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At least 181 records · Page 10Linked to original sources
PubMed2001

Insulin stimulates actin comet tails on intracellular GLUT4-containing compartments in differentiated 3T3L1 adipocytes.

Incubation of isolated GLUT4-containing vesicles with Xenopus oocyte extracts resulted in a guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) and sodium orthovanadate stimulation of actin comet tails. The in vitro actin-based GLUT4 vesicle motility was inhibited by both latrunculin B and a dominant-interfering N-WASP mutant, N-WASP/Delta VCA. Preparations of gently sheared (broken) 3T3L1 adipocytes also displayed GTP gamma S and sodium orthovanadate stimulation of actin comet tails on GLUT4 intracellular compartments. Furthermore, insulin pretreatment of intact adipocytes prior to gently shearing also resulted in a marked increase in actin polymerization and actin comet tailing on GLUT4 vesicles. In addition, the insulin stimulation of actin comet tails was completely inhibited by Clostridum difficile toxin B, demonstrating a specific role for a Rho family member small GTP-binding protein. Expression of N-WASP/Delta VCA in intact cells had little effect on adipocyte cortical actin but partially inhibited insulin-stimulated GLUT4 translocation. Taken together, these data demonstrate that insulin can induce GLUT4 vesicle actin comet tails that are necessary for the efficient translocation of GLUT4 from intracellular storage sites to the plasma membrane.

Actins↗
PubMed1997

The isolated comet tail pseudopodium of Listeria monocytogenes: a tail of two actin filament populations, long and axial and short and random.

Listeria monocytogenes is driven through infected host cytoplasm by a comet tail of actin filaments that serves to project the bacterium out of the cell surface, in pseudopodia, to invade neighboring cells. The characteristics of pseudopodia differ according to the infected cell type. In PtK2 cells, they reach a maximum length of approximately 15 microm and can gyrate actively for several minutes before reentering the same or an adjacent cell. In contrast, the pseudopodia of the macrophage cell line DMBM5 can extend to >100 microm in length, with the bacteria at their tips moving at the same speed as when at the head of comet tails in bulk cytoplasm. We have now isolated the pseudopodia from PtK2 cells and macrophages and determined the organization of actin filaments within them. It is shown that they possess a major component of long actin filaments that are more or less splayed out in the region proximal to the bacterium and form a bundle along the remainder of the tail. This axial component of filaments is traversed by variable numbers of short, randomly arranged filaments whose number decays along the length of the pseudopodium. The tapering of the tail is attributed to a grading in length of the long, axial filaments. The exit of a comet tail from bulk cytoplasm into a pseudopodium is associated with a reduction in total F-actin, as judged by phalloidin staining, the shedding of alpha-actinin, and the accumulation of ezrin. We propose that this transition reflects the loss of a major complement of short, random filaments from the comet, and that these filaments are mainly required to maintain the bundled form of the tail when its borders are not restrained by an enveloping pseudopodium membrane. A simple model is put forward to explain the origin of the axial and randomly oriented filaments in the comet tail.

Actins↗
PubMed2006

Rapid actin monomer-insensitive depolymerization of Listeria actin comet tails by cofilin, coronin, and Aip1.

Actin filaments in cells depolymerize rapidly despite the presence of high concentrations of polymerizable G actin. Cofilin is recognized as a key regulator that promotes actin depolymerization. In this study, we show that although pure cofilin can disassemble Listeria monocytogenes actin comet tails, it cannot efficiently disassemble comet tails in the presence of polymerizable actin. Thymus extracts also rapidly disassemble comet tails, and this reaction is more efficient than pure cofilin when normalized to cofilin concentration. By biochemical fractionation, we identify Aip1 and coronin as two proteins present in thymus extract that facilitate the cofilin-mediated disassembly of Listeria comet tails. Together, coronin and Aip1 lower the amount of cofilin required to disassemble the comet tail and permit even low concentrations of cofilin to depolymerize actin in the presence of polymerizable G actin. The cooperative activities of cofilin, coronin, and Aip1 should provide a biochemical basis for understanding how actin filaments can grow in some places in the cell while shrinking in others.

