Aqueous extract of bulbus Fritillaria cirrhosa induces cytokinesis failure by blocking furrow ingression in human colon epithelial NCM460 cells.

Bulbus Fritillariacirrhosa D. Don (BFC) has been widely used as an herbal medicament for respiratory diseases in China for over 2000 years. The ethnomedicinal effects of BFC have been scientifically verified, nevertheless its toxicity has not been completely studied. Previously, we have reported that the aqueous extract of BFC induces mitotic aberrations and chromosomal instability (CIN) in human colon epithelial NCM460 cells via dysfunctioning the mitotic checkpoint. Here, we extend this study and specifically focus on the influence of BFC on cytokinesis, the final step of cell division. One remarkable change in NCM460 cells following BFC treatment is the high incidence of binucleated cells (BNCs). More detailed investigation of the ana-telophases reveals that furrow ingression, the first stage of cytokinesis, is inhibited by BFC. Asynchronous cultures treatment demonstrates that furrow ingression defects induced by BFCs are highly associated with the formation of BNCs in ensuing interphase, indicating the BNCs phenotype after BFC treatment was resulted from cytokinesis failure. In line with this, the expression of genes involved in the regulation of furrow ingression is significantly de-regulated by BFC (e.g., LATS-1/2 and Aurora-B are upregulated, and YB-1 is downregulated). Furthermore, long-term treatment of BFC elucidates that the BNCs phenotype is transient and the loss of BNCs is associated with increased frequency of micronuclei and nuclear buds, two biomarkers of CIN. In supporting of these findings, the Nin Jiom Pei Pa Koa and Chuanbei Pipa Gao, two commercially available Chinese traditional medicines containing BFC, are able to induce multinucleation and CIN in NCM460 cells. Altogether, these data provide the first in vitro experimental evidence linking BFC to cytokinesis failure and suggest the resultant BNCs may be intermediates to produce CIN progenies.

Micronucleus Cytome Assays in Human Lymphocytes and Buccal Cells.

The micronucleus (MN) assay, applied in different surrogate tissues, is one of the best validated cytogenetic techniques for evaluating chromosomal damage in humans. The cytokinesis-block micronucleus cytome assay in peripheral blood lymphocytes (L-CBMNcyt) is the most frequently used method in biomonitoring human populations to evaluate DNA damage caused by exposure to genotoxic agents, micronutrient deficiency or excess and genetic instability. Furthermore, recent scientific evidence suggests an association between an increased MN frequency in lymphocytes and risk of cancer and other age-related degenerative diseases. The micronucleus cytome assay applied in buccal exfoliated cells (BMNCyt), provides a complementary method for measuring DNA damage and cytotoxic effects in an easily accessible tissue not requiring ex vivo/in vitro culture. The protocol for L-CBMNcyt described here, refers to the use of ex vivo whole blood method, involving 72 h of culture with the block of cytokinesis starting at 44 h. BMNCyt protocol reports the established method for sample collection, processing, slide preparation and scoring.

Automation of the in vitro micronucleus assay using the Imagestream® imaging flow cytometer.

The in vitro micronucleus (MN) assay is a well-established test for evaluating genotoxicity and cytotoxicity. The use of manual microscopy to perform the assay can be laborious and often suffers from user subjectivity and interscorer variability. Automated methods including slide-scanning microscopy and conventional flow cytometry have been developed to eliminate scorer bias and improve throughput. However, these methods possess several limitations such as lack of cytoplasmic visualization using slide-scanning microscopy and the inability to visually confirm the legitimacy of MN or storage of image data for re-evaluation using flow cytometry. The ImageStreamX® MK II (ISX) imaging flow cytometer has been demonstrated to overcome all of these limitations. The ISX combines the speed, statistical robustness, and rare event capture capability of conventional flow cytometry with high resolution fluorescent imagery of microscopy and possesses the ability to store all collected image data. This paper details the methodology developed to perform the in vitro MN assay in human lymphoblastoid TK6 cells on the ISX. High resolution images of micronucleated mono- and bi-nucleated cells as well as polynucleated cells can be acquired at a high rate of capture. All images can then be automatically identified, categorized and enumerated in the data analysis software that accompanies the ImageStream, allowing for the scoring of both genotoxicity and cytotoxicity. The results demonstrate that statistically significant increases in MN frequency when compared with solvent controls can be detected at varying levels of cytotoxicity following exposure to well-known aneugens and clastogens. This work demonstrates a fully automated method for performing the in vitro micronucleus assay on the ISX imaging flow cytometry platform. © 2018 The Author. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of ISAC.

