Adaptive Mutations in Replicase Transmembrane Subunits Can Counteract Inhibition of Equine Arteritis Virus RNA Synthesis by Cyclophilin Inhibitors.

Previously, the cyclophilin inhibitors cyclosporine (CsA) and alisporivir (ALV) were shown to inhibit the replication of diverse RNA viruses, including arteriviruses and coronaviruses, which both belong to the order Nidovirales In this study, we aimed to identify arterivirus proteins involved in the mode of action of cyclophilin inhibitors and to investigate how these compounds inhibit arterivirus RNA synthesis in the infected cell. Repeated passaging of the arterivirus prototype equine arteritis virus (EAV) in the presence of CsA revealed that reduced drug sensitivity is associated with the emergence of adaptive mutations in nonstructural protein 5 (nsp5), one of the transmembrane subunits of the arterivirus replicase polyprotein. Introduction of singular nsp5 mutations (nsp5 Q21R, Y113H, or A134V) led to an ∼2-fold decrease in sensitivity to CsA treatment, whereas combinations of mutations further increased EAV's CsA resistance. The detailed experimental characterization of engineered EAV mutants harboring CsA resistance mutations implicated nsp5 in arterivirus RNA synthesis. Particularly, in an in vitro assay, EAV RNA synthesis was far less sensitive to CsA treatment when nsp5 contained the adaptive mutations mentioned above. Interestingly, for increased sensitivity to the closely related drug ALV, CsA-resistant nsp5 mutants required the incorporation of an additional adaptive mutation, which resided in nsp2 (H114R), another transmembrane subunit of the arterivirus replicase. Our study provides the first evidence for the involvement of nsp2 and nsp5 in the mechanism underlying the inhibition of arterivirus replication by cyclophilin inhibitors.IMPORTANCE Currently, no approved treatments are available to combat infections with nidoviruses, a group of positive-stranded RNA viruses, including important zoonotic and veterinary pathogens. Previously, the cyclophilin inhibitors cyclosporine (CsA) and alisporivir (ALV) were shown to inhibit the replication of diverse nidoviruses (both arteriviruses and coronaviruses), and they may thus represent a class of pan-nidovirus inhibitors. In this study, using the arterivirus prototype equine arteritis virus, we have established that resistance to CsA and ALV treatment is associated with adaptive mutations in two transmembrane subunits of the viral replication machinery, nonstructural proteins 2 and 5. This is the first evidence for the involvement of specific replicase subunits of arteriviruses in the mechanism underlying the inhibition of their replication by cyclophilin inhibitors. Understanding this mechanism of action is of major importance to guide future drug design, both for nidoviruses and for other RNA viruses inhibited by these compounds.

Development of a Prodrug of Levofloxacin to Avoid Chelation with Al3+ and of Pemetrexed Dimedoxomil Esters for Oral Administration.

An ethoxycarbonyl 1-ethyl hemiacetal ester of levofloxacin (LVFX-EHE) avoids insoluble chelate formation with metal-containing drugs in the intestinal tract and is rapidly hydrolyzed to the parent drug. Furthermore, the minimum inhibitory concentration confirms that LVFX-EHE is less likely to cause pseudomembranous colitis because of less susceptibility to normal intestinal bacteria flora. Pemetrexed dimedoxomil, the prodrug of pemetrexed, was synthesized via reaction with medoxomil bromide after modification of L-glutamate with the tert-butyloxycarbonyl protecting group (BOC), followed by hydrolysis of the BOC moiety with trifluoroacetic acid (TFA) in CH2Cl2 at a temperature of 0°C for 2 h. A serum pemetrexed concentration of >2 µg/mL was observed after oral administration of pemetrexed dimedoxomil at a dose of 60 mg/kg to rats.

Characterization of prophages containing "evolved" Dit/Tal modules in the genome of Lactobacillus casei BL23.

