Chromosomal DNA Fragment Research Articles

English

A new gene organization related to carbohydrate metabolism of Citrobacter species has been identified on about 4.1 kb chromosomal DNA fragment. Sequence analysis revealed four open reading frames (ORFs), and gene orders were determined to be PTS system mannitol-specific transporter subunit EIICBA (MtlA), L-sorbose-1-phosphate reductase (SorE), class II fructose-1,6-biphosphatase (GlpX), and mannitol repressor (MtlR). The deduced amino acid sequences of these ORFs showed more than 80% identities to MtlA,SorE, GlpX, and MtlR of Escherichia coli, Shigella, and Salmonella enterica, respectively. The sorE gene was heterologously expressed in E. coli. The recombinant SorE clearly showed the L-sorbose-1-phosphate reductase activity.   Key words: Citrobactor sp., gene organization, carbohydrate catabolism, L-sorbose 1-phosphate reductase, gene expression.

Evidence for cell apoptosis suppressing white spot syndrome virus replication in Procambarus clarkii at high temperature

In shrimp, higher water temperatures (~32°C) can suppress the ability of white spot syndrome virus (WSSV) to replicate and cause mortality, but the mechanisms remain unclear. To investigate whether cell apoptosis might be involved, a Tdt-mediated dUTP nick-end label (TUNEL) method was used to assess levels of chromosomal DNA fragmentation in hepatopancreas and gill cells of Procambarus clarkii crayfish infected with WSSV and maintained at either 32 ± 1°C or 24 ± 1°C. Based on relative cell numbers with yellow-green colored TUNEL-positive nuclei, the apoptotic index was elevated in WSSV-infected crayfish maintained at 32°C. In gill tissue sections examined by transmission electron microscope, cells with nuclei displaying apoptotic bodies or marginated, condensed and fragmented chromatin without concurrent cell cytoplasm damage were also more prevalent. Flow cytometry sorting of annexin-stained cells showed apoptosis to be most prevalent in granular haemocytes, and assays for caspase-3 activity showed it to be most elevated in hepatopancreas tissue. Despite these indicators of cell apoptosis but consistent with WSSV replication being restricted at elevated temperatures, no increases in transcription of the viral anti-apoptosis genes ORF390 and ORF222 were detected by RT-PCR in shrimp maintained at 32°C, possibly due to the elevated levels of cellular apoptosis.

TiO2 particles induce apoptosis associated with chromosomal DNA fragmentation and without ROS production in L1210 leukemia cells

TiO2 particles induce apoptosis associated with chromosomal DNA fragmentation and without ROS production in L1210 leukemia cells

Ectopically Expressed Perforin-1 Is Proapoptotic in Tumor Cell Lines by Increasing Caspase-3 Activity and the Nuclear Translocation of Cytochrome c

Perforin-1 (PRF), a cytotoxic lymphocyte pore-forming protein, plays an important role in the action of cytotoxic T cells and natural killer cells in that it causes the lysis of abnormal body cells and the elimination of virus-infected cells and tumors. Upon degranulation, PRF inserts itself into the target cell’s plasma membrane, forming a pore. The subsequent translocation of pro-apoptotic granzymes (including granzyme B, A, M et al.) into the cytoplasm provides the proteases with access to numerous protein substrates that promote apoptosis after cleavage. These proteases are believed to be the main executioners of target cell apoptosis. Although the PRF and granzyme components are both critical to this process and in some way involved in inducing cell death in target cells, the inhibition of tumor growth could still be efficient in granzyme-deficient mice. It is unclear whether PRF alone can suppress tumors. In this study, we discovered that forced ectopic expression of PRF alone, in the absence of granzymes, could mediate cell death in cancer cells. Notably, transient expression of both full-length and truncated active-form PRF in human Hep G2, SK-BR-3, and HeLa cells was found to induce apparent cell growth inhibition and cell death, as evidenced by chromosome condensation and DNA fragmentation, increased caspase-3 activity, and the release of apoptosis inducing factor (AIF) and cytochrome c from the mitochondria. This PRF-induced cell death could be abrogated by pan-caspase inhibitor (Z-VAD) and mitochondria protector (TAT-BH4). The implication of these results is that ectopically expressed PRF has apoptosis-inducing abilities, and PRF alone is sufficient to induce apoptotic cell death in cells with ectopic expression. Taking this into consideration, our results suggest the possibility of using PRF as a pro-apoptotic gene for tumor therapeutics.