Actin Cytoskeleton↗
PubMed2006

A population of comets in the main asteroid belt.

Comets are icy bodies that sublimate and become active when close to the Sun. They are believed to originate in two cold reservoirs beyond the orbit of Neptune: the Kuiper Belt (equilibrium temperatures of approximately 40 kelvin) and the Oort Cloud (approximately 10 kelvin). We present optical data showing the existence of a population of comets originating in a third reservoir: the main asteroid belt. The main-belt comets are unlike the Kuiper Belt and Oort Cloud comets in that they likely formed where they currently reside and may be collisionally activated. The existence of the main-belt comets lends new support to the idea that main-belt objects could be a major source of terrestrial water.

Ice↗
PubMed2005

Abl kinases regulate actin comet tail elongation via an N-WASP-dependent pathway.

Microbial pathogens have evolved diverse strategies to modulate the host cell cytoskeleton to achieve a productive infection and have proven instrumental for unraveling the molecular machinery that regulates actin polymerization. Here we uncover a mechanism for Shigella flexneri-induced actin comet tail elongation that links Abl family kinases to N-WASP-dependent actin polymerization. We show that the Abl kinases are required for Shigella actin comet tail formation, maximal intracellular motility, and cell-to-cell spread. Abl phosphorylates N-WASP, a host cell protein required for actin comet tail formation, and mutation of the Abl phosphorylation sites on N-WASP impairs comet tail elongation. Furthermore, we show that defective comet tail formation in cells lacking Abl kinases is rescued by activated forms of N-WASP. These data demonstrate for the first time that the Abl kinases play a role in the intracellular motility and intercellular dissemination of Shigella and uncover a new role for Abl kinases in the regulation of pathogen motility.

Actins↗
PubMed2004

Migration of Jupiter-family comets and resonant asteroids to near-Earth space.

The orbital evolution of about 26,000 Jupiter-crossing objects and 1,500 resonant asteroids under the gravitational influence of planets was investigated. The rate of their collisions with terrestrial planets was estimated by computing the probabilities of collisions based on random-phase approximations and the orbital elements sampled with a 500 yr step. The Bulirsh-Stoer and a symplectic orbit integrator gave similar results for orbital evolution, but sometimes give different collision probabilities with the Sun. For orbits close to that of Comet 2P, the mean collision probabilities of Jupiter-crossing objects with terrestrial planets were greater by two orders of magnitude than for some other comets. For initial orbital elements close to those of Comets 2P, 10P, 44P, and 113P, a few objects (about 0.1%) entered Earth-crossing orbits with semi-major axes a < 2 AU and aphelion distances Q < 4.2 AU, and moved in such orbits for more than 1 Myr (up to tens or even hundreds of Myr). Some of them even got inner-Earth orbits (Q < 0.983 AU) and Aten orbits. A few comets achieve typical asteroidal orbits for hundreds of Myr. Most former trans-Neptunian objects that have typical near-Earth object orbits moved in such orbits for millions of years; thus, during most of this time they were extinct comet, if they did not disintegrate.

Journal Article↗
PubMed1999

Aflatoxin exposure and DNA damage in the comet assay in individuals from the Gambia, West Africa.

The single cell gel electrophoresis assay (Comet assay) was used to measure DNA damage in peripheral lymphocytes from a group of individuals from The Gambia in order to determine whether such damage could be associated with increased exposure to aflatoxin in this population. Responses obtained were correlated to responses previously obtained [1] in a cross-sectional study in the same individuals of various cytogenetic alterations [chromosomal aberrations, micronuclei (crest positive and negative staining), and sister chromatid exchanges], and aflatoxin-albumin adducts. Analysis of variance methods were used to assess the effects of smoking, GSTM1 genotype, sex, age, and smoking status. A comparison was also made between The Gambian individuals and a group of healthy, non-smoking volunteers in the United Kingdom where aflatoxin exposure would be expected to be low. From the earlier study [1], it was determined that the levels of the sister chromatid exchanges and micronuclei were higher in The Gambian group than in a European group where aflatoxin exposure was lower, but that there were no correlations between the adduct levels and the cytogenetic abnormalities at the individual level. In the present study, DNA damage as measured in the Comet assay was not significantly higher than in the healthy United Kingdom volunteers. In addition, there were no associations between cytogenetic damage, GSTM1 genotype, age, sex, lifestyle factors (smoking and aflatoxin exposure), and Comet response at the individual level. Comet response was higher in females than males in The Gambia if one outlier was excluded from analysis and not taking into account other sources of variability. It would appear that DNA damage as measured in the Comet assay in peripheral blood lymphocytes is not a sensitive genotoxic marker of aflatoxin exposure in this population.