Lactic acid bacteria (LAB) have many applications in food and industrial fermentations. Prophage induction and generation of new virulent phages is a risk for the dairy industry. We identified three complete prophages (PLE1, PLE2, and PLE3) in the genome of the well-studied probiotic strain Lactobacillus casei BL23. All of them have mosaic architectures with homologous sequences to Streptococcus, Lactococcus, Lactobacillus, and Listeria phages or strains. Using a combination of quantitative real-time PCR, genomics, and proteomics, we showed that PLE2 and PLE3 can be induced-but with different kinetics-in the presence of mitomycin C, although PLE1 remains as a prophage. A structural analysis of the distal tail (Dit) and tail associated lysin (Tal) baseplate proteins of these prophages and other L. casei/paracasei phages and prophages provides evidence that carbohydrate-binding modules (CBM) located within these "evolved" proteins may replace receptor binding proteins (RBPs) present in other well-studied LAB phages. The detailed study of prophage induction in this prototype strain in combination with characterization of the proteins involved in host recognition will facilitate the design of new strategies for avoiding phage propagation in the dairy industry.

Effects of Acyclovir, Foscarnet, and Ribonucleotides on Herpes Simplex Virus-1 DNA Polymerase: Mechanistic Insights and a Novel Mechanism for Preventing Stable Incorporation of Ribonucleotides into DNA.

We examined the impact of two clinically approved anti-herpes drugs, acyclovir and Forscarnet (phosphonoformate), on the exonuclease activity of the herpes simplex virus-1 DNA polymerase, UL30. Acyclovir triphosphate and Foscarnet, along with the closely related phosphonoacetic acid, did not affect exonuclease activity on single-stranded DNA. Furthermore, blocking the polymerase active site due to either binding of Foscarnet or phosphonoacetic acid to the E-DNA complex or polymerization of acyclovir onto the DNA also had a minimal effect on exonuclease activity. The inability of the exonuclease to excise acyclovir from the primer 3'-terminus results from the altered sugar structure directly impeding phosphodiester bond hydrolysis as opposed to inhibiting binding, unwinding of the DNA by the exonuclease, or transfer of the DNA from the polymerase to the exonuclease. Removing the 3'-hydroxyl or the 2'-carbon from the nucleotide at the 3'-terminus of the primer strongly inhibited exonuclease activity, although addition of a 2'-hydroxyl did not affect exonuclease activity. The biological consequences of these results are twofold. First, the ability of acyclovir and Foscarnet to block dNTP polymerization without impacting exonuclease activity raises the possibility that their effects on herpes replication may involve both direct inhibition of dNTP polymerization and exonuclease-mediated destruction of herpes DNA. Second, the ability of the exonuclease to rapidly remove a ribonucleotide at the primer 3'-terminus in combination with the polymerase not efficiently adding dNTPs onto this primer provides a novel mechanism by which the herpes replication machinery can prevent incorporation of ribonucleotides into newly synthesized DNA.

Toxicology of 3-epi-deoxynivalenol, a deoxynivalenol-transformation product by Devosia mutans 17-2-E-8.

Microbial detoxification of deoxynivalenol (DON) represents a new approach to treating DON-contaminated grains. A bacterium Devosia mutans 17-2-E-8 was capable of completely transforming DON into a major product 3-epi-DON and a minor product 3-keto-DON. Evaluation of toxicities of these DON-transformation products is an important part of hazard characterization prior to commercialization of the biotransformation application. Cytotoxicities of the products were demonstrated by two assays: a MTT bioassay assessing cell viability and a BrdU assay assessing DNA synthesis. Compared with DON, the IC50 values of 3-epi-DON and 3-keto-DON were respectively 357 and 3.03 times higher in the MTT bioassay, and were respectively 1181 and 4.54 times higher in the BrdU bioassay. Toxicological effects of 14-day oral exposure of the B6C3F1 mouse to DON and 3-epi-DON were also investigated. Overall, there were no differences between the control (free of toxin) and the 25 mg/kg bw/day or 100 mg/kg bw/day 3-epi-DON treatments in body and organ weights, hematology and organ histopathology. However, in mice exposed to DON (2 mg/kg bw/day), white blood cell numbers and serum immunoglobulin levels were altered relative to controls, and lesions were observed in adrenals, thymus, stomach, spleen and colon. Taken together, in vitro and in vivo studies indicate that 3-epi-DON is substantially less toxic than DON.

Nucleotides with altered hydrogen bonding capacities impede human DNA polymerase η by reducing synthesis in the presence of the major cisplatin DNA adduct.