Cell wall active antibiotics reduce chromosomal DNA fragmentation by peptidoglycan hydrolysis in Staphylococcus aureus

Lysostaphin digestion of peptidoglycan (PG) from Staphylococcus aureus resulted in chromosomal DNA fragmentation by released DNase, as directly visualized in situ on isolated nucleoids. Nevertheless, DNA digestion was partially prevented by previous incubation with antibiotics that inhibit PG synthesis. This inhibitory effect was much more remarkable with glycopeptides vancomycin and mainly teicoplanin than with beta-lactams cloxacillin and ceftazidime. Therefore, inhibition of PG chain elongation has a more significant inhibition of DNA degradation than inhibition of PG cross-linking, possibly due to a reduction in DNase storage at the cell wall.

Cytotoxic Effect of Aspartame (Diet Sweet) on the Histological and Genetic Structures of Female Albino Rats and Their Offspring

The present study evaluated the effect of aspartame intake on the histological and genetic structures of mother albino rats and their offspring. Sixty adult female albino rats and 180 of their offspring were equally divided into two groups (control and treated), each group divided into three subgroups. Each subgroup consisted of 10 pregnant rats and 30 of their offspring. The experimental design divided into three periods: (1) the gestation period (subgroup one), (2) the gestation period and three weeks after delivery (subgroup two) and (3) animals in the third subgroup treated as subgroup two then left till the end of the ninth week after delivery. Each pregnant rat in the treated subgroups was given a single daily dose of 1 mL aspartame solution (50.4 mg) by gastric gavage throughout the time intervals of experimental design. At the end of each experimental period for control and treated subgroups, the liver of half of both control and treated groups were subjected for histological study while the liver and bone marrow of the other halves were subjected for cytogenetic studies. Body weight of both groups were recorded individually twice weekly in the morning before offering the diet. The results revealed that the rats and their offspring in the subgroups of control animals showed increases in body weight, normal histological sections, low chromosomal aberration and low DNA fragmentation. The treated animals in the three subgroups rats and their offspring revealed decreases in body weight, high histological lesions, increases in the chromosomal aberration and DNA fragmentation compared with control groups. In conclusion, the consumption of aspartame leads to histopathological lesions in the liver and alterations of the genetic system in the liver and bone marrow of mother albino rats and their offspring. These toxicological changes were directly proportional to the duration of its administration and improved after its withdrawal.

Transferring chromosome DNA fragments from multiple donor cells into a host strain for yeast strain improvement

Based on a common biological phenomenon - homologous recombination - a novel method was developed by transferring chromosome DNA fragments extracted from multiple donor cells into a host strain. Through this method of transferring DNA fragments, foreign DNA fragments are introduced into one host cell and multiple positive traits from multiple strains may be integrated into the host strain. We first confirmed its feasibility in both prokaryotic and eukaryotic cells by selecting reverse mutants to prototrophy from auxotrophic strains through receiving chromosomal DNA fragments of wild-type parental strains. We then applied this method to Saccharomyces cerevisiae to improve its ethanol and temperature tolerance. We introduced donor chromosome DNA fragments from different S. cerevisiae strains with improvements in ethanol or temperature tolerance into a common strain S. cerevisiae and obtained a strain with much superior ethanol and temperature tolerance. The results showed that the Transferring DNA Fragments method provides a new way for strain breeding.

Molecular evidence of curcumin-induced apoptosis in the filarial worm Setaria cervi