Adolescent↗
PubMed2005

Organic matter in comets and cometary dust.

Comets are primitive conglomerates of the solar system containing a mixture of frozen gases, refractory grains, and carbonaceous particles rich in biogenic elements. The dramatic display of comets is mostly caused by a cloud of micrometer-sized dust particles that leave the comet nucleus when frozen gases sublimate as they approach the Sun. Analyses of cometary dust captured in the stratosphere together with data obtained from space missions to comets have revealed the presence of a great variety of organic molecules. Since substantial amounts of cometary dust were gently deposited on Earth, their organic content could have played a major role in prebiotic processes prior to the appearance of microorganisms. This review discusses the description and implications for life of the organic content of comets and cometary dust.

Cosmic Dust↗
PubMed1996

The comet assay for the detection of genotoxic damage in the earthworms: a promising tool for assessing the biological hazards of polluted sites.

The comet assay, a relatively new method for DNA strand break detection in individual cells, is becoming a major tool for environmental biomonitoring. One approach for assessing the possible environmental consequences of hazardous waste pollution involves the assessment of genotoxic damage (and other effects) in sentinel organism. The single cell gel electrophoresis (SCGE) technique or comet assay. because of its simplicity, sensitivity, and need for only small numbers of cells, has been suggested as an ideal technique for such studies. An important advantage of the technique is that it is applicable to any eukaryotic organism and cell type. Verschaeve et al. (1993) conducted a pilot study using alkaline comet assay to assess the extent of DNA damage in coelomic leucocytes (coelomocytes) of earthworms (Eisenia foetida) maintained in different soil samples as an indicator of soil pollution. The aim of our study was to evaluate the usefulness of monitoring single strand breaks in coelomocytes for assessing genotoxicity of pollutants in coke oven area. We exposed earthworms to samples of soils obtained from polluted areas of a coke oven. All samples gave a significantly higher comet tail moment that those obtained from worms kept in laboratory conditions (standard black earth = internal controls) and worms kept in soils from control areas (= external controls). Our results show that the comet assay applied to earthworm is very valuable for monitoring and detection of genotoxic compounds in terrestrial ecosystems.

Animals↗
PubMed1999

Comet assay and DNA flow cytometry analysis of staurosporine-induced apoptosis.

BACKGROUND: The ability of the comet assay to quantify DNA strand breaks and alkali labile sites has been widely demonstrated. In this study, this assay was tested for its ability to identify DNA fragmentation occurring during apoptosis in comparison with standard DNA flow cytometry analysis. METHODS: Staurosporine-induced apoptosis in CHO cells is an adequate model to study a rapid time- and dose-dependent appearance of this process. RESULTS: Nuclear staining with DAPI confirmed the induction of apoptosis with a typical chromatin condensation and fragmentation. Analysis of propidium-iodide- (PI) stained DNA by flow cytometry showed the presence of a pre-G1 peak, characteristic of apoptotic cells, 6 h after drug treatment. The detection of highly damaged cells (HDC) by the comet assay after 3 h treatment occurred earlier than the detection of apoptotic cells by flow cytometry. However, HDC were missed when the DNA fragmentation was too high, preventing accurate quantification of late apoptotic cells. CONCLUSIONS: The comet assay is more sensitive than standard DNA flow cytometry to detect early DNA fragmentation events occurring during apoptosis. However, the comet assay modified by omitting electrophoresis was necessary to quantify apoptotic fraction at later stages.

