...

  12. Cell-mediated immune responses differentiate infections with Brucella suis from Yersinia enterocolitica serotype O : 9 in pigs

    Riber, Ulla; Jungersen, Gregers


    serological Brucellosis reactions. While 36 of the 39 FPSR pigs were also FPSR in a second test, none of the pigs were test positive in whole blood IFN-gamma assay or Brucellergene OCB skin test. In conclusion, use of IFN-gamma assay and skin test as measurements of cell-mediated immune responses to non...

  13. Yersinia enterocolitica: Gıda Kaynaklı bir Patojen

    Özbaş, Z. Yeşim; Aytaç, S. Aykut


    In this review, a foodborn pathogen Yersinia enterocolitica was investigated. In addition to taxonomical and ecological characteristics of the bacterium, foods involved in Yersinia infections, pathogenicity and factors affecting the growth and survival of Yersinia in food were also discussed.

  14. Yersinia pestis and Yersinia pseudotuberculosis infection: a regulatory RNA perspective

    Martínez-Chavarría, Luary C.; Vadyvaloo, Viveka


    Yersinia pestis, responsible for causing fulminant plague, has evolved clonally from the enteric pathogen, Y. pseudotuberculosis, which in contrast, causes a relatively benign enteric illness. An ~97% nucleotide identity over 75% of their shared protein coding genes is maintained between these two pathogens, leaving much conjecture regarding the molecular determinants responsible for producing these vastly different disease etiologies, host preferences and transmission routes. One idea is that coordinated production of distinct factors required for host adaptation and virulence in response to specific environmental cues could contribute to the distinct pathogenicity distinguishing these two species. Small non-coding RNAs that direct posttranscriptional regulation have recently been identified as key molecules that may provide such timeous expression of appropriate disease enabling factors. Here the burgeoning field of small non-coding regulatory RNAs in Yersinia pathogenesis is reviewed from the viewpoint of adaptive colonization, virulence and divergent evolution of these pathogens. PMID:26441890

  15. SAMHD1: a new insight into HIV-1 restriction in myeloid cells

    Wu Li; St Gelais Corine


    Abstract Human myeloid-lineage cells are refractory to HIV-1 infection. The Vpx proteins from HIV-2 and sooty mangabey SIV render these cells permissive to HIV-1 infection through proteasomal degradation of a putative restriction factor. Two recent studies discovered the cellular protein SAMHD1 to be this restriction factor, demonstrating that Vpx induces proteasomal degradation of SAMHD1 and enhances HIV-1 infection in myeloid-lineage cells. SAMHD1 functions as a myeloid-cell-specific HIV-1 ...

  16. Inactive Doses and Protein Concentration of Gamma Irradiated Yersinia Enterocolitica

    Yersinia enterocolitica is one of bacteria which cause coliform mastitis in dairy cows. The bacteria could be inactivated by gamma irradiation as inactivated vaccine candidate. The experiment has been conducted to determine the inactive doses and the protein concentration of Yersinia enterocolitica Y3 which has been irradiated by gamma rays. The cells cultures were irradiated by gamma rays with doses of 0, 100, 200, 400, 600, 800, 1.000 and 1.500 Gy (doses rate was 1089,59 Gy/hours). The inactive dose was determined by the drop test method and the protein concentration of cells were determined by Lowry method. The results showed that the inactive doses occurred on 800 – 1500 Gy. The different irradiation doses of cell cultures showed the effect of gamma irradiation on the protein concentration that was random and has a significant effect on the protein concentration. (author)

  17. Genome Wide Search for Biomarkers to Diagnose Yersinia Infections.

    Kalia, Vipin Chandra; Kumar, Prasun


    Bacterial identification on the basis of the highly conserved 16S rRNA (rrs) gene is limited by its presence in multiple copies and a very high level of similarity among them. The need is to look for other genes with unique characteristics to be used as biomarkers. Fifty-one sequenced genomes belonging to 10 different Yersinia species were used for searching genes common to all the genomes. Out of 304 common genes, 34 genes of sizes varying from 0.11 to 4.42 kb, were selected and subjected to in silico digestion with 10 different Restriction endonucleases (RE) (4-6 base cutters). Yersinia species have 6-7 copies of rrs per genome, which are difficult to distinguish by multiple sequence alignments or their RE digestion patterns. However, certain unique combinations of other common gene sequences-carB, fadJ, gluM, gltX, ileS, malE, nusA, ribD, and rlmL and their RE digestion patterns can be used as markers for identifying 21 strains belonging to 10 Yersinia species: Y. aldovae, Y. enterocolitica, Y. frederiksenii, Y. intermedia, Y. kristensenii, Y. pestis, Y. pseudotuberculosis, Y. rohdei, Y. ruckeri, and Y. similis. This approach can be applied for rapid diagnostic applications. PMID:26543261

  18. Global gene expression profiling of Yersinia pestis replicating inside macrophages reveals the roles of a putative stress-induced operon in regulating type III secretion and intracellular cell division.

    Fukuto, Hana S; Svetlanov, Anton; Palmer, Lance E; Karzai, A Wali; Bliska, James B


    Yersinia pestis, the causative agent of plague, is a facultative intracellular pathogen. Previous studies have indicated that the ability of Y. pestis to survive inside macrophages may be critical during the early stages of plague pathogenesis. To gain insights into the biology of intracellular Y. pestis and its environment following phagocytosis, we determined the genome-wide transcriptional profile of Y. pestis KIM5 replicating inside J774.1 macrophage-like cells using DNA microarrays. At 1.5, 4, and 8 h postinfection, a total of 801, 464, and 416 Y. pestis genes were differentially regulated, respectively, compared to the level of gene expression of control bacteria grown in tissue culture medium. A number of stress-response genes, including those involved in detoxification of reactive oxygen species, as well as several metabolic genes involved in macromolecule synthesis, were significantly induced in intracellular Y. pestis, consistent with the presence of oxidative stress and nutrient starvation inside Yersinia-containing vacuoles. A putative stress-induced operon consisting of y2313, y2315, and y2316 (y2313-y2316), and a previously unidentified open reading frame, orfX, was studied further on the basis of its high level of intracellular expression. Mutant strains harboring either deletion, Deltay2313-y2316 or DeltaorfX, exhibited diverse phenotypes, including reduced effector secretion by the type III secretion system, increased intracellular replication, and filamentous morphology of the bacteria growing inside macrophages. The results suggest a possible role for these genes in regulating cell envelope characteristics in the intracellular environment. PMID:20566693

  19. Transfer of experimental allergic encephalomyelitis to bone marrow chimeras. Endothelial cells are not a restricting element

    The adoptive transfer of clinical and histopathologic signs of experimental allergic encephalomyelitis (EAE) requires MHC compatibility between cell donor and cell recipient. The results of adoptive transfer studies using F1 to parent bone marrow chimeras as recipients of parental-derived BP-sensitive spleen cells indicate that this restriction is not expressed at the level of the endothelial cell but is confined to the cells of bone marrow derivation. Furthermore, these results indicate that the development of EAE is not dependent on the activity of MHC-restricted cytotoxic cells


    It is well known that dietary energy restriction prolongs lifespan and enhances immune responsiveness in a wide range of laboratory animals. However, information on the applicability of these results to humans is limited. In this study we examined the effects of calorie restriction on T cell mediate...

  1. Restriction of Human Polyomavirus BK Virus DNA Replication in Murine Cells and Extracts

    Mahon, C.; Liang, B.; Tikhanovich, I.; et al


    BK virus (BKV) causes persistent and asymptomatic infections in most humans and is the etiologic agent of polyomavirus-associated nephropathy (PVAN) and other pathologies. Unfortunately, there are no animal models with which to study activation of BKV replication in the human kidney and the accompanying PVAN. Here we report studies of the restriction of BKV replication in murine cells and extracts and the cause(s) of this restriction. Upon infection of murine cells, BKV expressed large T anti...

  2. Proteomic Characterization of Host Response to Yersinia pestis

    Chromy, B; Perkins, J; Heidbrink, J; Gonzales, A; Murhpy, G; Fitch, J P; McCutchen-Maloney, S


    Host-pathogen interactions result in protein expression changes within both the host and the pathogen. Here, results from proteomic characterization of host response following exposure to Yersinia pestis, the causative agent of plague, and to two near neighbors, Y. pseudotuberculosis and Y. enterocolitica, are reported. Human monocyte-like cells were chosen as a model for macrophage immune response to pathogen exposure. Two-dimensional electrophoresis followed by mass spectrometry was used to identify host proteins with differential expression following exposure to these three closely related Yersinia species. This comparative proteomic characterization of host response clearly shows that host protein expression patterns are distinct for the different pathogen exposures, and contributes to further understanding of Y. pestis virulence and host defense mechanisms. This work also lays the foundation for future studies aimed at defining biomarkers for presymptomatic detection of plague.

  3. Lsd1 restricts the number of germline stem cells by regulating multiple targets in escort cells.

    Susan Eliazer


    Full Text Available Specialized microenvironments called niches regulate tissue homeostasis by controlling the balance between stem cell self-renewal and the differentiation of stem cell daughters. However the mechanisms that govern the formation, size and signaling of in vivo niches remain poorly understood. Loss of the highly conserved histone demethylase Lsd1 in Drosophila escort cells results in increased BMP signaling outside the cap cell niche and an expanded germline stem cell (GSC phenotype. Here we present evidence that loss of Lsd1 also results in gradual changes in escort cell morphology and their eventual death. To better characterize the function of Lsd1 in different cell populations within the ovary, we performed Chromatin immunoprecipitation coupled with massive parallel sequencing (ChIP-seq. This analysis shows that Lsd1 associates with a surprisingly limited number of sites in escort cells and fewer, and often, different sites in cap cells. These findings indicate that Lsd1 exhibits highly selective binding that depends greatly on specific cellular contexts. Lsd1 does not directly target the dpp locus in escort cells. Instead, Lsd1 regulates engrailed expression and disruption of engrailed and its putative downstream target hedgehog suppress the Lsd1 mutant phenotype. Interestingly, over-expression of engrailed, but not hedgehog, results in an expansion of GSC cells, marked by the expansion of BMP signaling. Knockdown of other potential direct Lsd1 target genes, not obviously linked to BMP signaling, also partially suppresses the Lsd1 mutant phenotype. These results suggest that Lsd1 restricts the number of GSC-like cells by regulating a diverse group of genes and provide further evidence that escort cell function must be carefully controlled during development and adulthood to ensure proper germline differentiation.

  4. Restriction of Rift Valley Fever Virus Virulence in Mosquito Cells

    Sonja R. Gerrard


    Full Text Available Arboviruses are maintained in a natural cycle that requires blood-sucking arthropod and vertebrate hosts. Arboviruses are believed to persistently infect their arthropod host without overt pathology and cause acute infection with viremia in their vertebrate host. We have focused on elucidating how a specific arbovirus, Rift Valley fever (RVF virus, causes cytopathic effect in cells derived from vertebrates and non-cytopathic infection in cells derived from arthropods. We demonstrate that the vertebrate virulence factor, NSs, is functional in arthropod cells but is expressed at significantly lower levels in infected arthropod versus infected vertebrate cells.

  5. Restrictive versus liberal transfusion strategy for red blood cell transfusion

    Holst, Lars B; Petersen, Marie W; Haase, Nicolai;


    titles and abstracts of trials identified, and relevant trials were evaluated in full text for eligibility. Two reviewers then independently extracted data on methods, interventions, outcomes, and risk of bias from included trials. random effects models were used to estimate risk ratios and mean...... trials, SilverPlatter Medline (1950 to date), SilverPlatter Embase (1980 to date), and Science Citation Index Expanded (1900 to present). Reference lists of identified trials and other systematic reviews were assessed, and authors and experts in transfusion were contacted to identify additional trials....... TRIAL SELECTION: Published and unpublished randomised clinical trials that evaluated a restrictive compared with a liberal transfusion strategy in adults or children, irrespective of language, blinding procedure, publication status, or sample size. DATA EXTRACTION: Two authors independently screened...

  6. Studies on the mechanism of the self restriction of T cell responses in radiation chimeras

    Recent experiments with murine radiation chimeras have shown that F1 T cells that mature in an H-2 homozygous thymus, as is the case in [F1 → Parent 1] chimeras, are restricted to recognizing foreign antigen in the context of Parent 1 H-2 antigens. Conflicting results on the stringency of self H-2 restriction of T cells from normal mice have suggested that the thymic restriction in chimeras may be due to active suppression of parent 2-restricted T cell clones. We have therefore conducted 3 sets of experiments to test for suppression of maturing T cells that could mediate thymic tutoring of H-2-restriction specificity in chimeras. In 2 sets of experiments, we found no evidence that suppressor cells could be exported from 1 thymus and act either intrathymically on thymocytes in a 2nd thymus or extrathymically on recent thymic emigrants. We believe current data support a role for the thymus in positive as well as negative selection of maturing thymocytes on the basis of self recognition, in the absence of any suppression. Our results do not support the concept that suppression is responsible for the difference in the degree of self preference in the T cells of chimeric mice relative to cell populations obtained from neonatally tolerant mice or from normal mice after acute negative selection


    Yu. Rud


    Full Text Available Purpose. The analysis of nucleotide sequences of the 16S rDNA gene of virulent strains of Yersinia ruckeri and to develop the method of molecular diagnostic of enteric redmouth disease. Methodology. By the method of CLUSTALW algorithm in MEGA software version 6.0 the nucleotide sequences of the 16S rDNA gene of virulent strains of Yersinia ruckeri were analysed. For development of molecular diagnostic of Y. ruckeri the method of polymerase chain reaction (PCR was used. Primer selection was carried out in software VectorNTI11 and on-line-service BLAST. The PCR products were investigated by the methods of sequencing and nucleotide analysis. Findings. Based on PCR assay the method of molecular diagnostic of enteric redmouth disease agent, bacterium Y. ruckeri was developed. It was shown that specific oligonucleotide primers generated PCR products in size of 600 base pairs. PCR products were investigated by the sequencing that showed right targeting of primers in reaction. Originality. Among high-conservative gene of 16S rDNA of Y. ruckeri the fragment of DNA was determined to which the specific primers for rapid diagnostic of virulent strains were selected. Practical Value. Rapid diagnostic of yersiniosis will allow to identify an agent of this infectious disease, bacterium Y. ruckeri, and to provide the prophylactic or medical measures in the fish farming of Ukraine.

  8. Endothelial progenitor cells display clonal restriction in multiple myeloma

    Dai Kezhi


    Full Text Available Abstract Background In multiple myeloma (MM, increased neoangiogenesis contributes to tumor growth and disease progression. Increased levels of endothelial progenitor cells (EPCs contribute to neoangiogenesis in MM, and, importantly, covary with disease activity and response to treatment. In order to understand the mechanisms responsible for increased EPC levels and neoangiogenic function in MM, we investigated whether these cells were clonal by determining X-chromosome inactivation (XCI patterns in female patients by a human androgen receptor assay (HUMARA. In addition, EPCs and bone marrow cells were studied for the presence of clonotypic immunoglobulin heavy-chain (IGH gene rearrangement, which indicates clonality in B cells; thus, its presence in EPCs would indicate a close genetic link between tumor cells in MM and endothelial cells that provide tumor neovascularization. Methods A total of twenty-three consecutive patients who had not received chemotherapy were studied. Screening in 18 patients found that 11 displayed allelic AR in peripheral blood mononuclear cells, and these patients were further studied for XCI patterns in EPCs and hair root cells by HUMARA. In 2 patients whose EPCs were clonal by HUMARA, and in an additional 5 new patients, EPCs were studied for IGH gene rearrangement using PCR with family-specific primers for IGH variable genes (VH. Results In 11 patients, analysis of EPCs by HUMARA revealed significant skewing (≥ 77% expression of a single allele in 64% (n = 7. In 4 of these patients, XCI skewing was extreme (≥ 90% expression of a single allele. In contrast, XCI in hair root cells was random. Furthermore, PCR amplification with VH primers resulted in amplification of the same product in EPCs and bone marrow cells in 71% (n = 5 of 7 patients, while no IGH rearrangement was found in EPCs from healthy controls. In addition, in patients with XCI skewing in EPCs, advanced age was associated with poorer clinical status

  9. Complement-Opsonized HIV-1 Overcomes Restriction in Dendritic Cells.

    Wilfried Posch


    Full Text Available DCs express intrinsic cellular defense mechanisms to specifically inhibit HIV-1 replication. Thus, DCs are productively infected only at very low levels with HIV-1, and this non-permissiveness of DCs is suggested to go along with viral evasion. We now illustrate that complement-opsonized HIV-1 (HIV-C efficiently bypasses SAMHD1 restriction and productively infects DCs including BDCA-1 DCs. Efficient DC infection by HIV-C was also observed using single-cycle HIV-C, and correlated with a remarkable elevated SAMHD1 T592 phosphorylation but not SAMHD1 degradation. If SAMHD1 phosphorylation was blocked using a CDK2-inhibitor HIV-C-induced DC infection was also significantly abrogated. Additionally, we found a higher maturation and co-stimulatory potential, aberrant type I interferon expression and signaling as well as a stronger induction of cellular immune responses in HIV-C-treated DCs. Collectively, our data highlight a novel protective mechanism mediated by complement opsonization of HIV to effectively promote DC immune functions, which might be in the future exploited to tackle HIV infection.

  10. Single Cell Analysis to locate the Restriction Point with respect to E2F Expression

    Pimienta, R.; Johnson, A.


    The restriction point is a G1-phase checkpoint that regulates passage through the cell cycle and is misregulated in all known types of cancer. The Rb-E2F switch is thought to be one of the most relevant molecular mechanisms which regulate the restriction point in mammalian cells. However, recent experiments have brought the timing of the restriction point into question. In previous studies, cells were analyzed as populations and this prevented an accurate determination of the restriction point. By creating and analyzing an E2F-GFP reporter in single cells, we can pinpoint the timing of E2F activation and determine whether it coincides with the restriction point. Using calcium phosphate and Fugene,we transfected human embryonic kidney (293T) cells with a CMV-GFP plasmid and an E2F-GFP reporter. Based on our results, it appears that calcium phosphate is more effective than Fugene at transfecting mammalian cells. The calcium phosphate transfection had 9.59% more fluorescent cells than Fugene. However, this result only occurred with the CMV-GFP plasmid and not the E2F-GFP reporter, which was not properly expressed in human embryonic kidney (293T) cells. We will continue troubleshooting to fix this reporter as we proceed with our research. Once the reporter is properly cloned, we will transfect it into retinal pigmented epithelial (RPE1-hTERT) cells using the calcium phosphate method. RPE1-hTERT cells are an immortalized with telomerase and are more close to normal cells than tumor-derived cell lines. Through this research we will better comprehend commitment to the mammalian cell cycle.

  11. Extrachromosomal recombination substrates recapitulate beyond 12/23 restricted VDJ recombination in nonlymphoid cells.

    Jung, David; Bassing, Craig H; Fugmann, Sebastian D; Cheng, Hwei-Ling; Schatz, David G; Alt, Frederick W


    V(D)J recombination occurs efficiently only between gene segments flanked by recombination signals (RSs) containing 12 and 23 base pair spacers (the 12/23 rule). A further limitation "beyond the 12/23 rule" (B12/23) exists at the TCRbeta locus and ensures Dbeta usage. Herein, we show that extrachromosomal V(D)J recombination substrates recapitulate B12/23 restriction in nonlymphoid cells. We further demonstrate that the Vbeta coding flank, the 12-RS heptamer/nonamer, and the 23-RS spacer each can significantly influence B12/23 restriction. Finally, purified core RAG1 and RAG2 proteins (together with HMG2) also reproduce B12/23 restriction in a cell-free system. Our findings indicate that B12/23 restriction of V(D)J recombination is cemented at the level of interactions between the RAG proteins and TCRbeta RS sequences. PMID:12530976

  12. Transcriptome changes associated with anaerobic growth in Yersinia intermedia (ATCC29909.

    Lavanya Babujee

    Full Text Available BACKGROUND: The yersiniae (Enterobacteriaceae occupy a variety of niches, including some in human and flea hosts. Metabolic adaptations of the yersiniae, which contribute to their success in these specialized environments, remain largely unknown. We report results of an investigation of the transcriptome under aerobic and anaerobic conditions for Y. intermedia, a non-pathogenic member of the genus that has been used as a research surrogate for Y. pestis. Y. intermedia shares characteristics of pathogenic yersiniae, but is not known to cause disease in humans. Oxygen restriction is an important environmental stimulus experienced by many bacteria during their life-cycles and greatly influences their survival in specific environments. How oxygen availability affects physiology in the yersiniae is of importance in their life cycles but has not been extensively characterized. METHODOLOGY/PRINCIPAL FINDINGS: Tiled oligonucleotide arrays based on a draft genome sequence of Y. intermedia were used in transcript profiling experiments to identify genes that change expression in response to oxygen availability during growth in minimal media with glucose. The expression of more than 400 genes, constituting about 10% of the genome, was significantly altered due to oxygen-limitation in early log phase under these conditions. Broad functional categorization indicated that, in addition to genes involved in central metabolism, genes involved in adaptation to stress and genes likely involved with host interactions were affected by oxygen-availability. Notable among these, were genes encoding functions for motility, chemotaxis and biosynthesis of cobalamin, which were up-regulated and those for iron/heme utilization, methionine metabolism and urease, which were down-regulated. CONCLUSIONS/SIGNIFICANCE: This is the first transcriptome analysis of a non-pathogenic Yersinia spp. and one of few elucidating the global response to oxygen limitation for any of the

  13. Vasodilator-stimulated phosphoprotein restricts cell-to-cell spread of Shigella flexneri at the cell periphery.

    Lee, Soo Young; Gertler, Frank B; Goldberg, Marcia B


    Shigella spp. are intracellular bacterial pathogens that cause diarrhoeal disease in humans. Shigella utilize the host actin cytoskeleton to enter cells, move through the cytoplasm of cells and pass into adjacent cells. Ena/VASP family proteins are highly conserved proteins that participate in actin-dependent dynamic cellular processes. We tested whether Ena/VASP family members VASP (vasodilator-stimulated phosphoprotein), Mena (mammalian-enabled) or EVL (Ena-VASP-like) contribute to Shigella flexneri spread through cell monolayers. VASP and EVL restricted cell-to-cell spread without significantly altering actin-based motility, whereas Mena had no effect on these processes. Phosphorylation of VASP on Ser153, Ser235 and Thr274 regulated its subcellular distribution and function. VASP derivatives that lack the Ena/VASP homology 1 (EVH1) domain or contain a phosphoablative mutation of Ser153 were defective in restricting S. flexneri spread, indicating that the EVH1 domain and phosphorylation on Ser153 are required for this process. The EVH1 domain and Ser153 of VASP were required for VASP localization to focal adhesions, and localization of VASP to focal adhesions and/or the leading edge was required for restriction of spread. The contribution of the EVH1 domain was from both the donor and the recipient cell, whereas the contribution of Ser153 phosphorylation was only from the donor cell. Thus, unlike host proteins characterized in Shigella pathogenesis that promote bacterial spread, VASP and EVL function to limit it. The ability of VASP and EVL to limit spread highlights the critical role of focal adhesion complexes and/or the leading edge in bacterial passage between cells. PMID:26358985

  14. Polyclonal B-cell response to stimulation with Escherichia coli lipopolysaccharide in dietary protein restriction.

    Malavé, I; Pocino, M


    The polyclonal B-cell response to Escherichia coli lipopolysaccharide was studied in C57BL/6 mice maintained after weaning on either a moderate protein-restricted diet with 8% casein or a normal diet. After in vitro or in vivo stimulation with the endotoxin, autoreactive and anti-hapten antibody-producing cells were quantitated by direct plaque assay, using bromelain-treated mouse erythrocytes and trinitrophenylated sheep erythrocytes as targets. Larger numbers of plaque-forming cells were ge...

  15. Passage-restricted differentiation potential of mesenchymal stem cells into cardiomyocyte-like cells

    Mesenchymal stem cells (MSCs) have limited ability to differentiate into cardiomyocytes and the factors affect this process are not fully understood. In this study, we investigated the passage (P)-related transdifferentiation potential of MSCs into cardiomyocyte-like cells and its relationship to the proliferation ability. After 5-azacytidine treatment, only P4 but not P1 and P8 rat bone marrow MSCs (rMSCs) showed formation of myotube and expressed cardiomyocyte-associated markers. The growth property analysis showed P4 rMSCs had a growth-arrest appearance, while P1 and P8 rMSCs displayed an exponential growth pattern. When the rapid proliferation of P1 and P8 rMSCs was inhibited by 5-bromo-2-deoxyuridine, a mitosis inhibitor, only P1, not P8 rMSCs, differentiated into cardiomyocyte-like cells after 5-azacytidine treatment. These results demonstrate that the differentiation ability of rMSCs into cardiomyocytes is in proliferation ability-dependent and passage-restricted patterns. These findings reveal a novel regulation on the transdifferentiation of MSCs and provide useful information for exploiting the clinical therapeutic potential of MSCs

  16. Interplay of Notch and FGF signaling restricts cell fate and MAPK activation in the Drosophila trachea.

    Ikeya, T; Hayashi, S


    The patterned branching in the Drosophila tracheal system is triggered by the FGF-like ligand Branchless that activates a receptor tyrosine kinase Breathless and the MAP kinase pathway. A single fusion cell at the tip of each fusion branch expresses the zinc-finger gene escargot, leads branch migration in a stereotypical pattern and contacts with another fusion cell to mediate fusion of the branches. A high level of MAP kinase activation is also limited to the tip of the branches. Restriction of such cell specialization events to the tip is essential for tracheal tubulogenesis. Here we show that Notch signaling plays crucial roles in the singling out process of the fusion cell. We found that Notch is activated in tracheal cells by Branchless signaling through stimulation of &Dgr; expression at the tip of tracheal branches and that activated Notch represses the fate of the fusion cell. In addition, Notch is required to restrict activation of MAP kinase to the tip of the branches, in part through the negative regulation of Branchless expression. Notch-mediated lateral inhibition in sending and receiving cells is thus essential to restrict the inductive influence of Branchless on the tracheal tubulogenesis. PMID:10498681

  17. Impact of HLA class I restricted T cells on HIV-1 disease progression

    Schellens, I.M.M.


    This thesis focuses on the impact of HLA class I restricted T cells on HIV-1 disease progression. It is generally accepted that cytotoxic T lymphocytes (CTL) play an important role in controlling HIV replication. In line with this, it has been well established that HLA class I alleles influence the

  18. Two mechanisms that account for major histocompatibility complex restriction of T cells

    Kranz, David M.


    In recent studies, two distinct mechanisms have been proposed to account for major histocompatibility complex (MHC) restriction of T-cell activity: (a) evolution-driven interactions between T-cell receptor (TCR) variable regions and MHC, and (b) a requirement for CD4 or CD8 binding to MHC to initiate signalling through the TCR complex. Both mechanisms are likely to be essential, but for different reasons.

  19. Delineation of Several DR-Restricted Tetanus Toxin T Cell Epitopes

    Demotz, Stephane; Lanzavecchia, Antonio; Eisel, Ulrich; Niemann, Heiner; Widmann, Christian; Corradin, Giampietro


    We have characterized five human T cell clones specific for tetanus toxin. The combination of different techniques allowed us to precisely map two T cell epitopes within fragments 830-843 and 1273-1284 of tetanus toxin, as formally demonstrated by the use of corresponding synthetic peptides. The three other T cell clones were specific for regions 2-602, 604-742, and 865-1315 of tetanus toxin, respectively. The five T cell clones were shown to be restricted to HLA-DR Ag. Furthermore, the allel...

  20. The Immunology of CD1- and MR1-Restricted T Cells.

    Mori, Lucia; Lepore, Marco; De Libero, Gennaro


    CD1- and MHC-related molecule-1 (MR1)-restricted T lymphocytes recognize nonpeptidic antigens, such as lipids and small metabolites, and account for a major fraction of circulating and tissue-resident T cells. They represent a readily activated, long-lasting population of effector cells and contribute to the early phases of immune response, orchestrating the function of other cells. This review addresses the main aspects of their immunological functions, including antigen and T cell receptor repertoires, mechanisms of nonpeptidic antigen presentation, and the current evidence for their participation in human and experimental diseases. PMID:26927205

  1. Calorie Restriction-Mediated Replicative Lifespan Extension in Yeast Is Non-Cell Autonomous

    Szu-Chieh Mei; Charles Brenner


    In laboratory yeast strains with Sir2 and Fob1 function, wild-type NAD+ salvage is required for calorie restriction (CR) to extend replicative lifespan. CR does not significantly alter steady state levels of intracellular NAD+ metabolites. However, levels of Sir2 and Pnc1, two enzymes that sequentially convert NAD+ to nicotinic acid (NA), are up-regulated during CR. To test whether factors such as NA might be exported by glucose-restricted mother cells to survive later generations, we develop...

  2. Lineage-restricted expression of homeobox-containing genes in human hematopoietic cell lines

    The authors investigated the role of homeobox-containing genes in human hematopoiesis because homeobox genes (i) control cell fate in the Drosophila embryo, (ii) are expressed in specific patterns in human embryos, and (iii) appear to function as transcription factors that control cell phenotype in other mammalian organs. Using four homeobox probes from the HOX2 locus and a previously undescribed homeobox cDNA (PL1), they screened mRNAs from 18 human leukemic cell lines representing erythroid, myeloid, and T- and B-cell lineages. Complex patterns of lineage-restricted expression are observed. No single homeobox gene is expressed in all types of hematopoietic cells, but each cell type exhibits homeobox gene expression. They have demonstrated (i) lineage-restricted expression of five homeobox genes in erythroid and monocytic cell lines; (ii) expression of additional homeobox genes in other cell lineages (HL-60 and lymphoid cells); (iii) expression of one homeobox gene in normal marrow cells; and (iv) modulation of expression during differentiation. These data suggest that these genes play a role in human hematopoietic development and lineage commitment

  3. APOBEC3G restricts early HIV-1 replication in the cytoplasm of target cells

    Cellular APOBEC3G (A3G) protein is packaged into human immunodeficiency virus type 1 (HIV-1) virions in producer cells yet restricts viral replication in target cells. To characterize this restriction in target cells, the effect of A3G on generating various HIV-1 cDNA products was measured by quantitative real-time PCR. A3G decreased cDNA products from Vif-deficient HIV-1, with minor effects on early reverse transcripts and larger declines in late reverse transcripts. However, the greatest decline was typically observed in nuclear 2-LTR circles. Moreover, the magnitude of these declines varied with A3G dose. Adding integration inhibitor did not stop the A3G-mediated loss in 2-LTR circles. Moreover, obstructing HIV-1 nuclear entry using vesicular stomatitis virus matrix protein did not stop the A3G-mediated decline in late reverse transcripts. Collectively, these data suggest that A3G has important restriction activity in the cytoplasm and progressively diminishes viral cytoplasmic and nuclear cDNA forms with increasing magnitude during restriction

  4. Cell-Specific Expression of Connexins and Evidence of Restricted Gap Junctional Coupling between Glial Cells and between Neurons

    Rash, John E.; Yasumura, Thomas; Dudek, F. Edward; NAGY, JAMES I.