Curcumin (diferuloyl methane) is a major curcuminoid from Curcuma longa that exhibits various pharmacological effects and has shown multiple beneficial activities. Our understanding of its anticarcinogenic and other activities occurring through curcumin-induced apoptosis in several cancer cells has greatly expanded in recent years. Lymphatic filariasis is a worldwide health problem causing global disability in humans and is caused by filarial nematodes. Development of efficient strategies to promote programmed cell death in filarial worms remains a key challenge for anti-filarial drug developing research and a crucial unmet medical need. In this study, we have taken molecular and biochemical approaches toward understanding the molecular basis for curcumin-mediated anti-filarial activity in the filarial nematode Setaria cervi. Results of MTT assay showed that curcumin causes a significant reduction in viability of Mf and adults and thus acts as a potent macro- and micro-filaricidal agent. Hoechst staining, TUNEL staining, showed several apoptotic nuclei in different parts of curcumin-treated adults. At 25 μM concentration it showed chromosomal DNA fragmentation in adult worms. Our results indicate that curcumin decreases protein and mRNA expression levels of anti-apoptotic gene ced-9 and enhances both the levels of pro-apoptotic genes ced-3 and ced-4 in a dose-dependent manner. All these observations ascertained the apoptogenicity of curcumin at a minimum concentration of 50 μM in this filarial worm. Furthermore, we showed that curcumin causes depletion of parasitic glutathione level, enhances the activities of glutathione S-transferase and superoxide dismutase and stimulates rapid generation of reactive oxygen species (ROS). Here, we present molecular evidence on curcumin-induced apoptosis in the filarial nematode S. cervi with probable involvement of ROS in a caspase-dependent manner.

Mutants of Burkholderia cenocepacia with a change in synthesis of N-acyl-homoserine lactones—Signal molecules of quorum sensing regulation

By means of plasposon mutagenesis, mutants of Burkholderia cenocepacia 370 with the change in production of N-acyl-homoserine lactones (AHL), signal molecules of the Quorum Sensing system of regulation, were obtained. To localize plasposon insertions in mutant strains, fragments of chromosomal DNA containing plasposons were cloned, adjacent DNA regions sequenced, and a search for homologous nucleotide sequences in the GeneBank was initiated. It has been shown that the insertion of plasposon into gene lon encoding lon proteinase drastically decreases AHL synthesis. Upon insertion of plasposon into gene pps encoding phosphoenolpyruvate-synthase, enhancement of AHL production is observed. In mutant carrying inactivated gene lon, a strong decline of extracellular protease activity, hemolytic, and chitinolytic activities was observed in comparison with the original strain; lipase activity was not changed in this mutant. Mutation in gene pps did not affect these properties of B. cenocepacia 370. Mutations in genes lon and pps reduced the virulence of bacteria upon infection of mice.

Differential viral propagation and induction of apoptosis by grouper iridovirus (GIV) in cell lines from three non-host species

Grouper iridovirus (GIV), belonging to the Ranavirus genus of the Iridoviridae family, was demonstrated to differentially express viral genes and induce apoptosis in three non-host fish cell lines rainbow trout monocyte/macrophage (RTS11), chinook salmon embryonic (CHSE-214) and fathead minnow Epithelioma papulosum cyprinid (EPC). These cells were challenged with GIV and virus entry into all three cell lines was confirmed by the expression of viral immediate early genes. The expression of the late major capsid protein gene was detected in CHSE-214 and EPC, but not in RTS11, suggesting an earlier termination in the viral replication cycle in RTS11. Approximately 12h after infection with GIV, cell death was prominent in all three non-host cell lines. Death was later confirmed to be apoptosis by the presence of chromosomal DNA fragmentation and phosphatidylserine externalization. To determine whether apoptosis was protein related or gene expression related, the three cell lines were challenged with heat-inactivated GIV and UV-treated GIV (GIVUV). The heat inactivation abolished apoptosis in all three cell lines, but each cell line responded differently to GIVUV. Relative to GIV, GIVUV caused no apoptosis in CHSE-214, decreased apoptosis in RTS11, and increased apoptosis in EPC. These results suggest that early GIV gene expression was needed for apoptosis in CHSE-214 but impeded apoptosis in EPC. At the cellular level, only EPC is a permissive host as EPC was the only cell line of the three capable of producing a moderate increase in virus titer. The three non-host cell lines present a good system for potentially identifying different components of GIV-induced apoptotic pathways in future studies.

