Structure-function analysis of the C-terminus of IcmT of Legionella pneumophila in pore formation-mediated egress from macrophages

We have recently shown an essential role of the 32 amino acids C-terminus domain of IcmT of Legionella pneumophila in bacterial egress from macrophages. Mutants expressing an IcmT protein with a truncation in the C-terminus, replicate intracellularly but are defective in pore formation-mediated egress. The C-terminus domain of IcmT is the only hydrophilic domain of IcmT that is predicted to be in the cytoplasm while the rest of the protein is in the cytoplasmic membrane. In order to characterize the structure-function of the C-terminus of IcmT in the pore-forming activity and bacterial egress, we constructed 10 icmT missense mutant alleles differing by a single amino acid in the C-terminus of icmT and introduced them into the null icmT mutant. The H58Q, W69L, R71I, R79I and R86I icmT mutant alleles showed significantly lower pore-forming activity as measured by hemolysis of sRBC. The Y59S, R68L and S77L mutant alleles showed significantly lower cytopathogenicity to U937 macrophages. All 10 mutant alleles enabled the icmT null mutant to replicate intracellularly as efficiently as icmT null mutant harboring the wild-type icmT. Seven of the icmT alleles enabled the icmT null mutant to egress from infected macrophages as efficiently as icmT null mutant harboring the wild-type icmT. The other 3 substitutions conferred a partial defect in hemolysis and two of them also conferred a defect in egress from macrophages. Thus, two amino acid residues in the C-terminus of IcmT are required for both pore formation and bacterial egress. However, certain single amino acid substitutions in the C-terminus reduce the pore-forming activity when tested in vitro, but may or may not have a detectable effect on egress of L. pneumophila from U937 macrophages.     

Glucose-induced regulation of novel iron transporters in vascular endothelial cell dysfunction

Increased iron indices have been associated with the development of diabetes and its complications. In the present study, we have investigated the glucose-induced alteration of iron transporters, divalent metal transporter-1 (DMT-1), iron regulated transporter protein-1 (IREG-1), and transferrin receptor (TfR), in endothelial cell iron accumulation and oxidative stress. Cells were exposed to high glucose levels and subjected to gene expression, protein expression, iron measurement and assessment of oxidative stress. Our results show, for the first time, expression of DMT-1 and IREG-1 in vascular endothelial cells. Our data further indicates upregulation of DMT-1 and IREG-1 mRNA and protein in response to high levels of glucose. TfR, however, exhibited a modest decrease in response to high levels of glucose. Increased expression of DMT-1 and IREG-1 was associated with iron accumulation and oxidative stress. Furthermore, our results show differential expression of iron transporters with treatment of high glucose-exposed cells with two different iron chelators. In conclusion, our study suggests that glucose-induced alteration of iron transporters may arbitrate iron accumulation and oxidative stress in endothelial cells.     

The Francisella tularensis pathogenicity island protein IglC and its regulator MglA are essential for modulating phagosome biogenesis and subsequent bacterial escape into the cytoplasm

The Francisella tularensis subsp. novicida-containing phagosome (FCP) matures into a late endosome-like stage that acquires the late endosomal marker LAMP-2 but does not fuse to lysosomes, for the first few hours after bacterial entry. This modulation in phagosome biogenesis is followed by disruption of the phagosome and bacterial escape into the cytoplasm where they replicate. Here we examined the role of the Francisella pathogenicity island (FPI) protein IglC and its regulator MglA in the intracellular fate of F. tularensis subsp. novicida within human macrophages. We show that F. tularensis mglA and iglC mutant strains are defective for survival and replication within U937 macrophages and human monocyte-derived macrophages (hMDMs). The defect in intracellular replication of both mutants is associated with a defect in disruption of the phagosome and failure to escape into the cytoplasm. Approximately, 80-90% of the mglA and iglC mutants containing phagosomes acquire the late endosomal/lysosomal marker LAMP-2 similar to the wild-type (WT) strain. Phagosomes harbouring the mglA or iglC mutants acquire the lysosomal enzyme Cathepsin D, which is excluded from the phagosomes harbouring the WT strain. In hMDMs in which the lysosomes are preloaded with BSA-gold or Texas Red Ovalbumin, phagosomes harbouring the mglA or the iglC mutants acquire both lysosomal tracers. We conclude that the FPI protein IglC and its regulator MglA are essential for modulating phagosome biogenesis and subsequent bacterial escape into the cytoplasm. Therefore, acquisition of the FPI, within which iglC is contained, is essential for the pathogenic evolution of F. tularensis to evade lysosomal fusion within human macrophages and cause tularemia. This is the first example of specific virulence factors of F. tularensis that are essential for evasion of fusion of the FCP to lysosomes.     