    The transmembrane connexin proteins of gap junctions link extracellularly to form channels for cell-to-cell exchange of ions and small molecules. Two primary hypotheses of gap junction coupling in the CNS are the following: (1) generalized coupling occurs between neurons and glia, with some connexins expressed in both neurons and glia, and (2) intercellular junctional coupling is restricted to specific coupling partners, with different connexins expressed in each cell type. There is consensus...

  5. CD1d-restricted peripheral T cell lymphoma in mice and humans.

    Bachy, Emmanuel; Urb, Mirjam; Chandra, Shilpi; Robinot, Rémy; Bricard, Gabriel; de Bernard, Simon; Traverse-Glehen, Alexandra; Gazzo, Sophie; Blond, Olivier; Khurana, Archana; Baseggio, Lucile; Heavican, Tayla; Ffrench, Martine; Crispatzu, Giuliano; Mondière, Paul; Schrader, Alexandra; Taillardet, Morgan; Thaunat, Olivier; Martin, Nadine; Dalle, Stéphane; Le Garff-Tavernier, Magali; Salles, Gilles; Lachuer, Joel; Hermine, Olivier; Asnafi, Vahid; Roussel, Mikael; Lamy, Thierry; Herling, Marco; Iqbal, Javeed; Buffat, Laurent; Marche, Patrice N; Gaulard, Philippe; Kronenberg, Mitchell; Defrance, Thierry; Genestier, Laurent


    Peripheral T cell lymphomas (PTCLs) are a heterogeneous entity of neoplasms with poor prognosis, lack of effective therapies, and a largely unknown pathophysiology. Identifying the mechanism of lymphomagenesis and cell-of-origin from which PTCLs arise is crucial for the development of efficient treatment strategies. In addition to the well-described thymic lymphomas, we found that p53-deficient mice also developed mature PTCLs that did not originate from conventional T cells but from CD1d-restricted NKT cells. PTCLs showed phenotypic features of activated NKT cells, such as PD-1 up-regulation and loss of NK1.1 expression. Injections of heat-killed Streptococcus pneumonia, known to express glycolipid antigens activating NKT cells, increased the incidence of these PTCLs, whereas Escherichia coli injection did not. Gene expression profile analyses indicated a significant down-regulation of genes in the TCR signaling pathway in PTCL, a common feature of chronically activated T cells. Targeting TCR signaling pathway in lymphoma cells, either with cyclosporine A or anti-CD1d blocking antibody, prolonged mice survival. Importantly, we identified human CD1d-restricted lymphoma cells within Vδ1 TCR-expressing PTCL. These results define a new subtype of PTCL and pave the way for the development of blocking anti-CD1d antibody for therapeutic purposes in humans. PMID:27069116

  6. Cytotoxic T lymphocyte responses in allogeneic radiation bone marrow chimeras. The chimeric host strictly dictates the self-repertoire of Ia-restricted T cells but not H-2K/D-restricted T cells

    The present report has used fully H-2 allogeneic radiation bone marrow chimeras to assess the role of host restriction elements in determining the self-specificity of Ia- and H-2K/D-restricted T cells that participate in the generation of trinitrophenyl (TNP)-specific cytotoxic T lymphocytes (CTL). It was demonstrated that there exists a stringent requirement for the recognition of host thymic-type Ia determinants, but there exists only a preference for host thymic-type H-2K/D determinants. Indeed, once the stringent requirement for recognition of host Ia determinants was fulfilled, anti-TNP CTL were generated in response to TNP-modified stimulators that expressed either donor-type or host-type H-2K/D determinants. The CTL that were generated in response to TNP-modified donor-type stimulators were shown to be specific for TNP and restricted to the non-thymic H-2K/D determinants of the chimeric donor. Thus, these results demonstrate in a single immune response that the thymic hypothesis accurately predicts the self-specificity expressed by Ia-restricted T cells, but does not fully account for the self-specificity expressed by H-2K/D-restricted T cells. These results are consistent with the concept that H-2K/D-restricted T cells, but not Ia-restricted T cells, can differentiate into functional competence either intrathymically or extra-thymically. The results demonstrate that the generation of anti-TNP CTL responses involve two parallel sets of major histocompatibility complex-restricted cell interactions, an Ia-restricted TH-accessory cell interaction required for TH cell activation, and an H-2K/D-restricted pCTL-stimulator cell interaction required for pCTL stimulation. The interaction between activated TH cells and stimulated pCTL is mediated, at least in part, by nonspecific soluble helper factors

  7. Regulation and expression of Lcr plasmid-mediated peptides in pesticinogenic Yersinia pestis

    It is shown in this thesis that cells of Lcr+, Pst- Y. pestis KIM are able to express Yops at levels comparable to that of Lcr+ Yersinia pseudotuberculosis. Pulse-chase radiolabeling with 35S-methionine was used to demonstrate that Lcr+, Pst+ Y. pestis synthesized at least 11 distinct peptides during the low calcium response and that seven of the labeled peptides were rapidly degraded. These seven peptides were stably expressed in Lcr+, Pst- Y. pestis and were of identical molecular weights as the Yops expressed by that strain. Radiolabeled fragments of low molecular weight accumulated in the extracellular medium of Pst+ cultures and were assumed to be stable degradation fragments derived from Yops. It was also shown that the set of stable peptides, including V antigen, were made during restriction by both Pst+ and Pst- Y. pestis KIM and were located primarily within the cytoplasm. Those radiolabeled peptides which underwent proteolytic degradation in Pst+ Y. pestis were localized to the outer membrane and extracellular medium in the Pst- strain. It is concluded that the failure of Lcr+, Pst+ Y. pestis to express Yops is the result of post-translational degradation and is not a block in the synthesis of Yops

  8. Demonstration of a Novel HIV-1 Restriction Phenotype from a Human T Cell Line

    Han, Yanxing; Xiaojun WANG; Dang, Ying; Zheng, Yong-Hui


    Background Although retroviruses may invade host cells, a productive infection can be established only after the virus counteracts inhibition from different types of host restriction factors. Fv1, APOBEC3G/F, TRIM5α, ZAP, and CD317 inhibit the replication of different retroviruses by interfering with viral uncoating, reverse transcription, nuclear import, RNA stability, and release. In humans, although APOBEC3G/3F and CD317 block HIV-1 replication, their antiviral activities are neutralized b...

  9. Why drivers use cell phones and support legislation to restrict this practice.

    Sanbonmatsu, David M; Strayer, David L; Behrends, Arwen A; Ward, Nathan; Watson, Jason M


    The use of cell phones while driving is ubiquitous, particularly in countries where the practice is legal. However, surveys indicate that most drivers favor legislation to limit the use of mobile devices during the operation of a vehicle. A study was conducted to understand this inconsistency between what drivers do and what they advocate for others. Participants completed a survey about their driving attitudes, abilities, and behaviors. Following previous research, drivers reported using cell phones for benefits such as getting work done. The hypocrisy of using cell phones while advocating restrictions appears to stem from differences in the perceived safety risks of self vs. others' use of cell phones. Many if not most drivers believe they can drive safely while using mobile devices. However, they lack confidence in others' ability to drive safely while distracted and believe that others' use of cell phones is dangerous. The threat to public safety of others' usage of mobile devices was one of the strongest independent predictors of support for legislation to restrict cell phone use. PMID:27035396

  10. Restriction of intestinal stem cell expansion and the regenerative response by YAP.

    Barry, Evan R; Morikawa, Teppei; Butler, Brian L; Shrestha, Kriti; de la Rosa, Rosemarie; Yan, Kelley S; Fuchs, Charles S; Magness, Scott T; Smits, Ron; Ogino, Shuji; Kuo, Calvin J; Camargo, Fernando D


    A remarkable feature of regenerative processes is their ability to halt proliferation once an organ's structure has been restored. The Wnt signalling pathway is the major driving force for homeostatic self-renewal and regeneration in the mammalian intestine. However, the mechanisms that counterbalance Wnt-driven proliferation are poorly understood. Here we demonstrate in mice and humans that yes-associated protein 1 (YAP; also known as YAP1)--a protein known for its powerful growth-inducing and oncogenic properties--has an unexpected growth-suppressive function, restricting Wnt signals during intestinal regeneration. Transgenic expression of YAP reduces Wnt target gene expression and results in the rapid loss of intestinal crypts. In addition, loss of YAP results in Wnt hypersensitivity during regeneration, leading to hyperplasia, expansion of intestinal stem cells and niche cells, and formation of ectopic crypts and microadenomas. We find that cytoplasmic YAP restricts elevated Wnt signalling independently of the AXIN-APC-GSK-3β complex partly by limiting the activity of dishevelled (DVL). DVL signals in the nucleus of intestinal stem cells, and its forced expression leads to enhanced Wnt signalling in crypts. YAP dampens Wnt signals by restricting DVL nuclear translocation during regenerative growth. Finally, we provide evidence that YAP is silenced in a subset of highly aggressive and undifferentiated human colorectal carcinomas, and that its expression can restrict the growth of colorectal carcinoma xenografts. Collectively, our work describes a novel mechanistic paradigm for how proliferative signals are counterbalanced in regenerating tissues. Additionally, our findings have important implications for the targeting of YAP in human malignancies. PMID:23178811

  11. Restriction specificity of virus-specific cytotoxic T cells from thymectomised irradiated bone marrow chimeras reconstituted with thymus grafts

    Adult-thymectomised lethally irradiated mice A that were reconstituted with T-cell-depleted bone marrow cells of (A X B)F1 origin plus fetal thymus grafts of (B X C)F1 origin generated virus-specific T cells restricted to B alone; adult-thymectomised and lethally irradiated (A X B)F1 mice that were reconstituted with T-cell depleted bone marrow cells of (A X B)F1 origin plus fetal thymus grafts of A and of B origin generated virus-specific T cells restricted to A or to B. These results do not reveal obvious suppressive influences of host or stem-cell origin that might have explained results obtained with various irradiated bone marrow or thymus chimeras, they indicate that the thymus' influence on maturing T cells is one of the limiting steps in the selection of T cells' restriction specificities. (Auth.)


    V. V. Evseeva


    Full Text Available Plague has been the cause of three pandemics and has led to the death of millions of people. Plague is a typical zoonosis caused by Yersinia pestis that circulates in populations of wild rodents inhabiting natural plague foci on all continents except for Australia. Transmission of plague is provided by flea bites. Circulation of Y. pestis in natural plague foci is supported by a numerous of pathogenicity factors. This review explores one of them, plasminogen activator Pla. This protein is one of representatives of omptins, a family of enterobacterial outer membrane proteases that are responsible for colonization of specific organs or even infection generalization as a result of successful overcoming of the host innate immunity. The review reflects the history of its discovery and studying of its genetic control, biosynthesis, isolation and purification, physicochemical properties. Highly purified preparations of plasminogen activator are deficient in enzymatic activities but renaturation in the presence of Y. pestis lipooligosaccharide restores enzymatic properties of Pla. This pathogenicity factor is absent in representatives of the most ancient phylogenetic group of the plague pathogen, bv. caucasica, while the ancestor of other groups of Y. pestis subsp. microtus obtained in result of horizontal transfer Pla isoform with characteristics similar to properties of omptins from the less virulent enterobacteria. After that in the course of microevolution the “classic” isoform of Pla with increased protease activity was selected that is typical of all highly virulent for humans strains of Y. pestis subsp. pestis. The “classic” isoform of Pla Y. pestis is functionally similar to mammalian plasminogen activators transforming plasminogen into plasmin with the help of limited proteolysis. Pla protease activating plasminogen and also degrading the main plasmin inhibitor — α2-antiplasmin and, respectively, determining Y. pestis ability to lyse

  13. Radioprotective effect of calorie restriction in Hela cells and SD rats

    Objective: To explore the effect of low calorie metabolism on the survival of HeLa cells exposed to X-rays, and the influence of starvation on the antioxidative factors in the blood of rats after irradiation. Methods: MTT method was used to evaluate the impact of different concentration glucose on the proliferation of HeLa cells. Colony formation assay was employed to detect the influence of glucose (1, 5, 10 and 25 mmol/L) on radiosensitivity of HeLa cells. Flow cytometry assay was used to analyze distribution of cell cycle and apoptosis. 60 male SD rats were randomly divided into 6 groups with 10 rats each. Rats in every two groups were fed ad libitum, fasted for 24 h and fasted for 48 h, respectively. Rats in one group of each approach were respectively exposed to whole-body X-rays at 11 Gy. At 2 h after irradiation,all of rats were sacrificed and their venous blood was collected. Elisa kits were used to detect superoxide dismutase (SOD) and total antioxidant capacity (T-AOC). Results: An increased viability was observed in HeLa cells treated with the glucose at low concentration (<25 mmol/L), while HeLa cell growth was inhibited by glucose at doses of >25 mmol/L. Relevant to cells treated with 1 mmoL/L glucose, SERs (sensitive enhancement ratio) in cells exposed to 5, 10 and 25 mmol/L glucose were 1.07, 1.10 and 1.23,respectively. A reduction of G2/M and S arrests and apoptosis caused by 6 Gy X-ray irradiation were observed [(49.68 ±1.88)% and (35.54±1.45)% at G2/M phase, (16.88 ±1.22)% and (10.23 ±1.65)% at S phase, t=10.42, 5.61, P<0.05] and in the cells treated with 1 mmol/L glucose compared with cells treated with 25 mmol/L glucose [(25.50 ± 0.95)% and (7.56 ± 1.07)%, t=21.72, P<0.05].Without irradiation, calorie restriction exhibited a negligible influence on SOD and T-AOC in rats. However, after 11 Gy irradiation, compared with rats fed ad libitum, the levels of SOD and T-AOC were significantly increased in rats with calorie restriction (t=40.32, 42

  14. Expression of hermes gene is restricted to the ganglion cells in the retina.

    Piri, Natik; Kwong, Jacky M K; Song, Min; Caprioli, Joseph


    The RNA binding protein with multiple splicing 2, or hermes, is a member of the RRM (RNA recognition motif) family of RNA-binding proteins. In this study, we show that the hermes gene is expressed in the rat retina, and its expression is restricted to the ganglion cell layer. Double in situ hybridization with riboprobes corresponding to the hermes gene and Thy-1, the RGC marker in the retina, showed that the majority of the Thy-1 positive cells in the ganglion cell layer were also hermes positive. This was also shown by co-localization of the hermes in situ hybridization signals with the retrogradely labeled RGCs. Our observations suggest that hermes is expressed in the majority, if not all, of RGCs and is not restricted to only certain RGC types. Hermes in situ hybridization signals were not detected in the retinal sections of optic nerve transected animals, which are characterized by rapid and specific RGC degeneration. The dramatic reduction of the hermes mRNA level in axotomized retinas was also observed by semi-quantitative RT-PCR. The specific expression of hermes in retinal ganglion cells qualifies this gene as a potential RGC marker in the retina. Outside the retina, hermes is expressed in the heart, liver, and kidney, and to a lesser degree in the cerebellum, cortex, lung, and small intestine. PMID:16870336

  15. Self-recognition specificity expressed by T cells from nude mice. Absence of detectable Ia-restricted T cells in nude mice that do exhibit self-K/D-restricted T cell responses

    The presence in athymic nude mice of precursor T cells with self-recognition specificity for either H-2 K/D or H-2 I region determinants was investigated. Chimeras were constructed of lethally irradiated parental mice receiving a mixture of F1 nude mouse (6-8 wk old) spleen and bone marrow cells. The donor inoculum was deliberately not subjected to any T cell depletion procedure, so that any potential major histocompatibility complex-committed precursor T cells were allowed to differentiate and expand in the normal parental recipients. 3 mo after reconstitution, the chimeras were immunized with several protein antigens in complete Freund's adjuvant in the footpads and their purified draining lymph node T cells tested 10 d later for ability to recognize antigen on antigen-presenting cells of either parental haplotype. Also, their spleen and lymph node cells were tested for ability to generate a cytotoxic T lymphocyte (CTL) response to trinitrophenyl (TNP)-modified stimulator cells of either parental haplotype. It was demonstrated that T cell proliferative responses of these F1(nude)----parent chimeras were restricted solely to recognizing parental host I region determinants as self and expressed the Ir gene phenotype of the host. In contrast, CTL responses could be generated (in the presence of interleukin 2) to TNP-modified stimulator cells of either parental haplotype. Thus these results indicate that nude mice which do have CTL with self-specificity for K/D region determinants lack proliferating T cells with self-specificity for I region determinants. These results provide evidence for the concepts that development of the I region-restricted T cell repertoire is strictly an intrathymically determined event and that young nude mice lack the unique thymic elements responsible for education of I region-restricted T cells

  16. Investigating the ?Trojan Horse? Mechanism of Yersinia pestis Virulence

    McCutchen-Maloney, S L; Fitch, J P


    Yersinia pestis, the etiological agent of plague, is a Gram-negative, highly communicable, enteric bacterium that has been responsible for three historic plague pandemics. Currently, several thousand cases of plague are reported worldwide annually, and Y. pestis remains a considerable threat from a biodefense perspective. Y. pestis infection can manifest in three forms: bubonic, septicemic, and pneumonic plague. Of these three forms, pneumonic plague has the highest fatality rate ({approx}100% if left untreated), the shortest intervention time ({approx}24 hours), and is highly contagious. Currently, there are no rapid, widely available vaccines for plague and though plague may be treated with antibiotics, the emergence of both naturally occurring and potentially engineered antibiotic resistant strains makes the search for more effective therapies and vaccines for plague of pressing concern. The virulence mechanism of this deadly bacterium involves induction of a Type III secretion system, a syringe-like apparatus that facilitates the injection of virulence factors, termed Yersinia outer membrane proteins (Yops), into the host cell. These virulence factors inhibit phagocytosis and cytokine secretion, and trigger apoptosis of the host cell. Y. pestis virulence factors and the Type III secretion system are induced thermally, when the bacterium enters the mammalian host from the flea vector, and through host cell contact (or conditions of low Ca{sup 2+} in vitro). Apart from the temperature increase from 26 C to 37 C and host cell contact (or low Ca{sup 2+} conditions), other molecular mechanisms that influence virulence induction in Y. pestis are largely uncharacterized. This project focused on characterizing two novel mechanisms that regulate virulence factor induction in Y. pestis, immunoglobulin G (IgG) binding and quorum sensing, using a real-time reporter system to monitor induction of virulence. Incorporating a better understanding of the mechanisms of virulence

  17. Ebola virus mediated infectivity is restricted in canine and feline cells.

    Han, Ziying; Bart, Stephen M; Ruthel, Gordon; Vande Burgt, Nathan H; Haines, Kathleen M; Volk, Susan W; Vite, Charles H; Freedman, Bruce D; Bates, Paul; Harty, Ronald N


    Ebolaviruses and marburgviruses belong to the Filoviridae family and often cause severe, fatal hemorrhagic fever in humans and non-human primates. The magnitude of the 2014 outbreak in West Africa and the unprecedented emergence of Ebola virus disease (EVD) in the United States underscore the urgency to better understand the dynamics of Ebola virus infection, transmission and spread. To date, the susceptibility and possible role of domestic animals and pets in the transmission cycle and spread of EVD remains unclear. We utilized infectious VSV recombinants and lentivirus pseudotypes expressing the EBOV surface glycoprotein (GP) to assess the permissiveness of canine and feline cells to EBOV GP-mediated entry. We observed a general restriction in EBOV-mediated infection of primary canine and feline cells. To address the entry mechanism, we used cells deficient in NPC1, a host protein implicated in EBOV entry, and a pharmacological blockade of cholesterol transport, to show that an NPC1-dependent mechanism of EBOV entry is conserved in canine and feline cells. These data demonstrate that cells of canine and feline origin are susceptible to EBOV GP mediated infection; however, infectivity of these cells is reduced significantly compared to controls. Moreover, these data provide new insights into the mechanism of EBOV GP mediated entry into cells of canine and feline origin. PMID:26711035

  18. Glucose restriction can extend normal cell lifespan and impair precancerous cell growth through epigenetic control of hTERT and p16 expression

    Li, Yuanyuan; Liang LIU; Tollefsbol, Trygve O.


    Cancer cells metabolize glucose at elevated rates and have a higher sensitivity to glucose reduction. However, the precise molecular mechanisms leading to different responses to glucose restriction between normal and cancer cells are not fully understood. We analyzed normal WI-38 and immortalized WI-38/S fetal lung fibroblasts and found that glucose restriction resulted in growth inhibition and apoptosis in WI-38/S cells, whereas it induced lifespan extension in WI-38 cells. Moreover, in WI-3...

  19. Lsd1 Restricts the Number of Germline Stem Cells by Regulating Multiple Targets in Escort Cells

    Eliazer, Susan; Palacios, Victor; Wang, Zhaohui; Kollipara, Rahul K.; Kittler, Ralf; Buszczak, Michael


    Specialized microenvironments called niches regulate tissue homeostasis by controlling the balance between stem cell self-renewal and the differentiation of stem cell daughters. However the mechanisms that govern the formation, size and signaling of in vivo niches remain poorly understood. Loss of the highly conserved histone demethylase Lsd1 in Drosophila escort cells results in increased BMP signaling outside the cap cell niche and an expanded germline stem cell (GSC) phenotype. Here we pre...

  20. Mouse T-cells restrict replication of human immunodeficiency virus at the level of integration

    Goffinet Christine


    Full Text Available Abstract Background The development of an immunocompetent, genetically modified mouse model to study HIV-1 pathogenesis and to test antiviral strategies has been hampered by the fact that cells from native mice do not or only inefficiently support several steps of the HIV-1 replication cycle. Upon HIV-1 infection, mouse T-cell lines fail to express viral proteins, but the underlying replication barrier has thus far not been unambiguously identified. Here, we performed a kinetic and quantitative assessment of consecutive steps in the early phase of the HIV-1 replication cycle in T-cells from mice and humans. Results Both T-cell lines and primary T-cells from mice harbor a severe post-entry defect that is independent of potential species-specTR transactivation. Reverse transcription occurred efficiently following VSV-G-mediated entry of virions into mouse T-cells, and abundant levels of 2-LTR circles indicated successful nuclear import of the pre-integration complex. To probe the next step in the retroviral replication cycle, i.e. the integration of HIV-1 into the host cell genome, we established and validated a nested real-time PCR to specifically quantify HIV-1 integrants exploiting highly repetitive mouse B1 elements. Importantly, we demonstrate that the frequency of integrant formation is diminished 18- to > 305-fold in mouse T-cell lines compared to a human counterpart, resulting in a largely abortive infection. Moreover, differences in transgene expression from residual vector integrants, the transcription off which is cyclin T1-independent, provided evidence for an additional, peri-integrational deficit in certain mouse T-cell lines. Conclusion In contrast to earlier reports, we find that mouse T-cells efficiently support early replication steps up to and including nuclear import, but restrict HIV-1 at the level of chromosomal integration.

  1. [Yersinia pestis as a dangerous biological weapon].

    Grygorczuk, Sambor; Hermanowska-Szpakowicz, Teresa


    Plague is an infectious disease caused by the Yersinia pestis microorganism, which is transmitted to the human host from a natural reservoir (different rodent species) by a flea bite. Plague is still encountered in humans in the areas of its enzootic prevalence in local rodent populations. Infection by flea bite results in a bubonic or septicemic plague, possibly complicated by secondary pneumonia. The person with pneumonic symptoms may be a source of a droplet-borne inhalatory infection for other people who consequently develop primary pneumonic plague. Despite a clinical form, plague is a severe infection characterized by a short incubation period, rapid onset and quick progress with mortality exceeding 50% if not treated properly. The pneumonic plague is associated with a particularly rapid progress and the mortality rate of almost 100% if not treated properly. As Yersinia pestis can be easily obtained and cultured and is highly pathogenic for humans, it poses a serious threat of being used for bioterrorism purposes. Artificially created aerosol containing plague bacilli can cause numerous and almost simultaneous cases of primary pulmonic plague in an exposed population. Persons exposed would most likely develop severe pneumonia with rapidly progressing respiratory and circulatory failure. The use of the Yersinia pestis strains resistant to antibiotics typically applied cannot be excluded. PMID:12474416

  2. The response regulator PhoP negatively regulates Yersinia pseudotuberculosis and Yersinia pestis biofilms

    Sun, Yi-Cheng; Koumoutsi, Alexandra; Darby, Creg


    A few Yersinia pseudotuberculosis strains form biofilms on the head of the nematode Caenorhabditis elegans, but numerous others do not. We show that a widely used Y. pseudotuberculosis strain, YPIII, is biofilm positive because of a mutation in phoP, which encodes the response regulator of a two-component system. For two wild-type Y. pseudotuberculosis that do not make biofilms on C. elegans, deletion of phoP was sufficient to produce robust biofilms. In Yersinia pestis, a phoP mutant made mo...

  3. Performance comparison of CPCs with and without exit angle restriction for concentrating radiation on solar cells

    Highlights: • CPCs with and without exit angle restriction based PV modules were fabricated and tested. • Performance of both CPVs was investigated and compared. • Results showed that CPV-65 performed slightly but insignificantly better as compared to CPV-90. - Abstract: To perform this comparison, the compound parabolic concentrator with a restricted exit angle of 65° (CPC-65) and the one without exit angle restriction (CPC-90) were fabricated and tested for concentrating radiation on multi-crystalline solar cells. Both CPC-65 and CPC-90 are identical in the acceptance half-angle (20°) and geometrical concentration factor (2×). Theoretical calculations showed that CPC-90 based PV system (CPV-90) annually concentrated about 3–5% more radiation on solar cells as compared to CPC-65 based PV system (CPV-65). For CPV-65, all radiation would arrive on the solar cells at the incidence angle less than 65°, but for CPV-90, about 8–10% of annual collectible radiation would arrive on solar cells at the incidence angle larger than 65°. Measurements at outdoor conditions showed that the CPV-65 performed slightly better than CPV-90 in terms of short-circuit current and power output as the projection incidence angle of solar rays on the cross-section of CPC-troughs (θp) less than the acceptance half-angle, otherwise the CPV-90 did better. Compared to CPV-90, the power output at maximum power points from CPV-65 were slightly higher, and increases of 2.1%, 5.4% and 8.17% were measured for θp = 0°, 10° and 16°, respectively. Analysis indicated that effect of solar flux distribution over solar cells on power output of both CPVs was almost identical and insignificant, and the CPV-65 performed slightly but insignificantly better than the CPV-90 in terms of annual power output except in areas with poor solar resources where the annual power output from both systems was almost identical

  4. Global Expression Studies of Yersinia Pestis Pathogenicity

    Garcia, E; Motin, V; Brubaker, R; Fitch, P


    The aim of these studies continues to be the investigation into the molecular mechanisms that underlie the virulence process in Yersinia pestis. In particular, the focus of this work centers on the identification of novel genes and pathways responsible for the pathogenic properties of this organism. In spite of more than four decades of intense investigation in this field, the dilemma as to what makes Y. pestis such a virulent and lethal pathogen remains unanswered. The method being employed makes use microarray technology (DNA chip) that enables the examination of the global activities of the whole complement of genes in this pathogen. Two primary resources available to the investigators (one directly obtained from a separate CBNP-funded project) make these studies possible: (1) Whole genome comparisons of the genes in Y. pestis and its near neighbors with attenuated or non pathogenic characteristics, and (2) the ability to duplicate in vitro, conditions that mimic the infection process of this pathogen. This year we have extended our studies from the original work of characterizing the global transcriptional regulation in Y. pestis triggered during temperature transition from 26 C to 37 C (roughly conditions found in the flea vector and the mammalian host, respectively) to studies of regulation encountered during shift between growth from conditions of neutral pH to acidic pH (the latter conditions, those mimic the environment found inside macrophages, a likely environment found by these cells during infection.). For this work, DNA arrays containing some 5,000 genes (the entire genome of Y. pestis plus those genes found uniquely in the enteropathogen, and near neighbor, Y. pseudotuberculosis) are used to monitor the simultaneous expression levels of each gene of known and unknown function in Y. pestis. Those genes that are up-regulate under the experimental conditions represent genes potentially involved in the pathogenic process. The ultimate role in

  5. Restricted mobility of specific functional groups reduces anti-cancer drug activity in healthy cells

    Martins, Murillo L.; Ignazzi, Rosanna; Eckert, Juergen; Watts, Benjamin; Kaneno, Ramon; Zambuzzi, Willian F.; Daemen, Luke; Saeki, Margarida J.; Bordallo, Heloisa N.


    The most common cancer treatments currently available are radio- and chemo-therapy. These therapies have, however, drawbacks, such as, the reduction in quality of life and the low efficiency of radiotherapy in cases of multiple metastases. To lessen these effects, we have encapsulated an anti-cancer drug into a biocompatible matrix. In-vitro assays indicate that this bio-nanocomposite is able to interact and cause morphological changes in cancer cells. Meanwhile, no alterations were observed in monocytes and fibroblasts, indicating that this system might carry the drug in living organisms with reduced clearance rate and toxicity. X-rays and neutrons were used to investigate the carrier structure, as well as to assess the drug mobility within the bio-nanocomposite. From these unique data we show that partial mobility restriction of active groups of the drug molecule suggests why this carrier design is potentially safer to healthy cells.

  6. A novel lineage restricted, pericyte-like cell line isolated from human embryonic stem cells.

    Greenwood-Goodwin, Midori; Yang, Jiwei; Hassanipour, Mohammad; Larocca, David


    Pericytes (PCs) are endothelium-associated cells that play an important role in normal vascular function and maintenance. We developed a method comparable to GMP quality protocols for deriving self-renewing perivascular progenitors from the human embryonic stem cell (hESC), line ESI-017. We identified a highly scalable, perivascular progenitor cell line that we termed PC-A, which expressed surface markers common to mesenchymal stromal cells. PC-A cells were not osteogenic or adipogenic under standard differentiation conditions and showed minimal angiogenic support function in vitro. PC-A cells were capable of further differentiation to perivascular progenitors with limited differentiation capacity, having osteogenic potential (PC-O) or angiogenic support function (PC-M), while lacking adipogenic potential. Importantly, PC-M cells expressed surface markers associated with pericytes. Moreover, PC-M cells had pericyte-like functionality being capable of co-localizing with human umbilical vein endothelial cells (HUVECs) and enhancing tube stability up to 6 days in vitro. We have thus identified a self-renewing perivascular progenitor cell line that lacks osteogenic, adipogenic and angiogenic potential but is capable of differentiation toward progenitor cell lines with either osteogenic potential or pericyte-like angiogenic function. The hESC-derived perivascular progenitors described here have potential applications in vascular research, drug development and cell therapy. PMID:27109637

  7. Escargot Restricts Niche Cell to Stem Cell Conversion in the Drosophila Testis

    Justin Voog


    Full Text Available Stem cells reside within specialized microenvironments, or niches, that control many aspects of stem cell behavior. Somatic hub cells in the Drosophila testis regulate the behavior of cyst stem cells (CySCs and germline stem cells (GSCs and are a primary component of the testis stem cell niche. The shutoff (shof mutation, characterized by premature loss of GSCs and CySCs, was mapped to a locus encoding the evolutionarily conserved transcription factor Escargot (Esg. Hub cells depleted of Esg acquire CySC characteristics and differentiate as cyst cells, resulting in complete loss of hub cells and eventually CySCs and GSCs, similar to the shof mutant phenotype. We identified Esg-interacting proteins and demonstrate an interaction between Esg and the corepressor C-terminal binding protein (CtBP, which was also required for maintenance of hub cell fate. Our results indicate that niche cells can acquire stem cell properties upon removal of a single transcription factor in vivo.