Plagiochiline A Inhibits Cytokinetic Abscission and Induces Cell Death.

We previously reported on the isolation and biological activities of plagiochiline A (1), a 2,3-secoaromadendrane-type sesquiterpenoid from the Peruvian medicinal plant, Plagiochila disticha. This compound was found to have antiproliferative effects on a variety of solid tumor cell lines, as well as several leukemia cell lines. Other researchers have also noted the cytotoxicity of plagiochiline A (isolated from different plant species), but there are no prior reports regarding the mechanism for this bioactivity. Here, we have evaluated the effects of plagiochiline A on cell cycle progression in DU145 prostate cancer cells. A cell cycle analysis indicated that plagiochiline A caused a significant increase in the percentage of cells in the G2/M phase when compared with control cells. When cells were stained and observed by fluorescence microscopy to examine progress through the mitotic phase, we found a significant increase in the proportion of cells with features of late cytokinesis (cells connected by intercellular bridges) in the plagiochiline A-treated samples. These results suggest that plagiochiline A inhibits cell division by preventing completion of cytokinesis, particularly at the final abscission stage. We also determined that plagiochiline A reduces DU145 cell survival in clonogenic assays and that it induces substantial cell death in these cells.

Novel Cell-Killing Mechanisms of Hydroxyurea and the Implication toward Combination Therapy for the Treatment of Fungal Infections.

We have previously reported that an erg11 mutation affecting ergosterol synthesis and a hem13 mutation in the heme synthesis pathway significantly sensitize the fission yeast Schizosaccharomyces pombe to hydroxyurea (HU) (1, 2). Here we show that treatment with inhibitors of Erg11 and heme biosynthesis phenocopies the two mutations in sensitizing wild-type cells to HU. Importantly, HU synergistically interacts with the heme biosynthesis inhibitor sampangine and several Erg11 inhibitors, the antifungal azoles, in causing cell lethality. Since the synergistic drug interactions are also observed in the phylogenetically divergent Saccharomyces cerevisiae and the opportunistic fungal pathogen Candida albicans, the synergism is likely conserved in eukaryotes. Interestingly, our genetic data for S. pombe has also led to the discovery of a robust synergism between sampangine and the azoles in C. albicans Thus, combinations of HU, sampangine, and the azoles can be further studied as a new method for the treatment of fungal infections.

Gibberellin Induces Diploid Pollen Formation by Interfering with Meiotic Cytokinesis.

The plant hormone gibberellic acid (GA) controls many physiological processes, including cell differentiation, cell elongation, seed germination, and response to abiotic stress. In this study, we report that exogenous treatment of flowering Arabidopsis (Arabidopsis thaliana) plants with GA specifically affects the process of male meiotic cytokinesis leading to meiotic restitution and the production of diploid (2n) pollen grains. Similar defects in meiotic cell division and reproductive ploidy stability occur in Arabidopsis plants depleted of RGA and GAI, two members of the DELLA family that function as suppressor of GA signaling. Cytological analysis of the double rga-24 gai-t6 mutant revealed that defects in male meiotic cytokinesis are not caused by alterations in meiosis I (MI or meiosis II (MII) chromosome dynamics, but instead result from aberrations in the spatial organization of the phragmoplast-like radial microtubule arrays (RMAs) at the end of meiosis II. In line with a role for GA in the genetic regulation of the male reproductive system, we additionally show that DELLA downstream targets MYB33 and MYB65 are redundantly required for functional RMA biosynthesis and male meiotic cytokinesis. By analyzing the expression of pRGA::GFP-RGA in the wild-type Landsberg erecta background, we demonstrate that the GFP-RGA protein is specifically expressed in the anther cell layers surrounding the meiocytes and microspores, suggesting that appropriate GA signaling in the somatic anther tissue is critical for male meiotic cell wall formation and thus plays an important role in consolidating the male gametophytic ploidy consistency.

