ROC1, a Homolog of APC11, Represents a Family of Cullin Partners with an Associated Ubiquitin Ligase Activity

AbstractWe have identified two highly conserved RING finger proteins, ROC1 and ROC2, that are homologous to APC11, a subunit of the anaphase-promoting complex. ROC1 and ROC2 commonly interact with all cullins while APC11 specifically interacts with APC2, a cullin-related APC subunit. Yeast ROC1 encodes an essential gene whose reduced expression resulted in multiple, elongated buds and accumulation of Sic1p and Cln2p. ROC1 and APC11 immunocomplexes can catalyze isopeptide ligations to form polyubiquitin chains in an E1- and E2-dependent manner. ROC1 mutations completely abolished their ligase activity without noticeable changes in associated proteins. Ubiquitination of phosphorylated IκBα can be catalyzed by the ROC1 immunocomplex in vitro. Hence, combinations of ROC/APC11 and cullin proteins potentially constitute a wide variety of ubiquitin ligases

Compact Cryogenic Source of Periodic Hydrogen and Argon Droplet Beams for Relativistic Laser-Plasma Generation

We present a cryogenic source of periodic streams of micrometer-sized hydrogen and argon droplets as ideal mass-limited target systems for fundamental intense laser-driven plasma applications. The highly compact design combined with a high temporal and spatial droplet stability makes our injector ideally suited for experiments using state-of-the-art high-power lasers in which a precise synchronization between the laser pulses and the droplets is mandatory. We show this by irradiating argon droplets with multi-Terawatt pulses.Comment: To be published in Review of Scientific Instrument

The Separation of Salicylate Metabolites By Paper Electrophoresis

The technique of paper electrophoresis was employed to develop a method for the separation of the end-products of salicylate metabolism. A phthalate buffer with a pH of 3.2 and an ionic strength of 0.0125 to 0.05 produced the most satisfactory separation of the metabolites. It was found that the end-products, i.e., salicylic, acetyl salicylic, salicyluric, and gentisic acids, could best be detected by the fluorescent spots they exhibited when the paper strips were viewed under ultraviolet light. The sensitivity of the method was such that as little as one microgram of salicylic acid could be detected as a fluorescent spot after electrophoretic separation

The Role of Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 in the p38/TNF-α Pathway of Systemic and Cutaneous Inflammation

Mitogen-activated protein kinase-activated protein kinase 2 (MK2) is a downstream molecule of p38, involved in the production of TNF-α, a key cytokine, and an established drug target for many inflammatory diseases. We investigated the role of MK2 in skin inflammation to determine its drug target potential. MK2 deficiency significantly decreased plasma TNF-α levels after systemic endotoxin application. Deficient mice showed decreased skin edema formation in chronic 2-O-tetradecanoylphorbol-13-acetate (TPA)-induced irritative dermatitis and in subacute 2,4-dinitrofluorobenzene (DNFB)-induced contact hypersensitivity. Surprisingly, MK2 deficiency did not inhibit edema formation in subacute 2,4-dinitrochlorobenzene (DNCB)-induced contact allergy and even increased TNF-α and IL-1β levels as well as granulocyte infiltration in diseased ears. Ear inflammation in this model, however, was inhibited by TNF-α neutralization as it was in the subacute DNFB model. MK2 deficiency also did not show anti-inflammatory effects in acute DNFB-induced contact hypersensitivity, whereas the p38 inhibitor, SB203580, ameliorated skin inflammation supporting a pathophysiological role of p38. When evaluating possible mechanisms, we found that TNF-α production in MK2-deficient spleen cells was strongly diminished after TLR stimulation but less affected after T-cell receptor stimulation. Our data suggest that MK2, in contrast to its downstream effector molecule, TNF-α, has a rather elusive role in T-cell-dependent cutaneous inflammation

Akt Suppresses Apoptosis by Stimulating the Transactivation Potential of the RelA/p65 Subunit of NF-kappa B

It is well established that cell survival signals stimulated by growth factors, cytokines, and oncoproteins are initiated by phosphoinositide 3-kinase (PI3K)- and Akt-dependent signal transduction pathways. Oncogenic Ras, an upstream activator of Akt, requires NF-κB to initiate transformation, at least partially through the ability of NF-κB to suppress transformation-associated apoptosis. In this study, we show that oncogenic H-Ras requires PI3K and Akt to stimulate the transcriptional activity of NF-κB. Activated forms of H-Ras and MEKK stimulate signals that result in nuclear translocation and DNA binding of NF-κB as well as stimulation of the NF-κB transactivation potential. In contrast, activated PI3K or Akt stimulates NF-κB-dependent transcription by stimulating transactivation domain 1 of the p65 subunit rather than inducing NF-κB nuclear translocation via IκB degradation. Inhibition of IκB kinase (IKK), using an IKKβ dominant negative protein, demonstrated that activated Akt requires IKK to efficiently stimulate the transactivation domain of the p65 subunit of NF-κB. Inhibition of endogenous Akt activity sensitized cells to H-Ras(V12)-induced apoptosis, which was associated with a loss of NF-κB transcriptional activity. Finally, Akt-transformed cells were shown to require NF-κB to suppress the ability of etoposide to induce apoptosis. Our work demonstrates that, unlike activated Ras, which can stimulate parallel pathways to activate both DNA binding and the transcriptional activity of NF-κB, Akt stimulates NF-κB predominantly by upregulating of the transactivation potential of p65

Towards personalized treatment of prostate cancer: PSMA I&T, a promising prostate-specific membrane antigen-targeted theranostic agent