Animals↗
PubMed2001

Assessment of DNA damage in lymphocytes of workers exposed to X-radiation using the micronucleus test and the comet assay.

The mutagenic and carcinogenic effects of genotoxic agents on exposed people have constituted an increasing concern. Therefore, the objective of this work was to assess DNA damage in lymphocytes of workers exposed to X-radiation using the cytokinesis-blocked micronucleus test and the comet assay (single-cell gel electrophoresis), and to compare these two techniques in the monitoring of exposed populations. The cytokinesis-blocked micronucleus test and the comet assay were employed in the monitoring of 22 workers occupationally exposed to X-radiation in a hospital in southern Brazil. The frequency of dicentric bridges was also measured. The results of both assays and the frequency of dicentric bridges revealed a significant increase in genetic effects on the cells of exposed individuals. Age was significantly correlated with micronucleus frequency and damage index in the comet assay. The concomitant analysis of dicentric bridges when determining micronucleus frequency does not require much extra work, and may serve as a reference to the type of mutagenic effect (clastogenic or aneugenic). The combination of the alkaline comet assay with the cytokinesis-blocked micronucleus test appears to be very informative for the monitoring of populations chronically exposed to genotoxic agents.

Adult↗
PubMed2003

Detection of ghost cells in the standard alkaline comet assay is not a good measure of apoptosis.

The single cell gel electrophoresis assay, or Comet assay, is a powerful tool for measurement of DNA strands breaks, oxidative damage, and alkali labile sites, and the assay was recently modified to detect DNA cross-links. It has also been proposed as a measure of apoptosis since apoptotic cells are suspected to result in total migration of the DNA from the nucleus into the tail. Cells with this appearance are called ghost cells, clouds, hedgehogs, or NDCN (nondetectable cell nuclei). The aim of this study was to determine if ghost cells can be used to measure apoptosis in the standard alkaline comet assay. To answer this question, we made use of two cell lines: CTLL-2 cells that can enter apoptosis upon addition of apoptosis stimuli or IL-2 deprivation, and CTLL-2 bcl2 cells that are protected from apoptosis due to the overexpression of the apoptosis inhibitor gene bcl2. The two cell lines were treated with cytotoxins (nongenotoxic apoptosis inducers, nongenotoxic necrotic agents) or genotoxins. They were also subjected to growth factor withdrawal, which induced apoptosis in the CTLL-2 cell line. The level of apoptosis was measured by the Annexin V-FITC method in parallel with performing the Comet assay. The results obtained in the two cell lines suggest that apoptotic or necrotic death does not correlate well with the detection of ghost cells, presumably because these cells are lost upon electrophoresis. A variant of the alkaline Comet assay that was performed without electrophoresis (halo method) was able to efficiently detect cells undergoing apoptosis, but it was unable to clearly distinguish between apoptosis and genotoxic damage.

Animals↗
PubMed2006

Variability in fecal water genotoxicity, determined using the Comet assay, is independent of endogenous N-nitroso compound formation attributed to red meat consumption.

Red meat consumption causes a dose-dependent increase in fecal apparent total N-nitroso compounds (ATNC). The genotoxic effects of these ATNCs were investigated using two different Comet assay protocols to determine the genotoxicity of fecal water samples. Fecal water samples were obtained from two studies of a total of 21 individuals fed diets containing different amounts of red meat, protein, heme, and iron. The first protocol incubated the samples with HT-29 cells for 5 min at 4 degrees C, whereas the second protocol used a longer exposure time of 30 min and a higher incubation temperature of 37 degrees C. DNA strand breaks were quantified by the tail moment (DNA in the comet tail multiplied by the comet tail length). The results of the two Comet assay protocols were significantly correlated (r = 0.35, P = 0.003), however, only the second protocol resulted in detectable levels of DNA damage. Inter-individual effects were variable and there was no effect on fecal water genotoxicity by diet (P > 0.20), mean transit time (P = 0.588), or weight (P = 0.705). However, there was a highly significant effect of age (P = 0.019). There was no significant correlation between concentrations of ATNCs in fecal homogenates and fecal water genotoxicity (r = 0.04, P = 0.74). ATNC levels were lower in fecal water samples (272 microg/kg) compared to that of fecal homogenate samples (895 microg/kg) (P < 0.0001). Failure to find dietary effects on fecal water genotoxicity may therefore be attributed to individual variability and low levels of ATNCs in fecal water samples.

