Influence of SOS repair on the specificity of radiation mutagenesis in bacteriophage ?X174

Summary The ochre mutant oc9 of bacteriophage X174 was irradiated with -rays and the revertants were assayed on unirradiated and UV-irradiated host bacteria carrying an amber suppressor. The yield of revertants (amber+wild type) was higher on UV-irradiated than on unirradiated bacteria, showing that -irradiated X174 was subjected to W-mutagenesis.For oc9 -irradiated in the presence of oxygen the fraction of amber mutants among the revertants was lower when mutants were scored on UV-irradiated bacteria than when assayed on unirradiated indicator cells. The same fraction of ambers was obtained when mutants were assayed on unirradiated and UV-irradiated samples of a recA indicator strain. UV-irradiated X174 showed a similar phenomenon. These results suggest that the specificity with regard to insertion of bases opposite radiation damage in X174 DNA is different for host cells in which SOS repair has been induced and cells in which SOS repair is not operative.     

Dissociation of intracellular lysosomal rupture from the cell death caused by silica

The relationship between intracellular lysosomal rupture and cell death caused by silica was studied in P388d(1) macrophages. After 3 h of exposure to 150 μg silica in medium containing 1.8 mM Ca(2+), 60 percent of the cells were unable to exclude trypan blue. In the absence of extracellular Ca(2+), however, all of the cells remained viable. Phagocytosis of silica particles occurred to the same extent in the presence or absence of Ca(2+). The percentage of P388D(1) cells killed by silica depended on the dose and the concentration of Ca(2+) in the medium. Intracellular lyosomal rupture after exposure to silica was measured by acridine orange fluorescence or histochemical assay of horseradish peroxidase. With either assay, 60 percent of the cells exposed to 150 μg silica for 3 h in the presence of Ca(2+) showed intracellular lysosomal rupture, was not associated with measureable degradation of total DNA, RNA, protein, or phospholipids or accelerated turnover of exogenous horseradish peroxidase. Pretreatment with promethazine (20 μg/ml) protected 80 percent of P388D(1) macrophages against silica toxicity although lysosomal rupture occurred in 60-70 percent of the cells. Intracellular lysosomal rupture was prevented in 80 percent of the cells by pretreatment with indomethacin (5 x 10(-5)M), yet 40-50 percent of the cells died after 3 h of exposure to 150 μg silica in 1.8 mM extracellular Ca(2+). The calcium ionophore A23187 also caused intracellular lysosomal rupture in 90-98 percent of the cells treated for 1 h in either the presence or absence of extracellular Ca(2+). With the addition of 1.8 mM Ca(2+), 80 percent of the cells was killed after 3 h, whereas all of the cells remained viable in the absence of Ca(2+). These experiments suggest that intracellular lysosomal rupture is not causally related to the cell death cause by silica or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Cell death is dependent on extracellular Ca(2+) and may be mediated by an influx of these ions across the plasma membrane permeability barrier damaged directly by exposure to these toxins.     

Binding of radiation-induced phenylalanine radicals to DNA: influence on the biological activity of the DNA and on its sensitivity to the induction of breaks by gamma-rays.

When an aqueous solution of double-stranded DNA of bacteriophage PM2 containing phenylalanine and saturated with N2O is irradiated with gamma-rays, radiation-induced phenylalanine radicals are bound covalently. Under the conditions used, about 25 phenylalanine molecules may be bound per lethal hit. For single-stranded PM2 DNA, most of the phenylalanine radicals bound are non-lethal. Evidence is presented that, in double-stranded DNA, an appreciable fraction of the single-strand breaks is induced by phenylalanine radicals. Radiation products of phenylalanine and the phenylalanine bound to the DNA decrease the sensitivity of the DNA to the induction of single-strand breaks. There are indications that the high efficiency of protection by radiation products of phenylalanine is due to their positive charge, which will result in a relatively high concentrations fo these compounds in the vicinity of the negatively-charged DNA molecules.     

Flow cytometry and FACS applied to filamentous fungi

Flow cytometry is an automated, laser- or impedance-based, high throughput method that allows very rapid analysis of multiple chemical and physical characteristics of single cells within a cell population. It is an extremely powerful technology that has been used for over four decades with filamentous fungi. Although single cells within a cell population are normally analysed rapidly on a cell-by-cell basis using the technique, flow cytometry can also be used to analyse cell (e.g. spore) aggregates or entire microcolonies. Living or fixed cells can be stained with a wide range of fluorescent reporters to label different cell components or measure different physiological processes. Flow cytometry is also suited for measurements of cell size, interaction, aggregation or shape using non-labelled cells by means of analysing their light scattering characteristics. Fluorescence-activated cell sorting (FACS) is a specialized form of flow cytometry that provides a method for sorting a heterogeneous mixture of cells into two or more containers based upon the fluorescence and/or light scattering properties of each cell. The major advantage of analysing cells by flow cytometry over microscopy is the speed of analysis: thousands of cells can be analysed per second or sorted in minutes. Drawbacks of flow cytometry are that specific cells cannot be followed in time and normally spatial information relating to individual cells is lacking. A big advantage over microscopy is when using FACS, cells with desired characteristics can be sorted for downstream experimentation (e.g. for growth, infection, enzyme production, gene expression assays or ‘omics’ approaches). In this review, we explain the basic concepts of flow cytometry and FACS, define its advantages and disadvantages in comparison with microscopy, and describe the wide range of applications in which these powerful technologies have been used with filamentous fungi.     

