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Featured researches published by Arie Dagan.


Journal of Biological Chemistry | 2000

Structure-Activity Relationship Study of Antimicrobial Dermaseptin S4 Showing the Consequences of Peptide Oligomerization on Selective Cytotoxicity

Rina Feder; Arie Dagan; Amram Mor

To understand how peptide organization in aqueous solution might affect the activity of antimicrobial peptides, the potency of various dermaseptin S4 analogs was assessed against human red blood cells (RBC), protozoa, and several Gram-negative bacteria. Dermaseptin S4 had weak antibacterial activity but potent hemolytic or antiprotozoan effects. K4K20-S4 was 2–3-fold more potent against protozoa and RBC, yet K4K20-S4 was more potent by 2 orders of magnitude against bacteria. K4-S4 had similar behavior as K4K20-S4, but K20-S4 and analogous negative charge substitutions were as active as dermaseptin S4 or had reduced activity. Binding experiments suggested that potency enhancement was not the result of increased affinity to target cells. In contrast, potency correlated well with aggregation properties. Fluorescence studies indicated that K20-S4 and all negative charge substitutions were as aggregated as dermaseptin S4, whereas K4-S4 and K4K20-S4 were clearly less aggregated. Overall, the data indicated that N-terminal domain interaction between dermaseptin S4 monomers is responsible for the peptides oligomerization in solution and, hence, for its limited spectrum of action. Moreover, bell-shaped dose-response profiles obtained with bacteria but not with protozoa or RBC implied that aggregation can have dramatic consequences on antibacterial activity. Based on these results, we tested the feasibility of selectivity reversal in the activity of dermaseptin S4. Tampering with the composition of the hydrophobic domains by reducing hydrophobicity or by increasing the net positive charge affected dramatically the peptides activity and resulted in various analogs that displayed potent antibacterial activity but reduced hemolytic activity. Among these, maximal antibacterial activity was displayed by a 15-mer version that was more potent by 2 orders of magnitude compared with native dermaseptin S4. These results emphasize the notion that peptide-based antibiotics represent a highly modular synthetic antimicrobial system and provide indications of how the peptides physico-chemical properties affect potency and selectivity.


The FASEB Journal | 2000

Infusion of recombinant human acid sphingomyelinase into Niemann-Pick disease mice leads to visceral, but not neurological, correction of the pathophysiology

Silvia R.P. Miranda; Xingxuan He; Calogera M. Simonaro; Shimon Gatt; Arie Dagan; Robert J. Desnick; Edward H. Schuchman

An inherited deficiency of acid sphingomyelinase (ASM) activity results in the Type A and B forms of Niemann‐Pick disease (NPD). Using the ASM‐deficient mouse model (ASMKO) of NPD, we evaluated the efficacy of enzyme replacement therapy (ERT) for the treatment of this disorder. Recombinant human ASM (rhASM) was purified from the media of overexpressing Chinese Hamster ovary cells and i.v. injected into 16 five‐month‐old ASMKO mice at doses of 0.3, 1, 3, or 10 mg/kg every other day for 14 days (7 injections). On day 16, the animals were killed and the tissues were analyzed for their sphingomyelin (SPM) content. Notably, the SPM levels were markedly reduced in the hearts, livers, and spleens of these animals, and to a lesser degree in the lungs. Little or no substrate depletion was found in the kidneys or brains. Based on these results, three additional 5‐month‐old ASMKO ani‐mals were injected every other day with 5 mg/kg for 8 days (4 injections) and killed on day 10 for histological analysis. Consistent with the biochemical results, marked histological improvements were observed in the livers, spleens, and lungs, indicating a reversal of the disease pathology. A group of 10 ASMKO mice were then i.v. injected once a week with 1 mg/kg rhASM for 15 wk, starting at 3 wk of age. Although anti‐rhASM antibodies were produced in these mice, the antibodies were not neutralizing and no adverse effects were observed from this treatment. Weight gain and rota‐rod performance were slightly improved in the treated animals as compared with ASMKO control animals, but significant neurological deficits were still observed and their life span was not extended by ERT. In contrast with these CNS results, striking histological and biochemical improvements were found in the reticuloendothelial system organs (livers, spleens, and lungs). These studies indicate that ERT should be an effective therapeutic approach for Type B NPD, but is unlikely to prevent the severe neurodegeneration associated with Type A NPD.—Miranda, S. R. P., He, X., Simonaro, C. M., Gatt, S., Dagan, A., Desnick, R. J., Schuchman, E. H. Infusion of recombinant human acid sphingomyelinase into Niemann‐Pick disease mice leads to visceral, but not neurological, correction of the pathophysiology. FASEB J. 14, 1988–1995 (2000)


Chemistry and Physics of Lipids | 1984

Preparation and characterization of well defined D-erythro sphingomyelins.