Application of the lymphocyte Cytokinesis-Block Micronucleus Assay to populations exposed to petroleum and its derivatives: Results from a systematic review and meta-analysis.

The lymphocyte cytokinesis-block micronucleus (CBMN) assay is applied in many different in vivo biomonitoring studies of human exposure to genotoxic chemicals. Among extensively chemicals investigated, we identified petroleum and its derivatives, in particular benzene and the most common mixture of benzene, toluene, and xylene. Although conflicting results have been reported on the effects of benzene exposure, the number of positive findings in independent studies suggests that occupational exposure to benzene causes DNA damage in peripheral blood lymphocytes. To assess current evidence on this hypothesis, we conducted a meta-analysis. Our aim was to evaluate the effect of benzene exposure on genetic damage, quantified using the CBMN assay on individuals occupationally exposed to petroleum and its derivatives. Statistical analyses were conducted using the rmeta package from the free Software Environment for Statistical Computing R. Combined study results indicated that benzene exposure is associated with an increased level of genetic damage in peripheral blood lymphocytes, as reflected by an increased MN frequency. The summary mean difference in MN frequency between exposed and unexposed individuals was 1.64 (95% CI: 0.80-2.47). Overall, this finding points to MN frequency as a sensitive biomarker which could be used to evaluate genetic damage induced by occupational - industrial or environmental - exposure to benzene. This review also identified some important knowledge gaps as well as the need of large, well-designed studies. In particular, it is fundamental to accurately characterize the investigated population, including dietary habits and genetic variability which could modulate MN frequency in both exposed individuals and unexposed controls. In conclusion, according to present findings the use of the CBMN assay in biomonitoring studies could provide objective evidence to guide prioritization of preventive interventions in subjects occupationally exposed to petroleum derivatives, and in particular benzene.

Cytotoxic and genotoxic activity of some Helleborus species.

Despite their known toxic properties, various Helleborus species are used as medicaments in folk medicine to treat some diseases and health conditions. As the main mechanism of many cytostatic drugs is based on their cytotoxic activity, there is potential for the toxicity of hellebore to be used in anticancer therapy. This study tested the geno- and cytotoxic effects of extracts of three hellebore taxa (Helleborus odorus, Helleborus multifidus and Helleborus hercegovinus) on meristemic onion (Alliumcepa L.) cells and human lymphocytes. Treatments with Helleborus extracts induced cytotoxic and cytostatic effects in meristemic onion cells as well as in cultivated cytokinesis-blocked human lymphocytes. Cytokinesis-block micronucleus cytome assay indicated that treatments with hellebore extracts induce genotoxic effects in human lymphocytes, and that the significant mechanism of their antiproliferative activity is apoptosis induction.

Rho-GTPase effector ROCK phosphorylates cofilin in actin-meditated cytokinesis during mouse oocyte meiosis.

During oocyte meiosis, a spindle forms in the central cytoplasm and migrates to the cortex. Subsequently, the oocyte extrudes a small body and forms a highly polarized egg; this process is regulated primarily by actin. ROCK is a Rho-GTPase effector that is involved in various cellular functions, such as stress fiber formation, cell migration, tumor cell invasion, and cell motility. In this study, we investigated possible roles for ROCK in mouse oocyte meiosis. ROCK was localized around spindles after germinal vesicle breakdown and was colocalized with cytoplasmic actin and mitochondria. Disrupting ROCK activity by RNAi or an inhibitor resulted in cell cycle progression and polar body extrusion failure. Time-lapse microscopy showed that this may have been due to spindle migration and cytokinesis defects, as chromosomes segregated but failed to extrude a polar body and then realigned. Actin expression at oocyte membranes and in cytoplasm was significantly decreased after these treatments. Actin caps were also disrupted, which was confirmed by a failure to form cortical granule-free domains. The mitochondrial distribution was also disrupted, which indicated that mitochondria were involved in the ROCK-mediated actin assembly. In addition, the phosphorylation levels of Cofilin, a downstream molecule of ROCK, decreased after disrupting ROCK activity. Thus, our results indicated that a ROCK-Cofilin-actin pathway regulated meiotic spindle migration and cytokinesis during mouse oocyte maturation.

Bisabolangelone inhibits dendritic cell functions by blocking MAPK and NF-κB signaling.

