Cellular mono(ADP-ribosyl) transferase inhibits protein synthesis

AbstractA reticulocyte translation system was depleted of functional EF-2 by treatment with diphtheria toxin (DT) fragment A and NAD. After dialysis to remove NAD, the system was reconstituted using preparation of EF-2 derived from pyBHK cells. Untreated and reconstituted lysates permitted similar rates of translation. As expected, when DT-treated EF-2 was used to reconstitute the system, no translation occurred. Furthermore EF-2, reacting with the endogenous ADP-ribosyl transferase from pyBHK cells, was also unable to restore protein synthesis in the reconstituted system. These studies suggest that eukaryotic cellular ADP-ribosyl transferases may play a role in regulating protein synthesis

Urotensin II-induced signaling involved in proliferation of vascular smooth muscle cells

The urotensin II receptor, bound by the ligand urotensin II, generates second messengers, ie, inositol triphosphate and diacylglycerol, which stimulate the subsequent release of calcium (Ca2+) in vascular smooth muscle cells. Ca2+ influx leads to the activation of Ca2+-dependent kinases (CaMK) via calmodulin binding, resulting in cellular proliferation. We hypothesize that urotensin II signaling in pulmonary arterial vascular smooth muscle cells (Pac1) and primary aortic vascular smooth muscle cells (PAVSMC) results in phosphorylation of Ca2+/calmodulin-dependent kinases leading to cellular proliferation. Exposure of Pac1 cultures to urotensin II increased intracellular Ca2+, subsequently activating Ca2+/calmodulin-dependent kinase kinase (CaMKK), and Ca2+/calmodulin-dependent kinase Type I (CaMKI), extracellular signal-regulated kinase (ERK 1/2), and protein kinase D. Treatment of Pac1 and PAVSMC with urotensin II increased proliferation as measured by 3H-thymidine uptake. The urotensin II-induced increase in 3H-thymidine incorporation was inhibited by a CaMKK inhibitor. Taken together, our results demonstrate that urotensin II stimulation of smooth muscle cells leads to a Ca2+/calmodulin-dependent kinase-mediated increase in cellular proliferation

Quinolone signaling in the cell-to-cell communication system of Pseudomonas aeruginosa

Numerous species of bacteria use an elegant regulatory mechanism known as quorum sensing to control the expression of specific genes in a cell-density dependent manner. In Gram-negative bacteria, quorum sensing systems function through a cell-to-cell signal molecule (autoinducer) that consists of a homoserine lactone with a fatty acid side chain. Such is the case in the opportunistic human pathogen Pseudomonas aeruginosa, which contains two quorum sensing systems (las and rhl) that operate via the autoinducers, N-(3-oxododecanoyl)-L-homoserine lactone and N-butyryl-Lhomoserine lactone. The study of these signal molecules has shown that they bind to and activate transcriptional activator proteins that specifically induce numerous P. aeruginosa virulence genes. We report here that P. aeruginosa produces another signal molecule, 2-heptyl-3-hydroxy-4-quinolone, which has been designated as the Pseudomonas quinolone signal. It was found that this unique cell-to-cell signal controlled the expression of lasB, which encodes for the major virulence factor, LasB elastase. We also show that the synthesis and bioactivity of Pseudomonas quinolone signal were mediated by the P. aeruginosa las and rhl quorum sensing systems, respectively. The demonstration that 2-heptyl-3- hydroxy-4-quinolone can function as an intercellular signal sheds light on the role of secondary metabolites and shows that P. aeruginosa cell-to-cell signaling is not restricted to acyl-homoserine lactones. Originally published Proc. Natl. Acad. Sci, Vol. 96, No. 20, Sep. 199

Cytotoxicity and the effect on the inflammation response of thyme oil and thymol: evaluation in human macrophage cells

The essential oil of Thymus vulgaris L. (Lamiaceae), thyme oil, shows a great variability of its composition with six main chemotypes recognized up to now: geraniol, linalool, g-terpineol, carvacrol, thymol, and trans-thujan-4-ol/terpinen-4-ol types. Due to this large chemical diversity, the subject of several investigations was to identify and determine their properties, including their potential effect on inflammation. In our previous microbiological study, this essential oil showed a significant antibacterial activity against bacteria of the respiratory tract [1].                The present research focuses on the evaluation of its cytotoxic and antiinflammatory effect in the case of the U937 human monocyte/macrophage cell line. Thyme oil composition was determined by GC/MS. Bürker chamber was used for cell counting and flow-cytometry to evaluate cellular toxicity (using 7-AAD). Then a qPCR method was used to determine the expression of TNFα mRNA.                The main component of the tested sample of thyme oil was thymol (38.7%) that showed a concentration-dependent cytotoxicity. Non-toxic dilutions showed preventive antiinflammatory potential

Phase I clinical study of the recombinant antibody toxin scFv(FRP5)-ETA specific for the ErbB2/HER2 receptor in patients with advanced solid malignomas

