GADD45 induction of a G2/M cell cycle checkpoint

G1/S and G2/M cell cycle checkpoints maintain genomic stability in eukaryotes in response to genotoxic stress. We report here both genetic and functional evidence of a Gadd45-mediated G2/M checkpoint in human and murine cells. Increased expression of Gadd45 via microinjection of an expression vector into primary human fibroblasts arrests the cells at the G2/M boundary with a phenotype of MPM2 immunopositivity, 4n DNA content and, in 15% of the cells, centrosome separation. The Gadd45-mediated G2/M arrest depends on wild-type p53, because no arrest was observed either in p53-null Li–Fraumeni fibroblasts or in normal fibroblasts coexpressed with p53 mutants. Increased expression of cyclin B1 and Cdc25C inhibited the Gadd45-mediated G2/M arrest in human fibroblasts, indicating that the mechanism of Gadd45-mediated G2/M checkpoint is at least in part through modulation of the activity of the G2-specific kinase, cyclin B1/p34cdc2. Genetic and physiological evidence of a Gadd45-mediated G2/M checkpoint was obtained by using GADD45-deficient human or murine cells. Human cells with endogenous Gadd45 expression reduced by antisense GADD45 expression have an impaired G2/M checkpoint after exposure to either ultraviolet radiation or methyl methanesulfonate but are still able to undergo G2 arrest after ionizing radiation. Lymphocytes from gadd45-knockout mice (gadd45 −/−) also retained a G2/M checkpoint initiated by ionizing radiation and failed to arrest at G2/M after exposure to ultraviolet radiation. Therefore, the mammalian genome is protected by a multiplicity of G2/M checkpoints in response to specific types of DNA damage.

The ATM homologue MEC1 is required for phosphorylation of replication protein A in yeast

Replication protein A (RPA) is a highly conserved single-stranded DNA-binding protein, required for cellular DNA replication, repair, and recombination. In human cells, RPA is phosphorylated during the S and G2 phases of the cell cycle and also in response to ionizing or ultraviolet radiation. Saccharomyces cerevisiae exhibits a similar pattern of cell cycle-regulated RPA phosphorylation, and our studies indicate that the radiation-induced reactions occur in yeast as well. We have examined yeast RPA phosphorylation during the normal cell cycle and in response to environmental insult, and have demonstrated that the checkpoint gene MEC1 is required for the reaction under all conditions tested. Through examination of several checkpoint mutants, we have placed RPA phosphorylation in a novel pathway of the DNA damage response. MEC1 is similar in sequence to human ATM, the gene mutated in patients with ataxia-telangiectasia (A-T). A-T cells are deficient in multiple checkpoint pathways and are hypersensitive to killing by ionizing radiation. Because A-T cells exhibit a delay in ionizing radiation-induced RPA phosphorylation, our results indicate a functional similarity between MEC1 and ATM, and suggest that RPA phosphorylation is involved in a conserved eukaryotic DNA damage-response pathway defective in A-T.

Epidermal trans-urocanic acid and the UV-A-induced photoaging of the skin

The premature photoaging of the skin is mediated by the sensitization of reactive oxygen species after absorption of ultraviolet radiation by endogenous chromophores. Yet identification of UV-A-absorbing chromophores in the skin that quantitatively account for the action spectra of the physiological responses of photoaging has remained elusive. This paper reports that the in vitro action spectrum for singlet oxygen generation after excitation of trans-urocanic acid mimics the in vivo UV-A action spectrum for the photosagging of mouse skin. The data presented provide evidence suggesting that the UV-A excitation of trans-urocanic acid initiates chemical processes that result in the photoaging of skin.

Mass extinctions and supernova explosions.

In a recent contribution to this journal Ellis and Schramm [Ellis, J. & Schramm, D. N. (1995) Proc. Natl. Acad. Sci. USA 92, 235-238] claim that supernova explosions can cause massive biological extinctions as a result of strongly enhanced stratospheric NOx (NO + NO2) production by accompanying galactic cosmic rays. They suggested that these NOx productions which would last over several centuries and occur once every few hundred million years would result in ozone depletions of about 95%, leading to vastly increased levels of biologically damaging solar ultraviolet radiation. Our detailed model calculations show, however, substantially smaller ozone depletions ranging from at most 60% at high latitudes to below 20% at the equator.

