Quantitative analysis of DNA-damage response factors after sequential ion microirradiation

Several proteins are known to form foci at DNA sites damaged by ionizing radiation. We study DNA damage response by immunofluorescence microscopy after microirradiation of cells with energetic ions. By using microirradiation, it is possible to irradiate different regions on a single dish at different time-points and to differentiate between cells irradiated earlier and later. This allows to directly compare immunofluorescence intensities in both subsets of cells with little systematic error because both subsets are cultivated and stained under identical conditions. In addition, by using irradiation patterns such as crossing lines, it is possible to irradiate individual cells twice and to differentiate between immunofluorescence signals resulting from the cellular response to the earlier and to the later irradiation event. Here, we describe the quantitative evaluation of immunofluorescence intensities after sequential irradiation.     

Recruitment of resting stages may induce blooms of Microcystis at low N:P ratios

Some species of cyanobacteria form resting stages at the sedimentsurface when environmental conditions become unfavourable. Asconditions turn more favourable, these resting stages hatchto the water phase, where the cells grow, reproduce, and sometimesform blooms. Since blooms of cyanobacteria have become an increasingthreat to inland and brackish waters, it is important to assessthe mechanisms and processes involved in the initiation of suchblooms. One such mechanism is recruitment from the sedimentsurface. Potential factors regulating the recruitment of restingstages include variations in nutrient concentrations and ratios,as well as variations in grazing. To investigate how the recruitmentof Microcystis responds to different levels of these factors,we performed an enclosure experiment (zooplankton abundanceswere regulated by predation from fish). We found that recruitmentand growth were most pronounced at the second highest nutrientconcentration (average concentrations were 498 µg l^-1of dissolved nitrogen and 134 µg l^-1 of total phosphorus),while no direct response to different grazing levels was detected.We also found that resting stages can be important for initiatingand sustaining blooms. The environmental conditions most importantin regulating the recruitment rate from resting stages correspondedto the requirements of the plankton cells, namely high nutrientaddition and low N:P ratio.     

An immunohistochemical approach to monitor the prolactin-induced activation of the JAK2/STAT5 pathway in pancreatic islets of Langerhans.

This study examined whether an immunohistochemical method examining the subcellular localization of STAT5 could be used to characterize the activation of the JAK2/STAT5 pathway by prolactin (PRL) in intact cells or tissues. In the Ins-1 beta-cell line, STAT5A and STAT5B were distributed almost equally in the cytoplasm and the nucleus in unstimulated cells. STAT5A was also detected along the border of cells and in the perinuclear region. After exposure to PRL, the redistribution from the cytoplasm to the nucleus was much higher for STAT5B compared to STAT5A. This translocation represented 12% of the STAT5A and 22% of the STAT5B originally located in the cytoplasm before stimulation. In isolated rat islets of Langerhans, PRL stimulated the nuclear translocation of both STAT5A and STAT5B only in beta-cells. The expression of the PRL receptor only by beta-cells was confirmed with a rabbit polyclonal antiserum raised against the rat PRL receptor. It was estimated that 4% of STAT5A and 9% of STAT5B originally located in the cytoplasm was translocated to the nucleus after stimulation. The presence of a functional JAK2/STAT5 signaling pathway in all islet cells was demonstrated by the nuclear translocation of STAT5B in all islet cells (i.e., alpha-, beta-, and delta-cells) after stimulation with fetal calf serum. The nuclear translocation and tyrosine phosphorylation of STAT5B was biphasic, with an initial peak within 30 min, a nadir between 1 and 3 hr, and prolonged activation after 4 hr. In contrast, the tyrosine phosphorylation of STAT5A was also biphasic but its nuclear translocation peaked within 30 min and was then reduced to a level slightly above that observed before PRL stimulation. This method is able to detect changes in STAT5 activation as small as 2% of the total cell content. These observations demonstrate the utility of this approach for studying the activation of STAT5 in a mixed population of cells within tissues or organs. In addition, the dose response for the nuclear translocation of STAT5B in normal beta-cells was similar to those for changes in proliferation and insulin secretion in isolated rat islets. Therefore, the subcellular localization can be used to monitor the activation of STAT5 and it may be a key event in the upregulation of the pancreatic islets of Langerhans during pregnancy.     

Oxygen enriched air supply in Escherichia coli processes: production of biomass and recombinant human growth hormone

In order to investigate the impact of high oxygen and carbon dioxide concentrations, Escherichia coli was grown in batch cultivations where the air supply was enriched with either oxygen or carbon dioxide. The effect of elevated concentrations of oxygen and carbon dioxide on stochiometric and kinetic constants was studied this way. The maximum growth rate was significantly reduced, the production of acetic acid and the biomass yield coefficient on glucose increased in cultures with carbon dioxide enriched air, compared to reference cultivations and cultivations with oxygen enriched air. The application of oxygen enriched air was studied in high cell density cultivations of Escherichia coli. Two production processes were chosen to investigate the impact of oxygen enrichment. Biomass concentration, specific growth rate, yield coefficient, respiration, mixed acid fermentation products and the product yield and quality for the recombinant product were investigated. First, a process for the production of biomass was investigated. Exponential growth could proceed for a longer time and higher growth rates could be maintained with oxygen enriched air supply. However, a higher specific oxygen consumption rate per glucose was measured after the start of the oxygen enrichment, indicating higher maintenance and consequently the growth rate and yield coefficient decreased drastically in the end of the process. Second, a process for the production of recombinant human growth hormone (rhGH) was investigated. Although the glucose feed rate and all medium components were doubled, the amount of produced biomass could only be increased by 77% when oxygen enriched air (40% oxygen) supply was applied. This was due to a decreased yield coefficient of biomass per glucose. The total amount of produced product was decreased by almost 50% compared to the control, although less proteolytically degraded variants were produced.     

Dynamic Nef and Nef dynamics: how structure could explain the complex activities of this small HIV protein.

The Nef protein of the human immunodeficiency virus is as important for disease progression as it is perplexing in its plethora of target molecules and functions. In this article, it is proposed that the complex biology of Nef is regulated through conformational changes of the protein that are triggered by cellular location and specific interactions as Nef traffics through the infected cell.     

New method for determination of fecal sterols in urine using non-chlorinated solvents

A new method has been developed to determine a number of sterols in urine using non-chlorinated solvents, namely methyl tert.-butyl ether and methanol (the MTB method). The method involves liquid–liquid extraction, saponification, reextraction, silylation and final identification and quantification by GC–FID. The sterols determined were coprostanol, epicoprostanol, cholesterol and dihydrocholesterol. 5α-Cholestane was used as internal standard. The limit of detection for the sterols in this experiment was 2 μg l⁻¹ urine. Recovery of coprostanol over the range 5–100 μg l⁻¹ urine by this method was between 80 and 92%.