Leptin-deficient (ob/ob) mice are protected from T cell-mediated hepatotoxicity: role of tumor necrosis factor alpha and IL-18.
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The role of leptin was investigated in two models of T cell-mediated hepatitis: the administration of Con A or of Pseudomonas aeruginosa exotoxin A (PEA). In both models, leptin-deficient (ob/ob) mice were protected from liver damage and showed lower induction of tumor necrosis factor (TNF) alpha and IL-18 compared with their lean littermates. Neutralization of TNF-alpha reduced induction of IL-18 by either Con A (70% reduction) or PEA (40% reduction). Pretreatment of lean mice with either soluble TNF receptors or with an anti-IL-18 antiserum significantly reduced Con A- and PEA-induced liver damage. The simultaneous neutralization of TNF-alpha and IL-18 fully protected the mice against liver toxicity. However, neutralization of either IL-18 or TNF-alpha did not inhibit Con A-induced production of IFN-gamma. Thymus atrophy and alterations in the number of circulating lymphocytes and monocytes were observed in ob/ob mice. Exogenous leptin replacement restored the responsiveness of ob/ob mice to Con A and normalized their lymphocyte and monocyte populations. These results demonstrate that leptin deficiency leads to reduced production of TNF-alpha and IL-18 associated with reduced T cell-mediated hepatotoxicity. In addition, both TNF-alpha and IL-18 appear to be essential mediators of T cell-mediated liver injury. (+info)
TraG from RP4 and TraG and VirD4 from Ti plasmids confer relaxosome specificity to the conjugal transfer system of pTiC58.
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Plasmid conjugation systems are composed of two components, the DNA transfer and replication system, or Dtr, and the mating pair formation system, or Mpf. During conjugal transfer an essential factor, called the coupling protein, is thought to interface the Dtr, in the form of the relaxosome, with the Mpf, in the form of the mating bridge. These proteins, such as TraG from the IncP1 plasmid RP4 (TraG(RP4)) and TraG and VirD4 from the conjugal transfer and T-DNA transfer systems of Ti plasmids, are believed to dictate specificity of the interactions that can occur between different Dtr and Mpf components. The Ti plasmids of Agrobacterium tumefaciens do not mobilize vectors containing the oriT of RP4, but these IncP1 plasmid derivatives lack the trans-acting Dtr functions and TraG(RP4). A. tumefaciens donors transferred a chimeric plasmid that contains the oriT and Dtr genes of RP4 and the Mpf genes of pTiC58, indicating that the Ti plasmid mating bridge can interact with the RP4 relaxosome. However, the Ti plasmid did not mobilize transfer from an IncQ relaxosome. The Ti plasmid did mobilize such plasmids if TraG(RP4) was expressed in the donors. Mutations in traG(RP4) with defined effects on the RP4 transfer system exhibited similar phenotypes for Ti plasmid-mediated mobilization of the IncQ vector. When provided with VirD4, the tra system of pTiC58 mobilized plasmids from the IncQ relaxosome. However, neither TraG(RP4) nor VirD4 restored transfer to a traG mutant of the Ti plasmid. VirD4 also failed to complement a traG(RP4) mutant for transfer from the RP4 relaxosome or for RP4-mediated mobilization from the IncQ relaxosome. TraG(RP4)-mediated mobilization of the IncQ plasmid by pTiC58 did not inhibit Ti plasmid transfer, suggesting that the relaxosomes of the two plasmids do not compete for the same mating bridge. We conclude that TraG(RP4) and VirD4 couples the IncQ but not the Ti plasmid relaxosome to the Ti plasmid mating bridge. However, VirD4 cannot couple the IncP1 or the IncQ relaxosome to the RP4 mating bridge. These results support a model in which the coupling proteins specify the interactions between Dtr and Mpf components of mating systems. (+info)
Distribution of IS1358 and linkage to rfb-related genes in Vibrio anguillarum.