Human DNA polymerase η (hPol η) contributes to anticancer drug resistance by catalyzing the replicative bypass of DNA adducts formed by the widely used chemotherapeutic agent cis-diamminedichloroplatinum (cisplatin). A chemical basis for overcoming bypass-associated resistance requires greater knowledge of how small molecules influence the hPol η-catalyzed bypass of DNA adducts. In this study, we demonstrated how synthetic nucleoside triphosphates act as hPol η substrates and characterized their influence on hPol η-mediated DNA synthesis over unmodified and platinated DNA. The single nucleotide incorporation efficiency of the altered nucleotides varied by more than 10-fold and the higher incorporation rates appeared to be attributable to the presence of an additional hydrogen bond between incoming dNTP and templating base. Finally, full-length DNA synthesis in the presence of increasing concentrations of synthetic nucleotides reduced the amount of DNA product independent of the template, representing the first example of hPol η inhibition in the presence of a platinated DNA template.

A prodrug strategy for the oral delivery of a poorly soluble HCV NS5B thumb pocket 1 polymerase inhibitor using self-emulsifying drug delivery systems (SEDDS).

A prodrug approach was developed to address the low oral bioavailability of a poorly soluble (<0.1µg/mL in pH 6.8 buffer) but highly permeable thumb pocket 1 HCV NS5B polymerase inhibitor. Bioconversion rates of structurally diverse prodrug derivatives were evaluated in a panel of in vitro assays using microsomes, from either liver or intestinal tissues, simulated intestinal fluids, simulated gastric fluids or plasma. In vivo bioconversion of promising candidates was evaluated following oral administration to rats. The most successful strategy involved modification of the parent drug carboxylic acid moiety to glycolic amide esters which improved solubility in lipid-based self-emulsifying drug delivery systems (SEDDS). Crystalline prodrug analog 36 (mp 161°C) showed good solubility in individual SEDDS components (up to 80mg/mL) compared to parent 2 (<3mg/mL; mp 267°C) and cross-species bioconversions which correlated with in vitro stability in liver microsomes.

Genetic requirements for sensitivity of bacteriophage t7 to dideoxythymidine.

We previously reported that the presence of dideoxythymidine (ddT) in the growth medium selectively inhibits the ability of bacteriophage T7 to infect Escherichia coli by inhibiting phage DNA synthese (N. Q. Tran, L. F. Rezende, U. Qimron, C. C. Richardson, and S. Tabor, Proc. Natl. Acad. Sci. U. S. A. 105:9373-9378, 2008, doi:10.1073/pnas.0804164105). In the presence of T7 gene 1.7 protein, ddT is taken up into the E. coli cell and converted to ddTTP. ddTTP is incorporated into DNA as ddTMP by the T7 DNA polymerase, resulting in chain termination. We have identified the pathway by which exogenous ddT is converted to ddTTP. The pathway consists of ddT transport by host nucleoside permeases and phosphorylation to ddTMP by the host thymidine kinase. T7 gene 1.7 protein phosphorylates ddTMP and ddTDP, resulting in ddTTP. A 74-residue peptide of the gene 1.7 protein confers ddT sensitivity to the same extent as the 196-residue wild-type gene 1.7 protein. We also show that cleavage of thymidine to thymine and deoxyribose-1-phosphate by the host thymidine phosphorylase greatly increases the sensitivity of phage T7 to ddT. Finally, a mutation in T7 DNA polymerase that leads to discrimination against the incorporation of ddTMP eliminates ddT sensitivity.

Inhibition of Mn(2+)-induced error-prone DNA synthesis with Cd(2+) and Zn(2+).

Bivalent metal cations are key components in the reaction of DNA synthesis. They are necessary for all DNA polymerases, being involved as cofactors in catalytic mechanisms of nucleotide polymerization. It is also known that in the presence of Mn2+ the accuracy of DNA synthesis is considerably decreased. The findings of this work show that Cd2+ and Zn2+ selectively inhibit the Mn2+-induced error-prone DNA polymerase activity in extracts of cells from human and mouse tissues. Moreover, these cations in low concentrations also can efficiently inhibit the activity of homogeneous preparations of DNA polymerase iota (Pol ι), which is mainly responsible for the Mn2+-induced error-prone DNA polymerase activity in cell extracts. Using a primary culture of granular cells from postnatal rat cerebellum, we show that low concentrations of Cd2+ significantly increase cell survival in the presence of toxic Mn2+ doses. Thus, we have shown that in some cases low concentrations of Cd2+ can display a positive influence on cells, whereas it is widely acknowledged that this metal is not a necessary microelement and is toxic for organisms.

Self resistance to the atypical cationic antimicrobial peptide edeine of Brevibacillus brevis Vm4 by the N-acetyltransferase EdeQ.

