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Infection with the SARS (Severe Acute Respiratory Syndrome)-associated coronavirus results in respiratory failure probably by immunological mechanisms in 10% of patients. Laboratory markers that predict subsequent respiratory failure would therefore be useful in patient management.
We describe the clinical course, hematologic parameters, lymphocyte subpopulations and markers of inflammation in two patients with SARS, i.e., one man with diabetes mellitus and one pregnant woman, infected by the same viral isolate.
The patient with underlying diabetes mellitus developed respiratory failure after admission in the second week of the illness while the second patient developed only a mild disease without respiratory failure. Subsequent respiratory dysfunction was associated with lown umbers of Natural Killer (NK) cells at presentation and elevated CRP levels during the illness.
NK cells and CRP levels at the end of the first week of the disease might be related to subsequent respiratory dysfunction and may link underlying conditions to disease severity.
SUMO proteins are ubiquitin-related modifiers implicated in the regulation of gene transcription, cell cycle, DNA repair, and protein localization. The molecular mechanisms by which the sumoylation of target proteins regulates diverse cellular functions remain poorly understood. Here we report isolation and characterization of SUMO1- and SUMO2-binding motifs. Using yeast two-hybrid system, bioinformatics, and NMR spectroscopy we define a common SUMO-interacting motif (SIM) and map its binding surfaces on SUMO1 and SUMO2. This motif forms a beta-strand that could bind in parallel or antiparallel orientation to the beta2-strand of SUMO due to the environment of the hydrophobic core. A negative charge imposed by a stretch of neighboring acidic amino acids and/or phosphorylated serine residues determines its specificity in binding to distinct SUMO paralogues and can modulate the spatial orientation of SUMO-SIM interactions.
Cyclic GMP-dependent protein kinases protein kinase G (PKG) Iα and PKGIβ are major mediators of cGMP signaling in the cardiovascular system. PKGIα is present in the heart, although its role in protection against ischemia/reperfusion injury is not known. We investigated the direct effect of PKGIα against necrosis and apoptosis following simulated ischemia (SI) and reoxygenation (RO) in cardiomyocytes. Adult rat cardiomyocytes were infected with adenoviral vectors containing hPKGIα or catalytically inactive mutant hPKGIαK390A. After 24 h, the cells were subjected to 90 min of SI and 2 h RO for necrosis (trypan blue exclusion and lactate dehydrogenase release) or 18 h RO for apoptosis studies. To evaluate the role of KATP channels, subgroups of cells were treated with 5-hydroxydecanoate (100 μm), HMR1098 (30 μm), or glibenclamide (50 μm), the respective blockers of mitochondrial, sarcolemmal, or both types of KATP channels prior to SI. The necrosis observed in 33.7 ± 1.6% of total myocytes in the SI-RO control group was reduced to 18.6 ± 0.8% by PKGIα (mean ± S.E., n = 7, p < 0.001). The apoptosis observed in 17.9 ± 1.3% of total myocytes in the SI-RO control group was reduced to 6.0 ± 0.6% by PKGIα (mean ± S.E., n = 7, p < 0.001). In addition, PKGIα inhibited the activation of caspase-3 after SI-RO in myocytes. Myocytes infected with the inactive PKGIαK390A mutant showed no protection. PKGIα enhanced phosphorylation of Akt, ERK1/2, and JNK, increased Bcl-2, inducible nitric-oxide synthase, endothelial nitric-oxide synthase, and decreased Bax expression. 5-Hydroxydecanoate and glibenclamide abolished PKGIα-mediated protection against necrosis and apoptosis. However, HMR1098, had no effect. A scavenger of reactive oxygen species, as well as inhibitors of phosphatidylinositol 3-kinase, ERK, JNK1, and NOS, also blocked PKGIα-mediated protection against necrosis and apoptosis. These results show that opening of mitochondrial KATP channels and generation of reactive oxygen species, in association with phosphorylation of Akt, ERK, and JNK, and increased expression of NOS and Bcl-2, play an essential role in the protective effect of PKGIα.