Avaliação de elementos potencialmente tóxicos em tintas

Neste trabalho são apresentados os resultados da determinação, por ICP OES, de elementos tóxicos e/ou potencialmente tóxicos (AI, Cr, Ni, Cu, Cd, As, Co e Pb) em tintas de acabamento imobiliário à base de água ou de solvente orgânico. Foram desenvolvidas e comparadas método de digestão de amostras utilizando diferentes misturas ácidas em bombas de decomposição, em fomo de microondas (sistema com radiação focalizada e com cavidade) e método de cinzas. O Método de digestão utilizando fomo de microondas com cavidade permitiu solubilização rápida e eficiente de todos os tipos de tintas testados, em tempo inferior a 35 minutos. As método apresentaram valores aceitáveis para a maioria dos elementos nos testes de adição e recuperação dos analitos. Os resíduos resultantes da digestão foram avaliados por MEV-EDS e não apresentaram os elementos estudados, comprovando a eficiência da metodologia. Mercúrio foi determinado usando um Analisador Direto de Mercúrio (DMA) e apresentou valores entre 43,0 ± 4,5 e 188 ± 9 µg Kg-1, valor considerado baixo quando comparado ao limite de 100 mg Kg-1, estabelecido na norma NRR 10004 para disposição de resíduos sólidos sem instalações especiais. O estudo da migração dos elementos para o ambiente após a exposição da tinta a agentes \"agressores\" , como raios ultravioletas e umidade, foi realizado usando câmara do tipo \"Weather-Ometer\" (envelhecimento acelerado). A avaliação dos resultados foi feita por MEV EDS e ICP OES. As micrografias de MEV mostraram que houve mudança na morfologia do polímero que foi submetido ao intemperismo acelerado. Os resultados obtidos não foram conclusivos quanto à migração dos analitos em função da baixa razão entre as massas degradada e não degradada das amostras. Uma proposta de metodologia para a avaliação por ICP OES das tintas e da disponibilidade de elementos tóxicos e potencialmente tóxicos, baseada na lixiviação de amostras secas em ambiente controlado é apresentada. São mostrados resultados de lixiviação de AI, Cr, Ni, Cu, Cd, As, Co e Pb com vários extratores e tempos diferentes de extração. Os resultados mostram que ocorre a migração de alguns elementos para as soluções estudadas e que, dos extratores avaliados, a chuva ácida apresentou maior potencial de lixiviação.

Versuche mit der medizinischen Quarzlampe bezüglich ihrer Wirkung und Anwendung bei Hunden.

Inaug.-diss.--Hannover, 1911.

Interactive effects of MC1R and OCA2 on melanoma risk phenotypes

The relationships between MC1R gene variants and red hair, skin reflectance, degree of freckling and nevus count were investigated in 2331 adolescent twins, their sibs and parents in 645 twin families. Penetrance of each MC1R variant allele was consistent with an allelic model where effects were multiplicative for red hair but additive for skin reflectance. Of nine MC1R variant alleles assayed, four common alleles were strongly associated with red hair and fair skin (Asp84Glu, Arg151Cys, Arg160Trp and Asp294His), with a further three alleles having low penetrance (Val60Leu, Val92Met and Arg163Gln). These variants were separately combined for the purposes of this analysis and designated as strong 'R' (OR=63.3; 95% CI 31.9-139.6) and weak 'r ' (OR=5.1; 95% CI 2.5-11.3) red hair alleles. Red-haired individuals are predominantly seen in the R/R and R/r groups with 67.1 and 10.8%, respectively. To assess the interaction of the brown eye color gene OCA2 on the phenotypic effects of variant MC1R alleles we included eye color as a covariate, and also genotyped two OCA2 SNPs (Arg305Trp and Arg419Gln), which were confirmed as modifying eye color. MC1R genotype effects on constitutive skin color, freckling and mole count were modified by eye color, but not genotype for these two OCA2 SNPs. This is probably due to the association of these OCA2 SNPs with brown/green not blue eye color. Amongst individuals with a R/R genotype (but not R/r), those who also had brown eyes had a mole count twice that of those with blue eyes. This suggests that other OCA2 polymorphisms influence mole count and remain to be described.

Analyzing checkpoint controls in human skin

A short-term whole-skin organ culture model has been established to enable the investigation of cell cycle perturbations in epidermal layer cells following exposure to ultraviolet radiation (UVR). This model affords the opportunity to manipulate the growth and nutrient conditions, and to perform detailed biochemical and immunohistochemical analysis of skin cells in their normal epidermal layer microenvironment. The use of this model is described in this chapter.