Toxicological effects of arsenic exposure in a freshwater teleost fish, Channa punctatus

High concentration of arsenic in groundwater in the north-eastern states of India has become a major cause of concern. Inorganic arsenic of geological origin is found in groundwater used as drinking-water in several parts of the world. Arsenic is used in various industries and agriculture and excessive arsenic finds its way into lakes and rivers. Since arsenic is a known human carcinogen, the epidemiological studies are extremely important for this metal. Fish are ideal organisms to work with in toxicogenomics studies due to the strong power of fish models to establish biomarkers of exposure. Since, fishes respond to toxicants in a similar way as higher vertebrates, they can be used to screen for chemicals that are potentially teratogenic and carcinogenic to humans. Also fish absorbs dissolved or available metals and therefore can serve as indicator of metal pollution. The objectives of this work were to examine the toxicological effect and molecular changes caused by a heavy metal sodium arsenite (NaAsO 2 ), in Channa punctatus. When fishes were exposed to NaAsO 2 , it induced death of the fishes in a concentration-dependent manner. 100 ppm, the highest concentration tested in this study, was found to be the deadliest and it induced death of the fishes within 18 to 20 h. As the concentration lowered the survival time increased dose dependently. Liver is the target organ of arsenic toxicity. Fish liver plays an important role in uptake, accumulation, bio-transformation and excretion of arsenic. We investigated the effect of NaAsO 2 on fish liver. NaAsO 2 was found to cause liver chromosomal DNA fragmentation and expression of certain proteins. Key words: DNA Fragmentation, sodium arsenite, Channa punctatus.

Activation of p53/ATM-dependent DNA damage signaling pathway by shiga toxin in mammalian cells

In this report, we studied the role of DNA damage signaling pathway in shiga toxin (STX)-induced mammalian cell death. Shiga toxin 1 exhibited cytotoxic activity in different mammalian cells such as HeLa cells, mouse embryo fibroblasts, and Caco-2 cells (a human intestinal primary fibroblast cell line). STX-1 was found to induce the release of cytochrome c from the mitochondria, nuclear condensation, and fragmentation of chromosomal DNA. STX-1 activated DNA damage signaling as determined by induction of H2AX phosphorylation and cleavage of PARP. Inhibition of caspase-3 reduced STX-1-induced phosphorylation of H2AX and nuclear condensation. It was also found that STX-1-induced p53 expression, and activated ATM in mammalian cells. STX-1-induced nuclear condensation significantly reduced in p53-, and ATM-knockout cells suggesting an involvement of p53 and ATM in transducing signals produced by STX in inducing apoptosis in mammalian cells. This is the first demonstration of involvement of ATM/p53 in STX-inducing mammalian cell death.

Synthetic Coprisin Analog Peptide, D-CopA3 has Antimicrobial Activity and Pro-Apoptotic Effects in Human Leukemia Cells

Recently, we reported that the synthetic Coprisin analog peptide 9-mer dimer CopA3 (consisted of all-L amino acid sequence) was designed based on a defensin-like peptide, Coprisin isolated from Copris tripartitus. The 9-mer dimer CopA3 (L-CopA3) had antibacterial activity and induced apoptosis in human leukemia cells via a caspase-independent pathway. In this study, all of amino acid sequences of L-CopA3 were modified to all D-form amino acids (D-CopA3) to develop a more effective antimicrobial peptide. We investigated whether D-CopA3 had antimicrobial activities against pathogenic microorganisms and proapoptotic effects in human leukemia cells (U937, Jurkat, and AML-2). The synthetic peptide D-CopA3 had antimicrobial activities against various pathogenic bacteria and yeast fungus with MIC values in the 4~64 microM range. Moreover, D-CopA3 caused cell growth inhibition, and increased the chromosomal DNA fragmentation and the expression of inflammatory cytokines, TNF-alpha and IL1-beta, transcripts in human leukemia cells. The all-D amino acid peptide D-CopA3 proved as effective as the L-CopA3 reported previously. These results provide the basis for developing D-CopA3 as a new antibiotic peptide.

α-Lipoic acid attenuates vascular calcification via reversal of mitochondrial function and restoration of Gas6/Axl/Akt survival pathway.