  8. Escargot restricts niche cell to stem cell conversion in the Drosophila testis.

    Voog, Justin; Sandall, Sharsti L; Hime, Gary R; Resende, Luís Pedro F; Loza-Coll, Mariano; Aslanian, Aaron; Yates, John R; Hunter, Tony; Fuller, Margaret T; Jones, D Leanne


    Stem cells reside within specialized microenvironments, or niches, that control many aspects of stem cell behavior. Somatic hub cells in the Drosophila testis regulate the behavior of cyst stem cells (CySCs) and germline stem cells (GSCs) and are a primary component of the testis stem cell niche. The shutoff (shof) mutation, characterized by premature loss of GSCs and CySCs, was mapped to a locus encoding the evolutionarily conserved transcription factor Escargot (Esg). Hub cells depleted of Esg acquire CySC characteristics and differentiate as cyst cells, resulting in complete loss of hub cells and eventually CySCs and GSCs, similar to the shof mutant phenotype. We identified Esg-interacting proteins and demonstrate an interaction between Esg and the corepressor C-terminal binding protein (CtBP), which was also required for maintenance of hub cell fate. Our results indicate that niche cells can acquire stem cell properties upon removal of a single transcription factor in vivo. PMID:24794442

  9. Generation of MHC class I-restricted cytotoxic T cell lines and clones against colonic epithelial cells from ulcerative colitis.

    Yonamine, Y; Watanabe, M; Kinjo, F; Hibi, T


    We established CTL lines and clones against colonic epithelial cells from PBLs of patients with ulcerative colitis by continuous stimulation with HLA-A locus-matched colonic epithelial cell lines. We developed a nonradioactive europium release cytotoxicity assay to detect CTLs. PBLs from 3 of 12 patients but not from any of 14 normal controls who shared at least one haplotype of HLA-A locus with two colonic epithelial cell lines, CW2 and ACM, showed increased cytotoxicity against these lines. Three CTL lines established from the PBLs of patients showed increased cytotoxicity against HLA-A locus-matched CW2 or ACM but not against matched lung or esophagus cell lines. The phenotypes of CTL lines were alpha beta-TCR+ CD3+ CD8+ CD16-. The CTL line MS showed increased cytotoxicity against freshly isolated colonic epithelial cells but not against cells with a different HLA-A locus. Two CTL clones were generated from MS and clone 3-2, expressing CD3+ CD8+ CD4- CD56-, showed high MHC class I-restricted cytotoxicity against the colonic epithelial cells. These results indicated that CTLs against colonic epithelial cells may contribute to epithelial cell damage in ulcerative colitis. PMID:10080107

  10. Nod2 mediates susceptibility to Yersinia pseudotuberculosis in mice.

    Ulrich Meinzer

    Full Text Available Nucleotide oligomerisation domain 2 (NOD2 is a component of the innate immunity known to be involved in the homeostasis of Peyer patches (PPs in mice. However, little is known about its role during gut infection in vivo. Yersinia pseudotuberculosis is an enteropathogen causing gastroenteritis, adenolymphitis and septicaemia which is able to invade its host through PPs. We investigated the role of Nod2 during Y. pseudotuberculosis infection. Death was delayed in Nod2 deleted and Crohn's disease associated Nod2 mutated mice orogastrically inoculated with Y. pseudotuberculosis. In PPs, the local immune response was characterized by a higher KC level and a more intense infiltration by neutrophils and macrophages. The apoptotic and bacterial cell counts were decreased. Finally, Nod2 deleted mice had a lower systemic bacterial dissemination and less damage of the haematopoeitic organs. This resistance phenotype was lost in case of intraperitoneal infection. We concluded that Nod2 contributes to the susceptibility to Y. pseudotuberculosis in mice.

  11. Impassable YscP Substrates and Their Impact on the Yersinia enterocolitica Type III Secretion Pathway▿

    Riordan, Kelly E.; Sorg, Joseph A.; Berube, Bryan J.; Schneewind, Olaf


    Yersinia type III machines secrete protein substrates across the bacterial envelope and, following assembly of their secretion needles, transport effector Yops into host cells. According to their destination during type III secretion, early, middle, and late secretion substrates can be distinguished; however, the signals and mechanisms whereby these proteins are recognized and transported by the secretion machine are not understood. Here, we examine several hybrids between secretion substrate...

  12. Susceptibility of chemostat-grown Yersinia enterocolitica and Klebsiella pneumoniae to chlorine dioxide.

    Harakeh, M S; Berg, J D; Hoff, J C; Matin, A.


    The resistance of bacteria to antimicrobial agents could be influenced by growth environment. The susceptibility of two enteric bacteria, Yersinia enterocolitica and Klebsiella pneumoniae, to chlorine dioxide was investigated. These organisms were grown in a defined medium in a chemostat and the influence of growth rate, temperature, and cell density on the susceptibility was studied. All inactivation experiments were conducted with a dose of 0.25 mg of chlorine dioxide per liter in phosphate...

  13. Activation of individual L1 retrotransposon instances is restricted to cell-type dependent permissive loci

    Philippe, Claude; Vargas-Landin, Dulce B; Doucet, Aurélien J; van Essen, Dominic; Vera-Otarola, Jorge; Kuciak, Monika; Corbin, Antoine; Nigumann, Pilvi; Cristofari, Gaël


    LINE-1 (L1) retrotransposons represent approximately one sixth of the human genome, but only the human-specific L1HS-Ta subfamily acts as an endogenous mutagen in modern humans, reshaping both somatic and germline genomes. Due to their high levels of sequence identity and the existence of many polymorphic insertions absent from the reference genome, the transcriptional activation of individual genomic L1HS-Ta copies remains poorly understood. Here we comprehensively mapped fixed and polymorphic L1HS-Ta copies in 12 commonly-used somatic cell lines, and identified transcriptional and epigenetic signatures allowing the unambiguous identification of active L1HS-Ta copies in their genomic context. Strikingly, only a very restricted subset of L1HS-Ta loci - some being polymorphic among individuals - significantly contributes to the bulk of L1 expression, and these loci are differentially regulated among distinct cell lines. Thus, our data support a local model of L1 transcriptional activation in somatic cells, governed by individual-, locus-, and cell-type-specific determinants. DOI: PMID:27016617

  14. Bone marrow-derived thymic antigen-presenting cells determine self-recognition of Ia-restricted T lymphocytes

    The authors previously have demonstrated that in radiation-induced bone marrow chimeras, T-cell self-Ia restriction specificity appeared to correlate with the phenotype of the bone marrow-derived antigen-presenting (or dendritic) cell in the thymus during T-cell development. However, these correlations were necessarily indirect because of the difficulty in assaying thymic function directly by adult thymus transplant, which has in the past been uniformly unsuccessful. They now report success in obtaining functional T cells from nude mice grafted with adult thymuses reduced in size by treatment of the thymus donor with anti-thymocyte globulin and cortisone. When (B10 Scn X B10.D2)F1 nude mice (I-Ab,d) are given parental B10.D2 (I-Ad) thymus grafts subcutaneously, their T cells are restricted to antigen recognition in association with I-Ad gene products but not I-Ab gene products. Furthermore, thymuses from (B10 X B10.D2)F1 (I-Ab,d)----B10 (I-Ab) chimeras transplanted 6 months or longer after radiation (a time at which antigen-presenting cell function is of donor bone marrow phenotype) into (B10 X B10.D2)F1 nude mice generate T cells restricted to antigen recognition in association with both I-Ad and I-Ab gene products. Thymuses from totally allogeneic bone marrow chimeras appear to generate T cells of bone marrow donor and thymic host restriction specificity. Thus, when thymus donors are radiation-induced bone marrow chimeras, the T-cell I-region restriction of the nude mice recipients is determined at least in part by the phenotype of the bone marrow-derived thymic antigen presenting cells or dendritic cells in the chimeric thymus

  15. Cbl enforces Vav1 dependence and a restricted pathway of T cell development.

    Jeffrey Chiang

    Full Text Available Extensive studies of pre-TCR- and TCR-dependent signaling have led to characterization of a pathway deemed essential for efficient T cell development, and comprised of a cascade of sequential events involving phosphorylation of Lck and ZAP-70, followed by phosphorylation of LAT and SLP-76, and subsequent additional downstream events. Of interest, however, reports from our lab as well as others have indicated that the requirements for ZAP-70, LAT, and SLP-76 are partially reversed by inactivation of c-Cbl (Cbl, an E3 ubiquitin ligase that targets multiple molecules for ubiquitination and degradation. Analysis of signaling events in these Cbl knockout models, including the recently reported analysis of SLP-76 transgenes defective in interaction with Vav1, suggested that activation of Vav1 might be a critical event in alternative pathways of T cell development. To extend the analysis of signaling requirements for thymic development, we have therefore assessed the effect of Cbl inactivation on the T cell developmental defects that occur in Vav1-deficient mice. The defects in Vav1-deficient thymic development, including a marked defect in DN3-DN4 transition, were completely reversed by Cbl inactivation, accompanied by enhanced phosphorylation of PLC-γ1 and ERKs in response to pre-TCR/TCR cross-linking of Vav1⁻/⁻Cbl⁻/⁻ DP thymocytes. Taken together, these results suggest a substantially modified paradigm for pre-TCR/TCR signaling and T cell development. The observed consensus pathways of T cell development, including requirements for ZAP-70, LAT, SLP-76, and Vav1, appear to reflect the restriction by Cbl of an otherwise much broader set of molecular pathways capable of mediating T cell development.

  16. Cell cycle restriction by histone H2AX limits proliferation of adult neural stem cells

    Fernando, R. N.; Eleuteri, B.; Abdelhady, S.; Nussenzweig, A; Andang, M; Ernfors, P.


    Adult neural stem cell proliferation is dynamic and has the potential for massive self-renewal yet undergoes limited cell division in vivo. Here, we report an epigenetic mechanism regulating proliferation and self-renewal. The recruitment of the PI3K-related kinase signaling pathway and histone H2AX phosphorylation following GABAA receptor activation limits subventricular zone proliferation. As a result, NSC self-renewal and niche size is dynamic and can be directly modulated in both directio...

  17. Laser ablation of persistent twist cells in Drosophila: muscle precursor fate is not segmentally restricted

    Farrell, E. R.; Keshishian, H.


    In Drosophila the precursors of the adult musculature arise during embryogenesis. These precursor cells have been termed Persistent Twist Cells (PTCs), as they continue to express the transcription factor Twist after that gene ceases expression elsewhere in the mesoderm. In the larval abdomen, the PTCs are associated with peripheral nerves in stereotypic ventral, dorsal, and lateral clusters, which give rise, respectively, to the ventral, dorsal, and lateral muscle fiber groups of the adult. We tested the developmental potential of the PTCs by using a microbeam laser to ablate specific clusters in larvae. We found that the ablation of a single segmental PTC cluster does not usually result in the deletion of the corresponding adult fibers of that segment. Instead, normal or near normal numbers of adult fibers can form after the ablation. Examination of pupae following ablation showed that migrating PTCs from adjacent segments are able to invade the affected segment, replenishing the ablated cells. However, the ablation of homologous PTCs in multiple segments does result in the deletion of the corresponding adult muscle fibers. These data indicate that the PTCs in an abdominal segment can contribute to the formation of muscle fibers in adjacent abdominal segments, and thus are not inherently restricted to the formation of muscle fibers within their segment of origin.

  18. Cytotoxic effect of lapatinib is restricted to human papillomavirus-positive head and neck squamous cell carcinoma cell lines

    Fumagalli I


    Full Text Available Ingrid Fumagalli,1,2 Delphine Dugue,1 Jean Emmanuel Bibault,1,2 Céline Clémenson,1 Marie Catherine Vozenin,1 Michele Mondini,1,* Eric Deutsch1,3,*1Inserm U1030, Molecular Radiotherapy, LABEX LERMIT, Gustave Roussy, University Paris XI, Villejuif, France; 2Radiation Therapy Department, Oscar Lambret Comprehensive Cancer Center, Lille, France; 3Department of Radiation Oncology, Gustave Roussy, University Paris XI, Villejuif, France*These authors share senior authorshipBackground: Lapatinib is a dual epidermal growth factor receptor (EGFR and HER2 inhibitor. Overexpression of these receptors is frequently observed in head and neck squamous cell carcinoma (HNSCC. As growing proportion of HNSCC is characterized by human papillomavirus (HPV infection, we aimed at evaluating the efficacy of lapatinib as function of HPV status in HNSCC cell lines.Methods: Two HPV-positive and two HPV-negative HNSCC cell lines were used. Proliferation, cell cycle, and Annexin V assays were performed to test their sensitivity to lapatinib. Combination of lapatinib and ionizing radiation was evaluated with clonogenic survival assays. Akt, EGFR and HER2, and E6/E7 expression and activation were analyzed by immunoblotting and quantitative reverse transcription polymerase chain reaction.Results: Lapatinib reduced E6 and E7 expression and Akt phosphorylation, inhibited cell proliferation and induced cell death in HPV-positive cell lines. An additive effect of lapatinib with radiation was observed in these cells. Lapatinib had no effect on HPV-negative cells.Conclusion: Lapatinib efficacy restricted to the HPV-positive cells suggests that HPV status could be a potential marker for enhanced response to lapatinib in HNSCC.Keywords: HPV, lapatinib, ionizing radiation, EGFR, HER2, tyrosine kinase inhibitor

  19. Epitopes associated with the MHC restriction site of T cells. II. Somatic generation of Iat epitopes on T cells in radiation bone marrow chimeras

    Asano, Y.; Tada, T.


    We described in this paper systematic alterations in the expression of unique I region controlled epitopes on helper T cells (Th) in chimeras according to the changes in their H-2 restriction specificity. Taking advantage of the reactivity of monoclonal antibodies (anti-Iat) putatively specific for the epitopes indirectly controlled by I region and expressed in association with the Iak restriction site of Th, we examined the alterations of these epitopes on Th cells from various bone marrow chimeras. Iatk epitopes were physiologically expressed on Iak-restricted but not on Iab-restricted Th cells in (H-2k X H-2b)F1 mice. In the chimeric condition, the H-2k-restricted Th of B6----F1 chimera acquired the expression of Iatk even though B6 Th is unable to express Iatk when developed under the physiologic condition. Iatk are also found on Th of fully allogeneic chimera of B6----C3H, whereas Th cells of C3H----B6 completely lost the Iatk expression. These results indicate that Iat epitopes originally defined as unique I region-controlled determinants selectively expressed on T cells are not encoded by the I region genes but are associated with the T cell receptor that sees the self Ia. The epitopes undergo the adaptive alterations according to the acquisition of a new MHC restriction. This is the first example to demonstrate the epitope associated with T cell receptor which undergo the systematic adaptive differentiation.

  20. Epitopes associated with the MHC restriction site of T cells. II. Somatic generation of Iat epitopes on T cells in radiation bone marrow chimeras

    We described in this paper systematic alterations in the expression of unique I region controlled epitopes on helper T cells (Th) in chimeras according to the changes in their H-2 restriction specificity. Taking advantage of the reactivity of monoclonal antibodies (anti-Iat) putatively specific for the epitopes indirectly controlled by I region and expressed in association with the Iak restriction site of Th, we examined the alterations of these epitopes on Th cells from various bone marrow chimeras. Iatk epitopes were physiologically expressed on Iak-restricted but not on Iab-restricted Th cells in (H-2k X H-2b)F1 mice. In the chimeric condition, the H-2k-restricted Th of B6----F1 chimera acquired the expression of Iatk even though B6 Th is unable to express Iatk when developed under the physiologic condition. Iatk are also found on Th of fully allogeneic chimera of B6----C3H, whereas Th cells of C3H----B6 completely lost the Iatk expression. These results indicate that Iat epitopes originally defined as unique I region-controlled determinants selectively expressed on T cells are not encoded by the I region genes but are associated with the T cell receptor that sees the self Ia. The epitopes undergo the adaptive alterations according to the acquisition of a new MHC restriction. This is the first example to demonstrate the epitope associated with T cell receptor which undergo the systematic adaptive differentiation

  1. The role of Ia molecules in the activation of T lymphocytes. III. Antigen-specific, Ia-restricted, interleukin 2-producing T cell hybridomas with detectable affinity for the restricting I-A molecule


    Antigen-specific I region-restricted, interleukin 2-producing T cell hybridomas were produced by fusing GAT-specific T cell blasts with BW5147. Two antigen-specific phenotypes were identified, one autoreactive and one nonautoreactive. All of the antigen-specific and autoreactive clones were H-2 restricted, mapping to the IA subregion by genetic analysis and monoclonal antibody inhibition. Both the antigen- specific and autoreactive stimulation are the property of a single cell and required no...

  2. The G1/S Specific Cyclin D2 Is a Regulator of HIV-1 Restriction in Non-proliferating Cells.

    Badia, Roger; Pujantell, Maria; Riveira-Muñoz, Eva; Puig, Teresa; Torres-Torronteras, Javier; Martí, Ramón; Clotet, Bonaventura; Ampudia, Rosa M; Vives-Pi, Marta; Esté, José A; Ballana, Ester


    Macrophages are a heterogeneous cell population strongly influenced by differentiation stimuli that become susceptible to HIV-1 infection after inactivation of the restriction factor SAMHD1 by cyclin-dependent kinases (CDK). Here, we have used primary human monocyte-derived macrophages differentiated through different stimuli to evaluate macrophage heterogeneity on cell activation and proliferation and susceptibility to HIV-1 infection. Stimulation of monocytes with GM-CSF induces a non-proliferating macrophage population highly restrictive to HIV-1 infection, characterized by the upregulation of the G1/S-specific cyclin D2, known to control early steps of cell cycle progression. Knockdown of cyclin D2, enhances HIV-1 replication in GM-CSF macrophages through inactivation of SAMHD1 restriction factor by phosphorylation. Co-immunoprecipitation experiments show that cyclin D2 forms a complex with CDK4 and p21, a factor known to restrict HIV-1 replication by affecting the function of the downstream cascade that leads to SAMHD1 deactivation. Thus, we demonstrate that cyclin D2 acts as regulator of cell cycle proteins affecting SAMHD1-mediated HIV-1 restriction in non-proliferating macrophages. PMID:27541004

  3. Interdisciplinary Evaluation of Broadly-Reactive HLA Class II Restricted Epitopes Eliciting HIV-Specific CD4+T Cell Responses

    Buggert, M.; Norström, M.; Lundegaard, Claus; Lund, Ole; Nielsen, Morten; Karlsson, A. C.


    Background: CD4+ T cells orchestrate immune protection by ‘‘helping’’ other cells of our immune system to clear viral infections. It is well known that the preferential infection and depletion of CD4+ T cells contributes to hampered systemic T cell help following HIV infection. However, the...... functional and immunodominant discrepancies of CD4+ T cell responses targeting promiscuous MHC II restricted HIV epitopes remains poorly defined. Thus, utilization of interdisciplinary approaches might aid revealing broadly- reactive peptides eliciting CD4 + T cell responses. Methods: We utilized the novel...... bioinformatic prediction program NetMHCIIpan to select 64 optimized MHC II restricted epitopes located in the HIV Gag, Pol, Env, Nef and Tat regions. The epitopes were selected to cover the global diversity of the virus (multiple subtypes) and the human immune system(diverse MHC II types). Optimized...

  4. Selective host range restriction of goat cells for recombinant murine leukemia virus and feline leukemia virus type A.

    Fischinger, P J; Thiel, H J; Blevins, C S; Dunlop, N M


    We isolated a strain of normal goat fibroblasts which was uniquely selective in that it allowed the replication of xenotropic murine leukemia virus but not polytropic recombinant murine leukemia virus. In addition, feline leukemia virus type A replication was severely diminished in these goat cells, whereas feline leukemia virus type B and feline endogenous RD114-CCC viruses replicated efficiently. No other known cells exhibit this pattern of virus growth restriction. These goat cells allow t...

  5. T cell determinants of myelin basic protein include a unique encephalitogenic I-E-restricted epitope for Lewis rats


    The major encephalitogenic epitope for Lewis rats is the 72-89 sequence of guinea pig basic protein (GP-BP) or rat basic protein (Rt-BP). T cells responsive to this epitope are I-A restricted and preferentially express the V alpha 2:V beta 8 gene combination in their TCR. In this work, we describe for the first time the delayed appearance of T cells specific for additional discrete determinant of BP, the nonencephalitogenic 55-68 sequence of GP-BP restricted by I-A, and the encephalitogenic 8...

  6. Is nectar reabsorption restricted by the stalk cells of floral and extrafloral nectary trichomes?

    Cardoso-Gustavson, P; Davis, A R


    Reabsorption is a phase of nectar dynamics that occurs concurrently with secretion; it has been described in floral nectaries that exude nectar through stomata or unicellular trichomes, but has not yet been recorded in extrafloral glands. Apparently, nectar reabsorption does not occur in multicellular secretory trichomes (MST) due to the presence of lipophilic impregnations - which resemble Casparian strips - in the anticlinal walls of the stalk cells. It has been assumed that these impregnations restrict solute movement within MST to occur unidirectionally and exclusively by the symplast, thereby preventing nectar reflux toward the underlying nectary tissues. We hypothesised that reabsorption is absent in nectaries possessing MST. The fluorochrome lucifer yellow (LYCH) was applied to standing nectar of two floral and extrafloral glands of distantly related species, and then emission spectra from nectary sections were systematically analysed using confocal microscopy. Passive uptake of LYCH via the stalk cells to the nectary tissues occurred in all MST examined. Moreover, we present evidence of nectar reabsorption in extrafloral nectaries, demonstrating that LYCH passed the stalk cells of MST, although it did not reach the deepest nectary tissues. Identical (control) experiments performed with neutral red (NR) demonstrated no uptake of this stain by actively secreting MST, whereas diffusion of NR did occur in plasmolysed MST of floral nectaries at the post-secretory phase, indicating that nectar reabsorption by MST is governed by stalk cell physiology. Interestingly, non-secretory trichomes failed to reabsorb nectar. The role of various nectary components is discussed in relation to the control of nectar reabsorption by secretory trichomes. PMID:24987788

  7. Tombusvirus-yeast interactions identify conserved cell-intrinsic viral restriction factors

    Zsuzsanna eSasvari


    Full Text Available To combat viral infections, plants possess innate and adaptive immune pathways, such as RNA silencing, R gene and recessive gene-mediated resistance mechanisms. However, it is likely that additional cell-intrinsic restriction factors (CIRF are also involved in limiting plant virus replication. This review discusses novel CIRFs with antiviral functions, many of them RNA-binding proteins or affecting the RNA binding activities of viral replication proteins. The CIRFs against tombusviruses have been identified in yeast (Saccharomyces cerevisiae, which is developed as an advanced model organism. Grouping of the identified CIRFs based on their known cellular functions and subcellular localization in yeast reveals that TBSV replication is limited by a wide variety of host gene functions. Yeast proteins with the highest connectivity in the network map include the well-characterized Xrn1p 5’-3’ exoribonuclease, Act1p actin protein and Cse4p centromere protein. The protein network map also reveals an important interplay between the pro-viral Hsp70 cellular chaperone and the antiviral co-chaperones, and possibly key roles for the ribosomal or ribosome-associated factors. We discuss the antiviral functions of selected CIRFs, such as the RNA binding nucleolin, ribonucleases, WW-domain proteins, single- and multi-domain cyclophilins, TPR-domain co-chaperones and cellular ion pumps. These restriction factors frequently target the RNA-binding region in the viral replication proteins, thus interfering with the recruitment of the viral RNA for replication and the assembly of the membrane-bound viral replicase. Although many of the characterized CIRFs act directly against TBSV, we propose that the TPR-domain co-chaperones function as guardians of the cellular Hsp70 chaperone system, which is subverted efficiently by TBSV for viral replicase assembly in the absence of the TPR-domain co-chaperones.

  8. Antiviral T cell competence and restriction specificity of mixed allogeneic (P1 + P2----P1) irradiation chimeras

    Mixed irradiation bone marrow chimeras were prepared by reconstituting lethally irradiated C57BL/10 (B10) or B10.D2 mice with T cell-depleted bone marrow cells of B10 plus B10.D2 origin. These chimeras were healthy and survived well under conventional housing conditions and after experimental laboratory infections. Of a total of 17 chimeras tested, 2 died spontaneously or from the injected virus. Twelve of fifteen chimeras mounted a measurable cytotoxic T cell response to virus. Despite approximately equal percentages of B10 and B10.D2 lymphocytes in chimeras, cytotoxic T cell responses to vaccinia virus and lymphocytic choriomeningitis virus were mediated variably by either syngeneic or allogeneic donor lymphocytes; thus the H-2 type of effector T cells frequently did not correspond to the 50:50 distribution of spleen or peripheral blood lymphocytes. Cytotoxic responses were restricted exclusively to recipient H-2 type. All mixed chimeras examined were able to mount a good IgG response to vesicular stomatitis virus. These results confirm previous data suggesting that such mixed chimeras are healthy and immunocompetent and demonstrate strict recipient-determined restriction specificity of effector T cells; they also suggest that if T help is necessary for induction of virus-specific cytotoxic T cells, it does not require host-restricted interactions between helper T cells and precursor cytotoxic T cells

  9. Features of T cells causing H-2-restricted lethal graft-vs.-host disease across minor histocompatibility barriers

    Evidence is presented that T cells that produce lethal graft-vs.-host disease (GVHD) to minor histocompatibility antigens (minor HA) comprise discrete subgroups of H-2K- and H-2D-restricted T cells; double negative selection of T cells in irradiated H-2 recombinant mice was used to separate these two subgroups. No evidence could be found that I-restricted T cells contributed to GVHD, either as effector cells or helper cells. The (unprimed) precursor cells for GVHD expressed the Thy-1+, Lyt-1+/-2, Ia- phenotype. Studies in which H-2-semiallogeneic bone marrow chimeras were used as hosts for negative selection suggested that presentation of minor HA to T cells during the induction phase is controlled by marrow-derived cells; indirect evidence was obtained that these latter cells can ''process'' minor HA presented on H-2 different cells and thereby render the antigens immunogenic. Studies in which minor HA-different, H-2-compatible chimeras were re-irradiated and then injected with donor-vs.-host T cells suggested that the effector phase of lethal GVHD involves contact of antigen on non-marrow-derived cells

  10. Recent emergence of new variants of Yersinia pestis in Madagascar.

    Guiyoule, A; Rasoamanana, B; Buchrieser, C; Michel, P; Chanteau, S; Carniel, E


    Yersinia pestis, the causative agent of plague, has been responsible for at least three pandemics. During the last pandemic, which started in Hong Kong in 1894, the microorganism colonized new, previously unscathed geographical areas where it has become well established. The aim of this longitudinal study was to investigate the genetic stability of Y. pestis strains introduced into a new environment just under a century ago and to follow the epidemiology of any new genetic variant detected. In the present study, 187 strains of Y. pestis isolated between 1939 and 1996 from different regions of Madagascar and responsible mainly for human cases of bubonic and pneumonic plague were studied. Our principal genotyping method was rRNA gene profiling (ribotyping), which has previously been shown to be an effective scheme for typing Y. pestis strains of different geographical origins. We report that all studied Y. pestis strains isolated in Madagascar before 1982 were of classical ribotype B, the ribotype attributed to the Y. pestis clone that spread around the world during the third pandemic. In 1982, 1983, and 1994, strains with new ribotypes, designated R, Q, and T, respectively, were isolated on the high-plateau region of the island. Analysis of other genotypic traits such as the NotI genomic restriction profiles and the EcoRV plasmid restriction profiles revealed that the new variants could also be distinguished by specific genomic and/or plasmid profiles. A follow-up of these new variants indicated that strains of ribotypes Q and R have become well established in their ecosystem and have a tendency to spread to new geographical areas and supplant the original classical strain. PMID:9350742

  11. Yersinia Virulence Depends on Mimicry of Host Rho-Family Nucleotide Dissociation Inhibitors

    Prehna,G.; Ivanov, M.; Blisha, J.; Stebbins, C.


    Yersinia spp. cause gastroenteritis and the plague, representing historically devastating pathogens that are currently an important biodefense and antibiotic resistance concern. A critical virulence determinant is the Yersinia protein kinase A, or YpkA, a multidomain protein that disrupts the eukaryotic actin cytoskeleton. Here we solve the crystal structure of a YpkA-Rac1 complex and find that YpkA possesses a Rac1 binding domain that mimics host guanidine nucleotide dissociation inhibitors (GDIs) of the Rho GTPases. YpkA inhibits nucleotide exchange in Rac1 and RhoA, and mutations that disrupt the YpkA-GTPase interface abolish this activity in vitro and impair in vivo YpkA-induced cytoskeletal disruption. In cell culture experiments, the kinase and the GDI domains of YpkA act synergistically to promote cytoskeletal disruption, and a Y. pseudotuberculosis mutant lacking YpkA GDI activity shows attenuated virulence in a mouse infection assay. We conclude that virulence in Yersinia depends strongly upon mimicry of host GDI proteins by YpkA.

  12. Plasmid regulation and temperature-sensitive behavior of the Yersinia pestis penicillin-binding proteins.

    Ferreira, R. C.; Park, J. T.; Ferreira, L. C.


    Six major bands corresponding to penicillin-binding proteins (PBPs) with molecular weights ranging from 43,000 to 97,000 were detected in cell envelopes of Yersinia pestis EV76 grown at 28 degrees C. When cells were transferred to 37 degrees C and incubated for extended periods of time, the amounts of all PBPs, except for PBP2, were gradually reduced in cell envelopes of a strain carrying a 75-kb virulence-associated plasmid (as measured by penicillin-binding capacity), whereas in a strain cu...