Rivka Cohen; Yechezkel Barenholz; Shimon Gatt; Arie Dagan

A simple semisynthetic procedure for the preparation of various D-erythro sphingomyelins (SPMs), differing in their acyl chains, is described. They were prepared by one-step condensation of the desired free fatty acid with sphingosyl phosphorylcholine (SPC) using dicyclohexylcarbodiimide. The D-erythro SPMs were obtained in high purity, high yields and resemble bovine brain SPM in their chromatographic behavior, infrared, circular dichroism (CD) and proton NMR (PMR) spectra as well as in their rate of hydrolysis by Staphylococcus aureus sphingomyelinase. Multilamellar vesicles can be prepared from the semisynthetic SPMs. Their thermotropic behavior is dependent mainly on the acyl chain though it is also affected by the heterogeneity of the sphingosine base composition. Intact sealed small unilamellar vesicles (SUV) cannot be prepared from a single semisynthetic saturated SPM but can be prepared from their mixtures. This acylation procedure can also be applied for preparing simple neutral glycosphingolipids. The sphingolipids prepared by this method can be used to study metabolism, enzymology and physicochemical properties of D-erythro well defined simple sphingolipids.


Biochimica et Biophysica Acta | 1999

Characterization of human acid sphingomyelinase purified from the media of overexpressing Chinese hamster ovary cells.

Xingxuan He; Silvia R.P. Miranda; Xiaoling Xiong; Arie Dagan; Shimon Gatt; Edward H. Schuchman

A rapid purification method was developed to isolate milligram quantities of human acid sphingomyelinase from the media of overexpressing Chinese hamster ovary cells. The purified, recombinant enzyme (rhASM) had physical and kinetic characteristics that were consistent with those reported for the non-recombinant enzyme, including an acidic pH optimum and sensitivity to sulfhydryl reducing reagents and the zinc specific chelator, 1, 10-phenanthroline. A novel assay using fluorescently conjugated sphingomyelin was developed to explore the substrate binding properties of rhASM. Substrate binding required a fatty acid chain length of at least six carbons and the presence of the phosphocholine headgroup on sphingomyelin. Substrate binding also required an acidic pH, and was inhibited by pretreatment of the enzyme with sulfhydral reducing reagents or 1,10-phenanthroline. rhASM was rapidly internalized by cultured skin fibroblasts from Niemann-Pick disease (NPD) patients, and approximately 50% of this uptake was dependent on the mannose 6-phosphate receptor system. Studies using FITC-labeled rhASM revealed that by 1 h the internalized enzyme was localized to acidic compartments and could degrade sphingomyelin, the first demonstration that a lysosomal sphingolipid hydrolase can be fluorescently labeled and retain its biological activity. Intravenous injection of rhASM into ASM knock-out mice showed that the t(1/2) in the plasma was less than 5 min, and that the majority of the injected enzyme was taken up by the liver, followed by the spleen. Thus, these studies lay the foundation for future structure/function investigations of ASM, further investigations into this enzymes role in ceramide mediated signal transduction, and the evaluation of enzyme replacement therapy for NPD using the mouse model.


Antimicrobial Agents and Chemotherapy | 2000

Antimalarial Activities of Dermaseptin S4 Derivatives

Miriam Krugliak; Rina Feder; Vadim Y. Zolotarev; Leonid Gaidukov; Arie Dagan; Hagai Ginsburg; Amram Mor