Vascular calcification is prevalent in patients with chronic kidney disease and leads to increased cardiovascular morbidity and mortality. Although several reports have implicated mitochondrial dysfunction in cardiovascular disease and chronic kidney disease, little is known about the potential role of mitochondrial dysfunction in the process of vascular calcification. This study investigated the effect of α-lipoic acid (ALA), a naturally occurring antioxidant that improves mitochondrial function, on vascular calcification in vitro and in vivo. Calcifying vascular smooth muscle cells (VSMCs) treated with inorganic phosphate (Pi) exhibited mitochondrial dysfunction, as demonstrated by decreased mitochondrial membrane potential and ATP production, the disruption of mitochondrial structural integrity and concurrently increased production of reactive oxygen species. These Pi-induced functional and structural mitochondrial defects were accompanied by mitochondria-dependent apoptotic events, including release of cytochrome c from the mitochondria into the cytosol, subsequent activation of caspase-9 and -3, and chromosomal DNA fragmentation. Intriguingly, ALA blocked the Pi-induced VSMC apoptosis and calcification by recovery of mitochondrial function and intracellular redox status. Moreover, ALA inhibited Pi-induced down-regulation of cell survival signals through the binding of growth arrest-specific gene 6 (Gas6) to its cognate receptor Axl and subsequent Akt activation, resulting in increased survival and decreased apoptosis. Finally, ALA significantly ameliorated vitamin D3-induced aortic calcification and mitochondrial damage in mice. Collectively, the findings suggest ALA attenuates vascular calcification by inhibiting VSMC apoptosis through two distinct mechanisms; preservation of mitochondrial function via its antioxidant potential and restoration of the Gas6/Axl/Akt survival pathway.

In vivo Comparative Studies on Antigenotoxicity of Date Palm (Phoenix Dactylifera L.) Pits Extract Against DNA Damage Induced by N-Nitroso-N-methylurea in Mice.

Aqueous extract of the date palm (Phoenix dactylifera L.) pits was prepared and its antigenotoxic activity was evaluated against N-Nitroso-N-methylurea (NMU) induced mutagenic effect in mice, using chromosome aberration (CA), micronuclei (MN) and DNA fragmentation assays as experimental end points in male mice. Date pits extract (DPE) was given orally to mice at the dose 25 mg/25 g mouse for successive five days in a week up to four consecutive weeks. NMU was used as mutagen and was given intraperitoneal (i.p) injection at single dose 80 mg/kg b.w., 24 hr after last dose of DPE in pre-treatment regimen and 24 hr before the first dose of DPE in the post-treatment regimen. Mice were scarified after one, two and seven days after the end of treatment. The results have shown that pre-and post-treatment regimens of DPE were significantly restored the DNA damage induced by NMU, as revealed by lowering of the occurrence of CAs and MN in bone marrow cells and inhibition of hepatic DNA fragmentation. These findings suggested that DPE produced their inhibitory activity either by desmutagenic or bioantimutagenic manner in pre-and post-treatment regimens respectively.

The use of RIVET to identify avian pathogenic Escherichia coli (APEC) genes induced in vivo during infection in chickens

