Ectopic expression of H2AX protein promotes TrkA-induced cell death via modulation of TrkA tyrosine-490 phosphorylation and JNK activity upon DNA damage

We previously reported that TrkA overexpression causes accumulation of γH2AX proteins in the cytoplasm, subsequently leading to massive cell death in U2OS cells. To further investigate how cytoplasmic H2AX is associated with TrkA-induced cell death, we established TrkA-inducible cells stably expressing GFP-tagged H2AX. We found that TrkA co-localizes with ectopically expressed GFP-H2AX proteins in the cytoplasm, especially at the juxta-nuclear membranes, which supports our previous results about a functional connection between TrkA and γH2AX in TrkA-induced cell death. γH2AX production from GFP-H2AX proteins was significantly increased when TrkA was overexpressed. Moreover, ectopic expression of H2AX activated TrkA-mediated signal pathways via up-regulation of TrkA tyrosine-490 phosphorylation. In addition, suppression of TrkA tyrosine-490 phosphorylation under a certain condition was removed by ectopic expression of H2AX, indicating a functional role of H2AX in the maintenance of TrkA activity. Indeed, TrkA-induced cell death was highly elevated by ectopic H2AX expression, and it was further accelerated by DNA damage via JNK activation. These all results suggest that cytoplasmic H2AX could play an important role in TrkA-mediated cell death by modulating TrkA upon DNA damage.     

Acetyltransferase p300 regulates NBS1-mediated DNA damage response

The role of p300 in DNA damage response is unclear. To understand how ATM-dependent phosphorylation of p300 affects its function in response to DNA damage, we present evidence that S106 of p300, which is phosphorylated by ATM, regulates stability of NBS1 and recruitment into damaged DNA, possibly leading to regulation of DNA repair. Non-phosphorylatable p300 (S106A) destabilized NBS1 and decreased NBS1–p300 interaction. The recruitment of NBS1 into damaged DNA was impaired in the presence of S106A. Also, a dominant negative p300 lacking enzymatic activity induced destabilization of NBS1, suggesting that its acetyltransferase is required for NBS1 stability. These results indicate that phosphorylation of p300 can regulate NBS1-mediated DNA damage response, and that these events occur in an acetylation-dependent manner.

Structured summary

MINT-8058074, MINT-8058083: p300 (uniprotkb:Q09472) physically interacts (MI:0915) with NBS1 (uniprotkb:O60934) by anti bait coimmunoprecipitation (MI:0006)MINT-8058111: p300 (uniprotkb:Q09472) and NBS1 (uniprotkb:O60934) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-8058657: p300 (uniprotkb:Q09472) physically interacts (MI:0915) with NBS1 (uniprotkb:O60934) by two hybrid (MI:0018)MINT-8058093: p300 (uniprotkb:Q09472) physically interacts (MI:0915) with NBS1 (uniprotkb:O60934) by anti tag coimmunoprecipitation (MI:0007)     

Dual effects of estrogen on vascular smooth muscle cells: receptor-mediated proliferative vs. metabolite-induced pro-senescent actions

Objective

To investigate the mechanism for the dual effects of estrogen on vascular smooth muscle cells (VSMCs).

Methods

Cultured rat VSMCs were exposed to gradient concentrations (10⁻⁹-10⁻⁵ M) of 17β-estradiol (E₂) with or without pre-administration of a broad-spectrum CYP450 inhibitor 1-aminobenzotriazole (ABT) (10 × 10⁻⁶ M) and an estrogen receptor (ER) antagonist ICI 182,780 (10⁻⁶ M), respectively. The growth, cell cycle progression, premature senescence, estrogen metabolites, reactive oxygen species (ROS) and DNA damage of the cells were analyzed with cell counting assay, flow cytometry, Western blot, liquid chromatography-mass spectrometry and comet assay, respectively.

Results

E₂ in its physiological levels from 10⁻⁹ M to 10⁻⁸ M had a concentration-dependent promoting effect on growth of VSMCs. However, when the concentration increased over 10⁻⁸ M, the growth-promoting effect gradually reversed to a growth-inhibiting action. When the activity of CYP450s was blocked by ABT, the growth-promoting effect of E₂ increased and did not reverse at high concentrations. Whereas when the ERs were blocked by ICI 182,780, E₂ showed a pure growth-inhibiting effect. The E₂ metabolites 2- and 4-hydroxyestradiols accumulated with the increase of E₂ over 10⁻⁸ M, which accompanied by increased ROS, DNA damage and cellular senescence. All of these changes were eliminated by block of CYP450s, indicating that the VSMC growth inhibition by E₂ is due to an increased production of ROS from accumulated E₂ metabolites which induces DNA damage, leading to VSMC premature senescence.

Conclusion

The complex effect of E₂ is due to two opposite actions: one ER-mediated and proliferative, and the other estrogen metabolite-induced and pro-senescent.     