  13. Insights into the genome evolution of Yersinia pestis through whole genome comparison with Yersinia pseudotuberculosis

    Souza, B; Stoutland, P; Derbise, A; Georgescu, A; Elliott, J; Land, M; Marceau, M; Motin, V; Hinnebusch, J; Simonet, M; Medigue, C; Dacheux, D; Chenal-Francisque, V; Regala, W; Brubaker, R R; Carniel, E; Chain, P; Verguez, L; Fowler, J; Garcia, E; Lamerdin, J; Hauser, L; Larimer, F


    Yersinia pestis, the causative agent of plague, is a highly uniform clone that diverged recently from the enteric pathogen Yersinia pseudotuberculosis. Despite their close genetic relationship, they differ radically in their pathogenicity and transmission. Here we report the complete genomic sequence of Y. pseudotuberculosis IP32953 and its use for detailed genome comparisons to available Y. pestis sequences. Analyses of identified differences across a panel of Yersinia isolates from around the world reveals 32 Y. pestis chromosomal genes that, together with the two Y. pestis-specific plasmids, represent the only new genetic material in Y. pestis acquired since the divergence from Y. pseudotuberculosis. In contrast, 149 new pseudogenes (doubling the previous estimate) and 317 genes absent from Y. pestis were detected, indicating that as many as 13% of Y. pseudotuberculosis genes no longer function in Y. pestis. Extensive IS-mediated genome rearrangements and reductive evolution through massive gene loss, resulting in elimination and modification of pre-existing gene expression pathways appear to be more important than acquisition of new genes in the evolution of Y. pestis. These results provide a sobering example of how a highly virulent epidemic clone can suddenly emerge from a less virulent, closely related progenitor.

  14. Direct ex vivo detection of HLA-DR3-restricted cytomegalovirus- and Mycobacterium tuberculosis-specific CD4+ T cells.

    Bronke, Corine; Palmer, Nanette M; Westerlaken, Geertje H A; Toebes, Mireille; van Schijndel, Gijs M W; Purwaha, Veenu; van Meijgaarden, Krista E; Schumacher, Ton N M; van Baarle, Debbie; Tesselaar, Kiki; Geluk, Annemieke


    In order to detect epitope-specific CD4+ T cells in mycobacterial or viral infections in the context of human class II major histocompatibility complex protein human leukocyte antigen (HLA)-DR3, two HLA-DR3 tetrameric molecules were successfully produced. One contained an immunodominant HLA-DR3-restricted T-cell epitope derived from the 65-kDa heat-shock protein of Mycobacterium tuberculosis, peptide 1-13. For the other tetramer, we used an HLA-DR3-restricted T-cell epitope derived from cytomegalovirus (CMV) pp65 lower matrix protein, peptide 510-522, which induced high levels of interferon (IFN)-gamma-producing CD4+ T cells in three of four HLA-DR3-positive CMV-seropositive individuals up to 0.84% of CD4+ T cells by intracellular cytokine staining. In peripheral blood mononuclear cells from M. tuberculosis-exposed, Mycobacterium bovis bacille Calmette-Guérin (BCG)-vaccinated, or CMV-seropositive individuals, we were able to directly detect with both tetramers epitope-specific T cells up to 0.62% and 0.45% of the CD4+ T-cell population reactive to M. tuberculosis and CMV, respectively. After a 6-day culture with peptide p510-522, the frequency of CMV-specific tetramer-binding T cells was expanded up to 9.90% tetramer+ CFSElow (5,6-carboxyfluorescein diacetate succinimidyl ester) cells within the CD4+ T-cell population, further confirming the specificity of the tetrameric molecules. Thus, HLA-DR3/peptide tetrameric molecules can be used to investigate HLA-DR3-restricted antigen-specific CD4+ T cells in clinical disease or after vaccination. PMID:16360834

  15. Identification of Yersinia spp. with the API 20E system.

    Archer, J R; Schell, R F; Pennell, D R; Wick, P D


    The ability of the API 20E system to identify 105 clinical isolates of Yersinia spp. was compared with those of conventional biochemical tests at 28 and 37 degrees C. Elimination of the Voges-Proskauer test (recorded as a negative result) increased the percentage of correct identifications for Yersinia spp. from 66 to 93% when the API 20E strips were incubated at 28 degrees C.

  16. Ferric Enterochelin Transport in Yersinia enterocolitica: Molecular and Evolutionary Aspects

    Schubert, S.; Fischer, D; Heesemann, J


    Yersinia enterocolitica is well equipped for siderophore piracy, encompassing the utilization of siderophores such as ferrioxamine, ferrichrome, and ferrienterochelin. In this study, we report on the molecular and functional characterization of the Yersinia fep-fes gene cluster orthologous to the Escherichia coli ferrienterochelin transport genes (fepA, fepDGC, and fepB) and the esterase gene fes. In vitro transcription-translation analysis identified polypeptides of 30 and 35 kDa encoded by ...

  17. Human GATA-3: a lineage-restricted transcription factor that regulates the expression of the T cell receptor alpha gene.

    Ho, I C; Vorhees, P; Marin, N; Oakley, B K; Tsai, S F; Orkin, S H; Leiden, J. M.


    In addition to its role in the recognition of foreign antigens, the T cell receptor (TCR) alpha gene serves as a model system for studies of developmentally-regulated, lineage-specific gene expression in T cells. TCR alpha gene expression is restricted to cells of the TCR alpha/beta+ lineage, and is controlled by a T cell-specific transcriptional enhancer located 4.5 kb 3' to the C alpha gene segment. The TCR alpha enhancer contains four nuclear protein binding sites called T alpha 1-T alpha ...

  18. Antenatal taurine reduces cerebral cell apoptosis in fetal rats with intrauterine growth restriction*

    Jing Liu; Xiaofeng Wang; Ying Liu; Na Yang; Jing Xu; Xiaotun Ren


    From pregnancy to parturition, Sprague-Dawley rats were daily administered a low protein diet to establish a model of intrauterine growth restriction. From the 12th day of pregnancy, 300 mg/kg rine was daily added to food until spontaneous delivery occurred. Brain tissues from normal neo-natal rats at 6 hours after delivery, neonatal rats with intrauterine growth restriction, and neonatal rats with intrauterine growth restriction undergoing taurine supplement were obtained for further experiments. The terminal deoxyribonucleotidyl transferase (TdT)-mediated biotin-16-dUTP nick-end labeling assay revealed that the number of apoptotic cel s in the brain tissue of neonatal rats with intrauterine growth restriction significantly increased. Taurine supplement in pregnant rats reduced cel apoptosis in brain tissue from neonatal rats with intrauterine growth restriction. nohistochemical staining revealed that taurine supplement increased glial cel line-derived neuro-trophic factor expression and decreased caspase-3 expression in the cerebral cortex of intrauterine growth-restricted fetal rats. These results indicate that taurine supplement reduces cel apoptosis through the glial cel line-derived neurotrophic factor-caspase-3 signaling pathway, resulting in a protective effect on the intrauterine growth-restricted fetal rat brain.

  19. Requirement of cAMP signaling for Schwann cell differentiation restricts the onset of myelination.

    Ketty Bacallao

    Full Text Available Isolated Schwann cells (SCs respond to cAMP elevation by adopting a differentiated post-mitotic state that exhibits high levels of Krox-20, a transcriptional enhancer of myelination, and mature SC markers such as the myelin lipid galactocerebroside (O1. To address how cAMP controls myelination, we performed a series of cell culture experiments which compared the differentiating responses of isolated and axon-related SCs to cAMP analogs and ascorbate, a known inducer of axon ensheathment, basal lamina formation and myelination. In axon-related SCs, cAMP induced the expression of Krox-20 and O1 without a concomitant increase in the expression of myelin basic protein (MBP and without promoting axon ensheathment, collagen synthesis or basal lamina assembly. When cAMP was provided together with ascorbate, a dramatic enhancement of MBP expression occurred, indicating that cAMP primes SCs to form myelin only under conditions supportive of basal lamina formation. Experiments using a combination of cell permeable cAMP analogs and type-selective adenylyl cyclase (AC agonists and antagonists revealed that selective transmembrane AC (tmAC activation with forskolin was not sufficient for full SC differentiation and that the attainment of an O1 positive state also relied on the activity of the soluble AC (sAC, a bicarbonate sensor that is insensitive to forskolin and GPCR activation. Pharmacological and immunological evidence indicated that SCs expressed sAC and that sAC activity was required for morphological differentiation and the expression of myelin markers such as O1 and protein zero. To conclude, our data indicates that cAMP did not directly drive myelination but rather the transition into an O1 positive state, which is perhaps the most critical cAMP-dependent rate limiting step for the onset of myelination. The temporally restricted role of cAMP in inducing differentiation independently of basal lamina formation provides a clear example of the

  20. MicroRNA inhibition fine-tunes and provides robustness to the restriction point switch of the cell cycle

    del Rosario, Ricardo C. H.; Damasco, Joseph Ray Clarence G.; Aguda, Baltazar D.


    The restriction point marks a switch in G1 from growth factor-dependent to growth factor-independent progression of the cell cycle. The proper regulation of this switch is important for normal cell processes; aberrations could result in a number of diseases such as cancer, neurodegenerative disorders, stroke and myocardial infarction. To further understand the regulation of the restriction point, we extended a mathematical model of the Rb-E2F pathway to include members of the microRNA cluster miR-17-92. Our mathematical analysis shows that microRNAs play an essential role in fine-tuning and providing robustness to the switch. We also demonstrate how microRNA regulation can steer cells in or out of cancer states. PMID:27610602

  1. Crystallization of the class IV adenylyl cyclase from Yersinia pestis

    The class IV adenylyl cyclase from Y. pestis has been crystallized in an orthorhombic form suitable for structure determination. The class IV adenylyl cyclase from Yersinia pestis has been cloned and crystallized in both a triclinic and an orthorhombic form. An amino-terminal His-tagged construct, from which the tag was removed by thrombin, crystallized in a triclinic form diffracting to 1.9 Å, with one dimer per asymmetric unit and unit-cell parameters a = 33.5, b = 35.5, c = 71.8 Å, α = 88.7, β = 82.5, γ = 65.5°. Several mutants of this construct crystallized but diffracted poorly. A non-His-tagged native construct (179 amino acids, MW = 20.5 kDa) was purified by conventional chromatography and crystallized in space group P212121. These crystals have unit-cell parameters a = 56.8, b = 118.6, c = 144.5 Å, diffract to 3 Å and probably have two dimers per asymmetric unit and VM = 3.0 Å3 Da−1. Both crystal forms appear to require pH below 5, complicating attempts to incorporate nucleotide ligands into the structure. The native construct has been produced as a selenomethionine derivative and crystallized for phasing and structure determination

  2. Identification of H-2d Restricted T Cell Epitope of Foot-and-mouth Disease Virus Structural Protein VP1

    Zhang Zhong-Wang


    Full Text Available Abstract Background Foot-and-mouth disease (FMD is a highly contagious and devastating disease affecting livestock that causes significant financial losses. Therefore, safer and more effective vaccines are required against Foot-and-mouth disease virus(FMDV. The purpose of this study is to screen and identify an H-2d restricted T cell epitope from the virus structural protein VP1, which is present with FMD. We therefore provide a method and basis for studying a specific FMDV T cell epitope. Results A codon-optimized expression method was adopted for effective expression of VP1 protein in colon bacillus. We used foot-and-mouth disease standard positive serum was used for Western blot detection of its immunogenicity. The VP1 protein was used for immunizing BALB/c mice, and spleen lymphocytes were isolated. Then, a common in vitro training stimulus was conducted for potential H-2Dd, H-2Kd and H-2Ld restricted T cell epitope on VP1 proteins that were predicted and synthesized by using a bioinformatics method. The H-2Kd restricted T cell epitope pK1 (AYHKGPFTRL and the H-2Dd restricted T cell epitope pD7 (GFIMDRFVKI were identified using lymphocyte proliferation assays and IFN-γ ELISPOT experiments. Conclusions The results of this study lay foundation for studying the FMDV immune process, vaccine development, among other things. These results also showed that, to identify viral T cell epitopes, the combined application of bioinformatics and molecular biology methods is effective.

  3. A hybrid approach for predicting promiscuous MHC class I restricted T cell epitopes

    Manoj Bhasin; G P S Raghava


    In the present study, a systematic attempt has been made to develop an accurate method for predicting MHC class I restricted T cell epitopes for a large number of MHC class I alleles. Initially, a quantitative matrix (QM)-based method was developed for 47 MHC class I alleles having at least 15 binders. A secondary artificial neural network (ANN)-based method was developed for 30 out of 47 MHC alleles having a minimum of 40 binders. Combination of these ANN- and QM-based prediction methods for 30 alleles improved the accuracy of prediction by 6% compared to each individual method. Average accuracy of hybrid method for 30 MHC alleles is 92.8%. This method also allows prediction of binders for 20 additional alleles using QM that has been reported in the literature, thus allowing prediction for 67 MHC class I alleles. The performance of the method was evaluated using jack-knife validation test. The performance of the methods was also evaluated on blind or independent data. Comparison of our method with existing MHC binder prediction methods for alleles studied by both methods shows that our method is superior to other existing methods. This method also identifies proteasomal cleavage sites in antigen sequences by implementing the matrices described earlier. Thus, the method that we discover allows the identification of MHC class I binders (peptides binding with many MHC alleles) having proteasomal cleavage site at C-terminus. The user-friendly result display format (HTML-II) can assist in locating the promiscuous MHC binding regions from antigen sequence. The method is available on the web at and its mirror site is available at

  4. Identification of the Immunodominant H-2Kk-Restricted Cytotoxic T-Cell Epitope in the Borna Disease Virus Nucleoprotein

    Schamel, Karin; Staeheli, Peter; Hausmann, Jürgen


    Borna disease virus (BDV)-induced immunopathology in mice is most prominent in strains carrying the major histocompatibility complex H-2k allele and is mediated by CD8+ T cells that are directed against the viral nucleoprotein p40. We now identified the highly conserved octamer peptide TELEISSI, located between amino acid residues 129 and 136 of BDV p40, as a potent H-2Kk-restricted cytotoxic T-cell (CTL) epitope. When added to the culture medium of L929 target cells, TELEISSI conferred sensi...

  5. Efficacious early antiviral activity of HIV Gag- and Pol-specific HLA-B 2705-restricted CD8+ T cells

    Payne, Rebecca P; Kløverpris, Henrik; Sacha, Jonah B;


    The association between HLA-B 2705 and the immune control of human immunodeficiency virus type 1 (HIV-1) has previously been linked to the targeting of the HLA-B 2705-restricted Gag epitope KRWIILGLNK (KK10) by CD8(+) T cells. In order to better define the mechanisms of the HLA-B 2705 immune...... control of HIV, we first characterized the CD8(+) T-cell responses of nine highly active antiretroviral therapy (HAART)-naïve B 2705-positive subjects. Unexpectedly, we observed a strong response to an HLA-B 2705-restricted Pol epitope, KRKGGIGGY (KY9), in 8/9 subjects. The magnitude of the KY9 response...... respective CD8(+) T-cell response. By comparing inhibitions of viral replication by CD8(+) T cells specific for the Gag KK10, Pol KY9, and Vpr VL9 HLA-B 2705-restricted epitopes, we observed a consistent hierarchy of antiviral efficacy (Gag KK10 > Pol KY9 > Vpr VL9). This hierarchy was associated with early...

  6. Kinetics of expression of interleukin 2 receptors on class I and class II restricted murine T cell clones

    The kinetics of interleukin 2 receptor (IL-2R) expression has been examined on various class I and class II restricted, influenza specific murine T cell clones. Expression and relative levels of IL-2R were examined by Fluorescence Activated Cell Sorter analysis utilizing 3 anti-murine IL-2R monoclonal antibodies. Receptor expression was analyzed by scatchard analysis using radiolabeled recombinant human interleukin 2 to access the number of high and low affinity IL-2R per cell as well as the affinity of binding. The clones tested bound all 3 monoclonal antibodies and were inhibited in an IL-2 dependent proliferation assay by the addition of the antibodies to the culture. There was, however, differing degrees of inhibition ranging up to 99%, depending on the clone and the antibody used. IL-2R expression was detectable as early as 4-6 hours after antigenic stimulation of quiescent cells. After maximal levels of receptors were expressed, which was about 24 hours after stimulation, expression of IL-2R decreased with time on all clones examined (both class I and class II restricted). Differing rates of receptor loss is seen however, with some class II restricted clones retaining relatively high levels of receptors

  7. Structural Requirements for Recognition of the Human Immunodeficiency Virus Type 1 Core during Host Restriction in Owl Monkey Cells

    Forshey, Brett M.; Shi, Jiong; Aiken, Christopher


    Human immunodeficiency virus type 1 (HIV-1) infection of simian cells is restricted at an early postentry step by host factors whose mechanism of action is unclear. These factors target the viral capsid protein (CA) and attenuate reverse transcription, suggesting that they bind to the HIV-1 core and interfere with its uncoating. To identify the relevant binding determinants in the capsid, we tested the capacity of viruses containing Gag cleavage site mutations and amino acid substitutions in ...

  8. Rapid identification and typing of Yersinia pestis and other Yersinia species by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF mass spectrometry

    Drancourt Michel


    Full Text Available Abstract Background Accurate identification is necessary to discriminate harmless environmental Yersinia species from the food-borne pathogens Yersinia enterocolitica and Yersinia pseudotuberculosis and from the group A bioterrorism plague agent Yersinia pestis. In order to circumvent the limitations of current phenotypic and PCR-based identification methods, we aimed to assess the usefulness of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF protein profiling for accurate and rapid identification of Yersinia species. As a first step, we built a database of 39 different Yersinia strains representing 12 different Yersinia species, including 13 Y. pestis isolates representative of the Antiqua, Medievalis and Orientalis biotypes. The organisms were deposited on the MALDI-TOF plate after appropriate ethanol-based inactivation, and a protein profile was obtained within 6 minutes for each of the Yersinia species. Results When compared with a 3,025-profile database, every Yersinia species yielded a unique protein profile and was unambiguously identified. In the second step of analysis, environmental and clinical isolates of Y. pestis (n = 2 and Y. enterocolitica (n = 11 were compared to the database and correctly identified. In particular, Y. pestis was unambiguously identified at the species level, and MALDI-TOF was able to successfully differentiate the three biotypes. Conclusion These data indicate that MALDI-TOF can be used as a rapid and accurate first-line method for the identification of Yersinia isolates.

  9. Maximizing Plasmid Stability and Production of Released Proteins in Yersinia enterocolitica

    Li, Huaiyu; Bhaduri, Saumya; Magee, Wayne E.


    Virulent serotypes of Yersinia enterocolitica carry a plasmid (pYV) encoding a family of proteins that are released into the medium and whose expression is temperature and calcium regulated. The plasmid is easily lost from cells during their growth in the laboratory. We have used sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting with a monoclonal antibody (3.2C) that is specific for a 25-kDa released protein to show that 32°C is the lowest temperature at which pla...

  10. Preformed purified peptide/major histocompatibility class I complexes are potent stimulators of class I-restricted T cell hybridomas

    Stryhn, A; Pedersen, L O; Ortiz-Navarrete, V;


    A panel of antigen-specific, major histocompatibility complex class I-restricted T cell hybridomas has been generated to examine the capacity of peptide/class I complexes to stimulate T cells at the molecular level. Peptide/class I complexes were generated in detergent solution, purified and...... quantitated. Latex particles were subsequently coated with known amounts of preformed complexes and used to stimulate the T cell hybridomas. Stimulation was specific, i.e. only the appropriate peptide/class I combination were stimulatory, and quite sensitive, i.e. as little as 300 complexes per bead could be...... detected by the T cells. Preformed complexes were about 500,000 times more potent than free peptide in terms of T cell stimulation, demonstrating the physiological relevancy of the biochemically generated complexes. Surprisingly, the majority (including the most sensitive of the hybridomas) had lost CD8...

  11. Ferric enterochelin transport in Yersinia enterocolitica: molecular and evolutionary aspects.

    Schubert, S; Fischer, D; Heesemann, J


    Yersinia enterocolitica is well equipped for siderophore piracy, encompassing the utilization of siderophores such as ferrioxamine, ferrichrome, and ferrienterochelin. In this study, we report on the molecular and functional characterization of the Yersinia fep-fes gene cluster orthologous to the Escherichia coli ferrienterochelin transport genes (fepA, fepDGC, and fepB) and the esterase gene fes. In vitro transcription-translation analysis identified polypeptides of 30 and 35 kDa encoded by fepC and fes, respectively. A frameshift mutation within the fepA gene led to expression of a truncated polypeptide of 40 kDa. The fepD, fepG, and fes genes of Y. enterocolitica were shown to complement corresponding E. coli mutants. Insertional mutagenesis of fepD or fes genes abrogates enterochelin-supported growth of Y. enterocolitica on iron-chelated media. In contrast to E. coli, the fep-fes gene cluster in Y. enterocolitica consists solely of genes required for uptake and utilization of enterochelin (fep) and not of enterochelin synthesis genes such as entF. By Southern hybridization, fepDGC and fes sequences could be detected in Y. enterocolitica biotypes IB, IA, and II but not in biotype IV strains, Yersinia pestis, and Yersinia pseudotuberculosis strains. According to sequence alignment data and the coherent structure of the Yersinia fep-fes gene cluster, we suggest early genetic divergence of ferrienterochelin uptake determinants among species of the family Enterobacteriaceae. PMID:10515929

  12. Genotyping of human and porcine Yersinia enterocolitica, Yersinia intertmedia, and Yersinia bercovieri strains from Switzerland by amplified fragment length polymorphism analysis

    Kuehni-Boghenbor, Kathrin; On, Stephen L.W.; Kokotovic, Branko; Baumgartner, Andreas; Wassenaar, Trudy M.; Wittwer, Matthias; Bissig-Choisat, Beatrice; Frey, Joachim


    In this study, 231 strains of Yersinia enterocolitica, 25 strains of Y. intermedia, and 10 strains of Y. bercovieri from human and porcine sources (including reference strains) were analyzed using amplified fragment length polymorphism (AFLP), a whole-genome fingerprinting method for subtyping ba...

  13. Synergistic effects of dicloxacillin or clavulanic acid in combination with penicillin G or cephalothin against Yersinia enterocolitica.

    Jimenez-Valera, M; Ruiz-Bravo, A; Ramos-Cormenzana, A.


    Cultures of Yersinia enterocolitica grown at 22 degrees C produced beta-lactamases, whereas cultures grown at 37 degrees C produced these enzymes much less effectively. Both dicloxacillin and clavulanic acid inhibited the beta-lactamase activity of bacterial crude extracts and potentiated the activity of penicillin G or cephalothin against 14 Y. enterocolitica strains. It appeared that the beta-lactamase activity present in Y. enterocolitica cells grown at 37 degrees C was great enough to pla...

  14. Vitamin B-6 restriction impairs fatty acid synthesis in cultured human hepatoma (HepG2) cells

    Zhao, Mei; Ralat, Maria A.; Da Silva, Vanessa; Garrett, Timothy J; Melnyk, Stephan; James, S. Jill; Gregory, Jesse F.


    Vitamin B-6 deficiency has been reported to alter n-6 and n-3 fatty acid profiles in plasma and tissue lipids; however, the mechanisms underlying such metabolic changes remain unclear. The objective of this study was to determine the effects of vitamin B-6 restriction on fatty acid profiles and fatty acid synthesis in HepG2 cells. Cells were cultured for 6 wk in media with four different vitamin B-6 concentrations (10, 20, 50, and 2,000 nM added pyridoxal, representing deficient, marginal, ad...

  15. HDAC6-mediated EGFR stabilization and activation restrict cell response to sorafenib in non-small cell lung cancer cells.

    Wang, Zhihao; Hu, Pengchao; Tang, Fang; Xie, Conghua


    Sorafenib is a multi-targeted kinase inhibitor and has been the subject of extensive clinical research in advanced non-small cell lung cancer (NSCLC). However, sorafenib fails to improve overall survival of patients with advanced NSCLC. The molecular mechanisms that account for this phenomenon are unclear. Here we show that sorafenib treatment stabilizes epidermal growth factor receptor (EGFR) and activates EGFR pathway. Moreover, this is partly mediated by stabilization of histone deacetylase 6 (HDAC6), which has been shown to regulate EGFR endocytic trafficking and degradation. Overexpression of HDAC6 confers resistance to sorafenib in NSCLC cells. Inhibition of HDAC6 with selective inhibitors synergizes with sorafenib to kill NSCLC cells via inhibition of sorafenib-mediated EGFR pathway activation. Taken together, our findings might partly explain the failure of Phase III trial of sorafenib in improving overall survival of advanced NSCLC patients and bear possible implications for the improvement on the efficacy of sorafenib in treatment of NSCLC. PMID:27090797

  16. Subcellular proteomic analysis of host-pathogen interactions using human monocytes exposed to Yersinia pestis and Yersinia pseudotuberculosis

    Zhang, C G; Gonzales, A D; Choi, M W; Chromy, B A; Fitch, J P; McCutchen-Maloney, S L


    Yersinia pestis, the etiological agent of plague, is of concern to human health both from an infectious disease and a civilian biodefense perspective. While Y. pestis and Y. pseudotuberculosis share more than 90% DNA homology, they have significantly different clinical manifestations. Plague is often fatal if untreated, yet Y. pseudotuberculosis causes severe intestinal distress and is rarely fatal. A better understanding of host response to these closely related pathogens may help explain the different mechanisms of virulence and pathogenesis that result in such different clinical outcomes. The aim of this study was to characterize host protein expression changes in human monocyte-like U937 cells after exposure to Y. pestis and Y. pseudotuberculosis. In order to gain global proteomic coverage of host response, proteins from cytoplasmic, nuclear and membrane fractions of host cells were studied by 2-dimensional differential gel electrophoresis (2-D DIGE) and relative protein expression differences were quantitated. Differentially expressed proteins, with at least 1.5 fold expression changes and p values of 0.01 or less, were identified by MALDI-MS or LC/MS/MS. With these criteria, differential expression was detected in 16 human proteins after Y. pestis exposure and 13 human proteins after Y. pseudotuberculosis exposure, of which only two of the differentially expressed proteins identified were shared between the two exposures. Proteins identified in this study are reported to be involved in a wide spectrum of cellular functions and host defense mechanisms including apoptosis, cytoskeletal rearrangement, protein synthesis and degradation, DNA replication and transcription, metabolism, protein folding, and cell signaling. Notably, the differential expression patterns observed can distinguish the two pathogen exposures from each other and from unexposed host cells. The functions of the differentially expressed proteins identified provide insight on the different

  17. Peripheral selection of V delta 1+ cells with restricted T cell receptor delta gene junctional repertoire in the peripheral blood of healthy donors


    To characterize the T cell receptor (TCR) repertoire expressed by the V delta 1+ gamma/delta T cell population, we have studied the V delta 1-J delta 1 junctional sequences from peripheral blood samples of healthy donors. We show that, surprisingly, this repertoire is restricted in most healthy adults, with a donor-specific and relatively stable pattern, whereas this repertoire remains unrestricted in infants, and is similar to that of thymocytes. These data contrast with the general assumpti...

  18. Resistant Yersinia Enterocolitica Peritonitis in a Peritoneal Dialysis Patient

    Mehmet ÖZDEN


    Full Text Available Peritonitis is an important problem of peritoneal dialysis patients. Although Gram (+ bacteria are more frequent, Gram (- bacteria can rarely be the cause of peritonitis. Yersinia enterocolitica is a Gram (- enteric bacteria that especially causes gastrointestinal infections via contaminated food intake in immunosuppressive situations such as haemochromatosis, desferroxamine therapy or chronic hepatitis. We report a 51-year-old man who had been undergoing SAPD because of chronic renal failure for 2 years. We evaluated his peritoneal fl uid because of symptoms of peritonitis. His peritoneal cellular composition was consistent with peritonitis and after the microbiological culture of the fl uid we determined Yersinia enterocolitica as the pathogenic agent. The peritonitis persisted despite the administration of long term combined antibiotherapy and we removed the peritoneal catheter. In conclusion, Yersinia enterocolitica peritonitis is resistant to conventional antibiotic therapy and an indication for catheter removal.

  19. GABA agonist promoted formation of low affinity GABA receptors on cerebellar granule cells is restricted to early development

    Belhage, B; Hansen, Gert Helge; Schousboe, A;


    The ability of the GABA receptor agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) to promote formation of low affinity GABA receptors on cerebellar granule cells was tested using primary cultures of these neurons. Granule cells were exposed to THIP (150 microM) for 6 hr after......, respectively, 4, 7, 10 and 14 days in culture. It was found that THIP treatment of 4- and 7-day-old cultures led to formation of low affinity GABA receptors, whereas such receptors could not be detected after THIP treatment in the older cultures (10 and 14 days) in spite of the fact that these cultured granule...... cells expressed a high density of high affinity GABA receptors. It is concluded that the ability of THIP to promote formation of low affinity GABA receptors on cerebellar granule cells is restricted to an early developmental period....

  20. Non-major histocompatibility complex-restricted cytotoxic activity of blood mononuclear cells stimulated with secreted mycobacterial proteins and other mycobacterial antigens

    Ravn, P; Pedersen, B K


    Several observations indicate that non-major histocompatibility complex (MHC)-restricted cytotoxicity, mediated for example by natural killer cells and lymphokine-activated killer cells, may serve as an important antimicrobial defense mechanism. The purpose of the present study was to investigate...... the influences of different mycobacterial antigens on non-MHC-restricted cytotoxicity and further to investigate the ways by which various lymphocyte subpopulations contribute to the development of this cytotoxicity. Non-MHC-restricted cytotoxicity was induced following stimulation of mononuclear...... interferon. The CD4+ cells proliferated and expressed interleukin-2 receptors following stimulation with mycobacterial antigens.Depletion studies after antigen stimulation showed that the cytotoxic effector cells were CD16+ CD56+ and CD4-; the CD4+ cells alone did not mediate non-MHC-restricted cytotoxicity...

  1. Lung Injury Combined with Loss of Regulatory T Cells Leads to De Novo Lung-Restricted Autoimmunity.

    Chiu, Stephen; Fernandez, Ramiro; Subramanian, Vijay; Sun, Haiying; DeCamp, Malcolm M; Kreisel, Daniel; Perlman, Harris; Budinger, G R Scott; Mohanakumar, Thalachallour; Bharat, Ankit


    More than one third of patients with chronic lung disease undergoing lung transplantation have pre-existing Abs against lung-restricted self-Ags, collagen type V (ColV), and k-α1 tubulin (KAT). These Abs can also develop de novo after lung transplantation and mediate allograft rejection. However, the mechanisms leading to lung-restricted autoimmunity remain unknown. Because these self-Ags are normally sequestered, tissue injury is required to expose them to the immune system. We previously showed that respiratory viruses can induce apoptosis in CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs), the key mediators of self-tolerance. Therefore, we hypothesized that lung-tissue injury can lead to lung-restricted immunity if it occurs in a setting when Tregs are impaired. We found that human lung recipients who suffer respiratory viral infections experienced a decrease in peripheral Tregs. Pre-existing lung allograft injury from donor-directed Abs or gastroesophageal reflux led to new ColV and KAT Abs post respiratory viral infection. Similarly, murine parainfluenza (Sendai) respiratory viral infection caused a decrease in Tregs. Intratracheal instillation of anti-MHC class I Abs, but not isotype control, followed by murine Sendai virus infection led to development of Abs against ColV and KAT, but not collagen type II (ColII), a cartilaginous protein. This was associated with expansion of IFN-γ-producing CD4(+) T cells specific to ColV and KAT, but not ColII. Intratracheal anti-MHC class I Abs or hydrochloric acid in Foxp3-DTR mice induced ColV and KAT, but not ColII, immunity, only if Tregs were depleted using diphtheria toxin. We conclude that tissue injury combined with loss of Tregs can lead to lung-tissue-restricted immunity. PMID:27194786

  2. mTORC1 and SIRT1 Cooperate to Foster Expansion of Gut Adult Stem Cells during Calorie Restriction.

