Stable Isotope-Guided Metabolomics Reveals Polar-Functionalized Fatty-Acylated RiPPs from Streptomyces.

Ribosomally synthesized and posttranslationally modified peptides (RiPPs) with polar-functionalized fatty acyl groups are a rarely found untapped class of natural products. Although polar-functionalized fatty-acylated RiPPs (PFARs) have potential as antimicrobial agents, the repertoire is still limited. Therefore, expanding the chemical space is expected to contribute to the development of pharmaceutical agents. In this study, we performed genome mining and stable isotope-guided comparative metabolomics to discover new PFAR natural products. We focused on the feature that PFARs incorporate l-arginine or l-lysine as the starter unit of the fatty acyl group and fed 13C6,15N4-l-arginine or 13C6,15N2-l-lysine to bacterial cultures. Metabolites were extracted and compared with those extracted from nonlabeled l-arginine or l-lysine fed cultures. We identified putative PFARs and successfully isolated solabiomycin A and B from Streptomyces lydicus NBRC 13 058 and albopeptin B from Streptomyces nigrescens HEK616, which contained a sulfoxide group in the labionin moiety. The gene disruption experiment indicated that solS, which encodes a putative flavin adenine dinucleotide (FAD)-nicotinamide adenine dinucleotide (phosphate) (NAD(P))-binding protein, is involved in the sulfoxidation of aryl sulfides. The solabiomycins showed antibacterial activity against Gram-positive bacteria, including Mycobacterium tuberculosis H37Rv with a minimum 95% inhibitory concentration (MIC95) of 3.125 µg/mL, suggesting their potential as antituberculosis agents.

Discharge of suspended solids and radiocesium into stream water in a forested watershed before and after line thinning with spur road construction.

In an experimental watershed located around 120 km southwest of the Fukushima Daiichi Nuclear Power Plant with a drainage area of 59.9 ha, suspended solids (SS) and radioactive cesium discharge from a forested headwater catchment were monitored before and after line thinning. The lower slopes in the experimental watershed were covered with plantation conifer trees (Japanese cedar), while the upper slopes were covered with deciduous trees. In 2012, line thinning was carried out at a thinning rate of 35% across 17% of the northeastern part of the watershed and across the remaining part in 2013. Spur roads were constructed along all tributaries without water, and logged trees were dragged and grappled using forestry machinery and transported along these roads to timber yards using forwarder-type forestry vehicles. A V-notch weir and a water level gauge were installed at the watershed outlet and stream water was sampled twice a month during base flow, whereas during flood flow, stream water samples of 1 L were collected every hour using an automatic water sampler. These samples were filtered through 0.5 µm glass fiber filters to measure the SS concentration. SS concentration data was collected for 21 floods before thinning and for 37 floods after thinning. A time-integrated SS sampler was installed in the stream close to the weir and SS samples were collected every two or three months to measure their Cs-137 concentrations. SS concentrations before (from July 2010 to August 2012) and after thinning (from October 2013 to December 2018) were compared, where the maximum SS concentrations before and after thinning were 211 and 790 mg L-1, respectively. It was discovered that some SS concentrations during flood flow were higher after carrying out thinning than before. Some ΣLss values (specific cumulative load of SS in a flood event) also showed the same results as the SS concentrations. Thus, it was clear that SS discharge immediately increases after thinning, but as it increases Cs-137 export is limited. This is related to a change in SS source brought about by the process of thinning, a decrease with time in the Cs-137 concentration in organic solid expected from that in litter, and a regrowth of vegetation on spur roads, protecting them against soil erosion. Therefore, it was concluded that thinning does not drastically increase Cs-137 export from a forested watershed.

Does maternal autoantibody that transfer to newborn cause disease?

Autoimmune pulmonary alveolar proteinosis (aPAP) is associated with excess amount of granulocyte-macrophage colony-stimulating factor (GM-CSF) autoantibody (GMAb) in the lung and blood. We experienced a female case with severe aPAP who could continue her pregnancy under home oxygen therapy and delivered a newborn by caesarean section. Maternal serum GMAb remained high level for up to one year after the delivery, although aPAP entered remission by whole lung lavage. While the newborn oxygen saturation as well as serum Krebs von den Lungen-6 and surfactant protein-D levels had been normal until one year. As GMAb likely transfer to the newborn and might cause the same disease, we carefully monitored both maternal and the newborn serum GMAb levels after the birth for up to one year. We confirmed that GMAb passively transferred to the newborn circulation but rapidly decreased exponentially to the cut-off level. It is suggested that this rapid decrease might prevent the newborn from developing aPAP.