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The insertion sequence IS1358 is linked to the rfb regions of both Vibrio cholerae O1 and O139, and its location was suggestive of a role in generating new combinations of rfb genes. This provoked an examination of the distribution and localization of IS1358 in Vibrio anguillarum. S11358 was widely distributed in a number of V. anguillarum serogroups. In particular, when cosmid clones of V. anguillarum O1 were screened with IS1358 and subsequently subcloned and sequenced, it was found that rfb-like genes were linked to this region. Furthermore, when the previously identified genes virA and virB from V. anguillarum O1, now known to be involved in LPS biosynthesis, were used as probes, it was discovered that they too are present on the same large EcoRI fragment as IS1358. This clearly indicated that IS1358 was linked to the rfb region of V. anguillarum O1. Further analysis of the location of IS1358 in other serotypes indicated that V. anguillarum O2 also has IS1358 associated with rfb-like genes. In V. anguillarum O2 there is more than one copy of IS1358, suggesting that this element is a site for recombination, gene duplication or that it may be capable of transposition. Following this latter premise, IS1358 elements from a variety of V. anguillarum strains have been cloned and sequenced. Only those strains with multiple copies of IS1358 produce a full-length putative transposase, as shown by protein overexpression, further strengthening the argument that the element is transposing within these strains. (+info)
Recombinant toxins that bind to the urokinase receptor are cytotoxic without requiring binding to the alpha(2)-macroglobulin receptor.
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The alpha(2-)macroglobulin receptor (alpha(2)MR) has been reported to mediate the internalization of the urokinase plasminogen activator receptor (uPAR) via ligand binding to both receptors. To target malignant uPAR-expressing cells and to determine whether uPAR can internalize without ligand binding to alpha(2)MR, we engineered two recombinant toxins, ATF-PE38 and ATF-PE38KDEL. Each consists of the amino-terminal fragment (ATF) of human urokinase and a truncated form of Pseudomonas exotoxin (PE) devoid of domain Ia, which binds alpha(2)MR. ATF-PE38 and ATF-PE38KDEL were cytotoxic toward malignant uPAR-bearing cells, with IC(50) values as low as 0.02 ng/ml (0.3 pM). Cytotoxicity could be blocked using either recombinant urokinase or free ATF, indicating that the cytotoxicity of the recombinant toxins was specific. Radiolabeled ATF-PE38 had high affinity for uPAR (K(d) = 0.4-8 nM) on a variety of different malignant cell types and internalized at a rate similar to that of ATF. The cytotoxicity was not diminished by receptor-associated protein, which binds and shields the alpha(2)MR from other proteins, or by incubation with phorbol myristate acetate, which is known to decrease the number of alpha(2)MRs in U937 cells or by antibodies to alpha(2)MR. Therefore, these recombinant toxins appear to internalize via uPAR without association with the alpha(2)MR. (+info)
Interleukin-13 fusion cytotoxin as a potent targeted agent for AIDS-Kaposi's sarcoma xenograft.
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Clinically advanced and rapidly progressive AIDS-associated Kaposi sarcoma (AIDS-KS) tumors require an aggressive tumor-directed therapy. We have observed that AIDS-KS cells express high levels of receptors for immune regulatory cytokine, interleukin-13 (IL-13). Two tumorigenic AIDS-KS cell lines, KS Y-1 and KS-imm, expressed 4560 and 9480 IL-13 binding sites per cell with an affinity (kd) of approximately 0.9 and 3.7 nmol/L, respectively. IL-13 cytotoxin IL13-PE38QQR, consisting of human IL-13 and a derivative of Pseudomonas exotoxin, is specifically cytotoxic to KS tumor cells. Systemic and loco regional administration of IL13-PE38QQR in immunodeficient mice with established human KS tumors produced remarkable antitumor activity. Three intratumoral (IT) injections of IL-13 toxin (250 microg/kg per dose) on alternate days (qod) or 5 daily (qd) IT injections with lower doses (50 or 100 microg/kg per dose) resulted in a complete regression of established subcutaneous tumors in most animals. Daily IT treatment with 250 microg/kg of IL-13 toxin in another KS-derived cell line also produced complete responses. Twice daily intraperitoneal injections of IL13-PE38QQR (25 or 50 microg/kg per dose) for 10 days (total injections = 20) also completely eradicated KS Y-1 tumors. Intravenous administration of IL13-PE38QQR also suppressed tumor growth; however, complete responses were not observed. All animals tolerated the therapeutic doses of IL-13 toxin without any visible signs of toxicity. The efficacy of receptor-directed IL13-PE38QQR therapy in mice warrants further exploration of this drug for AIDS-KS treatment. (+info)
Genetic and environmental factors affecting T-pilin export and T-pilus biogenesis in relation to flagellation of Agrobacterium tumefaciens.