Event Abstract Back to Event The use of RIVET to identify avian pathogenic Escherichia coli (APEC) genes induced in vivo during infection in chickens Huruma N. Tuntufye1* 1 Catholic University of Leuven, Faculty of Bioscience Engineering, Belgium H. N. TUNTUFYE1, 2, S. LEBEER3 and B. M. GODDEERIS1 1Department of Biosystems, Faculty of Bioscience Engineering, Katholieke Universiteit Leuven, Kasteelpark Arenberg 30, B-3001, Heverlee, Belgium 2Department of Veterinary Microbiology and Parasitology, Sokoine University of Agriculture, P.O.Box 3019, ChuoKikuu, Morogoro, Tanzania 3Department of Microbial and Molecular Systems, Faculty of Bioscience Engineering, Katholieke Universiteit, Kasteelpark Arenberg23, 3001, Heverlee, Belgium Avian pathogenic Escherichia coli (APEC) are a group of E.coli strains causing systemic disease in poultry known as avian colibacillosis. The disease manifests itself initially with septicaemia then either sudden death or localized multiple organ inflammation. The disease is associated with major economic losses to the poultry industry worldwide. Host and bacterial factors influencing and/or responsible for carriage and systemic translocation of APEC inside the host are poorly understood. Identification of such factors could help in the understanding of its pathogenesis and subsequently development of control strategies. In this study RIVET strategy (Camilli et al., 1994) was developed and used to isolate host-induced APEC promoters in order to investigate APEC pathogenesis in chicken. Random chromosomal DNA fragments from APEC genome were transcriptionally fused upstream to a promoterless cre gene to create an APEC RIVET library in a promoter trap plasmid. The reporter strain was constructed by integrating the loxP sites (in direct orientation) flanking the neomycin resistance marker (neo) for positive selection and streptomycin sensitivity gene (rpsL) for negative selection into APEC genome. Fused active promoters cause expression of Cre recombinase which subsequently cause recombination of the two loxP sites, deleting the cassette and permanently changing the bacterial phenotype such that could be detected after gene expression had ceased. The APEC RIVET library was pre-selected on kanamycin and ampicillin antibiotics to eliminate in vitro active promoters. The bacteria were then administered in chicken host via intra-tracheal route. After screening, 147 kanamycin sensitive clones were isolated from chicken indicating that the loxP-rpsL-neo-loxP (LoxP) cassette could be deleted as a result of in vivo active promoters. PCR analysis and sequencing demonstrated a range of insert sizes (1-2kb) suggesting that the screening is functional and the plasmid could stably be maintained in the bacteria even after infection. Further analysis of the DNA fragments' sequences revealed that 27 different APEC genes were induced inside the chicken during infection. The in vivo induced genes include metabolic genes, virulence genes and genes with prophage functions, regulatory functions and unknown functions. With these results APEC RIVET library could be adapted and the strategy showed to be functional for the screening of host-induced APEC genes/ promoters in chickens. Keywords: APEC, Chickens, RIVET strategy Conference: ECMIS - E. coli and the Mucosal Immune System : Interaction, Modulation and Vaccination, Ghent, Belgium, 2 Jul - 5 Jul, 2011. Presentation Type: Oral Presentation Topic: Infection and innate immunity, immunosupression and/or immunostimulation Citation: Tuntufye HN (2012). The use of RIVET to identify avian pathogenic Escherichia coli (APEC) genes induced in vivo during infection in chickens. Front. Immunol. Conference Abstract: ECMIS - E. coli and the Mucosal Immune System : Interaction, Modulation and Vaccination. doi: 10.3389/conf.fimmu.2012.01.00009 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 09 Jan 2012; Published Online: 12 Jan 2012. * Correspondence: Mr. Huruma N Tuntufye, Catholic University of Leuven, Faculty of Bioscience Engineering, Heverlee, 3001, Belgium, Huruma.Tuntufye@biw.kuleuven.be Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Huruma N Tuntufye Google Huruma N Tuntufye Google Scholar Huruma N Tuntufye PubMed Huruma N Tuntufye Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Chemopreventive effect of cactus Opuntia ficus indica on oxidative stress and genotoxicity of aflatoxin B1.

BackgroundAflatoxin B1 (AFB1) is potent hepatotoxic and hepatocarcinogenic agent. In aflatoxicosis, oxidative stress is a common mechanism contributing to initiation and progression of hepatic damage. The aim of this work was to evaluate the hepatoprotective effect of cactus cladode extract (CCE) on aflatoxin B1-induced liver damage in mice by measuring malondialdehyde (MDA) level, the protein carbonyls generation and the heat shock proteins Hsp 70 and Hsp 27 expressions in liver. We also looked for an eventual protective effect against AFB1-induced genotoxicity as determined by chromosome aberrations test, SOS Chromotest and DNA fragmentation assay. We further evaluated the modulation of p53, bax and bcl2 protein expressions in liver.MethodsAdult, healthy balbC (20-25 g) male mice were pre-treated by intraperitonial administration of CCE (50 mg/Kg.b.w) for 2 weeks. Control animals were treated 3 days a week for 4 weeks by intraperitonial administration of 250 μg/Kg.b.w AFB1. Animals treated by AFB1 and CCE were divided into two groups: the first group was administrated CCE 2 hours before each treatment with AFB1 3 days a week for 4 weeks. The second group was administrated without pre-treatment with CCE but this extract was administrated 24 hours after each treatment with AFB1 3 days a week for 4 weeks.ResultsOur results clearly showed that AFB1 induced significant alterations in oxidative stress markers. In addition, it has a genotoxic potential and it increased the expression of pro apoptotic proteins p53 and bax and decreased the expression of bcl2. The treatment of CCE before or after treatment with AFB1, showed (i) a total reduction of AFB1 induced oxidative damage markers, (ii) an anti-genotoxic effect resulting in an efficient prevention of chromosomal aberrations and DNA fragmentation compared to the group treated with AFB1 alone (iii) restriction of the effect of AFB1 by differential modulation of the expression of p53 which decreased as well as its associated genes such as bax and bcl2.ConclusionWe concluded that CCE might have a hepatoprotective effect against aflatoxicosis in mice, probably acting by promoting the antioxidant defence systems.

