Saccharomyces cerevisiae has been reported to die, under certain conditions, from programmed cell death with apoptotic markers. One of the most important markers is chromosomal DNA fragmentation as indicated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining. We found TUNEL staining in S. cerevisiae to be a consequence of both single-and double-strand DNA breaks, whereas in situ ligation specifically stained double-strand DNA breaks. Cells treated with hydrogen peroxide or acetic acid staining positively for TUNEL assay stained negatively for in situ ligation, indicating that DNA damage in both cases mainly consists of single-strand DNA breaks. Pulsed field gel electrophoresis of chromosomal DNA from cells dying from hydrogen peroxide, acetic acid, or hyperosmotic shock revealed DNA breakdown into fragments of several hundred kilobases, consistent with the higher order chromatin degradation preceding DNA laddering in apoptotic mammalian cells. DNA fragmentation was associated with death by treatment with 10 mM hydrogen peroxide but not 150 mM and was absent if cells were fixed with formaldehyde to eliminate enzyme activity before hydrogen peroxide treatment. These observations are consistent with a process that, like mammalian apoptosis, is enzyme dependent, degrades chromosomal DNA, and is activated only at low intensity of death stimuli. INTRODUCTIONProgrammed cell death (PCD) is an active form of cell death in which the cell uses specialized cellular machinery to commit suicide. PCD is found in many different cells of diverse organisms, the purpose being removal of damaged cells or cells representing a threat to the integrity of the organism. Such cells are, for example, virus-infected cells or cancerous cells. PCD is also a normal part of development of multicellular organisms and also necessary for the maintenance of cellular homeostasis. Apoptosis, the most common form of PCD in eukaryotes, is associated with a number of characteristic markers depending on cell type and organism. The most common are cell shrinkage and development of bubble-like blebs on the surface (Kerr et al., 1972). The phospholipid phosphatidylserine, which is normally hidden on the inside of the plasma membrane, can be exposed on the cell surface (Fadok et al., 1992). There is also a two-step degradation of chromatin. The first step, sometimes referred to as higher order chromatin fragmentation, produces large fragments, 50 -300 kilobases (kb) (Ucker et al., 1992;Oberhammer et al., 1993;Rusnak et al., 1996), perhaps reflecting chromatin higher order structure (Filipski et al., 1990). The second step is the formation of the characteristic internucleosomal DNA ladder of fragments differing in length by ϳ200 base pairs (Wyllie, 1980). A large portion of the fragments are blunt double-stranded DNA fragments (Staley et al., 1997;Didenko et al., 2003;Liu et al., 2003). Internucleosomal DNA laddering is often, but not always associated with apoptosis; some types of cells only undergo higher order chromatin fragm...
BackgroundRecent advances in synthetic biology have provided tools to efficiently construct complex DNA molecules which are an important part of many molecular biology and biotechnology projects. The planning of such constructs has traditionally been done manually using a DNA sequence editor which becomes error-prone as scale and complexity of the construction increase. A human-readable formal description of cloning and assembly strategies, which also allows for automatic computer simulation and verification, would therefore be a valuable tool.ResultsWe have developed pydna, an extensible, free and open source Python library for simulating basic molecular biology DNA unit operations such as restriction digestion, ligation, PCR, primer design, Gibson assembly and homologous recombination. A cloning strategy expressed as a pydna script provides a description that is complete, unambiguous and stable. Execution of the script automatically yields the sequence of the final molecule(s) and that of any intermediate constructs. Pydna has been designed to be understandable for biologists with limited programming skills by providing interfaces that are semantically similar to the description of molecular biology unit operations found in literature.ConclusionsPydna simplifies both the planning and sharing of cloning strategies and is especially useful for complex or combinatorial DNA molecule construction. An important difference compared to existing tools with similar goals is the use of Python instead of a specifically constructed language, providing a simulation environment that is more flexible and extensible by the user.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-015-0544-x) contains supplementary material, which is available to authorized users.
This study investigated the inclusion levels of defatted maize germ (DMG) in the diet of horses by determining the apparent digestibility coefficients of the nutrients, and the physicochemical parameters of feaces and blood samples. Four adult horses, weighing 483.3 ± 27.5 kg average, arranged in 4x4 Latin square were used. The energy provided by the diets came from hay, 50% (Jiggs hay), and from the concentrate, 50%, with increasing DMG levels (0, 10, 20 and 30%). Digestibility was determined using samples from total feaces collection. The characteristics of color, consistency, pH and buffering capacity (BC) 5 and 6 of the feaces were determined daily during the collection days. The blood samples were collected by venipuncture of the jugular, with vacuum tubes, to determine the concentration of glucose, triglycerides, cholesterol and insulin using biochemical kits and the concentration of short-chain fatty acids, by gas chromatography. The diets did not affect significantly (P> 0.05) the digestibility of nutrients, fecal pH (mean 6.7), buffering capacity at pH 5 (22.7) and pH 6 (6.9), and fecal concentration of short-chain fatty acids. Likewise, diets did not affect significantly (P> 0.05) the blood concentrations of glucose, insulin, cholesterol, and total short-chain fatty acids. However, diets changed significantly (P <0.05) the blood concentrations of triglycerides, described by the equation ŷ = 30.62 + 0.26x. The triglycerides, glucose and insulin variables also changed significantly (P <0.05) over time, according to the following equations: ŷ = 32.30 + 0.87x; ŷ = 85.93 + 3.17x -0.61x²; and y = 3.19 + 1.38x -0.22x², respectively. The defatted maize germ can be included up to 30% of the concentrate (up to 9.6% of the diet) without changing the diet apparent digestibility coefficient, and feaces and blood physicochemical parameters. This ingredient can be an alternative for high fiber diets and can contribute to formulating diets with lower glycemic and insulinemic indexes.
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