Scintillometric determination of DNA repair in human cell lines: A critical appraisal

The ability of a variety of chemical and physical agents to stimulate DNA repair synthesis in human cell cultures was tested by a simplified scintillometric procedure, with the use of hydroxyurea (HU) to suppress DNA replicative synthesis. After incubation with [³H]thymidine, the radioactivity incorporated in to DNA was determined in controls (C) and treated (T) cultures and in the corresponding HU series (C_HU, T_HU). The ratios T_HU/C_HU and T_HU/T:C_HU/C, indicating absolute and relative increases of DNA radioactivity, were calculated. When both ratios were significantly higher than 1, they were taken as indices of DNA repair stimulation, whereas, no stimulation in inferred when both of them are ?1. The scintillometric estimate of DNA repair was always in agreement with the autoradiographic observations, so that the procedure adopted can be used as a rapid test for screening investigations.Agents giving a relative but no an absolute increase of DNA radioactivity are generally not inducers of repair synthesis as estimated by autoradiography. However, the same scintillometric results are also occasionally observed with DNA repair inducers, such as methyl methanesulphonate (MMS) and ethyl methanesulphonate (EMS), owing to alterations of thymidine pool radioactivity. These chemicals, besides affecting the levels of labelled precursors in the intracellular pool in the T series, differently modified the increase of pool radioactivity which is a regular effect of HU. With such chemicals, DNA repair synthesis can be detected only after normalization of th DNA radioactivity on the basis of pool alterations.The quantitative value of the autoradiographic estimate of DNA repair is also affected by the changes in the radioactivity of the thymidine pool although autoradiography retains its qualitative value.Dimethylnitrosamine, mitomycin C and potassium dichromate, described by other authors as inducers of DNA repair, also gave negative results by the scintillometric procedure after normalization of DNA radioactivities. However, in our hands, these agents were unable to stimulated repair synthesis, according to the results of autoradiography and isopynic centrifugation.The proposed scintillometric procedure is effective in indicating false negative inducers of DNA repair, not giving rise to false positives.     

Arsenic trioxide and ascorbic acid interfere with the BCL2 family genes in patients with myelodysplastic syndromes: an ex-vivo study

ABSTRACT: BACKGROUND: Arsenic Trioxide (ATO) is effective in about 20% of patients with myelodysplasia (MDS); its mechanisms of action have already been evaluated in vitro, but the in vivo activity is still not fully understood. Since ATO induces apoptosis in in vitro models, we compared the expression of 93 apoptotic genes in patients' bone marrow before and after ATO treatment. For this analysis, we selected 12 patients affected by MDS who received ATO in combination with Ascorbic Acid in the context of the Italian clinical trial NCT00803530, EudracT Number 2005-001321-28. METHODS: Real-time PCR quantitative assays for genes involved in apoptosis were performed using TaqMan(R) Assays in 384-Well Microfluidic Cards "TaqMan(R) Human Apoptosis Array". Quantitative RT-PCR for expression of EVI1 and WT1 genes was also performed. Gene expression values (Ct) were normalized to the median expression of 3 housekeeping genes present in the card (18S, ACTB and GAPDH). RESULTS: ATO treatment induced up-regulation of some pro-apoptotic genes, such as HRK, BAK1, CASPASE-5, BAD, TNFRSF1A, and BCL2L14 and down-regulation of ICEBERG. In the majority of cases with stable disease, apoptotic gene expression profile did not change, whereas in cases with advanced MDS more frequently pro-apoptotic genes were upregulated. Two patients achieved a major response: in the patient with refractory anemia the treatment down-regulated 69% of the pro-apoptotic genes, whereas 91% of the pro-apoptotic genes were up-regulated in the patient affected by refractory anemia with excess of blasts-1. Responsive patients showed a higher induction of BAD than those with stable disease. Finally, WT1 gene expression was down-regulated by the treatment in responsive cases. CONCLUSIONS: These results represent the basis for a possible association of ATO with other biological compounds able to modify the apoptotic pathways, such as inhibitors of the BCL2 family.     

An update on North American boar stud practices

This survey included 44 boar studs from Canada and the USA with a total of approximately 10,000 boars. Studs with 51-500 boars accounted for 84% of respondents. More than 90% of boars were housed in stalls. Evaporative and mechanical cooling systems predominated and boars were typically fed based on body condition. The predominant age of boars was 1-2 years with annual culling rates between 20 and 70%. The primary reasons for culling included genetic improvement, semen quality and feet and leg issues. Collection occurred commonly on Mondays and Thursdays and boars were rested 3-7 days between collections. The average sperm produced per boar per week was 51-150 billions and resulted in 21-40 doses per boar per week. Most studs collected boars using double gloves and disposable cups or liners and used pre-warmed containers. Ejaculate pooling was practiced by >60% of studs. Evaluation of semen for motility was performed with 0-5min of warming in extender with viewing at 100-400x magnification. Concentration estimation occurred by photometer and CASA for 88% of studs. Ejaculate discard occurred for reasons of poor motility, abnormal sperm and bacteria. Most studs retained extended samples for 3-7 days for quality control. Discard rates were most common between 1 and 10% and were related to individual boar and season. Doses of semen contained 2-4 billion sperms, with final sperm numbers adjusted for fertile sperm and packaged as doses in tubes and bags with 60-100mL.     

Plasma membrane glycosphingolipids (GSLs) of the human lymphoblastoid cell-line BRI 8 and differences between the GSLs of BRI 8 cells and those of peripheral lymphocytes.

A cyclic undecapeptide, Wy-40,770, has been synthesized by a combination of solid phase and conventional peptide synthesis methodology. The compound inhibits the release of growth hormone without significantly affecting glucagon levels in rats. Wy-40,770 shows growth hormone release inhibiting activity for four hours after s.c. injection.     