Coral reefs in a century of rapid environmental change

Coral reefs are the most diverse marine ecosystem and embrace possibly millions of plant, animal and protist species. Mutualistic symbioses are a fundamental feature of coral reefs that have been used to explain their structure, biodiversity and existence. Complex inter-relationships between hosts, habitats and symbionts belie closely coupled nutrient and community dynamics that create the circumstances for something from nothing (or the oasis in a nutrient desert). The flip side of these dynamics is a close dependency between species, which results in a series of non-linear relationships as conditions change. These responses are being highlighted as anthropogenic influences increase across the world's tropical and subtropical coastlines. Caribbean as well as Indo-Pacific coral populations are now in a serious decline in many parts of the world. This has resulted in a significant reorganization of how coral reef ecosystems function. Among the spectra of changes brought about by humans is rapid climate change. Mass coral bleaching - the loss of the dinoflagellate symbionts from reef-building corals - and mortality has affected the world's coral reefs with increasing frequency and intensity since the late 1970s. Mass bleaching events, which often cover thousands of square kilometres of coral reefs, are triggered by small increases (+1-3degreesC) in water temperature. These increases in sea temperature are often seen during warm phase weather conditions (e.g. ENSO) and are increasing in size and magnitude. The loss of living coral cover (e.g. 16% globally in 1998, an exceptionally warm year) is resulting in an as yet unspecified reduction in the abundance of a myriad of other species. Projections from general circulation models (GCM) used to project changes in global temperature indicate that conditions even under the mildest greenhouse gas emission scenarios may exceed the thermal tolerances of most reef-building coral communities. Research must now explore key issues such as the extent to which the thermal tolerances of corals and their symbionts are dynamic if bleaching and disease are linked; how the loss of high densities of reef-building coral will affect other dependent species; and, how the loss of coral populations will affect the millions of people globally who depend on coral reefs for their daily survival.

Cell death and degeneration in the symbiotic dinoflagellates of the coral Stylophora pistillata during bleaching

Rising sea temperatures are increasing the incidences of mass coral bleaching (the dissociation of the coral-algal symbiosis) and coral mortality. In this study, the effects of bleaching (induced by elevated light and temperature) on the condition of symbiotic dinoflagellates (Symbiodinium sp.) within the tissue of the hard coral Stylophora pistillata (Esper) were assessed using a suite of techniques. Bleaching of S. pistillata was accompanied by declines in the maximum potential quantum yield of photosynthesis (F-v/F-m, measured using pulse amplitude modulated [PAM] fluorometry), an increase in the number of Sytox-green-stained algae (indicating compromised algal membrane integrity and cell death), an increase in 2',7'-dichlorodihydrofluroscein diacetate (H(2)DCFDA)stained algae (indicating increased oxidative stress), as well as ultrastructural changes (vacuolisation, losses of chlorophyll, and an increase in accumulation bodies). Algae expelled from S. pistillata exhibited a complete disorganisation of cellular contents; expelled cells contained only amorphous material. In situ samples taken during a natural mass coral bleaching event on the Great Barrier Reef in February 2002 also revealed a high number of Sytox-labelled algae cells in symbio. Dinoflagellate degeneration during bleaching seems to be similar to the changes resulting from senescence-phase cell death in cultured algae. These data support a role for oxidative stress in the mechanism of coral bleaching and highlight the importance of algal degeneration during the bleaching of a reef coral.

Testing the 'photoinhibition' model of coral bleaching using chemical inhibitors

Explants of the hard coral Seriatopora hystrix were exposed to sublethal concentrations of the herbicide diuron DCMU (N'-(3,4-dichlorophenyl,-N,N-dimethylurea)) and the heavy metal copper. Pulse amplitude modulated (PAM) chlorophyll fluorescence techniques were used to assess the effects on the photosynthetic efficiency of the algal symbionts in the tissue (in Symbio), and chlorophyll fluorescence and counts of symbiotic algae (normalised to surface area) were used to assess the extent of coral bleaching. At 30 mug DCMU l(-1), there was a reduction in both the maximum effective quantum yield (DeltaF/F-m') and maximum potential quantum yield (F-v/F-m) of the algal symbionts in symbio. Corals subsequently lost their algal symbionts and discoloured (bleached), especially on their upper sunlight-exposed surfaces. At the same DCMU concentration but under low light (5% of growth irradiance), there was a marked reduction in DeltaF/F-m' but only a slight reduction in F-v/F-m and slight loss of algae. Loss of algal symbionts was also noted after a 7 d exposure to concentrations as low as 10 mug DCMU l(-1) under normal growth irradiance, and after 14 d exposure to 10 mug DCMU l(-1) under reduced irradiance. Collectively the results indicate that DCMU-induced bleaching is caused by a light-dependent photoinactivation of algal symbionts, and that bleaching occurs when F-v/F-n, (measured 2 h after sunset) is reduced to a value of less than or equal to 0.6. Elevated copper concentrations (60 mug Cu l(-1) for 10 h) also induced a rapid bleaching in S. hystrix but without affecting the quantum yield of the algae in symbio. Tests with isolated algae indicated that substantially higher concentrations (300 mug Cu l(-1) for 8 h) were needed to significantly reduce the quantum yield. Thus, copper-induced bleaching occurs without affecting the algal photosynthesis and may be related to effects on the host (animal). It is argued that warm-water bleaching of corals resembles both types of chemically induced bleaching, suggesting the need for an integrated model of coral bleaching involving the effect of temperature on both host (coral) and algal symbionts.