ISO-1 binding to the tautomerase active site of MIF inhibits its pro-inflammatory activity and increases survival in severe sepsis

MIF is a proinflammatory cytokine that has been implicated in the pathogenesis of sepsis, arthritis, and other inflammatory diseases. Antibodies against MIF are effective in experimental models of inflammation, and there is interest in strategies to inhibit its deleterious cytokine activities. Here we identify a mechanism of inhibiting MIF pro-inflammatory activities by targeting MIF tautomerase activity. We designed small molecules to inhibit this tautomerase activity; a lead molecule, "ISO-1 ((S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester)," significantly inhibits the cytokine activity in vitro. Moreover, ISO-1 inhibits tumor necrosis factor release from macrophages isolated from LPStreated wild type mice but has no effect on cytokine release from MIFdeficient macrophages. The therapeutic importance of the MIF inhibition by ISO-1 is demonstrated by the significant protection from sepsis, induced by cecal ligation and puncture in a clinically relevant time frame. These results identify ISO-1 as the first small molecule inhibitor of MIF proinflammatory activities with therapeutic implications and indicate the potential of the MIF active site as a novel target for therapeutic interventions in human sepsis.     

Marrow stromal cells and osteoclast precursors differentially contribute to TNF-alpha-induced osteoclastogenesis in vivo

The marrow stromal cell is the principal source of the key osteoclastogenic cytokine receptor activator of NF-kappaB (RANK) ligand (RANKL). To individualize the role of marrow stromal cells in varying states of TNF-alpha-driven osteoclast formation in vivo, we generated chimeric mice in which wild-type (WT) marrow, immunodepleted of T cells and stromal cells, is transplanted into lethally irradiated mice deleted of both the p55 and p75 TNFR. As control, similarly treated WT marrow was transplanted into WT mice. Each group was administered increasing doses of TNF-alpha. Exposure to high-dose cytokine ex vivo induces exuberant osteoclastogenesis irrespective of in vivo TNF-alpha treatment or whether the recipient animals possess TNF-alpha-responsive stromal cells. In contrast, the osteoclastogenic capacity of marrow treated with lower-dose TNF-alpha requires priming by TNFR-bearing stromal cells in vivo. Importantly, the osteoclastogenic contribution of cytokine responsive stromal cells in vivo diminishes as the dose of TNF-alpha increases. In keeping with this conclusion, mice with severe inflammatory arthritis develop profound osteoclastogenesis and bone erosion independent of stromal cell expression of TNFR. The direct induction of osteoclast recruitment by TNF-alpha is characterized by enhanced RANK expression and sensitization of precursor cells to RANKL. Thus, osteolysis attending relatively modest elevations in ambient TNF-alpha depends upon responsive stromal cells. Alternatively, in states of severe periarticular inflammation, TNF-alpha may fully exert its bone erosive effects by directly promoting the differentiation of osteoclast precursors independent of cytokine-responsive stromal cells and T lymphocytes.     

