Hairy chinch bug (Hemiptera: Lygaeidae) damage, population density, and movement in relation to the incidence of perennial ryegrass infected by Neotyphodium endophytes

Studies examined hairy chinch bug, Blissus leucopterus hirtus Montandon, damage, population density, and movement in stands of perennial ryegrass, Lolium perenne L., containing various proportions of endophyte infected plants (E+). Our main objective was to determine the utility of mixtures containing E+ for management of chinch bugs. Chinch bug damage and population density decreased linearly as the proportion of E+ increased. This trend held true even when chinch bug populations were extremely high. Chinch bug nymphs emigrated more quickly from stands containing 100% E+ than they did from stands containing 50 or 0% E+, whereas adult chinch bug emigration was relatively unaffected by the proportion of E+. Our results indicate that turfgrass mixtures containing E+ can reduce chinch bug damage and population density.     

The degree of ultraviolet light damage to DNA containing iododeoxyuridine or bromodeoxyuridine is dependent on the DNA sequence.

The sequence selectivity of 300 nm ultraviolet light damage to DNA containing bromodeoxyuridine or iododeoxyuridine was examined on DNA sequencing gels. This was accomplished using a system where an M13 template was employed to direct synthesis of DNA in which thymidine was fully substituted with bromodeoxyuridine or iododeoxyuridine. The sites of damage corresponded to the positions of analogue incorporation. The extent of damage varied considerably at different sites of cleavage and ranged from the undetectable to over fifteen times the limit of detection (as assessed by laser densitometer scans). Strong damage sites had the "consensus" sequence CTT while sites of no detectable damage had the "consensus" sequence GTR. Bromodeoxyuridine and iododeoxyuridine had the same sites of damage although the extent of damage varied at different sites and bromodeoxyuridine damage was slightly greater than iododeoxyuridine. DNA containing thymidine was not damaged to any detectable level in this system with 300 nm ultraviolet light. The use of three closely related DNA sequences as targets for damage confirmed that (1) the sites of analogue incorporation are the cause of ultraviolet damage; and (2) that the neighbouring DNA sequence is an important parameter in determining the extent of damage. It is proposed that the microstructure of DNA--in particular the distance between the 5-carbon of the pyrimidine base (which is attached to the halogen) and hydrogen on the 2' carbon of the 5'-deoxyribose--ultimately determines the degree of cleavage with large distances giving a small degree of damage and smaller distances a large degree of damage.     

Differential repair of DNA damage in specific nucleotide sequences in monkey cells.

An immunological method was developed that isolates DNA fragments containing bromouracil in repair patches from unrepaired DNA using a monoclonal antibody that recognizes bromouracil. Cultured monkey cells were exposed to either UV light or the activated carcinogen aflatoxin B1 and excision repair of damage in DNA fragments containing the integrated and transcribed E. coli gpt gene was compared to that in the genome overall. A more rapid repair, of both UV and AFB1 damage was observed in the DNA fragments containing the E. coli gpt genes. The more efficient repair of UV damage was not due to a difference in the initial level of pyrimidine dimers as determined with a specific UV endonuclease. Consistent with previous observations using different methodology, repair of UV damage in the alpha sequences was found to occur at the same rate as that in the genome overall, while repair of AFB1 damage was deficient in alpha DNA. The preferential repair of damage in the gpt gene may be related to the functional state of the sequence and/or to alterations produced in the chromatin conformation by the integration of plasmid sequences carrying the gene.     

Whole cell yeast biotransformations in two-phase systems: Effect of solvent on product formation and cell structure

Summary Biotransformation of benzaldehyde and pyruvate to (R)-phenylacetyl carbinol bySaccharomyces cerevisiae was investigated in two-phase aqueous-organic reaction media. With hexane as organic solvent, maximum biotransformation activity was observed with a moisture content of 10%. Of the organic solvents tested, highest biotransformation activities were observed with hexane and hexadecane, and lowest activities occurred with chloroform and toluene. Biocatalyst samples from biphasic media containing hexane, decane and toluene manifested no apparent cell structural damage when examined using scanning electron microscopy. In contrast, cellular biocatalyst recovered from two-phase systems containing chloroform, butylacetate and ethylacetate exhibited damage in the form of cell puncturing after different incubation periods. Phospholipids were detected in reaction media from biocatalytic systems which exhibited cell damage in electron micrographs. Phospholipid release was much lower in the two-phase systems containing toluene or hexane or in 100% aqueous biocatalytic system.     