Bisabolangelone (BISA), isolated from the roots of Angelica koreana, has many pharmacological activities, such as anti-tumor, anti-microbial, antioxidant, and anti-inflammatory activities. In this study, we investigated the anti-inflammatory mechanisms of BISA in dendritic cells (DCs), which play an essential role in innate and adaptive immune responses. BISA attenuated the production of pro-inflammatory cytokines including interleukin (IL)-12, IL-1ß, and tumor necrosis factor-alpha (TNF-α), migration to macrophage inflammatory protein-3 beta, and allo-T cell activating ability of DCs. In addition, BISA affected endocytosis of DCs. Molecular studies showed that BISA suppressed MAPK phosphorylation and nuclear translocation of NF-κB p50/p65. Taken together, our data suggest that BISA inhibited DC functions by blocking MAPK and NF-κB signaling.

Black tea extract: a supplementary antioxidant in radiation-induced damage to DNA and normal lymphocytes.

Myriad research has contributed significantly toward the understanding and identification of health benefits stemming from tea polyphenols and many other naturally occurring flavonoids present in fruits and vegetables. These flavonoids are known to mitigate reactive oxygen species-induced damage by scavenging them. In this study, hot-water black tea extract rich in flavonoids is evaluated as a supplementary antioxidant. The antioxidant efficacy of black tea extract was investigated by evaluating radioprotection conferred to pBR322 DNA, calf thymus DNA, and normal lymphocytes during gamma irradiation. The protection was measured by gel electrophoresis, fluorimetric study, cell viability assay, cytokinesis-blocked micronuclei assay, and comet assay. The 2,2-diphenyl-1-picrylhydrazyl scavenging ability of the tea extract used increased in a dose-dependent manner (IC50: 182.45 µg/mL). Positive correlation of radioprotection with antioxidant activity of black tea extract was observed in all systems. Maximum protection against radiation-induced damage was observed in pBR322 DNA and calf thymus DNA at ≥200 µg/mL of black tea extract. At a dose of black tea extract as low as 5 µg/mL, efficient radioprotection was observed in normal lymphocytes, which is encouraging and can be tested in the future as a natural antioxidant supplement during radiotherapy.

Chemical genoprotection: reducing biological damage to as low as reasonably achievable levels.

OBJECTIVES: The aim of this study was to evaluate the antioxidant substances present in the human diet with an antimutagenic protective capacity against genotoxic damage induced by exposure to X-rays in an attempt to reduce biological damage to as low a level as reasonably possible. METHODS: Ten compounds were assessed using the lymphocyte cytokinesis-block micronucleus (MN) cytome test. The compounds studied were added to human blood at 25 µM 5 min before exposure to irradiation by 2 Gy of X-rays. RESULTS: The protective capacity of the antioxidant substances assessed was from highest to lowest according to the frequency of the MN generated by X-ray exposure: rosmarinic acid = carnosic acid = δ-tocopherol = l-acid ascorbic = apigenin = amifostine (P < 0.001) > green tea extract = diosmine = rutin = dimetylsulfoxide (P < 0.05) > irradiated control. The reduction in genotoxic damage with the radiation doses administered reached 58%, which represents a significant reduction in X-ray-induced chromosomal damage (P < 0.001). This degree of protection is greater than that obtained with amifostine, a radioprotective compound used in radiotherapy and which is characterised by its high toxicity. CONCLUSION: Several antioxidant substances, common components of the human diet and lacking toxicity, offer protection from the biological harm induced by ionizing radiation. Administering these protective substances to patients before radiological exploration should be considered, even in the case of small radiation doses and regardless of the biological damage expected.

Mangiferin attenuates methylmercury induced cytotoxicity against IMR-32, human neuroblastoma cells by the inhibition of oxidative stress and free radical scavenging potential.