Isotopic evidence for seasonality of microbial internal nitrogen cycles in a temperate forested catchment with heavy snowfall.

The Hokuriku district of central Japan receives high levels of precipitation during winter, largely in the form of snow. This study aimed to elucidate the internal nitrogen dynamics in this temperate forested region with heavy snowfall using the triple oxygen and nitrogen isotopic compositions of NO3-. The isotopic compositions of NO3- in atmospheric depositions (P and Tf), with terrestrial components of the soil layer (A0, S25, S55, and S90), ground water (G), and output (St) were measured from 2015 to 2016 in a forested catchment located in the southern area of the Ishikawa Prefecture, Japan. Seasonal distributions of Δ17O(NO3-) showed a decreasing trend from the inputs to outputs of the ecosystem. We found relatively constant Δ17O(NO3-) values in the output components (G and St), but found highly fluctuating Δ17O(NO3-) values resulting from the seasonal variations in the nitrification activity within soil waters. Specifically, we observed a lower nitrifying activity in the top soil layer throughout cold periods, presumably due to the input of cold melted snow water. The general trend of increasing δ15N(NO3-) value from the input to output components, with the changes in denitrification hotspots from shallow to deeper soil layer, can be observed between warm and cold periods. Thus, the seasonal changes of hotspots related to microbial nitrification and denitrification could be noted due to the seasonal changes in the isotopic compositions of nitrate. The estimated ecosystem-scale gross nitrification and denitrification rates are low; however, the output components are relatively stable with low concentrations of nitrate, indicating that the plant uptake of nitrogen most probably occurs at greater rates and scales in this forested ecosystem. Future nitrogen deposition and the vulnerable dynamics of snow melting are likely to have impactful consequences on such localities.

Assay system development to measure the concentration of sargramostim with high specificity in patients with autoimmune pulmonary alveolar proteinosis after single-dose inhalation.

During a clinical trial of a Saccharomyces cerviciae-derived recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF), sargramostim, in patients with autoimmune pulmonary alveolar proteinosis (aPAP), we conducted a pharmacokinetic study of single-dose sargramostim inhalation. Several problems were encountered whereby sargramostim formed an immune-complex with GM-CSF autoantibodies (GMAbs) immediately after entering the body; thus, we could not measure the concentration of sargramostim using a commercial high sensitivity enzyme-linked immunosorbent assay (ELISA). Moreover, the ELISA could not discriminate inhaled sargramostim from intrinsic GM-CSF. To solve these problems, we developed a novel ELISA system with a capture antibody that is specific for sargramostim and a detection antibody capable of binding with GM-CSF. This system quantified the serum sargramostim concentration, but not E. coli-, CHO-, or HEK293T-derived human recombinant GM-CSF. Using this system, serum pharmacokinetics were estimated in five patients after inhalation of 250 µg sargramostim, with a mean Cmax of 9.7 ±â€¯2.85 pg/ml at a Tmax of 2 ±â€¯1.22 h.

[Interlaboratory study on migration test of cadmium and lead for food contact articles].

An interlaboratory study was performed to evaluate a migration test method of cadmium (Cd) and lead (Pb), based on the Japanese Food Sanitation Law for glassware, ceramicware, enamelware and metal cans. Seventeen laboratories participated, and quantified Cd and Pb in eight test solutions as blind duplicates using flame atomic absorption spectrometry (AAS), graphite furnace atomic absorption spectrometry (GF-AAS), inductively coupled plasma-optical emission spectrometry (ICP-OES) or induced coupled plasma-mass spectrometry (ICP-MS). Statistical analysis revealed that the trueness, repeatability (RSDr) and reproducibility (RSDr) were 93-105%, 0.7-8.4% and 2.6-19.3% by using AAS, ICP-OES and ICP-MS (internal standard method). The performance of these methods is sufficient for testing specifications. However, some of the RSDr values exceeded 10% in GF-AAS, and careful control of accuracy is required.

Low concentrations of recombinant granulocyte macrophage-colony stimulating factor derived from Chinese hamster ovary cells augments long-term bioactivity with delayed clearance in vitro.