Expression and purification of recombinant human receptor for advanced glycation endproducts in Escherichia coli

The receptor for advanced glycation endproducts (RAGE) is a multiligand receptor that binds a variety of structurally and functionally unrelated ligands, including advanced glycation endproducts (AGEs), amyloid fibrils, amphoterin, and members of the S100 family of proteins. The receptor has been implicated in the pathology of diabetes as well as in inflammatory processes and tumor cell metastasis. For the present study, the extracellular region of RAGE (exRAGE) was expressed as a soluble, C-terminal hexahistidine-tagged fusion protein in the periplasmic space of Escherichia coli. Proper processing and folding of the purified protein, predicted to contain three immunoglobulin-type domains, was supported by the results of electrospray mass spectroscopy and circular dichroism experiments. Sedimentation velocity experiments showed that exRAGE was primarily monomeric in solution. Binding to several RAGE ligands, including AGE-BSA, immunoglobulin light chain amyloid fibrils, and glycosaminoglycans, was demonstrated using pull-down, dot-blot, or enzyme-linked microplate assays. Using surface plasmon resonance, the interaction of exRAGE with AGE-BSA was shown to fit a two-site model, with KD values of 88 nM and 1.4 microM. The E. coli-derived exRAGE did not bind the advanced glycation endproduct Nepsilon-(carboxymethyl)lysine, as reported for the cellular receptor, and the possible role of RAGE glycosylation in recognition of this ligand is discussed. This new RAGE construct will facilitate detailed studies of RAGE-ligand interactions and provides a platform for preparation of site-directed mutants for future structure/function studies.     

A comprehensive nomenclature for serine proteases with homology to tissue kallikreins

The human kallikrein locus on chromosome 19q13.3-13.4 contains kallikrein 1--the tissue kallikrein--and 14 related serine proteases. Recent investigations into their function and evolution have indicated that the present nomenclature for these proteins is inadequate or insufficient. Here we present a new nomenclature in which proteins without proven kininogenase activity are denoted kallikrein-related peptidase. Names are also given to the unique rodent proteins that are closely related to kallikrein 1.     

Clinical trials of vascular brachytherapy for in-stent restenosis: update

Vascular brachytherapy has become the therapeutic strategy of choice for in-stent restenosis. Clinical trials evaluating the effectiveness and safety of this technology have enrolled nearly 4000 patients using both gamma and beta emitters. At present, ongoing controversies include optimal dosimetry, whether beta emitters are as effective as gamma and whether centering delivery systems perform better than non-centering systems. Complications of brachytherapy such as edge effect, late thrombosis and late restenosis have received increasing attention. This review provides an update of the current status of clinical trials utilizing vascular brachytherapy to prevent the recurrence of in-stent restenosis.     

Genomic organization, mapping, tissue expression, and hormonal regulation of trypsin-like serine protease (TLSP PRSS20), a new member of the human kallikrein gene family

The cDNA for the trypsin-like serine protease gene (TLSP, HGMW-approved symbol PRSS20) has been recently identified. TLSP is expressed in brain and skin tissues but little else is known about this new serine protease gene. In this paper, we describe the complete genomic organization and precise mapping of the TLSP gene. This gene spans 5.3 kb of genomic sequence on chromosome 19q13.3-q13. 4. The gene consists of six exons, the first of which is untranslated. All splice junctions follow the GT/AG rule, and the intron phases are identical to those of other kallikrein-like genes, including zyme (PRSS9), NES1 (PRSSL1), and neuropsin (PRSS19). Fine-mapping of the area indicates that TLSP lies downstream from the PSA, zyme, neuropsin, and NES1 genes. Significant sequence homologies were found between TLSP and other human kallikreins. Furthermore, there is conservation of the catalytic triad (histidine, aspartic acid, serine) and of the number of coding exons (five; the same in all members of the kallikrein gene family). We thus suggest that TLSP is a new member of the human kallikrein gene family. TLSP is expressed in many tissues including cerebellum, prostate, salivary glands, stomach, lung, thymus, small intestine, spleen, liver, and uterus. TLSP expression appears to be regulated by steroid hormones in the breast carcinoma cell line BT-474.