Structural Maintenance of Chromosomes Flexible Hinge Domain Containing 1 (SMCHD1) Promotes Non-homologous End Joining and Inhibits Homologous Recombination Repair upon DNA Damage

Structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1) has been shown to be involved in gene silencing and DNA damage. However, the exact mechanisms of how SMCHD1 participates in DNA damage remains largely unknown. Here we present evidence that SMCHD1 recruitment to DNA damage foci is regulated by 53BP1. Knocking out SMCHD1 led to aberrant γH2AX foci accumulation and compromised cell survival upon DNA damage, demonstrating the critical role of SMCHD1 in DNA damage repair. Following DNA damage induction, SMCHD1 depletion resulted in reduced 53BP1 foci and increased BRCA1 foci, as well as less efficient non-homologous end joining (NHEJ) and elevated levels of homologous recombination (HR). Taken together, these results suggest an important function of SMCHD1 in promoting NHEJ and repressing HR repair in response to DNA damage.     

Effect of supplementing fava bean (Vicia faba L.) on ulcerative colitis and colonic mucosal DNA content in rats fed a high-sucrose diet

Ulcerative colitis (UC) is an inflammatory bowel disease with high morbidity. Acetic acid-induced damage of colonic mucosa in rats is a commonly used experimental animal model of UC. This research aimed to explore for the first time the ameliorative effect of dietary supplementation with fava bean on the incidence of UC in rats fed with sucrose containing diet. Rats were divided into five groups as follows: G1, control healthy rats; G2, colitic rats; G3, colitic rats fed diets containing 30% sucrose, G4, healthy rats fed diets containing 30% sucrose and G5, colitic rats fed diets containing 30% sucrose supplemented with dried ground fava bean. Colonic injury and inflammation were evaluated through a disturbance of oxidative biomarkers, a significant increase in inflammatory biomarkers and inflammatory cytokines, and histological abnormalities in colonic tissues accompanied by colonic mucosal DNA damage. Colitic rats fed on sucrose containing diet demonstrated additional histological, biochemical, and DNA alterations in colonic mucosa of rats. Dietary supplementation with dried ground fava bean significantly corrected the impaired oxidative and inflammatory biomarker levels and modulated histological features and DNA alterations. Finally, fava bean attenuated the oxidative damage and colonic injury induced by acetic acid, which confirmed its high anti-oxidant and anti-incendiary properties.     

Effect of polyunsaturated fatty acids on diphenyl hydantoin-induced genetic damage in vitro and in vivo

Phenytoin sodium/diphenyl hydantoin (DPH) is used by a major segment of epileptics and neuro surgery patients with head injury to prevent seizures. DPH is a known mutagen, carcinogen, and teratogen. Essential fatty acids (EFAs) are critical for various tissues and were reported to act as anti-mutagenic agents. In the present study we assessed the effect of five EFAs on DPH-induced genetic damage both in vitro and in vivo. DPH induced significant genetic damage. Of all the EFAs (linoleic acid, α-linolenic acid, gamma-linolenic acid, arachidonic acid, dihomo-gamma-linolenic acid, and eicosapentaenoic acid) studied, all except eicosapentaenoic acid showed significant decrease in DPH induced genetic damage as assessed by micronucleus (MN) test. However, gamma-linolenic acid (GLA) was found to be the most effective in reducing the number of MN containing lymphocytes both in vitro and in vivo to control values. EFAs when tested alone produced insignificant increase in the amount of genetic damage but when tested in combination with DPH the number of micronuclei containing lymphocytes was reduced; but the DNA ladder pattern, an indication of DNA damage, was increased. This apparently paradoxical action of EFAs, especially of GLA, suggests that, in all probability, fatty acids induce apoptosis of cells that harbor significant DNA damage. Based on these results we suggest that GLA functions as a unique endogenous molecule that protects cells from accumulating genetic damage.     

Changes in chromatin structure and mobility in living cells at sites of DNA double-strand breaks

The repair of DNA double-strand breaks (DSBs) is facilitated by the phosphorylation of H2AX, which organizes DNA damage signaling and chromatin remodeling complexes in the vicinity of the lesion. The disruption of DNA integrity induces an alteration of chromatin architecture that has been proposed to activate the DNA damage transducing kinase ataxia telangiectasia mutated. However, little is known about the physical properties of damaged chromatin. In this study, we use a photoactivatable version of GFP-tagged histone H2B to examine the mobility and structure of chromatin containing DSBs in living cells. We find that chromatin containing DSBs exhibits limited mobility but undergoes an energy-dependent local expansion immediately after DNA damage. The localized expansion observed in real time corresponds to a 30-40% reduction in the density of chromatin fibers in the vicinity of DSBs, as measured by energy-filtering transmission electron microscopy. The observed opening of chromatin occurs independently of H2AX and ATM. We propose that localized adenosine triphosphate-dependent decondensation of chromatin at DSBs establishes an accessible subnuclear environment that facilitates DNA damage signaling and repair.     