To date, the biological activity of granulocyte macrophage-colony stimulating factor (GM-CSF) has been investigated by using mostly Escherichia coli- or yeast cell-derived recombinant human GM-CSF (erhGM-CSF and yrhGM-CSF, respectively). However, Chinese hamster ovary cell-derived recombinant human GM-CSF (crhGM-CSF), as well as natural human GM-CSF, is a distinct molecule that includes modifications by complicated oligosaccharide moieties. In the present study, we reevaluated the bioactivity of crhGM-CSF by comparing it with those of erhGM-CSF and yrhGM-CSF. The effect of short-term stimulation (0.5h) on the activation of neutrophils/monocytes or peripheral blood mononuclear cells (PBMCs) by crhGM-CSF was lower than those with erhGM-CSF or yrhGM-CSF at low concentrations (under 60pM). Intermediate-term stimulation (24h) among the different rhGM-CSFs with respect to its effect on the activation of TF-1 cells, a GM-CSF-dependent cell line, or PBMCs was not significantly different. In contrast, the proliferation/survival of TF-1 cells or PBMCs after long-term stimulation (72-168h) was higher at low concentrations of crhGM-CSF (15-30pM) than that of cells treated with other GM-CSFs. The proportion of apoptotic TF-1 cells after incubation with crhGM-CSF for 72h was lower than that of cells incubated with other rhGM-CSFs. These effects were attenuated by desialylation of crhGM-CSF. Clearance of crhGM-CSF but not desialylated-crhGM-CSF by both TF-1 cells and PBMCs was delayed compared with that of erhGM-CSF or yrhGM-CSF. These results suggest that sialylation of oligosaccharide moieties delayed the clearance of GM-CSF, thus eliciting increased long-term bioactivity in vitro.

Secondary pulmonary alveolar proteinosis complicating myelodysplastic syndrome results in worsening of prognosis: a retrospective cohort study in Japan.

BACKGROUND: Secondary pulmonary alveolar proteinosis (sPAP) is a very rare lung disorder comprising approximately 10% of cases of acquired PAP. Hematological disorders are the most common underlying conditions of sPAP, of which 74% of cases demonstrate myelodysplastic syndrome (MDS). However, the impact of sPAP on the prognosis of underlying MDS remains unknown. The purpose of this study was to evaluate whether development of sPAP worsens the prognosis of MDS. METHODS: Thirty-one cases of sPAP and underlying MDS were retrospectively classified into mild and severe cases consisting of very low-/low-risk groups and intermediate-/high-/very high-risk groups at the time of diagnosis of MDS, according to the prognostic scoring system based on the World Health Organization classification. Next, we compared the characteristics, disease duration, cumulative survival, and prognostic factors of the groups. RESULTS: In contrast to previous reports on the prognosis of MDS, we found that the cumulative survival probability for mild MDS patients was similar to that in severe MDS patients. This is likely due to the poor prognosis of patients with mild MDS, whose 2-year survival rate was 46.2%. Notably, 75% and 62.5% of patients who died developed fatal infectious diseases and exacerbation of PAP, respectively, suggesting that the progression of PAP per se and/or PAP-associated infection contributed to poor prognosis. The use of corticosteroid therapy and a diffusing capacity of the lung for carbon monoxide of less than 44% were predictive of poor prognosis. CONCLUSION: Development of sPAP during the course of MDS may be an important adverse risk factor in prognosis of patients with mild MDS.

[Performance comparison of material tests for cadmium and lead in food contact plastics].

Based on the Japanese Food Sanitation Law, the performances of official and alternative material test methods for cadmium (Cd) and lead (Pb) in food contact plastics were compared. Nineteen laboratories participated to an interlaboratory study, and quantified Cd and Pb in three PVC pellets. in the official method, a sample is digested with H2SO4, taken up in HCl, and evaporated to dryness on a water bath, then measured by atomic absorption spectrometry (AAS) or inductively coupled plasma-optical emission spectrometry (ICP-OES). Statistical treatment revealed that the trueness, repeatability (RSDr) and reproducibility (RSDr) were 86-95%, 3.1-9.4% and 8.6-22.1%, respectively. The values of the performance parameters fulfilled the requirements , and the performances met the test specifications. The combination of evaporation to dryness on a hot plate and measurement by AAS or ICP-OES is applicable as an alternative method. However, the trueness and RSDr were inferior to those of the official method. The performance parameters obtained by using the microwave digestion method (MW method) to prepare test solution were better than those of the official method. Thus, the MW method is available as an alternative method. Induced coupled plasma-mass spectrometry (ICP-MS) is also available as an alternative method. However, it is necessary to ensure complete digestion of the sample.