ABSTRACT The hemolytic antimicrobial peptide dermaseptin S4 was recently shown to exert antimalarial activity. In this study, we attempted to understand the underlying mechanism(s) and identify derivatives with improved antimalarial activity. A number of dermaseptin S4 derivatives inhibited parasite growth with a 50% inhibitory concentration (IC50) in the micromolar range. Among these, the substituted S4 analog K4K20-S4 was the most potent (IC50 = 0.2 μM), while its shorter version, K4-S4(1–13)a, retained a considerable potency (IC50 = 6 μM). Both K4K20-S4 and K4-S4(1–13)a inhibited growth of the parasites more at the trophozoite stage than at the ring stage. Significant growth inhibition was observed after as little as 1 min of exposure to peptides and proceeded with nearly linear kinetics. The peptides selectively lysed infected red blood cells (RBC) while having a weaker effect on noninfected RBC. Thus, K4K20-S4 lysed trophozoites at concentrations similar to those that inhibited their proliferation, but trophozoites were >30-fold more susceptible than normal RBC to the lytic effect of K4K20-S4, the most hemolytic dermaseptin. The same trend was observed with K4-S4(1–13)a. The d isomers of K4K20-S4 or K4-S4(1–13)a were as active as the l counterparts, indicating that antimalarial activity of these peptides, like their membrane-lytic activity, is not mediated by specific interactions with a chiral center. Moreover, dissipation of transmembrane potential experiments with infected cells indicated that the peptides induce damage in the parasites plasma membrane. Fluorescence confocal microscopy analysis of treated infected cells also indicated that the peptide is able to find its way through the complex series of membranes and interact directly with the intracellular parasite. Overall, the data showed that dermaseptins exert antimalarial activity by lysis of infected cells. Dermaseptin derivatives are also able to disrupt the parasite plasma membrane without harming that of the host RBC.


Antimicrobial Agents and Chemotherapy | 2002

In Vitro Antiplasmodium Effects of Dermaseptin S4 Derivatives

Arie Dagan; Leah Efron; Leonid Gaidukov; Amram Mor; Hagai Ginsburg

ABSTRACT The 13-residue dermaseptin S4 derivative K4S4(1-13)a (P) was previously shown to kill intraerythrocytic malaria parasites through the lysis of the host cells. In this study, we have sought peptides that will kill the parasite without lysing the erythrocyte. To produce such peptides, 26 compounds of variable structure and size were attached to the N terminus of P and screened for antiplasmodium and hemolytic activities in cultures of Plasmodium falciparum. Results from this screen indicated that increased hydrophobicity results in amplified antiplasmodium effect, irrespective of the linearity or bulkiness of the additive. However, increased hydrophobicity also was generally associated with increased hemolysis, with the exception of two derivatives: propionyl-P (C3-P) and isobutyryl-P (iC4-P). Both acyl-peptides were more effective than P, with 50% growth inhibition at 3.8, 4.3, and 7.7 μM, respectively. The antiparasitic effect was time dependent and totally irreversible, implying a cytotoxic effect. The peptides were also investigated in parallel for their ability to inhibit parasite growth and to induce hemolysis in infected and uninfected erythrocytes. Whereas the dose dependence of growth inhibition and hemolysis of infected cells overlapped when cells were treated with P, the acyl-peptides exerted 50% growth inhibition at concentrations that did not cause hemolysis. Noticeably, the acyl derivatives, but not P, were able to dissipate the parasite plasma membrane potential and cause depletion of intraparasite potassium under nonhemolytic conditions. These results clearly demonstrate that the acyl-peptides can affect parasite viability in a manner that is dissociated from lysis of the host cell. Overall, the data indicate the potential usefulness of this strategy for development of selective peptides as investigative tools and eventually as antimalarial agents.


Chemical Communications | 1999

Sensing and amplification of oligonucleotide-DNA interactions by means of impedance spectroscopy: a route to a Tay–Sachs sensor

Amos Bardea; Fernando Patolsky; Arie Dagan; Itamar Willner

A three-component oligonucleotide/DNA layered assembly on a Au-electrode acts as a specific biosensor for the analysis of the Tay–Sachs mutant by means of Faradaic impedance spectroscopy.


Antimicrobial Agents and Chemotherapy | 2005

Effects of Acyl versus Aminoacyl Conjugation on the Properties of Antimicrobial Peptides

Inna Radzishevsky; Shahar Rotem; Fadia Zaknoon; Leonid Gaidukov; Arie Dagan; Amram Mor