Mangiferin (MGN), a C-glucosylxanthone was investigated for its ability to protect against methylmercury (MeHg) induced neurotoxicity by employing IMR-32 (human neuroblastoma) cell line. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and clonogenic cell survival assays confirmed the efficacy of MGN supplementation in attenuating MeHg-induced cytotoxicity. Pre-treatment with MGN significantly (p<0.01) inhibited MeHg-induced DNA damage (micronuclei, olive tail moment and % tail DNA) thereby demonstrating MGN's antigenotoxic potential. Also, pre-treatment with MGN significantly reduced MeHg-induced oxidative stress, intra-cellular Ca(2+) influx and inhibited depolarization of mitochondrial membrane. MGN pre-treated cells demonstrated a significant (p<0.05) increase in the GSH and GST levels followed by a significant (p<0.05) decrease in malondialdehyde (MDA) formation. In addition, inhibition of MeHg induced apoptotic cell death by MGN was demonstrated by microscopic, Annexin-V FITC and DNA fragmentation assays and further confirmed by western blot analysis. The present findings indicated the protective effect of MGN against MeHg induced toxicity, which may be attributed to its anti-genotoxic, anti-apoptotic and anti-lipid peroxidative potential plausibly because of its free radical scavenging ability, which reduced the oxidative stress and in turn facilitated the down-regulation of mitochondrial apoptotic signalling pathways.

Dynamin inhibitors induce caspase-mediated apoptosis following cytokinesis failure in human cancer cells and this is blocked by Bcl-2 overexpression.

BACKGROUND: The aim of both classical (e.g. taxol) and targeted anti-mitotic agents (e.g. Aurora kinase inhibitors) is to disrupt the mitotic spindle. Such compounds are currently used in the clinic and/or are being tested in clinical trials for cancer treatment. We recently reported a new class of targeted anti-mitotic compounds that do not disrupt the mitotic spindle, but exclusively block completion of cytokinesis. This new class includes MiTMAB and OcTMAB (MiTMABs), which are potent inhibitors of the endocytic protein, dynamin. Like other anti-mitotics, MiTMABs are highly cytotoxic and possess anti-proliferative properties, which appear to be selective for cancer cells. The cellular response following cytokinesis failure and the mechanistic pathway involved is unknown. RESULTS: We show that MiTMABs induce cell death specifically following cytokinesis failure via the intrinsic apoptotic pathway. This involves cleavage of caspase-8, -9, -3 and PARP, DNA fragmentation and membrane blebbing. Apoptosis was blocked by the pan-caspase inhibitor, ZVAD, and in HeLa cells stably expressing the anti-apoptotic protein, Bcl-2. This resulted in an accumulation of polyploid cells. Caspases were not cleaved in MiTMAB-treated cells that did not enter mitosis. This is consistent with the model that apoptosis induced by MiTMABs occurs exclusively following cytokinesis failure. Cytokinesis failure induced by cytochalasin B also resulted in apoptosis, suggesting that disruption of this process is generally toxic to cells. CONCLUSION: Collectively, these data indicate that MiTMAB-induced apoptosis is dependent on both polyploidization and specific intracellular signalling components. This suggests that dynamin and potentially other cytokinesis factors are novel targets for development of cancer therapeutics.

Characterization of AKT independent effects of the synthetic AKT inhibitors SH-5 and SH-6 using an integrated approach combining transcriptomic profiling and signaling pathway perturbations.

BACKGROUND: Signal transduction processes mediated by phosphatidyl inositol phosphates affect a broad range of cellular processes such as cell cycle progression, migration and cell survival. The protein kinase AKT is one of the major effectors in this signaling network. Chronic AKT activation contributes to oncogenic transformation and tumor development. Therefore, analogs of phosphatidyl inositol phosphates (PIAs) were designed as new small drugs to block AKT activity for cancer treatment. Here we characterize the biological effects of the PIAs SH-5 and SH-6 in colorectal cancer cell lines. METHODS: Serum-starved or serum-supplemented human colorectal cancer cell lines SW480, HT29 and HCT116 were exposed to SH-5 and SH-6. AKT activation was determined by western blotting. Cell viability was assessed using a colorimetric XTT-based assay, apoptosis and cell cycle changes were monitored by FACS analysis. The dynamics of cell morphology alterations was evaluated by confocal and time-lapse microscopy. Transcriptional changes due to inhibitor treatment were analyzed using Affymetrix HG-U133A microarrays and RT-PCR. RESULTS: While the PIAs clearly reduce AKT phosphorylation in serum starved cells, we did not observe a significant reduction under serum supplemented conditions, giving us the opportunity to analyze AKT independent effects of these compounds. Both inhibitors induce broadly the same morphological alterations, in particular changes in cell shape and formation of intracellular vesicles. Moreover, we observed the induction of binucleated cells specifically in the SW480 cell line. Gene expression analysis revealed transcriptional alterations, which are mostly cell line specific. In accordance to the phenotype we found a gene group associated with mitosis and spindle organization down regulated in SW480 cells, but not in the other cell lines. A bioinformatics analysis using the Connectivity Map linked the gene expression pattern of the inhibitor treated SW480 cells to PKC signaling. Using confocal laser scanning microscopy and time lapse recording we identified a specific defect in the last step of the cytokinesis as responsible for the binucleation. CONCLUSIONS: The PIAs SH-5 and SH-6 impinge on additional cellular targets apart from AKT in colorectal cancer cells. The effects are mostly cell line specific and have an influence at the outcome of the treatment. In view of potential clinical trials it will be necessary to take these diverse effects into consideration to optimize patient treatment.