Multi-walled carbon nanotubes (MWCNT): Induction of DNA damage in plant and mammalian cells

Increasing use of multiwalled carbon nanotubes (MWCNT) necessitates an improved understanding of their potential impact on environment health. In the present study we evaluated the genotoxicity of MWCNT on plant and mammalian test systems. Genotoxic responses such as chromosomal aberrations and DNA strand breakages were studied in Allium cepa, human lymphocytes, mouse bone marrow cells and pBR322 plasmid DNA. Results showed that MWCNT could cause chromosomal aberrations, DNA fragmentation and apoptosis in Allium root cells that could be correlated with the internalization of MWCNT in the plant cells. In human lymphocytes significant genotoxic response was observed at the concentration 2μg/ml. Higher concentrations led to a decrease in values of the tail DNA percent that may be due to the formation of crosslinks. Annexin V-FITC–PI staining indicated only a small percentage of cells were undergoing apoptosis. Genotoxic effects were shown by micronuclei (MN) frequencies in experiments on mouse bone marrow cells. In the cell free DNA system (plasmid pBR322), a strong correlation between DNA strand break and concentration was observed. Based on the findings of the present study MWCNT may have significant impact on genomic activities.

Mitotic Catastrophe Occurs in the Absence of Apoptosis in p53-Null Cells with a Defective G1 Checkpoint

Cell death occurring during mitosis, or mitotic catastrophe, often takes place in conjunction with apoptosis, but the conditions in which mitotic catastrophe may exhibit features of programmed cell death are still unclear. In the work presented here, we studied mitotic cell death by making use of a UV-inactivated parvovirus (adeno-associated virus; AAV) that has been shown to induce a DNA damage response and subsequent death of p53-defective cells in mitosis, without affecting the integrity of the host genome. Osteosarcoma cells (U2OSp53DD) that are deficient in p53 and lack the G1 cell cycle checkpoint respond to AAV infection through a transient G2 arrest. We found that the infected U2OSp53DD cells died through mitotic catastrophe with no signs of chromosome condensation or DNA fragmentation. Moreover, cell death was independent of caspases, apoptosis-inducing factor (AIF), autophagy and necroptosis. These findings were confirmed by time-lapse microscopy of cellular morphology following AAV infection. The assays used readily revealed apoptosis in other cell types when it was indeed occurring. Taken together the results indicate that in the absence of the G1 checkpoint, mitotic catastrophe occurs in these p53-null cells predominantly as a result of mechanical disruption induced by centrosome overduplication, and not as a consequence of a suicide signal.

Knockout and pullout recombineering for naturally transformable Burkholderia thailandensis and Burkholderia pseudomallei

Phage λ-Red proteins are powerful tools for pulling and knocking out chromosomal fragments but have been limited to the γ-proteobacteria. Procedures are described here to easily knock out (KO) and pull out (PO) chromosomal DNA fragments from naturally transformable Burkholderia thailandensis and Burkholderia pseudomallei. This system takes advantage of published compliant counterselectable and selectable markers (sacB, pheS, gat and the arabinose-utilization operon) and λ-Red mutant proteins. pheS-gat (KO) or oriT-ColE1ori-gat-ori1600-rep (PO) PCR fragments are generated with flanking 40- to 45-bp homologies to targeted regions incorporated on PCR primers. One-step recombination is achieved by incubation of the PCR product with cells expressing λ-Red proteins and subsequent selection on glyphosate-containing medium. This procedure takes ~10 d and is advantageous over previously published protocols: (i) smaller PCR products reduce primer numbers and amplification steps, (ii) PO fragments suitable for downstream manipulation in Escherichia coli are obtained and (iii) chromosomal KO increases flexibility for downstream processing.