Edeines are atypical cationic peptides produced by Brevibacillus brevis Vm4 with broad-spectrum antimicrobial activity. These linear nonribosomal peptides bind to the 30S ribosomal subunit and block t-RNA binding to the P-site. To identify the mechanism of high-level self-resistance in the producing organism, the B. brevis Vm4 genome was sequenced and the edeine biosynthetic cluster discovered. A potential edeine-modifying enzyme, EdeQ, showed similarity to spermidine N-acetyltransferases. EdeQ was purified and shown to convert edeine to N-acetyledeine, which is inactive against cells in vivo and against cell-free extracts. Unexpectedly, tandem mass spectroscopy and nuclear magnetic resonance demonstrate that N-acylation occurs on the free amine of the internal diaminopropionic acid rather than the N-terminal spermidine polyamine. Acetylation of edeine by EdeQ abolishes its ability to inhibit translation, thus conferring resistance to the antibiotic in the producing organism.

Interaction study of ciprofloxacin with human telomeric DNA by spectroscopy and molecular docking.

The interaction of ciprofloxacin (CIP) with human telomeric DNA was studied in vitro using multi-spectroscopy and molecular modeling methods. The hypochromic effect with a red shift in ultraviolet (UV) absorption indicated the occurrence of the interaction between CIP and DNA. The fluorescence quenching of CIP was observed with the addition of DNA and was proved to be the static quenching. The binding constant was found to be 9.62×10(4) L mol(-1). Electrospray ionization mass spectrometry (ESI-MS) result further confirmed the formation of 1:1 non-covalent complex between DNA and CIP. Combined with the UV melting results, circular dichroism (CD) results confirmed the existence of groove binding mode, as well as conformational changes of DNA. Molecular docking studies illustrated the visual display of the CIP binding to the GC region in the minor groove of DNA. Specific hydrogen bonds and van der Waals forces were demonstrated as main acting forces between CIP and guanine bases of DNA.

Role of Smad proteins in resistance to BMP-induced growth inhibition in B-cell lymphoma.

Bone morphogenetic protein (BMP) expression and signaling are altered in a variety of cancers, but the functional impact of these alterations is uncertain. In this study we investigated the impact of expression of multiple BMPs and their signaling pathway components in human B-cell lymphoma. BMP messages, in particular BMP7, were detected in normal and malignant B cells. Addition of exogenous BMPs inhibited DNA synthesis in most lymphoma cell lines examined, but some cell lines were resistant. Tumor specimens from three out of five lymphoma patients were also resistant to BMPs, as determined by no activation of the BMP effectors Smad1/5/8. We have previously shown that BMP-7 potently induced apoptosis in normal B cells, which was in contrast to no or little inhibitory effect of this BMP in the lymphoma cells tested. BMP-resistance mechanisms were investigated by comparing sensitive and resistant cell lines. While BMP receptors are downregulated in many cancers, we documented similar receptor levels in resistant and sensitive lymphoma cells. We found a positive correlation between activation of Smad1/5/8 and inhibition of DNA synthesis. Gene expression analysis of two independent data sets showed that the levels of inhibitory Smads varied across different B-cell lymphoma. Furthermore, stable overexpression of Smad7 in two different BMP-sensitive cell lines with low endogenous levels of SMAD7, rendered them completely resistant to BMPs. This work highlights the role of Smads in determining the sensitivity to BMPs and shows that upregulation of Smad7 in cancer cells is sufficient to escape the negative effects of BMPs.

Phenotypic enhancement of thymidylate synthetase pathway inhibitors following ablation of Neil1 DNA glycosylase/lyase.