INTRODUCTION: ScFv(FRP5)-ETA is a recombinant antibody toxin with binding specificity for ErbB2 (HER2). It consists of an N-terminal single-chain antibody fragment (scFv), genetically linked to truncated Pseudomonas exotoxin A (ETA). Potent antitumoral activity of scFv(FRP5)-ETA against ErbB2-overexpressing tumor cells was previously demonstrated in vitro and in animal models. Here we report the first systemic application of scFv(FRP5)-ETA in human cancer patients. METHODS: We have performed a phase I dose-finding study, with the objective to assess the maximum tolerated dose and the dose-limiting toxicity of intravenously injected scFv(FRP5)-ETA. Eighteen patients suffering from ErbB2-expressing metastatic breast cancers, prostate cancers, head and neck cancer, non small cell lung cancer, or transitional cell carcinoma were treated. Dose levels of 2, 4, 10, 12.5, and 20 μg/kg scFv(FRP5)-ETA were administered as five daily infusions each for two consecutive weeks. RESULTS: No hematologic, renal, and/or cardiovascular toxicities were noted in any of the patients treated. However, transient elevation of liver enzymes was observed, and considered dose limiting, in one of six patients at the maximum tolerated dose of 12.5 μg/kg, and in two of three patients at 20 μg/kg. Fifteen minutes after injection, peak concentrations of more than 100 ng/ml scFv(FRP5)-ETA were obtained at a dose of 10 μg/kg, indicating that predicted therapeutic levels of the recombinant protein can be applied without inducing toxic side effects. Induction of antibodies against scFv(FRP5)-ETA was observed 8 days after initiation of therapy in 13 patients investigated, but only in five of these patients could neutralizing activity be detected. Two patients showed stable disease and in three patients clinical signs of activity in terms of signs and symptoms were observed (all treated at doses ≥ 10 μg/kg). Disease progression occurred in 11 of the patients. CONCLUSION: Our results demonstrate that systemic therapy with scFv(FRP5)-ETA can be safely administered up to a maximum tolerated dose of 12.5 μg/kg in patients with ErbB2-expressing tumors, justifying further clinical development

Engineered Toxins “Zymoxins” Are Activated by the HCV NS3 Protease by Removal of an Inhibitory Protein Domain

The synthesis of inactive enzyme precursors, also known as “zymogens,” serves as a mechanism for regulating the execution of selected catalytic activities in a desirable time and/or site. Zymogens are usually activated by proteolytic cleavage. Many viruses encode proteases that execute key proteolytic steps of the viral life cycle. Here, we describe a proof of concept for a therapeutic approach to fighting viral infections through eradication of virally infected cells exclusively, thus limiting virus production and spread. Using the hepatitis C virus (HCV) as a model, we designed two HCV NS3 protease-activated “zymogenized” chimeric toxins (which we denote “zymoxins”). In these recombinant constructs, the bacterial and plant toxins diphtheria toxin A (DTA) and Ricin A chain (RTA), respectively, were fused to rationally designed inhibitor peptides/domains via an HCV NS3 protease-cleavable linker. The above toxins were then fused to the binding and translocation domains of Pseudomonas exotoxin A in order to enable translocation into the mammalian cells cytoplasm. We show that these toxins exhibit NS3 cleavage dependent increase in enzymatic activity upon NS3 protease cleavage in vitro. Moreover, a higher level of cytotoxicity was observed when zymoxins were applied to NS3 expressing cells or to HCV infected cells, demonstrating a potential therapeutic window. The increase in toxin activity correlated with NS3 protease activity in the treated cells, thus the therapeutic window was larger in cells expressing recombinant NS3 than in HCV infected cells. This suggests that the “zymoxin” approach may be most appropriate for application to life-threatening acute infections where much higher levels of the activating protease would be expected

Identification of Small Molecule Inhibitors of Pseudomonas aeruginosa Exoenzyme S Using a Yeast Phenotypic Screen

Pseudomonas aeruginosa is an opportunistic human pathogen that is a key factor in the mortality of cystic fibrosis patients, and infection represents an increased threat for human health worldwide. Because resistance of Pseudomonas aeruginosa to antibiotics is increasing, new inhibitors of pharmacologically validated targets of this bacterium are needed. Here we demonstrate that a cell-based yeast phenotypic assay, combined with a large-scale inhibitor screen, identified small molecule inhibitors that can suppress the toxicity caused by heterologous expression of selected Pseudomonas aeruginosa ORFs. We identified the first small molecule inhibitor of Exoenzyme S (ExoS), a toxin involved in Type III secretion. We show that this inhibitor, exosin, modulates ExoS ADP-ribosyltransferase activity in vitro, suggesting the inhibition is direct. Moreover, exosin and two of its analogues display a significant protective effect against Pseudomonas infection in vivo. Furthermore, because the assay was performed in yeast, we were able to demonstrate that several yeast homologues of the known human ExoS targets are likely ADP-ribosylated by the toxin. For example, using an in vitro enzymatic assay, we demonstrate that yeast Ras2p is directly modified by ExoS. Lastly, by surveying a collection of yeast deletion mutants, we identified Bmh1p, a yeast homologue of the human FAS, as an ExoS cofactor, revealing that portions of the bacterial toxin mode of action are conserved from yeast to human. Taken together, our integrated cell-based, chemical-genetic approach demonstrates that such screens can augment traditional drug screening approaches and facilitate the discovery of new compounds against a broad range of human pathogens