    Igarashi, Masaki; Guarente, Leonard


    Longevity-promoting caloric restriction is thought to trigger downregulation of mammalian target of rapamycin complex 1 (mTORC1) signaling and upregulation of SIRT1 activity with associated health benefits. Here, we show that mTORC1 signaling in intestinal stem cells (ISCs) is instead upregulated during calorie restriction (CR). SIRT1 deacetylates S6K1, thereby enhancing its phosphorylation by mTORC1, which leads to an increase in protein synthesis and an increase in ISC number. Paneth cells in the ISC niche secrete cyclic ADP ribose that triggers SIRT1 activity and mTORC1 signaling in neighboring ISCs. Notably, the mTOR inhibitor rapamycin, previously reported to mimic effects of CR, abolishes this expansion of ISCs. We suggest that Paneth cell signaling overrides any direct nutrient sensing in ISCs to sculpt the observed response to CR. Moreover, drugs that modulate pathways important in CR may exert opposing effects on different cell types. PMID:27345368

  3. Restrictive cardiomyopathy

    Cardiomyopathy - restrictive; Infiltrative cardiomyopathy ... In a case of restrictive cardiomyopathy, the heart muscle is normal size or slightly enlarged. Most of the time, it also pumps normally. However, it does not ...

  4. Genome-scale reconstruction of the metabolic network in Yersinia pestis, strain 91001

    Navid, A; Almaas, E


    The gram-negative bacterium Yersinia pestis, the aetiological agent of bubonic plague, is one the deadliest pathogens known to man. Despite its historical reputation, plague is a modern disease which annually afflicts thousands of people. Public safety considerations greatly limit clinical experimentation on this organism and thus development of theoretical tools to analyze the capabilities of this pathogen is of utmost importance. Here, we report the first genome-scale metabolic model of Yersinia pestis biovar Mediaevalis based both on its recently annotated genome, and physiological and biochemical data from literature. Our model demonstrates excellent agreement with Y. pestis known metabolic needs and capabilities. Since Y. pestis is a meiotrophic organism, we have developed CryptFind, a systematic approach to identify all candidate cryptic genes responsible for known and theoretical meiotrophic phenomena. In addition to uncovering every known cryptic gene for Y. pestis, our analysis of the rhamnose fermentation pathway suggests that betB is the responsible cryptic gene. Despite all of our medical advances, we still do not have a vaccine for bubonic plague. Recent discoveries of antibiotic resistant strains of Yersinia pestis coupled with the threat of plague being used as a bioterrorism weapon compel us to develop new tools for studying the physiology of this deadly pathogen. Using our theoretical model, we can study the cell's phenotypic behavior under different circumstances and identify metabolic weaknesses which may be harnessed for the development of therapeutics. Additionally, the automatic identification of cryptic genes expands the usage of genomic data for pharmaceutical purposes.

  5. Monocyte-derived macrophages exhibit distinct and more restricted HIV-1 integration site repertoire than CD4(+) T cells.

    Kok, Yik Lim; Vongrad, Valentina; Shilaih, Mohaned; Di Giallonardo, Francesca; Kuster, Herbert; Kouyos, Roger; Günthard, Huldrych F; Metzner, Karin J


    The host genetic landscape surrounding integrated HIV-1 has an impact on the fate of the provirus. Studies analysing HIV-1 integration sites in macrophages are scarce. We studied HIV-1 integration site patterns in monocyte-derived macrophages (MDMs) and activated CD4(+) T cells derived from seven antiretroviral therapy (ART)-treated HIV-1-infected individuals whose cells were infected ex vivo with autologous HIV-1 isolated during the acute phase of infection. A total of 1,484 unique HIV-1 integration sites were analysed. Their distribution in the human genome and genetic features, and the effects of HIV-1 integrase polymorphisms on the nucleotide selection specificity at these sites were indistinguishable between the two cell types, and among HIV-1 isolates. However, the repertoires of HIV-1-hosting gene clusters overlapped to a higher extent in MDMs than in CD4(+) T cells. The frequencies of HIV-1 integration events in genes encoding HIV-1-interacting proteins were also different between the two cell types. Lastly, HIV-1-hosting genes linked to clonal expansion of latently HIV-1-infected CD4(+) T cells were over-represented in gene hotspots identified in CD4(+) T cells but not in those identified in MDMs. Taken together, the repertoire of genes targeted by HIV-1 in MDMs is distinct from and more restricted than that of CD4(+) T cells. PMID:27067385

  6. Monocyte-derived macrophages exhibit distinct and more restricted HIV-1 integration site repertoire than CD4+ T cells

    Kok, Yik Lim; Vongrad, Valentina; Shilaih, Mohaned; Di Giallonardo, Francesca; Kuster, Herbert; Kouyos, Roger; Günthard, Huldrych F.; Metzner, Karin J.


    The host genetic landscape surrounding integrated HIV-1 has an impact on the fate of the provirus. Studies analysing HIV-1 integration sites in macrophages are scarce. We studied HIV-1 integration site patterns in monocyte-derived macrophages (MDMs) and activated CD4+ T cells derived from seven antiretroviral therapy (ART)-treated HIV-1-infected individuals whose cells were infected ex vivo with autologous HIV-1 isolated during the acute phase of infection. A total of 1,484 unique HIV-1 integration sites were analysed. Their distribution in the human genome and genetic features, and the effects of HIV-1 integrase polymorphisms on the nucleotide selection specificity at these sites were indistinguishable between the two cell types, and among HIV-1 isolates. However, the repertoires of HIV-1-hosting gene clusters overlapped to a higher extent in MDMs than in CD4+ T cells. The frequencies of HIV-1 integration events in genes encoding HIV-1-interacting proteins were also different between the two cell types. Lastly, HIV-1-hosting genes linked to clonal expansion of latently HIV-1-infected CD4+ T cells were over-represented in gene hotspots identified in CD4+ T cells but not in those identified in MDMs. Taken together, the repertoire of genes targeted by HIV-1 in MDMs is distinct from and more restricted than that of CD4+ T cells. PMID:27067385

  7. Yersinia outer protein YopE affects the actin cytoskeleton in Dictyostelium discoideum through targeting of multiple Rho family GTPases

    Vlahou, Georgia


    Abstract Background All human pathogenic Yersinia species share a virulence-associated type III secretion system that translocates Yersinia effector proteins into host cells to counteract infection-induced signaling responses and prevent phagocytosis. Dictyostelium discoideum has been recently used to study the effects of bacterial virulence factors produced by internalized pathogens. In this study we explored the potential of Dictyostelium as model organism for analyzing the effects of ectopically expressed Yersinia outer proteins (Yops). Results The Yersinia pseudotuberculosis virulence factors YopE, YopH, YopM and YopJ were expressed de novo within Dictyostelium and their effects on growth in axenic medium and on bacterial lawns were analyzed. No severe effect was observed for YopH, YopJ and YopM, but expression of YopE, which is a GTPase activating protein for Rho GTPases, was found to be highly detrimental. GFP-tagged YopE expressing cells had less conspicuous cortical actin accumulation and decreased amounts of F-actin. The actin polymerization response upon cAMP stimulation was impaired, although chemotaxis was unaffected. YopE also caused reduced uptake of yeast particles. These alterations are probably due to impaired Rac1 activation. We also found that YopE predominantly associates with intracellular membranes including the Golgi apparatus and inhibits the function of moderately overexpressed RacH. Conclusion The phenotype elicited by YopE in Dictyostelium can be explained, at least in part, by inactivation of one or more Rho family GTPases. It further demonstrates that the social amoeba Dictyostelium discoideum can be used as an efficient and easy-to-handle model organism in order to analyze the function of a translocated GAP protein of a human pathogen.

  8. Yersinia outer protein YopE affects the actin cytoskeleton in Dictyostelium discoideum through targeting of multiple Rho family GTPases

    Rivero Francisco


    Full Text Available Abstract Background All human pathogenic Yersinia species share a virulence-associated type III secretion system that translocates Yersinia effector proteins into host cells to counteract infection-induced signaling responses and prevent phagocytosis. Dictyostelium discoideum has been recently used to study the effects of bacterial virulence factors produced by internalized pathogens. In this study we explored the potential of Dictyostelium as model organism for analyzing the effects of ectopically expressed Yersinia outer proteins (Yops. Results The Yersinia pseudotuberculosis virulence factors YopE, YopH, YopM and YopJ were expressed de novo within Dictyostelium and their effects on growth in axenic medium and on bacterial lawns were analyzed. No severe effect was observed for YopH, YopJ and YopM, but expression of YopE, which is a GTPase activating protein for Rho GTPases, was found to be highly detrimental. GFP-tagged YopE expressing cells had less conspicuous cortical actin accumulation and decreased amounts of F-actin. The actin polymerization response upon cAMP stimulation was impaired, although chemotaxis was unaffected. YopE also caused reduced uptake of yeast particles. These alterations are probably due to impaired Rac1 activation. We also found that YopE predominantly associates with intracellular membranes including the Golgi apparatus and inhibits the function of moderately overexpressed RacH. Conclusion The phenotype elicited by YopE in Dictyostelium can be explained, at least in part, by inactivation of one or more Rho family GTPases. It further demonstrates that the social amoeba Dictyostelium discoideum can be used as an efficient and easy-to-handle model organism in order to analyze the function of a translocated GAP protein of a human pathogen.

  9. Prevalence, characterization, and antimicrobial resistance of Yersinia species and Yersinia enterocolitica isolated from raw milk in farm bulk tanks.

    Jamali, Hossein; Paydar, Mohammadjavad; Radmehr, Behrad; Ismail, Salmah


    The aims of this study were to investigate the prevalence and to characterize and determine the antibiotic resistance of Yersinia spp. isolates from raw milk. From September 2008 to August 2010, 446 raw milk samples were obtained from farm bulk milk tanks in Varamin, Iran. Yersinia spp. were detected in 29 (6.5%) samples, out of which 23 (79.3%), 5 (17.2%), and 1 (3.4%) were isolated from cow, sheep, and goat raw milk, respectively. The most common species isolated was Yersinia enterocolitica (65.5%), followed by Yersinia frederiksenii (31%), and Yersinia kristensenii (3.4%). Of the 19 Y. enterocolitica isolates, 14 (73.7%) were grouped into bioserotype 1A/O:9, 4 (21.1%) belonged to bioserotype 1B:O8, 1 (5.3%) belonged to bioserotype 4/O:3, and 1 isolate (biotype 1A) was not typable. All the isolates of biotypes 1B and 4harbored both the ystA and ail genes. However, all the isolates of biotype 1A were only positive for the ystB gene. The tested Yersinia spp. showed the highest percentages of resistance to tetracycline (48.3%), followed by ciprofloxacin and cephalothin (each 17.2%), ampicillin (13.8%), streptomycin (6.9%), and amoxicillin and nalidixic acid (each 3.4%). All of the tested isolates demonstrated significant sensitivity to gentamicin and chloramphenicol. Recovery of potentially pathogenic Y. enterocolitica from raw milk indicates high risks of yersiniosis associated with consumption of raw milk. PMID:25497824

  10. Methods to recover the narrow Dicke sub-Doppler feature in evacuated wall-coated cells without restrictions on cell size

    Robinson, H. G.


    The hyperfine resonance observed in evacuated wall-coated cells with dimensions lambda/2 (lambda is the hyperfine resonance wavelength) consists of a narrow Dicke sub-Doppler linewidth feature, the spike, superimposed on a broad pedestal. The hydrogen maser provides a classic example of this lineshape. As cell size is increased, an effect unique to evacuated wall-coated cells occurs. Certain combinations of microwave field distribution and cell size result in a lineshape having a pedestal with a small spike feature or only the broad pedestal with no spike. Such conditions are not appropriate for atomic frequency standard applications. The cause of the evacuated wall-coated cell lineshape is reviewed and methods to recover the narrow spike feature without restrictions on cell size is discussed. One example is a cell with dimensions having equal volumes of exposure to opposite phases of the microwave magnetic field.

  11. Interactions of opsonized immune complexes with whole blood cells: binding to erythrocytes restricts complex uptake by leucocyte populations

    Nielsen, C H; Svehag, S E; Marquart, H V; Leslie, R G


    respect to IC binding, the main contributors being B cells. E initially inhibited and then later enhanced the IC binding to lymphocytes, suggesting that E promote B cell uptake of C3d,g-covered IC via CR2. Our findings, that E can restrict the IC uptake by circulating leucocytes, and that an IC......The binding of opsonized, fluorescein-labelled bovine serum albumin (BSA)/rabbit anti-BSA complexes (IC) to washed human whole blood cells and isolated leucocytes in the presence of autologous serum was investigated by flow cytometry. In the presence of erythrocytes (E), the IC-binding to...... granulocytes (PMN), monocytes and lymphocytes was inhibited by up to 46%, 61% and 48%, respectively, depending on the incubation time and the IC-concentration tested. The E-mediated inhibition of the binding to PMN was found to correlate with the average numbers of CR1 per E during the initial 15 min of...

  12. Interactions of opsonized immune complexes with whole blood cells: binding to erythrocytes restricts complex uptake by leucocyte populations

    Nielsen, C H; Svehag, S E; Marquart, H V;


    The binding of opsonized, fluorescein-labelled bovine serum albumin (BSA)/rabbit anti-BSA complexes (IC) to washed human whole blood cells and isolated leucocytes in the presence of autologous serum was investigated by flow cytometry. In the presence of erythrocytes (E), the IC-binding to...... granulocytes (PMN), monocytes and lymphocytes was inhibited by up to 46%, 61% and 48%, respectively, depending on the incubation time and the IC-concentration tested. The E-mediated inhibition of the binding to PMN was found to correlate with the average numbers of CR1 per E during the initial 15 min of...... respect to IC binding, the main contributors being B cells. E initially inhibited and then later enhanced the IC binding to lymphocytes, suggesting that E promote B cell uptake of C3d,g-covered IC via CR2. Our findings, that E can restrict the IC uptake by circulating leucocytes, and that an IC...

  13. Methionine restriction inhibits chemically-induced malignant transformation in the BALB/c 3T3 cell transformation assay.

    Nicken, Petra; Empl, Michael T; Gerhard, Daniel; Hausmann, Julia; Steinberg, Pablo


    High consumption of red meat entails a higher risk of developing colorectal cancer. Methionine, which is more frequently a component of animal proteins, and folic acid are members of the one carbon cycle and as such important players in DNA methylation and cancer development. Therefore, dietary modifications involving altered methionine and folic acid content might inhibit colon cancer development. In the present study, the BALB/c 3T3 cell transformation assay was used to investigate whether methionine and folic acid are able to influence the malignant transformation of mouse fibroblasts after treatment with the known tumour initiator 3-methylcholanthrene. Three different methionine concentrations (representing a -40%, a "normal" and a +40% cell culture medium concentration, respectively) and two different folic acid concentrations (6 and 20 μM) were thereby investigated. Methionine restriction led to a decrease of type III foci, while enhancement of both methionine and folic acid did not significantly increase the cell transformation rate. Interestingly, the focus-lowering effect of methionine was only significant in conjunction with an elevated folic acid concentration. In summary, we conclude that the malignant transformation of mouse fibroblasts is influenced by methionine levels and that methionine restriction could be a possible approach to reduce cancer development. PMID:27427305

  14. Proteomic Characterization of Yersinia pestis Virulence

    Chromy, B; Murphy, G; Gonzales, A; Fitch, J P; McCutchen-Maloney, S L


    Yersinia pestis, the etiological agent of plague, functions via the Type III secretion mechanism whereby virulence factors are induced upon interactions with a mammalian host. Here, the Y. pestis proteome was studied by two-dimensional differential gel electrophoresis (2-D DIGE) under physiologically relevant growth conditions mimicking the calcium concentrations and temperatures that the pathogen would encounter in the flea vector and upon interaction with the mammalian host. Over 4100 individual protein spots were detected of which hundreds were differentially expressed in the entire comparative experiment. A total of 43 proteins that were differentially expressed between the vector and host growth conditions were identified by mass spectrometry. Expected differences in expression were observed for several known virulence factors including catalase-peroxidase (KatY), murine toxin (Ymt), plasminogen activator (Pla), and F1 capsule antigen (Caf1), as well as putative virulence factors. Chaperone proteins and signaling molecules hypothesized to be involved in virulence due to their role in Type III secretion were also identified. Other differentially expressed proteins not previously reported to contribute to virulence are candidates for more detailed mechanistic studies, representing potential new virulence determinants. For example, several sugar metabolism proteins were differentially regulated in response to lower calcium and higher temperature, suggesting these proteins, while not directly connected to virulence, either represent a metabolic switch for survival in the host environment or may facilitate production of virulence factors. Results presented here contribute to a more thorough understanding of the virulence mechanism of Y. pestis through proteomic characterization of the pathogen under induced virulence.

  15. Restricted movement of lipid and aqueous dyes through pores formed by influenza hemagglutinin during cell fusion


    The fusion of cells by influenza hemagglutinin (HA) is the best characterized example of protein-mediated membrane fusion. In simultaneous measurements of pairs of assays for fusion, we determined the order of detectable events during fusion. Fusion pore formation in HA-triggered cell-cell fusion was first detected by changes in cell membrane capacitance, next by a flux of fluorescent lipid, and finally by flux of aqueous fluorescent dye. Fusion pore conductance increased by small steps. A re...

  16. Effects of immobilisation and caloric restriction on antioxidant parameters and T-cell apoptosis in healthy young men

    Ellinger, S.; Arendt, B. M.; Boese, A.; Juschus, M.; Schaefer, S.; Stoffel-Wagner, B.; Goerlich, R.

    Background: Astronauts are exposed to oxidative stress due to radiation and microgravity, which might impair immune functions. Effects of hypocaloric nutrition as often observed in astronauts on oxidative stress and immune functions are not clear. We investigated, if microgravity, simulated by 6 Head-down tilt (HDT) and caloric restriction (-25%, fat reduced) with adequate supply of micronutrients affect DNA-damage in peripheral leukocytes, antioxidant parameters in plasma, and T-cell apoptosis. Material & Methods: 10 healthy male non-smokers were subjected to 4 different interventions (normocaloric diet or caloric restriction (CR) in upright position (UP) or HDT) for 14 days each (cross-over). DNA-damage in peripheral leukocytes (Comet Assay), trolox equivalent antioxidant capacity (TEAC) and uric acid in plasma were measured before, after 5, 10, and 13 days of intervention, and after 2 days recovery. T-cell apoptosis (Annexin V binding test) was assessed before and after intervention. Results: Preliminary results show that only endogenous, but not ex vivo H2O2-induced DNA strand breaks were reduced by CR compared to normocaloric diet. In upright position, endogenous DNA strand breaks decreased continuously during CR, reaching significance after recovery. During HDT, caloric restriction seems to counteract a temporary increase in DNA strand breaks observed in subjects receiving normocaloric diet. TEAC was reduced during HDT compared to UP in subjects under caloric restriction. An increase in plasma uric acid related to intervention occurred only after 5 days HDT in CR vs. normocaloric diet. T-cell apoptosis was not affected by any kind of intervention. Conclusion: Neither HDT nor CR with sufficient supply of micronutrients seem to induce oxidative stress or T-cell apoptosis in healthy young men. In contrast, CR might prevent endogenous DNA-damage in peripheral leukocytes. As DNA-damage is a risk factor for carcinogenesis, protective effects of energy reduction are

  17. Entry and release of transmissible gastroenteritis coronavirus are restricted to apical surfaces of polarized epithelial cells

    Rossen, J W; Bekker, C P; Voorhout, W F; Strous, G J; van der Ende, A; Rottier, P J


    The transmissible gastroenteritis coronavirus (TGEV) infects the epithelial cells of the intestinal tract of pigs, resulting in a high mortality rate in piglets. This study shows the interaction of TGEV with a porcine epithelial cell line. To determine the site of viral entry, LLC-PK1 cells were gro

  18. Restricted distribution of mrg-1 mRNA in C. elegans primordial germ cells through germ granule-independent regulation.

    Miwa, Takashi; Takasaki, Teruaki; Inoue, Kunio; Sakamoto, Hiroshi


    The chromodomain protein MRG-1 is an essential maternal factor for proper germline development that protects germ cells from cell death in C. elegans. Unlike germ granules, which are exclusively segregated to the germline blastomeres at each cell division from the first cleavage of the embryo, MRG-1 is abundant in all cells in early embryos and is then gradually restricted to the primordial germ cells (PGCs) by the morphogenesis stage. Here, we show that this characteristic spatiotemporal expression pattern is dictated by the mrg-1 3'UTR and is differentially regulated at the RNA level between germline and somatic cells. Asymmetric segregation of germ granules is not necessary to localize MRG-1 to the PGCs. We found that MES-4, an essential chromatin regulator in germ cells, also accumulates in the PGCs in a germ granule-independent manner. We propose that C.elegans PGCs have a novel mechanism to accumulate at least some chromatin-associated proteins that are essential for germline immortality. PMID:26537333

  19. Reversibility of β-Cell-Specific Transcript Factors Expression by Long-Term Caloric Restriction in db/db Mouse

    Chunjun Sheng


    Full Text Available Type 2 diabetes (T2D is characterized by β-cell dedifferentiation, but underlying mechanisms remain unclear. The purpose of the current study was to explore the mechanisms of β-cell dedifferentiation with and without long-term control of calorie intake. We used a diabetes mouse model (db/db to analyze the changes in the expression levels of β-cell-specific transcription factors (TFs and functional factors with long-term caloric restriction (CR. Our results showed that chronic euglycemia was maintained in the db/db mice with long-term CR intervention, and β-cell dedifferentiation was significantly reduced. The expression of Glut2, Pdx1, and Nkx6.1 was reversed, while MafA expression was significantly increased with long-term CR. GLP-1 pathway was reactivated with long-term CR. Our work showed that the course of β-cell dedifferentiation can intervene by long-term control of calorie intake. Key β-cell-specific TFs and functional factors play important roles in maintaining β-cell differentiation. Targeting these factors could optimize T2D therapies.

  20. Tuning nano electric field to affect restrictive membrane area on localized single cell nano-electroporation

    Santra, Tuhin Subhra; Wang, Pen-Cheng; Chang, Hwan-You; Tseng, Fan-Gang


    Interaction of electric field with biological cells is an important phenomenon for field induced drug delivery system. We demonstrate a selective and localized single cell nano-electroporation (LSCNEP) by applying an intense electric field on a submicron region of the single cell membrane, which can effectively allow high efficient molecular delivery but low cell damage. The delivery rate is controlled by adjusting transmembrane potential and manipulating membrane status. Thermal and ionic influences are deteriorated from the cell membrane by dielectric passivation. Either reversible or irreversible by LSCNEP can fully controlled with potential applications in medical diagnostics and biological studies.

  1. The effect of aging and caloric restriction on murine CD8+ T cell chemokine receptor gene expression

    Mo RuRan


    Full Text Available Abstract Background The mechanism explaining the increased disease susceptibility in aging is not well understood. CD8+ T cells are crucial in anti-viral and anti-tumor responses. Although the chemokine system plays a critical role in CD8+ T cell function, very little is known about the relationship between aging and the T cell chemokine system. Results In this study we have examined the effect of aging on murine CD8+ T cell chemokine receptor gene expression. Freshly isolated splenic CD8+ T cells from old C57BL/6 mice were found to have higher CCR1, CCR2, CCR4, CCR5 and CXCR5, and lower CCR7 gene expression compared to their younger cohort. Anti-CD3/anti-CD28 stimulation elicited a similar robust chemokine receptor response from young and old CD8+ T cells. Western blot analyses confirmed elevated protein level of CCR4 and CCR5 in aged CD8+ T cells. Increases in T cell CCR1 and CCR5 expression also correlate to increased in vitro chemotaxis response to macrophage-inflammatory protein-1 α(MIP-1α. Finally, caloric restriction selectively prevents the loss of CD8+ T cell CCR7 gene expression in aging to the level that is seen in young CD8+ T cells. Conclusion These findings are consistent with the notion that aging exists in a state of low grade pro-inflammatory environment. In addition, our results provide a potential mechanism for the reported aging-associated impaired T cell lymphoid homing and allograft response, and reduced survival in sepsis.

  2. Molecular cloning of cecropin B responsive endonucleases in Yersinia ruckeri

    We have previously demonstrated that Yersinia ruckeri resists cecropin B in an inducible manner. In this study, we sought to identify the molecular changes responsible for the inducible cecropin B resistance of Y. ruckeri. Differences in gene expression associated with the inducible resistance were ...

  3. A restricted set of apical proteins recycle through the trans-Golgi network in MDCK cells.

    Brändli, A W; Simons, K


    Sorting of newly synthesized proteins destined for the apical plasma membrane takes place in the trans-Golgi network (TGN) in MDCK cells. This process is most likely receptor mediated and requires components that recycle between both compartments. We have developed an assay to detect apical proteins that recycle through the sialyltransferase-containing TGN. Cell surface glycoproteins were exogalactosylated apically using a mutant cell line derived from MDCK, MDCKII-RCAr. The mutant exhibits i...

  4. Infected site-restricted Foxp3+ natural regulatory T cells are specific for microbial antigens

    Suffia, Isabelle J.; Reckling, Stacie K.; Piccirillo, Ciriaco A; Goldszmid, Romina S.; Belkaid, Yasmine


    Natural regulatory T (T reg) cells are involved in control of the immune response, including response to pathogens. Previous work has demonstrated that the repertoire of natural T reg cells may be biased toward self-antigen recognition. Whether they also recognize foreign antigens and how this recognition contributes to their function remain unknown. Our studies addressed the antigenic specificity of natural T reg cells that accumulate at sites of chronic infection with Leishmania major in mi...

  5. Neuroblastoma and pre-B lymphoma cells share expression of key transcription factors but display tissue restricted target gene expression

    Transcription factors are frequently involved in the process of cellular transformation, and many malignancies are characterized by a distinct genetic event affecting a specific transcription factor. This probably reflects a tissue specific ability of transcription factors to contribute to the generation of cancer but very little is known about the precise mechanisms that governs these restricted effects. To investigate this selectivity in target gene activation we compared the overall gene expression patterns by micro-array analysis and expression of target genes for the transcription factor EBF in lymphoma and neuroblastoma cells by RT-PCR. The presence of transcription factors in the different model cell lines was further investigated by EMSA analysis. In pre-B cells mb-1 and CD19 are regulate by EBF-1 in collaboration with Pax-5 and E-proteins. We here show that neuroblastoma cells express these three, for B cell development crucial transcription factors, but nevertheless fail to express detectable levels of their known target genes. Expression of mb-1 could, however, be induced in neuroblastoma cells after disruption of the chromatin structure by treatment with 5-azacytidine and Trichostatin A. These data suggest that transcription factors are able to selectively activate target genes in different tissues and that chromatin structure plays a key role in the regulation of this activity

  6. Severe cell reduction in the future brain cortex in human growth-restricted fetuses and infants

    Samuelsen, Grethe B; Pakkenberg, Bente; Bogdanović, Nenad;


    controls. The daily increase in brain cells in the future cortex was only half of that of the controls. In the 3 other developmental zones, no significant differences in cell numbers could be demonstrated. CONCLUSIONS: IUGR in humans is associated with a severe reduction in cortical growth and a...... estimated in 9 severely affected IUGR fetuses and 15 controls using the optical fractionator. Cell numbers were estimated within 4 developmental zones. The gestational ages were 19-41 weeks. RESULTS: The total cell number in the future cortex was significantly reduced in the IUGR fetuses, compared with...

  7. Feedback, receptor clustering, and receptor restriction to single cells yield large Turing spaces for ligand-receptor-based Turing models

    Kurics, Tamás; Menshykau, Denis; Iber, Dagmar


    Turing mechanisms can yield a large variety of patterns from noisy, homogenous initial conditions and have been proposed as patterning mechanism for many developmental processes. However, the molecular components that give rise to Turing patterns have remained elusive, and the small size of the parameter space that permits Turing patterns to emerge makes it difficult to explain how Turing patterns could evolve. We have recently shown that Turing patterns can be obtained with a single ligand if the ligand-receptor interaction is taken into account. Here we show that the general properties of ligand-receptor systems result in very large Turing spaces. Thus, the restriction of receptors to single cells, negative feedbacks, regulatory interactions among different ligand-receptor systems, and the clustering of receptors on the cell surface all greatly enlarge the Turing space. We further show that the feedbacks that occur in the FGF10-SHH network that controls lung branching morphogenesis are sufficient to result in large Turing spaces. We conclude that the cellular restriction of receptors provides a mechanism to sufficiently increase the size of the Turing space to make the evolution of Turing patterns likely. Additional feedbacks may then have further enlarged the Turing space. Given their robustness and flexibility, we propose that receptor-ligand-based Turing mechanisms present a general mechanism for patterning in biology.

  8. Multiple tissue-specific isoforms of sulfatide activate CD1d-restricted type II NKT cells

    Blomqvist, Maria; Rhost, Sara; Teneberg, Susann;


    relevant isoforms C24:1 and C24:0, major constituents of the myelin sheet of the nervous system, and C16:0, prominent in the pancreatic islet beta-cells. The most potent sulfatide isoform was lysosulfatide (lacking a fatty acid). Shortened fatty acid chain length (C24:1 versus C18:1), or saturation of the...... mixture of sulfatide isoforms, i.e. sulfatide molecules with different long-chain bases and fatty acid chain lengths and saturation. Here, we demonstrate that sulfatide-specific CD1d-restricted murine NKT hybridomas recognized several different sulfatide isoforms. These included the physiologically...... long fatty acid (C24:0), resulted in reduced stimulatory capacity, and fatty acid hydroxylation abolished the response. Moreover, sulfatide was not responsible for the natural autoreactivity toward splenocytes by XV19 T hybridoma cells. Our results reveal a promiscuity in the recognition of sulfatide...