Paracrine effects of oocyte secreted factors and stem cell factor on porcine granulosa and theca cells <Emphasis Type="Italic">in vitro</Emphasis>

Oocyte control of granulosa and theca cell function may be mediated by several growth factors via a local feedback loop(s) between these cell types. This study examined both the role of oocyte-secreted factors on granulosa and thecal cells, cultured independently and in co-culture, and the effect of stem cell factor (SCF); a granulosa cell derived peptide that appears to have multiple roles in follicle development. Granulosa and theca cells were isolated from 2–6 mm healthy follicles of mature porcine ovaries and cultured under serum-free conditions, supplemented with: 100 ng/ml LR3 IGF-1, 10 ng/ml insulin, 100 ng/ml testosterone, 0–10 ng/ml SCF, 1 ng/ml FSH (granulosa), 0.01 ng/ml LH (theca) or 1 ng/ml FSH and 0.01 ng/ml LH (co-culture) and with/without oocyte conditioned medium (OCM) or 5 oocytes. Cells were cultured in 96 well plates for 144 h, after which viable cell numbers were determined. Medium was replaced every 48 h and spent medium analysed for steroids.     

Bacterial luciferase reporters: the Swiss army knife of molecular biology

Bioluminescence is a process during which light in the visible spectrum is emitted as a consequence of an enzymatic reaction catalyzed by luciferases. Luciferases have been identified mainly in marine organisms and are used for several biological purposes include camouflage, repulsion, attraction, communication and illumination. Some of the currently known luciferases have become indispensible tools in modern molecular biology and are used for diverse applications such as autoinducer-1 activity assays, promoter test assays in both prokaryotes and eukaryotes, imaging of bacterial infections in live animals, in vivo activity assays genes involved in host response and disease and monitoring of bacterial contaminations of food products. With the present review, the authors intend to give an overview on the currently used bacterial luciferase reporter systems, their methodologies and applications and compare them to other reporter systems.     

Phylogenetic analysis of the tenascin gene family: evidence of origin early in the chordate lineage

Background  

Tenascins are a family of glycoproteins found primarily in the extracellular matrix of embryos where they help to regulate cell proliferation, adhesion and migration. In order to learn more about their origins and relationships to each other, as well as to clarify the nomenclature used to describe them, the tenascin genes of the urochordate Ciona intestinalis, the pufferfish Tetraodon nigroviridis and Takifugu rubripes and the frog Xenopus tropicalis were identified and their gene organization and predicted protein products compared with the previously characterized tenascins of amniotes.     

18F-2-deoxy-2-fluoro-D-glucose positron emission tomography, computed tomography, and magnetic resonance imaging for the detection of experimental colorectal liver metastases

During the treatment of colorectal liver metastases, evaluation of treatment efficacy is of the utmost importance for decision making. The aim of the present study was to explore the ability of preclinical imaging modalities to detect experimental liver metastases. Nine male Wag/Rij rats underwent a laparotomy with intraportal injection of CC531 tumor cells. On days 7, 10, and 14 after tumor induction, sequential positron emission tomography (PET), computed tomography (CT), and magnetic resonance imaging (MRI) scans were acquired of each rat. At each time point, three rats were euthanized and the metastases in the liver were documented histologically. Topographically, the liver was divided into eight segments and the image findings were compared on a segment-by-segment basis with the histopathologic findings. Sixty-four liver segments were analyzed, 20 of which contained tumor deposits. The overall sensitivity of PET, CT, and MRI was 30%, 25%, and 20%, respectively. For the detection of tumors with a histologic diameter exceeding 1 mm (n = 8), the sensitivity of PET, CT, and MRI was 63%, 38%, and 38%, respectively. The overall specificity of PET, CT, and MRI was 98%, 100%, and 93%, respectively. This study showed encouraging detectability and sensitivity for preclinical imaging of small liver tumors and provides valuable information on the imaging techniques for designing future protocols.     

Induction of mutations in bacteriophage T7 by gamma-rays: no influence of the SOS repair pathway

Under conditions where the reversion of an amber mutant of bacteriophage lambda by gamma-rays is enhanced by subjecting the irradiated phage to SOS repair, gamma-ray-induced reversion of two T7 ambers is not influenced by this error-prone bacterial repair system. The survival of T7 gamma-irradiated under anoxic conditions is somewhat enhanced by SOS repair, whereas the survival of phage irradiated under oxygen is not affected.