Oxygen effect in heat-induced DNA damage

The major kinds of heat-induced damage to DNA (depurination, guanine oxidation to 8-oxoguanine, cytosine deamination to uracil) were shown to depend in their extent on the oxygen content in solution. Formation of hydrogen peroxide in water upon heating was enhanced in the presence of D₂O and decreased by various scavengers of singlet oxygen, corroborating the involvement of ¹O₂ in the thermal generation of reactive oxygen species. The aggregate data indicate that all kinds of heat-induced DNA damage in solution arise through this common mechanism.     

Competition effect in DNA damage response

We have built an ion-microbeam for studies of the nuclear topography and kinetics of double-strand break repair at the single cell level. Here, we show that a first and a second, delayed single ion exposure at different nuclear sites led to comparable accumulations of phospho-ATM, γ-H2AX and Mdc1 at both earlier (e) and later (l) microirradiated sites. In contrast, accumulations of 53BP1 and the recombination protein Rad51 were strongly reduced at l-sites. This apparent competition effect is accompanied by a reduced amount of 53BP1 in undamaged areas of the irradiated nuclei. We suggest that a critically limited pool size combined with strong binding at irradiated sites leads to the exhaustion of unbound factors freely roaming the nuclear space. The undersupply of these factors at l-sites requires in addition a long-lasting binding at e-sites or a weaker binding at l-sites. The observed effects suggest that DNA damage response at individual nuclear sites depends on the time course of damage load. This may have implications for therapeutic radiation treatments. Christoph Greubel, Volker Hable and Guido A. Drexler contributed equally to this work.     

Bigger mothers are better mothers: disentangling size-related prenatal and postnatal maternal effects

Despite a vast literature on the factors controlling adult size, few studies have investigated how maternal size affects offspring size independent of direct genetic effects, thereby separating prenatal from postnatal influences. I used a novel experimental design that combined a cross-fostering approach with phenotypic manipulation of maternal body size that allowed me to disentangle prenatal and postnatal maternal effects. Using the burying beetle Nicrophorus vespilloides as model organism, I found that a mother''s body size affected egg size as well as the quality of postnatal maternal care, with larger mothers producing larger eggs and raising larger offspring than smaller females. However, with respect to the relative importance of prenatal and postnatal maternal effects on offspring growth, only the postnatal effects were important in determining offspring body size. Thus, prenatal effects can be offset by the quality of postnatal maternal care. This finding has implications for the coevolution of prenatal and postnatal maternal effects as they arise as a consequence of maternal body size. In general, my study provides evidence that there can be transgenerational phenotypic plasticity, with maternal size determining offspring size leading to a resemblance between mothers and their offspring above and beyond any direct genetic effects.     

The effect of chromatin decondensation on DNA damage and repair

The effects of chromatin compaction on X-radiation-induced cell killing and the induction and repair of DNA damage were studied in Chinese hamster ovary cells deprived of isoleucine for 24 h (Ile- cells) and compared to untreated controls. The results show that chromatin is decondensed in Ile- cells; i.e., in Ile- cells the nuclear area occupied by heterochromatin decreased 30-fold over control cells, both the rate and limit of micrococcal nuclease digestion were greater for Ile- cells, and 14.2% more propidium iodide was intercalated into the Ile- cell chromatin. The X-ray-induced cytotoxicity did not change in Ile- cells versus the control cells (D0 = 0.99 Gy) nor did the X-ray-induced DNA damage. However, the repair of DNA damage produced by 10 Gy proceeded with different kinetics in Ile- cells when compared to the controls. The initial rate of DNA damage repair was slower in Ile- cells by a factor of 2 compared to controls (the time required to rejoin 50% of the lesions was 6 versus 3 min, respectively). However, after 2 h of repair no DNA damage was detected in either group. Therefore, we conclude that this decondensation of chromatin, per se, does not directly modify the induction or ultimate repair of DNA damage by X radiation or cell clonogenicity and thus does not appear to be a primary factor in cell survival.     

Y-family DNA polymerases and their role in tolerance of cellular DNA damage

The past 15 years have seen an explosion in our understanding of how cells replicate damaged DNA and how this can lead to mutagenesis. The Y-family DNA polymerases lie at the heart of this process, which is commonly known as translesion synthesis. This family of polymerases has unique features that enable them to synthesize DNA past damaged bases. However, as they exhibit low fidelity when copying undamaged DNA, it is essential that they are only called into play when they are absolutely required. Several layers of regulation ensure that this is achieved.     