  9. A novel envelope mediated post entry restriction of murine leukaemia virus in human cells is Ref1/TRIM5α independent

    McKnight Áine


    Full Text Available Abstract Background 'Intrinsic' resistance to retroviral infection was first recognised with the Friend virus susceptibility gene (Fv1, which determines susceptibility to murine leukaemia virus (MLV infection in different murine species. Similarly, the tripartite motif (TRIM family of proteins determine lentiviral restriction in a primate host-species specific manner. For example rhesus TRIM5α (rhTRIM5α can potently restrict HIV-1 infection while human TRIM5α (huTRIM5α only has a mild effect on SIVmac and HIV-1 infectivity (Lv1. Human TRIM5α is able to restrict MLV-N virus replication, but is ineffective against MLV-B or MLV-NB virus infection. Lv2 restriction of some HIV-2 viruses is seen in human cells. Like Lv1, Lv2 is a post-entry restriction factor, whose viral determinants have been mapped to the viral capsid (CA. Unlike Lv1, however, Lv2 is determined by envelope (Env in addition to CA. Here we present evidence of a novel Env determined post entry restriction to infection in human cells of pseudotyped MLV-B and MLV-NB cores. Results We generated retroviral vectors pseudotyped with various gamma and lentiviral Envs on MLV-B and -NB CAs containing a green fluorescent protein (GFP reporter. Flow cytometry was used to determine transduction efficiencies in NP2/CD4/CXCR4 (glioma cell line stably transduced with the HIV receptors and HeLa/CD4 cell lines. The HeLa/CD4 cell line restricted both MLV CAs in an Env dependent manner, compared to NP2/CD4/CXCR4 cells. Quantitative polymerase chain reaction (QT-PCR analysis of reverse transcription (RT transcripts demonstrates that this restriction occurs at a post entry and RT level. siRNA knockdown of huTRIM5α ruled out a direct role for this cellular component in mediating this restriction. We describe a previously unobserved Env determined restriction of MLV-B and MLV-NB CAs in HeLa/CD4 cells when pseudotyped with HIV-2 and RD114 Envs, but not gibbon ape leukaemia virus (GALV, HIV-1 or

  10. Down-regulation of the cyprinid herpesvirus-3 annotated genes in cultured cells maintained at restrictive high temperature.

    Ilouze, Maya; Dishon, Arnon; Kotler, Moshe


    Cyprinid herpesvirus-3 (CyHV-3) is a member of the Alloherpesviridae, in the order Herpesvirales. It causes a fatal disease in carp and koi fish. The disease is seasonal and is active when water temperatures ranges from 18 to 28 °C. Little is known about how and where the virus is preserved between the permissive seasons. The hallmark of the herpesviruses is their ability to become latent, persisting in the host in an apparently inactive state for varying periods of time. Hence, it could be expected that CyHV-3 enter a latent period. CyHV-3 has so far been shown to persist in fish maintained under restrictive temperatures, while shifting the fish to permissive conditions reactivates the virus. Previously, we demonstrated that cultured cells infected with CyHV-3 at 22 °C and subsequently transferred to a restrictive temperature of 30 °C preserve the virus for 30 days. The present report shows that cultured carp cells maintained and exposed to CyHV-3 at 30 °C are abortively infected; that is, autonomous viral DNA synthesis is hampered and the viral genome is not multiplied. Under these conditions, 91 of the 156 viral annotated ORFs were initially transcribed. These transcripts were down-regulated and gradually shut off over 18 days post-infection, while two viral transcripts encoded by ORFs 114 and 115 were preserved in the infected cells for 18 days p.i. These experiments, carried out in cultured cells, suggest that fish could be infected at a high non-permissive temperature and harbor the viral genome without producing viral particles. PMID:22841492

  11. Yersinia pestis Requires Host Rab1b for Survival in Macrophages.

    Michael G Connor


    Full Text Available Yersinia pestis is a facultative intracellular pathogen that causes the disease known as plague. During infection of macrophages Y. pestis actively evades the normal phagosomal maturation pathway to establish a replicative niche within the cell. However, the mechanisms used by Y. pestis to subvert killing by the macrophage are unknown. Host Rab GTPases are central mediators of vesicular trafficking and are commonly targeted by bacterial pathogens to alter phagosome maturation and killing by macrophages. Here we demonstrate for the first time that host Rab1b is required for Y. pestis to effectively evade killing by macrophages. We also show that Rab1b is specifically recruited to the Yersinia containing vacuole (YCV and that Y. pestis is unable to subvert YCV acidification when Rab1b expression is knocked down in macrophages. Furthermore, Rab1b knockdown also altered the frequency of association between the YCV with the lysosomal marker Lamp1, suggesting that Rab1b recruitment to the YCV directly inhibits phagosome maturation. Finally, we show that Rab1b knockdown also impacts the pH of the Legionella pneumophila containing vacuole, another pathogen that recruits Rab1b to its vacuole. Together these data identify a novel role for Rab1b in the subversion of phagosome maturation by intracellular pathogens and suggest that recruitment of Rab1b to the pathogen containing vacuole may be a conserved mechanism to control vacuole pH.

  12. Lipid raft association restricts CD44-ezrin interaction and promotion of breast cancer cell migration.

    Donatello, Simona


    Cancer cell migration is an early event in metastasis, the main cause of breast cancer-related deaths. Cholesterol-enriched membrane domains called lipid rafts influence the function of many molecules, including the raft-associated protein CD44. We describe a novel mechanism whereby rafts regulate interactions between CD44 and its binding partner ezrin in migrating breast cancer cells. Specifically, in nonmigrating cells, CD44 and ezrin localized to different membranous compartments: CD44 predominantly in rafts, and ezrin in nonraft compartments. After the induction of migration (either nonspecific or CD44-driven), CD44 affiliation with lipid rafts was decreased. This was accompanied by increased coprecipitation of CD44 and active (threonine-phosphorylated) ezrin-radixin-moesin (ERM) proteins in nonraft compartments and increased colocalization of CD44 with the nonraft protein, transferrin receptor. Pharmacological raft disruption using methyl-β-cyclodextrin also increased CD44-ezrin coprecipitation and colocalization, further suggesting that CD44 interacts with ezrin outside rafts during migration. Conversely, promoting CD44 retention inside lipid rafts by pharmacological inhibition of depalmitoylation virtually abolished CD44-ezrin interactions. However, transient single or double knockdown of flotillin-1 or caveolin-1 was not sufficient to increase cell migration over a short time course, suggesting complex crosstalk mechanisms. We propose a new model for CD44-dependent breast cancer cell migration, where CD44 must relocalize outside lipid rafts to drive cell migration. This could have implications for rafts as pharmacological targets to down-regulate cancer cell migration.

  13. Restricted ultraviolet mutational spectrum in a shuttle vector propagated in xeroderma pigmentosum cells

    A shuttle vector plasmid, pZ189, carrying a bacterial suppressor tRNA marker gene, was treated with ultraviolet radiation and propagated in cultured skin cells from a patient with the skin-cancer-prone, DNA repair-deficient disease xeroderma pigmentosum and in repair-proficient cells. After replication in the human cells, progeny plasmids were purified. Plasmid survival and mutations inactivating the marker gene were scored by transforming an indicator strain of Escherichia coli carrying a suppressible amber mutation in the beta-galactosidase gene. Plasmid survival in the xeroderma pigmentosum cells was less than that of pZ189 harvested from repair-proficient human cells. The point-mutation frequency in the 150-base-pair tRNA marker gene increased up to 100-fold with ultraviolet dose. Sequence analysis of 150 mutant plasmids revealed that mutations were infrequent at potential thymine-thymine dimer sites. Ninety-three percent of the mutant plasmids from the xeroderma pigmentosum cells showed G X C----A X T transitions, compared to 73% in the normal cells (P less than 0.002). There were significantly fewer transversions (P less than 0.002) (especially G X C----T X A) and multiple base substitutions (P less than 0.00001) than when pZ189 was passaged in repair-proficient cells. The subset of mutational changes that are common to ultraviolet-treated plasmids propagated in both repair-proficient and xeroderma pigmentosum skin cells may be associated with the development of ultraviolet-induced skin cancer in humans

  14. Fast cellular automata with restricted inter-cell communication: computational capacity

    Kutrib, Martin; Malcher, Andreas


    A d-dimensional cellular automaton with sequential input mode is a d-dimensional grid of interconnected interacting finite automata. The distinguished automaton at the origin, the communication cell, is connected to the outside world and fetches the input sequentially. Often in the literature this model is referred to as iterative array. We investigate d-dimensional iterative arrays and one-dimensional cellular automata operating in real and linear time, whose inter-cell communicati...

  15. Drosophila Rabex-5 restricts Notch activity in hematopoietic cells and maintains hematopoietic homeostasis

    Reimels, Theresa A.; Cathie M. Pfleger


    ABSTRACT Hematopoietic homeostasis requires the maintenance of a reservoir of undifferentiated blood cell progenitors and the ability to replace or expand differentiated blood cell lineages when necessary. Multiple signaling pathways function in these processes, but how their spatiotemporal control is established and their activity is coordinated in the context of the entire hematopoietic network are still poorly understood. We report here that loss of the gene Rabex-5 in Drosophila causes se...

  16. Human CD1-restricted T cell recognition of lipids from pollens.

    Agea, Elisabetta; Russano, Anna; Bistoni, Onelia; Mannucci, Roberta; Nicoletti, Ildo; Corazzi, Lanfranco; Postle, Anthony D; De Libero, Gennaro; Porcelli, Steven A; Spinozzi, Fabrizio


    Plant pollens are an important source of environmental antigens that stimulate allergic responses. In addition to acting as vehicles for foreign protein antigens, they contain lipids that incorporate saturated and unsaturated fatty acids, which are necessary in the reproduction of higher plants. The CD1 family of nonpolymorphic major histocompatibility complex-related molecules is highly conserved in mammals, and has been shown to present microbial and self lipids to T cells. Here, we provide evidence that pollen lipids may be recognized as antigens by human T cells through a CD1-dependent pathway. Among phospholipids extracted from cypress grains, phosphatidyl-choline and phosphatidyl-ethanolamine were able to stimulate the proliferation of T cells from cypress-sensitive subjects. Recognition of phospholipids involved multiple cell types, mostly CD4(+) T cell receptor for antigen (TCR)alphabeta(+), some CD4(-)CD8(-) TCRgammadelta(+), but rarely Valpha24i(+) natural killer-T cells, and required CD1a(+) and CD1d(+) antigen presenting cell. The responding T cells secreted both interleukin (IL)-4 and interferon-gamma, in some cases IL-10 and transforming growth factor-beta, and could provide help for immunoglobulin E (IgE) production. Responses to pollen phospholipids were maximally evident in blood samples obtained from allergic subjects during pollinating season, uniformly absent in Mycobacterium tuberculosis-exposed health care workers, but occasionally seen in nonallergic subjects. Finally, allergic, but not normal subjects, displayed circulating specific IgE and cutaneous weal and flare reactions to phospholipids. PMID:16009719

  17. Engraftment potential of human fetal hematopoietic cells in NOD/SCID mice is not restricted to mitotically quiescent cells.

    Wilpshaar, Jannine; Bhatia, Mickie; Kanhai, Humphrey H H; Breese, Robert; Heilman, Doug K; Johnson, Cynthia S; Falkenburg, J H Frederik; Srour, Edward F


    During fetal development, there is a continued demand for large numbers of primitive and mature hematopoietic cells. This demand may require that all potential hematopoietic stem cells (HSCs) migrate effectively to emerging hematopoietic sites and subsequently contribute to blood cell production, regardless of their cell cycle status. We recently established that umbilical cord blood cells in the G(1) phase of the cell cycle have a repopulating potential similar to cells in G(0), suggesting that cycling prenatal and neonatal HSCs may have the same functional capabilities described for quiescent, but not cycling, cells from adult sources. To establish the relationship between cell cycle status and hematopoietic potential at early stages of human ontogeny, the in vivo engraftment potential of mitotically defined fetal liver (FL) and fetal bone marrow (FBM) cells were examined in NOD/SCID recipients. Following transplantation of the same numbers of G(0), G(1), or S/G(2)+M CD34(+) cells from FL, equivalent percentages of recipient mice were chimeric (55%, 60%, and 60%, respectively). FBM-derived CD34(+) cells in all phases of the cell cycle engrafted in conditioned recipients and sustained human hematopoiesis, albeit at lower levels than their FL-derived counterparts. Multilineage differentiation was evident in all transplanted mice independent of the source or cell cycle status of graft cells. In addition, levels of chimerism in mice transplanted with fetal blood-derived G(0) or G(1) CD34(+) lineage-depleted cells were similar. These results support the assertion that mitotically quiescent and cycling fetal hematopoietic cells contain marrow-repopulating stem cells capable of multilineage engraftment in NOD/SCID mouse recipients. PMID:12070016

  18. Pdx1 inactivation restricted to the intestinal epithelium in mice alters duodenal gene expression in enterocytes and enteroendocrine cells.

    Chen, Chin; Fang, Rixun; Davis, Corrine; Maravelias, Charalambos; Sibley, Eric


    Null mutant mice lacking the transcription factor pancreatic and duodenal homeobox 1 (Pdx1) are apancreatic and survive only a few days after birth. The role of Pdx1 in regulating intestinal gene expression has therefore yet to be determined in viable mice with normal pancreatic development. We hypothesized that conditional inactivation of Pdx1 restricted to the intestinal epithelium would alter intestinal gene expression and cell differentiation. Pdx1(flox/flox);VilCre mice with intestine-specific Pdx1 inactivation were generated by crossing a transgenic mouse strain expressing Cre recombinase, driven by a mouse villin 1 gene promoter fragment, with a mutant mouse strain homozygous for loxP site-flanked Pdx1. Pdx1 protein is undetectable in all epithelial cells in the intestinal epithelium of Pdx1(flox/flox);VilCre mice. Goblet cell number and mRNA abundance for mucin 3 and mucin 13 genes in the proximal small intestine are comparable between Pdx1(flox/flox);VilCre and control mice. Similarly, Paneth cell number and expression of Paneth cell-related genes Defa1, Defcr-rs1, and Mmp7 in the proximal small intestine remain statistically unchanged by Pdx1 inactivation. Although the number of enteroendocrine cells expressing chromogranin A/B, gastric inhibitory polypeptide (Gip), or somatostatin (Sst) is unaffected in the Pdx1(flox/flox);VilCre mice, mRNA abundance for Gip and Sst is significantly reduced in the proximal small intestine. Conditional Pdx1 inactivation attenuates intestinal alkaline phosphatase (IAP) activity in the duodenal epithelium, consistent with an average 91% decrease in expression of the mouse enterocyte IAP gene, alkaline phosphatase 3 (a novel Pdx1 target candidate), in the proximal small intestine following Pdx1 inactivation. We conclude that Pdx1 is necessary for patterning appropriate gene expression in enterocytes and enteroendocrine cells of the proximal small intestine. PMID:19808654

  19. Restricting nonclassical MHC genes coevolve with TRAV genes used by innate-like T cells in mammals.

    Boudinot, Pierre; Mondot, Stanislas; Jouneau, Luc; Teyton, Luc; Lefranc, Marie-Paule; Lantz, Olivier


    Whereas major histocompatibility class-1 (MH1) proteins present peptides to T cells displaying a large T-cell receptor (TR) repertoire, MH1Like proteins, such as CD1D and MR1, present glycolipids and microbial riboflavin precursor derivatives, respectively, to T cells expressing invariant TR-α (iTRA) chains. The groove of such MH1Like, as well as iTRA chains used by mucosal-associated invariant T (MAIT) and natural killer T (NKT) cells, respectively, may result from a coevolution under particular selection pressures. Herein, we investigated the evolutionary patterns of the iTRA of MAIT and NKT cells and restricting MH1Like proteins: MR1 appeared 170 Mya and is highly conserved across mammals, evolving more slowly than other MH1Like. It has been pseudogenized or independently lost three times in carnivores, the armadillo, and lagomorphs. The corresponding TRAV1 gene also evolved slowly and harbors highly conserved complementarity determining regions 1 and 2. TRAV1 is absent exclusively from species in which MR1 is lacking, suggesting that its loss released the purifying selection on MR1. In the rabbit, which has very few NKT and no MAIT cells, a previously unrecognized iTRA was identified by sequencing leukocyte RNA. This iTRA uses TRAV41, which is highly conserved across several groups of mammals. A rabbit MH1Like gene was found that appeared with mammals and is highly conserved. It was independently lost in a few groups in which MR1 is present, like primates and Muridae, illustrating compensatory emergences of new MH1Like/Invariant T-cell combinations during evolution. Deciphering their role is warranted to search similar effector functions in humans. PMID:27170188

  20. Prevalence of Yersinia Species in Traditional and Commercial Dairy Products in Isfahan Province, Iran

    Rahimi, Ebrahim; Sepehri, Sara; Safarpoor Dehkordi, Farhad; Shaygan, Shima; Momtaz, Hassan


    Background: Yersinia species, especially Yersinia enterocolitica, are considered as the most prevalent milk-borne pathogens. Several serological and molecular techniques have been developed for rapid and safe diagnosis of yersiniosis. Objectives: This study was carried out to assess the prevalence rate of Yersinia species, especially Y. enterocolitica, in milk and dairy products in Isfahan province, Iran. Materials and Methods: A total of 285 commercial and traditional dairy products as well ...

  1. Recognition of a subregion of human proinsulin by class I-restricted T cells in type 1 diabetic patients.

    Toma, Andréa; Haddouk, Samy; Briand, Jean-Paul; Camoin, Luc; Gahery, Hanne; Connan, Francine; Dubois-Laforgue, Danielle; Caillat-Zucman, Sophie; Guillet, Jean-Gérard; Carel, Jean-Claude; Muller, Sylviane; Choppin, Jeannine; Boitard, Christian


    Proinsulin is a key autoantigen in type 1 diabetes. Evidence in the mouse has underscored the importance of the insulin B chain region in autoimmunity to pancreatic beta cells. In man, a majority of proteasome cleavage sites are predicted by proteasome cleavage algorithms within this region. To study CD8+ T cell responses to the insulin B chain and adjacent C peptide, we selected 8- to 11-mer peptides according to proteasome cleavage patterns obtained by digestion of two peptides covering proinsulin residues 28 to 64. We studied their binding to purified HLA class I molecules and their recognition by T cells from diabetic patients. Peripheral blood mononuclear cells from 17 of 19 recent-onset and 12 of 13 long-standing type 1 diabetic patients produced IFN-gamma in response to proinsulin peptides as shown by using an ELISPOT assay. In most patients, the response was against several class I-restricted peptides. Nine peptides were recognized within the proinsulin region covering residues 34 to 61. Four yielded a high frequency of recognition in HLA-A1 and -B8 patients. Three peptides located in the proinsulin region 41-51 were shown to bind several HLA molecules and to be recognized in a high percentage of diabetic patients. PMID:16030147

  2. A multidirectional non-cell autonomous control and a genetic interaction restricting tobacco etch virus susceptibility in Arabidopsis.

    Suresh Gopalan

    Full Text Available BACKGROUND: Viruses constitute a major class of pathogens that infect a variety of hosts. Understanding the intricacies of signaling during host-virus interactions should aid in designing disease prevention strategies and in understanding mechanistic aspects of host and pathogen signaling machinery. METHODOLOGY/PRINCIPAL FINDINGS: An Arabidopsis mutant, B149, impaired in susceptibility to Tobacco etch virus (TEV, a positive strand RNA virus of picoRNA family, was identified using a high-throughput genetic screen and a counterselection scheme. The defects include initiation of infection foci, rate of cell-to-cell movement and long distance movement. CONCLUSIONS/SIGNIFICANCE: The defect in infectivity is conferred by a recessive locus. Molecular genetic analysis and complementation analysis with three alleles of a previously published mutant lsp1 (loss of susceptibility to potyviruses indicate a genetic interaction conferring haploinsufficiency between the B149 locus and certain alleles of lsp1 resulting in impaired host susceptibility. The pattern of restriction of TEV foci on leaves at or near the boundaries of certain cell types and leaf boundaries suggest dysregulation of a multidirectional non-cell autonomous regulatory mechanism. Understanding the nature of this multidirectional signal and the molecular genetic mechanism conferring it should potentially reveal a novel arsenal in the cellular machinery.

  3. miRNA Profiles of Monocyte-Lineage Cells Are Consistent with Complicated Roles in HIV-1 Restriction

    Janice E. Clements


    Full Text Available Long-lived HIV-1 reservoirs include tissue macrophages. Monocyte-derived macrophages are more susceptible to infection and more permissive to HIV-1 replication than monocytes for reasons that may include the effects of different populations of miRNAs in these two cell classes. Specifically, miRs-28-3p, -150, -223, -198, and -382 exert direct or indirect negative effects on HIV-1 and are reportedly downmodulated during monocyte-to-macrophage differentiation. Here, new experimental results are presented along with reviews and analysis of published studies and publicly available datasets, supporting a broader role of miRNAs in HIV-1 restriction than would be suggested by a simple and uniform downregulation of anti-HIV miRNAs during monocyte-to-macrophage differentiation. Although miR-223 is downregulated in macrophages, other putatively antiviral miRNAs are more abundant in macrophages than in monocytes or are rare and/or variably present in both cell classes. Our analyses point to the need for further studies to determine miRNA profiles of monocytes and macrophages, including classic and newly identified subpopulations; examine the sensitivity of miRNA profiling to cell isolation and differentiation protocols; and characterize rigorously the antiviral effects of previously reported and novel predicted miRNA-HIV-1 interactions in cell-specific contexts.

  4. Glucose restriction decreases telomerase activity and enhances its inhibitor response on breast cancer cells: possible extra-telomerase role of BIBR 1532

    Wardi, Layal; Alaaeddine, Nada; Raad, Issam; Sarkis, Riad; Serhal, Rim; Khalil, Charbel; Hilal, George


    Background Considerable progress has been made to understand the association between lifestyle and diet in cancer initiation and promotion. Because excessive glucose consumption is a key metabolic hallmark of cancer cells, glucose restriction (GR) decreases the proliferation, and promotes the differentiation and transformation of cancer cells to quiescent cells. The immortality of cancerous cells is largely assured by telomerase, which is an interesting target for inhibition by BIBR 1532. In ...

  5. XIAP Restricts TNF- and RIP3-Dependent Cell Death and Inflammasome Activation

    Monica Yabal


    Full Text Available X-linked inhibitor of apoptosis protein (XIAP has been identified as a potent regulator of innate immune responses, and loss-of-function mutations in XIAP cause the development of the X-linked lymphoproliferative syndrome type 2 (XLP-2 in humans. Using gene-targeted mice, we show that loss of XIAP or deletion of its RING domain lead to excessive cell death and IL-1β secretion from dendritic cells triggered by diverse Toll-like receptor stimuli. Aberrant IL-1β secretion is TNF dependent and requires RIP3 but is independent of cIAP1/cIAP2. The observed cell death also requires TNF and RIP3 but proceeds independently of caspase-1/caspase-11 or caspase-8 function. Loss of XIAP results in aberrantly elevated ubiquitylation of RIP1 outside of TNFR complex I. Virally infected Xiap−/− mice present with symptoms reminiscent of XLP-2. Our data show that XIAP controls RIP3-dependent cell death and IL-1β secretion in response to TNF, which might contribute to hyperinflammation in patients with XLP-2.

  6. Extra-Low-Frequency Magnetic Fields alter Cancer Cells through Metabolic Restriction

    Li, Ying


    Background: Biological effects of extra-low-frequency (ELF) magnetic fields (MF) have lacked a credible mechanism of interaction between MFs and living material. Objectives: Examine the effect of ELF-MFs on cancer cells. Methods: Five cancer cell lines were exposed to ELF-MFs within the range of 0.025 to 5 microT, and the cells were examined for karyotype changes after 6 days. Results: All cancer cells lines lost chromosomes from MF exposure, with a mostly flat dose-response. Constant MF exposures for three weeks allow a rising return to the baseline, unperturbed karyotypes. From this point, small MF increases or decreases are again capable of inducing karyotype contractions. Our data suggests that the karyotype contractions are caused by MF interference with mitochondria's ATP synthase (ATPS), compensated by the action of AMP-activated Protein Kinase (AMPK). The effects of MFs are similar to those of the ATPS inhibitor oligomycin. They are amplified by metformin, an AMPK stimulator, and attenuated by resisti...

  7. ERG promotes the maintenance of hematopoietic stem cells by restricting their differentiation

    Knudsen, Kasper Jermiin; Rehn, Matilda Carolina; Hasemann, Marie Sigurd;


    The balance between self-renewal and differentiation is crucial for the maintenance of hematopoietic stem cells (HSCs). Whereas numerous gene regulatory factors have been shown to control HSC self-renewal or drive their differentiation, we have relatively few insights into transcription factors...

  8. Non-major histocompatibility complex-restricted cytotoxic activity of blood mononuclear cells stimulated with secreted mycobacterial proteins and other mycobacterial antigens

    Ravn, P; Pedersen, B K


    Several observations indicate that non-major histocompatibility complex (MHC)-restricted cytotoxicity, mediated for example by natural killer cells and lymphokine-activated killer cells, may serve as an important antimicrobial defense mechanism. The purpose of the present study was to investigate...

  9. Food Restriction-Induced Changes in Gonadotropin-Inhibiting Hormone Cells are Associated with Changes in Sexual Motivation and Food Hoarding, but not Sexual Performance and Food Intake.

    Klingerman, Candice M; Williams, Wilbur P; Simberlund, Jessica; Brahme, Nina; Prasad, Ankita; Schneider, Jill E; Kriegsfeld, Lance J


    We hypothesized that putative anorectic and orexigenic peptides control the motivation to engage in either ingestive or sex behaviors, and these peptides function to optimize reproductive success in environments where energy fluctuates. Here, the putative orexigenic peptide, gonadotropin-inhibiting hormone (GnIH, also known as RFamide-related peptide-3), and the putative anorectic hormones leptin, insulin, and estradiol were examined during the course of food restriction. Groups of female Syrian hamsters were restricted to 75% of their ad libitum food intake or fed ad libitum for 4, 8, or 12 days. Two other groups were food-restricted for 12 days and then re-fed ad libitum for 4 or 8 days. After testing for sex and ingestive behavior, blood was sampled and assayed for peripheral hormones. Brains were immunohistochemically double-labeled for GnIH and the protein product of the immediate early gene, c-fos, a marker of cellular activation. Food hoarding, the number of double-labeled cells, and the percent of GnIH-Ir cells labeled with Fos-Ir were significantly increased at 8 and 12 days after the start of food restriction. Vaginal scent marking and GnIH-Ir cell number significantly decreased after the same duration of restriction. Food hoarding, but not food intake, was significantly positively correlated with cellular activation in GnIH-Ir cells. Vaginal scent marking was significantly negatively correlated with cellular activation in GnIH-Ir cells. There were no significant effects of food restriction on plasma insulin, leptin, estradiol, or progesterone concentrations. In the dorsomedial hypothalamus (DMH) of energetically challenged females, strong projections from NPY-Ir cells were found in close apposition to GnIH-Ir cells. Together these results are consistent with the idea that metabolic signals influence sexual and ingestive motivation via NPY fibers that project to GnIH cells in the DMH. PMID:22649396

  10. Food restriction-induced changes in gonadotropin-inhibiting hormone-immunoreactive cells are associated with sexual motivation and food hoarding, but not sexual performance and food intake



    Full Text Available We hypothesized that putative anorectic and orexigenic peptides control the motivation to engage in either ingestive or sex behaviors, and these peptides function to optimize reproductive success in environments where energy fluctuates. Here, the putative orexigenic peptide, gonadotropin-inhibiting hormone (GnIH, also known as RFamide-related peptide-3 and the putative anorectic hormones leptin, insulin and estradiol were examined during the course of food restriction. Groups of female Syrian hamsters were restricted to 75% of their ad libitum food intake or fed ad libitum for 4, 8, or 12 days. Two other groups were food restricted for 12 days and then re-fed ad libitum for 4 or 8 days. After testing for sex and ingestive behavior, blood was sampled and assayed for peripheral hormones. Brains were immunohistochemically double-labeled for GnIH and the protein product of the immediate early gene, c-fos, a marker of cellular activation. Food hoarding, the number of double-labeled cells, and the percent of GnIH-Ir cells labeled with Fos-Ir were significantly increased at 8 and 12 days after the start of food restriction. Vaginal scent marking and GnIH-Ir cell number significantly decreased after the same duration of restriction. Food hoarding, but not food intake, was significantly positively correlated with cellular activation in GnIH-Ir cells. Vaginal scent marking was significantly negatively correlated with cellular activation in GnIH-Ir cells. There were no significant effects of food restriction on plasma insulin, leptin, estradiol, or progesterone concentrations. In the dorsomedial hypothalamus (DMH of energetically-challenged females, strong projections from NPY-Ir cells were found in close apposition to GnIH-Ir cells. Together these results are consistent with the idea that metabolic signals influence sexual and ingestive motivation via NPY fibers that project to GnIH cells in the DMH.

  11. Initial presentation of CNS-restricted acute lymphoblastic B cell leukaemia as peripheral polyneuropathy.

    Piovezani Ramos, Guilherme; Villasboas Bisneto, Jose C; Chen, Dong; Pardanani, Animesh


    We report a case of a 58-year-old woman who presented with a 1-month course of progressive lower and upper extremity weakness in addition to binocular diplopia. Diagnostic lumbar puncture revealed atypical lymphoid cells with 28% blasts. Immunophenotype was consistent with B cell acute lymphoblastic leukaemia (B-ALL). Further work up showed no systemic involvement but extensive thoracolumbar-sacral leptomeningeal disease. The patient was treated with several courses of intrathecal and systemic chemotherapy followed by craniospinal irradiation for consolidation. There was initial steady improvement in neurological symptoms and leptomeningeal disease, the latter being ascertained through radiological studies and cerebrospinal fluid examination. After 10 months of response, the patient relapsed with central nervous system (CNS) and systemic disease. B-ALL is a rare precursor lymphoid neoplasm that generally presents with systemic disease. While CNS involvement is not uncommon, isolated involvement of this compartment without systemic disease is exceedingly rare. PMID:27095809

  12. SCS macrophages suppress melanoma by restricting tumor-derived vesicle-B cell interactions.

    Pucci, Ferdinando; Garris, Christopher; Lai, Charles P; Newton, Andita; Pfirschke, Christina; Engblom, Camilla; Alvarez, David; Sprachman, Melissa; Evavold, Charles; Magnuson, Angela; von Andrian, Ulrich H; Glatz, Katharina; Breakefield, Xandra O; Mempel, Thorsten R; Weissleder, Ralph; Pittet, Mikael J


    Tumor-derived extracellular vesicles (tEVs) are important signals in tumor-host cell communication, yet it remains unclear how endogenously produced tEVs affect the host in different areas of the body. We combined imaging and genetic analysis to track melanoma-derived vesicles at organismal, cellular, and molecular scales to show that endogenous tEVs efficiently disseminate via lymphatics and preferentially bind subcapsular sinus (SCS) CD169(+) macrophages in tumor-draining lymph nodes (tdLNs) in mice and humans. The CD169(+) macrophage layer physically blocks tEV dissemination but is undermined during tumor progression and by therapeutic agents. A disrupted SCS macrophage barrier enables tEVs to enter the lymph node cortex, interact with B cells, and foster tumor-promoting humoral immunity. Thus, CD169(+) macrophages may act as tumor suppressors by containing tEV spread and ensuing cancer-enhancing immunity. PMID:26989197

  13. Drosophila Pez acts in Hippo signaling to restrict intestinal stem cell proliferation

    Poernbacher, Ingrid; Baumgartner, Roland; Marada, Suresh K;


    The conserved Hippo signaling pathway acts in growth control and is fundamental to animal development and oncogenesis. Hippo signaling has also been implicated in adult midgut homeostasis in Drosophila. Regulated divisions of intestinal stem cells (ISCs), giving rise to an ISC and an enteroblast...... (EB) that differentiates into an enterocyte (EC) or an enteroendocrine (EE) cell, enable rapid tissue turnover in response to intestinal stress. The damage-related increase in ISC proliferation requires deactivation of the Hippo pathway and consequential activation of the transcriptional coactivator...... pathway activity specifically in the fly midgut epithelium. Thus, Pez displays a tissue-specific requirement and functions as a negative upstream regulator of Yki in the regulation of ISC proliferation....