Light chain (κ/λ) ratio of GM-CSF autoantibodies is associated with disease severity in autoimmune pulmonary alveolar proteinosis.

Previous studies demonstrated that antigranulocyte colony-stimulating factor autoantibody (GMAb) was consistently present in patients with autoimmune pulmonary alveolar proteinosis (aPAP), and, thus, represented candidature as a reliable diagnostic marker. However, our large cohort study suggested that the concentration of this antibody was not correlated with disease severity in patients. We found that the κ/λ ratio of GMAb was significantly correlated with the degree of hypoxemia. The proportion of λ-type GMAb per total λ-type IgG was significantly higher in severely affected patients than those in mildly affected patients, but the proportion of κ-type was unchanged. The κ/λ ratio was significantly correlated with both KL-6 and SP-D, which have been previously reported as disease severity markers. Thus, the light chain isotype usage of GMAb may not only be associated with the severity of aPAP, but may also represent a useful disease severity marker.

Osteoplastic ameloblastoma: a case report and literature review.

Ameloblastoma with bone formation is rare. We report a case of a 55-year-old woman with ameloblastoma accompanied by prominent osteoplasia. Histopathological examination exhibited an abundant stromal component between tumor nests. Therefore, she was diagnosed as the desmoplastic variant, except for the numerous bone trabeculae. The distinction between new bone formation and invasion of the bone marrow poses a problem. A thin rim of fibrous bone that can be accentuated by Masson-trichrome staining suggests the former.

Microvesicle-mediated RNA molecule delivery system using monocytes/macrophages.

Microvesicles (MVs) and exosomes, which are shed from cells as a cell-to-cell communication tool, are possible vehicles for navigating RNA molecules to body tissues. It is considered that intravenous injection of such MVs or exosomes from patients would not cause severe not-self and toxic reactions. Previously, we found that macrophages take up liposome-entrapped RNA molecules, some of which remain undegraded in the cells. Here, we demonstrate that transfected RNA molecules in human monocytic leukemia THP-1 cells were shed from THP-1 macrophages as contents in MVs during incubation in serum-free medium, which shedding was shown by biochemical analyses such as quantitative reverse transcription (qRT)-PCR, expression of TSG101 (a membrane-associated exosomal protein), and immunoelectron microscopic study. More chemically modified RNA molecules (miR-143BPs) entrapped by MVs (MV-miR-143BPs) were secreted from THP-1 macrophages after miR-143BP transfection compared with the amount after transfection with nonmodified miR-143 transfection. Furthermore, we show that the THP-1 macrophages, which were transfected with the miR-143BP ex vivo, secreted MV-miR-143BPs in xenografted nude mice after intravenous injection, because miR-143 levels were significantly increased in the serum, tumor, and kidney of the host animals. These data suggest that some of the transfected miR-143BPs were secreted from THP-1 macrophages as MV-RNAs both in vitro and in vivo.

Membrane-bound acid pyrophosphatase from Sulfolobus tokodaii, a thermoacidophilic archaeon: heterologous expression of the gene and characterization of the product.

Membranes of Sulfolobus tokodaii, a thermoacidophilic archaeon that grows optimally at pH 2-3, 75-80 degrees C, show the ability to hydrolyze PPi with an optimum pH of 2-3. This acid PPase is proposed to be a dolicholpyrophosphatase that participates in glycoprotein biosynthesis. In the present study, the archaeal membranes hydrolyzed isopentenylpyrophosphate and geranylpyrophosphate, compounds related to dolicholpyrophosphate, at pH 3. However, the dolicholpyrophosphate-binding antibiotic bacitracin failed to inhibit the acid PPase. To investigate further the function and structure of the acid PPase, the gene was cloned and heterologously expressed in Escherichia coli. The membranes from recombinant E. coli showed PPase activity with similar pH and temperature dependence, substrate specificity, and kinetic parameters to those reported for Sulfolobus membranes. The acid PPase was solubilized and purified to electrophoretic homogeneity from the recombinant E. coli. The purified enzyme showed similar K(m) values for PPi, ATP, and ADP to the membrane-bound enzyme. Lipids from the Sulfolobus membranes enhanced the activity to about threefold. Studies involving deletion mutants indicated that basic amino acids in the N-terminal (Arg2 and Lys3), as well as the residues (4th-69th) possibly twice-spanning the membrane, are essential for integration of the enzyme into membranes.

