Image analysis and in vivo imaging as tools for investigation of productivity dynamics in anthocyanin-producing cell cultures of Daucus carota

An anthocyanin-producing suspension culture of Daucus carota (L.) cv. Flakkese was used as model system to study secondary metabolite production in cell culture at the individual cell level. An approach was set up in which growth and production of anthocyanins were investigated using a combination of biochemical analysis, image (colour) analysis and in vivo imaging. This novel approach was used to segment the culture in different subpopulations and dissect the productive process in the cell culture grown under two different conditions, known to differ mainly for oxygen supply and mixing intensity (volume of 50 ml or 20 ml in 250 ml flasks). The 20 ml batch cultures gave a higher content and yield of anthocyanins, which depended on a complex balance between events that positively or negatively affected anthocyanin production. A model is proposed in which the different ability of cells to respond to environmental stimuli and stress depends on the different amount of anthocyanins accumulated within cells.     

Stability of the Plasma Membrane in Saccharomyces rouxii and Its Relationship to Glucose Tolerance

The stability of spheroplasts from the osmotrophic yeast Saccharomyces rouxii was studied in buffered solutions of mannitol and glucose. The plasma membranes from cells grown in high glucose concentrations were more stable to osmotic lysis than were membranes from cells grown in lower glucose concentrations. Mannitol was a better osmotic stabilizer than glucose, except when the cells were grown in a high glucose concentration. Spheroplasts from a glucose tolerant-deficient mutant were much less stable than the corresponding spheroplasts from the parent strain, especially when suspended in glucose solutions. These results suggest an involvement of the plasma membrane in the glucose-tolerant mechanism of S. rouxii.     

Dose-Dependent Cytokinetic Changes Following 1-β-D-Arabinofuranosylcytosine and Hydroxyurea In L1210 and S-180 In Vivo

DNA synthesis inhibition and recovery in L1210 and S-180 ascites tumors following 1-β-D-arabinofuranosylcytosine (Ara-C) and hydroxyurea (HU) were measured autoradiographically as a basis for optimizing drug schedules. Tumor bearing mice, 10⁶ cells day O, were treated on day 4 with 20, 200 or 2000 mg/kg Ara-C or 50, 300 or 1800 mg/kg HU. At various intervals following drug, [³H]thymidine was administered i.p. and mice were killed 1 hr later. Tumor cells were analyzed for labeling index (LI) and grain count (GC) to determine the percentage of cells in S phase and the distribution of DNA synthesis rates among the labeled cells, respectively. Following each dose of HU, DNA synthesis was inhibited completely. Recovery of LI was rapid and approached control values by 6 hr. Following each dose of Ara-C, DNA synthesis was inhibited completely for at least 6 hr. Recovery of LI was first noted 6 hr following 20 mg/kg Ara-C and 9 hr following 200 mg/kg. Following both doses the LI reached 100% of the control value by 26 hr. GC analysis indicated that following Ara-C treatment, DNA synthesis was reinitiated first with cells with low GC from 6 to 12 hr followed by cells with increasing GC from 12 to 20 hr. the labeling intensity reached control values by 20 hr and an ‘overshoot’ occurred by 26 hr. These data suggest that the recovery of DNA synthesis rate is a gradual process. Survival data for mice receiving two doses of Ara-C indicated that the optimal interval for retreatment following the lower dose of Ara-C occurred by 6 hr as compared to 12–16 hr for the higher dose. These times coincided in both instances with recovery of LI to 33–50% of control values. Early recovery of LI may be the best method currently available for estimating the optimal time for retreatment with an S phase specific drug.     

The selenoprotein GPX4 is essential for mouse development and protects from radiation and oxidative damage insults

Lipid peroxidation has been implicated in a variety of pathophysiological processes, including inflammation, atherogenesis, neurodegeneration, and the ageing process. Phospholipid hydroperoxide glutathione peroxidase (GPX4) is the only major antioxidant enzyme known to directly reduce phospholipid hydroperoxides within membranes and lipoproteins, acting in conjunction with alpha tocopherol (vitamin E) to inhibit lipid peroxidation. Here we describe the generation and characterization of GPX4-deficient mice by targeted disruption of the murine Gpx4 locus through homologous recombination in embryonic stem cells. Gpx4(-/-) embryos die in utero by midgestation (E7.5) and are associated with a lack of normal structural compartmentalization. Gpx4(+/-) mice display reduced levels of Gpx4 mRNA and protein in various tissues. Interestingly, cell lines derived from Gpx4(+/-) mice are markedly sensitive to inducers of oxidative stress, including gamma-irradiation, paraquat, tert-butylhydroperoxide, and hydrogen peroxide, as compared to cell lines derived from wild-type control littermates. Gpx4(+/-) mice also display reduced survival in response to gamma-irradiation. Our observations establish GPX4 as an essential antioxidant enzyme in mice and suggest that it performs broad functions as a component of the mammalian antioxidant network.     

Variation in Prokaryotic Community Composition as a Function of Resource Availability in Tidal Creek Sediments

In anaerobic coastal sediments, hydrolytic and/or fermentative bacteria degrade polymeric material and produce labile intermediates, which are used by terminal metabolizers to complete the conversion of organic material to CO₂. We used molecular approaches to evaluate the response of two bacterial terminal metabolizer groups from a coastal tidal creek sediments, sulfate reducers and methanogens, to controlled changes in carbon resource supply. Tidal creek sediment bioreactors were established in April and August 2004. For each date, intact sediment sections were continuously supplied with flowthrough seawater that was either unamended or amended with the high-molecular-weight polysaccharide dextran. Biogeochemical data indicate that the activity of fermenting bacteria and the terminal metabolizers was limited by organic carbon supply during both experiments, with a significant increase in net volatile fatty acid (VFA) production and rates of sulfate reduction and methanogenesis following dextran addition. Community composition (measured by using terminal restriction fragment length polymorphism analysis, and functional gene [dsrA, mcrA] clone libraries) changed from April to August. However, community composition was not different between amended and unamended cores within each month, despite the change in resource level. Moreover, there was no relationship between community richness and evenness with resource level. This lack of variation in community composition with C addition could be attributed to the dynamic environment these sediment communities experience in situ. Fluctuations in VFA concentrations are most likely very high, so that the dominant bacterial species must be able to outcompete other species at both high and low resource levels.     

Frequency of chromosomal abnormalities in embryos from superovulated merino ewes

Chromosomal analysis was carried out on 48 Day 2-7 embryos collected from superovulated Merino ewes. Three embryos had abnormal chromosome complements (1 X 1N, 1 X 1N/2N, 1 X 3N), yielding an incidence of 6.25% abnormal embryos. It is concluded that superovulation does not cause an increase in the incidence of chromosomal abnormalities in embryos of Merino sheep.