CXCR4 chemokine receptor mediates prostate tumor cell adhesion through alpha5 and beta3 integrins
(2006)
The mechanisms leading to prostate cancer metastasis are not understood completely. Although there is evidence that the CXC chemokine receptor (CXCR) 4 and its ligand CXCL12 may regulate tumor dissemination, their role in prostate cancer is controversial. We examined CXCR4 expression and functionality, and explored CXCL12-triggered adhesion of prostate tumor cells to human endothelium or to extracellular matrix proteins laminin, collagen, and fibronectin. Although little CXCR4 was expressed on LNCaP and DU-145 prostate tumor cells, CXCR4 was still active, enabling the cells to migrate toward a CXCL12 gradient. CXCL12 induced elevated adhesion to the endothelial cell monolayer and to immobilized fibronectin, laminin, and collagen. Anti-CXCR4 antibodies or CXCR4 knock out significantly impaired CXCL12-triggered tumor cell binding. The effects observed did not depend on CXCR4 surface expression level. Rather, CXCR4-mediated adhesion was established by alpha5 and beta3 integrin subunits and took place in the presence of reduced p38 and p38 phosphorylation. These data show that chemoattractive mechanisms are involved in adhesion processes of prostate cancer cells, and that binding of CXCL12 to its receptor leads to enhanced expression of alpha5 and beta3 integrins. The findings provide a link between chemokine receptor expression and integrin-triggered tumor dissemination.
The genome, antigens of human cytomegalovirus (HCMV) are frequently found in prostatic carcinoma. However, whether this infection is causative or is an epiphenomenon is not clear. We therefore investigated the ability of HCMV to promote metastatic processes, defined by tumor cell adhesion to the endothelium, extracellular matrix proteins. Experiments were based on the human prostate tumor cell line PC3, either infected with the HCMV strain Hi (HCMVHi) or transfected with cDNA encoding the HCMV-specific immediate early protein IEA1 (UL123) or IEA2 (UL122). HCMVHi upregulated PC3 adhesion to the endothelium, to the extracellular matrix proteins collagen, laminin, fibronectin. The process was accompanied by enhancement of β1-integrin surface expression, elevated levels of integrin-linked kinase, phosphorylation of focal adhesion kinase. IEA1 or IEA2 did not modulate PC3 adhesion or β1-integrin expression. Based on this in vitro model, we postulate a direct association between HCMV infection, prostate tumor transmigration, which is not dependent on IEA proteins. Integrin overexpression, combined with the modulation of integrin-dependent signalling, seems to be, at least in part, responsible for a more invasive PC3Hi tumor cell phenotype. Elevated levels of c-myc found in IEA1-transfected or IEA2-transfected PC3 cell populations might promote further carcinogenic processes through accelerated cell proliferation.
Proton pumping respiratory complex I (NADH:ubiquinone oxidoreductase) is a major component of the oxidative phosphorylation system in mitochondria and many bacteria. In mammalian cells it provides 40% of the proton motive force needed to make ATP. Defects in this giant and most complicated membrane-bound enzyme cause numerous human disorders. Yet the mechanism of complex I is still elusive. A group exhibiting redox-linked protonation that is associated with iron-sulfur cluster N2 of complex I has been proposed to act as a central component of the proton pumping machinery. Here we show that a histidine in the 49-kDa subunit that resides near iron-sulfur cluster N2 confers this redox-Bohr effect. Mutating this residue to methionine in complex I from Yarrowia lipolytica resulted in a marked shift of the redox midpoint potential of iron-sulfur cluster N2 to the negative and abolished the redox-Bohr effect. However, the mutation did not significantly affect the catalytic activity of complex I and protons were pumped with an unchanged stoichiometry of 4 H+/2e−. This finding has significant implications on the discussion about possible proton pumping mechanism for complex I.