Protective effect on human lymphocytes of some flavonoids isolated from two Achillea species.

This study was conducted to elucidate the in vitro protective effect of five flavonoids [apigenin (1), apigenin-7-O-glucoside (2), centaureidin (3), jaceidin (4) and quercetin (5)] against chromosomal damage in mitogen-induced human lymphocytes. Using the Cytochalasin-B blocked micronucleus (CBMN) assay, in which the biomarker of chromosome breakage and/or chromosome loss is the elevated frequency of micronucleus (MN) in binucleated (BN) cells, the presence of flavonoid 2 in minimal concentration (3 microg/mL) gave a 35.5% decrease in the frequency of MN when compared with control human lymphocytes. The same concentration of flavonoids 1, 3 and 4, reduced the MN frequency by 24.4%, 28.0% and 28.0%, respectively. Higher concentrations (6 microg/mL and 10 microg/mL) seemed less effective. Flavonoid 5 (3 microg/mL) induced a slight decrease in MN frequency (5%), while higher doses (6 microg/mL and 10 microg/mL) provoked an increase of DNA damage. The comparable values for the cytokinesis-block proliferation index (CBPI) of the tested flavonoids and positive control suggested an inhibitory effect on lymphocyte proliferation. In the DPPH scavenging assay, flavonoids 1-4 demonstrated modest activity, in a dose-dependent manner, compared with the synthetic antioxidants BHT and Trolox, while 5 exhibited comparably high antioxidative activity.

Areca nut-induced micronuclei and cytokinesis failure in Chinese hamster ovary cells is related to reactive oxygen species production and actin filament deregulation.

Epidemiological studies have shown a strong association between environmental exposure to betel quid (BQ) and oral cancer. Areca nut (AN), an ingredient of BQ, contains genotoxic and mutagenic compounds. In this study, we found that AN extract (ANE) inhibited the growth of Chinese hamster ovary cells (CHO-K1) in a dose- and time-dependent manner. Intracellular reactive oxygen species (ROS) levels and micronuclei (MN) frequency were significantly increased following ANE treatment in CHO-K1 cells. Addition of catalase markedly inhibited ANE-induced MN formation, indicating that ANE-induced genotoxicity was correlated with intracellular H(2)O(2). Incubation of CHO-K1 cells with ANE (400-800 microg/ml) for 24 hr caused G2/M arrest, and prolonged exposure to ANE (800 microg/ml) significantly induced cell death. Surprisingly, ANE itself caused cytokinesis failure and subsequent increase in binucleated cell formation. Coexposure to catalase (2,000 U/ml) and ANE (800 microg/ml) reduced the generation of binucleated cells, indicating that ANE-induced cytokinesis failure was associated with oxidative stress. Following prolonged exposure to ANE, an accumulation of hyperploid/aneuploid cells concomitant with bi-, micro- or multinucleated cells was found. In summary, our results demonstrate that ANE exposure to CHO-K1 cells caused increased MN frequency, G2/M arrest, cytokinesis failure, and an accumulation of hyperploid/aneuploid cells. These events are associated with an increase in intracellular H(2)O(2) level and actin filament disorganization.

Effects of essential oils from Cymbopogon citratus (DC) Stapf., Lippia sidoides Cham., and Ocimum gratissimum L. on growth and ultrastructure of Leishmania chagasi promastigotes.