Oxygen effect in heat-mediated damage to DNA

The effects of gassing conditions in DNA solution on the major types of heat-mediated DNA damage (depurination of DNA, generation of 8-oxoguanine, cytosine deamination with the formation of uracil) have been studied by ELISA, column liquid chromatography, and spectrophotometry. It was found that the number of DNA lesions depends on oxygen concentration in solution; i.e., the oxygen effect takes place. The heat-induced generation of hydrogen peroxide in water increased after the addition of D20 and decreased by the action of various 1O2 quenchers, suggesting that singlet oxygen is involved in the heat-induced production of reactive oxygen species (ROS) in water. The data obtained favor the hypothesis that all the types of heat-induced damage to DNA are due to a common mechanism associated with the heat-mediated generation of reactive oxygen species in solution.     

Ubiquitin-binding domains and their role in the DNA damage response

The modification of eukaryotic proteins by covalent attachment of ubiquitin is a versatile signaling event with a wide range of possible consequences. Canonical poly-ubiquitination by Lys-48 linked chains usually destines a protein for degradation by the proteasome. By contrast, attachment of a single ubiquitin or ubiquitin chains linked through Lys-63 or Lys-6 serves a non-proteolytic role. Over the last years, evidence has accumulated that several nuclear proteins become ubiquitinated in response to DNA damage. Typically, these proteins carry mono-ubiquitin or non-classical ubiquitin chains and are localized close to the site of DNA damage. Of particular interest are PCNA and the variant histone H2AX, two key proteins whose ubiquitination serves to recruit factors needed by the cell to cope with the damage. A prerequisite for docking effector proteins to the site of the lesion is the detection of a specific ubiquitin modification, a process that can be mediated by a range of dedicated ubiquitin-binding domains (UBDs). As the same types of ubiquitin modification are involved in entirely different processes, the recognition of the ubiquitin mark has to go along with the recognition of the modified protein. Thus, ubiquitin-binding domains gain their specificity through combination with other recognition domains and motifs. This review discusses ubiquitin-binding domains relevant to the DNA damage response, including their binding mode, their specificity, and their interdependence with other factors. For several repair pathways, current knowledge of the events downstream of the ubiquitin mark is sketchy. A closer look at orphan UBD proteins might lead to the identification of missing pieces in the DNA response puzzle.     

The chromolipoids: Dependance of histochemical characteristics on the degree of polymerization and their differentiation from DNA

Summary Chromolipoid pigments prepared in vitro were found to be a mixture of compounds of different degrees of polymerization and different histochemical characteristics. Some fractions give positive Feulgen nucleal reaction, stain with methyl-green and absorb ultraviolet light, thus closely mimicking DNA. Ways of differentiating such chromolipoids from DNA are pointed out.Similar chromolipoids have been produced in rats by injecting subcutaneously cod liver oil, or by chronic poisoning by lead.With 4 Figures in the Text, of which 2 in ColourThis study is part of a thesis submitted by Mr.S. Shoshan to the Hebrew University Hadassah Medical School in partial fulfilment of the requirements for the title of Master of Medical Sciences.     

Motherless mothers revisited: Rhesus maternal behavior and rearing history

Classic studies have demonstrated that isolation- and peer-rearing in infancy can result in overt deficits in maternal behavior in rhesus monkeys, although nonmother-reared monkeys may become adequate caretakers if allowed experience with infants. Maternal styles in normally-reared mothers have been reported to vary widely. In the present study, the range of maternal proficiency of nonabusive, nonrejecting nonmother-reared mothers was examined. The behavior of mother-reared, peer-reared, and isolate-reared multiparous mothers was observed and compared for the first six months following parturition. Both peer-reared and isolate-reared mothers exhibited differences in maternal behavior from mother-reared mothers. Inasmuch as variations in maternal behavior may impact on the infant, investigators should be aware of the rearing histories of females used as breeders in the laboratory.     

The DNA damage response: sensing and signaling

The protein kinases ATM and ATR are central components of the checkpoint mechanisms that signal the presence of damaged DNA and stalled replication forks. Recent studies have provided important new insights into how these kinases work together with their regulatory subunits, DNA repair proteins and adaptor proteins to sense abnormal DNA structures and implement the appropriate DNA damage response. These advances have provided a more detailed understanding of the interface between damaged DNA and the checkpoint sensor proteins.     