Responsiveness to dobutamine stimulation in patients with left ventricular apical ballooning syndrome.

To investigate the underlying mechanisms of the left ventricular (LV) apical ballooning syndrome, we evaluated the functional responsiveness to dobutamine stimulation in patients with the syndrome. Over a 22-month period, 11 consecutive patients with the apical ballooning syndrome were referred to our institution. All 11 patients were women and 57 to 85 years of age (mean 73 +/- 10). Among them, 10 patients underwent low-dose dobutamine echocardiography within 24 hours after admission (17 +/- 8 hours). Echocardiography was repeated in the convalescent phase (48 +/- 33 days) to assess functional outcome. In the resting state, all patients showed akinetic wall motion in the midportion of the left ventricle and apical left ventricle. After low-dose dobutamine infusion, akinetic wall motion detected at rest did not show any improvement despite the hypercontractile basal LV wall. In the convalescent phase, LV dysfunction was not observed on echocardiography in all 11 patients. The LV apical ballooning syndrome has a unique feature that reversible dysfunction lacks functional amelioration during dobutamine administration. In conclusion, this finding suggests that the pathophysiologic mechanisms of the syndrome appear to be distinct from those of myocardial stunning after transient ischemia, and catecholamine-mediated cardiac toxicity may play a role in the development of the syndrome.

Effect of Smad7 expression on metastasis of mouse mammary carcinoma JygMC(A) cells.

BACKGROUND: Transforming growth factor beta (TGF-beta) facilitates metastasis during the advanced stages of cancer. Smad6, Smad7, and c-Ski block signaling by the TGF-beta superfamily proteins through different modes of action. We used adenovirus-mediated gene transfer of these natural inhibitors in a mouse model of breast cancer to examine the roles of TGF-beta superfamily signaling in tumor growth and metastasis. METHODS: We systemically administered, by intravenous injection, adenoviruses (AdCMV) containing the mouse cDNAs for Smad7, Smad6, c-Ski, the c-Ski mutant c-Ski (ARPG), or LacZ (control) to nude mice (>19 mice/group) bearing tumors derived from mouse mammary carcinoma JygMC(A) cells, which spontaneously metastasize to lung and liver, and examined their effects on survival and metastasis. High-throughput western blotting analysis was used to examine the expression levels for 47 signal transduction proteins in JygMC(A) cells and primary tumors. We also investigated the proliferation, migration, and invasion of JygMC(A) cells that stably overexpressed Smad6 or Smad7. Nonparametric comparisons were done by Kruskal-Wallis H statistic and Wilcoxon's rank sum tests. Parametric comparisons were done by one-way analysis of variance or two-sided unpaired Student's t tests. All statistical tests were two-sided. RESULTS: Control mice bearing tumors derived from JygMC(A) cells showed many metastases to the lung and liver; all animals died by 50 days after cell inoculation. By contrast, mice treated with AdCMV-Smad7 or AdCMV-c-Ski demonstrated a dramatic decrease in metastasis and statistically significantly longer survival than control mice (Smad7 versus LacZ: medium survival = 55 days versus 41 days, difference = 14 days [95% confidence interval {CI} = 6 days to 22 days], P < .001), whereas mice treated with AdCMV-Smad6 or AdCMV-c-Ski (ARPG) did not. Expression of Smad7 in JygMC(A) cells was associated with increased expression of major components of adherens and tight junctions, including E-cadherin, decreased expression of N-cadherin, and decreases in the migratory and invasive abilities of the JygMC(A) cells. CONCLUSION: Smad7 inhibits metastasis, possibly by regulating cell-cell adhesion. Systemic expression of Smad7 may be a novel strategy for the prevention of metastasis of advanced cancers.

Thermus kawarayensis sp. nov., a new member of the genus Thermus, isolated from Japanese hot springs.