  14. Condensin II subunit dCAP-D3 restricts retrotransposon mobilization in Drosophila somatic cells.

    Andrew T Schuster


    Full Text Available Retrotransposon sequences are positioned throughout the genome of almost every eukaryote that has been sequenced. As mobilization of these elements can have detrimental effects on the transcriptional regulation and stability of an organism's genome, most organisms have evolved mechanisms to repress their movement. Here, we identify a novel role for the Drosophila melanogaster Condensin II subunit, dCAP-D3 in preventing the mobilization of retrotransposons located in somatic cell euchromatin. dCAP-D3 regulates transcription of euchromatic gene clusters which contain or are proximal to retrotransposon sequence. ChIP experiments demonstrate that dCAP-D3 binds to these loci and is important for maintaining a repressed chromatin structure within the boundaries of the retrotransposon and for repressing retrotransposon transcription. We show that dCAP-D3 prevents accumulation of double stranded DNA breaks within retrotransposon sequence, and decreased dCAP-D3 levels leads to a precise loss of retrotransposon sequence at some dCAP-D3 regulated gene clusters and a gain of sequence elsewhere in the genome. Homologous chromosomes exhibit high levels of pairing in Drosophila somatic cells, and our FISH analyses demonstrate that retrotransposon-containing euchromatic loci are regions which are actually less paired than euchromatic regions devoid of retrotransposon sequences. Decreased dCAP-D3 expression increases pairing of homologous retrotransposon-containing loci in tissue culture cells. We propose that the combined effects of dCAP-D3 deficiency on double strand break levels, chromatin structure, transcription and pairing at retrotransposon-containing loci may lead to 1 higher levels of homologous recombination between repeats flanking retrotransposons in dCAP-D3 deficient cells and 2 increased retrotransposition. These findings identify a novel role for the anti-pairing activities of dCAP-D3/Condensin II and uncover a new way in which dCAP-D3/Condensin

  15. Mitochondrial Hormesis links nutrient restriction to improved metabolism in fat cell

    Barbato, Daniele Lettieri; Tatulli, Giuseppe; Aquilano, Katia; Ciriolo, Maria R.


    Fasting promotes longevity by reprogramming metabolic and stress resistance pathways. However, although the impact on adipose tissue physiology through hormonal inputs is well established, the direct role of fasting on adipose cells is poorly understood. Herein we show that white and beige adipocytes, as well as mouse epididymal and subcutaneous adipose depots, respond to nutrient scarcity by acquiring a brown-like phenotype. Indeed, they improve oxidative metabolism through modulating the ex...

  16. E47 regulates hematopoietic stem cell proliferation and energetics but not myeloid lineage restriction

    Yang, Qi; Esplin, Brandt; Borghesi, Lisa


    The immune system is replenished by self-renewing hematopoietic stem cells (HSCs) that produce multipotent progenitors (MPPs) with little renewal capacity. E-proteins, the widely expressed basic helix-loop-helix transcription factors, contribute to HSC and MPP activity, but their specific functions remain undefined. Using quantitative in vivo and in vitro approaches, we show that E47 is dispensable for the short-term myeloid differentiation of HSCs but regulates their long-term capabilities. ...

  17. Dexamethasone-induced cell death is restricted to specific molecular subgroups of multiple myeloma

    Kervoëlen, Charlotte; Ménoret, Emmanuelle; Gomez-Bougie, Patricia; Bataille, Régis; Godon, Catherine; Marionneau-Lambot, Séverine; Moreau, Philippe; Pellat-Deceunynck, Catherine; Amiot, Martine


    Due to its cytotoxic effect in lymphoid cells, dexamethasone is widely used in the treatment of multiple myeloma (MM). However, only a subset of myeloma patients responds to high-dose dexamethasone. Despite the undeniable anti-myeloma benefits of dexamethasone, significant adverse effects have been reported. We re-evaluate the anti-tumor effect of dexamethasone according to the molecular heterogeneity of MM. We demonstrated that the pro-death effect of dexamethasone is related to the genetic ...

  18. Oncogenic transformation of rat lung epithelioid cells by SV40 DNA and restriction enzyme fragments

    Rat epithelioid lung cells were transformed with various preparations of SV40 DNA using the Ca2+-precipitation technique. The amount of SV40 genetic information integrated into transformed clones was evaluated by DNA-DNA renaturation kinetics. The growth properties on plastic and in soft-agar were examined, as well as the ability to induce tumors in syngeneic newborn animals or in adult nude mice. One particular transformed line, which had received the HpaII/BamHIA (59 per cent) fragment, was found to contain about 3 integrated copies of this fragment per cell and no significant amount of the HpaII/BamHIB (41 per cent fragment). This line which grew to high saturatio densities and efficiently formed clones in low serum on plastic, produced tumors in both syngeneic rats and nude mice. Thus the HpaII/BamHIA fragment, which mainly includes early viral information, was sufficient to impart these properties to rat epithelioid lung cells. (author)

  19. Activation of Nonclassical CD1d-Restricted NK T Cells Induces Airway Hyperreactivity in β2-Microglobulin-Deficient Mice1

    Meyer, Everett H.; Pichavant, Muriel; Akbari, Omid; Yasumi, Takahiro; Savage, Paul B.; DeKruyff, Rosemarie H.; Umetsu, Dale T.


    Allergic asthma is characterized by Th2-driven eosinophilic airway inflammation and by a central feature called airway hyperreactivity (AHR), development of which requires the presence of classical type I invariant NK T (iNKT) cells. Allergen-induced AHR, however, develops in β2-microglobulin (β2m)−/− mice, which lack classical iNKT cells, suggesting that in some situations iNKT cells may be dispensable for the development of AHR. In contrast, our studies now suggest that a CD1d-restricted, NK1.1+ noninvariant TCR NKT cell population is present in β2m−/− mice and is responsible for the development of AHR but not for Th2 responses. Furthermore, treatment of β2m−/− mice with anti-CD1d mAb or anti-NK1.1 mAb unexpectedly abolished allergen-induced AHR. The CD1-restricted NKT cells in these mice, which failed to respond to α-galactosylceramide and which therefore were not classical type I iNKT cells, appear to represent an NKT cell subset restricted by a β2m-independent form of CD1d. These results indicate that, although classical type I iNKT cells are normally required for the development of AHR, under different circumstances other NKT cell subsets, including nonclassical NKT cells, may substitute for classical iNKT cells and induce AHR. PMID:18802058

  20. Restricted Airspace

    Federal Laboratory Consortium — Redstone Technical Test Center has restricted airspace up to 30,000 feet ASL. Airspace encompasses R-2104 (Redstone). Airspace is used extensively for airborne/UAV...

  1. Restrictive glycosylphosphatidylinositol anchor synthesis in cwh6/gpi3 yeast cells causes aberrant biogenesis of cell wall proteins.

    Vossen, J.H.; Müller, W. H.; Lipke, P N; Klis, F. M.


    We previously reported that the defects in the Saccharomyces cerevisiae cwh6 Calcofluor white-hypersensitive cell wall mutant are caused by a mutation in SPT14/GPI3, a gene involved in glycosylphosphatidylinositol (GPI) anchor biosynthesis. Here we describe the effect of cwh6/spt14/gpi3 on the biogenesis of cell wall proteins. It was found that the release of precursors of cell wall proteins from the endoplasmic reticulum (ER) was retarded. This was accompanied by proliferation of ER structur...


    M. Soltan Dallal


    Full Text Available Biotypes, serotypes and lysotypes of 38 Yersinia isolated from 48 water samples were studied. These strains belonged to Y.enterocolitica, Y.frederiksenii, Y.kristensenii and Y. intermedia. Except 23.7% of non-serotypable strains, ten different serotypes were isolated of which 0:6 and 0:10 kl were the most frequent. The serotypes 0:3, 0:8, 0:9 responsible for almost all registered cases of yersiniosis in man were not detected. However, a few types 0:5, 0:6, 0:10 kl isolated rarely from specimens (urine of feces of patients were found. These serotypes can be used for correlation with Yersinia and yersiniosis in man.

  3. Pseudogene accumulation might promote the adaptive microevolution of Yersinia pestis

    Tong, Zongzhong; Zhou, Dongsheng; Song, Yajun; Zhang, Ling; Pei, Decui; Han, Yanping; Pang, Xin; Li, Min; Cui, Baizhong; Wang, Jin; Guo, Zhaobiao; Qi, Zhizhen; Jin, Lixia; Zhai, Junhui; Du, Zongmin; Wang, Jun; Wang, Xiaoyi; Yu, Jun; Wang, Jian; Huang, Peitang; Yang, Huanming; Yang, Ruifu

    Plague is a natural focus-based disease, and for better understanding of this disease it is crucial to determine the molecular mechanisms of its pathogen, Yersinia pestis, for adapting to different foci. Gene inactivation, loss and acquisition are the main mechanisms that contribute to a pathogen....... pestis from different natural plague foci in China based on pseudogene profiles. Twenty-four mutations that led to inactivation in the corresponding genes were analysed, and a PCR-based screening method was employed to investigate the distribution of these mutations among Y. pestis isolates from...... different foci and also among seven strains of Yersinia pseudotuberculosis. It was found that Y. pestis isolates from the same focus had identical mutation profiles, and 260 isolates of Y. pestis were divided into eight genotypes, while Y. pseudotuberculosis harboured wild-type alleles for all the mutations...

  4. Isolation of pathogenic yersiniae from wild animals in Bulgaria.

    Nikolova, S; Tzvetkov, Y; Najdenski, H; Vesselinova, A


    Pathogenic Yersinia strains were isolated between December 1998 and April 1999 from 37 wild animals: rabbit (Lepus europeus), boar (Sus scrofa scrofa), asiatic jackal (Canis aureus), red fox (Vulpes vulpes), mouflon (Ovis musimon), european river otter (Lutra lutra), beech marten (Martes foina), polecat (Musleta putorius) and wild cat (Felis silvestris). It was established that among the wild animals Y. enterocolitica strains of serotype 0:3 predominated, accompanied by Y. pseudotuberculosis strains of serotype 0:3. In one sample from asiatic jackal and one sample from rabbit, Y. enterocolitica serotype 0:8 was isolated. Yersinia enterocolitica and Y. pseudotuberculosis strains were isolated from tonsils and tongues as well as from the viscera--lung, liver, heart, spleen, kidney and lymph nodes, mainly in young animals (1-2 years of age). The results showed that wild animals are a possible natural reservoir for pathogenic Y. enterocolitica and Y. pseudotuberculosis and are included in the epidemiological chain of yersinioses. PMID:11393816


    M.M. Soltan-Dallal


    Full Text Available Yersiniosis is a common infection in human and animals. Yersinia enterocolitica infection has been associated with a variety of clinical manifestations. This bacterium has been isolated from water, meat, milk environment, animals and so on. Our studies have been accomplished about isolation of Y. enterocolitica from vegetables such as lettuce, carrot, celery, parsley, tomato, cabbage, radish, mushroom and leek. Four methods for isolating were: enrichment with ITC broth, enrichment with PSB broth, enrichment with PSB medium with KOH 0.25% and cold enrichment for 14 d at 4 C and used from PSB medium with KOH0.25%. Two cases of Yersinia were isolated from 110 samples of raw vegetables. One case of Y. enterocolotica enterocolitica was isolated from lettuce and the other case was Y.fredriksenli from parsley. Biochemical and virulent tests demonstrated environmental strains but not pathogenic.

  6. Restricted mobility of specific functional groups reduces anti-cancer drug activity in healthy cells

    Longo Martins, Murillo; Ignazzi, Rosanna; Eckert, Juergen;


    The most common cancer treatments currently available are radio- and chemo-therapy. These therapies have, however, drawbacks, such as, the reduction in quality of life and the low efficiency of radiotherapy in cases of multiple metastases. To lessen these effects, we have encapsulated an anti-cancer...... drug into a biocompatible matrix. In-vitro assays indicate that this bio-nanocomposite is able to interact and cause morphological changes in cancer cells. Meanwhile, no alterations were observed in monocytes and fibroblasts, indicating that this system might carry the drug in living organisms with...

  7. Mitochondrial Hormesis links nutrient restriction to improved metabolism in fat cell.

    Barbato, Daniele Lettieri; Tatulli, Giuseppe; Aquilano, Katia; Ciriolo, Maria R


    Fasting promotes longevity by reprogramming metabolic and stress resistance pathways. However, although the impact on adipose tissue physiology through hormonal inputs is well established, the direct role of fasting on adipose cells is poorly understood. Herein we show that white and beige adipocytes, as well as mouse epididymal and subcutaneous adipose depots, respond to nutrient scarcity by acquiring a brown-like phenotype. Indeed, they improve oxidative metabolism through modulating the expression of mitochondrial- and nuclear-encoded oxidative phosphorylation genes as well as mitochondrial stress defensive proteins (UCP1, SOD2). Such adaptation is placed in a canonical mitohormetic response that proceeds via mitochondrial reactive oxygen species ((mt)ROS) production and redistribution of FoxO1 transcription factor into nucleus. Nuclear FoxO1 ((n)FoxO1) mediates retrograde communication by inducing the expression of mitochondrial oxidative and stress defensive genes. Collectively, our findings describe an unusual white/beige fat cell response to nutrient availability highlighting another health-promoting mechanism of fasting. PMID:26540513

  8. Influence of dietary nucleotide restriction on bacterial sepsis and phagocytic cell function in mice.

    Kulkarni, A D; Fanslow, W C; Drath, D B; Rudolph, F B; Van Buren, C T


    Although enzyme defects in purine metabolism have revealed the importance of these substrates to maintenance of a normal immune response, the role of exogenous nucleotides on the cells that mediate the host defense system has remained largely unexplored. Recent investigations have revealed that dietary nucleotides are vital to the maintenance of cell-mediated responses to antigen stimulation. To test the influence of dietary nucleotide deprivation on resistance to infection, Balb/c mice were maintained on chow, a nucleotide-free (NF) diet, or an NF diet repleted with adenine, uracil, or RNA. Mice on the NF diet suffered 100% mortality following intravenous challenge with Staphylococcus aureus, while chow-fed and RNA- or uracil-repleted mice demonstrated significantly greater resistance to this bacterial challenge. Macrophages from mice on the NF diet had decreased phagocytic activity as measured by uptake of radiolabeled bacteria compared with mice maintained on the NF diet supplemented with adenine, uracil, or RNA. No change in S aureus antibody response was noted on the various diets. Although the mechanism of this suppression of nonspecific immunity remains unclear, provision of nucleotides to defined diets appears vital to maintain host resistance to bacterial challenge. PMID:3947217

  9. Microbiota restricts trafficking of bacteria to mesenteric lymph nodes by CX(3)CR1(hi) cells.

    Diehl, Gretchen E; Longman, Randy S; Zhang, Jing-Xin; Breart, Beatrice; Galan, Carolina; Cuesta, Adolfo; Schwab, Susan R; Littman, Dan R


    The intestinal microbiota has a critical role in immune system and metabolic homeostasis, but it must be tolerated by the host to avoid inflammatory responses that can damage the epithelial barrier separating the host from the luminal contents. Breakdown of this regulation and the resulting inappropriate immune response to commensals are thought to lead to the development of inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. We proposed that the intestinal immune system is instructed by the microbiota to limit responses to luminal antigens. Here we demonstrate in mice that, at steady state, the microbiota inhibits the transport of both commensal and pathogenic bacteria from the lumen to a key immune inductive site, the mesenteric lymph nodes (MLNs). However, in the absence of Myd88 or under conditions of antibiotic-induced dysbiosis, non-invasive bacteria were trafficked to the MLNs in a CCR7-dependent manner, and induced both T-cell responses and IgA production. Trafficking was carried out by CX(3)CR1(hi) mononuclear phagocytes, an intestinal-cell population previously reported to be non-migratory. These findings define a central role for commensals in regulating the migration to the MLNs of CX(3)CR1(hi) mononuclear phagocytes endowed with the ability to capture luminal bacteria, thereby compartmentalizing the intestinal immune response to avoid inflammation. PMID:23334413

  10. Expression and Association of the Yersinia pestis Translocon Proteins, YopB and YopD, Are Facilitated by Nanolipoprotein Particles.

    Coleman, Matthew A; Cappuccio, Jenny A; Blanchette, Craig D; Gao, Tingjuan; Arroyo, Erin S; Hinz, Angela K; Bourguet, Feliza A; Segelke, Brent; Hoeprich, Paul D; Huser, Thomas; Laurence, Ted A; Motin, Vladimir L; Chromy, Brett A


    Yersinia pestis enters host cells and evades host defenses, in part, through interactions between Yersinia pestis proteins and host membranes. One such interaction is through the type III secretion system, which uses a highly conserved and ordered complex for Yersinia pestis outer membrane effector protein translocation called the injectisome. The portion of the injectisome that interacts directly with host cell membranes is referred to as the translocon. The translocon is believed to form a pore allowing effector molecules to enter host cells. To facilitate mechanistic studies of the translocon, we have developed a cell-free approach for expressing translocon pore proteins as a complex supported in a bilayer membrane mimetic nano-scaffold known as a nanolipoprotein particle (NLP) Initial results show cell-free expression of Yersinia pestis outer membrane proteins YopB and YopD was enhanced in the presence of liposomes. However, these complexes tended to aggregate and precipitate. With the addition of co-expressed (NLP) forming components, the YopB and/or YopD complex was rendered soluble, increasing the yield of protein for biophysical studies. Biophysical methods such as Atomic Force Microscopy and Fluorescence Correlation Spectroscopy were used to confirm that the soluble YopB/D complex was associated with NLPs. An interaction between the YopB/D complex and NLP was validated by immunoprecipitation. The YopB/D translocon complex embedded in a NLP provides a platform for protein interaction studies between pathogen and host proteins. These studies will help elucidate the poorly understood mechanism which enables this pathogen to inject effector proteins into host cells, thus evading host defenses. PMID:27015536

  11. Yersinia enterocolitica : Genes involved in cold-adaptation

    Goverde, R.L.J.


    It is known from the literature that: -The application of chilling as a means of food preservation has frequently resulted in food borne infections with psychrotrophic micro-organisms, such as Yersinia enterocolitica, Listeria monocytogenes and Aeromonas hydrophila; - The injurious effect on human health of an infection with Y. enterocolitica should not be underestimated because of the risk of serious post-infective complications; - Almost all micro-organisms respond to an abrupt temperature ...

  12. Haemochromatosis and aldosterone deficiency presenting with Yersinia pseudotuberculosis septicaemia.

    Conway, S. P.; Dudley, N; Sheridan, P.; Ross, H.


    A 50 year old man presented with a pyrexial illness following a holiday abroad. Yersinia pseudotuberculosis was isolated from blood culture. Response to appropriate antibiotic therapy was prompt and complete, but full recovery was complicated by an episode of hyperkalaemia, hyponatraemia and the passing of large volumes of dilute urine. Three years previously he had developed diabetes mellitus and hypogonadotrophic hypogonadism. Investigation on recovery showed underlying haemochromatosis and...

  13. Yersinia pestis genome sequencing identifies patterns of global phylogenetic diversity.

    Morelli, Giovanna; Song, Yajun; Mazzoni, Camila J.; Eppinger, Mark; Roumagnac, Philippe; Wagner, David M.; Feldkamp, Mirjam; Kusecek, Barica; Vogler, Amy J.; Li, Yanjun; Cui, Yujun; Thomson, Nicholas R.; Jombart, Thibaut; Leblois, Raphael; Lichtner, Peter


    Plague is a pandemic human invasive disease caused by the bacterial agent Yersinia pestis. We here report a comparison of 17 whole genomes of Y. pestis isolates from global sources. We also screened a global collection of 286 Y. pestis isolates for 933 SNPs using Sequenom MassArray SNP typing. We conducted phylogenetic analyses on this sequence variation dataset, assigned isolates to populations based on maximum parsimony and, from these results, made inferences regarding historical transmiss...

  14. Recent emergence of new variants of Yersinia pestis in Madagascar.

    Guiyoule, A; Rasoamanana, B; Buchrieser, C.; Michel, P.; Chanteau, S.; Carniel, E


    Yersinia pestis, the causative agent of plague, has been responsible for at least three pandemics. During the last pandemic, which started in Hong Kong in 1894, the microorganism colonized new, previously unscathed geographical areas where it has become well established. The aim of this longitudinal study was to investigate the genetic stability of Y. pestis strains introduced into a new environment just under a century ago and to follow the epidemiology of any new genetic variant detected. I...

  15. Epidemiology and comparative analysis of Yersinia in Ireland

    Ringwood, Tamara


    Yersiniosis is an acute or chronic enteric zoonosis caused by enteropathogenic Yersinia species. Although yersiniosis is predominantly associated with gastroenteric forms of infection, extraintestinal forms are often reported from the elderly or patients with predisposing factors. Yersiniosis is often reported in countries with cold and mild climates (Northern and Central Europe, New Zealand and North of Russian Federation). The Irish Health Protection Surveillance Centre (HPSC) currently rec...

  16. Listeria monocytogenes, Yersinia enterocolitica and Salmonella enteritidis in Quail Eggs

    ERDOĞRUL, Özlem


    The aim of this study was to determine the presence of Listeria monocytogenes, Yersinia enterocolitica and Salmonella enteritidis in 123 liquid whole quail eggs. The method suggested by USDA-FSIS was used for the isolation and identification of L. monocytogenes. S. enteritidis was identified and sero-grouped by co-agglutination test and slide agglutination test. Y. enterocolitica was isolated in Trypticase-Soy Broth, with bile-oxalate-sorbose medium for enrichment. Both enrichment cultures w...

  17. MR1-restricted MAIT cells display ligand discrimination and pathogen selectivity through distinct T cell receptor usage

    Gold, Marielle C.; McLaren, James E.; Reistetter, Joseph A.;


    line with this interpretation, MAIT cell clones with distinct TCRs responded differentially to a riboflavin metabolite. These results suggest that MAIT cells can discriminate between pathogen-derived ligands in a clonotype-dependent manner, providing a basis for adaptive memory via recruitment of...

  18. MR1-restricted MAIT cells display ligand discrimination and pathogen selectivity through distinct T cell receptor usage

    Gold, Marielle C.; McLaren, James E.; Reistetter, Joseph A.;


    line with this interpretation, MAIT cell clones with distinct TCRs responded differentially to a riboflavin metabolite. These results suggest that MAIT cells can discriminate between pathogen-derived ligands in a clonotype-dependent manner, providing a basis for adaptive memory via recruitment of...

  19. Study of the Behavior of Some Yersinia enterocolitica Strains Susceptible to Disinfectants and Antibiotics Isolated from Swine

    Ciceronis Cumpanasoiu; Cristian Emil Cumpanasoiu; Emil Tirziu; Radu Valentin Gros; Bianca Cumpanasoiu; Adia Carmen Tirziu


    The species from Yersinia genus are widespread in nature, they could be isolated from warm-blooded and cold-bloodedanimals, from foods, water and soil. Among genus species, Yersinia enterocolitica is most frequently isolated fromhuman and animals. The resistance of Yersinia enterocolitica is similar to Yersinia pseudotuberculosis. Moreover, itresists to refrigeration temperature (+4°C). It is susceptible to streptomycin, chloramphenicol, tetracyclines, polymyxin Band colistin sulfate. Also, Y...

  20. Diet restriction inhibits apoptosis and HMGB1 oxidation and promotes inflammatory cell recruitment during acetaminophen hepatotoxicity.

    Antoine, Daniel James; Williams, Dominic P; Kipar, Anja; Laverty, Hugh; Park, B Kevin


    Acetaminophen (APAP) overdose is a major cause of acute liver failure and serves as a paradigm to elucidate mechanisms, predisposing factors and therapeutic interventions. The roles of apoptosis and inflammation during APAP hepatotoxicity remain controversial. We investigated whether fasting of mice for 24 h can inhibit APAP-induced caspase activation and apoptosis through the depletion of basal ATP. We also investigated in fasted mice the critical role played by inhibition of caspase-dependent cysteine 106 oxidation within high mobility group box-1 protein (HMGB1) released by ATP depletion in dying cells as a mechanism of immune activation. In fed mice treated with APAP, necrosis was the dominant form of hepatocyte death. However, apoptosis was also observed, indicated by K18 cleavage, DNA laddering and procaspase-3 processing. In fasted mice treated with APAP, only necrosis was observed. Inflammatory cell recruitment as a consequence of hepatocyte death was observed only in fasted mice treated with APAP or fed mice cotreated with a caspase inhibitor. Hepatic inflammation was also associated with loss in detection of serum oxidized-HMGB1. A significant role of HMGB1 in the induction of inflammation was confirmed with an HMGB1-neutralizing antibody. The differential response between fasted and fed mice was a consequence of a significant reduction in basal hepatic ATP, which prevented caspase processing, rather than glutathione depletion or altered APAP metabolism. Thus, the inhibition of caspase-driven apoptosis and HMGB1 oxidation by ATP depletion from fasting promotes an inflammatory response during drug-induced hepatotoxicity/liver pathology. PMID:20811657

  1. Controlled meal frequency without caloric restriction alters peripheral blood mononuclear cell cytokine production

    Longo Dan L


    Full Text Available Abstract Background Intermittent fasting (IF improves healthy lifespan in animals by a mechanism involving reduced oxidative damage and increased resistance to stress. However, no studies have evaluated the impact of controlled meal frequency on immune responses in human subjects. Objective A study was conducted to establish the effects of controlled diets with different meal frequencies, but similar daily energy intakes, on cytokine production in healthy male and female subjects. Design In a crossover study design with an intervening washout period, healthy normal weight middle-age male and female subjects (n = 15 were maintained for 2 months on controlled on-site one meal per day (OMD or three meals per day (TMD isocaloric diets. Serum samples and peripheral blood mononuclear cells (PBMCs culture supernatants from subjects were analyzed for the presence of inflammatory markers using a multiplex assay. Results There were no significant differences in the inflammatory markers in the serum of subjects on the OMD or TMD diets. There was an increase in the capacity of PBMCs to produce cytokines in subjects during the first month on the OMD or TMD diets. Lower levels of TNF-α, IL-17, MCP-1 and MIP-1β were produced by PBMCs from subjects on the OMD versus TMD diet. Conclusions PBMCs of subjects on controlled diets exhibit hypersensitivities to cellular stimulation suggesting that stress associated with altered eating behavior might affect cytokine production by immune cells upon stimulation. Moreover, stimulated PBMCs derived from healthy individuals on a reduced meal frequency diet respond with a reduced capability to produce cytokines.

  2. Human TRAV1-2-negative MR1-restricted T cells detect S. pyogenes and alternatives to MAIT riboflavin-based antigens.

    Meermeier, Erin W; Laugel, Bruno F; Sewell, Andrew K; Corbett, Alexandra J; Rossjohn, Jamie; McCluskey, James; Harriff, Melanie J; Franks, Tamera; Gold, Marielle C; Lewinsohn, David M


    Mucosal-associated invariant T (MAIT) cells are thought to detect microbial antigens presented by the HLA-Ib molecule MR1 through the exclusive use of a TRAV1-2-containing TCRα. Here we use MR1 tetramer staining and ex vivo analysis with mycobacteria-infected MR1-deficient cells to demonstrate the presence of functional human MR1-restricted T cells that lack TRAV1-2. We characterize an MR1-restricted clone that expresses the TRAV12-2 TCRα, which lacks residues previously shown to be critical for MR1-antigen recognition. In contrast to TRAV1-2(+) MAIT cells, this TRAV12-2-expressing clone displays a distinct pattern of microbial recognition by detecting infection with the riboflavin auxotroph Streptococcus pyogenes. As known MAIT antigens are derived from riboflavin metabolites, this suggests that TRAV12-2(+) clone recognizes unique antigens. Thus, MR1-restricted T cells can discriminate between microbes in a TCR-dependent manner. We postulate that additional MR1-restricted T-cell subsets may play a unique role in defence against infection by broadening the recognition of microbial metabolites. PMID:27527800

  3. Deciphering the acylation pattern of Yersinia enterocolitica lipid A.

    Mar Reinés

    Full Text Available Pathogenic bacteria may modify their surface to evade the host innate immune response. Yersinia enterocolitica modulates its lipopolysaccharide (LPS lipid A structure, and the key regulatory signal is temperature. At 21°C, lipid A is hexa-acylated and may be modified with aminoarabinose or palmitate. At 37°C, Y. enterocolitica expresses a tetra-acylated lipid A consistent with the 3'-O-deacylation of the molecule. In this work, by combining genetic and mass spectrometric analysis, we establish that Y. enterocolitica encodes a lipid A deacylase, LpxR, responsible for the lipid A structure observed at 37°C. Western blot analyses indicate that LpxR exhibits latency at 21°C, deacylation of lipid A is not observed despite the expression of LpxR in the membrane. Aminoarabinose-modified lipid A is involved in the latency. 3-D modelling, docking and site-directed mutagenesis experiments showed that LpxR D31 reduces the active site cavity volume so that aminoarabinose containing Kdo(2-lipid A cannot be accommodated and, therefore, not deacylated. Our data revealed that the expression of lpxR is negatively controlled by RovA and PhoPQ which are necessary for the lipid A modification with aminoarabinose. Next, we investigated the role of lipid A structural plasticity conferred by LpxR on the expression/function of Y. enterocolitica virulence factors. We present evidence that motility and invasion of eukaryotic cells were reduced in the lpxR mutant grown at 21°C. Mechanistically, our data revealed that the expressions of flhDC and rovA, regulators controlling the flagellar regulon and invasin respectively, were down-regulated in the mutant. In contrast, the levels of the virulence plasmid (pYV-encoded virulence factors Yops and YadA were not affected in the lpxR mutant. Finally, we establish that the low inflammatory response associated to Y. enterocolitica infections is the sum of the anti-inflammatory action exerted by pYV-encoded YopP and the

  4. Amelioration of neuronal cell death in a spontaneous obese rat model by dietary restriction through modulation of ubiquitin proteasome system.