Perinatal depression and DNA methylation of oxytocin-related genes: a study of mothers and their children

The present study investigated the association of perinatal depression (PD) with differential methylation of 3 genomic regions among mother and child dyads: exon 3 within the oxytocin receptor (OXTR) gene and 2 intergenic regions (IGR) between the oxytocin (OXT) and vasopressin (AVP) genes. Maternal PD was assessed at 5 time-points during pregnancy and postpartum. Four groups were established based on Edinburgh Postnatal Depression Scale (EPDS) cut-off scores: no PD, prenatal or postpartum depressive symptoms only and persistent PD (depressive symptoms both prenatally and postpartum). Salivary DNA was collected from mothers and children at the final time-point, 2.9 years postpartum. Mothers with persistent PD had significantly higher overall OXTR methylation than the other groups and this pattern extended to 16/22 individual CpG sites. For the IGR, only the region closer to the AVP gene (AVP IGR) showed significant differential methylation, with the persistent PD group displaying the lowest levels of methylation overall, but not for individual CpG sites. These results suggest that transient episodes of depression may not be associated with OXTR hypermethylation. Validation studies need to confirm the downstream biological effects of AVP IGR hypomethylation as it relates to persistent PD. Differential methylation of the OXTR and IGR regions was not observed among children exposed to maternal PD. The consequences of OXTR hypermethylation and AVP IGR hypomethylation found in mothers with persistent PDS may not only impact the OXT system, but may also compromise maternal behavior, potentially resulting in negative outcomes for the developing child.     

Low-energy diet in atopic dermatitis patients: clinical findings and DNA damage

Undernutrition without malnutrition (low-energy diet) increases maximum longevity, reduces the incidence of several cancers and delays their onset, in animal studies. It has also been demonstrated by experimental study that caloric restriction provides a beneficial effect on various inflammatory diseases. In this study, we offered a low-energy diet to patients with atopic dermatitis (AD). Nineteen adult patients (5 males and 14 females aged 15 to 36 years) were enrolled in the study which lasted 8 weeks. The energy intake was 55% of nutritional requirements; protein was 75%, calcium 180%, iron 130%, vitamin A 105%, vitamin C 250% and vitamin E 110% of the daily requirement. No patient experienced adverse reaction, and none dropped out of the trial. Body weight, body mass index (BMI), and systolic blood pressure had decreased significantly by the end of study. The SCORAD (scoring atopic dermatitis) index, which combines objective (extent and intensity of lesions) and subjective (daytime pruritus and sleep loss) criteria, was reduced significantly. In 11 patients with severe AD, there was a significant reduction in oxidative DNA damage. The change in the inflammatory intensity score and the change in BMI caused by energy restriction showed a significant positive correlation. The change in oxidative DNA damage levels and the change in BMI showed a positive correlation. These results clarify the relationship between weight loss and the improvement of AD. It may be hypothesized that this low-energy diet which included several additional nutrients has a possibility to reduce inflammatory symptoms of patients with AD.     

The degree of DNA homology in enterococci

Studies carried out by the method of DNA-DNA hybridization have confirmed that Enterococcus faecalis, S. faecium and S. durans are independent species. As revealed in these studies, S. faecalis are homogeneous, genetically related and form an independent, sharply defined species, while S. faecium form a heterogeneous species. Mobile enterococci with yellow pigmentation must not be classified with both S. faecalis and S. faecium.     

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.     

Clustered DNA damage on subcellular level: effect of scavengers

Clustered DNA damages are induced by ionizing radiation, particularly of high linear energy transfer (LET). Compared to isolated DNA damage sites, their biological effects can be more severe. We investigated a clustered DNA damage induced by high LET radiation (C 290 MeV u^?1 and Fe 500 MeV u^?1) in pBR322 plasmid DNA. The plasmid is dissolved in pure water or in aqueous solution of one of the three scavengers (coumarin-3-carboxylic acid, dimethylsulfoxide, and glycylglycine). The yield of double strand breaks (DSB) induced in the DNA plasmid-scavenger system by heavy ion radiation was found to decrease with increasing scavenging capacity due to reaction with hydroxyl radical, linearly with high correlation coefficients. The yield of non-DSB clusters was found to occur twice as much as the DSB. Their decrease with increasing scavenging capacity had lower linear correlation coefficients. This indicates that the yield of non-DSB clusters depends on more factors, which are likely connected to the chemical properties of individual scavengers.     