A long-rod-shaped thermophilic microorganism, strain KW11, was isolated from a hot springs located in the Kawarayu, Gunma, Japan. Cloning and preliminary sequence analysis of 16S rDNA showed that this isolate belongs to the genus Thermus. The cells were 10-20 microm long, about 0.8 microm in diameter, and produced no pigment in contrast with most of the Thermus species previously reported. KW11 was an aerobic heterotroph and grew at temperatures ranging from 40-73 degrees C, with optimal growth occurring at 68 degrees C. The pH range for growth was from 5.8-8.9, with optimal growth around pH 7. KW11 was sensitive to ampicillin, penicillin G, kanamycin, and streptomycin. The G+C content of DNA was 69 mol%. The main fatty acids were 16:0 (52.9%), iso-15:0 (22.1%), and iso-17:0 (15.6%). The 16S rDNA sequence of KW11 showed 96.0, 95.8, and 95.4% similarity with the sequences of T. aquaticus, T. igniterrae, and T. thermophilus, respectively, and less than 95% with other Thermus species. The physiological differences and phylogenetic evidence indicated that strain KW11 represents T. kawarayensis, a novel species of the genus Thermus. The type strain is isolate KW11T (JCM12314, DSM16200).

Hot-water extracts from adzuki beans (Vigna angularis) suppress not only the proliferation of KATO III cells in culture but also benzo(a)pyrene-induced tumorigenesis in mouse forestomatch.

Treatment of human stomach cancer KATO III cells with hot-water extracts from adzuki beans led to their growth inhibition as well as apoptosis induction. There are morphological changes in the cultured cells treated with the extracts, by which DNA fragmentation characteristic of apoptosis was actualized both concentration- and time-dependently. In contrast, N-acetyl-L-cysteine suppressed such DNA fragmentation, implying that the extracts from adzuki beans might exert antitumorigenicity via active oxygen-induced apoptosis. In order to verify this hypothesis in animal experiment, the 40% ethanol fraction of hot-water extracts was examined for its preventive effect against benzo(a)pyrene-induced tumorigenesis in the forestomach of A/J mice, given as drinking water containing the above fraction at 0.5-2.0% levels. Consequently, forestomach cancer has turned out to be reduced by 36-62% in tumor weight relative to the control. These results suggest that the fraction of hot-water adzuki extracts may serve as a nutrapharmaceutical or functional food available for cancer prevention.

Reclassification of Sulfolobus hakonensis Takayanagi et al. 1996 as Metallosphaera hakonensis comb. nov. based on phylogenetic evidence and DNA G+C content.

The taxonomic status of Sulfolobus hakonensis Takayanagi et al. 1996 was re-evaluated by fresh determinations of the 16S rDNA sequence and G+C content of the genomic DNA of the type strain, HO1-1(T). The 16S rDNA sequence of strain HO1-1(T) showed 98 % similarity to those of two Metallosphaera species and only

Marked prevention of tumor growth and metastasis by a novel immunosuppressive agent, FTY720, in mouse breast cancer models.

FTY720 is a unique immunosuppressive agent that exerts its activity by inducing apoptosis in lymphocytes. We conducted the present study to investigate the effects of FTY720 on cancer growth and metastasis, as well as its mechanism of action. In vitro treatment with FTY720 induced dramatic cancer cell apoptosis in a mouse breast cancer cell line, JygMC(A). Electron microscopy revealed distinct changes on the cell surface with decreased filopodias and microvilli in cancer cells treated with FTY720 at 2 microM and clear evidence of apoptosis at 10 microM. Interestingly, the effect of FTY720 was significantly less in the normal fibroblasts than in the cancer cells, indicating greater susceptibility of cancer cells to the agent. We then tested the in vivo effect of FTY720 in a mouse breast cancer model created by inoculating JygMC(A) cells (s.c.) in the flank region of BALB/c-nu/nu mice at three different dosages (2, 5, and 10 mg/kg/day; n = 30/group). Tumor growth was markedly suppressed at a dosage of 5 mg/kg or more without notable side effects. In addition, tumor metastasis, which was dramatically evident in control mice, was significantly prevented even at a low dose (2 mg/kg/day), resulting in a significant prolongation of animal survival. These data led us to additionally investigate the mechanism of action, especially the prevention of metastasis at a low dose. FTY720 treatment at 2 microM caused a remarkable cytoskeletal change with deformed and decreased filopodias in cancer cells. In addition, it significantly decreased the ability of cancer cells to adhere and migrate to extracellular matrix components, and markedly reduced the expression of integrins on the cancer cell surface. These results indicate that FTY720 is a potent anticancer agent that induces cancer cell apoptosis and is markedly effective for prevention of metastasis. The changes of cellular structure with reduction of integrin expression may be one of its underlying mechanisms of action.