Eco1 is important for DNA damage repair in S. cerevisiae

The cohesin network has an essential role in chromosome segregation, but also plays a role in DNA damage repair. Eco1 is an acetyltransferase that targets subunits of the cohesin complex and is involved in both the chromosome segregation and DNA damage repair roles of the network. Using budding yeast as a model system, we find that mutations in Eco1, including a genocopy of a human Roberts syndrome allele, do not cause gross defects in chromosome cohesion. We examined how mitotic and meiotic DNA damage repair is affected by mutations in Eco1. Strains containing mutations in Eco1 are sensitive to DNA damaging agents that cause double-strand breaks, such as Xrays and bleomycin. While meiotic crossing over is relatively unaffected in strains containing the Roberts mutation, reciprocal mitotic crossovers occur with extremely low frequency in this mutant background. Our results suggest that Eco1 promotes the reciprocal exchange of chromosome arms and maintenance of heterozygosity during mitosis.     

Liquid holding recovery in stationary and log phase cultures of diploid yeast exposed to gamma and alpha radiations

Summary The kinetics of liquid-holding recovery (LHR) in diploid yeast after gamma and alpha irradiation is studied. In case of stationary phase culture the rate and extent of LHR is found to be greater for gamma-ray-induced damage than for alpha-ray-induced damage. At 10% survival level, the half-time for recovery is 5.2 h for gamma-ray damage and 12 h for alpha-ray damage. Further, while the recovery factor for alpha damage reaches saturation at 5% survival level, that for gamma damage continues to increase as survival level decreases. Oxygen is required for the recovery process during LH after gamma irradiation. The cells can recover to the same extent from both oxygen-dependent and oxygen-independent components of damage. Log phase cells containing a high per cent of budding cells, however, exhibit negative liquid holding effect after gamma irradiation.     

Action of 1,1-dimethylhydrazine on bacterial cells is determined by hydrogen peroxide

Seven different recombinant bioluminescent strains of Escherichia coli containing, respectively, the promoters katG and soxS (responsive to oxidative damage), recA (DNA damage), fabA (membrane damage), grpE, and rpoE (protein damage) and lac (constitutive expression) fused to the bacterial operon from Photorhabdus luminescens, were used to describe the mechanism of toxicity of 1,1-dimethylhydrazine (1,1-DMH) on bacteria, as well as to determine whether bacteria can sensitively detect the presence of this compound. A clear response to 1,1-DMH was observed only in E. coli carrying the katG'::lux, soxS'::lux, and recA'::lux-containing constructs. Preliminary treatment with catalase of the medium containing 1,1-DMH completely diminished the stress-response of the P(katG), P(recA), and P(soxS) promoters. In the strain E. coli (pXen7), which contains a constitutive promoter, the level of cellular toxicity caused by the addition of 1,1-DMH was dramatically reduced in the presence of catalase. It is suggested that the action of 1,1-DMH on bacterial cells is determined by hydrogen peroxide, which is formed in response to reduction of the air oxygen level.     

Resistance of insecticide-treated foam board insulation against the eastern subterranean termite and the Formosan subterranean termite (Isoptera: Rhinotermitidae)

Three foam board types, one untreated control, one containing 2,000 ppm disodium octaborate tetrahydrate (DOT), and one containing 1,000 ppm deltamethrin, were exposed to field populations of the eastern subterranean termite, Reticulitermes flavipes (Kollar), and the Formosan subterranean termite, Coptotermes formosanus Shiraki. There was no significant difference in termite damage between foam boards treated with 2,000 ppm DOT and the untreated control. Form boards containing 1,000 ppm deltamethrin were not damaged by R. flavipes, whereas only minor damage occurred after exposure to C. formosanus.     

Enzymatic properties of Escherichia coli and human 7,8-dihydro-8-oxoguanine DNA glycosylases.