Prostate-specific membrane antigen (PSMA) is a well-established target for nuclear imaging and therapy of prostate cancer (PCa). Radiolabeled small-molecule PSMA inhibitors are excellent candidates for PCa theranostics-they rapidly and efficiently localize in tumor lesions. However, high tracer uptake in kidneys and salivary glands are major concerns for therapeutic applications. Here, we present the preclinical application of PSMA I&T, a DOTAGA-chelated urea-based PSMA inhibitor, for SPECT/CT imaging and radionuclide therapy of PCa. 111In-PSMA I&T showed dose-dependent uptake in PSMA-expressing tumors, kidneys, spleen, adrenals, lungs and salivary glands. Coadministration of 2-(phosphonomethyl)pentane-1,5-dioic acid (2-PMPA) efficiently reduced PSMA-mediated renal uptake of 111In-PSMA I&T, with the highest tumor/kidney radioactivity ratios being obtained using a dose of 50 nmol 2-PMPA. SPECT/CT clearly visualized subcutaneous tumors and sub-millimeter intraperitoneal metastases; however, high renal and spleen uptake in control mice (no 2-PMPA) interfered with visualization of metastases in the vicinity of those organs. Coadministration of 2-PMPA increased the tumor-to-kidney absorbed dose ratio during 177Lu-PSMA I&T radionuclide therapy. Hence, at equivalent absorbed dose to the tumor (36 Gy), coinjection of 2-PMPA decreased absorbed dose to the kidneys from 30 Gy to 12 Gy. Mice injected with 177Lu-PSMA I&T only, showed signs of nephrotoxicity at 3 months after therapy, whereas mice injected with 177Lu-PSMA I&T + 2-PMPA did not. These data indicate that PSMA I&T is a promising theranostic tool for PCa. PSMA-specific uptake in kidneys can be successfully tackled using blocking agents such as 2-PMPA

IL-10 inhibits transcription elongation of the human TNF gene in primary macrophages

IL-10 plays a central nonredundant role in limiting inflammation in vivo. However, the mechanisms involved remain to be resolved. Using primary human macrophages, we found that IL-10 inhibits selected inflammatory genes, primarily at a level of transcription. At the TNF gene, this occurs not through an inhibition of RNA polymerase II (Pol II) recruitment and transcription initiation but through a mechanism targeting the stimulation of transcription elongation by cyclin-dependent kinase (CDK) 9. We demonstrated an unanticipated requirement for a region downstream of the TNF 3′ untranslated region (UTR) that contains the nuclear factor κB (NF-κB) binding motif (κB4) both for induction of transcription by lipopolysaccharide (LPS) and its inhibition by IL-10. IL-10 not only inhibits the recruitment of RelA to regions containing κB sites at the TNF gene but also to those found at other LPS-induced genes. We show that although IL-10 elicits a general block in RelA recruitment to its genomic targets, the gene-specific nature of IL-10’s actions are defined through the differential recruitment of CDK9 and the control of transcription elongation. At TNF, but not NFKBIA, the consequence of RelA recruitment inhibition is a loss of CDK9 recruitment, preventing the stimulation of transcription elongation

Modulation of the NF-κB Pathway by Bordetella pertussis Filamentous Hemagglutinin

Background Filamentous hemagglutinin (FHA) is a cell-associated and secreted adhesin produced by Bordetella pertussis with pro-apoptotic and pro-inflammatory activity in host cells. Given the importance of the NF-κB transcription factor family in these host cell responses, we examined the effect of FHA on NF-κB activation in macrophages and bronchial epithelial cells, both of which are relevant cell types during natural infection. Methodology/Principal Findings Exposure to FHA of primary human monocytes and transformed U-937 macrophages, but not BEAS-2B epithelial cells, resulted in early activation of the NF-κB pathway, as manifested by the degradation of cytosolic IκBα, by NF-κB DNA binding, and by the subsequent secretion of NF-κB-regulated inflammatory cytokines. However, exposure of macrophages and human monocytes to FHA for two hours or more resulted in the accumulation of cytosolic IκBα, and the failure of TNF-α to activate NF-κB. Proteasome activity was attenuated following exposure of cells to FHA for 2 hours, as was the nuclear translocation of RelA in BEAS-2B cells. Conclusions These results reveal a complex temporal dynamic, and suggest that despite short term effects to the contrary, longer exposures of host cells to this secreted adhesin may block NF-κB activation, and perhaps lead to a compromised immune response to this bacterial pathogen

Near-infrared molecular imaging of tumors via chemokine receptors CXCR4 and CXCR7

The chemokine CXCL12/SDF-1 and its receptors CXCR4 and CXCR7 play a major role in tumor invasion, proliferation and metastasis. Since both receptors are overexpressed on distinct tumor cells and on the tumor vasculature, we evaluated their potential as targets for detection of cancers by molecular imaging. We synthesized conjugates of CXCL12 and the near-infrared (NIR) fluorescent dye IRDye®800CW, tested their selectivity, sensitivity and biological activity in vitro and their feasibility to visualize tumors in vivo. Purified CXCL12-conjugates detected in vitro as low as 500 A764 human glioma cells or MCF-7 breast cancer cells that express CXCR7 alone or together with CXCR4. Binding was time- and concentration-dependent, and the label could be competitively displaced by the native peptide. Control conjugates with bovine serum albumin or lactalbumin failed to label the cells. In mice, the conjugate distributed rapidly. After 1–92 h, subcutaneous tumors of human MCF-7 and A764 cells in immunodeficient mice were detected with high sensitivity. Background was observed in particular in liver within the first 24 h, but also skull and hind limbs yielded some background. Overall, fluorescent CXCL12-conjugates are sensitive and selective probes to detect solid and metastatic tumors by targeting tumor cells and tumor vasculature