When it is too hot for photosynthesis: heat-induced instability of photosynthesis in relation to respiratory burst, cell permeability changes and H₂O₂ formation

Photosynthesis rate (A_n) becomes unstable above a threshold temperature, and the recovery upon return to low temperature varies because of reasons not fully understood. We investigated responses of A_n, dark respiration and chlorophyll fluorescence to supraoptimal temperatures of varying duration and kinetics in Phaseolus vulgaris asking whether the instability of photosynthesis under severe heat stress is associated with cellular damage. Cellular damage was assessed by Evans blue penetration (enhanced membrane permeability) and by H₂O₂ generation [3,3′‐diaminobenzidine 4HCl (DAB)‐staining]. Critical temperature for dark fluorescence (F₀) rise (T_F) was at 46–48 °C, and a burst of respiration was observed near T_F. However, A_n was strongly inhibited already before T_F was reached. Membrane permeability increased with temperature according to a switch‐type response, with enhanced permeability observed above 48 °C. Experiments with varying heat pulse lengths and intensities underscored the threshold‐type loss of photosynthetic function, and indicated that the degree of photosynthetic deterioration and cellular damage depended on accumulated heat‐dose. Beyond the ‘point of no return’, propagation of cellular damage and reduction of photosynthesis continued upon transfer to lower temperatures and photosynthetic recovery was slow or absent. We conclude that instability of photosynthesis under severe heat stress is associated with time‐dependent propagation of cellular lesions.     

Studying post depositional damage on Acheulian bifaces using 3-D scanning

In this study, we explore post-depositional damage observed on Acheulian bifacial tools by comparing two assemblages: a collection of archaeological handaxes which shows pronounced damage marks associated with high energy water accumulation system, and an experimental assemblage that was rolled and battered in a controlled simulation experiment. Scanning the two assemblages with a precise 3-D optical scanner and subjecting the measured surfaces to the same mathematical analysis enabled the development of quantitative measures assessing and comparing the degree of damage observed on archaeological and experimental tools. The method presented here enables the definition of morphological patterns typically resulting from battering and different from intentional controlled knapping. The most important kinds of damage included the formation of deep, random ‘notch-like’ scars on the lateral edges and substantial degrees of damage to the tip of the tools, but minimal damage to the artifact's butt. Quantifying the degree of damage and its location and morphological characters allows us to present a method by which post depositional damage on archaeological tools can be measured.     

Assessing deer densities and impacts at the appropriate level for management: a review of methodologies for use beyond the site scale

• 1 Population density alone is unlikely to be a good predictor of the impacts of deer on their environment. The assessment of management requirements should therefore be based on assessment of deer impacts, alongside estimates of density.
• 2 Both density and impacts need to be monitored at a landscape scale, and there is a need to develop appropriate methodologies that allow managers to consider the current and likely future impact of deer.
• 3 The relevant scale for assessment (and management) varies both with deer species and context of impact, but should always encompass at least the estimated biological range of the population of deer present in an area. Some impacts (e.g. deer–vehicle collisions, and risks of disease transmission) may need to be assessed at a wider regional level.
• 4 In this review we consider various approaches available for assessing: absolute or relative animal abundance; impacts of ungulates on agriculture, forestry, amenity woodlands and other conservation sites; impacts on public safety (e.g. through road traffic accidents) and on humans or livestock through potential spread of disease.
• 5 In each case the advantages and disadvantages of a variety of methods are considered, before recommendations are made for methodologies which are sufficiently accurate, sufficiently robust and sufficiently practical to be favoured in a management context.
• 6 To address impacts at the landscape scale requires management policies that integrate information on both positive and negative impacts of deer in order to ensure appropriate and holistic management. We present a decision‐support framework suitable for use within the UK, using inputs from a variety of possible impact types to assist managers and forewarn of situations where current management may need to be modified.

     

外源24-表油菜素内酯对盐胁迫下茄子种子萌发和幼苗生理特性的影响

研究了150mmol·L^-1NaCI胁迫下,外源24-表油菜素内酯（EBR）对茄子种子萌发、幼苗生长和生理特性的影响。结果表明,0．05mg·L^-1外源EBR显著缓解150mmol·L^-1NaCI胁迫伤害,使茄子种子发芽率提高了8．23％,发芽势提高15．91％,发芽指数提高了17．23％,活力指数提高了44．29％;幼苗株高、根长和植株鲜重分别提高了56．67％、23．83％和56．68％;抗氧化酶（SOD、POD、CAT和APX）活性分别增加了13．75％、24．00％、28．64％和21．46％,脯氨酸和可溶性糖含量分别提高30．96％和23．66％;MDA含量、Ｏ产生速率分别降低了29．58％和14．80％。表明0．05mg·L^-1外源EBR能显著促进盐胁迫下茄子种子萌发和幼苗生长,明显缓解叶片氧化损伤,增强茄子的耐盐能力。