Activation by diazoxide and inhibition by 5-hydroxydecanoate are the hallmarks of mitochondrial ATP-sensitive K+ (K(ATP)) channels. Opening of these channels is thought to trigger cytoprotection (preconditioning) through the generation of reactive oxygen species. However, we found that diazoxide-induced oxidation of the widely used reactive oxygen species indicator 2',7'-dichlorodihydrofluorescein in isolated liver and heart mitochondria was observed in the absence of ATP or K+ and therefore independent of K(ATP) channels. The response was blocked by stigmatellin, implying a role for the cytochrome bc1 complex (complex III). Diazoxide, though, did not increase hydrogen peroxide (H2O2) production (quantitatively measured with Amplex Red) in intact mitochondria, submitochondrial particles, or purified cytochrome bc1 complex. We confirmed that diazoxide inhibited succinate oxidation, but it also weakly stimulated state 4 respiration even in K+-free buffer, excluding a role for K(ATP) channels. Furthermore, we have shown previously that 5-hydroxydecanoate is partially metabolized, and we hypothesized that fatty acid metabolism may explain the ability of this putative mitochondrial K(ATP) channel blocker to inhibit diazoxide-induced flavoprotein fluorescence, commonly used as an assay of K(ATP) channel activity. Indeed, consistent with our hypothesis, we found that decanoate inhibited diazoxide-induced flavoprotein oxidation. Taken together, our data question the "mitochondrial K(ATP) channel" hypothesis of preconditioning. Diazoxide did not evoke superoxide (which dismutates to H2O2) from the respiratory chain by a direct mechanism, and the stimulatory effects of this compound on mitochondrial respiration and 2',7'-dichlorodihydrofluorescein oxidation were not due to the opening of K(ATP) channels.
Prostaglandin (PG) E2 (PGE2) plays a predominant role in promoting colorectal carcinogenesis. The biosynthesis of PGE2 is accomplished by conversion of the cyclooxygenase (COX) product PGH2 by several terminal prostaglandin E synthases (PGES). Among the known PGES isoforms, microsomal PGES type 1 (mPGES-1) and type 2 (mPGES-2) were found to be overexpressed in colorectal cancer (CRC); however, the role and regulation of these enzymes in this malignancy are not yet fully understood. Here, we report that the cyclopentenone prostaglandins (CyPGs) 15-deoxy-Δ12,14-PGJ2 and PGA2 downregulate mPGES-2 expression in the colorectal carcinoma cell lines Caco-2 and HCT 116 without affecting the expression of any other PGES or COX. Inhibition of mPGES-2 was subsequently followed by decreased microsomal PGES activity. These effects were mediated via modulation of the cellular thiol-disulfide redox status but did not involve activation of the peroxisome proliferator-activated receptor γ or PGD2 receptors. CyPGs had antiproliferative properties in vitro; however, this biological activity could not be directly attributed to decreased PGES activity because it could not be reversed by adding PGE2. Our data suggest that there is a feedback mechanism between PGE2 and CyPGs that implicates mPGES-2 as a new potential target for pharmacological intervention in CRC.
High tumor interstitial fluid pressure (TIFP) is a characteristic of most solid tumors. TIFP may hamper adequate uptake of macromolecular therapeutics in tumor tissue. In addition, TIFP generates mechanical forces affecting the tumor cortex, which might influence the growth parameters of tumor cells. This seems likely as, in other tissues (namely, blood vessels or the skin), mechanical stretch is known to trigger proliferation. Therefore, we hypothesize that TIFP-induced stretch modulates proliferation-associated parameters. Solid epithelial tumors (A431 and A549) were grown in Naval Medical Research Institute nude mice, generating a TIFP of about 10 mm Hg (A431) or 5 mm Hg (A549). Tumor drainage of the central cystic area led to a rapid decline of TIFP, together with visible relaxation of the tumor cortex. It was found by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis that TIFP lowering yields a decreased phosphorylation of proliferation-associated p44/42 mitogen-activated protein kinase and tumor relaxation. In confirmation, immunohistochemical staining showed a decrease of tumor-associated proliferation marker Ki-67 after TIFP lowering. These data suggest that the mechanical stretch induced by TIFP is a positive modulator of tumor proliferation.
The use of neutralizing antibodies is one of the most successful methods to interfere with receptor-ligand interactions in vivo. In particular blockade of soluble inflammatory mediators or their corresponding cellular receptors was proven an effective way to regulate inflammation and/or prevent its negative consequences. However, one problem that comes along with an effective neutralization of inflammatory mediators is the general systemic immunomodulatory effect. It is therefore important to design a treatment regimen in a way to strike at the right place and at the right time in order to achieve maximal effects with minimal duration of immunosuppression or hyperactivation. In this review we reflect on two examples of how short time administration of such neutralizing antibodies can block two distinct inflammatory consequences of viral infection. First, we review recent findings that blockade of IL-10/IL-10R interaction can resolve chronic viral infection and second, we reflect on how neutralization of the chemokine CXCL10 can abrogate virus-induced type 1 diabetes.