Localisation of Epithelial Cells Capable of Holoclone Formation In Vitro and Direct Interaction with Stromal Cells in the Native Human Limbal Crypt

Limbal epithelial stem cells (LESCs) are essential to maintain the transparent ocular surface required for vision. Despite great advances in our understanding of ocular stem cell biology over the last decade, the exact location of the LESC niche remains unclear. In the present study we have used in vitro clonal analysis to confirm that limbal crypts provide a niche for the resident LESCs. We have used high-resolution imaging of the basal epithelial layer at the limbus to identify cells with a morphology consistent with stem cells that were only present within the basal layer of the limbal crypts. These cells are proximal to limbal stromal cells suggesting direct cell-to-cell interaction. Serial block-face scanning electron microscopy (SBFSEM) confirmed that the putative LESCs are indeed in direct contact with cells in the underlying stroma, a contact that is facilitated by focal basement membrane interruptions. Limbal mesenchymal cells previously identified in the human limbus collocate in the crypt-rich limbal stromal area in the vicinity of LESCs and may be involved in the cell-to-cell contact revealed by SBFSEM. We also observed a high population of melanocytes within the basal layer of the limbal crypts. From these observations we present a three dimensional reconstruction of the LESC niche in which the stem cell is closely associated and maintained by both dendritic pigmented limbal melanocytes and elongated limbal stromal cells.     

Application of a Life Cycle Model for European Union Policy‐Driven Waste Management Decision Making in Emerging Economies

Solid waste life cycle modeling has predominantly focused on developed countries, but there are significant opportunities to assist developing and transition economies to minimize the environmental impact of solid waste management (SWM). Serbia is representative of a transition country and most (92%) of its waste is landfilled. As a Candidate European Union (EU) country, Serbia is expected to implement SWM strategies that meet EU directives. The Solid Waste Life‐Cycle Optimization Framework (SWOLF) was used to evaluate scenarios that meet EU goals by 2030. Scenarios included combinations of landfills, anaerobic digestion, composting, material recovery facilities (MRFs), waste‐to‐energy (WTE) combustion, and the use of refuse‐derived fuel in cement kilns. Each scenario was evaluated with and without separate collection of recyclables. Modeled impacts included cost, climate change, cumulative fossil energy demand, acidification, eutrophication, photochemical oxidation, total eco‐toxicity, and total human toxicity. Trade‐offs among the scenarios were evaluated because no scenario performed best in every category. In general, SWM strategies that incorporated processes that recover energy and recyclable materials performed well across categories, whereas scenarios that did not include energy recovery performed poorly. Emissions offsets attributable to energy recovery and reduced energy requirements associated with remanufacturing of recovered recyclables had the strongest influence on the results. The scenarios rankings were robust under parametric sensitivity analysis, except when the marginal electricity fuel source changed from coal to natural gas. Model results showed that the use of existing infrastructure, energy recovery, and efficient recovery of recyclables from mixed waste can reduce environmental emissions at relatively low cost.     

Non‐adaptive phenotypic plasticity: the effects of terrestrial and aquatic herbicides on larval salamander morphology and swim speed

Phenotypic plasticity, although ubiquitous, may not always be advantageous. Non‐adaptive plasticity is likely to occur in response to novel environmental stress. Anthropogenic contaminants, such as herbicides, are novel stressors that are not present in the evolutionary history of most species. We investigated the pattern and consequences of phenotypic plasticity induced by four glyphosate‐based herbicides (two terrestrial and two aquatic) in larvae of the spotted salamander, Ambystoma maculatum, by determining (1) whether the herbicides induced different morphologies; (2) if different morphologies translated to differences in burst swim performance; and (3) how induced individuals performed relative to non‐induced controls. Different herbicide formulations led to the production of significantly different head and tail morphologies, and tail morphology correlated with fastest escape speed. However, escape speed did not vary among treatments. In addition, three out of four herbicide treatments experienced accelerated growth rates, in terms of the lateral size of tails, although the tail shapes were either similar to preliminary controls or intermediate between preliminary and final controls. These observations suggest that herbicide‐induced morphology is a case of non‐adaptive phenotypic plasticity, and that there is potentially a trade‐off between growth and development for larvae exposed to different formulations. Understanding the functional significance of induced phenotypes is important for determining their importance in shaping an organism's ecological interactions and evolutionary trajectories. Furthermore, under different conditions, the morphological changes that we observed in response to exposure to herbicides might affect salamander fitness and influence population dynamics.     

Intraspecific adaptive radiation: Competition,ecological opportunity,and phenotypic diversification within species

Intraspecific variation in resource‐use traits can have profound ecological and evolutionary implications. Among the most striking examples are resource polymorphisms, where alternative morphs that utilize different resources evolve within a population. An underappreciated aspect of their evolution is that the same conditions that favor resource polymorphism—competition and ecological opportunity—might foster additional rounds of diversification within already existing morphs. We examined these issues in spadefoot toad tadpoles that develop into either a generalist "omnivore" or a specialist "carnivore" morph. Specifically, we assessed the morphological diversity of tadpoles from natural ponds and experimentally induced carnivores reared on alternative diets. We also surveyed natural ponds to determine if the strength of intramorph competition and the diversity and abundance of dietary resources (measures of ecological opportunity) influenced the diversity of within‐morph variation. We found that five omnivore and four carnivore types were present in natural ponds; alternative diets led to shape differences, some of which mirrored variation in the wild; and both competition and ecological opportunity were associated with enhanced morphological diversity in natural ponds. Such fine‐scale intraspecific variation might represent an underappreciated form of biodiversity and might constitute a crucible of evolutionary innovation and diversification.