Paradoxical effect of diphenyleneiodonium in inducing DNA damage and apoptosis

Diphenyleneiodonium (DPI) is often used as a molecular tool in unravelling redox-sensitive cellular events involving NADPH oxidase. However, to better understand unexpected actions of DPI, it was ascertained if DPI affects cellular DNA. DPI induced single-strand breaks in DNA of HCT-116 cells, although this only slightly increased GADD153 expression. Nevertheless, after sustaining DNA damage, the DPI-treated cells subsequently had features characteristic of apoptosis, such as translocated membrane phospholipid and nuclei containing condensed chromatin. Paradoxically, DPI attenuated the DNA damage and overall ROS production caused by sodium deoxycholate (DOC), although DPI did not inhibit DOC-induced generation of mitochondrial [image omitted] . Furthermore, DPI prevented the occurrence of apoptosis caused by DOC. However, other known chemical inhibitors of NADPH oxidase did not produce the same results as DPI in negating the effects of DOC. Collectively, these disparate findings suggest that DPI can act not in accord with conventional wisdom depending on the experimental conditions.     

Elevated plasma corticosterone decreases yolk testosterone and progesterone in chickens: linking maternal stress and hormone-mediated maternal effects

Despite considerable research on hormone-mediated maternal effects in birds, the underlying physiology remains poorly understood. This study investigated a potential regulation mechanism for differential accumulation of gonadal hormones in bird eggs. Across vertebrates, glucocorticoids can suppress reproduction by downregulating gonadal hormones. Using the chicken as a model species, we therefore tested whether elevated levels of plasma corticosterone in female birds influence the production of gonadal steroids by the ovarian follicles and thus the amount of reproductive hormones in the egg yolk. Adult laying hens of two different strains (ISA brown and white Leghorn) were implanted subcutaneously with corticosterone pellets that elevated plasma corticosterone concentrations over a period of nine days. Steroid hormones were subsequently quantified in plasma and yolk. Corticosterone-implanted hens of both strains had lower plasma progesterone and testosterone levels and their yolks contained less progesterone and testosterone. The treatment also reduced egg and yolk mass. Plasma estrogen concentrations decreased in white Leghorns only whereas in both strains yolk estrogens were unaffected. Our results demonstrate for the first time that maternal plasma corticosterone levels influence reproductive hormone concentrations in the yolk. Maternal corticosterone could therefore mediate environmentally induced changes in yolk gonadal hormone concentrations. In addition, stressful situations experienced by the bird mother might affect the offspring via reduced amounts of reproductive hormones present in the egg as well as available nutrients for the embryo.     

The effect of ancient DNA damage on inferences of demographic histories

The field of ancient DNA (aDNA) is casting new light on many evolutionary questions. However, problems associated with the postmortem instability of DNA may complicate the interpretation of aDNA data. For example, in population genetic studies, the inclusion of damaged DNA may inflate estimates of diversity. In this paper, we examine the effect of DNA damage on population genetic estimates of ancestral population size. We simulate data using standard coalescent simulations that include postmortem damage and show that estimates of effective population sizes are inflated around, or right after, the sampling time of the ancestral DNA sequences. This bias leads to estimates of increasing, and then decreasing, population sizes, as observed in several recently published studies. We reanalyze a recently published data set of DNA sequences from the Bison (Bison bison/Bison priscus) and show that the signal for a change in effective population size in this data set vanishes once the effects of putative damage are removed. Our results suggest that population genetic analyses of aDNA sequences, which do not accurately account for damage, should be interpreted with great caution.     

The effect of two cryopreservation methods on human sperm DNA damage

Several methods are currently available for selection when conducting sperm cryopreservation, however, these methods might cause different degrees of damage on sperm DNA. The aim of the this study is to compare the effects of storage at −80 °C (in ultra-low temperature refrigerator) and at −196 °C (in liquid nitrogen) on sperm DNA damage, thus to provide a reference for choosing the right method according to different aims. We randomly collected 28 semen samples from college students of Chongqing city. The samples stored at −80 °C were neat semen samples and the samples stored at −196 °C were mixed with additional cryoprotectants. Each sample was subjected to two freezing-thawing cycles, and the sperm DNA damage levels of fresh and thawed samples were measured by single cell gel electrophoresis (SCGE) and sperm chromatin structure assay (SCSA). Both SCGE and SCSA assays showed cryopreservation induced significant damage to sperm DNA. However, storage at −196 °C lead to more severe damage to sperm DNA than storage at −80 °C measured by SCSA. Sperm DNA damage increased simultaneously with the higher frequency of freezing-thawing cycles. We concluded that storage of neat semen samples at −80 °C had milder damage to sperm DNA than storage at −196 °C mixed with cryoprotectants. To avoid additional sperm DNA damage, repeated freezing and thawing should be prevented.