Ammonia↗
PubMed2006

Radiation-induced DNA damage and repair in peripheral blood mononuclear cells from Nijmegen breakage syndrome patients and carriers assessed by the Comet assay.

Nijmegen breakage syndrome (NBS) patients and carriers are predisposed to malignancy and are often treated with X-irradiation. In the present study, the single-cell gel electrophoresis (Comet) assay was used to examine radiation-induced DNA damage and repair in peripheral blood mononuclear cells from NBS patients (n=13) and carriers (n=36) of six unrelated families. Cells from apparently healthy donors (n=10) and from breast cancer patients with normal clinical radiosensitivity (n=10) served as controls. Cells were irradiated with 5 Gy of X-rays and assayed for initial DNA damage and for residual DNA damage after 40 min of repair; the kinetics of DNA repair also was estimated. In addition, the nuclear area of unirradiated cells was extracted from the Comet data. The initial radiation-induced DNA fragmentation indicated that cells from members of two out of six NBS families were significantly more sensitive to X-irradiation than cells from the controls. Cells from four NBS families had longer DNA repair half-time values, while cells from five NBS families had significantly increased residual DNA damage following repair. The mean nuclear area of unirradiated cells processed in the Comet assay was 1.3-fold higher in cells from all NBS families than in the controls (P<0.05). Notably, the Comet assay parameters (initial and residual DNA damage and the repair kinetics) of irradiated NBS cells predicted the carrier status of the majority (86%) of blindly tested individuals. The prediction of NBS status was higher if the nuclear area of unirradiated cells was used as the endpoint. The results of this study suggest that the impaired radiation response of NBS cells should be taken into account if radiotherapy of NBS patients and carriers is required.

Adolescent↗
PubMed2006

DNA damage in Pakistani pesticide-manufacturing workers assayed using the Comet assay.

The production and use of chemical pesticides has increased in recent years. Although the increased use of pesticides may benefit agriculture, they are also the potential source of environmental pollution, and exposure to pesticides can have negative consequences for human health. In the present study, we have assessed DNA damage in blood leukocytes from 29 Pakistani pesticide-factory workers and 35 controls of similar age and smoking history. The workers were exposed to various mixtures of organophosphates, carbamates, and pyrethroids. DNA damage was measured with the single cell gel electrophoresis (SCGE) assay or Comet assay, using the mean comet tail length (microm) as the DNA damage metric. Exposed workers had significantly longer comet tail lengths than the controls (mean +/- SD 19.98 +/- 2.87 vs. 7.38 +/- 1.48, P < 0.001). Of the possible confounding factors, smokers had significantly longer mean comet tail lengths than nonsmokers and exsmokers for both the workers (21.48 +/- 2.58 vs.18.37 +/- 2.28, P < 0.001) and the controls (8.86 +/- 0.56 vs. 6.79 +/- 1.31, P < 0.001), while age had a minimal effect on DNA damage (P > 0.05 and P < 0.05 for workers and controls, respectively). The results of this study indicate that occupational exposure to pesticides causes DNA damage.

Adult↗
PubMed2002

Assessment of chemotherapy-induced DNA damage in peripheral blood leukocytes of cancer patients using the alkaline comet assay.