Inhibition of thymidine biosynthesis is a clinically-validated therapeutic approach for multiple cancers. Inhibition of thymidylate synthetase (TS) leads to a decrease in cellular TTP levels, replication stress and increased genomic incorporation of uridine (dUMP). Thus, inhibitors of this pathway (such as methotrexate) can drive a multitude of downstream cell cycle checkpoint and DNA repair processes. Genomic dUMP is recognized by the base excision repair (BER) pathway. Using a synthetic lethal siRNA-screening approach, we systematically screened for components of BER that, when ablated, enhanced methotrexate response in a high content γ-H2A.X bioassay. We observed specific ablation of the mixed function DNA glycosylase/lyase Neil1 phenotypically enhanced several inhibitors of thymidine biosynthesis, as well as cellular phenotypes downstream of gemcitabine, cytarabine and clofarabine exposure. These synthetic lethal interactions were associated with significantly enhanced accumulation of γ-H2A.X and improved growth inhibition. Significantly, following TS pathway inhibition, addition of exogenous dTTP complemented the primary Neil1 γ-H2A.X phenotypes. Similarly, co-depletion of Neil1 with Cdc45, Cdc6, Cdc7 or DNA polymerase ß (PolB) suppressed Neil1 phenotypes. Conversely, co-depletion of Neil1 with the Rad17, Rad9 ATR, ATM and DNA-PK checkpoint/sensor proteins appears primarily epistatic to Neil1. These data suggest Neil1 may be a critical mediator of BER of incorporated dUMP following TS pathway inhibition.

Cdc42 regulates microtubule-dependent Golgi positioning.

The molecular mechanisms underlying cytoskeleton-dependent Golgi positioning are poorly understood. In mammalian cells, the Golgi apparatus is localized near the juxtanuclear centrosome via dynein-mediated motility along microtubules. Previous studies implicate Cdc42 in regulating dynein-dependent motility. Here we show that reduced expression of the Cdc42-specific GTPase-activating protein, ARHGAP21, inhibits the ability of dispersed Golgi membranes to reposition at the centrosome following nocodazole treatment and washout. Cdc42 regulation of Golgi positioning appears to involve ARF1 and a binding interaction with the vesicle-coat protein coatomer. We tested whether Cdc42 directly affects motility, as opposed to the formation of a trafficking intermediate, using a Golgi capture and motility assay in permeabilized cells. Disrupting Cdc42 activation or the coatomer/Cdc42 binding interaction stimulated Golgi motility. The coatomer/Cdc42-sensitive motility was blocked by the addition of an inhibitory dynein antibody. Together, our results reveal that dynein and microtubule-dependent Golgi positioning is regulated by ARF1-, coatomer-, and ARHGAP21-dependent Cdc42 signaling.

Human metapneumovirus M2-2 protein inhibits viral transcription and replication.

M2-2 protein of human metapneumovirus (HMPV) is encoded by one of two overlapping open reading frames within M2 mRNA. The precise function of HMPV M2-2 protein remains unknown. We here examined effect of M2-2 protein on HMPV transcription and replication using a minigenome construct and monitoring luciferase reporter gene expression. The minigenome assays demonstrated that M2-2 protein inhibited both transcription and RNA replication. The inhibitory function of M2-2 protein was completely abrogated by removal of eight or four amino acids from its N- or C-terminus, respectively, demonstrating importance of both short terminal sequences for maintaining its functional structure. Immunoprecipitation experiments revealed interaction of M2-2 protein with L protein, which might be involved in inhibition of HMPV transcription and replication. Prior accumulation of intracellular M2-2 protein severely restrained HMPV from replicating. Thus inherent viral control of the M2-2 gene expression in infected cells seems to be essential for efficient HMPV replication.

ZO-1 stabilizes the tight junction solute barrier through coupling to the perijunctional cytoskeleton.

ZO-1 binds numerous transmembrane and cytoplasmic proteins and is required for assembly of both adherens and tight junctions, but its role in defining barrier properties of an established tight junction is unknown. We depleted ZO-1 in MDCK cells using siRNA methods and observed specific defects in the barrier for large solutes, even though flux through the small claudin pores was unaffected. This permeability increase was accompanied by morphological alterations and reorganization of apical actin and myosin. The permeability defect, and to a lesser extent morphological changes, could be rescued by reexpression of either full-length ZO-1 or an N-terminal construct containing the PDZ, SH3, and GUK domains. ZO-2 knockdown did not replicate either the permeability or morphological phenotypes seen in the ZO-1 knockdown, suggesting that ZO-1 and -2 are not functionally redundant for these functions. Wild-type and knockdown MDCK cells had differing physiological and morphological responses to pharmacologic interventions targeting myosin activity. Use of the ROCK inhibitor Y27632 or myosin inhibitor blebbistatin increased TER in wild-type cells, whereas ZO-1 knockdown monolayers were either unaffected or changed in the opposite direction; paracellular flux and myosin localization were also differentially affected. These studies are the first direct evidence that ZO-1 limits solute permeability in established tight junctions, perhaps by forming a stabilizing link between the barrier and perijunctional actomyosin.