Oxidative damage to DNA generates aberrant guanine bases such as 2,6-diamino-4-hydroxy-formamido-pyrimidine (Fapy) and 7,8-dihydro-8-oxoguanine (8-oxoG). Although synthetic oligonucleotides containing a single 8-oxoG have been widely used to study enzymatic processing of this lesion, the synthesis of oligonucleotides containing Fapy as a unique lesion has not been achieved to date. In this study, an oligonucleotide containing a single 2,6-diamino-4-hydroxy-5-(N-methyl)formamido-pyrimidine (me-Fapy, a methylated derivative of Fapy) was prepared by a DNA polymerase reaction and the subsequent alkali treatment. The repair activity of Fpg and hOGG1 proteins were compared using oligonucleotide substrates containing me-Fapy and 8-oxoG.     

Mechanism of radiosensitization by halogenated pyrimidines: effect of BrdU on radiation induction of DNA and chromosome damage and its correlation with cell killing

The effect of BrdU incorporation on cell radiosensitivity as well as on the induction of DNA double-strand breaks (DSB) and chromosome damage by radiation was studied in CHO cells. Induction of DNA DSB was measured by the nonunwinding filter elution technique and damage at the chromosome level was visualized and scored in G1 cells using the technique of premature chromosome condensation. The results indicated an increase in the radiosensitivity of cells grown in the presence of BrdU. Although sensitization was observed both in cells irradiated in the exponential phase and in cells irradiated in the plateau phase of growth, the degree of sensitization was greater in exponentially growing cells for the same degree of thymidine replacement by BrdU in the DNA. It is hypothesized that this indicates the possible importance of chromatin structure at the time of irradiation and/or the importance of chromatin conformation changes after irradiation in the expression of radiation-induced potentially lethal damage in cells containing BrdU. Incorporation of BrdU affected both the slope and the width of the shoulder of the survival curve and increased the induction of DNA and chromosome damage per unit absorbed dose. The increase observed in the slope of the survival curve was quantitatively similar to the increase observed in damage induction at the DNA and the chromosome level, suggesting a cause-effect relationship between these phenomena. Reduction in the width of the shoulder did not correlate with the increase in the induction of DNA and chromosome damage, suggesting that different phenomena, probably related to enhanced fixation of radiation-induced potentially lethal damage in cells containing BrdU, underlie its modulation.     

Eco1 is important for DNA damage repair in S. cerevisiae

The cohesin network has an essential role in chromosome segregation, but also plays a role in DNA damage repair. Eco1 is an acetyltransferase that targets subunits of the cohesin complex and is involved in both the chromosome segregation and DNA damage repair roles of the network. Using budding yeast as a model system, we find that mutations in Eco1, including a genocopy of a human Roberts syndrome allele, do not cause gross defects in chromosome cohesion. We examined how mitotic and meiotic DNA damage repair is affected by mutations in Eco1. Strains containing mutations in Eco1 are sensitive to DNA damaging agents that cause double-strand breaks, such as X-rays and bleomycin. While meiotic crossing over is relatively unaffected in strains containing the Roberts mutation, reciprocal mitotic crossovers occur with extremely low frequency in this mutant background. Our results suggest that Eco1 promotes the reciprocal exchange of chromosome arms and maintenance of heterozygosity during mitosis.Key words: cohesin, recombination, double-strand break, acetyltransferase, Roberts syndrome     

Clustered DNA damage induced by gamma radiation in human fibroblasts (HF19), hamster (V79-4) cells and plasmid DNA is revealed as Fpg and Nth sensitive sites

The signature DNA lesion induced by ionizing radiation is clustered DNA damage. Gamma radiation-induced clustered DNA damage containing base lesions was investigated in plasmid DNA under cell mimetic conditions and in two cell lines, V79-4 (hamster) and HF19 (human), using bacterial endonucleases Nth (endonuclease III) and Fpg (formamidopyrimidine DNA glycosylase). Following irradiation with ⁶⁰Co γ-rays, induction of double-strand breaks (DSB) and clustered DNA damage, revealed as DSB by the proteins, was determined in plasmid using the plasmid-nicking assay and in cells by either conventional pulsed field gel electrophoresis or a hybridization assay, in which a 3 Mb restriction fragment of the X chromosome is used as a radioactive labeled probe. Enzyme concentrations (30–60 ng/µg DNA) were optimized to minimize visualization of background levels of endogenous DNA damage and DSB produced by non-specific cutting by Fpg and Nth in cellular DNA. ⁶⁰Co γ- radiation produces a 1.8-fold increase in the yields of both types of enzyme sensitive sites, visualized as DSB compared with that of prompt DSB in plasmid DNA. In mammalian cells, the increase in yields of clustered DNA damage containing either Fpg or Nth sensitive sites compared with that of prompt DSB is 1.4–2.0- and 1.8-fold, respectively. Therefore, clustered DNA damage is induced in cells by sparsely ionizing radiation and their yield is significantly greater than that of prompt DSB.     