The alkaline comet assay was employed to assess the pre- and post-treatment levels of in vivo DNA damage in peripheral blood leukocytes of cancer patients. During the study all patients were given antineoplastic drugs, mainly as polychemotherapy. To quantify the DNA damage, two different comet parameters were evaluated: the tail length and the tail moment. Our results indicate marked interindividual variations between baseline DNA damage in peripheral blood leukocytes recorded among cancer patients prior to the chemotherapy. After intravenous administration of various antineoplastic drugs, a significantly increased level of DNA damage in all cancer patients compared to their pre-treatment values was recorded The highest level of DNA damage was seen following administration of 5-fluorouracil, adriamycin, and cisplatin (FAP protocol). The results indicate that administration of antineoplastic drugs in standard protocols is accompanied by significant DNA damage in peripheral blood leukocytes. In order to diminish the potential risks of developing second neoplasms, a continuous biomonitoring of cancer patients after the ending of chemotherapy becomes important. Despite their limitations, present results confirm the usefulness of the alkaline comet assay as a sensitive biomarker of exposure that enables rapid and simple detection of primary DNA damage in peripheral blood leukocytes of cancer patients. Together with standard cytogenetic endpoints, the comet assay provides a powerful technique for the routine detection of critical DNA lesions produced after administration of antineoplastic drugs in the clinical settings.

Adult↗
PubMed2006

Alkaline comet assay study with breast cancer patients: evaluation of baseline and chemotherapy-induced DNA damage in non-target cells.

The sensitivity of the alkaline comet assay for the evaluation of baseline and treatment-induced DNA damage in white blood cells of breast cancer patients receiving adjuvant chemotherapy according to three conventional anthracycline- and cyclophosphamide-containing protocols was investigated. Additionally, baseline DNA damage in cancer patients was compared with the levels of DNA damage recorded in healthy women. Altogether 30 patients with diagnosed breast cancer and 30 female blood donors with no known familial history of breast cancer participated in the study. Alkaline comet assay was performed according to standard protocol and DNA migration in peripheral blood leukocytes was measured by a computer-based image analysis system. For each subject the frequency of "damaged" cells, i.e., long-tailed nuclei (LTN) with tail length exceeding 95th percentile for the considered parameter among controls, is also reported. Breast cancer patients had significantly increased background levels of DNA damage in their peripheral blood leukocytes as compared to healthy women. Prior to the chemotherapy, a majority of patients showed a statistically significant increase in the number of LTN compared to healthy blood donors. Marked interindividual variations in baseline DNA damage among patients were recorded, some of them related to the disease stage and status. The present study confirmed the alkaline comet assay as a sensitive technique able to detect significantly elevated DNA migration in blood cells of patients already one hour after completion of the first cycle of chemotherapy. Administration of antineoplastic drugs in three chemotherapy protocols studied induced a similar increase of primary DNA damage in nontarget cells. The evaluation of the LTN frequencies indicates the best response to the protocol containing cyclophosphamide, methotrexate and 5-fluorouracil (CMF). Our results point to the significance of simultaneous evaluation of DNA migration and frequency of LTN in the same subject and approved the use of alkaline comet assay as a suitable method for the routine detection of critical DNA lesions produced after administration of antineoplastic drugs in the clinical settings.

Adult↗
PubMed2006

Genotoxicity of cadmium in marine diatom Chaetoceros tenuissimus using the alkaline Comet assay.

Genotoxic effects of cadmium on phytoplankton Chaetoceros tenuissimus have been evaluated using DNA damage by Comet assay. Cadmium concentrations ranging from 2.4 to 10 mg/l were used to evaluate the effects. Results showed that as the concentration of Cd increased growth of the diatom decreased. Alkaline single-cell gel electrophoresis (Comet assay) method, which is highly sensitive in detection of DNA damage in eukaryotic cells, was used to observe genomic changes in marine diatom cells. DNA damage was measured as percent number of comets and normal cells. 65% cells were found to be damaged at 10 mg/l concentration of Cd as compared to 23% in 2.4 mg/l and only 5% in controls. More than 50% apoptotic cells were observed on 8th day at 10 mg/l and 12th day at 7.5 mg/l concentrations. At lower Cd concentrations (4.5 mg/l and below) the damage was below 30% till the last day. This suggested that higher Cd levels have early damaging effects on cell nuclear material and that % injury increases with advancement of exposure period. One advantage of use of C. tenuissimus is the ease with which it can be cultured in a defined medium. C. tenuissimus diatom can be used as an in vivo model for ecogenotoxicity assessment using the Comet assay.

Animals↗