Effect of hydroxyurea on the promoter occupancy profiles of tumor suppressor p53 and p73.

BACKGROUND: The p53 tumor suppressor and its related protein, p73, share a homologous DNA binding domain, and mouse genetics studies have suggested that they have overlapping as well as distinct biological functions. Both p53 and p73 are activated by genotoxic stress to regulate an array of cellular responses. Previous studies have suggested that p53 and p73 independently activate the cellular apoptotic program in response to cytotoxic drugs. The goal of this study was to compare the promoter-binding activity of p53 and p73 at steady state and after genotoxic stress induced by hydroxyurea. RESULTS: We employed chromatin immunoprecipitation, the NimbleGen promoter arrays and a model-based algorithm for promoter arrays to identify promoter sequences enriched in anti-p53 or anti-p73 immunoprecipitates, either before or after treatment with hydroxyurea, which increased the expression of both p53 and p73 in the human colon cancer cell line HCT116-3(6). We calculated a model-based algorithm for promoter array score for each promoter and found a significant correlation between the promoter occupancy profiles of p53 and p73. We also found that after hydroxyurea treatment, the p53-bound promoters were still bound by p73, but p73 became associated with additional promoters that that did not bind p53. In particular, we showed that hydroxyurea induces the binding of p73 but not p53 to the promoter of MLH3, which encodes a mismatch repair protein, and causes an up-regulation of the MLH3 mRNA. CONCLUSION: These results suggest that hydroxyurea exerts differential effects on the promoter-binding functions of p53 and p73 and illustrate the power of model-based algorithm for promoter array in the analyses of promoter occupancy profiles of highly homologous transcription factors.

The 3'-untranslated region of ADAMTS1 regulates its mRNA stability.

ADAMTS1 (a disintegrin and metalloproteinase with thrombospondin motifs 1) is an inflammatory-induced gene. We have previously reported that ADAMTS1 was strongly but transiently expressed in the infarcted heart. In this study, we investigated whether a 3'-untranslated region (UTR) affects the mRNA stability of this gene. When stimulated with tissue necrosis factor (TNF)-alpha, the expression level of ADAMTS1 mRNA rapidly increased, but the induction of ADAMTS1 mRNA peaked at 6h after stimulation, after which the expression levels of ADAMTS1 mRNA decreased. The 3'-UTR ADAMTS1 mRNA contains multiple adenine and uridine-rich elements, suggesting that the 3'-UTR may regulate gene stability. The addition of actinomycin D, an RNA synthesis inhibitor, demonstrated the decay of induced ADAMTS1 mRNA by TNF-alpha. Furthermore, a region containing multiple AUUUA motifs within the ADAMTS1 3'-UTR destabilized transfected Enhanced Green Fluorescence Protein (EGFP) mRNA expression. These results demonstrated that the ADAMTS1 3'-UTR may regulate the expression of ADAMTS1 mRNA.

Phosphatidylcholine hydroperoxide-induced THP-1 cell adhesion to intracellular adhesion molecule-1.

The accumulation of phosphatidylcholine hydroperoxide (PCOOH), a primary oxidation product of phosphatidylcholine (PC), in blood plasma and tissues has been observed in various pathological conditions, including atherosclerosis. However, the biological roles of PCOOH in these conditions remain unknown. To estimate the atherogenicity of PCOOH, we evaluated the effect of PCOOH on THP-1 monocytic cell adherence to immobilized vascular endothelial cell adhesion molecules. THP-1 cell adhesion to intracellular adhesion molecule-1 (ICAM-1) was dose-dependently increased by addition of PCOOH. Phosphatidylcholine hydroxide (a hydroxyl analog of PCOOH) also induced THP-1 cell adhesion to ICAM-1, whereas nonoxidized PC, sn-2 truncated PCs, and other hydroperoxide compounds did not affect the adhesion. In the PCOOH-treated cells, obvious protruding F-actin-rich membrane structures were formed, and lymphocyte function-associated antigen-1 (LFA-1) was localized to the protruding structures. Cytochalasin D, an actin polymerization inhibitor, suppressed the PCOOH-induced cell adhesion to ICAM-1 and the membrane protrusions. These results indicate that PCOOH evokes LFA-1-mediated cell adhesion to ICAM-1 via actin cytoskeletal organization, and the mechanism may participate in monocyte adherence to the arterial wall in the initiation of atherosclerosis.