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The T pilus, primarily composed of cyclic T-pilin subunits, is essential for the transmission of the Ti-plasmid T-DNA from Agrobacterium tumefaciens to plant cells. Although the virB2 gene of the 11-gene virB operon was previously demonstrated to encode the full-length propilin, and other genes of this operon have been implicated as members of a conserved transmembrane transport apparatus, the role of each virB gene in T-pilin synthesis and transport and T-pilus biogenesis remained undefined. In the present study, each virB gene was examined and was found to be unessential for T-pilin biosynthesis, except virB2, but was determined to be essential for the export of the T-pilin subunits and for T-pilus formation. We also find that the genes of the virD operon are neither involved in T-pilin export nor T-pilus formation. Critical analysis of three different virD4 mutants also showed that they are not involved in T-pilus biogenesis irrespective of the A. tumefaciens strains used. With respect to the environmental effects on T-pilus biogenesis, we find that T pili are produced both on agar and in liquid culture and are produced at one end of the A. tumefaciens rod-shaped cell in a polar manner. We also report a novel phenomenon whereby flagellum production is shut down under conditions which turn on T-pilus formation. These conditions are the usual induction with acetosyringone at pH 5.5 of Ti-plasmid vir genes. A search of the vir genes involved in controlling this biphasic reaction in induced A. tumefaciens cells revealed that virA on the Ti plasmid is involved and that neither virB nor virD genes are needed for this reaction. The biphasic reaction therefore appears to be mediated through a two-component signal transducing system likely involving an unidentified vir gene in A. tumefaciens. (+info)
The N- and C-terminal portions of the Agrobacterium VirB1 protein independently enhance tumorigenesis.
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Genetic transformation of plants by Agrobacterium tumefaciens is mediated by a virulence (vir)-specific type IV secretion apparatus assembled from 11 VirB proteins and VirD4. VirB1, targeted to the periplasm by an N-terminal signal peptide, is processed to yield VirB1*, comprising the C-terminal 73 amino acids. The N-terminal segment, which shares homology with chicken egg white lysozyme as well as lytic transglycosylases, may provide local lysis of the peptidoglycan cell wall to create channels for transporter assembly. Synthesis of VirB1* followed by its secretion to the exterior of the cell suggests that VirB1* may also have a role in virulence. In the present study, we provide evidence for the dual roles of VirB1 in tumorigenesis as well as the requirements for processing and secretion of VirB1*. Complementation of a virB1 deletion strain with constructs expressing either the N-terminal lysozyme-homologous region or VirB1* results in tumors intermediate in size between those induced by a wild-type strain and a virB1 deletion strain, suggesting that each domain has a unique role in tumorigenesis. The secretion of VirB1* translationally fused to the signal peptide indicates that processing and secretion are not coupled. When expressed independently of all other vir genes, VirB1 was processed and VirB1* was secreted. When restricted to the cytoplasm by deletion of the signal peptide, VirB1 was neither processed nor secreted and did not restore virulence to the virB1 deletion strain. Thus, factors that mediate processing of VirB1 and secretion of VirB1* are localized in the periplasm or outer membrane and are not subject to vir regulation. (+info)
Transferred DNA (T-DNA)-associated proteins of Agrobacterium tumefaciens are exported independently of virB.
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The transfer of T-DNA from Agrobacterium to plant cells is mediated by a system which involves the virB operon of the Ti plasmid. We report that VirE2 and VirD2, two T-DNA-associated proteins, as well as VirF, a protein known to be secreted into plant cells, are present in the periplasm and supernatant fractions of growing cells of Agrobacterium as are VirJ and ChvE, two known periplasmic proteins. Two cytoplasmic proteins, Ros and chloramphenicol acetyl transferase, and a VirE2green fluorescent protein construct were not detected in the above fraction. Export of VirE2 into the culture supernatant did not require any Ti plasmid genes, except for VirE1, a specific chaperone for VirE2. The levels of the VirE2 and VirD2 proteins in the supernatant increased significantly when cells were grown at 19 degrees C as compared with 28 degrees C. When Agrobacterium expressed the oncogenic suppressive activity protein (Osa), VirE2 and VirF proteins could not be detected in the supernatant or the periplasm and the level of VirD2 was greatly reduced. However, oncogenic suppressive activity protein did not block the accumulation of VirJ and ChvE in the periplasm. Our data suggest that VirD2, VirE2, and VirF are transported across the cytoplasmic membrane by a specific pathway, independent of virB. Thus, transfer of the T-complex of Agrobacterium may take place in two steps, the first mediated by an unidentified pathway and the second by the virB system. (+info)