ABSTRACT To investigate the importance of increased hydrophobicity at the amino end of antimicrobial peptides, a dermaseptin derivative was used as a template for a systematic acylation study. Through a gradual increase of the acyl moiety chain length, hydrophobicity was monitored and further modulated by acyl conversion to aminoacyl. The chain lengths of the acyl derivatives correlated with a gradual increase in the peptides global hydrophobicity and stabilization of its helical structure. The effect on cytolytic properties, however, fluctuated for different cells. Whereas acylation gradually enhanced hemolysis of human red blood cells and antiprotozoan activity against Leishmania major, bacteria displayed a more complex behavior. The gram-positive organism Staphylococcus aureus was most sensitive to intermediate acyl chains, while longer acyls gradually led to a total loss of activity. All acyl derivatives were detrimental to activity against Escherichia coli, namely, but not solely, because of peptide aggregation. Although aminoacyl derivatives behaved essentially similarly to the nonaminated acyls, they displayed reduced hydrophobicity, and consequently, the long-chain acyls enhanced activity against all microorganisms (e.g., by up to 12-fold for the aminolauryl derivative) but were significantly less hemolytic than their acyl counterparts. Acylation also enhanced bactericidal kinetics and peptide resistance to plasma proteases. The similarities and differences upon acylation of MSI-78 and LL37 are presented and discussed. Overall, the data suggest an approach that can be used to enhance the potencies of acylated short antimicrobial peptides by preventing hydrophobic interactions that lead to self-assembly in solution and, thus, to inefficacy against cell wall-containing target cells.


Molecular Cell | 2013

Structural Switch of Lysyl-tRNA Synthetase between Translation and Transcription

Yifat Ofir-Birin; Pengfei Fang; Steven P. Bennett; Hui-Min Zhang; Jing Wang; Inbal Rachmin; Ryan Shapiro; Jing Song; Arie Dagan; Jorge Pozo; Sunghoon Kim; Alan G. Marshall; Paul Schimmel; Xiang-Lei Yang; Hovav Nechushtan; Ehud Razin; Min Guo

Lysyl-tRNA synthetase (LysRS), a component of the translation apparatus, is released from the cytoplasmic multi-tRNA synthetase complex (MSC) to activate the transcription factor MITF in stimulated mast cells through undefined mechanisms. Here we show that Ser207 phosphorylation provokes a new conformer of LysRS that inactivates its translational function but activates its transcriptional function. The crystal structure of an MSC subcomplex established that LysRS is held in the MSC by binding to the N terminus of the scaffold protein p38/AIMP2. Phosphorylation-created steric clashes at the LysRS domain interface disrupt its binding grooves for p38/AIMP2, releasing LysRS and provoking its nuclear translocation. This alteration also exposes the C-terminal domain of LysRS to bind to MITF and triggers LysRS-directed production of the second messenger Ap(4)A that activates MITF. Thus our results establish that a single conformational change triggered by phosphorylation leads to multiple effects driving an exclusive switch of LysRS function from translation to transcription.


Biochimica et Biophysica Acta | 2003

Synthetic, non-natural sphingolipid analogs inhibit the biosynthesis of cellular sphingolipids, elevate ceramide and induce apoptotic cell death

Arie Dagan; Chunbo Wang; Eitan Fibach; Shimon Gatt

Numerous studies have demonstrated the participation of sphingolipids in signal transduction and regulation of cell growth. Several cellular stress agents have been shown to elevate ceramide, the basic precursor of all sphingolipids, initiating a cascade of events leading to arrest of the cell cycle, apoptosis and cell death. Aiming at inhibiting metabolic pathways of sphingolipid metabolism that might lead to an increase of cellular ceramide, we have synthesized non-natural analogs of ceramide, sphingosine and trimethylsphingosine. When the respective analogs were applied to HL60 human myeloid leukemic cells they inhibited the biosynthesis of sphingomyelin (SPM) and glycosphingolipids and induced apoptosis that led to cell death. A fluorescent procedure which has been developed for quantifying the biosynthesis of cellular ceramide indicated an increase in the ceramide content following an incubation with the synthetic analogs. These results suggest that the newly synthesized sphingolipid analogs might be valuable for potential application as a therapeutic modality in leukemia and other malignancies.

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Shimon Gatt

Hebrew University of Jerusalem

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Itamar Willner

Hebrew University of Jerusalem

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Saul Yedgar

Hebrew University of Jerusalem

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Edward H. Schuchman

Icahn School of Medicine at Mount Sinai

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Mordecai Rabinovitz

Hebrew University of Jerusalem

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Ron Blonder

Hebrew University of Jerusalem

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Vered Agmon

Hebrew University of Jerusalem

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Yael Cohen

Hebrew University of Jerusalem

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Silvia R.P. Miranda

Icahn School of Medicine at Mount Sinai

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Azalia Riklin

Hebrew University of Jerusalem

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