    Shruthi, Karnam; Reddy, S Sreenivasa; Reddy, P Yadagiri; Shivalingam, Potula; Harishankar, Nemani; Reddy, G Bhanuprakash


    Dietary restriction (DR) has been shown to increase longevity, delay onset of aging, reduce DNA damage and oxidative stress and prevent age-related decline of neuronal activity. We previously reported the role of altered ubiquitin proteasome system (UPS) in the neuronal cell death in a spontaneous obese rat model (WNIN/Ob rat). In this study, we investigated the effect of DR on obesity-induced neuronal cell death in a rat model. Two groups of 40-day-old WNIN/Ob rats were either fed ad libitum (Ob) or pair-fed with lean. The lean phenotype of WNIN/Ob rats served as ad libitum control. These animals were maintained for 6.5months on their respective diet regime. At the end of the study, cerebral cortex was collected and markers of UPS, endoplasmic reticulum (ER) stress and autophagy were analyzed by quantitative real-time polymerase chain reaction, immunoblotting and immunohistochemistry. Chymotrypsin-like activity of proteasome was assayed by the fluorimetric method. Apoptotic cells were analyzed by TUNEL assay. DR improved metabolic abnormalities in obese rats. Alterations in UPS (up-regulation of UCHL1, down-regulation of UCHL5, declined proteasomal activity), increased ER stress, declined autophagy and increased expression of α-synuclein, p53 and BAX were observed in obese rats and DR alleviated these changes in obese rats. Further, DR decreased TUNEL-positive cells. In conclusion, DR in obese rats could not only restore the metabolic abnormalities but also preserved neuronal health in the cerebral cortex by preventing alterations in the UPS. PMID:27260470

  5. Propranolol Restricts the Mobility of Single EGF-Receptors on the Cell Surface before Their Internalization

    Otero, Carolina; Linke, Max; Sanchez, Paula; González, Alfonso; Schaap, Iwan A. T.


    The epidermal growth factor receptor is involved in morphogenesis, proliferation and cell migration. Its up-regulation during tumorigenesis makes this receptor an interesting therapeutic target. In the absence of the ligand, the inhibition of phosphatidic acid phosphohydrolase activity by propranolol treatment leads to internalization of empty/inactive receptors. The molecular events involved in this endocytosis remain unknown. Here, we quantified the effects of propranolol on the mobility of single quantum-dot labelled receptors before the actual internalization took place. The single receptors showed a clear stop-and-go motion; their diffusive tracks were continuously interrupted by sub-second stalling events, presumably caused by transient clustering. In the presence of propranolol we found that: i) the diffusion rate reduced by 22 %, which indicates an increase in drag of the receptor. Atomic force microscopy measurements did not show an increase of the effective membrane tension, such that clustering of the receptor remains the likely mechanism for its reduced mobility. ii) The receptor got frequently stalled for longer periods of multiple seconds, which may signal the first step of the internalization process. PMID:24349439

  6. Bacteriemia y absceso hepático causado por Yersinia enterocolitica Bacteremia and hepatic abscess caused by Yersinia enterocolitica

    A. Navascués


    Full Text Available Yersinia enterocolitica es un cocobacilo gram negativo de amplia distribución mundial cuyo reservorio natural se encuentra en una gran variedad de animales. La transmisión a los humanos se realiza principalmente a través de la vía fecal-oral aunque también se han descrito casos de transmisión a través de transfusiones sanguíneas. Su aislamiento se realiza habitualmente dentro de un cuadro gastrointestinal y rara vez produce trastornos extraintestinales como bacteriemia, abscesos, manifestaciones cutáneas, etc. Éstos se han asociado a diferentes enfermedades de base como alteraciones del metabolismo del hierro, diabetes mellitus, alcoholismo, malnutrición, tumores, terapia inmunosupresora y cirrosis. Presentamos el caso de un paciente diabético que desarrolló bacteriemia asociada a abscesso hepático por Yersinia enterocolitica.Yersinia enterocolitica is a Gram-negative coccobacillus that is distributed world-wide and whose natural reservoire is found in a great variety of animals. Transmission to humans mainly occurs through the faecal-oral path although cases have been described of transmission through blood transfusions. It is isolated within a gastro-intestinal clinical picture and it rarely produces extra-intestinal disorders such as bacteraemia, abscesses, cutaneous signs, etc. The latter have been associated with different underlying diseases such as alterations of the iron metabolism, diabetes mellitus, alcoholism, malnutrition, tumours, immunosuppressant therapy and cirrhosis. We present the case of a diabetic patient who developed bacteraemia associated with hepatic abscess due to Yersinia enterocolitica.

  7. Purification and characterization of Yersinia enterocolitica and Yersinia pestis LcrV–cholera toxin A2/B chimeras

    Tinker, Juliette K.; Chadwick T Davis; Arlian, Britni M.


    Yersinia pestis is a virulent human pathogen and potential biological weapon. Despite a long history of research on this organism, there is no licensed vaccine to protect against pneumonic forms of Y. pestis disease. In the present study, plasmids were constructed to express cholera toxin A2/B chimeric molecules containing the LcrV protective antigen from Y. enterocolitica and Y. pestis. These chimeras were expressed and purified to high yields from the supernatant of transformed E. coli. Wes...

  8. A rapid method for the nonselective enumeration of Yersinia enterocolitica, a foodborne pathogen associated with pork.

    Wang, Haoran; Palmer, Jon; Flint, Steve


    An impedance method was developed as a rapid, convenient method to enumerate pure culture of Yersinia enterocolitica. Cultures were incubated in trypticase soy broth (TSB) at 30°C. The BacTrac™ 4000 microorganism growth analyser was used to detect impedance change of TSB representing bacteria cell numbers in the samples. Good correlations with standard plate counts were obtained (r(2)>0.95). This method is also reliable to enumerate Y. enterocolitica growing in biofilms attached to stainless steel. Compared with a standard plate count which involves dislodging biofilms from surfaces, this method is more convenient saving both time and effort. Therefore, it will be useful to study the conditions required for the biofilm growth and control of Y. enterocolitica that could be applied to the pork industry. PMID:26613189

  9. Booster immersion vaccination using diluted Yersinia ruckeri bacterin confers protection against ERM in rainbow trout

    Chettri, Jiwan Kumar; Mohammad, Rezkar Jaafar; Skov, Jakob;


    A single immersion vaccination of rainbow trout fry using a Yersinia ruckeri bacterin confers immunity to reinfection but only for a shorter period. A longer protective period is needed in practical trout farming and we have shown that booster vaccination prolongs immunity. Due to economic...... considerations and management practices it is not possible to immersion vaccinate large trout (20–30 g) with the recommended high bacterin concentration. We here demonstrate that booster vaccination using dilutions of the bacterin (1:100, 1:1000 and 1:2000) with increased exposure time (1 h, 2 h) confers a...... such a practice will not challenge farm management and economy. Increased antibody levels were recorded after challenge of vaccinated fish but not after immersion vaccination alone which suggests that immersion induces priming of memory cells....

  10. Induction of DNA double-strand breaks by restriction enzymes in X-ray-sensitive mutant Chinese hamster ovary cells measured by pulsed-field gel electrophoresis

    This investigation was designed to determine whether the cytotoxic effects of different restriction endonucleases are related to the number and type of DNA double-strand breaks (DSBs) they produce. Chinese hamster ovary (CHO) K1 and xrs-5 cells, a radiosensitive mutant of CHO K1, were exposed to restriction endonucleases HaeIII, HinfI, PvuII and BamHI by electroporation. These enzymes represent both blunt and sticky end cutters with differing recognition sequence lengths. The number of DSBs was measured by pulsed-field gel electrophoresis (PFGE). Two forms of PFGE were employed: asymmetric field-inversion gel electrophoresis (AFIGE) for measuring the kinetics of DNA breaks by enzyme digestion and clamped homogeneous gel electrophoresis (CHEF) for examining the size distributions of damaged DNA. The amount of DNA damage induced by exposure to all four restriction enzymes was significantly greater in xrs-5 compared to CHO K1 cells, consistent with the reported DSB repair deficiency in these cells. Since restriction endonucleases produce DSBs alone as opposed to the various types of DNA damage induced by X rays, these results confirm that the repair defect in this mutant involves the rejoining of DSBs. Although the cutting frequency was directly related to the length of the recognition sequence for four restriction enzymes, there was no simple correlation between the cytotoxic effect and the amount of DNA damage produced by each enzyme in either cell line. This finding suggests that the type or nature of the cutting sequence itself may play a role in restriction enzyme-induced cell killing. 32 refs., 6 figs., 3 tabs

  11. Failure to detect a DNA repair-related defect in the transfection of ataxia-telangiectasia cells by enzymatically restricted plasmid

    Two SV40-transformed human fibroblast cell lines were transfected with plasmids in which double-strand breaks had been introduced by restriction enzymes, within or near the selected gene. Restriction of pSV2gpt with KpnI reduced the frequency of transfection more in the ionizing radiation-sensitive ataxia-telangiectasia line AT5BIVA than in the resistant line MRC5V1. When the related plasmid pSV2neo was restricted with SmaI, the reduction in transfection was less in the ataxia-telangiectasia than in the normal cells. The apparent defect in transfection of AT5BIVA by pSV2gpt appeared to be a result of the unusual sensitivity of the repair-deficient recipient to the selective agent. Loss of potential transfectants is exacerbated when transient gene expression is reduced by restriction of the plasmid. It is suggested that a reduction in yield of transfectants with restricted plasmid in ataxia-telangiectasia cells cannot readily be used as evidence of a defect in DNA repair. The results are also relevant to standard transfection experiments; they emphasize the importance of optimizing selection when transient expression may be reduced, to ensure that potential transfectants are not killed by the selection regime. (author)

  12. Oxygen restriction increases the infective potential of Listeria monocytogenes in vitro in Caco-2 cells and in vivo in guinea pigs

    Licht Tine


    Full Text Available Abstract Background Listeria monocytogenes has been implicated in several food borne outbreaks as well as sporadic cases of disease. Increased understanding of the biology of this organism is important in the prevention of food borne listeriosis. The infectivity of Listeria monocytogenes ScottA, cultivated with and without oxygen restriction, was compared in vitro and in vivo. Fluorescent protein labels were applied to allow certain identification of Listeria cells from untagged bacteria in in vivo samples, and to distinguish between cells grown under different conditions in mixed infection experiments. Results Infection of Caco-2 cells revealed that Listeria cultivated under oxygen-restricted conditions were approximately 100 fold more invasive than similar cultures grown without oxygen restriction. This was observed for exponentially growing bacteria, as well as for stationary-phase cultures. Oral dosage of guinea pigs with Listeria resulted in a significantly higher prevalence (p Listeria in fecal samples was observed after dosage with oxygen-restricted bacteria. These differences were seen after challenge with single Listeria cultures, as well as with a mixture of two cultures grown with and without oxygen restriction. Conclusion Our results show for the first time that the environmental conditions to which L. monocytogenes is exposed prior to ingestion are decisive for its in vivo infective potential in the gastrointestinal tract after passage of the gastric barrier. This is highly relevant for safety assessment of this organism in food.

  13. Production of a new non-specific nuclease from Yersinia enterocolitica subsp. palearctica: optimization of induction conditions using response surface methodology

    Fang, Xiu-Juan; Tang, Zhen-Xing; Li, Zhen-Hua; Zhang, Zhi-Liang; Shi, Lu-E


    A new non-specific nuclease from Yersinia enterocolitica subsp. palearctica (Y. NSN) was expressed in Escherichia coli (E. coli) BL 21 StarTM (DE3)plysS. Induction conditions, including isopropyl-β-D-thiogalactoside (IPTG) concentration, cell density (OD600), induction time and induction temperature, were optimized using response surface methodology. Statistical analysis of the results revealed that induction temperature and all the quadratic terms of variables had significant effects on enzy...

  14. Discerning Viable from Nonviable Yersinia pestis pgm- and Bacillus anthracis Sterne using Propidium Monoazide in the Presence of White Powders

    Hess, Becky M.; Kaiser, Brooke LD; Sydor, Michael A.; Wunschel, David S.; Bruckner-Lea, Cindy J.; Hutchison, Janine R.


    ABSTRACT Aims To develop and optimize an assay to determine viability status of Bacillus anthracis Sterne and Yersinia pestis pgm- strains in the presence of white powders by coupling propidium monoazide (PMA) treatment with real-time PCR (qPCR) analysis. Methods and Results PMA selectively enters nonviable cells and binds DNA, thereby increasing qPCR assay cycle threshold (CT) values compared to untreated samples. Dye concentration, cell number and fitness, incubation time, inactivation methods, and assay buffer were optimized for B. anthracis Sterne and Y. pestis pgm-. Differences in CT values in nonviable cells compared to untreated samples were consistently > 9 for both B. anthracis Sterne vegetative cells and Y. pestis pgm- in the presence and absence of three different white powders. Our method eliminates the need for a DNA extraction step prior to detection by qPCR. Conclusions The developed assay enables simultaneous identification and viability assessment for B. anthracis Sterne and Y. pestis pgm- under laboratory conditions, even in the presence of white powders. Eliminating the DNA extraction step that is typically used reduces total assay time and labor requirements for sample analysis. Significance and Impact of the Study The method developed for simultaneous detection and viability assessment for B. anthracis and Y. pestis can be employed in forming decisions about the severity of a biothreat event or the safety of food. Keywords Bacillus anthracis, Yersinia pestis, Propidium Monoazide, qPCR, White Powders, Rapid Viability Detection

  15. 'Add, stir and reduce': Yersinia spp. as model bacteria for pathogen evolution.

    McNally, Alan; Thomson, Nicholas R; Reuter, Sandra; Wren, Brendan W


    Pathogenic species in the Yersinia genus have historically been targets for research aimed at understanding how bacteria evolve into mammalian pathogens. The advent of large-scale population genomic studies has greatly accelerated the progress in this field, and Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica have once again acted as model organisms to help shape our understanding of the evolutionary processes involved in pathogenesis. In this Review, we highlight the gene gain, gene loss and genome rearrangement events that have been identified by genomic studies in pathogenic Yersinia species, and we discuss how these findings are changing our understanding of pathogen evolution. Finally, as these traits are also found in the genomes of other species in the Enterobacteriaceae, we suggest that they provide a blueprint for the evolution of enteropathogenic bacteria. PMID:26876035

  16. The study of responses to 'model' DNA breaks induced by restriction endonucleases in cells and cell-free systems: achievements and difficulties

    The use of restriction endonucleases (RE) as a means of implicating DNA double-strand breaks (dsb) in cellular responses is reviewed. The introduction of RE into cells leads to many of the responses known to be characteristic of radiation damage -cell killing, chromosomal aberration, oncogenic transformation, gene mutation and amplification. Additionally, radiosensitive cell lines are hypersensitive to RE, including those from the human disorder ataxia-telangiectasia. However, quantitation of response and comparisons of the effectiveness of different RE are difficult, partly because of unknown activity and lifetime of RE in the cell. Re-induced dsb have also been used to reveal molecular mechanisms of repair and misrepair at specific sites in DNA. Dsb have been implicated in recombination processes including those leading to illegitimate rejoining (formation of deletions and rearrangements) at short sequence features in DNA. Also model dsb act as a signal to activate other cellular processes, which may influence or indirectly cause some responses, including cell death. In these signalling responses the detailed chemistry at the break site may not be very important, perhaps explaining why there is considerable overlap in responses to RE and to ionizing radiations. (author)

  17. Completion of hepatitis C virus replication cycle in heterokaryons excludes dominant restrictions in human non-liver and mouse liver cell lines.

    Anne Frentzen


    Full Text Available Hepatitis C virus (HCV is hepatotropic and only infects humans and chimpanzees. Consequently, an immunocompetent small animal model is lacking. The restricted tropism of HCV likely reflects specific host factor requirements. We investigated if dominant restriction factors expressed in non-liver or non-human cell lines inhibit HCV propagation thus rendering these cells non-permissive. To this end we explored if HCV completes its replication cycle in heterokaryons between human liver cell lines and non-permissive cell lines from human non-liver or mouse liver origin. Despite functional viral pattern recognition pathways and responsiveness to interferon, virus production was observed in all fused cells and was only ablated when cells were treated with exogenous interferon. These results exclude that constitutive or virus-induced expression of dominant restriction factors prevents propagation of HCV in these cell types, which has important implications for HCV tissue and species tropism. In turn, these data strongly advocate transgenic approaches of crucial human HCV cofactors to establish an immunocompetent small animal model.

  18. Caloric Restriction reduces inflammation and improves T cell-mediated immune response in obese mice but concomitant consumption of curcumin/piperine adds no further benefit

    Obesity is associated with low-grade inflammation and impaired immune response. Caloric restriction (CR) has been shown to inhibit inflammatory response and enhance cell-mediated immune function. Curcumin, the bioactive phenolic component of turmeric spice, is proposed to have anti-obesity and anti-...

  19. Perforin-2 Protects Host Cells and Mice by Restricting the Vacuole to Cytosol Transitioning of a Bacterial Pathogen.

    McCormack, Ryan; Bahnan, Wael; Shrestha, Niraj; Boucher, Justin; Barreto, Marcella; Barrera, Carlos M; Dauer, Edward A; Freitag, Nancy E; Khan, Wasif N; Podack, Eckhard R; Schesser, Kurt


    The host-encoded Perforin-2 (encoded by the macrophage-expressed gene 1,Mpeg1), which possesses a pore-forming MACPF domain, reduces the viability of bacterial pathogens that reside within membrane-bound compartments. Here, it is shown that Perforin-2 also restricts the proliferation of the intracytosolic pathogenListeria monocytogenes Within a few hours of systemic infection, the massive proliferation ofL. monocytogenesinPerforin-2(-/-)mice leads to a rapid appearance of acute disease symptoms. We go on to show in culturedPerforin-2(-/-)cells that the vacuole-to-cytosol transitioning ofL. monocytogenesis greatly accelerated. Unexpectedly, we found that inPerforin-2(-/-)macrophages,Listeria-containing vacuoles quickly (≤15 min) acidify, and that this was coincident with greater virulence gene expression, likely accounting for the more rapid translocation ofL. monocytogenesto its replicative niche in the cytosol. This hypothesis was supported by our finding that aL. monocytogenesstrain expressing virulence factors at a constitutively high level replicated equally well inPerforin-2(+/+)andPerforin-2(-/-)macrophages. Our findings suggest that the protective role of Perforin-2 against listeriosis is based on it limiting the intracellular replication of the pathogen. This cellular activity of Perforin-2 may derive from it regulating the acidification ofListeria-containing vacuoles, thereby depriving the pathogen of favorable intracellular conditions that promote its virulence gene activity. PMID:26831467

  20. Rapid Antigen Processing and Presentation of a Protective and Immunodominant HLA-B*27-restricted Hepatitis C Virus-specific CD8+ T-cell Epitope

    Julia Schmidt; Iversen, Astrid K N; Stefan Tenzer; Emma Gostick; Price, David A.; Volker Lohmann; Ute Distler; Paul Bowness; Hansjörg Schild; Blum, Hubert E.; Paul Klenerman; Christoph Neumann-Haefelin; Robert Thimme


    HLA-B*27 exerts protective effects in hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infections. While the immunological and virological features of HLA-B*27-mediated protection are not fully understood, there is growing evidence that the presentation of specific immunodominant HLA-B*27-restricted CD8+ T-cell epitopes contributes to this phenomenon in both infections. Indeed, protection can be linked to single immunodominant CD8+ T-cell epitopes and functional constraints on e...

  1. In vitro intracellular trafficking of virulence antigen during infection by Yersinia pestis.

    Tracy L DiMezzo

    Full Text Available Yersinia pestis, the causative agent of plague, encodes several essential virulence factors on a 70 kb plasmid, including the Yersinia outer proteins (Yops and a multifunctional virulence antigen (V. V is uniquely able to inhibit the host immune response; aid in the expression, secretion, and injection of the cytotoxic Yops via a type III secretion system (T3SS-dependent mechanism; be secreted extracellularly; and enter the host cell by a T3SS-independent mechanism, where its activity is unknown. To elucidate the intracellular trafficking and target(s of V, time-course experiments were performed with macrophages (MPhis infected with Y. pestis or Y. pseudotuberculosis at intervals from 5 min to 6 h. The trafficking pattern was discerned from results of parallel microscopy, immunoblotting, and flow cytometry experiments. The MPhis were incubated with fluorescent or gold conjugated primary or secondary anti-V (antibodies [Abs] in conjunction with organelle-associated Abs or dyes. The samples were observed for co-localization by immuno-fluorescence and electron microscopy. For fractionation studies, uninfected and infected MPhis were lysed and subjected to density gradient centrifugation coupled with immunoblotting with Abs to V or to organelles. Samples were also analyzed by flow cytometry after lysis and dual-staining with anti-V and anti-organelle Abs. Our findings indicate a co-localization of V with (1 endosomal proteins between 10-45 min of infection, (2 lysosomal protein(s between 1-2 h of infection, (3 mitochondrial proteins between 2.5-3 h infection, and (4 Golgi protein(s between 4-6 h of infection. Further studies are being performed to determine the specific intracellular interactions and role in pathogenesis of intracellularly localized V.

  2. Structure of a complex of uridine phosphorylase from Yersinia pseudotuberculosis with the modified bacteriostatic antibacterial drug determined by X-ray crystallography and computer analysis

    Balaev, V. V.; Lashkov, A. A., E-mail:; Gabdoulkhakov, A. G.; Seregina, T. A.; Dontsova, M. V.; Mikhailov, A. M. [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation)


    Pseudotuberculosis and bubonic plague are acute infectious diseases caused by the bacteria Yersinia pseudotuberculosis and Yersinia pestis. These diseases are treated, in particular, with trimethoprim and its modified analogues. However, uridine phosphorylases (pyrimidine nucleoside phosphorylases) that are present in bacterial cells neutralize the action of trimethoprim and its modified analogues on the cells. In order to reveal the character of the interaction of the drug with bacterial uridine phosphorylase, the atomic structure of the unligated molecule of uridine-specific pyrimidine nucleoside phosphorylase from Yersinia pseudotuberculosis (YptUPh) was determined by X-ray diffraction at 1.7 Å resolution with high reliability (R{sub work} = 16.2, R{sub free} = 19.4%; r.m.s.d. of bond lengths and bond angles are 0.006 Å and 1.005°, respectively; DPI = 0.107 Å). The atoms of the amino acid residues of the functionally important secondary-structure elements—the loop L9 and the helix H8—of the enzyme YptUPh were located. The three-dimensional structure of the complex of YptUPh with modified trimethoprim—referred to as 53I—was determined by the computer simulation. It was shown that 53I is a pseudosubstrate of uridine phosphorylases, and its pyrimidine-2,4-diamine group is located in the phosphate-binding site of the enzyme YptUPh.

  3. Structure of a complex of uridine phosphorylase from Yersinia pseudotuberculosis with the modified bacteriostatic antibacterial drug determined by X-ray crystallography and computer analysis

    Pseudotuberculosis and bubonic plague are acute infectious diseases caused by the bacteria Yersinia pseudotuberculosis and Yersinia pestis. These diseases are treated, in particular, with trimethoprim and its modified analogues. However, uridine phosphorylases (pyrimidine nucleoside phosphorylases) that are present in bacterial cells neutralize the action of trimethoprim and its modified analogues on the cells. In order to reveal the character of the interaction of the drug with bacterial uridine phosphorylase, the atomic structure of the unligated molecule of uridine-specific pyrimidine nucleoside phosphorylase from Yersinia pseudotuberculosis (YptUPh) was determined by X-ray diffraction at 1.7 Å resolution with high reliability (Rwork = 16.2, Rfree = 19.4%; r.m.s.d. of bond lengths and bond angles are 0.006 Å and 1.005°, respectively; DPI = 0.107 Å). The atoms of the amino acid residues of the functionally important secondary-structure elements—the loop L9 and the helix H8—of the enzyme YptUPh were located. The three-dimensional structure of the complex of YptUPh with modified trimethoprim—referred to as 53I—was determined by the computer simulation. It was shown that 53I is a pseudosubstrate of uridine phosphorylases, and its pyrimidine-2,4-diamine group is located in the phosphate-binding site of the enzyme YptUPh

  4. Self-recognition specificity expressed by T cells from nude mice. Absence of detectable Ia-restricted T cells in nude mice that do exhibit self-K/D-restricted T cell responses


    The presence in athymic nude mice of precursor T cells with self- recognition specificity for either H-2 K/D or H-2 I region determinants was investigated. Chimeras were constructed of lethally irradiated parental mice receiving a mixture of F1 nude mouse (6-8 wk old) spleen and bone marrow cells. The donor inoculum was deliberately not subjected to any T cell depletion procedure, so that any potential major histocompatibility complex-committed precursor T cells were allowed to differentiate ...

  5. Improved procedure for recovery of Yersinia enterocolitica from meats.

    Doyle, M P; Hugdahl, M B


    A 1- to 3-day enrichment-KOH postenrichment procedure was evaluated and found to be as effective in recovering Yersinia enterocolitica from meats as a 14- to 21-day cold enrichment procedure, with or without KOH postenrichment. The shortened procedure consists of enriching 1.0- and 25-g samples of meat in phosphate-buffered saline (pH 7.2) at 25 degrees C. After incubation (48 and 72 h for 1.0-g samples and 24 and 48 h for 25-g samples); 0.5-ml portions of enrichment culture were treated with...

  6. Molecular mapping of interactions between a Mycobacterium leprae-specific T cell epitope, the restricting HLA-DR2 molecule, and two specific T cell receptors.

    Anderson, D C; van Schooten, W C; Janson, A; Barry, M E; de Vries, R R


    A systematic series of 89 single residue substitution analogs of the Mycobacterium leprae 65-kDa protein-derived peptide LQAAPALDKL were tested for stimulation of two HLA-DR2 restricted 65 kDa-reactive T cell clones from a tuberculoid leprosy patient. Some analogs with substitutions outside a "core" region showed enhanced stimulation of the T cell clones. This core region of seven or eight residues was essential for recognition, whereas substitution of amino acids outside this region did not affect T cell recognition although these residues could not be omitted. Thus these core residues interact directly with the presenting HLA-DR2 molecule and/or the TCR. Except for analogs of position 419 for clone 2B6, the majority of the nonstimulatory substitution analogs did not inhibit the presentation of LQAAPALDKL and thus probably failed to bind to the HLA-DR2 molecule. Unless all of the core residues are physically involved in binding to DR2, substitution at a position not directly involved in binding appears to have an influence on other residues that do bind to the DR2 molecule. Active peptide analogs with two or more internal prolines suggest that not all analogs need be helical for activity with clone 2F10. PMID:1690768

  7. Caloric restriction.

    Speakman, John R; Mitchell, Sharon E


    Restricting the intake of calories has been practiced as a method for increasing both the length and quality of life for over 500 years. Experimental work confirming the success of this approach in animals has accumulated over the last 100 years. Lifelong caloric restriction (CR) may extend life by up to 50% in rodents, with progressively less impact the later in life it is started. This effect is matched by profound impacts on age related diseases including reduced risk of cancer, neurodegenerative disorders, autoimmune disease, cardiovascular disease and type II diabetes mellitus. The disposable soma theory of ageing suggests that CR evolved as a somatic protection response to enable animals to survive periods of food shortage. The shutdown of reproductive function during CR is consistent with this suggestion, but other features of the phenomenon are less consistent with this theory, and some have suggested that in rodents it may be mostly an artifact of domestication. CR induces profound effects on animals at all levels from the transcriptome to whole animal physiology and behavior. Animals under CR lose weight which is disproportionately contributed to by white adipose tissue. Generally animals on CR change their activity patterns so that they are more active prior to food delivery each day but total activity may be unchanged or reduced. Considerable debate has occurred over the effects of CR on resting metabolic rate (RMR). Total RMR declines, but as body mass and body composition also change it is unclear whether metabolism at the tissue level also declines, is unchanged or even increases. Body temperature universally decreases. Hunger is increased and does not seem to abate even with very long term restriction. Circulating adipokines are reduced reflecting the reduction in white adipose tissue (WAT) mass under restriction and there is a large reduction in circulating insulin and glucose levels. There are profound tissue level changes in metabolism with a

  8. Yersinia spp. Identification Using Copy Diversity in the Chromosomal 16S rRNA Gene Sequence.

    Hao, Huijing; Liang, Junrong; Duan, Ran; Chen, Yuhuang; Liu, Chang; Xiao, Yuchun; Li, Xu; Su, Mingming; Jing, Huaiqi; Wang, Xin


    API 20E strip test, the standard for Enterobacteriaceae identification, is not sufficient to discriminate some Yersinia species for some unstable biochemical reactions and the same biochemical profile presented in some species, e.g. Yersinia ferderiksenii and Yersinia intermedia, which need a variety of molecular biology methods as auxiliaries for identification. The 16S rRNA gene is considered a valuable tool for assigning bacterial strains to species. However, the resolution of the 16S rRNA gene may be insufficient for discrimination because of the high similarity of sequences between some species and heterogeneity within copies at the intra-genomic level. In this study, for each strain we randomly selected five 16S rRNA gene clones from 768 Yersinia strains, and collected 3,840 sequences of the 16S rRNA gene from 10 species, which were divided into 439 patterns. The similarity among the five clones of 16S rRNA gene is over 99% for most strains. Identical sequences were found in strains of different species. A phylogenetic tree was constructed using the five 16S rRNA gene sequences for each strain where the phylogenetic classifications are consistent with biochemical tests; and species that are difficult to identify by biochemical phenotype can be differentiated. Most Yersinia strains form distinct groups within each species. However Yersinia kristensenii, a heterogeneous species, clusters with some Yersinia enterocolitica and Yersinia ferderiksenii/intermedia strains, while not affecting the overall efficiency of this species classification. In conclusion, through analysis derived from integrated information from multiple 16S rRNA gene sequences, the discrimination ability of Yersinia species is improved using our method. PMID:26808495

  9. In vitro and in vivo analyses of a genetically—restricted antigen specific factor from mixed cell cultures of macrophage,T and B lymphocytes



    An immunostimulatory factor was identified to be secreted by antigen-pulsed macrophages.This factor was able to induce the generation of antigen specific T helper lymphocytes in vitro as well as in vivo.Further in vitro experiments testing for the genetic restriction of this factor indicated that it is a geneticallyrestricted antigen specific factor (ASF).The Cunningham plaque assay was used to quantify the generation of T helper lymphocytes by measuring the number of plaque forming cells after sequential incubations of antigen-qulsed macrophages with T lymphocytes,and then spleen cells,and finally the TNP-coated sheep red blood cells.

  10. Outbreak of gastroenteritis caused by Yersinia pestis in Afghanistan.

    Leslie, T; Whitehouse, C A; Yingst, S; Baldwin, C; Kakar, F; Mofleh, J; Hami, A S; Mustafa, L; Omar, F; Ayazi, E; Rossi, C; Noormal, B; Ziar, N; Kakar, R


    Plague, which is most often caused by the bite of Yersinia pestis-infected fleas, is a rapidly progressing, serious disease that can be fatal without prompt antibiotic treatment. In late December 2007, an outbreak of acute gastroenteritis occurred in Nimroz Province of southern Afghanistan. Of the 83 probable cases of illness, 17 died (case fatality 20·5%). Being a case was associated with consumption or handling of camel meat (adjusted odds ratio 4·4, 95% confidence interval 2·2-8·8, P<0·001). Molecular testing of patient clinical samples and of tissue from the camel using PCR/electrospray ionization-mass spectrometry revealed DNA signatures consistent with Yersinia pestis. Confirmatory testing using real-time PCR and immunological seroconversion of one of the patients confirmed that the outbreak was caused by plague, with a rare gastrointestinal presentation. The study highlights the challenges of identifying infectious agents in low-resource settings; it is the first reported occurrence of plague in Afghanistan. PMID:20663260