p53 is required for nuclear but not mitochondrial DNA damage-induced degeneration

While the consequences of nuclear DNA damage have been well studied, the exact consequences of acute and selective mitochondrial DNA (mtDNA) damage are less understood. DNA damaging chemotherapeutic drugs are known to activate p53-dependent apoptosis in response to sustained nuclear DNA damage. While it is recognized that whole-cell exposure to these drugs also damages mtDNA, the specific contribution of mtDNA damage to cellular degeneration is less clear. To examine this, we induced selective mtDNA damage in neuronal axons using microfluidic chambers that allow for the spatial and fluidic isolation of neuronal cell bodies (containing nucleus and mitochondria) from the axons (containing mitochondria). Exposure of the DNA damaging drug cisplatin selectively to only the axons induced mtDNA damage in axonal mitochondria, without nuclear damage. We found that this resulted in the selective degeneration of only the targeted axons that were exposed to DNA damage, where ROS was induced but mitochondria were not permeabilized. mtDNA damage-induced axon degeneration was not mediated by any of the three known axon degeneration pathways: apoptosis, axon pruning, and Wallerian degeneration, as Bax-deficiency, or Casp3-deficiency, or Sarm1-deficiency failed to protect the degenerating axons. Strikingly, p53, which is essential for degeneration after nuclear DNA damage, was also not required for degeneration induced with mtDNA damage. This was most evident when the p53-deficient neurons were globally exposed to cisplatin. While the cell bodies of p53-deficient neurons were protected from degeneration in this context, the axons farthest from the cell bodies still underwent degeneration. These results highlight how whole cell exposure to DNA damage activates two pathways of degeneration; a faster, p53-dependent apoptotic degeneration that is triggered in the cell bodies with nuclear DNA damage, and a slower, p53-independent degeneration that is induced with mtDNA damage.Subject terms: Cell biology, Neuroscience     

Quantitative analysis of mitochondrial flocculent densities in rat hepatocytes during normothermic and hypothermic ischemia in vitro

The development of flocculent densities in mitochondria as a sign of irreversible cell injury in rat hepatocytes has been studied by quantitative electron microscopy during in vitro ischemia under both normothermic (37 degrees C) and hypothermic (4 degrees C) conditions. At 37 degrees C flocculent densities first appear after 1 h ischemia; at this stage they are small in diameter (170 nm) and occur in only 8% of mitochondria. After 1.5 hour ischemia, flocculent densities increase in diameter (207 nm) and are seen in 37% of mitochondria. Death of the majority of hepatocytes seems to occur between 1.5 and 2 h ischemia since at this stage the percentage of mitochondria containing flocculent densities reaches a maximum (48%). However, flocculent densities continue to increase in size (to 337 nm diam.) up to between 2 and 4 h ischemia (the prenecrotic phase). In contrast, at 4 degrees C signs of ischemic damage to hepatocytes are considerably delayed. Flocculent densities of comparable size and frequency to those observed after 1 h ischemia at 37 degrees C are not seen till as late as 4 days at 4 degrees C. At the latter temperature, only after 7 days ischemia a substantial rise (to about 25%) in the proportion of mitochondria containing flocculent densities occurs. A further slow increase in size and in the percentage of mitochondria containing densities occurs up to 14 days ischemia at 4 degrees C. It is concluded that the development of flocculent densities may be used only as a parameter of irreversible damage in cells with a sufficient number of mitochondria, such as hepatocytes, under normothermic conditions. With ischemia at 4 degrees C, possibly due to a different protein denaturation pattern, the development of flocculent densities is of much less value as an indication of irreversible cell damage and cannot, therefore, be considered as a reliable sign of cellular damage in organs stored at 4 degrees C for transplantation purposes.     

Effect of protective agents on amount and repair of sublethal freeze--thaw damage in mammalian cells.

Glycerol, DMSO, and HES are able to reduce by a factor of 2 the sublethal damage produced in mammalian cells after one freeze-thaw cycle. When sublethal freeze-thaw damage is already present, DMSO and HES are able to prevent about half of this damage from becoming lethal when a second freeze-thaw cycle is applied. Glycerol is only able to do this if dilution shock is avoided by thawing the cells into medium containing glycerol. The cells can repair 100% of this sublethal damage and do so in 2–3 hr at 37 °C in suspension. The data imply that the sites protected by DMSO, HES, and glycerol are the same as the sites repaired by the cells. The results also suggest that cells stop progressing in the cell cycle while repairing sublethal freeze-thaw damage.