The current therapy for leishmaniasis, which affects annually about 2 million people, is far from satisfactory. All available drugs require parenteral administration and are potentially toxic. Plant essential oils have been traditionally used in folk medicine and appear as valuable alternative source for chemotherapeutic compounds. In this study, we demonstrated the effect of essential oils from Cymbopogon citratus, Lippia sidoides, and Ocimum gratissimum on growth and ultrastructure of Leishmania chagasi promastigote forms. Steam distillation was used to isolate the essential oils, and their constituents were characterized by gas chromatography coupled to mass spectrometry and nuclear magnetic resonance. All essential oils showed in vitro inhibitory action on L. chagasi promastigotes growth in a dose-dependent way, with IC(50)/72 h of 45, 89, and 75 microg/mL for C. citratus, L. sidoides, and O. gratissimum, respectively. Drastic morphological alterations were observed in all essential oil-treated parasites, including cell swelling, accumulation of lipid droplets in the cytoplasm, and increase of acidocalcisome volume. Furthermore, aberrant-shaped cells with multi-septate body were observed by scanning electron microscopy, suggesting an additional effect on cytokinesis. Taken together, our data show that these essential oils affect the parasite viability being the C. citratus essential oil the most effective against L. chagasi.

Functional characterization of the fission yeast phosphatidylserine synthase gene, pps1, reveals novel cellular functions for phosphatidylserine.

To investigate the contributions of phosphatidylserine to the growth and morphogenesis of the rod-shaped fission yeast Schizosaccharomyces pombe, we have characterized the single gene in this organism, pps1, encoding a predicted phosphatidylserine synthase. S. pombe pps1Delta mutants grow slowly in rich medium and are inviable in synthetic minimal medium. They do not produce detectable phosphatidylserine in vivo and possess negligible in vitro phosphatidylserine synthase activity, indicating that pps1 encodes the major phosphatidylserine synthase activity in S. pombe. Supplementation of growth medium with ethanolamine partially suppresses the growth-defective phenotype of pps1Delta cells, reflecting the likely importance of phosphatidylserine as a precursor for phosphatidylethanolamine in S. pombe. In medium lacking ethanolamine, pps1Delta mutants exhibit striking cell morphology, cytokinesis, actin cytoskeleton, and cell wall remodeling and integrity defects. Overexpression of pps1 likewise leads to defects in cell morphology and cytokinesis, thus implicating phosphatidylserine as a dosage-dependent regulator of these processes. During log-phase growth, green fluorescent protein-Pps1p fusion proteins are concentrated at the cell and nuclear peripheries as well as presumptive endoplasmic reticulum membranes, while in stationary-phase cells, they are redistributed to unusual cytoplasmic structures of unknown origin. Moreover, stationary-phase pps1Delta cultures retain very poor viability relative to wild-type S. pombe cells, even in medium containing ethanolamine, demonstrating a role for phosphatidylserine in the physiological adaptations required for stationary-phase survival. Our findings reveal novel cellular functions for phosphatidylserine and emphasize the usefulness of S. pombe as a model organism for elucidating potentially conserved biological and molecular functions of this phospholipid.

Cerulenin-mediated apoptosis is involved in adenine metabolic pathway.

Cerulenin, a fatty acid synthase (FAS) inhibitor, induces apoptosis of variety of tumor cells. To elucidate mode of action by cerulenin, we employed the proteomics approach using Schizosaccharomyces pombe. The differential protein expression profile of S. pombe revealed that cerulenin modulated the expressions of proteins involved in stresses and metabolism, including both ade10 and adk1 proteins. The nutrient supplementation assay demonstrated that cerulenin affected enzymatic steps transferring a phosphoribosyl group. This result suggests that cerulenin accumulates AMP and p-ribosyl-s-amino-imidazole carboxamide (AICAR) and reduces other necessary nucleotides, which induces feedback inhibition of enzymes and the transcriptional regulation of related genes in de novo and salvage adenine metabolic pathway. Furthermore, the deregulation of adenine nucleotide synthesis may interfere ribonucleotide reductase and cause defects in cell cycle progression and chromosome segregation. In conclusion, cerulenin induces apoptosis through deregulation of adenine nucleotide biosynthesis resulting in nuclear division defects in S. pombe.